Sample records for cellobiose

  1. Study of cellobiose fermentation by a Dekkera strain

    Energy Technology Data Exchange (ETDEWEB)

    Blondin, B.; Ratomahenina, R.; Arnaud, A.; Galzy, P.


    The Dekkera intermedia strain studied has the ability to ferment cellobiose. The ethanol concentration obtained was 75 g/L from 180 g/L cellobiose (80% of all theoretical yield). The fermentation of more concentrated solutions of cellobiose did not proceed well. (Refs. 13).

  2. Characterization of cellobiose fermentations to ethanol by yeasts

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    Freer, S.N.; Detroy, R.W.


    Twenty-two different yeasts were screened for their ability to ferment both glucose and cellobiose. The fermentation characteristics of Candida lusitaniae (NRRL Y-5394) and C. wickerhamii (NRRL Y-2563) were selected for further study because their initial rate of ethanol production from cellobiose was faster than the other test cultures. C. lusitaniae produced 44 g/L ethanol from 90 g/L cellobiose after 5-7 days. When higher carbohydrate concentrations were employed, fermentation ceased when the ethanol concentration reached 45-60 g/L. C. lusitaniae exhibited barely detectable levels of BETA-glucosidase, even though the culture actively fermented cellobiose. C. wickerhamii produced ethanol from cellobiose at a rate equivalent to C. lusitaniae; however, once the ethanol concentration reached 20 g/L, fermentation ceased. Using p-nitrophenyl-BETA-D-glucopyranoside (pNPG) as substrate, BETA-glucosidase (3-5 U/mL) was detected when C. wickerhamii was grown anaerobically on glucose or cellobiose. About 35% of the BETA-glucosidase activity was excreted into the medium. The cell-associated activity was highest against pNPG and salicin. Approximately 100-fold less activity was detected with cellobiose as substrate. When employing these organisms in a simultaneous saccharification-fermentation of avicel, using Trichoderma reesei cellulase as the saccharifying agent, 10-30% more ethanol was produced by the two yeasts capable of fermenting cellobiose than by the control, Saccharomyces cerevisiae.

  3. Mechanism of cellobiose inhibition in cellulose hydrolysis by cellobiohydrolase

    Institute of Scientific and Technical Information of China (English)

    ZHAO Yue; WU Bin; YAN Baixu; GAO Peiji


    An experimental study of cellobiose inhibition in cellulose hydrolysis by synergism of cellobiohydrolyse I and endoglucanase I is presented. Cellobiose is the structural unit of cellulose molecules and also the main product in enzymatic hydrolysis of cellulose. It has been identified that cellobiose can strongly inhibit hydrolysis reaction of cellulase, whereas it has no effect on the adsorption of cellulase on cellulose surface. The experimental data of FT-IR spectra, fluorescence spectrum and circular dichroism suggested that cellobiose can be combined with tryptophan residue located near the active site of cellobiohydrolase and then form steric hindrance, which prevents cellulose molecule chains from diffusing into active site of cellulase. In addition, the molecular conformation of cellobiohydrolase changes after cellobiose binding, which also causes most of the non-productive adsorption. Under these conditions, microfibrils cannot be separated from cellulose chains, thus further hydrolysis of cellulose can hardly proceed.

  4. A New Way to Produce Cellobiose Carbonates Using Green Chemistry. (United States)

    Khiari, R; Brochier-Salon, M-C; Mhenni, M F; Mauret, E; Belgacem, M N


    The preparation of cellulose derivatives using green (i.e., environmentally friendly) reagents would improve sustainability and reduce concerns arising from the use of non-green reagents. The objective of this work was to prepare cellobiose carbonate using a green reagent, dimethyl carbonate. The carbonation reaction was carried out in the presence of ethanolic potassium hydroxide solution and dimethyl carbonate for 6 h at a range of temperatures (25-70 °C). A cellobiose derivative was successfully prepared with a recovered yield of more than 70 % and characterized by FTIR and NMR spectroscopy techniques. The presence of a grafted disaccharide with a degree of substitution higher than 2 was determined by (13) C NMR analysis. The spectra of the prepared cellobiose carbonate exhibited peaks that were associated with cellulose molecules (C1 -C6 ) and corresponded to carbonate functions at around 159.4 ppm.

  5. Conformational analysis of cellobiose by electronic structure theories (United States)

    Adiabatic phi/psi maps for cellobiose were prepared with B3LYP density functional theory. A mixed basis set was used for minimization, followed with 6-31+G(d) single-point calculations, with and without SMD continuum solvation. Different arrangements of the exocyclic groups (3starting geometries) we...

  6. Roles of starting geometries in quantum mechanics studies of cellobiose (United States)

    A relaxed HF/6 31G(d) energy surface was constructed for the fraction of phi,psi space that contains most geometries from crystals of molecules similar to cellobiose. Two regions around other minima were examined with unconstrained B3LYP/6 31+G(d) minimizations, as were two sub regions covered by th...

  7. Novel Functions and Regulation of Cryptic Cellobiose Operons in Escherichia coli.

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    Vinuselvi Parisutham

    Full Text Available Presence of cellobiose as a sole carbon source induces mutations in the chb and asc operons of Escherichia coli and allows it to grow on cellobiose. We previously engineered these two operons with synthetic constitutive promoters and achieved efficient cellobiose metabolism through adaptive evolution. In this study, we characterized two mutations observed in the efficient cellobiose metabolizing strain: duplication of RBS of ascB gene, (β-glucosidase of asc operon and nonsense mutation in yebK, (an uncharacterized transcription factor. Mutations in yebK play a dominant role by modulating the length of lag phase, relative to the growth rate of the strain when transferred from a rich medium to minimal cellobiose medium. Mutations in ascB, on the other hand, are specific for cellobiose and help in enhancing the specific growth rate. Taken together, our results show that ascB of the asc operon is controlled by an internal putative promoter in addition to the native cryptic promoter, and the transcription factor yebK helps to remodel the host physiology for cellobiose metabolism. While previous studies characterized the stress-induced mutations that allowed growth on cellobiose, here, we characterize the adaptation-induced mutations that help in enhancing cellobiose metabolic ability. This study will shed new light on the regulatory changes and factors that are needed for the functional coupling of the host physiology to the activated cryptic cellobiose metabolism.

  8. Lactose fermentation by engineered Saccharomyces cerevisiae capable of fermenting cellobiose. (United States)

    Liu, Jing-Jing; Zhang, Guo-Chang; Oh, Eun Joong; Pathanibul, Panchalee; Turner, Timothy L; Jin, Yong-Su


    Lactose is an inevitable byproduct of the dairy industry. In addition to cheese manufacturing, the growing Greek yogurt industry generates excess acid whey, which contains lactose. Therefore, rapid and efficient conversion of lactose to fuels and chemicals would be useful for recycling the otherwise harmful acid whey. Saccharomyces cerevisiae, a popular metabolic engineering host, cannot natively utilize lactose. However, we discovered that an engineered S. cerevisiae strain (EJ2) capable of fermenting cellobiose can also ferment lactose. This finding suggests that a cellobiose transporter (CDT-1) can transport lactose and a β-glucosidase (GH1-1) can hydrolyze lactose by acting as a β-galactosidase. While the lactose fermentation by the EJ2 strain was much slower than the cellobiose fermentation, a faster lactose-fermenting strain (EJ2e8) was obtained through serial subcultures on lactose. The EJ2e8 strain fermented lactose with a consumption rate of 2.16g/Lh. The improved lactose fermentation by the EJ2e8 strain was due to the increased copy number of cdt-1 and gh1-1 genes. Looking ahead, the EJ2e8 strain could be exploited for the production of other non-ethanol fuels and chemicals from lactose through further metabolic engineering.

  9. Homolactic fermentation from glucose and cellobiose using Bacillus subtilis

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    Martinez Alfredo


    Full Text Available Abstract Backgroung Biodegradable plastics can be made from polylactate, which is a polymer made from lactic acid. This compound can be produced from renewable resources as substrates using microorganisms. Bacillus subtilis is a Gram-positive bacterium recognized as a GRAS microorganism (generally regarded as safe by the FDA. B. subtilis produces and secretes different kind of enzymes, such as proteases, cellulases, xylanases and amylases to utilize carbon sources more complex than the monosaccharides present in the environment. Thus, B. subtilis could be potentially used to hydrolyze carbohydrate polymers contained in lignocellulosic biomass to produce chemical commodities. Enzymatic hydrolysis of the cellulosic fraction of agroindustrial wastes produces cellobiose and a lower amount of glucose. Under aerobic conditions, B. subtilis grows using cellobiose as substrate. Results In this study, we proved that under non-aerated conditions, B. subtilis ferments cellobiose to produce L-lactate with 82% of the theoretical yield, and with a specific rate of L-lactate production similar to that one obtained fermenting glucose. Under fermentative conditions in a complex media supplemented with glucose, B. subtilis produces L-lactate and a low amount of 2,3-butanediol. To increase the L-lactate production of this organism, we generated the B subtilis CH1 alsS- strain that lacks the ability to synthesize 2,3-butanediol. Inactivation of this pathway, that competed for pyruvate availability, let a 15% increase in L-lactate yield from glucose compared with the parental strain. CH1 alsS- fermented 5 and 10% of glucose to completion in mineral medium supplemented with yeast extract in four and nine days, respectively. CH1 alsS- produced 105 g/L of L-lactate in this last medium supplemented with 10% of glucose. The L-lactate yield was up to 95% using mineral media, and the optical purity of L-lactate was of 99.5% since B. subtilis has only one gene (lctE that

  10. Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum

    NARCIS (Netherlands)

    Nakai, H.; Abou Hachem, M.; Petersen, B.O.; Westphal, Y.; Mannerstedt, K.; Baumann, M.J.; Dilokpimol, A.; Schols, H.A.; Duus, J.O.; Svensson, B.


    Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from alpha-D-glucose 1-phosphate as donor with gluc

  11. Characterization of cellobiose fermentations to ethanol by yeasts. [Candida lusitaniae and C. wickerhamii

    Energy Technology Data Exchange (ETDEWEB)

    Freer, S.N.; Detroy, R.W.


    Twenty-two different yeasts were screened for their ability to ferment both glucose and cellobiose. The fermentation characteristics of Candida lusitaniae (NRRL Y-5394) and C. wickerhamii (NRRL Y-2563) were selected for further study because their initial rate of ethanol production from cellobiose was faster than the other test culture. C. lusitaniae produced 44 g/L ethanol from 90 g/L cellobiose after 5-7 days. When carbohydrate concentrations were employed, fermentation ceased when the ethanol concentration reached 45-60 g/L. C. lusitaniae exhibited barely detectable levels of beta-glucosidase, even though the culture actively fermented cellobiose. C. wickerhamii produced ethanol from cellobiose at a rate equivalent to C. lusitaniae; however, once the ethanol concentration reached 20 g/L, fermentation ceased. Using p-nitrophenyl-beta-D-glucopyranoside (pNPG) as substrate, beta- glucosidase (3-5 U/mL) was detected when C. wickerhamii was grown anaerobically on glucose or cellobiose. About 35% of the beta-glucosidase activity was excreted into the medium. The cell-associated activity was highest against pNPG and salicin. Approximately 100-fold less activity was detected with cellobiose as substrate. When employing these organisms in a simultaneous saccharification-fermentation of Avicel, using Trichoderma reesei cellulase as the saccharifying agent 10-30% more ethanol was produced by the two yeasts capable of fermenting cellobiose than by the control, Saccharomyces cerevisiae. (Refs. 27).

  12. Rapidly calculated density functional theory (DFT) relaxed Iso-potential Phi Si Maps: Beta-cellobiose (United States)

    New cellobiose Phi-H/Si-H maps are rapidly generated using a mixed basis set DFT method, found to achieve a high level of confidence while reducing computer resources dramatically. Relaxed iso-potential maps are made for different conformational states of cellobiose, showing how glycosidic bond dihe...

  13. Harvesting biohydrogen from cellobiose from sulfide or nitrite-containing wastewaters using Clostridium sp. R1. (United States)

    Ho, Kuo-Ling; Lee, Duu-Jong


    Harvesting biohydrogen from inhibiting wastewaters is of practical interest since the toxicity of compounds in a wastewater stream commonly prevents the bioenergy content being recovered. The isolated Clostridium sp. R1 is utilized to degrade cellobiose in sulfide or nitrite-containing medium for biohydrogen production. The strain can effectively degrade cellobiose free of severe inhibitory effects at up to 200 mgl(-1) sulfide or to 5 mgl(-1) nitrite, yielding hydrogen at >2.0 mol H2 mol(-1) cellobiose. Principal metabolites of cellobiose fermentation are acetate and butyrate, with the concentration of the former increases with increasing sulfide and nitrite concentrations. The isolated strain can yield hydrogen from cellobiose in sulfide-laden wastewaters. However, the present of nitrite significantly limit the efficiency of the biohydrogen harvesting process.

  14. Identification and functional characterisation of cellobiose and lactose transport systems in Lactococcus lactis IL1403. (United States)

    Kowalczyk, Magdalena; Cocaign-Bousquet, Muriel; Loubiere, Pascal; Bardowski, Jacek


    Physiological, biochemical and macroarray analyses of Lactococcus lactis IL1403 and its ccpA and bglR single and double mutants engaged in lactose and beta-glucosides catabolism were performed. The kinetic analysis indicated the presence of different transport systems for salicin and cellobiose. The control of salicin catabolism was found to be mediated by the transcriptional regulator BglR and the CcpA protein. The transcriptional analysis by macroarray technology of genes from the PEP:PTS regions showed that several genes, like ybhE, celB, ptcB and ptcA, were expressed at higher levels both in wild type cells exposed to cellobiose and in the ccpA mutant. We also demonstrated that in L. lactis IL1403 cultured on medium with cellobiose and lactose as carbon sources, after the first phase of cellobiose consumption and then co-metabolism of the two sugars, when cellobiose is exhausted the strain uses lactose as the only carbon source. These data could indicate that lactose and cellobiose are transported by a unique system-a PTS carrier induced by the presence of cellobiose, and negatively controlled by the CcpA regulator.

  15. Aqueous gel formation from sodium salts of cellobiose lipids. (United States)

    Imura, Tomohiro; Yamamoto, Shuhei; Yamashita, Chikako; Taira, Toshiaki; Minamikawa, Hiroyuki; Morita, Tomotake; Kitamoto, Dai


    Cellobiose lipids (CLs) are asymmetric bolaform biosurfactants, which are produced by Cryptococcus humicola JCM 10251 and have fungicidal activity. In this study, the sodium salts of CLs (CLNa) were prepared to improve aqueous solubility of the CLs, and their surface and gelation properties in aqueous solutions were examined by surface tension, rheology, and freeze-fracture transmission electron microscopy (FF-TEM) measurements. The surface tension measurements revealed that the CLNa have high surface activity: CMC1 and γCMC1 are 0.1 mg/mL and 34.7 mN/m, respectively. It was also found that the CLNa form giant micelles above their CMC, whose average size is 116.6 ± 31.9 nm. Unlike conventional surfactants, the surface tension reduced further with an increase in concentration and the aqueous solution became viscous at the minimum gelation concentration (MGC: 5.0 mg/mL). In rheological studies, the obtained gels proved to be rather soft and their sol-gel temperature was found to be approximately 50℃. FF-TEM observation of the gels showed 3D supramolecular structures with an entangled fibrous network. Since the present CLNa aqueous gels have a degree of fungicidal activity, they could be useful for novel multifunctional soft materials applicable to the food and cosmetic industries.

  16. Biochemical genetics of the cryptic gene system for cellobiose utilization in Escherichia coli K12. (United States)

    Kricker, M; Hall, B G


    The cellobiose catabolic system of Escherichia coli K12 is being used to study the role of cryptic genes in microbial evolution. Wild-type E. coli K12 do not utilize the beta-glucoside sugars, arbutin, salicin and cellobiose. A Cel+ (cellobiose utilizing) mutant which grows on cellobiose, arbutin, and salicin was isolated previously from wild-type E. coli K12. Biochemical assays indicate that a cel structural gene (celT) specifies a single transport protein that is a beta-glucoside specific enzyme of the phosphoenolpyruvate-dependent phosphotransferase system. The transport protein phosphorylates beta-glucosides at the expense of phosphoenolpyruvate. A single phosphoglucosidase, specified by celH, hydrolyzes phosphorylated cellobiose, arbutin, and salicin. The genes of the cel system are expressed constitutively in the Cel+ mutant, whereas they are not expressed at a detectable level in the wild-type strain. The transport and hydrolase genes are simultaneously silenced or simultaneously expressed and thus constitute an operon. Cel+ strains which fail to utilize one or more beta-glucosides express the transport system at a lower level than do Cel+ strains which grow on all three beta-glucosides. Other strains inducibly express a gene which specifies transport of arbutin but not the other beta-glucosides. The arbutin transport gene, arbT, maps outside of the cel locus.

  17. Electrochemical evidence of self-substrate inhibition as functions regulation for cellobiose dehydrogenase from Phanerochaete chrysosporium. (United States)

    Stoica, Leonard; Ruzgas, Tautgirdas; Gorton, Lo


    The reaction mechanism of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium, adsorbed on graphite electrodes, was investigated by following its catalytic reaction with cellobiose registered in both direct and mediated electron transfer modes between the enzyme and the electrode. A wall-jet flow through amperometric cell housing the CDH-modified graphite electrode was connected to a single line flow injection system. In the present study, it is proven that cellobiose, at concentrations higher than 200 microM, competes for the reduced state of the FAD cofactor and it slows down the transfer of electrons to any 2e(-)/H(+) acceptors or further to the heme cofactor, via the internal electron transfer pathway. Based on and proven by electrochemical results, a kinetic model of substrate inhibition is proposed and supported by the agreement between simulation of plots and experimental data. The implications of this kinetic model, called pseudo-ping-pong mechanism, on the possible functions CDH are also discussed. The enzyme exhibits catalytic activity also for lactose, but in contrast to cellobiose, this sugar does not inhibit the enzyme. This suggests that even if some other substrates are coincidentally oxidized by CDH, however, they do not trigger all the possible natural functions of the enzyme. In this respect, cellobiose is regarded as the natural substrate of CDH.

  18. Lactic acid production from cellobiose and xylose by engineered Saccharomyces cerevisiae. (United States)

    Turner, Timothy L; Zhang, Guo-Chang; Oh, Eun Joong; Subramaniam, Vijay; Adiputra, Andrew; Subramaniam, Vimal; Skory, Christopher D; Jang, Ji Yeon; Yu, Byung Jo; Park, In; Jin, Yong-Su


    Efficient and rapid production of value-added chemicals from lignocellulosic biomass is an important step toward a sustainable society. Lactic acid, used for synthesizing the bioplastic polylactide, has been produced by microbial fermentation using primarily glucose. Lignocellulosic hydrolysates contain high concentrations of cellobiose and xylose. Here, we constructed a recombinant Saccharomyces cerevisiae strain capable of fermenting cellobiose and xylose into lactic acid. Specifically, genes (cdt-1, gh1-1, XYL1, XYL2, XYL3, and ldhA) coding for cellobiose transporter, β-glucosidase, xylose reductase, xylitol dehydrogenase, xylulokinase, and lactate dehydrogenase were integrated into the S. cerevisiae chromosomes. The resulting strain produced lactic acid from cellobiose or xylose with high yields. When fermenting a cellulosic sugar mixture containing 10 g/L glucose, 40 g/L xylose, and 80 g/L cellobiose, the engineered strain produced 83 g/L of lactic acid with a yield of 0.66 g lactic acid/g sugar (66% theoretical maximum). This study demonstrates initial steps toward the feasibility of sustainable production of lactic acid from lignocellulosic sugars by engineered yeast.

  19. Improved ethanol production by engineered Saccharomyces cerevisiae expressing a mutated cellobiose transporter during simultaneous saccharification and fermentation. (United States)

    Lee, Won-Heong; Jin, Yong-Su


    Although simultaneous saccharification and fermentation (SSF) of cellulosic biomass can offer efficient hydrolysis of cellulose through alleviating feed-back inhibition of cellulases by glucose, supplementation of β-glucosidase is necessary because most fermenting microorganisms cannot utilize cellobiose. Previously, we observed that SSF of cellulose by an engineered Saccharomyces cerevisiae expressing a cellobiose transporter (CDT-1) and an intracellular β-glucosidase (GH1-1) without β-glucosidase could not be performed as efficiently as the traditional SSF with extracellular β-glucosidase. However, we improved the ethanol production from SSF of cellulose by employing a further engineered S. cerevisiae expressing a mutant cellobiose transporter [CDT-1 (F213L) exhibiting higher VMAX than CDT-1] and GH1-1 in this study. Furthermore, limitation of cellobiose formation by reducing the amounts of cellulases mixture in SSF could lead the further engineered strain to produce ethanol considerably better than the parental strain with β-glucosidase. Probably, better production of ethanol by the further engineered strain seemed to be due to a higher affinity to cellobiose, which might be attributed to not only 2-times lower Monod constant (KS) for cellobiose than KS of the parental strain for glucose but also 5-times lower KS than Michaelis-Menten constant (KM) of the extracellular β-glucosidase for glucose. Our results suggest that modification of the cellobiose transporter in the engineered yeast to transport lower level of cellobiose enables a more efficient SSF for producing ethanol from cellulose.

  20. Cellobiose-Mediated Gene Expression in Streptococcus pneumoniae : A Repressor Function of the Novel GntR-Type Regulator BguR

    NARCIS (Netherlands)

    Shafeeq, Sulman; Kuipers, Oscar P.; Kloosterman, Tomas G.; Leite, Luciana C.C.


    The human pathogen Streptococcus pneumoniae has the ability to use the carbon-and energy source cellobiose due to the presence of a cellobiose-utilizing gene cluster (cel locus) in its genome. This system is regulated by the cellobiose-dependent transcriptional activator CelR, which has been previou

  1. Simultaneous hydrolysis and hydrogenation of cellobiose to sorbitol in molten salt hydrate media

    NARCIS (Netherlands)

    Li, J.; Soares, H.S.M.P.; Moulijn, J.A.; Makkee, M.


    The hydrolysis and hydrogenation of cellobiose (4-O-b-D-glucopyranosyl-D-glucose) in ZnCl2_4H2O solvent was studied to optimize the conditions for conversion of lignocellulose (the most abundant renewable resource) into sorbitol (D-glucitol). Water at neutral pH does not allow hydrolysis of cellobio

  2. Improved isolation of Vibro vulnificus from seawater and sediment with cellobiose-colistin agar

    DEFF Research Database (Denmark)

    Høi, L.; Dalsgaard, Inger; Dalsgaard, A.


    An improved selective medium, cellobiose-colistin (CC) agar, gave a significantly higher (P agar, In a total of 446 alkaline peptone water preenrichments amended...... with polymyxin B, V. vulnificus was isolated from 154 preenrichments (35%) with mCPC agar and from 179 preenrichments (40%) with CC agar. CC agar gave a higher plating efficiency of V. vulnificus cells than did cellobiose-polymyxin B-colistin (CPC) agar, mCPC agar, or thiosulfate-citrate-bile salts-sucrose (TCBS......) agar; the only significant difference was observed with TCBS agar, which gave much lower plating efficiencies than the other selective media. Determination of MICs demonstrated that the concentrations of colistin and polymyxin B in CPC agar inhibit growth of a proportion of V. vulnificus strains....

  3. Direct isopropanol production from cellobiose by engineered Escherichia coli using a synthetic pathway and a cell surface display system. (United States)

    Soma, Yuki; Inokuma, Kentaro; Tanaka, Tsutomu; Ogino, Chiaki; Kondo, Akihiko; Okamoto, Masahiro; Hanai, Taizo


    Efficient bio-production from lignocellulosic biomass is required for the purpose of developing an inexpensive, practical bio-refinery process. As one approach to address this problem, we genetically engineered Escherichia coli to produce isopropanol directly from cellobiose via the cellobiose degradation by Beta-Glucosidase (BGL) on the cell surface. First, we investigated the cellobiose consumption of two E. coli strains with the BGL protein from Thermobifida fusca YX (Tfu0937) fused to the anchor protein Blc (Tfu0937/Blc) using different fusion sites. Next, we introduced the synthetic pathway for isopropanol production into those strains and compared their isopropanol production in the presence of glucose. Based on the results of these assays, TA212/pTA411, which was introduced Tfu-Blc fused protein expression system and the synthetic pathway for isopropanol production, was selected for the directly isopropanol production from cellobiose. TA212/pTA411 produced 69.0±11.6mM isopropanol at 21h of fermentation, whereas TA212/pTA147, which did not introduced the BGL/anchor fused protein but was introduced the synthetic pathway for isopropanol production, showed no cellobiose consumption and no isopropanol production during fermentation. To our knowledge, this is the first report of the production of a bio-product from cellobiose using E. coli.

  4. An evaluation of cellulose saccharification and fermentation with an engineered Saccharomyces cerevisiae capable of cellobiose and xylose utilization. (United States)

    Fox, Jerome M; Levine, Seth E; Blanch, Harvey W; Clark, Douglas S


    Commercial-scale cellulosic ethanol production has been hindered by high costs associated with cellulose-to-glucose conversion and hexose and pentose co-fermentation. Simultaneous saccharification and fermentation (SSF) with a yeast strain capable of xylose and cellobiose co-utilization has been proposed as a possible avenue to reduce these costs. The recently developed DA24-16 strain of Saccharomyces cerevisiae incorporates a xylose assimilation pathway and a cellodextrin transporter (CDT) that permit rapid growth on xylose and cellobiose. In the current work, a mechanistic kinetic model of cellulase-catalyzed hydrolysis of cellulose was combined with a multi-substrate model of microbial growth to investigate the ability of DA24-16 and improved cellobiose-consuming strains to obviate the need for exogenously added β-glucosidase and to assess the impact of cellobiose utilization on SSF and separate hydrolysis and fermentation (SHF). Results indicate that improved CDT-containing strains capable of growing on cellobiose as rapidly as on glucose produced ethanol nearly as rapidly as non-CDT-containing yeast supplemented with β-glucosidase. In producing 75 g/L ethanol, SSF with any strain did not result in shorter residence times than SHF with a 12 h saccharification step. Strains with improved cellobiose utilization are therefore unlikely to allow higher titers to be reached more quickly in SSF than in SHF.

  5. Glucose, cellobiose, lactose and raffinose used as chiral stationary phases in HPLC

    Institute of Scientific and Technical Information of China (English)

    Jian Yu Wang; Feng Zhao; Mei Zhang; Ya Peng; Li Ming Yuan


    This paper presents the enantioseparation using glucose,cellobiose,lactose and raffinose as chiral selector bonded to silica gel via an arm in HPLC.Surprisingly,they also possess high enantioseparation selectivity,may be used in normal-phase and reversedphase mode.and there is a big chiral discriminating complementary.This work indicates that oligosacchafides could soon become very attractive as a new class of chiral stationary phase for HPLC.

  6. Co-fermentation of glucose, xylose and/or cellobiose by yeast (United States)

    Jeffries, Thomas W.; Willis, Laura B.; Long, Tanya M.; Su, Yi-Kai


    Provided herein are methods of using yeast cells to produce ethanol by contacting a mixture comprising xylose with a Spathaspora yeast cell under conditions suitable to allow the yeast to ferment at least a portion of the xylose to ethanol. The methods allow for efficient ethanol production from hydrolysates derived from lignocellulosic material and sugar mixtures including at least xylose and glucose or xylose, glucose and cellobiose.

  7. Pt nanocatalysts supported on reduced graphene oxide for selective conversion of cellulose or cellobiose to sorbitol. (United States)

    Wang, Ding; Niu, Wenqi; Tan, Minghui; Wu, Mingbo; Zheng, Xuejun; Li, Yanpeng; Tsubaki, Noritatsu


    Pt nanocatalysts loaded on reduced graphene oxide (Pt/RGO) were prepared by means of a convenient microwave-assisted reduction approach with ethylene glycol as reductant. The conversion of cellulose or cellobiose into sorbitol was used as an application reaction to investigate their catalytic performance. Various metal nanocatalysts loaded on RGO were compared and RGO-supported Pt exhibited the highest catalytic activity with 91.5 % of sorbitol yield from cellobiose. The catalytic performances of Pt nanocatalysts supported on different carbon materials or on silica support were also compared. The results showed that RGO was the best catalyst support, and the yield of sorbitol was as high as 91.5 % from cellobiose and 58.9 % from cellulose, respectively. The improvement of catalytic activity was attributed to the appropriate Pt particle size and hydrogen spillover effect of Pt/RGO catalyst. Interestingly, the size and dispersion of supported Pt particles could be easily regulated by convenient adjustment of the microwave heating temperature. The catalytic performance was found to initially increase and then decrease with increasing particle size. The optimum Pt particle size was 3.6 nm. These findings may offer useful guidelines for designing novel catalysts with beneficial catalytic performance for biomass conversion.

  8. Production of Glycolipid Biosurfactants, cellobiose lipids, by Cryptococcus humicola JCM 1461 and their interfacial properties. (United States)

    Morita, Tomotake; Ishibashi, Yuko; Fukuoka, Tokuma; Imura, Tomohiro; Sakai, Hideki; Abe, Masahiko; Kitamoto, Dai


    Cryptococcus humicola JCM 1461 efficiently produced cellobiose lipids (CLs), bolaform biosurfactants. The main product was identified as 16-O-(2″,3″,4″,6'-tetra-O-acetyl-β-cellobiosyl)-2-hydroxyhexadecanoic acid. The production yield of CLs reached 13.1 g/L under the intermittent feeding of glucose. The critical micelle concentrations (CMC) of the main product at pH 4.0 and 7.0 were 3.3×10(-5) M and 4.1×10(-4) M respectively.

  9. Cellulose and cellobiose. Adventures of a wandering organic chemist in theoretical chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Baluyut, John [Iowa State Univ., Ames, IA (United States)


    The energies arising from the rotation of free hydroxyl groups in the central glucose residue of a cellulose crystalline assembly, calculated using RHF, DFT, and FMO2/MP2 methods, will be presented. In addition, interactions of this central glucose residue with some of the surrounding residues (selected on the basis of the interaction strengths) are analyzed. The mechanism of acid-catalyzed hydrolysis of cellobiose, which is the repeating unit of cellulose. Energies corresponding to the different steps of this mechanism calculated using RHF and DFT are compared with those previously reported using molecular dynamics calculations and with experimental data.

  10. Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum. (United States)

    Nakai, Hiroyuki; Hachem, Maher Abou; Petersen, Bent O; Westphal, Yvonne; Mannerstedt, Karin; Baumann, Martin J; Dilokpimol, Adiphol; Schols, Henk A; Duus, Jens Ø; Svensson, Birte


    Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α-d-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of α-d-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming β-glucosyl disaccharides with β-(1→4)- regioselectivity from five monosaccharides as well as branched β-glucosyl trisaccharides with β-(1→4)-regioselectivity from three (1→6)-linked disaccharides. CtCDP showed strict β-(1→4)-regioselectivity and catalysed linear chain extension of the three β-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two β-glucosyl oligosaccharide product series to represent novel compounds, i.e. β-D-glucopyranosyl-[(1→4)-β-D-glucopyranosyl](n)-(1→2)-D-glucopyranose, and β-D-glucopyranosyl-[(1→4)-β-D-glucopyranosyl](n)-(1→3)-D-glucopyranose (n = 1-7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the C1 hydroxyl of β-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of C1 substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637-Lys648 in CtCDP blocks binding of C6-linked disaccharides as

  11. Distinct Anaerobic Bacterial Consumers of Cellobiose-Derived Carbon in Boreal Fens with Different CO2/CH4 Production Ratios. (United States)

    Juottonen, Heli; Eiler, Alexander; Biasi, Christina; Tuittila, Eeva-Stiina; Yrjälä, Kim; Fritze, Hannu


    Northern peatlands in general have high methane (CH4) emissions, but individual peatlands show considerable variation as CH4 sources. Particularly in nutrient-poor peatlands, CH4 production can be low and exceeded by carbon dioxide (CO2) production from unresolved anaerobic processes. To clarify the role anaerobic bacterial degraders play in this variation, we compared consumers of cellobiose-derived carbon in two fens differing in nutrient status and the ratio of CO2 to CH4 produced. After [(13)C]cellobiose amendment, the mesotrophic fen produced equal amounts of CH4 and CO2 The oligotrophic fen had lower CH4 production but produced 3 to 59 times more CO2 than CH4 RNA stable-isotope probing revealed that in the mesotrophic fen with higher CH4 production, cellobiose-derived carbon was mainly assimilated by various recognized fermenters of Firmicutes and by Proteobacteria The oligotrophic peat with excess CO2 production revealed a wider variety of cellobiose-C consumers, including Firmicutes and Proteobacteria, but also more unconventional degraders, such as Telmatobacter-related Acidobacteria and subphylum 3 of Verrucomicrobia Prominent and potentially fermentative Planctomycetes and Chloroflexi did not appear to process cellobiose-C. Our results show that anaerobic degradation resulting in different levels of CH4 production can involve distinct sets of bacterial degraders. By distinguishing cellobiose degraders from the total community, this study contributes to defining anaerobic bacteria that process cellulose-derived carbon in peat. Several of the identified degraders, particularly fermenters and potential Fe(III) or humic substance reducers in the oligotrophic peat, represent promising candidates for resolving the origin of excess CO2 production in peatlands.

  12. Activity Coefficients for NaBr in Aqueous D-Trehalose/D-Cellobiose Solutions at 298.15 K

    Institute of Scientific and Technical Information of China (English)

    ZHUO,Kelei; WANG,Wenhao; CHEN,Yujuan; WANG,Jianji


    The ionic mean activity coefficients of NaBr in trehalose-water and cellobiose-water mixtures have been experimentally determined at 298.15 K from electrochemical potential measurements by an electrochemical cell conraining two ion selective electrodes (ISE),together with the activity coefficients of the disaccharides.The parameters C1 representing the pair interactions between the disaccharides and NaBr have been obtained.The interactions of NaBr with trehaiose,cellobiose and glucose were compared and discussed in terms of their difference in the stereo-structures.

  13. Fast pyrolysis of 13C-labeled cellobioses: gaining insights into the mechanisms of fast pyrolysis of carbohydrates. (United States)

    Degenstein, John C; Murria, Priya; Easton, Mckay; Sheng, Huaming; Hurt, Matt; Dow, Alex R; Gao, Jinshan; Nash, John J; Agrawal, Rakesh; Delgass, W Nicholas; Ribeiro, Fabio H; Kenttämaa, Hilkka I


    A fast-pyrolysis probe/tandem mass spectrometer combination was utilized to determine the initial fast-pyrolysis products for four different selectively (13)C-labeled cellobiose molecules. Several products are shown to result entirely from fragmentation of the reducing end of cellobiose, leaving the nonreducing end intact in these products. These findings are in disagreement with mechanisms proposed previously. Quantum chemical calculations were used to identify feasible low-energy pathways for several products. These results provide insights into the mechanisms of fast pyrolysis of cellulose.

  14. Low molecular weight gelators based on biosurfactants, cellobiose lipids by Cryptococcus humicola. (United States)

    Imura, Tomohiro; Kawamura, Daisuke; Ishibashi, Yuko; Morita, Tomotake; Sato, Shun; Fukuoka, Tokuma; Kikkawa, Yoshihiro; Kitamoto, Dai


    Cellobiose lipids (CLs) are bolaform glycolipid biosurfactants, which are produced from natural resources by a yeast strain and show fungicidal activity. In this study, the gelation properties of CL in solvents were investigated by several techniques including rheology and atomic force microscopy (AFM). The yeast CL was found to gelate 6 out of 26 solvents. Although it did not provide gels in ethanol or 1, 3-butanediol which are widely used for cosmetic industries, we succeeded in producing gels by mixing ethanol or 1, 3-butanediol with water. AFM observation of the gels on a silicon substrate provided 3D supramolecular structures with an entangled fibrous network. Moreover, it was also found that some of fibrous structures were twisted helical ribbons. This should be due to the cellobiose backbone having several chiral functional groups. The sol-gel phase transition temperatures for gels in mixed ethanol/water and 1, 3-butanediol/water systems were below 100°C, indicating that the gels can be obtained with rather mild preparation conditions. The present CL gels would be useful for novel multifunctional materials applicable to various industries.

  15. Oxygen-limited cellobiose fermentation and the characterization of the cellobiase of an industrial Dekkera/Brettanomyces bruxellensis strain. (United States)

    Reis, Alexandre Libanio Silva; de Fátima Rodrigues de Souza, Raquel; Baptista Torres, Rochane Regina Neves; Leite, Fernanda Cristina Bezerra; Paiva, Patrícia Maria Guedes; Vidal, Esteban Espinosa; de Morais, Marcos Antonio


    The discovery of a novel yeast with a natural capacity to produce ethanol from lignocellulosic substrates (second-generation ethanol) is of great significance for bioethanol technology. While there are some yeast strains capable of assimilating cellobiose in aerobic laboratory conditions, the predominant sugar in the treatment of lignocellulosic material, little is known about this ability in real industrial conditions. Fermentations designed to simulate industrial conditions were conducted in synthetic medium with glucose, sucrose, cellobiose and hydrolyzed pre-treated cane bagasse as a different carbon source, with the aim of further characterizing the fermentation capacity of a promising Dekkera bruxellensis yeast strain, isolated from the bioethanol process in Brazil. As a result, it was found (for the first time in oxygen-limiting conditions) that the strain Dekkera bruxellensis GDB 248 could produce ethanol from cellobiose. Moreover, it was corroborated that the cellobiase activity characterizes the enzyme candidate in semi-purified extracts (β-glucosidase). In addition, it was demonstrated that GDB 248 strain had the capacity to produce a higher acetic acid concentration than ethanol and glycerol, which confirms the absence of the Custer effect with this strain in oxygen-limiting conditions. Moreover, it is also being suggested that D. bruxellensis could benefit Saccharomyces cerevisiae and outcompete it in the industrial environment. In this way, it was confirmed that D. bruxellensis GDB 248 has the potential to produce ethanol from cellobiose, and is a promising strain for the fermentation of lignocellulosic substrates.

  16. ClaR--a novel key regulator of cellobiose and lactose metabolism in Lactococcus lactis IL1403. (United States)

    Aleksandrzak-Piekarczyk, Tamara; Stasiak-Różańska, Lidia; Cieśla, Jarosław; Bardowski, Jacek


    In a number of previous studies, our group has discovered an alternative pathway for lactose utilization in Lactococcus lactis that, in addition to a sugar-hydrolyzing enzyme with both P-β-glucosidase and P-β-galactosidase activity (BglS), engages chromosomally encoded components of cellobiose-specific PTS (PTS(Cel-Lac)), including PtcA, PtcB, and CelB. In this report, we show that this system undergoes regulation via ClaR, a novel activator protein from the RpiR family of transcriptional regulators. Although RpiR proteins are widely distributed among lactic acid bacteria, their roles have yet to be confirmed by functional assays. Here, we show that ClaR activity depends on intracellular cellobiose-6-phosphate availability, while other sugars such as glucose or galactose have no influence on it. We also show that ClaR is crucial for activation of the bglS and celB expression in the presence of cellobiose, with some limited effects on ptcA and ptcB activation. Among 190 of carbon sources tested, the deletion of claR reduces L. lactis growth only in lactose- and/or cellobiose-containing media, suggesting a narrow specificity of this regulator within the context of sugar metabolism.

  17. Cloning of cellobiose phosphoenolpyruvate-dependent phosphotransferase genes: Functional expression in recombinant Escherichia coli and identification of a putative binding region for disaccharides

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Xiaokuang; Davis, F.C.; Ingram, L.O. [Univ. of Florida, Gainesville, FL (United States); Hespell, R.B. [USDA Agricultural Research Service, Peoria, IL (United States)


    Genomic libraries from nine cellobiose-metabolizing bacteria were screened for cellobiose utilization. Positive clones were recovered from six libraries, all of which encode phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) proteins. Clones from Bacillus subtilis, Butyrivibrio fibrisolvens, and Klebsiella oxytoca allowed the growth of recombinant Escherichia coli in cellobiose-M9 minimal medium. The K. oxytoca clone, pLOI1906, exhibited an unusually broad substrate range (cellobiose, arbutin, salicin, and methylumbelliferyl derivatives of glucose, cellobiose, mannose, and xylose) and was sequenced. The insert in this plasmid encoded the carboxy-terminal region of a putative regulatory protein, cellobiose permease (single polypeptide), and phospho-{beta}-glucosidase, which appear to form an operon (casRAB). Subclones allowed both casA and casB to be expressed independently, as evidenced by in vitro complementation. An analysis of the translated sequences from the EIIC domains of cellobiose, aryl-{beta}-glucoside, and other disaccharide permeases allowed the identification of a 50-amino-acid conserved region. A disaccharide consensus sequence is proposed for the most conserved segment (13 amino acids), which may represent part of the EIIC active site for binding and phosphorylation. 63 refs., 4 figs., 4 tabs.

  18. Synergistic effects of cellobiose dehydrogenase and manganese-dependent peroxidases during lignin degradation

    Institute of Scientific and Technical Information of China (English)


    The synergistic effects of cellobiose dehydrogenase (CDH) and manganese-dependent peroxidases (MnP) on the degradation of kraft pulp cellulolytic enzyme lignin (CEL) were investigated. Addition of CDH significantly increased the amount of water-soluble products reduced from CEL by MnP. CDH facilitated the reduction of the contents of methoxyl, carboxyl, phenolic hydroxyl and total hydroxyl groups of CEL by MnP. 1H-NMR analysis showed that addition of CDH also decreased further the amount of protons of CEL degraded by MnP. The results proved for the first time that CDH could promote degradation of lignin by MnP and suggest that CDH could not only promote degradation of cellulose but also is an important part of the lignin biodegradation system.

  19. Third-generation biosensor for lactose based on newly discovered cellobiose dehydrogenase. (United States)

    Stoica, Leonard; Ludwig, Roland; Haltrich, Dietmar; Gorton, Lo


    The present paper describes the principle and characteristics of a biosensor for lactose based on a third-generation design involving cellobiose dehydrogenase. As resulted from a previous comparative study (submitted manuscript), the novelty of this lactose biosensor is based on highly efficient direct electron transfer between two newly discovered cellobiose dehydrogenases (CDH), from the white rot fungi Trametes villosa and Phanerochaete sordida, and a solid spectrographic graphite electrode. CDH was immobilized on the electrode surface (0.073 cm2) by simple physical adsorption, and the CDH-modified electrode was next inserted into a wall-jet amperometric cell connected on-line to a flow injection setup (0.5 mL x min(-1)). The P. sordida CDH-based lactose biosensor, proved to be the better one, has a detection limit for lactose of 1 microM, a sensitivity of 1100 microA x mM(-1) x cm(-2), a response time of 4 s (the time required to obtain the maximum peak current), and a linear range from 1 to 100 microM lactose (correlation coefficient 0.998). The simplicity of construction and analytical characteristics make this CDH-based lactose biosensor an excellent alternative to previous lactose biosensors reported in the literature or commercially available. The CDH-lactose sensor was used to quantify the content of lactose in pasteurized milk, buttermilk, and low-lactose milk, using the standard addition method. No effects of the samples matrixes were observed. The operational stability of the sensor was tested for 11 h by continuous injection of 100 microM lactose (290 injections). The final signal of the sensor was maintained at 98% of its initial signal, with a low standard deviation of 1.72 (RSD 2.41%).

  20. The Differential Proteome of the Probiotic Lactobacillus acidophilus NCFM Grown on the Potential Prebiotic Cellobiose Shows Upregulation of Two beta-Glycoside Hydrolases

    DEFF Research Database (Denmark)

    van Zanten, Gabriella Christina; Sparding, Nadja; Majumder, Avishek;


    Probiotics, prebiotics, and combinations there of, that is, synbiotics, are known to exert beneficial health effects in humans; however interactions between pro-and prebiotics remain poorly understood at the molecular level. The present study describes changes in abundance of different proteins...... of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) when grown on the potential prebiotic cellobiose as compared to glucose. Cytosolic cell extract proteomes after harvest at late exponential phase of NCFM grown on cellobiose or glucose were analyzed by two dimensional difference gel electrophoresis....... Many of these proteins were associated with energy metabolism, including the cellobiose related glycoside hydrolases phospho-β-glucosidase (LBA0881) and phospho-β-galactosidase II (LBA0726). The data provide insight into the utilization of the candidate prebiotic cellobiose by the probiotic bacterium...

  1. Vapor pressures and heats of sublimation of crystalline β-cellobiose from classical molecular dynamics simulations with quantum mechanical corrections. (United States)

    Wohlert, Jakob


    In this paper, we report the calculation of the enthalpy of sublimation, Δ(sub)H, as a function of temperature of crystalline β-cellobiose from molecular dynamics (MD) simulations, using two popular carbohydrate force fields. Together with the entropy difference between the solid and the vapor, ΔS, evaluated at atmospheric pressure, Δ(sub)H gives the vapor pressure of cellobiose over the solid phase as a function of T. It is found that when quantum mechanical corrections to the enthalpy calculated from the distribution of normal modes is applied both force fields give Δ(sub)H close to experiments. The entropy change, ΔS, which is calculated within a harmonic approximation becomes too small, leading to vapor pressures that are too low. These findings are relevant to MD simulations of crystalline carbohydrates in general, e.g., native cellulose.

  2. Assessment of the joint effect of laccase and cellobiose dehydrogenase on the decolouration of different synthetic dyes. (United States)

    Enayatzamir, Kheirghadam; Tabandeh, Fatemeh; Yakhchali, Bagher; Alikhani, Hossein A; Rodríguez Couto, Susana


    In this paper the efficiency of the combined action of laccase and cellobiose dehydrogenase (CDH) to decolourise different synthetic dyes such as Remazol Brilliant Blue R (RBBR), Methyl Green (MG), Direct Violet (DV), Ponceau Xylidine (PX), Bismark Brown (BB) and Poly R-478 (PR) was assessed. It was found that the use of CDH could be a promising alternative to the utilisation of the expensive and poisonous chemical mediators such as HOBT although much research on this topic remains still to be done.

  3. [Isolation and characterization of a cellobiose dehydrogenase formed by a asporogenic mycelial fungus INBI 2-26(-)]. (United States)

    Karapetian, K N; Iachkova, S N; Vasil'chenko, L G; Borzykh, M N; Rabinovich, M L


    A nonsporulating fungus isolated from dioxine-containing tropical soils forms cellobiose dehydrogenase, when grown in media supplemented by a source of cellulose. The enzyme purified to homogeneity by SDS-PAGE (yield, 43%) had an M(r) of 95 kDa; its pH optimum was in the range 5.5-7.0; more than 50% activity was retained at pH 4.0-8.0 (citrate-phosphate buffer). The absorption spectrum of the enzyme in the visible range had the characteristic appearance of flavocytochrome proteins. Cellobiose dehydrogenase oxidized cellobiose and lactose (the respective K(M) values at pH 6.0 equaled 4.5 +/- 1.5 and 56 microM) in the presence of dichlorophenolindophenol (K(M) app = 15 +/- 3 microM at pH 6.0) taken as an electron acceptor. Other sugars were barely if at all oxidized by the enzyme. Neither ethyl-beta-D-cellobioside, heptobiose, nor chitotriose inhibited the enzymatic oxidation of lactose, even under the conditions of 100-fold molar excess. The enzyme was weakly inhibited by sodium azide dichlorophenolindophenol reduction and exhibited affinity to amorphous cellulose. At 55 degrees C and pH 6.0 (optimum stability), time to half-maximum inactivation equaled 99 min. The enzyme reduced by cellobiose was more stable than the nonreduced form. Conversely, the presence of an oxidizer (dichlorophenolindophenol) decreased the stability eight times at pH 6.0. In addition, the enzyme acted as a potent reducer of the single-electron acceptor cytochrome c3+ (K(M) app = 15 microM at pH 6.0).

  4. Novel cellobiose 2-epimerases for the production of epilactose from milk ultrafiltrate containing lactose. (United States)

    Krewinkel, Manuel; Kaiser, Jana; Merz, Michael; Rentschler, Eva; Kuschel, Beatrice; Hinrichs, Jörg; Fischer, Lutz


    A selected number of enzymes have recently been assigned to the emerging class of cellobiose 2-epimerases (CE). All CE convert lactose to the rare sugar epilactose, which is regarded as a new prebiotic. Within this study, the gene products of 2 potential CE genes originating from the mesophilic bacteria Cellulosilyticum lentocellum and Dysgonomonas gadei were recombinantly produced in Escherichia coli and purified by chromatography. The enzymes have been identified as novel CE by sequence analysis and biochemical characterizations. The biochemical characterizations included the determination of the molecular weight, the substrate spectrum, and the kinetic parameters, as well as the pH and temperature profiles in buffer and food matrices. Both identified CE epimerize cellobiose and lactose into the C2 epimerization products glucosylmannose and epilactose, respectively. The epimerization activity for lactose was maximal at pH 8.0 or 7.5 and 40°C in defined buffer systems for the CE from C. lentocellum and the CE from D. gadei, respectively. In addition, biotransformations of the foodstuff milk ultrafiltrate containing lactose were demonstrated. The CE from D. gadei was produced in a stirred-tank reactor (12 L) and purified using an automatic system. Enzyme production and purification in this scale indicates that a future upscaling of CE production is possible. The bioconversions of lactose in milk ultrafiltrate were carried out either in a batch process or in a continuously operated enzyme membrane reactor (EMR) process. Both processes ran at an industrially relevant low temperature of 8°C to reduce undesirable microbial growth. The enzyme was reasonably active at the low process temperature because the CE originated from a mesophilic organism. An epilactose yield of 29.9% was achieved in the batch process within 28 h of operation time. In the continuous EMR process, the epilactose yield in the product stream was lower, at 18.5%. However, the enzyme productivity

  5. Fungal laccase, cellobiose dehydrogenase, and chemical mediators: combined actions for the decolorization of different classes of textile dyes. (United States)

    Ciullini, Ilaria; Tilli, Silvia; Scozzafava, Andrea; Briganti, Fabrizio


    Dyes belonging to the mono-, di-, tri- and poly-azo as well as anthraquinonic and mono-azo Cr-complexed classes, chosen among the most utilized in textile applications, were employed for a comparative enzymatic decolorization study using the extracellular crude culture extracts from the white rot fungus Funalia (Trametes) trogii grown on different culture media and activators able to trigger different levels of expression of oxidizing enzymes: laccase and cellobiose dehydrogenase. Laccase containing extracts were capable to decolorize some dyes from all the different classes analyzed, whereas the recalcitrant dyes were subjected to the combined action of laccase and the chemical mediator HBT, or laccase plus cellobiose dehydrogenase. Correlations among the decolorization degree of the various dyes and their electronic and structural diversities were rationalized and discussed. The utilization of cellobiose dehydrogenase in support to the activity of laccase for the decolorization of azo textile dyes resulted in substantial increases in decolorization for all the refractory dyes proving to be a valid alternative to more expensive and less environmentally friendly chemical treatments of textile dyes wastes.

  6. Efficient production of lactulose from whey powder by cellobiose 2-epimerase in an enzymatic membrane reactor. (United States)

    Wu, Lingtian; Xu, Cen; Li, Sha; Liang, Jinfeng; Xu, Hong; Xu, Zheng


    In this study, the gene encoding cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) was successfully expressed in Bacillus subtilis WB800. After the fermentation medium optimization, the activity of recombinant strain was 4.5-fold higher than the original medium in a 7.5L fermentor. The optimal catalytic pH and temperature of crude CsCE were 7.0 and 80°C, respectively. An enzymatic synthesis of lactulose was developed using cheese-whey lactose as its substrate. The maximum conversion rate of whey powder obtained was 58.5% using 7.5 U/mL CsCE. The enzymatic membrane reactor system exhibited a great operational stability, confirmed with the higher lactose conversion (42.4%) after 10 batches. To our best knowledge, this is the first report of lactulose synthesis in food grade strain, which improve the food safety, and we not only realize the biological production of lactulose, but also make good use of industrial waste, which have positive impact on environment.

  7. Quantifying the release of lactose from polymer matrix tablets with an amperometric biosensor utilizing cellobiose dehydrogenase. (United States)

    Knöös, Patrik; Schulz, Christopher; Piculell, Lennart; Ludwig, Roland; Gorton, Lo; Wahlgren, Marie


    The release of lactose (hydrophilic) from polymer tablets made with hydrophobically modified poly(acrylic acid) (HMPAA) have been studied and compared to the release of ibuprofen, a hydrophobic active substance. Lactose is one of the most used excipients for tablets, but lactose release has not been widely studied. One reason could be a lack of good analytical tools. A novel biosensor with cellobiose dehydrogenase (CDH) was used to detect the lactose release, which has a polydiallyldimethylammonium chloride (PDADMAC) layer that increases the response. A sample treatment using polyethylenimine (PEI) was developed to eliminate possible denaturants. The developed methodology provided a good approach to detect and quantify the released lactose. The release was studied with or without the presence of a model amphiphilic substance, sodium dodecyl sulphate (SDS), in the release medium. Ibuprofen showed very different release rates in the different media, which was attributed to hydrophobic interactions between the drug, the HMPAA and the SDS in the release medium. The release of hydrophilic lactose, which did not associate to any of the other components, was rapid and showed only minor differences. The new methodology provides a useful tool to further evaluate tablet formulations by a relatively simple set of experiments.

  8. Heterologous expression of Pycnoporus cinnabarinus cellobiose dehydrogenase in Pichia pastoris and involvement in saccharification processes

    Directory of Open Access Journals (Sweden)

    Bey Mathieu


    Full Text Available Abstract Background Cellobiose dehydrogenase (CDH is an extracellular hemoflavoenzyme produced by lignocellulose-degrading fungi including Pycnoporus cinnabarinus. We investigated the cellulolytic system of P. cinnabarinus, focusing on the involvement of CDH in the deconstruction of lignocellulosic biomass. Results First, P. cinnabarinus growth conditions were optimized for CDH production. Following growth under cellulolytic conditions, the main components secreted were cellulases, xylanases and CDH. To investigate the contribution of P. cinnabarinus secretome in saccharification processes, the Trichoderma reesei enzymatic cocktail was supplemented with the P. cinnabarinus secretome. A significant enhancement of the degradation of wheat straw was observed with (i the production of a large amount of gluconic acid, (ii increased hemicellulose degradation, and (iii increased overall degradation of the lignocellulosic material. P. cinnabarinus CDH was heterologously expressed in Pichia pastoris to obtain large amounts of pure enzyme. In a bioreactor, the recombinant CDH (rCDH expression level reached 7800 U/L. rCDH exhibited values of biochemical parameters similar to those of the natural enzyme, and was able to bind cellulose despite the absence of a carbohydrate-binding module (CBM. Following supplementation of purified rCDH to T. reesei enzymatic cocktail, formation of gluconic acid and increased hemicellulose degradation were observed, thus confirming the previous results observed with P. cinnabarinus secretome. Conclusions We demonstrate that CDH offers an attractive tool for saccharification process enhancement due to gluconic acid production from raw lignocellulosic material.

  9. Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps (United States)

    Brady, Sonia K.; Sreelatha, Sarangapani; Feng, Yinnian; Chundawat, Shishir P. S.; Lang, Matthew J.


    Cellobiohydrolase 1 from Trichoderma reesei (TrCel7A) processively hydrolyses cellulose into cellobiose. Although enzymatic techniques have been established as promising tools in biofuel production, a clear understanding of the motor's mechanistic action has yet to be revealed. Here, we develop an optical tweezers-based single-molecule (SM) motility assay for precision tracking of TrCel7A. Direct observation of motility during degradation reveals processive runs and distinct steps on the scale of 1 nm. Our studies suggest TrCel7A is not mechanically limited, can work against 20 pN loads and speeds up when assisted. Temperature-dependent kinetic studies establish the energy requirements for the fundamental stepping cycle, which likely includes energy from glycosidic bonds and other sources. Through SM measurements of isolated TrCel7A domains, we determine that the catalytic domain alone is sufficient for processive motion, providing insight into TrCel7A's molecular motility mechanism.

  10. The Differential Proteome of the Probiotic Lactobacillus acidophilus NCFM Grown on the Potential Prebiotic Cellobiose Shows Upregulation of Two β-Glycoside Hydrolases

    Directory of Open Access Journals (Sweden)

    Gabriella C. van Zanten


    Full Text Available Probiotics, prebiotics, and combinations thereof, that is, synbiotics, are known to exert beneficial health effects in humans; however interactions between pro- and prebiotics remain poorly understood at the molecular level. The present study describes changes in abundance of different proteins of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM when grown on the potential prebiotic cellobiose as compared to glucose. Cytosolic cell extract proteomes after harvest at late exponential phase of NCFM grown on cellobiose or glucose were analyzed by two dimensional difference gel electrophoresis (2D-DIGE in the acidic (pH 4–7 and the alkaline (pH 6–11 regions showing a total of 136 spots to change in abundance. Proteins were identified by MS or MS/MS from 81 of these spots representing 49 unique proteins and either increasing 1.5–13.9-fold or decreasing 1.5–7.8-fold in relative abundance. Many of these proteins were associated with energy metabolism, including the cellobiose related glycoside hydrolases phospho-β-glucosidase (LBA0881 and phospho-β-galactosidase II (LBA0726. The data provide insight into the utilization of the candidate prebiotic cellobiose by the probiotic bacterium NCFM. Several of the upregulated or downregulated identified proteins associated with utilization of cellobiose indicate the presence of carbon catabolite repression and regulation of enzymes involved in carbohydrate metabolism.

  11. Comparison of transcriptional profiles of Clostridium thermocellum grown on cellobiose and pretreated yellow poplar using RNA-Seq

    Directory of Open Access Journals (Sweden)

    Hui eWei


    Full Text Available The anaerobic, thermophilic bacterium, Clostridium thermocellum, secretes multi-protein enzyme complexes, termed cellulosomes, which synergistically interact with the microbial cell surface and efficiently disassemble plant cell wall biomass. C. thermocellum has also been considered a potential consolidated bioprocessing (CBP organism due to its ability to produce the biofuel products, hydrogen and ethanol. We found that C. thermocellum fermentation of pretreated yellow poplar (PYP produced 30% and 39% of ethanol and hydrogen product concentrations, respectively, compared to fermentation of cellobiose. RNA-seq was used to analyze the transcriptional profiles of these cells. The PYP-grown cells taken for analysis at the late stationary phase showed 1211 genes up-regulated and 314 down-regulated by more than 2-fold compared to the cellobiose-grown cells. These affected genes cover a broad spectrum of specific functional categories. The transcriptional analysis was further validated by sub-proteomics data taken from the literature; as well as by quantitative reverse transcription-PCR (qRT-PCR analyses of selected genes. Specifically, 47 cellulosomal protein-encoding genes, genes for 4 pairs of SigI-RsgI for polysaccharide sensing, 7 cellodextrin ABC transporter genes, and a set of NAD(PH hydogenase and alcohol dehydrogenase genes were up-regulated for cells growing on PYP compared to cellobiose. These genes could be potential candidates for future studies aimed at gaining insight into the regulatory mechanism of this organism as well as for improvement of C. thermocellum in its role as a CBP organism.

  12. Conversion of cellulose and cellobiose into sorbitol catalyzed by ruthenium supported on a polyoxometalate/metal-organic framework hybrid. (United States)

    Chen, Jinzhu; Wang, Shengpei; Huang, Jing; Chen, Limin; Ma, Longlong; Huang, Xing


    Cellulose and cellobiose were selectively converted into sorbitol over water-tolerant phosphotungstic acid (PTA)/metal- organic-framework-hybrid-supported ruthenium catalysts, Ru-PTA/MIL-100(Cr), under aqueous hydrogenation conditions. The goal was to investigate the relationship between the acid/metal balance of bifunctional catalysts Ru-PTA/MIL-100(Cr) and their performance in the catalytic conversion of cellulose and cellobiose into sugar alcohols. The control of the amount and strength of acid sites in the supported PTA/MIL-100(Cr) was achieved through the effective control of encapsulated-PTA loading in MIL-100(Cr). This design and preparation method led to an appropriately balanced Ru-PTA/MIL-100(Cr) in terms of Ru dispersion and hydrogenation capacity on the one hand, and acid site density of PTA/MIL-100(Cr) (responsible for acid-catalyzed hydrolysis) on the other hand. The ratio of acid site density to the number of Ru surface atoms (nA /nRu ) of Ru-PTA/MIL-100(Cr) was used to monitor the balance between hydrogenation and hydrolysis functions; the optimum balance between the two catalytic functions, that is, 8.84sorbitol of 57.9% at complete conversion of cellulose, and 97.1% yield in hexitols with a selectivity for sorbitol of 95.1% at complete conversion of cellobiose) were obtained using a Ru-PTA/MIL-100(Cr) catalyst with loadings of 3.2 wt % for Ru and 16.7 wt % for PTA. This research thus opens new perspectives for the rational design of acid/metal bifunctional catalysts for biomass conversion.

  13. Evaluation of cellobiose dehydrogenase and laccase containing culture fluids of Termitomyces sp. OE147 for degradation of Reactive blue 21

    Directory of Open Access Journals (Sweden)

    Rishabh Gangwar


    Full Text Available This study evaluates culture filtrate, rich in cellobiose dehydrogenase and laccases, of Termitomyces sp. OE 147, in decolouration and degradation of Reactive blue (RB 21. About 35% decolouration was achieved at low volumes of the culture supernatant without addition of external redox mediators. An optimized dye to culture fluid ratio (75 ppm: 0.1 ml at a pH of 4–5 resulted in removal of colour by 60%. The degradation products of RB21 were analysed by Electron Spray Ionization-Mass Spectrometry and several small molecules (of m/z 106–199 were detected. These were concluded to be o-Xylene, 2,3-Dihydro-1H-isoindole, Isoindole-1,3-dione, 2,Benzenesulfonyl-ethanol, (4-Hydroxy-phenyl-sulfamic acid, 2,3-Dihydro-1H-isoindole-5-sulfonic acid and proposed to result from joint action of cellobiose dehydrogenase, laccase, peroxidases and unidentified oxidoreductases present in the culture fluids. Based on the products formed and the known reactions of these enzymes, a degradation pathway was proposed for RB21. The culture fluid was also effective in decolouration (by about 50% and detoxification (by ∼25% of the combined effluent collected from a local mill indicating a treatment process that bypasses use of H2O2 and toxic mediators.

  14. Synthesis of bis-cellobiose and bis-glucose derivatives of azacrown macrocycles as hosts in complexes with acetylsalicylic acid and 4-acetamidophenol. (United States)

    Pintal, Michalina; Kryczka, Bogusław; Marsura, Alain; Porwański, Stanisław


    Two new C2 symmetric bis-cellobiose and bis-glucose azacrown derivatives were prepared according to the one-step procedure using azacrown ethers and azidosaccharides. Their complexes with aspirin and paracetamol were studied with the use of proton NMR spectroscopy. It was found that these pseudocryptands bind aspirin and paracetamol but each one in a different manner.

  15. A Sensitive Method Using 4-Methylumbelliferyl-(beta)-Cellobiose as a Substrate To Measure (1,4)-(beta)-Glucanase Activity in Sediments

    NARCIS (Netherlands)

    Boschker, H.T.S.; Cappenberg, T.E.


    A sensitive method to measure (1,4)-beta-glucanase activity in organic matter-rich sediments, using 4-methylumbelliferyl-beta- cellobiose as a substrate, is described. beta-Glucosidases, which were also able to hydrolyze this substrate, were inhibited with D-glucono-delta-lactone. The produced 4- me

  16. Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes

    DEFF Research Database (Denmark)

    Andric, Pavle; Meyer, Anne S.; Jensen, Peter Arendt;


    Achievement of efficient enzymatic degradation of cellulose to glucose is one of the main prerequisites and one of the main challenges in the biological conversion of lignocellulosic biomass to liquid fuels and other valuable products. The specific inhibitory interferences by cellobiose and gluco...

  17. Simultaneous utilization of cellobiose, xylose, and acetic acid from lignocellulosic biomass for biofuel production by an engineered yeast platform. (United States)

    Wei, Na; Oh, Eun Joong; Million, Gyver; Cate, Jamie H D; Jin, Yong-Su


    The inability of fermenting microorganisms to use mixed carbon components derived from lignocellulosic biomass is a major technical barrier that hinders the development of economically viable cellulosic biofuel production. In this study, we integrated the fermentation pathways of both hexose and pentose sugars and an acetic acid reduction pathway into one Saccharomyces cerevisiae strain for the first time using synthetic biology and metabolic engineering approaches. The engineered strain coutilized cellobiose, xylose, and acetic acid to produce ethanol with a substantially higher yield and productivity than the control strains, and the results showed the unique synergistic effects of pathway coexpression. The mixed substrate coutilization strategy is important for making complete and efficient use of cellulosic carbon and will contribute to the development of consolidated bioprocessing for cellulosic biofuel. The study also presents an innovative metabolic engineering approach whereby multiple substrate consumption pathways can be integrated in a synergistic way for enhanced bioconversion.

  18. Polyethyleneimine as a promoter layer for the immobilization of cellobiose dehydrogenase from Myriococcum thermophilum on graphite electrodes. (United States)

    Schulz, Christopher; Ludwig, Roland; Gorton, Lo


    Cellobiose dehydrogenase (CDH) is a promising enzyme for the construction of biofuel cell anodes and biosensors capable of oxidizing aldoses as cellobiose as well as lactose and glucose and with the ability to connect to an electrode through a direct electron transfer mechanism. In the present study, we point out the beneficial effect of a premodification of spectrographic graphite electrodes with the polycation polyethyleneimine (PEI) prior to adsorption of CDH from Myriococcum thermophilum (MtCDH). The application of PEI shifts the pH optimum of the response of the MtCDH modified electrode from pH 5.5 to 8. The catalytic currents to lactose were increased up to 140 times, and the K(M)(app) values were increased up to 9 times. The previously investigated, beneficial effect of divalent cations on the activity of CDH was also present for graphite/PEI/MtCDH electrodes but was less pronounced. Polarization curves revealed a second unexpected catalytic wave for graphite/PEI/MtCDH electrodes especially pronounced at pH 8. Square wave voltammetric studies revealed the presence of an unknown redox functionality present at 192 mV vs Ag|AgCl (0.1 M KCl) at pH 8, probably originating from an oxidized adenosine derivative. Adenosine is a structural part of the flavin adenine dinucleotide (FAD) cofactor of the dehydrogenase domain of CDH. It is suggested that for some enzyme molecules FAD leaks out from the active site, adsorbs onto graphite, and is oxidized on the electrode surface into a product able to mediate the electron transfer between CDH and the electrode. PEI is suggested and discussed to act in several manners by (a) increasing the surface loading of the enzyme, (b) possibly increasing the electron transfer rate between CDH and the electrode, and (c) facilitating the creation or immobilization of redox active adenosine derivatives able to additionally mediate the electron transfer between CDH and the electrode.

  19. The Differential Proteome of the Probiotic Lactobacillus acidophilus NCFM Grown on the Potential Prebiotic Cellobiose Shows Upregulation of Two beta-Glycoside Hydrolases

    DEFF Research Database (Denmark)

    van Zanten, Gabriella Christina; Sparding, Nadja; Majumder, Avishek


    Probiotics, prebiotics, and combinations there of, that is, synbiotics, are known to exert beneficial health effects in humans; however interactions between pro-and prebiotics remain poorly understood at the molecular level. The present study describes changes in abundance of different proteins...... (2D-DIGE) in the acidic (pH 4-7) and the alkaline (pH 6-11) regions showing a total of 136 spots to change in abundance. Proteins were identified by MS or MS/MS from 81 of these spots representing 49 unique proteins and either increasing 1.5-13.9-fold or decreasing 1.5-7.8-fold in relative abundance....... Many of these proteins were associated with energy metabolism, including the cellobiose related glycoside hydrolases phospho-β-glucosidase (LBA0881) and phospho-β-galactosidase II (LBA0726). The data provide insight into the utilization of the candidate prebiotic cellobiose by the probiotic bacterium...

  20. A novel bio-electronic tongue using cellobiose dehydrogenase from different origins to resolve mixtures of various sugars and interfering analytes



    A novel application of cellobiose dehydrogenase (CDH) as sensing element for a Bioelectronic Tongue (BioET) system has been tested. In this work CDHs from various fungi, which exhibit different substrate specificities, were used to discriminate between lactose and glucose in presence of the interfering matrix compound Ca²⁺ in various mixtures. This work exploits the advantage of an electronic tongue system with practically zero pre-treatment of samples and operation at low voltages in a direc...

  1. Characterization of Electrical Current-Generation Capabilities from Thermophilic Bacterium Thermoanaerobacter pseudethanolicus Using Xylose, Glucose, Cellobiose, or Acetate with Fixed Anode Potentials. (United States)

    Lusk, Bradley G; Khan, Qaiser Farid; Parameswaran, Prathap; Hameed, Abdul; Ali, Naeem; Rittmann, Bruce E; Torres, Cesar I


    Thermoanaerobacter pseudethanolicus 39E (ATCC 33223), a thermophilic, Fe(III)-reducing, and fermentative bacterium, was evaluated for its ability to produce current from four electron donors-xylose, glucose, cellobiose, and acetate-with a fixed anode potential (+ 0.042 V vs SHE) in a microbial electrochemical cell (MXC). Under thermophilic conditions (60 °C), T. pseudethanolicus produced high current densities from xylose (5.8 ± 2.4 A m(-2)), glucose (4.3 ± 1.9 A m(-2)), and cellobiose (5.2 ± 1.6 A m(-2)). It produced insignificant current when grown with acetate, but consumed the acetate produced from sugar fermentation to produce electrical current. Low-scan cyclic voltammetry (LSCV) revealed a sigmoidal response with a midpoint potential of -0.17 V vs SHE. Coulombic efficiency (CE) varied by electron donor, with xylose at 34.8% ± 0.7%, glucose at 65.3% ± 1.0%, and cellobiose at 27.7% ± 1.5%. Anode respiration was sustained over a pH range of 5.4-8.3, with higher current densities observed at higher pH values. Scanning electron microscopy showed a well-developed biofilm of T. pseudethanolicus on the anode, and confocal laser scanning microscopy demonstrated a maximum biofilm thickness (Lf) greater than ~150 μm for the glucose-fed biofilm.

  2. Expression of a codon-optimized β-glucosidase from Cellulomonas flavigena PR-22 in Saccharomyces cerevisiae for bioethanol production from cellobiose. (United States)

    Ríos-Fránquez, Francisco Javier; González-Bautista, Enrique; Ponce-Noyola, Teresa; Ramos-Valdivia, Ana Carmela; Poggi-Varaldo, Héctor Mario; García-Mena, Jaime; Martinez, Alfredo


    Bioethanol is one of the main biofuels produced from the fermentation of saccharified agricultural waste; however, this technology needs to be optimized for profitability. Because the commonly used ethanologenic yeast strains are unable to assimilate cellobiose, several efforts have been made to express cellulose hydrolytic enzymes in these yeasts to produce ethanol from lignocellulose. The C. flavigenabglA gene encoding β-glucosidase catalytic subunit was optimized for preferential codon usage in S. cerevisiae. The optimized gene, cloned into the episomal vector pRGP-1, was expressed, which led to the secretion of an active β-glucosidase in transformants of the S. cerevisiae diploid strain 2-24D. The volumetric and specific extracellular enzymatic activities using pNPG as substrate were 155 IU L(-1) and 222 IU g(-1), respectively, as detected in the supernatant of the cultures of the S. cerevisiae RP2-BGL transformant strain growing in cellobiose (20 g L(-1)) as the sole carbon source for 48 h. Ethanol production was 5 g L(-1) after 96 h of culture, which represented a yield of 0.41 g g(-1) of substrate consumed (12 g L(-1)), equivalent to 76% of the theoretical yield. The S. cerevisiae RP2-BGL strain expressed the β-glucosidase extracellularly and produced ethanol from cellobiose, which makes this microorganism suitable for application in ethanol production processes with saccharified lignocellulose.

  3. A novel bio-electronic tongue using different cellobiose dehydrogenases to resolve mixtures of various sugars and interfering analytes. (United States)

    Cipri, Andrea; Schulz, Christopher; Ludwig, Roland; Gorton, Lo; Del Valle, Manel


    A novel application of cellobiose dehydrogenase (CDH) as sensing element for a Bioelectronic Tongue (BioET) system has been tested. In this work CDHs from various fungi, which exhibit different substrate specificities, were used to discriminate between lactose and glucose in presence of the interfering matrix compound Ca(2+) in various mixtures. This work exploits the advantage of an electronic tongue system with practically zero pre-treatment of samples and operation at low voltages in a direct electron transfer mode. The Artificial Neural Network (ANN) used in the BioET system to interpret the voltammetric data was able to provide a correct prediction of the concentrations of the analytes considered. Correlation coefficients in the comparison of obtained vs. expected concentrations were highly significant, especially for lactose (R(2)=0.975) and Ca(2+) (R(2)=0.945). This BioET application has a high potential especially for the food and dairy industry and also, if further miniaturised in screen printed format, for its in-situ use.

  4. Membrane-less biofuel cell based on cellobiose dehydrogenase (anode)/laccase (cathode) wired via specific os-redox polymers

    Energy Technology Data Exchange (ETDEWEB)

    Stoica, L.; Dimcheva, N.; Ackermann, Y.; Guschin, D.A. [Analytische Chemie-Elektoanalytik und Sensorik, Ruhr-Universitaet Bochum (Germany); Karnicka, K.; Kulesza, P.J. [Department of Chemistry, University of Warsaw (Poland); Rogalski, J. [Department of Biochemistry, Maria Curie Sklodowska University, Lublin (Poland); Haltrich, D.; Ludwig, R. [Department of Food Sciences and Technology, Division of Food Biotechnology, BOKU-University of Natural Resources and Applied Life Sciences, Vienna Vienna (Austria); Gorton, L. [Department of Analytical Chemistry, Lund University (Sweden); Schuhmann, W.


