Production of xylan-degrading enzymes by a Trichoderma harzianum strain

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Cacais André O.Guerreiro

2001-01-01

Full Text Available Trichoderma harzianum strain 4 produced extracellular xylan-degrading enzymes, namely beta-xylanase, beta-xylosidase and alpha-arabinofuranosidase, when grown in liquid medium cultures containing oat spelt xylan as inducer. Cellulase activity was not detected. The pattern of xylan-degrading enzymes induction was influenced by the form of xylan present in the medium. They were detected in different incubation periods. Electrophoretic separation of the proteins from liquid culture filtrates by SDS-PAGE showed a variety of bands with high and low molecular weights.

Altering the selection capabilities of common cloning vectors via restriction enzyme mediated gene disruption

Science.gov (United States)

2013-01-01

Background The cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice, two phase cloning experiments and mutagenesis experiments, issues that result in additional unnecessary cloning steps, in which the DNA needs to be subcloned into a vector with a suitable selectable marker. Results We have used restriction enzyme mediated gene disruption to modify the selectable marker gene of a given vector by cloning a different selectable marker gene into the original marker present in that vector. Cloning a new selectable marker into a pre-existing marker was found to change the selection phenotype conferred by that vector, which we were able to demonstrate using multiple commonly used vectors and multiple resistance markers. This methodology was also successfully applied not only to cloning vectors, but also to expression vectors while keeping the expression characteristics of the vector unaltered. Conclusions Changing the selectable marker of a given vector has a number of advantages and applications. This rapid and efficient method could be used for co-expression of recombinant proteins, optimisation of two phase cloning procedures, as well as multiple genetic manipulations within the same host strain without the need to remove a pre-existing selectable marker in a previously genetically modified strain. PMID:23497512

Using heavy-ion mutagenesis technology to select cellulose enzyme vitality of mutants of Aspergillium niger

International Nuclear Information System (INIS)

Tang Jiahui; Yang Fumin; Wang Shuyang

2012-01-01

In order to improve the cellulose ion beam at 20, 40, 60, 80, 100, 120Gy and 140 enzyme vitality of Aspergillus niger (=AS3.316), heavy Gy doses was used for inducing mutation. Higher cellulose enzyme vitality strains were screened through the primary screening and secondary screening. The result showed that 5 mutants T2-1, T3-1, T5-1, T6-3, T6-4 were selected, and T6-4 had the highest cellulose enzyme activity. The activity of filter paper cellulose enzyme, endo-glucanase, exo-glucanase and 13-glucosidase of T6-4 was 61.3, 116.2, 29.9 U/mL and 35.9 U/mL respectively. Compared with the original A. niger (=AS3.316), the cellulose enzyme activity was increased by 3.5, 3.78, 2.76 and 2.52 times in turn. The activity of cellulose enzyme of the rest mutants sorted from strong to the weak were T6-3T5-1T3-1T2-1. The dose at 120 Gy showed the best mutagenesis effect. Mutants had different degree of changes in the genetic stability, but overall, the performance showed relatively stable

Identification of enzymes and quantification of metabolic fluxes in the wild type and in a recombinant Aspergillus oryzae strain

DEFF Research Database (Denmark)

Pedersen, Henrik; Carlsen, Morten; Nielsen, Jens Bredal

1999-01-01

Two alpha-amylase-producing strains of Aspergillus oryzae, a wild-type strain and a recombinant containing additional copies of the alpha-amylase gene, were characterized,vith respect to enzyme activities, localization of enzymes to the mitochondria or cytosol, macromolecular composition...

Evaluation of Leishmania (Leishmania chagasi strains isolated from dogs originating from two visceral leishmaniasis-endemic areas in Brazil using multilocus enzyme electrophoresis

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Carlos Eduardo Ribeiro Coutinho

2011-10-01

Full Text Available INTRODUCTION: Domestic dogs are the most important reservoir in the peridomestic transmission cycle of Leishmania (Leishmania chagasi. The genetic variability of subpopulations of this parasite circulating in dogs has not been thoroughly analyzed in Brazil, even though this knowledge has important implications in the clinical-epidemiological context. METHODS: The objective of this study was to evaluate and compare the phenotypic variability of 153 L. chagasi strains isolated from dogs originating from the municipalities of Rio de Janeiro (n = 57 and Belo Horizonte (n = 96, where the disease is endemic. Strains isolated only from intact skin were selected and analyzed by multilocus enzyme electrophoresis using nine enzyme systems (6PG, GPI, NH1 and NH2, G6P, PGM, MDH, ME, and IDHNADP. RESULTS: The electrophoretic profile was identical for all isolates analyzed and was the same as that of the L. chagasi reference strain (MHOM/BR/74/PP75. Phenetic analysis showed a similarity index of one for all strains, with the isolates sharing 100% of the characteristics analyzed. CONCLUSIONS: The results demonstrate that the L. chagasi populations circulating in dogs from Rio de Janeiro and Belo Horizonte belong to a single zymodeme.

Improvement of Aspergillus niger 55, a raw corn meal saccharifying enzyme hyperproducer, through mutation and selective screening techniques

International Nuclear Information System (INIS)

Oh, S.H.; O, P.S.

1991-01-01

Mutation experiments were performed to select the mutant of Aspergillus niger 55, which had lost almost all the ability to produce transglucosidases but retained that of high productivity of raw meal saccharifying enzyme, by means of successive induction with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG), ultraviolet(UV) light, and γ-rays. Also, we used the mutant enrichment techniques, such as liquid culture-filtration procedure and differential heat sensitivity of conidia, in order to increase the possibility of obtaining a mutant. The glucoamylase productivity of mutant PFST-38 was 11 times higher than that of the parent strain. The mutant PFST-38 was morphologically identical to the parent strain, except for the size of conidia, the tendency to form conidia and the length of conidiophore. Asp. niger mutant PFST-38 appeared to be useful for the submerged production of the raw corn meal saccharifying enzyme

Diversity of Ligninolytic Enzymes and Their Genes in Strains of the Genus Ganoderma: Applicable for Biodegradation of Xenobiotic Compounds?

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Giselle Torres-Farradá

2017-05-01

Full Text Available White-rot fungi (WRF and their ligninolytic enzymes (laccases and peroxidases are considered promising biotechnological tools to remove lignin related Persistent Organic Pollutants from industrial wastewaters and contaminated ecosystems. A high diversity of the genus Ganoderma has been reported in Cuba; in spite of this, the diversity of ligninolytic enzymes and their genes remained unexplored. In this study, 13 native WRF strains were isolated from decayed wood in urban ecosystems in Havana (Cuba. All strains were identified as Ganoderma sp. using a multiplex polymerase chain reaction (PCR-method based on ITS sequences. All Ganoderma sp. strains produced laccase enzymes at higher levels than non-specific peroxidases. Native-PAGE of extracellular enzymatic extracts revealed a high diversity of laccase isozymes patterns between the strains, suggesting the presence of different amino acid sequences in the laccase enzymes produced by these Ganoderma strains. We determined the diversity of genes encoding laccases and peroxidases using a PCR and cloning approach with basidiomycete-specific primers. Between two and five laccase genes were detected in each strain. In contrast, only one gene encoding manganese peroxidase or versatile peroxidase was detected in each strain. The translated laccases and peroxidases amino acid sequences have not been described before. Extracellular crude enzymatic extracts produced by the Ganoderma UH strains, were able to degrade model chromophoric compounds such as anthraquinone and azo dyes. These findings hold promises for the development of a practical application for the treatment of textile industry wastewaters and also for bioremediation of polluted ecosystems by well-adapted native WRF strains.

Diversity of Ligninolytic Enzymes and Their Genes in Strains of the Genus Ganoderma: Applicable for Biodegradation of Xenobiotic Compounds?

Science.gov (United States)

Torres-Farradá, Giselle; Manzano León, Ana M.; Rineau, François; Ledo Alonso, Lucía L.; Sánchez-López, María I.; Thijs, Sofie; Colpaert, Jan; Ramos-Leal, Miguel; Guerra, Gilda; Vangronsveld, Jaco

2017-01-01

White-rot fungi (WRF) and their ligninolytic enzymes (laccases and peroxidases) are considered promising biotechnological tools to remove lignin related Persistent Organic Pollutants from industrial wastewaters and contaminated ecosystems. A high diversity of the genus Ganoderma has been reported in Cuba; in spite of this, the diversity of ligninolytic enzymes and their genes remained unexplored. In this study, 13 native WRF strains were isolated from decayed wood in urban ecosystems in Havana (Cuba). All strains were identified as Ganoderma sp. using a multiplex polymerase chain reaction (PCR)-method based on ITS sequences. All Ganoderma sp. strains produced laccase enzymes at higher levels than non-specific peroxidases. Native-PAGE of extracellular enzymatic extracts revealed a high diversity of laccase isozymes patterns between the strains, suggesting the presence of different amino acid sequences in the laccase enzymes produced by these Ganoderma strains. We determined the diversity of genes encoding laccases and peroxidases using a PCR and cloning approach with basidiomycete-specific primers. Between two and five laccase genes were detected in each strain. In contrast, only one gene encoding manganese peroxidase or versatile peroxidase was detected in each strain. The translated laccases and peroxidases amino acid sequences have not been described before. Extracellular crude enzymatic extracts produced by the Ganoderma UH strains, were able to degrade model chromophoric compounds such as anthraquinone and azo dyes. These findings hold promises for the development of a practical application for the treatment of textile industry wastewaters and also for bioremediation of polluted ecosystems by well-adapted native WRF strains. PMID:28588565

Improvement of selected strains through gamma irradiation for enhanced lipolytic potential

International Nuclear Information System (INIS)

Iftikhar, T.; Mubashir, N.; Hussain, Y.; Abbas, S.Q.; Ashraf, I.

2010-01-01

The purpose of the present investigation was to enhance the production of industrially important enzyme lipase by subjecting the wild lipase producing fungal strains i.e. Aspergillus niger, Rhizopus microsporus and Penicillium atrovenetum to various doses of gamma irradiation (20, 40, 60, 80, 100, 120, 140 and 160 Gy). The isolation and lipolytic activity of selected mutant derived strains is described in this paper. Among all the mutants tested, MBL-5 obtained at 140Gy of Aspergillus niger strain showed highest extracellular lipase activity (13.75 +- 0.15 U mL/sup -1/) while MBL-1 Rhizopus microsporus at the rate 20Gy showed the lowest activity i.e., 1.06 +- 0.11 U mL/sup -1/. A range of pH 3, 5, 7, 9 and 11 was used to check the lipolytic potential of various mutants along with their wild type. It was observed that MBL-5 (Aspergillus niger) and MBL-2 (Rhizopus microsporus) showed enhanced extracellular lipase activity at pH 11 while MBL-3 (Penicillium atrovenetum) showed the highest extracellular lipase activity 22.53 +- 0.21 U mL/sup -1/ at pH 9. It indicates a possible role for the MBL-2, MBL-3 and MBL-5 mutant strains in the detergent industry for the development of eco-friendly technologies. (author)

Evaluation of Lactobacillus strains for selected probiotic properties.

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Turková, Kristýna; Mavrič, Anja; Narat, Mojca; Rittich, Bohuslav; Spanová, Alena; Rogelj, Irena; Matijašić, Bojana Bogovič

2013-07-01

Eleven strains of Lactobacillus collected in the Culture Collection of Dairy Microorganisms (CCDM) were evaluated for selected probiotic properties such as survival in gastrointestinal fluids, antimicrobial activity, and competition with non-toxigenic Escherichia coli O157:H7 for adhesion on Caco-2 cells. The viable count of lactobacilli was reduced during 3-h incubation in gastric fluid followed by 3-h incubation in intestinal fluid. All strains showed antimicrobial activity and the three most effective strains inhibited the growth of at least 16 indicator strains. Antimicrobial metabolites of seven strains active against Lactobacillus and Clostridium indicator strains were found to be sensitive to proteinase K and trypsin, which indicates their proteinaceous nature. The degree of competitive inhibition of non-toxigenic E. coli O157:H7 adhesion on the surface of Caco-2 cells was strain-dependent. A significant decrease (P strains were selected for additional studies of antimicrobial activity, i.e., Lactobacillus gasseri CCDM 215, Lactobacillus acidophilus CCDM 149, and Lactobacillus helveticus CCDM 82.

Optimization of liquid-state fermentation conditions for the glyphosate degradation enzyme production of strain Aspergillus oryzae by ultraviolet mutagenesis.

Science.gov (United States)

Fu, Gui-Ming; Li, Ru-Yi; Li, Kai-Min; Hu, Ming; Yuan, Xiao-Qiang; Li, Bin; Wang, Feng-Xue; Liu, Cheng-Mei; Wan, Yin

2016-11-16

This study aimed to obtain strains with high glyphosate-degrading ability and improve the ability of glyphosate degradation enzyme by the optimization of fermentation conditions. Spore from Aspergillus oryzae A-F02 was subjected to ultraviolet mutagenesis. Single-factor experiment and response surface methodology were used to optimize glyphosate degradation enzyme production from mutant strain by liquid-state fermentation. Four mutant strains were obtained and named as FUJX 001, FUJX 002, FUJX 003, and FUJX 004, in which FUJX 001 gave the highest total enzyme activity. Starch concentration at 0.56%, GP concentration at 1,370 mg/l, initial pH at 6.8, and temperature at 30°C were the optimum conditions for the improved glyphosate degradation endoenzyme production of A. oryzae FUJX 001. Under these conditions, the experimental endoenzyme activity was 784.15 U/100 ml fermentation liquor. The result (784.15 U/100 ml fermentation liquor) was approximately 14-fold higher than that of the original strain. The result highlights the potential of glyphosate degradation enzyme to degrade glyphosate.

Controlled Autolysis and Enzyme Release in a Recombinant Lactococcal Strain Expressing the Metalloendopeptidase Enterolysin A

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Hickey, Rita M.; Ross, R. Paul; Hill, Colin

2004-01-01

This study concerns the exploitation of the lytic enzyme enterolysin A (EntL), produced by Enterococcus faecalis strain DPC5280, to elicit the controlled autolysis of starter lactococci. EntL, a cell wall metalloendopeptidase secreted by some E. faecalis strains, can kill a wide range of gram-positive bacteria, including lactococci. The controlled expression of entL, which encodes EntL, was achieved using a nisin-inducible expression system in a lactococcal host. Zymographic analysis of EntL activity demonstrated that active enzyme is produced by the recombinant lactococcal host. Indeed, expression of EntL resulted in almost complete autolysis of the host strain 2 h after induction with nisin. Model cheese experiments using a starter strain in addition to the inducible enterolysin-producing strain showed a 27-fold increase in activity with respect to the release of lactate dehydrogenase in the strain overexpressing EntL, demonstrating the potential of EntL production in large-scale cheese production systems. Indeed, the observation that a wide range of lactic bacteria are sensitive to EntL suggests that EntL-induced autolysis has potential applications with a variety of lactic acid bacteria and could be a basis for probiotic delivery systems. PMID:15006800

Quality parameters and RAPD-PCR differentiation of commercial baker's yeast and hybrid strains.

Science.gov (United States)

El-Fiky, Zaki A; Hassan, Gamal M; Emam, Ahmed M

2012-06-01

Baker's yeast, Saccharomyces cerevisiae, is a key component in bread baking. Total of 12 commercial baker's yeast and 2 hybrid strains were compared using traditional quality parameters. Total of 5 strains with high leavening power and the 2 hybrid strains were selected and evaluated for their alpha-amylase, maltase, glucoamylase enzymes, and compared using random amplified polymorphic DNA (RAPD). The results revealed that all selected yeast strains have a low level of alpha-amylase and a high level of maltase and glucoamylase enzymes. Meanwhile, the Egyptian yeast strain (EY) had the highest content of alpha-amylase and maltase enzymes followed by the hybrid YH strain. The EY and YH strains have the highest content of glucoamylase enzyme almost with the same level. The RAPD banding patterns showed a wide variation among commercial yeast and hybrid strains. The closely related Egyptian yeast strains (EY and AL) demonstrated close similarity of their genotypes. The 2 hybrid strains were clustered to Turkish and European strains in 1 group. The authors conclude that the identification of strains and hybrids using RAPD technique was useful in determining their genetic relationship. These results can be useful not only for the basic research, but also for the quality control in baking factories. © 2012 Institute of Food Technologists®

Multilocus enzyme electrophoresis on agarose gel as an aid to the identification of entomopathogenic Bacillus sphaericus strains.

Science.gov (United States)

Zahner, V; Rabinovitch, L; Cavados, C F; Momen, H

1994-04-01

Sixty strains of Bacillus sphaericus, including 31 insect pathogens were studied by multilocus enzyme electrophoresis and were classified into 44 zymovars (electrophoretic types). Among the entomopathogenic strains, 11 belong to the same zymovar (Z59) indicating a widespread frequent genotype. Bands of enzyme activity were not detected among the strains for the loci GPI (E.C.5.3.1.9), G6P (E.C.1.1.1.49), 6PG (E.C.1.1.1.44) and ME (E.C.1.1.1.40). The enzymatic loci NP (E.C.2.4.2.1) and ACON (E.C.4.2.1.3) were monomorphic while the other enzymes, MDH (E.C.1.1.1.37), LeDH (E.C.1.4.1.9), ADH (E.C.1.4.1.1), EST (E.C.3.1.1.1), PEP-2 (E.C.3.4.11.1), PEP-3 (E.C.3.4.11) and PEP-D (E.C. 3.4.13.9) were polymorphic. The genetic variation in the non-insect pathogenic group seemed to be greater than in the entomopathogenic group. This latter group appears to be distinct from other strains of these species. All insect pathogens were recovered in the same phenetic cluster and a diagnostic allele is reported for the identification of entomopathogenic strains.

Screening of probiotic activities of forty-seven strains of Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans.

Science.gov (United States)

Jacobsen, C N; Rosenfeldt Nielsen, V; Hayford, A E; Møller, P L; Michaelsen, K F; Paerregaard, A; Sandström, B; Tvede, M; Jakobsen, M

1999-11-01

The probiotic potential of 47 selected strains of Lactobacillus spp. was investigated. The strains were examined for resistance to pH 2.5 and 0.3% oxgall, adhesion to Caco-2 cells, and antimicrobial activities against enteric pathogenic bacteria in model systems. From the results obtained in vitro, five strains, Lactobacillus rhamnosus 19070-2, L. reuteri DSM 12246, L. rhamnosus LGG, L. delbrueckii subsp. lactis CHCC 2329, and L. casei subsp. alactus CHCC 3137, were selected for in vivo studies. The daily consumption by 12 healthy volunteers of two doses of 10(10) freeze-dried bacteria of the selected strains for 18 days was followed by a washout period of 17 days. Fecal samples were taken at days 0 and 18 and during the washout period at days 5 and 11. Lactobacillus isolates were initially identified by API 50CHL and internal transcribed spacer PCR, and their identities were confirmed by restriction enzyme analysis in combination with pulsed-field gel electrophoresis. Among the tested strains, L. rhamnosus 19070-2, L. reuteri DSM 12246, and L. rhamnosus LGG were identified most frequently in fecal samples; they were found in 10, 8, and 7 of the 12 samples tested during the intervention period, respectively, whereas reisolations were less frequent in the washout period. The bacteria were reisolated in concentrations from 10(5) to 10(8) cells/g of feces. Survival and reisolation of the bacteria in vivo appeared to be linked to pH tolerance, adhesion, and antimicrobial properties in vitro.

Effect of Additives on the Selectivity and Reactivity of Enzymes.

Science.gov (United States)

Liang, Yi-Ru; Wu, Qi; Lin, Xian-Fu

2017-01-01

Enzymes have been widely used as efficient, eco-friendly, and biodegradable catalysts in organic chemistry due to their mild reaction conditions and high selectivity and efficiency. In recent years, the catalytic promiscuity of many enzymes in unnatural reactions has been revealed and studied by chemists and biochemists, which has expanded the application potential of enzymes. To enhance the selectivity and activity of enzymes in their natural or promiscuous reactions, many methods have been recommended, such as protein engineering, process engineering, and media engineering. Among them, the additive approach is very attractive because of its simplicity to use and high efficiency. In this paper, we will review the recent developments about the applications of additives to improve the catalytic performances of enzymes in their natural and promiscuous reactions. These additives include water, organic bases, water mimics, cosolvents, crown ethers, salts, surfactants, and some particular molecular additives. © 2017 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Extracellular enzyme activities during lignocellulose degradation by Streptomyces spp.: a comparative study of wild-type and genetically manipulated strains

International Nuclear Information System (INIS)

Ramachandra, M.; Crawford, D.L.; Pometto, A.L. III

1987-01-01

The wild-type ligninolytic actinomycete Streptomyces viridosporus T7A and two genetically manipulated strains with enhanced abilities to produce a water-soluble lignin degradation intermediate, an acid-precipitable polymeric lignin (APPL), were grown on lignocellulose in solid-state fermentation cultures. Culture filtrates were periodically collected, analyzed for APPL, and assayed for extracellular lignocellulose-catabolizing enzyme activities. Two APPL-overproducing strains, UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10, had higher and longer persisting peroxidase, esterase, and endoglucanase activities than did the wild-type strain T7A. Results implicated one or more of these enzymes in lignin solubilization. Only mutant T7A-81 had higher xylanase activity than the wild type. The peroxidase was induced by both lignocellulose and APPL. This extracellular enzyme has some similarities to previously described ligninases in fungi. This is the first report of such an enzyme in Streptomyces spp. Four peroxidase isozymes were present, and all catalyzed the oxidation of 3,4-dihydroxyphenylalanine, while one also catalyzed hydrogen peroxide-dependent oxidation of homoprotocatechuic acid and caffeic acid. Three constitutive esterase isozymes were produced which differed in substrate specificity toward α-naphthyl acetate and α-naphthyl butyrate. Three endoglucanase bands, which also exhibited a low level of xylanase activity, were identified on polyacrylamide gels as was one xylanase-specific band. There were no major differences in the isoenzymes produced by the different strains. The probable role of each enzyme in lignocellulose degradation is discussed

Influence of protoplast fusion between two Trichoderma spp. on extracellular enzymes production and antagonistic activity.

Science.gov (United States)

Hassan, Mohamed M

2014-11-02

Biological control plays a crucial role in grapevine pathogens disease management. The cell-wall degrading enzymes chitinase, cellulase and β-glucanase have been suggested to be essential for the mycoparasitism activity of Trichoderma species against grapevine fungal pathogens. In order to develop a useful strain as a single source of these vital enzymes, it was intended to incorporate the characteristics of two parental fungicides tolerant mutants of Trichoderma belonging to the high chitinase producing species T. harzianum and the high cellulase producing species T. viride , by fusing their protoplasts. The phylogeny of the parental strains was carried out using a sequence of the 5.8S-ITS region. The BLAST of the obtained sequence identified these isolates as T. harzianum and T. viride . Protoplasts were isolated using lysing enzymes and were fused using polyethylene glycol. The fused protoplasts have been regenerated on protoplast regeneration minimal medium supplemented with two selective fungicides. Among the 40 fast growing fusants, 17 fusants were selected based on their enhanced growth on selective media for further studies. The fusant strains were growing 60%-70% faster than the parents up to third generation. All the 17 selected fusants exhibited morphological variations. Some fusant strains displayed threefold increased chitinase enzyme activity and twofold increase in β-glucanase enzyme activity compared to the parent strains. Most fusants showed powerful antagonistic activity against Macrophomin aphaseolina , Pythium ultimum and Sclerotium rolfsii pathogens. Fusant number 15 showed the highest inhibition percentage (92.8%) against M. phaseolina and P. ultimum, while fusant number 9 showed the highest inhibition percentage (98.2%) against the growth of S. rolfsii. A hyphal intertwining and degradation phenomenon was observed by scanning electron microscope. The Trichoderma antagonistic effect against pathogenic fungal mycelia was due to the

Evaluation of Strains of Metarhizium anisopliae and Beauveria bassiana against Spodoptera litura on the Basis of Their Virulence, Germination Rate, Conidia Production, Radial Growth and Enzyme Activity.

Science.gov (United States)

Petlamul, Wanida; Prasertsan, Poonsuk

2012-06-01

Ten strains of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana were evaluated to find the most effective strain for optimization studies. The first criterion tested for strain selection was the mortality (> 50%) of Spodoptera litura larvae after inoculation of the fungus for 4 days. Results on several bioassays revealed that B. bassiana BNBCRC showed the most virulence on mortality S. litura larvae (80% mortality). B. bassiana BNBCRC also showed the highest germination rate (72.22%). However, its conidia yield (7.2 × 10(8) conidia/mL) was lower than those of B. bassiana B 14841 (8.3 × 10(8) conidia/mL) and M. anisopliae M6 (8.2 × 10(8) conidia/mL). The highest accumulative radial growth was obtained from the strain B14841 (37.10 mm/day) while the strain BNBCRC showed moderate radial growth (24.40 mm/day). M. anisopliae M6 possessed the highest protease activity (145.00 mU/mL) while M. anisopliae M8 possessed the highest chitinase activity (20.00 mU/mL) during 96~144 hr cultivation. Amongst these criteria, selection based on virulence and germination rate lead to the selection of B. bassiana BNBCRC. B. bassiana B14841 would be selected if based on growth rate while M. anisopliae M6 and M8 possessed the highest enzyme activities.

Selection of cellulolytic fungi isolated from diverse substrates

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Mônica Caramez Triches Damaso

2012-08-01

Full Text Available The aim of the present work was to select filamentous fungi isolated from diverse substrates to obtain the strains with potential to produce the hydrolytic enzymes. From a total of 215 strains, seven strains from the soils, six from the plants and one from sugarcane bagasse were selected and identified as belonging to the Trichoderma, Penicillium and Aspergillus genera. The best hydrolytic activities obtained by semi-solid fermentation using these strains were approximately: 35; 1; 160; 170 and 120 U/gdm (CMCase, FPase, β-glucosidase, xylanase and polygalacturonase, respectively, demonstrating their potential to synthesize the enzymes compared with the results reported in the literature.

Selected soil enzymes: Examples of their potential roles in the ...

African Journals Online (AJOL)

Soil enzymes regulate ecosystem functioning and in particular play a key role in nutrient cycling. In this review we briefly summarise potential roles of selected enzymes such as amylase, arylsulphatases, -glucosidase, cellulose, chitinase, dehydrogenase, phosphatase, protease and urease in the ecosystem. We also ...

Deletion of creB in Aspergillus oryzae increases secreted hydrolytic enzyme activity.

Science.gov (United States)

Hunter, A J; Morris, T A; Jin, B; Saint, C P; Kelly, J M

2013-09-01

Aspergillus oryzae has been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes.

Isolation and screening of strains producing high amounts of rutin degrading enzymes from Fagopyrum tataricum seeds.

Science.gov (United States)

Zheng, Ya-Di; Luo, Qing-Lin; Zhou, Mei-Liang; Wang, De-Zhou; Zhang, Ye-Dong; Shao, Ji-Rong; Zhu, Xue-Mei; Tang, Yu

2013-02-01

The rutin degrading enzyme (RDE) was isolated and purified from tartary buckwheat seeds. The RDE was purified about 11.34-fold and its final yield was 3.5%, which was very low, due to our purification strategy of giving priority to purity over yield. The RDE molecular weight was estimated to be about 60 kDa. When rutin was used as substrate, an optimal enzyme activity was seen at around pH 5.0 and 40 °C. Strains isolation strategy characterized by the use of rutin as sole carbon source in enrichment cultures was used to isolate RDE-producing strains. Then the active strains were identified by morphology characterization and 18s rDNA-ITS (Internal Transcribed Spacer) gene sequencing. Three isolates coded as B3, W2, Y2 were successfully isolated from fusty Fagopyrum tataricum flour cultures. Strain B3 possessed the highest unit activity among these three strains, and its total activity reached up to 171.0 Unit. The active isolate (B3) could be assigned to Penicillium farinosum. When the Penicillium farinosum strains were added to tartary buckwheat flour cultures at pH 5.0, 30 °C after 5 days fermentation, the quercetin production raised up to 1.78 mg/l, almost 5.1 times higher than the fermentation without the above active strains. Hence, a new approach was available to utilize microorganism-aided fermentation for effective quercetin extraction from Fagopyrum tataricum seeds. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural characterization of bioengineered α-D-glucans produced by mutant glucansucrase GTF180 enzymes of lactobacillus reuteri strain 180

NARCIS (Netherlands)

Leeuwen, S.S. van; Kralj, S.; Eeuwema, W.; Gerwig, G.J.; Dijkhuizen, L.; Kamerling, J.P.

2009-01-01

Mutagenesis of specific amino acid residues of the glucansucrase (GTF180) enzyme from Lactobacillus reuteri strain 180 yielded 12 mutant enzymes that produced modified exopolysaccharides (mEPSs) from sucrose. Ethanol-precipitated and purified mEPSs were subjected to linkage analysis, Smith

Structural Characterization of Bioengineered alpha-D-Glucans Produced by Mutant Glucansucrase GTF180 Enzymes of Lactobacillus reuteri Strain 180

NARCIS (Netherlands)

van Leeuwen, Sander S.; Kralj, Slavko; Eeuwema, Wieger; Gerwig, Gerrit J.; Dijkhuizen, Lubbert; Kamerling, Johannis P.

Mutagenesis of specific amino acid residues of the glucansucrase (GTF180) enzyme from Lactobacillus reuteri strain 180 yielded 12 mutant enzymes that produced modified exopolysaccharides (mEPSs) from sucrose. Ethanol-precipitated and purified mEPSs were subjected to linkage analysis, Smith

Beyond Iron: Iridium-Containing P450 Enzymes for Selective Cyclopropanations of Structurally Diverse Alkenes

International Nuclear Information System (INIS)

Key, Hanna M.; Dydio, Paweł; Liu, Zhennan

2017-01-01

Enzymes catalyze organic transformations with exquisite levels of selectivity, including chemoselectivity, stereoselectivity, and substrate selectivity, but the types of reactions catalyzed by enzymes are more limited than those of chemical catalysts. Thus, the convergence of chemical catalysis and biocatalysis can enable enzymatic systems to catalyze abiological reactions with high selectivity. Recently, we disclosed artificial enzymes constructed from the apo form of heme proteins and iridium porphyrins that catalyze the insertion of carbenes into a C-H bond. Here, we postulated that the same type of Ir(Me)-PIX enzymes could catalyze the cyclopropanation of a broad range of alkenes with control of multiple modes of selectivity. Here, we report the evolution of artificial enzymes that are highly active and highly stereoselective for the addition of carbenes to a wide range of alkenes. These enzymes catalyze the cyclopropanation of terminal and internal, activated and unactivated, electron-rich and electron-deficient, conjugated and nonconjugated alkenes. In particular, Ir(Me)-PIX enzymes derived from CYP119 catalyze highly enantio- and diastereoselective cyclopropanations of styrene with ±98% ee, > 70:1 dr, > 75% yield, and ~10,000 turnovers (TON), as well as 1,2-disubstituted styrenes with up to 99% ee, 35:1 dr, and 54% yield. Moreover, Ir(Me)-PIX enzymes catalyze cyclopropanation of internal, unactivated alkenes with up to 99% stereoselectivity, 76% yield, and 1300 TON. They also catalyze cyclopropanation of natural products with diastereoselectivities that are complementary to those attained with standard transition metal catalysts. Finally, Ir(Me)-PIX P450 variants react with substrate selectivity that is reminiscent of natural enzymes; they react preferentially with less reactive internal alkenes in the presence of more reactive terminal alkenes. Altogether, the studies reveal the suitability of Ir-containing P450s to combine the broad reactivity and

Selection Finder (SelFi: A computational metabolic engineering tool to enable directed evolution of enzymes

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Neda Hassanpour

2017-06-01

Full Text Available Directed evolution of enzymes consists of an iterative process of creating mutant libraries and choosing desired phenotypes through screening or selection until the enzymatic activity reaches a desired goal. The biggest challenge in directed enzyme evolution is identifying high-throughput screens or selections to isolate the variant(s with the desired property. We present in this paper a computational metabolic engineering framework, Selection Finder (SelFi, to construct a selection pathway from a desired enzymatic product to a cellular host and to couple the pathway with cell survival. We applied SelFi to construct selection pathways for four enzymes and their desired enzymatic products xylitol, D-ribulose-1,5-bisphosphate, methanol, and aniline. Two of the selection pathways identified by SelFi were previously experimentally validated for engineering Xylose Reductase and RuBisCO. Importantly, SelFi advances directed evolution of enzymes as there is currently no known generalized strategies or computational techniques for identifying high-throughput selections for engineering enzymes.

Selection and evaluation of Rhizobial strains of Vigna radiata L ...

African Journals Online (AJOL)

STORAGESEVER

2008-10-20

Oct 20, 2008 ... Selection and evaluation of Rhizobial strains of Vigna radiata L. beneficial to ... This study aimed to select suitable strains that can be used as inoculants to enhance legume production and simultaneously reduce the use of ... contributor to natural or biological N2 fixation and allows legumes to grow in the ...

Hydrolytic enzyme activities in shiitake mushroom (Lentinula edodes) strains cultivated on coffee pulp.

Science.gov (United States)

Mata, Gerardo; Salmones, Dulce; Pérez-Merlo, Rosalía

Hydrolytic enzyme production (cellulases, laminarinases and xylanases) was studied in cultures of Lentinula edodes on sterilized coffee pulp. Samples of substrate colonized by mycelia were taken after 7, 14, 21, 28 and 35 days of incubation at 25°C (W1 to W5) and during the fruiting period at different stages: formation of primordia (PF), first harvest (H) and one week after the first harvest (PH). The enzymatic activity was lower during the early mycelial growth and showed higher levels during the formation and development of fruiting bodies. During the reproductive stage of the fungus, the samples were subjected to a soaking treatment; however, it was not possible to relate this soaking treatment to the increase in enzyme production. The levels of enzymatic activity suggest that secretion of the studied enzymes does not influence the adaptability of the strains to the substrate. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

The role of detoxifying enzymes in the resistance of the cowpea aphid (Aphis craccivora Koch to thiamethoxam

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Abdallah Ibrahim Saleh

2016-01-01

Full Text Available The cowpea aphid (Aphis craccivora Koch is considered a serious insect pest attacking several crops. We carried out biochemical studies to elucidate the role of the metabolising enzymes in conferring resistance to thiamethoxam, in two strains (resistant and susceptible of the cowpea aphid. Bioassay experiments showed that the thiamethoxam selected strain developed a 48 fold resistance after consecutive selection with thiamethoxam for 12 generations. This resistant strain also exhibited cross-resistance to the tested carbamates; pirimicarb and carbosulfan, organophosphorus (malathion, fenitrothion, and chlorpyrifos-methyl, and the neonicotinoid (acetamiprid. Synergism studies have indicated that S,S,S-tributyl phosphorotrithioate (DEF, a known inhibitor for esterases, increased thiamethoxam toxicity 5.58 times in the resistant strain compared with the susceptible strain. Moreover, the biochemical determination revealed that carboxylestersae activity was 30 times greater in the resistant strain than in the susceptible strain. In addition, the enzyme activity of glutathione S-transferase (GST and mixed function oxidases (mfo increased only in the resistant strain 3.7 and 2.7 times, respectively, in relation to the susceptible (the control. Generally, our results suggest that the higher activity of the detoxifying enzymes, particularly carboxylesterase, in the resistant strain of the cowpea aphid, apparently have a significant role in endowing resistance to thiamethoxam, although additional mechanisms may contribute.

EFSA CEF Panel (EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids), 2014. Scientific Opinion on lipase from a genetically modified strain of Aspergillus oryzae (strain NZYM-FL)

DEFF Research Database (Denmark)

Poulsen, Morten; Hallas-Møller, Torben; Binderup, Mona-Lise

The food enzyme considered in this opinion is a lipase (triacylglycerol lipase; EC 3.1.1.3) produced with a genetically modified strain of Aspergillus oryzae. The genetic modifications do not raise safety concern. The food enzyme contains neither the production organism nor recombinant DNA...

Nitrate-Dependent Degradation of Acetone by Alicycliphilus and Paracoccus Strains and Comparison of Acetone Carboxylase Enzymes ▿

Science.gov (United States)

Dullius, Carlos Henrique; Chen, Ching-Yuan; Schink, Bernhard

2011-01-01

A novel acetone-degrading, nitrate-reducing bacterium, strain KN Bun08, was isolated from an enrichment culture with butanone and nitrate as the sole sources of carbon and energy. The cells were motile short rods, 0.5 to 1 by 1 to 2 μm in size, which gave Gram-positive staining results in the exponential growth phase and Gram-negative staining results in the stationary-growth phase. Based on 16S rRNA gene sequence analysis, the isolate was assigned to the genus Alicycliphilus. Besides butanone and acetone, the strain used numerous fatty acids as substrates. An ATP-dependent acetone-carboxylating enzyme was enriched from cell extracts of this bacterium and of Alicycliphilus denitrificans K601T by two subsequent DEAE Sepharose column procedures. For comparison, acetone carboxylases were enriched from two additional nitrate-reducing bacterial species, Paracoccus denitrificans and P. pantotrophus. The products of the carboxylase reaction were acetoacetate and AMP rather than ADP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of cell extracts and of the various enzyme preparations revealed bands corresponding to molecular masses of 85, 78, and 20 kDa, suggesting similarities to the acetone carboxylase enzymes described in detail for the aerobic bacterium Xanthobacter autotrophicus strain Py2 (85.3, 78.3, and 19.6 kDa) and the phototrophic bacterium Rhodobacter capsulatus. Protein bands were excised and compared by mass spectrometry with those of acetone carboxylases of aerobic bacteria. The results document the finding that the nitrate-reducing bacteria studied here use acetone-carboxylating enzymes similar to those of aerobic and phototrophic bacteria. PMID:21841031

PRODUCTION OF FIBRINOLYTIC ENZYME (NATTOKINASE) FROM BACILLUS SP.

OpenAIRE

Padma Singh, Rekha Negi*, Vani Sharma, Alka Rani, Pallavi and Richa Prasad

2018-01-01

During present study Nattokinase which is a novel fibrinolytic enzyme was produced by Bacillus sp. To screen and extract nattokinase enzyme from Bacillus sp. were isolated from soil of different agricultural field by serial dilution method. Out of 10 isolate, one strain i.e. B3 produced nattokinase on screening medium. B3 was identified by biochemical characterization. The caseinolytic activity of Nattokinase was 0.526 U/ml and the selected isolate Bacillus sp. could produce active nattokinas...

Bacillus cereus strain MCN as a debriding agent

Science.gov (United States)

Dalton, H. P.; Haynes, B. W.; Stone, L. L.

1978-01-01

Biologically active means are effective for rapidly removing scar tissue caused by burns or corrosive agents. Specially selected strain of bacteria applied to injury site releases enzymes which are active against eschar. These bacteria tend to locate between eschar and unburned tissue, thus providing optimal cell surface area arrangement for enzyme dispersal. Procedure may prove especially useful in treatment of disaster casualties under relatively primitive conditions.

Differential expression of glutathione s-transferase enzyme in different life stages of various insecticide-resistant strains of Anopheles stephensi: a malaria vector.

Science.gov (United States)

Sanil, D; Shetty, V; Shetty, N J

2014-06-01

Interest in insect glutathione s-transferases (GSTs) has primarily focused on their role in insecticide resistance. These play an important role in biotransformation and detoxification of many different xenobiotic and endogenous substances including insecticides. The GST activity among 10 laboratory selected insecticide resistant and susceptible/control strains of Anopheles stephensi was compared using the substrates 1-chloro-2,4-dinitrobenzene (CDNB). The difference in the GST activities of different life stages of diverse insecticide resistant strains was compared and presented. About 100 larvae, pupae, adult males, adult females and eggs (100 μg in total weight) were collected and used for the experiment. The extracts were prepared from each of the insecticide-resistant strains and control. Protein contents of the enzyme homogenate and GST activities were determined. Deltamethrin and cyfluthrin-resistant strains of An. stephensi showed significantly higher GST activity. Larvae and pupae of DDT-resistant strain showed peak GST activity followed by the propoxur-resistant strain. On contrary, the GST activity was found in reduced quantity in alphamethrin, bifenthrin, carbofuran and chloropyrifos resistant strains. Adults of either sexes showed higher GST activity in mosquito strain resistant to organophosphate group of insecticides namely, temephos and chloropyrifos. The GST activity was closely associated with almost all of the insecticides used in the study, strengthening the fact that one of the mechanisms associated with resistance includes an increase of GST activity. This comparative data on GST activity in An. stephensi can be useful database to identify possible underlying mechanisms governing insecticide-resistance by GSTs.

[Diversity and enzyme-producing activity of culturable halophilic bacteria in Daishan Saltern of East China].

Science.gov (United States)

Yang, Dan-Dan; Li, Qian; Huang, Jing-Jing; Chen, Min

2012-11-01

Soil and saline water samples were collected from the Daishan Saltern of East China, and the halophilic bacteria were isolated and cultured by using selective media, aimed to investigate the diversity and enzyme-producing activity of culturable halophilic bacteria in saltern environment. A total of 181 strains were isolated by culture-dependent method. Specific primers were used to amplify the 16S rRNA gene of bacteria and archaea. The operation taxonomy units (OTUs) were determined by ARDRA method, and the representative strain of each OTU was sequenced. The phylogenetic position of all the isolated strains was determined by 16S rRNA sequencing. The results showed that the isolated 181 strains displayed 21 operational taxonomic units (OTUs), of which, 12 OTUs belonged to halophilic bacteria, and the others belonged to halophilic archaea. Phylogenetic analysis indicated that there were 7 genera presented among the halophilic bacteria group, and 4 genera presented among the halophilic archaea group. The dominant halophilic strains were of Halomonas and Haloarcula, with 46.8% in halophilic bacteria and 49.1% in halophilic archaea group, respectively. Enzyme-producing analysis indicated that most strains displayed enzyme-producing activity, including the activities of producing amylase, proteinase and lipase, and the dominant strains capable of enzyme-producing were of Haloarcula. Our results showed that in the environment of Daishan Saltern, there existed a higher diversity of halophilic bacteria, being a source sink for screening enzyme-producing bacterial strains.

Bioassay and Molecular Screening of Pectinase Enzyme in halophilic bacteria from Salt Lake, Iran

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Zohre Nasrollahzadeh

2018-06-01

Discussion and conclusion: Quantitative evaluation showed that production and activity of pectinase enzyme in R2S25 strain increased simultaneously with increasing the growth of selected strain in logarithmic phase. Molecular study also showed that the genuses of Martelella, Aeromocrobium, Planococcus, Marinobacter, Virgibacillus, Kocuria and Micrococcus contain the pectinase gene.

Selected Enzyme Inhibitory Effects of Euphorbia characias Extracts

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Antonella Fais

2018-01-01

Full Text Available Extracts of aerial part of Euphorbia characias were examined to check potential inhibitors for three selected enzymes involved in several metabolic disorders. Water and ethanol extracts from leaves and flowers showed in vitro inhibitory activity toward α-amylase, α-glucosidase, and xanthine oxidase. IC50 values were calculated for all the extracts and the ethanolic extracts were found to exert the best effect. In particular, for the α-glucosidase activity, the extracts resulted to be 100-fold more active than the standard inhibitor. The inhibition mode was investigated by Lineweaver-Burk plot analysis. E. characias extracts display different inhibition behaviors toward the three enzymes acting as uncompetitive, noncompetitive, and mixed-type inhibitors. Moreover, ethanolic extracts of E. characias showed no cytotoxic activity and exhibited antioxidant capacity in a cellular model. The LC-DAD metabolic profile was also performed and it showed that leaves and flowers extracts contain high levels of quercetin derivatives. The results suggest that E. characias could be a promising source of natural inhibitors of the enzymes involved in carbohydrate uptake disorders and oxidative stress.

[Hydrogen production and enzyme activity of acidophilic strain X-29 at different C/N ratio].

Science.gov (United States)

Li, Qiu-bo; Xing, De-feng; Ren, Nan-qi; Zhao, Li-hua; Song, Ye-ying

2006-04-01

Some fermentative bacteria can produce hydrogen by utilizing carbohydrate and other kinds of organic compounds as substrates. Hydrogen production was also determined by both the limiting of growth and related enzyme activity in energy metabolism. Carbon and nitrogen are needed for the growth and metabolism of microorganisms. In addition, the carbon/nitrogen (C/N) ratio can influence the material metabolized and the energy produced. In order to improve the hydrogen production efficiency of the bacteria, we analyzed the effect of different C/N ratios on hydrogen production and the related enzyme activities in the acidophilic strain X-29 using batch test. The results indicate that the differences in the metabolism level and enzyme activity are obvious at different C/N ratios. Although the difference in liquid fermentative products produced per unit of biomass is not obvious, hydrogen production is enhanced at a specifically determined ratio. At a C/N ratio of 14 the accumulative hydrogen yield of strain X-29 reaches the maximum, 2210.9 mL/g. At different C/N ratios, the expression of hydrogenase activity vary; the activity of hydrogenase decrease quickly after reaching a maximum along with the fermentation process, but the time of expression is short. The activity of alcohol dehydrogenase (ADH) tend to stabilize after reaching a peak along with the fermentation process, the difference in expression activity is little, and the expression period is long at different C/N ratios. At a C/N ratio of 14 hydrogenase and ADH reach the maximum 2.88 micromol x (min x mg)(-1) and 33.2 micromol x (min x mg)(-1), respectively. It is shown that the C/N ratio has an important effect on enhancing hydrogen production and enzyme activity.

Bi-enzyme L-arginine-selective amperometric biosensor based on ammonium-sensing polyaniline-modified electrode.

Science.gov (United States)

Stasyuk, Nataliya; Smutok, Oleh; Gayda, Galina; Vus, Bohdan; Koval'chuk, Yevgen; Gonchar, Mykhailo

2012-01-01

A novel L-arginine-selective amperometric bi-enzyme biosensor based on recombinant human arginase I isolated from the gene-engineered strain of methylotrophic yeast Hansenula polymorpha and commercial urease is described. The biosensing layer was placed onto a polyaniline-Nafion composite platinum electrode and covered with a calcium alginate gel. The developed sensor revealed a good selectivity to L-arginine. The sensitivity of the biosensor was 110 ± 1.3 nA/(mM mm(2)) with the apparent Michaelis-Menten constant (K(M)(app)) derived from an L-arginine (L-Arg) calibration curve of 1.27 ± 0.29 mM. A linear concentration range was observed from 0.07 to 0.6mM, a limit of detection being 0.038 mM and a response time - 10s. The developed biosensor demonstrated good storage stability. A laboratory prototype of the proposed amperometric biosensor was applied to the samples of three commercial pharmaceuticals ("Tivortin", "Cytrarginine", "Aminoplazmal 10% E") for L-Arg testing. The obtained L-Arg-content values correlated well with those declared by producers. Copyright © 2012 Elsevier B.V. All rights reserved.

Optimization of enzyme parameters for fermentative production of biorenewable fuels and chemicals

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Ping Liu

2012-10-01

Full Text Available Microbial biocatalysts such as Escherichia coli and Saccharomyces cerevisiae have been extensively subjected to Metabolic Engineering for the fermentative production of biorenewable fuels and chemicals. This often entails the introduction of new enzymes, deletion of unwanted enzymes and efforts to fine-tune enzyme abundance in order to attain the desired strain performance. Enzyme performance can be quantitatively described in terms of the Michaelis-Menten type parameters Km, turnover number kcat and Ki, which roughly describe the affinity of an enzyme for its substrate, the speed of a reaction and the enzyme sensitivity to inhibition by regulatory molecules. Here we describe examples of where knowledge of these parameters have been used to select, evolve or engineer enzymes for the desired performance and enabled increased production of biorenewable fuels and chemicals. Examples include production of ethanol, isobutanol, 1-butanol and tyrosine and furfural tolerance. The Michaelis-Menten parameters can also be used to judge the cofactor dependence of enzymes and quantify their preference for NADH or NADPH. Similarly, enzymes can be selected, evolved or engineered for the preferred cofactor preference. Examples of exporter engineering and selection are also discussed in the context of production of malate, valine and limonene.

OPTIMIZATION OF ENZYME PARAMETERS FOR FERMENTATIVE PRODUCTION OF BIORENEWABLE FUELS AND CHEMICALS

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Laura R. Jarboe

2012-10-01

Full Text Available Microbial biocatalysts such as Escherichia coli and Saccharomyces cerevisiae have been extensively subjected to Metabolic Engineering for the fermentative production of biorenewable fuels and chemicals. This often entails the introduction of new enzymes, deletion of unwanted enzymes and efforts to fine-tune enzyme abundance in order to attain the desired strain performance. Enzyme performance can be quantitatively described in terms of the Michaelis-Menten type parameters Km, turnover number kcat and Ki, which roughly describe the affinity of an enzyme for its substrate, the speed of a reaction and the enzyme sensitivity to inhibition by regulatory molecules. Here we describe examples of where knowledge of these parameters have been used to select, evolve or engineer enzymes for the desired performance and enabled increased production of biorenewable fuels and chemicals. Examples include production of ethanol, isobutanol, 1-butanol and tyrosine and furfural tolerance. The Michaelis-Menten parameters can also be used to judge the cofactor dependence of enzymes and quantify their preference for NADH or NADPH. Similarly, enzymes can be selected, evolved or engineered for the preferred cofactor preference. Examples of exporter engineering and selection are also discussed in the context of production of malate, valine and limonene.

Molecular basis of cyclooxygenase enzymes (COXs) selective inhibition

Science.gov (United States)

Limongelli, Vittorio; Bonomi, Massimiliano; Marinelli, Luciana; Gervasio, Francesco Luigi; Cavalli, Andrea; Novellino, Ettore; Parrinello, Michele

2010-01-01

The widely used nonsteroidal anti-inflammatory drugs block the cyclooxygenase enzymes (COXs) and are clinically used for the treatment of inflammation, pain, and cancers. A selective inhibition of the different isoforms, particularly COX-2, is desirable, and consequently a deeper understanding of the molecular basis of selective inhibition is of great demand. Using an advanced computational technique we have simulated the full dissociation process of a highly potent and selective inhibitor, SC-558, in both COX-1 and COX-2. We have found a previously unreported alternative binding mode in COX-2 explaining the time-dependent inhibition exhibited by this class of inhibitors and consequently their long residence time inside this isoform. Our metadynamics-based approach allows us to illuminate the highly dynamical character of the ligand/protein recognition process, thus explaining a wealth of experimental data and paving the way to an innovative strategy for designing new COX inhibitors with tuned selectivity. PMID:20215464

Selective distribution of enzymes in a microfluidic reactor

DEFF Research Database (Denmark)

Daugaard, Anders Egede; Pereira Rosinha Grundtvig, Ines; Krühne, Ulrich

Off stoichiometric thiol-ene mixtures are well suited for preparation of microfluidic devices with highly functional surfaces. Here a two stage process employing first thiol-ene chemistry (TEC) to prepare two opposite parts of a microfluidic system with a 30x30 mm reactor and subsequently a thiol......-epoxy bonding was used to prepare a fully sealed microfluidic system. The reactor was surface functionalized in-situ with allyl glycidyl ether in different patterns (half-reactor, full-reactor, checkerboard structures) on the surface to provide a controlled distribution of epoxides. The method additionally...... enables the selective immobilization on either top-side or bottom-side or both sides of the reactor. Thereafter horseradish peroxidase was immobilized on the surface and activity tests illustrated how this distribution of the enzyme on the surface could be used to optimize the activity of the enzyme...

In Vitro Assessment of Bioactivities of Lactobacillus Strains as Potential Probiotics for Humans and Chickens.

Science.gov (United States)

Shokryazdan, P; Jahromi, M F; Liang, J B; Sieo, C C; Kalavathy, R; Idrus, Z; Ho, Y W

2017-11-01

Twelve previously isolated Lactobacillus strains were investigated for their in vitro bioactivities, including bile salt hydrolase (BSH), cholesterol-reducing and antioxidant activities, cytotoxic effects against cancer cells, enzyme activity, and biogenic amine production. Among them, only 4 strains showed relatively high BSH activity, whereas the rest exhibited low BSH activity. All 12 strains showed cholesterol-reducing and antioxidant activities, especially in their intact cells, which in most of the cases, the isolated strains were stronger in these activities than the tested commercial reference strains. None of the tested strains produced harmful enzymes (β-glucosidase and β-glucuronidase) or biogenic amines. Among the 12 strains, 3 strains were tested for their cytotoxic effects against 3 cancer cell lines, which exhibited strong cytotoxic effects, and they also showed selectivity in killing cancer cells when compared to normal cells. Hence, all 12 Lactobacillus strains could be considered good potential probiotic candidates because of their beneficial functional bioactivities. The Lactobacillus strains tested in this study could be considered good potential probiotic candidates for food/feed industry because of their beneficial functional bioactivities such as good cholesterol-reducing ability, high antioxidant activity, and good and selective cytotoxic effect against cancer cells. © 2017 Institute of Food Technologists®.

Private selective sweeps identified from next-generation pool-sequencing reveal convergent pathways under selection in two inbred Schistosoma mansoni strains.

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Julie A J Clément

Full Text Available BACKGROUND: The trematode flatworms of the genus Schistosoma, the causative agents of schistosomiasis, are among the most prevalent parasites in humans, affecting more than 200 million people worldwide. In this study, we focused on two well-characterized strains of S. mansoni, to explore signatures of selection. Both strains are highly inbred and exhibit differences in life history traits, in particular in their compatibility with the intermediate host Biomphalaria glabrata. METHODOLOGY/PRINCIPAL FINDINGS: We performed high throughput sequencing of DNA from pools of individuals of each strain using Illumina technology and identified single nucleotide polymorphisms (SNP and copy number variations (CNV. In total, 708,898 SNPs were identified and roughly 2,000 CNVs. The SNPs revealed low nucleotide diversity (π = 2 × 10(-4 within each strain and a high differentiation level (Fst = 0.73 between them. Based on a recently developed in-silico approach, we further detected 12 and 19 private (i.e. specific non-overlapping selective sweeps among the 121 and 151 sweeps found in total for each strain. CONCLUSIONS/SIGNIFICANCE: Functional annotation of transcripts lying in the private selective sweeps revealed specific selection for functions related to parasitic interaction (e.g. cell-cell adhesion or redox reactions. Despite high differentiation between strains, we identified evolutionary convergence of genes related to proteolysis, known as a key virulence factor and a potential target of drug and vaccine development. Our data show that pool-sequencing can be used for the detection of selective sweeps in parasite populations and enables one to identify biological functions under selection.

Selection of tannase-producing Aspergillus niger strains

OpenAIRE

Pinto,Gustavo A.S.; Leite,Selma G.F.; Terzi,Selma C.; Couri,Sonia

2001-01-01

The aim of this work was to select strains of Aspergillus niger for tannase production. Growth of colonies in plates with tannic acid-containing medium indicated their ability to synthesize tannase. Tannase activity was also measured in solid-state fermentation. A. niger 11T25A5 was the best tannase producer (67.5 U.g-1/72 hours of fermentation).

Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang.

Science.gov (United States)

Kim, W; Choi, K; Kim, Y; Park, H; Choi, J; Lee, Y; Oh, H; Kwon, I; Lee, S

1996-01-01

Bacillus sp. strain CK 11-4, which produces a strongly fibrinolytic enzyme, was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce. The fibrinolytic enzyme (CK) was purified from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH were 70 degrees C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 14 amino acids of the N-terminal sequence of CK are Ala-Gin-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp. This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of fibrinolytic activity that was about eight times higher than that of subtilisin Carlsberg. The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys-pNA). A similar result was also obtained from fibrin plate analysis. PMID:8779587

Model for how type I restriction enzymes select cleavage sites in DNA

International Nuclear Information System (INIS)

Studier, F.W.; Bandyopadhyay, P.K.