    A membrane-free biofuel cell (BFC) is reported based on enzymes wired to graphite electrodes by means of Os-complex modified redox polymers. For the anode cellobiose dehydrogenase (CDH) is used as a biocatalyst whereas for the cathode a laccase was applied. This laccase is a high-potential laccase and hence able to reduce O{sub 2} to H{sub 2}O at a formal potential higher than +500 mV versus Ag/AgCl. In order to establish efficient electrochemical contact between the enzymes and graphite electrodes electrodeposition polymers containing Os-complex with specifically designed monomer compositions and formal potentials of the coordinatively bound Os-complex were synthesised and used to wire the enzymes to the electrodes. The newly designed CDH/Os-redox polymer anode was characterised at different pH values and optimised with respect to the nature of the polymer and the enzyme-to-polymer ratio. The resulting BFC was evaluated running on {beta}-lactose as a fuel and air/O{sub 2} as an oxidising agent. The power output, the maximum current density and the electromotor force (E{sub emf}) were found to be affected by the pH value, resulting in a maximum power output of 1.9 {mu}W cm{sup -2} reached at pH 4.3, a maximum current density of about 13 {mu}A cm{sup -2} at pH 3.5, and the highest E{sub emf} approaching 600 mV at pH 4.0. (Abstract Copyright [2009], Wiley Periodicals, Inc.)

  5. Acetone-butanol fermatative production using cellobiose%纤维二糖发酵生产丙酮丁醇

    Institute of Scientific and Technical Information of China (English)

    熊莲; 潘微; 陈新德; 唐绿蓉; 马隆龙


    分别考察C.acetobutylicum 810705、810706以不同浓度的麸皮和玉米粉添加物作为营养元素,纤维二糖直接进行丙酮丁醇(ABE)发酵的结果,发现2株菌对于玉米粉和麸皮的浓度变化趋势一致,C.acetobutylicum 810706转化率较高.纤维二糖ABE发酵工艺条件表明:玉米粉添加量为总糖含量的30%、底物糖质量浓度60 g/L,pH 6.5、温度35℃时,C.acetobutylicum 810706转化率达到37.38%,总溶剂质量浓度22.43 g/L,比葡萄糖、木糖ABE发酵转化率高.模拟纤维素酶水解产物配制混合糖培养基,其溶剂转化率较单独的葡萄糖、木糖发酵的转化率高,为34.95%.对比纤维素酶水解条件,C.acetobutylicum 810706具有优良的纤维素酶水解同步糖化ABE发酵能力.%Fermantative production of acetone-butanol by C. Acetobutylicum 810705 and C. Acetobutylicum 810706 was studied from cellobiose with different additive amounts of bran and corn starch. The same variation tendency of solvent concentration and substrate conversion was testified, and substrate conversion of C. Acetobutylicum 810706 was a little higher. Corn starch was proved to be better nutritional supplements than bran, and the optimum additive ratio was 30% of total sugar. Based on the optimization of culture conditions, C. Acetobutylicum 810706 achieved a 37. 38% substrate conversion and 22. 43 g/L solvent concerntration, and it was better than fermantative production of acetone-butanol from glucose and xylose while cultured at 35 ℃ with 60 g/L sugar concentration of substrate and initial pH 6. 5. Mixed sugar substrate prepared by imitating the concentration of cellulose enzymatic hydrolysis was fermented. 34. 95% of substrate conversion was obtained, and it was higher than any single sugar. Results showed that C. Acetobutylicum 810706 had excellent potential of cellulose saccharified by enzymatic hydrolysis and acetone-butanol fermentation simultaneously.

  6. Discrimination among eight modified michaelis-menten kinetics models of cellulose hydrolysis with a large range of substrate/enzyme ratios: inhibition by cellobiose. (United States)

    Bezerra, Rui M F; Dias, Albino A


    The kinetics of exoglucanase (Cel7A) from Trichoderma reesei was investigated in the presence of cellobiose and 24 different enzyme/Avicel ratios for 47 h, in order to establish which of the eight available kinetic models best explained the factors involved. The heterogeneous catalysis was studied and the kinetic parameters were estimated employing integrated forms of Michaelis-Menten equations through the use of nonlinear least squares. It was found that cellulose hydrolysis follows a model that takes into account competitive inhibition by cellobiose (final product) with the following parameters: Km = 3.8 mM, Kic = 0.041 mM, kcat = 2 h-1 (5.6 x 10-4 s-1). Other models, such as mixed type inhibition and those incorporating improvements concerning inhibition by substrate and parabolic inhibition, increased the modulation performance very slightly. The results support the hypothesis that nonproductive enzyme substrate complexes, parabolic inhibition, and enzyme inactivation (Selwyn test) are not the principal constraints in enzymatic cellulose hydrolysis. Under our conditions, the increment in hydrolysis was not significant for substrate/enzyme ratios <6.5.

  7. Fermentation of succinic acid using cellobiose from cellulose hydrolysates%利用纤维素水解液中的纤维二糖发酵制备丁二酸

    Institute of Scientific and Technical Information of China (English)

    徐蓉; 奚永兰; 张九花; 戴文宇; 万月佳; 陈可泉; 姜岷


    Cellobiose was often found in cellulose hydrolysates. In this study, the ability to use cellobiose to produce succinic acid by A.succinogenes NJ113 and using cellobiose prepared from sugarcane bagasse cellulose hydrolysates as a carbon source to produce succinic acid were investigated. A final succinic acid concentration of 23.51 g/L with a yield of 67.17% was achieved from an initial cellobiose concentration of 35 g/L via batch fermentation. In batch fermentation with 18 g/L of cellobiose and 17 g/L of other sugars from sugarcane bagasse cellulose hydrolysates, a succinic acid concentration of 20.00 g/L was obtained, with a yield of 64.73%. This study suggested that A. succinogenes had a strong ability to convert cellobiose into succinic acid and cellobiose from cellulose hydrolysate could be a potential carbon source for economical and efficient succinic acid production by A. succinogenes.%纤维素水解液中通常含有纤维二糖.本文考察了Actinobacillus succinogenes NJ 113利用纤维二糖厌氧发酵生产丁二酸的能力,并利用蔗渣纤维素制备纤维二糖作为碳源用于厌氧发酵生产丁二酸.3L发酵罐厌氧发酵结果显示:以35 g/L纤维二糖作为碳源发酵制备丁二酸,其产量为23.51 g/L,产率达到67.17%;用含有18g/L纤维二糖和17 g/L其它糖类的蔗渣纤维素水解液作为碳源发酵制备丁二酸,丁二酸的产量和产率分别为20.00 g/L和64.73%.因此,Actinobacillus succinogenes NJ 113具有较强的利用纤维二糖生产丁二酸的能力,而且利用废弃的纤维素制备纤维二糖作为碳源高效、经济地发酵制备丁二酸具有可行性.

  8. Production of lactose-free galacto-oligosaccharide mixtures: comparison of two cellobiose dehydrogenases for the selective oxidation of lactose to lactobionic acid. (United States)

    Maischberger, Thomas; Nguyen, Thu-Ha; Sukyai, Prakit; Kittl, Roman; Riva, Sergio; Ludwig, Roland; Haltrich, Dietmar


    Galacto-oligosaccharides, complex mixtures of various sugars, are produced by transgalactosylation from lactose using beta-galactosidase and are of great interest for food and feed applications because of their prebiotic properties. Most galacto-oligosaccharide preparations currently available in the market contain a significant amount of monosaccharides and lactose. The mixture of galacto-oligosaccharides (GalOS) in this study produced from lactose using recombinant beta-galactosidase from Lactobacillus reuteri contains 48% monosaccharides, 26.5% lactose and 25.5% GalOS. To remove efficiently both monosaccharides and lactose from this GalOS mixture containing significant amounts of prebiotic non-lactose disaccharides, a biocatalytic approach coupled with subsequent chromatographic steps was used. Lactose was first oxidised to lactobionic acid using fungal cellobiose dehydrogenases, and then lactobionic acid and monosaccharides were removed by ion-exchange and size-exclusion chromatography. Two different cellobiose dehydrogenases (CDH), originating from Sclerotium rolfsii and Myriococcum thermophilum, were compared with respect to their applicability for this process. CDH from S. rolfsii showed higher specificity for the substrate lactose, and only few other components of the GalOS mixture were oxidised during prolonged incubation. Since these sugars were only converted once lactose oxidation was almost complete, careful control of the CDH-catalysed reaction will significantly reduce the undesired oxidation, and hence subsequent removal, of any GalOS components. Removal of ions and monosaccharides by the chromatographic steps gave an essentially pure GalOS product, containing less than 0.3% lactose and monosaccharides, in a yield of 60.3%.

  9. Gold nanoparticles/water-soluble carbon nanotubes/aromatic diamine polymer composite films for highly sensitive detection of cellobiose dehydrogenase gene

    Energy Technology Data Exchange (ETDEWEB)

    Zeng Guangming, E-mail: [College of Environmental Science and Engineering, Hunan University, Changsha 410082 (China); Key Laboratory of Environmental Biology and Pollution Control (Hunan University), Ministry of Education, Changsha 410082 (China); Li Zhen, E-mail: [College of Environmental Science and Engineering, Hunan University, Changsha 410082 (China); Key Laboratory of Environmental Biology and Pollution Control (Hunan University), Ministry of Education, Changsha 410082 (China); Tang Lin; Wu Mengshi; Lei Xiaoxia; Liu Yuanyuan; Liu Can; Pang Ya; Zhang Yi [College of Environmental Science and Engineering, Hunan University, Changsha 410082 (China); Key Laboratory of Environmental Biology and Pollution Control (Hunan University), Ministry of Education, Changsha 410082 (China)


    Highlights: > Gold nanoparticles/multiwalled carbon nanotubes/poly (1,5-naphthalenediamine) modified electrode was fabricated. > The sensor was applied for the detection of cellobiose dehydrogenase genes. > An effective method to distribute MWCNTs and attach to the electrode was proposed. > The composite films greatly improved the sensitivity and enhanced the DNA immobilization. > The DNA biosensor exhibited fairly high sensitivity and quite low detection limit. - Abstract: An electrochemical sensor based on gold nanoparticles (GNPs)/multiwalled carbon nanotubes (MWCNTs)/poly (1,5-naphthalenediamine) films modified glassy carbon electrode (GCE) was fabricated. The effectiveness of the sensor was confirmed by sensitive detection of cellobiose dehydrogenase (CDH) gene which was extracted from Phanerochaete chrysosporium using polymerase chain reaction (PCR). The monomer of 1,5-naphthalenediamine was electropolymerized on the GCE surface with abundant free amino groups which enhanced the stability of MWCNTs modified electrode. Congo red (CR)-functionalized MWCNTs possess excellent conductivity as well as high solubility in water which enabled to form the uniform and stable network nanostructures easily and created a large number of binding sites for electrodeposition of GNPs. The continuous GNPs together with MWCNTs greatly increased the surface area, conductivity and electrocatalytic activity. This electrode structure significantly improved the sensitivity of sensor and enhanced the DNA immobilization and hybridization. The thiol modified capture probes were immobilized onto the composite films-modified GCE by a direct formation of thiol-Au bond and horseradish peroxidase-streptavidin (HRP-SA) conjugates were labeled to the biotinylated detection probes through biotin-streptavidin bond. Scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to investigate the film assembly and DNA hybridization processes

  10. Examining the role of phosphate in glycosyl transfer reactions of Cellulomonas uda cellobiose phosphorylase using D-glucal as donor substrate. (United States)

    Wildberger, Patricia; Brecker, Lothar; Nidetzky, Bernd


    Cellobiose phosphorylase from Cellulomonas uda (CuCPase) is shown to utilize D-glucal as slow alternative donor substrate for stereospecific glycosyl transfer to inorganic phosphate, giving 2-deoxy-α-D-glucose 1-phosphate as the product. When performed in D(2)O, enzymatic phosphorolysis of D-glucal proceeds with incorporation of deuterium in equatorial position at C-2, implying a stereochemical course of reaction where substrate becomes protonated from below its six-membered ring through stereoselective re side attack at C-2. The proposed catalytic mechanism, which is supported by results of docking studies, involves direct protonation of D-glucal by the enzyme-bound phosphate, which then performs nucleophilic attack on the reactive C-1 of donor substrate. When offered D-glucose next to D-glucal and phosphate, CuCPase produces 2-deoxy-β-D-glucosyl-(1→4)-D-glucose and 2-deoxy-α-D-glucose 1-phosphate in a ratio governed by mass action of the two acceptor substrates present. Enzymatic synthesis of 2-deoxy-β-D-glucosyl-(1→4)-D-glucose is effectively promoted by catalytic concentrations of phosphate, suggesting that catalytic reaction proceeds through a quaternary complex of CuCPase, D-glucal, phosphate, and D-glucose. Conversion of D-glucal and phosphate presents a convenient single-step synthesis of 2-deoxy-α-D-glucose 1-phosphate that is difficult to prepare chemically.

  11. A simple and sensitive method for lactose detection based on direct electron transfer between immobilised cellobiose dehydrogenase and screen-printed carbon electrodes

    Energy Technology Data Exchange (ETDEWEB)

    Safina, Gulnara, E-mail: [Department of Analytical Chemistry/Biochemistry, Lund University, Box 124, 221 00 Lund (Sweden); Ludwig, Roland [Department of Analytical Chemistry/Biochemistry, Lund University, Box 124, 221 00 Lund (Sweden); Research Centre Applied Biocatalysis, Petersgasse 18, 8010 Graz (Austria); Gorton, Lo, E-mail: [Department of Analytical Chemistry/Biochemistry, Lund University, Box 124, 221 00 Lund (Sweden)


    A rapid and simple approach of lactose analysis is proposed based on 3rd generation amperometric biosensors employing cellobiose dehydrogenase (CDH) from Trametes villosa or Phanerochaete sordida immobilised on screen-printed carbon electrodes (SPCEs). After optimisation of the working conditions of the biosensors - pH of the carrier buffer, flow rate and applied potential - the sensors were able to detect lactose in a concentration range between 0.5-200 {mu}M and 0.5-100 {mu}M employing T. villosa and P. sordida CDH, respectively. The limit of detection is 250 nM (90 {mu}g/L) for both. Biosensors based on SPCEs modified with multiwalled carbon nanotubes showed a higher sensitivity than unmodified SPCEs. Cross-linking with glutaraldehyde or poly(ethyleneglycol)diglycidyl ether improved not only the stability but also the analytical response. The developed sensor has been successfully applied for the determination of lactose in dairy (milk with different percentages of fat, lactose-free milk and yogurt) with a good reproducibility (RSD = 1.5-2.2%). No sample preparation except a simple dilution process is needed. The biosensor is easy to make and operate, is inexpensive and reveals a high sensitivity and reliability.

  12. Differential decolorization of textile dyes in mixtures and the joint effect of laccase and cellobiose dehydrogenase activities present in extracellular extracts from Funalia trogii. (United States)

    Tilli, Silvia; Ciullini, Ilaria; Scozzafava, Andrea; Briganti, Fabrizio


    The largest part of the bio-decolorization investigations have been performed to date on a single dye without exploring the behavior in complex mixtures as the real dyeing baths. Therefore, mixtures of dyes belonging to azo and anthraquinonic classes, chosen among the most utilized in textile wool dyeing, were employed for comparative enzymatic decolorization studies using the extracellular extracts from the white rot fungus Funalia trogii, to understand how the concomitant presence of more than one dye could influence their degradation course and yield. Fungal extracts containing laccase activity only were capable to partially decolorize dyes mixtures from the different classes analyzed. The deconvolution of the decolorization with time allowed to monitor the degradation of the single dyes in the mixtures evidencing a time dependent differential decolorization not observed for the singles alone. Some dyes in the blend were in fact decolorized only when the most easily converted dyes were largely transformed. These experiments would allow to help the dyeing factories in the selection of the most readily degraded dyes. Since F. trogii grown on different media and activators shows diverse levels of expression of the redox enzymes laccase and cellobiose dehydrogenase (CDH), the dyes mixtures recalcitrant to decolorization by laccase activity alone, were subjected to the combined action of extracts containing laccase and CDH. The use of CDH, in support to the activity of laccase, resulted in substantial decolorization increases (>84%) for all the refractory dyes mixtures.

  13. Improved DET communication between cellobiose dehydrogenase and a gold electrode modified with a rigid self-assembled monolayer and green metal nanoparticles: The role of an ordered nanostructuration. (United States)

    Bollella, P; Mazzei, F; Favero, G; Fusco, G; Ludwig, R; Gorton, L; Antiochia, R


    Efficient direct electron transfer (DET) between cellobiose dehydrogenase from Corynascus thermophilus (CtCDH) and a novel gold electrode platform, obtained by covalent linking of green AuNPs and AgNPs modified with a dithiol self-assembled monolayer, consisting of biphenyl-4,4'-dithiol (BPDT), was presented. The green AuNPs and AgNPs were synthesized using quercetin as reducing agent at room temperature. TEM experiments showed that the AuNPs and AgNPs were circular in shape with an average diameter of 5 and 8nm, respectively. Cyclic voltammetry of CtCDH immobilized onto the AuNPs/BPDT/AuE and the AgNPs/BPDT/AuE electrode platforms were carried out and compared with naked AuE, BPDT/AuE, AuNPs/AuE, and AgNPs/AuE. A pair of well-defined redox waves in neutral pH solution due to efficient DET of CtCDH was present with both MNPs/BPDT/AuE platforms. No DET communication was found with platforms without MNPs linked to BPDT. The apparent heterogeneous electron transfer rate constants (kS) of CtCDH were calculated to be 21.5±0.8s(-1) and 10.3±0.7s(-1), for the AuNPs/BPDT/AuE and the AgNPs/BPDT/AuE platforms, respectively. The modified electrodes were successively used to develop an eco-friendly biosensor for lactose detection. The CtCDH/AuNPs/BPDT/AuE based biosensor showed the best analytical performances with an excellent stability, a detection limit of 3µM, a linear range between 5 and 400µM and a sensitivity of 27.5±2.5µAcm(-2)mM(-1). Such performances were favorably compared with other lactose biosensors reported in literature. The biosensor was successively tested to quantify lactose content in real milk and cream samples. No significant interference present in the sample matrices was observed.

  14. Genetic characterization of the CcpA-dependent, cellobiose-specific PTS system comprising CelB, PtcB and PtcA that transports lactose in Lactococcus lactis IL1403. (United States)

    Aleksandrzak-Piekarczyk, Tamara; Polak, Jolanta; Jezierska, Beata; Renault, Pierre; Bardowski, Jacek


    Lactose metabolism is one of the most important areas of research on Lactic Acid Bacteria (LAB). In rapidly acidifying industrial Lactococcus lactis strains, lactose is transported by a lactose-specific phosphotransferase system (PTS) encoded by a plasmid. However, an alternative lactose catabolic pathway was evidenced in the plasmid-cured, and thus initially lactose-negative L. lactis IL1403. We showed that in this strain the chromosomally-encoded cellobiose-specific PTS system comprising the celB, ptcB and ptcA genes is also able to transport lactose. By expression studies in the wild type IL1403 strain and IBB550, its ccpA-deficient derivative, we demonstrated that celB, ptcB and ptcA are tightly regulated by the general catabolite repression system, whereas celB additionally requires the presence of cellobiose to be fully induced. The comparison of expression levels of sugar catabolic genes indicated that the efficiency of CcpA-mediated catabolic repression depends on conservation of the cre sequence, and that in the case of perfect matching with the cre consensus, CcpA still drives a strong repression even under non-repressing conditions.

  15. Thermodynamics of the Hydrolysis Reactions of 1,4-beta-D-xylobiose, 1,4-beta-D-xylotriose, D-cellobiose, and D-Maltose

    Energy Technology Data Exchange (ETDEWEB)

    Tewari, Y. B.; Lang, B. E.; Decker, S. R.; Goldberg, R. N.


    Microcalorimetry and high-performance liquid chromatography have been used to conduct a thermodynamic investigation of the following reactions: (1) 1,4-{beta}-Dxylobiose(aq)+H{sub 2}O(I)=2D-xylose(aq); (2) 1,4-{beta}-D-xylotriose+2H{sub 2}O(I)=3D-xylose(aq); (3) D-maltose(aq)+H{sub 2}O(I)=2{alpha}-D-glucose(aq); and (4) D-cellobiose(aq)+H{sub 2}O(I)=2{alpha}-D-glucose(aq). The results of the equilibrium measurements were K = (1.46 {+-} 0.15) {center_dot} 10{sup 3} for reaction (1) and K = (551 {+-} 34) for reaction (3). Although it was not possible to measure directly a value for the equilibrium constant for reaction (4), it was possible to obtain the value K = 657 for this reaction via a thermochemical pathway calculation. The results of the calorimetric measurements were standard enthalpies of reaction {Delta}{sub r}H{sup o} = (0.12 {+-} 0.26) kJ {center_dot} mol{sup -1} for reaction (1) and {Delta}{sub r}H{sup o} = -(0.06 {+-} 0.18) kJ {center_dot} mol{sup -1} for reaction (2). It is noted that values of {Delta}{sub r}H{sup o} for reactions (1) and (2) are equal to each other within their respective experimental errors. This fact is consistent with earlier observations that, for reactions involving the making/breaking of N saccharide linkages, the assignment of characteristic values of {Delta}{sub r}H{sup o}/N or {Delta}{sub r}G{sup o}/N or {Delta}{sub r}S{sup o}/N for a specified linkage, is accurate in predicting the values of {Delta}{sub r}H{sup o}, {Delta}{sub r}G{sup o}, and {Delta}{sub r}S{sup o} for reactions involving saccharides that contain multiples or combinations of such linkages. Also, the values of the standard entropy changes {Delta}{sub r}S{sup o} for the hydrolysis reactions (3) and (4) fall into the range of values {l_brace}(32 to 48) J {center_dot} K{sup -1} {center_dot} mol{sup -1}{r_brace} previously noted for the hydrolysis of six-carbon disaccharides. In order to tie the results of this study into the thermochemical literature, a reaction

  16. Thermodynamics of the hydrolysis reactions of 1,4-{beta}-D-xylobiose, 1,4-{beta}-D-xylotriose, D-cellobiose, and D-maltose

    Energy Technology Data Exchange (ETDEWEB)

    Tewari, Yadu B. [Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, MD 20876 (United States)], E-mail:; Lang, Brian E. [Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, MD 20876 (United States)], E-mail:; Decker, Stephen R. [National Renewable Energy Laboratory, Chemical and Biosciences Center, 1617 Cole Boulevard, Golden, CO 80401 (United States)], E-mail:; Goldberg, Robert N. [Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, MD 20876 (United States); Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, MD 21250 (United States)], E-mail:


    Microcalorimetry and high-performance liquid chromatography have been used to conduct a thermodynamic investigation of the following reactions: (1)1,4-{beta}-D-xylobiose(aq)+H{sub 2}O(l)=2D-xylose(aq), (2)1,4-{beta}-D-xylotriose+2H{sub 2}O(l)=3D-xylose(aq), (3)D-maltose(aq)+H{sub 2}O(l)=2{alpha}-D-glucose(aq), (4)D-cellobiose(aq)+H{sub 2}O(l)=2{alpha}-D-glucose(aq). The results of the equilibrium measurements were K = (1.46 {+-} 0.15) . 10{sup 3} for reaction (1) and K = (551 {+-} 34) for reaction (3). Although it was not possible to measure directly a value for the equilibrium constant for reaction (4), it was possible to obtain the value K = 657 for this reaction via a thermochemical pathway calculation. The results of the calorimetric measurements were standard enthalpies of reaction {delta}{sub r}H{sup 0} = (0.12 {+-} 0.26) kJ . mol{sup -1} for reaction (1) and {delta}{sub r}H{sup 0} = -(0.06 {+-} 0.18) kJ . mol{sup -1} for reaction (2). It is noted that values of {delta}{sub r}H{sup 0} for reactions (1) and (2) are equal to each other within their respective experimental errors. This fact is consistent with earlier observations that, for reactions involving the making/breaking of N saccharide linkages, the assignment of characteristic values of {delta}{sub r}H{sup 0}/N or {delta}{sub r}G{sup 0}/N or {delta}{sub r}S{sup 0}/N for a specified linkage, is accurate in predicting the values of {delta}{sub r}H{sup 0}, {delta}{sub r}G{sup 0}, and {delta}{sub r}S{sup 0} for reactions involving saccharides that contain multiples or combinations of such linkages. Also, the values of the standard entropy changes {delta}{sub r}S{sup 0} for the hydrolysis reactions (3) and (4) fall into the range of values {l_brace}(32 to 48) J . K{sup -1} . mol{sup -1}){r_brace} previously noted for the hydrolysis of six-carbon disaccharides. In order to tie the results of this study into the thermochemical literature, a reaction catalog of related property values was created. Selected

  17. Synbiotic Lactobacillus acidophilus NCFM and cellobiose does not affect human gut bacterial diversity but increases abundance of lactobacilli, bifidobacteria and branched-chain fatty acids: a randomized, double-blinded cross-over trial. (United States)

    van Zanten, Gabriella C; Krych, Lukasz; Röytiö, Henna; Forssten, Sofia; Lahtinen, Sampo J; Abu Al-Soud, Waleed; Sørensen, Søren; Svensson, Birte; Jespersen, Lene; Jakobsen, Mogens


    Probiotics, prebiotics, and combinations thereof, that is synbiotics, have been reported to modulate gut microbiota of humans. In this study, effects of a novel synbiotic on the composition and metabolic activity of human gut microbiota were investigated. Healthy volunteers (n = 18) were enrolled in a double-blinded, randomized, and placebo-controlled cross-over study and received synbiotic [Lactobacillus acidophilus NCFM (10(9) CFU) and cellobiose (5 g)] or placebo daily for 3 weeks. Fecal samples were collected and lactobacilli numbers were quantified by qPCR. Furthermore, 454 tag-encoded amplicon pyrosequencing was used to monitor the effect of synbiotic on the composition of the microbiota. The synbiotic increased levels of Lactobacillus spp. and relative abundances of the genera Bifidobacterium, Collinsella, and Eubacterium while the genus Dialister was decreased (P < 0.05). No other effects were found on microbiota composition. Remarkably, however, the synbiotic increased concentrations of branched-chain fatty acids, measured by gas chromatography, while short-chain fatty acids were not affected.

  18. Direct Catalytic Conversion of Cellobiose into Sorbitol in Extremely Low Acid over Ruthenium Catalysts%超低酸性环境中Ru/C催化纤维二糖一步法制备山梨醇的研究

    Institute of Scientific and Technical Information of China (English)

    李计彪; 武书彬; 张军


    以Ru/C为催化剂,在超低酸环境和氢气气氛下,将纤维二糖一步转化制备山梨醇.考察了反应温度、反应时间以及催化剂用量对山梨醇产率的影响.实验结果表明,在0.05%H3PO4环境,Ru/C催化剂用量15%,转速为600 r/min,反应温度为458 K以及3 MPa氢气条件下反应1h,产物山梨醇的产率最高,可达到87.1%.同时,催化剂重复利用研究表明Ru/C是一种较理想的氢化反应催化剂,可重复利用且催化效率较高.%Ru/C catalyst and extremely low acid were employed for one-step conversion of cellobiose into sorbitol in the presence of hydrogen. Effects of reaction temperature, reaction time and catalyst usage on sorbitol yield were discussed. Experimental results showed that the highest sorbitol yield of 87. 1% was obtained at reaction temperature of 458 K and catalyst usage of 15 % for 1 h under 3 MPa H2 with the stirring rate of 600 r/min. Meanwhile, catalyst recycling showed that Ru/C was an ideal hydro-genation catalyst with high catalytic efficiency, which could be recycled several times.

  19. Kinetics of the Oxidation of D-Glucose and Cellobiose by Acidic Solution of N-Bromoacetamide Using Transition Metal Complex Species,[RuCl3(H2O)2OH]-,as Catalyst

    Institute of Scientific and Technical Information of China (English)

    SINGH,Ashok Kumar; SRIVASTAVA,Jaya; SRIVASTAVA,Shalini; RAHMANI,Shahla


    The kinetics of Ru(Ⅲ)-catalyzed and Hg(Ⅱ)-co-catalyzed oxidation of D-glucose (Glc) and cellobiose (Cel) by N-bromoacetamide (NBA) in the presence of perchloric acid at 40 ℃ have been investigated.The reactions exhibit the first order kinetics with respect to NBA,but tend towards the zeroth order to higher NBA.The reactions are the first order with respect to Ru(Ⅲ) and are fractional positive order with respect to [reducing sugar].Positive effect of Cl- and Hg(Oac)2 on the rate of reaction is also evident in the oxidation of both reducing sugars.A negative effect of variation of H+ and acetamide was observed whereas the ionic strength (μ) of the medium had no influence on the oxidation rate.The rate of reaction decreased with the increase in dielectric constant and this enabled the computation of dAB,the size of the activated complex.Various activation parameters have been evaluated and suitable explanation for the formation of the most reactive activated complex has been given.The main products of the oxidation are the corresponding arabinonic acid and formic acid.HOBr and [RuCl3(H2O)2OH]- were postulated as the reactive species of oxidant and catalyst respectively.A common mechanism,consistent with the kinetic data and supported by the observed effect of ionic strength,dielectric constant and multiple regression analysis,has been proposed.Formation of complex species such as [RuCl3·S·(H2O)OH]- and RuCl3·S·OHgBr·OH during the course of reaction was fully supported by kinetic and spectral evidences.

  20. Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum

    DEFF Research Database (Denmark)

    Nakai, Hiroyuki; Abou Hachem, Maher; Petersen, Bent O.;


    . Structure analysis by NMR and ESI-MS confirmed two β-glucosyl oligosaccharide product series to represent novel compounds, i.e. β-d-glucopyranosyl-[(1→4)-β-d-glucopyranosyl]n-(1→2)-d-glucopyranose, and β-d-glucopyranosyl-[(1→4)-β-d-glucopyranosyl]n-(1→3)-d-glucopyranose (n = 1–7). Multiple sequence...

  1. Lactic acid production from Cellobiose and xylose by engineered Saccharomyces cerevisiae (United States)

    Efficient and rapid production of value-added chemicals from lignocellulosic biomass is an important step towards a sustainable society. Lactic acid, used for synthesizing the bioplastic polylactide, has been produced by microbial fermentation using primarily glucose. Lignocellulosic hydrolysates co...

  2. Lower-cost cellulosic ethanol production using cellobiose fermenting yeast Clavispora NRRL Y-50464 (United States)

    For ethanol production from cellulosic materials, there are generally two major steps needed including enzymatic hydrolysis to break down biomass sugars and microbial fermentation to convert available simple sugars into ethanol. It often requires two different kinds of microorganisms since ethanolog...

  3. Production, characterization and purification of fungal beta-glucosidases and their action in the hydrolys of amygdalin, cellobiose and p-nitrophenyl-beta-glucopiranoside


    Carolina Casagrande Bedani


    Resumo: As ß-glicosidases (ß-D-glucosídeo glucohidrolase, EC catalisam a hidrólise de dissacarídeos e glicosídeos conjugados a partir da extremidade não redutora. A enzima ß-glicosidase apresenta inúmeras aplicações na indústria de alimentos e farmacêutica, atuando na hidrólise da celobiose em glicose, no processo de conversão da celulose em glicose em combinação com outras enzimas celulolíticas; na liberação de compostos de aroma em sucos de frutas e vinho; na hidrólise de composto...

  4. The Differential Proteome of the Probiotic Lactobacillus acidophilus NCFM Grown on the Potential Prebiotic Cellobiose Shows Upregulation of Two beta-Glycoside Hydrolases

    DEFF Research Database (Denmark)

    van Zanten, Gabriella Christina; Sparding, Nadja; Majumder, Avishek;


    (2D-DIGE) in the acidic (pH 4-7) and the alkaline (pH 6-11) regions showing a total of 136 spots to change in abundance. Proteins were identified by MS or MS/MS from 81 of these spots representing 49 unique proteins and either increasing 1.5-13.9-fold or decreasing 1.5-7.8-fold in relative abundance...

  5. Synbiotic Lactobacillus acidophilus NCFM and cellobiose does not affect human gut bacterial diversity but increases abundance of lactobacilli, bifidobacteria and branched-chain fatty acids: a randomized, double-blinded cross-over trial

    DEFF Research Database (Denmark)

    van Zanten, Gabriella Christina; Krych, Lukasz; Roytio, Henna


    Probiotics, prebiotics, and combinations thereof, that is synbiotics, have been reported to modulate gut microbiota of humans. In this study, effects of a novel synbiotic on the composition and metabolic activity of human gut microbiota were investigated. Healthy volunteers (n=18) were enrolled i...... pyrosequencing was used to monitor the effect of synbiotic on the composition of the microbiota. The synbiotic increased levels of Lactobacillus spp. and relative abundances of the genera Bifidobacterium, Collinsella, and Eubacterium while the genus Dialister was decreased (P...

  6. [superscript 1]H NMR Spectroscopy-Based Configurational Analysis of Mono- and Disaccharides and Detection of ß-Glucosidase Activity: An Undergraduate Biochemistry Laboratory (United States)

    Periyannan, Gopal R.; Lawrence, Barbara A.; Egan, Annie E.


    A [superscript 1]H NMR spectroscopy-based laboratory experiment explores mono- and disaccharide structural chemistry, and the enzyme-substrate specificity of glycosidic bond cleavage by ß-glucosidase towards cellobiose (ß-linked gluco-disaccharide) and maltose (a-linked gluco-disaccharide). Structural differences between cellobiose, maltose, and…

  7. NCBI nr-aa BLAST: CBRC-ACAR-01-0026 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ACAR-01-0026 ref|YP_535807.1| Cellobiose PTS, EIIC [Lactobacillus salivarius subsp. saliva...rius UCC118] gb|ABD99724.1| Cellobiose PTS, EIIC [Lactobacillus salivarius subsp. salivarius UCC118] YP_535807.1 0.010 26% ...

  8. Degradation of cellulosic biomass and its subsequent utilization for the production of chemical feedstocks. Progress report, June 1-August 31, 1978

    Energy Technology Data Exchange (ETDEWEB)

    Wang, D.I.C.; Cooney, C.L.; Demain, A.L.; Gomez, R.F.; Sinskey, A.J.


    Studies concerning the cellobiose properties of Clostridium thermocellum were started to determine if the cellulose degradation end products can be enhanced for glucose (with a subsequent decrease in cellobiose). Implications of preliminary studies indicate that the cells or the enzyme(s) responsible for converting cellobiose to glucose can be manipulated environmentally and genetically to increase the final yield of glucose. The second area of effort is to the production of chemical feedstocks. Three fermentations have been identified for exploration. Preliminary reports on acrylic acid acetone/butanol, and acetic acid production by C. propionicum, C. acetobutylicum, and C. thermoaceticum, respectively, are included. (DMC)

  9. Transcriptional Analysis of Prebiotic Uptake and Catabolism by Lactobacillus acidophilus NCFM

    DEFF Research Database (Denmark)

    Andersen, Joakim Mark; Barrangou, Rodolphe; Abou Hachem, Maher


    ) transporters. PTS systems were upregulated primarily by di- and tri-saccharides such as cellobiose, isomaltose, isomaltulose, panose and gentiobiose, while ABC transporters were upregulated by raffinose, Polydextrose, and stachyose. A single GPH transporter was induced by lactitol and galactooligosaccharides...

  10. Identification of xanthans isolated from sugarcane juices obtained from scalded plants infected by Xanthomonas albilineans. (United States)

    Fontaniella, Blanca; Rodríguez, C W; Piñón, Dolores; Vicente, C; Legaz, María-Estrella


    The exudate gum produced by Xanthomonas albilineans, a specific sugarcane pathogen, has been isolated from juices of diseased sugarcane stalks, hydrolyzed with hydrochloric acid, and the hydrolysate analyzed by capillary electrophoresis. Sucrose. cellobiose, mannose, glucose, glucose-1-P and glucuronic acid were identified as the major components of the polysaccharide isolated from diseased stalks. Juices from healthy stalks contained maltose instead of cellobiose. The chemical nature of this polysaccharide is discussed.

  11. Cryptococcus socialis sp. nov. and Cryptococcus consortionis sp. nov., Antarctic basidioblastomycetes (United States)

    Vishniac, H. S.


    New yeasts from the Ross Desert (dry valley area) of Antarctica include Cryptococcus socialis sp. nov. and Cryptococcus consortionis sp. nov. Cryptococcus socialis MYSW A801-3aY1 (= ATCC 56685) requires no vitamins, assimilates L-arabinose, cellobiose, D-glucuronate, maltose, melezitose, raffinose, soluble starch, sucrose, and trehalose, and may be distinguished from all other basidioblastomycetes by the combination of amylose production, cellobiose assimilation, and failure to utilize nitrate, D-galactose, myo-inositol, and mannitol. Its guanine-plus-cytosine content is 56 mol%. Cryptococcus consortionis MYSW A801-3aY92 (= ATCC 56686) requires thiamine, assimilates L-arabinose, D-glucuronate, 2-ketogluconate, salicin, succinate, sucrose, trehalose, and D-xylose, and may be distinguished from all other basidioblastomycetes by the combination of amylose production and failure to utilize nitrate, cellobiose, D-galactose, myo-inositol, and mannitol. Its guanine-plus-cytosine content is 56 mol%.

  12. Yeast surface display of dehydrogenases in microbial fuel-cells. (United States)

    Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital


    Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems.

  13. Kinetic characterization of a glycoside hydrolase family 44 xyloglucanase/endoglucanase from Ruminococcus flavefaciens FD-1. (United States)

    Warner, Christopher D; Go, Rica M; García-Salinas, Carolina; Ford, Clark; Reilly, Peter J


    Two forms of Ruminococcus flavefaciens FD-1 endoglucanase B, a member of glycoside hydrolase family 44, one with only a catalytic domain and the other with a catalytic domain and a carbohydrate binding domain (CBM), were produced. Both forms hydrolyzed cellotetraose, cellopentaose, cellohexaose, carboxymethylcellulose (CMC), birchwood and larchwood xylan, xyloglucan, lichenan, and Avicel but not cellobiose, cellotriose, mannan, or pullulan. Addition of the CBM increased catalytic efficiencies on both CMC and birchwood xylan but not on xyloglucan, and it decreased rates of cellopentaose and cellohexaose hydrolysis. Catalytic efficiencies were much higher on xyloglucan than on other polysaccharides. Hydrolysis rates increased with increasing cellooligosaccharide chain length. Cellotetraose hydrolysis yielded only cellotriose and glucose. Hydrolysis of cellopentaose gave large amounts of cellotetraose and glucose, somewhat more of the former than of the latter, and much smaller amounts of cellobiose and cellotriose. Cellohexaose hydrolysis yielded much more cellotetraose than cellobiose and small amounts of glucose and cellotriose, along with a low and transient amount of cellopentaose.

  14. Production of β-Glucosidase from a Newly Isolated Aspergillus Species Using Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    Pilanee Vaithanomsat


    Full Text Available A newly isolated fungus Aspergillus niger SOI017 was shown to be a good producer of β-glucosidase from all isolated fungal strains. Fermentation condition (pH, cellobiose concentration, yeast extract concentration, and ammonium sulfate concentration was optimized for producing the enzyme in shake flask cultures. Response surface methodology was used to investigate the effects of 4 fermentation parameters (yeast extract concentration, cellobiose concentration, ammonium sulfate concentration, and pH on β-glucosidase enzyme production. Production of β-glucosidase was most sensitive to the culture medium, especially the nitrogen source yeast extract. The optimized medium for producing maximum β-glucosidase specific activity consisted of 0.275% yeast extract, 1.125% cellobiose, and 2.6% ammonium sulfate at a pH value of 3.

  15. Utilization of immobilized B-glucosidase in the enzymatic hydrolysis of cellulose

    Energy Technology Data Exchange (ETDEWEB)

    Issacs, S.H.; Wilke, C.R.


    ..beta..-glucosidase obtained from Aspergillus phoenicis was immobilized onto phenol formaldehyde resin using glutaraldehyde as a fixing agent, and kinetic characteristics such as pH optimum, temperature stability and Michaelis-Menton constants were determined. Three experiments were performed where a batch hydrolysis of a cellulosic source was carried out with a recycle stream through an immobilized ..beta..-glucosidase column in order to continuously remove cellobiose. The first two experiments using pretreated corn stover as the substrate showed no increase in hydrolysis over that of a control system, presumably because the cellobiose production was too low for cellobiose inhibition to occur. The third experiment, using Solka Floc as the substrate, which produced as high as 8.8 grams per liter of cellobiose, showed only a slight increase in soluble sugar production over that of the control system. Since the current process indicates the use of corn stover or a similar substrate, it does not appear useful to include an immobilized enzyme reactor in this manner. Since the fermentation part of the process cannot use cellobiose to produce ethanol, the use of the immobilized ..beta..-glucosidase reactor to convert the cellobiose to glucose may have economic significance by increasing the ethanol yield in this fashion. A computer program was produced in order to simulate a fixed-bed reactor with diffusion limitations and to determine the cost per pound of glucose for a given reactor design. Use of the immobilized enzyme system results in a savings of 0.53 cents per pound of glucose, which results in a corresponding savings of 7.2 cents per gallon of ethanol upon subsequent fermentation of the hydrolyzate.

  16. Cryptococcus friedmannii, a new species of yeast from the Antarctic (United States)

    Vishniac, H. S.


    Cryptococcus friedmannii Vishniac sp. nov. from an Antarctic cryptoendolithic community is a psychrophilic basidioblastomycete characterized by cream-colored colonies of cells with smooth, layered walls, budding monopolarly, producing amylose and extracellular proteinase, utilizing nitrate and D-alanine (inter alia) as nitrogen sources and L-arabinose, arbutin, cellobiose, D-glucuronate, maltose, melezitose, salicin, soluble starch, trehalose, and D-xylose as carbon sources. This species differs from all other basidiomycetous yeasts in possessing the following combination of characters: amylose production (positive), assimilation of cellobiose (positive), D-galactose (negative), myo-inositol (negative), D-mannitol (negative), and sucrose (negative).

  17. Supplementation of the diet of dairy cows with trehalose results in milk with low lipid peroxide and high antioxidant content. (United States)

    Aoki, N; Furukawa, S; Sato, K; Kurokawa, Y; Kanda, S; Takahashi, Y; Mitsuzumi, H; Itabashi, H


    The objective of this study was to investigate the effect of dietary supplementation with the disaccharides trehalose and cellobiose on antioxidant activity in rumen fluid, blood, and milk of dairy cows. Nine Holstein dairy cows housed in a free-stall barn were divided into 3 groups, with each group receiving a different dietary treatment (a control diet, a 1% trehalose-supplemented diet, or a 1% cellobiose-supplemented diet) following a 3x3 Latin square design. Feed intake and milk production increased in cows receiving the trehalose-supplemented diet compared with those receiving the control and cellobiose-supplemented diets. The total protozoa numbers in the rumen fluid of cows fed trehalose- or cellobiose-supplemented diets were greater than those of the control group. The C18:0 and C18:1 fatty acid content was increased in the milk of cows fed the trehalose-supplemented diet compared with that of the control group, and the C18:3n-3 fatty acid content in the milk of cows fed the cellobiose-supplemented diet was less than that of the control group. Plasma biochemical parameters were unchanged among the different treatments. In rumen fluid, 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and superoxide dismutase activity were increased 2h after feeding in cows receiving the cellobiose-supplemented diet compared with the control group, and the concentration of thiobarbituric acid reactive substances in the rumen fluid of cows fed the cellobiose-supplemented diet was decreased. In contrast, the values of these parameters measured in the milk of cows fed the cellobiose-supplemented diet were no different from those of control cows. Dietary supplementation with trehalose did, however, bring about an improvement of the oxidative status of milk and blood in these animals compared with controls. These results provide the first evidence supporting the use of dietary disaccharides to decrease lipid peroxide levels and increase the antioxidant content of dairy

  18. Enzymes coimmobilized with microorganisms for the microbial conversion of nonmetabolizable substrates

    Energy Technology Data Exchange (ETDEWEB)

    Haegerdal, B.


    EtOH is produced from cellobiose and from lactose by bakers' yeast coimmobilized on Ca alginate with Beta-glucosidase and lactase respectively. The maximum EtOH yield was 2.2%, or 80% of theoretical, when a 5% cellobiose solution was passed through a column containing 2-mm diameter beads with the immobilized enzyme and yeast cells. The EtOH yield was 66% of theoretical when acid whey permeate, containing 4.5% lactose, was passed over the column containing immobilized yeast cells and lactase.

  19. Unprecedented selectivity in molecular recognition of carbohydrates by a metal-organic framework. (United States)

    Yabushita, Mizuho; Li, Peng; Bernales, Varinia; Kobayashi, Hirokazu; Fukuoka, Atsushi; Gagliardi, Laura; Farha, Omar K; Katz, Alexander


    Metal-organic framework (MOF) material NU-1000 adsorbs dimers cellobiose and lactose from aqueous solution, in amounts exceeding 1250 mg gNU-1000(-1) while completely excluding the adsorption of the monomer glucose, even in a competitive mode with cellobiose. The MOF also discriminates between dimers consisting of α and β linkages, showing no adsorption of maltose. Electronic structure calculations demonstrate that key to this selective molecular recognition is the number of favorable CH-π interactions made by the sugar with pyrene units of the MOF.

  20. Studies on cellulose degradation by a Thermoactinimyces Sp

    Energy Technology Data Exchange (ETDEWEB)


    Progress in studies on the mechanism of cellulose degradation by Thermoactinomyces is reported. Two pure cellulosic substrates AVICEL and SOLKA FLOC were used in the experiments. A low substituted carboxymethylcellulose (Hercules 4M CMC), cellobiose, and glucose were also used as growth substrates. Results indicate that glucose is not inhibitory to growth up to 1% concetrations, and that cellobiose may not be a good inducer of the cellobiase enzyme activity. Production of biomass and soluble protein was found to be 50% greater on crystalline AVICEL than on the amorphous SOLKA FLOC, even though approximately the same amount and rate of cellulose degradation occurred. A model for cellulose digestion is presented. (JGB)

  1. Comparison of a commercial biochemical kit and an oligonucleotide probe for identification of environmental isolates of Vibrio vulnificus

    DEFF Research Database (Denmark)

    Dalsgaard, A.; Dalsgaard, Inger; Høi, L.;


    Methods for the identification and isolation of environmental isolates of Vibrio vulnificus were evaluated. Alkaline peptone water supplemented with polymyxin B and colistin-polymyxin B- cellobiose agar were employed for the isolation of suspected V. vulnificus from water, sediment and shellfish ...

  2. Direct ethanol production from starch, wheat bran and rice straw by the white rot fungus Trametes hirsuta

    NARCIS (Netherlands)

    Okamoto, Kenji; Nitta, Yasuyuki; Maekawa, Nitaro; Yanase, Hideshi


    The white rot fungus Trametes hirsuta produced ethanol from a variety of hexoses: glucose, mannose, cellobiose and maltose, with yields of 0.49. 0.48, 0.47 and 0.47 g/g of ethanol per sugar utilized, respectively. In addition, this fungus showed relatively favorable xylose consumption and ethanol pr

  3. Microbial Biosensors for Selective Detection of Disaccharides (United States)

    Seven microbial strains were screened for their ability to detect disaccharides as components of Clark-type oxygen biosensors. Sensors responded to varying degrees to maltose, cellobiose, sucrose, and melibiose, but none responded strongly to lactose. Although microbial sensors are relatively nons...

  4. Alternative lactose catabolic pathway in Lactococcus lactis IL1403

    NARCIS (Netherlands)

    Aleksandrzak-Piekarczyk, T; Kok, J; Renault, P; Bardowski, J


    In this study, we present a glimpse of the diversity of Lactococcus lactis subsp. lactis IL1403 beta-galactosidase phenotype-negative mutants isolated by negative selection on solid media containing cellobiose or lactose and X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), and we identif

  5. Improved radioimmunotherapy of hematologic malignancies. Progress report, November 1, 1993--October 31, 1994

    Energy Technology Data Exchange (ETDEWEB)

    Press, O.W.


    This report summaries progress made during the time interval between November 1, 1993 and October 31, 1994 and briefly describes studies on the metabolism of antibodies targeting B cell antigens, retention of labeled antibodies by human B cell lymphocytes, and tissue distribution of Chloramine T and tyramine cellobiose labeled antibodies in mice harboring a human erythroleukemia tumor transplant.

  6. Comparisons of experiment with cellulose models based on electronic structure and empirical force field theories (United States)

    Studies of cellobiose conformations with HF/6-31G* and B3LYP/6-31+G*quantum theory [1] gave a reference for studies with the much faster empirical methods such as MM3, MM4, CHARMM and AMBER. The quantum studies also enable a substantial reduction in the number of exo-cyclic group orientations that...

  7. Revisiting the Brønsted acid catalysed hydrolysis kinetics of polymeric carbohydrates in ionic liquids by in situ ATR-FTIR spectroscopy

    DEFF Research Database (Denmark)

    Kunov-Kruse, Andreas Jonas; Riisager, Anders; Shunmugavel, Saravanamurugan;


    A new versatile method to measure rates and determine activation energies for the Brønsted acid catalysed hydrolysis of cellulose and cellobiose (and other polymeric carbohydrates) in ionic liquids is demonstrated by following the C–O stretching band of the glycoside bond with in situ ATR-FTIR. A...

  8. Two major facilitator superfamily sugar transporters from Trichoderma reesei and their roles in induction of cellulase biosynthesis. (United States)

    Zhang, Weixin; Kou, Yanbo; Xu, Jintao; Cao, Yanli; Zhao, Guolei; Shao, Jing; Wang, Hai; Wang, Zhixing; Bao, Xiaoming; Chen, Guanjun; Liu, Weifeng


    Proper perception of the extracellular insoluble cellulose is key to initiating the rapid synthesis of cellulases by cellulolytic Trichoderma reesei. Uptake of soluble oligosaccharides derived from cellulose hydrolysis represents a potential point of control in the induced cascade. In this study, we identified a major facilitator superfamily sugar transporter Stp1 capable of transporting cellobiose by reconstructing a cellobiose assimilation system in Saccharomyces cerevisiae. The absence of Stp1 in T. reesei resulted in differential cellulolytic response to Avicel versus cellobiose. Transcriptional profiling revealed a different expression profile in the Δstp1 strain from that of wild-type strain in response to Avicel and demonstrated that Stp1 somehow repressed induction of the bulk of major cellulase and hemicellulose genes. Two other putative major facilitator superfamily sugar transporters were, however, up-regulated in the profiling. Deletion of one of them identified Crt1 that was required for growth and enzymatic activity on cellulose or lactose, but was not required for growth or hemicellulase activity on xylan. The essential role of Crt1 in cellulase induction did not seem to rely on its transporting activity because the overall uptake of cellobiose or sophorose by T. reesei was not compromised in the absence of Crt1. Phylogenetic analysis revealed that orthologs of Crt1 exist in the genomes of many filamentous ascomycete fungi capable of degrading cellulose. These data thus shed new light on the mechanism by which T. reesei senses and transmits the cellulose signal and offers potential strategies for strain improvement.

  9. Characterization of atypical Candida tropicalis and other uncommon clinical yeast isolates.


    Schlitzer, R L; Ahearn, D G


    Clinical yeast isolates representing alpha-glucoside-deficient variants of Candida tropicalis, C. lusitaniae, atypical C. albicans, and Saccharomyces cerevisiae were characterized. Additional physiological tests, including cellobiose fermentation, rhamnose assimilation, and triphenyl tetrazolium chloride reduction, are recommended for the detection and presumptive identification of uncommon Candida spp. in the clinical laboratory.

  10. Photoinduced Biohydrogen Production from Biomass

    Directory of Open Access Journals (Sweden)

    Yutaka Amao


    Full Text Available Photoinduced biohydrogen production systems, coupling saccharaides biomass such as sucrose, maltose, cellobiose, cellulose, or saccharides mixture hydrolysis by enzymes and glucose dehydrogenase (GDH, and hydrogen production with platinum colloid as a catalyst using the visible light-induced photosensitization of Mg chlorophyll-a (Mg Chl-a from higher green plant or artificial chlorophyll analog, zinc porphyrin, are introduced.

  11. Production of aromatic hydrocarbons via catalytic pyrolysis of biomass over fe-modified HZSM-5 zeolites (United States)

    Iron modified HZSM-5 catalysts were prepared by partial ion exchange of NH4ZSM-5 with Fe (II) at three different loadings (1.4, 2.8 and 4.2 wt%), and their effectiveness for producing aromatic hydrocarbons from cellulose, cellobiose, lignin and switchgrass by catalytic pyrolysis were screened using ...

  12. The Role of Product Inhibition as a Yield-Determining Factor in Enzymatic High-Solid Hydrolysis of Pretreated Corn Stover

    DEFF Research Database (Denmark)

    Nymand Olsen, Søren; Borch, Kim; Cruys-Bagger, Nicolaj


    . The results suggest that the solid effect is mainly controlled by product inhibition under the given experimental conditions (washed pretreated corn stover as substrate). Cellobiose was found to be approximately 15 times more inhibitory than glucose on a molar scale. However, considering that glucose...

  13. Visualizing Structure and Dynamics of Disaccharide Simulations

    Energy Technology Data Exchange (ETDEWEB)

    Matthews, J. F.; Beckham, G. T.; Himmel, M. E.; Crowley, M. F.


    We examine the effect of several solvent models on the conformational properties and dynamics of disaccharides such as cellobiose and lactose. Significant variation in timescale for large scale conformational transformations are observed. Molecular dynamics simulation provides enough detail to enable insight through visualization of multidimensional data sets. We present a new way to visualize conformational space for disaccharides with Ramachandran plots.

  14. Covalent Immobilization of β-Glucosidase on Magnetic Particles for Lignocellulose Hydrolysis

    DEFF Research Database (Denmark)

    Alftrén, Johan; Hobley, Timothy John


    β-Glucosidase hydrolyzes cellobiose to glucose and is an important enzyme in the consortium used for hydrolysis of cellulosic and lignocellulosic feedstocks. In the present work, β-glucosidase was covalently immobilized on non-porous magnetic particles to enable re-use of the enzyme. It was found...

  15. Utilization of individual cellodextrins by three predominant ruminal cellulolytic bacteria.


    Shi, Y; Weimer, P J


    Growth of the ruminal bacteria Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and R. albus 7 followed Monod kinetics with respect to concentrations of individual pure cellodextrins (cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose). Under the conditions tested, R. flavefaciens FD-1 possesses the greatest capacity to compete for low concentrations of these cellodextrins.

  16. Approaches to new derivatives of cellulose as designed pharmaceutical excipients

    Directory of Open Access Journals (Sweden)

    Schwarz Brigitte


    Full Text Available Recently, our group initiated a synthetic program directed at new derivatives of cellulose intended as novel pharmaceutical excipients. With several notable exceptions, the attempted regioselective introduction of chemical functionality into natural cellulose by direct chemical modification will result in heterogeneous products that are difficult to characterize and the preparation of which is insufficiently reproduceable. Approaches to the chemical polymerization of appropriate glucose monomers are available, leading to a degree of polymerization in the order of 100. However, the nature of these processes does not readily lend itself to the preparation of products comprising regularly arranged protecting groups in defined positions. We have chosen a mixed organic chemical-enzyme catalyzed approach based on a procedure of Kobayashi, Shoda, Donnelly and Church. Fluoride derivatives of cellobiose may be polymerized, under catalysis by cellobiose hydrolase, to form cellulose oligosaccharides of different chain lengths. We describe the chemical syntheses of cellobiose fluoride derivatives comprising protecting groups in defined positions of the reducing or nonreducing glucose moieties of cellobiose. Such derivatives may be polymerized to afford cellulose derivatives with protecting groups on alternate glucose units. The processing of these protected cellulose derivatives to afford novel biomimetic polymers will be described.

  17. The anaerobic fungus Neocallimastix sp. strain L2 : Growth and production of (Hemi)cellulolytic enzymes on a range of carbohydrate substrates

    NARCIS (Netherlands)

    Dijkerman, R; Ledeboer, J; op den Camp, H.J M; Prins, R.A; van der Drift, C


    The anaerobic fungus Neocallimastix sp. strain L2, isolated from the feces of a Ilama, was tested for growth on a range of soluble and insoluble carbohydrate substrates. The fungus was able to ferment glucose, cellobiose, fructose, lactose, maltose, sucrose, soluble starch, inulin, filter paper cell

  18. Isolation and characterization of a ß-glucosidase from a Clavispora strain with potential applications in bioethanol production from cellulosic materials (United States)

    We previously reported on a new yeast strain of Clavispora sp. NRRL Y-50464 that is capable of utilizing cellobiose as sole source of carbon and energy by producing sufficient native ß-glucosidase enzyme activity without further enzyme supplementation for cellulosic ethanol production using simultan...

  19. Regulation of carbon catabolism in Lactococcus lactis.

    NARCIS (Netherlands)

    Aleksandrzak, T; Kowalczyk, M; Kok, J; Bardowski, J; Bielecki, S; Tramper, J; Polak, J


    The Lactococcus lactis IL1403 is a lactose negative, plasmid free strain. Nevertheless, it is able to hydrolyze lactose in the presence of cellobiose. In this work we describe identification of a gene involved in this process. The gene was found to be homologous to the sugar catabolism regulator, cc

  20. Fast and reliable production, purification and characterization of heat-stable, bifunctional enzyme chimeras. (United States)

    Neddersen, Mara; Elleuche, Skander


    Degradation of complex plant biomass demands a fine-regulated portfolio of glycoside hydrolases. The LE (LguI/Eco81I)-cloning approach was used to produce two enzyme chimeras CB and BC composed of an endoglucanase Cel5A (C) from the extreme thermophilic bacterium Fervidobacterium gondwanense and an archaeal β-glucosidase Bgl1 (B) derived from a hydrothermal spring metagenome. Recombinant chimeras and parental enzymes were produced in Escherichia coli and purified using a two-step affinity chromatography approach. Enzymatic properties revealed that both chimeras closely resemble the parental enzymes and physical mixtures, but Cel5A displayed lower temperature tolerance at 100°C when fused to Bgl1 independent of the conformational order. Moreover, the determination of enzymatic performances resulted in the detection of additive effects in case of BC fusion chimera. Kinetic measurements in combination with HPLC-mediated product analyses and site-directed mutation constructs indicated that Cel5A was strongly impaired when fused at the N-terminus, while activity was reduced to a slighter extend as C-terminal fusion partner. In contrast to these results, catalytic activity of Bgl1 at the N-terminus was improved 1.2-fold, effectively counteracting the slightly reduced activity of Cel5A by converting cellobiose into glucose. In addition, cellobiose exhibited inhibitory effects on Cel5A, resulting in a higher yield of cellobiose and glucose by application of an enzyme mixture (53.1%) compared to cellobiose produced from endoglucanase alone (10.9%). However, the overall release of cellobiose and glucose was even increased by catalytic action of BC (59.2%). These results indicate possible advantages of easily produced bifunctional fusion enzymes for the improved conversion of complex polysaccharide plant materials.

  1. Hydrogen production from cellulose in a two-stage process combining fermentation and electrohydrogenesis

    KAUST Repository

    Lalaurette, Elodie


    A two-stage dark-fermentation and electrohydrogenesis process was used to convert the recalcitrant lignocellulosic materials into hydrogen gas at high yields and rates. Fermentation using Clostridium thermocellum produced 1.67 mol H2/mol-glucose at a rate of 0.25 L H2/L-d with a corn stover lignocellulose feed, and 1.64 mol H2/mol-glucose and 1.65 L H2/L-d with a cellobiose feed. The lignocelluose and cellobiose fermentation effluent consisted primarily of: acetic, lactic, succinic, and formic acids and ethanol. An additional 800 ± 290 mL H2/g-COD was produced from a synthetic effluent with a wastewater inoculum (fermentation effluent inoculum; FEI) by electrohydrogensis using microbial electrolysis cells (MECs). Hydrogen yields were increased to 980 ± 110 mL H2/g-COD with the synthetic effluent by combining in the inoculum samples from multiple microbial fuel cells (MFCs) each pre-acclimated to a single substrate (single substrate inocula; SSI). Hydrogen yields and production rates with SSI and the actual fermentation effluents were 980 ± 110 mL/g-COD and 1.11 ± 0.13 L/L-d (synthetic); 900 ± 140 mL/g-COD and 0.96 ± 0.16 L/L-d (cellobiose); and 750 ± 180 mL/g-COD and 1.00 ± 0.19 L/L-d (lignocellulose). A maximum hydrogen production rate of 1.11 ± 0.13 L H2/L reactor/d was produced with synthetic effluent. Energy efficiencies based on electricity needed for the MEC using SSI were 270 ± 20% for the synthetic effluent, 230 ± 50% for lignocellulose effluent and 220 ± 30% for the cellobiose effluent. COD removals were ∼90% for the synthetic effluents, and 70-85% based on VFA removal (65% COD removal) with the cellobiose and lignocellulose effluent. The overall hydrogen yield was 9.95 mol-H2/mol-glucose for the cellobiose. These results show that pre-acclimation of MFCs to single substrates improves performance with a complex mixture of substrates, and that high hydrogen yields and gas production rates can be achieved using a two-stage fermentation and MEC

  2. Enhanced enzymatic cellulose degradation by cellobiohydrolases via product removal

    DEFF Research Database (Denmark)

    Ahmadi Gavlighi, Hassan; Meyer, Anne S.; Mikkelsen, Jørn Dalgaard


    Product inhibition by cellobiose decreases the rate of enzymatic cellulose degradation. The optimal reaction conditions for two Emericella (Aspergillus) nidulans-derived cellobiohydrolases I and II produced in Pichia pastoris were identified as CBHI: 52 °C, pH 4.5–6.5, and CBHII: 46 °C, pH 4.......8. The optimum in a mixture of the two was 50 °C, pH 4.9. An almost fourfold increase in enzymatic hydrolysis yield was achieved with intermittent product removal of cellobiose with membrane filtration (2 kDa cut-off): The conversion of cotton cellulose after 72 h was ~19 % by weight, whereas the conversion...

  3. Activation energies of fragmentations of disaccharides by tandem mass spectrometry. (United States)

    Kuki, Ákos; Nagy, Lajos; Szabó, Katalin E; Antal, Borbála; Zsuga, Miklós; Kéki, Sándor


    A simple multiple collision model for collision induced dissociation (CID) in quadrupole was applied for the estimation of the activation energy (E(o)) of the fragmentation processes for lithiated and trifluoroacetated disaccharides, such as maltose, cellobiose, isomaltose, gentiobiose, and trehalose. The internal energy-dependent rate constants k(E(int)) were calculated using the Rice-Ramsperger-Kassel-Marcus (RRKM) or the Rice-Ramsperger-Kassel (RRK) theory. The E(o) values were estimated by fitting the calculated survival yield (SY) curves to the experimental ones. The calculated E(o) values of the fragmentation processes for lithiated disaccharides were in the range of 1.4-1.7 eV, and were found to increase in the order trehalose < maltose < isomaltose < cellobiose < gentiobiose.

  4. Efficient simultaneous saccharification and fermentation of agricultural residues by Saccharomyces cerevisiae and Candida shehatae. The D-xylose fermenting yeast. (United States)

    Palnitkar, S S; Lachke, A H


    Simultaneous Saccharification and Fermentation (SSF) experiments were carried out on agricultural residues using culture filtrate of Sclerotium rolfsii, which produces high levels of cellulases and hemicellulases for the saccharification of rice straw and bagasse, and Candida shehatae--the D-xylose fermenting yeast, and Saccharomyces cerevisiae, both separately and in coculture, for fermenting the released sugars. The coculture system showed efficient utilization of hydrolyzed sugars with 30-38% and 10-13% increase in ethanol production as compared to C. shehatae and S. cerevisiae, respectively, when cultivated separately. SSF simulation studies were carried out using standard sugar mixtures of glucose, xylose, and cellobiose. Both organisms could not use cellobiose, whereas glucose was used preferentially. C. shehatae was capable of utilizing xylose in the presence of glucose.

  5. Cellulose hydrolysis by Trichoderma reesei cellulases: studies on adsorption, sugar production and synergism of cellobiohydrolase I,II and endoglucanase II

    Energy Technology Data Exchange (ETDEWEB)

    Medve, J.


    Three major cellulases have been purified by ion-exchange chromatography in an FPLC system. Microcrystalline cellulose (Avicel) was hydrolyzed by the single enzymes and by equimolar mixtures of CBH I-CBH II and CBH I-EG II. Enzyme adsorption was followed indirectly by selectively quantifying the enzymes in the supernatant by ion-exchange chromatography in an FPLC system. The (synergistic) production of small, soluble sugars (glucose, cellobiose and cellotriose) by the enzymes was followed by HPLC. 76 refs

  6. Observations and Measurements of Planktonic Bioluminescence in and Around a Milky Sea (United States)


    produced by plankton subjected to mechanical stimulation) can be observed from breaking wave crests and swimming shoals of fish . The Arabian Sea is...identification: growth at 4 ’C, growth at 35 ’C, ainylase, lipase , gelatinase. growth on maltose, cellobiose, gluconate, BIOLUMINESCENCE IN MILKY SEA 57...neofluar oil -immersion objective. BIOLUMINESCENCE MEASUREMENTS Surface-water bioluminescence Surface-water bioluminescence was measured continuously during

  7. Enzymatic synthesis of beta-glucosylglycerol using a continuous-flow microreactor containing thermostable beta-glycoside hydrolase CelB immobilized on coated microchannel walls. (United States)

    Schwarz, Alexandra; Thomsen, Malene S; Nidetzky, Bernd


    beta-Glucosylglycerol (betaGG) has potential applications as a moisturizing agent in cosmetic products. A stereochemically selective method of its synthesis is kinetically controlled enzymatic transglucosylation from a suitable donor substrate to glycerol as acceptor. Here, the thermostable beta-glycosidase CelB from Pyrococcus furiosus was used to develop a microstructured immobilized enzyme reactor for production of betaGG under conditions of continuous flow at 70 degrees C. Using CelB covalently attached onto coated microchannel walls to give an effective enzyme activity of 30 U per total reactor working volume of 25 microL, substrate conversion and formation of transglucosylation product was monitored in dependence of glucosyl donor (2-nitrophenyl-beta-D-glucoside (oNPGlc), 3.0 or 15 mM; cellobiose, 250 mM), the concentration of glycerol (0.25-1.0 M), and the average residence time (0.2-90 s). Glycerol caused a concentration-dependent decrease in the conversion of the glucosyl donor via hydrolysis and strongly suppressed participation of the substrate in the reaction as glucosyl acceptor. The yields of betaGG were > or =80% and approximately 60% based on oNPGlc and cellobiose converted, respectively, and maintained up to near exhaustion of substrate (> or =80%), giving about 120 mM (30 g/L) of betaGG from the reaction of cellobiose and 1 M glycerol. The structure of the transglucosylation products, 1-O-beta-D-glucopyranosyl-rac-glycerol (79%) and 2-O-beta-D-glucopyranosyl-sn-glycerol (21%), was derived from NMR analysis of the product mixture of cellobiose conversion. The microstructured reactor showed conversion characteristics similar to those for a batchwise operated stirred reactor employing soluble CelB. The advantage of miniaturization to the microfluidic format lies in the fast characterization of full reaction time courses for a range of process conditions using only a minimum amount of enzyme.

  8. Use of rapid auxanographic procedures for recognition of an atypical Candida.


    Baker, J. G.; Salkin, I F; Pincus, D. H.; D'Amato, R F


    An atypical Candida which can cause diagnostic problems in clinical laboratories has recently been characterized. Assimilation patterns of 29 clinical isolates of an atypical Candida were obtained by the API 30C (Analytab Products, Plainview, N.Y.)., Uni-Yeast-Tek (Flow Laboratories, Inc., Rockville, Md.), and dye pour-plate auxanographic methods. The low frequency of assimilation of cellobiose, sucrose, and melezitose noted in all of these procedures permitted the early recognition of the at...

  9. Thermal Diffusion of Oligosaccharide Solutions: The Role of Chain Length and Structure



    We investigated the chain length dependence of the thermodiffusion behavior of oligosaccharides by the infrared thermal diffusion forced Rayleigh scattering (IR-TDFRS) technique. Three disaccharides, sucrose, cellobiose and maltose, two trisaccharides, melezitose and raffinose, and a tetrasaccharide, stachyose, have been studied. We determined the thermal diffusion (D(T)), mass diffusion (D), and Soret (S(T)) coefficient as a function of temperature and concentration. While monosaccharides in...

  10. Kinetics and Regulation Studies of the Production of β-Galactosidase from Kluyveromyces marxianus Grown on Different Substrates



    Lactose-intolerance is manifested in 50 % of the world’s population. This can be remediated by removing lactose from the diet or converting it into glucose and galactose with β-galactosidase (EC In this work, batch production of this enzyme in the presence of lactose, galactose, cellobiose, xylose, arabinose, sucrose and glucose was investigated using Kluyveromyces marxianus in shake flask culture studies. Substrate type and temperature were the independent variables that directly ...

  11. Functional Studies of β-Glucosidases of Cytophaga hutchinsonii and Their Effects on Cellulose Degradation (United States)

    Bai, Xinfeng; Wang, Xifeng; Wang, Sen; Ji, Xiaofei; Guan, Zhiwei; Zhang, Weican; Lu, Xuemei


    Cytophaga hutchinsonii can rapidly digest crystalline cellulose without free cellulases or cellulosomes. Its cell-contact cellulose degradation mechanism is unknown. In this study, the four β-glucosidase (bgl) genes in C. hutchinsonii were singly and multiply deleted, and the functions of these β-glucosidases in cellobiose and cellulose degradation were investigated. We found that the constitutively expressed BglB played a key role in cellobiose utilization, while BglA which was induced by cellobiose could partially make up for the deletion of bglB. The double deletion mutant ΔbglA/bglB lost the ability to digest cellobiose and could not thrive in cellulose medium, indicating that β-glucosidases were important for cellulose degradation. When cultured in cellulose medium, a small amount of glucose accumulated in the medium in the initial stage of growth for the wild type, while almost no glucose accumulated for ΔbglA/bglB. When supplemented with a small amount of glucose, ΔbglA/bglB started to degrade cellulose and grew in cellulose medium. We inferred that glucose might be essential for initiating cellulose degradation, and with additional glucose, C. hutchinsonii could partially utilize cellulose without β-glucosidases. We also found that there were both cellulose binding cells and free cells when cultured in cellulose. Since direct contact between C. hutchinsonii cells and cellulose is necessary for cellulose degradation, we deduced that the free cells which were convenient to explore new territory in the environment might be fed by the adherent cells which could produce cello-oligosaccharide and glucose into the environment. This study enriched our knowledge of the cellulolytic pathway of C. hutchinsonii. PMID:28210251

  12. Cellulose digestion in heterotmes indicola, Wasmann and Coptotermes HIEMI Wasmann

    Directory of Open Access Journals (Sweden)

    J. N. Misra


    Full Text Available High activities of cellulose and cellobiase have been found in the gut extracts of the worker caste of two species of Heterotermes indicola, Wasmann and Coptotermes heimi, Wasmann. The properties of the two enzymes from H. Indicola have been investigated. It has been found that the soldier caste of these termites is capable of splitting cellobiose while incapable of breaking down cellulose into simpler sugars.

  13. Validation of Inhibition Effect in the Cellulose Hydrolysis: a Dynamic Modelling Approach

    DEFF Research Database (Denmark)

    Morales Rodriguez, Ricardo; Tsai, Chien-Tai; Meyer, Anne S.;


    Enzymatic hydrolysis is one of the main steps in the processing of bioethanol from lignocellulosic raw materials. However, complete understanding of the underlying phenomena is still under development. Hence, this study has focused on validation of the inhibition effects in the cellulosic biomass...... hydrolysis employing a dynamic mathematical model. A systematic framework for parameter estimation is used for model validation, which helps overcome the problem of parameter correlation. Data sets obtained from carefully designed enzymatic cellulose and cellobiose hydrolysis experiments, were used...

  14. Cloning of a Gene Cluster from Cellvibrio mixtus which Codes for Cellulase, Chitinase, Amylase, and Pectinase



    The soil isolate Cellvibrio mixtus UQM2294 degraded a variety of polysaccharides including microcrystalline cellulose. Among 6,000 cosmid clones carrying C. mixtus DNA, constructed in Escherichia coli with pHC79, 50 expressed the ability to degrade one or more of the following substrates: carboxymethyl cellulose, chitin, pectin (polygalacturonic acid), cellobiose, and starch. These degradative genes are encoded in a single 94.1-kilobase segment of the C. mixtus genome; a preliminary order of ...

  15. Cellulolytic Activity of Clostridium acetobutylicum


    Lee, Song F.; Forsberg, Cecil W.; Gibbins, L N


    Clostridium acetobutylicum NRRL B527 and ATCC 824 exhibited extracellular and cell-bound endoglucanase and cellobiase activities during growth in a chemically defined medium with cellobiose as the sole source of carbohydrate. For both strains, the endoglucanase was found to be mainly extracellular (70 to 90%) during growth in continuous or batch cultures with the pH maintained at 5.2, whereas the cellobiase was mainly cell associated (60 to 90%). During continuous cultivation of strain B527 w...

  16. Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples.


    Berlemont, Renaud; Delsaute, Maud; Pipers, Delphine; D'Amico, Salvino; Feller, Georges; Galleni, Moreno; Power, Pablo


    In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carb...

  17. Deletion of a gene cluster for [Ni-Fe] hydrogenase maturation in the anaerobic hyperthermophilic bacterium Caldicellulosiruptor bescii identifies its role in hydrogen metabolism. (United States)

    Cha, Minseok; Chung, Daehwan; Westpheling, Janet


    The anaerobic, hyperthermophlic, cellulolytic bacterium Caldicellulosiruptor bescii grows optimally at ∼80 °C and effectively degrades plant biomass without conventional pretreatment. It utilizes a variety of carbohydrate carbon sources, including both C5 and C6 sugars, released from plant biomass and produces lactate, acetate, CO2, and H2 as primary fermentation products. The C. bescii genome encodes two hydrogenases, a bifurcating [Fe-Fe] hydrogenase and a [Ni-Fe] hydrogenase. The [Ni-Fe] hydrogenase is the most widely distributed in nature and is predicted to catalyze hydrogen production and to pump protons across the cellular membrane creating proton motive force. Hydrogenases are the key enzymes in hydrogen metabolism and their crystal structure reveals complexity in the organization of their prosthetic groups suggesting extensive maturation of the primary protein. Here, we report the deletion of a cluster of genes, hypABFCDE, required for maturation of the [Ni-Fe] hydrogenase. These proteins are specific for the hydrogenases they modify and are required for hydrogenase activity. The deletion strain grew more slowly than the wild type or the parent strain and produced slightly less hydrogen overall, but more hydrogen per mole of cellobiose. Acetate yield per mole of cellobiose was increased ∼67 % and ethanol yield per mole of cellobiose was decreased ∼39 %. These data suggest that the primary role of the [Ni-Fe] hydrogenase is to generate a proton gradient in the membrane driving ATP synthesis and is not the primary enzyme for hydrogen catalysis. In its absence, ATP is generated from increased acetate production resulting in more hydrogen produced per mole of cellobiose.

  18. Induction of cellulose in Schizophyllum commune: thiocellobiose as a new inducer.



    Several mono-, di, tetra-, and polysaccharides were screened for their ability to induced cellulase production by the tetrapolar hymenomycete Schizophyllum commune. Out of 21 carbohydrates screened, 4 (thiocellobiose, carboxymethylcellulose, cellobiose, and xylan) induced all three enzymes tested (carboxymethylcellulase, beta-glucosidase, and xylanase). The inducing effect increased with rising concentrations of the inducers up to a certain value, beyond which there was either a leveling off ...

  19. Genomics of aerobic cellulose utilization systems in actinobacteria.

    Directory of Open Access Journals (Sweden)

    Iain Anderson

    Full Text Available Cellulose degrading enzymes have important functions in the biotechnology industry, including the production of biofuels from lignocellulosic biomass. Anaerobes including Clostridium species organize cellulases and other glycosyl hydrolases into large complexes known as cellulosomes. In contrast, aerobic actinobacteria utilize systems comprised of independently acting enzymes, often with carbohydrate binding domains. Numerous actinobacterial genomes have become available through the Genomic Encyclopedia of Bacteria and Archaea (GEBA project. We identified putative cellulose-degrading enzymes belonging to families GH5, GH6, GH8, GH9, GH12, GH48, and GH51 in the genomes of eleven members of the actinobacteria. The eleven organisms were tested in several assays for cellulose degradation, and eight of the organisms showed evidence of cellulase activity. The three with the highest cellulase activity were Actinosynnema mirum, Cellulomonas flavigena, and Xylanimonas cellulosilytica. Cellobiose is known to induce cellulolytic enzymes in the model organism Thermobifida fusca, but only Nocardiopsis dassonvillei showed higher cellulolytic activity in the presence of cellobiose. In T. fusca, cellulases and a putative cellobiose ABC transporter are regulated by the transcriptional regulator CelR. Nine organisms appear to use the CelR site or a closely related binding site to regulate an ABC transporter. In some, CelR also regulates cellulases, while cellulases are controlled by different regulatory sites in three organisms. Mining of genome data for cellulose degradative enzymes followed by experimental verification successfully identified several actinobacteria species which were not previously known to degrade cellulose as cellulolytic organisms.

  20. Synthesis of multifunctional bioresponsive polymers for the management of chronic wounds. (United States)

    Nyanhongo, Gibson S; Sygmund, Christoph; Ludwig, Roland; Prasetyo, Endry Nugroho; Guebitz, Georg M


    Novel multifunctional bioresponsive gelatin and alginate based hydrogels with in-built antioxidant regenerating system and antimicrobial properties were successfully synthesized. These hydrogels are based on the versatile reactions catalyzed by cellobiose dehydrogenase (CDH). CDH uses cellobiose and cello-oligosacharides as electron donors to reduce oxidized phenolic antioxidants, quinones, or molecular oxygen to H₂O₂ (a well-known antimicrobial agent). The antioxidant regenerating system consisting of CDH and cellobiose increased the ability of catechol to quench nitric oxide (NO), superoxide (O₂⁻) and hydroxyl radicals (OH•) in solution and when incorporated into hydrogels. The CDH loaded into the hydrogels free of oxidized phenolic antioxidants and quinones reduced molecular to H₂O₂ resulting in the complete inhibition of the growth of Stapylococcus aeureus, Bacillus subtilis, Pseudomonas putida, Escherichia coli and Cellulomonasmicrobium cellulans. This study therefore presents a new concept for synthesizing multifunctional bioresponsive chronic wound dressing polymers with in-built continuous antioxidant system able to continuously quench [reactive oxygen species (ROS) and reactive nitrogen species (RNOS)], and antimicrobial properties able to prevent microbial colonization of wound.

  1. Fundamental study of the mechanism and kinetics of cellulose hydrolysis by acids and enzymes. Final report, June 1, 1978-January 31, 1981

    Energy Technology Data Exchange (ETDEWEB)

    Gong, C.S.; Chang, M.


    There are three basic enzymes (e.g., endoglucanase (C/sub x/), exoglucanase (C/sub 1/) and cellobiase) comprising the majority of extracellular cellulase enzymes produced by the cellulolytic mycelial fungi, Trichoderma reesei, and other cellulolytic microorganisms. The enzymes exhibited different mode of actions in respect to the hydrolysis of cellulose and cellulose derived oligosaccharides. In combination, these enzymes complimented each other to hydrolyze cellulose to its basic constituent, glucose. The kinetics of cellobiase were developed on the basis of applying the pseudo-steady state assumption to hydrolyze cellobiose to glucose. The results indicated that cellobiase was subjected to end-product inhibition by glucose. The kinetic modeling of exoglucanase (C/sub 1/) with respect to cellodextrins was studied. Both glucose and cellobiose were found to be inhibitors of this enzyme with cellobiose being a stronger inhibitor than glucose. Similarly, endoglucanase (C/sub x/) is subject to end-product inhibition by glucose. Crystallinity of the cellulose affects the rate of hydrolysis by cellulases. Hence, the changes in crystallinity of cellulose in relation to chemical pretreatment and enzyme hydrolysis was compared. The study of cellulase biosynthesis resulted in the conclusion that exo- and endo-glucanases are co-induced while cellobiase is synthesized independent of the other two enzymes. The multiplicity of cellulase enzymes are the end results of post-translational modification during and/or after the secretion of enzymes into growth environment.

  2. Extracellular gluco-oligosaccharide degradation by Caulobacter crescentus. (United States)

    Presley, Gerald N; Payea, Matthew J; Hurst, Logan R; Egan, Annie E; Martin, Brandon S; Periyannan, Gopal R


    The oligotrophic bacterium Caulobacter crescentus has the ability to metabolize various organic molecules, including plant structural carbohydrates, as a carbon source. The nature of β-glucosidase (BGL)-mediated gluco-oligosaccharide degradation and nutrient transport across the outer membrane in C. crescentus was investigated. All gluco-oligosaccharides tested (up to celloheptose) supported growth in M2 minimal media but not cellulose or CM-cellulose. The periplasmic and outer membrane fractions showed highest BGL activity, but no significant BGL activity was observed in the cytosol or extracellular medium. Cells grown in cellobiose showed expression of specific BGLs and TonB-dependent receptors (TBDRs). Carbonyl cyanide 3-chlorophenylhydrazone lowered the rate of cell growth in cellobiose but not in glucose, indicating potential cellobiose transport into the cell by a proton motive force-dependent process, such as TBDR-dependent transport, and facilitated diffusion of glucose across the outer membrane via specific porins. These results suggest that C. crescentus acquires carbon from cellulose-derived gluco-oligosaccharides found in the environment by extracellular and periplasmic BGL activity and TBDR-mediated transport. This report on extracellular degradation of gluco-oligosaccharides and methods of nutrient acquisition by C. crescentus supports a broader suite of carbohydrate metabolic capabilities suggested by the C. crescentus genome sequence that until now have not been reported.

  3. The electronic nature of the 1,4-β-glycosidic bond and its chemical environment: DFT insights into cellulose chemistry. (United States)

    Loerbroks, Claudia; Rinaldi, Roberto; Thiel, Walter


    The molecular understanding of the chemistry of 1,4-β-glucans is essential for designing new approaches to the conversion of cellulose into platform chemicals and biofuels. In this endeavor, much attention has been paid to the role of hydrogen bonding occurring in the cellulose structure. So far, however, there has been little discussion about the implications of the electronic nature of the 1,4-β-glycosidic bond and its chemical environment for the activation of 1,4-β-glucans toward acid-catalyzed hydrolysis. This report sheds light on these central issues and addresses their influence on the acid hydrolysis of cellobiose and, by analogy, cellulose. The electronic structure of cellobiose was explored by DFT at the BB1 K/6-31++G(d,p) level. Natural bond orbital (NBO) analysis was performed to grasp the key bonding concepts. Conformations, protonation sites, and hydrolysis mechanisms were examined. The results for cellobiose indicate that cellulose is protected against hydrolysis not only by its supramolecular structure, as currently accepted, but also by its electronic structure, in which the anomeric effect plays a key role.

  4. Purification and characterization of beta-glucosidase of Alcaligenes faecalis. (United States)

    Han, Y W; Srinivasan, V R


    A cellobiose-utilizing bacterium isolated from sugar cane bagasse and identified as a strain of Alcaligenes faecalis (ATCC 21400) produced an inducible beta-glucoside-splitting enzyme. The enzyme was purified by a series of streptomycin and ammonium sulfate fractionations and by Sephadex and diethylaminoethyl column chromatography. The final preparation was purified 130-fold, with a recovery of about 10% of the initial enzyme activity. The enzyme had a wide pH range, with optimal activity at pH 6.0 to 7.0. The enzyme was stable in solution at pH 6.5 to 7.8 when kept at 30 C for 2 hr, but it was destroyed by temperatures above 55 C. At 58 and 60 C, the time required to inactivate 90% of the initial activity was 16 and 6.5 min, respectively. An activation energy of 9,500 cal/mole and a K(m) of 1.25 x 10(-4)m were obtained by using p-nitrophenyl beta-glucoside as a substrate. The K(i) value and hydrolysis of cellobiose by the enzyme indicated a high affinity of the enzyme for the cellobiose. The enzyme had its specificity on beta-glucosidic linkage and the rate of hydrolisis of glucosides depended upon the nature of the aglycon moiety. The inactivation studies showed the presence of sulfhydryl groups in the enzyme. The activity of the enzyme was easily destroyed by the Cu(++) and Hg(++) ions. The Michaelis-Menton relationship and the rate of heat inactivation indicated the presence of one type of noninteracting active site in the bacterial beta-glucosidase. Molecular weight of the enzyme was estimated by gel filtration (Sephadex G-200) and sucrose density gradient, and a value of 120,000 to 160,000 was obtained.

  5. [Studies of the genetic regulation of the Thermomonospora cellulase complex]. Progress report, June 1, 1990--January 10, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, D.B.


    The goals of this project are to determine the molecular mechanisms regulating cellulose synthesis in the soil bacterium Thermomonosporafusca and to determine the molecular mechanism by which T.fusca cellulases degrade crystalline cellulose. We have determined a structure for the T.fusca E{sub 2} catalytic subunit (E{sub 2}-30) by x-ray crystallography. This structure is quite similar to that of T.reesei CBHU but there are a number of differences. One is that the E{sub 2} active site is in a cleft while that of CBHII is in a tunnel. This is an expected result since E{sub 2} is an endocellulase. Large amounts of homogenous E{sub 5} catalytic subunit have been prepared and attempts to crystallize it are underway. Crystals of E{sub 2}-30 were soaked in cellobiose and modified crystals detracted well, however difference Fourier analysis showed many changes, so that we could not localize cellobiose in the 3-D structure of E{sub 2}-30. This implies that binding of cellobiose causes a significant change in the structure of E{sub 2}-30. The stereochemistry of the cleavage catalyzed by E{sub l}, E{sub 2} and E{sub 5} was determined in collaboration with Dr. Stephen Withers and E{sub 1} and 2 inverted the glycoside linkage while E{sub 5} does not. The entire E{sub l} and E{sub 4} genes have been induced into Streptomyces lividans where they are expressed at a high level and the E{sub l} and E{sub 4} are completely secreted into the medium. Studies on the synergism between the exocellulase E{sub 3} and the endocellulases E{sub 2} or E{sub 5} show that both exo and endocellulase activities are stimulated when they are assayed together.

  6. (Studies of the genetic regulation of the Thermomonospora cellulase complex)

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, D.B.


    The goals of this project are to determine the molecular mechanisms regulating cellulose synthesis in the soil bacterium Thermomonosporafusca and to determine the molecular mechanism by which T.fusca cellulases degrade crystalline cellulose. We have determined a structure for the T.fusca E{sub 2} catalytic subunit (E{sub 2}-30) by x-ray crystallography. This structure is quite similar to that of T.reesei CBHU but there are a number of differences. One is that the E{sub 2} active site is in a cleft while that of CBHII is in a tunnel. This is an expected result since E{sub 2} is an endocellulase. Large amounts of homogenous E{sub 5} catalytic subunit have been prepared and attempts to crystallize it are underway. Crystals of E{sub 2}-30 were soaked in cellobiose and modified crystals detracted well, however difference Fourier analysis showed many changes, so that we could not localize cellobiose in the 3-D structure of E{sub 2}-30. This implies that binding of cellobiose causes a significant change in the structure of E{sub 2}-30. The stereochemistry of the cleavage catalyzed by E{sub l}, E{sub 2} and E{sub 5} was determined in collaboration with Dr. Stephen Withers and E{sub 1} and 2 inverted the glycoside linkage while E{sub 5} does not. The entire E{sub l} and E{sub 4} genes have been induced into Streptomyces lividans where they are expressed at a high level and the E{sub l} and E{sub 4} are completely secreted into the medium. Studies on the synergism between the exocellulase E{sub 3} and the endocellulases E{sub 2} or E{sub 5} show that both exo and endocellulase activities are stimulated when they are assayed together.

  7. Degradation of cellulosic biomass and its subsequent utilization for the production of chemical feedstocks. Progress report, June 1, 1977--August 31, 1977

    Energy Technology Data Exchange (ETDEWEB)

    Wang, D.I.C.; Cooney, C.L.; Demain, A.L.; Gomez, R.F.; Sinskey, A.J.


    Studies on the microbial degradation of cellulose biomass continues to be centered around Clostridium thermocellum. The effect of surfactants on growth and cellulase production by C. thermocellum was investigated. The effect of pH on growth and reducing sugar accumulation rate of Clostridium thermocellum on solka floc was evaluated. Activity of extracellular cellulase of Clostridium thermocellum ATCC 27405 was examined using TNP--CMC and Avicel as substrates. The pH optima are 5 and 4.5, respectively. Hydrolysis of either substrate is not inhibited by cellobiose, xylose, or glucose. The enzyme appears to be quite stable under reaction conditions at 60/sup 0/C. Thus far, regulation studies indicate that CMCase formation is not repressed by cellobiose. The search for plasmids in C. thermocellum was continued. The presence of plasmids was confirmed by cesium chloride ethidium bromide gradient centrifugation and electron microscopy. Two plasmids were detected, one with an approximate molecular weight of 1 x 10/sup 6/ daltons. Studies on the fermentation of lactic acid to propionic acid showed the pathway in C. propionicum to be simpler than in M. elsdenii and hence more amenable to manipulation for acrylate production. Using Lactobacillius delbrueckii, it was possible to convert glucose, cellobiose, and cellulose hydrolysates to lactic acid rapidly and quantitatively. Fermentations of C. acetobutylicum growing in soluble media were performed. Detailed studies of Clostridium thermoaceticum have shown that pH is the primary limiting factor in the production of acetic acid. pH-controlled fermentations indicated accumulations of over 30 gm/l of acetic acid.

  8. Formate synthesis by Clostridium thermocellum during anaerobic fermentation. (United States)

    Sparling, Richard; Islam, Rumana; Cicek, Nazim; Carere, Carlo; Chow, Herman; Levin, David B


    We have detected formate synthesis by Clostridium thermocellum 27405 cultured in both cellobiose and alpha-cellulose. While formate synthesis has been reported for one strain of C. thermocellum (strain I-1-B), numerous studies of C. thermocellum 27405 fermentation, conducted under different growth conditions, failed to detect the presence of formate. Thus, the status of formate synthesis as a fermentation end product by C. thermocellum has been uncertain. Formate synthesis competes with the synthesis of hydrogen (H2) as a fermentation end product, and thus would negatively impact H2 yields in processes designed to generate H2 from biomass. Understanding the mechanism of formate synthesis is the first step in devising means of mitigating its production. Transcription of putative pfl, fnr, and adhE genes, encoding pyruvate formate-lyase (PFL), PFL-activating enzyme (PFL-AE), and alcohol dehydrogenase E (ADH-E) enzymes, respectively, were detected by reverse transcriptase polymerase chain reactions using total RNA extracted from stationary phase C. thermocellum cultured on cellobiose. The PCR products observed correspond to the expected amplicon sizes. Nucleotide sequence analysis of the cloned PCR products followed by BLAST analyses confirmed their identity. Formate production was detected throughout growth, and PFL enzyme activity was detected in late log and stationary phase (OD600 = 0.7 and 0.9, respectively) in extracts of C. thermocellum cultured on cellobiose. BLAST analyses revealed that C. thermocellum PFL and PFL-AE have greater amino acid sequence identity with equivalent enzymes from Bacillus and Thermocynechococcus species than with other Clostridium species, but C. thermocellum ADH-E has greater amino acid sequence identity with Clostridium species.

  9. A novel biochemical route for fuels and chemicals production from cellulosic biomass.

    Directory of Open Access Journals (Sweden)

    Zhiliang Fan

    Full Text Available The conventional biochemical platform featuring enzymatic hydrolysis involves five key steps: pretreatment, cellulase production, enzymatic hydrolysis, fermentation, and product recovery. Sugars are produced as reactive intermediates for subsequent fermentation to fuels and chemicals. Herein, an alternative biochemical route is proposed. Pretreatment, enzymatic hydrolysis and cellulase production is consolidated into one single step, referred to as consolidated aerobic processing, and sugar aldonates are produced as the reactive intermediates for biofuels production by fermentation. In this study, we demonstrate the viability of consolidation of the enzymatic hydrolysis and cellulase production steps in the new route using Neurospora crassa as the model microorganism and the conversion of cellulose to ethanol as the model system. We intended to prove the two hypotheses: 1 cellulose can be directed to produce cellobionate by reducing β-glucosidase production and by enhancing cellobiose dehydrogenase production; and 2 both of the two hydrolysis products of cellobionate--glucose and gluconate--can be used as carbon sources for ethanol and other chemical production. Our results showed that knocking out multiple copies of β-glucosidase genes led to cellobionate production from cellulose, without jeopardizing the cellulose hydrolysis rate. Simulating cellobiose dehydrogenase over-expression by addition of exogenous cellobiose dehydrogenase led to more cellobionate production. Both of the two hydrolysis products of cellobionate: glucose and gluconate can be used by Escherichia coli KO 11 for efficient ethanol production. They were utilized simultaneously in glucose and gluconate co-fermentation. Gluconate was used even faster than glucose. The results support the viability of the two hypotheses that lay the foundation for the proposed new route.

  10. [L'application des radioisotopes a la chromatographie sur colonnes de celluloses substituees-IV L'analyse du mercure et du zinc dans le bismuth]. (United States)

    Muzzarelli, R A; Marcotrigiano, G


    The Chromatographic behaviour of nanogram amounts of bismuth has been studied by radioisotope techniques on cellobiose, cellulose and seven substituted celluloses. All celluloses in ethyl ether adsorb bismuth, provided that it is as nitrate, and that excess of nitric acid is avoided. Bismuth can be eluted with thiocyanate in ether-methanol or with hydrochloric acid in methanol, depending on the retention strength of the various functional groups of celluloses. A very simple method of separation of bismuth from mercury over a wide range of concentration is presented.

  11. High ethanol producing derivatives of Thermoanaerobacter ethanolicus (United States)

    Ljungdahl, Lars G.; Carriera, Laura H.


    Derivatives of the newly discovered microorganism Thermoanaerobacter ethanolicus which under anaerobic and thermophilic conditions continuously ferment substrates such as starch, cellobiose, glucose, xylose and other sugars to produce recoverable amounts of ethanol solving the problem of fermentations yielding low concentrations of ethanol using the parent strain of the microorganism Thermoanaerobacter ethanolicus are disclosed. These new derivatives are ethanol tolerant up to 10% (v/v) ethanol during fermentation. The process includes the use of an aqueous fermentation medium, containing the substrate at a substrate concentration greater than 1% (w/v).

  12. A Dynamic Model for Cellulosic Biomass Hydrolysis: a Comprehensive Analysis and Validation of Hydrolysis and Product Inhibition Mechanisms

    DEFF Research Database (Denmark)

    Tsai, Chien Tai; Morales Rodriguez, Ricardo; Sin, Gürkan;


    product inhibitors such as glucose, cellobiose and xylose) to test the hydrolysis and product inhibition mechanisms of the model. A nonlinear least squares method was used to identify the model and estimate kinetic parameters based on the experimental data. The suitable mathematical model for industrial...... of cellulose hydrolysis behaviour over a broad range of substrate concentrations (50–150 g/L) and enzyme loadings (15.8–31.6 and 1–5.9 mg protein/g cellulose for Celluclast and Novozyme 188, respectively) was possible. This is the first study introducing transglycosylation into the semimechanistic model...

  13. Kinetics of Cellobiohydrolase (Cel7A) Variants with Lowered Substrate Affinity

    DEFF Research Database (Denmark)

    Kari, Jeppe; Olsen, Johan Pelck; Borch, Kim;


    Cellobiohydrolases are exo-active glycosyl hydrolases that processively convert cellulose to soluble sugars, typically cellobiose. They effectively break down crystalline cellulose and make up a major component in industrial enzyme mixtures used for deconstruction of lignocellulosic biomass...... design of enzymes with improved industrial applicability. To elucidate the role of on- and off-rates, respectively, on the overall kinetics, we have expressed a variant in which a tryptophan residue (Trp-38) in the middle of the active tunnel has been replaced with an alanine. This mutation weakens...

  14. Optimization of IC Separation Based on Isocratic-to-Gradient Retention Modeling in Combination with Sequential Searching or Evolutionary Algorithm

    Directory of Open Access Journals (Sweden)

    Šime Ukić


    Full Text Available Gradient ion chromatography was used for the separation of eight sugars: arabitol, cellobiose, fructose, fucose, lactulose, melibiose, N-acetyl-D-glucosamine, and raffinose. The separation method was optimized using a combination of simplex or genetic algorithm with the isocratic-to-gradient retention modeling. Both the simplex and genetic algorithms provided well separated chromatograms in a similar analysis time. However, the simplex methodology showed severe drawbacks when dealing with local minima. Thus the genetic algorithm methodology proved as a method of choice for gradient optimization in this case. All the calculated/predicted chromatograms were compared with the real sample data, showing more than a satisfactory agreement.

  15. sp³ -linked amorphous carbon with sulfonic acid groups as a heterogeneous acid catalyst. (United States)

    Suganuma, Satoshi; Nakajima, Kiyotaka; Kitano, Masaaki; Hayashi, Shigenobu; Hara, Michikazu


    SO₃H-bearing amorphous carbon prepared from polyvinyl chloride (PVC) is studied as a heterogeneous Brønsted acid catalyst. Sulfonation of partially carbonized PVC produces amorphous carbon consisting of small SO₃H-bearing carbon sheets linked by sp³ -based aliphatic hydrocarbons. This carbon material exhibits much higher catalytic performance in the hydrolysis of cellobiose than conventional heterogeneous Brønsted acid catalysts with SO₃H groups, including SO₃H-bearing amorphous carbon derived from cellulose. This can be attributed to a high density of SO₃H groups and the fast diffusion of reactants and products enabled by a flexible carbon network.

  16. Conversion of celluloses to proteins. [Use of Cellulomonas and Alcaligenes faecalis

    Energy Technology Data Exchange (ETDEWEB)

    De Leon, C.A.; Joson, L.M.


    Cellulosic wastes in the form of sugar cane bagasse was converted to single-cell protein by the action of Cellulomonas species subsequent to hydrolysis by NaOH and(or) autoclaving. As an aid to the process, in order to decompose the inhibitory cellobiose, Alcaligenes faecalis was introduced along with the Cellulomonas. Fungal enzyme production was greatest at the onset of fermentation, while the bacterial cellulases were produced over a more prolonged period, after an initial lag period. Biomass yield showed no correlation with enzyme production. The single-cell protein obtained comprised mainly lysine, methionine, cysteine, glycine, and valine.

  17. Isolation and characterization of Caldicellulosiruptor lactoaceticus sp. nov., an extremely thermophilic, cellulolytic, anaerobic bacterium

    DEFF Research Database (Denmark)

    Mladenovska, Zuzana; Mathrani, Indra M.; Ahring, Birgitte Kiær


    activity. The G + C content of the cellular DNA of strain 6A was 35.2 +/- 0.8 mol%. Complete 16S rDNA sequence analysis showed that strain 6A was phylogenetically related to Caldicellulosiruptor saccharolyticus. It is proposed that the isolated bacterium be named Caldicellulosiruptor lactoaceticus sp. nov....... and ethanol occurred as minor fermentation products. Only a restricted number of carbon sources (cellulose, xylan, starch, pectin, cellobiose, xylose, maltose and lactose) were used as substrates. During growth on Avicel, the bacterium produced free cellulases with carboxymethylcellulase and avicelase...

  18. Deciphering the signaling mechanisms of the plant cell wall degradation machinery in Aspergillus oryzae

    DEFF Research Database (Denmark)

    Udatha, D. B. R. K. Gupta; Topakas, Evangelos; Salazar, Margarita Pena;


    . oryzae genome were only partially explained by the chemical similarity of the enzyme inducers. Genes encoding enzymes that have attracted considerable interest such as cellobiose dehydrogenases and copper-dependent polysaccharide mono-oxygenases presented a substrate-specific induction. Several homology...... in 2360 reactions in the genome scale metabolic network of A. oryzae, was performed through a two-step molecular docking against the binding pockets of the transcription factors AoXlnR and AoAmyR. A total of six metabolites viz., sulfite (H2SO3), sulfate (SLF), uroporphyrinogen III (UPGIII), ethanolamine...

  19. Location, formation and biosynthetic regulation of cellulases in the gliding bacteria Cytophaga hutchinsonii

    Directory of Open Access Journals (Sweden)

    Elijah Johnson


    Full Text Available An analysis of the recently published genome sequence of Cytophagahutchinsonii revealed an unusual collection of genes for an organism that can attackcrystalline cellulose. Consequently, questions were being raised by cellulase scientists, as towhat mechanism this organism uses to degrade its insoluble substrates. Cellulose, being ahighly polymeric compound and insoluble in water, cannot enter the cell walls ofmicroorganisms. Cellulose-degrading enzymes have therefore to be located on the surface ofthe cell wall or released extracellularly. The location of most cellulase enzymes has beenstudied. However, basic information on C. hutchinsonii cellulases is almost non-existent. Inthe present study, the location, formation and biosynthetic regulation of cellulases in C.hutchinsonii were demonstrated on different substrates. Various fractions isolated from C.hutchinsonii after cell rupture were assayed for carboxymethyl-cellulase activity (CMC.The cellulases were found to be predominantly cell-free during active growth on solka-flok,although 30% of activity was recorded on cell-bound enzymes. Relatively little CM-cellulase was formed when cells were grown on glucose and cellobiose. Apparently glucoseor labile substrates such as cellobiose seem to repress the formation of CM-cellulase. Thesefindings should provide some insight into possible hydrolysis mechanisms by C.hutchinsonii.

  20. Characterization of a multi-function processive endoglucanase CHU_2103 from Cytophaga hutchinsonii. (United States)

    Zhang, Cong; Wang, Ying; Li, Zhe; Zhou, Xiangru; Zhang, Weican; Zhao, Yue; Lu, Xuemei


    Cytophaga hutchinsonii is a Gram-negative gliding bacterium which can efficiently degrade crystalline cellulose by an unknown strategy. Genomic analysis suggests the C. hutchinsonii genome lacks homologs to an obvious exoglucanase that previously seemed essential for cellulose degradation. One of the putative endoglucanases, CHU_2103, was successfully expressed in Escherichia coli JM109 and identified as a processive endoglucanase with transglycosylation activity. It could hydrolyze carboxymethyl cellulose (CMC) into cellodextrins and rapidly decrease the viscosity of CMC. When regenerated amorphous cellulose (RAC) was degraded by CHU_2103, the ratio of the soluble to insoluble reducing sugars was 3.72 after 3 h with cellobiose and cellotriose as the main products, indicating that CHU_2103 was a processive endoglucanase. CHU_2103 could degrade cellodextrins of degree of polymerization ≥3. It hydrolyzed p-nitrophenyl β-D-cellodextrins by cutting glucose or cellobiose from the non-reducing end. Meanwhile, some larger-molecular-weight cellodextrins could be detected, indicating it also had transglycosylation activity. Without carbohydrate-binding module (CBM), CHU_2103 could bind to crystalline cellulose and acted processively on it. Site-directed mutation of CHU_2103 demonstrated that the conserved aromatic amino acid W197 in the catalytic domain was essential not only for its processive activity, but also its cellulose binding ability.

  1. Ethanol Production from Various Sugars and Cellulosic Biomass by White Rot Fungus Lenzites betulinus. (United States)

    Im, Kyung Hoan; Nguyen, Trung Kien; Choi, Jaehyuk; Lee, Tae Soo


    Lenzites betulinus, known as gilled polypore belongs to Basidiomycota was isolated from fruiting body on broadleaf dead trees. It was found that the mycelia of white rot fungus Lenzites betulinus IUM 5468 produced ethanol from various sugars, including glucose, mannose, galactose, and cellobiose with a yield of 0.38, 0.26, 0.07, and 0.26 g of ethanol per gram of sugar consumed, respectively. This fungus relatively exhibited a good ethanol production from xylose at 0.26 g of ethanol per gram of sugar consumed. However, the ethanol conversion rate of arabinose was relatively low (at 0.07 g of ethanol per gram sugar). L. betulinus was capable of producing ethanol directly from rice straw and corn stalks at 0.22 g and 0.16 g of ethanol per gram of substrates, respectively, when this fungus was cultured in a basal medium containing 20 g/L rice straw or corn stalks. These results indicate that L. betulinus can produce ethanol efficiently from glucose, mannose, and cellobiose and produce ethanol very poorly from galactose and arabinose. Therefore, it is suggested that this fungus can ferment ethanol from various sugars and hydrolyze cellulosic materials to sugars and convert them to ethanol simultaneously.

  2. Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors (United States)

    Meyer, Andreas; Pellaux, René; Potot, Sébastien; Becker, Katja; Hohmann, Hans-Peter; Panke, Sven; Held, Martin


    Microcompartmentalization offers a high-throughput method for screening large numbers of biocatalysts generated from genetic libraries. Here we present a microcompartmentalization protocol for benchmarking the performance of whole-cell biocatalysts. Gel capsules served as nanolitre reactors (nLRs) for the cultivation and analysis of a library of Bacillus subtilis biocatalysts. The B. subtilis cells, which were co-confined with E. coli sensor cells inside the nLRs, converted the starting material cellobiose into the industrial product vitamin B2. Product formation triggered a sequence of reactions in the sensor cells: (1) conversion of B2 into flavin mononucleotide (FMN), (2) binding of FMN by a RNA riboswitch and (3) self-cleavage of RNA, which resulted in (4) the synthesis of a green fluorescent protein (GFP). The intensity of GFP fluorescence was then used to isolate B. subtilis variants that convert cellobiose into vitamin B2 with elevated efficiency. The underlying design principles of the assay are general and enable the development of similar protocols, which ultimately will speed up the optimization of whole-cell biocatalysts.

  3. Whole-Cell Biocatalysis for Producing Ginsenoside Rd from Rb1 Using Lactobacillus rhamnosus GG. (United States)

    Ku, Seockmo; You, Hyun Ju; Park, Myeong Soo; Ji, Geun Eog


    Ginsenosides are the major active ingredients in ginseng used for human therapeutic plant medicines. One of the most well-known probiotic bacteria among the various strains on the functional food market is Lactobacillus rhamnosus GG. Biocatalytic methods using probiotic enzymes for producing deglycosylated ginsenosides such as Rd have a growing significance in the functional food industry. The addition of 2% cellobiose (w/v) to glucose-free de Man-Rogosa-Sharpe broths notably induced β-glucosidase production from L. rhamnosus GG. Enzyme production and activity were optimized at a pH, temperature, and cellobiose concentration of 6.0, 40°C, and 2% (w/v), respectively. Under these controlled conditions, β-glucosidase production in L. rhamnosus GG was enhanced by 25-fold. Additionally, whole-cell homogenates showed the highest β-glucosidase activity when compared with disrupted cell suspensions; the cell disruption step significantly decreased the β-glucosidase activity. Based on the optimized enzyme conditions, whole-cell L. rhamnosus GG was successfully used to convert ginsenoside Rb1 into Rd.

  4. Assimilation of organic and inorganic nutrients by Erica root fungi from the fynbos ecosystem. (United States)

    Bizabani, Christine; Dames, Joanna Felicity


    Erica dominate the fynbos ecosystem, which is characterized by acidic soils that are rich in organic matter. The ericaceae associate with ericoid mycorrhizal (ERM) fungi for survival. In this study fungal biomass accumulation in vitro was used to determine nutrient utilisation of various inorganic and organic substrates. This is an initial step towards establishment of the ecological roles of typical ERM fungi and other root fungi associated with Erica plants, with regard to host nutrition. Meliniomyces sp., Acremonium implicatum, Leohumicola sp., Cryptosporiopsis erica, Oidiodendron maius and an unidentified Helotiales fungus were selected from fungi previously isolated and identified from Erica roots. Sole nitrogen sources ammonium, nitrate, arginine and Bovine Serum Albumin (BSA) were tested. Meliniomyces and Leohumicola species were able to utilise BSA effectively. Phosphorus nutrition was tested using orthophosphate, sodium inositol hexaphosphate and DNA. Most isolates preferred orthophosphate. Meliniomyces sp. and A. implicatum were able to accumulate significant biomass using DNA. Carbon utilisation was tested using glucose, cellobiose, carboxymethylcellulose, pectin and tannic acid substrates. All fungal isolates produced high biomass on glucose and cellobiose. The ability to utilize organic nutrient sources in culture, illustrates their potential role of these fungi in host nutrition in the fynbos ecosystem.

  5. Ethanol production from high cellulose concentration by the basidiomycete fungus Flammulina velutipes. (United States)

    Maehara, Tomoko; Ichinose, Hitomi; Furukawa, Takanori; Ogasawara, Wataru; Takabatake, Koji; Kaneko, Satoshi


    Ethanol production by Flammulina velutipes from high substrate concentrations was evaluated. F. velutipes produces approximately 40-60 g l(-1) ethanol from 15% (w/v) D-glucose, D-fructose, D-mannose, sucrose, maltose, and cellobiose, with the highest conversion rate of 83% observed using cellobiose as a carbon source. We also attempted to assess direct ethanol fermentation from sugarcane bagasse cellulose (SCBC) by F. velutipes. The hydrolysis rate of 15% (w/v) SCBC with commercial cellulase was approximately 20%. In contrast, F. velutipes was able to produce a significant amount of ethanol from 15% SCBC with the production of β-glucosidase, cellobohydrolase, and cellulase, although the addition of a small amount of commercial cellulase to the culture was required for the conversion. When 9 mg g(-1) biomass of commercial cellulase was added to cultures, 0.36 g of ethanol was produced from 1 g of cellulose, corresponding to an ethanol conversion rate of 69.6%. These results indicate that F. velutipes would be useful for consolidated bioprocessing of lignocellulosic biomass to bioethanol.

  6. Establishment and metabolic analysis of a model microbial community for understanding trophic and electron accepting interactions of subsurface anaerobic environments

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    Yang Zamin K


    Full Text Available Abstract Background Communities of microorganisms control the rates of key biogeochemical cycles, and are important for biotechnology, bioremediation, and industrial microbiological processes. For this reason, we constructed a model microbial community comprised of three species dependent on trophic interactions. The three species microbial community was comprised of Clostridium cellulolyticum, Desulfovibrio vulgaris Hildenborough, and Geobacter sulfurreducens and was grown under continuous culture conditions. Cellobiose served as the carbon and energy source for C. cellulolyticum, whereas D. vulgaris and G. sulfurreducens derived carbon and energy from the metabolic products of cellobiose fermentation and were provided with sulfate and fumarate respectively as electron acceptors. Results qPCR monitoring of the culture revealed C. cellulolyticum to be dominant as expected and confirmed the presence of D. vulgaris and G. sulfurreducens. Proposed metabolic modeling of carbon and electron flow of the three-species community indicated that the growth of C. cellulolyticum and D. vulgaris were electron donor limited whereas G. sulfurreducens was electron acceptor limited. Conclusions The results demonstrate that C. cellulolyticum, D. vulgaris, and G. sulfurreducens can be grown in coculture in a continuous culture system in which D. vulgaris and G. sulfurreducens are dependent upon the metabolic byproducts of C. cellulolyticum for nutrients. This represents a step towards developing a tractable model ecosystem comprised of members representing the functional groups of a trophic network.

  7. Effect of substrate loading on hydrogen production during anaerobic fermentation by Clostridium thermocellum 27405. (United States)

    Islam, Rumana; Cicek, Nazim; Sparling, Richard; Levin, David


    We have investigated hydrogen (H2) production by the cellulose-degrading anaerobic bacterium, Clostridium thermocellum. In the following experiments, batch-fermentations were carried out with cellobiose at three different substrate concentrations to observe the effects of carbon-limited or carbon-excess conditions on the carbon flow, H2-production, and synthesis of other fermentation end products, such as ethanol and organic acids. Rates of cell growth were unaffected by different substrate concentrations. H2, carbon dioxide (CO2), acetate, and ethanol were the main products of fermentation. Other significant end products detected were formate and lactate. In cultures where cell growth was severely limited due to low initial substrate concentrations, hydrogen yields of 1 mol H2/mol of glucose were obtained. In the cultures where growth ceased due to carbon depletion, lactate and formate represented a small fraction of the total end products produced, which consisted mainly of H2, CO2, acetate, and ethanol throughout growth. In cultures with high initial substrate concentrations, cellobiose consumption was incomplete and cell growth was limited by factors other than carbon availability. H2-production continued even in stationary phase and H2/CO2 ratios were consistently greater than 1 with a maximum of 1.2 at the stationary phase. A maximum specific H2 production rate of 14.6 mmol g dry cell(-1) h(-1) was observed. As cells entered stationary phase, extracellular pyruvate production was observed in high substrate concentration cultures and lactate became a major end product.

  8. Effect of carbon substrates on rock phosphate solubilization by bacteria from composts and macrofauna. (United States)

    Hameeda, B; Reddy, Y Harish Kumar; Rupela, O P; Kumar, G N; Reddy, Gopal


    Five of the 207 isolates from different composts, farm waste compost (FWC), rice straw compost (RSC), Gliricidia vermicompost (GVC), and macrofauna, showed rock phosphate (RP) solubilization in buffered medium in plate culture. When tested in RP broth medium, all five strains, Enterobacter cloacae EB 27, Serratia marcescens EB 67, Serratia sp. EB 75, Pseudomonas sp. CDB 35, and Pseudomonas sp. BWB 21, showed gluconic acid production and solubilized RP. Based on cellulose-degrading and P-solubilizing ability, two strains were selected for further studies. In the presence of different carbon sources, both strains showed a drop in pH and solubilized RP. P released was maximum with glucose (1212 and 522 micromol) and minimum with cellobiose (455 and 306 micromol) by S. marcescens EB 67 and Pseudomonas sp. CDB 35, respectively. Glucose dehydrogenase (GDH) activity was 63 and 77% with galactose and 35 and 46% with cellobiose when compared to glucose (100%) by EB 67 and CDB 35, respectively. Both strains solubilized RP in the presence of different crop residues. EB 67 and CDB 35 showed maximum cellulase activity (0.027 units) in the presence of rice straw and a mixture of rice straw and root. P solubilized from RP in the presence of pigeonpea root was 134 and 140 micromol with EB 67 and CDB 35. Significantly, these bacteria isolated from composts and macrofauna solubilized rock phosphate in the presence of various pure carbon substrates and crop residues and their importance in soil/rhizosphere conditions is discussed.

  9. Hydrolysis and transglycosylation activity of a thermostable recombinant beta-glycosidase from Sulfolobus acidocaldarius. (United States)

    Park, Ah-Reum; Kim, Hye-Jung; Lee, Jung-Kul; Oh, Deok-Kun


    We expressed a putative beta-galactosidase from Sulfolobus acidocaldarius in Escherichia coli and purified the recombinant enzyme using heat treatment and Hi-Trap ion-exchange chromatography. The resultant protein gave a single 57-kDa band by SDS-PAGE and had a specific activity of 58 U/mg. The native enzyme existed as a dimer with a molecular mass of 114 kDa by gel filtration. The maximum activity of this enzyme was observed at pH 5.5 and 90 degrees C. The half-lives of the enzyme at 70, 80, and 90 degrees C were 494, 60, and 0.2 h, respectively. The hydrolytic activity with p-nitrophenyl(pNP) substrates followed the order p-nitrophenyl-beta-D-fucopyranoside > pNP-beta-D-glucopyranoside > pNP-beta-D-galactopyranoside > pNP-beta-D-mannopyranoside > pNP-beta-D-xylopyranoside, but not toward aryl-alpha-glycosides or pNP-beta-L-arabinofuranoside. Thus, the enzyme was actually a beta-glycosidase. The beta-glycosidase exhibited transglycosylation activity with pNP-beta-D-galactopyranoside, pNP-beta-D-glucopyranoside, and pNP-beta-D-fucopyranoside in decreasing order of activity, in the reverse order of its hydrolytic activity. The hydrolytic activity was higher toward cellobiose than toward lactose, but the transglycosylation activity was lower with cellobiose than with lactose.

  10. Oligosaccharide synthesis in Fibrobacter succinogenes S85 and its modulation by the substrate. (United States)

    Nouaille, Régis; Matulova, Maria; Delort, Anne-Marie; Forano, Evelyne


    In this article we compared the metabolism of phosphorylated and unphosphorylated oligosaccharides (cellodextrins and maltodextrins) in Fibrobacter succinogenes S85 resting cells incubated with the following substrates: glucose; cellobiose; a mixture of glucose and cellobiose; and cellulose. Intracellular and extracellular media were analysed by (1)H-NMR and by TLC. The first important finding is that no cellodextrins were found to accumulate in the extracellular media of cells, regardless of the substrate; this contrasts to what is generally reported in the literature. The second finding of this work is that maltodextrins of degree of polymerization > 2 are synthesized regardless of the substrate, and can be used by the bacteria. Maltotriose plays a key role in this metabolism of maltodextrin. Maltodextrin-1-phosphate was detected in all the incubations, and a new metabolite, corresponding to a phosphorylated glucose derivative, was produced in the extracellular medium when cells were incubated with cellulose. The accumulation of these phosphorylated sugars increased with the degree of polymerization of the substrate.


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    Full Text Available The use of nanotechnology in drug delivery is spreading rapidly. The nanocarriers have been used for the enhanced delivery of a range of drugs. The present study was aimed at investigating the application of ceramic nanoparticles called as aquasomes for the delivery of drug, piroxicam. Piroxicam belongs to oxicam group of NSAID’s, commonly used for the treatment of arthritis. It is a BCS class II drug, with low solubility. There is a need to improve the dissolution property of piroxicam in order to enhance its therapeutic efficacy. Ceramic Nanoparticles were prepared by colloidal precipitation method. The ceramic core was coated with polysaccharide, cellobiose, followed by adsorption of drug. The drug loaded nanoparticles were evaluated for size, entrapment efficiency and drug release profile. The SEM studies indicated that the formed particles were with nanometric dimensions (185 nm. 21% drug loading was observed and more than 95% drug release was observed within 135 min in 0.1N HCl compared with pure drug which released 89% in 90 mins. In vitro dissolution studies indicated that the piroxicam ceramic nanoparticles released the drug in a controlled manner. Anti-nociceptive and anti-inflammatory studies were performed with piroxicam cellobiose aquasomes. Paw edema method was employed for assessing anti-inflammatory effect. The anti-inflammatory activity of aquasome formulation showed quicker effect up to 3 h compared to pure piroxicam.

  12. The Putative Cellodextrin Transporter-like Protein CLP1 Is Involved in Cellulase Induction in Neurospora crassa* (United States)

    Cai, Pengli; Wang, Bang; Ji, Jingxiao; Jiang, Yongsheng; Wan, Li; Tian, Chaoguang; Ma, Yanhe


    Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, cellodextrin transporter-like protein 1 (CLP1; NCU05853), a putative cellodextrin transporter-like protein that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain Δ3βG formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with Δ3βG. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators. PMID:25398875

  13. Heterologously expressed Aspergillus aculeatus β-glucosidase in Saccharomyces cerevisiae is a cost-effective alternative to commercial supplementation of β-glucosidase in industrial ethanol production using Trichoderma reesei cellulases. (United States)

    Treebupachatsakul, Treesukon; Nakazawa, Hikaru; Shinbo, Hideaki; Fujikawa, Hiroki; Nagaiwa, Asami; Ochiai, Nobuhiro; Kawaguchi, Takashi; Nikaido, Mitsuru; Totani, Kazuhide; Shioya, Koki; Shida, Yosuke; Morikawa, Yasushi; Ogasawara, Wataru; Okada, Hirofumi


    Trichoderma reesei is a filamentous organism that secretes enzymes capable of degrading cellulose to cellobiose. The culture supernatant of T. reesei, however, lacks sufficient activity to convert cellobiose to glucose using β-glucosidase (BGL1). In this study, we identified a BGL (Cel3B) from T. reesei (TrCel3B) and compared it with the active β-glucosidases from Aspergillus aculeatus (AaBGL1). AaBGL1 showed higher stability and conversion of sugars to ethanol compared to TrCel3B, and therefore we chose to express this recombinant protein for use in fermentation processes. We expressed the recombinant protein in the yeast Saccharomyces cerevisiae, combined it with the superb T. reesei cellulase machinery and used the combination in a simultaneous saccharification and fermentation (SSF) process, with the hope that the recombinant would supplement the BGL activity. As the sugars were processed, the yeast immediately converted them to ethanol, thereby eliminating the problem posed by end product inhibition. Recombinant AaBGL1 activity was compared with Novozyme 188, a commercially available supplement for BGL activity. Our results show that the recombinant protein is as effective as the commercial supplement and can process sugars with equal efficiency. Expression of AaBGL1 in S. cerevisiae increased ethanol production effectively. Thus, heterologous expression of AaBGL1 in S. cerevisiae is a cost-effective and efficient process for the bioconversion of ethanol from lignocellulosic biomass.

  14. Structure-activity relationships in carbohydrates revealed by their hydration. (United States)

    Maugeri, Laura; Busch, Sebastian; McLain, Sylvia E; Pardo, Luis Carlos; Bruni, Fabio; Ricci, Maria Antonietta


    One of the more intriguing aspects of carbohydrate chemistry is that despite having very similar molecular structures, sugars have very different properties. For instance, there is a sensible difference in sweet taste between glucose and trehalose, even though trehalose is a disaccharide that comprised two glucose units, suggesting a different ability of these two carbohydrates to bind to sweet receptors. Here we have looked at the hydration of specific sites and at the three-dimensional configuration of water molecules around three carbohydrates (glucose, cellobiose, and trehalose), combining neutron diffraction data with computer modelling. Results indicate that identical chemical groups can have radically different hydration patterns depending on their location on a given molecule. These differences can be linked with the specific activity of glucose, cellobiose, and trehalose as a sweet substance, as building block of cellulose fiber, and as a bioprotective agent, respectively. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editors: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.

  15. Cellulolytic Activity of Clostridium acetobutylicum. (United States)

    Lee, S F; Forsberg, C W; Gibbins, L N


    Clostridium acetobutylicum NRRL B527 and ATCC 824 exhibited extracellular and cell-bound endoglucanase and cellobiase activities during growth in a chemically defined medium with cellobiose as the sole source of carbohydrate. For both strains, the endoglucanase was found to be mainly extracellular (70 to 90%) during growth in continuous or batch cultures with the pH maintained at 5.2, whereas the cellobiase was mainly cell associated (60 to 90%). During continuous cultivation of strain B527 with cellobiose as the limiting nutrient, maximum production of the endoglucanase and cellobiase occurred at pH values of 5.2 and 4.8, respectively. In the carbon-limited continuous cultures, strain 824 produced similar levels of endoglucanase, cellobiosidase, and cellobiase activities regardless of the carbon source used. However, in ammonium- or phosphate-limited cultures, with an excess of glucose, only 1/10 of the endoglucanase was produced, and neither cellobiosidase nor cellobiase activities were detectable. A crude extracellular enzyme preparation from strain B527 hydrolyzed carboxymethylcellulose and phosphoric acid-swollen cellulose readily and microcrystalline cellulose (A vicel) to a lesser extent. Glucose accounted for more than 90% of the reducing sugar produced by the hydrolysis of acid-swollen cellulose and Avicel. Strain B527 did not grow in medium with acid-swollen cellulose as the sole source of carbohydrate, although it grew readily on the products obtained by hydrolyzing the cellulose in vitro with a preparation of extracellular cellulase derived from the same organism.

  16. Role of the components of the cellulase complex on hydrolysis of insoluble cellulose

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    Klyosov, A.A.; Goldstein, G.C.; Kude, J.; Meyer, D.


    The kinetics of the hydrolysis of microcrystalline cellulose (MC) by a Trichoderma reesei cellulase complex and by the individual endoglucanase (pI 4.4-5.2) and cellobiohydrolase (pI 4.0-4.2) has been studied. A flow chart for the enzymatic hydrolysis of the cellulose has been revealed, which formed a basis for a computer simulation of the kinetic regularities observed. As a result of it, the values of the catalytic rate constants for the individual stages of the enzymatic degradation of MC have been calculated. Then, the synergistic behaviour of endoglucanase and cellobiohydrolase in the hydrolysis of MC has been described both quantitatively and graphically. The relative efficiency of the individual stages for the MC hydrolysis in terms of glucose and cellobiose formation for cellulase complexes of various composition has been calculated. It was quantitatively shown that cellobiohydrolase plays the key role in the MC hydrolysis by T. reesei cellulase preparations, because it gives up to 80% glucose and up to 80-90% cellobiose in the presence of endoglucanase which in turn plays a relatively minor role in a direct formation of both soluble products of the hydrolysis.

  17. Functional and structural analyses of a 1,4-β-endoglucanase from Ganoderma lucidum. (United States)

    Liu, Guizhi; Li, Qian; Shang, Na; Huang, Jian-Wen; Ko, Tzu-Ping; Liu, Weidong; Zheng, Yingying; Han, Xu; Chen, Yun; Chen, Chun-Chi; Jin, Jian; Guo, Rey-Ting


    Ganoderma lucidum is a saprotrophic white-rot fungus which contains a rich set of cellulolytic enzymes. Here, we screened an array of potential 1,4-β-endoglucanases from G. lucidum based on the gene annotation library and found that one candidate gene, GlCel5A, exhibits CMC-hydrolyzing activity. The recombinant GlCel5A protein expressed in Pichia pastoris is able to hydrolyze CMC and β-glucan but not xylan and mannan. The enzyme exhibits optimal activity at 60°C and pH 3-4, and retained 50% activity at 80 and 90°C for at least 15 and 10min. The crystal structure of GlCel5A and its complex with cellobiose, solved at 2.7 and 2.86Å resolution, shows a classical (β/α)8 TIM-barrel fold as seen in other members of glycoside hydrolase family 5. The complex structure contains a cellobiose molecule in the +1 and +2 subsites, and reveals the interactions with the positive sites of the enzyme. Collectively, the present work provides the first comprehensive characterization of an endoglucanase from G. lucidum that possesses properties for industrial applications, and strongly encourages further studying in the cellulolytic enzyme system of G. lucidum.

  18. Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples. (United States)

    Berlemont, Renaud; Delsaute, Maud; Pipers, Delphine; D'Amico, Salvino; Feller, Georges; Galleni, Moreno; Power, Pablo


    In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carboxymethyl cellulose as a substrate. Moreover, the study of pH and the thermal dependence of the hydrolytic activity shows that RBcel1 was active from pH 6 to pH 9 and remained significantly active when temperature decreased (18% of activity at 10 degrees C). It is interesting that RBcel1 was able to synthetize non-reticulated cellulose using cellobiose as a substrate. Moreover, by a combination of bioinformatics and enzyme analysis, the physiological relevance of the RBcel1 protein and its mesophilic homologous Pst_2494 protein from P. stutzeri, A1501, was established as the key enzymes involved in the production of cellulose by bacteria. In addition, RBcel1 and Pst_2494 are the two primary enzymes belonging to the GH5 family involved in this process.

  19. Characterization of Clostridium thermocellum strains with disrupted fermentation end-product pathways

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    Van Der Veen, Douwe [ORNL; Lo, Jonathan [Dartmouth College; Brown, Steven D [ORNL; Johnson, Courtney M [ORNL; Tschaplinski, Timothy J [ORNL; Martin, Madhavi Z [ORNL; Engle, Nancy L [ORNL; Van den Berg, Robert A [Katholieke University Leuven, Belgium; Argyros, Aaron [Mascoma Corporation; Caiazza, Nicky [Mascoma Corporation; Guss, Adam M [ORNL; Lynd, Lee R [Thayer School of Engineering at Dartmouth


    Clostridium thermocellum is a thermophilic, cellulolytic anaerobe that is a candidate microorganism for industrial biofuels production. Strains with mutations in genes associated with production of L-lactate (Dldh) and/or acetate (Dpta) were characterized to gain insight into the intracellular processes that convert cellobiose to ethanol and other fermentation end-products. Cellobiose-grown cultures of the Dldh strain had identical biomass accumulation, fermentation end-products, transcription profile, and intracellular metabolite concentrations compared to its parent strain (DSM1313 Dhpt Dspo0A). The Dpta-deficient strain grew slower and had 30 % lower final biomass concentration compared to the parent strain, yet produced 75% more ethanol. A Dldh Dpta double-mutant strain evolved for faster growth had a growth rate and ethanol yield comparable to the parent strain, whereas its biomass accumulation was comparable to Dpta. Free amino acids were secreted by all examined strains, with both Dpta strains secreting higher amounts of alanine, valine, isoleucine, proline, glutamine, and threonine. Valine concentration for Dldh Dpta reached 5 mM by the end of growth, or 2.7 % of the substrate carbon utilized. These secreted amino acid concentrations correlate with increased intracellular pyruvate concentrations, up to sixfold in the Dpta and 16-fold in the Dldh Dpta strain. We hypothesize that the deletions in fermentation end-product pathways result in an intracellular redox imbalance, which the organism attempts to relieve, in part by recycling NADP* through increased production of amino acids.

  20. Characterization of Clostridium thermocellum strains with disrupted fermentation end product pathways

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    Van Der Veen, Douwe [ORNL; Lo, Jonathan [Dartmouth College; Brown, Steven D [ORNL; Johnson, Courtney M [ORNL; Tschaplinski, Timothy J [ORNL; Martin, Madhavi Z [ORNL; Engle, Nancy L [ORNL; Argyros, Aaron [Mascoma Corporation; Van den Berg, Robert A [Katholieke University Leuven, Belgium; Caiazza, Nicky [Mascoma Corporation; Guss, Adam M [ORNL; Lynd, Lee R [Thayer School of Engineering at Dartmouth


    Clostridium thermocellum is a thermophilic, cellulolytic anaerobe that is a candidate microorganism for industrial biofuels production. Strains with mutations in genes associated with production of Llactate ( ldh) and/or acetate ( pta) were characterized to gain insight into the intracellular processes that convert cellobiose to ethanol and other fermentation end products. Cellobiose-grown cultures of the ldh strain had identical biomass accumulation, fermentation end products, transcription profile and intracellular metabolite concentrations compared to its parent strain (DSM1313 hpt spo0A). The pta-deficient strain grew slower and had 30% lower final biomass concentration compared to the parent strain, yet produced 75% more ethanol. A ldh pta double mutant strain evolved for faster growth had growth rate and ethanol yield comparable to the parent strain, whereas its biomass accumulation was comparable to pta. Free amino acids were secreted by all examined strains, with both pta strains secreting higher amounts of alanine, valine, isoleucine, proline, glutamine, and threonine. Valine concentration for ldh pta reached 5 mM by the end of growth, or 2.7% of the substrate carbon utilized. These secreted amino acid concentrations correlate with increased intracellular pyruvate concentrations, up to 6-fold in the pta and 16-fold in the ldh pta strain. We hypothesize that the deletions in fermentation end product pathways result in an intracellular redox imbalance, which the organism attempts to relieve, in part by recycling NADP+ through increased production of amino acids.

  1. Analysis and optimization of triacylglycerol synthesis in novel oleaginous Rhodococcus and Streptomyces strains isolated from desert soil. (United States)

    Röttig, Annika; Hauschild, Philippa; Madkour, Mohamed H; Al-Ansari, Ahmed M; Almakishah, Naief H; Steinbüchel, Alexander


    As oleaginous microorganisms represent an upcoming novel feedstock for the biotechnological production of lipids or lipid-derived biofuels, we searched for novel, lipid-producing strains in desert soil. This was encouraged by the hypothesis that neutral lipids represent an ideal storage compound, especially under arid conditions, as several animals are known to outlast long periods in absence of drinking water by metabolizing their body fat. Ten lipid-accumulating bacterial strains, affiliated to the genera Bacillus, Cupriavidus, Nocardia, Rhodococcus and Streptomyces, were isolated from arid desert soil due to their ability to synthesize poly(β-hydroxybutyrate), triacylglycerols or wax esters. Particularly two Streptomyces sp. strains and one Rhodococcus sp. strain accumulate significant amounts of TAG under storage conditions under optimized cultivation conditions. Rhodococcus sp. A27 and Streptomyces sp. G49 synthesized approx. 30% (w/w) fatty acids from fructose or cellobiose, respectively, while Streptomyces isolate G25 reached a cellular fatty acid content of nearly 50% (w/w) when cultivated with cellobiose. The stored triacylglycerols were composed of 30-40% branched fatty acids, such as anteiso-pentadecanoic or iso-hexadecanoic acid. To date, this represents by far the highest lipid content described for streptomycetes. A biotechnological production of such lipids using (hemi)cellulose-derived raw material could be used to obtain sustainable biodiesel with a high proportion of branched-chain fatty acids to improve its cold-flow properties and oxidative stability.

  2. Boosting dark fermentation with co-cultures of extreme thermophiles for biohythane production from garden waste. (United States)

    Abreu, Angela A; Tavares, Fábio; Alves, Maria Madalena; Pereira, Maria Alcina


    Proof of principle of biohythane and potential energy production from garden waste (GW) is demonstrated in this study in a two-step process coupling dark fermentation and anaerobic digestion. The synergistic effect of using co-cultures of extreme thermophiles to intensify biohydrogen dark fermentation is demonstrated using xylose, cellobiose and GW. Co-culture of Caldicellulosiruptor saccharolyticus and Thermotoga maritima showed higher hydrogen production yields from xylose (2.7±0.1molmol(-1) total sugar) and cellobiose (4.8±0.3molmol(-1) total sugar) compared to individual cultures. Co-culture of extreme thermophiles C. saccharolyticus and Caldicellulosiruptor bescii increased synergistically the hydrogen production yield from GW (98.3±6.9Lkg(-1) (VS)) compared to individual cultures and co-culture of T. maritima and C. saccharolyticus. The biochemical methane potential of the fermentation end-products was 322±10Lkg(-1) (CODt). Biohythane, a biogas enriched with 15% hydrogen could be obtained from GW, yielding a potential energy generation of 22.2MJkg(-1) (VS).

  3. From Vibrational Spectroscopy to Force Fields and Structures of Saccharides: New Computational Algorithms and Applications

    Energy Technology Data Exchange (ETDEWEB)

    Pincu, Madeleine [UCI; Gerber, Robert Benny [Professor, UCI, Chemistry Dept.


    This work was undertaken with the main objective to investigate basic reactions that take place in relatively simple saccharides (mono-saccharides and cellobiose - the building block of cellulose) , in isolation and in cluster with few water molecules or with (gas-phase) clusters of few waters and ionic compounds (salt, isolated ions like H{sup +} or OH{sup -}). Within the context of this work, different potentials were investigated; among them, were the PM3 semi empirical potential, DFT/BLYP and a new hybrid potential constructed from MP2 for the harmonic part and from adjusted Hartree-Fock anharmonic interactions (VSCF-PT2). These potentials were evaluated by comparison with experimental data from published sources and from several collaborating groups. The findings show excellent agreement between experiments and predictions with the hybrid VSCF-PT2 potential and very good agreement with predictions obtained from dynamics with dispersion corrected DFT/BLYP potential. Investigation of hydration of cellobiose, was another topic of interest. Guided by a hydration motif demonstrated by our experimental collaborators (team of Prof J.P. Simons), we demonstrated large energetic and structural differences between the two species of cellobiose: cis and trans. The later, which is dominant in solid and liquid phases, is higher in energy in the gas-phase and compared to pure water, it does not disturb as much the network of H bonds. In contrast, the cis species exhibits asymmetric hydration in cluster with up to 25 waters, indicating that it has surfactant properties. Another highlight of this research effort was the successful first time spectrometric and spectroscopic study of a gas-phase protonated sugar derivative (alpha-D-Galactopyranoside) and its interpretation by Ab Initio molecular dynamics (AIMD) simulations. The findings demonstrate the formation of a motif in which a proton bridges between two Oxygen atoms (belonging to OH groups) at the sugar; The vibrational

  4. Psychrotolerant Anaerobes from Lake Podprudnoe, Antarctica and Penguin Spheniscus demersus Colony, South Africa (United States)

    Guisler, Melissa; Pikuta, Elena V.; Townsend, Alisa; Hoover, Richard B.


    The study of a sample collected from a wind-made ice sculpture near Lake Podprudnoe, Antarctica led to the isolation of the psychrotolerant strain ISLP-3. Cells of the new isolate are vibrio-shaped that measure 0.5 x 1.0-3.0 micron in size. Growth occurs within the temperature range 5-35 C with the optimum at 22 C. Salinity range for growth is 0-2 % NaCl with the optimum at 0.25 %. The new isolate grows within a pH range from 6.0 to 9.5 with the optimum at 7.5. Strain ISLP-3 is saccharolytic, growing on the following substrates: D-glucose, D-ribose, D-fructose, D-arabinose, maltose, sucrose, D-trehalose, D-mannose, D-cellobiose, lactose, starch, chitin, triethylamine, N-acetylglucosamine, and urea. The best growth occurred on D-cellobiose. An environmental sample of pond water near a colony of the endemic species of African penguins, Spheniscus demersus, was collected in February 2008 and delivered directly to the Astrobiology laboratory at NSSTC. The microbiological study of this sample led to the isolation of two psychrotolerant strains ARHSd-7G and ARHSd-9G. Both strains are strictly anaerobic bacteria and are able to grow at high pH and low temperatures. The cells of strain ARHSd-7G are motile, vibrio-shaped, spore-forming cells. Optimal growth of this strain occurs at 30 C, 3 % NaCl, and pH 8.9. The isolate ARHSd-7G combines sugarlytic and proteolytic metabolisms, growing on some proteolysis products including peptone and yeast extract and a number of sugars. The second isolate, ARHSd-9G, exhibits thin, elongated rods that measure 0.4 x 3-5 micron. The cells are motile and spore-forming. Optimal growth of strain ARHSd-9G occurs at 30 C, 1.75 % NaCl, and pH 8.5. The strain ARHSd-9G is sugarlytic, growing well on substrates such as D-glucose, sucrose, D-cellobiose, maltose, fructose, D-mannose, and trehalose (the only exception is positive growth on yeast extract). In this report, the physiological and morphological characteristics of the novel psychrotolerant

  5. Psychrotolerant anaerobes from Lake Podprudnoye, Antarctica and penguin Spheniscus demersus colony, South Africa (United States)

    Guisler, Melissa; Pikuta, Elena V.; Townsend, Alisa; Hoover, Richard B.


    The study of a sample collected from a wind-made ice sculpture near Lake Podprudnoe, Antarctica led to the isolation of the psychrotolerant strain ISLP-3. Cells of the new isolate are vibrio-shaped that measure 0.5 x 1.0-3.0 μm in size. Growth occurs within the temperature range 5-35ºC with the optimum at 22 °C. Salinity range for growth is 0-2 % NaCl with the optimum at 0.25 %. The new isolate grows within a pH range from 6.0 to 9.5 with the optimum at 7.5. Strain ISLP-3 is saccharolytic, growing on the following substrates: D-glucose, D-ribose, D-fructose, D-arabinose, maltose, sucrose, D-trehalose, D-mannose, D-cellobiose, lactose, starch, chitin, triethylamine, N-acetylglucosamine, and urea. The best growth occurred on D-cellobiose. An environmental sample of pond water near a colony of the endemic species of African penguins, Spheniscus demersus, was collected in February 2008 and delivered directly to the Astrobiology laboratory at NSSTC. The microbiological study of this sample led to the isolation of two psychrotolerant strains ARHSd-7G and ARHSd-9G. Both strains are strictly anaerobic bacteria and are able to grow at high pH and low temperatures. The cells of strain ARHSd-7G are motile, vibrio-shaped, spore-forming cells. Optimal growth of this strain occurs at 30 ºC, 3 % NaCl, and pH 8.9. The isolate ARHSd-7G combines sugarlytic and proteolytic metabolisms, growing on some proteolysis products including peptone and yeast extract and a number of sugars. The second isolate, ARHSd-9G, exhibits thin, elongated rods that measure 0.4 x 3-5 μm. The cells are motile and spore-forming. Optimal growth of strain ARHSd-9G occurs at 30 ºC, 1.75 % NaCl, and pH 8.5. The strain ARHSd-9G is sugarlytic, growing well on substrates such as D-glucose, sucrose, D-cellobiose, maltose, fructose, D-mannose, and trehalose (the only exception is positive growth on yeast extract). In this report, the physiological and morphological characteristics of the novel

  6. A highly efficient β-glucosidase from the buffalo rumen fungus Neocallimastix patriciarum W5

    Directory of Open Access Journals (Sweden)

    Chen Hsin-Liang


    Full Text Available Abstract Background Cellulose, which is the most abundant renewable biomass on earth, is a potential bio-resource of alternative energy. The hydrolysis of plant polysaccharides is catalyzed by microbial cellulases, including endo-β-1,4-glucanases, cellobiohydrolases, cellodextrinases, and β-glucosidases. Converting cellobiose by β-glucosidases is the key factor for reducing cellobiose inhibition and enhancing the efficiency of cellulolytic enzymes for cellulosic ethanol production. Results In this study, a cDNA encoding β-glucosidase was isolated from the buffalo rumen fungus Neocallimastix patriciarum W5 and is named NpaBGS. It has a length of 2,331 bp with an open reading frame coding for a protein of 776 amino acid residues, corresponding to a theoretical molecular mass of 85.1 kDa and isoelectric point of 4.4. Two GH3 catalytic domains were found at the N and C terminals of NpaBGS by sequence analysis. The cDNA was expressed in Pichia pastoris and after protein purification, the enzyme displayed a specific activity of 34.5 U/mg against cellobiose as the substrate. Enzymatic assays showed that NpaBGS was active on short cello-oligosaccharides from various substrates. A weak activity in carboxymethyl cellulose (CMC digestion indicated that the enzyme might also have the function of an endoglucanase. The optimal activity was detected at 40°C and pH 5 ~ 6, showing that the enzyme prefers a weak acid condition. Moreover, its activity could be enhanced at 50°C by adding Mg2+ or Mn2+ ions. Interestingly, in simultaneous saccharification and fermentation (SSF experiments using Saccharomyces cerevisiae BY4741 or Kluyveromyces marxianus KY3 as the fermentation yeast, NpaBGS showed advantages in cell growth, glucose production, and ethanol production over the commercial enzyme Novo 188. Moreover, we showed that the KY3 strain engineered with the NpaNGS gene can utilize 2 % dry napiergrass as the sole carbon source to produce 3.32 mg

  7. Glucose-tolerant β-glucosidase retrieved from the metagenome

    Directory of Open Access Journals (Sweden)

    Taku eUchiyama


    Full Text Available β-glucosidases (BGLs hydrolyze cellooligosaccharides to glucose and play a crucial role in the enzymatic saccharification of cellulosic biomass. Despite their significance for the production of glucose, most identified BGLs are commonly inhibited by low (~mM concentrations of glucose. Therefore, BGLs that are insensitive to glucose inhibition have great biotechnological merit. We applied a metagenomic approach to screen for such rare glucose-tolerant BGLs. A metagenomic library was created in Escherichia coli (approximately 10,000 colonies and grown on LB agar plates containing 5-bromo-4-chloro-3-indolyl-β-D-glucoside, yielding 828 positive (blue colonies. These were then arrayed in 96-well plates, grown in LB, and secondarily screened for activity in the presence of 10% (w/v glucose. Seven glucose-tolerant clones were identified, each of which contained a single bgl gene. The genes were classified into two groups, differing by two nucleotides. The deduced amino acid sequences of these genes were identical (452 aa and found to belong to the glycosyl hydrolase family 1. The recombinant protein (Ks5A7 was overproduced in E. coli as a C-terminal 6 × His-tagged protein and purified to apparent homogeneity. The molecular mass of the purified Ks5A7 was determined to be 54 kDa by SDS-PAGE, and 160 kDa by gel filtration analysis. The enzyme was optimally active at 45°C and pH 5.0–6.5 and retained full or 1.5–2-fold enhanced activity in the presence of 0.1–0.5 M glucose. It had a low KM (78 µM with p-nitrophenyl β-D-glucoside; 0.36 mM with cellobiose and high Vmax (91 µmol min-1 mg-1 with p-nitrophenyl β-D-glucoside; 155 µmol min-1 mg-1 with cellobiose among known glucose-tolerant BGLs and was free from substrate (0.1 M cellobiose inhibition. The efficient use of Ks5A7 in conjunction with Trichoderma reesei cellulases in enzymatic saccharification of alkaline-treated rice straw was demonstrated by increased production of glucose.

  8. Nitrogen and Sulfur Requirements for Clostridium thermocellum and Caldicellulosiruptor bescii on Cellulosic Substrates in Minimal Nutrient Media

    Energy Technology Data Exchange (ETDEWEB)

    Kridelbaugh, Donna M [ORNL; Nelson, Josh C [ORNL; Engle, Nancy L [ORNL; Tschaplinski, Timothy J [ORNL; Graham, David E [ORNL


    Growth media for cellulolytic Clostridium thermocellum and Caldicellulosiruptor bescii bacteria usually contain excess nutrients that would increase costs for consolidated bioprocessing for biofuel production and create a waste stream with nitrogen, sulfur and phosphate. C. thermocellum was grown on crystalline cellulose with varying concentrations of nitrogen and sulfur compounds, and growth rate and alcohol production response curves were determined. Both bacteria assimilated sulfate in the presence of ascorbate reductant, increasing the ratio of oxidized to reduced fermentation products. From these results, a low ionic strength, defined minimal nutrient medium with decreased nitrogen, sulfur, phosphate and vitamin supplements was developed for the fermentation of cellobiose, cellulose and acid-pretreated Populus. Carbon and electron balance calculations indicate the unidentified residual fermentation products must include highly reduced molecules. Both bacterial populations were maintained in co-cultures with substrates containing xylan or hemicellulose in defined medium with sulfate and basal vitamin supplements.

  9. A new highly efficient beta-glucosidase from the novel species, Aspergillus saccharolyticus

    DEFF Research Database (Denmark)

    Sørensen, Annette

    , combined with Celluclast 1.5L, substitute the use of Novozym 188 in hydrolysis of pretreated biomass. In the wake of this, focus was placed on beta-glucosidases. A screening for beta-glucosidase activity was conducted, using wheat bran as substrate in simple submerged fermentation, testing selected strains...... mainly opens up the cell wall structure and partly hydrolyzes hemicellulose, so that cellulose is the main target for enzyme hydrolysis. Beta-glucosidases (EC play an essential role in efficient hydrolysis of cellulose. By hydrolysis of cellobiose, beta-glucosidases relieve inhibiting...... preparations Novozym 188 (mainly beta-glucosidase activity) and Celluclast 1.5L (mainly cellobiohydrolase and endoglucanase activity) (Novozymes A/S) have been used in combination for hydrolysis of pretreated biomass, and recently complete enzyme cocktails have been launched, Cellic CTec (Novozymes A...