1988-01-01

Under appropriate conditions, digestion of phage T7 DNA by the type I restriction enzyme EcoK produces an orderly progression of discrete DNA fragments. All details of the fragmentation pattern can be explained on the basis of the known properties of type I enzymes, together with two further assumptions: (i) in the ATP-stimulated translocation reaction, the enzyme bound at the recognition sequence translocates DNA toward itself from both directions simultaneously; and (ii) when translocation causes neighboring enzymes to meet, they cut the DNA between them. The kinetics of digestion at 37 degree C indicates that the rate of translocation of DNA from each side of a bound enzyme is about 200 base pairs per second, and the cuts are completed within 15-25 sec of the time neighboring enzymes meet. The resulting DNA fragments each contain a single recognition site with an enzyme (or subunit) remaining bound to it. At high enzyme concentrations, such fragments can bu further degraded, apparently by cooperation between the specifically bound and excess enzymes. This model is consistent with a substantial body of previous work on the nuclease activity of EcoB and EcoK, and it explains in a simple way how cleavage sites are selected

Characterization of Selected Lactobacillus Strains for Use as Probiotics

OpenAIRE

Song, Minyu; Yun, Bohyun; Moon, Jae-Hak; Park, Dong-June; Lim, Kwangsei; Oh, Sejong

2015-01-01

The aim of this study was to evaluate the functional properties of lactic acid bacteria from various sources and to identify strains for use as probiotics. Ten Lactobacillus strains were selected and their properties such as bile tolerance, acid resistance, cholesterol assimilation activity, and adherence to HT-29 cells were assessed to determine their potential as probiotics. Lactobacillus sp. JNU 8829, L. casei MB3, L. sakei MA9, L. sakei CH8, and L. acidophilus M23 were found to show full ...

Identification of thermophilic bacterial strains producing thermotolerant hydrolytic enzymes from manure compost.

Science.gov (United States)

Charbonneau, David M; Meddeb-Mouelhi, Fatma; Boissinot, Maurice; Sirois, Marc; Beauregard, Marc

2012-03-01

Ten thermophilic bacterial strains were isolated from manure compost. Phylogenetic analysis based on 16S rRNA genes and biochemical characterization allowed identification of four different species belonging to four genera: Geobacillus thermodenitrificans, Bacillus smithii, Ureibacillus suwonensis and Aneurinibacillus thermoaerophilus. PCR-RFLP profiles of the 16S-ITS-23S rRNA region allowed us to distinguish two subgroups among the G. thermodenitrificans isolates. Isolates were screened for thermotolerant hydrolytic activities (60-65°C). Thermotolerant lipolytic activities were detected for G. thermodenitrificans, A. thermoaerophilus and B. smithii. Thermotolerant protease, α-amylase and xylanase activities were also observed in the G. thermodenitrificans group. These species represent a source of potential novel thermostable enzymes for industrial applications.

Determining the safety of enzymes used in animal feed.

Science.gov (United States)

Pariza, Michael W; Cook, Mark

2010-04-01

The purpose of this paper is to provide guidance for evaluating the safety of enzyme preparations used in animal feed. Feed enzymes are typically added to animal feed to increase nutrient bioavailability by acting on feed components prior to or after consumption, i.e., within the gastrointestinal tract. In contrast, food processing enzymes are generally used during processing and then inactivated or removed prior to consumption. The enzymes used in both applications are almost always impure mixtures of active enzyme and other metabolites from the production strain, hence similar safety evaluation procedures for both are warranted. We propose that the primary consideration should be the safety of the production strain and that the decision tree mechanism developed previously for food processing enzymes (Pariza and Johnson, 2001) is appropriate for determining the safety of feed enzymes. Thoroughly characterized non-pathogenic, non-toxigenic microbial strains with a history of safe use in enzyme manufacture are also logical candidates for generating safe strain lineages, from which additional strains may be derived via genetic modification by traditional and non-traditional strategies. For new feed enzyme products derived from a safe strain lineage, it is important to ensure a sufficiently high safety margin for the intended use, and that the product complies with appropriate specifications for chemical and microbial contamination. Copyright 2009 Elsevier Inc. All rights reserved.

Alkaline thermostable pectinase enzyme from Aspergillus niger strain MCAS2 isolated from Manaslu Conservation Area, Gorkha, Nepal.

Science.gov (United States)

Khatri, Bhim Prakash; Bhattarai, Tribikram; Shrestha, Sangita; Maharjan, Jyoti

2015-01-01

Pectinase enzymes are one of the commercially important enzymes having great potential in various industries especially in food industry. Pectinases accounts for 25 % of global food enzymes produced and their market is increasing day by day. Therefore, the exploration of microorganism with novel characteristics has always been the focus of the research. Microorganism dwelling in unique habitat may possess unique characteristics. As such, a pectinase producing fungus Aspergillus niger strain MCAS2 was isolated from soil of Manaslu Conservation Area (MCA), Gorkha, Nepal. The optimum production of pectinase enzyme was observed at 48 h of fermentation. The pectinase enzyme was partially purified by cold acetone treatment followed by Sephadex G-75 gel filtration chromatography. The partially purified enzyme exhibited maximum activity 60 U/mg which was almost 8.5-fold higher than the crude pectinase. The approximate molecular weight of the enzyme was found to be 66 kDa as observed from SDS-PAGE. The pectinase enzyme was active at broad range of temperature (30-70 °C) and pH (6.2-9.2). Optimum temperature and pH of the pectinase enzyme were 50 °C and 8.2 respectively. The enzyme was stable up to 70 °C and about 82 % of pectinase activity was still observed at 100 °C. The thermostable and alkaline nature of this pectinase can meet the demand of various industrial processes like paper and pulp industry, in textile industry, fruit juice industry, plant tissue maceration and wastewater treatment. In addition, the effect of different metal ions on pectinase activity was also studied.

The potential of halophilic and halotolerant bacteria for the production of antineoplastic enzymes: L-asparaginase and L-glutaminase.

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Shirazian, Pejman; Asad, Sedigheh; Amoozegar, Mohammad Ali

2016-01-01

L-asparaginase and L-glutaminase can be effectively used for the treatment of patients who suffer from accute lymphoblastic leukemia and tumor cells. Microbial sources are the best source for the bulk production of these enzymes. However, their long-term administration may cause immunological responses, so screening for new enzymes with novel properties is required. Halophilic and halotolerant bacteria with novel enzymatic characteristics can be considered as a potential source for production of enzymes with different immunological properties. In this study, L-asparaginase and L-glutaminase production by halophilic bacteria isolated from Urmia salt lake was studied. Out of the 85 isolated halophilic and halotolerant bacterial strains, 16 (19 %) showed L-asparaginase activity and 3 strains (3.5 %) showed L-glutaminase activity. Strains with the highest activities were selected for further studies. Based on 16S rDNA sequence analysis, it was shown that the selected isolates for L-asparaginase and L-glutaminase production belong to the genus Bacillus and Salicola, respectively. Both enzymes were produced extracellularly. The strain with the most L-asparaginase production did not show L-glutaminase production which is medically important. The effects of key parameters including temperature, initial pH of the solution, and concentrations of glucose, asparagine or glutamine, and sodium chloride were evaluated by means of response surface methodology (RSM) to optimize enzymes production. Under the obtained optimal conditions, L-asparaginase and L-glutaminase production was increased up to 1.5 (61.7 unit/mL) and 2.6 fold (46.4 unit/mL), respectively.

GROWTH AND ENZYME PRODUCTION DURING CONTINUOUS CULTURES OF A HIGH AMYLASE-PRODUCING VARIANT OF Aspergillus Oryzae

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T.C. Zangirolami

2002-03-01

Full Text Available Growth and product formation by a selected variant of Aspergillus oryzae showing high alpha-amylase production was studied in continuous cultivations carried out at six different specific growth rates, using glucose as the growth-limiting nutrient. The analysis of the steady-state data revealed that the variant and wild-type strains were similar with respect to glucose uptake system and stoichiometric coefficients. However, the variant was capable of maintaining an enzyme production as high as 40 FAUgDW-1h-1 at a dilution rate of 0.2 h-1, while the wild-type strain reached a maximum specific alpha-amylase production rate of 17 FAUgDW-1h-1 at a dilution rate of 0.1 h-1. Using a morphologically structured model originally proposed for the wild-type strain, it was possible to describe enzyme production, biomass formation and glucose consumption after modification of a few parameters to adjust the model to the characteristics of the selected variant.

Thermotolerant and mesophylic fungi from sugarcane bagasse and their prospection for biomass-degrading enzyme production

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Bruna Silveira Lamanes dos Santos

2015-09-01

Full Text Available Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3, A. sydowii (SBCM7 and A. fumigatus (SBC4. The best β-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g. For β-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g and 4.0 (573 U/g, respectively. All these crude enzymes presented stability around pH 3.0–8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors.

Investigation of lactic acid bacterial strains for meat fermentation and the product's antioxidant and angiotensin-I-converting-enzyme inhibitory activities.

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Takeda, Shiro; Matsufuji, Hisashi; Nakade, Koji; Takenoyama, Shin-Ichi; Ahhmed, Abdulatef; Sakata, Ryoichi; Kawahara, Satoshi; Muguruma, Michio

2017-03-01

In the lactic acid bacteria (LAB) strains screened from our LAB collection, Lactobacillus (L.) sakei strain no. 23 and L. curvatus strain no. 28 degraded meat protein and tolerated salt and nitrite in vitro. Fermented sausages inoculated strains no. 23 and no. 28 showed not only favorable increases in viable LAB counts and reduced pH, but also the degradation of meat protein. The sausages fermented with these strains showed significantly higher antioxidant activity than those without LAB or fermented by each LAB type strain. Angiotensin-I-converting-enzyme (ACE) inhibitory activity was also significantly higher in the sausages fermented with strain no. 23 than in those fermented with the type strain. Higher ACE inhibitory activity was also observed in the sausages fermented with strain no. 28, but did not differ significantly from those with the type strain. An analysis of the proteolysis and degradation products formed by each LAB in sausages suggested that those bioactivities yielded fermentation products such as peptides. Therefore, LAB starters that can adequately ferment meat, such as strains no. 23 and no. 28, should contribute to the production of bioactive compounds in meat products. © 2016 Japanese Society of Animal Science.

SELEKSI DAN IDENTIFIKASI BAKTERI ENDOFIT POTENSIAL PENGHASIL ENZIM PROTEASE DARI TAMAN NASIONAL GUNUNG HALIMUN - (The Selection and Identification of Potential Endophyte Bacteria as Protease Enzyme Producer from Halimun Mount National Park

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Ruth Melliawati

2016-12-01

Full Text Available Endophytic bacteria have an equal chance to bacteria that live outside the plant tissue as potential bacteria. The selection has done towards 326 bacterial endophyte isolates. This research aimed to find and identify proteolytic potential isolates. The proteolytic selection of endophytic bacteria had done using solid skim milk. The capability of endophytic bacteria to agglomerate milk was tested using liquid skim milk which incubated for 7 days at room temperature. Enzyme production of four selected isolates was made through fermentation in GYS medium. The results showed that 86 isolates have proteolytic potential. Isolate HL.29B.63 had highest protease enzymes activity (65.918 U/mL. Medium optimization was able to increase the enzyme activity into 89.94% (125.04 U/mL. The analysis used 16s rDNA showed that isolate HL.29B.63 was Bacillus amyloliquefacient subs. plantarum strain FZB42.Keywords: endophytic bacteria, fermentation, identification, protease, selection ABSTRAKBakteri endofit mempunyai peluang yang sama dengan bakteri yang hidup diluar jaringan tanaman sebagai bakteri potensial. Seleksi dilakukan terhadap 326 isolat bakteri endofit. Tujuan penelitian ini adalah mencari isolat yang berpotensi proteolitik dan mengidentifikasinya. Seleksi proteolitik terhadap bakteri endofitik menggunakan skim milk padat. Uji kemampuan bakteri endofitik dalam menggumpalkan susu menggunakan medium skim milk cair yang diinkubasi selama 7 hari pada suhu ruang. Produksi enzim terhadap empat isolat terseleksi dilakukan melalui fermentasi dalam medium GYS. Hasilnya menunjukkan bahwa 86 isolat mempunyai potensi proteolitik. Isolat HL.29B.63 mempunyai aktif enzim protease tertinggi (65,918 U/mL. Optimasi medium dapat meningkatkan aktivitas enzim sebesar 89,94% (125,04 U/mL. Analisis menggunakan 16s rDNA menunjukkan bahwa isolat HL.29B.63 adalah Bacillus amyloliquefaciens subs. plantarum strain FZB42.Kata kunci: bakteri endofit, fermentasi, identifikasi, protease

Design and Validation of a Cyclic Strain Bioreactor to Condition Spatially-Selective Scaffolds in Dual Strain Regimes

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J. Matthew Goodhart

2014-03-01

Full Text Available The objective of this study was to design and validate a unique bioreactor design for applying spatially selective, linear, cyclic strain to degradable and non-degradable polymeric fabric scaffolds. This system uses a novel three-clamp design to apply cyclic strain via a computer controlled linear actuator to a specified zone of a scaffold while isolating the remainder of the scaffold from strain. Image analysis of polyethylene terephthalate (PET woven scaffolds subjected to a 3% mechanical stretch demonstrated that the stretched portion of the scaffold experienced 2.97% ± 0.13% strain (mean ± standard deviation while the unstretched portion experienced 0.02% ± 0.18% strain. NIH-3T3 fibroblast cells were cultured on the PET scaffolds and half of each scaffold was stretched 5% at 0.5 Hz for one hour per day for 14 days in the bioreactor. Cells were checked for viability and proliferation at the end of the 14 day period and levels of glycosaminoglycan (GAG and collagen (hydroxyproline were measured as indicators of extracellular matrix production. Scaffolds in the bioreactor showed a seven-fold increase in cell number over scaffolds cultured statically in tissue culture plastic petri dishes (control. Bioreactor scaffolds showed a lower concentration of GAG deposition per cell as compared to the control scaffolds largely due to the great increase in cell number. A 75% increase in hydroxyproline concentration per cell was seen in the bioreactor stretched scaffolds as compared to the control scaffolds. Surprisingly, little differences were experienced between the stretched and unstretched portions of the scaffolds for this study. This was largely attributed to the conditioned and shared media effect. Results indicate that the bioreactor system is capable of applying spatially-selective, linear, cyclic strain to cells growing on polymeric fabric scaffolds and evaluating the cellular and matrix responses to the applied strains.

Selection of autochthonous Oenococcus oeni strains according to their oenological properties and vinification results.

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Ruiz, Patricia; Izquierdo, Pedro Miguel; Seseña, Susana; Palop, María Llanos

2010-02-28

The goal of this study is to carry out a characterization of 84 Oenococcus oeni strains isolated from Tempranillo wine samples taken at the cellars in Castilla-La Mancha, in order to select those showing the highest potential as oenological starter cultures. Various oenological properties were analyzed and the ability of some of these strains to grow and undergo MLF in simulated laboratory microvinifications was tested. Twenty-two strains were selected on the basis of fermentation assays and the eight that produced the best results in the chemical analysis of the wines were chosen for further assays. None of the eight strains was either able to produce biogenic amines or displayed tannase or anthocyanase activities. On the other hand all presented activity against p-NP-beta Glucopyranoside, p-NP-alpha Glucopyranoside and p-NP-beta xylopyranoside. Randomly Amplified Polymorphic DNA (RAPD)-PCR was used to determine the colonizing ability of the inoculated strains. C22L9 and D13L13 strains showed the highest implantation values. On the basis of this characterization, two strains have been selected which are suitable as starter cultures for MLF of Tempranillo wine. Use of these strains will ensure that MLF proceeds successfully and gives retention of the organoleptic characteristics of wines made in Castilla-La Mancha. (c) 2009 Elsevier B.V. All rights reserved.

Improvement in antioxidant activity, angiotensin-converting enzyme inhibitory activity and in vitro cellular properties of fermented pepino milk by Lactobacillus strains containing the glutamate decarboxylase gene.

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Chiu, Tsai-Hsin; Tsai, Shwu-Jene; Wu, Tsung-Yen; Fu, Szu-Chieh; Hwang, Yi-Ting

2013-03-15

The purpose of this study was to evaluate the functional potential of fermented pepino extract (PE) milk by Lactobacillus strains containing the glutamate decarboxylase (GAD) gene. Three Lactobacillus strains were selected, including L. brevis BCRC 12310, L. casei BCRC 14082 and L. salivarius subsp. salivarius BCRC 14759. The contents of free amino acids, total phenolics content, total carotenoids and the associated functional and antioxidant abilities were analyzed, including angiotensin-converting enzyme (ACE) inhibition activity, 1,1-diphenyl-2-picylhydrazyl (DPPH) radical-scavenging ability and oxygen radical absorbance capacity (ORAC). Cell proliferation of fermented PE milk was also evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Compared to the unfermented PE, fermented PE milk from Lactobacillus strains with the GAD gene showed higher levels of total phenolics, γ-aminobutyric acid, ACE inhibitory activity, DPPH, and ORAC. The viability of human promyelocytic leukemia cells (HL-60) determined by the MTT method decreased significantly when the cells were incubated with the PE and the fermented PE milk extracts. The consumption of fermented PE milk from Lactobacillus strains with the GAD gene is expected to benefit health. Further application as a health food is worthy of investigation. © 2012 Society of Chemical Industry. © 2012 Society of Chemical Industry.

Differential Selectivity of the Escherichia coli Cell Membrane Shifts the Equilibrium for the Enzyme-Catalyzed Isomerization of Galactose to Tagatose▿

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Kim, Jin-Ha; Lim, Byung-Chul; Yeom, Soo-Jin; Kim, Yeong-Su; Kim, Hye-Jung; Lee, Jung-Kul; Lee, Sook-Hee; Kim, Seon-Won; Oh, Deok-Kun

2008-01-01

An Escherichia coli galactose kinase gene knockout (ΔgalK) strain, which contains the l-arabinose isomerase gene (araA) to isomerize d-galactose to d-tagatose, showed a high conversion yield of tagatose compared with the original galK strain because galactose was not metabolized by endogenous galactose kinase. In whole cells of the ΔgalK strain, the isomerase-catalyzed reaction exhibited an equilibrium shift toward tagatose, producing a tagatose fraction of 68% at 37°C, whereas the purified l-arabinose isomerase gave a tagatose equilibrium fraction of 36%. These equilibrium fractions are close to those predicted from the measured equilibrium constants of the isomerization reaction catalyzed in whole cells and by the purified enzyme. The equilibrium shift in these cells resulted from the higher uptake and lower release rates for galactose, which is a common sugar substrate, than for tagatose, which is a rare sugar product. A ΔmglB mutant had decreased uptake rates for galactose and tagatose, indicating that a methylgalactoside transport system, MglABC, is the primary contributing transporter for the sugars. In the present study, whole-cell conversion using differential selectivity of the cell membrane was proposed as a method for shifting the equilibrium in sugar isomerization reactions. PMID:18263746

Differential selectivity of the Escherichia coli cell membrane shifts the equilibrium for the enzyme-catalyzed isomerization of galactose to tagatose.

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Kim, Jin-Ha; Lim, Byung-Chul; Yeom, Soo-Jin; Kim, Yeong-Su; Kim, Hye-Jung; Lee, Jung-Kul; Lee, Sook-Hee; Kim, Seon-Won; Oh, Deok-Kun

2008-04-01

An Escherichia coli galactose kinase gene knockout (DeltagalK) strain, which contains the l-arabinose isomerase gene (araA) to isomerize d-galactose to d-tagatose, showed a high conversion yield of tagatose compared with the original galK strain because galactose was not metabolized by endogenous galactose kinase. In whole cells of the DeltagalK strain, the isomerase-catalyzed reaction exhibited an equilibrium shift toward tagatose, producing a tagatose fraction of 68% at 37 degrees C, whereas the purified l-arabinose isomerase gave a tagatose equilibrium fraction of 36%. These equilibrium fractions are close to those predicted from the measured equilibrium constants of the isomerization reaction catalyzed in whole cells and by the purified enzyme. The equilibrium shift in these cells resulted from the higher uptake and lower release rates for galactose, which is a common sugar substrate, than for tagatose, which is a rare sugar product. A DeltamglB mutant had decreased uptake rates for galactose and tagatose, indicating that a methylgalactoside transport system, MglABC, is the primary contributing transporter for the sugars. In the present study, whole-cell conversion using differential selectivity of the cell membrane was proposed as a method for shifting the equilibrium in sugar isomerization reactions.

Overproduction of ligninolytic enzymes

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Elisashvili, Vladimir; Kachlishvili, Eva; Torok, Tamas

2014-06-17

Methods, compositions, and systems for overproducing ligninolytic enzymes from the basidiomycetous fungus are described herein. As described, the method can include incubating a fungal strain of Cerrena unicolor IBB 303 in a fermentation system having growth medium which includes lignocellulosic material and then cultivating the fungal strain in the fermentation system under conditions wherein the fungus expresses the ligninolytic enzymes. In some cases, the lignocellulosic material is mandarin peel, ethanol production residue, walnut pericarp, wheat bran, wheat straw, or banana peel.

Characterization of Selected Lactobacillus Strains for Use as Probiotics.

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Song, Minyu; Yun, Bohyun; Moon, Jae-Hak; Park, Dong-June; Lim, Kwangsei; Oh, Sejong

2015-01-01

The aim of this study was to evaluate the functional properties of lactic acid bacteria from various sources and to identify strains for use as probiotics. Ten Lactobacillus strains were selected and their properties such as bile tolerance, acid resistance, cholesterol assimilation activity, and adherence to HT-29 cells were assessed to determine their potential as probiotics. Lactobacillus sp. JNU 8829, L. casei MB3, L. sakei MA9, L. sakei CH8, and L. acidophilus M23 were found to show full tolerance to the 0.3% bile acid. All strains without L. acidophilus M23 were the most acid-tolerant strains. After incubating the strains at pH 2.5 for 2 h, their viability decreased by 3 Log cells. Some strains survived at pH 2.5 in the presence of pepsin and 0.3% bile acid. Lactobacillus sp. JNU 8829, L. acidophilus KU41, L. acidophilus M23, L. fermentum NS2, L. plantarum M13, and L. plantarum NS3 were found to reduce cholesterol levels by >50% in vitro. In the adhesion assay, Lactobacillus sp. JNU 8829, L. casei MB3, L. sakei MA9, and L. sakei CH8 showed higher adhesion activities after 2 h of co-incubation with the intestinal cells. The results of this comprehensive analysis shows that this new probiotic strain named, Lactobacillus sp. JNU 8829 could be a promising candidate for dairy products.

Antagonistic Activity of Lactobacillus reuteri Strains on the Adhesion Characteristics of Selected Pathogens.

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Singh, Tejinder P; Kaur, Gurpreet; Kapila, Suman; Malik, Ravinder K

2017-01-01

Adhesion ability of probiotics is the key factor that decides their colonization in the gastrointestinal tract and potential to inhibit pathogens. Therefore, adhesion ability can be considered as a key determinant for probiotic efficacy. Presents study documents the antagonistic activity of viable/untreated, Lithium chloride (LiCl) treated or heat-killed forms of eight probiotic Lactobacillus reuteri strains on the adhesion characteristics of selected pathogens. All strains investigated were able to adhere to Caco-2 cells. L. reuteri strains tested were able to inhibit and displace ( P strain L. reuteri LR6 showed the strongest adhesion and pathogen inhibition ability among the eight L. reuteri strains tested. In addition, the abilities to inhibit and to displace adhered pathogens depended on both the probiotic and the pathogen strains tested suggesting the involvement of various mechanisms. The adhesion and antagonistic potential of the probiotic strains were significantly decreased upon exposure to 5 M LiCl, showing that surface molecules, proteinaceous in nature, are involved. The heat-killed forms of the probiotic L. reuteri strains also inhibited the attachment of selected pathogens to Caco-2 cells. In conclusion, in vitro assays showed that L. reuteri strains, as viable or heat-killed forms, are adherent to Caco-2 cells and are highly antagonistic to pathogens tested in which surface associated proteins play an important role.

Comparative analysis of response to selection with three insecticides in the dengue mosquito Aedes aegypti using mRNA sequencing.

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David, Jean-Philippe; Faucon, Frédéric; Chandor-Proust, Alexia; Poupardin, Rodolphe; Riaz, Muhammad Asam; Bonin, Aurélie; Navratil, Vincent; Reynaud, Stéphane

2014-03-05

Mosquito control programmes using chemical insecticides are increasingly threatened by the development of resistance. Such resistance can be the consequence of changes in proteins targeted by insecticides (target site mediated resistance), increased insecticide biodegradation (metabolic resistance), altered transport, sequestration or other mechanisms. As opposed to target site resistance, other mechanisms are far from being fully understood. Indeed, insecticide selection often affects a large number of genes and various biological processes can hypothetically confer resistance. In this context, the aim of the present study was to use RNA sequencing (RNA-seq) for comparing transcription level and polymorphism variations associated with adaptation to chemical insecticides in the mosquito Aedes aegypti. Biological materials consisted of a parental susceptible strain together with three child strains selected across multiple generations with three insecticides from different classes: the pyrethroid permethrin, the neonicotinoid imidacloprid and the carbamate propoxur. After ten generations, insecticide-selected strains showed elevated resistance levels to the insecticides used for selection. RNA-seq data allowed detecting over 13,000 transcripts, of which 413 were differentially transcribed in insecticide-selected strains as compared to the susceptible strain. Among them, a significant enrichment of transcripts encoding cuticle proteins, transporters and enzymes was observed. Polymorphism analysis revealed over 2500 SNPs showing > 50% allele frequency variations in insecticide-selected strains as compared to the susceptible strain, affecting over 1000 transcripts. Comparing gene transcription and polymorphism patterns revealed marked differences among strains. While imidacloprid selection was linked to the over transcription of many genes, permethrin selection was rather linked to polymorphism variations. Focusing on detoxification enzymes revealed that permethrin

Iron-Dependent Enzyme Catalyzes the Initial Step in Biodegradation of N-Nitroglycine by Variovorax sp. Strain JS1663.

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Mahan, Kristina M; Zheng, Hangping; Fida, Tekle T; Parry, Ronald J; Graham, David E; Spain, Jim C

2017-08-01

Nitramines are key constituents of most of the explosives currently in use and consequently contaminate soil and groundwater at many military facilities around the world. Toxicity from nitramine contamination poses a health risk to plants and animals. Thus, understanding how nitramines are biodegraded is critical to environmental remediation. The biodegradation of synthetic nitramine compounds such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has been studied for decades, but little is known about the catabolism of naturally produced nitramine compounds. In this study, we report the isolation of a soil bacterium, Variovorax sp. strain JS1663, that degrades N -nitroglycine (NNG), a naturally produced nitramine, and the key enzyme involved in its catabolism. Variovorax sp. JS1663 is a Gram-negative, non-spore-forming motile bacterium isolated from activated sludge based on its ability to use NNG as a sole growth substrate under aerobic conditions. A single gene ( nnlA ) encodes an iron-dependent enzyme that releases nitrite from NNG through a proposed β-elimination reaction. Bioinformatics analysis of the amino acid sequence of NNG lyase identified a PAS (Per-Arnt-Sim) domain. PAS domains can be associated with heme cofactors and function as signal sensors in signaling proteins. This is the first instance of a PAS domain present in a denitration enzyme. The NNG biodegradation pathway should provide the basis for the identification of other enzymes that cleave the N-N bond and facilitate the development of enzymes to cleave similar bonds in RDX, nitroguanidine, and other nitramine explosives. IMPORTANCE The production of antibiotics and other allelopathic chemicals is a major aspect of chemical ecology. The biodegradation of such chemicals can play an important ecological role in mitigating or eliminating the effects of such compounds. N -Nitroglycine (NNG) is produced by the Gram-positive filamentous soil bacterium Streptomyces noursei This study reports the

Effect of gamma irradiation and environmental factors on the production of extracellular cellulase enzyme by trichoderma Spp. using banana waste under solid state bio processing

International Nuclear Information System (INIS)

El-Shafey, H.M.; Matar, Z.A.I.; Ghanem, S.M.A.

2007-01-01

Fungal strains were isolated from degraded banana waste including leaves, pseudo stems and skins. Many isolated strains showed cellulolytic activities using the plate screening medium. The hyper cellulolytic isolates were selected on the basis of the diameter of the hydrolysis zone surrounding the colonies and identified to the genus level. The identified strains were found to belong to one of the genera Trichoderma, Aspergillus, Pleurotus or Penicillium. The strain with the larger diameter of the hydrolysis zone was found to belong to the genus Trichoderma. It was further identified to be Trichoderma harzianum, which was selected to be studied. Banana waste including leaves and pseudo stems were inoculated by the selected fungus and the production of the carboxymethyl cellulase (CMCase) and filter paper cellulase (FPCase) was followed during changes of the growth conditions under solid state fermentation. It was found that the two enzymes shared the same incubation temperature (25 degree C) and incubation period (18 days) for the maximum enzyme production. The gamma radiation dose of 1.5 KGy increased the production of CMCase produced on leaves by 4.0% and on pseudo stems by 5.6% and the production of FPCase produced on leaves by 2.4% and on pseudo stems by 2.3%. The results also suggest that FPCase and CMCase enzymes produced on leaves were higher than those produced from pseudo stems and the level of CMCase enzyme produced was higher than that of FPCase

Characterization of poly(L-lactide)-degrading enzyme produced by thermophilic filamentous bacteria Laceyella sacchari LP175.

Science.gov (United States)

Hanphakphoom, Srisuda; Maneewong, Narisara; Sukkhum, Sukhumaporn; Tokuyama, Shinji; Kitpreechavanich, Vichien

2014-01-01

Eleven strains of poly(L-lactide) (PLLA)-degrading thermophilic bacteria were isolated from forest soils and selected based on clear zone formation on an emulsified PLLA agar plate at 50°C. Among the isolates, strain LP175 showed the highest PLLA-degrading ability. It was closely related to Laceyella sacchari, with 99.9% similarity based on the 16S rRNA gene sequence. The PLLA-degrading enzyme produced by the strain was purified to homogeneity by 48.1% yield and specific activity of 328 U·mg-protein-1 with a 15.3-fold purity increase. The purified enzyme was strongly active against specific substrates such as casein and gelatin and weakly active against Suc-(Ala)₃-pNA. Optimum enzyme activity was exhibited at a temperature of 60°C with thermal stability up to 50°C and a pH of 9.0 with pH stability in a range of 8.5-10.5. Molecular weight of the enzyme was approximately 28.0 kDa, as determined by gel filtration and SDS-PAGE. The inhibitors phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetate (EDTA), and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) strongly inhibited enzyme activity, but the activity was not inhibited by 1 mM 1,10-phenanthroline (1,10-phen). The N-terminal amino acid sequences had 100% homology with thermostable serine protease (thermitase) from Thermoactinomyces vulgaris. The results obtained suggest that the PLLA-degrading enzyme produced by L. sacchari strain LP175 is serine protease.

Diversity and characterization of ramie-degumming strains

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Shengwen Duan

2012-04-01

Full Text Available Ramie (Boehmeria nivea and Boehmeria tenacissima is a widely used fiber crop. Traditional water retting or chemical boiling method performed in order to extract ramie fiber seriously pollute the environment and severely damage the fiber, so biological method is the general trend of the fiber-extracting industry. Some strains (687, involving 26 genera and 43 species, were collected from the three samples, which produce hydrolyzed circles in the selective culture medium in order to detect the degumming effect and to compare the enzyme activity. Among these strains, 13 of them did not produce cellulase and had a ramie decreasing weight rate above 25 %, which were regarded as efficient ramie-degumming strains named from R1 to R13. R1 to R13 belonged to Amycolata autotrobutylicun, Bacillus subtilis, Clostridium acetobutylicum, Bacillus subtilis, Rhizobium leguminosarum, Bacteroides finegoldii, Streptomyces lividans, Bacillus amyloliquefaciens, Clostridium acetobutylicum, Pseudomonas brassicacearum, Bacillus pumilus, Bacillus licheniformis, Pectobacterium wasabiae respectively. Bacteroides sp., Rhizobium sp. and Pseudomonas sp. were firstly reported to be used in ramie-degumming. At the same time, the pectinase was the key enzyme in the ramie-degumming process.

Molybdenum reduction to molybdenum blue in Serratia sp. Strain DRY5 is catalyzed by a novel molybdenum-reducing enzyme.

Science.gov (United States)

Shukor, M Y; Halmi, M I E; Rahman, M F A; Shamaan, N A; Syed, M A

2014-01-01

The first purification of the Mo-reducing enzyme from Serratia sp. strain DRY5 that is responsible for molybdenum reduction to molybdenum blue in the bacterium is reported. The monomeric enzyme has an apparent molecular weight of 105 kDalton. The isoelectric point of this enzyme was 7.55. The enzyme has an optimum pH of 6.0 and maximum activity between 25 and 35°C. The Mo-reducing enzyme was extremely sensitive to temperatures above 50°C (between 54 and 70°C). A plot of initial rates against substrate concentrations at 15 mM 12-MP registered a V max for NADH at 12.0 nmole Mo blue/min/mg protein. The apparent K m for NADH was 0.79 mM. At 5 mM NADH, the apparent V max and apparent K m values for 12-MP of 12.05 nmole/min/mg protein and 3.87 mM, respectively, were obtained. The catalytic efficiency (k cat/K m ) of the Mo-reducing enzyme was 5.47 M(-1) s(-1). The purification of this enzyme could probably help to solve the phenomenon of molybdenum reduction to molybdenum blue first reported in 1896 and would be useful for the understanding of the underlying mechanism in molybdenum bioremediation involving bioreduction.

Molybdenum Reduction to Molybdenum Blue in Serratia sp. Strain DRY5 Is Catalyzed by a Novel Molybdenum-Reducing Enzyme

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M. Y. Shukor

2014-01-01

Full Text Available The first purification of the Mo-reducing enzyme from Serratia sp. strain DRY5 that is responsible for molybdenum reduction to molybdenum blue in the bacterium is reported. The monomeric enzyme has an apparent molecular weight of 105 kDalton. The isoelectric point of this enzyme was 7.55. The enzyme has an optimum pH of 6.0 and maximum activity between 25 and 35°C. The Mo-reducing enzyme was extremely sensitive to temperatures above 50°C (between 54 and 70°C. A plot of initial rates against substrate concentrations at 15 mM 12-MP registered a Vmax for NADH at 12.0 nmole Mo blue/min/mg protein. The apparent Km for NADH was 0.79 mM. At 5 mM NADH, the apparent Vmax and apparent Km values for 12-MP of 12.05 nmole/min/mg protein and 3.87 mM, respectively, were obtained. The catalytic efficiency (kcat/Km of the Mo-reducing enzyme was 5.47 M-1 s-1. The purification of this enzyme could probably help to solve the phenomenon of molybdenum reduction to molybdenum blue first reported in 1896 and would be useful for the understanding of the underlying mechanism in molybdenum bioremediation involving bioreduction.

Effect of chlorpyrifos and enrofloxacin on selected enzymes in rats.

Science.gov (United States)

Barski, D; Spodniewska, A

2018-03-01

This study examined the effect of chlorpyrifos and/or enrofloxacin on the activity of acetylcholinesterase (AChE) in the blood and brain, and the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum. The experiment was conducted on Wistar strain rats. Chlorpyrifos was administered with a stomach tube at a dose of 0.04 LD50 for 28 days and enrofloxacin at a dose of 5 mg/kg bw for 5 consecutive days. The experiment found that enrofloxacin changed the activity of the enzymes under study only to a small extent. At the dose applied in the experiment, chlorpyrifos decreased the activity of AChE significantly, both in blood and in the brain, and increased the activity of ALT and AST in rat serum. The administration of chlorpyrifos in combination with enrofloxacin changed the activity of the enzymes under study only slightly. A weaker, but longer, inhibition of AChE activity in both blood and the brain was observed in this group compared to the animals exposed only to chlorpyrifos. However, although enrofloxacin, like chlorpyrifos, increases the activity of ALT and AST in serum, their combined administration did not increase the hepatotoxic effect. Copyright© by the Polish Academy of Sciences.

Mutant strain screening by 60Co γ-rays irradiation and its cellulase enzyme produce condition

International Nuclear Information System (INIS)

Song Andong; Su Lijuan; Xie Hui; Qu Yinbo; Yang Ming

2008-01-01

A mutant strain A50 with high cellulase activity was induced and isolated by using 60 Co γ-rays irradiation from the initial Penicillium decumbens A10. The optimum fermentation conditions of A50 were investigated through orthogonal designing experiment, the major carbon resource 5%, the ratio between wheat bran and corn straw 1:1, the concentration of glucose as supplemental carbon 0.1%, the concentration of (NH 4 ) 2 HPO 4 as supplemental nitrogen resource 0.2%, the initial pH of liquid medium 5.0, the inoculated amount for fermentation 10% and the concentration of Tween-80 0.1%, 30 ml initial media filled in the 300 ml flask with culture condition of 32 degree C and 200 r/min. Under the optimum conditions mentioned above, the highest activities of cellulase and filter paper enzyme were 27.28 and 1.98IU/ml at 60 h fermentation, respectively, which was 33.2% and 45.59% higher than those of the initial strain. (authors)

Actinomycete enzymes and activities involved in straw saccharification

Energy Technology Data Exchange (ETDEWEB)

McCarthy, A J; Ball, A S [Liverpool Univ. (UK). Dept. of Genetics and Microbiology

1990-01-01

This research programme has been directed towards the analysis of actinomycete enzyme systems involved in the degradation of plant biomass (lignocellulose.) The programme was innovative in that a novel source of enzymes was systematically screened and wheat straw saccharifying activity was the test criterion. Over 200 actinomycete strains representing a broad taxonomic range were screened. A range of specific enzyme activities were involved and included cellulase, xylanase, arabinofuranosidase, acetylesterase, {beta}-xylosidase and {beta}-glucosidase. Since hemicellulose (arabinoxylan) was the primary source of sugar, xylanases were characterized. The xylan-degrading systems of actinomycetes were complex and nonuniform, with up to six separate endoxylanases identified in active strains. Except for microbispora bispora, actinomycetes were found to be a poor source of cellulase activity. Evidence for activity against the lignin fraction of straw was produced for a range of actinomycete strains. While modification reactions were common, cleavage of inter-monomer bonds, and utilization of complex polyphenolic compounds were restricted to two strains: Thermomonospora mesophila and Streptomyces badius. Crude enzyme preparations from actinomycetes can be used to generate sugar, particularly pentoses, directly from cereal straw. The potential for improvements in yield rests with the formulation to cooperative enzyme combinations from different strains. The stability properties of enzymes from thermophilic strains and the general neutral to alkali pH optima offer advantages in certain process situations. Actinomycetes are a particularly rich source of xylanases for commercial application and can rapidly solubilise a lignocarbohydrate fraction of straw which may have both product and pretreatment potential. 31 refs., 4 figs., 5 tabs.

Molecular characterization of the probiotic strain Bacillus cereus var. toyoi NCIMB 40112 and differentiation from food poisoning strains.

Science.gov (United States)

Klein, Günter

2011-07-01

Bacillus cereus var. toyoi strain NCIMB 40112 (Toyocerin), a probiotic authorized in the European Union as feed additive for swine, bovines, poultry, and rabbits, was characterized by DNA fingerprinting applying pulsed-field gel electrophoresis and multilocus sequence typing and was compared with reference strains (of clinical and environmental origins). The probiotic strain was clearly characterized by pulsed-field gel electrophoresis using the restriction enzymes Apa I and Sma I resulting in unique DNA patterns. The comparison to the clinical reference strain B. cereus DSM 4312 was done with the same restriction enzymes, and again a clear differentiation of the two strains was possible by the resulting DNA patterns. The use of the restriction enzymes Apa I and Sma I is recommended for further studies. Furthermore, multilocus sequence typing analysis revealed a sequence type (ST 111) that was different from all known STs of B. cereus strains from food poisoning incidents. Thus, a strain characterization and differentiation from food poisoning strains for the probiotic strain was possible. Copyright ©, International Association for Food Protection

Secretion of laccase and manganese peroxidase by Pleurotus strains cultivate in solid-state using Pinus spp. sawdust

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Marli Camassola

2013-01-01

Full Text Available Pleurotus species secrete phenol oxidase enzymes: laccase (Lcc and manganese peroxidase (MnP. New genotypes of these species show potential to be used in processes aiming at the degradation of phenolic compounds, polycyclic aromatic hydrocarbons and dyes. Hence, a screening of some strains of Pleurotus towards Lcc and MnP production was performed in this work. Ten strains were grown through solid-state fermentation on a medium based on Pinus spp. sawdust, wheat bran and calcium carbonate. High Lcc and MnP activities were found with these strains. Highest Lcc activity, 741 ± 245 U gdm-1 of solid state-cultivation medium, was detected on strain IB11 after 32 days, while the highest MnP activity occurred with strains IB05, IB09, and IB11 (5,333 ± 357; 4,701 ± 652; 5,999 ± 1,078 U gdm-1, respectively. The results obtained here highlight the importance of further experiments with lignocellulolytic enzymes present in different strains of Pleurotus species. Such results also indicate the possibility of selecting more valuable strains for future biotechnological applications, in soil bioremediation and biological biomass pre-treatment in biofuels production, for instance, as well as obtaining value-added products from mushrooms, like phenol oxidase enzymes.

[Comparative study of aromatic ring meta-cleavage enzymes in Pseudomonas strains with plasmid and chromosomal genetic control of the catabolism of biphenyl and m-toluate].

Science.gov (United States)

Selifonov, S A; Starozoĭtov, I I

1990-12-01

It was shown that two different enzymes of aromatic ring oxidative meta-cleavage (2,3-dihydroxybiphenyl-1,2-dioxygenase), DBO and catechol-2,3-dioxygenase, C230) function in Pseudomonas strains with a plasmid and chromosomal genetic control of biphenyl and toluate catabolism. A comparative analysis of DBO's and C230's expressed by the pBS241 biphenyl degradative plasmid in P. putida BS893, pBS311 in P. putida U83, chromosomal genes in P. putida BF and C230 from P. putida PaW160 (pWWO) was carried out. It was found that the DBO's of all strains under study are highly specialized enzymes in respect of 2,3-dihydroxybiphenyl cleavage and are also able to cleave 3-methyl-catechol and catechol (but not 4-methylcatechol) at low rates. In contrast with DBO's, in Pseudomonas strains the substrate specificities of all C230's are variable. The C230's expressed by the D-plasmids pBS241 and pBC311 have a moderate affinity for catechol, 3-methyl- and 4-methylcatechol, but are unable to cleave 2,3-dihydroxybiphenyl. The C230 which is encoded by the chromosomal structure gene from P. putida BF is very similar to C230 which codes for the TOL-plasmid pWWO. These plasmid differ from C230's expressed by biphenyl D-plasmids due to their capability to cleave 2,3-dihydroxybiphenyl in addition to catechol cleavage. All DBO's and C230's under study possess a number of properties that are typical for the enzymes having an oxidative meta-cleaving effect. The different roles of these enzymes in biphenyl and toluate catabolism in Pseudomonas strains are discussed.

Expression of lignocellulolytic enzymes in Pichia pastoris

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Mellitzer Andrea

2012-05-01

Full Text Available Abstract Background Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes. Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic proteins due to several advantages. Results In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes we could develop strains capable of secreting gram quantities of enzyme per liter in fed-batch cultivations. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. Conclusion In our experiments we could clearly show the importance of gene optimization and strain characterization for successfully improving secretion levels. We also present a basic guideline how to correctly interpret the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris.

Cellulase production by a strain of Myrothecium sp

Energy Technology Data Exchange (ETDEWEB)

Kassim, E A

1982-01-01

A selected strain of Myrothecium sp. was grown on various carbon sources. Cellulose was found to be the highest inducer of cellulase. CMC resulted in a moderate yield. Cellobiose resulted in a low yield. Glucose, lactose, maltose and soluble starch resulted in negligible amounts. Sucrose, glycerol and salicin were extremely unsuitable. Continuous addition of glucose or cellobiose during fermentation to cellulosic culture media reduced cellulase production, whereas addition of the entire amount of glucose or cellobiose at the beginning did not affect the enzyme production. The enzyme was precipitated from the culture filtrate with ammonium sulfate giving crude cellulase, 3854 units/g. The culture filtrate was concentrated to a one-tenth volume, 97 units/ml. The purified cellulase was prepared by dialysis 6700 units/g of enzyme precipitate.

Coccolithophores: Functional Biodiversity, Enzymes and Bioprospecting

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Michael J. Allen

2011-04-01

Full Text Available Emiliania huxleyi is a single celled, marine phytoplankton with global distribution. As a key species for global biogeochemical cycling, a variety of strains have been amassed in various culture collections. Using a library consisting of 52 strains of E. huxleyi and an ‘in house‘ enzyme screening program, we have assessed the functional biodiversity within this species of fundamental importance to global biogeochemical cycling, whilst at the same time determining their potential for exploitation in biocatalytic applications. Here, we describe the screening of E. huxleyi strains, as well as a coccolithovirus infected strain, for commercially relevant biocatalytic enzymes such as acid/alkali phosphodiesterase, acid/alkali phosphomonoesterase, EC1.1.1-type dehydrogenase, EC1.3.1-type dehydrogenase and carboxylesterase.

The activity of detoxifying enzymes in the infective juveniles of Heterorhabditis bacteriophora strains: Purification and characterization of two acetylcholinesterases.

Science.gov (United States)

Mohamed, Magda A; Mahdy, El-Sayed M E; Ghazy, Abd-El-Hady M; Ibrahim, Nihal M; El-Mezayen, Hatem A; Ghanem, Manal M E

2016-02-01

The infectivity and detoxifying enzyme activities including glutathione-S-transferase (GST), acetylcholinesterase (AChE) and carboxylesterase (CaE) are investigated in the infective juveniles (IJs) of six different strains of Heterorhabditis bacteriophora as a biocontrol agent against insect pests. The specific activities ranged from 10.8-29.8 and 50-220units/mg protein for GST and AChE, respectively; and from 24.7-129 and 22.6-77.3units/mg protein for CaE as estimated by P-nitrophenyl and α-naphthyl acetates, respectively. H. bacteriophora EM2 strain has the highest infectivity and the highest enzymatic activities as well. AChE is the predominant detoxifying enzyme that might imply its major role in the detoxification of insecticide(s). The isoenzyme pattern demonstrated two major slow-moving isoforms in all EPN strains examined. Purification of two AChE isoforms, AChEAII and AChEBI, from H. bacteriophora EM2 strain is performed by ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and chromatography on DEAE-Sepharose. AChEAII and AChEBII have specific activities of 1207 and 1560unit/mg protein, native molecular weights of 180 and 68kDa, and are found in dimeric and monomeric forms, respectively. Both isoforms showed optimum activity at pH8.5 and 35°C. AChEBI exhibited higher thermal stability and higher activation energy than AChEAII. The enzymatic activities of purified AChEs are completely inhibited by Hg(+2) and Ni(+2) and greatly enhanced by Mn(+2). The substrate specificity, the relative efficiency of substrates hydrolysis, substrate inhibition and inhibition by BW284C51, but not by iso-OMPA, clearly indicated that they are true AChEs; their properties are compared with those recorded for insects as target hosts for H. bacteriophora EM2. Copyright © 2015 Elsevier Inc. All rights reserved.

Assessing the effect of selection with deltamethrin on biological parameters and detoxifying enzymes in Aedes aegypti (L.).

Science.gov (United States)

Alvarez-Gonzalez, Leslie C; Briceño, Arelis; Ponce-Garcia, Gustavo; Villanueva-Segura, O Karina; Davila-Barboza, Jesus A; Lopez-Monroy, Beatriz; Gutierrez-Rodriguez, Selene M; Contreras-Perera, Yamili; Rodriguez-Sanchez, Iram P; Flores, Adriana E

2017-11-01

Resistance to insecticides through one or several mechanisms has a cost for an insect in various parameters of its biological cycle. The present study evaluated the effect of deltamethrin on detoxifying enzymes and biological parameters in a population of Aedes aegypti selected for 15 generations. The enzyme activities of alpha- and beta-esterases, mixed-function oxidases and glutathione-S-transferases were determined during selection, along with biological parameters. Overexpression of mixed-function oxidases as a mechanism of metabolic resistance to deltamethrin was found. There were decreases in percentages of eggs hatching, pupation and age-specific survival and in total survival at the end of the selection (F 16 ). Although age-specific fecundity was not affected by selection with deltamethrin, total fertility, together with lower survival, significantly affected gross reproduction rate, gradually decreasing due to deltamethrin selection. Similarly, net reproductive rate and intrinsic growth rate were affected by selection. Alterations in life parameters could be due to the accumulation of noxious effects or deleterious genes related to detoxifying enzymes, specifically those coding for mixed-function oxidases, along with the presence of recessive alleles of the V1016I and F1534C mutations, associating deltamethrin resistance with fitness cost in Ae. aegypti. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Selection of Yeast Strains for Tequila Fermentation Based on Growth Dynamics in Combined Fructose and Ethanol Media.

Science.gov (United States)

Aldrete-Tapia, J A; Miranda-Castilleja, D E; Arvizu-Medrano, S M; Hernández-Iturriaga, M

2018-02-01

The high concentration of fructose in agave juice has been associated with reduced ethanol tolerance of commercial yeasts used for tequila production and low fermentation yields. The selection of autochthonous strains, which are better adapted to agave juice, could improve the process. In this study, a 2-step selection process of yeasts isolated from spontaneous fermentations for tequila production was carried out based on analysis of the growth dynamics in combined conditions of high fructose and ethanol. First, yeast isolates (605) were screened to identify strains tolerant to high fructose (20%) and to ethanol (10%), yielding 89 isolates able to grow in both conditions. From the 89 isolates, the growth curves under 8 treatments of combined fructose (from 20% to 5%) and ethanol (from 0% to 10%) were obtained, and the kinetic parameters were analyzed with principal component analysis and k-means clustering. The resulting yeast strain groups corresponded to the fast, medium and slow growers. A second clustering of only the fast growers led to the selection of 3 Saccharomyces strains (199, 230, 231) that were able to grow rapidly in 4 out of the 8 conditions evaluated. This methodology differentiated strains phenotypically and could be further used for strain selection in other processes. A method to select yeast strains for fermentation taking into account the natural differences of yeast isolates. This methodology is based on the cell exposition to combinations of sugar and ethanol, which are the most important stress factors in fermentation. This strategy will help to identify the most tolerant strain that could improve ethanol yield and reduce fermentation time. © 2018 Institute of Food Technologists®.

Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Characterization of Biocatalytically Active Bacterial Strains and of Cytochrome P450 Monooxygenase Enzymes and Their Genes

Science.gov (United States)

Jungmann, Volker; Molnár, István; Hammer, Philip E.; Hill, D. Steven; Zirkle, Ross; Buckel, Thomas G.; Buckel, Dagmar; Ligon, James M.; Pachlatko, J. Paul

2005-01-01

4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined. PMID:16269732

NUCLEOTIDE SEQUENCING AND TRANSCRIPTIONAL MAPPING OF THE GENES ENCODING BIPHENYL DIOXYGENASE, A MULTICOM- PONENT POLYCHLORINATED-BIPHENYL-DEGRADING ENZYME IN PSEUDOMONAS STRAIN LB400

Science.gov (United States)

The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. Six open reading frames were identified, four of which are homologous to the components of toluene dioxy...

The orotate transporter oroP from Lactococcus lactis can be used both as a very efficient, food-grade selection and counter-selection marker for strain construction in many different organisms

DEFF Research Database (Denmark)

Defoor, Els Marie Celine; Martinussen, Jan

frame oroP on pDBORO necessary for the uptake of orotate was identified. A number of industrial important strains like Lactococcus lactis, Bacillus subtilus, and Bacillus licheniformis have been shown to be unable to metabolize orotate. If the oroP gene was introduced into these strains they acquired...... the ability to utilize orotate. If the strains had a pyrimidine requirement, the oroP gene could function as a selectable marker when growing in the presence of orotate as sole pyrimidine source. In an otherwise resistant strain, oroP was shown to sensitize the strain towards the analog 5-Fluoroorotate....... It was shown that strains who have lost the oroP gene could easily be selected in the presence of 5-Fluoroorotate, thus being an efficient counter-selection marker. pyrimidine-requiring strain (pyr B, C or D) orotate negative Counter-selection marker: wild-type strain! Fluoro-orotate resistant Functional...

Cellulolytic enzyme expression and simultaneous conversion of lignocellulosic sugars into ethanol and xylitol by a new Candida tropicalis strain.

Science.gov (United States)

Mattam, Anu Jose; Kuila, Arindam; Suralikerimath, Niranjan; Choudary, Nettem; Rao, Peddy V C; Velankar, Harshad Ravindra

2016-01-01

Lignocellulosic ethanol production involves major steps such as thermochemical pretreatment of biomass, enzymatic hydrolysis of pre-treated biomass and the fermentation of released sugars into ethanol. At least two different organisms are conventionally utilized for producing cellulolytic enzymes and for ethanol production through fermentation, whereas in the present study a single yeast isolate with the capacity to simultaneously produce cellulases and xylanases and ferment the released sugars into ethanol and xylitol has been described. A yeast strain isolated from soil samples and identified as Candida tropicalis MTCC 25057 expressed cellulases and xylanases over a wide range of temperatures (32 and 42 °C) and in the presence of different cellulosic substrates [carboxymethylcellulose and wheat straw (WS)]. The studies indicated that the cultivation of yeast at 42 °C in pre-treated hydrolysate containing 0.5 % WS resulted in proportional expression of cellulases (exoglucanases and endoglucanases) at concentrations of 114.1 and 97.8 U g(-1) ds, respectively. A high xylanase activity (689.3 U g(-1) ds) was also exhibited by the yeast under similar growth conditions. Maximum expression of cellulolytic enzymes by the yeast occurred within 24 h of incubation. Of the sugars released from biomass after pretreatment, 49 g L(-1) xylose was aerobically converted into 15.8 g L(-1) of xylitol. In addition, 25.4 g L(-1) glucose released after the enzymatic hydrolysis of biomass was fermented by the same yeast to obtain an ethanol titer of 7.3 g L(-1). During the present study, a new strain of C. tropicalis was isolated and found to have potential for consolidated bioprocessing (CBP) applications. The strain could grow in a wide range of process conditions (temperature, pH) and in the presence of lignocellulosic inhibitors such as furfural, HMF and acetic acid. The new yeast produced cellulolytic enzymes over a wide temperature range and in the presence of

Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes.