  10. Membrane-based recovery of glucose from enzymatic hydrolysis of ionic liquid pretreated cellulose. (United States)

    Abels, Christian; Thimm, Kristof; Wulfhorst, Helene; Spiess, Antje Christine; Wessling, Matthias


    In this work, a membrane-based downstream process for the recovery of glucose from cellulose hydrolysis is described and evaluated. The cellulose is pretreated with the ionic liquid 1,3-dimethyl-imidazolium dimethylphosphate to reduce its crystallinity. After enzymatic conversion of cellulose to glucose the hydrolysate is filtered with an ultrafiltration membrane to remove residual particulates and enzymes. Nanofiltration is applied to purify the glucose from molecular intermediates, such as cellobiose originating from the hydrolysis reaction. Finally, the ionic liquid is removed from the hydrolysate via electrodialysis. Technically, these process steps are feasible. An economic analysis of the process reveals that the selling price of glucose from this production process is about 2.75 €/kg which is too high as compared to the current market price.

  11. Identification and molecular modeling of a family 5 endocellulase from Thermus caldophilus GK24, a cellulolytic strain of Thermus thermophilus

    Directory of Open Access Journals (Sweden)

    Dae-Sil Lee


    Full Text Available The genome of T. caldophilus GK24 was recently sequenced and annotated as 14contigs, equivalent to 2.3 mega basepairs (Mbp of DNA. In the current study, we identifieda unique 13.7 kbp DNA sequence, which included the endocellulase gene of T. caldophilusGK24, which did not appear to be present in the complete genomic sequence of the closelyrelated species T. thermophilus HB27 and HB8. Congo-red staining revealed a uniquephenotype of cellulose degradation by strain GK24 that was distinct from other closelyrelated Thermus strains. The results showed that strain GK24 is an aerobic, thermophilic,cellulolytic eubacterium which belongs to the group T. thermophilus. In order to understandthe mechanism of production of cellobiose in T. caldophilus GK24, a three-dimensionalmodel of the endocellulase, TcCel5A, was generated based on known crystal structures.Using this model, we carried out a flexible cellotetraose docking study.

  12. A snapshot into the uptake and utilization of potential oligosaccharide prebiotics by probiotic lactobacilli and bifidobacteria as accessed by transcriptomics, functional genomics, and recombinant protein characterization

    DEFF Research Database (Denmark)

    Andersen, Joakim Mark

    with uptake and catabolism of potential prebiotics by the probiotics Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04 as model organisms, using DNA whole genome microarrays and by in silico pathway re-construction to identify key genes for further functional analysis by gene......Microorganisms that when administered in sufficient amounts exert a beneficial effect to the host are defined as probiotics. The positive clinical effects of probiotics, mainly belonging to the Bifidobacterium and Lactobacillus genera in treatments of irritated bowel disorders, gut infections......-galacto-oligosaccharides, cellobiose, gentiobiose, isomaltose, panose, raffinose, stachyose and selected strain-specific potential prebiotics – L. acidophilus NCFM: barley ß-glucan hydrolysate, lactitol, isomaltulose and polydextrose, while for B. lactis Bl-04: maltotriose, melibiose, xylobiose and xylo-oligosaccharides were used...

  13. Ultrastructural studies of the termite (Odontotermes obesus) gut microflora and its cellulolytic properties. (United States)

    Paul, J; Saxena, S; Varma, A


    The major gut microflora colonizing the hind gut of a higher termite,Odontotermes obesus, included morphologically diverse bacteria, both coccoid and rod-shaped, along with spirochaetes, pseudomonads and actinomycetes. Flagellated protozoa were totally absent. When the gut extract was inoculated on plates containing carboxymethyl cellulose or cellobiose, higher numbers of bacteria grew than on plates without cellulosic sources. The gut homogenate exhibited strong hydrolytic activity when carboxymethyl cellulose,p-nitrophenyl-β-D-glucoside or xylan were used as substrate, indicating the role of gut microbiota in the process of cellulose and hemicellulose digestion. Activities were highest in the hind gut, and the paunch was probably the major site of polysaccharide digestion in this higher termite.In vitro cultivation of some of the isolates revealed both cellulase and xylanase activities. To our knowledge, this is the first report on ultrastructural studies of the higher termiteOdontotermes obesus.


    Directory of Open Access Journals (Sweden)

    Ruchi Agrawal


    Full Text Available The bioethanol production from lignocellulosic biomass has attracted wide interest globally in last decade. One of the main reasons for the high cost of bioethanol production from lignocellulosic biomass is the expensive enzymes involved in enzymatic hydrolysis of cellulose (cellulase. The utilization of agro-industrial waste as a potential substrate for producing enzymes may serve a dual purpose of reducing the environmental pollution along with producing a high value commercial product. Twelve different agro-industrial wastes were evaluated for extracellular cellobiose or β-glucosidase production by a mutant of Bacillus subtilis on solid state fermentations (SSF. The Citrus sinensis peel waste was found to be the most suitable substrate with highest BGL titre (35 U/gds. Optimum incubation time, inoculum size, moisture content and volume of buffer for enzyme extraction were 72 h, 40 % v/w, 10 mL and 20 mL respectively.

  15. Comparative data on effects of leading pretreatments and enzyme loadings and formulations on sugar

    Energy Technology Data Exchange (ETDEWEB)

    Wyman, Charles [Univ. of California, Riverside, CA (United States); Balan, Venkatech [Michigan State Univ., East Lansing, MI (United States); Dale, Bruce E. [Michigan State Univ., East Lansing, MI (United States); Elander, Richard [National Renewable Energy Lab. (NREL), Golden, CO (United States); Falls, Matthew [Texas A & M Univ., College Station, TX (United States); Hames, Bonnie [Ceres Corporation, Thousand Oaks, CA (United States); Holtzapple, Mark [Texas A & M Univ., College Station, TX (United States); Ladisch, Michael R. [Purdue Univ., West Lafayette, IN (United States); Lee, Y. Y. [Auburn Univ., AL (United States); Mosier, Nathan [Purdue Univ., West Lafayette, IN (United States); Pallapolu, Venkata R. [Auburn Univ., AL (United States); Shi, Jian [Univ. of California, Riverside, CA (United States); Warner, Ryan E. [Genencor, Palo Alto, CA (United States)


    Dilute sulfuric acid (DA), sulfur dioxide (SO2), liquid hot water (LHW), soaking in aqueous ammonia (SAA), ammonia fiber expansion (AFEX), and lime pretreatments were applied to Alamo, Dacotah, and Shawnee switchgrass. Application of the same analytical methods and material balance approaches facil-itated meaningful comparisons of glucose and xylose yields from combined pretreatment and enzymatic hydrolysis. Use of a common supply of cellulase, beta-glucosidase, and xylanase also eased comparisons. All pretreatments enhanced sugar recovery from pretreatment and subsequent enzymatic hydrolysis substantially compared to untreated switchgrass. Adding beta-glucosidase was effective early in enzy-matic hydrolysis while cellobiose levels were high but had limited effect on longer term yields at the enzyme loadings applied. Adding xylanase improved yields most for higher pH pretreatments where more xylan was left in the solids. Harvest time had more impact on performance than switchgrass variety, and microscopy showed changes in different features could impact performance by different pretreatments.

  16. Detection of Extracellular Enzyme Activity in Penicillium using Chromogenic Media. (United States)

    Yoon, Ji Hwan; Hong, Seung Beom; Ko, Seung Ju; Kim, Seong Hwan


    A total of 106 Penicillium species were tested to examine their ability of degrading cellobiose, pectin and xylan. The activity of β-glucosidase was generally strong in all the Penicillium species tested. P. citrinum, P. charlesii, P. manginii and P. aurantiacum showed the higher ability of producing β-glucosidase than other tested species. Pectinase activity was detected in 24 Penicillium species. P. paracanescens, P. sizovae, P. sartoryi, P. chrysogenum, and P. claviforme showed strong pectinase activity. In xylanase assay, 84 Penicillium species showed activity. Strong xylanase activity was detected from P. megasporum, P. sartoryi, P. chrysogenum, P. glandicola, P. discolor, and P. coprophilum. Overall, most of the Penicillium species tested showed strong β-glucosidase activity. The degree of pectinase and xylanase activity varied depending on Penicillium species.

  17. A process for energy-efficient high-solids fed-batch enzymatic liquefaction of cellulosic biomass. (United States)

    Cardona, M J; Tozzi, E J; Karuna, N; Jeoh, T; Powell, R L; McCarthy, M J


    The enzymatic hydrolysis of cellulosic biomass is a key step in the biochemical production of fuels and chemicals. Economically feasible large-scale implementation of the process requires operation at high solids loadings, i.e., biomass concentrations >15% (w/w). At increasing solids loadings, however, biomass forms a high viscosity slurry that becomes increasingly challenging to mix and severely mass transfer limited, which limits further addition of solids. To overcome these limitations, we developed a fed-batch process controlled by the yield stress and its changes during liquefaction of the reaction mixture. The process control relies on an in-line, non-invasive magnetic resonance imaging (MRI) rheometer to monitor real-time evolution of yield stress during liquefaction. Additionally, we demonstrate that timing of enzyme addition relative to biomass addition influences process efficiency, and the upper limit of solids loading is ultimately limited by end-product inhibition as soluble glucose and cellobiose accumulate in the liquid phase.

  18. Extracellular cellulolytic enzyme system of Aspergillus japonicus: Pt. 2. Purification and characterization of an inducible extracellular. beta. -glucosidase

    Energy Technology Data Exchange (ETDEWEB)

    Sanyal, Arunik; Kundu, R.K.; Dube, S.; Dube, D.K.


    A high molecular weight ..beta..-glucosidase (mol. wt. > 240 000 daltons) was isolated from the culture filtrate of Aspergillus japonicus and was finally purified to 86-fold by alcohol precipitation, gel filtration and ion exchange chromatography on Whatman DE-52. An apparently homogeneous form of the enzyme appeared in the polyacrylamide gel electrophoresis. It is capable of utilizing cellobiose, salicin, o-nitrophenyl-..beta..-D-glucoside (ONPG), methyl-..beta..-D-glucoside and amygdalin effectively as substrates but not arbutin, esculin hydrate and phloridzin. No metal ion is required for its catalytic activity. Hg/sup ++/ and p-chloromercuricbenzoate (PCMB) are strong inhibitors for the enzyme. Nojirimycin and glucono-delta-lactone are two competitive inhibitors of the same enzyme, and nojirimycin is the more potent of the two.

  19. An efficient process for lactic acid production from wheat straw by a newly isolated Bacillus coagulans strain IPE22. (United States)

    Zhang, Yuming; Chen, Xiangrong; Luo, Jianquan; Qi, Benkun; Wan, Yinhua


    A thermophilic lactic acid (LA) producer was isolated and identified as Bacillus coagulans strain IPE22. The strain showed remarkable capability to ferment pentose, hexose and cellobiose, and was also resistant to inhibitors from lignocellulosic hydrolysates. Based on the strain's promising features, an efficient process was developed to produce LA from wheat straw. The process consisted of biomass pretreatment by dilute sulfuric acid and subsequent SSCF (simultaneous saccharification and co-fermentation), while the operations of solid-liquid separation and detoxification were avoided. Using this process, 46.12 g LA could be produced from 100g dry wheat straw with a supplement of 10 g/L corn steep liquid powder at the cellulase loading of 20 FPU (filter paper activity units)/g cellulose. The process by B. coagulans IPE22 provides an economical route to produce LA from lignocellulose.

  20. A direct electron transfer-based glucose/oxygen biofuel cell operating in human serum

    Energy Technology Data Exchange (ETDEWEB)

    Coman, V.; Gorton, L. [Department of Analytical Chemistry/Biochemistry, Lund University, 22100 Lund (Sweden); Ludwig, R. [Research Centre Applied Biocatalysis, 8010 Graz (Austria); Department of Food Sciences and Technology, BOKU-University of Natural Resources and Applied Life Sciences, 1190 Wien (Austria); Harreither, W.; Haltrich, D. [Department of Food Sciences and Technology, BOKU-University of Natural Resources and Applied Life Sciences, 1190 Wien (Austria); Ruzgas, T. [Biomedical Laboratory Science, Health and Society, Malmoe University, 20506 Malmoe (Sweden); Laboratory of Chemical Enzymology, A.N. Bach Institute of Biochemistry, 119071 Moscow (Russian Federation); Shleev, S.


    We report on the fabrication and characterisation of the very first direct electron transfer-based glucose/oxygen biofuel cell (BFC) operating in neutral glucose-containing buffer and human serum. Corynascus thermophilus cellobiose dehydrogenase and Myrothecium verrucaria bilirubin oxidase were used as anodic and cathodic bioelements, respectively. The following characteristics of the mediator-, separator- and membrane-less, a priori, non-toxic and simple miniature BFC, was obtained: an open-circuit voltage of 0.62 and 0.58 V, a maximum power density of ca. 3 and 4 {mu}W cm{sup -2} at 0.37 and 0.19 V of cell voltage, in phosphate buffer and human serum, respectively. (Abstract Copyright [2010], Wiley Periodicals, Inc.)

  1. Ectoine production from lignocellulosic biomass-derived sugars by engineered Halomonas elongata. (United States)

    Tanimura, Kosuke; Nakayama, Hideki; Tanaka, Tsutomu; Kondo, Akihiko


    In this study, the water-retaining cyclic amino acid ectoine was produced from a variety of sugars, including glucose, xylose, cellobiose, and glucose/xylose mixture using engineered Halomonas elongata. When grown on xylose as the sole carbon source, H. elongata produced 333 mmol/kg fresh cell weight (FW) of ectoine, which was 1.4-fold higher than that produced from glucose. To improve ectoine production, an ectD deficient H. elongata mutant was constructed. The engineered H. elongata produced 377 mmol/kg FW of ectoine from a glucose/xylose mixture. Ectoine was also produced from rice straw hydrolysate. These results show that H. elongata can produce ectoine from a variety of sugars derived from lignocellulosic biomass and thus has tremendous potential as a host for producing useful compounds from biomass resources.

  2. Hydrolysis of a mixture of saccharides by cellulase from Aspergillus niger and its application for visible-light-induced hydrogen gas production system using Mg chlorophyll-a and platinum nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Amao, Yutaka; Hirakawa, Takamasa [Department of Applied Chemistry, Oita University, Dannoharu 700, Oita 870-1192 (Japan)


    Cellulase obtained from Aspergillus niger was used to hydrolyze a mixture of saccharides containing sucrose, maltose, and cellobiose; the reduced form of nicotinamide-adenine dinucleotide (NAD{sup +}), which is NADH, was produced during hydrolysis of the mixture of saccharides in the presence of NAD{sup +} and glucose dehydrogenase (GDH). We have developed a visible-light-induced enzymatic biohydrogen production system involving the combination of cellulase-mediated hydrolysis of the mixture of saccharides and hydrogen production by platinum nanoparticles using photosensitization of Mg chlorophyll-a (Mg Chl-a). Continuous production of hydrogen gas was observed when the reaction mixture containing saccharides, cellulase, GDH, NAD{sup +}, Mg Chl-a, methylviologen (MV{sup 2+}, an electron donor), and platinum nanoparticles was irradiated by visible light. After 120 min of irradiation, the amount of hydrogen produced from the mixture of saccharides was approximately 2.8 {mu}mol. (author)


    Institute of Scientific and Technical Information of China (English)

    ZHANG Lina; LIU Haiqing; ZHENG Lianshuang; ZHANG Jiayao; DU Yumin; LIU Weili


    The biodegradability of Aspergillus niger (A. niger), Mucor (M-305) and Trichoderma (T-311) strains on regenerated cellulose films in media was investigated. The results showed that T-311 strain isolated from soil adhered on the cellulose film fragments has stronger degradation effect on the cellulose film than A. niger strain. The weights, molecular weights and tensile strengths of the cellulose films in both shake culture and solid media decreased with incubation time, accompanied by producing CO2 and saccharides. HPLC, IR and released CO2 analysis indicated that the biodegradation products of the regenerated cellulose films mainly contain oligosaccharides, cellobiose, glucose, arabinose, erythrose, glycerose,glycerol, ethanal, formaldehyde and organic acid, the end products were CO2 and water.After a month, the films were completely decomposed by fungi in the media at 30℃.


    Directory of Open Access Journals (Sweden)

    Keikhosro Karimi


    Full Text Available This article reviews developments in the technology for ethanol produc-tion from lignocellulosic materials by “enzymatic” processes. Several methods of pretreatment of lignocelluloses are discussed, where the crystalline structure of lignocelluloses is opened up, making them more accessible to the cellulase enzymes. The characteristics of these enzymes and important factors in enzymatic hydrolysis of the cellulose and hemicellulose to cellobiose, glucose, and other sugars are discussed. Different strategies are then described for enzymatic hydrolysis and fermentation, including separate enzymatic hydrolysis and fermentation (SHF, simultaneous saccharification and fermentation (SSF, non-isothermal simultaneous saccharification and fermentation (NSSF, simultaneous saccharification and co-fermentation (SSCF, and consolidated bioprocessing (CBP. Furthermore, the by-products in ethanol from lignocellulosic materials, wastewater treatment, commercial status, and energy production and integration are reviewed.

  5. Simultaneous saccharification and fermentation of cellulose in a CSTR type fermenter by thermotolerat Kluyveromyces marxianus

    Energy Technology Data Exchange (ETDEWEB)

    Campos, I. del; Ballesteros, I.; Ballesteros, M.


    Production of ethanol from lignocellulose biomass could provide a renewable source of premium transportation fuels that could reduce or eliminate the European dependence on mounting imports od petroleum from unstable sources. Enzymatic hydrolysis of cellulose is a key step in the bioconversion of lifnocellulosic biomass to fuel ethanol. the reaction is characterised by the synergistic effect of multi-enzyme systems and the adsorption of enzyme to the substrate is needed. The several end-product inhibition of cellulase action by the accumulation of both cellobiose and glucose can greatly limit concentration, yield, and reaction rate for enzymatic hydrolysis. This bottleneck can be overcome by the simultaneous saccharification and fermentation (SSF) Process, in which the enzymatic hydrolysis and yeast fermentation steps are combined in one vessel. In the SSP process the end-product inhibition of the enzyme is eliminated, therefore can be achieved high yields, rates and concentrations of ethanol. (Author)

  6. The type of carbohydrates specifically selects microbial community structures and fermentation patterns. (United States)

    Chatellard, Lucile; Trably, Eric; Carrère, Hélène


    The impact on dark fermentation of seven carbohydrates as model substrates of lignocellulosic fractions (glucose, cellobiose, microcrystalline cellulose, arabinose, xylose, xylan and wheat straw) was investigated. Metabolic patterns and bacterial communities were characterized at the end of batch tests inoculated with manure digestate. It was found that hydrogen production was linked to the sugar type (pentose or hexose) and the degree of polymerisation. Hexoses produced less hydrogen, with a specific selection of lactate-producing bacterial community structures. Maximal hydrogen production was five times higher on pentose-based substrates, with specific bacterial community structures producing acetate and butyrate as main metabolites. Low hydrogen amounts accumulated from complex sugars (cellulose, xylan and wheat straw). A relatively high proportion of the reads was affiliated to Ruminococcaceae suggesting an efficient hydrolytic activity. Knowing that the bacterial community structure is very specific to a particular substrate offers new possibilities to design more efficient H2-producing biological systems.

  7. 一株分离自西藏高山草甸土壤的肠球菌的分离鉴定及代谢特征%Isolation and characterization of an Enterococcus strain from Tibetan alpine meadow soil

    Institute of Scientific and Technical Information of China (English)

    杨磊; 邓宇; 张辉; 刁青云


    [目的]了解高海拔低温缺氧条件下土壤环境中微生物生理生化特性及代谢产物[方法]采用Hungate厌氧操作技术从西藏纳木措高山草甸土壤中分离到一株肠球菌C J-1.通过生理生化特征分析和16S rRNA基因序列的系统发育学分析确定该菌株的系统发育学地位.[结果]菌株C J-1为兼性厌氧的革兰氏阳性菌,菌体呈不规则球形,大小为直径约1 μm-1.5 μm,无鞭毛,不运动,成串珠型或成对排列.生长温度范围为10℃-50℃(最适温度为25℃) ;pH范围为5.0-8.5(最适pH为7.0) ;NaC1浓度为0% -7%(最适NaC1浓度为5%).能够利用葡萄糖、核糖、松三糖以及纤维二糖等多种碳水化合物,发酵纤维二糖的产物是乳酸、乙酸、丙酸、CO2、H2以及少量的丁酸.菌株CJ-1的(G+C)Mo1%为39.2%.与Enterococcus aquimarinus( CCM7283)的相似性达95.9%.菌株可利用纤维二糖,其在沼气发酵过程中充当中间代谢物.[结论]菌株CJ-1为耐低温肠球菌,可以降解纤维二糖等,且适应性较强,在发酵过程中具有重要意义,将其暂定命名为Enterococcus namtsoensis,模式菌株DSM23475T(=ACCC 00521T).%We studied the physiological, biochemical properties and metabolism of Enterococcus strain CJ-1 from high-altitude soil in Namtso, Tibet. [ Methods] Strain CJ-1T was isolated from the soil of Namtso alpine meadow soil by Hungate anaerobic technique. Through physiological, biochemical and phylogenetic analysis, we identified the strain CJ-1. [Results] Strain CJ-1 was Gram-positive and facultative anaerobe, 1-1.5 μm in diameter. CJ-1T was atrichia nonmotile cocci, and always occurred in pairs. CJ-1T occurred in the presence of 0% -7% NaCl (optimum at 5% ) , pH 5. 0 - 8. 5 (optimum 7. 0) and temperature between 10℃ and 50℃ (optimum at 25℃ ). CJ-1 could metabolize many carbon sources including cellobiose, melezitose and ribose. Metabolites of cellobiose were lactic acid, acetic acid, propionic acid

  8. An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose

    DEFF Research Database (Denmark)

    Cruys-Bagger, Nicolaj; Guilin, Ren; Tatsumi, Hirosuke;


    An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi-crystalline and amorphous......) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH-biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the β-anomer of cello-oligosaccharides and the approach were validated against HPLC....... It is suggested that quantitative, real-time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose...

  9. Cellulase Inhibition by High Concentrations of Monosaccharides

    DEFF Research Database (Denmark)

    Hsieh, Chia-Wen; Cannella, David; Jørgensen, Henning;


    Biological degradation of biomass on an industrial scale culminates in high concentrations of end products. It is known that the accumulation of glucose and cellobiose, end products of hydrolysis, inhibit cellulases and decrease glucose yields. Aside from these end products, however, other...... monosaccharides such as mannose and galactose (stereoisomers of glucose) decrease glucose yields as well. NMR relaxometry measurements showed direct correlations between the initial T 2 of the liquid phase in which hydrolysis takes place and the total glucose production during cellulose hydrolysis, indicating...... that low free water availability contributes to cellulase inhibition. Of the hydrolytic enzymes involved, those acting on the cellulose substrate, that is, exo- and endoglucanases, were the most inhibited. The β -glucosidases were shown to be less sensitive to high monosaccharide concentrations except...

  10. Complete genome sequence of the gliding, heparinolytic Pedobacter saltans type strain (113T)

    Energy Technology Data Exchange (ETDEWEB)

    Liolios, Konstantinos [U.S. Department of Energy, Joint Genome Institute; Sikorski, Johannes [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Lu, Megan [Los Alamos National Laboratory (LANL); Nolan, Matt [U.S. Department of Energy, Joint Genome Institute; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Hammon, Nancy [U.S. Department of Energy, Joint Genome Institute; Deshpande, Shweta [U.S. Department of Energy, Joint Genome Institute; Cheng, Jan-Fang [U.S. Department of Energy, Joint Genome Institute; Tapia, Roxanne [Los Alamos National Laboratory (LANL); Han, Cliff [Los Alamos National Laboratory (LANL); Goodwin, Lynne A. [Los Alamos National Laboratory (LANL); Pitluck, Sam [U.S. Department of Energy, Joint Genome Institute; Huntemann, Marcel [U.S. Department of Energy, Joint Genome Institute; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Pagani, Ioanna [U.S. Department of Energy, Joint Genome Institute; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Ovchinnikova, Galina [U.S. Department of Energy, Joint Genome Institute; Pati, Amrita [U.S. Department of Energy, Joint Genome Institute; Chen, Amy [U.S. Department of Energy, Joint Genome Institute; Palaniappan, Krishna [U.S. Department of Energy, Joint Genome Institute; Land, Miriam L [ORNL; Hauser, Loren John [ORNL; Brambilla, Evelyne-Marie [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Kotsyurbenko, Oleg [Technical University of Braunschweig; Rohde, Manfred [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Tindall, Brian [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Abt, Birte [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Goker, Markus [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Detter, J. Chris [U.S. Department of Energy, Joint Genome Institute; Woyke, Tanja [U.S. Department of Energy, Joint Genome Institute; Bristow, James [U.S. Department of Energy, Joint Genome Institute; Eisen, Jonathan [U.S. Department of Energy, Joint Genome Institute; Markowitz, Victor [U.S. Department of Energy, Joint Genome Institute; Hugenholtz, Philip [U.S. Department of Energy, Joint Genome Institute; Klenk, Hans-Peter [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute


    Pedobacter saltans Steyn et al. 1998 is one of currently 32 species in the genus Pedobacter within the family Sphingobacteriaceae. The species is of interest for its isolated location in the tree of life. Like other members of the genus P. saltans is heparinolytic. Cells of P. saltans show a peculiar gliding, dancing motility and can be distinguished from other Pedobacter strains by their ability to utilize glycerol and the inability to assimilate D-cellobiose. The ge- nome presented here is only the second completed genome sequence of a type strain from a member of the family Sphingobacteriaceae to be published. The 4,635,236 bp long genome with its 3,854 protein-coding and 67 RNA genes consists of one chromosome, and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  11. A pyranose dehydrogenase-based biosensor for kinetic analysis of enzymatic hydrolysis of cellulose by cellulases

    DEFF Research Database (Denmark)

    Cruys-Bagger, Nicolaj; Badino, Silke Flindt; Tokin, Radina Naytchova


    A novel electrochemical enzyme biosensor was developed for real-time detection of cellulase activity when acting on their natural insoluble substrate, cellulose. The enzyme biosensor was constructed with pyranose dehydrongease (PDH) from Agaricus meleagris that was immobilized on the surface...... of a carbon paste electrode, which contained the mediator 2,6-dichlorophenolindophenol (DCIP). An oxidation current of the reduced form of DCIP, DCIPH2, produced by the PDH-catalyzed reaction with either glucose or cellobiose, was recorded under constant-potential amperometry at +0.25 V (vs. Ag/AgCl). The PDH-biosensor...... was shown to be anomer unspecific and it can therefore be used in kinetic studies over broad time-scales of both retaining- and inverting cellulases (in addition to enzyme cocktails). The biosensor was used for real-time measurements of the activity of the inverting cellobiohydrolase Cel6A from Hypocrea...

  12. Emerging synbiotics and their effect on the composition and functionality of the human gut microbiota

    DEFF Research Database (Denmark)

    van Zanten, Gabriella Christina

    , may be used to change the composition and activity of the human GM and thereby potentially affect the host health beneficially. In this PhD study it was hypothesized that emerging synbiotics have the potential of modulating the human GM composition as well as the functionality. To gain the beneficial...... effects of both probiotics and prebiotics, they may be combined to synbiotics. The definition of synbiotics implies that the prebiotic should be specific to the probiotic in question. Therefore, a library of 37 emerging prebiotics, comprising carbohydrates with a large range of degrees of polymerization...... emerging prebiotics were selected for further investigation using a four-stage semi-continuous model system of the human colon. The selected combinations were NCFM with isomaltulose, cellobiose, raffinose and oat β-glucan hydrolyzed with endo-1,3-β-glucanase (OBGH), respectively and Bl-04 with melibiose...

  13. Numerical prediction of kinetic model for enzymatic hydrolysis of cellulose using DAE-QMOM approach (United States)

    Jamil, N. M.; Wang, Q.


    Bioethanol production from lignocellulosic biomass consists of three fundamental processes; pre-treatment, enzymatic hydrolysis, and fermentation. In enzymatic hydrolysis phase, the enzymes break the cellulose chains into sugar in the form of cellobiose or glucose. A currently proposed kinetic model for enzymatic hydrolysis of cellulose that uses population balance equation (PBE) mechanism was studied. The complexity of the model due to integrodifferential equations makes it difficult to find the analytical solution. Therefore, we solved the full model of PBE numerically by using DAE-QMOM approach. The computation was carried out using MATLAB software. The numerical results were compared to the asymptotic solution developed in the author's previous paper and the results of Griggs et al. Besides confirming the findings were consistent with those references, some significant characteristics were also captured. The PBE model for enzymatic hydrolysis process can be solved using DAE-QMOM method. Also, an improved understanding of the physical insights of the model was achieved.

  14. Probiotic activity of lignocellulosic enzyme as bioactivator for rice husk degradation (United States)

    Lamid, Mirni; Al-Arif, Anam; Warsito, Sunaryo Hadi


    The utilization of lignocellulosic enzyme will increase nutritional value of rice husk. Cellulase consists of C1 (β-1, 4-glucan cellobiohydrolase or exo-β-1,4glucanase), Cc (endo-β-1,4-glucanase) and component and cellobiose (β-glucocidase). Hemicellulase enzyme consists of endo-β-1,4-xilanase, β-xilosidase, α-L arabinofuranosidase, α-D-glukuronidaseand asetil xilan esterase. This research aimed to study the activity of lignocellulosic enzyme, produced by cows in their rumen, which can be used as a bioactivator in rice husk degradation. This research resulted G6 and G7 bacteria, producing xylanase and cellulase with the activity of 0.004 U mL-1 and 0.021 U mL-1; 0.003 ( U mL-1) and 0.026 (U mL-1) respectively.

  15. A kinetics modeling study on the inhibition of glucose on cellulosome of Clostridium thermocellum. (United States)

    Zhang, Pengcheng; Wang, Buyun; Xiao, Qunfang; Wu, Shan


    A simplified kinetics model was built to study the inhibition of glucose on cellulosome of Clostridium thermocellum. Suitable reaction conditions were adopted to evaluate the model. The model was evaluated at different temperatures and further with various activated carbon additions as adsorbent for glucose. Investigation results revealed that the model could describe the hydrolysis kinetics of cellulose by cellulosome quite well. Glucose was found to be an inhibitor for cellulosome based on the kinetics analysis. Inhibition increased with the increase in temperature. Activated carbon as adsorbent could lower the inhibition. Parameters in the model were further discussed based on the experiment. The model might also be used to describe the strong inhibition of cellobiose on cellulosome. Saccharification of cellulose by both cellulosome and C. thermocellum could be enhanced efficiently by activated carbon addition.


    Directory of Open Access Journals (Sweden)

    Maria Dimarogona


    Full Text Available Enzymatic degradation of plant biomass has attracted intensive research interest for the production of economically viable biofuels. Here we present an overview of the recent findings on biocatalysts implicated in the oxidative cleavage of cellulose, including polysaccharide monooxygenases (PMOs or LPMOs which stands for lytic PMOs, cellobiose dehydrogenases (CDHs and members of carbohydrate-binding module family 33 (CBM33. PMOs, a novel class of enzymes previously termed GH61s, boost the efficiency of common cellulases resulting in increased hydrolysis yields while lowering the protein loading needed. They act on the crystalline part of cellulose by generating oxidized and non-oxidized chain ends. An external electron donor is required for boosting the activity of PMOs. We discuss recent findings concerning their mechanism of action and identify issues and questions to be addressed in the future.

  17. Preparation and characterization of. beta. -D-glucosidase immobilized in calcium alginate

    Energy Technology Data Exchange (ETDEWEB)

    Krasniak, S. R.; Smith, R. D.


    Enzymatic hydrolysis of biomass to produce glucose may become feasible if an inexpensive method to reuse the enzyme can be found. This study investigated one such method whereby ..beta..-D-glucosidase (E.C. was immobilized in calcium alginate gel spheres, which were shown to catalyze the hydrolysis of cellobiose to glucose. There was a loss of 49% of the enzyme from the alginate slurry during gelation. After gelation, in the stable gel spheres, there was a 37% retention of the enzyme activity that was actually immobilized. The reason for the loss in activity was investigated and may be caused by inhibition of the enzyme within the sphere by the calcium cations and the alginate anions also present. Mass transfer effects were minimal in this system and were not responsible for the activity loss.

  18. A new family-3 glycoside hydrolase from Penicillium oxalicum BL 3005 catalyzing tyrosol glucosylation to form salidroside. (United States)

    Yang, Xue-Peng; Wang, Fang-Fang; Yan, Ji; Ma, Ke; Mao, Duo-Bin


    A glycoside hydrolase from Penicillium oxalicum BL 3005 was purified to apparent homogeneity. Its molecular mass was estimated to be 90 kDa by SDS-PAGE. The enzyme was identified to be a new member of family-3 by peptide sequence. High transglycosylation activity was found in the hydrolytic reaction of cellobiose. In the reaction, salidroside (4-hydroxyphenethyl O-β-d-glucopyranoside) was formed by adding tyrosol as the glycosyl acceptor. The optimum reaction pH and temperature were pH 6.5 and 55 °C, respectively. The maximum yield of salidroside was almost 20 g/L. These results indicated that the β-glucosidase of P. oxalicum can be considered as a very promising catalyst for the synthesis of salidroside.


    Directory of Open Access Journals (Sweden)

    Nurul Kartini Abu Bakar,


    Full Text Available Utilization of oil palm empty fruit bunch (OPEFB for bioethanol production with crude cellulase cocktails from locally isolated fungi was studied. Enzymatic saccharification of alkaline pretreated OPEFB was done using different cellulase enzyme preparations. Crude cellulase cocktails from Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 produced 8.37 g/L reducing sugars with 0.17 g/g yield. Production of bioethanol from OPEFB hydrolysate using Baker’s yeast produced approximately 0.59 g/L ethanol, corresponding to 13.8% of the theoretical yield. High reducing sugars concentration in the final fermentation samples resulted from accumulation of non-fermentable sugars such as xylose and cellobiose that were not consumed by the yeast. The results obtained support the possible utilization of OPEFB biomass for bioethanol production in the future.

  20. The Metabolic Inhibition Model Which Predicts the Intestinal Absorbability and Metabolizability of Drug: Theory and Experiment

    Directory of Open Access Journals (Sweden)

    Mizuma Takashi


    Full Text Available The intestinal absorption of analgesic peptides (leucine enkephalin and kyotorphin and modified peptides in rat were studied. Although these peptides were not absorbed, the absorbability (absorption clearance of these peptides were increased in the presence of peptidase inhibitors. In order to kinetically analyze these phenomena, we proposed the metabolic inhibition model, which incorporated the metabolic clearance (metabolizability with the absorption clearance. Metabolic activity was determined with intestinal homogenates. The higher the metabolic clearance was, the lower was the absorption clearance. The relationships between the absorption clearance and the metabolic clearance of the experimental data as well as of the theoretical values were hyperbolic. This model predicted the maximum absorption clearances of cellobiose-coupled leucine enkephalin (0.654 &mgr;l/min/cm and kyotorphin (0.247 &mgr;l/min/cm. Details of the experimental methods are described.

  1. A Streamlined Strategy for Biohydrogen Production with Halanaerobium hydrogeniformans, an Alkaliphilic Bacterium. (United States)

    Begemann, Matthew B; Mormile, Melanie R; Sitton, Oliver C; Wall, Judy D; Elias, Dwayne A


    Biofuels are anticipated to enable a shift from fossil fuels for renewable transportation and manufacturing fuels, with biohydrogen considered attractive since it could offer the largest reduction of global carbon budgets. Currently, lignocellulosic biohydrogen production remains inefficient with pretreatments that are heavily fossil fuel-dependent. However, bacteria using alkali-treated biomass could streamline biofuel production while reducing costs and fossil fuel needs. An alkaliphilic bacterium, Halanaerobiumhydrogeniformans, is described that is capable of biohydrogen production at levels rivaling neutrophilic strains, but at pH 11 and hypersaline conditions. H. hydrogeniformans ferments a variety of 5- and 6-carbon sugars derived from hemicellulose and cellulose including cellobiose, and forms the end products hydrogen, acetate, and formate. Further, it can also produce biohydrogen from switchgrass and straw pretreated at temperatures far lower than any previously reported and in solutions compatible with growth. Hence, this bacterium can potentially increase the efficiency and efficacy of biohydrogen production from renewable biomass resources.

  2. A Streamlined Strategy for Biohydrogen Production with an Alkaliphilic Bacterium

    Energy Technology Data Exchange (ETDEWEB)

    Elias, Dwayne A [ORNL; Wall, Judy D. [University of Missouri; Mormile, Dr. Melanie R. [Missouri University of Science and Technology; Begemann, Matthew B [University of Wisconsin, Madison


    Biofuels are anticipated to enable a shift from fossil fuels for renewable transportation and manufacturing fuels, with biohydrogen considered attractive since it could offer the largest reduction of global carbon budgets. Currently, biohydrogen production remains inefficient and heavily fossil fuel-dependent. However, bacteria using alkali-treated biomass could streamline biofuel production while reducing costs and fossil fuel needs. An alkaliphilic bacterium, Halanaerobium strain sapolanicus, is described that is capable of biohydrogen production at levels rivaling neutrophilic strains, but at pH 11 and hypersaline conditions. H. sapolanicus ferments a variety of 5- and 6- carbon sugars derived from hemicellulose and cellulose including cellobiose, and forms the end products hydrogen and acetate. Further, it can also produce biohydrogen from switchgrass and straw pretreated at temperatures far lower than any previously reported and in solutions compatible with growth. Hence, this bacterium can potentially increase the efficiency and efficacy of biohydrogen production from renewable biomass resources.

  3. A Streamlined Strategy for Biohydrogen Production with Halanaerobium hydrogeniformans, an Alkaliphilic Bacterium

    Directory of Open Access Journals (Sweden)

    Matthew eBegemann


    Full Text Available Biofuels are anticipated to enable a shift from fossil fuels for renewable transportation and manufacturing fuels, with biohydrogen considered attractive since it could offer the largest reduction of global carbon budgets. Currently, lignocellulosic biohydrogen production remains inefficient with pretreatments that are heavily fossil fuel-dependent. However, bacteria using alkali-treated biomass could streamline biofuel production while reducing costs and fossil fuel needs. An alkaliphilic bacterium, Halanaerobium hydrogeniformans, is described that is capable of biohydrogen production at levels rivaling neutrophilic strains, but at pH 11 and hypersaline conditions. H. hydrogeniformans ferments a variety of 5- and 6- carbon sugars derived from hemicellulose and cellulose including cellobiose, and forms the end products hydrogen, acetate and formate. Further, it can also produce biohydrogen from switchgrass and straw pretreated at temperatures far lower than any previously reported and in solutions compatible with growth. Hence, this bacterium can potentially increase the efficiency and efficacy of biohydrogen production from renewable biomass resources.

  4. Metabolic engineering of Caldicellulosiruptor bescii yields increased hydrogen production from lignocellulosic biomass

    Energy Technology Data Exchange (ETDEWEB)

    Cha, Minseok [University of Georgia, Athens, GA; Chung, Daehwan [University of Georgia, Athens, GA; Elkins, James G [ORNL; Guss, Adam M [ORNL; Westpheling, Janet [University of Georgia, Athens, GA


    Background: Members of the anaerobic thermophilic bacterial genus Caldicellulosiruptor are emerging candidates for consolidated bioprocessing (CBP) because they are capable of efficiently growing on biomass without conventional pretreatment. C. bescii produces primarily lactate, acetate and hydrogen as fermentation products, and while some Caldicellulosiruptor strains produce small amounts of ethanol C. bescii does not, making it an attractive background to examine the effects of metabolic engineering. The recent development of methods for genetic manipulation has set the stage for rational engineering of this genus for improved biofuel production. Here, we report the first targeted gene deletion, the gene encoding lactate dehydrogenase (ldh), for metabolic engineering of a member of this genus. Results: A deletion of the C. bescii L-lactate dehydrogenase gene (ldh) was constructed on a non-replicating plasmid and introduced into the C. bescii chromosome by marker replacement. The resulting strain failed to produce detectable levels of lactate from cellobiose and maltose, instead increasing production of acetate and H2 by 21-34% relative to the wild type and pyrFA parent strains. The same phenotype was observed on a real-world substrate switchgrass (Panicum virgatum). Furthermore, the ldh deletion strain grew to a higher maximum optical density than the wild type on maltose and cellobiose, consistent with the prediction that the mutant would gain additional ATP with increased acetate production. Conclusions: Deletion of ldh in C. bescii is the first use of recently developed genetic methods for metabolic engineering of these bacteria. This deletion resulted in a redirection of electron flow from production of lactate to acetate and hydrogen. New capabilities in metabolic engineering combined with intrinsic utilization of lignocellulosic materials position these organisms to provide a new paradigm for consolidated bioprocessing of fuels and other products from

  5. Paracellular absorption: a bat breaks the mammal paradigm.

    Directory of Open Access Journals (Sweden)

    Enrique Caviedes-Vidal

    Full Text Available Bats tend to have less intestinal tissue than comparably sized nonflying mammals. The corresponding reduction in intestinal volume and hence mass of digesta carried is advantageous because the costs of flight increase with load carried and because take-off and maneuverability are diminished at heavier masses. Water soluble compounds, such as glucose and amino acids, are absorbed in the small intestine mainly via two pathways, the transporter-mediated transcellular and the passive, paracellular pathways. Using the microchiropteran bat Artibeus literatus (mean mass 80.6+/-3.7 g, we tested the predictions that absorption of water-soluble compounds that are not actively transported would be extensive as a compensatory mechanism for relatively less intestinal tissue, and would decline with increasing molecular mass in accord with sieve-like paracellular absorption. Using a standard pharmacokinetic technique, we fed, or injected intraperitoneally the metabolically inert carbohydrates L-rhamnose (molecular mass = 164 Da and cellobiose (molecular mass = 342 Da which are absorbed only by paracellular transport, and 3-O-methyl-D-glucose (3OMD-glucose which is absorbed via both mediated (active and paracellular transport. As predicted, the bioavailability of paracellular probes declined with increasing molecular mass (rhamnose, 90+/-11%; cellobiose, 10+/-3%, n = 8 and was significantly higher in bats than has been reported for laboratory rats and other mammals. In addition, absorption of 3OMD-glucose was high (96+/-11%. We estimated that the bats rely on passive, paracellular absorption for more than 70% of their total glucose absorption, much more than in non-flying mammals. Although possibly compensating for less intestinal tissue, a high intestinal permeability that permits passive absorption might be less selective than a carrier-mediated system for nutrient absorption and might permit toxins to be absorbed from plant and animal material in the

  6. Genomic, proteomic, and biochemical analyses of oleaginous Mucor circinelloides: evaluating its capability in utilizing cellulolytic substrates for lipid production.

    Directory of Open Access Journals (Sweden)

    Hui Wei

    Full Text Available Lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. However, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. We report the first comprehensive investigation of Mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids. Our genomic analysis revealed the existence of genes encoding 13 endoglucanases (7 of them secretory, 3 β-D-glucosidases (2 of them secretory and 243 other glycoside hydrolase (GH proteins, but not genes for exoglucanases such as cellobiohydrolases (CBH that are required for breakdown of cellulose to cellobiose. Analysis of the major PAGE gel bands of secretome proteins confirmed expression of two secretory endoglucanases and one β-D-glucosidase, along with a set of accessory cell wall-degrading enzymes and 11 proteins of unknown function. We found that M. circinelloides can grow on CMC (carboxymethyl cellulose and cellobiose, confirming the enzymatic activities of endoglucanases and β-D-glucosidases, respectively. The data suggested that M. circinelloides could be made usable as a consolidated bioprocessing (CBP strain by introducing a CBH (e.g. CBHI into the microorganism. This proposal was validated by our demonstration that M. circinelloides growing on Avicel supplemented with CBHI produced about 33% of the lipid that was generated in glucose medium. Furthermore, fatty acid methyl ester (FAME analysis showed that when growing on pre-saccharified Avicel substrates, it produced a higher proportion of C14 fatty acids, which has an interesting implication in that shorter fatty acid chains have characteristics that are ideal for use in jet fuel. This substrate-specific shift in FAME profile warrants further investigation.

  7. [Zeolite catalysis in conversion of cellulosics

    Energy Technology Data Exchange (ETDEWEB)

    Tsao, G.T.


    To transform biomass into fermentable substrate for yeast, we are using zeolites instead of enzymes to catalyze the two bottleneck reactions in biomass conversion, xylose isomerization and ceuobiose hydrolysis. The experimental results on these reactions carried out over various zeolites and other catalysts are presented herein. The advantages and disadvantages of using these catalysts over enzymes are also discussed. Heterogeneous solid catalysts other than zeolites has been employed for cellobiose-to-glucose hydrolysis. The size and shape selectivity that makes zeoutes unique for some reactions can add diffusional hindrance. We have spent some time screening various known solid acidic catalysts. We report that a class of cationic ion exchange resins in the acidified form (e.g. Amberlite) has worked well as an acidic catalyst in hydrolyzing cellobiose to glucose. Our experimental results, together with those obtained from a homogeneous acid catalyst (e.g. sulfuric acid) for comparison are provided. Having succeeded in finding an alternative solid acid catalyst for hydrolysis, we explored other solid resin or other homogeneous but non-enzyme catalyst to carry out the xylose-to-xylulose isomerization. A fairly extensive search has been made. We explored the use of sodium aluminates in the homogeneous phase isomerization of glucose to fructose and obtained a very high conversion of glucose to fructose with the final mixture containing 85% of fructose instead of the common 45%. Fructose apparently complexes with aluminates, and its equilibrium concentration is shifted to considerably higher values than permitted by simple glucose/fructose equilibrium. We have recently found a number of catalysts capable of promoting isomerization between aldoses and ketoses. One solid resin, known as polyvinyl pyridine (PVP), is able to convert xylose to xylulose at a pH below 7. Our usage of alternative isomerization catalysts, including PVP, are described.

  8. [Zeolite catalysis in conversion of cellulosics

    Energy Technology Data Exchange (ETDEWEB)

    Tsao, G.T.


    To transform biomass into fermentable substrate for yeast, we are using zeolites instead of enzymes to catalyze the two bottleneck reactions in biomass conversion, xylose isomerization and ceuobiose hydrolysis. The experimental results on these reactions carried out over various zeolites and other catalysts are presented herein. The advantages and disadvantages of using these catalysts over enzymes are also discussed. Heterogeneous solid catalysts other than zeolites has been employed for cellobiose-to-glucose hydrolysis. The size and shape selectivity that makes zeoutes unique for some reactions can add diffusional hindrance. We have spent some time screening various known solid acidic catalysts. We report that a class of cationic ion exchange resins in the acidified form (e.g. Amberlite) has worked well as an acidic catalyst in hydrolyzing cellobiose to glucose. Our experimental results, together with those obtained from a homogeneous acid catalyst (e.g. sulfuric acid) for comparison are provided. Having succeeded in finding an alternative solid acid catalyst for hydrolysis, we explored other solid resin or other homogeneous but non-enzyme catalyst to carry out the xylose-to-xylulose isomerization. A fairly extensive search has been made. We explored the use of sodium aluminates in the homogeneous phase isomerization of glucose to fructose and obtained a very high conversion of glucose to fructose with the final mixture containing 85% of fructose instead of the common 45%. Fructose apparently complexes with aluminates, and its equilibrium concentration is shifted to considerably higher values than permitted by simple glucose/fructose equilibrium. We have recently found a number of catalysts capable of promoting isomerization between aldoses and ketoses. One solid resin, known as polyvinyl pyridine (PVP), is able to convert xylose to xylulose at a pH below 7. Our usage of alternative isomerization catalysts, including PVP, are described.

  9. 转基因pYBGAl酵母发酵木质纤维素水解液制备燃料乙醇%Ethanol production from lignocellulose hydrolysate by yeast-pYBGA1

    Institute of Scientific and Technical Information of China (English)

    梁鲜香; 吉田孝; 金日光


    he glucose and cellobiose was fermented by using pYBGA1 recombinant yeast, study the effects of the temperature and concentration of yeast-pYBGA1 on the ferment ethanol rate by using cross experiments; the lig-nocellulosic was pretreated through steam explosion pretreatment method, the glucose and cellobiose content in the pretreatment hydrolysate was 50 % ; the pretreatment hydrolysate was fermented by using yeast-pYBGA1 to prepare the ethanol. Results shows that, the best fermentation temperature was 28℃ and the best to the ethanol productivity was 10~8 mL~(-1) ; the highest concentration of ethanol by direct fermentation of mixed sugar in ligno-cellulose-pretreatment hydrolysate is 6.8 g/L when the suger concentration is 20 g/L in the hydrolysate.%采用转基因pYBGA1酵母发酵葡萄糖及纤维二糖培养基,通过交叉实验研究了温度、酵母投入量对乙醇产率的影响;对木质纤维素进行爆破预处理,得到葡萄糖和纤维二糖的质量分数占50%预处理水解液,采用转基因pYBGA1酵母对预处理水解液发酵制备乙醇.结果表明,转基因pYBGA1酵母最佳发酵温度为28℃,最佳酵母投入量为10~8mL~(-1);当水解液中糖浓度为20g/L时,预处理水解液发酵得到乙醇的最大浓度可达6.8g/L.

  10. PGASO: A synthetic biology tool for engineering a cellulolytic yeast

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    Chang Jui-Jen


    Full Text Available Abstract Background To achieve an economical cellulosic ethanol production, a host that can do both cellulosic saccharification and ethanol fermentation is desirable. However, to engineer a non-cellulolytic yeast to be such a host requires synthetic biology techniques to transform multiple enzyme genes into its genome. Results A technique, named Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO, that employs overlapping oligonucleotides for recombinatorial assembly of gene cassettes with individual promoters, was developed. PGASO was applied to engineer Kluyveromycesmarxianus KY3, which is a thermo- and toxin-tolerant yeast. We obtained a recombinant strain, called KR5, that is capable of simultaneously expressing exoglucanase and endoglucanase (both of Trichodermareesei, a beta-glucosidase (from a cow rumen fungus, a neomycin phosphotransferase, and a green fluorescent protein. High transformation efficiency and accuracy were achieved as ~63% of the transformants was confirmed to be correct. KR5 can utilize beta-glycan, cellobiose or CMC as the sole carbon source for growth and can directly convert cellobiose and beta-glycan to ethanol. Conclusions This study provides the first example of multi-gene assembly in a single step in a yeast species other than Saccharomyces cerevisiae. We successfully engineered a yeast host with a five-gene cassette assembly and the new host is capable of co-expressing three types of cellulase genes. Our study shows that PGASO is an efficient tool for simultaneous expression of multiple enzymes in the kefir yeast KY3 and that KY3 can serve as a host for developing synthetic biology tools.

  11. The Hadal Amphipod Hirondellea gigas possessing a unique cellulase for digesting wooden debris buried in the deepest seafloor.

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    Hideki Kobayashi

    Full Text Available The Challenger Deep in the Mariana Trench is the deepest point in the ocean (10,994 m. Certain deep-sea animals can withstand the extreme pressure at this great depth. The amphipod Hirondellea gigas is a resident of the Challenger Deep. Amphipods are common inhabitants at great depths and serve as scavengers. However, there is relatively little information available regarding the physiology of H. gigas or this organism's ecological interactions in the hadopelagic zone. To understand the feeding behavior of this scavenger in the deepest oligotrophic hadal zone, we analyzed the digestive enzymes in whole-body extracts. We describe the detection of amylase, cellulase, mannanase, xylanase, and α-glycosidase activities that are capable of digesting plant-derived polysaccharides. Our identification of glucose, maltose, and cellobiose in the H. gigas extracts indicated that these enzymes function under great pressure in situ. In fact, the glucose content of H. gigas averaged 0.4% (w/dry-w. The purified H. gigas cellulase (HGcel converted cellulose to glucose and cellobiose at an exceptional molar ratio of 2:1 and efficiently produced glucose from dried wood, a natural cellulosic biomass, at 35 °C. The enzyme activity increased under a high hydrostatic pressure of 100 MPa at 2 °C, conditions equivalent to those found in the Challenger Deep. An analysis of the amino acid sequence of HGcel supported its classification as a family 31 glycosyl hydrolase. However, none of the enzymes of this family had previously been shown to possess cellulase activity. These results strongly suggested that H. gigas adapted to its extreme oligotrophic hadal oceanic environment by evolving digestive enzymes capable of digesting sunken wooden debris.

  12. Absorption of sugars in the Egyptian fruit bat (Rousettus aegyptiacus): a paradox explained. (United States)

    Tracy, Christopher R; McWhorter, Todd J; Korine, Carmi; Wojciechowski, Michał S; Pinshow, Berry; Karasov, William H


    Two decades ago D. J. Keegan reported results on Egyptian fruit bats (Rousettus aegyptiacus, Megachiroptera) that were strangely at odds with the prevailing understanding of how glucose is absorbed in the mammalian intestine. Keegan's in vitro tests for glucose transport against a concentration gradient and with phloridzin inhibition in fruit bat intestine were all negative, although he used several different tissue preparations and had positive control results with laboratory rats. Because glucose absorption by fruit bats is nonetheless efficient, Keegan postulated that the rapid glucose absorption from the fruit bat intestine is not through the enterocytes, but must occur via spaces between the cells. Thus, we hypothesized that absorption of water-soluble compounds that are not actively transported would be extensive in these bats, and would decline with increasing molecular mass in accord with sieve-like paracellular absorption. We did not presume from Keegan's studies that there is no Na(+)-coupled, mediated sugar transport in these bats, and our study was not designed to rule it out, but rather to quantify the level of possible non-mediated absorption. Using a standard pharmacokinetic technique, we fed, or injected intraperitonealy, the metabolically inert carbohydrates L-rhamnose (molecular mass=164 Da) and cellobiose (molecular mass=342 Da), which are absorbed by paracellular uptake, and 3-O-methyl-D-glucose (3OMD-glucose), a D-glucose analog that is absorbed via both mediated (active) and paracellular uptake. As predicted, the bioavailability of paracellular probes declined with increasing molecular mass (rhamnose, 62+/-4%; cellobiose, 22+/-4%) and was significantly higher in bats than has been reported for rats and other mammals. In addition, fractional absorption of 3OMd-glucose was high (91+/-2%). We estimated that Egyptian fruit bats rely on passive, paracellular absorption for the majority of their glucose absorption (at least 55% of 3OMD

  13. Escherichia fergusonii and Enterobacter taylorae, two new species of Enterobacteriaceae isolated from clinical specimens. (United States)

    Farmer, J J; Fanning, G R; Davis, B R; O'Hara, C M; Riddle, C; Hickman-Brenner, F W; Asbury, M A; Lowery, V A; Brenner, D J


    Escherichia fergusonii (formerly known as Enteric Group 10) and Enterobacter taylorae (formerly known as Enteric Group 19) are proposed as new species in the family Enterobacteriaceae. By DNA hybridization (32P, 60 degrees C, hydroxyapatite), strains of E. fergusonii were 90 to 97% related to the type strain (holotype) ATCC 35469. They were most closely related to Escherichia coli and more distantly related to species in other genera. E. fergusonii strains are positive for indole production, methyl red, lysine decarboxylase, ornithine decarboxylase, and motility. They ferment D-glucose with gas production and also ferment adonitol, L-arabinose, L-rhamnose, maltose, D-xylose, trehalose, cellobiose, and D-arabitol. They are negative for Voges-Proskauer, citrate utilization (17% positive), urea hydrolysis, phenylalanine deamination, arginine dihydrolase, growth in KCN, and fermentation of lactose, sucrose, myo-inositol, D-sorbitol, raffinose, and alpha-methyl-D-glucoside. By DNA hybridization (32P, 60 degrees C, hydroxyapatite), strains of E. taylorae were 84 to 95% related to the type strain (holotype) ATCC 35317. Their nearest relative was E. cloacae, to which they were 61% related. Other named species were more distantly related. Strains of E. taylorae are positive for Voges-Proskauer, citrate utilization, arginine dihydrolase, ornithine decarboxylase, motility, growth in KCN medium, and malonate utilization. They ferment D-glucose with gas production and also ferment D-mannitol, L-arabinose, L-rhamnose, maltose, D-xylose, trehalose, and cellobiose. They are negative for indole production, methyl red, H2S production on triple sugar-iron agar, urea hydrolysis, phenylalanine deamination, lysine decarboxylase, gelatin hydrolysis, and fermentation of adonitol, i-inositol, D-sorbitol, and raffinose. Both new species occur in human clinical specimens. Two strains of E. fergusonii were isolated from blood. Five stains of E. taylorae were isolated from blood, and one was

  14. Mesophilic and thermophilic conditions select for unique but highly parallel microbial communities to perform carboxylate platform biomass conversion.

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    Emily B Hollister

    Full Text Available The carboxylate platform is a flexible, cost-effective means of converting lignocellulosic materials into chemicals and liquid fuels. Although the platform's chemistry and engineering are well studied, relatively little is known about the mixed microbial communities underlying its conversion processes. In this study, we examined the metagenomes of two actively fermenting platform communities incubated under contrasting temperature conditions (mesophilic 40°C; thermophilic 55 °C, but utilizing the same inoculum and lignocellulosic feedstock. Community composition segregated by temperature. The thermophilic community harbored genes affiliated with Clostridia, Bacilli, and a Thermoanaerobacterium sp, whereas the mesophilic community metagenome was composed of genes affiliated with other Clostridia and Bacilli, Bacteriodia, γ-Proteobacteria, and Actinobacteria. Although both communities were able to metabolize cellulosic materials and shared many core functions, significant differences were detected with respect to the abundances of multiple Pfams, COGs, and enzyme families. The mesophilic metagenome was enriched in genes related to the degradation of arabinose and other hemicellulose-derived oligosaccharides, and the production of valerate and caproate. In contrast, the thermophilic community was enriched in genes related to the uptake of cellobiose and the transfer of genetic material. Functions assigned to taxonomic bins indicated that multiple community members at either temperature had the potential to degrade cellulose, cellobiose, or xylose and produce acetate, ethanol, and propionate. The results of this study suggest that both metabolic flexibility and functional redundancy contribute to the platform's ability to process lignocellulosic substrates and are likely to provide a degree of stability to the platform's fermentation processes.

  15. Functional analysis of hyperthermophilic endocellulase from Pyrococcus horikoshii by crystallographic snapshots. (United States)

    Kim, Han-Woo; Ishikawa, Kazuhiko


    A hyperthermophilic membrane-related β-1,4-endoglucanase (family 5, cellulase) of the archaeon Pyrococcus horikoshii was found to be capable of hydrolysing cellulose at high temperatures. The hyperthermophilic cellulase has promise for applications in biomass utilization. To clarify its detailed function, we determined the crystal structures of mutants of the enzyme in complex with either the substrate or product ligands. We were able to resolve different kinds of complex structures at 1.65-2.01 Å (1 Å=0.1 nm). The structural analysis of various mutant enzymes yielded a sequence of crystallographic snapshots, which could be used to explain the catalytic process of the enzyme. The substrate position is fixed by the alignment of one cellobiose unit between the two aromatic amino acid residues at subsites +1 and +2. During the enzyme reaction, the glucose structure of cellulose substrates is distorted at subsite -1, and the β-1,4-glucoside bond between glucose moieties is twisted between subsites -1 and +1. Subsite -2 specifically recognizes the glucose residue, but recognition by subsites +1 and +2 is loose during the enzyme reaction. This type of recognition is important for creation of the distorted boat form of the substrate at subsite -1. A rare enzyme-substrate complex was observed within the low-activity mutant Y299F, which suggested the existence of a trapped ligand structure before the formation by covalent bonding of the proposed intermediate structure. Analysis of the enzyme-substrate structure suggested that an incoming water molecule, essential for hydrolysis during the retention process, might be introduced to the cleavage position after the cellobiose product at subsites +1 and +2 was released from the active site.

  16. The Effect of pH Control on Acetone-Butanol-Ethanol Fermentation by Clostridium acetobutylicum ATCC 824 with Xylose and D-Glucose and D-Xylose Mixture

    Institute of Scientific and Technical Information of China (English)

    Wei Jiang; Zhiqiang Wen; Mianbin Wu; Hong Li; Jun Yang; Jianping Lin; Yijun Lin; Lirong Yang; Peilin Cen


    D-Glucose, L-arabinose, D-mannose, D-xylose, and cellobiose are saccharification products of lignocellulose and important carbon sources for industrial fermentation. The fermentation efficiency with each of the five sugars and the mixture of the two most dominant sugars, D-glucose and D-xylose, was evaluated for acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum ATCC 824. The utilization efficacy of the five reducing sugars was in the order of D-glucose, L-arabinose, D-mannose, D-xylose and cellobiose. D-Xylose, the second most abundant component in lignocellulosic hydrolysate, was used in the fermentation either as sole carbon source or mixed with glucose. The results indicated that maintaining pH at 4.8, the optimal pH value for solventogenesis, could increase D-xylose consumption when it was the sole carbon source. Different media con-taining D-glucose and D-xylose at different ratios (1:2, 1:5, 1.5:1, 2:1) were then attempted for the ABE fermenta-tion. When pH was at 4.8 and xylose concentration was five times that of glucose, a 256.9%increase in xylose utilization and 263.7%increase in solvent production were obtained compared to those without pH control. These results demonstrate a possible approach combining optimized pH control and D-glucose and D-xylose ratio to increase the fermentation efficiency of lignocellulosic hydrolysate.

  17. Modulation of ocular surface glycocalyx barrier function by a galectin-3 N-terminal deletion mutant and membrane-anchored synthetic glycopolymers.

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    Jerome Mauris

    Full Text Available BACKGROUND: Interaction of transmembrane mucins with the multivalent carbohydrate-binding protein galectin-3 is critical to maintaining the integrity of the ocular surface epithelial glycocalyx. This study aimed to determine whether disruption of galectin-3 multimerization and insertion of synthetic glycopolymers in the plasma membrane could be used to modulate glycocalyx barrier function in corneal epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Abrogation of galectin-3 biosynthesis in multilayered cultures of human corneal epithelial cells using siRNA, and in galectin-3 null mice, resulted in significant loss of corneal barrier function, as indicated by increased permeability to the rose bengal diagnostic dye. Addition of β-lactose, a competitive carbohydrate inhibitor of galectin-3 binding activity, to the cell culture system, transiently disrupted barrier function. In these experiments, treatment with a dominant negative inhibitor of galectin-3 polymerization lacking the N-terminal domain, but not full-length galectin-3, prevented the recovery of barrier function to basal levels. As determined by fluorescence microscopy, both cellobiose- and lactose-containing glycopolymers incorporated into apical membranes of corneal epithelial cells, independently of the chain length distribution of the densely glycosylated, polymeric backbones. Membrane incorporation of cellobiose glycopolymers impaired barrier function in corneal epithelial cells, contrary to their lactose-containing counterparts, which bound to galectin-3 in pull-down assays. CONCLUSIONS/SIGNIFICANCE: These results indicate that galectin-3 multimerization and surface recognition of lactosyl residues is required to maintain glycocalyx barrier function at the ocular surface. Transient modification of galectin-3 binding could be therapeutically used to enhance the efficiency of topical drug delivery.

  18. Isolation and characterization of a hydrogen- and ethanol-producing Clostridium sp. strain URNW. (United States)

    Ramachandran, Umesh; Wrana, Nathan; Cicek, Nazim; Sparling, Richard; Levin, David B


    Identification, characterization, and end-product synthesis patterns were analyzed in a newly identified mesophilic, anaerobic Clostridium sp. strain URNW, capable of producing hydrogen (H₂) and ethanol. Metabolic profiling was used to characterize putative end-product synthesis pathways of the Clostridium sp. strain URNW, which was found to grow on cellobiose; on hexose sugars, such as glucose, sucrose, and mannose; and on sugar alcohols, like mannitol and sorbitol. When grown in batch cultures on 2 g cellobiose·L⁻¹, Clostridium sp. strain URNW showed a cell generation time of 1.5 h, and the major end-products were H2, formate, carbon dioxide (CO₂), lactate, butyrate, acetate, pyruvate, and ethanol. The total volumetric H₂ production was 14.2 mmol·(L culture)⁻¹ and the total production of ethanol was 0.4 mmol·(L culture)⁻¹. The maximum yield of H₂ was 1.3 mol·(mol glucose equivalent)⁻¹ at a carbon recovery of 94%. The specific production rates of H₂, CO₂, and ethanol were 0.45, 0.13, and 0.003 mol·h⁻¹·(g dry cell mass)-1, respectively. BLAST analyses of 16S rDNA and chaperonin 60 (cpn60) sequences from Clostridium sp. strain URNW revealed a 98% nucleotide sequence identity with the 16S rDNA and cpn60 sequences from Clostridium intestinale ATCC 49213. Phylogenetic analyses placed Clostridium sp. strain URNW within the butyrate-synthesizing clostridia.

  19. Genetic and functional characterization of an extracellular modular GH6 endo-β-1,4-glucanase from an earthworm symbiont, Cellulosimicrobium funkei HY-13. (United States)

    Kim, Do Young; Lee, Min Ji; Cho, Han-Young; Lee, Jong Suk; Lee, Mi-Hwa; Chung, Chung Wook; Shin, Dong-Ha; Rhee, Young Ha; Son, Kwang-Hee; Park, Ho-Yong


    The gene (1608-bp) encoding a GH6 endo-β-1,4-glucanase (CelL) from the earthworm-symbiotic bacterium Cellulosimicrobium funkei HY-13 was cloned from its whole genome sequence, expressed recombinantly, and biochemically characterized. CelL (56.0 kDa) is a modular enzyme consisting of an N-terminal catalytic GH6 domain (from Val57 to Pro396), which is 71 % identical to a GH6 protein (accession no.: WP_034662937) from Cellulomonas sp. KRMCY2, together with a C-terminal CBM 2 domain (from Cys429 to Cys532). The highest catalytic activity of CelL toward carboxymethylcellulose (CMC) was observed at 50 °C and pH 5.0, and was relatively stable at a broad pH range of 4.0-10.0. The enzyme was capable of efficiently hydrolyzing the cellulosic polymers in the order of barley β-1,3-1,4-D-glucan > CMC > lichenan > Avicel > konjac glucomannan. However, cellobiose, cellotriose, p-nitrophenyl derivatives of mono- and disaccharides, or structurally unrelated carbohydrate polymers including β-1,3-D-glucan, β-1,4-D-galactomannan, and β-1,4-D-xylan were not susceptible to CelL. The enzymatic hydrolysis of cellopentaose resulted in the production of a mixture of 68.6 % cellobiose and 31.4 % cellotriose but barley β-1,3-1,4-D-glucan was 100 % degraded to cellotriose by CelL. The enzyme strongly bound to Avicel, ivory nut mannan, and chitin but showed relatively weak binding affinity to lichenan, lignin, or poly(3-hydroxybutyrate) granules.

  20. Comparative genomic hybridization analysis shows different epidemiology of chromosomal and plasmid-borne cpe-carrying Clostridium perfringens type A.

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    Päivi Lahti

    Full Text Available Clostridium perfringens, one of the most common causes of food poisonings, can carry the enterotoxin gene, cpe, in its chromosome or on a plasmid. C. perfringens food poisonings are more frequently caused by the chromosomal cpe-carrying strains, while the plasmid-borne cpe-positive genotypes are more commonly found in the human feces and environmental samples. Different tolerance to food processing conditions by the plasmid-borne and chromosomal cpe-carrying strains has been reported, but the reservoirs and contamination routes of enterotoxin-producing C. perfringens remain unknown. A comparative genomic hybridization (CGH analysis with a DNA microarray based on three C. perfringens type A genomes was conducted to shed light on the epidemiology of C. perfringens food poisonings caused by plasmid-borne and chromosomal cpe-carrying strains by comparing chromosomal and plasmid-borne cpe-positive and cpe-negative C. perfringens isolates from human, animal, environmental, and food samples. The chromosomal and plasmid-borne cpe-positive C. perfringens genotypes formed two distinct clusters. Variable genes were involved with myo-inositol, ethanolamine and cellobiose metabolism, suggesting a new epidemiological model for C. perfringens food poisonings. The CGH results were complemented with growth studies, which demonstrated different myo-inositol, ethanolamine, and cellobiose metabolism between the chromosomal and plasmid-borne cpe-carrying strains. These findings support a ubiquitous occurrence of the plasmid-borne cpe-positive strains and their adaptation to the mammalian intestine, whereas the chromosomal cpe-positive strains appear to have a narrow niche in environments containing degrading plant material. Thus the epidemiology of the food poisonings caused by two populations appears different, the plasmid-borne cpe-positive strains probably contaminating foods via humans and the chromosomal strains being connected to plant material.

  1. Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus--production of extracellular enzymes and characterization of the major cellulases. (United States)

    Valásková, Vendula; Baldrian, Petr


    Piptoporus betulinus is a common wood-rotting fungus parasitic for birch (Betula species). It is able to cause fast mass loss of birch wood or other lignocellulose substrates. When grown on wheat straw, P. betulinus caused 65% loss of dry mass within 98 days, and it produced endo-1,4-beta-glucanase (EG), endo-1,4-beta-xylanase, endo-1,4-beta-mannanase, 1,4-beta-glucosidase (BG), 1,4-beta-xylosidase, 1,4-beta-mannosidase and cellobiohydrolase activities. The fungus was not able to efficiently degrade crystalline cellulose. The major glycosyl hydrolases, endoglucanase EG1 and beta-glucosidase BG1, were purified. EG1 was a protein of 62 kDa with a pI of 2.6-2.8. It cleaved cellulose internally, produced cellobiose and glucose from cellulose and cellooligosaccharides, and also showed beta-xylosidase and endoxylanase activities. The K(m) for carboxymethylcellulose was 3.5 g l(-1), with the highest activity at pH 3.5 and 70 degrees C. BG1 was a protein of 36 kDa with a pI around 2.6. It was able to produce glucose from cellobiose and cellooligosaccharides, but also produced galactose, mannose and xylose from the respective oligosaccharides and showed some cellobiohydrolase activity. The K(m) for p-nitrophenyl-1,4-beta-glucoside was 1.8 mM, with the highest activity at pH 4 and 60 degrees C, and the enzyme was competitively inhibited by glucose (K(i)=5.8 mM). The fungus produced mainly beta-glucosidase and beta-mannosidase activity in its fruit bodies, while higher activities of endoglucanase, endoxylanase and beta-xylosidase were found in fungus-colonized wood.

  2. Engineering Cellulase Enzymes for Bioenergy (United States)

    Atreya, Meera Elizabeth

    methods. Protein engineering targets to improve cellulases include reducing enzyme inhibition, improving inter-enzyme synergy, and increasing enzyme thermotolerance. Ameliorating enzyme inhibition could improve catalytic activity and thus the speed of conversion from biomass to fermentable sugars. Improved enzyme synergy could reduce the enzyme loading required to achieve equivalent biomass conversion. Finally, thermostable enzymes could enable more biomass to be processed at a time, due to high temperatures decreasing the viscosity of biomass slurries. A high-temperature enzyme saccharification reaction could also decrease the risk of contamination in the resulting concentrated sugar solution. Throughout my PhD, I have explored research projects broadly across all of these topics, with the most success in addressing the issue of enzyme inhibition. Cellulase enzyme Cel7A is the most abundant cellulase employed by natural systems for cellulose hydrolysis. Cellobiohydrolase enzymes like Cel7A break down cellulose into cellobiose (two glucose molecules). Unfortunately, upon cleavage, this product molecule interferes with continued hydrolysis activity of Cel7A; the strong binding of cellobiose in the active site can obstruct the enzyme from processing down the cellulase chain. This phenomenon, known as product inhibition, is a bottleneck to efficient biomass breakdown. Using insights from computational protein modeling studies, I experimentally generated and tested mutant Cel7A enzymes for improved tolerance to cellobiose. Indeed, this strategy yielded Cel7A enzymes exhibiting reduced product inhibition, including some mutants completely impervious to cellobiose. The improvements in tolerance to cellobiose, however, resulted in an overall reduction of enzyme activity for the mutants tested. Nevertheless, my findings substantiated computational reports with experimental evidence and pinpointed an amino acid residue in the Cel7A product binding site that is of interest for

  3. Comparative kinetic analysis of two fungal β-glucosidases

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    Casanave Dominique


    Full Text Available Abstract Background The enzymatic hydrolysis of cellulose is still considered as one of the main limiting steps of the biological production of biofuels from lignocellulosic biomass. It is a complex multistep process, and various kinetic models have been proposed. The cellulase enzymatic cocktail secreted by Trichoderma reesei has been intensively investigated. β-glucosidases are one of a number of cellulolytic enzymes, and catalyze the last step releasing glucose from the inhibitory cellobiose. β-glucosidase (BGL1 is very poorly secreted by Trichoderma reesei strains, and complete hydrolysis of cellulose often requires supplementation with a commercial β-glucosidase preparation such as that from Aspergillus niger (Novozymes SP188. Surprisingly, kinetic modeling of β-glucosidases lacks reliable data, and the possible differences between native T. reesei and supplemented β-glucosidases are not taken into consideration, possibly because of the difficulty of purifying BGL1. Results A comparative kinetic analysis of β-glucosidase from Aspergillus niger and BGL1 from Trichoderma reesei, purified using a new and efficient fast protein liquid chromatography protocol, was performed. This purification is characterized by two major steps, including the adsorption of the major cellulases onto crystalline cellulose, and a final purification factor of 53. Quantitative analysis of the resulting β-glucosidase fraction from T. reesei showed it to be 95% pure. Kinetic parameters were determined using cellobiose and a chromogenic artificial substrate. A new method allowing easy and rapid determination of the kinetic parameters was also developed. β-Glucosidase SP188 (Km = 0.57 mM; Kp = 2.70 mM has a lower specific activity than BGL1 (Km = 0.38 mM; Kp = 3.25 mM and is also more sensitive to glucose inhibition. A Michaelis-Menten model integrating competitive inhibition by the product (glucose has been validated and is able to predict the

  4. Mating type and ploidy effect on the β-glucosidase activity and ethanol-producing performance of Saccharomyces cerevisiae with multiple δ-integrated bgl1 gene. (United States)

    Wang, Jianjun; Ma, Yuanyuan; Zhang, Kun; Yang, Huajun; Liu, Cheng; Zou, Shaolan; Hong, Jiefang; Zhang, Minhua


    In order to investigate the effect of mating type and ploidy on enzymatic activity and fermentation performance in yeast with multiple δ-integrated foreign genes, eight ploidy series strains were constructed. The initial haploid strain BGL-a was shown to contain about 19 copies of the bgl1 gene. In rich media containing 2% (w/v) sugar the specific activities of BGL-aα were lower than those of BGL-aa or BGL-αα, which indicates the existence of mating type effects. While the maximum OD660 decreased with rising ploidy, the biomass yield showed no significant difference between the eight strains and the specific activities (expressed as U/mL or U/mg DCW) showed little to no variation. When cellobiose was used as the carbon source and β-glucosidase substrate, β-glucosidase was expressed more quickly and at higher levels than in glucose-containing media. The maximum specific activitiy values obtained were 19.07U/mL and 19.39U/mL for BGL-αα and BGL-aa, repsectively. The anaerobic biomass and ethanol-producing performance in rich media containing 10% cellobiose showed no significant difference among the eight strains. Their maximal ethanol concentrations and corresponding yields ranged from 40.27 to 43.46g/L and 77.56 to 83.71%, respectively. When the acid- and alkali-pretreated corncob (10% solids content) was used, the diploid BGL-aα fermented the best. When urea was used as the only supplemented nutrient, the ethanol titer and yield were 35.65g/L and 83.69%, respectively, while a control experiment using industrial Angel yeast with exogenous β-glucosidase addition gave values of 37.93g/L and 89.04%. The combined effects of δ-integration of bgl1, ploidy and mating type result in BGL-aa or BGL-αα being the optimal choice for enzyme production and BGL-aα being more suitable for cellulosic ethanol fermentation. These results provide valuable information for future yeast breeding and utilization efforts.

  5. Stability of SG1 nitroxide towards unprotected sugar and lithium salts: a preamble to cellulose modification by nitroxide-mediated graft polymerization

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    Guillaume Moreira


    Full Text Available The range of applications of cellulose, a glucose-based polysaccharide, is limited by its inherently poor mechanical properties. The grafting of synthetic polymer chains by, for example, a “grafting from” process may provide the means to broaden the range of applications. The nitroxide-mediated polymerization (NMP method is a technique of choice to control the length, the composition and the architecture of the grafted copolymers. Nevertheless, cellulose is difficult to solubilize in organic media because of inter- and intramolecular hydrogen bonds. One possibility to circumvent this limitation is to solubilize cellulose in N,N-dimethylformamide (DMF or N,N-dimethylacetamide (DMA with 5 to 10 wt % of lithium salts (LiCl or LiBr, and carry out grafted polymerization in this medium. The stability of nitroxides such as SG1 has not been studied under these conditions yet, even though these parameters are of crucial importance to perform the graft modification of polysaccharide by NMP. The aim of this work is to offer a model study of the stability of the SG1 nitroxide in organic media in the presence of unprotected glucose or cellobiose (used as a model of cellulose and in the presence of lithium salts (LiBr or LiCl in DMF or DMA.Contrary to TEMPO, SG1 proved to be stable in the presence of unprotected sugar, even with an excess of 100 molar equivalents of glucose. On the other hand, lithium salts in DMF or DMA clearly degrade SG1 nitroxide as proven by electron-spin resonance measurements. The instability of SG1 in these lithium-containing solvents may be explained by the acidification of the medium by the hydrolysis of DMA in the presence of LiCl. This, in turn, enables the disproportionation of the SG1 nitroxide into an unstable hydroxylamine and an oxoammonium ion.Once the conditions to perform an SG1-based nitroxide-mediated graft polymerization from cellobiose have been established, the next stage of this work will be the modification of

  6. Clostridium hydrogeniformans sp. nov. and Clostridium cavendishii sp. nov., hydrogen-producing bacteria from chlorinated solvent-contaminated groundwater. (United States)

    Bowman, Kimberly S; Dupré, Rachael E; Rainey, Fred A; Moe, William M


    Four hydrogen-producing, aerotolerant, anaerobic bacterial strains isolated from chlorinated solvent-contaminated groundwater were characterized using a polyphasic approach. Three of the strains, designated BL-18, BL-19 and BL-20(T), were found to be identical in 16S rRNA gene sequences and in phenotypic properties. Cells of these strains are Gram-positive-staining, spore-forming, motile rods with peritrichous flagella. Growth occurred at 15-40 degrees C, pH 5.0-10.0 and at NaCl concentrations up to 5 % (w/v). Acid was produced in fermentation of cellobiose, fructose, galactose (weak), glucose, maltose and salicin. Products of fermentation in PYG medium were acetate, butyrate, ethanol, formate, carbon dioxide and hydrogen. Dominant cellular fatty acids when grown in PYG medium were C(13 : 0) iso, C(16 : 0), C(13 : 0) anteiso, C(15 : 0) iso and C(15 : 0) anteiso. The genomic DNA G+C content was 30.4 mol%. These isolates can be differentiated from their closest phylogenetic relative, the cluster I Clostridium species Clostridium frigidicarnis (97.2 % similar to the type strain in 16S rRNA gene sequence), on the basis of phenotypic and chemotaxonomic properties. The other strain characterized in this study, BL-28(T), was Gram-positive-staining with spore-forming, rod-shaped cells. Growth occurred at 15-46 degrees C, pH 6.0-8.5 and at NaCl concentrations up to 3 % (w/v). Acid was produced from cellobiose, dextran, fructose (weak), glucose, maltose, salicin and trehalose. End products of PYG fermentation included acetate, butyrate, pyruvate, carbon dioxide and hydrogen. Dominant cellular fatty acids from cells grown in PYG medium at 30 degrees C were C(14 : 0), C(14 : 0) dimethyl aldehyde, C(16 : 0) and C(12 : 0). The DNA G+C content was 28.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BL-28(T) falls within cluster I of the genus Clostridium, but with Clostridium with the names Clostridium hydrogeniformans sp. nov. and Clostridium

  7. Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail

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    Del Pozo Mercedes V


    Full Text Available Abstract Background A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product. Results In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45–55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using pNPbetaG as substrate, the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of pNPbetaG. LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions after 96–120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2 and 2–38000 fold higher (as compared with reported beta-glucosidases activity towards cello-oligosaccharides may account for its performance in supplementation assays. Conclusions The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of

  8. Comprehensive enzymatic analysis of the cellulolytic system in digestive fluid of the Sea Hare Aplysia kurodai. Efficient glucose release from sea lettuce by synergistic action of 45 kDa endoglucanase and 210 kDa ß-glucosidase.

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    Akihiko Tsuji

    Full Text Available Although many endo-ß-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and seaweeds. In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the sea hare (Aplysia kurodai were screened and characterized to determine how the sea hare obtains glucose from sea lettuce (Ulva pertusa. Four endo-ß-1,4-glucanases (21K, 45K, 65K, and 95K cellulase and 2 ß-glucosidases (110K and 210K were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21K cellulase, whereas 45K and 65K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210K ß-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU-ß-glucoside, 4MU-ß-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210K ß-glucosidase possesses lactase activity. Although ß-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210K ß-glucosidase. Kinetic analysis of the inhibition of 210K ß-glucosidase by D-glucono-1,5-lactone suggested the presence of 2 active sites similar to those of mammalian lactase-phlorizin hydrolase. Saccharification of sea lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K ß-glucosidase. Our results indicate that 45K cellulase and 210K

  9. Temperature dependence of the heat capacities in the solid state of 18 mono-, di-, and poly-saccharides

    Energy Technology Data Exchange (ETDEWEB)

    Hernandez-Segura, Gerardo O. [Laboratorio de Biofisicoquimica, Departamento de Fisicoquimica, Facultad de Quimica, Universidad Nacional Autonoma de Mexico, Mexico D.F. 04510 (Mexico); Campos, Myriam [Departamento de Quimica, Centro de Investigacion y Estudios Avanzados del I.P.N., Apdo. Postal 14-740, Mexico D.F. 07000 (Mexico); Costas, Miguel [Laboratorio de Biofisicoquimica, Departamento de Fisicoquimica, Facultad de Quimica, Universidad Nacional Autonoma de Mexico, Mexico D.F. 04510 (Mexico)], E-mail:; Torres, Luis A. [Departamento de Quimica, Centro de Investigacion y Estudios Avanzados del I.P.N., Apdo. Postal 14-740, Mexico D.F. 07000 (Mexico)], E-mail:


    The temperature dependence of the heat capacities in solid state C{sub p}(T) of 18 mono-, di-, and poly-saccharides has been determined using a power-compensation differential scanning calorimeter. The saccharides were {alpha}-D-xylose, D-ribose, 2-deoxy-D-ribose, methyl-{beta}-D-ribose, {alpha}-D-glucose, 2-deoxy-D-glucose, {alpha}-D-mannose, {beta}-D-fructose, {alpha}-D-galactose, methyl-{alpha}-D-glucose, sucrose, maltose monohydrate, {alpha}-lactose monohydrate, cellobiose, maltotriose, N-acetyl-D-glucosamine, {alpha}-cyclodextrin, and {beta}-cyclodextrin. The measurements were carried out at atmospheric pressure and from T = (288.15 to 358.15) K for 15 saccharides and from T = (288.15 to 328.15) K for D-ribose, 2-deoxy-D-ribose, and methyl-{beta}-D-ribose. The present results are compared against literature values both at single temperatures, where most of the data are available, and throughout a range of temperatures, i.e., for C{sub p}(T). The predictions of a recently published correlation for organic solids are briefly discussed. By grouping saccharides in subsets, our present results can be used to compare amongst saccharide isomers and to assess the effect of different chemical groups and molecular size.

  10. Comparative analysis of carbohydrate active enzymes in Clostridium termitidis CT1112 reveals complex carbohydrate degradation ability.

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    Riffat I Munir

    Full Text Available Clostridium termitidis strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, Nasutitermes lujae. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes, sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in C. termitidis, the recently sequenced strain CT1112 of C. termitidis was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in C. termitidis, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199 and carbohydrate binding modules (95 were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.

  11. Clostridium thermocellum ATCC27405 transcriptomic, metabolomic and proteomic profiles after ethanol stress

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    Yang Shihui


    Full Text Available Abstract Background Clostridium thermocellum is a candidate consolidated bioprocessing biocatalyst, which is a microorganism that expresses enzymes for both cellulose hydrolysis and its fermentation to produce fuels such as lignocellulosic ethanol. However, C. thermocellum is relatively sensitive to ethanol compared to ethanologenic microorganisms such as yeast and Zymomonas mobilis that are used in industrial fermentations but do not possess native enzymes for industrial cellulose hydrolysis. Results In this study, C. thermocellum was grown to mid-exponential phase and then treated with ethanol to a final concentration of 3.9 g/L to investigate its physiological and regulatory responses to ethanol stress. Samples were taken pre-shock and 2, 12, 30, 60, 120, and 240 min post-shock, and from untreated control fermentations for systems biology analyses. Cell growth was arrested by ethanol supplementation with intracellular accumulation of carbon sources such as cellobiose, and sugar phosphates, including fructose-6-phosphate and glucose-6-phosphate. The largest response of C. thermocellum to ethanol shock treatment was in genes and proteins related to nitrogen uptake and metabolism, which is likely important for redirecting the cells physiology to overcome inhibition and allow growth to resume. Conclusion This study suggests possible avenues for metabolic engineering and provides comprehensive, integrated systems biology datasets that will be useful for future metabolic modeling and strain development endeavors.

  12. Metabolic adaption of ethanol-tolerant Clostridium thermocellum.

    Directory of Open Access Journals (Sweden)

    Xinshu Zhu

    Full Text Available Clostridium thermocellum is a major candidate for bioethanol production via consolidated bioprocessing. However, the low ethanol tolerance of the organism dramatically impedes its usage in industry. To explore the mechanism of ethanol tolerance in this microorganism, systematic metabolomics was adopted to analyse the metabolic phenotypes of a C. thermocellum wild-type (WT strain and an ethanol-tolerant strain cultivated without (ET0 or with (ET3 3% (v/v exogenous ethanol. Metabolomics analysis elucidated that the levels of numerous metabolites in different pathways were changed for the metabolic adaption of ethanol-tolerant C. thermocellum. The most interesting phenomenon was that cellodextrin was significantly more accumulated in the ethanol-tolerant strain compared with the WT strain, although cellobiose was completely consumed in both the ethanol-tolerant and wild-type strains. These results suggest that the cellodextrin synthesis was active, which might be a potential mechanism for stress resistance. Moreover, the overflow of many intermediate metabolites, which indicates the metabolic imbalance, in the ET0 cultivation was more significant than in the WT and ET3 cultivations. This indicates that the metabolic balance of the ethanol-tolerant strain was adapted better to the condition of ethanol stress. This study provides additional insight into the mechanism of ethanol tolerance and is valuable for further metabolic engineering aimed at higher bioethanol production.

  13. Clostridium thermocellum ATCC27405 transcriptomic, metabolomic and proteomic profiles after ethanol stress

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Shihui [ORNL; Giannone, Richard J [ORNL; Dice, Lezlee T [ORNL; Yang, Zamin Koo [ORNL; Engle, Nancy L [ORNL; Tschaplinski, Timothy J [ORNL; Hettich, Robert {Bob} L [ORNL; Brown, Steven D [ORNL


    Clostridium thermocellum is a candidate consolidated bioprocessing biocatalyst, which is a microorganism that expresses enzymes for both cellulose hydrolysis and its fermentation to produce fuels such as lignocellulosic ethanol. However, C. thermocellum is relatively sensitive to ethanol compared to ethanologenic microorganisms such as yeast and Zymomonas mobilis that are used in industrial fermentations but do not possess native enzymes for industrial cellulose hydrolysis. In this study, C. thermocellum was grown to mid-exponential phase and then treated with ethanol to a final concentration of 3.9 g/L to investigate its physiological and regulatory responses to ethanol stress. Samples were taken pre-shock and 2, 12, 30, 60, 120, and 240 min post-shock, and from untreated control fermentations for systems biology analyses. Cell growth was arrested by ethanol supplementation with intracellular accumulation of carbon sources such as cellobiose, and sugar phosphates, including fructose-6-phosphate and glucose-6-phosphate. The largest response of C. thermocellum to ethanol shock treatment was in genes and proteins related to nitrogen uptake and metabolism, which is likely important for redirecting the cells physiology to overcome inhibition and allow growth to resume. This study suggests possible avenues for metabolic engineering and provides comprehensive, integrated systems biology datasets that will be useful for future metabolic modeling and strain development endeavors.

  14. Cloning and expression of cyclodextrin glycosyltransferase gene from Paenibacillus sp. T16 isolated from hot spring soil in northern Thailand. (United States)

    Charoensakdi, Ratiya; Murakami, Shuichiro; Aoki, Kenji; Rimphanitchayakit, Vichien; Limpaseni, Tipaporn


    Gene encoding cyclodextrin glycosyltransferase (CGTase), from thermotolerant Paenibacillus sp. T16 isolated from hot spring area in northern Thailand, was cloned and expressed in E. coli (JM109). The nucleotide sequences of both wild type and transformed CGTases consisted of 2139 bp open reading frame, 713 deduced amino acids residues with difference of 4 amino acid residues. The recombinant cells required 24 h culture time and a neutral pH for culture medium to produce compatible amount of CGTase compared to 72 h culture time and pH 10 for wild type. The recombinant and wild-type CGTases were purified by starch adsorption and phenyl sepharose column chromatography and characterized in parallel. Both enzymes showed molecular weight of 77 kDa and similar optimum pHs and temperatures with recombinant enzyme showing broader range. There were some significant difference in pH, temperature stability and kinetic parameters. The presence of high starch concentration resulted in higher thermostability in recombinant enzyme than the wild type. The recombinant enzyme was more stable at higher temperature and lower pH, with lower K(m) for coupling reaction using cellobiose and cyclodextrins as substrates.

  15. Free-living spirochetes from Cape Cod microbial mats detected by electron microscopy (United States)

    Teal, T. H.; Chapman, M.; Guillemette, T.; Margulis, L.


    Spirochetes from microbial mats and anaerobic mud samples collected in salt marshes were studied by light microscopy, whole mount and thin section transmission electron microscopy. Enriched in cellobiose-rifampin medium, selective for Spirochaeta bajacaliforniensis, seven distinguishable spirochete morphotypes were observed. Their diameters ranged from 0.17 micron to > 0.45 micron. Six of these morphotypes came from southwest Cape Cod, Massachusetts: five from Microcoleus-dominated mat samples collected at Sippewissett salt marsh and one from anoxic mud collected at School Street salt marsh (on the east side of Eel Pond). The seventh morphotype was enriched from anoxic mud sampled from the north central Cape Cod, at the Sandy Neck salt marsh. Five of these morphotypes are similar or identical to previously described spirochetes (Leptospira, Spirochaeta halophila, Spirochaeta bajacaliforniensis, Spirosymplokos deltaeiberi and Treponema), whereas the other two have unique features that suggest they have not been previously described. One of the morphotypes resembles Spirosymplokos deltaeiberi (the largest free-living spirochete described), in its large variable diameter (0.4-3.0 microns), cytoplasmic granules, and spherical (round) bodies with composite structure. This resemblance permits its tentative identification as a Sippewissett strain of Spirosymplokos deltaeiberi. Microbial mats samples collected in sterile Petri dishes and stored dry for more than four years yielded many organisms upon rewetting, including small unidentified spirochetes in at least 4 out of 100 enrichments.

  16. A novel thermophilic endoglucanase from a mesophilic fungus Fusarium oxysporum

    Institute of Scientific and Technical Information of China (English)

    LIU Shuyan; DUAN Xinyuan; LU Xuemei; GAO Peiji


    A novel thermophilic endoglucanase (EGt) was extracted from a mesophilic fungus (Fusarium oxysporum L19). We invoked conventional kinetic enzyme reactions using the sodium salt of carboxymethyl cellulose (CMC-Na) as substrate. EGt displayed optimal activity at 75℃ when kept running 30 min in the temperature range of 30―85℃. Thermal stability curve measured at 70℃ suggested that its half-life time is 15.1 min. The activity was enhanced in the presence of Co2+ or Mg2+ but inhibited by Pb2+ and Fe3+. Moreover, N-bromosuccinimide (NBS) modification resulted in a complete loss of EGt activity, suggesting that tryptophan residues 5 be involved in the enzyme active site. Amino acid composition analysis demonstrated that EGt contains more proline residues. EGt lacks activity towards p-nitrophenyl cellobiose (pNPC). The N-terminal amino acid sequence of EGt is SYRVPAANGFPNP- DASQEKQ, and the gene of EGt was sequenced and analyzed. Extensive sequence alignments failed to show any homology between EGt and any known endoglucanases. This is the first report addressing the thermal adaptation of a cellulolytic enzyme from the mesophilic fungus F. oxysporum. 5be the expression of multiple isoenzyme in an organism helps it adapt to complex living environments.

  17. Determination of the action modes of cellulases from hydrolytic profiles over a time course using fluorescence-assisted carbohydrate electrophoresis. (United States)

    Zhang, Qing; Zhang, Xiaomei; Wang, Peipei; Li, Dandan; Chen, Guanjun; Gao, Peiji; Wang, Lushan


    Fluorescence-assisted carbohydrate electrophoresis (FACE) is a sensitive and simple method for the separation of oligosaccharides. It relies on labeling the reducing ends of oligosaccharides with a fluorophore, followed by PAGE. Concentration changes of oligosaccharides following hydrolysis of a carbohydrate polymer could be quantitatively measured continuously over time using the FACE method. Based on the quantitative analysis, we suggested that FACE was a relatively high-throughput, repeatable, and suitable method for the analysis of the action modes of cellulases. On account of the time courses of their hydrolytic profiles, the apparent processivity was used to show the different action modes of cellulases. Cellulases could be easily differentiated as exoglucanases, β-glucosidases, or endoglucanases. Moreover, endoglucanases from the same glycoside hydrolases family had a variety of apparent processivity, indicating the different modes of action. Endoglucanases with the same binding capacities and hydrolytic activities had similar oligosaccharide profiles, which aided in their classification. The hydrolytic profile of Trichoderma reesei Cel12A, an endoglucanases from T. reesei, contained glucose, cellobiose, and cellotriose, which revealed that it may have a new glucosidase activity, corresponding to that of EC A hydrolysate study of a T. reesei Cel12A-N20A mutant demonstrated that the FACE method was sufficiently sensitive to detect the influence of a single-site mutation on enzymatic activity.

  18. The fungus gardens of leaf-cutter ants undergo a distinct physiological transition during biomass degradation

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Eric L.; Aylward, Frank O.; Kim, Young-Mo; Webb-Robertson, Bobbie-Jo M.; Nicora, Carrie D.; Hu, Zeping; Metz, Thomas O.; Lipton, Mary S.; Smith, Richard D.; Currie, Cameron R.; Burnum-Johnson, Kristin E.


    Leaf-cutter ants are dominant herbivores in ecosystems throughout the Neotropics. Rather than directly consuming the fresh foliar biomass they harvest, these ants use it to cultivate specialized fungus gardens. Although recent investigations have shed light on how plant biomass is degraded in fungus gardens, the cycling of nutrients that takes place in these specialized microbial ecosystems is still not well understood. Here, using metametabolomics and metaproteomics techniques, we examine the dynamics of nutrient turnover and biosynthesis in these gardens. Our results reveal that numerous free amino acids and sugars are depleted throughout the process of biomass degradation, indicating that easily accessible nutrients from plant material are readily consumed by microbes in these ecosystems. Accumulation of cellobiose and lignin derivatives near the end of the degradation process is consistent with previous findings of cellulases and laccases produced by Leucoagaricus gongylophorus, the fungus cultivated by leaf-cutter ants. Our results also suggest that ureides may be an important source of nitrogen in fungus gardens, especially during nitrogen-limiting conditions. No free arginine was detected in our metametabolomics experiments despite evidence that the host ants cannot produce this amino acid, suggesting that biosynthesis of this metabolite may be tightly regulated in the fungus garden. These results provide new insights into the dynamics of nutrient cycling that underlie this important ant-fungus symbiosis.

  19. High production of cellulose degrading endo-1,4-β-D-glucanase using bagasse as a substrate from Bacillus subtilis KIBGE HAS. (United States)

    Bano, Saeeda; Qader, Shah Ali Ul; Aman, Afsheen; Syed, Mohammad Noman; Durrani, Kamran


    Sugarcane bagasse is a cheap carbon source for endo-1,4-β-D-glucanase production as it is easily available as by-product from sugar industries. Fermentation conditions for endo-1,4-β-D-glucanase production by Bacillus subtilis KIBGE HAS were optimized by using un-treated sugarcane bagasse for induction of endo-1,4-β-D-glucanase and it was found that 2.0 g% bagasse in fermentation medium induced maximum endo-1,4-β-D-glucanase production. It was also found that when sugarcane bagasse was supplemented with different carbon sources, the results showed that lactose, xylose, maltose and sucrose favored endo-1,4-β-D-glucanase production, whereas cellobiose and fructose inhibit enzyme production. Maximum endo-1,4-β-D-glucanase production was obtained at 40 °C keeping the initial pH of the medium at 7.0 before sterilization. Maximum endo-1,4-β-D-glucanase production was obtained after 48 h incubation. Among different nitrogen sources, ammonium nitrate enhanced endo-1,4-β-D-glucanase production. The optimal temperature and pH for enzyme activity were 60 °C and 7.0, respectively.

  20. Quantum supercharger library: hyper-parallel integral derivatives algorithms for ab initio QM/MM dynamics. (United States)

    Renison, C Alicia; Fernandes, Kyle D; Naidoo, Kevin J


    This article describes an extension of the quantum supercharger library (QSL) to perform quantum mechanical (QM) gradient and optimization calculations as well as hybrid QM and molecular mechanical (QM/MM) molecular dynamics simulations. The integral derivatives are, after the two-electron integrals, the most computationally expensive part of the aforementioned calculations/simulations. Algorithms are presented for accelerating the one- and two-electron integral derivatives on a graphical processing unit (GPU). It is shown that a Hartree-Fock ab initio gradient calculation is up to 9.3X faster on a single GPU compared with a single central processing unit running an optimized serial version of GAMESS-UK, which uses the efficient Schlegel method for s- and l-orbitals. Benchmark QM and QM/MM molecular dynamics simulations are performed on cellobiose in vacuo and in a 39 Å water sphere (45 QM atoms and 24843 point charges, respectively) using the 6-31G basis set. The QSL can perform 9.7 ps/day of ab initio QM dynamics and 6.4 ps/day of QM/MM dynamics on a single GPU in full double precision. © 2015 Wiley Periodicals, Inc.

  1. How Molecular Evolution Technologies can Provide Bespoke Industrial Enzymes: Application to Biofuels Comment les technologies d’évolution moléculaire peuvent fournir des enzymes industrielles sur mesure : application aux biocarburants

    Directory of Open Access Journals (Sweden)

    Fourage L.


    Full Text Available Enzymatic hydrolysis of lignocellulose is one of the major bottlenecks in the development of biological conversion of lignocellulosic biomass to biofuels. One of the most efficient organisms for the production of cellulolytic enzymes is the fungus Trichoderma reesei, mainly thanks to its high secretion capacity. The conversion of cellulose to glucose involves three types of cellulases working in synergy: endoglucanases (EC randomly cleave 13-1,4 glycosidic linkages of cellulose, cellobiohydrolases (EC attack cellulose chain ends to produce cellobiose dimers which are converted into glucose by the 13-glucosidases (EC 3.2.1 21. Unexpectedly, the amount of l3-glucosidase (BGLI from T. reesei hyperproducing strains represents a very low percentage of the total secreted proteins. A suboptimal content of this enzyme limits the performance of commercial cellulase preparations as cellobiose represents the main inhibitor of the cellulolysis reaction by cellobiohydrolases. This bottleneck can be alleviated either by overexpressing the f3-glucosidase in T. reesei or optimized its specific activity. After giving a brief overview of the main available technologies, this example will be used to illustrate the potential of directed evolution technologies to devolop enzymes tailored to fit industrial needs. We describe the L-ShuffiingTM strategy implemented with three parental genes originating from microbial biodiversity leading to identification of an efficient 13-glucosidase showing a 242 fold increase in specific activity for the pNPGIc substrate compared to WT (Wild Type Cel3a beta-glucosidase of T. reesei. After expression of the best improved 13-glucosidase in T. reesei and secretion of a new enzymatic cocktail, improvement of the glucosidase activity allows a 4-fold decrease of cellulase loading for the saccharification of an industrial pretreated biomass compared to the parental cocktail. L’hydrolyse enzymatique de la lignocellulose

  2. Involvement of Acetobacter orientalis in the production of lactobionic acid in Caucasian yogurt ("Caspian Sea yogurt") in Japan. (United States)

    Kiryu, T; Kiso, T; Nakano, H; Ooe, K; Kimura, T; Murakami, H


    Lactobionic acid was first found in a Caucasian fermented milk product popularly known as "Caspian Sea yogurt" in Japan. The presence of lactobionic acid in the fermented milk was indicated by the results of both high-performance anion-exchange chromatographic analysis with pulsed amperometric detection and mass spectrometric analysis. Thereafter, the acid was purified from the yogurt and analyzed by nuclear magnetic resonance. A substantial amount of lactobionic acid was found to be accumulated in the upper layer of the yogurt, especially within 10 mm from the surface. A total of 45 mg of lactobionic acid per 100 g of the upper yogurt layer was collected after 4 d of fermentation. The annual intake of lactobionic acid in individuals consuming 100 g of the yogurt every day would be 0.5 to 1.0 g. A lactose-oxidizing bacterium was isolated from the fermented milk and was identified as Acetobacter orientalis. Washed A. orientalis cells oxidized monosaccharides such as d-glucose at considerable rates, although their activities for substrates such as lactose, maltose, and cellobiose were much lower. When A. orientalis cells were cultivated in cow's milk, they exhibited lactose-oxidizing activity, suggesting that this bacterium was the main organism involved in the production of lactobionic acid in the yogurt.

  3. Possibilities for recycling cellulases after use in cotton processing: part I: Effects of end-product inhibition, thermal and mechanical deactivation, and cellulase depletion by adsorption. (United States)

    Azevedo, Helena; Bishop, David; Cavaco-Paul, Artur


    Preliminary recycling experiments with cellulase enzymes after cotton treatments at 50 degrees C showed that activity remaining in the treatment liquors was reduced by about 80% after five recycling steps. The potential problems of end-product inhibition, thermal and mechanical deactivation, and the loss of some components of the cellulase complex by preferential and or irreversible adsorption to cotton substrates were studied. End-product inhibition studies showed that the build-up of cellobiose and glucose would be expected to cause no more than 40% activity loss after five textile treatment cycles. Thermal and mechanical treatments of cellulases suggested that the enzymes start to be deactivated at 60 degrees C and agitation levels similar to those used in textile processing did not cause significant enzyme deactivation. Analysis of cellulase solutions, by fast protein liquid chromatography, before and after adsorption on cotton fabrics, suggested that the cellobiohydrolase II (Cel6A) content of the cellulase complex was reduced, relative to the other components, by preferential adsorption. This would lead to a marked reduction in activity after several treatment cycles and top-up with pure cellobiohydrolase II would be necessary unless this component is easily recoverable from the treated fabric.

  4. How Cellulose Elongates--A QM/MM Study of the Molecular Mechanism of Cellulose Polymerization in Bacterial CESA. (United States)

    Yang, Hui; Zimmer, Jochen; Yingling, Yaroslava G; Kubicki, James D


    The catalytic mechanism of bacterial cellulose synthase was investigated by using a hybrid quantum mechanics and molecular mechanics (QM/MM) approach. The Michaelis complex model was built based on the X-ray crystal structure of the cellulose synthase subunits BcsA and BcsB containing a uridine diphosphate molecule and a translocating glucan. Our study identified an SN2-type transition structure corresponding to the nucleophilic attack of the nonreducing end O4 on the anomeric carbon C1, the breaking of the glycosidic bond C1-O1, and the transfer of proton from the nonreducing end O4 to the general base D343. The activation barrier found for this SN2-type transition state is 68 kJ/mol. The rate constant of polymerization is estimated to be ∼8.0 s(-1) via transition state theory. A similar SN2-type transition structure was also identified for a second glucose molecule added to the growing polysaccharide chain, which aligned with the polymer 180° rotated compared to the initially added unit. This study provides detailed insights into how cellulose is extended by one glucose molecule at a time and how the individual glucose units align into cellobiose repeating units.

  5. Genomic insights into the carbohydrate catabolism of Cairneyella variabilis gen. nov. sp. nov., the first reports from a genome of an ericoid mycorrhizal fungus from the southern hemisphere. (United States)

    Midgley, David J; Rosewarne, Carly P; Greenfield, Paul; Li, Dongmei; Vockler, Cassandra J; Hitchcock, Catherine J; Sawyer, Nicole A; Brett, Robyn; Edwards, Jacqueline; Pitt, John I; Tran-Dinh, Nai


    This paper describes a novel species of ericoid mycorrhizal fungus from Australia, Cairneyella variabilis, Midgley and Tran-Dinh, gen. nov. sp. nov. The genome of C. variabilis was sequenced and a draft genome assembled. The draft genome of C. variabilis is 52.4 Mbp in length, and to our knowledge, this is the first study to present a genome of an ericoid mycorrhizal fungus from the southern hemisphere. Using the SignalP and dbCAN bioinformatic pipelines, a study of the catabolic potential of C. variabilis was undertaken and showed genes for an array of degradative enzymes, most of which appear to be secreted from the hyphae, to access a suite of different carbon sources. Isolates of C. variabilis have been previously shown to utilise cellulose, carboxymethyl cellulose (CMC), cellobiose, xylan, pectin, starch and tannic acid for growth, and in the current study, putative enzymes for these processes were revealed. These enzymes likely play key roles in nutrient cycling and other edaphic processes in heathland environments. ITS phylogenetic analyses showed C. variabilis to be distinct from the fungi of the "Hymenoscyphus ericae aggregate".

  6. Changes in soil microbial functional diversity and biochemical characteristics of tree peony with amendment of sewage sludge compost. (United States)

    Huang, Xiangdong; Xue, Dong; Xue, Lian


    A greenhouse experiment was conducted to investigate the impact of sewage sludge compost application on functional diversity of soil microbial communities, based on carbon source utilization, and biochemical characteristics of tree peony (Paeonia suffruticosa). Functional diversity was estimated with incubations in Biolog EcoPlates and well color development was used as the functional trait for carbon source utilization. The average well color development and Shannon index based on the carbon source utilization pattern in Biolog EcoPlates significantly increased with the increasing sludge compost application in the range of 0-45%, with a decreasing trend above 45%. Principal component analysis of carbon source utilization pattern showed that sludge compost application stimulated the utilization rate of D-cellobiose and α-D-lactose, while the utilization rate of β-methyl-D-glucoside, L-asparagine, L-serine, α-cyclodextrin, γ-hydroxybutyric acid, and itaconic acid gradually increased up to a sludge compost amendment dosage of 45% and then decreased above 45%. The chlorophyll content, antioxidase (superoxide dismutase, catalase, and peroxidase) activities, plant height, flower diameter, and flower numbers per plant of tree peony increased significantly with sludge compost dosage, reaching a peak value at 45 %, and then decreased with the exception that activity of superoxide dismutase and catalase did not vary significantly.

  7. Rhynchophorus ferrugineus attack affects a group of compounds rather than rearranging Phoenix canariensis metabolic pathways. (United States)

    Giovino, Antonio; Martinelli, Federico; Saia, Sergio


    The red palm weevil (RPW; Rhynchophorus ferrugineus) is spreading worldwide and severely harming many palm species. However, most studies on RPW focused on insect biology, and little information is available about the plant response to the attack. In the present experiment, we used metabolomics to study the alteration of the leaf metabolome of Phoenix canariensis at initial (1st stage) or advanced (2nd stage) attack by RPW compared with healthy (unattacked) plants. The leaf metabolome significantly varied among treatments. At the 1st stage of attack, plants showed a reprogramming of carbohydrate and organic acid metabolism; in contrast, peptides and lipid metabolic pathways underwent more changes during the 2nd than 1st stage of attack. Enrichment metabolomics analysis indicated that RPW attack mostly affected a particular group of compounds rather than rearranging plant metabolic pathways. Some compounds selectively affected during the 1st rather than 2nd stage (e.g. phenylalanine; tryptophan; cellobiose; xylose; quinate; xylonite; idonate; and iso-threonate; cellobiotol and arbutine) are upstream events in the phenylpropanoid, terpenoid and alkaloid biosynthesis. These compounds could be designated as potential markers of initial RPW attack. However, further investigation is needed to determine efficient early screening methods of RPW attack based on the concentrations of these molecules.

  8. Amino acids interference on the quantification of reducing sugars by the 3,5-dinitrosalicylic acid assay mislead carbohydrase activity measurements. (United States)

    Teixeira, Ricardo Sposina Sobral; da Silva, Ayla Sant'Ana; Ferreira-Leitão, Viridiana Santana; da Silva Bon, Elba Pinto


    This study evaluated the interference of the amino acids tryptophan, cysteine, histidine, tyrosine, hydroxyproline, leucine, proline, serine, glycine, valine, glutamic acid, phenylalanine, and methionine on the measurement of reducing sugars using a phenol-free 3,5-dinitrosalicylic acid (DNS) reagent. It was found that in reaction mixtures containing 20mM of either tryptophan, cysteine, histidine, tyrosine, or hydroxyproline the measurement of 3.7 mM glucose was overestimated by 76%, 50%, 35%, 18%, and 10%, respectively. The amino acids valine, glutamic acid, and phenylalanine did not affect the DNS reaction, while methionine decreased the color development by 5%. The measurement of glucose, xylose, arabinose, and cellobiose at the 3.7-12.4 mM range in the presence of 20 mM cysteine resulted in an overestimated concentration of 34.8-50%. Enzymatic assays for measuring xylanolytic and filter paper activity (FPAse) were conducted in the presence of 20-60 mM cysteine, and compared to cysteine-free assays. In the presence of cysteine, the measured xylanase activity increased threefold and the FPAse activity increased twofold due to the overestimation of the reducing sugar concentrations in the assays. The interference from cysteine was reduced to a maximum of 8.6% when a DNS reagent containing phenol was used.

  9. Analysis of reducing carbohydrates by reductive tryptamine derivatization prior to micellar electrokinetic capillary chromatography. (United States)

    Andersen, Keld E; Bjergegaard, Charlotte; Sørensen, Hilmer


    A micellar electrokinetic capillary chromatography method for determination of low molecular weight carbohydrates (dp 1-2) with an unbound carbonyl group as in aldoses or other reducing carbohydrates has been developed. Reductive amination of aldoses on the carbonyl group using tryptamine introduced a chromophor system to the carbohydrates enabling their sensitive UV detection at 220 nm and identification based on the indole group using diode array detection. Twelve carbohydrates including pentoses (d-ribose, l-arabinose, and d-xylose), hexoses (d-glucose, d-mannose, and d-galactose), deoxy sugars (l-rhamnose and l-fucose), uronic acids (d-glucuronic acid and d-galacturonic acid), and disaccharides (cellobiose and melibiose) are included in the study, using d-thyminose (2-deoxy-d-ribose) as the internal standard. Detection of all 12 carbohydrates is performed within 30 min. Linearity with correlation coefficients from 0.9864 to 0.9992 was found in the concentration range of 25-2500 micromol/L for all carbohydrates; the relative standard deviation on the migration times was between 0.27 and 0.80 min, and limits of quantification and limits of determination were in the picomole range.

  10. Contribution of manganese peroxidase and laccase to dye decoloration by Trametes versicolor. (United States)

    Champagne, Paul-Philippe; Ramsay, Juliana Akit


    During dye decoloration by Trametes versicolor ATCC 20869 in modified Kirk's medium, manganese peroxidase (MnP) and laccase were produced, but not lignin peroxidase, cellobiose dehydrogenase or manganese-independent peroxidase. Purified MnP decolorized azo dyes [amaranth, reactive black 5 (RB5) and Cibacron brilliant yellow] in Mn(2+)-dependent reactions but did not decolorize an anthraquinone dye [Remazol brilliant blue R (RBBR)]. However, the purified laccase decolorized RBBR five to ten times faster than the azo dyes and the addition of a redox mediator, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), did not alter decoloration rates. Amaranth and RB5 were decolorized the most rapidly by MnP since they have a hydroxyl group in an ortho position and a sulfonate group in the meta position relative to the azo bond. During a typical batch decoloration with the fungal culture, the ratio of laccase:MnP was 10:1 to 20:1 (based on enzyme activity) and increased to greater than 30:1 after decoloration was complete. Since MnP decolorized amaranth about 30 times more rapidly than laccase per unit of enzyme activity, MnP should have contributed more to decoloration than laccase in batch cultures.

  11. Complete genome sequences of Geobacillus sp. Y412MC52, a xylan-degrading strain isolated from obsidian hot spring in Yellowstone National Park. (United States)

    Brumm, Phillip; Land, Miriam L; Hauser, Loren J; Jeffries, Cynthia D; Chang, Yun-Juan; Mead, David A


    Geobacillus sp. Y412MC52 was isolated from Obsidian Hot Spring, Yellowstone National Park, Montana, USA under permit from the National Park Service. The genome was sequenced, assembled, and annotated by the DOE Joint Genome Institute and deposited at the NCBI in December 2011 (CP002835). Based on 16S rRNA genes and average nucleotide identity, Geobacillus sp. Y412MC52 and the related Geobacillus sp. Y412MC61 appear to be members of a new species of Geobacillus. The genome of Geobacillus sp. Y412MC52 consists of one circular chromosome of 3,628,883 bp, an average G + C content of 52 % and one circular plasmid of 45,057 bp and an average G + C content of 45 %. Y412MC52 possesses arabinan, arabinoglucuronoxylan, and aromatic acid degradation clusters for degradation of hemicellulose from biomass. Transport and utilization clusters are also present for other carbohydrates including starch, cellobiose, and α- and β-galactooligosaccharides.

  12. Understanding the Role of Physical Properties of Cellulose on Its Hydrolyzability by Cellulases (United States)

    O'Dell, Patrick Jonathan

    Cellulose has long been explored as a potential feedstock for biofuel, however the recalcitrance of cellulose makes its conversion into biofuel much more challenging and economically unfavorable compared to well-established processes for converting starch or sugar feedstocks into biofuel. Enzymes capable of hydrolyzing cellulose into soluble sugars, glucose and cellobiose, have been found to work processively along cellulose microfibrils starting from reducing end groups. For this study, cellulose was produced and purified in-house from Gluconacetobacter xylinum cultures, and characterized by quantifying functional groups (aldehyde, ketone, and carboxyl groups) to determine the extent of oxidation of cellulose due to the processing steps. The main goal of this study was to look at the impacts of ultrasonication on cellulose's structure and the enzymatic hydrolyzability of cellulose. A completely randomized experimental design was used to test the effect of ultrasonication time and amplitude (intensity) on changes in cellulose fibril length, degree of polymerization, and rates and extents of hydrolysis. Results indicated that sonication time does significantly impact both the fibril length and average degree of polymerization of cellulose. The impact of ultrasonication on the hydrolyzability of cellulose by commercial cellulase and beta-glucosidase preparations could not be effectively resolved due to high variability in the experimental results. These studies serve as a basis for future studies understanding the role of cellulose microstructure in the mechanism of cellulase hydrolysis of cellulose.

  13. Molecular characterisation of Xanthomonas strains isolated from aroids in Mauritius. (United States)

    Khoodoo, M H R; Sahin, F; Donmez, M F; Fakim, Y Jaufeerally


    Mauritius is one of the largest world producers of Anthurium cut flowers but outbreaks of bacterial blight have never been reported on the island. This work was about the characterisation and identification of bacterial strains isolated from Anthurium andreanum, Dieffenbachia maculata and Aglaonema simplex in Mauritius. Fifteen strains, that showed the morphological properties of Xanthomonas on conventional media, were tested on two semi-selective media (Esculin-trehalose and cellobiose-starch). ELISA tests using a panel of monoclonal antibodies were carried out and three out of 15 strains reacted with a Xanthomonas-specific monoclonal antibody (MAb XII). Analysis using four sets of ribosomal primers revealed that the same three Mauritius strains shared conserved PCR products with reference xanthomonads including virulent strains of Xanthomonas axonopodis pv. dieffenbachiae (Xad). BIOLOG tests and the Sherlock Microbial Identification system (MIDI) identified these three new strains at the species level as X. axonopodis. The complementary tests that were carried out clearly confirmed that the three strains are xanthomonads and, moreover, a DNA probe which showed specificity to Xad strains suggested that the three Mauritius strains are non-virulent forms of the pathogen causing Anthurium blight.

  14. Thermal diffusion of oligosaccharide solutions: the role of chain length and structure. (United States)

    Blanco, Pablo; Kriegs, Hartmut; Arlt, Bastian; Wiegand, Simone


    We investigated the chain length dependence of the thermodiffusion behavior of oligosaccharides by the infrared thermal diffusion forced Rayleigh scattering (IR-TDFRS) technique. Three disaccharides, sucrose, cellobiose and maltose, two trisaccharides, melezitose and raffinose, and a tetrasaccharide, stachyose, have been studied. We determined the thermal diffusion (D(T)), mass diffusion (D), and Soret (S(T)) coefficient as a function of temperature and concentration. While monosaccharides in water accumulate at the cold side in the investigated temperature (20-50 degrees C) and concentration (0.2-0.6667 wt) range, oligosaccharides enrich on the warm side with decreasing temperature or increasing sugar concentration. Additionally, we determined the kinematic viscosity (nu), the density (rho), and the thermal expansion coefficient (alpha) of the mixtures in order to check the linear correlation between D(T) and the ratio alpha/nu, which has been recently found for aqueous solutions of monosaccharides and for alkane mixtures. Finally, we found that D(T) and D decay with increasing chain length of the oligosaccharides in the whole studied range of temperatures, in contradiction with experimental results for nonpolar mixtures and theoretical predictions.

  15. Metabolic engineering of bacteria for ethanol production

    Energy Technology Data Exchange (ETDEWEB)

    Ingram, L.O.; Gomez, P.F.; Lai, X.; Moniruzzaman, M.; Wood, B.E.; Yomano, L.P.; York, S.W. [Univ. of Florida, Gainesville, FL (United States). Dept. of Microbiology and Cell Science


    Technologies are available which will allow the conversion of lignocellulose into fuel ethanol using genetically engineered bacteria. Assembling these into a cost-effective process remains a challenge. The authors` work has focused primarily on the genetic engineering of enteric bacteria using a portable ethanol production pathway. Genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase have been integrated into the chromosome of Escherichia coli B to produce strain KO11 for the fermentation of hemicellulose-derived syrups. This organism can efficiently ferment all hexose and pentose sugars present in the polymers of hemicellulose. Klebsiella oxytoca M5A1 has been genetically engineered in a similar manner to produce strain P2 for ethanol production from cellulose. This organism has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. The optimal pH for cellulose fermentation with this organism is near that of fungal cellulases. The general approach for the genetic engineering of new biocatalysts has been most successful with enteric bacteria thus far. However, this approach may also prove useful with gram-positive bacteria which have other important traits for lignocellulose conversion. Many opportunities remain for further improvements in the biomass to ethanol processes.

  16. Enteric bacterial catalysts for fuel ethanol production

    Energy Technology Data Exchange (ETDEWEB)

    Ingram, L.O.; Aldrich, H.C.; Borges, A.C.C. [and others


    The technology is available to produce fuel ethanol from renewable lignocellulosic biomass. The current challenge is to assemble the various process options into a commercial venture and begin the task of incremental improvement. Current process designs for lignocellulose are far more complex than grain to ethanol processes. This complexity results in part from the complexity of the substrate and the biological limitations of the catalyst. Their work at the University of Florida has focused primarily on the genetic engineering of Enteric bacteria using genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase. These two genes have been assembled into a portable ethanol production cassette, the PET operon, and integrated into the chromosome of Escherichia coli B for use with hemicellulose-derived syrups. The resulting strain, KO11, produces ethanol efficiently from all hexose and pentose sugars present in the polymers of hemicellulose. By using the same approach, the authors integrated the PET operon into the chromosome of Klebsiella oxytoca to produce strain P2 for use in the simultaneous saccharification and fermentation (SSF) process for cellulose. Strain P2 has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes.

  17. Authenticity analysis of pear juice employing chromatographic fingerprinting. (United States)

    Willems, Jamie L; Low, Nicholas H


    Pear juice is predominately composed of carbohydrates/polyols (>95% of the total soluble solids), making it susceptible to adulteration by the addition of less expensive commercial sweeteners. In this research, the major carbohydrate and polyol (fructose, glucose, sucrose, and sorbitol) content of 32 pure pear juices representing five world producing regions and three years of production was determined. Additionally, methods employing oligosaccharide profiling to detect the debasing of these samples with four commercial sweeteners (HFCS 55 and 90, TIS, and HIS) were developed using capillary gas chromatography with flame ionization detection (CGC-FID) and high-performance liquid chromatography with pulsed amperometric detection (HPAE-PAD). Detection limits for the four commercial sweeteners ranged from 0.5 to 5.0% (v/v). In addition, the developed CGC-FID method could be used to (a) detect the addition of pear to apple juice via arbutin detection and (b) determine if a pear juice was produced using enzymatic liquefaction via the presence of O-β-d-glucopyranosyl-(1→4)-d-glucopyranose (cellobiose), all within a single chromatographic analysis.

  18. The use of a thermotolerant fermentative Kluyveromyces marxianus IMB3 yeast strain for ethanol production

    Energy Technology Data Exchange (ETDEWEB)

    Banat, I.M. [Univ. of the United Arab Emirates, Al-Ain (United Arab Emirates). Dept. of Biolology; Singh, D. [Haryana Agriculture Univ., Hisar (India). Dept. of Microbiology; Marchant, R. [Ulster Univ. (United Kingdom). School of Applied Biological and Chemical Sciences


    An investigation was carried out on the growth and ethanol production of a novel thermotolerant ethanol-producing Kluyveromyces marxianus IMB3 yeast strain. It grew aerobically on glucose, lactose, cellobiose, xylose and whey permeate and fermented all the above carbon sources to ethanol at 45 C. This strain was capable of growing under anaerobic chemostat fermentation conditions at 45 C and a dilution rate of 0.15 h{sup -1} and produced {<=}0.9 g/l biomass and 1.8% (v/v) ethanol. An increase in biomass (up to 10.0 g/l) and ethanol (up to 4.3% v/v at 45 C and 7.7% v/v at 40 C) were achieved by applying a continuous two-stage fermentation in sequence (one aerobic and one anerobic stage) or a two-stage anaerobic fermentation with cell recycling. Potential applications, involving alcohol production systems, for use in dairy and wood related industries, were discussed. (orig.)

  19. Direct hydrogen production from cellulosic waste materials with a single-step dark fermentation process

    Energy Technology Data Exchange (ETDEWEB)

    Magnusson, Lauren; Islam, Rumana; Levin, David; Cicek, Nazim [Department of Biosystems Engineering, University of Manitoba, Winnipeg, MB (Canada); Sparling, Richard [Department of Microbiology, University of Manitoba, Winnipeg, MB (Canada)


    Biohydrogen production from cellulosic waste materials using dark fermentation is a promising technology for producing renewable energy. The purpose of this study was to evaluate residual cellulosic materials generated from local sources for their H{sub 2} production potential without any pretreatment. Clostridium thermocellum ATCC 27405, a cellulolytic, thermophilic bacterium that has been shown to be capable of H{sub 2} production on both cellobiose and {alpha}-cellulose substrates, was used in simultaneous batch fermentation experiments with dried distillers grain (DDGs), barley hulls (BH) and fusarium head blight contaminated barley hulls (CBH) as the carbon source. Overall, the dried distillers grain produced the highest concentration of hydrogen gas at 1.27 mmol H{sub 2}/glucose equivalent utilized. CBH and BH produced 1.18 and 1.24 mmol H{sub 2}/glucose equivalent utilized, respectively. Overall, this study indicates that hydrogen derived from a variety of cellulosic waste biomass sources is a possible candidate for the development of sustainable energy. (author)

  20. Chironomid egg masses harbour the clinical species Aeromonas taiwanensis and Aeromonas sanarellii. (United States)

    Beaz-Hidalgo, Roxana; Shakèd, Tamar; Laviad, Sivan; Halpern, Malka; Figueras, María J


    Bacteria of the genus Aeromonas are found worldwide in aquatic environments and may produce human infections. In 2010, two new clinical species, Aeromonas sanarellii and Aeromonas taiwanensis, were described on the basis of one strain recovered from wounds of hospitalized patients in Taiwan. So far, only four environmental isolates of A. sanarellii and one of A. taiwanensis have been recorded from waste water in Portugal and an additional clinical strain of A. taiwanensis from the faeces of a patient with diarrhoea in Israel. In the present study, strains belonging to these two species were identified from chironomid egg masses from the same area in Israel by sequencing the rpoD gene. This represents a new environmental habitat for these novel species. The first data on the virulence genes and antibiotic susceptibility are provided. The isolates of these two new species possess multiple virulence genes and are sensitive to amikacin, aztreonam, cefepime, cefoxatime, ceftazidime, ciprofloxacin, gentamicin, piperacillin-tazobactam, tigecycline, tobramycin, trimethoprim-sulfamethoxazole and imipenem. The key phenotypic tests for the differentiation of these new species from their closest relative Aeromonas caviae included the utilization of citrate, growth at 45 °C in sheep blood agar and acid production of cellobiose.

  1. [Biosynthesis of cellulolytic enzymes and xylanase during submerged cultivation of the fungus Aspergillus terreus 17P]. (United States)

    Loginova, L G; Guzhova, E P; Ismanlova, D Iu; Burdenko, L G


    The fungus Aspergillus terreus 17P--producer of cellulolytic enzymes--was cultivated in the Biotec 10 l fermenter on the medium containing minced and heated (at 200 degrees) wheat straw aerated with a different rate. At the mixing rate of 350 rpm and aeration rate of 0.7 r/rpm on the fourth day the culture liquid was obtained whose filtrate contained an active complex of cellulolytic enzymes and xylanase: CI--3.4; APB--1.1, Cx--35.7, cellobiase--0.23, xylanase--73.8 units/ml. The fractionation of the culture liquid filtrate with ammonium sulphate showed that the fraction precipitated at an interval of saturation of 0.3--0.7 contained the largest portion of cellulolytic enzymes and xylanase. The isolated enzymic preparations had a cellulolytic and xylanase activity and contained lipase, pectinase, laminarinase. They also contained low quantities of amylase, protease, beta-1,4- and beta-1,6-glucanase. Enzymic hylrolysis by the Asp. terreus 17P preparation of straw yielded glucose and xylose, of cotton, Na-KMC, cellobiose--glucose, Xylane hydrolyzate contained xylose and arabinose.

  2. [Expression of the genes CelA and XylA isolated from a fragment of metagenomic DNA in Escherichia coli]. (United States)

    Shedova, E N; Lunina, N A; Berezina, O V; Zverlov, V V; Schwarz, V; Velikodvorskaia, G A


    The glycosyl hydrolase genes cel5A and xyl3A previously isolated by ourselves within a fragment of DNA from the methagenomic library of cow rumen microflora DNA were sub-cloned and expressed in E. coli. The recombinant proteins Cel5A and Xyl3A were purified and characterized. Cellulase Cel5A belongs to the Family 5 glycosyl hydrolases and is a one-module 38.2 kDa enzyme that hydrolyses the 1,4-glycoside bonds of soluble cellulose substrates and amorphous cellulose, showing its maximal activity (31200 u/mg) on lichenan, a soluble substrate with mixed (beta-1,3-1,4) bonds. The end product of the amorphous cellulose hydrolysis is cellobiose. Cel5A is inactive toward the crystal forms of cellulose. Cel5A is an endoglucanase capable of exohydrolysis. The molecular mass of beta-xylosidase Xyl3A belonging to the Family 3 glycosyl hydrolases is 83.7 kDa. The enzyme is active only on xylooligosaccharides, with the maximal activity shown on xylobiose, the end product of the reaction being xylose. No activity on xylane was hitherto observed. Recombinant Cel5A and Xyl3A are stable over a wide range of pH and temperatures, their maximal activity being observed at pH 6.5 and at 55 degrees C.

  3. Characterization of the newly isolated Geobacillus sp. T1, the efficient cellulase-producer on untreated barley and wheat straws. (United States)

    Assareh, Reza; Shahbani Zahiri, Hossein; Akbari Noghabi, Kambiz; Aminzadeh, Saeed; Bakhshi Khaniki, Gholamreza


    A thermophile cellulase-producing bacterium was isolated and identified as closely related to Geobacillus subterraneus. The strain, named Geobacillus sp. T1, was able to grow and produce cellulase on cellobiose, microcrystalline cellulose, carboxymethylcellulose (CMC), barley straw, wheat straw and Whatman No. 1 filter paper. However, barley and wheat straws were significantly better substrates for cellulase production. When Geobacillus sp. T1 was cultivated in the presence of 0.5% barley straw, 0.1% Tween 80 and pH 6.5 at 50°C, the maximum level of free cellulase up to 143.50 U/mL was produced after 24h. This cellulase (≈ 54 kDa) was most active at pH 6.5 and 70°C. The enzyme in citrate phosphate buffer (10mM) was stable at 60°C for at least 1h. Geobacillus sp. T1 with efficient growth and cellulase production on straws seems a potential candidate for conversion of agricultural biomass to fuels.

  4. Isobutanol production at elevated temperatures in thermophilic Geobacillus thermoglucosidasius. (United States)

    Lin, Paul P; Rabe, Kersten S; Takasumi, Jennifer L; Kadisch, Marvin; Arnold, Frances H; Liao, James C


    The potential advantages of biological production of chemicals or fuels from biomass at high temperatures include reduced enzyme loading for cellulose degradation, decreased chance of contamination, and lower product separation cost. In general, high temperature production of compounds that are not native to the thermophilic hosts is limited by enzyme stability and the lack of suitable expression systems. Further complications can arise when the pathway includes a volatile intermediate. Here we report the engineering of Geobacillus thermoglucosidasius to produce isobutanol at 50°C. We prospected various enzymes in the isobutanol synthesis pathway and characterized their thermostabilities. We also constructed an expression system based on the lactate dehydrogenase promoter from Geobacillus thermodenitrificans. With the best enzyme combination and the expression system, 3.3g/l of isobutanol was produced from glucose and 0.6g/l of isobutanol from cellobiose in G. thermoglucosidasius within 48h at 50°C. This is the first demonstration of isobutanol production in recombinant bacteria at an elevated temperature.

  5. [Engineering and metabolic characteristics of a Clostridium tyrobutyricum strain]. (United States)

    Yang, Guiqing; Liu, Gang; Yang, Changde


    Clostridium tyrobutyricum is suitable for simultaneous saccharification and fermentation of lignocellulosic. It can produce butyric acid, acetic acid as its main fermentation products from a wide variety of carbohydrates such as glucose, xylose, cellobiose and arabinose. In order to decrease acetic acid content and increase butyric acid content in C. tyrobutyricum, we replaced genes on the acetic acid fermentation pathway with genes on the butyric acid fermentation pathway. Three genes were selected. They were acetyl-CoA acetylrtansfers gene (thl) which is the key enzyme gene associated with acetic acid fermentation pathway from Clostridium acetobutylicum, erythromycin gene (em) from plasmid pIMP1 and phosphotransacetylase gene (pta) which is the key enzyme gene associated with butyric acid fermentation pathway from C. tyrobutyricum. We fused these genes with pUC19 to construct nonreplicative integrated plasmids pUC19-EPT. Then we transformed pUC19-EPT into C. tyrobutyricum through electroporation. The recombinant transformants grown on plates containing erythromycin were validated by PCR. A mutant whose pta gene was displaced by thl gene on the chromosome was selected. In the fermentation from glucose, the mutant's yield of butyric acid is 0.47, increased by 34% compared with wild type; and the yield of acetic acid is 0.05, decreased by 29% compared with wild type.

  6. High-titer lactic acid production from NaOH-pretreated corn stover by Bacillus coagulans LA204 using fed-batch simultaneous saccharification and fermentation under non-sterile condition. (United States)

    Hu, Jinlong; Zhang, Zhenting; Lin, Yanxu; Zhao, Shumiao; Mei, Yuxia; Liang, Yunxiang; Peng, Nan


    Lactic acid (LA) is an important chemical with various industrial applications. Non-food feedstock is commercially attractive for use in LA production; however, efficient LA fermentation from lignocellulosic biomass resulting in both high yield and titer faces technical obstacles. In this study, the thermophilic bacterium Bacillus coagulans LA204 demonstrated considerable ability to ferment glucose, xylose, and cellobiose to LA. Importantly, LA204 produces LA from several NaOH-pretreated agro stovers, with remarkably high yields through simultaneous saccharification and fermentation (SSF). A fed-batch SSF process conducted at 50°C and pH 6.0, using a cellulase concentration of 30 FPU (filter paper unit)/g stover and 10 g/L yeast extract in a 5-L bioreactor, was developed to produce LA from 14.4% (w/w) NaOH-pretreated non-sterile corn stover. LA titer, yield, and average productivity reached 97.59 g/L, 0.68 g/g stover, and 1.63 g/L/h, respectively. This study presents a feasible process for lignocellulosic LA production from abundant agro stovers.

  7. Supplementation with xylanase and β-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover

    Directory of Open Access Journals (Sweden)

    Qing Qing


    Full Text Available Abstract Background Hemicellulose is often credited with being one of the important physical barriers to enzymatic hydrolysis of cellulose, and acts by blocking enzyme access to the cellulose surface. In addition, our recent research has suggested that hemicelluloses, particularly in the form of xylan and its oligomers, can more strongly inhibit cellulase activity than do glucose and cellobiose. Removal of hemicelluloses or elimination of their negative effects can therefore become especially pivotal to achieving higher cellulose conversion with lower enzyme doses. Results In this study, cellulase was supplemented with xylanase and β-xylosidase to boost conversion of both cellulose and hemicellulose in pretreated biomass through conversion of xylan and xylo-oligomers to the less inhibitory xylose. Although addition of xylanase and β-xylosidase did not necessarily enhance Avicel hydrolysis, glucan conversions increased by 27% and 8% for corn stover pretreated with ammonia fiber expansion (AFEX and dilute acid, respectively. In addition, adding hemicellulase several hours before adding cellulase was more beneficial than later addition, possibly as a result of a higher adsorption affinity of cellulase and xylanase to xylan than glucan. Conclusions This key finding elucidates a possible mechanism for cellulase inhibition by xylan and xylo-oligomers and emphasizes the need to optimize the enzyme formulation for each pretreated substrate. More research is needed to identify advanced enzyme systems designed to hydrolyze different substrates with maximum overall enzyme efficacy.

  8. Relationship between autophagy and the intracellular degradation of asialoglycoproteins in cultured rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kindberg, G.M.; Refsnes, M.; Christoffersen, T.; Norum, K.R.; Berg, T.


    The relationship between autophagy and the intracellular distribution of endocytosed asialoorosomucoid was studied in cultured rat hepatocytes. Overt autophagy was induced by shifting the cells to a minimal salt medium. Incubation in minimal salt medium led to the formation of buoyant lysosomes at the expense of denser lysosomes manifested as a dual distribution of these organelles in Nycodenz gradients. Asialoorosomucoid was labeled with /sup 125/I-tyramine cellobiose. The labeled degradation products formed from this ligand are trapped at the site of degradation and may therefore serve as markers for the subgroup of lysosomes involved in the degradation. In control cells the degradation of the ligand was initiated in a light prelysosomal compartment and continued in denser lysosomes. In cells with high autophagic activity, the degradation of labeled asialoorosomucoid took place exclusively in a buoyant group of lysosomes. These results suggest that degradation of endocytosed ligand takes place in the same secondary lysosomes as substrate sequestered by autophagic mechanisms. These light lysosomes represent a subgroup of active lysosomes which are gradually recruited from dense bodies. Data are also presented that indicate that insulin may prevent the change in buoyant density brought about by incubation in deficient medium.

  9. Purification and characterization of vanilla bean (Vanilla planifolia Andrews) beta-D-glucosidase. (United States)

    Odoux, Eric; Chauwin, Audrey; Brillouet, Jean-Marc


    Vanilla bean beta-D-glucosidase was purified to apparent homogeneity by successive anion exchange, hydrophobic interaction, and size-exclusion chromatography. The enzyme is a tetramer (201 kDa) made up of four identical subunits (50 kDa). The optimum pH was 6.5, and the optimum temperature was 40 degrees C at pH 7.0. K(m) values for p-nitrophenyl-beta-D-glucopyranoside and glucovanillin were 1.1 and 20.0 mM, respectively; V(max) values were 4.5 and 5.0 The beta-D-glucosidase was competitively inhibited by glucono-delta-lactone and 1-deoxynojirimycin, with respective K(i) values of 670 and 152 microM, and not inhibited by 2 M glucose. The beta-D-glucosidase was not inhibited by N-ethylmaleimide and DTNB and fully inhibited by 1.5-2 M 2-mercaptoethanol and 1,4-dithiothreitol. The enzyme showed decreasing activity on p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, and p-nitrophenyl-beta-D-xylopyranoside. The enzyme was also active on prunasin, esculin, and salicin and inactive on cellobiose, gentiobiose, amygdalin, phloridzin, indoxyl-beta-D-glucopyranoside, and quercetin-3-beta-D-glucopyranoside.

  10. Styrene production from a biomass-derived carbon source using a coculture system of phenylalanine ammonia lyase and phenylacrylic acid decarboxylase-expressing Streptomyces lividans transformants. (United States)

    Fujiwara, Ryosuke; Noda, Shuhei; Tanaka, Tsutomu; Kondo, Akihiko


    To produce styrene from a biomass-derived carbon source, Streptomyces lividans was adopted as a host strain. The gene encoding ferulic acid decarboxylase from Saccharomyces cerevisiae (FDC1) was introduced into S. lividans, and the resulting S. lividans transformant successfully expressed FDC1 and converted trans-cinnamic acid (CA) to styrene. A key factor in styrene production using microbes is the recovery of volatile styrene. In the present study, we selected polystyrene resin beads XRD-4 as the absorbent agent to recover styrene produced using S. lividans transformants, which enabled recovery of styrene from the culture broth. For styrene production from biomass-derived carbon sources, S. lividans/FDC1 was cultured together with S. lividans/p-encP, which we previously reported as a CA-producing S. lividans strain. This coculture system combined with the recovery of styrene using XAD-4 allowed the production of styrene from glucose, cellobiose, or xylo-oligosaccharide, respectively.

  11. [Determination of sugars, organic acids and alcohols in microbial consortium fermentation broth from cellulose using high performance liquid chromatography]. (United States)

    Jiang, Yan; Fan, Guifang; Du, Ran; Li, Peipei; Jiang, Li


    A high performance liquid chromatographic method was established for the determination of metabolites (sugars, organic acids and alcohols) in microbial consortium fermentation broth from cellulose. Sulfate was first added in the samples to precipitate calcium ions in microbial consortium culture medium and lower the pH of the solution to avoid the dissociation of organic acids, then the filtrates were effectively separated using high performance liquid chromatography. Cellobiose, glucose, ethanol, butanol, glycerol, acetic acid and butyric acid were quantitatively analyzed. The detection limits were in the range of 0.10-2.00 mg/L. The linear correlation coefficients were greater than 0.999 6 in the range of 0.020 to 1.000 g/L. The recoveries were in the range of 85.41%-115.60% with the relative standard deviations of 0.22% -4.62% (n = 6). This method is accurate for the quantitative analysis of the alcohols, organic acids and saccharides in microbial consortium fermentation broth from cellulose.

  12. Direct Production of 5-Hydroxymethylfurfural via Catalytic Conversion of Simple and Complex Sugars over Phosphated TiO2. (United States)

    Atanda, Luqman; Shrotri, Abhijit; Mukundan, Swathi; Ma, Qing; Konarova, Muxina; Beltramini, Jorge


    A water-THF biphasic system containing N-methyl-2-pyrrolidone (NMP) was found to enable the efficient synthesis of 5-hydroxymethylfurfural (HMF) from a variety of sugars (simple to complex) using phosphated TiO2 as a catalyst. Fructose and glucose were selectively converted to HMF resulting in 98 % and 90 % yield, respectively, at 175 °C. Cellobiose and sucrose also gave rise to high HMF yields of 94 % and 98 %, respectively, at 180 °C. Other sugar variants such as starch (potato and rice) and cellulose were also investigated. The yields of HMF from starch (80-85 %) were high, whereas cellulose resulted in a modest yield of 33 %. Direct transformation of cellulose to HMF in significant yield (86 %) was assisted by mechanocatalytic depolymerization-ball milling of acid-impregnated cellulose. This effectively reduced cellulose crystallinity and particle size, forming soluble cello-oligomers; this is responsible for the enhanced substrate-catalytic sites contact and subsequent rate of HMF formation. During catalyst recyclability, P-TiO2 was observed to be reusable for four cycles without any loss in activity. We also investigated the conversion of the cello-oligomers to HMF in a continuous flow reactor. Good HMF yield (53 %) was achieved using a water-methyl isobutyl ketone+NMP biphasic system.

  13. Isolation of Homogeneous Polysaccharide Monooxygenases from Fungal Sources and Investigation of Their Synergism with Cellulases when Acting on Cellulose. (United States)

    Bulakhov, A G; Gusakov, A V; Chekushina, A V; Satrutdinov, A D; Koshelev, A V; Matys, V Yu; Sinitsyn, A P


    Lytic polysaccharide monooxygenases (PMO) discovered several years ago are enzymes classified as oxidoreductases. In nature, they participate in microbial degradation of cellulose together with cellulases that belong to the hydrolytic type of enzymes (class of hydrolases). Three PMO from ascomycetes - Thielavia terrestris, Trichoderma reesei, and Myceliophthora thermophila - were isolated and purified to homogeneous state using various types of chromatography. The first two enzymes are recombinant proteins heterologously expressed by the Penicillium verruculosum fungus, while the third is a native PMO secreted by M. thermophila. When acting on microcrystalline cellulose, all these PMOs displayed synergism with the cellulase complex of the P. verruculosum fungus. Replacing 10% of cellulases (by protein concentration) with PMO in the presence of 6.25 mM gallic acid or 2.5 µM of cellobiose dehydrogenase from M. thermophila, used as electron donors for PMO, resulted in the 17-31% increase in the yield of reducing sugars after 24-48 h of the enzymatic reaction.

  14. Inhibition effects of dilute-acid prehydrolysate of corn stover on enzymatic hydrolysis of Solka Floc. (United States)

    Kothari, Urvi D; Lee, Yoon Y


    Dilute-acid pretreatment liquor (PL) produced at NREL through a continuous screw-driven reactor was analyzed for sugars and other potential inhibitory components. Their inhibitory effects on enzymatic hydrolysis of Solka Floc were investigated. When the PL was mixed into the enzymatic hydrolysis reactor at 1:1 volume ratio, the glucan and xylan digestibility decreased by 63% and 90%, respectively. The tolerance level of the enzyme for each inhibitor was determined. Of the identified degradation components, acetic acid was found to be the strongest inhibitor for cellulase activity, as it decreased the glucan yield by 10% at 1 g/L. Among the sugars, cellobiose and glucose were found to be strong inhibitors to glucan hydrolysis, whereas xylose is a strong inhibitor to xylan hydrolysis. Xylo-oligomers inhibit xylan digestibility more strongly than the glucan digestibility. Inhibition by the PL was higher than that of the simulated mixture of the identifiable components. This indicates that some of the unidentified degradation components, originated mostly from lignin, are potent inhibitors to the cellulase enzyme. When the PL was added to a simultaneous saccharification and co-fermentation using Escherichia coli KO11, the bioprocess was severely inhibited showing no ethanol formation or cell growth.

  15. Exploring the microbiota dynamics related to vegetable biomasses degradation and study of lignocellulose-degrading bacteria for industrial biotechnological application (United States)

    Ventorino, Valeria; Aliberti, Alberto; Faraco, Vincenza; Robertiello, Alessandro; Giacobbe, Simona; Ercolini, Danilo; Amore, Antonella; Fagnano, Massimo; Pepe, Olimpia


    The aims of this study were to evaluate the microbial diversity of different lignocellulosic biomasses during degradation under natural conditions and to isolate, select, characterise new well-adapted bacterial strains to detect potentially improved enzyme-producing bacteria. The microbiota of biomass piles of Arundo donax, Eucalyptus camaldulensis and Populus nigra were evaluated by high-throughput sequencing. A highly complex bacterial community was found, composed of ubiquitous bacteria, with the highest representation by the Actinobacteria, Proteobacteria, Bacteroidetes and Firmicutes phyla. The abundances of the major and minor taxa retrieved during the process were determined by the selective pressure produced by the lignocellulosic plant species and degradation conditions. Moreover, cellulolytic bacteria were isolated using differential substrates and screened for cellulase, cellobiase, xylanase, pectinase and ligninase activities. Forty strains that showed multienzymatic activity were selected and identified. The highest endo-cellulase activity was seen in Promicromonospora sukumoe CE86 and Isoptericola variabilis CA84, which were able to degrade cellulose, cellobiose and xylan. Sixty-two percent of bacterial strains tested exhibited high extracellular endo-1,4-ß-glucanase activity in liquid media. These approaches show that the microbiota of lignocellulosic biomasses can be considered an important source of bacterial strains to upgrade the feasibility of lignocellulose conversion for the `greener' technology of second-generation biofuels.

  16. Enzymatic decolorization of spent textile dyeing baths composed by mixtures of synthetic dyes and additives. (United States)

    Ciullini, Ilaria; Gullotto, Antonella; Tilli, Silvia; Sannia, Giovanni; Basosi, Riccardo; Scozzafava, Andrea; Briganti, Fabrizio


    The effects of different components of real dyeing bath formulations, such as the equalizing and fixing additives-acids, salts, and surfactants-on the decolorization catalyzed by Funalia trogii enzymatic extracts, were investigated to understand their influence on the recalcitrance to biodegradation of this type of wastewater. The decolorization of selected dyes and dye mixtures after tissue dyeing was performed in the presence/absence of auxiliary compounds. All spent dyeing baths were enzymatically decolorized to different extents, by the addition of extracts containing laccase only or laccase plus cellobiose dehydrogenase. Whereas surfactant auxiliaries, in some instances, inhibit the decolorization of spent dyeing baths, in several occurrences the acid/salt additives favor the enzymatic process. In general, the complete spent dyeing formulations are better degraded than those containing the dyes only. The comparison of extracellular extracts obtained from spent straws from the commercial growth of Pleurotus sp. mushrooms with those from F. trogii reveals similar decolorization extents thus allowing to further reduce the costs of bioremediation.