Science.gov (United States)

Loder, Andrew J; Zeldes, Benjamin M; Garrison, G Dale; Lipscomb, Gina L; Adams, Michael W W; Kelly, Robert M

2015-10-01

n-Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. A synthetic pathway for n-butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled in vitro from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Recombinant versions of eight thermophilic enzymes (β-ketothiolase [Thl], 3-hydroxybutyryl-CoA dehydrogenase [Hbd], and 3-hydroxybutyryl-CoA dehydratase [Crt] from Caldanaerobacter subterraneus subsp. tengcongensis; trans-2-enoyl-CoA reductase [Ter] from Spirochaeta thermophila; bifunctional acetaldehyde dehydrogenase/alcohol dehydrogenase [AdhE] from Clostridium thermocellum; and AdhE, aldehyde dehydrogenase [Bad], and butanol dehydrogenase [Bdh] from Thermoanaerobacter sp. strain X514) were utilized to examine three possible pathways for n-butanol. These pathways differed in the two steps required to convert butyryl-CoA to n-butanol: Thl-Hbd-Crt-Ter-AdhE (C. thermocellum), Thl-Hbd-Crt-Ter-AdhE (Thermoanaerobacter X514), and Thl-Hbd-Crt-Ter-Bad-Bdh. n-Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, was used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to n-butanol was observed in vitro, but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to n-butanol is predicted, with a 19% increase in pathway flux. Thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic n-butanol production at elevated temperatures. Copyright © 2015, American Society for

Production of extracellular proteolytic enzymes by Beauveria bassiana

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Józefa Chrzanowska

2014-08-01

Full Text Available The production of proteolytic enzymes by two strains of Beauveria bassiana 278, B. bassiana 446 and one strain of Ascosphera apis 496 was analysed. It was demonstrated that the strain of B. bassiana 278 proved to be the best producer of basic and acid proteases. The influence of different environmental factors such as nitrogen and carbon sources on the production of extracellular hydrolytic enzymes was assessed. In addition the acid protease from B. bassiana was partially characterized.

Immunomagnetic separation combined with colony immunoblotting for selective enrichment and detection of piliated Lactobacillus rhamnosus strains.

Science.gov (United States)

Yang, Z Q; Wei, Y F; Rao, S Q; Gao, L; Yin, Y Q; Xue, F; Fang, W M; Gu, R X; Jiao, X A

2016-11-01

Piliated Lactobacillus rhamnosus (pLR) strains have attracted much attention owing to their excellent mucus adhering capacity and immunomodulatory effects. Here, we aimed to develop a rapid, sensitive method for isolating pLR strains in complex ecosystems using immunomagnetic separation (IMS) with colony immunoblotting (CIB). Magnetic nanobeads (diameter: 180 nm) conjugated with anti-pLR SpaA pilin antibodies (anti-SpaA) were prepared and used to preconcentrate pLR strains in samples, followed by confirmation with anti-SpaA-based CIB analysis. Under optimized experimental conditions, IMS-CIB selectively recovered pLR strains from 10 7  CFU ml -1 of faecal microbiota samples spiked with 2·9 × 10 1 to 2·4 × 10 6  CFU ml -1 of pLR strains. No positive colonies were detected in samples without addition of pLR strains. The detection limit of IMS-CIB was 29 CFU pLR ml -1 of faecal microbiota, which is much lower than that of CIB without IMS preconcentration (2·0 × 10 4  CFU ml -1 ). IMS-CIB allowed selective preconcentration of pLR strains in highly heterogeneous bacterial suspensions and direct detection of pLR colonies, which remained readily available for subsequent isolation. Our findings established an effective method for selective enrichment and detection of pLR strains. © 2016 The Society for Applied Microbiology.

Genotypic and Phenotypic Assessment of Hyaluronidase among Type Strains of a Select Group of Staphylococcal Species

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Mark E. Hart

2009-01-01

Full Text Available Hyaluronidases degrade hyaluronic acid, a major polysaccharide of the extracellular matrix of tissues, and are considered important for virulence in a number of Gram-positive and -negative bacteria. The purpose of the present study was to determine the prevalence of hyaluronidase among clinical strains of Staphylococcus aureus and among other Staphylococcus species. Spent media and chromosomal DNA were assessed for hyaluronidase activity and the absence or presence of a hyaluronidase gene (hysA by Southern analysis, respectively. All S. aureus strains examined exhibited at least one hybridizing band (half of the strains exhibited two or more hybridizing bands when probed for hysA and all but three of these strains produced hyaluronidase. In contrast, none of the type strains of 19 other species exhibited either hyaluronidase activity or hybridizing bands when probed for hysA. These data support the hypothesis that among members of the Staphylococcus genus only strains of S. aureus possess the enzyme hyaluronidase. This would suggest that hyaluronidase represents yet another potential virulence factor employed by S. aureus to cause disease and may represent a diagnostically important characteristic for distinguishing S. aureus from other members of this genus.

Chlorinated Electron Acceptor Abundance Drives Selection of Dehalococcoides mccartyi (D. mccartyi Strains in Dechlorinating Enrichment Cultures and Groundwater Environments

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Alfredo Pérez-de-Mora

2018-05-01

Full Text Available Dehalococcoides mccartyi (D. mccartyi strains differ primarily from one another by the number and identity of the reductive dehalogenase homologous catalytic subunit A (rdhA genes within their respective genomes. While multiple rdhA genes have been sequenced, the activity of the corresponding proteins has been identified in only a few cases. Examples include the enzymes whose substrates are groundwater contaminants such as trichloroethene (TCE, cis-dichloroethene (cDCE and vinyl chloride (VC. The associated rdhA genes, namely tceA, bvcA, and vcrA, along with the D. mccartyi 16S rRNA gene are often used as biomarkers of growth in field samples. In this study, we monitored an additional 12 uncharacterized rdhA sequences identified in the metagenome in the mixed D. mccartyi-containing culture KB-1 to monitor population shifts in more detail. Quantitative PCR (qPCR assays were developed for 15 D. mccartyi rdhA genes and used to measure population diversity in 11 different sub-cultures of KB-1, each enriched on different chlorinated ethenes and ethanes. The proportion of rdhA gene copies relative to D. mccartyi 16S rRNA gene copies revealed the presence of multiple distinct D. mccartyi strains in each culture, many more than the two strains inferred from 16S rRNA analysis. The specific electron acceptor amended to each culture had a major influence on the distribution of D. mccartyi strains and their associated rdhA genes. We also surveyed the abundance of rdhA genes in samples from two bioaugmented field sites (Canada and United Kingdom. Growth of the dominant D. mccartyi strain in KB-1 was detected at the United Kingdom site. At both field sites, the measurement of relative rdhA abundances revealed D. mccartyi population shifts over time as dechlorination progressed from TCE through cDCE to VC and ethene. These shifts indicate a selective pressure of the most abundant chlorinated electron acceptor, as was also observed in lab cultures. These

Research of angelica sinensis (Oliv) diels new strain DGA2000-02 selection

International Nuclear Information System (INIS)

Xie Hongmei; Li Wenjian; Hao Jifang; Liu Jing; Liu Xiaorui; Jing Yanming; Shang Hushan; Liu Rongqing; He Baogang; Wang Chunming; Zhang Guoli; Chen Shuzhen

2008-01-01

Angelica sinensis(Oliv)Diets new strain DGA2000-02 was selected successfully by Institute of Modern Physics, Chinese Academy of Sciences, Dryland Farming Research and Extension Center of Dingxi Prefecture, etc. According to the program of new strain selection, this new strain was selected for several year's after the dry seeds of Gansu Angelica sinensis(Oliv)Diels 90-01 was irradiated by ions of 55 MeV/u 40 Ar +15 . During the period of year 2005-2007, region experiments of Angelica sinensis (Oliv)Diels new strain DGA2000-02 were developed in Minxian, Weiyuan, Zhangxian and Longxi etc. Average yield of the fresh DGA2000-02 Angelica was 10 621.5 kg/hm 2 , and 15.0% production was increased more than control (for 1 386.0 kg/hm 2 of 90-01). The growth stage of the DGA2000-02 was 790 d, and it has deep purple stem and yellow-white root. The quality analysis results are as follows: total ash content is 4.2% and acidast ash content is 0.4%, 16% and 33.3% better than control, respectively; the lixivium is 61.4%, i.e., 4.4% more than the standard of Pharmacopoeia of People's Republic of China (2005 edition); the ferullc acid content is 0.148%, i.e., 2.96 times ligher than the standard. All these results showed that the quality of the DGA2000-02 was better significantly than both control and the standard. It can be grown appropriately at the high, cold and dankness regions at the altitude of 2000-2600 m and with a annual precipitation of 500-600 mm. (authors)

Preparation of progenin III from total steroidal saponins of Dioscorea nipponica Makino using a crude enzyme from Aspergillus oryzae strain.

Science.gov (United States)

Liu, Tingqiang; Yu, Hongshan; Liu, Chunying; Bao, Yongming; Hu, Xiangchun; Wang, Yuanhao; Liu, Bing; Fu, Yaoyao; Tang, Sihui; Jin, Fengxie

2013-05-01

Progenin III, one of the most active spirostanol saponins, is a potential candidate for anti-cancer therapy due to its strong antitumor activity and low hemolytic activity. However, the concentration of progenin III is extremely low in natural Dioscorea plants. In this paper, the progenin III production from total steroidal saponins of Dioscorea nipponica Makino was studied using the crude enzyme from Aspergillus oryzae DLFCC-38. The crude enzyme converting total steroidal saponins into progenin III was obtained from the A. oryzae DLFCC-38 culture. For enzyme production, the strain was cultured for 72 h at 30 °C with shaking at 150 rpm in 5 % (w/v) malt extract medium containing 2 % (v/v) extract of D. nipponica as the enzyme inducer. The crude enzyme converted total steroidal saponins into major progenin III with a high yield when the reaction was carried out for 9 h at 50 °C and pH 5.0 with the 20 mg/ml of substrate. In the preparation of progenin III, 117 g of crude progenin III was obtained from 160 g of substrate, and the crude product was purified with silica gel column to obtain 60.3 g progenin III of 93.4 % purity.

Screening of Probiotic Activities of Forty-Seven Strains of Lactobacillus spp. by In Vitro Techniques and Evaluation of the Colonization Ability of Five Selected Strains in Humans

OpenAIRE

Jacobsen, C. N.; Rosenfeldt Nielsen, V.; Hayford, A. E.; Møller, P. L.; Michaelsen, K. F.; Pærregaard, A.; Sandström, B.; Tvede, M.; Jakobsen, M.

1999-01-01

The probiotic potential of 47 selected strains of Lactobacillus spp. was investigated. The strains were examined for resistance to pH 2.5 and 0.3% oxgall, adhesion to Caco-2 cells, and antimicrobial activities against enteric pathogenic bacteria in model systems. From the results obtained in vitro, five strains, Lactobacillus rhamnosus 19070-2, L. reuteri DSM 12246, L. rhamnosus LGG, L. delbrueckii subsp. lactis CHCC 2329, and L. casei subsp. alactus CHCC 3137, were selected for in vivo studi...

Differences in Attack Avoidance and Mating Success between Strains Artificially Selected for Dispersal Distance in Tribolium castaneum.

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Kentarou Matsumura

Full Text Available Individuals of both dispersal and non-dispersal types (disperser and non-disperser are found in a population, suggesting that each type has both costs and benefits for fitness. However, few studies have examined the trade-off between the costs and benefits for the types. Here, we artificially selected for walking distance, i.e., an indicator of dispersal ability, in the red flour beetle Tribolium castaneum and established strains with longer (L-strains or shorter (S-strains walking distances. We then compared the frequency of predation by the assassin bug Amphibolus venator and the mating frequency of the selected strains. L-strain beetles suffered higher predation risk, than did S-strain beetles. L-strain males had significantly increased mating success compared to S-strain males, but females did not show a significant difference between the strains. The current results showed the existence of a trade-off between predation avoidance and mating success associated with dispersal types at a genetic level only in males. This finding can help to explain the maintenance of variation in dispersal ability within a population.

Plant Cell Protolytic Enzymes Activity under Exposure to Lectins of Endophytic and Epiphytic Azospirillum Strains

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S.A. Alen’kina

2016-05-01

Full Text Available We studied the ability of lectins isolated from the surface of the two strains of nitrogen-fixing soil bacteria of the genus Azospirillum, A. brasilense Sp7 (epiphytic and A. brasilense Sp245 (endophytic, to show have a regulating effect on the activity of pectinolytic enzymes in the roots of wheat seedlings. Research results showed that the lectins under study can cause the induction of the activity of polygalacturonase, pectinesterase, pectatlyase from the plant cell wall, thereby ensuring the bacteria penetration in the plant tissues, as well as the induction of plants responses which, being combined with growth-stimulating effect of bacteria, contributes to the formation of plants stability and productivity.

Relation Between Motility, Accelerated Aging and Gene Expression in Selected Drosophila Strains under Hypergravity Conditions

Science.gov (United States)

Serrano, Paloma; van Loon, Jack J. W. A.; Medina, F. Javier; Herranz, Raúl

2013-02-01

Motility and aging in Drosophila have proven to be highly modified under altered gravity conditions (both in space and ground simulation facilities). In order to find out how closely connected they are, five strains with altered geotactic response or survival rates were selected and exposed to an altered gravity environment of 2 g. By analysing the different motile and behavioural patterns and the median survival rates, we show that altered gravity leads to changes in motility, which will have a negative impact on the flies' survival. Previous results show a differential gene expression between sessile samples and adults and confirm that environmentally-conditioned behavioural patterns constrain flies' gene expression and life span. Therefore, hypergravity is considered an environmental stress factor and strains that do not respond to this new environment experience an increment in motility, which is the major cause for the observed increased mortality also under microgravity conditions. The neutral-geotaxis selected strain (strain M) showed the most severe phenotype, unable to respond to variations in the gravitational field. Alternatively, the opposite phenotype was observed in positive-geotaxis and long-life selected flies (strains B and L, respectively), suggesting that these populations are less sensitive to alterations in the gravitational load. We conclude that the behavioural response has a greater contribution to aging than the modified energy consumption in altered gravity environments.

Antagonistic Activity of Lactobacillus reuteri Strains on the Adhesion Characteristics of Selected Pathogens

OpenAIRE

Singh, Tejinder P.; Kaur, Gurpreet; Kapila, Suman; Malik, Ravinder K.

2017-01-01

Adhesion ability of probiotics is the key factor that decides their colonization in the gastrointestinal tract and potential to inhibit pathogens. Therefore, adhesion ability can be considered as a key determinant for probiotic efficacy. Presents study documents the antagonistic activity of viable/untreated, Lithium chloride (LiCl) treated or heat-killed forms of eight probiotic Lactobacillus reuteri strains on the adhesion characteristics of selected pathogens. All strains investigated were ...

Detection of phosphatase activity in aquatic and terrestrial cyanobacterial strains

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Babić Olivera B.

2013-01-01

Full Text Available Cyanobacteria, as highly adaptable microorganisms, are characterized by an ability to survive in different environmental conditions, in which a significant role belongs to their enzymes. Phosphatases are enzymes produced by algae in relatively large quantities in response to a low orthophosphate concentration and their activity is significantly correlated with their primary production. The activity of these enzymes was investigated in 11 cyanobacterial strains in order to determine enzyme synthesis depending on taxonomic and ecological group of cyanobacteria. The study was conducted with 4 terrestrial cyanobacterial strains, which belong to Nostoc and Anabaena genera, and 7 filamentous water cyanobacteria of Nostoc, Oscillatoria, Phormidium and Microcystis genera. The obtained results showed that the activity of acid and alkaline phosphatases strongly depended on cyanobacterial strain and the environment from which the strain originated. Higher activity of alkaline phosphatases, ranging from 3.64 to 85.14 μmolpNP/s/dm3, was recorded in terrestrial strains compared to the studied water strains (1.11-5.96 μmolpNP/s/dm3. The activity of acid phosphatases was higher in most tested water strains (1.67-6.28 μmolpNP/s/dm3 compared to the activity of alkaline phosphatases (1.11-5.96 μmolpNP/s/dm3. Comparing enzyme activity of nitrogen fixing and non-nitrogen fixing cyanobacteria, it was found that most nitrogen fixing strains had a higher activity of alkaline phosphatases. The data obtained in this work indicate that activity of phosphatases is a strain specific property. The results further suggest that synthesis and activity of phosphatases depended on eco-physiological characteristics of the examined cyanobacterial strains. This can be of great importance for the further study of enzymes and mechanisms of their activity as a part of cyanobacterial survival strategy in environments with extreme conditions. [Projekat Ministarstva nauke Republike

In vitro screening of selected probiotic properties of Lactobacillus strains isolated from traditional fermented cabbage and cucumber.

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Zielińska, Dorota; Rzepkowska, Anna; Radawska, Anna; Zieliński, Konrad

2015-02-01

Most important during probiotic selection are gastric acid and bile tolerance, the adhesion to the luminal epithelium to colonize the lower gastrointestinal tract of a human and safety for human consumption. The aim of this study was to evaluate the selected probiotic in vitro properties of Lactobacillus spp. Strains isolated from traditional fermented food. A total 38 strains were isolated from the pickled samples and 14 were identified as Lactobacillus spp. The survival of almost all strains after incubation at pH 2.5 did not change markedly, and remained at above 90 % (10(9) CFU/mL). The strains also exhibited a high survival rate at pH 3.5 (>90 %), whereas pH 1.5 all were died. Just four strains could survive 90 min. at pH 1.5 (survival rates of 81-94 % after 24 h, whereas after incubation in 2 and 4 % bile salt solution it was 59-94 %. All tested strains showed very good and good resistance to 0.4 % phenol addition, however only Lb. johnsonii K4 was able to multiply. The hydrophobic nature of the cell surface of the tested strains was moderated recording hydrophobicity of Lb. johnsonii K4 and Lb. rhamnosus K3 above 60 %. Safety evaluation excluded four of tested strains as candidate probiotics, according to antibiotic resistance patterns and certain metabolic activities. On the basis on the results 10 of the selected Lactobacillus strains are safe and can survive under gastrointestinal conditions, which requires them to future in vitro and in vivo probiotic studies.

Combination of selected enzymes with cetyltrimethylammonium bromide in biofilm inactivation, removal and regrowth

KAUST Repository

Araujo, Paula Alexandra Da Silva; Machado, Idalina; Meireles, Ana; Leiknes, TorOve; Mergulhã o, Filipe; Melo, Luí s F.; Simõ es, Manuel

2017-01-01

Enzymes are considered an innovative and environmentally friendly approach for biofilm control due to their lytic and dispersal activities. In this study, four enzymes (β-glucanase, α-amylase, lipase and protease) were tested separately and in combination with the quaternary ammonium compound cetyltrimethylammonium bromide (CTAB) to control flow-generated biofilms of Pseudomonas fluorescens. The four enzymes caused modest reduction of biofilm colony forming units (CFU). Protease, β-glucanase and α-amylase also caused modest biofilm removal. CTAB combined with either β-glucanase or α-amylase increased biofilm removal. Its combination with either β-glucanase or protease increased CFU reduction. However, CTAB−protease combination was antagonist in biofilm removal. Long-term effects in biofilm mass reduction were observed after protease exposure. In contrast, biofilms treated with β-glucanase were able to regrowth significantly after exposure. Moreover, short-term respirometry tests with planktonic cells were performed to understand the effects of enzymes and their combination with CTAB on P. fluorescens viability. Protease and lipase demonstrated antimicrobial action, while α-amylase increased bacterial metabolic activity. The combination of CTAB with either protease or α-amylase was antagonistic, decreasing the antimicrobial action of CTAB. The overall results demonstrate a modest effect of the selected enzymes in biofilm control, either when applied alone or each one in combination with CTAB. Total biofilm removal or CFU reduction was not achieved and, in some cases, the use of enzymes antagonized the effects of CTAB. The results also propose that complementary tests, to characterize biofilm integrity and microbial viability, are required when someone is trying to assess the role of novel biocide - enzyme mixtures for effective biofilm control.

Combination of selected enzymes with cetyltrimethylammonium bromide in biofilm inactivation, removal and regrowth

KAUST Repository

Araujo, Paula Alexandra Da Silva

2017-03-01

Enzymes are considered an innovative and environmentally friendly approach for biofilm control due to their lytic and dispersal activities. In this study, four enzymes (β-glucanase, α-amylase, lipase and protease) were tested separately and in combination with the quaternary ammonium compound cetyltrimethylammonium bromide (CTAB) to control flow-generated biofilms of Pseudomonas fluorescens. The four enzymes caused modest reduction of biofilm colony forming units (CFU). Protease, β-glucanase and α-amylase also caused modest biofilm removal. CTAB combined with either β-glucanase or α-amylase increased biofilm removal. Its combination with either β-glucanase or protease increased CFU reduction. However, CTAB−protease combination was antagonist in biofilm removal. Long-term effects in biofilm mass reduction were observed after protease exposure. In contrast, biofilms treated with β-glucanase were able to regrowth significantly after exposure. Moreover, short-term respirometry tests with planktonic cells were performed to understand the effects of enzymes and their combination with CTAB on P. fluorescens viability. Protease and lipase demonstrated antimicrobial action, while α-amylase increased bacterial metabolic activity. The combination of CTAB with either protease or α-amylase was antagonistic, decreasing the antimicrobial action of CTAB. The overall results demonstrate a modest effect of the selected enzymes in biofilm control, either when applied alone or each one in combination with CTAB. Total biofilm removal or CFU reduction was not achieved and, in some cases, the use of enzymes antagonized the effects of CTAB. The results also propose that complementary tests, to characterize biofilm integrity and microbial viability, are required when someone is trying to assess the role of novel biocide - enzyme mixtures for effective biofilm control.

Enzyme-linked immunosorbent assay and polymerase chain reaction performance using Mexican and Guatemalan discrete typing unit I strains of Trypanosoma cruzi.

Science.gov (United States)

Ballinas-Verdugo, Martha; Reyes, Pedro Antonio; Mejia-Dominguez, Ana; López, Ruth; Matta, Vivian; Monteón, Victor M

2011-12-01

Thirteen Trypanosoma cruzi isolates from different geographic regions of Mexico and Guatemala belonging to discrete typing unit (DTU) I and a reference CL-Brener (DTU VI) strain were used to perform enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). A panel of 57 Mexican serum samples of patients with chronic chagasic cardiopathy and asymptomatic infected subjects (blood bank donors) were used in this study. DNA from the above 14 T. cruzi strains were extracted and analyzed by PCR using different sets of primers designed from minicircle and satellite T. cruzi DNA. The chronic chagasic cardiopathy serum samples were easily recognized with ELISA regardless of the source of antigenic extract used, even with the CL-Brener TcVI, but positive serum samples from blood bank donors in some cases were not recognized by some Mexican antigenic extracts. On the other hand, PCR showed an excellent performance despite the set of primers used, since all Mexican and Guatemalan T. cruzi strains were correctly amplified. In general terms, Mexican, Guatemalan, and CL-Brener T. cruzi strains are equally good sources of antigen when using the ELISA test to detect Mexican serum samples. However, there are some strains with poor performance. The DTU I strains are easily detected using either kinetoplast or satellite DNA target designed from DTU VI strains.

Genetic relationships between clinical and non-clinical strains of Yersinia enterocolitica biovar 1A as revealed by multilocus enzyme electrophoresis and multilocus restriction typing

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Virdi Jugsharan S

2010-05-01

Full Text Available Abstract Background Genetic relationships among 81 strains of Y. enterocolitica biovar 1A isolated from clinical and non-clinical sources were discerned by multilocus enzyme electrophoresis (MLEE and multilocus restriction typing (MLRT using six loci each. Such studies may reveal associations between the genotypes of the strains and their sources of isolation. Results All loci were polymorphic and generated 62 electrophoretic types (ETs and 12 restriction types (RTs. The mean genetic diversity (H of the strains by MLEE and MLRT was 0.566 and 0.441 respectively. MLEE (DI = 0.98 was more discriminatory and clustered Y. enterocolitica biovar 1A strains into four groups, while MLRT (DI = 0.77 identified two distinct groups. BURST (Based Upon Related Sequence Types analysis of the MLRT data suggested aquatic serotype O:6,30-6,31 isolates to be the ancestral strains from which, clinical O:6,30-6,31 strains might have originated by host adaptation and genetic change. Conclusion MLEE revealed greater genetic diversity among strains of Y. enterocolitica biovar 1A and clustered strains in four groups, while MLRT grouped the strains into two groups. BURST analysis of MLRT data nevertheless provided newer insights into the probable evolution of clinical strains from aquatic strains.

High-Resolution Amplified Fragment Length Polymorphism Typing of Lactococcus lactis Strains Enables Identification of Genetic Markers for Subspecies-Related Phenotypes▿

Science.gov (United States)

Kütahya, Oylum Erkus; Starrenburg, Marjo J. C.; Rademaker, Jan L. W.; Klaassen, Corné H. W.; van Hylckama Vlieg, Johan E. T.; Smid, Eddy J.; Kleerebezem, Michiel

2011-01-01

A high-resolution amplified fragment length polymorphism (AFLP) methodology was developed to achieve the delineation of closely related Lactococcus lactis strains. The differentiation depth of 24 enzyme-primer-nucleotide combinations was experimentally evaluated to maximize the number of polymorphisms. The resolution depth was confirmed by performing diversity analysis on 82 L. lactis strains, including both closely and distantly related strains with dairy and nondairy origins. Strains clustered into two main genomic lineages of L. lactis subsp. lactis and L. lactis subsp. cremoris type-strain-like genotypes and a third novel genomic lineage rooted from the L. lactis subsp. lactis genomic lineage. Cluster differentiation was highly correlated with small-subunit rRNA homology and multilocus sequence analysis (MLSA) studies. Additionally, the selected enzyme-primer combination generated L. lactis subsp. cremoris phenotype-specific fragments irrespective of the genotype. These phenotype-specific markers allowed the differentiation of L. lactis subsp. lactis phenotype from L. lactis subsp. cremoris phenotype strains within the same L. lactis subsp. cremoris type-strain-like genomic lineage, illustrating the potential of AFLP for the generation of phenotype-linked genetic markers. PMID:21666014

Isolation and optimization of pectinase enzyme production one of useful industrial enzyme in Aspergillus niger, Rhizopus oryzae, Penicilium chrysogenum

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akram songol

2016-06-01

Full Text Available Introduction: Pectinase enzyme is one of the most important industrial enzymes which isolated from a wide variety of microorganisms such as bacteria and filamentous fungi. This enzyme has been usually used in the fruit and textile industry. In this study, the isolation and optimization of pectinase-producing fungi on decaying rotten fruits were studied. Materials and methods: Isolation and screening of pectinase producing fungi performed through plate culture on pectin medium and staining with Lugol's iodine solution. The best strains were identified by ITS1, 4 sequencing as Aspergillus fumigatus, Rhizopus oryzae, Penicilium chrysogenum. The enzyme production was optimized by application of the five factorial design, each at three levels. These factors are carbon sources (whey, glucose and stevia, ammonium sulfate, manganese sulfate, temperature, and pH. Pectinase concentration was measured by the Miller method. Results: The results indicate that optimum condition for enzyme production for three fungi strains was obtained at 32 °C, pH = 6, 3g / L manganese sulfate, 2.75g / L of ammonium sulfate and 10g / L of each carbon source. The best experiment in obtaining the optimum enzyme contained 1.328 mg / ml of glucose for Aspergillus niger 1.284 and 1.039 mg / ml of whey for Rhizopus oryzae and Penicilium chrysogenum. Molecular weight of enzyme was about 40 and 37 kDa which was obtained by SDS- PAGE. Discussion and conclusion: The results indicate that three strains could grow in a wide range of carbon source, pH and temperature, which could be a good candidate for industrial application.

A novel bi-enzyme electrochemical biosensor for selective and sensitive determination of methyl salicylate.

Science.gov (United States)

Fang, Yi; Umasankar, Yogeswaran; Ramasamy, Ramaraja P

2016-07-15

An amperometric sensor based on a bi-enzyme modified electrode was fabricated to detect methyl salicylate, a volatile organic compound released by pathogen-infected plants via systemic response. The detection is based on cascadic conversion reactions that result in an amperometric electrochemical signal. The bi-enzyme electrode is made of alcohol oxidase and horseradish peroxidase enzymes immobilized on to a carbon nanotube matrix through a molecular tethering method. Methyl salicylate undergoes hydrolysis to form methanol, which is consumed by alcohol oxidase to form formaldehyde while simultaneously reducing oxygen to hydrogen peroxide. The hydrogen peroxide will be further reduced to water by horseradish peroxidase, which results in an amperometric signal via direct electron transfer. The bi-enzyme biosensor was evaluated by cyclic voltammetry and constant potential amperometry using hydrolyzed methyl salicylate as the analyte. The sensitivity of the bi-enzyme biosensor as determined by cyclic voltammetry and constant potential amperometry were 112.37 and 282.82μAcm(-2)mM(-1) respectively, and the corresponding limits of detection were 22.95 and 0.98μM respectively. Constant potential amperometry was also used to evaluate durability, repeatability and interference from other compounds. Wintergreen oil was used for real sample study to establish the application of the bi-enzyme sensor for selective determination of plant pathogen infections. Copyright © 2016 Elsevier B.V. All rights reserved.

Mechanism of Excretion of a Bacterial Proteinase: Demonstration of Two Proteolytic Enzymes Produced by a Sarcina Strain (Coccus P)

Energy Technology Data Exchange (ETDEWEB)

SARNER, NITZA Z; BISSELL, MINA J; GIROLAMO, MARIO Di; GORINI, LUIGI

1970-06-29

A Sarcina strain (Coccus P) produces two proteolytic enzymes. One is found only extracellularly, is far more prevalent, and is actively excreted during exponential growth. It is the enzyme responsible for the known strong proteolytic activity of the cultures of this strain. A second protease is, however, produced which remains associated with the intact cells but is released by the protoplasts. The two enzymes appear unrelated in their derivation. Calcium ions play an essential role in preventing autodigestion of the excreted enzyme. Bacterial proteins are found outside the cell boundary as a consequence either of passive processes such as leakage or lysis or of active excretion. Under conditions in which leakage and lysis do not occur, as during exponential growth, the cell boundary is a barrier causing a complete separation of the bulk of the intracellular proteins from the one or very few extracellular proteins, with no trace of either type being detectable on the wrong side of the boundary. Since in bacteria there is no evidence of protein being produced other than internally, the separation into intraand extracellular proteins should occur after peptide chain formation. The question arises as to whether the structure of the cell boundary or that of the excreted proteins themselves determines this separation. Coccus P, a Sarcina closely related to Micrococcus lysodeikticus (3), produces an extracellular proteinase during the exponential phase of growth so that the process appears to be active excretion. The organism grows exponentially in a defined synthetic medium (12) to relatively high cell density (10{sup 9} cells/ml); therefore the mechanism of excretion can be studied over an extended period of time without the difficulties of changing growth rates. Coagulation of reconstituted skim milk provides a simple and sensitive assay for enzyme activity (I 1). The extracellular proteinase has also been purified and partially characterized (6-8). It has been shown

Genetic and phenotypic diversity of naturally isolated wild strains of Aspergillus niger with hyper glucose oxidase production

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MAHMOUD EL-HARIRI

2015-12-01

Full Text Available Glucose oxidase (GOx is the basic stone for many of biological industry worldwide. The improvement of GOx production basically depends on selection of hyper producer strain of Aspergillus niger. Selective isolation and screening for natural hyper producer strains of A. niger and sequence analysis of the GOD gene, which is responsible for production of the enzyme, are very effective approaches to investigate the naturally modified strains of A. niger with hyper productive capacity of GOx enzyme. The aims of the current study were selective isolation of naturally hyper GOx producing strains of A. niger and evaluation of their GOx activities under optimized parameters in the laboratory. Five wild Egyptian isolates of A. niger were screened for GOx and catalase activity using two types of modified basal liquid media. The GOx activity was evaluated by high throughout liquid phase system. The isolates showed a variable activity for GOx production ranged from 0 to 28.7 U.ml-1. One isolate coded Strain 7 was negative GOx producer on Vogel's broth medium in comparison to other isolates, while its GOx activity on Cazpek Dox was considered as positive (7.28 U.ml-1. It was concluded that GOx production is affected by three controllable factors – the basal media components, time of incubation, and the strain with its adaption to the media components‎. Also, the catalase activity was tested and it was produced with a different degree of variability, which may be reflected on GOx stability. GOD genes of these wild variant of A. niger were cloned and sequenced to determine intraspecies diversity of GOD between the wild variants. The comparison of isolated wild variants to other reference hyper GOx producer strains of A. niger was performed to determine if the GOD sequence analysis of these strains can be distinguished based on their GOx activity. This is the first report for isolation and detection of naturally A. niger hyper GOx-producer strains with

Selection of Bacillus thuringiensis strains toxic to cotton boll weevil (Anthonomus grandis, Coleoptera: Curculionidae) larvae.

Science.gov (United States)

Pérez, Melisa P; Sauka, Diego H; Onco, María I; Berretta, Marcelo F; Benintende, Graciela B

Preliminary bioassays with whole cultures (WC) of 124 Bacillus thuringiensis strains were performed with neonate larvae of Anthonomus grandis, a major cotton pest in Argentina and other regions of the Americas. Three exotic and four native strains were selected for causing more than 50% mortality. All of them were β-exotoxin producers. The native strains shared similar morphology of parasporal crystals, similar protein pattern and identical insecticidal gene profiles. These features resembled Lepidoptera-toxic strains. Furthermore, these strains showed a Rep-PCR pattern identical to lepidoptericidal strain HD-1, suggesting that these strains may belong to serovar kurstaki. However, some differences were observed in the plasmid profiles and in the production of β-exotoxin. To determine the culture fractions where the insecticidal metabolites were present, bioassays including resuspended spore-crystal pellets, filtered supernatants (FS) were compared with those of WC. Both fractions tested showed some level of insecticidal activity. The results may suggest that the main toxic factors can be found in FS and could be directly correlated with the presence of β-exotoxin. Based on the bioassays with FS and autoclaved FS, the participation of thermolabile virulence factors such as Cry1I in toxicity is neither discarded. In the selected strains, β-exotoxin would be the major associated virulence factor; therefore, their use in biological control of A. grandis should be restricted. Nevertheless, these strains could be the source of genes (e.g., cry1Ia) to produce transgenic cotton plants resistant to this pest. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain

International Nuclear Information System (INIS)

Miyanoiri, Yohei; Ishida, Yojiro; Takeda, Mitsuhiro; Terauchi, Tsutomu; Inouye, Masayori; Kainosho, Masatsune

2016-01-01

We recently developed a practical protocol for preparing proteins bearing stereo-selectively 13 C-methyl labeled leucines and valines, instead of the commonly used 13 C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and β-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,β-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,β-dihydroxy-α-methylvalerate to α-keto-β-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.

Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain

Energy Technology Data Exchange (ETDEWEB)

Miyanoiri, Yohei [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Ishida, Yojiro [Rutgers University-Robert Wood Johnson Medical School, Center for Advanced Biotechnology and Medicine (United States); Takeda, Mitsuhiro [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Terauchi, Tsutomu [Tokyo Metropolitan University, Graduate School of Science and Engineering (Japan); Inouye, Masayori [Rutgers University-Robert Wood Johnson Medical School, Center for Advanced Biotechnology and Medicine (United States); Kainosho, Masatsune, E-mail: kainosho@tmu.ac.jp [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan)

2016-06-15

We recently developed a practical protocol for preparing proteins bearing stereo-selectively {sup 13}C-methyl labeled leucines and valines, instead of the commonly used {sup 13}C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and β-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,β-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,β-dihydroxy-α-methylvalerate to α-keto-β-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.

Cellulase and xylanase productions by isolated Amazon Bacillus strains using soybean industrial residue based solid-state cultivation

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Heck Júlio X.

2002-01-01

Full Text Available In Brazil, a large amount of a fibrous residue is generated as result of soybean (Glycine max protein production. This material, which is rich in hemicellulose and cellulose, can be used in solid state cultivations for the production of valuable metabolites and enzymes. In this work, we studied the bioconversion of this residue by bacteria strains isolated from water and soil collected in the Amazon region. Five strains among 87 isolated bacteria selected for their ability to produce either celullases or xylanases were cultivated on the aforementioned residue. From strain BL62, identified as Bacillus subtilis, it was obtained a preparation showing the highest specific cellulase activity, 1.08 UI/mg protein within 24 hours of growth. Concerning xylanase, the isolate BL53, also identified as Bacillus subtilis, showed the highest specific activity for this enzyme, 5.19 UI/mg protein within 72 hours of cultivation. It has also been observed the production of proteases that were associated with the loss of cellulase and xylanase activities. These results indicated that the selected microorganisms, and the cultivation process, have great biotechnological potential.

The temperature--dependent expression of GST of Schistosoma japonicum (Philippine strain).

Science.gov (United States)

Cai, Z H; Song, G C; Xu, Y X; Liu, S X

1993-03-01

Obtained from pSj5, the cDNA gene encoding GST antigen of Schistosoma japonicum (Philippine strain) was ligated with efficient temperature-dependent PBV220 vector which was constructed in CAPM, and then introduced into host bacterium-DH5 alpha (E. coli) by transformation. Transformants were selected by ampicillin and recombinant clones were identified by restriction mapping. The result showed that recombinant clone 43 was the one carrying recombinant plasmid PBV 220 with the correct insertion of the gene fragment. The GST expression ability of clone 43 was investigated by GST enzymic activity assay and SDS-PAGE. A relatively high level of GST enzymic activity was expressed by this clone under the temperature-dependent condition, that is, cultured at 30 degrees C and expressed at 42 degrees C. A more strongly stained 26 kDa protein band was identified by SDS-PAGE. The result indicated that GST of S. japonicum (Philippine strain) could be expressed not only by IPTG induction, but also by the temperature-dependent method.

Development and selection of fungal and bacterial mutants using ionizing radiation and radioisotopes for improved enzyme production (cellulase and coagulase)

International Nuclear Information System (INIS)

Markov, K.I.

1975-01-01

Ultraviolet and gamma radiations, chemical mutagens, and combinations of chemical and physical mutagens were used in order to obtain mutants of Bacillus mesentericus and Trichoderma viridae with a higher production of coagulase and cellulase, respectively. It was possible to isolate mutant strains, with enzyme activity increased by a factor of 2 and 3

Novel enzymic hydrolytic dehalogenation of a chlorinated aromatic

International Nuclear Information System (INIS)

Scholten, J.D.; Chang, Kaihsuan; Dunaway-Mariano, D.; Babbitt, P.C.; Charest, H.; Sylvestre, M.

1991-01-01

Microbial enzyme systems may be used in the biodegradation of persistent environmental pollutants. The three polypeptide components of one such system, the 4-chlorobenzoate dehalogenase system, have been isolated, and the chemical steps of the 4-hydroxybenzoate-forming reaction that they catalyze have been identified. The genes contained within a 4.5-filobase Pseudomonas sp. strain CBS3 chromosomal DNA fragment that encode dehalogenase activity were selectively expressed in transformed Escherichia coli. Oligonucleotide sequencing revealed a stretch of homology between the 57-kilodalton (kD) polypeptide and several magnesium adenosine triphosphate (MgATP)-cleaving enzymes that allowed MgATP and coenzyme A (CoA) to be identified as the dehalogenase cosubstrate and cofactor, respectively. The dehalogenase activity arises from two components, a 4-chlorobenzoate:CoA ligase-dehalogenase (an αβ dimer of the 57- and 30-kD polypeptides) and a thioesterase (the 16-kD polypeptide)

Regioselective alkane hydroxylation with a mutant CYP153A6 enzyme

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Koch, Daniel J.; Arnold, Frances H.

2013-01-29

Cytochrome P450 CYP153A6 from Myobacterium sp. strain HXN1500 was engineered using in-vivo directed evolution to hydroxylate small-chain alkanes regioselectively. Mutant CYP153A6-BMO1 selectively hydroxylates butane and pentane at the terminal carbon to form 1-butanol and 1-pentanol, respectively, at rates greater than wild-type CYP153A6 enzymes. This biocatalyst is highly active for small-chain alkane substrates and the regioselectivity is retained in whole-cell biotransformations.

Fungal Screening on Olive Oil for Extracellular Triacylglycerol Lipases: Selection of a Trichoderma harzianum Strain and Genome Wide Search for the Genes

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Canseco-Pérez, Miguel Angel; Castillo-Avila, Genny Margarita; Islas-Flores, Ignacio; Apolinar-Hernández, Max M.; Rivera-Muñoz, Gerardo; Gamboa-Angulo, Marcela; Couoh-Uicab, Yeny

2018-01-01

A lipolytic screening with fungal strains isolated from lignocellulosic waste collected in banana plantation dumps was carried out. A Trichoderma harzianum strain (B13-1) showed good extracellular lipolytic activity (205 UmL−1). Subsequently, functional screening of the lipolytic activity on Rhodamine B enriched with olive oil as the only carbon source was performed. The successful growth of the strain allows us to suggest that a true lipase is responsible for the lipolytic activity in the B13-1 strain. In order to identify the gene(s) encoding the protein responsible for the lipolytic activity, in silico identification and characterization of triacylglycerol lipases from T. harzianum is reported for the first time. A survey in the genome of this fungus retrieved 50 lipases; however, bioinformatic analyses and putative functional descriptions in different databases allowed us to choose seven lipases as candidates. Suitability of the bioinformatic screening to select the candidates was confirmed by reverse transcription polymerase chain reaction (RT-PCR). The gene codifying 526309 was expressed when the fungus grew in a medium with olive oil as carbon source. This protein shares homology with commercial lipases, making it a candidate for further applications. The success in identifying a lipase gene inducible with olive oil and the suitability of the functional screening and bioinformatic survey carried out herein, support the premise that the strategy can be used in other microorganisms with sequenced genomes to search for true lipases, or other enzymes belonging to large protein families. PMID:29370083

Diverse effects of arsenic on selected enzyme activities in soil-plant-microbe interactions.

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Lyubun, Yelena V; Pleshakova, Ekaterina V; Mkandawire, Martin; Turkovskaya, Olga V

2013-11-15

Under the influence of pollutants, enzyme activities in plant-microbe-soil systems undergo changes of great importance in predicting soil-plant-microbe interactions, regulation of metal and nutrient uptake, and, ultimately, improvement of soil health and fertility. We evaluated the influence of As on soil enzyme activities and the effectiveness of five field crops for As phytoextraction. The initial As concentration in soil was 50mg As kg(-1) soil; planted clean soil, unplanted polluted soil, and unplanted clean soil served as controls. After 10 weeks, the growth of the plants elevated soil dehydrogenase activity relative to polluted but unplanted control soils by 2.4- and 2.5-fold for sorghum and sunflower (respectively), by 3-fold for ryegrass and sudangrass, and by 5.2-fold for spring rape. Soil peroxidase activity increased by 33% with ryegrass and rape, while soil phosphatase activity was directly correlated with residual As (correlation coefficient R(2)=0.7045). We conclude that soil enzyme activities should be taken into account when selecting plants for phytoremediation. Copyright © 2013 Elsevier B.V. All rights reserved.

Sexual selection, sexual isolation and pheromones in Drosophila melanogaster strains after long-term maintaining on different diets.

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Trajković, Jelena; Miličić, Dragana; Savić, Tatjana; Pavković-Lučić, Sofija

2017-07-01

Evolution of reproductive isolation may be a consequence of a variety of signals used in courtship and mate preferences. Pheromones play an important role in both sexual selection and sexual isolation. The abundance of pheromones in Drosophila melanogaster may depend on different environmental factors, including diet. The aim of this study was to ascertain to which degree principal pheromones affect sexual selection in D. melanogaster. We used D. melanogaster strains reared for 14 years on four substrates: standard cornmeal substrate and those containing tomato, banana and carrot. We have previously determined that long-term maintaining of these dietary strains resulted in differences in their cuticular hydrocarbons profile (CHs). In this work, we have tested the level of sexual selection and sexual isolation between aforementioned strains. We found that the high levels of cis-vaccenyl acetate, 7-pentacosene and 7,11-nonacosadiene in the strain reared on a substrate containing carrot affected the individual attractiveness and influenced sexual isolation between flies of this strain and flies reared on a substrate containing banana. Based on these results, long-term different diets, may contribute, to sexual behaviour of D. melanogaster via the effects of principal pheromones. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficacy of selected Pseudomonas strains for biocontrol of Rhizoctonia solani in potato

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Moncef MRABET

2014-01-01

Full Text Available Thirty seven bacterial isolates from faba bean (Vicia faba L. root-nodules were screened for their antagonistic activity against eight Rhizoctonia solani strains isolated from infected potato (Solanum tuberosum L. tubers. Two bacterial strains (designated as Kl.Fb14 and S8.Fb11 gave 50% in vitro inhibition of R. solani mycelial growth. 16S rDNA sequence analysis indicated that strain Kl.Fb14 exhibited 99.5% identity with Pseudomonas moraviensis, and that S8.Fb11 exhibited 99.8% identity with Pseudomonas reinekei. Greenhouse trials in soil showed that strain S8.Fb11 reduced the percentage of sclerotia on potato tubers and amounts of tuber infection for the potato cultivars Spunta and Nicola. In a field trial conducted in South Tunisia, infection with R. solani reduced potato yield by approximately 40% for ‘Spunta’ and 17% for ‘Nicola’; about 20% of the total tuber production was severely infected. However, when potato tubers were treated with strain S8.Fb11 prior to sowing, disease incidence was reduced to 6% of total production with low infection levels; potato yield was enhanced by about 6 kg per 10 m row in comparison to R. solani infected plants. The second selected Pseudomonas sp. (strain Kl.Fb14 did not affect either the levels of sclerotia on tubers or potato yield.

Environmental carbonate chemistry selects for phenotype of recently isolated strains of Emiliania huxleyi

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Rickaby, Rosalind E. M.; Hermoso, Michaël; Lee, Renee B. Y.; Rae, Benjamin D.; Heureux, Ana M. C.; Balestreri, Cecilia; Chakravarti, Leela; Schroeder, Declan C.; Brownlee, Colin

2016-05-01

Coccolithophorid algae, particularly Emiliania huxleyi, are prolific biomineralisers that, under many conditions, dominate communities of marine eukaryotic plankton. Their ability to photosynthesise and form calcified scales (coccoliths) has placed them in a unique position in the global carbon cycle. Contrasting reports have been made with regards to the response of E. huxleyi to ocean acidification. Therefore, there is a pressing need to further determine the fate of this key organism in a rising CO2 world. In this paper, we investigate the phenotype of newly isolated, genetically diverse, strains of E. huxleyi from UK Ocean Acidification Research Programme (UKOA) cruises around the British Isles, the Arctic, and the Southern Ocean. We find a continuum of diversity amongst the physiological and photosynthetic parameters of different strains of E. huxleyi morphotype A under uniform, ambient conditions imposed in the laboratory. This physiology is best explained by adaptation to carbonate chemistry in the former habitat rather than being prescribed by genetic fingerprints such as the coccolithophore morphology motif (CMM). To a first order, the photosynthetic capacity of each strain is a function of both aqueous CO2 availability, and calcification rate, suggestive of a link between carbon concentrating ability and calcification. The calcification rate of each strain is related linearly to the natural environmental [CO32-] at the site of isolation, but a few exceptional strains display low calcification rates at the highest [CO32-] when calcification is limited by low CO2 availability and/or a lack of a carbon concentrating mechanism. We present O2-electrode measurements alongside coccolith oxygen isotopic composition and the uronic acid content (UAC) of the coccolith associated polysaccharide (CAP), that act as indirect tools to show the differing carbon concentrating ability of the strains. The environmental selection revealed amongst our recently isolated strain

Determination of the Influence of Substrate Concentration on Enzyme Selectivity Using Whey Protein Isolate and Bacillus licheniformis Protease

NARCIS (Netherlands)

Butré, C.I.; Sforza, S.; Gruppen, H.; Wierenga, P.A.

2014-01-01

Increasing substrate concentration during enzymatic protein hydrolysis results in a decrease in hydrolysis rate. To test if changes in the mechanism of hydrolysis also occur, the enzyme selectivity was determined. The selectivity is defined quantitatively as the relative rate of hydrolysis of each

Kinetic characterization of glucose aerodehydrogenase from Aspergillus niger EMS-150-F after optimizing the dose of mutagen for enhanced production of enzyme

OpenAIRE

Umbreen, Huma; Zia, Muhammad Anjum; Rasul, Samreen

2013-01-01

In the present study enhanced production of glucose aerodehydrogenase from Aspergillus niger has been achieved after optimizing the dose of chemical mutagen ethyl methane sulfonate (EMS) that has not been reported earlier. Different doses of mutagen were applied and a strain was developed basing upon the best production. The selected strain Aspergillus niger EMS-150-F was optimized for nutrient requirements in order to produce enzyme through fermentation and the results showed the best yield ...

The selective interaction between silica nanoparticles and enzymes from molecular dynamics simulations.

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Xiaotian Sun

Full Text Available Nanoscale particles have become promising materials in many fields, such as cancer therapeutics, diagnosis, imaging, drug delivery, catalysis, as well as biosensors. In order to stimulate and facilitate these applications, there is an urgent need for the understanding of the interaction mode between the nano-particles and proteins. In this study, we investigate the orientation and adsorption between several enzymes (cytochrome c, RNase A, lysozyme and 4 nm/11 nm silica nanoparticles (SNPs by using molecular dynamics (MD simulation. Our results show that three enzymes are adsorbed onto the surfaces of both 4 nm and 11 nm SNPs during our MD simulations and the small SNPs induce greater structural stabilization. The active site of cytochrome c is far away from the surface of 4 nm SNPs, while it is adsorbed onto the surface of 11 nm SNPs. We also explore the influences of different groups (-OH, -COOH, -NH2 and CH3 coated onto silica nanoparticles, which show significantly different impacts. Our molecular dynamics results indicate the selective interaction between silicon nanoparticles and enzymes, which is consistent with experimental results. Our study provides useful guides for designing/modifying nanomaterials to interact with proteins for their bio-applications.

Enzymes produced by halotolerant spore-forming gram-positive bacterial strains isolated from a resting habitat (Restinga de Jurubatiba) in Rio de Janeiro, Brazil: focus on proteases.

Science.gov (United States)

D Santos, Anderson Fragoso; Pacheco, Clarissa Almeida; Valle, Roberta D Santos; Seldin, Lucy; D Santos, André Luis Souza

2014-12-01

The screening for hydrolases-producing, halotolerant, and spore-forming gram-positive bacteria from the root, rhizosphere, and non-rhizosphere soil of Blutaparon portulacoides, a plant found in the Restinga de Jurubatiba located at the northern region of Rio de Janeiro State, Brazil, resulted in the isolation of 22 strains. These strains were identified as Halobacillus blutaparonensis (n = 2), Oceanobacillus picturae (n = 5), and Oceanobacillus iheyensis (n = 15), and all showed the ability to produce different extracellular enzymes. A total of 20 isolates (90.9 %) showed activity for protease, 5 (22.7 %) for phytase, 3 (13.6 %) for cellulase, and 2 (9.1 %) for amylase. Some bacterial strains were capable of producing three (13.6 %) or two (9.1 %) distinct hydrolytic enzymes. However, no bacterial strain with ability to produce esterase and DNase was observed. The isolate designated M9, belonging to the species H. blutaparonensis, was the best producer of protease and also yielded amylase and phytase. This strain was chosen for further studies regarding its protease activity. The M9 strain produced similar amounts of protease when grown either without or with different NaCl concentrations (from 0.5 to 10 %). A simple inspection of the cell-free culture supernatant by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of three major alkaline proteases of 40, 50, and 70 kDa, which were fully inhibited by phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-phenylalanine chloromethyl ketone (TPCK) (two classical serine protease inhibitors). The secreted proteases were detected in a wide range of temperature (from 4 to 45 °C) and their hydrolytic activities were stimulated by NaCl (up to 10 %). The serine proteases produced by the M9 strain cleaved gelatin, casein, albumin, and hemoglobin, however, in different extensions. Collectively, these results suggest the potential use of the M9 strain in biotechnological

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

Science.gov (United States)

Castro-Fernandez, Víctor; Herrera-Morande, Alejandra; Zamora, Ricardo; Merino, Felipe; Gonzalez-Ordenes, Felipe; Padilla-Salinas, Felipe; Pereira, Humberto M; Brandão-Neto, Jose; Garratt, Richard C; Guixe, Victoria

2017-09-22

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales , Methanosarcinales , and Methanococcales , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Patterns of survival and volatile metabolites of selected Lactobacillus strains during long-term incubation in milk.

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Łaniewska-Trokenheim, Łucja; Olszewska, Magdalena; Miks-Krajnik, Marta; Zadernowska, Anna

2010-08-01

The focus of this study was to monitor the survival of populations and the volatile compound profiles of selected Lactobacillus strains during long-term incubation in milk. The enumeration of cells was determined by both the Direct Epifluorescent Filter Technique using carboxyfluorescein diacetate (CFDA) staining and the plate method. Volatile compounds were analysed by the gas-chromatography technique. All strains exhibited good survival in cultured milks, but Lactobacillus crispatus L800 was the only strain with comparable growth and viability in milk, assessed by plate and epifluorescence methods. The significant differences in cell numbers between plate and microscopic counts were obtained for L. acidophilus strains. The investigated strains exhibited different metabolic profiles. Depending on the strain used, 3 to 8 compounds were produced. The strains produced significantly higher concentrations of acetic acid, compared to other volatiles. Lactobacillus strains differed from one another in number and contents of the volatile compounds.

Biofortification of folates in white wheat bread by selection of yeast strain and process.

Science.gov (United States)

Hjortmo, Sofia; Patring, Johan; Jastrebova, Jelena; Andlid, Thomas

2008-09-30

We here demonstrate that folate content in yeast fermented food can be dramatically increased by using a proper (i) yeast strain and (ii) cultivation procedure for the selected strain prior to food fermentation. Folate levels were 3 to 5-fold higher in white wheat bread leavened with a Saccharomyces cerevisiae strain CBS7764, cultured in defined medium and harvested in the respiro-fermentative phase of growth prior to dough preparation (135-139 microg/100 dry matter), compared to white wheat bread leavened with commercial Baker's yeast (27-43 microg/100 g). The commercial Baker's yeast strain had been industrially produced, using a fed-batch process, thereafter compressed and stored in the refrigerator until bakings were initiated. This strategy is an attractive alternative to fortification of bread with synthetically produced folic acid. By using a high folate producing strain cultured a suitable way folate levels obtained were in accordance with folic acid content in fortified cereal products.

Angiotensin-converting enzyme inhibitory activity of Lactobacillus helveticus strains from traditional fermented dairy foods and antihypertensive effect of fermented milk of strain H9.

Science.gov (United States)

Chen, Yongfu; Liu, Wenjun; Xue, Jiangang; Yang, Jie; Chen, Xia; Shao, Yuyu; Kwok, Lai-yu; Bilige, Menghe; Mang, Lai; Zhang, Heping

2014-11-01

Hypertension is a major global health issue which elevates the risk of a large world population to chronic life-threatening diseases. The inhibition of angiotensin-converting enzyme (ACE) is an effective target to manage essential hypertension. In this study, the fermentation properties (titratable acidity, free amino nitrogen, and fermentation time) and ACE-inhibitory (ACEI) activity of fermented milks produced by 259 Lactobacillus helveticus strains previously isolated from traditional Chinese and Mongolian fermented foods were determined. Among them, 37 strains had an ACEI activity of over 50%. The concentrations of the antihypertensive peptides, Ile-Pro-Pro and Val-Pro-Pro, were further determined by ultra performance liquid chromatography with quadrupole-time-of-flight mass spectrometry. The change of ACEI activity of the fermented milks of 3 strains exhibiting the highest ACEI activity upon gastrointestinal protease treatment was assayed. Fermented milks produced by strain H9 (IMAU60208) had the highest in vitro ACEI activity (86.4 ± 1.5%), relatively short fermentation time (7.5 h), and detectable Val-Pro-Pro (2.409 ± 0.229 µM) and Ile-Pro-Pro (1.612 ± 0.114 µM) concentrations. Compared with the control, a single oral dose of H9-fermented milk significantly attenuated the systolic, diastolic, and mean blood pressure of spontaneously hypertensive rats (SHR) by 15 to 18 mmHg during the 6 to 12 h after treatment. The long-term daily H9-fermented milk intake over 7 wk exerted significant antihypertensive effect to SHR, but not normotensive rats, and the systolic and diastolic blood pressure were significantly lower, by 12 and 10 mmHg, respectively, compared with the control receiving saline. The feeding of H9-fermented milk to SHR resulted in a significantly higher weight gain at wk 7 compared with groups receiving saline, commercial yogurt, and captopril. Our study identified a novel probiotic L. helveticus strain originated from kurut sampled from Tibet

Complete genome sequence of N2-fixing model strain Klebsiella sp. nov. M5al, which produces plant cell wall-degrading enzymes and siderophores

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Zhili Yu

2018-03-01

Full Text Available The bacterial strain M5al is a model strain for studying the molecular genetics of N2-fixation and molecular engineering of microbial production of platform chemicals 1,3-propanediol and 2,3-butanediol. Here, we present the complete genome sequence of the strain M5al, which belongs to a novel species closely related to Klebsiella michiganensis. M5al secretes plant cell wall-degrading enzymes and colonizes rice roots but does not cause soft rot disease. M5al also produces siderophores and contains the gene clusters for synthesis and transport of yersiniabactin which is a critical virulence factor for Klebsiella pathogens in causing human disease. We propose that the model strain M5al can be genetically modified to study bacterial N2-fixation in association with non-legume plants and production of 1,3-propanediol and 2,3-butanediol through degradation of plant cell wall biomass.