  17. Isolation and characterization of a new cellulosome-producing Clostridium thermocellum strain. (United States)

    Tachaapaikoon, Chakrit; Kosugi, Akihiko; Pason, Patthra; Waeonukul, Rattiya; Ratanakhanokchai, Khanok; Kyu, Khin Lay; Arai, Takamitsu; Murata, Yoshinori; Mori, Yutaka


    The anaerobic thermophilic bacterium, Clostridium thermocellum, is a potent cellulolytic microorganism that produces large extracellular multienzyme complexes called cellulosomes. To isolate C. thermocellum organisms that possess effective cellulose-degrading ability, new thermophilic cellulolytic strains were screened from more than 800 samples obtained mainly from agriculture residues in Thailand using microcrystalline cellulose as a carbon source. A new strain, C. thermocellum S14, having high cellulose-degrading ability was isolated from bagasse paper sludge. Cellulosomes prepared from S14 demonstrated faster degradation of microcrystalline cellulose, and 3.4- and 5.6-fold greater Avicelase activity than those from C. thermocellum ATCC27405 and JW20 (ATCC31449), respectively. Scanning electron microscopic analysis showed that S14 had unique cell surface features with few protuberances in contrast to the type strains. In addition, the cellulosome of S14 was resistant to inhibition by cellobiose that is a major end product of cellulose hydrolysis. Saccharification tests conducted using rice straw soaked with sodium hydroxide indicated the cellulosome of S14 released approximately 1.5-fold more total sugars compared to that of ATCC27405. This newly isolated S14 strain has the potential as an enzyme resource for effective lignocellulose degradation.

  18. Synthesis of 5-hydroxymethylfurural from carbohydrates using large-pore mesoporous tin phosphate. (United States)

    Dutta, Arghya; Gupta, Dinesh; Patra, Astam K; Saha, Basudeb; Bhaumik, Asim


    A large-pore mesoporous tin phosphate (LPSnP-1) material has been synthesized hydrothermally by using Pluronic P123 as the structure-directing agent. The material is composed of aggregated nanoparticles of 10-15 nm in diameter and has a BET surface area of 216 m(2)  g(-1) with an average pore diameter of 10.4 nm. This pore diameter is twice as large as that of mesoporous tin phosphate materials synthesized through the surfactant-templating pathways reported previously. LPSnP-1 shows excellent catalytic activity for the conversion of fructose, glucose, sucrose, cellobiose, and cellulose to 5-hydroxymethylfurfural (HMF) in a water/methyl isobutyl ketone biphasic solvent to give maximum yields of HMF of 77, 50, 51, 39, and 32 mol %, respectively, under microwave-assisted heating at 423 K. Under comparable reaction conditions, LPSnP-1 gives 12 % more HMF yield than a small-pore mesoporous tin phosphate catalyst that has an identical framework composition. This confirms the beneficial role of large mesopores and nanoscale particle morphology in catalytic reactions that involve bulky natural carbohydrate molecules.

  19. Complete genome sequence of Enterococcus mundtii QU 25, an efficient L-(+)-lactic acid-producing bacterium. (United States)

    Shiwa, Yuh; Yanase, Hiroaki; Hirose, Yuu; Satomi, Shohei; Araya-Kojima, Tomoko; Watanabe, Satoru; Zendo, Takeshi; Chibazakura, Taku; Shimizu-Kadota, Mariko; Yoshikawa, Hirofumi; Sonomoto, Kenji


    Enterococcus mundtii QU 25, a non-dairy bacterial strain of ovine faecal origin, can ferment both cellobiose and xylose to produce l-lactic acid. The use of this strain is highly desirable for economical l-lactate production from renewable biomass substrates. Genome sequence determination is necessary for the genetic improvement of this strain. We report the complete genome sequence of strain QU 25, primarily determined using Pacific Biosciences sequencing technology. The E. mundtii QU 25 genome comprises a 3 022 186-bp single circular chromosome (GC content, 38.6%) and five circular plasmids: pQY182, pQY082, pQY039, pQY024, and pQY003. In all, 2900 protein-coding sequences, 63 tRNA genes, and 6 rRNA operons were predicted in the QU 25 chromosome. Plasmid pQY024 harbours genes for mundticin production. We found that strain QU 25 produces a bacteriocin, suggesting that mundticin-encoded genes on plasmid pQY024 were functional. For lactic acid fermentation, two gene clusters were identified-one involved in the initial metabolism of xylose and uptake of pentose and the second containing genes for the pentose phosphate pathway and uptake of related sugars. This is the first complete genome sequence of an E. mundtii strain. The data provide insights into lactate production in this bacterium and its evolution among enterococci.

  20. Studies on the Characteris of the Induction of Cotton Fiber Derived from Cotton Ovule Callus Cells%棉胚珠愈伤组织诱导成纤维实验系统的研究

    Institute of Scientific and Technical Information of China (English)

    汤清秀; 赵旌旌; 王隆华


    Cotton nbers were induced from cotton ovule callus cells by suspension culture,floatculture or filter paper bridge culture.h was found that float culture or filter paper bridge culture was bener than suspension culture.APM and actinomycin D inhibit the fibe elongation.Cellobiose promotes its development. Polarity affects its culture.%用棉花胚珠切块诱导愈伤组织,经悬浮振荡培养、漂浮培养、滤纸桥法等方法诱导成纤维细胞。发现漂浮培养和改进的滤纸桥法对纤维的诱导效果比悬浮振荡培养的效果好。微管解聚剂APM和核酸抑制剂抑制纤维的生长,纤维二糖有一定的促进作用。极性对纤维的生长有影响。

  1. Thermogladius calderae gen. nov., sp. nov., an anaerobic, hyperthermophilic crenarchaeote from a Kamchatka hot spring. (United States)

    Kochetkova, Tatiana V; Kublanov, Ilya V; Toshchakov, Stepan V; Osburn, Magdalena R; Novikov, Andrei A; Bonch-Osmolovskaya, Elizaveta A; Perevalova, Anna A


    An obligately anaerobic, hyperthermophilic, organoheterotrophic archaeon, strain 1633T, was isolated from a terrestrial hot spring of the Uzon Caldera (Kamchatka Peninsula, Russia). Cells were regular cocci, 0.5-0.9 μm in diameter, with one flagellum. The temperature range for growth was 80-95°C, with an optimum at 84°C. Strain 1633T grew on yeast extract, beef extract, peptone, cellulose and cellobiose. No growth was detected on other sugars or carbohydrates, organic acids, or under autotrophic conditions. The only detected growth products were CO2, acetate, and H2. Growth rate was stimulated by elemental sulfur, which was reduced to hydrogen sulfide. In silico calculated G+C content of strain 1633T genomic DNA was 55.64 mol%. 16S rRNA gene sequence analysis placed the strain 1633T together with the non-validly published "Thermogladius shockii" strain WB1 in a separate genus-level cluster within the Desulfurococcaceae family. ANI results revealed 75.72% identity between 1633T and WB1. Based on these results we propose a novel genus and species, for which the name Thermogladius calderae gen. nov., sp. nov. (type strain 1633T=DSM 22663T=VKM B-2946T) is proposed.

  2. A novel GH6 cellobiohydrolase from Paenibacillus curdlanolyticus B-6 and its synergistic action on cellulose degradation. (United States)

    Baramee, Sirilak; Teeravivattanakit, Thitiporn; Phitsuwan, Paripok; Waeonukul, Rattiya; Pason, Patthra; Tachaapaikoon, Chakrit; Kosugi, Akihiko; Sakka, Kazuo; Ratanakhanokchai, Khanok


    We recently discovered a novel glycoside hydrolase family 6 (GH6) cellobiohydrolase from Paenibacillus curdlanolyticus B-6 (PcCel6A), which is rarely found in bacteria. This enzyme is a true exo-type cellobiohydrolase which exhibits high substrate specificity on amorphous cellulose and low substrate specificity on crystalline cellulose, while this showed no activity on substitution substrates, carboxymethyl cellulose and xylan, distinct from all other known GH6 cellobiohydrolases. Product profiles, HPLC analysis of the hydrolysis products and a schematic drawing of the substrate-binding subsites catalysing cellooligosaccharides can explain the new mode of action of this enzyme which prefers to hydrolyse cellopentaose. PcCel6A was not inhibited by glucose or cellobiose at concentrations up to 300 and 100 mM, respectively. A good synergistic effect for glucose production was found when PcCel6A acted together with processive endoglucanase Cel9R from Clostridium thermocellum and β-glucosidase CglT from Thermoanaerobacter brockii. These properties of PcCel6A make it a suitable candidate for industrial application in the cellulose degradation process.

  3. Biotechnological applications of bacterial cellulases

    Directory of Open Access Journals (Sweden)

    Esther Menendez


    Full Text Available Cellulases have numerous applications in several industries, including biofuel production, food and feed industry, brewing, pulp and paper, textile, laundry, and agriculture.Cellulose-degrading bacteria are widely spread in nature, being isolated from quite different environments. Cellulose degradation is the result of a synergic process between an endoglucanase, an exoglucanase and a,β-glucosidase. Bacterial endoglucanases degrade ß-1,4-glucan linkages of cellulose amorphous zones, meanwhile exoglucanases cleave the remaining oligosaccharide chains, originating cellobiose, which is hydrolyzed by ß-glucanases. Bacterial cellulases (EC are comprised in fourteen Glycosil Hydrolase families. Several advantages, such as higher growth rates and genetic versatility, emphasize the suitability and advantages of bacterial cellulases over other sources for this group of enzymes. This review summarizes the main known cellulolytic bacteria and the best strategies to optimize their cellulase production, focusing on endoglucanases, as well as it reviews the main biotechnological applications of bacterial cellulases in several industries, medicine and agriculture.

  4. Caldicellulosiruptor obsidiansis sp. nov., an anaerobic, extremely thermophilic, cellulolytic bacterium isolated from Obsidian Pool, Yellowstone National Park

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton-Brehm, Scott [ORNL; Elkins, James G [ORNL; Phelps, Tommy Joe [ORNL; Keller, Martin [ORNL; Carroll, Sue L [ORNL; Allman, Steve L [ORNL; Podar, Mircea [ORNL; Mosher, Jennifer J [ORNL; Vishnivetskaya, Tatiana A [ORNL


    A novel, obligately anaerobic, extremely thermophilic, cellulolytic bacterium, designated OB47T, was isolated from Obsidian Pool, Yellowstone National Park, WY, USA. The isolate was a non-motile, non-spore forming, Gram-positive rod approximately 2 m long by 0.2 m wide and grew at temperatures between 55-85oC with the optimum at 78oC. The pH range for growth was 6.0-8.0 with values of near 7.0 being optimal. Growth on cellobiose produced the fastest specific growth rates at 0.75 hr-1. The organism also displayed fermentative growth on glucose, maltose, arabinose, fructose, starch, lactose, mannose, sucrose, galactose, xylose, arabinogalactan, Avicel, xylan, filter paper, processed cardboard, pectin, dilute acid-pretreated switchgrass and Populus. OB47T was unable to grow on mannitol, fucose, lignin, Gelrite, acetate, glycerol, ribose, sorbital, carboxymethylcellulose and casein. Yeast extract stimulated growth and thiosulfate, sulfate, nitrate, and sulfur were not reduced. Fermentation end products were mainly acetate, H2, and CO2 although lactate and ethanol were produced in 5 l batch fermentations. The G+C content of the DNA was 35 mol% and sequence analysis of the small subunit ribosomal RNA gene placed OB47T within the genus Caldicellulosiruptor. Based on its phylogenetic and phenotypic properties, the isolate is proposed to be designated Caldicellulosiruptor obsidiansis sp. nov. and OB47T is the type stain (ATCC = ____, JCM = ____).

  5. The Thermoanaerobacter glycobiome reveals mechanisms of pentose and hexose co-utilization in bacteria.

    Directory of Open Access Journals (Sweden)

    Lu Lin


    Full Text Available Thermoanaerobic bacteria are of interest in cellulosic-biofuel production, due to their simultaneous pentose and hexose utilization (co-utilization and thermophilic nature. In this study, we experimentally reconstructed the structure and dynamics of the first genome-wide carbon utilization network of thermoanaerobes. The network uncovers numerous novel pathways and identifies previously unrecognized but crucial pathway interactions and the associated key junctions. First, glucose, xylose, fructose, and cellobiose catabolism are each featured in distinct functional modules; the transport systems of hexose and pentose are apparently both regulated by transcriptional antiterminators of the BglG family, which is consistent with pentose and hexose co-utilization. Second, glucose and xylose modules cooperate in that the activity of the former promotes the activity of the latter via activating xylose transport and catabolism, while xylose delays cell lysis by sustaining coenzyme and ion metabolism. Third, the vitamin B₁₂ pathway appears to promote ethanologenesis through ethanolamine and 1, 2-propanediol, while the arginine deiminase pathway probably contributes to cell survival in stationary phase. Moreover, by experimentally validating the distinct yet collaborative nature of glucose and xylose catabolism, we demonstrated that these novel network-derived features can be rationally exploited for product-yield enhancement via optimized timing and balanced loading of the carbon supply in a substrate-specific manner. Thus, this thermoanaerobic glycobiome reveals novel genetic features in carbon catabolism that may have immediate industrial implications and provides novel strategies and targets for fermentation and genome engineering.

  6. Enzymatic hydrolysis of oil palm empty fruits bunch fiber using Celluclast® and Accellerase® BG for sugar production (United States)

    Salleh, Noor Shafryna; Murad, Abdul Munir Abdul


    In this work, the ability of commercial Trichoderma reesei cellulases preparation, Celluclast® or in combination with Accellerase®BG β-glucosidase to hydrolyse pretreated oil palm empty fruit bunch (OPEFB) was evaluated. Celluclast® alone hydrolyzed OPEFB to produce 2.41±0.44 mg glucose per gram OPEFB. However, the production of glucose was significantly improved with supplementation of Accellerase®BG (8.12±0.93 mg/g). This result suggested that the endoglucanases and exoglucanases in Celluclast® and β-glucosidase in Accellerase®BG able to work synergistically to increase the production of glucose from OPEFB. In addition, the production of xylose was also improved by 30% when the enzyme mixture was used. The result suggested that the mixture of Celluclast® with Accellerase®BG work synergistically to improve the production of sugars by removing the inhibition by cellobiose for complete cellulose hydrolysis. The production of glucose and xylose from OPEFB wastes showed the potential of this biomass as the source of renewable energy and fine chemicals production in Malaysia.

  7. Functional diversity of family 3 β-glucosidases from thermophilic cellulolytic fungus Humicola insolens Y1. (United States)

    Xia, Wei; Bai, Yingguo; Cui, Ying; Xu, Xinxin; Qian, Lichun; Shi, Pengjun; Zhang, Wei; Luo, Huiying; Zhan, Xiuan; Yao, Bin


    The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose. However, studies on the β-glucosidases from this fungus remain insufficient, especially on glycosyl hydrolase family 3 enzymes. In the present study, we analyzed the functional diversity of three distant family 3 β-glucosidases from Humicola insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastoris strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50-60 °C and pH 5.5-6.0) and high glucose tolerance, but differed in substrate specificities and kinetics. HiBgl3B was solely active towards aryl β-glucosides while HiBgl3A and HiBgl3C showed broad substrate specificities including both disaccharides and aryl β-glucosides. Of the three enzymes, HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Substitutions of three key residues Ile48, Ile278 and Thr484 of HiBgl3B to the corresponding residues of HiBgl3A conferred the enzyme activity towards sophorose, and vice versa. This study reveals the functional diversity of GH3 β-glucosidases as well as the key residues in recognizing +1 subsite of different substrates.

  8. Expression and characterization of a novel highly glucose-tolerant β-glucosidase from a soil metagenome

    Institute of Scientific and Technical Information of China (English)

    Jian Lu; Liqin Du; Yutuo Wei; Yuanyuan Hu; Ribo Huang


    A β-glucosidase gene unbgl1A was isolated by the functionbased screening of a metagenomic library and the enzyme protein was expressed in Escherichia coli,purified,and biochemically characterized.The enzyme Unbgl1A had a Km value of 2.09 ± 0.31 mM,and a Vmax value of 183.90 ± 9.61 μmol min-1 mg-1 under the optimal reaction conditions,which were pH 6.0 at 50℃.Unbgl1A can be activated by a variety of monosaccharides,disaccharides,and NaCl,and exhibits a high level of stability at high concentration of NaCl.Two prominent features for this enzyme are:(i) high glucose tolerance.It can be tolerant to glucose as high as 2000 mM,with Ki =1500 mM; (ii) high NaCl tolerance.Its activity is not affected by 600 mM NaCl.The enzyme showed transglucosylation activities resulting in the formation of cellotriose from cellobiose.These properties of Unbgl1A should have important practical implication in its potential applications for better industrial production of glucose or bioethanol started from lignocellulosic biomass.

  9. Improved radioimmunotherapy of hematologic malignancies. [Final report

    Energy Technology Data Exchange (ETDEWEB)

    Press, O.W.


    This research project proposes to develop novel new approaches of improving the radioimmunodetection and radioimmunotherapy of malignancies by augmenting retention of radioimmunoconjugates by tumor cells. The approaches shown to be effective in these laboratory experiments will subsequently be incorporated into out ongoing clinical trials in patients. Specific project objectives include: to study the rates of endocytosis, intracellular routing, and metabolic degradation of radiolabeled monoclonal antibodies targeting tumor-associated antigens on human leukemia and lymphoma cells; To examine the effects of lysosomotropic amines (e.g. chloroquine, amantadine), carboxylic ionophores (monensin, nigericin), and thioamides (propylthiouracil), on the retention of radiolabeled MoAbs by tumor cells; to examine the impact of newer radioiodination techniques (tyramine cellobiose, paraiodobenzoyl) on the metabolic degradation of radioiodinated antibodies; to compare the endocytosis, intracellular routing, and degradation of radioimmunoconjugates prepared with different radionuclides ({sup 131}Iodine, {sup 111}Indium, {sup 90}Yttrium, {sup 99m}Technetium, {sup 186}Rhenium); and to examine the utility of radioimmunoconjugates targeting oncogene products for the radioimmunotherapy and radioimmunoscintigraphy of cancer.

  10. Hydrolysis of bamboo fiber cellulose in formic acid

    Institute of Scientific and Technical Information of China (English)

    Yong SUN; Lu LIN; Haibo DENG; Hong PENG; Jiazhe LI; Runchang SUN; Shijie LIU


    Bamboo fiber dissolution and hydrolysis in formic acid were studied. After hydrolysis, formic acid can be recovered in a clean state and reused. Solid water-soluble sugars were obtained. After being dipped into the formic acid solution for 30 min, the bamboo fibers started to swell. After one hour, the bamboo fibers gradually started to dissolve in the formic acid solution. The color of the liquor/solution turned green and dark. In the end, the bamboo fibers became thoroughly dissolved in the liquor after four hours. There was a clear hierarch-ical tissue structure on the fiber surface, as observed by AFM before treatment. The differential structure disap-peared after 30 min of treatment. The fiber surface became plump and glossy. After six hours reaction at 60℃, the solid sugar mixture recovered contained glu-cose, cellobiose, cellotriose, cellotetrose, cellopentose and cellohexaose. A significant fraction of the sugar pro-ducts consisted of monomeric glucose. More than 54.5% of the bamboo fiber mass had been transformed into monomeric glucose.

  11. The fungus gardens of leaf-cutter ants undergo a distinct physiological transition during biomass degradation. (United States)

    Huang, Eric L; Aylward, Frank O; Kim, Young-Mo; Webb-Robertson, Bobbie-Jo M; Nicora, Carrie D; Hu, Zeping; Metz, Thomas O; Lipton, Mary S; Smith, Richard D; Currie, Cameron R; Burnum-Johnson, Kristin E


    Leaf-cutter ants are dominant herbivores in ecosystems throughout the Neotropics that feed on fungus gardens cultivated on fresh foliar biomass. Although recent investigations have shed light on how plant biomass is degraded in fungus gardens, the cycling of nutrients that takes place in these specialized microbial ecosystems is still not well understood. Here, using metabolomic and metaproteomic techniques, we examine the dynamics of nutrient turnover in these gardens. Our results reveal that numerous free amino acids and sugars are depleted throughout the process of biomass degradation, indicating that easily accessible nutrients from plant material are readily consumed by microbes in these ecosystems. Accumulation of cellobiose and lignin derivatives near the end of the degradation process is consistent with previous characterization of lignocellulases produced by the fungal cultivar of the ants. Our results also suggest that ureides may be an important source of nitrogen in fungus gardens, especially during nitrogen-limiting conditions. No free arginine was detected in our metabolomic experiments despite evidence that the host ants cannot produce this amino acid, suggesting that biosynthesis of this metabolite may be tightly regulated in fungus gardens. These results provide new insights into microbial community-level processes that underlie this important ant-fungus symbiosis.

  12. Temperature-mediated changes in microbial carbon use efficiency and 13C discrimination (United States)

    Lehmeier, Christoph A.; Ballantyne, Ford, IV; Min, Kyungjin; Billings, Sharon A.


    Understanding how carbon dioxide (CO2) flux from ecosystems feeds back to climate warming depends in part on our ability to quantify the efficiency with which microorganisms convert organic carbon (C) into either biomass or CO2. Quantifying ecosystem-level respiratory CO2 losses often also requires assumptions about stable C isotope fractionations associated with the microbial transformation of organic substrates. However, the diversity of organic substrates' δ13C and the challenges of measuring microbial C use efficiency (CUE) in their natural environment fundamentally limit our ability to project ecosystem C budgets in a warming climate. Here, we quantify the effect of temperature on C fluxes during metabolic transformations of cellobiose, a common microbial substrate, by a cosmopolitan microorganism growing at a constant rate. Biomass C specific respiration rate increased by 250 % between 13 and 26.5 °C, decreasing CUE from 77 to 56 %. Biomass C specific respiration rate was positively correlated with an increase in respiratory 13C discrimination from 4.4 to 6.7 ‰ across the same temperature range. This first demonstration of a direct link between temperature, microbial CUE, and associated isotope fluxes provides a critical step towards understanding δ13C of respired CO2 at multiple scales, and towards a framework for predicting future ecosystem C fluxes.

  13. Cost-effective simultaneous saccharification and fermentation of l-lactic acid from bagasse sulfite pulp by Bacillus coagulans CC17. (United States)

    Zhou, Jie; Ouyang, Jia; Xu, Qianqian; Zheng, Zhaojuan


    The main barriers to cost-effective lactic acid production from lignocellulose are the high cost of enzymes and the ineffective utilization of the xylose within the hydrolysate. In the present study, the thermophilic Bacillus coagulans strain CC17 was used for the simultaneous saccharification and fermentation (SSF) of bagasse sulfite pulp (BSP) to produce l-lactic acid. Unexpectedly, SSF by CC17 required approximately 33.33% less fungal cellulase than did separate hydrolysis and fermentation (SHF). More interestingly, CC17 can co-ferment cellobiose and xylose without any exogenous β-glucosidase in SSF. Moreover, adding xylanase could increase the concentration of lactic acid produced via SSF. Up to 110g/L of l-lactic acid was obtained using fed-batch SSF, resulting in a lactic acid yield of 0.72g/g cellulose. These results suggest that SSF using CC17 has a remarkable advantage over SHF and that a potentially low-cost and highly-efficient fermentation process can be established using this protocol.

  14. Improved radioimmunotherapy of hematologic malignancies

    Energy Technology Data Exchange (ETDEWEB)

    Press, O.W.


    This research project proposes to develop novel new approaches of improving the radioimmunodetection and radioimmunotherapy of malignancies by augmenting retention of radioimmunoconjugates by tumor cells. The approaches shown to be effective in these laboratory experiments will subsequently be incorporated into out ongoing clinical trials in patients. Specific project objectives include: to study the rates of endocytosis, intracellular routing, and metabolic degradation of radiolabeled monoclonal antibodies targeting tumor-associated antigens on human leukemia and lymphoma cells; To examine the effects of lysosomotropic amines (e.g. chloroquine, amantadine), carboxylic ionophores (monensin, nigericin), and thioamides (propylthiouracil), on the retention of radiolabeled MoAbs by tumor cells; to examine the impact of newer radioiodination techniques (tyramine cellobiose, paraiodobenzoyl) on the metabolic degradation of radioiodinated antibodies; to compare the endocytosis, intracellular routing, and degradation of radioimmunoconjugates prepared with different radionuclides ({sup 131}Iodine, {sup 111}Indium, {sup 90}Yttrium, {sup 99m}Technetium, {sup 186}Rhenium); and to examine the utility of radioimmunoconjugates targeting oncogene products for the radioimmunotherapy and radioimmunoscintigraphy of cancer.

  15. Mechanistic study on the cellulose dissolution in ionic liquids by density functional theory☆

    Institute of Scientific and Technical Information of China (English)

    Yingying Yao; Yao Li; Xiaomin Liu; Xiaochun Zhang; Jianji Wang; Xiaoqian Yao; Suojiang Zhang


    Ionic liquids (ILs) have attracted many attentions in the dissolution of cellulose due to their unique physicochem-ical properties as green solvents. However, the mechanism of dissolution is stil under debate. In this work, com-putational investigation for the mechanisms of dissolution of cellulose in [Bmim]Cl, [Emim]Cl and [Emim]OAc ILs was performed, and it was focused on the process of breakage of cel ulose chain and ring opening using cel obiose as a model molecule. The detailed mechanism and reaction energy barriers were computed for various possible pathways by density functional theoretical method. The key finding was that ILs catalyze the dissolution process by synergistic effect of anion and cation, which led to the cleavage of cellulose chain and formation of derivatives of cel ulose. The investigation on ring opening process of cellobiose suggested that carbene formed in ILs played an important role in the side reaction of cellulose, and it facilitated the formation of a covalent bond between cel-lulose and imidazolium core. These computation results may provide new perspective to understand and apply ILs for pretreatment of cellulose.

  16. Engineering of family-5 glycoside hydrolase (Cel5A from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.

    Directory of Open Access Journals (Sweden)

    Amar A Telke

    Full Text Available Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.

  17. Enzymatic hydrolysis of cellulose: Study of the process of recovery of cellulose glucides by the technique of hyperfiltration on polysulphonic membranes. Idrolisi enzimatica della cellulosa. Studio del processo di recupero dei glucidi da cellulasi con tecniche di ultrafiltrazione su membrane polisolfoniche

    Energy Technology Data Exchange (ETDEWEB)

    Pizzichini, M.; Fabiani, C.; Sperandei, M.


    Membrane separation technology can optimize some steps of cellulose enzymatic hydrolysis process. In order to continuously remove glucose and cellobiose in the permeate solution and recover the enzymes in the recycling stream, the separation by ultrafiltration of glucides from enzymes was studied. Celluclast enzyme supplied by Novo,in aqueous buffer solution at pH5 and concentration of 0.2-4% w/v range, was used as a feed. Glucides concentration was in the 0.02-0,95% w/v$range. A DDS UF System (Lab Unit-20) was employed with 16 flat membranes type GS81PP with cut off at 6000 dalton. During the separation test, a reduction in the permeate flux caused by protein deposition on the membrane surface was observed. Water washing of the membranes cleans all the membranes package and the original membranes permeability (80 1/sq. m/h at 4 bars) is recovered. Glucides can be quantitatively recovered by the UF process. However the high cellulase concentration may produce a slight enzyme inactivation (2-9%).

  18. Comparative analysis of carbohydrate active enzymes in Clostridium termitidis CT1112 reveals complex carbohydrate degradation ability. (United States)

    Munir, Riffat I; Schellenberg, John; Henrissat, Bernard; Verbeke, Tobin J; Sparling, Richard; Levin, David B


    Clostridium termitidis strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, Nasutitermes lujae. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes), sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in C. termitidis, the recently sequenced strain CT1112 of C. termitidis was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in C. termitidis, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199) and carbohydrate binding modules (95) were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.

  19. Biochemical conversion of sugarcane straw hemicellulosic hydrolyzate supplemented with co-substrates for xylitol production. (United States)

    Hernández-Pérez, A F; Costa, I A L; Silva, D D V; Dussán, K J; Villela, T R; Canettieri, E V; Carvalho, J A; Soares Neto, T G; Felipe, M G A


    Biotechnological production of xylitol is an attractive route to add value to a sugarcane biorefinery, through utilization of the hemicellulosic fraction of sugarcane straw, whose availability is increasing in Brazil. Herein, supplementation of the sugarcane straw hemicellulosic hydrolyzate (xylose 57gL(-1)) with maltose, sucrose, cellobiose or glycerol was proposed, and their effect as co-substrates on xylitol production by Candida guilliermondii FTI 20037 was studied. Sucrose (10gL(-1)) and glycerol (0.7gL(-1)) supplementation led to significant increase of 8.88% and 6.86% on xylose uptake rate (1.11gL(-1)h(-1) and 1.09gL(-1)), respectively, but only with sucrose, significant increments of 12.88% and 8.69% on final xylitol concentration (36.11gL(-1)) and volumetric productivity (0.75gL(-1)h(-1)), respectively, were achieved. Based on these results, utilization of complex sources of sucrose, derived from agro-industries, as nutritional supplementation for xylitol production can be proposed as a strategy for improving the yeast performance and reducing the cost of this bioprocess by replacing more expensive nutrients.

  20. Selective adhesion of wastewater bacteria to Pleurotus ostreatus mycelium in a trickle-bed bioreactor

    Directory of Open Access Journals (Sweden)

    Čeněk Novotný


    Full Text Available The work is focused on spontaneous colonization of fungal mycelium by invading microorganisms in a trickle-bed fungal bioreactor operating under semi-sterile conditions. Pleurotus ostreatus was grown under the flow of synthetic wastewater containing activated sludge bacteria and the microbial consortium developed in the reactor was characterized. Genotype and phenotype profile of the reactor-invading, bacterial consortium was clearly distinctive from that of the original activated sludge. The bacterial consortium from the reactor contained a higher portion of bacteria capable of cellobiose utilization and a small amount of bacteria with the ability to utilize benzoic acids. The invading bacteria had no effect on the dye decolorization performance of the fungal reactor. Five bacterial strains colonizing P. ostreatus reactor cultures were isolated and identified as species of the genera Pseudomonas and Bacillus. Except for Bacillus cereus all strains displayed a potential to inhibit fungal growth on solid media (14 to 51 % inhibition which was comparable or higher than that of the reference bacterial strains. The pH- and media composition-dependence of the growth inhibition was demonstrated.

  1. Purification and characterization of cold-active endo-1,4-β-glucanase produced by Pseudoalteromonas sp.AN545 from Antarctica

    Institute of Scientific and Technical Information of China (English)

    SHEN Jihong; KAN Guangfeng; SHI Cuijuan; LEI Zhenhuan; XIE Qiuju; QIAN Wenjia


    A bacterium hydrolyzing carboxymethylcellulose,isolated from Antarctic sea ice,was identified as Pseudoalteromonas sp.based on 16S rDNA gene sequences and named as Pseudoalteromonas sp.AN545.The extracellular endo-1,4-β-glucanase AN-1 was purified successively by ammonium sulfate precipitation,DEAE-Sepharose ion exchange chromatography and Sephadex G-75 gel filtration chromatography.The molecular mass of AN-1 was estimated to be 47.5 kDa utilizing SDS-PAGE and gel chromatography analysis.AN-1 could hydrolyze caboxymethylcellulose,avicel and β-glucan,but not cellobiose,xylan and p-Nitrophenyl-β-D-glucopyranoside.The optimal temperature and pH for the β-glucanase activity of AN-1 were determined to be at 30℃ and pH 6.0,respectively.AN-1 was stable at acidic solutions of pH 5.0-6.5 and temperatures below 30℃ for 1 h.Moreover,the specific activity was enhanced by Ca2+ and Mg2+,and inhibited by Cu2+.The kinetic parameters Michaelis constant (Km) and maximum velocity (Vmax) of AN-1 were 3.96 mg/mL and 6.06×10-2 mg/(min.mL),respectively.

  2. Evidence that the xylanase activity from Sulfolobus solfataricus Oalpha is encoded by the endoglucanase precursor gene (sso1354) and characterization of the associated cellulase activity. (United States)

    Maurelli, Luisa; Giovane, Alfonso; Esposito, Alessandra; Moracci, Marco; Fiume, Immacolata; Rossi, Mosè; Morana, Alessandra


    Sulfolobus solfataricus strain Oalpha was previously isolated for its ability to grow on minimal medium supplemented with xylan as a carbon source. The strain exhibited thermostable xylanase activity but several attempts to identify the gene encoding for the activity failed. Further studies showed that the xylanase displayed activity on carboxymethylcellulose (CMC) and the new activity was characterized. It exhibited an optimal temperature and pH of 95 degrees C and 3.5, respectively, and a half-life of 53 min at 95 degrees C. The enzyme, which was demonstrated to be glycosylated, hydrolyzed CMC in an endo-manner releasing cellobiose and other cello-oligomers. Analysis of the tryptic fragments by tandem mass spectrometry led to identification of the endoglucanase precursor, encoded by the sso1354 gene, as the protein possessing dual activity. The efficiency of the SSO1354 protein in degrading cellulosic and hemicellulosic fractions contained in agronomic residues was tested at low pH and high temperature. Cellulose and xylan were degraded to glucose and xylose at 90 degrees C, pH 4 by an enzyme mix consisting of SSO1354 and additional glycosyl hydrolases from S. solfataricus Oalpha. Given its role in saccharification processes requiring high temperatures and acidic environments, SSO1354 represents an interesting candidate for the utilization of agro-industrial waste for fuel production.

  3. Purification and characterization of a thermophilic 1,3-1,4-β-glucanase from Bacillus methylotrophicus S2 isolated from booklice. (United States)

    Niu, Qiuhong; Zhang, Guo; Zhang, Lin; Ma, Yali; Shi, Qian; Fu, Weiwei


    An extracellular 1,3-1,4-β-glucanase-producing strain S2 was isolated from booklice and identified as Bacillus methylotrophicus. Furthermore, a homogeneous extracellular 1,3-1,4-β-glucanase GCS2 was purified by ammonium sulfate precipitation and cation-exchange chromatography. The gene for the 1,3-1,4-β-glucanase was cloned, and the nucleotide sequence was determined. Characterization of the purified enzyme revealed the molecular mass of 26 kDa and the optimum activity at pH 7.5, 55°C. The purified enzyme can highly hydrolyze carboxymethylcellulose including oat gum, barley β-glucan, CMC and lichenan, while low activity on avicel, cellobiose, filter paper, p-nitrophenyl β-d-cellobioside, and p-nitrophenyl β-d-glucoside, but no activity against microcrystalline cellulose or salicin. The enzyme was stable at wide range of pHs 5-10 and still maintained above 60% activity at 70°C. The enzyme activity was stimulated by Trixon X-100. The property of the enzyme GCS2 makes this enzyme a broad prospect in brewing and commercial detergent industry. To our knowledge, this is the first report of a 1,3-1,4-β-glucanase from microbes associated with booklice.

  4. A novel optimization approach to estimating kinetic parameters of the enzymatic hydrolysis of corn stover

    Directory of Open Access Journals (Sweden)

    Fenglei Qi


    Full Text Available Enzymatic hydrolysis is an integral step in the conversion of lignocellulosic biomass to ethanol. The conversion of cellulose to fermentable sugars in the presence of inhibitors is a complex kinetic problem. In this study, we describe a novel approach to estimating the kinetic parameters underlying this process. This study employs experimental data measuring substrate and enzyme loadings, sugar and acid inhibitions for the production of glucose. Multiple objectives to minimize the difference between model predictions and experimental observations are developed and optimized by adopting multi-objective particle swarm optimization method. Model reliability is assessed by exploring likelihood profile in each parameter space. Compared to previous studies, this approach improved the prediction of sugar yields by reducing the mean squared errors by 34% for glucose and 2.7% for cellobiose, suggesting improved agreement between model predictions and the experimental data. Furthermore, kinetic parameters such as K2IG2, K1IG, K2IG, K1IA, and K3IA are identified as contributors to the model non-identifiability and wide parameter confidence intervals. Model reliability analysis indicates possible ways to reduce model non-identifiability and tighten parameter confidence intervals. These results could help improve the design of lignocellulosic biorefineries by providing higher fidelity predictions of fermentable sugars under inhibitory conditions.

  5. Lactobacillus arizonensis sp. nov., isolated from jojoba meal. (United States)

    Swezey, J L; Nakamura, L K; Abbott, T P; Peterson, R E


    Five strains of simmondsin-degrading, lactic-acid-producing bacteria were isolated from fermented jojoba meal. These isolates were facultatively anaerobic, gram-positive, non-motile, non-spore-forming, homofermentative, rod-shaped organisms. They grew singly and in short chains, produced lactic acid but no gas from glucose, and did not exhibit catalase activity. Growth occurred at 15 and 45 degrees C. All strains fermented cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannitol, D-mannose, melibiose, D-ribose, salicin, D-sorbitol, sucrose and trehalose. Some strains fermented L-(-)-arabinose and L-rhamnose. D-Xylose was not fermented and starch was not hydrolysed. The mean G+C content of the DNA was 48 mol%. Phylogenetic analyses of 16S rDNA established that the isolates were members of the genus Lactobacillus. DNA reassociation of 45% or less was obtained between the new isolates and the reference strains of species with G+C contents of about 48 mol%. The isolates were differentiated from other homofermentative Lactobacillus spp. on the basis of 16S rDNA sequence divergence, DNA relatedness, stereoisomerism of the lactic acid produced, growth temperature and carbohydrate fermentation. The data support the conclusion that these organisms represent strains of a new species, for which the name Lactobacillus arizonensis is proposed. The type strain of L. arizonensis is NRRL B-14768T (= DSM 13273T).

  6. Identifying and characterizing the most significant β-glucosidase of the novel species Aspergillus saccharolyticus

    Energy Technology Data Exchange (ETDEWEB)

    Sorensen, Anette; Ahring, Birgitte K.; Lubeck, Mette; Ubhayasekera, Wimal; Bruno, Kenneth S.; Culley, David E.; Lubeck, Peter S.


    A newly discovered fungal species, Aspergillus saccharolyticus, was found to produce a culture broth rich in beta-glucosidase activity. In this present work, the main beta-glucosidase of A. saccharolyticus responsible for the efficient hydrolytic activity was identified, isolated, and characterized. Ion exchange chromatography was used to fractionate the culture broth, yielding fractions with high beta-glucosidase activity and only one visible band on an SDS-PAGE gel. Mass spectrometry analysis of this band gave peptide matches to beta-glucosidases from aspergilli. Through a PCR approach using degenerate primers and genome walking, a 2919 base pair sequence encoding the 860 amino acid BGL1 polypeptide was determined. BGL1 of A. saccharolyticus has 91% and 82% identity with BGL1 from Aspergillus aculeatus and BGL1 from Aspergillus niger, respectively, both belonging to Glycoside hydrolase family 3. Homology modeling studies suggested beta-glucosidase activity with preserved retaining mechanism and a wider catalytic pocket compared to other beta-glucosidases. The bgl1 gene was heterologously expressed in Trichoderma reesei QM6a, purified, and characterized by enzyme kinetics studies. The enzyme can hydrolyze cellobiose, pNPG, and cellodextrins. The enzyme showed good thermostability, was stable at 50°C, and at 60°C it had a half-life of approximately 6 hours.

  7. Diversity and functional significance of cellulolytic microbes living in termite, pill-bug and stem-borer guts. (United States)

    Bashir, Zeenat; Kondapalli, Vamsi Krishna; Adlakha, Nidhi; Sharma, Anil; Bhatnagar, Raj K; Chandel, Girish; Yazdani, Syed Shams


    Arthropods living on plants are able to digest plant biomass with the help of microbial flora in their guts. This study considered three arthropods from different niches - termites, pill-bugs and yellow stem-borers - and screened their guts for cellulase producing microbes. Among 42 unique cellulase-producing strains, 50% belonged to Bacillaceae, 26% belonged to Enterobacteriaceae, 17% belonged to Microbacteriaceae, 5% belonged to Paenibacillaceae and 2% belonged to Promicromonosporaceae. The distribution of microbial families in the three arthropod guts reflected differences in their food consumption habits. Most of the carboxymethylcellulase positive strains also hydrolysed other amorphous substrates such as xylan, locust bean gum and β-D-glucan. Two strains, A11 and A21, demonstrated significant activity towards Avicel and p-nitrophenyl-β-D-cellobiose, indicating that they express cellobiohydrolase. These results provide insight into the co-existence of symbionts in the guts of arthropods and their possible exploitation for the production of fuels and chemicals derived from plant biomass.

  8. Engineering and Evolution of Saccharomyces cerevisiae to Produce Biofuels and Chemicals. (United States)

    Turner, Timothy L; Kim, Heejin; Kong, In Iok; Liu, Jing-Jing; Zhang, Guo-Chang; Jin, Yong-Su


    To mitigate global climate change caused partly by the use of fossil fuels, the production of fuels and chemicals from renewable biomass has been attempted. The conversion of various sugars from renewable biomass into biofuels by engineered baker's yeast (Saccharomyces cerevisiae) is one major direction which has grown dramatically in recent years. As well as shifting away from fossil fuels, the production of commodity chemicals by engineered S. cerevisiae has also increased significantly. The traditional approaches of biochemical and metabolic engineering to develop economic bioconversion processes in laboratory and industrial settings have been accelerated by rapid advancements in the areas of yeast genomics, synthetic biology, and systems biology. Together, these innovations have resulted in rapid and efficient manipulation of S. cerevisiae to expand fermentable substrates and diversify value-added products. Here, we discuss recent and major advances in rational (relying on prior experimentally-derived knowledge) and combinatorial (relying on high-throughput screening and genomics) approaches to engineer S. cerevisiae for producing ethanol, butanol, 2,3-butanediol, fatty acid ethyl esters, isoprenoids, organic acids, rare sugars, antioxidants, and sugar alcohols from glucose, xylose, cellobiose, galactose, acetate, alginate, mannitol, arabinose, and lactose.

  9. A thermostable Gloeophyllum trabeum xylanase with potential for the brewing industry. (United States)

    Wang, Xiaoyu; Luo, Huiying; Yu, Wangning; Ma, Rui; You, Shuai; Liu, Weina; Hou, Lingyu; Zheng, Fei; Xie, Xiangming; Yao, Bin


    A xylanase gene of glycoside hydrolase family 10, GtXyn10, was cloned from Gloeophyllum trabeum CBS 900.73 and expressed in Pichia pastoris GS115. Purified recombinant GtXyn10 exhibited significant activities to xylan (100.0%), lichenan (11.2%), glucan (15.2%) and p-nitrophenol-β-cellobiose (18.6%), demonstrated the maximum xylanase and glucanase activities at pH 4.5-5.0 and 75°C, retained stability over the pH range of 2.0-7.5 and at 70°C, and was resistant to pepsin and trypsin, most metal ions and SDS. Multiple sequence alignment and modeled-structure analysis identified a unique Gly48 in GtXyn10, and site-directed mutagenesis of Gly48 to Lys improved the temperature optimum up to 80°C. Under simulated mashing conditions, GtXyn10 (80U) reduced the mash viscosity by 12.8% and improved the filtration rate by 31.3%. All these properties above make GtXyn10 attractive for potential applications in the feed and brewing industries.

  10. Utilization of cellulosic waste from tequila bagasse and production of polyhydroxyalkanoate (PHA) bioplastics by Saccharophagus degradans. (United States)

    Alva Munoz, Luis Esteban; Riley, Mark R


    Utilization of wastes from agriculture is becoming increasingly important due to concerns of environmental impact. The goals of this work were to evaluate the ability of an unusual organism, Saccharophagus degradans (ATCC 43961), to degrade the major components of plant cell walls and to evaluate the ability of S. degradans to produce polyhydroxyalkanoates (PHAs, also known as bioplastics). S. degradans can readily attach to cellulosic fibers, degrade the cellulose, and utilize this as the primary carbon source. The growth of S. degradans was assessed in minimal media (MM) containing glucose, cellobiose, avicel, and bagasse with all able to support growth. Cells were able to attach to avicel and bagasse fibers; however, growth on these insoluble fibers was much slower and led to a lower maximal biomass production than observed with simple sugars. Lignin in MM alone did not support growth, but did support growth upon addition of glucose, although with an increased adaptation phase. When culture conditions were switched to a nitrogen depleted status, PHA production commences and extends for at least 48 h. At early stationary phase, stained inclusion bodies were visible and two chronologically increasing infrared light absorbance peaks at 1,725 and 1,741 cm(-1) confirmed the presence of PHAs. This work demonstrates for what we believe to be the first time, that a single organism can degrade insoluble cellulose and under similar conditions can produce and accumulate PHA. Additional work is necessary to more fully characterize these capabilities and to optimize the PHA production and purification.

  11. Improved electroporation procedure for genetic transformation of Dekkera/Brettanomyces bruxellensis. (United States)

    Miklenić, Marina; Žunar, Bojan; Štafa, Anamarija; Svetec, Ivan-Krešimir


    Yeast Dekkera/Brettanomyces bruxellensis is one of the most common contaminants in wine industry, but also one of the most promising candidates for large-scale bioethanol production. Brettanomyces bruxellensis not only produces and tolerates high ethanol concentrations, but can also ferment cellobiose and adapt to lignocellulose hydrolasate. Furthermore, genome sequences of several B. bruxellensis strains are available, and efforts have been made to develop tools for genetic transformation of this yeast. Previously, we reported a successful transformation using lithium acetate/PEG method and electroporation, however, with very low transformation efficiency (10-20 transformants μg(-1)). Here we describe an optimization of electroporation procedure which resulted in a significant increase of transformation efficiency (2.8 × 10(3) transformants μg(-1)). Several key transformation parameters were optimized including cell growth phase, density of cells in the transformation sample and electroporation settings. We determined that treating the cells with both lithium acetate (100 mM) and dithiothreitol (35 mM) synergistically improves transformation efficiency. Using the described procedure around 500 transformants can be obtained per transformation sample with 180 ng of non-homologous linear transforming fragment. Additionally, several transformants were obtained with less than 1 ng of DNA demonstrating that this procedure is adequate even when very limited amount of DNA is available.

  12. Structural changes of cellobiohydrolase I (1,4-β-D-glucan-cellobiohydrolase I, CBHI) and PNPC (p-nitro-phenyl-β-D-cellobioside) during the binding process

    Institute of Scientific and Technical Information of China (English)


    Conformational changes to 1,4-β-D-glucan cellobiohydrolase I (CBHI) in response to its binding with p-nitrophenyl β-D-cellobioside (PNPC) were analyzed by second-derivative fluorescence spectrometry at the saturation binding point. Irreversible changes to the configuration of PNPC during the course of the binding process were characterized by UV spectral analysis. Isothermal titration calorimetry (ITC) was used to determine the stoichiometry of binding (i.e. the number of molar binding sites) of PNPC to CBHI. Two points on the surface of the CBHI molecule interact with PNPC, and irreversible changes to the configuration of PNPC occur during its conversion to p-nitrophenyl (PNP). The ITC studies demonstrated that the binding of PNPC to CBHI is an irreversible process, in which heat is released, but where there is no reversible equilibrium between PNPC-CBHI and CBHI and PNPC. On the other hand, PNP and cellobiose need to be released from the PNPC-CBHI complex to facilitate the repeated binding of new PNPC molecules to the renewable CBHI molecules. Therefore, we speculate that the energy, which powers the configurational change of PNPC as it is converted to PNP, is generated from cyclic changes in the conformation of CBHI during the binding/de-sorption process. These new insights may provide a basis for a better understanding of the binding mechanism in enzyme-substrate interactions.

  13. Glycolipid biosurfactants: main properties and potential applications in agriculture and food industry. (United States)

    Mnif, Inès; Ghribi, Dhouha


    Glycolipids, consisting of a carbohydrate moiety linked to fatty acids, are microbial surface active compounds produced by various microorganisms. They are characterized by high structural diversity and have the ability to decrease the surface and interfacial tension at the surface and interface, respectively. Rhamnolipids, trehalolipids, mannosylerythritol lipids and cellobiose lipids are among the most popular glycolipids. They have received much practical attention as biopesticides for controlling plant diseases and protecting stored products. As a result of their antifungal activity towards phytopathogenic fungi and larvicidal and mosquitocidal potencies, glycolipid biosurfactants permit the preservation of plants and plant crops from pest invasion. Also, as a result of their emulsifying and antibacterial activities, glycolipids have great potential as food additives and food preservatives. Furthermore, the valorization of food byproducts via the production of glycolipid biosurfactant has received much attention because it permits the bioconversion of byproducts on valuable compounds and decreases the cost of production. Generally, the use of glycolipids in many fields requires their retention from fermentation media. Accordingly, different strategies have been developed to extract and purify glycolipids. © 2016 Society of Chemical Industry.

  14. Design of an Os Complex-Modified Hydrogel with Optimized Redox Potential for Biosensors and Biofuel Cells. (United States)

    Pinyou, Piyanut; Ruff, Adrian; Pöller, Sascha; Ma, Su; Ludwig, Roland; Schuhmann, Wolfgang


    Multistep synthesis and electrochemical characterization of an Os complex-modified redox hydrogel exhibiting a redox potential ≈+30 mV (vs. Ag/AgCl 3 M KCl) is demonstrated. The careful selection of bipyridine-based ligands bearing N,N-dimethylamino moieties and an amino-linker for the covalent attachment to the polymer backbone ensures the formation of a stable redox polymer with an envisaged redox potential close to 0 V. Most importantly, the formation of an octahedral N6-coordination sphere around the Os central atoms provides improved stability concomitantly with the low formal potential, a low reorganization energy during the Os(3+/2+) redox conversion and a negligible impact on oxygen reduction. By wiring a variety of enzymes such as pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase, flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase and the FAD-dependent dehydrogenase domain of cellobiose dehydrogenase, low-potential glucose biosensors could be obtained with negligible co-oxidation of common interfering compounds such as uric acid or ascorbic acid. In combination with a bilirubin oxidase-based biocathode, enzymatic biofuel cells with open-circuit voltages of up to 0.54 V were obtained.

  15. Cellulose fermentation by nitrogen-fixing anaerobic bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Canale-Parola, E.


    In anaerobic natural environments cellulose is degraded to methane, carbon dioxide and other products by the combined activities of many diverse microorganisms. We are simulating processes occurring in natural environments by constructing biologically-defined, stable, heterogeneous bacterial communities (consortia) that we use as in vitro systems for quantitative studies of cellulose degradation under conditions of combined nitrogen deprivation. These studies include the investigation of (i) metabolic interactions among members of cellulose-degrading microbial populations, and (ii) processes that regulate the activity or biosynthesis of cellulolytic enzymes. In addition, we are studying the sensory mechanisms that, in natural environments, may enable motile cellulolytic bacteria to migrate toward cellulose. This part of our work includes biochemical characterization of the cellobiose chemoreceptor of cellulolytic bacteria. Finally, an important aspect of our research is the investigation of the mechanisms by which multienzyme complexes of anaerobic bacteria catalyze the depolymerization of crystalline cellulose and of other plant cell wall polysacchaddes. The research will provide fundamental information on the physiology and ecology of cellulose-fermenting, N{sub 2}-fixing bacteria, and on the intricate processes involved in C and N cycling in anaerobic environments. Furthermore, the information will be valuable for the development of practical applications, such as the conversion of plant biomass (e.g., agricultural, forestry and municipal wastes) to automotive fuels such as ethanol.

  16. Cellulase recycling after high-solids simultaneous saccharification and fermentation of combined pretreated corncob

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    Ruoyu eDu


    Full Text Available Despite the advantageous prospect of second-generation bioethanol, its final commercialization must overcome the primary cost impediment due to enzyme assumption. To solve this problem, this work achieves high-concentration ethanol fermentation and multi-round cellulase recycling through process integration. The optimal time and temperature of the re-adsorption process were determined by monitoring the adsorption kinetics of cellulases. Both glucose and cellobiose inhibited cellulase adsorption. After 96 h of ethanol fermentation, 40% of the initial cellulase remained in the broth, from which 62.5% of the cellulase can be recycled and reused in fresh substrate re-adsorption for 90 min. Under optimum conditions, i.e., pH 5.0, dry matter loading of 15 wt%, cellulase loading of 45 FPU/g glucan, two cycles of fermentation and re-adsorption can yield two-fold increased ethanol outputs and reduce enzyme costs by over 50%. The ethanol concentration in each cycle can be achieved at levels greater than 40 g/L.

  17. Influence of forage phenolics on ruminal fibrolytic bacteria and in vitro fiber degradation. (United States)

    Varel, V H; Jung, H J


    In vitro cultures of ruminal microorganisms were used to determine the effect of cinnamic acid and vanillin on the digestibility of cellulose and xylan. Cinnamic acid and vanillin depressed in vitro dry matter disappearance of cellulose 14 and 49%, respectively, when rumen fluid was the inoculum. The number of viable Bacteroides succinogenes cells, the predominant cellulolytic organism, was threefold higher for fermentations which contained vanillin than for control fermentations. When xylan replaced cellulose as the substrate, a 14% decrease in the digestibility of xylan was observed with vanillin added; however, the number of viable xylanolytic bacteria cultured from the batch fermentation was 10-fold greater than that of control fermentations. The doubling time of B. succinogenes was increased from 2.32 to 2.58 h when vanillin was added to cellobiose medium, and absorbance was one-half that of controls after 18 h. The growth rate of Ruminococcus albus and Ruminococcus flavefaciens was inhibited more by p-coumaric acid than by vanillin, although no reduction of final absorbance was observed in their growth cycles. Vanillin, and to a lesser extent cinnamic acid, appeared to prevent the attachment of B. succinogenes cells to cellulose particles, but did not affect dissociation of cells from the particles. B. succinogenes, R. albus, R. flavefaciens, and Butyrivibrio fibrisolvens all modified the parent monomers cinnamic acid, p-coumaric acid, ferulic acid, and vanillin, with B. fibrisolvens causing the most extensive modification. These results suggest that phenolic monomers can inhibit digestibility of cellulose and xylan, possibly by influencing attachment of the fibrolytic microorganisms to fiber particles. The reduced bacterial attachment to structural carbohydrates in the presence of vanillin may generate more free-floating fibrolytic organisms, thus giving a deceptively higher viable count.

  18. Lactococcus lactis metabolism and gene expression during growth on plant tissues. (United States)

    Golomb, Benjamin L; Marco, Maria L


    Lactic acid bacteria have been isolated from living, harvested, and fermented plant materials; however, the adaptations these bacteria possess for growth on plant tissues are largely unknown. In this study, we investigated plant habitat-specific traits of Lactococcus lactis during growth in an Arabidopsis thaliana leaf tissue lysate (ATL). L. lactis KF147, a strain originally isolated from plants, exhibited a higher growth rate and reached 7.9-fold-greater cell densities during growth in ATL than the dairy-associated strain L. lactis IL1403. Transcriptome profiling (RNA-seq) of KF147 identified 853 induced and 264 repressed genes during growth in ATL compared to that in GM17 laboratory culture medium. Genes induced in ATL included those involved in the arginine deiminase pathway and a total of 140 carbohydrate transport and metabolism genes, many of which are involved in xylose, arabinose, cellobiose, and hemicellulose metabolism. The induction of those genes corresponded with L. lactis KF147 nutrient consumption and production of metabolic end products in ATL as measured by gas chromatography-time of flight mass spectrometry (GC-TOF/MS) untargeted metabolomic profiling. To assess the importance of specific plant-inducible genes for L. lactis growth in ATL, xylose metabolism was targeted for gene knockout mutagenesis. Wild-type L. lactis strain KF147 but not an xylA deletion mutant was able to grow using xylose as the sole carbon source. However, both strains grew to similarly high levels in ATL, indicating redundancy in L. lactis carbohydrate metabolism on plant tissues. These findings show that certain strains of L. lactis are well adapted for growth on plants and possess specific traits relevant for plant-based food, fuel, and feed fermentations.

  19. Effective Production of Sorbitol and Mannitol from Sugars Catalyzed by Ni Nanoparticles Supported on Aluminium Hydroxide

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    Rodiansono Rodiansono


    Full Text Available Effective production of hexitols (sorbitol and mannitol was achieved from sugars by means of nickel nanoparticles supported on aluminium hydroxide (NiNPs/AlOH catalyst. NiNPs/AlOH catalyst was prepared by a simple and benign environmentally procedure using less amount of sodium hydroxide. ICP-AES and XRD analyses confirmed that the NiNPs/AlOH catalysts comprised a large amount of remained aluminium hydroxide (i.e. bayerite and gibbsite. The presence of aluminium hydroxide caused a high dispersion Ni metal species. The average Ni crystallite sizes that derived from the Scherrer`s equation for former R-Ni and NiNPs/AlOH were 8.6 nm and 4.1 nm, respectively. The catalyst exhibited high activity and selectivity both hydrogenolysis of disaccharides (sucrose and cellobiose and monosaccharides (glucose, fructose, and xylose at 403 K for 24 h. The NiNPs/AlOH catalyst was found to be reusable for at least five consecutive runs without any significant loss of activity and selectivity. © 2013 BCREC UNDIP. All rights reservedReceived: 21st December 2012; Revised: 7th February 2013; Accepted: 10th February 2013[How to Cite: Rodiansono, R., Shimazu, S. (2013. Effective Production of Sorbitol and Mannitol from Sug-ars Catalyzed by Ni Nanoparticles Supported on Aluminium Hydroxide. Bulletin of Chemical Reaction Engineering & Catalysis, 8 (1: 40-46. (doi:10.9767/bcrec.8.1.4290.40-46][Permalink/DOI:] | View in  |

  20. Difference analysis of the enzymatic hydrolysis performance of acid-catalyzed steam-exploded corn stover before and after washing with water. (United States)

    Zhu, Junjun; Shi, Linli; Zhang, Lingling; Xu, Yong; Yong, Qiang; Ouyang, Jia; Yu, Shiyuan


    The difference in the enzymatic hydrolysis yield of acid-catalyzed steam-exploded corn stover (ASC) before and after washing with water reached approximately 15 % under the same conditions. The reasons for the difference in the yield between ASC and washed ASC (wASC) were determined through the analysis of the composition of ASC prehydrolyzate and sugar concentration of enzymatic hydrolyzate. Salts produced by neutralization (CaSO4, Na2SO4, K2SO4, and (NH4)2SO4), sugars (polysaccharides, oligosaccharides, and monosaccharides), sugar-degradation products (weak acids and furans), and lignin-degradation products (ethyl acetate extracts and nine main lignin-degradation products) were back-added to wASC. Results showed that these products, except furans, exerted negative effect on enzymatic hydrolysis. According to the characteristics of acid-catalyzed steam explosion pretreatment, the five sugar-degradation products' mixture and salts [Na2SO4, (NH4)2SO4] showed minimal negative inhibition effect on enzymatic hydrolysis. By contrast, furans demonstrated a promotion effect. Moreover, soluble sugars, such as 13 g/L xylose (decreased by 6.38 %), 5 g/L cellobiose (5.36 %), 10 g/L glucose (3.67 %), as well as lignin-degradation products, and ethyl acetate extracts (4.87 %), exhibited evident inhibition effect on enzymatic hydrolysis. Therefore, removal of soluble sugars and lignin-degradation products could effectively promote the enzymatic hydrolysis performance.

  1. Occurrence of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus in the clam Ruditapes philippinarum (Adams & Reeve, 1850 from Emilia Romagna and Sardinia, Italy

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    Pier Luca Passalacqua


    Full Text Available Marine vibrios, Vibrio parahaemolyticus, V. vulnificus and V. cholerae are responsible of the majority of food-borne human infections by consumption of bivalve shellfish. The aim of the present study was to ascertain the occurrence of these bacteria, and their potential pathogenicity, in the Manila clam R. philippinarum from Emilia Romagna (ER and Sardinia (SR regions, Italy. Isolation was performed on CHROMagarTM vibrio with subculture on (thiosulfate-citrate-bile salts-sucrose Agar and m-modified-cellobiose-polymyxin bcolistin (-CPC Agar. Suspected strains were purified, biochemically characterized and genotyped by simplex polymerase chain reaction (PCR for the specie-specific and pathogenic gene markers: V. parahaemolyticus (toxRP, tdh and trh; V. vulnificus (vvhA, hsp, vcgC, vcgE, CPS operon allele 1, CPS operon allele 2, 16s-rRNA operon allele A, 16s-rRNA operon allele B; V. cholerae (toxRC, hlya, tcpI, tcpA, ctxA, ctxB, stn/sto. Moreover a multiplex PCR was applied to the SR bivalve shellfish, for the simultaneous detection of the three targets directly on homogenate samples, targeting the species-specific gene for V. cholerae (toxRC, V. parahaemolyticus (toxRP and V. vulnificus (vvhA. As a result of phenotyping and genotyping of isolates, bivalve shellfish from ER resulted positive for V. parahaemolyticus (27.8% and V. vulnificus (10.1%, but negative for V. cholerae. Shellfish from SR resulted positive for V. parahaemolyticus (30.3%, V. vulnificus (6.1% and V. cholerae (3%. No significant differences emerged between the two areas (P>0.05.

  2. Ethanol and xylitol production from glucose and xylose at high temperature by Kluyveromyces sp. IIPE453. (United States)

    Kumar, Sachin; Singh, Surendra P; Mishra, Indra M; Adhikari, Dilip K


    A yeast strain Kluyveromyces sp. IIPE453 (MTCC 5314), isolated from soil samples collected from dumping sites of crushed sugarcane bagasse in Sugar Mill, showed growth and fermentation efficiency at high temperatures ranging from 45 degrees C to 50 degrees C. The yeast strain was able to use a wide range of substrates, such as glucose, xylose, mannose, galactose, arabinose, sucrose, and cellobiose, either for growth or fermentation to ethanol. The strain also showed xylitol production from xylose. In batch fermentation, the strain showed maximum ethanol concentration of 82 +/- 0.5 g l(-1) (10.4% v/v) on initial glucose concentration of 200 g l(-1), and ethanol concentration of 1.75 +/- 0.05 g l(-1) as well as xylitol concentration of 11.5 +/- 0.4 g l(-1) on initial xylose concentration of 20 g l(-1) at 50 degrees C. The strain was capable of simultaneously using glucose and xylose in a mixture of glucose concentration of 75 g l(-1) and xylose concentration of 25 g l(-1), achieving maximum ethanol concentration of 38 +/- 0.5 g l(-1) and xylitol concentration of 14.5 +/- 0.2 g l(-1) in batch fermentation. High stability of the strain was observed in a continuous fermentation by feeding the mixture of glucose concentration of 75 g l(-1) and xylose concentration of 25 g l(-1) by recycling the cells, achieving maximum ethanol concentration of 30.8 +/- 6.2 g l(-1) and xylitol concentration of 7.35 +/- 3.3 g l(-1) with ethanol productivity of 3.1 +/- 0.6 g l(-1) h(-1) and xylitol productivity of 0.75 +/- 0.35 g l(-1) h(-1), respectively.

  3. Extraction, partial characterization, and storage stability of β-glucosidase from propolis. (United States)

    Zhang, Cui-Ping; Zheng, Huo-Qing; Hu, Fu-Liang


    Extraction and assay conditions for β-glucosidase from propolis were optimized. Highest enzyme activity was obtained in a citric acid-disodium hydrogen phosphate buffer at pH 6.0 with 2.5% insoluble polyvinylpyrrolidone at incubation temperature of 57 °C. β-Glucosidase activities were found in all freshly harvested propolis while β-glucosidase activities were scarcely present in the randomly bought propolis. Propolis was stored at -20 °C and 4 °C for 3 mo with almost no loss of β-glucosidase activity, but at room temperature the activity decreased exponentially with the increase of storage time. These results indicated that the activity of β-glucosidase could be a candidate for propolis-freshness index. β-Glucosidase from propolis was capable of hydrolyzing p-nitrophenyl-β-D-glucoside and p-nitrophenyl-β-D-galactoside, but lacked activity toward p-nitrophenyl-β-D-glucuronide, p-nitrophenyl-β-D-cellobioside, amygdalin, cellobiose, and gentiobiose. These results were consistent with the hypothesis that flavonoid glucosides were hydrolyzed by β-glucosidase during propolis collection and processing and provided a possible explanation for why some flavonoid biosides (that is, rutin and isorhamnetin-3-O-rutinoside) exist in propolis. Practical Application: β-Glucosidase activity was detected and partial characterization of the enzyme was determined in propolis. The enzyme activity decreased exponentially with the increase of storage time at room temperature, which suggested that the activity of β-glucosidase could be regarded as a freshness index of propolis. The research will be useful for studying the chemical constituents of propolis.

  4. Genome-scale metabolic analysis of Clostridium thermocellum for bioethanol production

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    Brooks J Paul


    Full Text Available Abstract Background Microorganisms possess diverse metabolic capabilities that can potentially be leveraged for efficient production of biofuels. Clostridium thermocellum (ATCC 27405 is a thermophilic anaerobe that is both cellulolytic and ethanologenic, meaning that it can directly use the plant sugar, cellulose, and biochemically convert it to ethanol. A major challenge in using microorganisms for chemical production is the need to modify the organism to increase production efficiency. The process of properly engineering an organism is typically arduous. Results Here we present a genome-scale model of C. thermocellum metabolism, iSR432, for the purpose of establishing a computational tool to study the metabolic network of C. thermocellum and facilitate efforts to engineer C. thermocellum for biofuel production. The model consists of 577 reactions involving 525 intracellular metabolites, 432 genes, and a proteomic-based representation of a cellulosome. The process of constructing this metabolic model led to suggested annotation refinements for 27 genes and identification of areas of metabolism requiring further study. The accuracy of the iSR432 model was tested using experimental growth and by-product secretion data for growth on cellobiose and fructose. Analysis using this model captures the relationship between the reduction-oxidation state of the cell and ethanol secretion and allowed for prediction of gene deletions and environmental conditions that would increase ethanol production. Conclusions By incorporating genomic sequence data, network topology, and experimental measurements of enzyme activities and metabolite fluxes, we have generated a model that is reasonably accurate at predicting the cellular phenotype of C. thermocellum and establish a strong foundation for rational strain design. In addition, we are able to draw some important conclusions regarding the underlying metabolic mechanisms for observed behaviors of C. thermocellum

  5. ToMI-FBA: A genome-scale metabolic flux based algorithm to select optimum hosts and media formulations for expressing pathways of interest

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    Hadi Nazem-Bokaee


    Full Text Available The Total Membrane Influx constrained Flux Balance Analysis (ToMI-FBA algorithm was developed in this research as a new tool to help researchers decide which microbial host and medium formulation are optimal for expressing a new metabolic pathway. ToMI-FBA relies on genome-scale metabolic flux modeling and a novel in silico cell membrane influx constraint that specifies the flux of atoms (not molecules into the cell through all possible membrane transporters. The ToMI constraint is constructed through the addition of an extra row and column to the stoichiometric matrix of a genome-scale metabolic flux model. In this research, the mathematical formulation of the ToMI constraint is given along with four case studies that demonstrate its usefulness. In Case Study 1, ToMI-FBA returned an optimal culture medium formulation for the production of isobutanol from Bacillus subtilis. Significant levels of L-valine were recommended to optimize production, and this result has been observed experimentally. In Case Study 2, it is demonstrated how the carbon to nitrogen uptake ratio can be specified as an additional ToMI-FBA constraint. This was investigated for maximizing medium chain length polyhydroxyalkanoates (mcl-PHA production from Pseudomonas putida KT2440. In Case Study 3, ToMI-FBA revealed a strategy of adding cellobiose as a means to increase ethanol selectivity during the stationary growth phase of Clostridium acetobutylicum ATCC 824. This strategy was also validated experimentally. Finally, in Case Study 4, B. subtilis was identified as a superior host to Escherichia coli, Saccharomyces cerevisiae, and Synechocystis PCC6803 for the production of artemisinate.

  6. Crystal structure of the heme-IsdC complex, the central conduit of the Isd iron/heme uptake system in Staphylococcus aureus. (United States)

    Sharp, Katherine H; Schneider, Sabine; Cockayne, Alan; Paoli, Max


    Pathogens such as Staphylococcus aureus require iron to survive and have evolved specialized proteins to steal heme from their host. IsdC is the central conduit of the Isd (iron-regulated surface determinant) multicomponent heme uptake machinery; staphylococcal cell-surface proteins such as IsdA, IsdB, and IsdH are thought to funnel their molecular cargo to IsdC, which then mediates the transfer of the iron-containing nutrient to the membrane translocation system IsdDEF. The structure of the heme-IsdC complex reveals a novel heme site within an immunoglobulin-like domain and sheds light on its binding mechanism. The folding topology is reminiscent of the architecture of cytochrome f, cellobiose dehydrogenase, and ethylbenzene dehydrogenase; in these three proteins, the heme is bound in an equivalent position, but interestingly, IsdC features a distinct binding pocket with the ligand located next to the hydrophobic core of the beta-sandwich. The iron is coordinated with a tyrosine surrounded by several non-polar side chains that cluster into a tightly packed proximal side. On the other hand, the distal side is relatively exposed with a short helical peptide segment that acts as a lip clasping onto almost half of the porphyrin plane. This structural feature is argued to play a role in the mechanism of binding and release by switching to an open conformation and thus loosening the interactions holding the heme. The structure of the heme-IsdC complex provides a template for the understanding of other proteins, such as IsdA, IsdB, and IsdH, that contain the same heme-binding module as IsdC, known as the NEAT (near transporter) domain.

  7. Kallotenue papyrolyticum gen. nov., sp. nov., a cellulolytic and filamentous thermophile that represents a novel lineage (Kallotenuales ord. nov., Kallotenuaceae fam. nov.) within the class Chloroflexia

    Energy Technology Data Exchange (ETDEWEB)

    Cole, Jesse; Gieler, Brandon; Heisler, Devon; Palisoc, Maryknoll; Williams, Amanda; Dohnalkova, Alice; Ming, Hong; Yu, Tian T.; Dodsworth, Jeremy A.; Li, Wen J.; Hedlund, Brian P.


    Several closely-related, thermophilic, and cellulolytic bacterial strains, designated JKG1T, JKG2, JKG3, JKG4, and JKG5, were isolated from a cellulolytic enrichment (corn stover) incubated in the water column of Great Boiling Spring, NV. Strain JKG1T had cells of a diameter of 0.7 - 0.9 μm and length of ~2.0 μm that formed non-branched multicellular filaments reaching >300 μm. Spores were not formed and dense liquid cultures were red. The temperature range for growth was 45-65 °C, with an optimum of 55 °C. The pH range for growth was 5.6-9.0, with an optimum of 7.5. JKG1T grew as an aerobic heterotroph, utilizing glucose, sucrose, xylose, arabinose, cellobiose, carboxymethylcellulose, filter paper, microcrystalline cellulose, xylan, starch, casamino acids, tryptone, peptone, yeast extract, acetate, citrate, lactate, pyruvate, and glycerol as sole carbon sources, and was not observed to photosynthesize. The cells stained Gram-negative. Phylogenetic analysis using 16S rRNA gene sequences placed the new isolates in the class Chloroflexia, but distant from other cultivated members, with the highest sequence identity of 82.5% to Roseiflexus castenholzii. The major quinone was menaquinone-9; no ubiquinones were detected. The major cellular fatty acids (>5%) were C18:0, anteiso-C17:0, iso-C18:0, and iso-C17:0. C16:0, iso-C16:0, and C17:0. The peptidoglycan amino acids were alanine, ornithine, glutamic acid, serine, and asparagine. Whole-cell sugars included mannose, rhamnose, glucose, galactose, ribose, arabinose, and xylose. Morphological, phylogenetic, and chemotaxonomic results suggest that JKG1T is representative of a new lineage within the class Chloroflexia, which we propose to designate Kallotenue papyrolyticum gen. nov., sp. nov., Kallotenuaceae fam. nov., Kallotenuales ord. nov.

  8. Doprinos istraživanju slobodnih i glikozidno vezanih isparljivih spojeva od 2001. do 2006.

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    Jerković, I.


    Full Text Available This review paper presents a contribution to the research of free and glycosidically bound volatiles in the 2001-2006 period obtained by work in the Department of Organic Chemistry, Faculty of Chemistry and Technology, University of Split, Croatia. Many terpene structures, phenylpropane derivatives (Fig. 6, as well as aliphatic hydrocarbons, alcohols, carbonyls, fatty acids and others were identified (Fig. 5. The chemotypes of Origanum vulgare ssp. hirtum, Artemisia absinthium and Myrtus communis were determined and some uninvestigated or insufficiently investigated plants (Micromeria juliana, Sequaoiadendron giganteum, Populus nigra, Prunus mahaleb, Ailanthus altissima and others were analyzed. A newly developed method of co-distillation enabled isolation of unstable volatile compounds without formation of artefacts (Fig. 1.The volatile aglycones liberated by enzymatic hydrolysis of the corresponding glycosides were aliphatic and phenylpropanoic derivatives, as well as monoterpenes, sesquiterpenes and others (Fig. 7. Only partial similarity was observed in the composition of volatile aglycones and corresponding free volatiles of the same plant.Furthermore, glucosides of ubiquitous monoterpene and aliphatic alcohols and phenols were prepared by Koenigs-Knorr glucosylation (Fig. 8, by enzymatic condensation and transglucosidation from cellobiose (Fig. 11 and by direct glucosylation with FeCl3 (Fig. 13. The products were characterized by GC-MS analysis of prepared tetraacetyl glucosides. Fragment ion characteristics of the aglucone moiety were present in all mass spectra, along with the fragments obtained from acetylated glucose (Fig. 12 and Fig. 14. Acetylated glucosides were separable on the HP-101 column, Fig. 9 (even diastereomeric tetraacetyl ß-glucosides of enantiomeric alcohols, Fig. 10.