Determination of Volatile Organic Compounds in Selected Strains of Cyanobacteria

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Ivan Milovanović

2015-01-01

Full Text Available Microalgal biomass can be used in creating various functional food and feed products, but certain species of microalgae and cyanobacteria are known to produce various compounds causing off-flavour. In this work, we investigated selected cyanobacterial strains of Spirulina, Anabaena, and Nostoc genera originating from Serbia, with the aim of determining the chemical profile of volatile organic compounds produced by these organisms. Additionally, the influence of nitrogen level during growth on the production of volatile compounds was investigated for Nostoc and Anabaena strains. In addition, multivariate techniques, namely, principal component analysis (PCA and hierarchical cluster analysis (HCA, were used for making distinction among different microalgal strains. The results show that the main volatile compounds in these species are medium chain length alkanes, but other odorous compounds such as 2-methylisoborneol (0.51–4.48%, 2-pentylfuran (0.72–8.98%, β-cyclocitral (0.00–1.17%, and β-ionone (1.15–2.72% were also detected in the samples. Addition of nitrogen to growth medium was shown to negatively affect the production of 2-methylisoborneol, while geosmin was not detected in any of the analyzed samples, which indicates that the manipulation of growth conditions may be useful in reducing levels of some unwanted odor-causing components.

[Potentialization of antibiotics by lytic enzymes].

Science.gov (United States)

Brisou, J; Babin, P; Babin, R

1975-01-01

Few lytic enzymes, specially papaine and lysozyme, acting on the membrane and cell wall structures facilitate effects of bacitracine, streptomycine and other antibiotics. Streptomycino resistant strains became sensibles to this antibiotic after contact with papaine and lysozyme. The results of tests in physiological suspensions concern only the lytic activity of enzymes. The results on nutrient medium concern together lytic, and antibiotic activities.

Isolation, characterization and transcriptome analysis of a novel Antarctic Aspergillus sydowii strain MS-19 as a potential lignocellulosic enzyme source.

Science.gov (United States)

Cong, Bailin; Wang, Nengfei; Liu, Shenghao; Liu, Feng; Yin, Xiaofei; Shen, Jihong

2017-05-30

applications of the novel Antarctic Aspergillus sydowii strain MS-19 as a potential lignocellulosic enzyme source.

Antimicrobial Resistance of Staphylococcal Strains Isolated from Various Pathological Products

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Laura-Mihaela SIMON

2010-12-01

Full Text Available Background: The optimal choice of antimicrobial therapy is an important problem in hospital environment in which the selection of resistant and virulent strains easy occurs. S. aureus and especially MRSA(methicillin-resistant S. aureus creates difficulties in both treatment and prevention of nosocomial infections. Aim: The purpose of this study is to determine the sensitivity and the resistance to chemotherapy of staphylococci strains isolated from various pathological products. Material and Method: We identified Staphylococccus species after morphological appearance, culture properties, the production of coagulase, hemolisines and the enzyme activity. The susceptibility tests were performed on Mueller-Hinton medium according to CLSI (Clinical and Laboratory Standards Institute. Results: The strains were: MSSA (methicillin-susceptible S. aureus (74%, MRSA (8%, MLS B (macrolides, lincosamides and type B streptogramines resistance (12% and MRSA and MLS B (6%. MRSA strains were more frequently isolated from sputum. MRSA associated with the MLS B strains were more frequently isolated from pus. MLS B strains were more frequently isolated from sputum and throat secretions. All S. aureus strains were susceptible to vancomycin and teicoplanin. Conclusions: All staphylococcal infections require resistance testing before treatment. MLS B shows a high prevalence among strains of S. aureus. The association between MLS B and MRSA remains a major problem in Romania.

The genomic landscape shaped by selection on transposable elements across 18 mouse strains.

Science.gov (United States)

Nellåker, Christoffer; Keane, Thomas M; Yalcin, Binnaz; Wong, Kim; Agam, Avigail; Belgard, T Grant; Flint, Jonathan; Adams, David J; Frankel, Wayne N; Ponting, Chris P

2012-06-15

Transposable element (TE)-derived sequence dominates the landscape of mammalian genomes and can modulate gene function by dysregulating transcription and translation. Our current knowledge of TEs in laboratory mouse strains is limited primarily to those present in the C57BL/6J reference genome, with most mouse TEs being drawn from three distinct classes, namely short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs) and the endogenous retrovirus (ERV) superfamily. Despite their high prevalence, the different genomic and gene properties controlling whether TEs are preferentially purged from, or are retained by, genetic drift or positive selection in mammalian genomes remain poorly defined. Using whole genome sequencing data from 13 classical laboratory and 4 wild-derived mouse inbred strains, we developed a comprehensive catalogue of 103,798 polymorphic TE variants. We employ this extensive data set to characterize TE variants across the Mus lineage, and to infer neutral and selective processes that have acted over 2 million years. Our results indicate that the majority of TE variants are introduced though the male germline and that only a minority of TE variants exert detectable changes in gene expression. However, among genes with differential expression across the strains there are twice as many TE variants identified as being putative causal variants as expected. Most TE variants that cause gene expression changes appear to be purged rapidly by purifying selection. Our findings demonstrate that past TE insertions have often been highly deleterious, and help to prioritize TE variants according to their likely contribution to gene expression or phenotype variation.

Development of a rapid screening protocol for selection of strains resistant to spray drying and storage in dry powder.

Science.gov (United States)

Reimann, S; Grattepanche, F; Baggenstos, C; Rezzonico, E; Berger, B; Arigoni, F; Lacroix, C

2010-06-01

An efficient screening method for selection of Bifidobacterium longum strains resistant to spray drying and storage was developed based on randomly amplified polymorphic DNA (RAPD) for identification of the best survivors in mixed strains bacterial preparations. Three different primers were used to generate RAPD profiles of 22 B. longum strains. All strains were distinguished according to their RAPD profiles except for the strain NCC2705 and its H(2)O(2) resistant derivative variant. The 22 strains were grouped in 3 batches of 7, 7 and 8 strains and subjected to spray drying and storage at 30 and 37 °C under anaerobic conditions. Batch survival rates after spray drying reached 17.1±4.4%. Strains showing the highest prevalence and/or resistance to storage at 37 °C were selected from individual batches for subsequent spray drying and storage testing. After 67 days of storage, NCC572 was identified as the dominant strain in powder. The stability of strain NCC572 was confirmed by performing single spray drying and storage tests. Out of 22 B. longum strains, a robust strain was identified by combining RAPD with a simultaneous screening test for survival under spray drying and storage. The method allowed a fast screening of B. longum strains in mixture for resistance to spray drying and storage compared to traditional screening procedures carried out with individual strains, in the same conditions. This approach could be applied to other stress conditions.

ligninolytic enzymes of the fungus isolated from soil contaminated

African Journals Online (AJOL)

FUTE

aimed at isolating lignin degrading fungi from soil contaminated with cow dung ... strain was screened for production of ligninolytic enzymes using Rhemazol Brilliant blue R ... put in airtight plastic bag and carried out to ..... Enzyme Microbial.

Role of some selected Bifidobacterium strains in modulating immunosenescence of aged albino rats

Directory of Open Access Journals (Sweden)

Hanan A. El-Bakry

2013-10-01

Full Text Available Probiotic administration has been associated with enhanced immune function in elderly subjects. However, approaches for selection of an “ideal” strain of bifidobacteria are still difficult. The aim of the present study is to investigate the possible modulatory effects of three strains of Bifidobacterium species (Bifidobacterium adolescentis ATCC 15704, Bifidobacterium breve ATCC 15700 and Bifidobacterium longum ATCC 15707 on haematological and immunological parameters of aged albino rats corresponding to normal adult ones. The animals were divided into six groups; three groups of aged rats were fed yoghurt inoculated with one of the Bifidobacterium strains; one group of aged rats was fed yoghurt alone (control aged; two groups of adult and aged rats were provided with normal diet and assigned as normal groups. The total leucocyte count was significantly increased in the three bifidobacteria-treated aged groups as compared with both normal and control aged rats. Serum IgA level was considerably increased in all treated rats. On the contrary, serum IgE level was significantly decreased in rats supplemented with yoghurt inoculated with B. adolescentis or B. breve. Both B. adolescentis and B. breve groups showed significant enhanced production of TNF-α. Furthermore, the production of cytokine IL-8 was significantly increased in the B. adolescentis group. Interestingly, it was apparent that only B. adolescentis had the most pronounced effect on aged rats to regain nearly normal values as measured in normal adult rats. Conclusively, the present work indicates that dietary consumption of selected bifidobacteria strains may have a particular application in the elderly especially in terms of immunomodulation.

Selection and production of insoluble xylan hydrolyzing enzyme by ...

African Journals Online (AJOL)

Jane

2011-03-07

Mar 7, 2011 ... The effect of pH and temperature on the enzyme activity and stability of crude enzyme produced by T. lanuginosus THKU 56 were investigated. To study the effect of pH on activity, the reaction mixture of 0.5 ml of enzyme and 0.5 ml of 1% insoluble oat spelt xylan in 50 mM buffers with various pH values ...

Biodegradation of methyl red by Bacillus sp. strain UN2: decolorization capacity, metabolites characterization, and enzyme analysis.

Science.gov (United States)

Zhao, Ming; Sun, Peng-Fei; Du, Lin-Na; Wang, Guan; Jia, Xiao-Ming; Zhao, Yu-Hua

2014-05-01

Azo dyes are recalcitrant and refractory pollutants that constitute a significant menace to the environment. The present study is focused on exploring the capability of Bacillus sp. strain UN2 for application in methyl red (MR) degradation. Effects of physicochemical parameters (pH of medium, temperature, initial concentration of dye, and composition of the medium) were studied in detail. The suitable pH and temperature range for MR degradation by strain UN2 were respectively 7.0-9.0 and 30-40 °C, and the optimal pH value and temperature were respectively 8.0 and 35 °C. Mg(2+) and Mn(2+) (1 mM) were found to significantly accelerate the MR removal rate, while the enhancement by either Fe(3+) or Fe(2+) was slight. Under the optimal degradation conditions, strain UN2 exhibited greater than 98 % degradation of the toxic azo dye MR (100 ppm) within 30 min. Analysis of samples from decolorized culture flasks confirmed biodegradation of MR into two prime metabolites: N,N'dimethyl-p-phenyle-nediamine and 2-aminobenzoic acid. A study of the enzymes responsible for the biodegradation of MR, in the control and cells obtained during (10 min) and after (30 min) degradation, showed a significant increase in the activities of azoreductase, laccase, and NADH-DCIP reductase. Furthermore, a phytotoxicity analysis demonstrated that the germination inhibition was almost eliminated for both the plants Triticum aestivum and Sorghum bicolor by MR metabolites at 100 mg/L concentration, yet the germination inhibition of parent dye was significant. Consequently, the high efficiency of MR degradation enables this strain to be a potential candidate for bioremediation of wastewater containing MR.

Enzyme immunoassay for rabies antibody in hybridoma culture fluids and its application to differentiation of street and laboratory strains of rabies virus.

OpenAIRE

Smith, J S; Sumner, J W; Roumillat, L F

1984-01-01

A rapid and sensitive enzyme immunoassay is described for detecting rabies antibody in hybridoma culture fluids. Glass fiber filter disks were used to immobilize gamma-irradiated mouse neuroblastoma cells infected with street or laboratory strains of rabies virus. Bound rabies-specific antibody was detected by reaction with horseradish peroxidase-labeled goat anti-mouse immunoglobulin G. The assay was performed in a 96-well filtration device developed by Cleveland et al. (J. Clin. Microbiol. ...

Production, optimization and characterization of fibrinolytic enzyme by Bacillus subtilis RJAS19.

Science.gov (United States)

Kumar, D J Mukesh; Rakshitha, R; Vidhya, M Annu; Jennifer, P Sharon; Prasad, Sandip; Kumar, M Ravi; Kalaichelvan, P T

2014-04-01

The present study aimed at the production, purification and characterization of fibrinolytic nattokinase enzyme from the bacteria isolated from natto food. For the purpose, a fibrinolytic bacterium was isolated and identified as Bacillus subtilis based on 16S rDNA sequence analysis. The strain was employed for the production and optimization of fibrinolytic enzyme. The strain showed better enzyme production during 72nd h of incubation time with 50 degrees C at the pH 9. The lactose and peptone were found to be increasing the enzyme production rate. The enzyme produced was purified and also characterized with the help of SDS-PAGE analysis. The activity and stability profile of the purified enzyme was tested against different temperature and pH. The observations suggesting that the potential of fibrinolytic enzyme produced by Bacillus subtilis RJAS 19 for its applications in preventive medicines.

Combining selected immunomodulatory Propionibacterium freudenreichii and Lactobacillus delbrueckii strains: Reverse engineering development of an anti-inflammatory cheese.

Science.gov (United States)

Plé, Coline; Breton, Jérôme; Richoux, Romain; Nurdin, Marine; Deutsch, Stéphanie-Marie; Falentin, Hélène; Hervé, Christophe; Chuat, Victoria; Lemée, Riwanon; Maguin, Emmanuelle; Jan, Gwénaël; Van de Guchte, Maarten; Foligné, Benoit

2016-04-01

Inflammatory bowel disease (IBD) constitutes a growing public health concern in western countries. Bacteria with anti-inflammatory properties are lacking in the dysbiosis accompanying IBD. Selected strains of probiotic bacteria with anti-inflammatory properties accordingly alleviate symptoms and enhance treatment of ulcerative colitis in clinical trials. Such properties are also found in selected strains of dairy starters such as Propionibacterium freudenreichii and Lactobacillus delbrueckii (Ld). We thus investigated the possibility to develop a fermented dairy product, combining both starter and probiotic abilities of both lactic acid and propionic acid bacteria, designed to extend remissions in IBD patients. We developed a single-strain Ld-fermented milk and a two-strain P. freudenreichii and Ld-fermented experimental pressed cheese using strains previously selected for their anti-inflammatory properties. Consumption of these experimental fermented dairy products protected mice against trinitrobenzenesulfonic acid induced colitis, alleviating severity of symptoms, modulating local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages. As a control, the corresponding sterile dairy matrix failed to afford such protection. This work reveals the probiotic potential of this bacterial mixture, in the context of fermented dairy products. It opens new perspectives for the reverse engineering development of anti-inflammatory fermented foods designed for target populations with IBD, and has provided evidences leading to an ongoing pilot clinical study in ulcerative colitis patients. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Selection of Trichogramma strains and determination of number of parasitoids to be released to control Gymnandrosoma aurantianum Lima (Lepidoptera, Tortricidae)

International Nuclear Information System (INIS)

Molina, Rosa Maria da Silva; Parra, Jose Roberto Postali

2006-01-01

In order to select Trichogramma strains to control G. aurantianum, the biological characteristics of 13 Trichogramma species/strains were evaluated. After selection, the ideal number of T. pretiosum (G18 strain) to be released per G. aurantianum egg was determined in greenhouse tests. The selection test for species/strains was carried out in an incubator adjusted to 25 +- 1 deg C, RH 70 +- 10%, and a 14 h photo phase. The ideal number of parasitoids was estimated in cages covered with a piece of voile-type fabric. The cycle duration for the 13 Trichogramma species/strains varied from 10.2 to 11.9 days. The Atp strain (T. atopovirilia) showed greater parasitism capacity, with an average of 23.3 parasitized eggs and 77.5% parasitism in 24 hours. The G18 strain (T. pretiosum) came next, with an average of 16.8 parasitized eggs and 56.1% parasitism during the same time interval. The emergency, longevity of males, and sex ratio for the 13 species/strains were similar. The number of adults emerged per egg was 1.8 for strains G11 (T. pretiosum) and Br10 (T. bruni), which were different from the G3 strain (T. pretiosum) only, with 1.3 adults per egg. With regard to female longevity, distinct values were observed only between T. pretiosum strains Tp and L2, with 6.3 and 9.3 days, respectively. Under greenhouse conditions, the estimated ratio of 36 T. pretiosum parasitoids per G. aurantianum egg allowed the highest percentage of parasitism (89%). Therefore, Trichogramma spp. has a potential to control G. aurantianum, as long as a large number of parasitoids is released per unit area. (author)

Survival of Lactobacillus rhamnosus strains inoculated in cheese matrix during simulated human digestion.

Science.gov (United States)

Pitino, Iole; Randazzo, Cinzia L; Cross, Kathryn L; Parker, Mary L; Bisignano, Carlo; Wickham, Martin S J; Mandalari, Giuseppina; Caggia, Cinzia

2012-08-01

Survival of probiotic bacteria during transit through the gastrointestinal (GI) tract is influenced by a number of environmental variables including stomach acidity, bile salts, digestive enzymes and food matrix. This study assessed survival of seven selected Lactobacillus rhamnosus strains delivered within a model cheese system to the human upper GI tract using a dynamic gastric model (DGM). Good survival rates for all tested strains were recorded during both simulated gastric and duodenal digestion. Strains H12, H25 and N24 demonstrated higher survival capacities during gastric digestion than L. rhamnosus GG strain used as control, with H12 and N24 continuing to grow during duodenal digestion. Strains L. rhamnosus F17, N24 and R61 showed adhesion properties to both HT-29 and Caco-2 cells. The ability to attach to the cheese matrix during digestion was confirmed by scanning electron microscopy, also indicating production of extracellular polysaccharides as a response to acid stress. Copyright © 2012 Elsevier Ltd. All rights reserved.

Selection of bacteriocin producer strains of lactic acid bacteria from a dairy environment.

Science.gov (United States)

Lasagno, M; Beoleito, V; Sesma, F; Raya, R; Font de Valdez, G; Eraso, A

2002-01-01

Two strains showing bacteriocin production were selected from a total of 206 lactic acid bacteria isolated from samples of milk, milk serum, whey and homemade cheeses in Southern Cordoba, Argentina. This property was detected by means of well diffusion assays. The strains were identified as Enterococcus hirae and Enterococcus durans. The protein nature of those substances was proved by showing their sensitivity to type IV and XXV proteases, papaine, trypsin, pepsin and K proteinase. The bacteriocins inhibited the growth of Listeria monocytogenes, Bacillus cereus, Clostridium perfringes and two strains of Staphylococcus aureus, an A-enterotoxin and a B-enterotoxin producers. All of these bacteria are common pathogens usually associated with food borne diseases (ETA). These lactic acid bacteria or their bacteriocins could be suitable candidates for food preservation and specially useful in the our regional dairy industry.

Resistance to Diamide Insecticides in Plutella xylostella (Lepidoptera: Plutellidae): Comparison Between Lab-Selected Strains and Field-Collected Populations.

Science.gov (United States)

Qin, Chao; Wang, Cheng-Hua; Wang, Ying-Ying; Sun, Shi-Qing; Wang, Huan-Huan; Xue, Chao-Bin

2018-04-02

Diamondback moth, Plutella xylostella (L.; Lepidoptera: Plutellidae), is an important pest of crucifers worldwide. The extensive use of diamide insecticides has led to P. xylostella resistance and this presents a serious threat to vegetable production. We selected chlorantraniliprole (Rf) and flubendiamide (Rh) resistance strains of P. xylostella with resistance ratios of 684.54-fold and 677.25-fold, respectively. The Rf and Rh strains underwent 46 and 36 generations of lab-selection for resistance, respectively. Low cross resistance of Rh to cyantraniliprole was found. Cross resistance to chlorfenapyr, tebufenozid, and indoxacarb was not found in Rf and Rh strains. The P. xylostella ryanodine receptor gene (PxRyR) transcripts level in the Rf and Rh strains was up-regulated. Except for Rf34 and Rh36, PxRyR expression in all generations of Rf and Rh selection gradually increased with increasing resistance. Two resistant populations were field-collected from Guangzhou Baiyun (Rb) and Zengcheng (Rz) and propagated for several generations without exposure to any pesticide had higher PxRyR expression than the susceptible strain (S). In the S strain, PxRyR expression was not related to the resistance ratio. Gene sequencing found that the RyR 4946 gene site was glycine (G) in the S, Rf, and Rh strains, and was glutamate (E) with 70% and 80% frequency in the Rb and Rz populations, respectively. The 4946 gene site was substituted by valine (V) with the frequency of 30% and 20% in Rb and Rz populations, respectively. These results increase the understanding of the mechanisms of diamide insecticide resistance in P. xylostella.

Assessment of hemolytic activity, enzyme production and bacteriocin characterization of Bacillus subtilis LR1 isolated from the gastrointestinal tract of fish.

Science.gov (United States)

Banerjee, Goutam; Nandi, Ankita; Ray, Arun Kumar

2017-01-01

In the present investigation, probiotic potential (antagonistic activity, enzyme production, hemolytic activity, biosafety, antibiotic sensitivity and bile tolerance level) of Bacillus subtilis LR1 was evaluated. Bacteriocin produced by the bacterial strain B. subtilis LR1 isolated from the gastrointestinal tract of Labeo rohita was purified and characterized. The molecular weight of the purified bacteriocin was ~50 kDa in 12 % Native PAGE and showed inhibitory activity against four fish pathogens such as Bacillus mycoides, Aeromonas salmonicida, Pseudomonas fluorescens and Aeromonas hydrophila. The purified bacteriocin was maximally active at temperature 40 °C and pH 7.0, while none of the tested surfactants affect the bacteriocin activity. Extracellular enzyme activity of the selected bacterial strain was also evaluated. Amylase activity was estimated to be highest (38.23 ± 1.15 µg of maltose liberated mg -1  protein ml -1 of culture filtrate) followed by cellulase and protease activity. The selected bacterium was sensitive to most of the antibiotics used in this experiment, can tolerate 0.25 % bile salt and non-hemolytic in nature. Finally, the efficiency of the proposed probiotic candidate was evaluated in in vivo condition. It was detected that the bacterial strain can effectively reduce bacterial pathogenicity in Indian major carps.

Control of biofouling by xanthine oxidase on seawater reverse osmosis membranes from a desalination plant: enzyme production and screening of bacterial isolates from the full-scale plant.

Science.gov (United States)

Nagaraj, V; Skillman, L; Li, D; Xie, Z; Ho, G

2017-07-01

Control of biofouling on seawater reverse osmosis (SWRO) membranes is a major challenge as treatments can be expensive, damage the membrane material and often biocides do not remove the polymers in which bacteria are embedded. Biological control has been largely ignored for biofouling control. The objective of this study was to demonstrate the effectiveness of xanthine oxidase enzyme against complex fouling communities and then identify naturally occurring bacterial strains that produce the free radical generating enzyme. Initially, 64 bacterial strains were isolated from different locations of the Perth Seawater Desalination Plant. In our preceding study, 25/64 isolates were selected from the culture collection as models for biofouling studies, based on their prevalence in comparison to the genomic bacterial community. In this study, screening of these model strains was performed using a nitroblue tetrazolium assay in the presence of hypoxanthine as substrate. Enzyme activity was measured by absorbance. Nine of 25 strains tested positive for xanthine oxidase production, of which Exiguobacterium from sand filters and Microbacterium from RO membranes exhibited significant levels of enzyme production. Other genera that produced xanthine oxidase were Marinomonas, Pseudomonas, Bacillus, Pseudoalteromonas and Staphylococcus. Strain variations were observed between members of the genera Microbacterium and Bacillus. Xanthine oxidase, an oxidoreductase enzyme that generates reactive oxygen species, is endogenously produced by many bacterial species. In this study, production of the enzyme by bacterial isolates from a full-scale desalination plant was investigated for potential use as biological control of membrane fouling in seawater desalination. We have previously demonstrated that free radicals generated by a commercially available xanthine oxidase in the presence of a hypoxanthine substrate, effectively dispersed biofilm polysaccharides on industrially fouled membranes

21 CFR 184.1150 - Bacterially-derived protease enzyme preparation.

Science.gov (United States)

2010-04-01

... filtrate resulting from a pure culture fermentation of a nonpathogenic and nontoxigenic strain of Bacillus subtilis or B. amyloliquefaciens. The preparation is characterized by the presence of the enzymes..._federal_regulations/ibr_locations.html. In addition, antibiotic activity is absent in the enzyme...

Flavourzyme, an Enzyme Preparation with Industrial Relevance: Automated Nine-Step Purification and Partial Characterization of Eight Enzymes.

Science.gov (United States)

Merz, Michael; Eisele, Thomas; Berends, Pieter; Appel, Daniel; Rabe, Swen; Blank, Imre; Stressler, Timo; Fischer, Lutz

2015-06-17

Flavourzyme is sold as a peptidase preparation from Aspergillus oryzae. The enzyme preparation is widely and diversely used for protein hydrolysis in industrial and research applications. However, detailed information about the composition of this mixture is still missing due to the complexity. The present study identified eight key enzymes by mass spectrometry and partially by activity staining on native polyacrylamide gels or gel zymography. The eight enzymes identified were two aminopeptidases, two dipeptidyl peptidases, three endopeptidases, and one α-amylase from the A. oryzae strain ATCC 42149/RIB 40 (yellow koji mold). Various specific marker substrates for these Flavourzyme enzymes were ascertained. An automated, time-saving nine-step protocol for the purification of all eight enzymes within 7 h was designed. Finally, the purified Flavourzyme enzymes were biochemically characterized with regard to pH and temperature profiles and molecular sizes.

Genetic engineering of industrial Saccharomyces cerevisiae strains using a selection/counter-selection approach.

Science.gov (United States)

Kutyna, Dariusz R; Cordente, Antonio G; Varela, Cristian

2014-01-01

Gene modification of laboratory yeast strains is currently a very straightforward task thanks to the availability of the entire yeast genome sequence and the high frequency with which yeast can incorporate exogenous DNA into its genome. Unfortunately, laboratory strains do not perform well in industrial settings, indicating the need for strategies to modify industrial strains to enable strain development for industrial applications. Here we describe approaches we have used to genetically modify industrial strains used in winemaking.

Broad specificity dioxygenase enzymes and the bioremediation of hazardous aromatic pollutants

International Nuclear Information System (INIS)

Bonus, P.A.; Nies, L.

1996-01-01

The release of aromatic compounds to the environment is a major source of global pollution. In particular, the contamination of soil and groundwater with benzene, toluene, and xylenes (BTX) is the most ubiquitous form of aromatic pollution. The major source of BTX contamination is the release of gasoline and other petroleum products. This research focused on the improvement of bioremediation of BTX through a better understanding of broad specificity dioxygenase enzymes produced by soil and sediment bacteria. The investigation utilized pure bacterial strains isolated on biphenyl, naphthalene, or toluene. These isolated aerobic bacteria were then used to investigate the specificity of the initial enzymatic attack on aromatic compounds including BTX and polychlorinated biphenyls (PCBs). The enzymatic specificity and competency of the five isolates selected for study were determined through the use of growth tests and two rapid assay techniques. The growth tests were conducted on mineral agar plates or in liquid cultures, and they were used to determine substrate specificity. In addition, rapid assays for both BTX and PCBs were carried out using various growth substrates. These assays allowed further clarification of the specificity of the dioxygenase enzymes involved in aromatic degradation. Preliminary results of the PCB assay show that biphenyl and naphthalene isolated organisms grown on biphenyl, benzoate, naphthalene, and succinate maintain production of broad specificity dioxygenase enzymes able to degrade PCBs. Likewise, the BTX assay confirms that biphenyl and naphthalene selected organisms grown on their respective selection substrates completely degrade BTX including all three xylene isomers. In comparison, the toluene selected organism that was studied was unable to degrade PCBs, but it was able to degrade all BTX constituents

A multi-phase approach to select new wine yeast strains with enhanced fermentative fitness and glutathione production.

Science.gov (United States)

Bonciani, Tommaso; De Vero, Luciana; Mezzetti, Francesco; Fay, Justin C; Giudici, Paolo

2018-03-01

The genetic improvement of winemaking yeasts is a virtually infinite process, as the design of new strains must always cope with varied and ever-evolving production contexts. Good wine yeasts must feature both good primary traits, which are related to the overall fermentative fitness of the strain, and secondary traits, which provide accessory features augmenting its technological value. In this context, the superiority of "blind," genetic improvement techniques, as those based on the direct selection of the desired phenotype without prior knowledge of the genotype, was widely proven. Blind techniques such as adaptive evolution strategies were implemented for the enhancement of many traits of interest in the winemaking field. However, these strategies usually focus on single traits: this possibly leads to genetic tradeoff phenomena, where the selection of enhanced secondary traits might lead to sub-optimal primary fermentation traits. To circumvent this phenomenon, we applied a multi-step and strongly directed genetic improvement strategy aimed at combining a strong fermentative aptitude (primary trait) with an enhanced production of glutathione (secondary trait). We exploited the random genetic recombination associated to a library of 69 monosporic clones of strain UMCC 855 (Saccharomyces cerevisiae) to search for new candidates possessing both traits. This was achieved by consecutively applying three directional selective criteria: molybdate resistance (1), fermentative aptitude (2), and glutathione production (3). The strategy brought to the selection of strain 21T2-D58, which produces a high concentration of glutathione, comparable to that of other glutathione high-producers, still with a much greater fermentative aptitude.

In vitro profiling of antimethicillin-resistant Staphylococcus aureus activity of thymoquinone against selected type and clinical strains.

Science.gov (United States)

Hariharan, P; Paul-Satyaseela, M; Gnanamani, A

2016-03-01

This study explores antimethicillin-resistant Staphylococcus aureus (MRSA) activity of a bioactive phytochemical constituent, thymoquinone obtained from the medicinal herb, Nigella sativa Linn. Based on initial assessment on crude extract of seeds of Nigella sativa Linn, the pure active constituent was employed in the study. A total of 99 MRSA strains which comprised of 40 types and 59 clinical strains were selected for the study. Minimum inhibitory concentration (MIC), bactericidal activity, postantibiotic effect (PAE) and propensity to select resistant mutants were determined using standard protocols. Results revealed that thymoquinone exhibited MIC in the range of 8-16 μg ml(-1) and MIC90 of 16 μg ml(-1) against MRSA strains. It was bactericidal to MRSA by demonstrating >3 log kill. It showed a longer PAE of 3·2 ± 0·2 h. Upon exposure to high-density inoculum of MRSA, it did not select resistant mutants. Transmission electron microscopy of thymoquinone-treated MRSA showed no lysis but damage to cell wall and cell membrane which corroborated well with the salt tolerance and bacteriolysis assays. In conclusion, MIC90 , bactericidal property, longer PAE, absence of resistant mutant selection and damages in cell membrane and cell wall imply a promising anti-MRSA activity of thymoquinone. This is the first detailed report on anti-MRSA activity of thymoquinone. The assessment was made with both type and clinical strains. Thymoquinone may be a potential lead compound which can be further optimized to discover novel anti-MRSA agents. © 2016 The Society for Applied Microbiology.

The Presence of Biomarker Enzymes of Selected Scleractinian Corals of Palk Bay, Southeast Coast of India

Science.gov (United States)

Anithajothi, R.; Duraikannu, K.; Umagowsalya, G.; Ramakritinan, C. M.

2014-01-01

The health and existence of coral reefs are in danger by an increasing range of environmental and anthropogenic impacts. The causes of coral reef decline include worldwide climate change, shoreline development, habitat destruction, pollution, sedimentation and overexploitation. These disasters have contributed to an estimated loss of 27% of the reefs. If the current pressure continues unabated, the estimated loss of coral reef will be about 60% by the year 2030. Therefore, the present study was aimed to analyze the enzymes involved in stress induced by coral pathogen and its resistance. We focused on the enzymes involved in melanin synthesis pathway (phenoloxidase (PO) and peroxidases (POD)) and free radical scavenging enzymes (super oxide dismutase (SOD), catalase (CAT)) and glutathione peroxidase (Gpx) in selected scleractinian corals such as Acropora formosa, Echinopora lamellosa, Favia favus, Favites halicora, Porites sp., and Anacropora forbesi. Overall, PO activity of coral was significantly lower than that of zooxanthellae except for Favia favus. Coral colonies with lower PO and POD activities are prone to disease. Maximum antioxidant defensive enzymes were observed in Favia favus followed by Echinopora lamellose. It is concluded that assay of these enzymes can be used as biomarkers for identifying the susceptibility of corals towards coral bleaching induced by pathogen. PMID:25215288

The Presence of Biomarker Enzymes of Selected Scleractinian Corals of Palk Bay, Southeast Coast of India

Directory of Open Access Journals (Sweden)

R. Anithajothi

2014-01-01

Full Text Available The health and existence of coral reefs are in danger by an increasing range of environmental and anthropogenic impacts. The causes of coral reef decline include worldwide climate change, shoreline development, habitat destruction, pollution, sedimentation and overexploitation. These disasters have contributed to an estimated loss of 27% of the reefs. If the current pressure continues unabated, the estimated loss of coral reef will be about 60% by the year 2030. Therefore, the present study was aimed to analyze the enzymes involved in stress induced by coral pathogen and its resistance. We focused on the enzymes involved in melanin synthesis pathway (phenoloxidase (PO and peroxidases (POD and free radical scavenging enzymes (super oxide dismutase (SOD, catalase (CAT and glutathione peroxidase (Gpx in selected scleractinian corals such as Acropora formosa, Echinopora lamellosa, Favia favus, Favites halicora, Porites sp., and Anacropora forbesi. Overall, PO activity of coral was significantly lower than that of zooxanthellae except for Favia favus. Coral colonies with lower PO and POD activities are prone to disease. Maximum antioxidant defensive enzymes were observed in Favia favus followed by Echinopora lamellose. It is concluded that assay of these enzymes can be used as biomarkers for identifying the susceptibility of corals towards coral bleaching induced by pathogen.

The presence of biomarker enzymes of selected Scleractinian corals of Palk Bay, southeast coast of India.

Science.gov (United States)

Anithajothi, R; Duraikannu, K; Umagowsalya, G; Ramakritinan, C M

2014-01-01

The health and existence of coral reefs are in danger by an increasing range of environmental and anthropogenic impacts. The causes of coral reef decline include worldwide climate change, shoreline development, habitat destruction, pollution, sedimentation and overexploitation. These disasters have contributed to an estimated loss of 27% of the reefs. If the current pressure continues unabated, the estimated loss of coral reef will be about 60% by the year 2030. Therefore, the present study was aimed to analyze the enzymes involved in stress induced by coral pathogen and its resistance. We focused on the enzymes involved in melanin synthesis pathway (phenoloxidase (PO) and peroxidases (POD)) and free radical scavenging enzymes (super oxide dismutase (SOD), catalase (CAT)) and glutathione peroxidase (Gpx) in selected scleractinian corals such as Acropora formosa, Echinopora lamellosa, Favia favus, Favites halicora, Porites sp., and Anacropora forbesi. Overall, PO activity of coral was significantly lower than that of zooxanthellae except for Favia favus. Coral colonies with lower PO and POD activities are prone to disease. Maximum antioxidant defensive enzymes were observed in Favia favus followed by Echinopora lamellose. It is concluded that assay of these enzymes can be used as biomarkers for identifying the susceptibility of corals towards coral bleaching induced by pathogen.

Establishment and characterization of a hypocatalasemic mouse cell strain

International Nuclear Information System (INIS)

Utsumi, Hiroshi; Tano, Keizo; Hashimoto, Mitsumasa W.; Kodama, Seiji; Watanabe, Hiromitsu

1998-01-01

Contact-inhibited catalase-deficient fibroblast cell strain has been established from the homozygous hypocatalasemic C3H/Cs b mutant mouse. This cell strain has low level of catalase enzyme activity and has normal level of enzyme activities of both glutathione peroxidase and superoxide dismutase. Catalase-deficient C3H/Cs b mutant cell strain is markedly more sensitive to the toxicity of hydrogen peroxide compared to wild-type C3H/Cs a cell strain. In addition, mutant cell strain is sensitive to X-rays and near-UV compared to wild-type cell strain, but shows the same sensitivities to topoisomerase II inhibitors, adriamycin and 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA), and the DNA cross-linking agents, cis-diamminedichloroplatinum (II) (cis-Pt) and trans-diamminedichloroplatinum (II) (trans-Pt). These cell strains will be of use in the study of the roles which catalase plays in the intracellular prevention of DNA damage induced by oxidative stress. (author)

Digestive enzyme as benchmark for insecticide resistance development in Culex pipiens larvae to chemical and bacteriologic insecticides.

Science.gov (United States)

Kamel, Nashwa H; Bahgat, Iman M; El Kady, Gamal A

2013-04-01

This work monitored changes in some digestive enzymes (trypsin and aminopeptidase) associated with the building up of resistance in Cx. pipiens larvae to two chemical insecticides (methomyl and/or malathion) and one biological insecticide (Bacillus thuringiensis-H14 or B.t H 14). The LC50 value of methomyl for both field- and the 12th generation (F12) of the selected strain was 1.789 ppm and 8.925 ppm respectively. The LC50 value of malathion for both field and the F12 of the selected strain was 0.082 ppm and 0.156 ppm respectively, and those of B.t H14 of field strain and the F12 was 2.550ppm & 2.395ppm respectively. The specific activity of trypsin enzyme in control susceptible colony was 20.806 +/- 0.452micromol/min/mg protein; but at F4 and F8 for malathion and methomyl treated larvae were 10.810 +/- 0.860 & 15.616+/-0.408 micromol/min/mg protein, respectively. Trypsin activity of F12 in treated larvae with B.t.H14 was 2.097 +/- 0.587 microiol/min/mg protein. Aminopeptidase specific activity for susceptible control larvae was 173.05 +/- 1.3111 micromol/min/mg protein. This activity decreased to 145.15 +/- 4.12, 152.497 +/- 6.775 & 102.04 +/- 3.58a micromol/min/mg protein after larval (F 12) treatment with methomyl, malathion and B.t H 14 respectively.

The study of variability and strain selection in Streptomyces atroolivaceus. III

International Nuclear Information System (INIS)

Blumauerova, M.; Lipavska, H.; Stajner, K.; Vanek, Z.

1976-01-01

Mutants of Streptomyces atroolivaceus blocked in the biosynthesis of mithramycin were isolated both by natural selection and after treatment with mutagenic factors (UV and gamma rays, nitrous acid). Both physical factors were more effective than nitrous acid. The selection was complicated by the high instability of isolates, out of which 20 to 80%=. (depending on their origin) reversed spontaneously to the parent type. Primary screening (selection of morphological variants and determination of their activity using the method of agar blocks) made it possible to detect only potentially non-productive strains; however, the final selection always had to be made under submerged conditions. Fifty-four stable non-productive mutants were divided, according to results of the chromatographic analysis, into five groups differing in the production of the six biologically inactive metabolites. The mutants did not accumulate chromomycinone, chromocyclomycin and chromocyclin. On mixed cultivation none of the pairs of mutants was capable of the cosynthesis of mithramycin or of new compounds differing from standard metabolites. Possible causes of the above results are discussed. (author)

Characterization and identification of enzyme-producing microflora isolated from the gut of sea cucumber Apostichopus japonicus

Science.gov (United States)

Li, Fenghui; Gao, Fei; Tan, Jie; Fan, Chaojing; Sun, Huiling; Yan, Jingping; Chen, Siqing; Wang, Xiaojun

2016-01-01

Gut microorganisms play an important role in the digestion of their host animals. The purpose of this research was to isolate and assess the enzyme-producing microbes from the Apostichopus japonicus gut. Thirty-nine strains that can produce at least one of the three digestive enzymes (protease, amylase, and cellulase) were qualitatively screened based on their extracellular enzyme-producing abilities. The enzyme-producing strains clustered into eight groups at the genetic similarity level of 100% by analyzing the restriction patterns of 16S rDNA amplified with Mbo I. Phylogenetic analysis revealed that 37 strains belonged to the genus Bacillus and two were members of the genus Virgibacillus. Enzyme-producing capability results indicate that the main enzyme-producing microflora in the A. japonicus gut was Bacillus, which can produce protease, amylase, and cellulase. Virgibacillus, however, can only produce protease. The high enzyme-producing capability of the isolates suggests that the gut microbiota play an important role in the sea cucumber digestive process.

Growth kinetic and fuel quality parameters as selective criterion for screening biodiesel producing cyanobacterial strains.

Science.gov (United States)

Gayathri, Manickam; Shunmugam, Sumathy; Mugasundari, Arumugam Vanmathi; Rahman, Pattanathu K S M; Muralitharan, Gangatharan

2018-01-01

The efficiency of cyanobacterial strains as biodiesel feedstock varies with the dwelling habitat. Fourteen indigenous heterocystous cyanobacterial strains from rice field ecosystem were screened based on growth kinetic and fuel parameters. The highest biomass productivity was obtained in Nostoc punctiforme MBDU 621 (19.22mg/L/day) followed by Calothrix sp. MBDU 701 (13.43mg/L/day). While lipid productivity and lipid content was highest in Nostoc spongiaeforme MBDU 704 (4.45mg/L/day and 22.5%dwt) followed by Calothrix sp. MBDU 701 (1.54mg/L/day and 10.75%dwt). Among the tested strains, Nostoc spongiaeforme MBDU 704 and Nostoc punctiforme MBDU 621 were selected as promising strains for good quality biodiesel production by Preference Ranking Organization Method for Enrichment Evaluation (PROMETHEE) and Graphical Analysis for Interactive Assistance (GAIA) analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Selection of yeast starter culture strains for the production of marula fruit wines and distillates.

Science.gov (United States)

Fundira, M; Blom, M; Pretorius, I S; van Rensburg, P

2002-03-13

Juice of the Sclerocarya birrea subsp. caffra (marula) fruit was fermented by indigenous microflora and different commercial Saccharomyces cerevisiae yeast strains at different temperatures, namely, 15 and 30 degrees C. Volatile acids, esters, and higher alcohols were quantified in the wine and distillates, and the results were interpreted using a multivariate analysis of variance and an average linkage cluster analysis. Significant differences between 15 and 30 degrees C and also among yeasts with respect to volatile compounds were observed. Yeast strains VIN7 and FC consistently produced wines and final distillates significantly different from the other strains. A panel of tasters and marula and brandy producers was asked to select wines and distillates that had an acceptable and typical marula "nose". They were also asked to detect the differences among wines and distillates fermented with the same yeast strain at different temperatures.

Detection of molecular markers by comparative sequence analysis of enzymes from mycobacteria species

International Nuclear Information System (INIS)

Asad, S.; Hussain, M.; Siddiqua, A.; Ain, Q.U.

2014-01-01

Mycobacterial species are one of the most important pathogens and among these members of non-tuberculous mycobacteria (NTM) and mycobacterial tuberculousis complex (MTC) are the causative agent of a relatively milder form of Tuberculosis. Traditional methods for identification of these groups of pathogens are time consuming, lack specificity and sensitivity and furthermore lead to the misidentification due to high similarity index. Therefore, more rapid, specific and cost-effective methods are required for the accurate identification of Mycobacterium species in routine diagnostics. In our study, we identified molecular markers in order to differentiate closely related cousin species of genus Mycobacterium including M. bovis, M. avium, M. leprae and M. tuberculosis. The nucleotide sequences of selected unique markers, i.e., enzymes (used previously in various biochemical tests for the identification of M. species) were selected and their ORFs were retrieved and selected functional proteins of respective biosynthetic pathways were compared in-silico. Result suggested that the variations in nucleotide sequences of the selected enzymes can be directly used for M. species discrimination in one step PCR test. We believe that the in-silico identification and storage of these distinctive characteristics of individual M. species will help in more precise recognition of pathogenic strains and hence specie specific targeted therapy. (author)

GROWTH AND ENZYME PRODUCTION DURING CONTINUOUS CULTURES OF A HIGH AMYLASE-PRODUCING VARIANT OF Aspergillus Oryzae

OpenAIRE

Zangirolami,T.C.; Carlsen,M.; Nielsen,J.; Jørgensen,S.B.

2002-01-01

Growth and product formation by a selected variant of Aspergillus oryzae showing high alpha-amylase production was studied in continuous cultivations carried out at six different specific growth rates, using glucose as the growth-limiting nutrient. The analysis of the steady-state data revealed that the variant and wild-type strains were similar with respect to glucose uptake system and stoichiometric coefficients. However, the variant was capable of maintaining an enzyme production as high a...

High strain amount in recessed junctions induced by selectively deposited boron-doped SiGe layers

International Nuclear Information System (INIS)

Radamson, H.H.; Kolahdouz, M.; Ghandi, R.; Ostling, M.

2008-01-01

This work presents the selective epitaxial growth (SEG) of Si 1-x Ge x (x = 0.15-0.315) layers with high amount of boron (1 x 10 20 -1 x 10 21 cm -3 ) in recessed or unprocessed (elevated) openings for source/drain applications in CMOS has been studied. The influence of the growth rate and strain on boron incorporation has been studied. A focus has been made on the strain distribution and boron incorporation in SEG of SiGe layers

[Comparison on agronomy and quality characters of selective strain of Schizonepeta tenuifolia].

Science.gov (United States)

Cao, Liang; Jin, Yue; Wei, Jianhe; Chu, Qinglong; Zhao, Runhuai; Wang, Weiquan

2009-05-01

With the purpose of selecting adequate quality and high production of Schizonepeta tenuifolia, the comparative experiments were carried out on different strain of S. tenuifolia in 2007. The test fields were divided into blocks randomly, and the agronomy characters were investigated in harvest time; the content of volatile oil was measured by steam distillation and the pulegone were determined by HPLC. The yield of S4 was 18.63% and 29.99% higher than that of CK1 and CK2, respectively. The contents of volatile oil and pulegone were also higher than those of CK and other strains in this test. S4 shows the advantages of high production, strong disease resistance and high active components. S4 would be extended as the good breed in production.

Photoreactivating enzyme from Escherichia coli

International Nuclear Information System (INIS)

Snapka, R.M.; Fuselier, C.O.

1977-01-01

Escherichia coli photoreactivating enzyme (PRE) has been purified in large amounts from an E.coli strain lysogenic for a defective lambda bacteriophage carrying the phr gene. The resulting enzyme had a pH optimum of 7.2 and an ionic strength optimum of 0.18. It consisted of an apoprotein and cofactor, both of which were necessary for catalytic activity. The apoprotein had a monomer molecular weight of 35,200 and showed stable aggregates under denaturing conditions. The amino acid analysis of the E.coli enzyme was very similar to that of the photoreactivating enzyme from orchid seedlings (Cattelya aurantiaca). Both had arginine at the amino terminus. The cofactor, like the holoenzyme, showed absorption, magnetic circular dichroism, and emission properties indicative of an adenine moiety. Although the isolated enzyme had an action spectrum which peaked at about 360 nm, neither the cofactor, apoenzyme nor holoenzyme showed any detectable absorption between 300 and 400 nm. (author)

Photoreactivating enzyme from Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Snapka, R M; Fuselier, C O [California Univ., Irvine (USA)

1977-05-01

Escherichia coli photoreactivating enzyme (PRE) has been purified in large amounts from an E.coli strain lysogenic for a defective lambda bacteriophage carrying the phr gene. The resulting enzyme had a pH optimum of 7.2 and an ionic strength optimum of 0.18. It consisted of an apoprotein and cofactor, both of which were necessary for catalytic activity. The apoprotein had a monomer molecular weight of 35,200 and showed stable aggregates under denaturing conditions. The amino acid analysis of the E.coli enzyme was very similar to that of the photoreactivating enzyme from orchid seedlings (Cattelya aurantiaca). Both had arginine at the amino terminus. The cofactor, like the holoenzyme, showed absorption, magnetic circular dichroism, and emission properties indicative of an adenine moiety. Although the isolated enzyme had an action spectrum which peaked at about 360 nm, neither the cofactor, apoenzyme nor holoenzyme showed any detectable absorption between 300 and 400 nm.

Lignocellulolytic enzyme production of Pleurotus ostreatus growth in agroindustrial wastes

Directory of Open Access Journals (Sweden)

José Maria Rodrigues da Luz

2012-12-01

Full Text Available The mushroom Pleurotus ostreatus has nutritional and medicinal characteristics that depend on the growth substrate. In nature, this fungus grows on dead wood, but it can be artificially cultivated on agricultural wastes (coffee husks, eucalyptus sawdust, corncobs and sugar cane bagasse. The degradation of agricultural wastes involves some enzyme complexes made up of oxidative (laccase, manganese peroxidase and lignin peroxidase and hydrolytic enzymes (cellulases, xylanases and tanases. Understanding how these enzymes work will help to improve the productivity of mushroom cultures and decrease the potential pollution that can be caused by inadequate discharge of the agroindustrial residues. The objective of this work was to assess the activity of the lignocellulolytic enzymes produced by two P. ostreatus strains (PLO 2 and PLO 6. These strains were used to inoculate samples of coffee husks, eucalyptus sawdust or eucalyptus bark add with or without 20 % rice bran. Every five days after substrate inoculation, the enzyme activity and soluble protein concentration were evaluated. The maximum activity of oxidative enzymes was observed at day 10 after inoculation, and the activity of the hydrolytic enzymes increased during the entire period of the experiment. The results show that substrate composition and colonization time influenced the activity of the lignocellulolytic enzymes.

Selection of oleuropein-degrading lactic acid bacteria strains isolated from fermenting Moroccan green olives

Energy Technology Data Exchange (ETDEWEB)

Ghabbour, N.; Lamzira, Z.; Thonart, P.; Cidalia, P.; Markaouid, M.; Asehraoua, A.

2011-07-01

A total of 177 strains of lactic acid bacteria (LAB) were isolated from early-stage Moroccan Picholine green olive fermentation, including Lactobacillus plantarum (44.63%), Lactobacillus pentosus (25.99%), Lactobacillus brevis (9.61%) and Pediococcus pentosaceus (19.77%). All the isolates were screened for their tolerance to olive leaf extract and oleuropein. Most of the isolates (85.3%) were found able to degrade oleuropein, when evaluated by either oleuropein or 5-Bromo-4-chloro-3-indolyl {beta}-D-glucuronide (X-Gluc) as substrates. The biodegradation capacity of the selected strains of each species was confirmed by HPLC analysis. (Author).

Fermentation of Apple Juice with a Selected Yeast Strain Isolated from the Fermented Foods of Himalayan Regions and Its Organoleptic Properties.

Science.gov (United States)

Kanwar, S S; Keshani

2016-01-01

Twenty-three Saccharomyces cerevisiae strains isolated from different fermented foods of Western Himalayas have been studied for strain level and functional diversity in our department. Among these 23 strains, 10 S. cerevisiae strains on the basis of variation in their brewing traits were selected to study their organoleptic effect at gene level by targeting ATF1 gene, which is responsible for ester synthesis during fermentation. Significant variation was observed in ATF1 gene sequences, suggesting differences in aroma and flavor of their brewing products. Apple is a predominant fruit in Himachal Pradesh and apple cider is one of the most popular drinks all around the world hence, it was chosen for sensory evaluation of six selected yeast strains. Organoleptic studies and sensory analysis suggested Sc21 and Sc01 as best indigenous strains for soft and hard cider, respectively, indicating their potential in enriching the local products with enhanced quality.

Isolation and Fatty Acid Profile of Selected Microalgae Strains from the Red Sea for Biofuel Production

Directory of Open Access Journals (Sweden)

Khalid M. Abu-Salah

2013-05-01

Full Text Available The isolation of lipid-rich autochthonous strains of microalgae is a crucial stage for the development of a microalgae-based biofuel production plant, as these microalgae already have the necessary adaptations to withstand competition, predation and the temperatures observed at each production site. This is particularly important in extreme climates such as in Saudi Arabia. Resorting to fluorescence activated cell sorting (FACS we screened for and isolated several microalgal strains from samples collected from the Red Sea. Relying on the fluorescence of BODIPY 505/515 (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diazasindacene and growth performance, four promising candidates were identified and the total lipid content and fatty acid profile was assessed for biofuels production. Selected isolates were classified as chlorophytes, belonging to three different genera: Picochlorum, Nannochloris and Desmochloris. The lipid contents were assessed microscopically by means of BODIPY 505/515-associated fluorescence to detect intracellular lipid bodies, which revealed several lipid drops in all selected strains. This result was confirmed by lipid gravimetric determination, which demonstrated that all strains under study presented inner cell lipid contents ranging from 20% to 25% of the biomass dry weight. Furthermore, the fatty acid methyl esters profile of all strains seems ideal for biodiesel production due to a low degree of polyunsaturated fatty acid methyl esters and high amount of palmitic and oleic acids.

Enzymic hydrolysis of cellulosic wastes to glucose

Energy Technology Data Exchange (ETDEWEB)

Spano, L A; Medeiros, J; Mandels, M

1976-01-01

An enzymic process for the conversion of cellulose to glucose is based on the use of a specific enzyme derived from mutant strains of the fungus trichoderma viride which is capable of reacting with the crystalline fraction of the cellulose molecule. The production and mode of action of the cellulase complex produced during the growth of trichoderma viride is discussed as well as the application of such enzymes for the conversion of cellulosic wastes to crude glucose syrup for use in production of chemical feedstocks, single-cell proteins, fuels, solvents, etc.

Synthesis of non-steroidal anti-inflammatory drug analogues for selective studies on the COX-II enzyme

International Nuclear Information System (INIS)

Fleming, S.A.; Ridges, M.D.; Jensen, A.W.

1996-01-01

Synthesis of the azido substituted non-steroidal anti-inflammatory drug 2-(2,6-dichloroanilino)phenylacetic acid and isotope labeling of this compound have been performed and are described. Initial evaluation of the binding ability and photoreactivity indicates that this compound has potential for photoaffinity labeling as well as enzyme selectivity studies. (author)

High strain amount in recessed junctions induced by selectively deposited boron-doped SiGe layers

Energy Technology Data Exchange (ETDEWEB)

Radamson, H.H. [School of Information and Communication Technology, KTH (Royal Institute of Technology) Isafjordsg. 22-26, Electrum 229, 16640 Kista (Sweden)], E-mail: rad@kth.se; Kolahdouz, M.; Ghandi, R.; Ostling, M. [School of Information and Communication Technology, KTH (Royal Institute of Technology) Isafjordsg. 22-26, Electrum 229, 16640 Kista (Sweden)

2008-12-05

This work presents the selective epitaxial growth (SEG) of Si{sub 1-x}Ge{sub x} (x = 0.15-0.315) layers with high amount of boron (1 x 10{sup 20}-1 x 10{sup 21} cm{sup -3}) in recessed or unprocessed (elevated) openings for source/drain applications in CMOS has been studied. The influence of the growth rate and strain on boron incorporation has been studied. A focus has been made on the strain distribution and boron incorporation in SEG of SiGe layers.

Selective Inhibition of Steroidogenic Enzymes by Ketoconazole in Rat Ovary Cells

Directory of Open Access Journals (Sweden)

Michael Gal

2014-01-01

Full Text Available Objective Ketoconazole (KCZ is an anti-fungal agent extensively used for clinical applications related to its inhibitory effects on adrenal and testicular steroidogenesis. Much less information is available on the effects of KCZ on synthesis of steroid hormones in the ovary. The present study aimed to characterize the in situ effects of KCZ on steroidogenic enzymes in primary rat ovary cells. Methods Following the induction of folliculogenesis in gonadotropin treated rats, freshly prepared ovarian cells were incubated in suspension for up to four hours while radiolabeled steroid substrates were added and time dependent generation of their metabolic products was analyzed by thin layer chromatography (TLC. Results KCZ inhibits the P450 steroidogenic enzymes in a selective and dose dependent manner, including cholesterol side-chain cleavage cytochrome P450 (CYP11A1/P450scc, the 17α-hydroxylase activity of CYP17A1/P450c17, and CYP19A1/P450arom, with IC 50 values of 0.3, 1.8, and 0.3 μg/mL (0.56, 3.36, and 0.56 μM, respectively. Unaffected by KCZ, at 10 μg/mL, were the 17,20 lyase activity of CYP17A1, as well as five non-cytochrome steroidogenic enzymes including 3β-hydroxysteroid dehydrogenase-δ 5-4 isomerase type 1 (3βHSD1, 5α-reductase, 20α-hydroxysteroid dehydrogenase (20α-HSD, 3α-hydroxysteroid dehydrogenase (3α-HSD, and 17β-hydroxysteroid dehydrogenase type 1 (17HSD1. Conclusion These findings map the effects of KCZ on the ovarian pathways of progestin, androgen, and estrogen synthesis. Hence, the drug may have a potential use as an acute and reversible modulator of ovarian steroidogenesis in pathological circumstances.

Strain selection, biomass to biofuel conversion, and resource colocation have strong impacts on the economic performance of algae cultivation sites

Directory of Open Access Journals (Sweden)

Erik R. Venteris

2014-09-01

Full Text Available Decisions involving strain selection, biomass to biofuel technology, and the location of cultivation facilities can strongly influence the economic viability of an algae-based biofuel enterprise. We summarize our past results in a new analysis to explore the relative economic impact of these design choices. Our growth model is used to predict average biomass production for two saline strains (Nannocloropsis salina, Arthrospira sp., one fresh to brackish strain (Chlorella sp., DOE strain 1412, and one freshwater strain (order Sphaeropleales. Biomass to biofuel conversion is compared between lipid extraction (LE and hydrothermal liquefaction (HTL technologies. National-scale models of water, CO2 (as flue gas, land acquisition, site leveling, construction of connecting roads, and transport of HTL oil to existing refineries are used in conjunction with estimates of fuel value (from HTL to prioritize and select from 88,692 unit farms (UF, 405 ha in pond area, a number sufficient to produce 136E+9 L yr-1 of renewable diesel (36 billion gallons yr-1. Strain selection and choice of conversion technology have large economic impacts, with differences between combinations of strains and biomass to biofuel technologies being up to $10 million dollars yr-1 UF-1. Results based on the most productive strain, HTL-based fuel conversion, and resource costs show that the economic potential between geographic locations within the selection can differ by up to $4 million yr-1 UF-1, with 1.8 BGY of production possible from the most cost-effective sites. The local spatial variability in site rank is extreme, with very high and low sites within 10s of km of each other. Colocation with flue gas sources has a strong influence on rank, but the most costly resource component varies from site to site. The highest rank UFs are located predominantly in Florida and Texas, but most states south of 37°N latitude contain promising locations.

Strain selection, biomass to biofuel conversion, and resource colocation have strong impacts on the economic performance of algae cultivation sites

Energy Technology Data Exchange (ETDEWEB)

Venteris, Erik R.; Wigmosta, Mark S.; Coleman, Andre M.; Skaggs, Richard

2014-09-16

Decisions involving strain selection, biomass to biofuel technology, and the location of cultivation facilities can strongly influence the economic viability of an algae-based biofuel enterprise. In this contribution we summarize our past results in a new analysis to explore the relative economic impact of these design choices. We present strain-specific growth model results from two saline strains (Nannocloropsis salina, Arthrospira sp.), a fresh to brackish strain (Chlorella sp., DOE strain 1412), and a freshwater strain of the order Sphaeropleales. Biomass to biofuel conversion is compared between lipid extraction (LE) and hydrothermal liquefaction (HTL) technologies. National-scale models of water, CO2 (as flue gas), land acquisition, site leveling, construction of connecting roads, and transport of HTL oil to existing refineries are used in conjunction with estimates of fuel value (from HTL) to prioritize and select from 88,692 unit farms (UF, 405 ha in pond area), a number sufficient to produce 136E+9 L yr-1 of renewable diesel (36 billion gallons yr-1, BGY). Strain selection and choice of conversion technology have large economic impacts, with differences between combinations of strains and biomass to biofuel technologies being up to $10 million dollars yr-1 UF-1. Results based on the most productive species, HTL-based fuel conversion, and resource costs show that the economic potential between geographic locations within the selection can differ by up to $4 million yr-1 UF-1, with 2.0 BGY of production possible from the most cost-effective sites. The local spatial variability in site rank is extreme, with very high and low rank sites within 10s of km of each other. Colocation with flue gas sources has a strong influence on site rank, but the most costly resource component varies from site to site. The highest rank sites are located predominantly in Florida and Texas, but most states south of 37°N latitude contain promising locations. Keywords: algae

Strain Selection, Biomass to Biofuel Conversion, and Resource Colocation have Strong Impacts on the Economic Performance of Algae Cultivation Sites

Energy Technology Data Exchange (ETDEWEB)

Venteris, Erik R., E-mail: erik.venteris@pnl.gov; Wigmosta, Mark S.; Coleman, Andre M.; Skaggs, Richard L. [Pacific Northwest National Laboratory, Richland, WA (United States)

2014-09-16

Decisions involving strain selection, biomass to biofuel technology, and the location of cultivation facilities can strongly influence the economic viability of an algae-based biofuel enterprise. We summarize our past results in a new analysis to explore the relative economic impact of these design choices. Our growth model is used to predict average biomass production for two saline strains (Nannochloropsis salina and Arthrospira sp.), one fresh to brackish strain (Chlorella sp., DOE strain 1412), and one freshwater strain (order Sphaeropleales). Biomass to biofuel conversion is compared between lipid extraction and hydrothermal liquefaction (HTL) technologies. National-scale models of water, CO{sub 2} (as flue gas), land acquisition, site leveling, construction of connecting roads, and transport of HTL oil to existing refineries are used in conjunction with estimates of fuel value (from HTL) to prioritize and select from 88,692 unit farms (UF, 405 ha in pond area), a number sufficient to produce 136E + 9 L year{sup −1} of renewable diesel [36 billion gallons year{sup −1} (BGY)]. Strain selection and choice of conversion technology have large economic impacts, with differences between combinations of strains and biomass to biofuel technologies being up to $10 million year{sup −1} UF{sup −1}. Results based on the most productive strain, HTL-based fuel conversion, and resource costs show that the economic potential between geographic locations within the selection can differ by up to 4 million year{sup −1} UF{sup −1}, with 1.8 BGY of production possible from the most cost-effective sites. The local spatial variability in site rank is extreme, with very high and low sites within 10 kms of each other. Colocation with flue gas sources has a strong influence on rank, but the most costly resource component varies from site to site. The highest rank UFs are located predominantly in Florida and Texas, but most states south of 37°N latitude contain promising

Strain differentiation of polioviruses with monoclonal antibodies.

NARCIS (Netherlands)

A.D.M.E. Osterhaus (Albert); A.L. van Wezel; A.J.H. Stegmann; J.A.A.M. van Asten (Jack)

1984-01-01

textabstractPanels of monoclonal antibodies raised against different poliovirus type 1, 2 and 3 strains, were tested in a micro-neutralization test and in a micro-enzyme linked immunosorbent assay against a large number of poliovirus strains. The results were compared with those obtained with the

[Investigation of some virulence factors in Trichosporon spp. strains].

Science.gov (United States)

Demir, Feyza; Kuştimur, Semra

2014-10-01

The frequency of fungal infections have increased recently in parallel to prolonged survival of patients with chronical infections, common use of the broad-spectrum antibiotics and cytotoxic drugs and surgical interventions. Fungi such as Trichosporon, Fusarium and Geotrichum that were previously evaluated as contaminant/colonization, become important causes of morbidity and mortality especially in neutropenic patients. The aim of this study was to investigate the presence of virulence factors such as acid proteinase, phospholipase, esterase, coagulase and hemolytic activity among Trichosporon species. A total of 40 Trichosporon strains, of them 24 (60%) were T.asahii, 6 (15%) were T.inkin and 10 (25%) were the other species (one of each of T.aquatile, T.asteroides, T.coremiiforme, T.cutaneum, T.dermatis, T.faecale, T.japonicum, T.montevideense, T.mucoides, T.ovoides) were included in the study. Identification of the isolates was performed according to microscopic morphology (blastospores, arthrospores, pseudohyphae and true hyphae) on corn meal agar media, and carbohydrate assimilation patterns (API ID32C; bioMérieux, France). Secretory acid proteinase, phospholipase and esterase activities of the strains were evaluated by 1% bovine serum albumin containing agar, by egg yolk containing solid medium, and by Tween 80 containing solid medium, respectively. Hemolytic activity of the isolates were evaluated by 5-10% sheep blood Sabouraud dextrose agar. Coagulase enzyme activity was determined by using human and rabbit plasma. In our study, all of the 40 Trichosporon spp. strains were found negative in terms of acid proteinase and phospholipase enzyme activity, however all were positive for esterase enzyme activity. Hemolytic enzyme activity were identified in a total of 15 (37.5%) strains, being "+++" in three strains (2 T.asahii, 1 T.japonicum), and "++" in 12 isolates (9 T.asahii, 1 T.inkin, 1 T.asteroides, 1 T.mentevideense). Although 11 of those 15 positive

EFFECT OF AERO-/ANAEROBIOSIS ON DECARBOXYLASE ACTIVITY OF SELECTED LACTIC ACID BACTERIA

Directory of Open Access Journals (Sweden)

Stanislav Kráčmar

2010-05-01

Full Text Available Biogenic amines are undesirable compounds produced in foods mainly through bacterial decarboxylase activity. The aim of this study was to investigate some environmental conditions (particularly aero/anaerobiosis, sodium chloride concentration (0–2% w/w, and amount of lactose (0–1% w/w on the activity of tyrosine decarboxylase enzymes of selected six technological important Lactococcus lactis strains. The levels of parameters tested were chosen according to real situation in fermented dairy products technology (especially cheese-making. Tyramine was determined by the ion-exchange chromatography with post-column ninhydrine derivatization and spectrophotometric detection. Tyrosine decarboxylation occurred during the active growth phase. Under the model conditions used, oxygen availability had influence on tyramine production, anaerobiosis seemed to favour the enzyme activity because all L. lactis strains produced higher tyramine amount. doi:10.5219/43

Effect of strain, substrate surface and growth rate on B-doping in selectively grown SiGe layers

International Nuclear Information System (INIS)

Ghandi, R.; Kolahdouz, M.; Hallstedt, J.; Wise, R.; Wejtmans, Hans; Radamson, H.H.

2008-01-01

In this work, the role of strain and growth rate on boron incorporation in selective epitaxial growth (SEG) of B-doped Si 1-x Ge x (x = 0.15-0.25) layers in recessed or unprocessed (elevated) openings for source/drain applications in CMOS has been studied. A focus has been made on the strain distribution and B incorporation in SEG of SiGe layers

Effect of strain, substrate surface and growth rate on B-doping in selectively grown SiGe layers

Energy Technology Data Exchange (ETDEWEB)

Ghandi, R. [School of Information and Communication Technology, KTH (Royal Institute of Technology), Isafjordsg. 22-26, Electrum 229, 16640 Kista (Sweden)], E-mail: ghandi@kth.se; Kolahdouz, M.; Hallstedt, J. [School of Information and Communication Technology, KTH (Royal Institute of Technology), Isafjordsg. 22-26, Electrum 229, 16640 Kista (Sweden); Wise, R.; Wejtmans, Hans [Texas Instrument, 13121 TI Boulevard, Dallas, Tx 75243 (United States); Radamson, H.H. [School of Information and Communication Technology, KTH (Royal Institute of Technology), Isafjordsg. 22-26, Electrum 229, 16640 Kista (Sweden)

2008-11-03

In this work, the role of strain and growth rate on boron incorporation in selective epitaxial growth (SEG) of B-doped Si{sub 1-x}Ge{sub x} (x = 0.15-0.25) layers in recessed or unprocessed (elevated) openings for source/drain applications in CMOS has been studied. A focus has been made on the strain distribution and B incorporation in SEG of SiGe layers.

Tellurite and Tellurate Reduction by the Aerobic Anoxygenic Phototroph Erythromonas ursincola, Strain KR99 Is Carried out by a Novel Membrane Associated Enzyme

Directory of Open Access Journals (Sweden)

Chris Maltman

2017-04-01

Full Text Available Erythromonas ursincola, strain KR99 isolated from a freshwater thermal spring of Kamchatka Island in Russia, resists and reduces very high levels of toxic tellurite under aerobic conditions. Reduction is carried out by a constitutively expressed membrane associated enzyme, which was purified and characterized. The tellurite reductase has a molecular weight of 117 kDa, and is comprised of two subunits (62 and 55 kDa in a 1:1 ratio. Optimal activity occurs at pH 7.0 and 28 °C. Tellurite reduction has a Vmax of 5.15 µmol/min/mg protein and a Km of 3.36 mM. The enzyme can also reduce tellurate with a Vmax and Km of 1.08 µmol/min/mg protein and 1.44 mM, respectively. This is the first purified membrane associated Te oxyanion reductase.

Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

Science.gov (United States)

2012-01-01

Βackground The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains. Results A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production. Conclusions Co

Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

Directory of Open Access Journals (Sweden)

Krainer Florian W

2012-02-01

Full Text Available Abstract Βackground The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains. Results A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein

Process optimization for a potent wild and mutant strain of aspergillus niger for biosynthesis of amyloglucosidase

International Nuclear Information System (INIS)

Malik, S.; Haq, I.U.; Iftikhar, T.

2011-01-01

The present study is concerned with the selection of a potent strain of Aspergillus niger and optimization of the cultural conditions for the biosynthesis of amyloglucosidase. The cultural conditions were optimized for the enzyme production. Twenty percent (50/250ml flask) was found to be optimum volume of the medium. Optimum temperature was 30 deg. C after 72 h of incubation, with the initial pH of the medium 5.0. 2% Starch with 1% glucose as an additional carbon source gave maximum amyloglucosidase production Addition of 0.3% ammonium sulphate in the fermentation medium increased the enzyme production while 2% spore inoculum showed best amyloglucosidase production. (author)

Enzymic hydrolysis of starch in continuous alcoholic fermentation

Energy Technology Data Exchange (ETDEWEB)

Yarovenko, V.L.; Pykhova, S.V.; Ustinnikov, B.A.; Lazareva, A.N.; Makeev, D.M.

1965-01-01

Fermentations were conducted on a plant scale, using starch of various origins, e.g., potatoes, wheat, and other cereals, and as enzyme source a number of strains of Aspergillus oryzae, A. awamori, A. usamii, A. niger, A. batatae, and Bacillus mesentericus. The starches were broken down to a molecular weight between 900 and 1600; time requirements differed from those commonly known. Comparison of these enzymic actions to that of standard malt discloses that in breaking down a potato starch from a molecular weight of 268,000 to one of 1353 to 1556, the malt and A. Oryzae require 1 hour, whereas A. awamori and B. mesentericus require 18, and a different strain of A. awamori requires 24 hours.

Probiotic Potential of Lactobacillus Strains with Antifungal Activity Isolated from Animal Manure.

Science.gov (United States)

Ilavenil, Soundharrajan; Park, Hyung Soo; Vijayakumar, Mayakrishnan; Arasu, Mariadhas Valan; Kim, Da Hye; Ravikumar, Sivanesan; Choi, Ki Choon

2015-01-01

The aim of the study was to isolate and characterize the lactic acid bacteria (LAB) from animal manure. Among the thirty LAB strains, four strains, namely, KCC-25, KCC-26, KCC-27, and KCC-28, showed good cell growth and antifungal activity and were selected for further characterization. Biochemical and physiology properties of strains confirmed that the strains are related to the Lactobacillus sp.; further, the 16S rRNA sequencing confirmed 99.99% sequence similarity towards Lactobacillus plantarum. The strains exhibited susceptibility against commonly used antibiotics with negative hemolytic property. Strains KCC-25, KCC-26, KCC-27, and KCC-28 showed strong antifungal activity against Aspergillus fumigatus, Penicillium chrysogenum, Penicillium roqueforti, Botrytis elliptica, and Fusarium oxysporum, respectively. Fermentation studies noted that the strains were able to produce significant amount of lactic, acetic, and succinic acids. Further, the production of extracellular proteolytic and glycolytic enzymes, survival under low pH, bile salts, and gastric juice together with positive bile salt hydrolase (Bsh) activity, cholesterol lowering, cell surface hydrophobicity, and aggregation properties were the strains advantages. Thus, KCC-25, KCC-26, KCC-27, and KCC-28 could have the survival ability in the harsh condition of the digestive system in the gastrointestinal tract. In conclusion, novel L. plantarum KCC-25, KCC-26, KCC-27, and KCC-28 could be considered as potential antimicrobial probiotic strains.

Probiotic Potential of Lactobacillus Strains with Antifungal Activity Isolated from Animal Manure

Directory of Open Access Journals (Sweden)

Soundharrajan Ilavenil

2015-01-01

Full Text Available The aim of the study was to isolate and characterize the lactic acid bacteria (LAB from animal manure. Among the thirty LAB strains, four strains, namely, KCC-25, KCC-26, KCC-27, and KCC-28, showed good cell growth and antifungal activity and were selected for further characterization. Biochemical and physiology properties of strains confirmed that the strains are related to the Lactobacillus sp.; further, the 16S rRNA sequencing confirmed 99.99% sequence similarity towards Lactobacillus plantarum. The strains exhibited susceptibility against commonly used antibiotics with negative hemolytic property. Strains KCC-25, KCC-26, KCC-27, and KCC-28 showed strong antifungal activity against Aspergillus fumigatus, Penicillium chrysogenum, Penicillium roqueforti, Botrytis elliptica, and Fusarium oxysporum, respectively. Fermentation studies noted that the strains were able to produce significant amount of lactic, acetic, and succinic acids. Further, the production of extracellular proteolytic and glycolytic enzymes, survival under low pH, bile salts, and gastric juice together with positive bile salt hydrolase (Bsh activity, cholesterol lowering, cell surface hydrophobicity, and aggregation properties were the strains advantages. Thus, KCC-25, KCC-26, KCC-27, and KCC-28 could have the survival ability in the harsh condition of the digestive system in the gastrointestinal tract. In conclusion, novel L. plantarum KCC-25, KCC-26, KCC-27, and KCC-28 could be considered as potential antimicrobial probiotic strains.

Determination of Strain Rate Sensitivity of Micro-struts Manufactured Using the Selective Laser Melting Method

Science.gov (United States)

Gümrük, Recep; Mines, R. A. W.; Karadeniz, Sami

2018-03-01

Micro-lattice structures manufactured using the selective laser melting (SLM) process provides the opportunity to realize optimal cellular materials for impact energy absorption. In this paper, strain rate-dependent material properties are measured for stainless steel 316L SLM micro-lattice struts in the strain rate range of 10-3 to 6000 s-1. At high strain rates, a novel version of the split Hopkinson Bar has been developed. Strain rate-dependent materials data have been used in Cowper-Symonds material model, and the scope and limit of this model in the context of SLM struts have been discussed. Strain rate material data and the Cowper-Symonds model have been applied to the finite element analysis of a micro-lattice block subjected to drop weight impact loading. The model output has been compared to experimental results, and it has been shown that the increase in crush stress due to impact loading is mainly the result of strain rate material behavior. Hence, a systematic methodology has been developed to investigate the impact energy absorption of a micro-lattice structure manufactured using additive layer manufacture (SLM). This methodology can be extended to other micro-lattice materials and configurations, and to other impact conditions.

The dtd gene from Bacillus amyloliquefaciens encodes a putative D-tyrosyl-tRNATyr deacylase and is a selectable marker for Bacillus subtilis.

Science.gov (United States)

Geraskina, Natalia V; Butov, Ivan A; Yomantas, Yurgis A V; Stoynova, Nataliya V

2015-02-01

Genetically engineered microbes are of high practical importance due to their cost-effective production of valuable metabolites and enzymes, and the search for new selectable markers for genetic manipulation is of particular interest. Here, we revealed that the soil bacterium Bacillus amyloliquefaciens A50 is tolerant to the non-canonical amino acid D-tyrosine (D-Tyr), in contrast to the closely related Bacillus strain B. subtilis 168, which is a widely used "domesticated" laboratory strain. The gene responsible for resistance to D-Tyr was identified. The resistance was associated with the activity of a potential D-tyrosyl-tRNA(Tyr) deacylase. Orthologs of this enzyme are capable of hydrolyzing the ester bond and recycling misacetylated D-aminoacyl-tRNA molecules into free tRNAs and D-amino acids. This gene, yrvI (dtd), is applicable as a convenient, small selectable marker for non-antibiotic resistance selection in experiments aimed at genome editing of D-Tyr-sensitive microorganisms. Copyright © 2014 Elsevier GmbH. All rights reserved.

21 CFR 184.1250 - Cellulase enzyme preparation derived from Trichoderma longibrachiatum.

Science.gov (United States)

2010-04-01

... Trichoderma longibrachiatum. 184.1250 Section 184.1250 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT....1250 Cellulase enzyme preparation derived from Trichoderma longibrachiatum. (a) Cellulase enzyme preparation is derived from a nonpathogenic, nontoxicogenic strain of Trichoderma longibrachiatum (formerly T...

Erythrocyte phosphofructokinase in rat strains with genetically determined differences in 2,3-diphosphoglycerate levels.

Science.gov (United States)

Noble, N A; Tanaka, K R

1981-02-01

We have studied the erythrocyte enzyme phosphofructokinase (PFK) from two strains of Long-Evans rats with genetically determined differences in erythrocyte 2,3-diphosphoglycerate (DPG) levels. The DPG difference is due to two alleles at one locus. With one probable exception, the genotype at this locus is always associated with the hemoglobin (Hb) electrophoretic phenotype, due to a polymorphism at the III beta-globin locus. The enzyme PFK has been implicated in the DPG difference because glycolytic intermediate levels suggest that this enzyme has a higher in vivo activity in High-DPG strain rats, although the total PFK activity does not differ. We report here that partially purified erythrocyte PFK from Low-DPG strain cells is inhibited significantly more at physiological levels of DPG (P less than 0.01) than PFK from High-DPG strain erythrocytes. Citrate and adenosine triphosphate also inhibit the Low-DPG enzyme more than the High-DPG enzyme. Therefore, a structurally different PFK, with a greater sensitivity to inhibitors, may explain the lower DPG and ATP levels observed in Low-DPG strain animals. These data support a two-locus (Hb and PFK) hypothesis and provide a gene marker to study the underlying genetic and physiologic relationships of these loci.

Improving Aspergillus carbonarius crude enzymes for lignocellulose hydrolysis

DEFF Research Database (Denmark)

Hansen, Gustav Hammerich

and single enzyme supplementation. Fungal strains were screened in order to determine crude enzyme extracts that could be supplemented as boosters of A. carbonarius own crude enzyme extract, when applied in lignocellulose hydrolysis. The fungi originated from different environmental niches, which all had...... for their potential in hydrolysis of wheat straw both by application of monocultures and by supplementing to crude enzymes of A. carbonarius. For the crude enzymes from solid cultivations there were eight isolates that showed synergistic interaction resulting in doubling and tripling of the glucose release in wheat...... straw hydrolysis. A completely different profile of synergy was observed for crude enzymes from liquid cultivations, as there were only three isolates that enhanced glucose release. Only one of these three isolates had shown synergistic effects when cultivated in a solid medium. The screening...

The effects of stabilizing and directional selection on phenotypic and genotypic variation in a population of RNA enzymes.

Science.gov (United States)

Hayden, Eric J; Bratulic, Sinisa; Koenig, Iwo; Ferrada, Evandro; Wagner, Andreas

2014-02-01

The distribution of variation in a quantitative trait and its underlying distribution of genotypic diversity can both be shaped by stabilizing and directional selection. Understanding either distribution is important, because it determines a population's response to natural selection. Unfortunately, existing theory makes conflicting predictions about how selection shapes these distributions, and very little pertinent experimental evidence exists. Here we study a simple genetic system, an evolving RNA enzyme (ribozyme) in which a combination of high throughput genotyping and measurement of a biochemical phenotype allow us to address this question. We show that directional selection, compared to stabilizing selection, increases the genotypic diversity of an evolving ribozyme population. In contrast, it leaves the variance in the phenotypic trait unchanged.

Mimicking heme enzymes in the solid state: metal-organic materials with selectively encapsulated heme.

Science.gov (United States)

Larsen, Randy W; Wojtas, Lukasz; Perman, Jason; Musselman, Ronald L; Zaworotko, Michael J; Vetromile, Carissa M

2011-07-13

To carry out essential life processes, nature has had to evolve heme enzymes capable of synthesizing and manipulating complex molecules. These proteins perform a plethora of chemical reactions utilizing a single iron porphyrin active site embedded within an evolutionarily designed protein pocket. We herein report the first class of metal-organic materials (MOMs) that mimic heme enzymes in terms of both structure and reactivity. The MOMzyme-1 class is based upon a prototypal MOM, HKUST-1, into which catalytically active metalloporphyrins are selectively encapsulated in a "ship-in-a-bottle" fashion within one of the three nanoscale cages that exist in HKUST-1. MOMs offer unparalleled levels of permanent porosity and their modular nature affords enormous diversity of structures and properties. The MOMzyme-1 class could therefore represent a new paradigm for heme biomimetic catalysis since it combines the activity of a homogeneous catalyst with the stability and recyclability of heterogeneous catalytic systems within a single material.

Experimental induction of paromomycin resistance in antimony-resistant strains of L. donovani: outcome dependent on in vitro selection protocol.

Directory of Open Access Journals (Sweden)

Sarah Hendrickx

Full Text Available Paromomycin (PMM has recently been introduced for treatment of visceral leishmaniasis in India. Although no clinical resistance has yet been reported, proactive vigilance should be warranted. The present in vitro study compared the outcome and stability of experimental PMM-resistance induction on promastigotes and intracellular amastigotes. Cloned antimony-resistant L. donovani field isolates from India and Nepal were exposed to stepwise increasing concentrations of PMM (up to 500 µM, either as promastigotes or intracellular amastigotes. One resulting resistant strain was cloned and checked for stability of resistance by drug-free in vitro passage as promastigotes for 20 weeks or a single in vivo passage in the golden hamster. Resistance selection in promastigotes took about 25 weeks to reach the maximal 97 µM inclusion level that did not affect normal growth. Comparison of the IC(50 values between the parent and the selected strains revealed a 9 to 11-fold resistance for the Indian and 3 to 5-fold for the Nepalese strains whereby the resistant phenotype was also maintained at the level of the amastigote. Applying PMM pressure to intracellular amastigotes produced resistance after just two selection cycles (IC(50 = 199 µM compared to the parent strain (IC(50 = 45 µM. In the amastigote-induced strains/clones, lower PMM susceptibilities were seen only in amastigotes and not at all in promastigotes. This resistance phenotype remained stable after serial in vitro passage as promastigote for 20 weeks and after a single in vivo passage in the hamster. This study clearly demonstrates that a different PMM-resistance phenotype is obtained whether drug selection is applied to promastigotes or intracellular amastigotes. These findings may have important relevance to resistance mechanism investigations and the likelihood of resistance development and detection in the field.

Production of an extracellular thermohalophilic lipase from a moderately halophilic bacterium, Salinivibrio sp. strain SA-2.

Science.gov (United States)

Amoozegar, Mohammad Ali; Salehghamari, Ensieh; Khajeh, Khosro; Kabiri, Mahbube; Naddaf, Saied

2008-06-01

Fifty strains of moderately halophilic bacteria were isolated from various salty environments in Iran. A strain designated as SA-2 was shown to be the best producer of extracellular lipase and was selected for further studies. Biochemical and physiological characterization along with 16S rDNA sequence analysis placed SA-2 in the genus Salinivibrio. The optimum salt, pH, temperature and aeration for enzyme production were 0.1 M KCl, pH 8, 35 degrees C and 150 rpm, respectively. The enzyme production was synchronized bacterial growth and reached a maximum level during the early-stationary phase in the basal medium containing 1 M NaCl. Triacylglycerols enhanced lipase production, while carbohydrates had inhibitory effects on it. The maximum lipase activity was obtained at pH 7.5, 50 degrees C and CaCl(2) concentration of 0.01 M. The enzyme was stable at pH range of 7.5-8 and retained 90% of its activity at 80 degrees C for 30 min. Different concentrations of NaNO(3), Na(2)SO(4), KCl and NaCl had no affect on lipase stability for 3 h. These results suggest that the lipase secreted by Salinivibrio sp. strain SA-2 is industrially important from the perspective of its tolerance to a broad temperature range, its moderate thermoactivity and its high tolerance to a wide range of salt concentrations (0-3 M NaCl).

Kinetic characterization of glucose aerodehydrogenase from Aspergillus niger EMS-150-F after optimizing the dose of mutagen for enhanced production of enzyme

Directory of Open Access Journals (Sweden)

Huma Umbreen

2013-12-01

Full Text Available In the present study enhanced production of glucose aerodehydrogenase from Aspergillus niger has been achieved after optimizing the dose of chemical mutagen ethyl methane sulfonate (EMS that has not been reported earlier. Different doses of mutagen were applied and a strain was developed basing upon the best production. The selected strain Aspergillus niger EMS-150-F was optimized for nutrient requirements in order to produce enzyme through fermentation and the results showed the best yield at 2% corn steep liquor (CSL, 36 hours fermentation time, pH 5, 30°C temperature, 0.3% KH2PO4, 0.3% urea and 0.06% CaCO3. The enzyme was then purified and resulted in 57.88 fold purification with 52.12% recovery. On kinetic characterization, the enzyme showed optimum activity at pH 6 and temperature 30°C. The Michaelis-Menton constants (Km, Vmax, Kcat and Kcat/Km were 20 mM, 45.87 U mL-1, 1118.81 s-1 and 55.94 s-1 mM-1, respectively. The enzyme was found to be thermaly stable and the enthalpy and free energy showed an increase with increase in temperature and ΔS* was highly negative proving the enzyme from A. niger EMS-150-F resistant to temperature and showing a very little disorderliness.

The amyR-deletion strain of Aspergillus niger CICC2462 is a suitable host strain to express secreted protein with a low background.

Science.gov (United States)

Zhang, Hui; Wang, Shuang; Zhang, Xiang Xiang; Ji, Wei; Song, Fuping; Zhao, Yue; Li, Jie

2016-04-28

The filamentous fungus Aspergillus niger is widely exploited as an important expression host for industrial production. The glucoamylase high-producing strain A. niger CICC2462 has been used as a host strain for the establishment of a secretion expression system. It expresses recombinant xylanase, mannase and asparaginase at a high level, but some high secretory background proteins in these recombinant strains still remain, such as alpha-amylase and alpha-glucosidase; lead to a low-purity of fermentation products. The aim was to construct an A. niger host strain with a low background of protein secretion. The transcription factor amyR was deleted in A. niger CICC2462, and the results from enzyme activity assays and SDS-PAGE analysis showed that the glucoamylase and amylase activities of the ∆amyR strains were significantly lower than those of the wild-type strain. High-throughput RNA-sequencing and shotgun LC-MS/MS proteomic technology analysis demonstrated that the expression of amylolytic enzymes was decreased at both the transcriptional and translational levels in the ∆amyR strain. Interestingly, the ∆amyR strain growth rate better than the wild-type strain. Our findings clearly indicated that the ∆amyR strain of A. niger CICC2462 can be used as a host strain with a low background of protein secretion.

Studies on Nitrobenzene Metabolism by a Comamonas sp. Strain JS7651

National Research Council Canada - National Science Library

Gibson, David

2000-01-01

.... The nitrobenzene dioxygenase enzyme system shares high amino acid homology with other identified nitroarene dioxygenase enzymes, in particular the 2-nitrotoluene dioxygenase system from Pseudomonas sp. strain JS42...

Relation between motility, accelerated aging and gene expression in selected Drosophila strains under hypergravity conditions

NARCIS (Netherlands)

Serrano, P.; van Loon, J.J.W.A.; Javier Medina, F.; Herranz, R.

2013-01-01

Motility and aging in Drosophila have proven to be highly modified under altered gravity conditions (both in space and ground simulation facilities). In order to find out how closely connected they are, five strains with altered geotactic response or survival rates were selected and exposed to an

Fermentation of Apple Juice with a Selected Yeast Strain Isolated from the Fermented Foods of Himalayan Regions and Its Organoleptic Properties

OpenAIRE

Kanwar, S. S.; Keshani,

2016-01-01

Twenty-three Saccharomyces cerevisiae strains isolated from different fermented foods of Western Himalayas have been studied for strain level and functional diversity in our department. Among these 23 strains, 10 S. cerevisiae strains on the basis of variation in their brewing traits were selected to study their organoleptic effect at gene level by targeting ATF1 gene, which is responsible for ester synthesis during fermentation. Significant variation was observed in ATF1 gene sequences, sugg...

Selected soil enzymes: Examples of their potential roles in the ...

African Journals Online (AJOL)

SERVER

2008-02-05

Feb 5, 2008 ... Soil enzymes regulate ecosystem functioning and in particular play a key role in nutrient cycling. In ... A better understanding of the role of these soil enzyme- es activity ..... measure of any disruption caused by pesticides, trace.

Strain measurement based battery testing

Science.gov (United States)

Xu, Jeff Qiang; Steiber, Joe; Wall, Craig M.; Smith, Robert; Ng, Cheuk

2017-05-23

A method and system for strain-based estimation of the state of health of a battery, from an initial state to an aged state, is provided. A strain gauge is applied to the battery. A first strain measurement is performed on the battery, using the strain gauge, at a selected charge capacity of the battery and at the initial state of the battery. A second strain measurement is performed on the battery, using the strain gauge, at the selected charge capacity of the battery and at the aged state of the battery. The capacity degradation of the battery is estimated as the difference between the first and second strain measurements divided by the first strain measurement.

Optimization of pectinase enzyme production in Aspergillus fumigatus isolated from rotten fruits

Directory of Open Access Journals (Sweden)

2015-12-01

Full Text Available Introduction: Pectinase is one of the most important industrial enzymes which was isolated from a wide variety of microorganisms such as bacteria and filamentous fungi. This enzyme has been usually used in the juice and textile industry. In this study, the isolation and optimization of pectinase-producing fungi on decaying rotten fruits were studied. Materials and methods: Isolation and screening of pectinase producing fungi have been done by plate culture on pectin medium and staining with Lugol's iodine solution. The best strain was identified by method of Pitt and Hocking as Aspergillus fumigates. The enzyme production was optimized by application of the factorial design which involves five factors, each at three levels. Five factors were carbon sources (whey, sugar, stevia and ammonium sulfate, manganese sulfate, temperature, and pH. Pectinase concentration was measured by the Miller method. Results: The results showed that the optimum condition for enzyme production was at 32 °C, PH = 6 , 3g / L manganese sulfate, 2.75g / L of ammonium sulfate, 10g / L of each carbon source (whey, stevia, and glucose. Optimum of enzyme production was observed in the presence of 1.328 mg / ml of glucose. Molecular weight of enzyme was obtained about 40 kDa by SDS-PAGE. Discussion and conclusion: The results demonstrated that this strain could grow in a wide range of carbon sources, PH and temperature. This study indicates that this strain is a good candidate for use in industrial application.

Expression of Heterologous Cellulases in Thermotoga sp. Strain RQ2

Directory of Open Access Journals (Sweden)

Hui Xu

2015-01-01

Full Text Available The ability of Thermotoga spp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA and Csac_1078 (celB from Caldicellulosiruptor saccharolyticus were cloned into T. sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA and TM0070 (xynB, resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried by Thermotoga-E. coli shuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed in E. coli DH5α and T. sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. In E. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas in T. sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost in T. sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study have ever been expressed in Thermotoga, demonstrating the feasibility of using engineered Thermotoga spp. for efficient cellulose utilization.

CONCERNING THE HYGIENIC STUDY OF ENZYME PREPARATIONS PRODUCED BY MICROFUNGI AND THEIR POSSIBLE USE IN THE FOOD INDUSTRY

Science.gov (United States)

Conclusions: (1) Large doses of enzyme preparations of the fungi Trichothecium roseum, Aspergillus oryzae strain No. 476I, and Aspergillus awamori...extensive industrial testing in the brewing industry, as well as enzyme preparations of the fungi Aspergillus oryzae strain No. 476I and Aspergillus

[Biodiversity and enzymes of culturable facultative-alkaliphilic actinobacteria in saline-alkaline soil in Fukang, Xinjiang].

Science.gov (United States)

Zhang, Yongguang; Liu, Qing; Wang, Hongfei; Zhang, Daofeng; Chen, Jiyue; Zhang, Yuanming; Li, Wenjun

2014-02-04

In order to analyze the biodiversity of cultivable facultative-alkaliphilic actinobacteria and the enzymes they produced. Total 10 soil samples were collected from saline-alkaline environments of Fukang, Xinjiang province. Facultative-alkaliphilic actinobacteria strains were isolated and identified by 16S rRNA gene sequence analysis. Enzymes including amylase, proteinase, xylanase, and cellulase were detected. Total 116 facultative-alkaliphilic actinobacterial strains and 4 alkali-tolerant actinobacterial strains were isolated from the samples, and those strains were distributed within 22 genera in 13 families and 8 orders of actinobacteria based on their 16S rRNA gene sequence analysis. The ratio of non-predominant Streptomyces and Nocardiopsis strains were 53.3%. The positive rates of amylase, proteinase, xylanase and cellulase were 35.8, 37.6, 28.3 and 17.5%, respectively. Diverse facultative-alkaliphilic actinobacteria were discovered from saline-alkaline environments of Fukang. Facultative-alkaliphilic actinobacteria are a potential source for enzymes. The study would facilitate the knowledge of the diversity of facultative-alkaliphilic actinobacteria, and provide the technical basis for exploration of facultative-alkaliphilic actinobacteria resources.

Development of a commercial enzymes system for lignocellulosic biomass saccharification

Energy Technology Data Exchange (ETDEWEB)

Kumar, Manoj

2012-12-20

DSM Innovation Inc., in its four year effort was able to evaluate and develop its in-house DSM fungal cellulolytic enzymes system to reach enzyme efficiency mandates set by DoE Biomass program MYPP goals. DSM enzyme cocktail is uniquely active at high temperature and acidic pH, offering many benefits and product differentiation in 2G bioethanol production. Under this project, strain and process development, ratio optimization of enzymes, protein and genetic engineering has led to multitudes of improvement in productivity and efficiency making development of a commercial enzyme system for lignocellulosic biomass saccharification viable. DSM is continuing further improvement by additional biodiversity screening, protein engineering and overexpression of enzymes to continue to further lower the cost of enzymes for saccharification of biomass.

Canthaxanthin production with modified Mucor circinelloides strains.

Science.gov (United States)

Papp, Tamás; Csernetics, Arpád; Nagy, Gábor; Bencsik, Ottó; Iturriaga, Enrique A; Eslava, Arturo P; Vágvölgyi, Csaba

2013-06-01

Canthaxanthin is a natural diketo derivative of β-carotene primarily used by the food and feed industries. Mucor circinelloides is a β-carotene-accumulating zygomycete fungus and one of the model organisms to study the carotenoid biosynthesis in fungi. In this study, the β-carotene ketolase gene (crtW) of the marine bacterium Paracoccus sp. N81106 fused with fungal promoter and terminator regions was integrated into the M. circinelloides genome to construct stable canthaxanthin-producing strains. Different transformation methods including polyethylene glycol-mediated transformation with linear DNA fragments, restriction enzyme-mediated integration and Agrobacterium tumefaciens-mediated transformation were tested to integrate the crtW gene into the Mucor genome. Mitotic stability, site of integration and copy number of the transferred genes were analysed in the transformants, and several stable strains containing the crtW gene in high copy number were isolated. Carotenoid composition of selected transformants and effect of culturing conditions, such as temperature, carbon sources and application of certain additives in the culturing media, on their carotenoid content were analysed. Canthaxanthin-producing transformants were able to survive at higher growth temperature than the untransformed strain, maybe due to the effect of canthaxanthin on the membrane fluidity and integrity. With the application of glucose, trehalose, dihydroxyacetone and L-aspartic acid as sole carbon sources in minimal medium, the crtW-expressing M. circinelloides strain, MS12+pCA8lf/1, produced more than 200 μg/g (dry mass) of canthaxanthin.

An objective approach for Burkholderia pseudomallei strain selection as challenge material for medical countermeasures efficacy testing

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Kristopher E. Van Zandt

2012-09-01

Full Text Available Burkholderia pseudomallei is the causative agent of melioidosis, a rare disease of biodefense concern with high mortality and extreme difficulty in treatment. No human vaccines are available that protect against B. pseudomallei infection, and with the current limitations of antibiotic treatment, the development of new preventative and therapeutic interventions is crucial. Although clinical trials could be used to test the efficacy of new medical countermeasures (MCMs, the high mortality rates associated with melioidosis raises significant ethical issues concerning treating individuals with new compounds with unknown efficacies. The US Food and Drug Administration (FDA has formulated a set of guidelines for the licensure of new MCMs to treat diseases in which it would be unethical to test the efficacy of these drugs in humans. The FDA Animal Rule 21 CFR 314 calls for consistent, well-characterized B. pseudomallei strains to be used as challenge material in animal models. In order to facilitate the efficacy testing of new MCMs for melioidosis using animal models, we intend to develop a well-characterized panel of strains for use. This panel will comprise of strains that were isolated from human cases, have a low passage history, are virulent in animal models, and are well characterized phenotypically and genotypically. We have reviewed published and unpublished data on various B. pseudomallei strains to establish an objective method for selecting the strains to be included in the panel of B. pseudomallei strains with attention to five categories: animal infection models, genetic characterization, clinical and passage history, and availability of the strain to the research community. We identified 109 strains with data in at least one of the five categories, scored each strain based on the gathered data and identified 6 strains as candidate for a B. pseudomallei strain panel.

An objective approach for Burkholderia pseudomallei strain selection as challenge material for medical countermeasures efficacy testing.

Science.gov (United States)

Van Zandt, Kristopher E; Tuanyok, Apichai; Keim, Paul S; Warren, Richard L; Gelhaus, H Carl

2012-01-01

Burkholderia pseudomallei is the causative agent of melioidosis, a rare disease of biodefense concern with high mortality and extreme difficulty in treatment. No human vaccines are available that protect against B. pseudomallei infection, and with the current limitations of antibiotic treatment, the development of new preventative and therapeutic interventions is crucial. Although clinical trials could be used to test the efficacy of new medical countermeasures (MCMs), the high mortality rates associated with melioidosis raises significant ethical issues concerning treating individuals with new compounds with unknown efficacies. The US Food and Drug Administration (FDA) has formulated a set of guidelines for the licensure of new MCMs to treat diseases in which it would be unethical to test the efficacy of these drugs in humans. The FDA "Animal Rule" 21 CFR 314 calls for consistent, well-characterized B. pseudomallei strains to be used as challenge material in animal models. In order to facilitate the efficacy testing of new MCMs for melioidosis using animal models, we intend to develop a well-characterized panel of strains for use. This panel will comprise of strains that were isolated from human cases, have a low passage history, are virulent in animal models, and are well-characterized phenotypically and genotypically. We have reviewed published and unpublished data on various B. pseudomallei strains to establish an objective method for selecting the strains to be included in the panel of B. pseudomallei strains with attention to five categories: animal infection models, genetic characterization, clinical and passage history, and availability of the strain to the research community. We identified 109 strains with data in at least one of the five categories, scored each strain based on the gathered data and identified six strains as candidate for a B. pseudomallei strain panel.

Residual strain in the Nb-H system measured by selected area diffraction (SAD)

International Nuclear Information System (INIS)

Bulhoes, I.A.M.; Akune, K.; Pinatti, Dyonisio G.

1981-07-01

Various specimens of Nb were annealed in vacuum of 10 -3 torr for four hours at 1770 0 K. These speciments were doped with hydrogen up to 1000 ppm by weight and then were analyzed selected area diffraction. The line resolution of the electron channelling pattern was meassured for the specimens with different hydrogen content. These measurements, combined with the measurement of density, permitted one to estimate the residual strain caused by hydrogen. (Author) [pt

The effect of new probiotic strain Lactobacillus plantarum on counts of coliforms, lactobacilli and bacterial enzyme activities in rats exposed to N,N-dimethylhydrazine (chemical carcinogen

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Denisa Čokášová

2012-01-01

Full Text Available The aim of the present study was to evaluate the effect of the new probiotic strain Lactobacillus plantarum on chemically induced carcinogenesis in rats. Sprague dowley rats (n = 33 were divided into control and experimental groups and were fed a conventional laboratory diet. In the experimental group, rats were treated with the probiotic at the dose of 1 × 109 CFU (colony-forming units/ml. Two weeks after the beginning of the trial, N,N-dimethylhydrazine (chemical carcinogen injections were applied s.c. at the dose of 21 mg/kg b.w., 5 × weekly. At the end of the 8-month experimental period, faeces samples were taken from the rats and used for laboratory analysis. The counts of lactobacilli and coliforms and bacterial enzyme activity were determined. The probiotic strain L. plantarum as single species or in combination with oil (Lini oleum virginale decreased the count of total coliforms and increased lactobacilli in faeces of rats. Application of probiotic microorganisms significantly (P < 0.05 decreased the activities of bacterial enzymes (β-galactosidase and β-glucuronidase compared to the control group rats. The results of this study indicate that probiotic microorganisms could exert a preventive effect on colon carcinogenesis induced by N,N-dimethylhydrazine.

Evaluation of the organophosphorus hydrolase enzyme activity in creams and investigation of its stability

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Mariye Rajaie

2016-06-01

Full Text Available The main purpose of this project is investigation of the organophosphorus hydrolase (OPH enzyme activity in water in oil (w/o and oil in water (o/w creams and investigation of the OPH enzyme stability in formulated creams. OPH enzyme was extracted and purified from strain flavobacterium. The w/o and o/w creams were prepared using different formulations. In order to achieve an emulsion with maximum stability, appropriate percentage of the cream components was selected by studying different formulations and the physical and chemical stability of the produced cream were considered. 5Uenzyme/90gcream enzyme was used for each formulation. To measure the enzyme activity in creams, extraction method was used and enzyme activity was determined based on parathion hydrolysis. The thermal stability of OPH in both types of w/o and o/w creams was studied at 4 and 30  °C for various time periods. The average enzyme activity was about 0.0065 U/gcream and 0.018 U/gcream for w/o and o/w creams respectivly. According to the results, the relative activity at 4 °C was reduced to 50% after 26 and 45 days in w/o and o/w creams, respectivly. The results showed that the OPH enzyme activity in o/w cream was 2.6 times more than that of w/o cream, because of the higher hydrophobicity of o/w cream compared to w/o. The OPH enzyme stability in o/w cream was greater in comparison to w/o cream. The OPH enzyme was active for nearly 2 months on o/w creams at 4 °C .

Hydrocarbon degradation and plant colonization of selected bacterial strains isolated from the rhizsophere and plant interior of Italian ryegrass and Birdsfoot trefoil

Science.gov (United States)

Sohail, Y.; Andria, V.; Reichenauer, T. G.; Sessitsch, A.

2009-04-01

Hydrocarbon-degrading strains were isolated from the rhizosphere, root and shoot interior of Italian ryegrass (Lolium multiflorum var. Taurus), Birdsfoot trefoil (Lotus corniculatus var. Leo) grown in a soil contaminated with petroleum oil. Strains were tested regarding their phylogeny and their degradation efficiency. The most efficient strains were tested regarding their suitability to be applied for phytoremediation of diesel oils. Sterilized and non-sterilized agricultural soil, with and with out compost, were spiked with diesel and used for planting Italian ryegrass and birdsfoot trefoil. Four selected strains with high degradation activities, derived from the rhizosphere and plant interior, were selected for individual inoculation. Plants were harvested at flowering stage and plant biomass and hydrocarbon degradation was determined. Furthermore, it was investigated to which extent the inoculant strains were able to survive and colonize plants. Microbial community structures were analysed by 16S rRNA and alkB gene analysis. Results showed efficient colonization by the inoculant strains and improved degradation by the application of compost combined with inoculation as well as on microbial community structures will be presented.

Product-selective blot: a technique for measuring enzyme activities in large numbers of samples and in native electrophoresis gels

International Nuclear Information System (INIS)

Thompson, G.A.; Davies, H.M.; McDonald, N.

1985-01-01

A method termed product-selective blotting has been developed for screening large numbers of samples for enzyme activity. The technique is particularly well suited to detection of enzymes in native electrophoresis gels. The principle of the method was demonstrated by blotting samples from glutaminase or glutamate synthase reactions into an agarose gel embedded with ion-exchange resin under conditions favoring binding of product (glutamate) over substrates and other substances in the reaction mixture. After washes to remove these unbound substances, the product was measured using either fluorometric staining or radiometric techniques. Glutaminase activity in native electrophoresis gels was visualized by a related procedure in which substrates and products from reactions run in the electrophoresis gel were blotted directly into a resin-containing image gel. Considering the selective-binding materials available for use in the image gel, along with the possible detection systems, this method has potentially broad application

Screening and characterization of thermo-active enzymes of biotechnological interest produced by thermophilic Bacillus isolated from hot springs in Tunisia.

Science.gov (United States)

Thebti, Wajdi; Riahi, Yosra; Gharsalli, Rawand; Belhadj, Omrane

2016-01-01

As part of the contribution to the global efforts in research of thermostable enzymes being of industrial interest, we focus on the isolation of thermophilic bacteria from Tunisian hot springs. Among the collection of 161 strains of thermophilic Bacillus isolated from different samples of thermal water in Tunisia, 20% are capable of growing at 100°C and the rest grow at 70°C or above. Preliminary activity tests on media supplemented with enzyme-substrates confirmed that 35 strains produced amylases, 37 - proteases, 43 - cellulases, 31 - xylanases and 37 - mannanases. The study of the effect of temperature on enzyme activity led to determination of the optimal temperatures of activities that vary between 60 and 100°C. Several enzymes were active at high temperatures (80, 90 and 100°C) and kept their activity even at 110°C. Several isolated strains producing enzymes with high optimal temperatures of activity were described for the first time in this study. Both strains B62 and B120 are producers of amylase, protease, cellulase, xylanase, and mannanase. The sequencing of 16S DNA identified isolated strains as Geobacillus kaustophillus, Aeribacillus pallidus, Geobacillus galactosidasus and Geobacillus toebii.

Selected Lactobacillus strains isolated from sugary and milk kefir reduce Salmonella infection of epithelial cells in vitro.

Science.gov (United States)

Zavala, L; Golowczyc, M A; van Hoorde, K; Medrano, M; Huys, G; Vandamme, P; Abraham, A G

2016-09-01

The isolation of potentially probiotic strains and the subsequent study of their properties are very important steps to gain insight in the health benefits ascribed to sugary and milk kefir. The aim of the present study was to characterise fifteen Lactobacillus strains isolated from these beverages by determining some surface properties and their ability to antagonise enterocyte cell damage after Salmonella infection in vitro. Lactobacillus surface properties were determined by hydrophobicity, autoaggregation, and coaggregation assays with Salmonella. In addition, lactobacilli adhesion to Caco-2/TC-7 cells and the effect on Salmonella invasion were evaluated. Finally, the disassembly of F-actin cytoskeleton on intestinal epithelial cells was assayed in vitro when Salmonella infection was performed in the presence of selected Lactobacillus strains. Ten out of the 15 strains showed a high adhesion capacity to Caco-2/TC-7 cells. Most of the strains were hydrophilic and non-autoaggregating. Strains isolated from sugary kefir were non-coaggregating with Salmonella, while strains Lactobacillus paracasei CIDCA 83120, 83121, 83123, 83124, 8339, 83102 isolated from milk kefir were able to coaggregate after 1 h. L. paracasei CIDCA 8339 and Lactobacillus kefiri CIDCA 83102 were able to diminish Salmonella invasion to the enterocytes. An antagonistic effect on cytoskeleton disruption elicited by the pathogen was also demonstrated. Our results suggest that both strains isolated from milk kefir could be considered as appropriate probiotic candidates.

Impact of interplay between magnetic field, transformation strain, and coarsening on variant selection in L10-type FePd

International Nuclear Information System (INIS)

Ueshima, N.; Yasuda, H.; Yoshiya, M.; Fukuda, T.; Kakeshita, T.

2014-01-01

Variant selection of L1 0 -type ferromagnetic alloys has been numerically investigated using the phase-field modeling, to clarify the phenomena at greater temporal and spatial resolution and to reveal the underlying mechanism. The duration for which the external magnetic field is effective is found to be very short, and variant selection is significantly affected by not only direct response to the external magnetic field but also their interplay between the field, intrinsic transformation strain, and various thermodynamic energy components involved in the course of microstructure evolution. The detailed mechanism of the interplay was quantitatively analyzed in terms of the driving force for the variant selection, by partitioning it into the various energy components. Careful examination of the variant selection at the very early stage revealed that the slight difference in size and configuration of variants during disorder-to-order transition realized by the interplay between transformation strain and external field is essentially needed before proceeding to the latter stage of the variant selection driven by interface energy

Selective amyloid β oligomer assay based on abasic site-containing molecular beacon and enzyme-free amplification.

Science.gov (United States)

Zhu, Linling; Zhang, Junying; Wang, Fengyang; Wang, Ya; Lu, Linlin; Feng, Chongchong; Xu, Zhiai; Zhang, Wen

2016-04-15

Amyloid-beta (Aβ) oligomers are highly toxic species in the process of Aβ aggregation and are regarded as potent therapeutic targets and diagnostic markers for Alzheimer's disease (AD). Herein, a label-free molecular beacon (MB) system integrated with enzyme-free amplification strategy was developed for simple and highly selective assay of Aβ oligomers. The MB system was constructed with abasic site (AP site)-containing stem-loop DNA and a fluorescent ligand 2-amino-5,6,7-trimethyl-1,8-naphyridine (ATMND), of which the fluorescence was quenched upon binding to the AP site in DNA stem. Enzyme-free amplification was realized by target-triggered continuous opening of two delicately designed MBs (MB1 and MB2). Target DNA hybridization with MB1 and then MB2 resulted in the release of two ATMND molecules in one binding event. Subsequent target recycling could greatly amplify the detection sensitivity due to the greatly enhanced turn-on emission of ATMND fluorescence. Combining with Aβ oligomers aptamers, the strategy was applied to analyze Aβ oligomers and the results showed that it could quantify Aβ oligomers with high selectivity and monitor the Aβ aggregation process. This novel method may be conducive to improve the diagnosis and pathogenic study of Alzheimer's disease. Copyright © 2015 Elsevier B.V. All rights reserved.