  9. Degradation capability of the coastal environment adjacent to the Itata River in central Chile (36.5° S

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    P. Ampuero


    Full Text Available The response of the coastal ocean influenced by both river discharges and inputs of photosynthetically derived organic carbon product of upwelling, was evaluated by estimating rates of microbial hydrolysis of macromolecules with the goal of estimating the potential degradation capability of the coastal ecosystem off central Chile. Extracellular enzymatic activity (EEA in seawater was dominated by aminopeptidase activity on substrate L-leucine-4-methyl-7-coumarinylamide (MCA-leu (1.2 to 182 nmol l−1 h−1 followed by 4-methylumbelliferyl-ß-D-glucoside (MUF-glu (0.08–61 nmol l−1 h−1 and 4-methylumbelliferyl-ß-D-cellobiose (MUF-cel (0.15–7 nmol l−1 h−1, with the highest rates measured during spring-summer. In riverine waters, extracellular enzymatic hydrolysis remained within the range of 45 to 131 nmol l−1 h−1 for MCA-leu and ca. 20 nmol l−1 h−1 for glucosidic substrates, year-round. Contrary to the EEA observed for the marine water column, surface sediment extracellular enzymatic hydrolysis of MCA-leu (0.04 to 6.13 nmol g−1 dw h−1 was in the same order of magnitude as the rates observed for MUF-cel (0.004 to 5.1 nmol g−1 dw h−1 and MUF-glu (0.007 to 10.5 nmol g−1 dw h−1. Moreover, hydrolysis in sediments was characterized by higher rates during winter compared with spring-summer in the coastal and estuarine zone. The five years of data allowed us to evaluate the potential capability of microbial processing of organic carbon in the coastal area adjacent to the Itata river discharge where the increase in primary production in the productive seasons is accompanied by the increase in hydrolysis of macromolecules.

  10. Occurrence of Vibrio Parahaemolyticus, Vibrio Cholerae and Vibrio Vulnificus in the Clam Ruditapes Philippinarum (Adams & Reeve, 1850) from Emilia Romagna and Sardinia, Italy. (United States)

    Passalacqua, Pier Luca; Zavatta, Emanuele; Bignami, Giorgia; Serraino, Andrea; Serratore, Patrizia


    Marine vibrios, Vibrio parahaemolyticus, V. vulnificus and V. cholerae are responsible of the majority of food-borne human infections by consumption of bivalve shellfish. The aim of the present study was to ascertain the occurrence of these bacteria, and their potential pathogenicity, in the Manila clam R. philippinarum from Emilia Romagna (ER) and Sardinia (SR) regions, Italy. Isolation was performed on CHROMagar(TM) vibrio with subculture on (thiosulfate-citrate-bile salts-sucrose) Agar and m-modified-cellobiose-polymyxin b-colistin (-CPC) Agar. Suspected strains were purified, biochemically characterized and genotyped by simplex polymerase chain reaction (PCR) for the specie-specific and pathogenic gene markers: V. parahaemolyticus (toxRP, tdh and trh); V. vulnificus (vvhA, hsp, vcgC, vcgE, CPS operon allele 1, CPS operon allele 2, 16s-rRNA operon allele A, 16s-rRNA operon allele B; V. cholerae (toxRC, hlya, tcpI, tcpA, ctxA, ctxB, stn/sto). Moreover a multiplex PCR was applied to the SR bivalve shellfish, for the simultaneous detection of the three targets directly on homogenate samples, targeting the species-specific gene for V. cholerae (toxRC), V. parahaemolyticus (toxRP) and V. vulnificus (vvhA). As a result of phenotyping and genotyping of isolates, bivalve shellfish from ER resulted positive for V. parahaemolyticus (27.8%) and V. vulnificus (10.1%), but negative for V. cholerae. Shellfish from SR resulted positive for V. parahaemolyticus (30.3%), V. vulnificus (6.1%) and V. cholerae (3%). No significant differences emerged between the two areas (P>0.05).

  11. Occurrence of Vibrio Parahaemolyticus, Vibrio Cholerae and Vibrio Vulnificus in the Clam Ruditapes Philippinarum (Adams & Reeve, 1850) from Emilia Romagna and Sardinia, Italy (United States)

    Passalacqua, Pier Luca; Zavatta, Emanuele; Bignami, Giorgia; Serraino, Andrea


    Marine vibrios, Vibrio parahaemolyticus, V. vulnificus and V. cholerae are responsible of the majority of food-borne human infections by consumption of bivalve shellfish. The aim of the present study was to ascertain the occurrence of these bacteria, and their potential pathogenicity, in the Manila clam R. philippinarum from Emilia Romagna (ER) and Sardinia (SR) regions, Italy. Isolation was performed on CHROMagarTM vibrio with subculture on (thiosulfate-citrate-bile salts-sucrose) Agar and m-modified-cellobiose-polymyxin b-colistin (-CPC) Agar. Suspected strains were purified, biochemically characterized and genotyped by simplex polymerase chain reaction (PCR) for the specie-specific and pathogenic gene markers: V. parahaemolyticus (toxRP, tdh and trh); V. vulnificus (vvhA, hsp, vcgC, vcgE, CPS operon allele 1, CPS operon allele 2, 16s-rRNA operon allele A, 16s-rRNA operon allele B; V. cholerae (toxRC, hlya, tcpI, tcpA, ctxA, ctxB, stn/sto). Moreover a multiplex PCR was applied to the SR bivalve shellfish, for the simultaneous detection of the three targets directly on homogenate samples, targeting the species-specific gene for V. cholerae (toxRC), V. parahaemolyticus (toxRP) and V. vulnificus (vvhA). As a result of phenotyping and genotyping of isolates, bivalve shellfish from ER resulted positive for V. parahaemolyticus (27.8%) and V. vulnificus (10.1%), but negative for V. cholerae. Shellfish from SR resulted positive for V. parahaemolyticus (30.3%), V. vulnificus (6.1%) and V. cholerae (3%). No significant differences emerged between the two areas (P>0.05). PMID:27800436

  12. Process development studies for the production of. beta. -glucosidase from Aspergillus phoenicis

    Energy Technology Data Exchange (ETDEWEB)

    Howell, M.J.; Wilke, C.R.


    This work is concerned with the production of ..beta..-glucosidase from Aspergillus phoenicis for use in the enzymatic hydrolysis of cellulose. Kinetic growth data indicate that two distinct periods of growth exist. The observed growth kinetics result from a biochemical differentiation of the filament which is independent of the substrate concentration. The optimum temperature for cell mass and ..beta..-glucosidase production was found to be 30/sup 0/C. The optimum pH for ..beta..-glucosidase production is 5 and the highest specific cell growth rate was observed when the growth medium was controlled at pH 4.5. The most economical substrate was 0.75 g/l of Solka Floc, a spruce wood pulp, plus 0.25 g/l of Trichoderma viride cellulase, required because A. phoenicis does not produce all the enzymes required to solubilize cellulose. When freeze-dried A. phoenicis enzyme was added to the hydrolysis of acid treated corn stover by Tricoderma viride cellulase, the total sugar yield was increased by 4 g/l of hydrolysate over the yield of 20 g/l obtained without ..beta..-glucosidase addition. In addition, the cellobiose, which accounted for about 10% of the sugar concentration, was converted to glucose, a more widely useable product. Preliminary designs of several processes for the production of ..beta..-glucosidase were made. The most economical processes were continuous production schemes. Ball milling was the most cost effective method, but the use of an elevated temperature stage was economical enough to warrant further study. The cost of production of ..beta..-glucosidase was found to be too high to justify its addition to a process for enzymatically hydrolyzing cellulose at this time.

  13. Catalytic properties and mode of action of endo-(1-->3)-beta-D-glucanase and beta-D-glucosidase from the marine mollusk Littorina kurila. (United States)

    Pesentseva, Maria S; Kusaykin, Mikhail I; Anastyuk, Stanislav D; Sova, Victoria V; Zvyagintseva, Tatyana N


    A complex of the enzymes from the liver of the marine mollusk Littorina kurila that hydrolyzes laminaran was investigated. Two (1-->3)-beta-d-glucanases (G-I and G-II) were isolated. The molecular mass of G-I as estimated by gel-permeation chromatography and SDS-PAGE analysis was 32 and 40kDa, respectively. The G-II molecular mass according to SDS-PAGE analysis was about 200kDa. The pH optimum for both G-I and G-II was pH 5.4. The G-I had narrow substrate specificity and hydrolyzed only the (1-->3)-beta-d-glucosidic bonds in the mixed (1-->3),(1-->6)- and (1-->3),(1-->4)-beta-d-glucans down to glucose and glucooligosaccharides. This enzyme acted with retention of the anomeric configuration and catalyzed a transglycosylation reaction. G-I was classified as the glucan endo-(1-->3)-beta-d-glucosidase (EC G-II exhibited both exo-glucanase and beta-d-glucoside activities. This enzyme released from the laminaran glucose as a single product, but retained the anomeric center configuration and possessed transglycosylation activity. The hydrolysis rate of glucooligosaccharides by G-I decreased with an increase of the substrate's degree of polymerization. In addition to (1-->3)-beta-d-glucanase activity, the enzyme had the ability to hydrolyze p-nitrophenyl beta-d-glucoside and beta-d-glucobioses: laminaribiose, gentiobiose, and cellobiose, with the rate ratio of 50:12:1. G-II may correspond to beta-d-glucoside glucohydrolase (EC

  14. Improvement of xylanase production by Penicillium canescens 10-10c in solid-state fermentation

    Directory of Open Access Journals (Sweden)

    Thonart P.


    Full Text Available Among hemicellulases, xylanases are catalysts of considerable interest so as fundamental than applied point of view. However,it is paradoxical to note that the high cost of their production limits their use on a large scale. The use of purified xylan as culturesubstrate increases the production cost of the enzyme. Consequently, for commercial applications, it is advisable to developprocesses starting from inexpensive substrates. The purpose of this study is to optimise xylanases production in solid-statefermentation based on agricultural residues. The strain is Penicillium canescens 10-10c, selected in our laboratory for his abilityto produce xylanase activity free of cellulase. Assays concern optimization of different culture parameters in order to developin the future a solid-state fermentation reactor with soya oil cake. These parameters are: medium composition, temperatureincubation, induction and repression mechanisms. Soya oil cake in pellets (size > 10 mm gave a higher enzymatic activity.Great volume of culture medium reduced the enzymatic production. The presence of lactose, saccharose or starch of corn hasa positive effect on the production of xylanase while the presence of xylose, mannose, galactose, arabinose, cellobiose andpectin or methylcellulose reduces the production of xylanase. The sources of phosphorus (di-potassic and di-sodic enhancexylanase production. The enzymatic production obtained in Erlenmeyer flasks (250 ml after 7 days incubation at 30°C isabout 14 000 U.g-1 of carbon source. The nature of inoculum affects the enzymatic productivity. Indeed, better productivitywas obtained with inoculation by solid preculture (956 U.g-1 per day than liquid preculture (473 U.g-1 per day and sporessuspension (383 U.g-1 per day. These observed enzymatic activity levels are higher than those related in the literature, whichshows all the potentialities of this strain and this technique for the production of xylanase and allow to develop

  15. Proteinivorax tanatarense gen. nov., sp. nov., an anaerobic, haloalkaliphilic, proteolytic bacterium isolated from a decaying algal bloom, and proposal of Proteinivoraceae fam. nov. (United States)

    Kevbrin, Vadim; Boltyanskaya, Yulia; Zhilina, Tatjana; Kolganova, Tatjana; Lavrentjeva, Elena; Kuznetsov, Boris


    Two strains of a novel anaerobic, protein- and nucleoside-utilizing bacterium, Z-910(T) and Z-810, were isolated. The strains were spore-forming, mainly nonmotile rods, exhibiting positive Gram reaction with Gram-positive cell wall structure. The strains were mesophilic and haloalkaliphilic. Cultures used proteins and proteinaceous substrates as carbon, nitrogen, and energy sources. Both strains used also ribonucleosides, cellobiose, pyruvate, and glycerol. Ribose and nucleobases did not support growth. The fermentation products from all utilized substrates were identical but varied in content and included straight and branched acids, as well as hydrogen and ammonia. When grown on tryptone, strain Z-910(T) was able to reduce fumarate, dimethyl sulfoxide, thiosulfate, and elemental sulfur. Neither nitrate nor sulfate was reduced. The DNA G + C content of strain Z-910(T) was 32.2 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence similarity revealed that strains Z-910(T) and Z-810 represented a new branch within the order Clostridiales, with 90.2 % similarity to the nearest genus with a validly published name Anaerobranca gottschalkii DSM 13577(T). According to their physiological, chemotaxonomic, and phylogenetic properties, strains Z-910(T) and Z-810 represented a new genus and novel species, for which the name Proteinivorax tanatarense gen. nov., sp. nov. was proposed. Phylogenetic analysis showed that the genera Proteinivorax gen. nov. and Anaerobranca formed a separate cluster within the order Clostridiales. The family Proteinivoraceae fam. nov. comprising the genera Proteinivorax gen. nov. and Anaerobranca was therefore proposed within the order Clostridiales of the phylum Firmicutes with Proteinivorax as a type genus of the new family.

  16. CO 2 -fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Wei; Lin, Paul P.; Magnusson, Lauren; Warner, Lisa; Liao, James C.; Maness, Pin-Ching; Chou, Katherine J.


    Clostridium thermocellum can ferment cellulosic biomass to formate and other end products, including CO2. This organism lacks formate dehydrogenase (Fdh), which catalyzes the reduction of CO2 to formate. However, feeding the bacterium 13C-bicarbonate and cellobiose followed by NMR analysis showed the production of 13C-formate in C. thermocellum culture, indicating the presence of an uncharacterized pathway capable of converting CO2 to formate. Combining genomic and experimental data, we demonstrated that the conversion of CO2 to formate serves as a CO2 entry point into the reductive one-carbon (C1) metabolism, and internalizes CO2 via two biochemical reactions: the reversed pyruvate:ferredoxin oxidoreductase (rPFOR), which incorporates CO2 using acetyl-CoA as a substrate and generates pyruvate, and pyruvate-formate lyase (PFL) converting pyruvate to formate and acetyl-CoA. We analyzed the labeling patterns of proteinogenic amino acids in individual deletions of all five putative PFOR mutants and in a PFL deletion mutant. We identified two enzymes acting as rPFOR, confirmed the dual activities of rPFOR and PFL crucial for CO2 uptake, and provided physical evidence of a distinct in vivo 'rPFOR-PFL shunt' to reduce CO2 to formate while circumventing the lack of Fdh. Such a pathway precedes CO2 fixation via the reductive C1 metabolic pathway in C. thermocellum. These findings demonstrated the metabolic versatility of C. thermocellum, which is thought of as primarily a cellulosic heterotroph but is shown here to be endowed with the ability to fix CO2 as well.

  17. Effect of water on hydrolytic cleavage of non-terminal α-glycosidic bonds in cyclodextrins to generate monosaccharides and their derivatives in a dimethyl sulfoxide-water mixture. (United States)

    Kimura, Hiroshi; Hirayama, Masaki; Yoshida, Ken; Uosaki, Yasuhiro; Nakahara, Masaru


    Hydrolytic cleavage of the non-terminal α-1,4-glycosidic bonds in α-, β-, and γ-cyclodextrins and the anomeric-terminal one in d-maltose was investigated to examine how the cleavage rate for α-, β-, and γ-cyclodextrins is slower than that for d-maltose. Effects of water and temperature were studied by applying in situ (13)C NMR spectroscopy and using a dimethyl sulfoxide (DMSO)-water mixture over a wide range of water mole fraction, xw = 0.004-1, at temperatures of 120-180 °C. The cleavage rate constant for the non-anomeric glycosidic bond was smaller by a factor of 6-10 than that of the anomeric-terminal one. The glycosidic-bond cleavage is significantly accelerated through the keto-enol tautomerization of the anomeric-terminal d-glucose unit into the d-fructose one. The smaller the size of the cyclodextrin, the easier the bond cleavage due to the ring strain. The remarkable enhancement in the cleavage rate with decreasing water content was observed for the cyclodextrins and d-maltose as well as d-cellobiose. This shows the important effect of the solitary water whose hydrogen bonding to other water molecules is prohibited by the presence of the organic dipolar aprotic solvent, DMSO, and which has more naked partial charges and higher reactivity. A high 5-hydroxymethyl-2-furaldehyde (5-HMF) yield of 64% was attained in a non-catalytic conversion by tuning the water content to xw = 0.30, at which the undesired polymerization by-paths can be most effectively suppressed. This study provides a step toward designing a new optimal, earth-benign generation process of 5-HMF starting from biomass.

  18. Mining the Sinorhizobium meliloti transportome to develop FRET biosensors for sugars, dicarboxylates and cyclic polyols.

    Directory of Open Access Journals (Sweden)

    Alexandre Bourdès

    Full Text Available Förster resonance energy transfer (FRET biosensors are powerful tools to detect biologically important ligands in real time. Currently FRET bisosensors are available for twenty-two compounds distributed in eight classes of chemicals (two pentoses, two hexoses, two disaccharides, four amino acids, one nucleobase, two nucleotides, six ions and three phytoestrogens. To expand the number of available FRET biosensors we used the induction profile of the Sinorhizobium meliloti transportome to systematically screen for new FRET biosensors.Two new vectors were developed for cloning genes for solute-binding proteins (SBPs between those encoding FRET partner fluorescent proteins. In addition to a vector with the widely used cyan and yellow fluorescent protein FRET partners, we developed a vector using orange (mOrange2 and red fluorescent protein (mKate2 FRET partners. From the sixty-nine SBPs tested, seven gave a detectable FRET signal change on binding substrate, resulting in biosensors for D-quinic acid, myo-inositol, L-rhamnose, L-fucose, β-diglucosides (cellobiose and gentiobiose, D-galactose and C4-dicarboxylates (malate, succinate, oxaloacetate and fumarate. To our knowledge, we describe the first two FRET biosensor constructs based on SBPs from Tripartite ATP-independent periplasmic (TRAP transport systems.FRET based on orange (mOrange2 and red fluorescent protein (mKate2 partners allows the use of longer wavelength light, enabling deeper penetration of samples at lower energy and increased resolution with reduced back-ground auto-fluorescence. The FRET biosensors described in this paper for four new classes of compounds; (i cyclic polyols, (ii L-deoxy sugars, (iii β-linked disaccharides and (iv C4-dicarboxylates could be developed to study metabolism in vivo.

  19. An improved theoretical approach to the empirical corrections of density functional theory (United States)

    Lii, Jenn-Huei; Hu, Ching-Han


    An empirical correction to density functional theory (DFT) has been developed in this study. The approach, called correlation corrected atomization-dispersion (CCAZD), involves short- and long-range terms. Short-range correction consists of bond ( 1,2-) and angle ( 1,3-) interactions, which remedies the deficiency of DFT in describing the proto-branching stabilization effects. Long-range correction includes a Buckingham potential function aiming to account for the dispersion interactions. The empirical corrections of DFT were parameterized to reproduce reported Δ H f values of the training set containing alkane, alcohol and ether molecules. The Δ H f of the training set molecules predicted by the CCAZD method combined with two different DFT methods, B3LYP and MPWB1K, with a 6-31G* basis set agreed well with the experimental data. For 106 alkane, alcohol and ether compounds, the average absolute deviations (AADs) in Δ H f were 0.45 and 0.51 kcal/mol for B3LYP- and MPWB1K-CCAZD, respectively. Calculations of isomerization energies, rotational barriers and conformational energies further validated the CCAZD approach. The isomerization energies improved significantly with the CCAZD treatment. The AADs for 22 energies of isomerization reactions were decreased from 3.55 and 2.44 to 0.55 and 0.82 kcal/mol for B3LYP and MPWB1K, respectively. This study also provided predictions of MM4, G3, CBS-QB3 and B2PLYP-D for comparison. The final test of the CCAZD approach on the calculation of the cellobiose analog potential surface also showed promising results. This study demonstrated that DFT calculations with CCAZD empirical corrections achieved very good agreement with reported values for various chemical reactions with a small basis set as 6-31G*.

  20. Pectin-rich biomass as feedstock for fuel ethanol production

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, Meredith C.; Doran-Peterson, Joy [Georgia Univ., Athens, GA (United States). Dept. of Microbiology


    The USA has proposed that 30 % of liquid transportation fuel be produced from renewable resources by 2030 (Perlack and Stokes 2011). It will be impossible to reach this goal using corn kernel-based ethanol alone. Pectin-rich biomass, an under-utilized waste product of the sugar and juice industry, can augment US ethanol supplies by capitalizing on this already established feedstock. Currently, pectin-rich biomass is sold (at low value) as animal feed. This review focuses on the three most studied types of pectin-rich biomass: sugar beet pulp, citrus waste and apple pomace. Fermentations of these materials have been conducted with a variety of ethanologens, including yeasts and bacteria. Escherichia coli can ferment a wide range of sugars including galacturonic acid, the primary component of pectin. However, the mixed acid metabolism of E. coli can produce unwanted side products. Saccharomyces cerevisiae cannot naturally ferment galacturonic acid nor pentose sugars but has a homoethanol pathway. Erwinia chrysanthemi is capable of degrading many of the cell wall components of pectin-rich materials, including pectin. Klebsiella oxytoca can metabolize a diverse array of sugars including cellobiose, one degradation product of cellulose. However, both E. chrysanthemi and K. oxytoca produce side products during fermentation, similar to E. coli. Using pectin-rich residues from industrial processes is beneficial because the material is already collected and partially pretreated to facilitate enzymatic deconstruction of the plant cell walls. Using biomass already produced for other purposes is an attractive practice because fewer greenhouse gases (GHG) will be anticipated from land-use changes. (orig.)

  1. Transcriptional regulation of the carbohydrate utilization network in Thermotoga maritima

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    Dmitry A Rodionov


    Full Text Available Hyperthermophilic bacteria from the Thermotogales lineage can produce hydrogen by fermenting a wide range of carbohydrates. Previous experimental studies identified a large fraction of genes committed to carbohydrate degradation and utilization in the model bacterium Thermotoga maritima. Knowledge of these genes enabled comprehensive reconstruction of biochemical pathways comprising the carbohydrate utilization network. However, transcriptional factors (TFs and regulatory mechanisms driving this network remained largely unknown. Here, we used an integrated approach based on comparative analysis of genomic and transcriptomic data for the reconstruction of the carbohydrate utilization regulatory networks in 11 Thermotogales genomes. We identified DNA-binding motifs and regulons for 19 orthologous TFs in the Thermotogales. The inferred regulatory network in T. maritima contains 181 genes encoding TFs, sugar catabolic enzymes and ABC-family transporters. In contrast to many previously described bacteria, a transcriptional regulation strategy of Thermotoga does not employ global regulatory factors. The reconstructed regulatory network in T. maritima was validated by gene expression profiling on a panel of mono- and disaccharides and by in vitro DNA-binding assays. The observed upregulation of genes involved in catabolism of pectin, trehalose, cellobiose, arabinose, rhamnose, xylose, glucose, galactose, and ribose showed a strong correlation with the UxaR, TreR, BglR, CelR, AraR, RhaR, XylR, GluR, GalR, and RbsR regulons. Ultimately, this study elucidated the transcriptional regulatory network and mechanisms controlling expression of carbohydrate utilization genes in T. maritima. In addition to improving the functional annotations of associated transporters and catabolic enzymes, this research provides novel insights into the evolution of regulatory networks in Thermotogales.

  2. Application of lignocellulolytic fungi for bioethanol production from renewable biomass

    Directory of Open Access Journals (Sweden)

    Jović Jelena M.


    Full Text Available Pretreatment is a necessary step in the process of conversion of lignocellulosic biomass to ethanol; by changing the structure of lignocellulose, enhances enzymatic hydrolysis, but, often, it consumes large amounts of energy and/or needs an application of expensive and toxic chemicals, which makes the process economically and ecologically unfavourable. Application of lignocellulolytic fungi (from the class Ascomycetes, Basidiomycetes and Deuteromycetes is an attractive method for pre-treatment, environmentally friendly and does not require the investment of energy. Fungi produce a wide range of enzymes and chemicals, which, combined in a variety of ways, together successfully degrade lignocellulose, as well as aromatic polymers that share features with lignin. On the basis of material utilization and features of a rotten wood, they are divided in three types of wood-decay fungi: white rot, brown rot and soft rot fungi. White rot fungi are the most efficient lignin degraders in nature and, therefore, have a very important role in carbon recycling from lignified wood. This paper describes fungal mechanisms of lignocellulose degradation. They involve oxidative and hydrolytic mechanisms. Lignin peroxidase, manganese peroxidase, laccase, cellobiose dehydrogenase and enzymes able to catalyze formation of hydroxyl radicals (•OH such as glyoxal oxidase, pyranose-2-oxidase and aryl-alcohol oxidase are responsible for oxidative processes, while cellulases and hemicellulases are involved in hydrolytic processes. Throughout the production stages, from pre-treatment to fermentation, the possibility of their application in the technology of bioethanol production is presented. Based on previous research, the advantages and disadvantages of biological pre-treatment are pointed out.

  3. Enzymatic process of rice bran: a stabilized functional food with nutraceuticals and nutrients. (United States)

    S Vallabha, Vishwanath; Indira, T N; Jyothi Lakshmi, A; Radha, C; Tiku, Purnima Kaul


    Rice bran (RB), a byproduct of rice milling industry, is a rich source of nutraceuticals and nutrients. However its utility is limited due to the presence of lipase and lipoxygenase which initiates rancidity on milling. The aim of this investigation is to prevent oxidation of free fatty acids by enzymatic approach for its effective utilization. The enzymatic treatment comprised of alcalase treatment for complete inactivation of lipase along with reduction in lipoxygenase (LOX) activity and endoglucanase for improving the soluble fiber content. The enzyme treated rice bran was drum dried for further use. The nutraceutical molecules like γ-oryzanol, α-tocopherol and polyphenols were retained in the range of 68 to 110 % and the total antioxidant activity was improved. By the action of endoglucanase the complex carbohydrate was converted into glucose (72.28 %), cellobiose (18.36 %) and cellotriose (9.36 %). The prebiotic effect of enzyme treated rice bran was evaluated by the action of lactobacillus which was measured through the release of the short chain free fatty acids (SCFAs) analyzed by HPLC. The SCFAs; acetic acid and propionic acid increased by 1.72 folds and 2.12 folds respectively. B-complex vitamins showed maximum retention with vitamins like B1 (66.3 %), B2 (68.3 %) and B3 (55.0 %) after enzyme treatment. At different humidity levels, storage studies showed no change in LOX activity and also retained ubiquinol-10 in reduced state in enzyme treated RB for a period of 3 months. A stabilized RB has been developed enriched with short chain prebiotics and antioxidant molecules.

  4. Degradation of cellulosic biomass and its subsequent utilization for the production of chemical feedstocks. Progress report, March 1, 1977--May 31, 1977

    Energy Technology Data Exchange (ETDEWEB)

    Wang, D.I.C.; Cooney, C.L.; Demain, A.L.; Gomez, R.F.; Sinskey, A.J.


    The degradation of cellulosic biomass continues to focus on the anaerobic thermophile Clostridium thermocellum. When grown on crystalline cellulose (MN300) in batch culture, there is an initial rapid accumulation of reducing sugars but the sugars are rapidly metabolized in later times during the fermentation. When grown on Solka floc with periodic addition of the substrate, there is a continual accumulation of reducing sugars (xylose, glucose, and cellobiose) as well as ethanol and acetic acid during the entire course of the fermentation. In the presence of surfactant in the growth medium, there is an increased appearance of extracellular cellulases. A chemically defined medium is being developed for growth Cl. thermocellum in order to study the enzyme regulations. Lastly, a trinitrophenyl-carboxylmethyl cellulose substrate for determining cellulose activity appears to be a promising and rapid assay. Progress in the genetic manipulations has been cautious but promising. Preliminary evidence leads to optimistic projection on the presence of plasmids and bacteriophage in Cl. thermocellum. The production of chemical feedstocks continues to focus on acrylic acid, acetone/butanol and acetic acid. Studies with cell free extracts of Clostridium propionicum have shown the production and accumulation of acrylic acid from lactic acid. The use of electron acceptor in cell-free systems has shown effective prevention on the reduction of acrylic acid to propionic acid. Medium development and strain selection using available acetone/butanol producing Cl. acetobutylicum have been initiated. There is every indication that these strains are capable to produce mixed solvents close to the theoretical maximum yield. An accurate and rapid method for quantifying acetic acid was developed. This technique is being used to examine the pertinent parameters on the production of acetic acid by Clostridium thermoaceticum.

  5. Genome sequence of the plant growth promoting endophytic bacterium Enterobacter sp. 638.

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    Safiyh Taghavi


    Full Text Available Enterobacter sp. 638 is an endophytic plant growth promoting gamma-proteobacterium that was isolated from the stem of poplar (Populus trichocarpaxdeltoides cv. H11-11, a potentially important biofuel feed stock plant. The Enterobacter sp. 638 genome sequence reveals the presence of a 4,518,712 bp chromosome and a 157,749 bp plasmid (pENT638-1. Genome annotation and comparative genomics allowed the identification of an extended set of genes specific to the plant niche adaptation of this bacterium. This includes genes that code for putative proteins involved in survival in the rhizosphere (to cope with oxidative stress or uptake of nutrients released by plant roots, root adhesion (pili, adhesion, hemagglutinin, cellulose biosynthesis, colonization/establishment inside the plant (chemiotaxis, flagella, cellobiose phosphorylase, plant protection against fungal and bacterial infections (siderophore production and synthesis of the antimicrobial compounds 4-hydroxybenzoate and 2-phenylethanol, and improved poplar growth and development through the production of the phytohormones indole acetic acid, acetoin, and 2,3-butanediol. Metabolite analysis confirmed by quantitative RT-PCR showed that, the production of acetoin and 2,3-butanediol is induced by the presence of sucrose in the growth medium. Interestingly, both the genetic determinants required for sucrose metabolism and the synthesis of acetoin and 2,3-butanediol are clustered on a genomic island. These findings point to a close interaction between Enterobacter sp. 638 and its poplar host, where the availability of sucrose, a major plant sugar, affects the synthesis of plant growth promoting phytohormones by the endophytic bacterium. The availability of the genome sequence, combined with metabolome and transcriptome analysis, will provide a better understanding of the synergistic interactions between poplar and its growth promoting endophyte Enterobacter sp. 638. This information can be further

  6. Clostridium oryzae sp. nov., from soil of a Japanese rice field. (United States)

    Horino, Haruka; Ito, Miyuki; Tonouchi, Akio


    An obligately anaerobic bacterial strain designated KC3(T) was isolated from a rice straw-degrading culture, for which soil of a Japanese rice field was used as the inoculum. Cells of strain KC3(T) were determined to be non-cellulolytic, Gram-stain-positive, non-motile, ellipsoidal, spore-forming rods, 0.8-1×4-25 µm. Endospores were formed at a terminal position in elongated cells (12-25 µm, mean 15 µm). The temperature range for growth was 20-50 °C, with an optimum at 37 °C. The pH range for growth was 5.0-7.5, with an optimum at pH 6.0 (slightly acidophilic). Strain KC3(T) fermented cellobiose to lactate, butyrate, acetate, formate, hydrogen and carbon dioxide. The major cellular fatty acids (>10 %) were C14 : 0, C16 : 0 and C19 : 0 cyclo 11,12 dimethylacetal. The DNA G+C content of strain KC3(T) was 37.5 mol%. 16S rRNA gene sequence analysis revealed that strain KC3(T) shared low sequence similarity (Clostridium sensu stricto (Clostridium rRNA cluster I). Analyses of the DNA gyrase A and ATP synthase beta subunit sequences supported the affiliation of strain KC3(T) to the genus Clostridium sensu stricto. The evidence presented here indicates that strain KC3(T) represents a novel species of the genus Clostridium, for which the name Clostridium oryzae sp. nov. is proposed. The type strain of Clostridium oryzae is KC3(T) ( = DSM 28571(T) = NBRC 110163(T)).

  7. Clostridium amylolyticum sp. nov., isolated from H2-producing UASB granules. (United States)

    Song, Lei; Dong, Xiuzhu


    A Gram-stain-positive, strictly anaerobic, mesophilic, amylolytic, rod-shaped bacterium, designated strain SW408(T), was isolated from a laboratory-scale H(2)-producing upflow anaerobic sludge blanket reactor. The strain grew at 24-45 degrees C (no growth at or below 22 degrees C or at or above 47 degrees C), with optimum growth at 37 degrees C. The pH range for growth was 4.0-9.0 (no growth at or below pH 3.6 or at or above pH 9.3), with optimum growth at pH 7.0. Starch, cellobiose, glucose, fructose, galactose, lactose, maltose, mannose, ribose and sucrose supported growth. The major end products from glucose fermentation were ethanol, acetate, hydrogen and carbon dioxide. Abundant H(2) was produced from starch fermentation. The DNA G+C content was 33.1 mol% (T(m) method). Phylogenetic analysis based on 16S rRNA gene sequence analysis showed that the bacterium represents a previously unrecognized species within Clostridium rRNA cluster I and is most closely related to the type strain of Clostridium frigidicarnis (94.9% similarity). On the basis of phenotypic, genotypic and phylogenetic characteristics, strain SW408(T) was identified as a representative of a novel species of the genus Clostridium, for which the name Clostridium amylolyticum sp. nov. is proposed. The type strain is SW408(T) (=JCM 14823(T)=AS 1.5069(T)=CGMCC 1.5069(T)).

  8. An acidothermophilic functionally active novel GH12 family endoglucanase from Aspergillus niger HO: purification, characterization and molecular interaction studies. (United States)

    Rawat, Rekha; Kumar, Sunil; Chadha, Bhupinder Singh; Kumar, Dinesh; Oberoi, Harinder Singh


    Endoglucanase (EG) from Aspergillus niger HO was sequentially purified through ultrafiltration, ion exchange and size exclusion chromatography to homogeneity, with an overall recovery of 18 %. The purified EG was a monomeric protein with a molecular weight of about 55 kDa. The enzyme was optimally active at pH 3.5 and 70 °C with a half life (t1/2) of 3 h and Km value of 2.5 mg/ml. Metal ions, such as Ca(2+) and Co(2+) helped in enzyme induction, while Hg(2+) and Cu(2+) strongly inhibited the enzyme activity. Peptide mass fingerprinting results revealed that the purified EG is a novel enzyme that belongs to family 12 of glycoside hydrolase (GH12). Molecular docking studies indicated the presence of Glu116 and Glu204 as important determinant residues for the functional interaction with carboxymethylcellulose and showed hydrogen bonding with Asp99, Glu116, Glu204 and hydrophobic interactions with Trp22, Val58, Tyr61, Phe101, Met118, Trp120, Pro129, Ile130, Thr160 and Phe206. Hydrolysis of 2 % CMC with purified acidothermophilic EG at its optimum temperature and pH resulted in complete hydrolysis within 2 h yielding 18 % cellotriose, 72 % cellobiose and 10 % glucose as evident from HPLC analysis. In comparison to most of the EGs reported in literature, EG from A. niger HO exhibited higher thermostability. The acidothermophilic nature of this enzyme makes it potentially useful for industrial applications.

  9. Oxidized Cellulose with Different Carboxyl Content: Structure and Properties before and after Beating (United States)

    Vendula, Hejlová; Miloslav, Milichovský

    Our recent studies concentrated in investigating influence of beating oxidized cellulose, with different carboxyl content, on changing their basic properties (degree of polymerization, WRV - water resistant value and X-ray diffraction). Cellulose samples of oxidized cellulose were beated by toroidal beating machine. Cellulose consists of both amorphous and crystalline regions. Cellulose consists of linear chains of poly[ß-1,4-D- anhydroglucopyranose] (C6nH10n + 2O5n + 1 (n = degree of polymerization of glucose)), which crystallize through hydrogen bonding between the chains and has cellobiose as repeat unit. Oxidized cellulose is preparing by oxidation of cellulose in the C6 position of the glucopyranose units to carboxylic group (-COOH) and polyanhydroglukuronic acid (PAGA) is arised. An other option is oxidation with sodium hypochlorite with catalytic amounts of sodium bromide and 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) under various conditions. Beating and refining or mechanical treatment of fibers in water is an important step in using pulps for papermaking. It is an energy intensive process. The purpose of the treatment is to modify fiber properties to obtain the most desirable paper machine runnability and product properties. End of beating pulps was characterized by position, when all beated pulps under mixture passed through of riddle (about sizes mesh of 50). During beating of samples about different ratio of oxidation it was found, that samples with higher contents of COOH groups in starting pulp are characterized by a significantly lower specific beating energy consumption needed to achieving the same sizes of particles. X-ray analyse shows that for non-beated oxidized cellulose was perceptible high share amorphous contents compared with beated oxidized cellulose.

  10. Nanostructured cupric oxide electrode: An alternative to amperometric detection of carbohydrates in anion-exchange chromatography. (United States)

    Barragan, José T C; Kubota, Lauro T


    In this paper, a new and low cost copper/cupric oxide nanostructured electrode is presented as an alternative to the amperometric detection of carbohydrates in high-performance anion exchange chromatography. The modified copper electrodes were prepared by a simple and fast method which resulted in the obtainment of homogeneously distributed nanostructures adhered to the surface with controlled chemical nature. The results, when compared to conventional copper electrodes, exhibited considerable improvements in analytical results, including: 1) Better repeatability in consecutive glucose detections, in which the percent relative standard deviation improved from 15.1% to 0.279%. 2) Significant improvements in the stability of the baseline and a decrease of the stabilization time, going from several hours to approximately 15 min. 3) Considerable increase in the sensitivity towards glucose, from 5.02 nA min mg L(-1) to 25.5 nA min mg L(-1). 4) Improvements in the detectability with limits as low as 1.09 pmol. 5) Wide working range of concentrations (1 × 10(-2) to 1 × 10(4) mg L(-1)). 6) Good linearity with correlation coefficients greater than 0.998. 7) Possibility of detecting different molecules of carbohydrates (lactose, maltose, sucrose cellobiose, sorbitol, fructose, glucose, galactose, manose, arabitol, xylose, ribose and arabnose). In comparison to the electrode that is more employed for this type of application (gold electrode), the low cost, the possibility of detection at constant potential and the equivalent detection limits presented by the new electrode material introduced in this work emerge as characteristics that make this material a powerful alternative considering the detection of carbohydrates in anion exchange chromatography.

  11. Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme. (United States)

    Pellegrini, Vanessa O A; Serpa, Viviane Isabel; Godoy, Andre S; Camilo, Cesar M; Bernardes, Amanda; Rezende, Camila A; Junior, Nei Pereira; Franco Cairo, João Paulo L; Squina, Fabio M; Polikarpov, Igor


    Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

  12. Characterization of β-glucosidase from Aspergillus terreus and its application in the hydrolysis of soybean isoflavones* # (United States)

    Yan, Feng-ying; Xia, Wei; Zhang, Xiao-xu; Chen, Sha; Nie, Xin-zheng; Qian, Li-chun


    An extracellular β-glucosidase produced by Aspergillus terreus was identified, purified, characterized and was tested for the hydrolysis of soybean isoflavone. Matrix-assisted laser desorption/ionization with tandem time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) revealed the protein to be a member of the glycosyl hydrolase family 3 with an apparent molecular mass of about 120 kDa. The purified β-glucosidase showed optimal activity at pH 5.0 and 65 °C and was very stable at 50 °C. Moreover, the enzyme exhibited good stability over pH 3.0–8.0 and possessed high tolerance towards pepsin and trypsin. The kinetic parameters K m (apparent Michaelis-Menten constant) and V max (maximal reaction velocity) for p-nitrophenyl-β-D-glucopyranoside (pNPG) were 1.73 mmol/L and 42.37 U/mg, respectively. The K m and V max for cellobiose were 4.11 mmol/L and 5.7 U/mg, respectively. The enzyme efficiently converted isoflavone glycosides to aglycones, with a hydrolysis rate of 95.8% for daidzin, 86.7% for genistin, and 72.1% for glycitin. Meanwhile, the productivities were 1.14 mmol/(L·h) for daidzein, 0.72 mmol/(L·h) for genistein, and 0.19 mmol/(L·h) for glycitein. This is the first report on the application of A. terreus β-glucosidase for converting isoflavone glycosides to their aglycones in soybean products. PMID:27256679

  13. Metabolic pathways regulated by abscisic acid, salicylic acid and γ-aminobutyric acid in association with improved drought tolerance in creeping bentgrass (Agrostis stolonifera). (United States)

    Li, Zhou; Yu, Jingjin; Peng, Yan; Huang, Bingru


    Abscisic acid (ABA), salicylic acid (SA) and γ-aminobutyric acid (GABA) are known to play roles in regulating plant stress responses. This study was conducted to determine metabolites and associated pathways regulated by ABA, SA and GABA that could contribute to drought tolerance in creeping bentgrass (Agrostis stolonifera). Plants were foliar sprayed with ABA (5 μM), GABA (0.5 mM) and SA (10 μM) or water (untreated control) prior to 25 days drought stress in controlled growth chambers. Application of ABA, GABA or SA had similar positive effects on alleviating drought damages, as manifested by the maintenance of lower electrolyte leakage and greater relative water content in leaves of treated plants relative to the untreated control. Metabolic profiling showed that ABA, GABA and SA induced differential metabolic changes under drought stress. ABA mainly promoted the accumulation of organic acids associated with tricarboxylic acid cycle (aconitic acid, succinic acid, lactic acid and malic acid). SA strongly stimulated the accumulation of amino acids (proline, serine, threonine and alanine) and carbohydrates (glucose, mannose, fructose and cellobiose). GABA enhanced the accumulation of amino acids (GABA, glycine, valine, proline, 5-oxoproline, serine, threonine, aspartic acid and glutamic acid) and organic acids (malic acid, lactic acid, gluconic acid, malonic acid and ribonic acid). The enhanced drought tolerance could be mainly due to the enhanced respiration metabolism by ABA, amino acids and carbohydrates involved in osmotic adjustment (OA) and energy metabolism by SA, and amino acid metabolism related to OA and stress-defense secondary metabolism by GABA.

  14. Benzoic acid fermentation from starch and cellulose via a plant-like β-oxidation pathway in Streptomyces maritimus

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    Noda Shuhei


    Full Text Available Abstract Background Benzoic acid is one of the most useful aromatic compounds. Despite its versatility and simple structure, benzoic acid production using microbes has not been reported previously. Streptomyces are aerobic, Gram-positive, mycelia-forming soil bacteria, and are known to produce various kinds of antibiotics composed of many aromatic residues. S. maritimus possess a complex amino acid modification pathway and can serve as a new platform microbe to produce aromatic building-block compounds. In this study, we carried out benzoate fermentation using S. maritimus. In order to enhance benzoate productivity using cellulose as the carbon source, we constructed endo-glucanase secreting S. maritimus. Results After 4 days of cultivation using glucose, cellobiose, or starch as a carbon source, the maximal level of benzoate reached 257, 337, and 460 mg/l, respectively. S. maritimus expressed β-glucosidase and high amylase-retaining activity compared to those of S. lividans and S. coelicolor. In addition, for effective benzoate production from cellulosic materials, we constructed endo-glucanase-secreting S. maritimus. This transformant efficiently degraded the phosphoric acid swollen cellulose (PASC and then produced 125 mg/l benzoate. Conclusions Wild-type S. maritimus produce benzoate via a plant-like β-oxidation pathway and can assimilate various carbon sources for benzoate production. In order to encourage cellulose degradation and improve benzoate productivity from cellulose, we constructed endo-glucanase-secreting S. maritimus. Using this transformant, we also demonstrated the direct fermentation of benzoate from cellulose. To achieve further benzoate productivity, the L-phenylalanine availability needs to be improved in future.

  15. Genome-scale comparison and constraint-based metabolic reconstruction of the facultative anaerobic Fe(III-reducer Rhodoferax ferrireducens

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    Daugherty Sean


    Full Text Available Abstract Background Rhodoferax ferrireducens is a metabolically versatile, Fe(III-reducing, subsurface microorganism that is likely to play an important role in the carbon and metal cycles in the subsurface. It also has the unique ability to convert sugars to electricity, oxidizing the sugars to carbon dioxide with quantitative electron transfer to graphite electrodes in microbial fuel cells. In order to expand our limited knowledge about R. ferrireducens, the complete genome sequence of this organism was further annotated and then the physiology of R. ferrireducens was investigated with a constraint-based, genome-scale in silico metabolic model and laboratory studies. Results The iterative modeling and experimental approach unveiled exciting, previously unknown physiological features, including an expanded range of substrates that support growth, such as cellobiose and citrate, and provided additional insights into important features such as the stoichiometry of the electron transport chain and the ability to grow via fumarate dismutation. Further analysis explained why R. ferrireducens is unable to grow via photosynthesis or fermentation of sugars like other members of this genus and uncovered novel genes for benzoate metabolism. The genome also revealed that R. ferrireducens is well-adapted for growth in the subsurface because it appears to be capable of dealing with a number of environmental insults, including heavy metals, aromatic compounds, nutrient limitation and oxidative stress. Conclusion This study demonstrates that combining genome-scale modeling with the annotation of a new genome sequence can guide experimental studies and accelerate the understanding of the physiology of under-studied yet environmentally relevant microorganisms.

  16. A novel method to optimize culture conditions for biomass and sporulation of the entomopathogenic fungus Beauveria Bassiana IBC1201

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    Li Gao


    Full Text Available Biomass yields and sporulation of Beauveria bassiana was concerned on culture conditions, environmental factors and cultivation method. We optimized the best culture conditions for biomass yields of B. bassiana IBC1201 with the novel "two-stage" cultivation method as well as orthogonal matrix method. Firstly, we cultured spore suspension on the basal medium (sucrose 19.00 g, soy peptone 4.06 g, K2HPO4 1.00 g, KCl 0.50 g, MgSO4 0.50 g, FeSO4 0.10 g and 17.00 g Bactor for the first stage culture of 4 days under room condition. Then, we transferred them to another defined medium (Cellobiose 9.52 g, urea 1.70 g, ZnSO4•7H2O 0.05 g/L, MnSO4•H2O 0.005 g/L, CaCl2 1.00 g/L, CuSO4•5H2O 0.05 g/L and 17.00 g Bactor for more 4 days cultivation with the environmental factors combination of water potential -1.2 MPa /pH 3 /12 h light cycle/ 23 ℃ for biomass yields, and with the environmental factors combination of water potential -0.8 MPa /pH 3 /24 h light cycle/ 23 ℃ for spore yields. These results provided important information for mass production (including biomass and spore yields of this great potential biocontrol fungus.

  17. A novel method to optimize culture conditions for biomass and sporulation of the entomopathogenic fungus Beauveria bassiana IBC1201. (United States)

    Gao, Li


    Biomass yields and sporulation of Beauveria bassiana was concerned on culture conditions, environmental factors and cultivation method. We optimized the best culture conditions for biomass yields of B. bassiana IBC1201 with the novel "two-stage" cultivation method as well as orthogonal matrix method. Firstly, we cultured spore suspension on the basal medium (sucrose 19.00 g, soy peptone 4.06 g, K2HPO4 1.00 g, KCl 0.50 g, MgSO4 0.50 g, FeSO4 0.10 g and 17.00 g Bactor) for the first stage culture of 4 days under room condition. Then, we transferred them to another defined medium (Cellobiose 9.52 g, urea 1.70 g, ZnSO4•7H2O 0.05 g/L, MnSO4•H2O 0.005 g/L, CaCl2 1.00 g/L, CuSO4•5H2O 0.05 g/L and 17.00 g Bactor) for more 4 days cultivation with the environmental factors combination of water potential -1.2 MPa /pH 3 /12 h light cycle/23 ℃ for biomass yields, and with the environmental factors combination of water potential -0.8 MPa /pH 3 /24 h light cycle/23 ℃ for spore yields. These results provided important information for mass production (including biomass and spore yields) of this great potential biocontrol fungus.

  18. Population level analysis of evolved mutations underlying improvements in plant hemicellulose and cellulose fermentation by Clostridium phytofermentans.

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    Supratim Mukherjee

    Full Text Available The complexity of plant cell walls creates many challenges for microbial decomposition. Clostridium phytofermentans, an anaerobic bacterium isolated from forest soil, directly breaks down and utilizes many plant cell wall carbohydrates. The objective of this research is to understand constraints on rates of plant decomposition by Clostridium phytofermentans and identify molecular mechanisms that may overcome these limitations.Experimental evolution via repeated serial transfers during exponential growth was used to select for C. phytofermentans genotypes that grow more rapidly on cellobiose, cellulose and xylan. To identify the underlying mutations an average of 13,600,000 paired-end reads were generated per population resulting in ∼300 fold coverage of each site in the genome. Mutations with allele frequencies of 5% or greater could be identified with statistical confidence. Many mutations are in carbohydrate-related genes including the promoter regions of glycoside hydrolases and amino acid substitutions in ABC transport proteins involved in carbohydrate uptake, signal transduction sensors that detect specific carbohydrates, proteins that affect the export of extracellular enzymes, and regulators of unknown specificity. Structural modeling of the ABC transporter complex proteins suggests that mutations in these genes may alter the recognition of carbohydrates by substrate-binding proteins and communication between the intercellular face of the transmembrane and the ATPase binding proteins.Experimental evolution was effective in identifying molecular constraints on the rate of hemicellulose and cellulose fermentation and selected for putative gain of function mutations that do not typically appear in traditional molecular genetic screens. The results reveal new strategies for evolving and engineering microorganisms for faster growth on plant carbohydrates.

  19. Lactobacillus herbarum sp. nov., a species related to Lactobacillus plantarum. (United States)

    Mao, Yuejian; Chen, Meng; Horvath, Philippe


    Strain TCF032-E4 was isolated from a traditional Chinese fermented radish. It shares >99% 16S rRNA sequence identity with L. plantarum, L. pentosus and L. paraplantarum. This strain can ferment ribose, galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, lactose, melibiose, trehalose and gentiobiose. It cannot ferment sucrose, which can be used by L. pentosus, L. paraplantarum, L. fabifermentans, L. xiangfangensis and L. mudanjiangensis, as well as most of the L. plantarum strains (88.7%). TCF032-E4 cannot grow at temperature above 32 °C. This strain shares 78.2-83.6% pheS (phenylalanyl-tRNA synthetase alpha subunit) and 89.5-94.9% rpoA (RNA polymerase alpha subunit) sequence identity with L. plantarum, L. pentosus, L. paraplantarum, L. fabifermentans, L. xiangfangensis and L. mudanjiangensis. These results indicate that TCF032-E4 represents a distinct species. This hypothesis was further confirmed by whole-genome sequencing and comparison with available genomes of related species. The draft genome size of TCF032-E4 is approximately 2.9 Mb, with a DNA G+C content of 43.5 mol%. The average nucleotide identity (ANI) between TCF032-E4 and related species ranges from 79.0 to 81.1%, the highest ANI value being observed with L. plantarum subsp. plantarum ATCC 14917T. A novel species, Lactobacillus herbarum sp. nov., is proposed with TCF032-E4T ( = CCTCC AB2015090T = DSM 100358T) as the type strain.

  20. Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression

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    Rydzak Thomas


    Full Text Available Abstract Background Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. Results Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative

  1. Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

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    Li Yongchao


    for this bacterium, markerless targeted mutagenesis enables functional genomic research in C. cellulolyticum and rapid genetic engineering to significantly alter the mixture of fermentation products. The initial application of this system successfully engineered a strain with high ethanol productivity from cellobiose, cellulose and switchgrass.

  2. Global transcriptional analysis of spontaneous sakacin P-resistant mutant strains of Listeria monocytogenes during growth on different sugars.

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    Girum Tadesse Tessema

    Full Text Available Subclass IIa bacteriocins have strong antilisterial activity and can control the growth of Listeria monocytogenes in food. However, L. monocytogenes may develop resistance towards such bacteriocins. In this follow-up study, the transcriptomes of a high level (L502-1 and a low level (L502-6 spontaneous sakacin P-resistant mutant strain of L. monocytogenes were compared to the wild-type (L502. The growth of the resistant strains was reduced on mannose but not affected on cellobiose and the transcriptomics was performed during growth on these sugars. The mannose phosphotransferase system (PTS encoded by the mptACD operon (mpt is known for transporting mannose and also act as a receptor to class IIa bacteriocins. The mpt was repressed in L502-1 and this is in accordance with abolition of the bacteriocin receptor with resistance to class IIa bacteriocins. In contrast, the mpt was induced in L502-6. Despite the induction of the mpt, L502-6 showed 1,000 times more resistance phenotype and reduced growth on mannose suggesting the mannose-PTS may not be functional in L502-6. The microarray data suggests the presence of other transcriptional responses that may be linked to the sakacin P resistance phenotype particularly in L502-6. Most of commonly regulated genes encode proteins involved in transport and energy metabolism. The resistant strains displayed shift in general carbon catabolite control possibly mediated by the mpt. Our data suggest that the resistant strains may have a reduced virulence potential. Growth sugar- and mutant-specific responses were also revealed. The two resistant strains also displayed difference in stability of the sakacin P resistance phenotype, growth in the presence of both the lytic bacteriophage P100 and activated charcoal. Taken together, the present study showed that a single time exposure to the class IIa bacteriocin sakacin P may elicit contrasting phenotypic and transcriptome responses in L. monocytogenes possibly

  3. Asticcacaulis solisilvae sp. nov., isolated from forest soil. (United States)

    Kim, Seil; Gong, Gyeongtaek; Park, Tai Hyun; Um, Youngsoon


    An obligately aerobic, chemoheterotrophic, mesophilic prosthecate bacterium, designated strain CGM1-3EN(T), was isolated from the enrichment cultures of forest soil from Cheonggyesan Mountain, Republic of Korea. Cells were Gram-reaction-negative, motile rods (1.3-2.4 µm long by 0.30-0.75 µm wide) with single flagella. The strain grew at 10-37 °C (optimum 25-30 °C) and at pH 4.5-9.5 (optimum 5.0-7.0). The major cellular fatty acids were C16 : 0, C18 : 1ω7c 11-methyl, C12 : 1 3-OH and summed feature 8 (comprising C18 : 1ω7c/C18 : 1ω6c). The genomic DNA G+C content of strain CGM1-3EN(T) was 63.7 mol%. The closest phylogenetic neighbour to strain CGM1-3EN(T) was identified as Asticcacaulis biprosthecium DSM 4723(T) (97.2 % 16S rRNA gene sequence similarity) and the DNA-DNA hybridization value between strain CGM1-3EN(T) and A. biprosthecium DSM 4723(T) was less than 24.5 %. Strain CGM1-3EN(T) used d-glucose, d-fructose, sucrose, maltose, trehalose, d-mannose, d-mannitol, d-sorbitol, d-galactose, cellobiose, lactose, raffinose, fumarate, pyruvate, dl-alanine and glycerol as carbon sources. Based on data from the present polyphasic study, the forest soil isolate CGM1-3EN(T) is considered to represent a novel species of the genus Asticcacaulis, for which the name Asticcacaulis solisilvae sp. nov. is proposed. The type strain is CGM1-3EN(T) ( = AIM0088(T) = KCTC 32102(T) = JCM 18544(T)).

  4. Multisubstrate isotope labeling and metagenomic analysis of active soil bacterial communities. (United States)

    Verastegui, Y; Cheng, J; Engel, K; Kolczynski, D; Mortimer, S; Lavigne, J; Montalibet, J; Romantsov, T; Hall, M; McConkey, B J; Rose, D R; Tomashek, J J; Scott, B R; Charles, T C; Neufeld, J D


    Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon ((12)C) or stable-isotope-labeled ((13)C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the (13)C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. Importance: The ability to identify genes based on function, instead of sequence homology, allows the discovery of genes that would not be identified through sequence alone. This

  5. Ethanol from lignocellulosic wastes with utilization of recombinant bacteria. (United States)

    Katzen, R; Fowler, D E


    This article presents the advanced technology that has been developed by BioEnergy International of Gainesville, Florida, utilizing novel recombinant strains of bacteria developed by Lonnie Ingram of the University of Florida. The first commercial applications of these unique fermenting organisms convert 5-carbon sugars, as well as 6-carbon sugars, and oligomers of cellulose (e.g., cellobiose and cellotriose) directly to ethanol. The proposed systems that will be utilized for conversion of agricultural wastes, mixed waste papers, and pulp and paper mill waste in forthcoming commercial installations are now under design. This involves the extensive experience of Raphael Katzen Associates International, Inc. in acid hydrolysis, enzyme production, enzymatic hydrolysis, large-scale fermentation engineering, and distillation/dehydration. Specific examples of this advanced technology will be presented in different applications, namely: 1. Conversion of the hemicellulose content of sugar cane bagasse to 5-carbon sugars by mild-acid prehydrolysis, followed by fermentation of the 5-carbon sugar extract with recombinant Escherichia coli in a commercial installation soon to be under construction in Brazil. This unique process utilizes the surplus hemicellulose fraction of bagasse not required for steam and power generation to produce ethanol, additional to that from the original can juice, which has been converted by conventional sucrose fermentation to ethanol. The process also recovers and converts to ethanol the majority of sucrose normally lost with the bagasse fibers. Resultant beer is enriched in an innovative process to eliminate the need for incremental rectification capacity. 2. Application of this technology to mixed waste paper in Florida, with a moderate loading of newsprint (85% mechanical wood fiber), will involve a mild-acid prehydrolysis, the partial extraction of the 5-carbon sugars produced from hemicellulose as a feedstock for propagation of the recombinant

  6. Characterization of a GHF45 cellulase, AkEG21, from the common sea hare Aplysia kurodai (United States)

    Rahman, Mohammad; Inoue, Akira; Ojima, Takao


    The common sea hare Aplysia kurodai is known to be a good source for the enzymes degrading seaweed polysaccharides. Recently four cellulases, i.e., 95 kDa, 66 kDa, 45 kDa and 21 kDa enzymes, were isolated from A. kurodai (Tsuji et al., PLoS ONE, 8, e65418, 2013). The former three cellulases were regarded as glycosyl-hydrolase-family 9 (GHF9) enzymes, while the 21 kDa cellulase was suggested to be a GHF45 enzyme. The 21 kDa cellulase was significantly heat stable, and appeared to be advantageous in performing heterogeneous expression and protein-engineering study. In the present study, we determined some enzymatic properties of the 21 kDa cellulase and cloned its cDNA to provide the basis for the protein engineering study of this cellulase. The purified 21 kDa enzyme, termed AkEG21 in the present study, hydrolyzed carboxymethyl cellulose with an optimal pH and temperature at 4.5 and 40oC, respectively. AkEG21 was considerably heat-stable, i.e., it was not inactivated by the incubation at 55oC for 30 min. AkEG21 degraded phosphoric-acid-swollen cellulose producing cellotriose and cellobiose as major end products but hardly degraded oligosaccharides smaller than tetrasaccharide. This indicated that AkEG21 is an endolytic ?-1,4-glucanase (EC A cDNA of 1,013 bp encoding AkEG21 was amplified by PCR and the amino-acid sequence of 197 residues was deduced. The sequence comprised the initiation Met, the putative signal peptide of 16 residues for secretion and the catalytic domain of 180 residues, which lined from the N-terminus in this order. The sequence of the catalytic domain showed 47-62% amino-acid identities to those of GHF45 cellulases reported in other mollusks. Both the catalytic residues and the N-glycosylation residues known in other GHF45 cellulases were conserved in AkEG21. Phylogenetic analysis for the amino-acid sequences suggested the close relation between AkEG21 and fungal GHF45 cellulases.

  7. Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophila

    Directory of Open Access Journals (Sweden)

    Anthi Karnaouri


    Full Text Available The β-glucosidase gene bgl3a from Myceliophthora thermophila, member of the fungal glycosyl hydrolase (GH family 3, was cloned and expressed in Pichia pastoris. The mature β-glucosidase gene, which results after the excision of one intron and the secreting signal peptide, was placed under the control of the strong alcohol oxidase promoter (AOX1 in the plasmid pPICZαC. The recombinant enzyme (90 kDa was purified and characterized in order to evaluate its biotechnological potential. Recombinant P. pastoris efficiently secreted β-glucosidase into the medium and produced high level of enzymatic activity (41 U/ml after 192 h of growth, under methanol induction. MtBgl3a was able to hydrolyze low molecular weight substrates and polysaccharides containing β-glucosidic residues. The Km was found to be 0.39 mM on p-β-NPG and 2.64 mM on cellobiose. Optimal pH and temperature for the p-β-NPG hydrolysis were 5.0 and 70 °C. The β-glucosidase exhibits a half life of 143 min at 60 °C. Kinetic parameters of inhibition were determined for D-glucose, D-xylose and D-gluconic acid, indicating tolerance of the enzyme for these sugars and oxidized products. The recombinant enzyme was stimulated by short chain alcohols and has been shown to efficiently synthesize methyl-D-glucoside in the presence of methanol due to its transglycosylation activity. The stability of MtBgl3a in ethanol was prominent, and it retained most of its original activity after we exposed it to 50% ethanol for 6 h. The high catalytic performance, good thermal stability and tolerance to elevated concentrations of ethanol, D-xylose and D-glucose qualify this enzyme for use in the hydrolysis of lignocellulosic biomass for biofuel production, as part of an efficient complete multi-enzyme cocktail.

  8. Production and effect of aldonic acids during enzymatic hydrolysis of lignocellulose at high dry matter content

    Directory of Open Access Journals (Sweden)

    Cannella David


    Full Text Available Abstract Background The recent discovery of accessory proteins that boost cellulose hydrolysis has increased the economical and technical efficiency of processing cellulose to bioethanol. Oxidative enzymes (e.g. GH61 present in new commercial enzyme preparations have shown to increase cellulose conversion yields. When using pure cellulose substrates it has been determined that both oxidized and unoxidized cellodextrin products are formed. We report the effect of oxidative activity in a commercial enzyme mix (Cellic CTec2 upon overall hydrolysis, formation of oxidized products and impact on β-glucosidase activity. The experiments were done at high solids loadings using a lignocellulosic substrate simulating commercially relevant conditions. Results The Cellic CTec2 contained oxidative enzymes which produce gluconic acid from lignocellulose. Both gluconic and cellobionic acid were produced during hydrolysis of pretreated wheat straw at 30% WIS. Up to 4% of released glucose was oxidized into gluconic acid using Cellic CTec2, whereas no oxidized products were detected when using an earlier cellulase preparation Celluclast/Novozym188. However, the cellulose conversion yield was 25% lower using Celluclast/Novozym188 compared to Cellic CTec2. Despite the advantage of the oxidative enzymes, it was shown that aldonic acids could be problematic to the hydrolytic enzymes. Hydrolysis experiments revealed that cellobionic acid was hydrolyzed by β-glucosidase at a rate almost 10-fold lower than for cellobiose, and the formed gluconic acid was an inhibitor of the β-glucosidase. Interestingly, the level of gluconic acid varied significantly with temperature. At 50°C (SHF conditions 35% less gluconic acid was produced compared to at 33°C (SSF conditions. We also found that in the presence of lignin, no reducing agent was needed for the function of the oxidative enzymes. Conclusions The presence of oxidative enzymes in Cellic CTec2 led to the formation of

  9. Diversity and strain specificity of plant cell wall degrading enzymes revealed by the draft genome of Ruminococcus flavefaciens FD-1.

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    Margret E Berg Miller

    Full Text Available BACKGROUND: Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. METHODOLOGY/PRINCIPAL FINDINGS: The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb, and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs, polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs. Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315 exhibited the highest levels of up-regulation. CONCLUSIONS/SIGNIFICANCE: The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional

  10. 里氏木霉产纤维素酶的诱导和合成机理研究进展%Research progress of the mechanism of induction and synthesis of cellulase by Trichoderma reesei

    Institute of Scientific and Technical Information of China (English)

    李辉; 王义强; 陈介南; 张伟涛


    Cellulase is a kind of inducible enzyme.The inducer is required for cellulase expression.Cellulose, sophorose, lactose, cellobiose and sorbitol are common inducers.Trichoderma reesei mainly expresses three cellulases, endoglucanase, exoglucanase and β-1,4-glucosidase, in which the expression level of exoglucanase Ⅰ is highest, followed by endoglucanase and β-1,4-glucosidase.The synthesis and expression of cellulase is affected by active elements and inhibitory factors.In details, the combination of inducer and active element induces the synthesis and expression of cellulose.When the decomposition products of cellulase accumulate to a certain level, the intracellular inhibitory factor will combine with promoter to prevent the expression of cellulase.%纤维素酶是一类诱导酶,诱导剂对纤维素酶的表达是必需的.常用的诱导剂如纤维素、槐糖、乳糖、纤维二糖、山梨糖等.里氏木霉主要表达3种纤维素酶:内切葡聚糖酶、外切葡聚糖酶和β-1,4-葡萄糖苷酶,其中以外切葡聚糖苷酶Ⅰ的合成量最多,其次是内切葡聚糖苷酶和β-1,4-葡萄糖苷酶.纤维素酶的合成和表达是激活原件和抑制因子共同作用的结果:诱导物质和激活元件结合激活细胞内纤维素酶的合成和表达;当纤维素酶的分解产物达到一定水平时,细胞内的抑制因子和启动子结合,阻止纤维素酶的表达.

  11. Synthesis of branched dithiotrisaccharides via ring-opening reaction of sugar thiiranes. (United States)

    Repetto, Evangelina; Manzano, Verónica E; Uhrig, María Laura; Varela, Oscar


    Satisfactory procedures are described for the synthesis of 5,6- and 3,4-thiirane derivatives from the respective hexofuranose or hexopyranose epoxide precursors. The controlled ring-opening reaction of thiiranes by 1-thioaldoses was successfully accomplished to afford, regio- and stereoselectively, β-S-(1→4)-3,4-dithiodisaccharides. For instance, the regioselective attack of per-O-acetyl-1-thioglucose (16) to C-4 of 2-propyl 2,6-di-O-acetyl-3,4-epithio-α-D-galactopyranoside (14) gave the derivative of Glcp-β-S-(1→4)-3,4-dithioGlcp-O-iPr (17). This thiodisaccharide was accompanied by the (1→3)-disulfide 18, formed between 16 and 17, and the symmetric (3→3)-disulfide 19, which resulted from the oxidative dimerization of 17. However, the S-acetyl derivative of 17 could be obtained in good yield (62%) by LiAlH(4) reduction of the crude mixture 17-19, followed by acetylation. The same sequence of reactions starting from 14 and the 1-thiolate of Galp afforded the per-O,S-acetyl derivative of Galp-β-S-(1→4)-3,4-dithio-α-D-Glcp-O-iPr (23), which was selectively S-deacetylated to give 25. The dithiosaccharides 17 and 25 are 3,4-di-S-analogues of derivatives of the natural disaccharides cellobiose and lactose, respectively. The ring-opening reaction of 5,6-epithiohexofuranoses of D-galacto (8) or L-altro (11) configuration with 1-thioaldoses was also regio- and stereoselective to give the respective β-S-(1→6)-linked 5,6-dithiodisaccharides 26 or 29 in excellent yields. Glycosylation of the free thiol group of 17, 25, or 26, using trichloroacetimidates as glycosyl donors, led to the corresponding branched dithiotrisaccharides. Some of them are sulfur analogues of derivatives of branched trisaccharides found in natural polysaccharides.

  12. Effect of sodium acetate on the volumetric behaviour of some mono-, di-, and tri-saccharides in aqueous solutions over temperature range (288.15 to 318.15) K

    Energy Technology Data Exchange (ETDEWEB)

    Banipal, Parampaul K., E-mail: [Department of Chemistry, Guru Nanak Dev University, Amritsar 143 005 (India); Singh, Vickramjeet [Department of Chemistry, Guru Nanak Dev University, Amritsar 143 005 (India); Banipal, Tarlok S. [Department of Applied Chemistry, Guru Nanak Dev University, Amritsar 143 005 (India)


    The standard partial molar volumes, V{sub 2}{sup 0} at infinite dilution of eight monosaccharides [D(+)-xylose, D(-)-arabinose, D(-)-ribose, L(-)-sorbose, D(-)-fructose, D(+)-galactose, D(+)-glucose, and D(+)-mannose], six disaccharides [D(+)-cellobiose, sucrose, D(+)-melibiose, D(+)-lactose monohydrate, D(+)-trehalose dihydrate, and D(+)-maltose monohydrate] and two trisaccharides [D(+)-melizitose and D(+)-raffinose pentahydrate] (molalities of saccharides range from (0.03 to 0.12) mol . kg{sup -1}) have been determined in water and in (0.5, 1.0, 2.0, and 3.0) mol . kg{sup -1} aqueous sodium acetate solutions at temperatures, T = (288.15, 298.15, 308.15, and 318.15) K from density measurements using a vibrating-tube digital densimeter. From these results, corresponding standard partial molar volumes of transfer, DELTA{sub t}V{sub 2}{sup 0} have been determined for the transfer of various saccharides from water to aqueous solutions of sodium acetate. Positive values of DELTA{sub t}V{sub 2}{sup 0} were obtained for most of the saccharides, whose magnitude increase with the concentration of sodium acetate as well as temperature. However, negative DELTA{sub t}V{sub 2}{sup 0} values were observed for L(-)-sorbose, D(-)-fructose and D(+)-xylose at lower concentrations of co-solute. The negative magnitude of DELTA{sub t}V{sub 2}{sup 0} values decrease with rise of temperature from (288.15 to 318.15) K. Pair and higher order volumetric interaction coefficients have been determined by using McMillan-Mayer theory. Partial molar expansion coefficients, (partial derivV{sub 2}{sup 0}/partial derivT){sub p} and the second derivatives (partial deriv{sup 2}V{sub 2}{sup 0}/partial derivT{sup 2}){sub p} have also been estimated. These parameters have been utilized to understand various mixing effects in aqueous solutions due to the interactions between solute (saccharide) and co-solute (sodium acetate).

  13. Anaerovirgula multivorans gen. nov., sp. nov., a Novel Spore-Forming, Alkaliphilic Anaerobe Isolated from Owens Lake, California, USA (United States)

    Pikuta, Elena V.; Itoh, Takashi; Krader, Paul; Whitman, William B.; Hoover, Richard B.


    A novel, alkaliphilic, obligately anaerobic bacterium, strain SCAT, was isolated from mud sediments of a soda lake in California, USA. The rod-shaped cells were motile, Gram-positive, formed spores and were 0.4-0.5x2.5-5.0 micrometers in size. Growth occurred within the pH range 6.7-10.0 and was optimal at pH 8.5. The temperature range for growth was 10-45 degrees C, with optimal growth at 35 degrees C. NaCl was required for growth. Growth occurred at 0.5-9.0% (w/v) NaCl and was optimal at 1-2% (w/v). The novel isolate was a catalase-negative chemo-organoheterotroph that fermented sugars, proteolysis products, some organic and amino acids, glycerol, d-cellobiose and cellulose. It was also capable of growth by the Stickland reaction. Strain SCAT was sensitive to tetracycline, chloramphenicol, rifampicin and gentamicin, but it was resistant to ampicillin and kanamycin. The G+C content of the genomic DNA was 34.2 mol%. Major fatty acid components were C14:0, iso-C15:0, C16:1omega9c and C16:0. 16S rRNA gene sequence analysis of strain SCAT showed a similarity of approximately 97% with the type strains of Clostridium formicaceticum and Clostridium aceticum in clostridial cluster XI and a similarity of less than 94.2% to any other recognized Clostridium species and those of related genera in this cluster. Strain SCAT was clearly differentiated from C. formicaceticum and C. aceticum based on comparison of their phenotypic properties and fatty acid profiles, as well as low levels of DNA-DNA relatedness between strain SCAT and the type strains of these two species. Therefore, strain SCAT is considered to represent a novel species of a new genus, Anaerovirgula multivorans gen. nov., sp. nov., in clostridial cluster XI. The type strain is SCAT (=ATCC BAA-1084T=JCM 12857T=DSM 17722T=CIP 107910T).

  14. Biochemical studies on immobilized fungal β-glucosidase

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    S. A. Ahmed


    than that of the free enzyme by 7.69%. Sponge-immobilized β-glucosidase was repeatedly used to hydrolyze cellobiose (5 and 8 cycles with retained activity of 67.32 and 51.04%, respectively.