Algal bioremediation of waste waters from land-based aquaculture using ulva: selecting target species and strains.

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Rebecca J Lawton

Full Text Available The optimised reduction of dissolved nutrient loads in aquaculture effluents through bioremediation requires selection of appropriate algal species and strains. The objective of the current study was to identify target species and strains from the macroalgal genus Ulva for bioremediation of land-based aquaculture facilities in Eastern Australia. We surveyed land-based aquaculture facilities and natural coastal environments across three geographic locations in Eastern Australia to determine which species of Ulva occur naturally in this region and conducted growth trials at three temperature treatments on a subset of samples from each location to determine whether local strains had superior performance under local environmental conditions. DNA barcoding using the markers ITS and tufA identified six species of Ulva, with U. ohnoi being the most common blade species and U. sp. 3 the most common filamentous species. Both species occurred at multiple land-based aquaculture facilities in Townsville and Brisbane and multiple strains of each species grew well in culture. Specific growth rates of U. ohnoi and U. sp. 3 were high (over 9% and 15% day(-1 respectively across temperature treatments. Within species, strains of U. ohnoi had higher growth in temperatures corresponding to local conditions, suggesting that strains may be locally adapted. However, across all temperature treatments Townsville strains had the highest growth rates (11.2-20.4% day(-1 and Sydney strains had the lowest growth rates (2.5-8.3% day(-1. We also found significant differences in growth between strains of U. ohnoi collected from the same geographic location, highlighting the potential to isolate and cultivate fast growing strains. In contrast, there was no clearly identifiable competitive strain of filamentous Ulva, with multiple species and strains having variable performance. The fast growth rates and broad geographical distribution of U. ohnoi make this an ideal species to

Selective Activation of N,N'-Diacyl Rhodamine Pro-fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE).

Science.gov (United States)

Abney, Kristopher K; Ramos-Hunter, Susan J; Romaine, Ian M; Godwin, J Shawn; Sulikowski, Gary A; Weaver, Charles David

2018-04-21

This study reports the synthesis and testing of a family of rhodamine pro-fluorophores and an enzyme capable of converting pro-fluorophores to Rhodamine 110. We prepared a library of simple N,N'-diacyl rhodamines and investigated Porcine Liver Esterase (PLE) as an enzyme to activate rhodamine-based pro-fluorophores. A PLE-expressing cell line generated an increase in fluorescence rapidly upon pro-fluorophore addition demonstrating the rhodamine pro-fluorophores are readily taken up and fluorescent upon PLE-mediated release. Rhodamine pro-fluorophore amides trifluoroacetamide (TFAm) and proponamide (PAm) appeared to be the best substrates using a cell-based assay using PLE expressing HEK293. Our pro-fluorophore series showed diffusion into live cells and resisted endogenous hydrolysis. The use of our engineered cell line containing the exogenous enzyme PLE demonstrated the rigorousness of amide masking when compared to cells not containing PLE. This simple and selective pro-fluorophore rhodamine pair with PLE offers the potential to be used in vitro and in vivo fluorescence based assays. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genomic comparison of invasive and rare non-invasive strains reveals Porphyromonas gingivalis genetic polymorphisms

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Svetlana Dolgilevich

2011-03-01

Full Text Available Porphyromonas gingivalis strains are shown to invade human cells in vitro with different invasion efficiencies, varying by up to three orders of magnitude.We tested the hypothesis that invasion-associated interstrain genomic polymorphisms are present in P. gingivalis and that putative invasion-associated genes can contribute to P. gingivalis invasion.Using an invasive (W83 and the only available non-invasive P. gingivalis strain (AJW4 and whole genome microarrays followed by two separate software tools, we carried out comparative genomic hybridization (CGH analysis.We identified 68 annotated and 51 hypothetical open reading frames (ORFs that are polymorphic between these strains. Among these are surface proteins, lipoproteins, capsular polysaccharide biosynthesis enzymes, regulatory and immunoreactive proteins, integrases, and transposases often with abnormal GC content and clustered on the chromosome. Amplification of selected ORFs was used to validate the approach and the selection. Eleven clinical strains were investigated for the presence of selected ORFs. The putative invasion-associated ORFs were present in 10 of the isolates. The invasion ability of three isogenic mutants, carrying deletions in PG0185, PG0186, and PG0982 was tested. The PG0185 (ragA and PG0186 (ragB mutants had 5.1×103-fold and 3.6×103-fold decreased in vitro invasion ability, respectively.The annotation of divergent ORFs suggests deficiency in multiple genes as a basis for P. gingivalis non-invasive phenotype. Access the supplementary material to this article: Supplement, table (see Supplementary files under Reading Tools online.

Enzyme-lipid complex. Koso-shishitsu fukugotai

Energy Technology Data Exchange (ETDEWEB)

Okahata, Y; Ijiro, K [Tokyo Inst. of Technology., Tokyo (Japan)

1990-08-01

Enzyme, as unstable against organic solvent, being to be designed not to be quenched, organic solvent was tried to be made soluble by making enzyme-lipid complex. By mixing aqueous solution of enzyme with aqueous dispersion liquid of lipid, white powder was obtaind. Enzyme has monomolecular film through which reaction substance passes. Lipase-lipid complex, of which monomolecular film is qualified by hydrogen and other soft linkages, homogeneously dissolves in organic solvent and has a high activity, not given by the conventional qualification method. That activity being applied, asymmetrical esterificating reaction of alcohol could be done in organic solvent, containing high concentration reactive substance. While substance selectivity, not known in water, was obtained. Through reaction of amine with amino acid dielectrics in isooctane solvent by {alpha}-chymotrypsin-lipid complex, was indicated an exact substance selectivity. Enzyme-lipid complex dissolving in organic solvent, monomolecular film can be formed without being quenched on aqueous surface, which film can be utilized as sensor film. 10 refs., 5 figs. 1 tab.

Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88

DEFF Research Database (Denmark)

Andersen, Mikael Rørdam; Salazar, Margarita Pena; Schaap, Peter J.

2011-01-01

The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme......-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compel additional exploration. We therefore undertook whole-genome sequencing of the acidogenic A. niger wild-type strain (ATCC 1015) and produced a genome sequence of very high quality. Only 15...

Genotyping of Yersinia enterocolitica biotype 1A strains from clinical and nonclinical origins by pulsed-field gel electrophoresis.

Science.gov (United States)

Campioni, Fábio; Falcão, Juliana P

2014-06-01

Yersinia enterocolitica biotype 1A (B1A) strains are considered mainly nonpathogenic. However, some studies considered strains of this biotype to be the causal agents of infections in humans and animals. In South America, there are no studies that have compared clinical and nonclinical strains of B1A typed by pulsed-field gel electrophoresis (PFGE) and none that have compared the capability of different enzymes on typing these strains. This study typed 51 Y. enterocolitica B1A strains isolated in Brazil and Chile by PFGE, testing the enzymes XbaI, NotI, and XhoI. The resulting dendrograms discriminated the strains in 47, 40, and 49 pulsotypes generated by the cleavage with the enzymes XbaI, NotI, and XhoI, respectively. The majority of the strains were grouped independently of their clinical or nonclinical origins. The high discriminatory power of PFGE confirmed the heterogeneity of B1A strains but could not divide the strains studied into clusters that differed in the frequency of some virulence genes as observed in studies using other methodologies.

Effect of temperature and mixing speed on immobilization of crude enzyme from Aspergillus niger on chitosan for hydrolyzing cellulose

Science.gov (United States)

Hamzah, Afan; Gek Ela Kumala, P.; Ramadhani, Dwi; Maziyah, Nurul; Rahmah, Laila Nur; Soeprijanto, Widjaja, Arief

2017-05-01

Conversion of cellulose into reducing sugar through enzymatic hydrolysis has advantageous because it produces greater product yield, higher selectivity, require less energy, more moderate operating conditions and environment friendly. However, the nature of the enzyme that is difficult to separate and its expensive price become an obstacle. These obstacles can be overcome by immobilizing the enzyme on chitosan material so that the enzyme can be reused. Chitosan is chosen because it is cheap, inert, hydrophilic, and biocompatible. In this research, we use covalent attachment and combination between covalent attachment and cross-linking method for immobilizing crude enzyme. This research was focusing in study of Effect of temperature and mixing speed on Immobilization Enzyme From Aspergillus Niger on Chitosan For Hydrolyzing both soluble (Carboxymethylcellulose) and insoluble Cellulose (coconut husk). This Research was carried out by three main step. First, coconut husk was pre-treated mechanically and chemically, Second, Crude enzyme from Aspergillus niger strain was immobilized on chitosan in various immobilization condition. At last, the pre-treated coconut husk and Carboxymetylcellulose (CMC) were hydrolyzed by immobilized cellulose on chitosan for reducing sugar production. The result revealed that the most reducing sugar produced by immobilized enzyme on chitosan+GDA with immobilization condition at 30 °C and 125 rpm. Enzyme immobilized on chitosan cross-linked with GDA produced more reducing sugar from preteated coconut husk than enzyme immobilized on chitosan.

Factors influencing production of lipase under metal supplementation by bacterial strain, Bacillus subtilis BDG-8.

Science.gov (United States)

Dhevahi, B; Gurusamy, R

2014-11-01

Lipases are biocatalyst having wide applications in industries due to their versatile properties. In the present study, a lipolytic bacterial strain, Bacillus subtilis BDG-8 was isolated from an oil based industrial soil. The effect of selenium and nickel as a media supplement on enhancement of lipase production, was studied individually with the isolated strain by varying the concentration of selected metal. 60 μg l(-1) selenium enhanced lipase production to an enzyme activity measuring 7.8 U ml(-1) while 40 μgI(-1) nickel gave the maximum enzyme activity equivalent to 7.5 U ml(-1). However, nickel and selenium together at a range of concentration with an equal w/v ratio, at 60 μg l(-1) each, showed the maximum lipase activity of 8.5 U ml(-1). The effect of pH and temperature on lipase production showed maximum enzyme activity in the presence of each of the metals at pH 7 and 35°C among the other tested ranges. After optimisation of the parameters such as metal concentration, pH and temperature lipase production by Bacillus subtilis BDG-8 had increased several folds. This preliminary investigation may consequently lead as to various industrial applications such as treatment of wastewater contaminated with metal or oil with simultaneous lipase production.

Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost

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Amore Antonella

2012-12-01

Full Text Available Abstract Background The use of lignocellulosic materials for second generation ethanol production would give several advantages such as minimizing the conflict between land use for food and fuel production, providing less expensive raw materials than conventional agricultural feedstock, allowing lower greenhouse gas emissions than those of first generation ethanol. However, cellulosic biofuels are not produced at a competitive level yet, mainly because of the high production costs of the cellulolytic enzymes. Therefore, this study was aimed at discovering new cellulolytic microorganisms and enzymes. Results Different bacteria isolated from raw composting materials obtained from vegetable processing industry wastes were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. Four strains belonging to the actinomycetes group were selected on the basis of their phenotypic traits and cellulolytic activity on solid medium containing carboxymethylcellulose. The strain showing the highest cellulolytic activity was identified by 16S rRNA sequencing as belonging to Streptomyces genus and it was designated as Streptomyces sp. strain G12. Investigating the enzymes responsible for cellulase activity produced by Streptomyces G12 by proteomic analyses, two endoglucanases were identified. Gene coding for one of these enzymes, named CelStrep, was cloned and sequenced. Molecular analysis showed that the celstrep gene has an open reading frame encoding a protein of 379 amino acid residues, including a signal peptide of 37 amino acid residues. Comparison of deduced aminoacidic sequence to the other cellulases indicated that the enzyme CelStrep can be classified as a family 12 glycoside hydrolase. Heterologous recombinant expression of CelStrep was carried out in Escherichia coli, and the active recombinant enzyme was purified from culture supernatant and characterized. It catalyzes the hydrolysis of carboxymethylcellulose

Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost

Science.gov (United States)

2012-01-01

Background The use of lignocellulosic materials for second generation ethanol production would give several advantages such as minimizing the conflict between land use for food and fuel production, providing less expensive raw materials than conventional agricultural feedstock, allowing lower greenhouse gas emissions than those of first generation ethanol. However, cellulosic biofuels are not produced at a competitive level yet, mainly because of the high production costs of the cellulolytic enzymes. Therefore, this study was aimed at discovering new cellulolytic microorganisms and enzymes. Results Different bacteria isolated from raw composting materials obtained from vegetable processing industry wastes were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. Four strains belonging to the actinomycetes group were selected on the basis of their phenotypic traits and cellulolytic activity on solid medium containing carboxymethylcellulose. The strain showing the highest cellulolytic activity was identified by 16S rRNA sequencing as belonging to Streptomyces genus and it was designated as Streptomyces sp. strain G12. Investigating the enzymes responsible for cellulase activity produced by Streptomyces G12 by proteomic analyses, two endoglucanases were identified. Gene coding for one of these enzymes, named CelStrep, was cloned and sequenced. Molecular analysis showed that the celstrep gene has an open reading frame encoding a protein of 379 amino acid residues, including a signal peptide of 37 amino acid residues. Comparison of deduced aminoacidic sequence to the other cellulases indicated that the enzyme CelStrep can be classified as a family 12 glycoside hydrolase. Heterologous recombinant expression of CelStrep was carried out in Escherichia coli, and the active recombinant enzyme was purified from culture supernatant and characterized. It catalyzes the hydrolysis of carboxymethylcellulose following a Michaelis�

Global Phenotypic Characterization of Effects of Fluoroquinolone Resistance Selection on the Metabolic Activities and Drug Susceptibilities of Clostridium perfringens Strains

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Miseon Park

2014-01-01

Full Text Available Fluoroquinolone resistance affects toxin production of Clostridium perfringens strains differently. To investigate the effect of fluoroquinolone resistance selection on global changes in metabolic activities and drug susceptibilities, four C. perfringens strains and their norfloxacin-, ciprofloxacin-, and gatifloxacin-resistant mutants were compared in nearly 2000 assays, using phenotype microarray plates. Variations among mutant strains resulting from resistance selection were observed in all aspects of metabolism. Carbon utilization, pH range, osmotic tolerance, and chemical sensitivity of resistant strains were affected differently in the resistant mutants depending on both the bacterial genotype and the fluoroquinolone to which the bacterium was resistant. The susceptibilities to gentamicin and erythromycin of all resistant mutants except one increased, but some resistant strains were less susceptible to amoxicillin, cefoxitin, ceftriaxone, chloramphenicol, and metronidazole than their wild types. Sensitivity to ethidium bromide decreased in some resistant mutants and increased in others. Microarray analysis of two gatifloxacin-resistant mutants showed changes in metabolic activities that were correlated with altered expression of various genes. Both the chemical structures of fluoroquinolones and the genomic makeup of the wild types influenced the changes found in resistant mutants, which may explain some inconsistent reports of the effects of therapeutic use of fluoroquinolones on clinical isolates of bacteria.

Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions

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McClendon Shara D

2012-07-01

Full Text Available Abstract Background Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents. Results Thermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of ionic liquid pretreated switchgrass (Panicum virgatum revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails. Conclusions T. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for

Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions.

Science.gov (United States)

McClendon, Shara D; Batth, Tanveer; Petzold, Christopher J; Adams, Paul D; Simmons, Blake A; Singer, Steven W

2012-07-28

Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents. Thermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of ionic liquid pretreated switchgrass (Panicum virgatum) revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails. T. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for biomass deconstruction, without strain development or

Antimalarial activity of potential inhibitors of Plasmodium falciparum lactate dehydrogenase enzyme selected by docking studies.

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Julia Penna-Coutinho

Full Text Available The Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH has been considered as a potential molecular target for antimalarials due to this parasite's dependence on glycolysis for energy production. Because the LDH enzymes found in P. vivax, P. malariae and P. ovale (pLDH all exhibit ∼90% identity to PfLDH, it would be desirable to have new anti-pLDH drugs, particularly ones that are effective against P. falciparum, the most virulent species of human malaria. Our present work used docking studies to select potential inhibitors of pLDH, which were then tested for antimalarial activity against P. falciparum in vitro and P. berghei malaria in mice. A virtual screening in DrugBank for analogs of NADH (an essential cofactor to pLDH and computational studies were undertaken, and the potential binding of the selected compounds to the PfLDH active site was analyzed using Molegro Virtual Docker software. Fifty compounds were selected based on their similarity to NADH. The compounds with the best binding energies (itraconazole, atorvastatin and posaconazole were tested against P. falciparum chloroquine-resistant blood parasites. All three compounds proved to be active in two immunoenzymatic assays performed in parallel using monoclonals specific to PfLDH or a histidine rich protein (HRP2. The IC(50 values for each drug in both tests were similar, were lowest for posaconazole (<5 µM and were 40- and 100-fold less active than chloroquine. The compounds reduced P. berghei parasitemia in treated mice, in comparison to untreated controls; itraconazole was the least active compound. The results of these activity trials confirmed that molecular docking studies are an important strategy for discovering new antimalarial drugs. This approach is more practical and less expensive than discovering novel compounds that require studies on human toxicology, since these compounds are already commercially available and thus approved for human use.

Increased saccharification of kallar grass using ultrafiltrated enzyme from sporrotrichum thermophile

International Nuclear Information System (INIS)

Latif, F.; Rajoka, M.I.; Malik, K.A.

1991-01-01

The local wild type strain of sporotrichum thermophile when grown on untreated lingo cellulose was found to produce a greater level of B-glucosidase component along with other cellulase/xylanase components than most of the reported wild type potent strains. Culture filtrate obtained, when grown on 4% leptochloa fusca (kallar grass) was used as such and after concentration by ultrafiltration technique for saccharification purpose. Concentrated enzymes titre was increased to 1.2 and 4.0 U/ml for Fp-ase and B-glucosidase, respectively. There were losses in the enzyme titre obtained through ultrafiltration possibly due to adsorption on to the ultrafiltration membrane. Enzyme preparations used, saccharifide 5% kallar grass to 70, 55, 75 and 60% (theoretical basis) from cellulases of S. thermophile concentrate, dilute, T. reesei alone and in supplementation with B-glucosidase from A. niger, respectively. Analysis by HPLC revealed slightly higher glucose yield from S. thermophile enzyme preparations, whereas higher level of xylose was attained from T. reesei preparations. Rest of the sugars pooled as Oligo-sugars were found in almost similar concentrations. (author)

Enzymes from Higher Eukaryotes for Industrial Biocatalysis

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Zhibin Liu

2004-01-01

Full Text Available The industrial production of fine chemicals, feed and food ingredients, pharmaceuticals, agrochemicals and their respective intermediates relies on an increasing application of biocatalysis, i.e. on enzyme or whole-cell catalyzed conversions of molecules. Simple procedures for discovery, cloning and over-expression as well as fast growth favour fungi, yeasts and especially bacteria as sources of biocatalysts. Higher eukaryotes also harbour an almost unlimited number of potential biocatalysts, although to date the limited supply of enzymes, the high heterogeneity of enzyme preparations and the hazard of infectious contaminants keep some interesting candidates out of reach for industrial bioprocesses. In the past only a few animal and plant enzymes from agricultural waste materials were employed in food processing. The use of bacterial expression strains or non-conventional yeasts for the heterologous production of efficient eukaryotic enzymes can overcome the bottleneck in enzyme supply and provide sufficient amounts of homogenous enzyme preparations for reliable and economically feasible applications at large scale. Ideal enzymatic processes represent an environmentally friendly, »near-to-completion« conversion of (mostly non-natural substrates to pure products. Recent developments demonstrate the commercial feasibility of large-scale biocatalytic processes employing enzymes from higher eukaryotes (e.g. plants, animals and also their usefulness in some small-scale industrial applications.

Reconstructed Ancestral Enzymes Impose a Fitness Cost upon Modern Bacteria Despite Exhibiting Favourable Biochemical Properties.

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Hobbs, Joanne K; Prentice, Erica J; Groussin, Mathieu; Arcus, Vickery L

2015-10-01

Ancestral sequence reconstruction has been widely used to study historical enzyme evolution, both from biochemical and cellular perspectives. Two properties of reconstructed ancestral proteins/enzymes are commonly reported--high thermostability and high catalytic activity--compared with their contemporaries. Increased protein stability is associated with lower aggregation rates, higher soluble protein abundance and a greater capacity to evolve, and therefore, these proteins could be considered "superior" to their contemporary counterparts. In this study, we investigate the relationship between the favourable in vitro biochemical properties of reconstructed ancestral enzymes and the organismal fitness they confer in vivo. We have previously reconstructed several ancestors of the enzyme LeuB, which is essential for leucine biosynthesis. Our initial fitness experiments revealed that overexpression of ANC4, a reconstructed LeuB that exhibits high stability and activity, was only able to partially rescue the growth of a ΔleuB strain, and that a strain complemented with this enzyme was outcompeted by strains carrying one of its descendants. When we expanded our study to include five reconstructed LeuBs and one contemporary, we found that neither in vitro protein stability nor the catalytic rate was correlated with fitness. Instead, fitness showed a strong, negative correlation with estimated evolutionary age (based on phylogenetic relationships). Our findings suggest that, for reconstructed ancestral enzymes, superior in vitro properties do not translate into organismal fitness in vivo. The molecular basis of the relationship between fitness and the inferred age of ancestral LeuB enzymes is unknown, but may be related to the reconstruction process. We also hypothesise that the ancestral enzymes may be incompatible with the other, contemporary enzymes of the metabolic network.

Toxigenic genes, spoilage potential, and antimicrobial resistance of Bacillus cereus group strains from ice cream.

Science.gov (United States)

Arslan, Seza; Eyi, Ayla; Küçüksarı, Rümeysa

2014-02-01

Bacillus spp. can be recovered from almost every environment. It is also found readily in foods, where it may cause food spoilage and/or food poisoning due to its toxigenic and pathogenic nature, and extracellular enzymes. In this study, 29 Bacillus cereus group strains from ice cream were examined for the presence of following virulence genes hblC, nheA, cytK and ces genes, and tested for a range of the extracellular enzymes, and antimicrobial susceptibility. The strains were found to produce extracellular enzymes: proteolytic and lipolytic activity, gelatin hydrolysis and lecithinase production (100%), DNase production (93.1%) and amylase activity (93.1%). Of 29 strains examined, 24 (82.8%) showed hemolytic activity on blood agar. Beta-lactamase enzyme was only produced by 20.7% of B. cereus group. Among 29 B. cereus group from ice cream, nheA was the most common virulence gene detected in 44.8% of the strains, followed by hblC gene with 17.2%. Four (13.8%) of the 29 strains were positive for both hblC gene and nheA gene. Contrarily, cytK and ces genes were not detected in any of the strains. Antimicrobial susceptibility of ice cream isolates was tested to 14 different antimicrobial agents using the disc diffusion method. We detected resistance to penicillin and ampicillin with the same rate of 89.7%. Thirty-one percent of the strains were multiresistant to three or more antibiotics. This study emphasizes that the presence of natural isolates of Bacillus spp. harboring one or more enterotoxin genes, producing extracellular enzymes which may cause spoilage and acquiring antibiotic resistance might hold crucial importance in the food safety and quality. Copyright © 2013 Elsevier Ltd. All rights reserved.

Efficient Production of Enantiopure d-Lysine from l-Lysine by a Two-Enzyme Cascade System

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Xin Wang

2016-10-01

Full Text Available The microbial production of d-lysine has been of great interest as a medicinal raw material. Here, a two-step process for d-lysine production from l-lysine by the successive microbial racemization and asymmetric degradation with lysine racemase and decarboxylase was developed. The whole-cell activities of engineered Escherichia coli expressing racemases from the strains Proteus mirabilis (LYR and Lactobacillus paracasei (AAR were first investigated comparatively. When the strain BL21-LYR with higher racemization activity was employed, l-lysine was rapidly racemized to give dl-lysine, and the d-lysine yield was approximately 48% after 0.5 h. Next, l-lysine was selectively catabolized to generate cadaverine by lysine decarboxylase. The comparative analysis of the decarboxylation activities of resting whole cells, permeabilized cells, and crude enzyme revealed that the crude enzyme was the best biocatalyst for enantiopure d-lysine production. The reaction temperature, pH, metal ion additive, and pyridoxal 5′-phosphate content of this two-step production process were subsequently optimized. Under optimal conditions, 750.7 mmol/L d-lysine was finally obtained from 1710 mmol/L l-lysine after 1 h of racemization reaction and 0.5 h of decarboxylation reaction. d-lysine yield could reach 48.8% with enantiomeric excess (ee ≥ 99%.

Mead production: selection and characterization assays of Saccharomyces cerevisiae strains.

Science.gov (United States)

Pereira, Ana Paula; Dias, Teresa; Andrade, João; Ramalhosa, Elsa; Estevinho, Letícia M

2009-08-01

Mead is a traditional drink, which results from the alcoholic fermentation of diluted honey carried out by yeasts. However, when it is produced in a homemade way, mead producers find several problems, namely, the lack of uniformity in the final product, delayed and arrested fermentations, and the production of "off-flavours" by the yeasts. These problems are usually associated with the inability of yeast strains to respond and adapt to unfavourable and stressful growth conditions. The main objectives of this work were to evaluate the capacity of Saccharomyces cerevisiae strains, isolated from honey of the Trás-os-Montes (Northeast Portugal), to produce mead. Five strains from honey, as well as one laboratory strain and one commercial wine strain, were evaluated in terms of their fermentation performance under ethanol, sulphur dioxide and osmotic stress. All the strains showed similar behaviour in these conditions. Two yeasts strains isolated from honey and the commercial wine strain were further tested for mead production, using two different honey (a dark and a light honey), enriched with two supplements (one commercial and one developed by the research team), as fermentation media. The results obtained in this work show that S. cerevisiae strains isolated from honey, are appropriate for mead production. However it is of extreme importance to take into account the characteristics of the honey, and supplements used in the fermentation medium formulation, in order to achieve the best results in mead production.

Production of amylase enzyme from mangrove fungal isolates

African Journals Online (AJOL)

sunny

2014-11-12

Nov 12, 2014 ... The use of fungi as a source of industrially relevant enzymes led to an increased interest in the ..... Black colony with whitish margin, smooth, reverse light yellow. MSF- .... strain was found to grow luxuriantly in CDA media and.

The feasibility of enzyme targeted activation for amino acid/dipeptide monoester prodrugs of floxuridine; cathepsin D as a potential targeted enzyme.

Science.gov (United States)

Tsume, Yasuhiro; Amidon, Gordon L

2012-03-26

The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5'-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0-105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5'-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5'-O-L-phenylalanyl-L-tyrosylfloxuridine and 5'-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enzymes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5'-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs.

Selection of Leuconostoc strains isolated from artisanal Serrano Catarinense cheese for use as adjuncts in cheese manufacture.

Science.gov (United States)

Seixas, Felipe Nael; Rios, Edson Antônio; Martinez de Oliveira, André Luiz; Beloti, Vanerli; Poveda, Justa Maria

2018-08-01

Serrano Catarinense cheese is a raw bovine milk cheese produced in the region of Santa Catarina, Brazil. Twelve representative strains of Leuconostoc isolated from 20 samples of this artisanal cheese were selected and submitted for evaluation of the acidifying, proteolytic, autolytic, aminopeptidase and lipolytic activities, NaCl and acid resistance, production of dextran and biogenic amines and antimicrobial activity. The aim was to genetically and technologically characterize the Leuconostoc strains in order to use them in mixed starter cultures for cheese manufacture. Leuconostoc mesenteroides subsp. mesenteroides was the species that accounted for the largest proportion of isolates of Leuconostoc genus. Two leuconostoc isolates stood out in the acidifying activity, with reduction in pH of 1.12 and 1.04 units. The isolates showed low proteolytic and autolytic activity. Most of the isolates were dextran producers, presented good resistance to the salt and pH conditions of the cheese and showed antimicrobial activity against cheese pathogen bacteria, and none of them produced biogenic amines. These results allowed the selection of five strains (UEL 04, UEL 12, UEL 18, UEL 21 and UEL 28) as good candidates for use as adjunct cultures for cheese manufacture. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

Synthesis of enzymes connected with mycoparasitism by ectomycorrhizal fungi.

Science.gov (United States)

Mucha, Joanna; Dahm, Hanna; Strzelczyk, Edmund; Werner, Antoni

2006-03-01

The production of enzymes involved in mycoparasitism by several strains of ectomycorrhizal fungi: Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), S. bovinus (15-3), S. luteus (14-7) on different substrates such as colloidal chitin, mycelia of Trichoderma harzianum, T. virens and Mucor hiemalis was examined. Chitinases and beta-1,3-glucanases were assayed spectrophotometrically by measuring the amount of reducing sugars releasing from suitable substrate by means of Miller's method. Beta-glucosidases were determined by measuring the amount of p-nitrophenol released from p-nitrophenyl-beta-D-glucopyranoside. It was observed that A. muscaria (16-3) and L. laccata (9-12) biosynthesized the highest activity of enzymes in contrast to the strains of S. bovinus and S. luteus. The mycelium of T. harzianum turned out to be the best substrate for the induction of beta-1,3-glucanases and beta-glucosidases for both strains of L. laccata, although the difference in the induction of chitinases in the presence of mycelia of different species of Trichoderma was not indicated.

Scientific Opinion on Lipase from a Genetically Modified Strain of Aspergillus oryzae (strain NZYM-LH)

OpenAIRE

EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF)

2014-01-01

The food enzyme considered in this opinion is a lipase (triacylglycerol lipase; EC 3.1.1.3) produced with a genetically modified strain of Aspergillus oryzae. The genetic modifications do not raise safety concern. The food enzyme contains neither the production organism nor recombinant DNA. The lipase is intended to be used in a number of food manufacturing processes, such as in baking and other cereal-based processes. The dietary exposure was assessed on the basis of data retrieved from the ...

Management of Fusarium Wilt using mycolytic enzymes produced by ...

African Journals Online (AJOL)

Aghomotsegin

Trichoderma strain to manage the Fusarium wilt disease of Cicer aritenum under in vitro conditions. We also studied ... antibiosis, competition, parasitism and cell lysis can ideally be ... hydrolytic enzymes associated with fungal cell wall lysis,.

Co-immobilization of multiple enzymes by metal coordinated nucleotide hydrogel nanofibers: improved stability and an enzyme cascade for glucose detection.

Science.gov (United States)

Liang, Hao; Jiang, Shuhui; Yuan, Qipeng; Li, Guofeng; Wang, Feng; Zhang, Zijie; Liu, Juewen

2016-03-21

Preserving enzyme activity and promoting synergistic activity via co-localization of multiple enzymes are key topics in bionanotechnology, materials science, and analytical chemistry. This study reports a facile method for co-immobilizing multiple enzymes in metal coordinated hydrogel nanofibers. Specifically, four types of protein enzymes, including glucose oxidase, Candida rugosa lipase, α-amylase, and horseradish peroxidase, were respectively encapsulated in a gel nanofiber made of Zn(2+) and adenosine monophosphate (AMP) with a simple mixing step. Most enzymes achieved quantitative loading and retained full activity. At the same time, the entrapped enzymes were more stable against temperature variation (by 7.5 °C), protease attack, extreme pH (by 2-fold), and organic solvents. After storing for 15 days, the entrapped enzyme still retained 70% activity while the free enzyme nearly completely lost its activity. Compared to nanoparticles formed with AMP and lanthanide ions, the nanofiber gels allowed much higher enzyme activity. Finally, a highly sensitive and selective biosensor for glucose was prepared using the gel nanofiber to co-immobilize glucose oxidase and horseradish peroxidase for an enzyme cascade system. A detection limit of 0.3 μM glucose with excellent selectivity was achieved. This work indicates that metal coordinated materials using nucleotides are highly useful for interfacing with biomolecules.

Mumps Hoshino and Torii vaccine strains were distinguished from circulating wild strains.

Science.gov (United States)

Sawada, Akihito; Yamaji, Yoshiaki; Nakayama, Tetsuo

2013-06-01

Aseptic meningitis and acute parotitis have been observed after mumps vaccination. Mumps outbreaks have been reported in Japan because of low vaccine coverage, and molecular differentiation is required to determine whether these cases are vaccine associated. RT-nested PCR was performed in the small hydrophobic gene region, and viruses were differentiated by restriction fragment length polymorphism assay. A total of 584 nucleotides were amplified. The PCR product of the Hoshino strain was cut into two fragments (313 and 271 nucleotides) by MfeI; that of the Torii strain was digested with EcoT22I, resulting in 332- and 252-nucleotide fragments. Both strains were genotype B and had an XbaI site, resulting in two fragments: 299 and 285 nucleotides. Current circulating wild types were cut only by XbaI or MfeI. However, the MfeI site of the wild types was different from that of the Hoshino strain, resulting in 451- and 133-nucleotide fragments. Using three restriction enzymes, two mumps vaccine strains were distinguished from wild types, and this separation was applied to the identification of vaccine-related adverse events.

Comparative Genomics Revealed Genetic Diversity and Species/Strain-Level Differences in Carbohydrate Metabolism of Three Probiotic Bifidobacterial Species

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Toshitaka Odamaki

2015-01-01

Full Text Available Strains of Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium animalis are widely used as probiotics in the food industry. Although numerous studies have revealed the properties and functionality of these strains, it is uncertain whether these characteristics are species common or strain specific. To address this issue, we performed a comparative genomic analysis of 49 strains belonging to these three bifidobacterial species to describe their genetic diversity and to evaluate species-level differences. There were 166 common clusters between strains of B. breve and B. longum, whereas there were nine common clusters between strains of B. animalis and B. longum and four common clusters between strains of B. animalis and B. breve. Further analysis focused on carbohydrate metabolism revealed the existence of certain strain-dependent genes, such as those encoding enzymes for host glycan utilisation or certain membrane transporters, and many genes commonly distributed at the species level, as was previously reported in studies with limited strains. As B. longum and B. breve are human-residential bifidobacteria (HRB, whereas B. animalis is a non-HRB species, several of the differences in these species’ gene distributions might be the result of their adaptations to the nutrient environment. This information may aid both in selecting probiotic candidates and in understanding their potential function as probiotics.

Antifungal activity of plant essential oils and selected Pseudomonas strains against Phomopsis theicola

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Starović Mira

2017-01-01

Full Text Available Development of natural plant protection products as an alternative to synthetic fungicides is of significant importance regarding the environment. This study was carried out with an objective to investigate in vitro antifungal activities of several essential oils extracted from oregano, basil, myrtle and Turkish pickling herb, and the plant growth-promoting rhizobacteria in the genus Pseudomonas, against the phytopathogenic fungus Phomopsis theicola. Microdilution methods were used to determine the minimum inhibitory concentrations (MIC of selected antimicrobial essential oils (EOs. All EOs exhibited significant levels of antifungal activity against the tested fungal isolates. The oregano EO was found the most potent one (MIC - 5.5 µg/mL, followed by basil (MIC - 75.0µg/mL, myrtle (MIC - 775 µg/mL and Turkish pickling herb (MIC - 7750 µg/mL. Inhibition of Ph. theicola mycelial growth was observed for all tested Pseudomonas spp. strains. K113 and L1 strains were highly effective and achieved more than 60% of fungal growth inhibition using the overnight culture and more than 57% inhibition by applying cell-free supernatants of both strains. A future field trial with K113 and L1 cultures and cell-free supernatants, containing extracellular metabolites toward Ph. theicola, will estimate their effectiveness and applicability as an alternative to chemical protection of apple trees.

Novel concept of enzyme selective nicotinamide adenine dinucleotide (NAD)-modified inhibitors based on enzyme taxonomy from the diphosphate conformation of NAD.

Science.gov (United States)

Fujii, Mikio; Kitagawa, Yasuyuki; Iida, Shui; Kato, Keisuke; Ono, Machiko

2015-11-15

The dihedral angle θ of the diphosphate part of NAD(P) were investigated to distinguish the differences in the binding-conformation of NAD(P) to enzymes and to create an enzyme taxonomy. Furthermore, new inhibitors with fixed dihedral angles showed that enzymes could recognize the differences in the dihedral angle θ. We suggest the taxonomy and the dihedral angle θ are important values for chemists to consider when designing inhibitors and drugs that target enzymes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Embedded enzymes catalyse capture

Science.gov (United States)

Kentish, Sandra

2018-05-01

Membrane technologies for carbon capture can offer economic and environmental advantages over conventional amine-based absorption, but can suffer from limited gas flux and selectivity to CO2. Now, a membrane based on enzymes embedded in hydrophilic pores is shown to exhibit combined flux and selectivity that challenges the state of the art.

Extracellular Pectinase from a Novel Bacterium Chryseobacterium indologenes Strain SD and Its Application in Fruit Juice Clarification

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Karabi Roy

2018-01-01

Full Text Available Pectinase is one of the important enzymes of industrial sectors. Presently, most of the pectinases are of plant origin but there are only a few reports on bacterial pectinases. The aim of the present study was to isolate a novel and potential pectinase producing bacterium as well as optimization of its various parameters for maximum enzyme production. A total of forty bacterial isolates were isolated from vegetable dump waste soil using standard plate count methods. Primary screening was done by hydrolysis of pectin. Pectinase activity was determined by measuring the increase in reducing sugar formed by the enzymatic hydrolysis of pectin. Among the bacterial isolates, the isolate K6 exhibited higher pectinase activity in broth medium and was selected for further studies. The selected bacterial isolate K6 was identified as Chryseobacterium indologenes strain SD. The isolate was found to produce maximum pectinase at 37°C with pH 7.5 upon incubation for 72 hours, while cultured in production medium containing citrus pectin and yeast extract as C and N sources, respectively. During enzyme-substrate reaction phase, the enzyme exhibited its best activity at pH of 8.0 and temperature of 40°C using citrus pectin as substrate. The pectinase of the isolate showed potentiality on different types of fruit juice clarification.

Antimicrobial properties of indigenous Lactobacillus sakei strain

OpenAIRE

Vesković-Moračanin Slavica; Obradović D.; Velebit B.; Borović Branka; Škrinjar Marija; Turubatović L.

2010-01-01

The strain I 154 of Lactobacillus sakei has been isolated from traditionally fermented sausages in the course of the realization of the international project (INCO PROJECT No ICA4-CT-2002-10037). This strain exhibited the ability for bacteriocin production. Antimicrobial properties of the isolated bacteriocin (sakacine), its sensibility towards proteolytic enzymes, as well as the effect of increased to high temperatures on its stability have been examined in this work. Semi purified bacterioc...

Modification of γ-induced mutagenesis in Ames test-strains

International Nuclear Information System (INIS)

Basha, S.G.; Krasavin, E.A.; Kozubek, S.; Amirtaev, K.G.

1990-01-01

Glycerine and cysteamine protective effect on mutagenesis was studied in 3 strains of Salmonella typhimurium under γ-radiation. Glycerine modifying effect was shown to be not similar for various test-strains and depended on DNA injury nature. DNA complex injuries were shown to play significant role in mutagenesis of TA100 and TA102 strains. Absence of cysteamine modifying effect on γ-induced mutagenesis testified to cysteamine effect on enzyme balance. 20 refs.; 2 figs.; 1 tab

Production of lactic acid from sucrose: strain selection, fermentation, and kinetic modeling.

Science.gov (United States)

Lunelli, Betânia H; Andrade, Rafael R; Atala, Daniel I P; Wolf Maciel, Maria Regina; Maugeri Filho, Francisco; Maciel Filho, Rubens

2010-05-01

Lactic acid is an important product arising from the anaerobic fermentation of sugars. It is used in the pharmaceutical, cosmetic, chemical, and food industries as well as for biodegradable polymer and green solvent production. In this work, several bacterial strains were isolated from industrial ethanol fermentation, and the most efficient strain for lactic acid production was selected. The fermentation was conducted in a batch system under anaerobic conditions for 50 h at a temperature of 34 degrees C, a pH value of 5.0, and an initial sucrose concentration of 12 g/L using diluted sugarcane molasses. Throughout the process, pulses of molasses were added in order to avoid the cell growth inhibition due to high sugar concentration as well as increased lactic acid concentrations. At the end of the fermentation, about 90% of sucrose was consumed to produce lactic acid and cells. A kinetic model has been developed to simulate the batch lactic acid fermentation results. The data obtained from the fermentation were used for determining the kinetic parameters of the model. The developed model for lactic acid production, growth cell, and sugar consumption simulates the experimental data well.

Characterization of putative virulence factors of Serratia marcescens strain SEN for pathogenesis in Spodoptera litura.

Science.gov (United States)

Aggarwal, Chetana; Paul, Sangeeta; Tripathi, Vishwas; Paul, Bishwajeet; Khan, Md Aslam

2017-02-01

Two Serratia marcescens strains, SEN and ICC-4, isolated from diseased insect cadavers were observed to differ considerably in their virulence towards Spodoptera litura. The present study was aimed to characterize the possible virulence factors present in the virulent Serratia marcescens strain SEN. Both the S. marcescens strains were evaluated for the presence of various lytic enzymes such as chitinase, lipase, protease and phospholipase. The virulent S. marcescens strain SEN was observed to possess considerably higher activity of chitinase and protease enzymes; activity of phospholipase enzyme was also higher. Although, all the three toxin genes shlA, phlA and swr could be detected in both the S. marcescens strains, there was a higher expression of these genes in the virulent strain SEN. S. marcescens strain ICC-4 showed greater reduction in overall growth yield in the post-exponential phase in the presence of midgut juice and hemolymph of S. litura larvae, as compared to S. marcescens strain SEN. Proliferation of the S. marcescens strain SEN was also considerably higher in foregut, midgut and hemolymph of S. litura larvae, as compared to strain ICC-4. Peritrophic membrane treated with broth culture of the S. marcescens strain SEN showed higher damage as compared to strain ICC-4. The peritrophic membrane of larvae fed on diet treated with the virulent strain showed considerable damage while the peritrophic membrane of larvae fed on diet treated with the non-virulent strain showed no damage. This is the first report documenting the fate of ingested S. marcescens in S. litura gut and the relative expression of toxin genes from two S. marcescens strains differing in their virulence towards S. litura. Copyright © 2016 Elsevier Inc. All rights reserved.

Strain measurement technique

International Nuclear Information System (INIS)

1987-01-01

The 10 contributions are concerned with selected areas of application, such as strain measurements in wood, rubber/metal compounds, sets of strain measurements on buildings, reinforced concrete structures without gaps, pipes buried in the ground and measurements of pressure fluctuations. To increase the availability and safety of plant, stress analyses were made on gas turbine rotors with HT-DMS or capacitive HT-DMS (high temperature strain measurements). (DG) [de

Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan.

Science.gov (United States)

Asad, Saeed Ahmad; Tabassum, Ayesha; Hameed, Abdul; Hassan, Fayyaz Ul; Afzal, Aftab; Khan, Sabaz Ali; Ahmed, Rafiq; Shahzad, Muhammad

2015-01-01

This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan

Science.gov (United States)

Asad, Saeed Ahmad; Tabassum, Ayesha; Hameed, Abdul; Hassan, Fayyaz ul; Afzal, Aftab; Khan, Sabaz Ali; Ahmed, Rafiq; Shahzad, Muhammad

2015-01-01

Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract. PMID:26691463

Activity of selected hydrolytic enzymes in Allium sativum L. anthers.

Science.gov (United States)

Winiarczyk, Krystyna; Gębura, Joanna

2016-05-01

The aim of the study was to determine enzymatic activity in sterile Allium sativum anthers in the final stages of male gametophyte development (the stages of tetrads and free microspores). The analysed enzymes were shown to occur in the form of numerous isoforms. In the tetrad stage, esterase activity was predominant, which was manifested by the greater number of isoforms of the enzyme. In turn, in the microspore stage, higher numbers of isoforms of acid phosphatases and proteases were detected. The development of sterile pollen grains in garlic is associated with a high level of protease and acid phosphatase activity and lower level of esterase activities in the anther locule. Probably this is the first description of the enzymes activity (ACPH, EST, PRO) in the consecutives stages of cell wall formation which is considered to be one of the causes of male sterility in flowering plant. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Screening and characterization of endophytic Bacillus and Paenibacillus strains from medicinal plant Lonicera japonica for use as potential plant growth promoters

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Longfei Zhao

2015-12-01

Full Text Available Abstract A total of 48 endophytic bacteria were isolated from surface-sterilized tissues of the medicinal plant Lonicera japonica, which is grown in eastern China; six strains were selected for further study based on their potential ability to promote plant growth in vitro (siderophore and indoleacetic acid production. The bacteria were characterized by phylogenetically analyzing their 16S rRNA gene similarity, by examining their effect on the mycelial development of pathogenic fungi, by testing their potential plant growth-promoting characteristics, and by measuring wheat growth parameters after inoculation. Results showed that the number of endophytic bacteria in L. japonica varied among different tissues, but it remained relatively stable in the same tissues from four different plantation locations. Among the three endophytic strains, strains 122 and 124 both had high siderophore production, with the latter showing the highest phosphate solubilization activity (45.6 mg/L and aminocyclopropane-1-carboxylic acid deaminase activity (47.3 nmol/mg/h. Strain 170 had the highest indoleacetic acid (IAA production (49.2 mg/L and cellulase and pectinase activities. After inoculation, most of the six selected isolates showed a strong capacity to promote wheat growth. Compared with the controls, the increase in the shoot length, root length, fresh weight, dry weight, and chlorophyll content was most remarkable in wheat seedlings inoculated with strain 130. The positive correlation between enzyme (cellulose and pectinase activity and inhibition rate on Fusarium oxysporum, the IAA production, and the root length of wheat seedlings inoculated with each tested endophytic strain was significant in regression analysis. Deformity of pathogenic fungal mycelia was observed under a microscope after the interaction with the endophytic isolates. Such deformity may be directly related to the production of hydrolytic bacterial enzymes (cellulose and pectinase. The six

Substrate specificity of glucose dehydrogenase and carbon source utilization pattern of pantoea dispersa strain P2 and its radiation induced mutants

International Nuclear Information System (INIS)

Lee, Young Keun; Murugesan, Senthilkumar

2009-01-01

Mineral phosphate solubilizing pantoea dispersa strain P2 produced 5.5 mM and 42.6 mM of gluconic acid on 24 h and 72 h incubation, respectively. Strain P2 exhibited glucose dehydrogenase (GDH) specific activity of 0.32 IU mg -1 protein. We have studied the substrate specificity of GDH as well as carbon source utilization pattern of strain P2. GDH of strain P2 did not use ribose as substrate. Utilization of lactose with specific activity of 0.65 IU mg -1 protein indicated that the enzyme belongs to GDH type B isozyme. Arabinose, galactose, ribose, sucrose and xylose did not induce the synthesis of GDH enzyme while mannose induced the synthesis of GDH with highest specific activity of 0.58 IU mg -1 protein. Through radiation mutagenesis, the substrate specificity of GDH was modified in order to utilize side range of sugars available in root exudates. Ribose, originally not a substrate for GDH of strain P2 was utilized as substrate by mutants P2-M5 with specific activity of 0.44 and 0.57 IU mg -1 protein, respectively. Specific activity of GDH on the media containing lactose and galactose was also improved to 1.2 and 0.52 IU mg -1 protein in P2-M5 and P2-M6 respectively. Based on the carbon source availability in root exudate, the mutants can be selected and utilized as efficient biofertilizer under P-deficient soil conditions

Trypanosoma cruzi: strain selection by diferent schedules of mouse passage of an initially mixed infection

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Maria P. Deane

1984-12-01

Full Text Available From an initial double infection in mice, established by simultaneous and equivalent inocula of bloodstream forms of strains Y and F of Trypanosoma cruzi, two lines were derived by subinoculations: one (W passaged every week, the other (M every month. Through biological and biochemical methods only the Y strain was identified at the end of the 10th and 16th passages of line W and only the F strain at the 2nd and 4th passages of line M. The results illustrate strain selection through laboratory manipulation of initially mixed populations of T. cruzi.De uma infecção inicialmente dupla em camundongo, estabelecida por inóculo simultaneo e equivalente de formas sanguíneas das cepas Y e F de Trypanosoma cruzi, duas linhagens foram originadas por subinoculações: uma (W passada casa semana, a outra (M cada mês. Por métodos biológicos e bioquímicos apenas a cepa Y foi identificada ao fim a 10a. e 16a. passagens da linhagem W e apenas a cepa F na 2a. e 4a.passagens de linhagem M. Os resultados demonstram a seleção de cepas através de manipulação em laboratorio de populações inicialmente mistas de T. cruzi.

Highly efficient gene targeting in Aspergillus oryzae industrial strains under ligD mutation introduced by genome editing: Strain-specific differences in the effects of deleting EcdR, the negative regulator of sclerotia formation.

Science.gov (United States)

Nakamura, Hidetoshi; Katayama, Takuya; Okabe, Tomoya; Iwashita, Kazuhiro; Fujii, Wataru; Kitamoto, Katsuhiko; Maruyama, Jun-Ichi

2017-07-11

Numerous strains of Aspergillus oryzae are industrially used for Japanese traditional fermentation and for the production of enzymes and heterologous proteins. In A. oryzae, deletion of the ku70 or ligD genes involved in non-homologous end joining (NHEJ) has allowed high gene targeting efficiency. However, this strategy has been mainly applied under the genetic background of the A. oryzae wild strain RIB40, and it would be laborious to delete the NHEJ genes in many A. oryzae industrial strains, probably due to their low gene targeting efficiency. In the present study, we generated ligD mutants from the A. oryzae industrial strains by employing the CRISPR/Cas9 system, which we previously developed as a genome editing method. Uridine/uracil auxotrophic strains were generated by deletion of the pyrG gene, which was subsequently used as a selective marker. We examined the gene targeting efficiency with the ecdR gene, of which deletion was reported to induce sclerotia formation under the genetic background of the strain RIB40. As expected, the deletion efficiencies were high, around 60~80%, in the ligD mutants of industrial strains. Intriguingly, the effects of the ecdR deletion on sclerotia formation varied depending on the strains, and we found sclerotia-like structures under the background of the industrial strains, which have never been reported to form sclerotia. The present study demonstrates that introducing ligD mutation by genome editing is an effective method allowing high gene targeting efficiency in A. oryzae industrial strains.

Nicotinoprotein methanol dehydrogenase enzymes in Gram-positive methylotrophic bacteria

NARCIS (Netherlands)

Hektor, Harm J.; Kloosterman, Harm; Dijkhuizen, Lubbert

2000-01-01

A novel type of alcohol dehydrogenase enzyme has been characterized from Gram-positive methylotrophic (Bacillus methanolicus, the actinomycetes Amycolatopsis methanolica and Mycobacterium gastri) and non-methylotrophic bacteria (Rhodococcus strains). Its in vivo role is in oxidation of methanol and

Homology modelling and docking studies on Neuraminidase enzyme as a natural product target for combating influenza

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Nisha Singh

2017-10-01

Full Text Available Influenza remains to be dreadful with yearly epidemics and sudden pandemic outbreaks causing significant mortality, even in nations with the most advanced health care systems. Thus, there has been a long-standing interest to develop effective and safe antiviral agents to treat infected individuals. Attempt to identify suitable molecular targets as antiviral compounds have focused recently on the influenza virus neuraminidase (NA, a key enzyme in viral replication [1]. In this research, virtual screening was done on a total of 600 natural compounds from 22 ethno medicinal Indian herbs for activity against neuraminidase enzyme exploiting representative protein conformations selected from molecular dynamics simulations. Neuraminidase enzyme sequences from different existing strains available on National Center of Biotechnology Information [2] (NCBI protein database were aligned using Clustal W [3] and CLC workbench 10 [4] to find the conserved residues. Neuraminidase protein sequence from H1N1 strain available on NCBI was used to structure 3D target model predicted against dataset from Protein data bank using modeller [5]. The target model was validated on different parameter at SAVES Server [6]. Using this target model a pharmacophore model was developed using ligand based strategy exploiting the three known inhibitors. The docking parameters were validated by redocking Zanamivir to its co-complex 2009 H1N1 NA crystal structure (PDB ID: 3TI5 generating best pose with a RMSD value of 0.7543 A°. This model was then used for in silico analysis of a library of natural compounds from 22 ethno medicinal Indian herbs known to have antiviral activity taken downloaded from PubChem database and selected on the basis of drug likeliness. All the compounds were docked in the binding pocket of neuraminidase. Top compounds having binding affinity better than or comparable to the control drug Zanamivir were selected and analyzed for their ADME and toxicity

Effects of irradiation on enzymes in E. coli

Energy Technology Data Exchange (ETDEWEB)

Geyer, H.

1962-08-15

To determine the effects of irradiation on enzymes in Escherichia coli strain Crookes, the influence of x radiation on the content of the coenzyme pyridoxal phosphate was investigated. The method of pyridoxal phosphate assay used was based on the fact that E. coli is able to produce tryptophanase. Enzyme activity was measured by determination of indole produced from tryptophane. Doses of 10,000 and 80,000 r of x radiation were given to resting cells and growing cells. It was found that pyridoxal phosphate production and content were not infiuenced by irradiation. (H.M.G.)

Enzymes as Green Catalysts for Precision Macromolecular Synthesis.

Science.gov (United States)

Shoda, Shin-ichiro; Uyama, Hiroshi; Kadokawa, Jun-ichi; Kimura, Shunsaku; Kobayashi, Shiro

2016-02-24

The present article comprehensively reviews the macromolecular synthesis using enzymes as catalysts. Among the six main classes of enzymes, the three classes, oxidoreductases, transferases, and hydrolases, have been employed as catalysts for the in vitro macromolecular synthesis and modification reactions. Appropriate design of reaction including monomer and enzyme catalyst produces macromolecules with precisely controlled structure, similarly as in vivo enzymatic reactions. The reaction controls the product structure with respect to substrate selectivity, chemo-selectivity, regio-selectivity, stereoselectivity, and choro-selectivity. Oxidoreductases catalyze various oxidation polymerizations of aromatic compounds as well as vinyl polymerizations. Transferases are effective catalysts for producing polysaccharide having a variety of structure and polyesters. Hydrolases catalyzing the bond-cleaving of macromolecules in vivo, catalyze the reverse reaction for bond forming in vitro to give various polysaccharides and functionalized polyesters. The enzymatic polymerizations allowed the first in vitro synthesis of natural polysaccharides having complicated structures like cellulose, amylose, xylan, chitin, hyaluronan, and chondroitin. These polymerizations are "green" with several respects; nontoxicity of enzyme, high catalyst efficiency, selective reactions under mild conditions using green solvents and renewable starting materials, and producing minimal byproducts. Thus, the enzymatic polymerization is desirable for the environment and contributes to "green polymer chemistry" for maintaining sustainable society.

Lactobacillus casei and Lactobacillus fermentum Strains Isolated from Mozzarella Cheese: Probiotic Potential, Safety, Acidifying Kinetic Parameters and Viability under Gastrointestinal Tract Conditions.