  15. Mediatorless sugar/oxygen enzymatic fuel cells based on gold nanoparticle-modified electrodes. (United States)

    Wang, Xiaoju; Falk, Magnus; Ortiz, Roberto; Matsumura, Hirotoshi; Bobacka, Johan; Ludwig, Roland; Bergelin, Mikael; Gorton, Lo; Shleev, Sergey


    We report on the fabrication and characterisation of a gold-nanoparticle (AuNP)-based mediatorless sugar/oxygen biofuel cell (BFC) operating in neutral sugar-containing buffers and human physiological fluids, such as blood and plasma. First, Corynascus thermophilus cellobiose dehydrogenase (CtCDH) and Myrothecium verrucaria bilirubin oxidase (MvBOx), used as anodic and cathodic bioelements, respectively, were immobilised on gold electrodes modified with 20 nm AuNPs. Detailed characterisation and optimisation of a new CDH/AuNP-based bioanode were performed and the following fundamental parameters were obtained: (i) the redox potential of the haem-containing centre of the enzyme was measured to be 75 mV vs. NHE, (ii) the surface coverage of CtCDH was found to be 0.65 pmol cm(-2) corresponding to a sub-monolayer coverage of the thiol-modified AuNPs by the enzyme, (iii) a turnover number for CtCDH immobilised on thiol-modified AuNPs was calculated to be ca. 0.5 s(-1), and (iv) the maximal current densities as high as 40 μA cm(-2) were registered in sugar-containing neutral buffers. Second, both biomodified electrodes, namely the CtCDH/AuNP-based bioanode and the MvBOx/AuNP-based biocathode, were combined into a functional BFC and the designed biodevices were carefully investigated. The following characteristics of the mediator-, separator- and membrane-less, miniature BFC were obtained: in phosphate buffer; an open-circuit voltage of 0.68 V, a maximum power density of 15 μW cm(-2) at a cell voltage of 0.52 V and in human blood; an open-circuit voltage of 0.65 V, a maximum power density of 3 μW cm(-2) at a cell voltage of 0.45 V, respectively. The estimated half-lives of the biodevices were found to be >12, <8, and <2 h in a sugar-containing buffer, human plasma, and blood, respectively. The basic characteristics of mediatorless sugar/oxygen BFCs were significantly improved compared with previously designed biodevices, because of the usage of three-dimensional Au

  16. Modular system for assessment of glycosyl hydrolase secretion in Geobacillus thermoglucosidasius. (United States)

    Bartosiak-Jentys, Jeremy; Hussein, Ali H; Lewis, Claire J; Leak, David J


    The facultatively anaerobic, thermophilic bacterium Geobacillus thermoglucosidasius is being developed as an industrial micro-organism for cellulosic bioethanol production. Process improvement would be gained by enhanced secretion of glycosyl hydrolases. Here we report the construction of a modular system for combining promoters, signal peptide encoding regions and glycosyl hydrolase genes to facilitate selection of the optimal combination in G. thermoglucosidasius. Initially, a minimal three-part E. coli-Geobacillus sp. shuttle vector pUCG3.8 was constructed using Gibson isothermal DNA assembly. The three PCR amplicons contained the pMB1 E. coli origin of replication and multiple cloning site (MCS) of pUC18, the Geobacillus sp. origin of replication pBST1 and the thermostable kanamycin nucleotidyltransferase gene (knt), respectively. G. thermoglucosidasius could be transformed with pUCG3.8 at an increased efficiency [2.8×10(5) c.f.u. (µg DNA)(-1)] compared to a previously reported shuttle vector, pUCG18. A modular cassette for the inducible expression and secretion of proteins in G. thermoglucosidasius, designed to allow the simple interchange of parts, was demonstrated using the endoglucanase Cel5A from Thermotoga maritima as a secretion target. Expression of cel5A was placed under the control of a cellobiose-inducible promoter (Pβglu) together with a signal peptide encoding sequence from a G. thermoglucosidasius C56-YS93 endo-β-1,4-xylanase. The interchange of parts was demonstrated by exchanging the cel5A gene with the 3' region of a gene with homology to celA from Caldicellulosiruptor saccharolyticus and substituting Pβglu for the synthetic, constitutive promoter PUp2n38, which increased Cel5A activity five-fold. Cel5A and CelA activities were detected in culture supernatants indicating successful expression and secretion. N-terminal protein sequencing of Cel5A carrying a C-terminal FLAG epitope confirmed processing of the signal peptide sequence.

  17. Aspergillus nidulans alpha-galactosidase of glycoside hydrolase family 36 catalyses the formation of alpha-galacto-oligosaccharides by transglycosylation. (United States)

    Nakai, Hiroyuki; Baumann, Martin J; Petersen, Bent O; Westphal, Yvonne; Hachem, Maher Abou; Dilokpimol, Adiphol; Duus, Jens Ø; Schols, Henk A; Svensson, Birte


    The alpha-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic alpha-galactosidases and alpha-galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g.L(-1) culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with alpha-(1-->6) regioselectivity from 40 mm 4-nitrophenol alpha-d-galactopyranoside, melibiose or raffinose, resulting in a 37-74% yield of 4-nitrophenol alpha-D-Galp-(1-->6)-D-Galp, alpha-D-Galp-(1-->6)-alpha-D-Galp-(1-->6)-D-Glcp and alpha-D-Galp-(1-->6)-alpha-D-Galp-(1-->6)-D-Glcp-(alpha1-->beta2)-d-Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol alpha-D-galactopyranoside (40 mm), alpha-(1-->6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39-58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose, L-arabinose, L-fucose and L-rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose and L-rhamnose. Structural modelling using Thermotoga maritima GH36 alpha-galactosidase as the template and superimposition of melibiose from the complex with human GH27 alpha-galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred alpha-galactosyl to 6-OH of the terminal residue in the alpha-linked melibiose, maltose, trehalose, sucrose and turanose in 6-46% yield and the beta-linked lactose, lactulose and cellobiose in 28-38% yield. The product structures were identified using NMR and ESI-MS and five of the 13

  18. Rheological behaviour of some saccharides in aqueous potassium chloride solutions over temperature range (288.15 to 318.15) K

    Energy Technology Data Exchange (ETDEWEB)

    Banipal, Parampaul K., E-mail: [Department of Chemistry, Guru Nanak Dev University, Amritsar 143005 (India); Chahal, Amanpreet K.; Singh, Vickramjeet [Department of Chemistry, Guru Nanak Dev University, Amritsar 143005 (India); Banipal, Tarlok S. [Department of Applied Chemistry, Guru Nanak Dev University, Amritsar 143005 (India)


    The viscosities, {eta} of mono-, di-, tri-saccharides and methylglycosides, viz., D(+)-xylose (XYL), D(-)-arabinose (ARA), D(-)-ribose (RIB), D(-)-fructose (FRU), D(+)-galactose (GAL), D(+)-mannose (MAN), D(+)-glucose (GLU), D(+)-melibiose (MEL), D(+)-cellobiose (CEL), D(+)-lactose monohydrate (LAC), D(+)-maltose monohydrate (MAL), D(+)-trehalose dihydrate (TRE), sucrose (SUC), D(+)-raffinose pentahydrate (RAF), {alpha}-methyl-D(+)-glucoside ({alpha}-Me-GLU), methyl-{alpha}-D-xylopyranoside (Me-{alpha}-XYL), and methyl-{beta}-D-xylopyranoside (Me-{beta}-XYL) in water and in (0.5, 1.0, 2.0, and 3.0) mol . kg{sup -1} aqueous solutions of potassium chloride (KCl) have been determined at T = (288.15, 298.15, 308.15, and 318.15) K from efflux time measurements by using a capillary viscometer. Densities used to determine viscosities have been reported earlier. The viscosity data have been utilized to determine the viscosity B-coefficients employing the Jones-Dole equation at different temperatures. From these data, the viscosity B-coefficients of transfer, {Delta}{sub t}B have been estimated for the transfer of various saccharides/methylglycosides from water to aqueous potassium chloride solutions. The {Delta}{sub t}B values have been found to be positive, whose magnitude increases with the increase in concentration of potassium chloride in all cases. The dB/dT coefficients, pair, {eta}{sub AB} and triplet, {eta}{sub ABB} viscometric interaction coefficients have also been determined. Gibbs free energies of activation and related thermodynamic parameters of activation of viscous flow have been determined employing Feakin's transition-state theory. The signs and magnitudes of various parameters have been discussed in terms of solute-solute and solute-solvent interactions occurring in these solutions. The effect of substitution of -OH by methoxy group, -OCH{sub 3} has also been discussed.

  19. Effect of superfine comminution on reducing sugar components in corn stalk enzymatic hydrolysate%超微粉碎预处理对玉米秸杆酶解液中还原糖组分的影响

    Institute of Scientific and Technical Information of China (English)

    赵晓燕; 朱海涛; 张桂香; 张立金; 何磊


    The compositions of five different monosaccharides in corn stalk enzymatic hydrolysate were an-alyzed by HPLC.They were arabinose,mannose,galactose,glucose and fructose,in which glucose and fructose were the main compositions.The results showed that the reducing sugar composition contents in corn stalk powder with a particle >1 200 mesh was higher than that with a particle size 100 ~200 mesh. The content of arabinose,galactose,glucose,mannose and fructose in enzymatic hydrolysate with particle size of >1 200 mesh increased 48.38%、64.29%、37.63%、70.07% and 22.34%,respectively.In addition,three new unknown response peaks occurred in the chromatograms,which might be fucose,su-crose and cellobiose.%HPLC 分析了玉米秸杆微粉酶解液中5种单糖组成,主要包括阿拉伯糖、甘露糖、半乳糖、葡萄糖、果糖,其中以葡糖糖与果糖为主。结果表明:粒径大于1200目的玉米秸杆粉酶解液还原糖单糖组分与100~200目的相比,其含量明显高,阿拉伯糖、半乳糖、葡萄糖、甘露糖和果糖分别提高了48.38%、64.29%、37.63%、70.07%和22.34%。此外,在超微细秸杆粉的酶解液 HPLC 色谱图上出现三个新的未知响应峰,推测可能为岩藻糖、庶糖与纤维二糖。

  20. Temperature effect on high salinity depuration of Vibrio vulnificus and V. parahaemolyticus from the Eastern oyster (Crassostrea virginica). (United States)

    Larsen, A M; Rikard, F S; Walton, W C; Arias, C R


    Vibrio vulnificus (Vv) and Vibrio parahaemolyticus (Vp) are opportunistic human pathogens naturally associated with the Eastern oyster Crassostrea virginica. The abundances of both pathogens in oysters are positively correlated with temperature, thus ingestion of raw oysters during the warm summer months is a risk factor for contracting illness from these bacteria. Current post-harvest processing (PHP) methods for elimination of these pathogens are expensive and kill the oyster, changing their organoleptic properties and making them less appealing to some consumers. High salinity has proven effective in reducing Vv numbers in the wild and our research aims at developing an indoor recirculating system to reduce pathogenic Vibrios while maintaining the taste and texture of live oysters. The goal of this study was to determine the influence of temperature on the efficacy of high salinity depuration. Vv was enumerated as most probable number (MPN) per gram of oyster tissue using the FDA-approved modified cellobiose polymyxin colistin (mCPC) protocol and with an alternative Vibrio specific media CHROMagar™ Vibrio (CaV). CaV was also used to quantify Vp. Oysters were held at 35 psu for 10 days at three temperatures: low (20°C), mid (22.5°C) and high (25°C). There was no difference in MPN/g of Vv between media; however more Vv isolates were obtained from mCPC than CaV. There was no significant effect of temperature on reduction of Vv or Vp throughout depuration but there was a tendency for low temperatures to be less effective than the higher ones. High salinity resulted in a significant decrease in Vv by day 3 and again by day 10, and a decrease in Vp by day 3. Oyster condition indices were maintained throughout depuration and mortality was low (4% across three trials). Overall these results support the use of mCPC for Vv enumeration and demonstrate the promise of high salinity depuration for PHP of the Eastern oyster. The trend for lower temperatures to be less

  1. Genomic Analysis of Caldithrix abyssi, the Thermophilic Anaerobic Bacterium of the Novel Bacterial Phylum Calditrichaeota (United States)

    Kublanov, Ilya V.; Sigalova, Olga M.; Gavrilov, Sergey N.; Lebedinsky, Alexander V.; Rinke, Christian; Kovaleva, Olga; Chernyh, Nikolai A.; Ivanova, Natalia; Daum, Chris; Reddy, T.B.K.; Klenk, Hans-Peter; Spring, Stefan; Göker, Markus; Reva, Oleg N.; Miroshnichenko, Margarita L.; Kyrpides, Nikos C.; Woyke, Tanja; Gelfand, Mikhail S.; Bonch-Osmolovskaya, Elizaveta A.


    The genome of Caldithrix abyssi, the first cultivated representative of a phylum-level bacterial lineage, was sequenced within the framework of Genomic Encyclopedia of Bacteria and Archaea (GEBA) project. The genomic analysis revealed mechanisms allowing this anaerobic bacterium to ferment peptides or to implement nitrate reduction with acetate or molecular hydrogen as electron donors. The genome encoded five different [NiFe]- and [FeFe]-hydrogenases, one of which, group 1 [NiFe]-hydrogenase, is presumably involved in lithoheterotrophic growth, three other produce H2 during fermentation, and one is apparently bidirectional. The ability to reduce nitrate is determined by a nitrate reductase of the Nap family, while nitrite reduction to ammonia is presumably catalyzed by an octaheme cytochrome c nitrite reductase εHao. The genome contained genes of respiratory polysulfide/thiosulfate reductase, however, elemental sulfur and thiosulfate were not used as the electron acceptors for anaerobic respiration with acetate or H2, probably due to the lack of the gene of the maturation protein. Nevertheless, elemental sulfur and thiosulfate stimulated growth on fermentable substrates (peptides), being reduced to sulfide, most probably through the action of the cytoplasmic sulfide dehydrogenase and/or NAD(P)-dependent [NiFe]-hydrogenase (sulfhydrogenase) encoded by the genome. Surprisingly, the genome of this anaerobic microorganism encoded all genes for cytochrome c oxidase, however, its maturation machinery seems to be non-operational due to genomic rearrangements of supplementary genes. Despite the fact that sugars were not among the substrates reported when C. abyssi was first described, our genomic analysis revealed multiple genes of glycoside hydrolases, and some of them were predicted to be secreted. This finding aided in bringing out four carbohydrates that supported the growth of C. abyssi: starch, cellobiose, glucomannan and xyloglucan. The genomic analysis

  2. Effects of Moringa oleifera seed extract on rumen fermentation in vitro. (United States)

    Hoffmann, E M; Muetzel, S; Becker, K


    Moringa oleifera is a pantropical tree of the family Moringaceae. A previously undescribed property of an aqueous extract from the seeds of this plant is the modulation of ruminal fermentation patterns, especially protein degradation, as demonstrated in a short-term batch incubation system. Gas, short chain fatty acids (SCFA) and cellulolytic enzyme activities were determined as general fermentation parameters. A dot blot assay able to directly detect true protein in rumen fluid samples was used to quantify protein degradation. For complex substrates the interpretation of protein degradation profiles was amended by polyacrylamide gel electrophoresis (PAGE) of the samples. When incubated with pure carbohydrates at a concentration of 1 mg ml(-1), the extract reduced microbial degradation of the model protein, bovine serum albumin (BSA), such that its concentration was at least 40% above the control after 12 h of incubation. Total protein degradation was thus delayed by approximately 9 h. When fermented along with wheat straw, leaf protein (Rubisco) was almost entirely protected during 12 h of fermentation. The degradation of soy proteins was retarded by at least 4-6 h, depending on the protein band. There were strong side effects on the fermentation of pure cellulose (SCFA yield-60% after 12 h), whereas cellobiose and starch fermentation were less affected (-18 and -8%, respectively). When the complex substrates were fermented, SCFA yield was reduced by approximately 30% after 12 h. In our work we clearly demonstrate the efficacy of the new substance, which is neither a tannin nor a saponin, in an in vitro system, using pure as well as complex substrates. The properties shown in vitro for the crude extract suggest that it could have a positive effect on the protein metabolism of ruminants under intensive management and that negative side effects can be overcome by an optimized dosage. If the chemical nature of the active substance and its mechanism of action can be

  3. Production, purification and characterisation of alkali stable xylanase from Cellulosimicrobium sp. MTCC 10645

    Institute of Scientific and Technical Information of China (English)

    Rajashri D Kamble; Anandrao R Jadhav


    Objective: The aim of this experimental study was production, purification and characterization of alkali stable xylanase from locally isolated Cellulosimicrobium sp. MTCC 10645, which is an important industrial enzyme used in the pulp and paper industry. Methods: The enzyme was produced in Erlenmeyer flasks containing fresh basal salt medium supplemented with 1% oat spelt xylan. The enzyme was extracted and isolated using ammonium sulphate precipitation and dialysis. It was further purified using DEAE cellulose chromatography and purity was checked by SDS-PAGE. Effect of temperature and pH on activity and stability of enzyme was studied. The enzyme was laso studied for its substrate specificity and kinetic parameters. Results: The isolate was identified on the basis of cultural, morphological, physiological and biochemical properties as well as 16S rRNA sequencing. Among the carbon sources tested, birchwood xylan found prominent for increased level of xylanase i. e. 96.33 U/ml. The enzyme was purified by DEAE cellulose chromatography at NaCl concentration of 0.25 M and had a molecular mass of 78.0 kDa. Xylanase was purified sixteen fold with a specific activity of 246.6 U/mg. Xylanase activity was maximum at 50℃. The enzyme was thermostable retaining 8%of the original activity after incubation at 60℃ of 4 h. The enzyme was active over a pH range of 6.0-11.0, although its activity was optimal at pH 7.0. About 48.52% of the enzyme activity was retained after 4 h at pH 11.0. The enzyme was active on oat spelt and birchwood xylans but not on avicel, CMC, cellobiose, starch or p-nitrophenyl xylopyranoside. The xylanase had Km and Vmax values of 4.76 mg/ml and 232.5 μmol/min/mg, respectively when birchwood xylan used as substrate. Conclusions:The xylanase showed a unique pattern of xylan hydrolysis releasing a large amount of intermediate products (xylotriose and xylobiose) with small quantity of xylose. Some of these characteristics make this enzyme potentially

  4. Compounds Released from Biomass Deconstruction: Understanding Their Effect on Cellulose Enzyme Hydrolysis and Their Biological Activity (United States)

    Djioleu, Angele Mezindjou

    The effect of compounds produced during biomass pretreatment on cellulolytic enzyme was investigated. Liquid prehydrolyzates were prepared by pretreating switchgrass using 24 combinations of temperature, time, and sulfuric acid concentration based on a full factorial design. Temperature was varied from 140°C to 180°C; time ranged from 10 to 40 min; and the sulfuric acid concentrations were 0.5% or 1% (v/v). Identified products in the prehydrolyzates included xylose, glucose, hydroxymethylfurfural (HMF), furfural, acetic acid, formic acid, and phenolic compounds at concentration ranging from 0 to 21.4 g/L. Pretreatment conditions significantly affected the concentrations of compounds detected in prehydrolyzates. When assayed in the presence of switchgrass prehydrolyzates against model substrates, activities of cellulase, betaglucosidase, and exoglucanase, were significantly reduced by at least 16%, 31.8%, and 57.8%, respectively, as compared to the control. A strong positive correlation between inhibition of betaglucosidase and concentration of glucose, acetic acid, and furans in prehydrolyzate was established. Exoglucanase inhibition correlated with the presence of phenolic compounds and acetic acid. The prehydrolyzate, prepared at 160°C, 30 min, and 1% acid, was fractionated by centrifugal partition chromatography (CPC) into six fractions; the inhibition effect of these fractions on betaglucosidase and exoglucanase was determined. The initial hydrolysis rate of cellobiose by betaglucosidase was significantly reduced by the CPC sugar-rich fraction; however, exoglucanase was deactivated by the CPC phenolic-rich fraction. Finally, biological activities of water-extracted compounds from sweetgum bark and their effect on cellulase was investigated. It was determined that 12% of solid content of the bark extract could be accounted by phenolic compounds with gallic acid identified as the most concentrated phytochemical. Sweetgum bark extract inhibited Staphylococcus

  5. Factors that contribute to the mycoparasitism stimulus in Trichoderma atroviride strain P1

    Institute of Scientific and Technical Information of China (English)

    Woo S L; Lorito M; Formisano E; Fogliano V; Cosenza C; Mauro A; Turrà D; Soriente I; Ferraioli S; Scala F


    the presence of host fungi. The disease symptom development on bean leaves inoculated with Botrytis and Trichoderma spores was dearly reduced by the addition of the inducers, unless these molecules were not specifically inactivated. Finally,purified inducers added to liquid cultures of T. atroviride P1 stimulated the production of low MW antibiotics and metabolites which inhibited Botrytis spore germination. Mass spectrometry analysis (ESI-MS) of the inducers indicated the presence of hexose oligomers, like cellobiose, while MS/MS analysis by selective fragmentation of peaks in the spectrum demonstrated the presence of at least three distinct compounds that were biologically active.

  6. A time course analysis of the extracellular proteome of Aspergillus nidulans growing on sorghum stover

    Directory of Open Access Journals (Sweden)

    Saykhedkar Sayali


    Full Text Available Abstract Background Fungi are important players in the turnover of plant biomass because they produce a broad range of degradative enzymes. Aspergillus nidulans, a well-studied saprophyte and close homologue to industrially important species such as A. niger and A. oryzae, was selected for this study. Results A. nidulans was grown on sorghum stover under solid-state culture conditions for 1, 2, 3, 5, 7 and 14 days. Based on analysis of chitin content, A. nidulans grew to be 4-5% of the total biomass in the culture after 2 days and then maintained a steady state of 4% of the total biomass for the next 12 days. A hyphal mat developed on the surface of the sorghum by day one and as seen by scanning electron microscopy the hyphae enmeshed the sorghum particles by day 5. After 14 days hyphae had penetrated the entire sorghum slurry. Analysis (1-D PAGE LC-MS/MS of the secretome of A. nidulans, and analysis of the breakdown products from the sorghum stover showed a wide range of enzymes secreted. A total of 294 extracellular proteins were identified with hemicellulases, cellulases, polygalacturonases, chitinases, esterases and lipases predominating the secretome. Time course analysis revealed a total of 196, 166, 172 and 182 proteins on day 1, 3, 7 and 14 respectively. The fungus used 20% of the xylan and cellulose by day 7 and 30% by day 14. Cellobiose dehydrogenase, feruloyl esterases, and CAZy family 61 endoglucanases, all of which are thought to reduce the recalcitrance of biomass to hydrolysis, were found in high abundance. Conclusions Our results show that A. nidulans secretes a wide array of enzymes to degrade the major polysaccharides and lipids (but probably not lignin by 1 day of growth on sorghum. The data suggests simultaneous breakdown of hemicellulose, cellulose and pectin. Despite secretion of most of the enzymes on day 1, changes in the relative abundances of enzymes over the time course indicates that the set of enzymes

  7. Enzymic synthesis of alpha- and beta-D-glucosides of 1-deoxynojirimycin and their glycosidase inhibitory activities. (United States)

    Asano, N; Oseki, K; Kaneko, E; Matsui, K


    1-Deoxynojirimycin (1) is a potent inhibitor of mammalian and rice alpha-glucosidase. Several glucosides of 1 were synthesized by use of the native and immobilized enzyme and their effect on various enzymes was investigated. Transglucosylation reactions using rice alpha-glucosidase, yeast alpha- and beta-glucosidases purified from Rhodotorula lactosa were performed with maltose or cellobiose as a glucose donor and N-(benzyloxycarbonyl)-1-deoxynojirimycin (2) as an acceptor. The transglucosylation reaction using native rice alpha-glucosidase afforded 3-O-alpha-D-glucopyranosyl-N-(benzyloxycarbonyl)-1-deoxynojirimycin (4), 4-O-alpha-D-glucopyranosyl-N-(benzyloxycarbonyl)-1-deoxynojirimycin (5), and 2-O-alpha-D-glucopyranosyl-N-(benzyloxycarbonyl)-1-deoxynojirimycin (3) in yields of 40, 13, and 2%, respectively, after 30 min. The transglucosylation reaction using immobilized rice alpha-glucosidase was similar to that using the native enzyme. In the system using native yeast alpha-glucosidase, 3, 5, and 4 were formed in yields of 34, 13, and 6%, respectively, after 15 h. The immobilization of yeast alpha-glucosidase caused a significant decrease in transglucosylation activity. Yeast beta-glucosidase showed a high transglucosylation activity and incubation with the reaction system afforded 2-O-beta-D-glucopyranosyl-N-(benzyloxycarbonyl)-1-deoxynojirimycin (6) and 4-O-beta-D-glucopyranosyl-N-(benzyloxycarbonyl)-1-deoxynojirimycin (7) in yields of 69 and 3%, respectively, after 3 h. The transglucosylation reaction using immobilized yeast beta-glucosidase preferentially afforded 6 in a yield of 73% after 3 h. After removal of N-benzyloxycarbonyl group from the product glucosides, their glycosidase inhibitory activities were measured. 3-O-alpha-D-Glucopyranosyl-1-deoxynojirimycin (9) retained the potent inhibition of 1 against rat intestinal sucrase activity and was more effective than 1 against rice alpha-glucosidase. 4-O-alpha-D-Glucopyranosyl-1-deoxynojirimycin (10

  8. Description of Pseudomonas asuensis sp. nov. from biological soil crusts in the Colorado plateau, United States of America. (United States)

    Reddy, Gundlapally Sathyanarayana; Garcia-Pichel, Ferran


    A Gram-negative, aerobic, non spore-forming, non-motile, rod-shaped, yellow pigmented bacterium CP155-2(T) was isolated from a biological soil crusts sample collected in the Colorado plateau, USA and subjected to polyphasic taxonomic characterization. Strain CP155-2(T) contained summed feature 3 (C(16:1)ω5c/C(16:1)ω7c) and C(18:1)ω7c as major fatty acids and diphosphatidylglycerol (DPG) along with phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) as major polar lipids. Based on these characteristics CP155-2(T) was assigned to the genus Pseudomonas. Phylogenetic analysis based on 16S rRNA gene sequence further confirmed the affiliation of CP155-2(T) to the genus Pseudomonas and showed a 16S rRNA gene sequence similarity of less than 98.7% with already described species of the genus. Pseudomonas luteola, Pseudomonas zeshuii, and Pseudomonas duriflava were identified as the closest species of the genus Pseudomonas with 16S rRNA gene sequence similarities of 98.7%, 98.6%, and 96.9%, respectively. The values for DNA¨CDNA relatedness between CP155-2(T) and Pseudomonas luteola and Pseudomonas zeshuii were 23% and 14% respectively a value below the 70% threshold value, indicating that strain CP155-2(T) belongs to a novel taxon of the genus Pseudomonas lineage. The novel taxon status was strengthened by a number of phenotypic differences wherein CP155-2(T) was positive for oxidase, negative for gelatin hydrolysis, could utilize D-cellobiose, D-raffinose, L-rhamnose, D-sorbitol but not L-aspartic acid and L-glutamic acid. Based on the collective differences strain CP155-2(T) exhibited, it was identified as a novel species and the name Pseudomonas asuensis sp. nov. was proposed. The type strain of Pseudomonas asuensis sp. nov. is CP155-2(T) (DSM 17866(T) =ATCC BAA-1264(T) =JCM13501(T) =KCTC 32484(T)).

  9. Characterization of Cellulase Enzyme Inhibitors Formed During the Chemical Pretreatments of Rice Straw (United States)

    Rajan, Kalavathy

    Production of fuels and chemicals from a renewable and inexpensive resource such as lignocellulosic biomass is a lucrative and sustainable option for the advanced biofuel and bio-based chemical platform. Agricultural residues constitute the bulk of potential feedstock available for cellulosic fuel production. On a global scale, rice straw is the largest source of agricultural residues and is therefore an ideal crop model for biomass deconstruction studies. Lignocellulosic biofuel production involves the processes of biomass conditioning, enzymatic saccharification, microbial fermentation and ethanol distillation, and one of the major factors affecting its techno-economic feasibility is the biomass recalcitrance to enzymatic saccharification. Preconditioning of lignocellulosic biomass, using chemical, physico-chemical, mechanical and biological pretreatments, is often practiced such that biomass becomes available to downstream processing. Pretreatments, such as dilute acid and hot water, are effective means of biomass conversion. However, despite their processing importance, preconditioning biomass also results in the production of carbohydrate and lignin degradation products that are inhibitory to downstream saccharification enzymes. The saccharification enzyme cocktail is made up of endo-cellulase, exo-cellulase and beta-glucosidase enzymes, whose role is to cleave cellulose polymers into glucose monomers. Specifically, endo-cellulase and exo-cellulase enzymes cleave cellulose chains in the middle and at the end, resulting in cellobiose molecules, which are hydrolyzed into glucose by beta-glucosidase. Unfortunately, degradation compounds generated during pretreatment inhibit the saccharification enzyme cocktail. Various research groups have identified specific classes of inhibitors formed during biomass pretreatment and have studied their inhibitory effect on the saccharification cocktail. These various research groups prepared surrogate solutions in an attempt to

  10. 蒸气爆破预处理芦苇酶解工艺优化及产物分析%Hydrolysate Alalyze and Enzymatic Saccharification Conditions Optimization of Reed by Steam Explosion Pretreatment

    Institute of Scientific and Technical Information of China (English)

    朱作华; 蔡霞; 严理; 李智敏; 谢纯良; 胡镇修; 彭源德


    Reed (Phragmites australis)was a kind of alternative raw material for bio-ethanol production. The important parame-ters of influencing enzymatic sacharification for reed were screened by Plackett-Burman design. Furthermore, based on Box-Behnken design, the enzymatic saccharification conditions of reed by steam explosion pretreatment were optimized by response surface methodology, and a mathematial model of a second order quadratic equation was developed for reducing sugar conen-tration. The hydrolysate was alalysed by HPLC. The resutlts suggested that the three important parameters included dilute sul-phuric acid, Tween-80 and manganese sulfate concentration. The optimized technologial parameters were as follows: dilute sulphuric acid concentration 0.88%,、Tween-80 concentration 0.61%, manganese sulfate contration 0.26%. The reducing sugar concentration was up to 45.68 mg/mL. The results verified the validity of the mathematical model. The concentrations of glu-cose and xylose were 21.36 mg/mL and 16.62 mg/mL, respectly. Contents of arabinose, cellobiose and galactose were very low.%芦苇(Phragmites australis)是一种有潜力的能源作物,为优化芦苇酶解糖化工艺,应用Plackett-Burman试验设计筛选影响芦苇酶糖化的重要参数,通过Box-Behnken设计确定重要参数的最佳水平,应用高效液相色谱仪(HPLC)对糖化过程中的单糖种类及含量进行分析.结果表明,影响芦苇酶糖化的重要参数是H2SO4浓度、Tween-80和MnSO4浓度,最佳工艺参数为H2SO4浓度0.88%、表面活性剂Tween-80添加量0.61%、MnSO4添加量0.26%,在此条件下,8~10 h可以完成糖化,总还原糖浓度达到45.68 mg/mL,同时验证了数学模型的有效性, 液相分析表明糖化液中主要的糖种类为葡萄糖和木糖, 其含量分别为21.36、16.62 mg/mL,阿拉伯糖、纤维二糖和半乳糖含量较少.

  11. Isolation of anaerobic bacterial strains from cellulolytic bacterial com- munity WSC-9%纤维素分解复合菌系WSC-9中厌氧细菌的分离

    Institute of Scientific and Technical Information of China (English)

    温雪; 付博锐; 王彦杰; 高亚梅; 刘权; 晏磊; 王伟东


    The microbial community WSC-9 capable of degrading lignocellulose with high efficiency was enriched from composting of cattle manure and rice straw. The aims of this study were to isolate and identify the anaerobic bacterial strains from the cellulose-degrading bacterial community. An anaerobic, thermophilic and cellulolytic bacterium was isolated, which was named WSC-9-7. More than 47% of rice straw was degraded within 10 days of inoculation at 50 ° C by WSC-9-7. WSC-9-7 is a spore-forming and straight rod. Cellobiose, cellulose, filter paper and rice straw can be utilized as sole carbon and energy sources by WSC-9-7 in medium. On the basis of 16S rDNA gene sequence similarity, WSC-9-7 was mapped to the genus Clostridium. It is closely related to HAW-RM37-2-B-1600d-W (99%), and Clostridium islandicum AK1(98%). Clostridium islandicum AK1 was a new anaerobic, saccharolytic, thermophilic bacterium, isolated from hot spring in Iceland. HAW-RM37-2-B-1600d-W was uncultured clone from compost environmental samples. All of them were thermophilic and anaerobic bacterium. Hence, WSC-9-7 represents a novel species is proposed.%  复合菌系WSC-9是一组具高效稳定分解纤维素能力的细菌复合群体.为了研究其微生物组成,以纤维素分解情况为依据,分离复合菌系中具有纤维素分解能力的厌氧纯培养菌株,通过16S rDNA基因序列初步分析确定系统发育地位.从WSC-9中获得1株可有效降解纤维素的严格厌氧细菌WSC-9-7,50℃培养10 d,稻秆的总干重减少了47%.WSC-9-7为杆菌,产孢,能够利用纤维二糖、纤维素、滤纸、稻秆等.经数据库比对,与菌株HAW-RM37-2-B-1600d-W(FN563295)的相似性达到99%,与Clostridium islandicum AK1(EF088328)的相似性为98%.其中,Clostridium islandicum AK1厌氧且可以分解多糖类物质,获于冰岛的热泉;HAW-RM37-2-B-1600d-W在堆肥样品的克隆结果中获得,未获得纯培养.菌株WSC-9-7与这两株细菌均为嗜高温的严格

  12. 水生植物对沉积物微生物群落功能多样性的影响%Effects of Aquatic Macrophyte Planting on Functional Diversity of Microbial Community in Sediment

    Institute of Scientific and Technical Information of China (English)

    赵良元; 陶晶祥; 刘敏


    Differences in the functiona1 diversity of microbia1 community between rhizosphere sediment of Acorus calamus and non-rhizosphere sediment were investigated using Biolog-Eco microplates.Results showed that the growth of Acorus calamus could significantly increase the microbial activity and diversity in the rhizosphere sedi-ment.The total microorganism activity (AWCD,average well color development)and the diversity including the Shannon-Wiener index,Simpson index,richness index and Pielou evenness index of rhizosphere sediment was sig-nificantly higher than that of non-rhizosphere sediment.Microorganisms using sugar as the main carbon source was the main microorganism groups in the rhizosphere sediment of Acorus calamus,whereas microbes in the non-rhizo-sphere sediment mainly used ester and amine as carbon source.The carbon sources causing the metabolic differ-ences between non-rhizosphere sediment and rhizosphere sediment were D-Cellobiose,β-Methyl-D-Glucoside,Glu-cose-1-Phosphate,D-Galacturonic Acid,D-Glucosaminic Acid,Phenylethyl-amine,N-Acetyl-D-Glucosamine,D-Galactonic Acid y-Lactone,D-Mannitol and D,L-a-Glycerol.Analysis on the differences of functional diversity in microbial community between rhizosphere and nonrhizosphere sediment aims to reveal the mechanism of rhizosphere effect of aquatic macrophyte and provides scientific basis for improving the efficiency of pollutant removal by aquatic macrophyte.%为研究水生植物对沉积物微生物群落功能多样性的影响,采用Biolog-Eco微平板法分析非根际沉积物及菖蒲根际沉积物微生物群落功能多样性差异。结果表明:水生植物菖蒲的生长可大大提高沉积物中微生物的活性及多样性,菖蒲根际微生物总活性(AWCD)、微生物多样性指数(包括Shannon-Wiener 指数、丰富度指数及Pielou 均匀度指数)均显著高于非根际沉积物;菖蒲根际沉积物微生物主要是一些利用酯类及糖类作为碳源的微生物

  13. Biomass Conversion over Heteropoly Acid Catalysts

    KAUST Repository

    Zhang, Jizhe


    (Au/Cs2HPW12O40) that enabled the selective conversion of cellobiose to gluconic acid with a very high yield of 96.4% (Chapter II); we realized a direct oxidative conversion of cellulose to glycolic acid (yield of 49.3 %) in a water medium for the first time, by using a phosphomolybdic acid catalyst (chapter III); we found that a vanadium-substituted phosphomolybdic acid catalyst (H4PVMo11O40) is capable of converting various biomass-derived substrates to formic acid and acetic acid with high selectivity, and under optimized reaction conditions, high yield of formic acid (67.8%) can be obtained from cellulose (chapter IV); we discovered that the vanadium-substituted phosphomolybdic acids can also selectively oxidize glycerol, a low-cost by-product of biodiesel, to formic acid, and interestingly this conversion can be performed in highly concentration aqueous solution (glycerol: water = 50: 50), giving rise to exceptionally high conversion efficiency (chapter V). These results reveal that HPAs are useful and suitable catalysts for selective oxidation of biomass, and that the reaction pathway is largely determined by the type of addenda atom in the HPA catalyst. The optimization of the reaction conditions and processes in these systems are also discussed in this thesis.

  14. Heterologous Expression, Purification and Characterization of Thermo-stable 6-phosphate-β-glucosidase TteBglB%热稳定6-磷酸-β-葡萄糖苷酶TteBglB异源表达、分离纯化及酶学性质分析

    Institute of Scientific and Technical Information of China (English)

    刘晴; 张宇微; 张宇宏; 徐欣欣; 张伟; 刘波; 顾青


    6⁃phosphate⁃β⁃glucosidase (EC, an enzyme catalyzes 6⁃phosphate⁃glucoside compounds (such as 6⁃phosphate⁃cellobiose, 6⁃phosphate⁃cellulose oligosaccharides ) to produce 6⁃phosphate⁃glucose, plays an important role in carbon utilization for microbes. Thermoanaerobacter tengcongensis MB4, a strain of thermophilic anaerobic microorganism, is an excellent resource for thermo⁃stable protein. In this study, gene TtebglB was isolated from this strain and then heterologously expressed in E. coli BL21 ( DE3 ) . The optimum pH and temperature of TteBglB were pH 6.0 and 70℃, respectively, and the enzyme kept stable at a pH 4~10 at 70℃. Additionally, it was also confirmed that TteBglB belonged to glycosyl hydrolase family 1 ( GH1) and could act without Mn2+, Ni2+, Co2+, Fe2+ and other divalent metal ions. Using pNPβG6P as substrate, the values of Km , Kcat , and Vmax for this enzyme was 0.054 mmol/L, 81.47/min and 0.003 992 mmol/min, respectively, and specific activity of the enzyme reached to 18�093 U/mg.%6⁃磷酸⁃β⁃葡萄糖苷酶( EC催化6⁃磷酸-葡萄糖苷类化合物(如6⁃磷酸-纤维二糖、6⁃磷酸-纤维素寡糖)产生6⁃磷酸-葡萄糖而使纤维素完全分解,在微生物碳源利用过程中起重要作用。腾冲嗜热厌氧杆菌是一株嗜热厌氧微生物,提供了丰富的热稳定性蛋白基因资源。从该菌株中克隆编码6⁃磷酸⁃β⁃葡萄糖苷酶的基因TtebglB,并在E.coli BL21(DE3)进行了异源表达。结果表明,TteBglB催化反应的最适pH为6.0、最适温度为70℃,在pH 4~10或70℃时有着良好的稳定性;同时证实,TteBglB属于糖基水解酶家族1( GH1)成员,可不依赖Mn2+、Ni2+、Co2+或Fe2+等二价金属离子而发挥催化作用。以pNPβG6P为底物时,催化反应的Km为0.054 mmol/L,Kcat为81.47/min,Vmax为0.003992 mmol/min,酶比活为18.093 U/mg。

  15. Nanoconfinement Effects in Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Kung, Harold H. [Northwestern Univ., Evanston, IL (United States)


    neighboring Sn-O-Si bond. The resulting acidic silanol is active in epoxide ring opening and acetalization reactions. The open structure of the Sn center makes it accessible to larger molecules, including cellobiose which can be converted to 5-(hydroxymethyl)-furfural. The third structure is a support planted with functional group pairing of a known separation distance. Using a precursor molecule that contains a hydrolysable silyl ester bond, and making use of known chemistry to convert silanol groups into amino/pyridyl and phosphinyl groups, silica surfaces with carboxylic acid/silanol, carboxylic acid/amine, carboxylic acid/pyridine, and carboxylic acid/phosphine pairs can be constructed. The amino groups paired with carboxylic acid on such a surface is more active in the Henry reaction of 4-nitobenzaldehyde with nitromethane.

  16. Response of the grass-cutting ant Atta capiguara Gonçalves, 1944 (Hymenoptera: Formicidae to sugars and artificial sweeteners Resposta da saúva Atta capiguara Gonçalves, 1944 (Hymenoptera: Formicidae a açúcares e edulcorantes artificiais

    Directory of Open Access Journals (Sweden)

    Maria Aparecida Castellani Boaretto


    Full Text Available Using of toxic baits made of dehydrated citric pulp to control grass-cutting ants can lead to unsatisfactory results because of the low attractiveness of the substrate to worker ants. This work aimed to identify attractive substances, with potential for incorporation in a matrix of granulated baits for grass-cutting ants, among several kinds of sugars and substances used in artificial sweeteners. Experiments were carried out in mature nests of Atta capiguara (Hym.: Formicidae set in pasture. Studied substances were sucrose, fructose, soluble starch, raffinose, maltose, lactose, sorbose, cellobiose, arabinose, xylose, glucose, galactose, rhamnose, arabinose, melezitose, saccharine and cyclamate (at 5.0% w/v. Later, on maltose, xylose, sucrose, fructose and glucose solutions were included at 5.0%, 7.5%, 10.0% and 20.0% w/v, respectively. Cellulose rectangles were used as vehicle and number of rectangles carried into the colonies was evaluated. Carrying rates were very low with maximum means of 9.6% for lactose and 6.0% for arabinose and cyclamate, at the 5.0% concentration. No differences (P > 0.05 were observed relatively to the control (distilled water. No effects were detected for solution, concentration and for the interaction of these factors. Sugars and artificial sweeteners studied were not attractive to Atta capiguara workers, turning their inclusion as attractants in toxic ant baits not viable.O uso de iscas tóxicas, formuladas à base de polpa cítrica desidratada, para o controle de formigas cortadeiras de gramíneas pode levar a resultados insatisfatórios devido à baixa atratividade do substrato às operárias. Este trabalho foi realizado com o objetivo de identificar substâncias atrativas e com potencial para incorporação em matrizes de iscas granuladas para formigas cortadeiras de gramíneas, dentre diversos tipos de açúcares e edulcorantes artificiais. Os experimentos foram realizados em ninhos adultos de Atta capiguara Gon

  17. Fungal sporulation in two-stage cultivation%两步培养法测定真菌产孢营养需求

    Institute of Scientific and Technical Information of China (English)

    孙漫红; 高利; 刘杏忠; 王金利


    yeast extract were as carbon and nitrogen sources, respectively. It was revealed yeast extract supported abundant sporulation of T. viride by two-stage cultivation. Based on the new method, Z viride produced the most spores when the medium contained 2g carbon/L, C:N=10:1, and cellobiose/yeast as the carbon/nitrogen sources.

  18. Isolation and expeditious morphological, biochemical and kinetic characterization of propolis-tolerant ruminal bacteria Isolamento e caracterização expedita morfológica, bioquímica e cinética de bactérias ruminais tolerantes a própolis

    Directory of Open Access Journals (Sweden)

    Odimári Pricila Pires do Prado


    Full Text Available It was aimed in this work to evaluate bacterial strains tolerant to products based on propolis (LLOS through the isolation, morphological and biochemical characterization techniques in diets with roughage:concentrate ratio 100:0 and 50:50. For roughage diets, the products LLOSC1 and LLOSB3 were evaluated, and for 50:50% diets, the products LLOSC1, LLOSD1, LLOSA2, and LLOSC3, which differed in alcoholic concentrations (1, 2 and 3 and propolis (A, B, C and D concentrations. The ruminal liquid was anaerobically incubated at 39°C for 6 days in medium containing LLOS. After isolation, the strains were submitted to Gram staining and the bacterial growth was monitored by photospectrometer. It was evaluated the strain growth in the presence of the following subtracts: arabinose, cellulose, glucose, cellobiose, xylose, fructose, and lactose. In roughage diets, strains tolerant to LLOSC1 and LLOSB3 were similar to carbohydrates degradation, except lactose in which LLOSC1 was superior to strains tolerant to LLOSB3. For diets with 50:50 roughage:concentrate ratio, the products LLOSC3 and LLOSA2 stood out because they selected the highest number of strains able to degrade most of the tested carbohydrates. The results suggest that tolerance to propolis is higher in Gram-positive strains with several growth metabolic levels.Objetivou-se avaliar cepas bacterianas tolerantes a produtos à base de própolis pelas técnicas de isolamento, caracterização morfológica e bioquímica, em dietas com relação volumoso: concentrado de 100:0 e 50:50. Para dietas volumosas foram avaliados os produtos LLOSC1 e LLOSB3 e, para dietas 50:50% os produtos LLOSC1, LLOSD1, LLOSA2 e LLOSC3, diferentes quanto aos teores alcoólicos (1, 2 e 3 e as concentrações de própolis (A, B, C e D. O líquido ruminal foi incubado anaerobiamente a 39°C durante 6 dias em meio contendo LLOS. Após o isolamento, as cepas foram submetidas à coloração de Gram e o crescimento bacteriano foi

  19. Linking microbial carbon utilization with microbially-derived soil organic matter (United States)

    Kallenbach, Cynthia M.; Grandy, A. Stuart


    Soil microbial communities are fundamental to plant C turnover, as all C inputs eventually pass through the microbial biomass. In turn, there is increasing evidence that this biomass accumulates as a significant portion of stable soil organic matter (SOM) via physiochemical interactions with the soil matrix. However, when exploring SOM dynamics, these two processes are often regarded as discrete from one another, despite potentially important linkages between microbial C utilization and the fate of that biomass C as SOM. Specifically, if stable SOM is largely comprised of microbial products, we need to better understand the soil C inputs that influence microbial biomass production and microbial C allocation. Microbial physiology, such as microbial growth efficiency (MGE), growth rate and turnover have direct influences on microbial biomass production and are highly sensitive to resource quality. Therefore, the importance of resource quality on SOM accumulation may not necessarily be a function of resistance to decay but the degree to which it optimizes microbial biomass production. To examine the relationship between microbial C utilization and microbial contributions to SOM, an ongoing 15-mo incubation experiment was set up using artificial, initially C- and microbial-free soils. Soil microcosms were constructed by mixing sand with either kaolinite or montmorillonite clays followed with a natural soil microbial inoculum. For both soil mineral treatments, weekly additions of glucose, cellobiose, or syringol are carried out, with an additional treatment of plant leachate to serve as a reference. This simplified system allows us to determine 1) if, in absence of plant-derived C, chemically complex SOM similar to natural soils can accumulate through the production of microbial residues and 2) how differences in C utilization of simple substrates, varying in energy yields, influence the quantity and chemistry of newly formed SOM. Over the course of the incubation, MGE

  20. Genome Sequence of the Plant Growth Promoting Endophytic Bacterium Enterobacter sp. 638

    Energy Technology Data Exchange (ETDEWEB)

    Taghavi, S.; van der Lelie, D.; Hoffman, A.; Zhang, Y.-B.; Walla, M. D.; Vangronsveld, J.; Newman, L.; Monchy, S.


    Enterobacter sp. 638 is an endophytic plant growth promoting gamma-proteobacterium that was isolated from the stem of poplar (Populus trichocarpa x deltoides cv. H11-11), a potentially important biofuel feed stock plant. The Enterobacter sp. 638 genome sequence reveals the presence of a 4,518,712 bp chromosome and a 157,749 bp plasmid (pENT638-1). Genome annotation and comparative genomics allowed the identification of an extended set of genes specific to the plant niche adaptation of this bacterium. This includes genes that code for putative proteins involved in survival in the rhizosphere (to cope with oxidative stress or uptake of nutrients released by plant roots), root adhesion (pili, adhesion, hemagglutinin, cellulose biosynthesis), colonization/establishment inside the plant (chemiotaxis, flagella, cellobiose phosphorylase), plant protection against fungal and bacterial infections (siderophore production and synthesis of the antimicrobial compounds 4-hydroxybenzoate and 2-phenylethanol), and improved poplar growth and development through the production of the phytohormones indole acetic acid, acetoin, and 2,3-butanediol. Metabolite analysis confirmed by quantitative RT-PCR showed that, the production of acetoin and 2,3-butanediol is induced by the presence of sucrose in the growth medium. Interestingly, both the genetic determinants required for sucrose metabolism and the synthesis of acetoin and 2,3-butanediol are clustered on a genomic island. These findings point to a close interaction between Enterobacter sp. 638 and its poplar host, where the availability of sucrose, a major plant sugar, affects the synthesis of plant growth promoting phytohormones by the endophytic bacterium. The availability of the genome sequence, combined with metabolome and transcriptome analysis, will provide a better understanding of the synergistic interactions between poplar and its growth promoting endophyte Enterobacter sp. 638. This information can be further exploited to

  1. Analysis of monosaccharides in the saffron corm glycoconjugate by capillary electrophoresis%毛细管电泳法分析藏红花植物细胞多糖中单糖组成

    Institute of Scientific and Technical Information of China (English)

    马海宁; 华玉娟; 屠春燕; 袁丽红; 韦萍


    以对甲氧基苯胺为衍生试剂,采用毛细管电泳法分析了藏红花植物细胞多糖中的单糖组成.对衍生条件进行了优化,并对毛细管分离条件进行了系统的研究.衍生反应在醋酸含量9.5% (v/v)、80℃下反应2h的衍生产率最大,衍生产物紫外检测波长234 nm.在优化的毛细管电泳分离条件(未涂层熔融石英毛细管柱(60 cm(有效长度50 cm)×50μm),柱温25℃,电压20 kV,使用350 mmol/L硼酸电解液(pH 10.21),压力(3.447 5 kPa)进样5s)下,基线分离了11种结构相近的醛糖(来苏糖、木糖、核糖、葡萄糖、甘露糖、半乳糖、鼠李糖、纤维二糖、麦芽糖、乳糖)、酮糖(果糖)的衍生产物.应用该方法定量检测了藏红花植物细胞多糖水解物中糖的成分,各糖的回收率为94.3%~105.4%,相对标准偏差为3.3%~4.6%.%The monosaccharides in the saffron corm glycoconjugate were separated by capillary electrophoresis ( CE) coupled with pre-column derivatization. 4-Methoxyaniline was used as derivatization reagent. The derivatization and CE separation conditions were investigated. The ultraviolet detection wavelength was 234 nm. The maximum yield of this derivatization reaction was obtained under the presence of 9.5% (v/v) acetic acid at 80 t for 2 h. An uncoated fused-silica capillary of 50 μm I. D. And 50/60 cm length (effective length/total length) was employed, and a pressure injection (3. 447 5 kPa, 5 s) was applied. The baseline separation of 11 monosaccharides and disaccharides (lyxose, xylose, ribose, glucose, mannose, galactose, rhamnose, cellobiose, maltose, lactose, fructose) was reached at 25 t, 20 kV of separation voltage and with 350 mmol/L boric acid (pH 10. 21) as running buffer. The developed method has been successfully applied to quantitatively determine the components of saffron corm glycoconjugate , and the results showed that the recovery of each monosaccharide was in the range of 94. 3% - 105. 4%, the

  2. 稻草秸秆超低酸水解及水解产物分析%Hydrolysis of Rice Straw with Extremely Dilute Acid and Products Analysis

    Institute of Scientific and Technical Information of China (English)

    王为国; 李自豪; 覃远航; 吕仁亮; 张俊峰; 王存文


    The hydrolysis of rice straw with extremely dilute sulfuric acid(0.05%(w)) was conducted.It was found that the optimal solid content and stirring speed for the rice straw hydrolysis were 0.050 0 g/mL and 700 r/min,respectively.The change of the hydrolysis with time was investigated in the temperature range of 160-210 ℃.The results indicated that,in the hydrolysis,there was a single-peak of reducing sugar content at 160 ℃,in the temperature range of 170-200 ℃there was a double-peak,and a single-peak reappeared at 210 ℃.The reason was that the reducing sugar concentration from the hydrolysis of hemicellulose and cellulose showed two peaks,and the peaks superimposed or separated as the hydrolysis temperature changed.The hydrolysis products and solid residues were characterized by means of FTIR and HPLC.The results showed that,most of hemicellulose hydrolyzed and disappeared,the structure of the lignin was destroyed to a certain extent,the polymerization degree of the cellulose decreased greatly but its crystallinity was still high.The main hydrolysis products were cellotriose,cellobiose,glucose,xylose,methylglyoxal and some unknown compounds.%用0.05%(w)的稀硫酸对湖北省稻草秸秆进行水解,考察了混合原料中固体含量和搅拌转速对稻草秸秆水解的影响.实验结果表明,最佳秸秆颗粒的质量浓度为0.050 0 g/mL,最佳转速为700 r/min.研究了稻草秸秆在160~ 210℃内的水解趋势.研究结果表明,160℃时,稻草秸秆水解得到的还原糖含量随水解的进行呈单峰趋势;在170~ 200℃时呈双峰趋势;当温度达到210℃时又呈现单峰趋势,这与稻草秸秆中纤维素和半纤维素水解得到的两个还原糖峰随水解的进行呈现叠加和分离有关.表征固体残渣并分析水解产物组成的结果显示,水解后半纤维素大部分水解,木质素的结构在一定程度上被破坏,纤维素的聚合度急剧降低但结晶度仍较高;水解产物主要为

  3. Sugar-Based Ethanol Biorefinery: Ethanol, Succinic Acid and By-Product Production

    Energy Technology Data Exchange (ETDEWEB)

    Donal F. Day


    The work conducted in this project is an extension of the developments itemized in DE-FG-36-04GO14236. This program is designed to help the development of a biorefinery based around a raw sugar mill, which in Louisiana is an underutilized asset. Some technical questions were answered regarding the addition of a biomass to ethanol facility to existing sugar mills. The focus of this work is on developing technology to produce ethanol and valuable by-products from bagasse. Three major areas are addressed, feedstock storage, potential by-products and the technology for producing ethanol from dilute ammonia pre-treated bagasse. Sugar mills normally store bagasse in a simple pile. During the off season there is a natural degradation of the bagasse, due to the composting action of microorganisms in the pile. This has serious implications if bagasse must be stored to operate a bagasse/biorefinery for a 300+ day operating cycle. Deterioration of the fermentables in bagasse was found to be 6.5% per month, on pile storage. This indicates that long term storage of adequate amounts of bagasse for year-round operation is probably not feasible. Lignin from pretreatment seemed to offer a potential source of valuable by-products. Although a wide range of phenolic compounds were present in the effluent from dilute ammonia pretreatment, the concentrations of each (except for benzoic acid) were too low to consider for extraction. The cellulosic hydrolysis system was modified to produce commercially recoverable quantities of cellobiose, which has a small but growing market in the food process industries. A spin-off of this led to the production of a specific oligosaccharide which appears to have both medical and commercial implications as a fungal growth inhibitor. An alternate use of sugars produced from biomass hydrolysis would be to produce succinic acid as a chemical feedstock for other conversions. An organism was developed which can do this bioconversion, but the economics of

  4. Spathaspora passalidarum突变株U-30木糖乙醇发酵条件研究%Xylose-ethanol Fermentation Conditions for Spathaspora Passalidarum Mutant U-30

    Institute of Scientific and Technical Information of China (English)

    黄文连; 陈叶福; 付更新; 杨金龙; 郭学武; 肖冬光


    Spathaspora passalidarum是近来发现的一种能天然利用木糖产乙醇的酵母菌,其发酵木糖速度比葡萄糖快,而且能混合发酵木糖、葡萄糖和纤维二糖.主要从氮源种类及浓度、接种量、转速及装液量、初始pH值等方面对Spathaspora passalidarum突变株U-30木糖乙醇发酵进行了研究,得到的优化摇瓶培养基组成为:初始木糖浓度为100 g/L,氮源为15 g/L的玉米浆;发酵条件为:温度30℃、接种量7%(v/v)、初始pH5.5、摇床转速120 r/min、装液量100 mL/250 mL.在此优化发酵培养基和发酵条件下,U-30发酵液中的乙醇浓度为39.04 g/L,乙醇得率为0.40 g/g,乙醇生产速率为0.325 g/L·h.以摇瓶中所得到的发酵条件为基础,在5 L发酵罐进行初步放大实验,发酵时间明显缩短为72 h,乙醇浓度达到39.875 g/L,乙醇生产速率为0.553 g/L·h.%Spathaspora passalidarum, a recently isolated native xylose-fermenting yeast strain, can ferment xylose faster than glucose, and can co-ferment glucose, xylose, and cellobiose. In this study, the fermentation conditions of a Spathaspora passalidarum mutant U-30 were investi-gated, including xylose content, the type and the content of nitrogen source, inoculation volume, shaking rate, and initial pH. The optimum fer-mentation conditions were summed up as follows:xylose of 100 g/L, corn steep of 15 g/L, temperature at 30℃, inoculation size of 7%(v/v), initial pH of 5.5, shaking rate of 120 r/min, and liquid volume 100 mL/250 mL. Under the above conditions, 39.04 g/L of ethanol was obtained, ethanol yield was 0.40 g/g, xylose utilization rate was about 100%, and ethanol productivity was 0.325 g/L·h. Further, the optimum fermenta-tion conditions in the shake flask was enlarged to 5 L fermentor. And the results showed that the fermentation time significantly shortened to 72 h, ethanol concentration reached up to 39.875 g/L, and ethanol productivity was as high as 0.553 g/L·h.

  5. Electron Scattering by biomass molecular fragments (United States)

    Lima, Marco


    The replacement of fossil fuels by biofuels from renewable sources may not be a definite answer for greenhouse gas emissions problems, but it is a good step towards a sustainable energy strategy. Few per cent of ethanol is being mixed to gasoline in many countries and in some of them, like Brazil, a very aggressive program has been developed, using, in large scale, flex fuel engines that can run with any mixture of gasoline and ethanol, including 100% ethanol. Important points are how to produce ethanol in a sustainable way and with which technology? Biomass is a good candidate to enhance the first generation (produced from Corn in USA and from sugarcane in Brazil) production towards the so-called second-generation ethanol, since it has cellulose and hemicellulose as source of sugars. In order to liberate these sugars for fermentation, it is important to learn how to separate the main components. Chemical routes (acid treatment) and biological routes (enzymatic hydrolysis) are combined and used for these purposes. Atmospheric plasmas can be useful for attacking the biomass in a controlled manner and low energy electrons may have an important role in the process. Recently, we have been studying the interaction of electrons with lignin subunits (phenol, guaiacol, p-coumaryl alcohol), cellulose components, β-D-glucose and cellobiose (β(1-4) linked glucose dimer) and hemicellulose components [2] (β-D-xylose). We also obtained results for the amylose subunits α-D-glucose and maltose (α(1-4) linked glucose dimer). Altogether, the resonance spectra of lignin, cellulose and hemicellulose components establish a physical-chemical basis for electron-induced biomass pretreatment that could be applied to biofuel production. In order to describe a more realistic system (where molecules are ``wet''), we have obtained the shape resonance spectra of phenol-water clusters, as obtained previously from elastic electron scattering calculations. Our results, obtained in a simple

  6. 糠醛渣/木薯渣混合底物同步糖化发酵转化乙醇研究%Ethanol Production from Mixture of Furfural Residues and Cassava Residues by Simultaneous Saccharification and Fermentataion

    Institute of Scientific and Technical Information of China (English)

    吉骊; 赵鹏翔; 游艳芝; 卜令习; 蒋建新


    Simultaneous saccharification and fermentation ( SSF ) of furfural residues ( FR ) pretreated with green liquor and hydrogen peroxide ( GL-H2 O2 ) mixed with cassava residues ( CR ) with the addition of Sapindus mukurossi saponin to product ethanol was investigated in present study. The SSF was optimized by changing the substrate concentration, dosage of cellulase and the addition of saponin, and the corresponding concentrations of ethanol and by-products were analyzed. The results indicated that 56. 5% furfural lignin was removed after 5 g/L substrate was treated by 0. 6 g/g H2 O2 and 2 mL/g GL, at 80 ℃ for 3 h. The ethanol concentration after SSF was 56. 6 g/L, which was equivalent to the theoretical ethanol yield of 86. 3%, as the mixture of pretreated FR and cassava residues with a mass ratio of 2∶1 were fermented with the aid of 0. 5 g/L Sapindus mukurossi saponin, 12 FPU/g cellulose, and 15 IU/g cellobiose enzyme, for 120 h. Moreover, the amounts of cellulose and generation of by-products were declined when the Sapindus mukurossi saponin was added, and the fluctuation of glycerol production was minimized.%研究了糠醛渣( FR)经不同强度绿液-过氧化氢预处理脱木质素后,与木薯渣( CR)混合进行同步糖化发酵生产乙醇,通过改变原料底物浓度、纤维素酶用量和添加无患子表面活性剂来优化混合底物同步糖化发酵条件,并分析了发酵过程中乙醇和副产物的浓度变化。结果表明,在糠醛渣预处理条件为:底物质量浓度5 g/L、温度80℃、H2 O2用量为0.6 g/g、绿液用量为2 mL/g(以糠醛渣计)预处理时间3 h,在此条件下糠醛渣木质素脱除率可达56.5%。同步糖化发酵产乙醇条件为无患子皂素表面活性剂添加量0.5 g/L,纤维素酶用量12 FPU/g,纤维二糖酶用量15 IU/g,预处理后的糠醛渣与木薯渣混合作底物(质量比为2∶1),底物质量浓度200 g/L时,发酵120 h最终乙醇质量浓度可达56.6 g/L,乙醇得率为86

  7. Caracterização de bactérias dos gêneros Mima e Herella (Tribo Mimeae, DeBord, 1942: 1 Propriedade morfo-bioquímicas e sensibilidade aos antibióticos

    Directory of Open Access Journals (Sweden)

    Altair A. Zebral


    % lactose and irregularly utilize rhamnose and cellobiose; in the synthetic base media the strains produced acid from lactose and have the same sugar oxidations as described before. Mima polymorpha was oxidase-negative and failed to utilize the carbohidrates tested in either the complex nitrogenous media or the synthetic base media. Mima polymorpha var. oxidans was oxidase-positive and failed to utilize the carbohydrates tested. The Herellea vaginicola and Mima polymorpha was very susceptible to gabromycin, knamycin, neomycin, colistin and the former was very sensitive to chloranphenicol and rovamycin. Mima polymorpha var. oxidans presented high sensibility to kanamycin, neomycin, colistin, chloranphenicol and nalidixic acid. With the results obtained by the sensibility of strains to the 1 and 0,1 unity of penicillin by milliliter, it was impossible to separate the oxidase-positive and oxidase-negative strains in discordance with the data obtained by Baumann, Dodoroff & Stanier (1968a.

  8. Enzyme electrode configurations : for application in biofuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang Xiaoju


    ; their effects on the electrode performance were then investigated. It is proposed that the {eta}-{eta} interaction between the PSS{sup -} and the hydrophobic substrate-binding pocket in the vicinity of the T1 Cu site results in a favorable location of the conducting polymer chain of PEDOT-PSS close to the T1 Cu site and thus facilitates the DET of ThL within this particular architecture. The flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase from Glomerella cingulata (GcGDH) and cellobiose dehydrogense from Corynascus thermophuilus (CtCDH) have been studied to construct different enzyme electrode configurations as bioanodes towards biofuel cell applications. For GcGDH, six Os-containing polymers, whose redox potentials range across a broad potential window between +15 and +489 mV vs. NHE, were used to 'wire' the GcGDH on the graphite electrodes to catalyze the oxidation of glucose. The ratio of GcGDH:Os-polymer in the overall loading onto the electrode surface significantly affected the catalytic performance of the enzyme electrode on the glucose oxidation. Both the Os-polymer and the GcGDH:Os-polymer ratio were optimized for obtaining the maximum current density; a high current density of 493 {mu}A/cm{sup 2} for 30 mM glucose was produced by a GcGDH/Os c modified electrode. DET type biocatalysis of CtCDH on lactose (and glucose) oxidation was accomplished on Au nanoparticle (AuNP) structured electrode. The haem site in the CtCDH enzyme functions as a 'built-in' mediator for communicating the electron transfer between the FAD site and the AuNP surface. The redox potential of the haem site in CtCDH was determined to be E{sub 1/2} = -122 mV vs. Ag/AgCl/KCl(s) (75 mV vs. NHE). The CtCDH/AuNP/Au bioanode can generate a maximum current response for lactose with I{sub max} = 43.3{+-}1.5 ({mu}A/cm{sup 2}) or for glucose with I{sub max} = 31.2{+-}2.3 ({mu}A/cm{sup 2}). The DET type biocatalysis of CtCDH works most efficiently in a more neutral

  9. Kinetics of high-Level of ß-glucosidase production by a 2-deoxyglucose-resistant mutant of Humicola lanuginosa in submerged fermentation Cinética de produção de ß-glucosidase por um mutante de Hemicola lanuginosa resistente a 2-deoxiglucose em fermentação submersa

    Directory of Open Access Journals (Sweden)

    Syed Ali Imran Bokhari


    Full Text Available A 2-deoxyglucose-resistant mutant (M7 of Humicola lanuginosa was obtained by exposing conidia to γ-rays and permitting expression in broth containing 0.6% 2-deoxyglucose (DG and cellobiose (1% before plating on DG esculin-ferric ammonium citrate agar medium from which colonies showing faster and bigger blackening zones were selected. Kinetic parameters for enhanced ß-glucosidase (BGL synthesis by M7 were achieved when corncobs acted as the carbon source. The combination between corncobs and corn steep liquor was the best to support higher values of all product formation kinetic parameters. Effect of temperature on the kinetic and thermodynamic attributes of BGL production equilibrium in the wild organismand M7was studied using batch process at eight different temperatures in shake-flask studies. The best performance was found at 45ºC and 20 g L-1 corncobs in 64 h. Both growth and product formation (17.93 U mL-1 were remarkably high at 45ºC and both were coupled under optimum working conditions. Product yield of BGL from the mutant M7 (1556.5 U g-1 dry corncobs was significantly higher than the values reported on all fungal and bacterial systems. Mutation had thermo-stabilization influence on the organism and mutant required lower activation energy for growth and lower magnitudes of enthalpy and entropy for product formation than those demanded by the wild organism, other mesophilic and thermo-tolerant organisms. In the inactivation phase, the organisms needed lower values of activation energy, enthalpy and entropy for product formation equilibrium, confirming thermophilic nature of metabolic network possessed by the mutant organism.Um mutante de Hemicola lanuginosa resistente a 2-deoxiglucose(M7 foi obtido através de exposição de conídios a raios γ, permitindo a expressão em caldo contendo 0,6% de 2-deoxiglucose (DG e celobiose (1% antes da semeadura em ágar DG esculina citrato de ferro amoniacal, da qual foram selecionadas as col

  10. Research on the Nature of Thermophilic Anaerobic Ethanol Producer Thermo anaerobacter sp DF3 in Petroleum Reservoirs%一株产乙醇嗜热厌氧油藏微生物ThermoanaerobacterspDF3的性质研究

    Institute of Scientific and Technical Information of China (English)

    兰贵红; 邢钰; 曹毅; 乔代蓉; 邹长军; 邓宇; 张辉; 尹小波


    produced. Its growth temperature was 45-78 and 65 ℃ was the optimum. Many substances could be used as carbon sources, including glucose, xylose, fructose, ribose, mannose, arabinose, sucrose, galactose, lactose, cellobiose, melizitose, raffinose-, and starch. The similarity between strain DF3 and T. pseudoethanolicus achieved 99.7%. The main product of glucose and xylose fermentation was ethanol. After the culture plan was optimized,the final concentration of ethanol was 2.0 g/L. [Conclusion] It was proved through experiments that the strain DF3 was one of the strains with higher activity to produce ethanol at present and it could produce 2.0 g/L ethanol from xylose metabolization at 65 ℃. It has been demonstrated that DF3 is one of the known strains with high-production to produce ethanol,for example, 2.0 g/L ethano at 65℃. Presently, all the high-yield ethanol can be produced from metabolic xylose strains of metabolic xylose were isolat- ed by foreign countries, therefore, isolation of strain DF3 has provided an excellent original strain for studying ethanol production from lignocellulose in China.

  11. 高效协同酶解中性汽爆玉米秸秆的工艺优化%Optimization of enzymatic saccharification process for neutral steam exploded corn stover

    Institute of Scientific and Technical Information of China (English)

    钟健; 杨敬; 钞亚鹏; 武改红; 贾文娣; 张国青; 石家骥; 孙艳; 钱世钧


    Abstract: Corn stover is one of the major agricultural residues in China and could be potential source for ethanol production by saccharification process. But the problem in current process is higher cost for pretreatment and enzymatic saccharification of corn stover. In this paper, four kinds of cellulase candidates ( volume ratio 6 : 4) were optimized based on the best synergistic effect for the degradation of neutral steam exploded corn stover, and Trichoderma L8 and Aspergillus niger were filtered. Then, the effect of enzymes (xylanase, /?-glucosidase, /?-glucanase, laccase, manganese peroxidase) and non-enzyme factors (polyethyleneglycol (PEG) -4000, Tween-80, bovine serum albumin) on saccharification efficiency was evaluated to get a mixed enzyme system which could make steam exploded corn stove high efficient synergistic degradation, and to obtain suitable conditions of saccharification process. The results showed that only xylanase in selected enzymes had positive effects on the saccharification, while for non-enzyme factors polyethyleneglycol (PEG) -4000 and Tween-80 could enhance the saccharification rates. The optimized enzyme was a mixture of Trichoderma L8 cellulase, β-glucosidase from Aspergillus niger andxylase, and the optimized conditions of enzymatic degradation condition for one gram neutral steam exploded corn stover (10%, W/V) as follows: 10FPU cellulase, plus 1000IU xylanase and 0. 05 g PEG-4000. After incubation at 50℃ and 150 r ? Min-1 for 144 h, the final concentration of cellobiose, glucose, and xylose were 8. 4 g ? L-1, 25.1 g ? L-1, and 15. 5 g ? L-1, and the final conversion of total biomass, cellulose and hemicellulose were 71.1%, 81.5% and 55.3%, respectively. Observation of scanning electron microscope indicated that most the free fiber disappeared within 24 h. Some of the partially destroyed structures in the pretreatment process were also degested slowly with time prolonged.%玉米秸秆是我国主要的农业废弃物之一,

  12. Ethanol fermentation characteristics of thermotolerant Issatchenkia orientalis%耐高温东方伊萨酵母乙醇发酵特性

    Institute of Scientific and Technical Information of China (English)

    王风芹; 汪媛媛; 陶西; 张瑞; 谢慧; 宋安东


    As a renewable and clean energy, bio-ethanol has been received widespread attention in recent years. Ethanol fermentation at high temperature can reduce the consumption of cooling water and energy in the process of fermentation, it also solve the problems caused by saccharification and uncontrolled fermentation temperature, resulting in the simultaneous saccharification and fermentation of cellulosic ethanol. Therefore, the fermentation period can be shorten and the production cost be reduced. A thermotolerant yeast strain HN-1 isolated from rotting tobacco leaves was identified and its ethanol fermentation characteristics was investigated in this study. The phylogenetic analysis indicated that phylogenetic strain HN-1 behaved likeIssatchenkia orientalis.This strain useed glucose and D-fructose but could not use xylose, maltose, lactose, sucrose, starch and cellobiose as sole carbon source to produce ethanol. When HN-1 was cultivated in 50 g/L glucose liquid medium, the highest biomass was obtained at 35 and 38℃. Increasing the cultivation temperature to 42 and 45℃ decreased the biomass growth and intensified the cell death. There was no major difference between ethanol productions (21.43~23.12 g/L) fermented at 35~45℃ from 50 g/L glucose. When increasing the fermentation temperature, the ethanol productivity was increased from 0.31 g/(L·h) at 35℃ to 0.65 g/(L·h) at 45℃ after 24h fermentation. The fermentation time was shortened. When the fermentation was conducted at 38℃, 28.77, 43.21, 58.19 and 59.53 g/L ethanol were produced from 60, 90, 120 and 150 g/L glucose with yields of 0.457, 0.468, 0.460 and 0.386 g/g and productivities of 0.38, 0.39, 0.48 and 0.40 g/(L·h) The results also indicated that fed batch fermentation could not enhance the ethanol production. Strain HN-1 could utilize corn straw hydrolysate with 43.08 g/L glucose and 27.13 g/L xylose to produce 20.74 g/L ethanol. The glucose conversion rate was 0.468 g/g, which was 91.6% of the

  13. 甘蔗间作玉米对甘蔗根际微生物代谢功能多样性的影响%Effects of sugarcane and maize intercropping on sugarcane rhizosphere microbe metabolic function diversity

    Institute of Scientific and Technical Information of China (English)

    郑亚强; 李正跃; 陈斌; 张立敏; 杨进成; 杨坚; 高锐; 陈亮新; 董雪梅; 孙继红; 肖关丽


    Sugarcane-maize intercropping is an important planting pattern in sugarcane production area, which is in favor of sugarcane production benefit and land use efficiency increases, and pesticide control of sugarcane. However, the effects of sugarcane and maize intercropping on sugarcane rhizosphere microbe community have less been reported. In this study, a plot experiment was carried out in sugarcane fields in Yuanjiang and Longchuan Counties of Yunnan Province to investigate the microbe activities, metabolic function diversity and carbon sources utilization by using Biolog techniques. The results showed that compared with monocultured sugarcane at Longchuan County experimental site, Shannon index, Simpson index, McIntosh index and evenness index of Shannon and McIntosh of microbial communities in rhizosphere soils of intercropped sugarcane increased by 7.08%, 11.25%, 63.16%, 1.31% and 2.26%, respectively. Then, compared with sugarcane monoculture in Yuanjiang County, the above diversity indices increased by 10.58%, 48.40%, 43.42%, 0.20%, and 1.65%, respectively, in intercropped sugarcane treatment. This suggested that intercropping increased the metabolic function diversity of rhizosphere soil microbes. Although the utilization of carbon resources increased in intercropping systems, it was lower at Yuanjiang experimental site than that at Longchuan experimental site. Compared with monoculture, the utilization of carbohydrate, amino acids, polymeric substances, amines, carboxylic acids and phenolic acids increased by 141.71%, 50.53%, 62.38%, 92.82%, 43.21% and 6.30%, respectively, at Yuanjiang experimental site. Correspondingly, the variables increased, respectively by 42.90%, 51.50%, 33.30%, 42.64%, 16.72% and 24.47% at Longchuan experimental site. Moreover, the utilization of D-cellobiose, D,L-α-glycerol phosphate, D-galactonic acid-γ-lactone, L-serine, Glycyl-L-Glutamine and 2-Hydroxy benzoic acid increased by over 100% at both of Yuanjiang and Longchuan