Science.gov (United States)

de Souza, Bruna Maria Salotti; Borgonovi, Taís Fernanda; Casarotti, Sabrina Neves; Todorov, Svetoslav Dimitrov; Penna, Ana Lúcia Barretto

2018-03-14

The objective of this study was to evaluate the probiotic properties of Lactobacillus casei and Lactobacillus fermentum strains, as well as to select novel and safe strains for future development of functional fermented products. The in vitro auto-aggregation, co-aggregation, hydrophobicity, β-galactosidase production, survival to gastrointestinal tract (GIT), and antibiotic susceptibility were evaluated. The selected strains were additionally tested by the presence of genes encoding adhesion, aggregation and colonization, virulence factors, antibiotic resistance, and biogenic amine production, followed by the evaluation of acidifying kinetic parameters in milk, and survival of the strains under simulated GIT conditions during refrigerated storage of fermented milk. Most strains of both species showed high auto-aggregation; some strains showed co-aggregation ability with other lactic acid bacteria (LAB) and/or pathogens, and both species showed low hydrophobicity values. Seven L. casei and six L. fermentum strains produced β-galactosidase enzymes, and ten strains survived well the simulation of the GIT stressful conditions evaluated in vitro. All strains were resistant to vancomycin, and almost all the strains were resistant to kanamycin. L. casei SJRP38 and L. fermentum SJRP43 were distinguished among the other LAB strains by their higher probiotic potential. L. fermentum SJRP43 presented fewer genes related to virulence factors and antibiotic resistance and needed more time to reach the maximum acidification rate (V max ). The other kinetic parameters were similar. Both strains survived well (> 8 log 10 CFU/mL) to the GIT-simulated conditions when incorporated in fermented milk. Therefore, these strains presented promising properties for further applications in fermented functional products.

Production of certain hydrolytic enzymes by psychrophilic bacteria from the Antarctic krill, zooplankton and seawater

Digital Repository Service at National Institute of Oceanography (India)

Ramaiah, N.

of hydrolytic enzymes compared to those strains collected either from water or krill samples. Based on these results, the functional role of bacterial enzymes in relation to trophodynamics of euphausiids and their role in the post-harvest technology of krill...

Genetic diversity and symbiotic effectiveness of Bradyrhizobium strains nodulating selected annual grain legumes growing in Ethiopia.

Science.gov (United States)

Degefu, Tulu; Wolde-Meskel, Endalkachew; Rasche, Frank

2018-01-01

Vigna unguiculata, Vigna radiata and Arachis hypogaea growing in Ethiopia are nodulated by a genetically diverse group of Bradyrhizobium strains. To determine the genetic identity and symbiotic effectiveness of these bacteria, a collection of 36 test strains originating from the root nodules of the three hosts was investigated using multilocus sequence analyses (MLSA) of core genes including 16S rRNA, recA, glnII, gyrB, atpD and dnaK. Sequence analysis of nodA and nifH genes along with tests for symbiotic effectiveness using δ 15 N analysis were also carried out. The phylogenetic trees derived from the MLSA grouped most test strains into four well-supported distinct positions designated as genospecies I-IV. The maximum likelihood (ML) tree that was constructed based on the nodA gene sequences separated the entire test strains into two lineages, where the majority of the test strains were clustered on one of a well-supported large branch that comprise Bradyrhizobium species from the tropics. This clearly suggested the monophyletic origin of the nodA genes within the bradyrhizobia of tropical origin. The δ 15 N-based symbiotic effectiveness test of seven selected strains revealed that strains GN100 (δ 15 N=0.73) and GN102 (δ 15 N=0.79) were highly effective nitrogen fixers when inoculated to cowpea, thus can be considered as inoculants in cowpea production. It was concluded that Ethiopian soils are a hotspot for rhizobial diversity. This calls for further research to unravel as yet unknown bradyrhizobia nodulating legume host species growing in the country. In this respect, prospective research should also address the mechanisms of symbiotic specificity that could lead to high nitrogen fixation in target legumes.

Listeria monocytogenes Strains Underrepresented during Selective Enrichment with an ISO Method Might Dominate during Passage through Simulated Gastric Fluid and In Vitro Infection of Caco-2 Cells

Science.gov (United States)

Zilelidou, Evangelia; Karmiri, Christina-Vasiliki; Zoumpopoulou, Georgia; Mavrogonatou, Eleni; Kletsas, Dimitris; Tsakalidou, Effie; Papadimitriou, Konstantinos; Drosinos, Eleftherios

2016-01-01

ABSTRACT Various Listeria monocytogenes strains may contaminate a single food product, potentially resulting in simultaneous exposure of consumers to multiple strains. However, due to bias in strain recovery, L. monocytogenes strains isolated from foods by selective enrichment (SE) might not always represent those that can better survive the immune system of a patient. We investigated the effect of cocultivation in tryptic soy broth with 0.6% yeast extract (TSB-Y) at 10°C for 8 days on (i) the detection of L. monocytogenes strains during SE with the ISO 11290-1:1996/Amd 1:2004 protocol and (ii) the in vitro virulence of strains toward the Caco-2 human colon epithelial cancer cell line following exposure to simulated gastric fluid (SGF; pH 2.0)-HCl (37°C). We determined whether the strains which were favored by SE would be effective competitors under the conditions of challenges related to gastrointestinal passage of the pathogen. Interstrain competition of L. monocytogenes in TSB-Y determined the relative population of each strain at the beginning of SE. This in turn impacted the outcome of SE (i.e., favoring survival of competitors with better fitness) and the levels exposed subsequently to SGF. However, strong growth competitors could be outcompeted after SGF exposure and infection of Caco-2 cells by strains outgrown in TSB-Y and underdetected (or even missed) during enrichment. Our data demonstrate a preferential selection of certain L. monocytogenes strains during enrichments, often not reflecting a selective advantage of strains during infection. These findings highlight a noteworthy scenario associated with the difficulty of matching the source of infection (food) with the L. monocytogenes isolate appearing to be the causative agent during listeriosis outbreak investigations. IMPORTANCE This report is relevant to understanding the processes involved in selection and prevalence of certain L. monocytogenes strains in different environments (i.e., foods or

Comparative evaluation of the Ridascreen Verotoxin enzyme immunoassay for detection of Shiga-toxin producing strains of Escherichia coli (STEC) from food and other sources.

Science.gov (United States)

Beutin, L; Steinrück, H; Krause, G; Steege, K; Haby, S; Hultsch, G; Appel, B

2007-03-01

To evaluate the suitability of the commercially distributed Ridascreen Verotoxin enzyme immunoassay (EIA) for detection of known genetic types of the Vero (Shiga) toxins 1 (Stx1) and 2 (Stx2) families and to determine its relative sensitivity and specificity. The Ridascreen-EIA was compared with the Vero cell assay, a P(1)-glycoprotein receptor EIA and with stx gene-specific PCs for detection of Stx with 43 Shiga toxin-producing strains of Escherichia coli (STEC) reference strains and with 241 test strains. The Ridascreen-EIA detects strains producing Stx1 and variants Stx1c and Stx1d, as well as Stx2 and variants Stx2d1, Stx2d2, Stx2e, Stx2d, Stx2-O118 (Stx2d-ount), Stx2-NV206, Stx2f and Stx2g. The assay showed a relative sensitivity of 95.7% and a relative specificity of 98.7%. Some of the Stx2-O118-, Stx2e- and Stx2g-producing STEC were not detected with the Ridascreen-EIA probably because of low amount of toxin produced by these strains. The Ridascreen-EIA is able to detect all known types of Stx and is applicable for routine screening of bacterial isolates owing to its high specificity. It is less applicable for testing samples where low amounts of Stx are expected, such as mixed cultures and certain Stx2 variants. This study presents a first comprehensive evaluation of the Ridascreen-EIA, a rapid standardized STEC screening test for routine diagnostic laboratories. Data are presented on the type of the spectrum of Stx that are detected with this immunoassay and its advantages and limits for practical use.

Lactic acid bacteria affect serum cholesterol levels, harmful fecal enzyme activity, and fecal water content.

Science.gov (United States)

Lee, Do Kyung; Jang, Seok; Baek, Eun Hye; Kim, Mi Jin; Lee, Kyung Soon; Shin, Hea Soon; Chung, Myung Jun; Kim, Jin Eung; Lee, Kang Oh; Ha, Nam Joo

2009-06-11

Lactic acid bacteria (LAB) are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as lower cholesterol. Although present in many foods, most trials have been in spreads or dairy products. Here we tested whether Bifidobacteria isolates could lower cholesterol, inhibit harmful enzyme activities, and control fecal water content. In vitro culture experiments were performed to evaluate the ability of Bifidobacterium spp. isolated from healthy Koreans (20 approximately 30 years old) to reduce cholesterol-levels in MRS broth containing polyoxyethanylcholesterol sebacate. Animal experiments were performed to investigate the effects on lowering cholesterol, inhibiting harmful enzyme activities, and controlling fecal water content. For animal studies, 0.2 ml of the selected strain cultures (108 approximately 109 CFU/ml) were orally administered to SD rats (fed a high-cholesterol diet) every day for 2 weeks. B. longum SPM1207 reduced serum total cholesterol and LDL levels significantly (p water content, and reduced body weight and harmful intestinal enzyme activities. Daily consumption of B. longum SPM1207 can help in managing mild to moderate hypercholesterolemia, with potential to improve human health by helping to prevent colon cancer and constipation.

Proteomic analysis of nitrate-dependent acetone degradation by Alicycliphilus denitrificans strain BC

NARCIS (Netherlands)

Oosterkamp, M.J.; Boeren, S.; Atashgahi, S.; Plugge, C.M.; Schaap, P.J.; Stams, A.J.M.

2015-01-01

Alicycliphilus denitrificans strain BC grows anaerobically on acetone with nitrate as electron acceptor. Comparative proteomics of cultures of A. denitrificans strain BC grown on either acetone or acetate with nitrate was performed to study the enzymes involved in the acetone degradation pathway. In

The Feasibility of Enzyme Targeted Activation for Amino Acid/Dipeptide Monoester Prodrugs of Floxuridine; Cathepsin D as a Potential Targeted Enzyme

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Gordon L. Amidon

2012-03-01

Full Text Available The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5¢-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0–105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5¢-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5¢-O-L-phenylalanyl-L-tyrosylfloxuridine and 5¢-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enyzmes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5¢-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs.

Construction of efficient xylose utilizing Pichia pastoris for industrial enzyme production.

Science.gov (United States)

Li, Pengfei; Sun, Hongbing; Chen, Zao; Li, Yin; Zhu, Taicheng

2015-02-21

Cellulosic biomass especially agricultural/wood residues can be utilized as feedstock to cost-effectively produce fuels, chemicals and bulk industrial enzymes, which demands xylose utilization from microbial cell factories. While previous works have made significant progress in improving microbial conversion of xylose into fuels and chemicals, no study has reported the engineering of efficient xylose utilizing protein expression systems for the purpose of producing industrial enzymes. In this work, using Pichia pastoris as an example, we demonstrated the successful engineering of xylose metabolizing ability into of protein expression systems. A heterologous XI (xylose isomerase) pathway was introduced into P. pastoris GS115 by overexpressing the Orpinomyces spp. XI or/and the endogenous XK (xylulokinase) gene, and evolutionary engineering strategies were also applied. Results showed that the XI pathway could be functionally expressed in P. pastoris. After 50 generation of sequential batch cultivation, a set of domesticated recombinant P. pastoris strains with different performance metrics on xylose were obtained. One evolved strain showed the highest xylose assimilation ability, whose cell yield on xylose can even be comparable to that on glucose or glycerol. This strain also showed significantly increased β-mannanase production when cultured on xylose medium. Furthermore, transcription analysis of xylose pathway genes suggested that overexpression of XI and XK might be the key factors affecting effective xylose assimilation. To our best knowledge, this study is the first work demonstrating the construction of efficient xylose utilizing P. pastoris strains, thus providing a basis for using cellulosic biomass for bulk industrial enzyme production.

SELECTED PARAMETERS OF THE WORK OF SPEED LIMITER IN LINE STRAINING SYSTEM IN A FRICTIONAL LIFT

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Paweł Lonkwic

2014-03-01

Full Text Available The article presents the analysis of selected work parameters of speed limiter in line straining system. We analyzed the effect of changing the geometrical conditions of the new solution for the speed limiter in line straining system upon the working conditions in frictional lift braking system. Within the conducted simulations of the work of the system, which is responsible for lift braking with a tension with spring, a test bed was prepared, which simulated the work of tension-rope-limiter system. The tests were performed in the conditions reflecting the work of a lifting appliance. Analyzing the results obtained through empirical calculations, we can conclude that there is a possibility of applying the spring to eliminate the weight.

Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression

Science.gov (United States)

McDonald, Bradon R.; Takasuka, Taichi E.; Wendt-Pienkowski, Evelyn; Doering, Drew T.; Raffa, Kenneth F.; Fox, Brian G.; Currie, Cameron R.

2016-01-01

The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology. PMID:27276034

Exploiting fine-scale genetic and physiological variation of closely related microbes to reveal unknown enzyme functions.

Science.gov (United States)

Badur, Ahmet H; Plutz, Matthew J; Yalamanchili, Geethika; Jagtap, Sujit Sadashiv; Schweder, Thomas; Unfried, Frank; Markert, Stephanie; Polz, Martin F; Hehemann, Jan-Hendrik; Rao, Christopher V

2017-08-04

Polysaccharide degradation by marine microbes represents one of the largest and most rapid heterotrophic transformations of organic matter in the environment. Microbes employ systems of complementary carbohydrate-specific enzymes to deconstruct algal or plant polysaccharides (glycans) into monosaccharides. Because of the high diversity of glycan substrates, the functions of these enzymes are often difficult to establish. One solution to this problem may lie within naturally occurring microdiversity; varying numbers of enzymes, due to gene loss, duplication, or transfer, among closely related environmental microbes create metabolic differences akin to those generated by knock-out strains engineered in the laboratory used to establish the functions of unknown genes. Inspired by this natural fine-scale microbial diversity, we show here that it can be used to develop hypotheses guiding biochemical experiments for establishing the role of these enzymes in nature. In this work, we investigated alginate degradation among closely related strains of the marine bacterium Vibrio splendidus One strain, V. splendidus 13B01, exhibited high extracellular alginate lyase activity compared with other V. splendidus strains. To identify the enzymes responsible for this high extracellular activity, we compared V. splendidus 13B01 with the previously characterized V. splendidus 12B01, which has low extracellular activity and lacks two alginate lyase genes present in V. splendidus 13B01. Using a combination of genomics, proteomics, biochemical, and functional screening, we identified a polysaccharide lyase family 7 enzyme that is unique to V. splendidus 13B01, secreted, and responsible for the rapid digestion of extracellular alginate. These results demonstrate the value of querying the enzymatic repertoires of closely related microbes to rapidly pinpoint key proteins with beneficial functions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Positron emitter labeled enzyme inhibitors

International Nuclear Information System (INIS)

Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

1990-01-01

This invention involves a new strategy for imagining and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography

Characterisation of lactic acid bacteria in spontaneously fermented camel milk and selection of strains for fermentation of camel milk

DEFF Research Database (Denmark)

Fugl, Angelina June Brandt; Berhe, Tesfemariam; Kiran, Anil

2017-01-01

The microbial communities in spontaneously fermented camel milk from Ethiopia were characterised through metagenomic 16S rRNA sequencing and lactic acid bacteria were isolated with the goal of selecting strains suitable as starter cultures. The fermented camel milk microbiota was dominated either...... by Lactobacillales or by Enterobacteriaceae, depending on incubation temperature and the provider of the milk. Strains of species with a potential use as starter cultures i.e., Lactococcus lactis, Lactobacillus plantarum, and Pediococcus acidilactici, were isolated. Fast acidifiers of camel milk have been isolated...

Modelling Fungal Fermentations for Enzyme Production

DEFF Research Database (Denmark)

Albæk, Mads Orla; Gernaey, Krist; Hansen, Morten S.

We have developed a process model of fungal fed-batch fermentations for enzyme production. In these processes, oxygen transfer rate is limiting and controls the substrate feeding rate. The model has been shown to describe cultivations of both Aspergillus oryzae and Trichoderma reesei strains in 550......L stirred tank pilot plant reactors well. For each strain, 8 biological parameters are needed as well as a correlation of viscosity, as viscosity has a major influence on oxygen transfer. The parameters were measured averages of at least 9 batches for each strain. The model is successfully able...... to cover a wide range of process conditions (0.3-2 vvm of aeration, 0.2-10.0 kW/m3 of specific agitation power input, and 0.1-1.3 barg head space pressure). Uncertainty and sensitivity analysis have shown that the uncertainty of the model is mainly due to difficulties surrounding the estimation...

Targeted enzyme prodrug therapies.

Science.gov (United States)

Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C

2010-09-01

The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

Strategy for improving extracellular lipolytic activities by a novel thermotolerant Staphylococcus sp. strain

Directory of Open Access Journals (Sweden)

Cherif Slim

2011-11-01

Full Text Available Abstract Background Extracellular bacterial lipases received much attention for their substrate specificity and their ability to function under extreme environments (pH, temperature.... Many staphylococci produced lipases which were released into the culture medium. Reports of extracellular thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described. Results This study focused on novel strategies to increase extracellular lipolytic enzyme production by a novel Staphylococcus sp. strain ESW. The microorganism needed neutral or alkaline pH values between 7.0 and 12.0 for growth. For pH values outside this range, cell growth seemed to be significantly inhibited. Staphylococcus sp. culture was able to grow within a wide temperature range (from 30 to 55°C. The presence of oils in the culture medium leaded to improvements in cells growth and lipolytic enzyme activity. On the other hand, although chemical surfactants leaded to an almost complete inhibition of growth and lipolytic enzyme production, their addition along the culture could affect the location of the enzyme. In addition, our results showed that this novel Staphylococcus sp. strain produced biosurfactants simultaneously with lipolytic activity, when soapstock (The main co-product of the vegetable oil refining industry, was used as the sole carbon source. Conclusion A simultaneous biosurfactant and extracellular lipolytic enzymes produced bacterial strain with potential application in soap stock treatment

Characterization and monitoring of selected rhizobial strains ...

African Journals Online (AJOL)

SERVER

2007-06-18

Jun 18, 2007 ... Fax: +66-44-216345. fixing symbiosis with bacteria known as rhizobia. ... Rhizobial strains were cultured on Yeast-Malt extract agar contain- ing bromthymol blue ... Colony form-ing was observed every day as well as the ...

Industrially Important Carbohydrate Degrading Enzymes from Yeasts: Pectinases, Chitinases, and β-1,3-Glucanases

Science.gov (United States)

Gummadi, Sathyanarayana N.; Kumar, D. Sunil; Dash, Swati S.; Sahu, Santosh Kumar

Polysaccharide degrading enzymes are hydrolytic enzymes, which have a lot of industrial potential and also play a crucial role in carbon recycling. Pectinases, chitinases and glucanases are the three major polysaccharide degrading enzymes found abundantly in nature and these enzymes are mainly produced by fungal strains. Production of these enzymes by yeasts is advantageous over fungi, because the former are easily amenable to genetic manipulations and time required for growth and production is less than that of the latter. Several yeasts belonging to Saccharomyces, Pichia, Rhodotorula and Cryptococcus produce extracellular pectinases, glucanases and chitinases. This chapter emphasizes on the biological significance of these enzymes, their production and their industrial applications.

N- vs. C-Domain Selectivity of Catalytic Inactivation of Human Angiotensin Converting Enzyme by Lisinopril-Coupled Transition Metal Chelates

Science.gov (United States)

Hocharoen, Lalintip; Joyner, Jeff C.; Cowan, J. A.

2014-01-01

The N- and C-terminal domains of human somatic Angiotensin I Converting Enzyme (sACE-1) demonstrate distinct physiological functions, with resulting interest in the development of domain-selective inhibitors for specific therapeutic applications. Herein, the activity of lisinopril-coupled transition metal chelates were tested for both reversible binding and irreversible catalytic inactivation of sACE-1. C/N domain binding selectivity ratios ranged from 1 to 350, while rates of irreversible catalytic inactivation of the N- and C-domains were found to be significantly greater for the N-domain, suggesting a more optimal orientation of the M-chelate-lisinopril complexes within the active site of the N-domain of sACE-1. Finally, the combined effect of binding selectivity and inactivation selectivity was assessed for each catalyst (double-filter selectivity factors), and several catalysts were found to cause domain-selective catalytic inactivation. The results of this study demonstrate the ability to optimize the target selectivity of catalytic metallopeptides through both binding and orientation factors (double-filter effect). PMID:24228790

N- versus C-domain selectivity of catalytic inactivation of human angiotensin converting enzyme by lisinopril-coupled transition metal chelates.

Science.gov (United States)

Hocharoen, Lalintip; Joyner, Jeff C; Cowan, J A

2013-12-27

The N- and C-terminal domains of human somatic angiotensin I converting enzyme (sACE-1) demonstrate distinct physiological functions, with resulting interest in the development of domain-selective inhibitors for specific therapeutic applications. Herein, the activity of lisinopril-coupled transition metal chelates was tested for both reversible binding and irreversible catalytic inactivation of each domain of sACE-1. C/N domain binding selectivity ratios ranged from 1 to 350, while rates of irreversible catalytic inactivation of the N- and C-domains were found to be significantly greater for the N-domain, suggesting a more optimal orientation of M-chelate-lisinopril complexes within the active site of the N-domain of sACE-1. Finally, the combined effect of binding selectivity and inactivation selectivity was assessed for each catalyst (double-filter selectivity factors), and several catalysts were found to cause domain-selective catalytic inactivation. The results of this study demonstrate the ability to optimize the target selectivity of catalytic metallopeptides through both binding and catalytic factors (double-filter effect).

Selected wild strains of Agaricus bisporus produce high yields of mushrooms at 25°C.

Science.gov (United States)

Navarro, Pilar; Savoie, Jean-Michel

2015-01-01

To cultivate the button mushroom Agaricus bisporus in warm countries or during summer in temperate countries, while saving energy, is a challenge that could be addressed by using the biological diversity of the species. The objective was to evaluate the yield potential of eight wild strains previously selected in small scale experiments for their ability to produce mature fruiting bodies at 25°C and above. Culture units of 8 kg of compost were used. The yield expressed as weight or number per surface unit and earliness of fruiting were recorded during cultivation in climatic rooms at 17, 25 or 30°C. Only strains of A. bisporus var. burnettii were able to fruit at 30°C. At 25°C they produced the highest yields (27 kg m(-2)) and had best earliness. The yields at 25°C for the strains of A. bisporus var. bisporus ranged from 12 to 16 kg m(-2). The yield ratios 25°C/17°C ranged from 0.8 to 1.2. The variety burnettii originated in the Sonoran Desert in California showed adaptation for quickly producing fruiting bodies at high temperature when humidity conditions were favorable. Strains of the variety bisporus showed interesting potentials for their ability to produce mature fruiting bodies at higher temperature than present cultivars and might be used in breeding programs. Copyright © 2012 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

Research progress of nanoparticles as enzyme mimetics

Science.gov (United States)

Hu, XiaoNa; Liu, JianBo; Hou, Shuai; Wen, Tao; Liu, WenQi; Zhang, Ke; He, WeiWei; Ji, YingLu; Ren, HongXuan; Wang, Qi; Wu, XiaoChun

2011-10-01

Natural enzymes as biological catalysts possess remarkable advantages, especially their highly efficient and selective catalysis under mild conditions. However, most natural enzymes are proteins, thus exhibiting an inherent low durability to harsh reaction conditions. Artificial enzyme mimetics have been pursued extensively to avoid this drawback. Quite recently, some inorganic nanoparticles (NPs) have been found to exhibit unique enzyme mimetics. In addition, their much higher stability overcomes the inherent disadvantage of natural enzymes. Furthermore, easy mass-production and low cost endow them more benefits. As a new member of artificial enzyme mimetics, they have received intense attention. In this review article, major progress in this field is summarized and future perspectives are highlighted.

Experimental Strategy to Discover Microbes with Gluten-degrading Enzyme Activities.

Science.gov (United States)

Helmerhorst, Eva J; Wei, Guoxian

2014-05-05

Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement.

Characterization of a catalase-deficient strain of Neisseria gonorrhoeae: evidence for the significance of catalase in the biology of N. gonorrhoeae.

OpenAIRE

Johnson, S R; Steiner, B M; Cruce, D D; Perkins, G H; Arko, R J

1993-01-01

We obtained a catalase-deficient (Kat-) strain of Neisseria gonorrhoeae isolated from a patient who had been unsuccessfully treated with penicillin. Quantitative enzyme assays and electrophoresis of cell extracts on native polyacrylamide gels subsequently stained for catalase and peroxidase activities failed to detect both enzymes. The strain exhibited no growth anomalies or unusual requirements when grown under ordinary laboratory conditions. However, the Kat- strain proved extremely sensiti...

[Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

Science.gov (United States)

Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

2015-09-01

Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

Mixed submerged fermentation with two filamentous fungi for cellulolytic and xylanolytic enzyme production.

Science.gov (United States)

Garcia-Kirchner, O; Muñoz-Aguilar, M; Pérez-Villalva, R; Huitrón-Vargas, C

2002-01-01

The efficient saccharification of lignocellulosic materials requires the cooperative actions of different cellulase enzyme activities: exoglucanase, endoglucanase, beta-glucosidase, and xylanase. Previous studies with the fungi strains Aureobasidium sp. CHTE-18, Penicillium sp. CH-TE-001, and Aspergillus terreus CH-TE-013, selected mainly because of their different cellulolytic and xylanolytic activities, have demonstrated the capacity of culture filtrates of cross-synergistic action in the saccharification of native sugarcane bagasse pith. In an attempt to improve the enzymatic hydrolysis of different cellulosic materials, we investigated a coculture fermentation with two of these strains to enhance the production of cellulases and xylanases. The 48-h batch experimental results showed that the mixed culture of Penicillium sp. CH-TE-001 and A. terreus CH-TE-013 produced culture filtrates with high protein content, cellulase (mainly beta-glucosidase), and xylanase activities compared with the individual culture of each strain. The same culture conditions were used in a simple medium with mineral salts, corn syrup liquor, and sugarcane bagasse pith as the sole carbon source with moderate shaking at 29 degrees C. Finally, we compared the effect of the cell-free culture filtrates obtained from the mixed and single fermentations on the saccharification of different kinds of cellulosic materials.

Tannin degradation by a novel tannase enzyme present in some Lactobacillus plantarum strains.

Science.gov (United States)

Jiménez, Natalia; Esteban-Torres, María; Mancheño, José Miguel; de Las Rivas, Blanca; Muñoz, Rosario

2014-05-01

Lactobacillus plantarum is frequently isolated from the fermentation of plant material where tannins are abundant. L. plantarum strains possess tannase activity to degrade plant tannins. An L. plantarum tannase (TanBLp, formerly called TanLp1) was previously identified and biochemically characterized. In this study, we report the identification and characterization of a novel tannase (TanALp). While all 29 L. plantarum strains analyzed in the study possess the tanBLp gene, the gene tanALp was present in only four strains. Upon methyl gallate exposure, the expression of tanBLp was induced, whereas tanALp expression was not affected. TanALp showed only 27% sequence identity to TanBLp, but the residues involved in tannase activity are conserved. Optimum activity for TanALp was observed at 30°C and pH 6 in the presence of Ca(2+) ions. TanALp was able to hydrolyze gallate and protocatechuate esters with a short aliphatic alcohol substituent. Moreover, TanALp was able to fully hydrolyze complex gallotannins, such as tannic acid. The presence of the extracellular TanALp tannase in some L. plantarum strains provides them an advantage for the initial degradation of complex tannins present in plant environments.

The Enzyme Function Initiative†

Science.gov (United States)

Gerlt, John A.; Allen, Karen N.; Almo, Steven C.; Armstrong, Richard N.; Babbitt, Patricia C.; Cronan, John E.; Dunaway-Mariano, Debra; Imker, Heidi J.; Jacobson, Matthew P.; Minor, Wladek; Poulter, C. Dale; Raushel, Frank M.; Sali, Andrej; Shoichet, Brian K.; Sweedler, Jonathan V.

2011-01-01

The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily-specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include: 1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation); 2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia; 3) computational and bioinformatic tools for using the strategy; 4) provision of experimental protocols and/or reagents for enzyme production and characterization; and 5) dissemination of data via the EFI’s website, enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal and pharmaceutical efforts. PMID

The Enzyme Function Initiative.

Science.gov (United States)

Gerlt, John A; Allen, Karen N; Almo, Steven C; Armstrong, Richard N; Babbitt, Patricia C; Cronan, John E; Dunaway-Mariano, Debra; Imker, Heidi J; Jacobson, Matthew P; Minor, Wladek; Poulter, C Dale; Raushel, Frank M; Sali, Andrej; Shoichet, Brian K; Sweedler, Jonathan V

2011-11-22

The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic, we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include (1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation), (2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia, (3) computational and bioinformatic tools for using the strategy, (4) provision of experimental protocols and/or reagents for enzyme production and characterization, and (5) dissemination of data via the EFI's Website, http://enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal, and pharmaceutical efforts. �

Isolation of nitrite-degrading strains from Douchi and their application to degrade high nitrite in Jiangshui.

Science.gov (United States)

Guo, Xing; Liu, Bianfang; Gao, Lina; Zhou, Yuan; Shan, Yuanyuan; Lü, Xin

2018-06-01

Excessive nitrite in food is potentially harmful to human health because of its carcinogenic effects caused by nitroso-dervivatives. Douchi, which widely distributed throughout the country, is a traditional solid fermented soybean food with low nitrite content. In this study, bacterias which can degrade nitrite were isolated from Douchi and identified according to 16S rDNA sequence. Acinetobacter guillouiae, Acinetobacter bereziniae, Bacillus subtilis, Bacillus tequilensis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus aryabhattai and Bacillus methylotrophicus were selected. It was shown that all strains have nitrite degradation capability, in which 99.41 % nitrite can be degraded by Bacillus subtilis NDS1. The enzyme activities of these strains were determined at 24 h and 48 h, which corresponded to their nitrite degradation rates. The strains were firstly tried to inoculate in Jiangshui, which is a kind of traditional fermented vegetable in northwest China and often has high nitrite content. It was found that Bacillus subtilis NDS1, Bacillus tequilensis NDS3, Acinetobacter bereziniae NDS4, Bacillus subtilis NDS6, Bacillus subtilis NDS12 can degrade nitrite in Jiangshui more quickly, among which Acinetobacter bereziniae NDS4 degraded almost all nitrite in 48 h while it took 180 h for control. These results indicated that the selected strains have potential to become nitrite degradition agent in food. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Selection for chlorpyrifos resistance in Liriomyza sativae Blanchard: Cross-resistance patterns, stability and biochemical mechanisms.

Science.gov (United States)

Askari-Saryazdi, Ghasem; Hejazi, Mir Jalil; Ferguson, J Scott; Rashidi, Mohammad-Reza

2015-10-01

The vegetable leafminer (VLM), Liriomyza sativae (Diptera: Agromyzidae) is a serious pest of vegetable crops and ornamentals worldwide. In cropping systems with inappropriate management strategies, development of resistance to insecticides in leafminers is probable. Chlorpyrifos is a commonly used pesticide for controlling leafminers in Iran, but resistance to this insecticide in leafminers has not been characterized. In order to develop strategies to minimize resistance in the field and greenhouse, a laboratory selected chlorpyrifos resistant strain of L. sativae was used to characterize resistance and determine the rate of development and stability of resistance. Selecting for resistance in the laboratory after 23 generations yielded a chlorpyrifos resistant selected strain (CRSS) with a resistance ratio of 40.34, determined on the larval stage. CRSS exhibited no cross-resistance to other tested insecticides except for diazinon. Synergism and biochemical assays indicated that esterases (EST) had a key role in metabolic resistance to chlorpyrifos, but glutathione S-transferase (GST) and mixed function oxidase (MFO) were not mediators in this resistance. In CRSS acetylcholinesterase (AChE) was more active than the susceptible strain, Sharif (SH). AChE in CRSS was also less sensitive to inhibition by propoxur. The kinetics parameters (Km and Vmax) of AChE indicated that affinities and hydrolyzing efficiencies of this enzyme in CRSS were higher than SH. Susceptibility to chlorpyrifos in L. sativae was re-gained in the absence of insecticide pressure. Synergism, biochemical and cross-resistance assays revealed that overactivity of metabolic enzymes and reduction in target site sensitivity are probably joint factors in chlorpyrifos resistance. An effective insecticide resistance management program is necessary to prevent fast resistance development in crop systems. Copyright © 2015 Elsevier Inc. All rights reserved.

Does cypermethrin affect enzyme activity, respiration rate and walking behavior of the maize weevil (Sitophilus zeamais)?

Institute of Scientific and Technical Information of China (English)

Ronnie Von Santos Veloso; Eliseu José G.Pereira; Raul Narciso C.Guedes; Maria Goreti A.Oliveira

2013-01-01

Insecticides cause a range of sub-lethal effects on targeted insects,which are frequently detrimental to them.However,targeted insects are able to cope with insecticides within sub-lethal ranges,which vary with their susceptibility.Here we assessed the response of three strains of the maize weevil Sitophilus zeamais Motschulsky (Coleoptera:Curculionidae) to sub-lethal exposure to the pyrethoid insecticide cypermethrin.We expected enzyme induction associated with cypermethrin resistance since it would aid the resistant insects in surviving such exposure.Lower respiration rate and lower activity were also expected in insecticide-resistant insects since these traits are also likely to favor survivorship under insecticide exposure.Curiously though,cypermethrin did not affect activity of digestive and energy metabolism enzymes,and even reduced the activity of some enzymes (particularly for cellulase and cysteine-proteinase activity in this case).There was strain variation in response,which may be (partially) related to insecticide resistance in some strains.Sub-lethal exposure to cypermethrin depressed proteolytic and mainly cellulolytic activity in the exposed insects,which is likely to impair their fitness.However,such exposure did not affect respiration rate and walking behavior of the insects (except for the susceptible strain where walking activity was reduced).Walking activity varies with strain and may minimize insecticide exposure,which should be a concern,particularly if associated with (physiological) insecticide resistance.

Substrate specificity of glucose dehydrogenase and carbon source utilization pattern of pantoea dispersa strain P2 and its radiation induced mutants

Energy Technology Data Exchange (ETDEWEB)

Lee, Young Keun; Murugesan, Senthilkumar [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

2009-06-15

Mineral phosphate solubilizing pantoea dispersa strain P2 produced 5.5 mM and 42.6 mM of gluconic acid on 24 h and 72 h incubation, respectively. Strain P2 exhibited glucose dehydrogenase (GDH) specific activity of 0.32 IU mg{sup -1} protein. We have studied the substrate specificity of GDH as well as carbon source utilization pattern of strain P2. GDH of strain P2 did not use ribose as substrate. Utilization of lactose with specific activity of 0.65 IU mg{sup -1} protein indicated that the enzyme belongs to GDH type B isozyme. Arabinose, galactose, ribose, sucrose and xylose did not induce the synthesis of GDH enzyme while mannose induced the synthesis of GDH with highest specific activity of 0.58 IU mg{sup -1} protein. Through radiation mutagenesis, the substrate specificity of GDH was modified in order to utilize side range of sugars available in root exudates. Ribose, originally not a substrate for GDH of strain P2 was utilized as substrate by mutants P2-M5 with specific activity of 0.44 and 0.57 IU mg{sup -1} protein, respectively. Specific activity of GDH on the media containing lactose and galactose was also improved to 1.2 and 0.52 IU mg{sup -1} protein in P2-M5 and P2-M6 respectively. Based on the carbon source availability in root exudate, the mutants can be selected and utilized as efficient biofertilizer under P-deficient soil conditions.

The Dimerization Domain in DapE Enzymes Is required for Catalysis

OpenAIRE

Nocek, Boguslaw; Starus, Anna; Makowska-Grzyska, Magdalena; Gutierrez, Blanca; Sanchez, Stephen; Jedrzejczak, Robert; Mack, Jamey C.; Olsen, Kenneth W.; Joachimiak, Andrzej; Holz, Richard C.

2014-01-01

The emergence of antibiotic-resistant bacterial strains underscores the importance of identifying new drug targets and developing new antimicrobial compounds. Lysine and meso-diaminopimelic acid are essential for protein production and bacterial peptidoglycan cell wall remodeling and are synthesized in bacteria by enzymes encoded within dap operon. Therefore dap enzymes may serve as excellent targets for developing a new class of antimicrobial agents. The dapE-encoded N-succinyl-L,L-diaminopi...

Kinases of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae: an overview

Directory of Open Access Journals (Sweden)

Alexandre Melo Bailão

2007-01-01

Full Text Available Mycoplasma synoviae and Mycoplasma hyopneumoniae are wall-less eubacteria belonging to the class of Mollicutes. These prokaryotes have a reduced genome size and reduced biosynthetic machinery. They cause great losses in animal production. M. synoviae is responsible for an upper respiratory tract disease of chickens and turkeys. M. hyopneumoniae is the causative agent of enzootic pneumonia in pigs. The complete genomes of these organisms showed 17 ORFs encoding kinases in M. synoviae and 15 in each of the M. hyopneumoniae strain. Four kinase genes were restricted to the avian pathogen while three were specific to the pig pathogen when compared to each other. All deduced kinases found in the non pathogenic strain (J[ATCC25934] were also found in the pathogenic M. hyopneumoniae strain. The enzymes were classified in nine families composing five fold groups.

Succession of Selected Strains of Acetobacter pasteurianus and Other Acetic Acid Bacteria in Traditional Balsamic Vinegar

OpenAIRE

Gullo, M.; De Vero, L.; Giudici, P.

2009-01-01

The application of a selected Acetobacter pasteurianus strain for traditional balsamic vinegar production was assessed. Genomic DNA was extracted from biofilms after enrichment cultures on GYC medium (10% glucose, 1.0% yeast extract, 2.0% calcium carbonate) and used for PCR/denaturing gradient gel electrophoresis, 16S rRNA gene sequencing, and enterobacterial repetitive intergenic consensus/PCR sequencing. Results suggested that double-culture fermentation is suitable for traditional balsamic...

Selection by mating competitiveness improves the performance of Anastrepha ludens males of the genetic sexing strain Tapachula-7.

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Quintero-Fong, L; Toledo, J; Ruiz, L; Rendón, P; Orozco-Dávila, D; Cruz, L; Liedo, P

2016-10-01

The sexual performance of Anastrepha ludens males of the Tapachula-7 genetic sexing strain, produced via selection based on mating success, was compared with that of males produced without selection in competition with wild males. Mating competition, development time, survival, mass-rearing quality parameters and pheromone production were compared. The results showed that selection based on mating competitiveness significantly improved the sexual performance of offspring. Development time, survival of larvae, pupae and adults, and weights of larvae and pupae increased with each selection cycle. Differences in the relative quantity of the pheromone compounds (Z)-3-nonenol and anastrephin were observed when comparing the parental males with the F4 and wild males. The implications of this colony management method on the sterile insect technique are discussed.

Multi-enzyme Process Modeling

DEFF Research Database (Denmark)

Andrade Santacoloma, Paloma de Gracia

are affected (in a positive or negative way) by the presence of the other enzymes and compounds in the media. In this thesis the concept of multi-enzyme in-pot term is adopted for processes that are carried out by the combination of enzymes in a single reactor and implemented at pilot or industrial scale...... features of the process and provides the information required to structure the process model by using a step-by-step procedure with the required tools and methods. In this way, this framework increases efficiency of the model development process with respect to time and resources needed (fast and effective....... In this way the model parameters that drives the main dynamic behavior can be identified and thus a better understanding of this type of processes. In order to develop, test and verify the methodology, three case studies were selected, specifically the bi-enzyme process for the production of lactobionic acid...

In vitro sensitivity of Hungarian Actinobaculum suis strains to selected antimicrobials.

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Biksi, I; Major, Andrea; Fodor, L; Szenci, O; Vetési, F

2003-01-01

In vitro antimicrobial sensitivity of 12 Hungarian isolates and the type strain ATCC 33144 of Actinobaculum suis to different antimicrobial compounds was determined both by the agar dilution and by the disc diffusion method. By agar dilution, MIC50 values in the range of 0.05-3.125 micrograms/ml were determined for penicillin, ampicillin, ceftiofur, doxycycline, tylosin, pleuromutilins, chloramphenicol, florfenicol, enrofloxacin and lincomycin. The MIC50 value of oxytetracycline and spectinomycin was 6.25 and 12.5 micrograms/ml, respectively. For ofloxacin, flumequine, neomycin, streptomycin, gentamicin, nalidixic acid, nitrofurantoin and sulphamethoxazole + trimethoprim MIC50 values were in the range of 25-100 micrograms/ml. With the disc diffusion method, all strains were sensitive to penicillin, cephalosporins examined, chloramphenicol and florfenicol, tetracyclines examined, pleuromutilins, lincomycin and tylosin. Variable sensitivity was observed for fluoroquinolones (flumequine, enrofloxacin, ofloxacin), most of the strains were susceptible to marbofloxacin. Almost all strains were resistant to aminoglycosides but most of them were sensitive to spectinomycin. A strong correlation was determined for disc diffusion and MIC results (Spearman's rho 0.789, p < 0001). MIC values of the type strain and MIC50 values of other tested strains did not differ significantly. Few strains showed a partially distinct resistance pattern for erythromycin, lincomycin and ampicillin in both methods.

A benchmark of co-flow and cyclic deposition/etch approaches for the selective epitaxial growth of tensile-strained Si:P

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Hartmann, J. M.; Veillerot, M.; Prévitali, B.

2017-10-01

We have compared co-flow and cyclic deposition/etch processes for the selective epitaxial growth of Si:P layers. High growth rates, relatively low resistivities and significant amounts of tensile strain (up to 10 nm min-1, 0.55 mOhm cm and a strain equivalent to 1.06% of substitutional C in Si:C layers) were obtained at 700 °C, 760 Torr with a co-flow approach and a SiH2Cl2 + PH3 + HCl chemistry. This approach was successfully used to thicken the sources and drains regions of n-type fin-shaped Field Effect Transistors. Meanwhile, the (Si2H6 + PH3/HCl + GeH4) CDE process evaluated yielded at 600 °C, 80 Torr even lower resistivities (0.4 mOhm cm, typically), at the cost however of the tensile strain which was lost due to (i) the incorporation of Ge atoms (1.5%, typically) into the lattice during the selective etch steps and (ii) a reduction by a factor of two of the P atomic concentration in CDE layers compared to that in layers grown in a single step (5 × 1020 cm-3 compared to 1021 cm-3).

Tannin Degradation by a Novel Tannase Enzyme Present in Some Lactobacillus plantarum Strains

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Jiménez, Natalia; Esteban-Torres, María; Mancheño, José Miguel; de las Rivas, Blanca

2014-01-01

Lactobacillus plantarum is frequently isolated from the fermentation of plant material where tannins are abundant. L. plantarum strains possess tannase activity to degrade plant tannins. An L. plantarum tannase (TanBLp, formerly called TanLp1) was previously identified and biochemically characterized. In this study, we report the identification and characterization of a novel tannase (TanALp). While all 29 L. plantarum strains analyzed in the study possess the tanBLp gene, the gene tanALp was present in only four strains. Upon methyl gallate exposure, the expression of tanBLp was induced, whereas tanALp expression was not affected. TanALp showed only 27% sequence identity to TanBLp, but the residues involved in tannase activity are conserved. Optimum activity for TanALp was observed at 30°C and pH 6 in the presence of Ca2+ ions. TanALp was able to hydrolyze gallate and protocatechuate esters with a short aliphatic alcohol substituent. Moreover, TanALp was able to fully hydrolyze complex gallotannins, such as tannic acid. The presence of the extracellular TanALp tannase in some L. plantarum strains provides them an advantage for the initial degradation of complex tannins present in plant environments. PMID:24610854

Treatment of bran containing bread by baking enzymes; effect on the growth of probiotic bacteria on soluble dietary fiber extract in vitro.

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Saarinen, Markku T; Lahtinen, Sampo J; Sørensen, Jens F; Tiihonen, Kirsti; Ouwehand, Arthur C; Rautonen, Nina; Morgan, Andrew

2012-01-01

Different ways of treating bran by baking enzymes prior to dough making and the baking process were used to increase the amount of water-soluble dietary fiber (DF) in wheat bread with added bran. Soluble DF was extracted from the bread with water and separated from the digestible material with gastrointestinal tract enzymes and by solvent precipitation. The baking enzyme mixtures tested (xylanase and glucanase/cellulase, with and without lipase) increased the amounts of soluble arabinoxylan and protein resistant to digestion. The isolated fiber was used as a growth substrate for 11 probiotic and intestinal Bifidobacterium strains, for commensal strains of Bacteroides fragilis and Escherichia coli, and for potential intestinal pathogenic strains of E. coli O157:H7, Salmonella typhimurium, and Clostridium perfringens. Fermentation analyses indicated that the tested strains had varying capacity to grow in the presence of the extracted fiber. Of the tested probiotic strains B. longum species generally showed the highest ability to utilize the fiber extracts, although the potential pathogens tested also showed an ability to grow on these fiber extracts. In sum, the enzymes used to improve the baking process for high-fiber bread can also be used to produce in situ soluble fiber material, which in turn can exert prebiotic effects on certain potentially beneficial microbes.

Identification and isolation of bacteria containing OPH enzyme for biodegradation of organophosphorus pesticide diazinon from contaminated agricultural soil

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Sara Mobarakpoor

2015-04-01

Full Text Available Background: Organophosphorus insecticide diazinon has been widely used in agriculture and has the ability to transfer and accumulate in soil, water and animal tissues, and to induce toxicity in plants, animals and humans. In humans, diazinon inhibits nerve transmission by inactivating acetylcholinesterase enzyme. The present study was carried out to identify bacteria containing OPH enzyme for biodegradation of diazinon from contaminated agricultural soil. Methods: In this study, 8 contaminated agricultural soil samples that were exposed to pesticides, especially diazinon in the last two decades, were collected from the farms of Hamedan province. After preparing the media, for isolation of several bacterial strains containing OPH enzyme that are capable of biodegrading organophosphorus pesticides by diazinon enzymatic hydrolysis, bacterial genomic DNA extraction, plasmid product sequencing, phylogenetic sequence processing and phylogenetic tree drawing were carried out. Results: Eight bacterial strains, capable of secreting OPH enzyme, were isolated from soil samples, one of which named BS-1 with 86% similarity to Bacillus safensis displayed the highest organophosphate-hydrolyzing capability and can be used as a source of carbon and phosphorus. Conclusion: It can be concluded that the isolated bacterial strain identified in this study with OPH enzyme secretion has the potential for biodegradation of organophosphorus pesticides, especially diazinon in invitro conditions. Also, further studies such as the environmental stability and interaction, production strategies, safety, cost-benefit, environmental destructive parameters, and, toxicological, genetic and biochemical aspects are recommended prior to the application of bacterial strains in the field-scale bioremediation.

Cold-Adapted Enzymes

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Georlette, D.; Bentahir, M.; Claverie, P.; Collins, T.; D'amico, S.; Delille, D.; Feller, G.; Gratia, E.; Hoyoux, A.; Lonhienne, T.; Meuwis, M.-a.; Zecchinon, L.; Gerday, Ch.

In the last few years, increased attention has been focused on enzymes produced by cold-adapted micro-organisms. It has emerged that psychrophilic enzymes represent an extremely powerful tool in both protein folding investigations and for biotechnological purposes. Such enzymes are characterised by an increased thermosensitivity and, most of them, by a higher catalytic efficiency at low and moderate temperatures, when compared to their mesophilic counterparts. The high thermosensitivity probably originates from an increased flexibility of either a selected area of the molecular edifice or the overall protein structure, providing enhanced abilities to undergo conformational changes during catalysis at low temperatures. Structure modelling and recent crystallographic data have allowed to elucidate the structural parameters that could be involved in this higher resilience. It was demonstrated that each psychrophilic enzyme adopts its own adaptive strategy. It appears, moreover, that there is a continuum in the strategy of protein adaptation to temperature, as the previously mentioned structural parameters are implicated in the stability of thermophilic proteins. Additional 3D crystal structures, site-directed and random mutagenesis experiments should now be undertaken to further investigate the stability-flexibility-activity relationship.

Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering.

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Ninh, Pham Huynh; Honda, Kohsuke; Sakai, Takaaki; Okano, Kenji; Ohtake, Hisao

2015-01-01

In vitro reconstitution of an artificial metabolic pathway is an emerging approach for the biocatalytic production of industrial chemicals. However, several enzymes have to be separately prepared (and purified) for the construction of an in vitro metabolic pathway, thereby limiting the practical applicability of this approach. In this study, genes encoding the nine thermophilic enzymes involved in a non-ATP-forming chimeric glycolytic pathway were assembled in an artificial operon and co-expressed in a single recombinant Escherichia coli strain. Gene expression levels of the thermophilic enzymes were controlled by their sequential order in the artificial operon. The specific activities of the recombinant enzymes in the cell-free extract of the multiple-gene-expression E. coli were 5.0-1,370 times higher than those in an enzyme cocktail prepared from a mixture of single-gene-expression strains, in each of which a single one of the nine thermophilic enzymes was overproduced. Heat treatment of a crude extract of the multiple-gene-expression cells led to the denaturation of indigenous proteins and one-step preparation of an in vitro synthetic pathway comprising only a limited number of thermotolerant enzymes. Coupling this in vitro pathway with other thermophilic enzymes including the H2 O-forming NADH oxidase or the malate/lactate dehydrogenase facilitated one-pot conversion of glucose to pyruvate or lactate, respectively. © 2014 Wiley Periodicals, Inc.

Lignocellulose-Adapted Endo-Cellulase Producing Streptomyces Strains for Bioconversion of Cellulose-Based Materials.

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Ventorino, Valeria; Ionata, Elena; Birolo, Leila; Montella, Salvatore; Marcolongo, Loredana; de Chiaro, Addolorata; Espresso, Francesco; Faraco, Vincenza; Pepe, Olimpia

2016-01-01

Twenty-four Actinobacteria strains, isolated from Arundo donax, Eucalyptus camaldulensis and Populus nigra biomass during natural biodegradation and with potential enzymatic activities specific for the degradation of lignocellulosic materials, were identified by a polyphasic approach. All strains belonged to the genus Streptomyces ( S .) and in particular, the most highly represented species was Streptomyces argenteolus representing 50% of strains, while 8 strains were identified as Streptomyces flavogriseus (synonym S. flavovirens ) and Streptomyces fimicarius (synonyms Streptomyces acrimycini, Streptomyces baarnensis, Streptomyces caviscabies , and Streptomyces flavofuscus ), and the other four strains belonged to the species Streptomyces drozdowiczii, Streptomyces rubrogriseus, Streptomyces albolongus , and Streptomyces ambofaciens . Moreover, all Streptomyces strains, tested for endo and exo-cellulase, cellobiase, xylanase, pectinase, ligninase, peroxidase, and laccase activities using qualitative and semi-quantitative methods on solid growth medium, exhibited multiple enzymatic activities (from three to six). The 24 strains were further screened for endo-cellulase activity in liquid growth medium and the four best endo-cellulase producers ( S. argenteolus AE58P, S. argenteolus AE710A, S. argenteolus AE82P, and S. argenteolus AP51A) were subjected to partial characterization and their enzymatic crude extracts adopted to perform saccharification experiments on A. donax pretreated biomass. The degree of cellulose and xylan hydrolysis was evaluated by determining the kinetics of glucose and xylose release during 72 h incubation at 50°C from the pretreated biomass in the presence of cellulose degrading enzymes (cellulase and β-glucosidase) and xylan related activities (xylanase and β-xylosidase). The experiments were carried out utilizing the endo-cellulase activities from the selected S. argenteolus strains supplemented with commercial β-gucosidase and

Genotypic characterization of Azotobacteria isolated from Argentinean soils and plant-growth-promoting traits of selected strains with prospects for biofertilizer production.

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Rubio, Esteban Julián; Montecchia, Marcela Susana; Tosi, Micaela; Cassán, Fabricio Darío; Perticari, Alejandro; Correa, Olga Susana

2013-01-01

The genetic diversity among 31 putative Azotobacter isolates obtained from agricultural and non-agricultural soils was assessed using rep-PCR genomic fingerprinting and identified to species level by ARDRA and partial 16S rRNA gene sequence analysis. High diversity was found among the isolates, identified as A. chroococcum, A. salinestris, and A. armeniacus. Selected isolates were characterized on the basis of phytohormone biosynthesis, nitrogenase activity, siderophore production, and phosphate solubilization. Indole-3 acetic-acid (IAA), gibberellin (GA3) and zeatin (Z) biosynthesis, nitrogenase activity, and siderophore production were found in all evaluated strains, with variation among them, but no phosphate solubilization was detected. Phytohormones excreted to the culture medium ranged in the following concentrations: 2.2-18.2 μ g IAA mL(-1), 0.3-0.7 μ g GA3 mL(-1), and 0.5-1.2 μ g Z mL(-1). Seed inoculations with further selected Azotobacter strains and treatments with their cell-free cultures increased the number of seminal roots and root hairs in wheat seedlings. This latter effect was mimicked by treatments with IAA-pure solutions, but it was not related to bacterial root colonization. Our survey constitutes a first approach to the knowledge of Azotobacter species inhabiting Argentinean soils in three contrasting geographical regions. Moreover, this phenotypic characterization constitutes an important contribution to the selection of Azotobacter strains for biofertilizer formulations.

Characterization of cellulolytic enzymes and bioH2 production from anaerobic thermophilic Clostridium sp. TCW1.

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Lo, Yung-Chung; Huang, Chi-Yu; Cheng, Chieh-Lun; Lin, Chiu-Yue; Chang, Jo-Shu

2011-09-01

A thermophilic anaerobic bacterium Clostridium sp. TCW1 was isolated from dairy cow dung and was used to produce hydrogen from cellulosic feedstock. Extracellular cellulolytic enzymes produced from TCW1 strain were identified as endoglucanases (45, 53 and 70 kDa), exoglucanase (70 kDa), xylanases (53 and 60 kDa), and β-glucosidase (45 kDa). The endoglucanase and xylanase were more abundant. The optimal conditions for H2 production and enzyme production of the TCW1 strain were the same (60 °C, initial pH 7, agitation rate of 200 rpm). Ten cellulosic feedstock, including pure or natural cellulosic materials, were used as feedstock for hydrogen production by Clostridium strain TCW1 under optimal culture conditions. Using filter paper at 5.0 g/L resulted in the most effective hydrogen production performance, achieving a H2 production rate and yield of 57.7 ml/h/L and 2.03 mol H2/mol hexose, respectively. Production of cellulolytic enzyme activities was positively correlated with the efficiency of dark-H2 fermentation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Structure-based redesign of lysostaphin yields potent antistaphylococcal enzymes that evade immune cell surveillance

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Kristina Blazanovic

Full Text Available Staphylococcus aureus infections exert a tremendous burden on the health-care system, and the threat of drug-resistant strains continues to grow. The bacteriolytic enzyme lysostaphin is a potent antistaphylococcal agent with proven efficacy against both drug-sensitive and drug-resistant strains; however, the enzyme's own bacterial origins cause undesirable immunogenicity and pose a barrier to clinical translation. Here, we deimmunized lysostaphin using a computationally guided process that optimizes sets of mutations to delete immunogenic T cell epitopes without disrupting protein function. In vitro analyses showed the methods to be both efficient and effective, producing seven different deimmunized designs exhibiting high function and reduced immunogenic potential. Two deimmunized candidates elicited greatly suppressed proliferative responses in splenocytes from humanized mice, while at the same time the variants maintained wild-type efficacy in a staphylococcal pneumonia model. Overall, the deimmunized enzymes represent promising leads in the battle against S. aureus.

5-Fluorouracil-resistant strain of Methanobacterium thermoautortrophicum

International Nuclear Information System (INIS)

Nagle, D.P. Jr.; Teal, R.; Eisenbraun, A.

1987-01-01

Growth of Methanobacterium thermoautotrophicum Marburg is inhibited by the pyrimidine, 5-fluorouracil (FU). It was shown previously that methanogenesis is not inhibited to the same extent as growth. A spontaneously occurring FU-resistant strain (RTAE-1) was isolated from a culture of strain Marburg. The growth of both strains was inhibited by 5-fluorodeoxyuridine but not 5-fluorocytosine, and the wild type was more susceptible to inhibition by 5-azauracil and 6-azauracil than was strain RTAE-1. The cellular targets for the pyrimidine analogs are not known. When the accumulation of 14 C-labeled uracil or FU by the two strains was compared, the wilt type took up 15-fold more radiolabel per cell than did the FU-resistant strain. In the wild type, radiolabel from uracil was incorporated into the soluble pool, RNA, and DNA. The metabolism of uracil appeared to involve a uracil phosphoribosyltransferase activity. Strain Marburg extracts contained this enzyme, whereas FU-resistant strain RTAE-1 extracts had less than 1/10 as much activity. Although it is possible that a change in permeability to the compounds plays a role in the stable resistance of strain RTAE-1, the fact that it lacks the ability to metabolize pyrimidines to nucleotides is sufficient to account for its phenotype

5-Fluorouracil-resistant strain of Methanobacterium thermoautotrophicum.

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Nagle, D P; Teal, R; Eisenbraun, A

1987-09-01

Growth of Methanobacterium thermoautotrophicum Marburg is inhibited by the pyrimidine, 5-fluorouracil (FU). It was shown previously that methanogenesis is not inhibited to the same extent as growth. A spontaneously occurring FU-resistant strain (RTAE-1) was isolated from a culture of strain Marburg. The growth of both strains was inhibited by 5-fluorodeoxyuridine but not 5-fluorocytosine, and the wild type was more susceptible to inhibition by 5-azauracil and 6-azauracil than was strain RTAE-1. The cellular targets for the pyrimidine analogs are not known. When the accumulation of 14C-labeled uracil or FU by the two strains was compared, the wild type took up 15-fold more radiolabel per cell than did the FU-resistant strain. In the wild type, radiolabel from uracil was incorporated into the soluble pool, RNA, and DNA. The metabolism of uracil appeared to involve a uracil phosphoribosyltransferase activity. Strain Marburg extracts contained this enzyme, whereas FU-resistant strain RTAE-1 extracts had less than 1/10 as much activity. Although it is possible that a change in permeability to the compounds plays a role in the stable resistance of strain RTAE-1, the fact that it lacks the ability to metabolize pyrimidines to nucleotides is sufficient to account for its phenotype.

[Isolation and identification of rumen bacteria for cellulolytic enzyme production].

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Aihemaiti, Maierhaba; Zhen, Fan; Li, Yuezhong; Aibaidoula, Gulisimayi; Yimit, Wusiman

2013-05-04

We screened aerobic bacteria with cellulolytic activity from ruminal fluid of sheep, cattle and camel in Xinjiang. Fresh ruminal fluid was inoculated on sterilized sodium carboxymethylcellulose agar plates. Highly cellulolytic aerobic bacteria were screened out by using Congo red staining and liquid secondary screening culture media. The combination of morphological and biochemical test with 16SrDNA sequence analysis were used to classify the strains. Enzymatic activities of four strains with strong cellulose-decomposing abilities were studied under different culture conditions. Out 84 isolated cellulolytic strains, 40 exhibited strong abilities in decomposing cellulose. They are including 37 Gram-negative isolates and 3 Gram-positive strains. Identification of these 40 strains shows that they belong to 11 species of 6 genera, 16 strains in Stenotrophomonas maltophilia, 10 Ochrobactrum, 5 Sphingobacterium, 3 Microbacterium, 3 Paracoccus and 2 Pseudomonas. The results of the enzymatic studies of four strains with strong cellulolytic abilities indicates that the strains have the best enzyme producing property when straw powder was chosen as the carbon source; the pH at 5.5 -6.0 and temperature at 37 degrees C. The strains with highly cellulolytic abilities isolated from ruminal fluid show strong abilities in cellulose decomposition.

Lactic acid bacteria affect serum cholesterol levels, harmful fecal enzyme activity, and fecal water content

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Chung Myung

2009-06-01

Full Text Available Abstract Background Lactic acid bacteria (LAB are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as lower cholesterol. Although present in many foods, most trials have been in spreads or dairy products. Here we tested whether Bifidobacteria isolates could lower cholesterol, inhibit harmful enzyme activities, and control fecal water content. Methods In vitro culture experiments were performed to evaluate the ability of Bifidobacterium spp. isolated from healthy Koreans (20~30 years old to reduce cholesterol-levels in MRS broth containing polyoxyethanylcholesterol sebacate. Animal experiments were performed to investigate the effects on lowering cholesterol, inhibiting harmful enzyme activities, and controlling fecal water content. For animal studies, 0.2 ml of the selected strain cultures (108~109 CFU/ml were orally administered to SD rats (fed a high-cholesterol diet every day for 2 weeks. Results B. longum SPM1207 reduced serum total cholesterol and LDL levels significantly (p B. longum SPM1207 also increased fecal LAB levels and fecal water content, and reduced body weight and harmful intestinal enzyme activities. Conclusion Daily consumption of B. longum SPM1207 can help in managing mild to moderate hypercholesterolemia, with potential to improve human health by helping to prevent colon cancer and constipation.

INVERTASE FROM A CANDIDA STELLATA STRAIN ISOLATED FROM GRAPE: PRODUCTION AND PHYSICO-CHEMICAL CHARACTERIZATION

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Cristiane Abe Gargel

2014-08-01

Full Text Available Invertases are enzymes which hydrolyze the sucrose and are widely employed in food and pharmaceutical industries. In this work, the screening of autochthonous grape yeasts from Brazil was carried out in order to investigate their invertase production potential. Yeasts belonging to Saccharomyces, Hanseniaspora, Sporidiobolus, Issatchenkia, Candida, Cryptococcus and Pichia genera were analyzed by submerged fermentation (SbmF using sucrose as substrate. Among them, Candida stellata strain (N5 strain was selected as the best producer (10.6 U/ml after 48 hours of SbmF. This invertase showed optimal activity at pH 3.0 and 55°C, demonstrating appropriate characters for application in several industrial processes, which includes high temperatures and acid pHs. In addition, this invertase extract presented tolerance to low concentrations of ethanol, suggesting that it could also be suitable for application at the beginning of alcoholic fermentation. These data provide promising prospects of the use of this new invertase in food and ethanol industry.

Laccase Enzymes in Inocula Pleurotus spp

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Nora García-Oduardo

2017-01-01

Full Text Available The cultivation of edible and medicinal mushrooms Pleurotus has been aimed at promoting alternative management for agricultural products. This basidiomicete has been the subject of numerous studies because of its fruiting body constitutes a food, being a producer of enzymes with industrial interest and for its ability of biotransformation of lignocellulosic substrates. Pleurotus inocula in the established technology for growing edible and medicinal mushrooms in the CEBI Research- Production Plant were performed using sorghum or wheat. However, it is possible to expand the possibilities with other substrates. In this paper, the results of laccase enzymes production in inocula prepared with sorghum, corn and coffee pulp with two strains Pleurotus ostreatus CCEBI 3021 and Pleurotus ostreatus CCEBI 3024 are presented. The period of preparation of seed reaches 15-21 days, the measurements of laccase activity were performed in periods of seven days. Extraction of crude enzyme was performed in aqueous phase, the determination of the laccase enzyme activity, using guaiacol as substrate. The results obtained in this work with studies in previous work using sorghum as inocula are compared. It is found that higher yields are obtained laccase in coffee pulp. This study contributes to the theoretical knowledge and to provide alternatives for securing the production process of the plant.

Purification and characterization of an eggshell membrane decomposing protease from Pseudomonas aeruginosa strain ME-4.

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Cheng, Minyi; Takenaka, Shinji; Aoki, Shunsuke; Murakami, Shuichiro; Aoki, Kenji

2009-04-01

A bacterial strain, ME-4, isolated from farm soil and identified as Pseudomonas aeruginosa, grew well on a medium containing eggshell membrane (ESM). P. aeruginosa strain ME-4 decomposed the ESM by producing an extracellular protease able to solubilize it. The protease was purified to homogeneity from culture supernatant by fractionation with (NH(4))(2)SO(4), as well as CM52 cellulose and DE52 cellulose column chromatography, with a final yield of 47%. The molecular mass of the enzyme was 33 kDa. The isolated enzyme was a metalloprotease and was strongly inhibited by EDTA, o-phenanthroline, and phosphoramidon. The enzyme inhibited by these reagents was reactivated in the presence of several metal ions. The enzyme acted on various proteins and showed higher activity with collagen than collagenase from Clostridium histolyticum. Results of assays with the FRETS combinatorial libraries revealed that the enzyme preferred Ser at the P1 position and Lys at the P2 position. It also preferred hydrophobic amino acid residues at the P1' and P2' positions. The enzyme showed a much higher solubilization activity with the ESM substrate than commercially obtained enzymes. The enzyme decomposed ESM to produce water-soluble peptides, Val-Leu-Pro-Pro and (X)-Val-Pro-Pro, and a free amino acid, tryptophan.

Bacillus amyloliquefaciens SUBSP. plantarum PROBIOTIC STRAINS AS PROTEASE PRODUCERS

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E. V. Маtseliukh

2015-04-01

Full Text Available Proteases from probiotic strains of the genus Bacillus, just like the antibiotics, bacteriocins and other hydrolytic enzymes, are one of the main factors that determine their biological activity. The aim of this work was to study the synthesis and biochemical properties of proteases from two strains Bacillus amyloliquefaciens subsp. plantarum UCM B-5139 and UCM B-5140 that included in the probiotic Endosporin. The cultivation of strains was carried out in flasks under rotating for two days. The influence of physico-chemical parameters of the reaction medium on proteolytic activity was studied on partially purified protease preparations. Lytic activity was determined by turbidimetric method. On the second day of cultivation B. amyloliquefaciens subsp. plantarum UCM В-5139 and UCM В-5140 synthesized the metal-dependent peptidase and serine protease, respectively. The optimum conditions of their action were the following: temperature 37–40 °C and pH 6.5–7.0. Isolated proteases are able to lyse the living cells of Staphylococcus aureus and Candida albicans. Thus we demonstrated that B. amyloliquefaciens subsp. plantarum UCM B-5140 and UCM B-5139, included in the probiotic veterinary preparation Endosporin, produced proteolytic enzymes that hydrolyze the native insoluble proteins (elastin, fibrin and collagen. These enzymes belong to the group of neutral metal-dependent and serine proteases. They are active under physiological conditions against gram-positive bacteria and yeasts. The application of these proteases in biotechnology is considered.

Evaluation of MALDI-TOF mass spectrometry for the competitiveness analysis of selected indigenous cowpea (Vigna unguiculata L. Walp.) Bradyrhizobium strains from Kenya.

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Ndungu, Samuel Mathu; Messmer, Monika M; Ziegler, Dominik; Thuita, Moses; Vanlauwe, Bernard; Frossard, Emmanuel; Thonar, Cécile

2018-06-01

Cowpea N 2 fixation and yield can be enhanced by selecting competitive and efficient indigenous rhizobia. Strains from contrasting agro-ecologies of Kilifi and Mbeere (Kenya) were screened. Two pot experiments were established consisting of 13 Bradyrhizobium strains; experiment 1 (11 Mbeere + CBA + BK1 from Burkina Faso), experiment 2 (12 Kilifi + CBA). Symbiotic effectiveness was assessed (shoot biomass, SPAD index and N uptake). Nodule occupancy of 13 simultaneously co-inoculated strains in each experiment was analyzed by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) to assess competitiveness. Strains varied in effectiveness and competitiveness. The four most efficient strains were further evaluated in a field trial in Mbeere during the 2014 short rains. Strains from bacteroids of cowpea nodules from pot and field experiments were accurately identified as Bradyrhizobium by MALDI-TOF based on the SARAMIS™ database. In the field, abundant indigenous populations 7.10 × 10 3 rhizobia g -1 soil, outcompeted introduced strains. As revealed by MALDI-TOF, indigenous strains clustered into six distinct groups (I, II, III, IV, V and VI), group III were most abundant occupying 80% of nodules analyzed. MALDI-TOF was rapid, affordable and reliable to identify Bradyrhizobium strains directly from nodule suspensions in competition pot assays and in the field with abundant indigenous strains thus, its suitability for future competition assays. Evaluating strain competitiveness and then symbiotic efficacy is proposed in bioprospecting for potential cowpea inoculant strains.

Determination of yeast killer activity in fermenting sugarcane juice using selected ethanol-making strains

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Sandra Regina Ceccato-Antonini

2004-03-01

Full Text Available Twenty-four yeasts out of 342 isolated from the fermentative process showed killer activity and three of them were selected for the fermentative efficiency evaluation in batch system with cell recycle, flask and fermentor experiments. The selected three killer strains did not present similar results to those of pressed (baking yeast concerning ethanol (0.07-0.18; 0.12-0.20; 0.10-0.13; 0.22-0.25 g/g, respectively and biomass (0.19-0.26; 0.33-0.39; 0.13-0.27; 0.47-0.61 g/g, respectively yields and fermentative efficiency (12.3-36.3; 21.0-40.0; 19.3-26.3; 47.6-54.0 %, respectively in sugarcane juice, in flasks. In fermentor, similar behaviour was observed. However, the selected strains showed high cellular viability and killer activity (using cell-free filtrate along the fermentative cycles, in spite of the unfavourable conditions of the medium, like high pH variation of the medium (from 5.5-6.0 to 3.0-4.0, low aeration and higher temperature (30º C, which were not the ideal ones for the production/activity of killer toxins. A Pichia strain (CCA 510 showed the best results among the killer yeasts tested, exhibiting a killer activity against 92% of isolated fermentative yeasts of the process and against the pressed (baking ferment. It also demonstrated killer activity (using crude toxin preparation at higher temperatures (38ºC and low pH (4.0 after 72 hours of incubation, under proliferative and non-proliferative conditions. The results indicated that the killer activity should be a characteristic to be looked for in the strain selection for ethanolic fermentation, beside other important productivity-based characteristics, since it assure the permanence of the selected strain during the process.A atividade 'killer' poderia garantir às leveduras fermentativas uma vantagem competitiva sobre outras linhagens durante a fermentação etanólica, no entanto, pouco se sabe sobre o papel do sistema 'killer' nesse tipo de fermentação alcoólica. A sele

Pathway Construction in Corynebacterium glutamicum and Strain Engineering To Produce Rare Sugars from Glycerol.

Science.gov (United States)

Yang, Jiangang; Zhu, Yueming; Men, Yan; Sun, Shangshang; Zeng, Yan; Zhang, Ying; Sun, Yuanxia; Ma, Yanhe

2016-12-21

Rare sugars are valuable natural products widely used in pharmaceutical and food industries. In this study, we expected to synthesize rare ketoses from abundant glycerol using dihydroxyacetone phosphate (DHAP)-dependent aldolases. First, a new glycerol assimilation pathway was constructed to synthesize DHAP. The enzymes which convert glycerol to 3-hydroxypropionaldehyde and l-glyceraldehyde were selected, and their corresponding aldehyde synthesis pathways were constructed in vivo. Four aldol pathways based on different aldolases and phosphorylase were gathered. Next, three pathways were assembled and the resulting strains synthesized 5-deoxypsicose, 5-deoxysorbose, and 5-deoxyfructose from glucose and glycerol and produce l-fructose, l-tagatose, l-sorbose, and l-psicose with glycerol as the only carbon source. To achieve higher product titer and yield, the recombinant strains were further engineered and fermentation conditions were optimized. Fed-batch culture of engineered strains obtained 38.1 g/L 5-deoxypsicose with a yield of 0.91 ± 0.04 mol product per mol of glycerol and synthesized 20.8 g/L l-fructose, 10.3 g/L l-tagatose, 1.2 g/L l-sorbose, and 0.95 g/L l-psicose.

Novel nonsense mutation in the katA gene of a catalase-negative Staphylococcus aureus strain.

Science.gov (United States)

Lagos, Jaime; Alarcón, Pedro; Benadof, Dona; Ulloa, Soledad; Fasce, Rodrigo; Tognarelli, Javier; Aguayo, Carolina; Araya, Pamela; Parra, Bárbara; Olivares, Berta; Hormazábal, Juan Carlos; Fernández, Jorge

2016-01-01

We report the first description of a rare catalase-negative strain of Staphylococcus aureus in Chile. This new variant was isolated from blood and synovial tissue samples of a pediatric patient. Sequencing analysis revealed that this catalase-negative strain is related to ST10 strain, which has earlier been described in relation to S. aureus carriers. Interestingly, sequence analysis of the catalase gene katA revealed presence of a novel nonsense mutation that causes premature translational truncation of the C-terminus of the enzyme leading to a loss of 222 amino acids. Our study suggests that loss of catalase activity in this rare catalase-negative Chilean strain is due to this novel nonsense mutation in the katA gene, which truncates the enzyme to just 283 amino acids. Copyright © 2015 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Evaluation of possible occurrence of mutation in MMR repair system genes in resistant and sensitiveclinical strains of Mycobacterium tuberculosisby using sequencing method

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AmirPoyan Afzali

2016-07-01

Full Text Available Background:during recent years, the incidence and spread of drug resistance in Mycobacterium tuberculosis, the bacterium causing tuberculosis, has set this disease in World Health Organizationpriorities alignment of diseases like AIDS and hepatitis. Study of close examination of resistant and susceptible clinical strains genotypes is necessary to overcome drug resistance. Among the numerous repair systems, only there are limited number of encoding genes of DNA repair enzymes in Mycobacterium tuberculosis. Commonly these genes have been conserved and any changes among them likely increasethe mutation occurance due to the impossibility of correctionof spontaneous mutations insensitive strains of this bacteria.mut genes encodeDNA repairable enzymes.This study investigated the mutations in these genes and the effect of these mutations on tuberculosis drug resistance. Materials&Methods: In this study,of 29 available specimens,we were selected 8 susceptible strains and 21 resistantstrains andafter ordering appropriate primers and performing the proliferation reaction two types of amplicons produced which includingfragments of genes mut T2 and mut T4 and they were sent inorder to sequencing. Results:The results of chain reactionprimer represents an appropriate choice of primerswhich were investigated. Sequencing results showed that overall 73% of resistant strains that had been selected for study of mutT4gene, have no mutations in codons 48of mutT4 gene, and 70% of resistant strains have no GGA >>> CGA mutation at codon 58 of mutT2 gene. Conclusion: One of the strategies to overcome tuberculosis drug resistance is a close examination of genotypes of resistant and susceptible clinical strains. Results of this study was performedby examining changes in mut T2 and mut T4 gene sequence. The mutation in mut T2 always associated with mutation in mut T4, in this way, the first mutation may occurs in mut T4and after that, the second mutationmay occurs in mut T

Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris

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Simona Capone

2015-09-01

Full Text Available Horseradish peroxidase (HRP, conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris, the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1 was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins.

Selection and molecular characterization of cellulolytic-xylanolytic fungi from surface soil-biomass mixtures from Black Belt sites.

Science.gov (United States)

Okeke, Benedict C; Hall, Rosine W; Nanjundaswamy, Ananda; Thomson, M Sue; Deravi, Yasaman; Sawyer, Leah; Prescott, Andrew

2015-06-01

Plant biomass is an abundant renewable natural resource that can be transformed into chemical feedstocks. Enzymes used in saccharification of lignocellulosic biomass are a major part of biofuel production costs. A cocktail of cellulolytic and xylanolytic enzymes are required for optimal saccharification of biomass. Accordingly, thirty-two fungal pure cultures were obtained from surface soil-biomass mixtures collected from Black Belt sites in Alabama by culturing on lignocellulosic biomass medium. The fungal strains were screened for the coproduction of cellulolytic and xylanolytic enzymes. Strains that displayed promising levels of cellulolytic and xylanolytic enzymes were characterized by molecular analysis of DNA sequences from the large subunit and internal transcribed spacer (ITS) of their ribosomal RNA gene. Nucleotide sequence analysis revealed that two most promising isolates FS22A and FS5A were most similar to Penicillium janthinellum and Trichoderma virens. Production dynamics of cellulolytic and xylanolytic enzymes from these two strains were explored in submerged fermentation. Volumetric productivity after 120 h incubation was 121.08 units/L/h and 348 units/L/h for the filter paper cellulase and xylanase of strain FS22A, and 90.83 units/L/h and 359 units/L/h, respectively for strain FS5A. Assays with 10 times dilution of enzymes revealed that the activities were much higher than that observed in the crude culture supernatant. Additionally, both FS22A and FS5A also produced amylase in lignocellulose medium. The enzyme profiles of these strains and their activities suggest potential applications in cost effective biomass conversion and biodegradation. Copyright © 2015 Elsevier GmbH. All rights reserved.

Selection of Rhizobium strain from Wonogiri, Central Java on the growth of soybean (Glycine max L. on the sand sterile medium in greenhouse

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SRI PURWANINGSIH

2005-07-01

Full Text Available An experiment on the selection of Rhizobium strain from Wonogiri, Central Java on the growth of soybean (Glycine max L. on the sand sterile medium in green house. The aim of the experiment the selection and potency of the Rhizobium strain to increase the growth of soybean. The experiment was carried out in green house condition in Microbiology Division, Research Center for Biology-LIPI with sterile sand medium. The research design was Completely Randomized Design with three replications for each treatment. The Rhizobium strains used were 1 W (isolated from bean, Vigna radiata, 2 W (isolated from soybean, 3 W (isolated from bean, 4 W (isolated from soybean, 5 W (isolated from soybean, 6 W (isolated from peanut, Arachis hypogaea, 7 W (isolated from peanut, 8 W (isolated from peanut, the controls were uninoculated with Rhizobium strain and without urea fertilizer (K1, uninoculated and with urea fertilizer equal 100 kg/ha (K2. The plants were harvested after 50 days, the variable of investigation were the dry weight of canopy, roots, nodules root, total plants, number of nodules and ‘symbiotic capacity”. The results showed that all of experiment plant which be inoculated with Rhizobium able to form nodule. Strain of 2 W (isolated from soybean has given the best effects on the growth of soybean.

Effect of irradiation on protease production by a Philippine strain of Aspergillus oryzae (ahlburg) cohn

International Nuclear Information System (INIS)

Anglo, P.G.

1974-03-01

The Philippine strain of Aspergillus oryzae (ahlburg) cohn. was exposed to ultraviolet rays and ionizing radiation from cobalt-60 for the purpose of obtaining possible mutants or resistant strains which produce powerful proteolytic enzymes. Out of 58 isolates, only 3 gave significant proteolytic values (PV) high enough to merit further investigation. The isolates, G-10, G-110, and 23-110, were picked from plates exposed to gamma rays from cobalt-60. Optimum incubation temperature for these isolates for highest percentage of active protease was 24 0 -27 0 C. The isolates were found capable of producing active protease from the second day of incubation up to the fifth day, whereas the activity of the parent strain was retained the fourth day only. The isolates showed maximum digestive ability at 25 0 -55 0 C, giving proteolytic values of 833. The pH activity curves showed that the enzyme produced by the irradiated isolates G-10 and G-110 were very active at pH 9.0-10.0, and isolate 23-110 at pH 6.0-10.0. The parent strain revealed two pH optima, one at pH 7.5-8.5 and the other at pH 9.0-9.5. Crude enzyme powder gave activities comparable to alkalase and maxatase, commercial proteolytic enzymes imported from Belgium and Netherlands being used as component of laundry detergents by some manufacturing companies in the Philippines. The results obtained give valuable information for the commercial application of the enzyme. Since the organism can produce high yields of protease from copra meal, a by-product of the coconut industry, commerical feasibility may be envisioned in the near future

Revealing differences in metabolic flux distributions between a mutant strain and its parent strain Gluconacetobacter xylinus CGMCC 2955.

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Cheng Zhong

Full Text Available A better understanding of metabolic fluxes is important for manipulating microbial metabolism toward desired end products, or away from undesirable by-products. A mutant strain, Gluconacetobacter xylinus AX2-16, was obtained by combined chemical mutation of the parent strain (G. xylinus CGMCC 2955 using DEC (diethyl sulfate and LiCl. The highest bacterial cellulose production for this mutant was obtained at about 11.75 g/L, which was an increase of 62% compared with that by the parent strain. In contrast, gluconic acid (the main byproduct concentration was only 5.71 g/L for mutant strain, which was 55.7% lower than that of parent strain. Metabolic flux analysis indicated that 40.1% of the carbon source was transformed to bacterial cellulose in mutant strain, compared with 24.2% for parent strain. Only 32.7% and 4.0% of the carbon source were converted into gluconic acid and acetic acid in mutant strain, compared with 58.5% and 9.5% of that in parent strain. In addition, a higher flux of tricarboxylic acid (TCA cycle was obtained in mutant strain (57.0% compared with parent strain (17.0%. It was also indicated from the flux analysis that more ATP was produced in mutant strain from pentose phosphate pathway (PPP and TCA cycle. The enzymatic activity of succinate dehydrogenase (SDH, which is one of the key enzymes in TCA cycle, was 1.65-fold higher in mutant strain than that in parent strain at the end of culture. It was further validated by the measurement of ATPase that 3.53-6.41 fold higher enzymatic activity was obtained from mutant strain compared with parent strain.

Selective epitaxial growth properties and strain characterization of Si1- x Ge x in SiO2 trench arrays

Science.gov (United States)

Koo, Sangmo; Jang, Hyunchul; Ko, Dae-Hong

2017-04-01

In this study, we investigated the formation of a Si1- x Ge x fin structure in SiO2 trench arrays via an ultra-high-vacuum chemical-vapor deposition (UHV-CVD) selective epitaxial growth (SEG) process. Defect generation and microstructures of Si1- x Ge x fin structures with different Ge concentrations ( x = 0.2, 0.3 and 0.45) were examined. In addition, the strain evolution of a Si1- x Ge x fin structure was analyzed by using reciprocal space mapping (RSM). An (111) facet was formed from the Si1- x Ge x epi-layer and SiO2 trench wall interface to minimize the interface and the surface energy. The Si1- x Ge x fin structures were fully relaxed along the direction perpendicular to the trenches regardless of the Ge concentration. On the other hand, the fin structures were fully or partially strained along the direction parallel to the trenches depending on the Ge concentration: fully strained Si0.8Ge0.2 and Si0.7Ge0.3, and a Si0.55Ge0.45 strain-relaxed buffer. We further confirmed that the strain on the Si1- x Ge x fin structures remained stable after oxide removal and H2/N2 post-annealing.

Fructose 6-phosphate phosphoketolase activity in wild-type strains of Lactobacillus, isolated from the intestinal tract of pigs.

Science.gov (United States)

Bolado-Martínez, E; Acedo-Félix, E; Peregrino-Uriarte, A B; Yepiz-Plascencia, G

2012-01-01

Phosphoketolases are key enzymes of the phosphoketolase pathway of heterofermentative lactic acid bacteria, which include lactobacilli. In heterofermentative lactobacilli xylulose 5-phosphate phosphoketolase (X5PPK) is the main enzyme of the phosphoketolase pathway. However, activity of fructose 6-phosphate phosphoketolase (F6PPK) has always been considered absent in lactic acid bacteria. In this study, the F6PPK activity was detected in 24 porcine wild-type strains of Lactobacillus reuteri and Lactobacillus mucosae, but not in the Lactobacillus salivarius or in L. reuteri ATCC strains. The activity of F6PPK increased after treatment of the culture at low-pH and diminished after porcine bile-salts stress conditions in wild-type strains of L. reuteri. Colorimetric quantification at 505 nm allowed to differentiate between microbial strains with low activity and without the activity of F6PPK. Additionally, activity of F6PPK and the X5PPK gene expression levels were evaluated by real time PCR, under stress and nonstress conditions, in 3 L. reuteri strains. Although an exact correlation, between enzyme activity and gene expression was not obtained, it remains possible that the xpk gene codes for a phosphoketolase with dual substrate, at least in the analyzed strains of L. reuteri.

Expression of enzymes in yeast for lignocellulose derived oligomer CBP

Science.gov (United States)

McBride, John E.; Wiswall, Erin; Shikhare, Indraneel; Xu, Haowen; Thorngren, Naomi; Hau, Heidi H.; Stonehouse, Emily

2017-08-29

The present invention provides a multi-component enzyme system that hydrolyzes hemicellulose oligomers from hardwood which can be expressed, for example, in yeast such as Saccharomyces cerevisiae. In some embodiments, this invention provides for the engineering of a series of biocatalysts combining the expression and secretion of components of this enzymatic system with robust, rapid xylose utilization, and ethanol fermentation under industrially relevant process conditions for consolidated bioprocessing. In some embodiments, the invention utilizes co-cultures of strains that can achieve significantly improved performance due to the incorporation of additional enzymes in the fermentation system.

Decrease in Activities of Selected Rat Liver Enzymes following ...

African Journals Online (AJOL)

The effects of the chemical effluent from Soap and Detergent Industry on some rat liver enzymes were investigated. Chemical analyses of both the effluent and tap water which served as the control were carried out before various concentrations of the effluent (5%v/v, 25%v/v, 50%v/v and 100%v/v) were made. The effluent ...

Selecting the Best: Evolutionary Engineering of Chemical Production in Microbes

DEFF Research Database (Denmark)

Shepelin, Denis; Hansen, Anne Sofie Lærke; Lennen, Rebecca

2018-01-01

, we focus primarily on a more challenging problem-the use of evolutionary engineering for improving the production of chemicals in microbes directly. We describe recent developments in evolutionary engineering strategies, in general, and discuss, in detail, case studies where production of a chemical......Microbial cell factories have proven to be an economical means of production for many bulk, specialty, and fine chemical products. However, we still lack both a holistic understanding of organism physiology and the ability to predictively tune enzyme activities in vivo, thus slowing down rational...... engineering of industrially relevant strains. An alternative concept to rational engineering is to use evolution as the driving force to select for desired changes, an approach often described as evolutionary engineering. In evolutionary engineering, in vivo selections for a desired phenotype are combined...

Potential of Laceyella sacchari strain B42 crude xylanase in ...

African Journals Online (AJOL)

rajmac

2013-02-06

Feb 6, 2013 ... sacchari strain B42. Maximal xylanase production was achieved at the incubation period of 48 h with ... chemicals for pulp processing and bleaching. The major ... industrial enzyme with great biotechnological application.

Production of native-starch-degrading enzymes by a Bacillus firmus/lentus strain

NARCIS (Netherlands)

Wijbenga, Dirk-Jan; Beldman, Gerrit; Veen, Anko; Binnema, Doede

1991-01-01

A bacterium belonging to the Bacillus firmus/lentus-complex and capable of growth on native potato starch was isolated from sludge of a pilot plant unit for potato-starch production. Utilization of a crude enzyme preparation obtained from the culture fluid after growth of the microorganism on native

Mercury (II) removal by resistant bacterial isolates and mercuric (II) reductase activity in a new strain of Pseudomonas sp. B50A.

Science.gov (United States)

Giovanella, Patricia; Cabral, Lucélia; Bento, Fátima Menezes; Gianello, Clesio; Camargo, Flávio Anastácio Oliveira

2016-01-25

This study aimed to isolate mercury resistant bacteria, determine the minimum inhibitory concentration for Hg, estimate mercury removal by selected isolates, explore the mer genes, and detect and characterize the activity of the enzyme mercuric (II) reductase produced by a new strain of Pseudomonas sp. B50A. The Hg removal capacity of the isolates was determined by incubating the isolates in Luria Bertani broth and the remaining mercury quantified by atomic absorption spectrophotometry. A PCR reaction was carried out to detect the merA gene and the mercury (II) reductase activity was determined in a spectrophotometer at 340 nm. Eight Gram-negative bacterial isolates were resistant to high mercury concentrations and capable of removing mercury, and of these, five were positive for the gene merA. The isolate Pseudomonas sp. B50A removed 86% of the mercury present in the culture medium and was chosen for further analysis of its enzyme activity. Mercuric (II) reductase activity was detected in the crude extract of this strain. This enzyme showed optimal activity at pH 8 and at temperatures between 37 °C and 45 °C. The ions NH4(+), Ba(2+), Sn(2+), Ni(2+) and Cd(2+) neither inhibited nor stimulated the enzyme activity but it decreased in the presence of the ions Ca(2+), Cu(+) and K(+). The isolate and the enzyme detected were effective in reducing Hg(II) to Hg(0), showing the potential to develop bioremediation technologies and processes to clean-up the environment and waste contaminated with mercury. Copyright © 2015 Elsevier B.V. All rights reserved.

Decreasing Distortion Energies without Strain: Diazo-Selective 1,3-Dipolar Cycloadditions.

Science.gov (United States)

Gold, Brian; Aronoff, Matthew R; Raines, Ronald T

2016-07-15

The diazo group has attributes that complement those of the azido group for applications in chemical biology. Here, we use computational analyses to provide insights into the chemoselectivity of the diazo group in 1,3-dipolar cycloadditions. Dipole distortion energies are responsible for ∼80% of the overall energetic barrier for these reactions. Here, we show that diazo compounds, unlike azides, provide an opportunity to decrease that barrier substantially without introducing strain into the dipolarophile. The ensuing rate enhancement is due to the greater nucleophilic character of a diazo group compared to that of an azido group, which can accommodate decreased distortion energies without predistortion. The tuning of distortion energies with substituents in a diazo compound or dipolarophile can enhance reactivity and selectivity in a predictable manner. Notably, these advantages of diazo groups are amplified in water. Our findings provide a theoretical framework that can guide the design and application of both diazo compounds and azides in "orthogonal" contexts, especially for biological investigations.

Molecular relationships between closely related strains and species of nematodes

Science.gov (United States)

Butler, M. H.; Wall, S. M.; Luehrsen, K. R.; Fox, G. E.; Hecht, R. M.

1981-01-01

Electrophoretic comparisons have been made for 24 enzymes in the Bergerac and Bristol strains of Caenorhabditis elegans and the related species, Caenorhabditis briggsae. No variation was detected between the two strains of C. elegans. In contrast, the two species, C. elegans and C. briggsae exhibited electrophoretic differences in 22 of 24 enzymes. A consensus 5S rRNA sequence was determined for C. elegans and found to be identical to that from C. briggsae. By analogy with other species with relatively well established fossil records it can be inferred that the time of divergence between the two nematode species is probably in the tens of millions of years. The limited anatomical evolution during a time period in which proteins undergo extensive changes supports the hypothesis that anatomical evolution is not dependent on overall protein changes.

Decolorization and degradation of daunomycin by bjerkandera adusta R59 strain

Energy Technology Data Exchange (ETDEWEB)

Cho, N.S.; Belearz, A.; Ginalska, G.; Kornillowicz, K.; Cho, H.Y.; Ohga, S. [Kyushu University, Fukuoka (Japan)

2009-02-15

The ability of Bjerkandera adusta R59 strain to degrade anthraquinonic antibiotic (daunomycin) points on its possible aptitudes for decomposing of other anthraquinonic derivatives, e.g. lignocellulose subunits or metabolically related lipids, present in wood. This study was performed to investigate the possibility of B. adusta, R59 to synthesize enzymes participating in decay of wood compounds (including lignin, celluloses, hemicelluloses and lipids). Geotrichum-like strain, anamorphic stadium of B. adusta, white-rot. fungus, was isolated from soil. It was found to completely decolorize and degrade 10% daunomycin post-production effluent during 10 days of incubation at 26{sup o}C. R59 strain produces only small activities of lignolytic enzymes when grown on wheat straw or beech sawdust-containing media but in the presence of humic acids derived from brown coal synthesizes significant activities of laccase and lipase. This phenomenon was coupled with entering the idiophase by this fungus and appearance of aerial mycelium. The ability of B. adusta R59 strain to degrade humic acids from brown coal could be useful in constructing of new generation of biologically active filters for purification of humic acids-contaminated comestible waters.

molecular identification of rotavirus strains associated with diarrhea

African Journals Online (AJOL)

DR. AMINU

ABSTRACT. The study was carried out to determine the molecular characteristics of the rotavirus strains associated with diarrhea among children in Kwara state, Nigeria. A total of 150 stool samples were collected from diarrheic children. The stool samples were screened for rotavirus,using Enzyme linked Immunosorbent ...

Molecular identification of rotavirus strains associated with diarrhea ...

African Journals Online (AJOL)

The study was carried out to determine the molecular characteristics of the rotavirus strains associated with diarrhea among children in Kwara state, Nigeria. A total of 150 stool samples were collected from diarrheic children. The stool samples were screened for rotavirus,using Enzyme linked Immunosorbent assay (ELISA).

Tetracycline-resistant Escherichia coli strains are inherited from parents and persist in the infant's intestines in the absence of selective pressure.

Science.gov (United States)

Prelog, Martina; Grif, Katharina; Decristoforo, Cornelia; Würzner, Reinhard; Kiechl-Kohlendorfer, Ursula; Brunner, Andrea; Zimmerhackl, Lothar Bernd; Orth, Dorothea

2009-10-01

The study investigated tetracycline (TC), ampicillin (AMP), cefazolin (CEF), and trimethoprim (TMP) resistance in Escherichia coli (E. coli) in the feces of 21 infants up to 6 months of age and in their parents in the absence of selective antimicrobial pressure. Clonality of strains was assessed by pulsed-field gel electrophoresis. Three infants had resistant E. coli strains in their feces identical to the mothers' from week 1 on, which persisted over weeks. From week 2 on, in another four infants, persisting resistant E. coli were found, two of them identical to the mothers'. All of these persisting E. coli strains (except one family) showed at least resistance to TC. In infants, resistant E. coli strains inherited from their mothers tended to persist over months. Therefore, the persistence of resistant E. coli and their possible capacity to cause symptomatic infection or transfer its resistance genes to other bacteria deserves more attention.

Effect of rana galamensis–based diet on the activities of some enzymes and histopathology of selected tissues of albino rats

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Basiru Olaitan Ajiboye

2016-10-01

Full Text Available The effect of Rana galamensis-based diet on the activities of some enzymes and histopathology of selected tissues of albino rats was investigated for eight weeks. A total of sixteen albino rats weighing between 29.15 and 26.01g (21 days old were divided into two groups. The first group contains animals fed on casein-based diet (control; the second group was fed on Rana galamensis-based diet. The animals were fed with their appropriate diet on daily basis and on the eight weeks of the experiment the animals were sacrificed using diethyl ether as anesthesia, blood was collected by cardiac puncture and organs of interest were harvested. Thereafter, organ to body weight ratio, some biochemical parameters and histopathology examination were carried out. There was no significant difference (p >0.05 in the organ to body weight ratio of the animals fed on control and Rana galamensis-based diets. Also, there was no significant different (p >0.05 in the activities of all the enzymes (ALP [alkaline phosphatase], AST [asparate transaminase], ALT [alanine transaminase], and γGT [gamma glutamyl transferase] investigated in the selected tissues and serum of rats fed on Rana galamensis- based diet when compared with the control. In addition, histological examinations of hepatocyte's rats fed on Rana galamensis- based diet show normal architecture structure when compared with the control. The insignificant different in the activities of all the enzymes studies (ALP, AST, ALT and γGT indicated no organ damage, supported by the normal histology studies. The obtained results may imply that Rana galamensis is safe for consumption.  Normal 0 false false false EN-US X-NONE X-NONE

Altered Regulation of Escherichia coli Biotin Biosynthesis in BirA Superrepressor Mutant Strains

Science.gov (United States)

Chakravartty, Vandana

2012-01-01

Transcription of the Escherichia coli biotin (bio) operon is directly regulated by the biotin protein ligase BirA, the enzyme that covalently attaches biotin to its cognate acceptor proteins. Binding of BirA to the bio operator requires dimerization of the protein, which is triggered by BirA-catalyzed synthesis of biotinoyl-adenylate (biotinoyl-5′-AMP), the obligatory intermediate of the ligation reaction. Although several aspects of this regulatory system are well understood, no BirA superrepressor mutant strains had been isolated. Such superrepressor BirA proteins would repress the biotin operon transcription in vivo at biotin concentrations well below those needed for repression by wild-type BirA. We isolated mutant strains having this phenotype by a combined selection-screening approach and resolved multiple mutations to give several birA superrepressor alleles, each having a single mutation, all of which showed repression dominant over that of the wild-type allele. All of these mutant strains repressed bio operon transcription in vivo at biotin concentrations that gave derepression of the wild-type strain and retained sufficient ligation activity for growth when overexpressed. All of the strains except that encoding G154D BirA showed derepression of bio operon transcription upon overproduction of a biotin-accepting protein. In BirA, G154D was a lethal mutation in single copy, and the purified protein was unable to transfer biotin from enzyme-bound biotinoyl-adenylate either to the natural acceptor protein or to a biotin-accepting peptide sequence. Consistent with the transcriptional repression data, each of the purified mutant proteins showed increased affinity for the biotin operator DNA in electrophoretic mobility shift assays. Surprisingly, although most of the mutations were located in the catalytic domain, all of those tested, except G154D BirA, had normal ligase activity. Most of the mutations that gave superrepressor phenotypes altered residues

Effect of strain rate and temperature on mechanical properties of selected building Polish steels

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Moćko Wojciech

2015-01-01

Full Text Available Currently, the computer programs of CAD type are basic tool for designing of various structures under impact loading. Application of the numerical calculations allows to substantially reduce amount of time required for the design stage of such projects. However, the proper use of computer aided designing technique requires input data for numerical software including elastic-plastic models of structural materials. This work deals with the constitutive model developed by Rusinek and Klepaczko (RK applied for the modelling of mechanical behaviour of selected grades structural St0S, St3SX, 18GS and 34GS steels and presents here results of experimental and empirical analyses to describe dynamic elastic-plastic behaviours of tested materials at wide range of temperature. In order to calibrate the RK constitutive model, series of compression tests at wide range of strain rates, including static, quasi-static and dynamic investigations at lowered, room and elevated temperatures, were carried out using two testing stands: servo-hydraulic machine and split Hopkinson bar. The results were analysed to determine influence of temperature and strain rate on visco-plastic response of tested steels, and show good correlation with experimental data.

Genotypic Characterization of Azotobacteria Isolated from Argentinean Soils and Plant-Growth-Promoting Traits of Selected Strains with Prospects for Biofertilizer Production

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Esteban Julián Rubio

2013-01-01

Full Text Available The genetic diversity among 31 putative Azotobacter isolates obtained from agricultural and non-agricultural soils was assessed using rep-PCR genomic fingerprinting and identified to species level by ARDRA and partial 16S rRNA gene sequence analysis. High diversity was found among the isolates, identified as A. chroococcum, A. salinestris, and A. armeniacus. Selected isolates were characterized on the basis of phytohormone biosynthesis, nitrogenase activity, siderophore production, and phosphate solubilization. Indole-3 acetic-acid (IAA, gibberellin (GA3 and zeatin (Z biosynthesis, nitrogenase activity, and siderophore production were found in all evaluated strains, with variation among them, but no phosphate solubilization was detected. Phytohormones excreted to the culture medium ranged in the following concentrations: 2.2–18.2 μg IAA mL−1, 0.3–0.7 μg GA3 mL−1, and 0.5–1.2 μg Z mL−1. Seed inoculations with further selected Azotobacter strains and treatments with their cell-free cultures increased the number of seminal roots and root hairs in wheat seedlings. This latter effect was mimicked by treatments with IAA-pure solutions, but it was not related to bacterial root colonization. Our survey constitutes a first approach to the knowledge of Azotobacter species inhabiting Argentinean soils in three contrasting geographical regions. Moreover, this phenotypic characterization constitutes an important contribution to the selection of Azotobacter strains for biofertilizer formulations.

Isolation and Selection of Microalgal Strains from Natural Water Sources in Viet Nam with Potential for Edible Oil Production.

Science.gov (United States)

Thao, Tran Yen; Linh, Dinh Thi Nhat; Si, Vo Chi; Carter, Taylor W; Hill, Russell T

2017-06-23

fatty acids in these strains. Other strains had fatty acid compositions similar to that of palm oil. Several strains have been selected on the basis of their suitable fatty acid profiles and high lipid content for further chemical and physical characterization, toxicity and organoleptic tests of their oils, and for scale-up.

UV mutagenesis of aspergillus niger for enzyme production in submerged fermentation

International Nuclear Information System (INIS)

Irfan, M.; Syed, Q.; Javed, J.

2011-01-01

The present study was conducted to improve the enzyme production from Aspergillus niger using UV mutation. Submerged fermentation was carried out in 250 ml Erlenmeyer flask using Vogel's media at 30 deg. C for six days. Results of this study revealed that UV-mutation enhanced CM Case activity up to two times while FPase activity up to three times as compared to the parental strain. For avicelase, xylanase and fungal biomass production, UV radiation has slight effect as compared to parental strain. (author)

Determination of dehydrogenase activities involved in D-glucose oxidation in Gluconobacter and Acetobacter strains

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Florencia Sainz

2016-08-01

Full Text Available Acetic acid bacteria (AAB are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane bound dehydrogenases. In the present study, the enzyme activity of the membrane bound dehydrogenases (membrane-bound PQQ-glucose dehydrogenase (mGDH, D-gluconate dehydrogenase (GADH and membrane-bound glycerol dehydrogenase (GLDH involved in the oxidation of D-glucose and D-gluconic acid (GA was determined in six strains of three different species of AAB (three natural and three type strains. Moreover, the effect of these activities on the production of related metabolites (GA, 2-keto-D-gluconic acid (2KGA and 5-keto-D-gluconic acid (5KGA was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the A. malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h, which coincided with glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of G. oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition.Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter were

Improving the performance of electrochemical microsensors based on enzymes entrapped in a redox hydrogel

International Nuclear Information System (INIS)

Mitala, J.J.; Michael, A.C.

2006-01-01

Microsensors based on carbon fiber microelectrodes coated with enzyme-entrapping redox hydrogels facilitate the in vivo detection of substances of interest within the central nervous system, including hydrogen peroxide, glucose, choline and glutamate. The hydrogel, formed by cross-linking a redox polymer, entraps the enzymes and mediates electron transfer between the enzymes and the electrode. It is important that the enzymes are entrapped in their enzymatically active state. Should entrapment cause enzyme denaturation, the sensitivity and the selectivity of the sensor may be compromised. Synthesis of the redox polymer according to published procedures may yield a product that precipitates when added to aqueous enzyme solutions. Casting hydrogels from solutions that contain the precipitate produces microsensors with low sensitivity and selectivity, suggesting that the precipitation disrupts the structure of the enzymes. Herein, we show that a surfactant, sodium dodecyl sulfate (SDS), can prevent the precipitation and improve the sensitivity and selectivity of the sensors

Development of an indirect enzyme-linked immunosorbent assay (ELISA) to differentiate antibodies against wild-type porcine reproductive and respiratory syndrome from the vaccine strain TJM-F92 based on a recombinant Nsp2 protein.

Science.gov (United States)

Wang, X X; Wang, F X; Li, Z G; Wen, Y J; Wang, X; Song, N; Wu, H

2018-01-01

An accurate ELISA method to differentiate pigs infected with wild-type porcine reproductive and respiratory syndrome (PRRSV) strains from vaccinated ones would help to monitor PRRSV vaccination compliance. The recombinant protein GST-d120aa derived from the continuous deletion of 120 amino acids in the non-structural protein 2 region of the modified-live vaccine strain TJM-F92 was used to develop an indirect enzyme-linked immunosorbent assay (d120-ELISA) for differentiating serum antibodies against TJM-F92 from other PRRSV strains. At the optimized cut-off value which was calculated at an S/P of 0.25, it yielded a sensitivity of 90.7% and a specificity of 95.1%. Cross-reactivity tests suggested that the d120-ELISA was PRRSV-specific. Coefficient of variations of the repeatability tests ranged between 1.41-17.02%. The results suggest that the d120-ELISA is suitable for differentiating animals infected with wild-type strains from those immunized with MLV TJM-F92. Copyright © 2017. Published by Elsevier B.V.

Processing plant persistent strains of Listeria monocytogenes appear to have a lower virulence potential than clinical strains in selected virulence models

DEFF Research Database (Denmark)

Jensen, Anne; Thomsen, L.E.; Jørgensen, R.L.

2008-01-01

cell line, Caco-2; time to death in a nematode model, Caenorhabditis elegans and in a fruit fly model, Drosophila melanogaster and fecal shedding in a guinea pig model. All strains adhered to and grew in Caco-2 cells in similar levels. When exposed to 10(6) CFU/ml, two strains representing......% killed C elegans worms was longer (110 h) for the RAPD type 9 strains than for the other four strains (80 h). The Scott A strain and one RAPD type 9 strain were suspended in whipping cream before being fed to guinea pigs and the persistent RAPD type 9 strain was isolated from feces in a lower level...... to contaminate food products, and it is important to determine their virulence potential to evaluate risk to consumers. We compared the behaviour of food processing persistent and clinical L. monocytogenes strains in four virulence models: Adhesion, invasion and intracellular growth was studied in an epithelial...

Selected enzyme activities of urban heavy metal-polluted soils in the presence and absence of an oligochaete, Lampito mauritii (Kinberg)

International Nuclear Information System (INIS)

Sivakumar, S.; Nityanandi, D.; Barathi, S.; Prabha, D.; Rajeshwari, S.; Son, H.K.; Subbhuraam, C.V.

2012-01-01

Highlights: ► Soils samples were collected from five different electroplating industrial areas. ► Samples were incubated with and without earthworms for 45 days. ► All enzymes increased with duration of incubation expect phosphatase. - Abstract: Soils samples collected from five different areas (S1–S5) around electroplating industries in the city of Coimbatore were analysed for the activities of selected enzymes (cellulase, phosphatase, amylase, urease, and invertase) in the presence and absence of the earthworm Lampito mauritii (Kinberg). Heavy metal analysis of soils showed that chromium (<504 mg/kg) and copper (<28.1 mg/kg) contents were much higher than cadmium (<10.60 mg/kg) except in S5, where cadmium (10.6 mg/kg) was higher than the copper. Except for phosphatase, the activities of all enzymes increased with increasing period of incubation under laboratory conditions, both with and without earthworms. The results of the three-way ANOVA (effect of three factors- worms-with and without addition, soil and incubation time), however, showed that there was no significant difference between enzyme activities (with and without earthworm) and soil and incubation time for amylase and urease activity. Further, no significant difference was found between soils for cellulase activity and between all the above factors for urease activity. The results concluded that though the earthworms died at the end of the incubation period, the resultant increase or decrease in the enzymatic activity may be attributed to the metabolic activities of the worms during their lifetime in the experimental container. Also, the worms after death may have provided suitable substrate for the growth of the microorganisms thereby influencing enzyme activity.

Selected enzyme activities of urban heavy metal-polluted soils in the presence and absence of an oligochaete, Lampito mauritii (Kinberg)

Energy Technology Data Exchange (ETDEWEB)

Sivakumar, S., E-mail: ssivaphd@yahoo.com [Department of Health and Environment, Kosin University, Young Do Gu, Busan 606 701 (Korea, Republic of); Nityanandi, D.; Barathi, S.; Prabha, D. [Department of Environmental Sciences, Bharathiar University, Coimbatore 641 046 (India); Rajeshwari, S. [Department of Biotechnology, Karpagam University, Coimbatore 641 021 (India); Son, H.K. [Department of Health and Environment, Kosin University, Young Do Gu, Busan 606 701 (Korea, Republic of); Subbhuraam, C.V. [Department of Environmental Sciences, Bharathiar University, Coimbatore 641 046 (India)

2012-08-15

Highlights: Black-Right-Pointing-Pointer Soils samples were collected from five different electroplating industrial areas. Black-Right-Pointing-Pointer Samples were incubated with and without earthworms for 45 days. Black-Right-Pointing-Pointer All enzymes increased with duration of incubation expect phosphatase. - Abstract: Soils samples collected from five different areas (S1-S5) around electroplating industries in the city of Coimbatore were analysed for the activities of selected enzymes (cellulase, phosphatase, amylase, urease, and invertase) in the presence and absence of the earthworm Lampito mauritii (Kinberg). Heavy metal analysis of soils showed that chromium (<504 mg/kg) and copper (<28.1 mg/kg) contents were much higher than cadmium (<10.60 mg/kg) except in S5, where cadmium (10.6 mg/kg) was higher than the copper. Except for phosphatase, the activities of all enzymes increased with increasing period of incubation under laboratory conditions, both with and without earthworms. The results of the three-way ANOVA (effect of three factors- worms-with and without addition, soil and incubation time), however, showed that there was no significant difference between enzyme activities (with and without earthworm) and soil and incubation time for amylase and urease activity. Further, no significant difference was found between soils for cellulase activity and between all the above factors for urease activity. The results concluded that though the earthworms died at the end of the incubation period, the resultant increase or decrease in the enzymatic activity may be attributed to the metabolic activities of the worms during their lifetime in the experimental container. Also, the worms after death may have provided suitable substrate for the growth of the microorganisms thereby influencing enzyme activity.

Water Pollution, and Treatments Part III: Biodegradation of Oil in Refineries Waste Water and Oils Adsorbed in Agricultural Wastes by Selected Strains of Cyanobacteria

International Nuclear Information System (INIS)

El-Emary, M.M.; Ali, N.A.; Naguib, M.M.

2011-01-01

The main objective of this study is to determine the biological degradation of oil hydrocarbons and sulfur compounds of Marine Balayim crude oil and its refined products by selected indigenous Cyanobacteria strains. The oils used were Marine Balayim crude oil, skimmed oil and some refined products such as gasoline, kerosene, gas oil, fuel oil and petroleum coke. The selected organisms in the current study are the Blue-Green Algae Cyanobacteria, Oscillatoria limentica. This organism was collected from the hyper saline environment of the solar lake in Taba, Sinai, Egypt. The results obtained revealed that the utilization of such strains can be used for the bioremediation of oily waste water.

Utilization of cassava waste through fermentation technology

International Nuclear Information System (INIS)

Lotong, N.

1991-01-01

Over 400 isolates of molds were screened for raw starch digesting enzymes and aspergillus J8 ad Rhizopus N37 were selected for further investigations. Crude enzymes obtained from wheat bran was higher than from rice bran. Crude enzymes from Aspergillus is active at pH 4.0, whereas that from Rhizopus is active at pH 5.0. Aspergillus J8 gave higher yield of silage fermentation. Selection of yeast strain was accomplished, it was found that Saccharomyces cerevisiae SC90, the local commercial strain (non-flocculent) performed best in fermentation of cassava mash. Another strain AM12, a flocculent fusant strain derived from fusion between flocculent strain and sake brewing strain was comparable to that of commercial strain at normal temperature but performed better at higher temperature up to 40 deg C. It is unlikely that fuel alcohol produced from raw cassava will be able to compete with petroleum fuel at this moment. However, silage fermentation to increase nutritional quality of the silage through selected strains of microorganisms has a good prospect to pursue. (author)

Draft genome sequence of Streptomyces sp. strain F1, a potential source for glycoside hydrolases isolated from Brazilian soil

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Ricardo Rodrigues de Melo

Full Text Available ABSTRACT Here, we show the draft genome sequence of Streptomyces sp. F1, a strain isolated from soil with great potential for secretion of hydrolytic enzymes used to deconstruct cellulosic biomass. The draft genome assembly of Streptomyces sp. strain F1 has 69 contigs with a total genome size of 8,142,296 bp and G + C 72.65%. Preliminary genome analysis identified 175 proteins as Carbohydrate-Active Enzymes, being 85 glycoside hydrolases organized in 33 distinct families. This draft genome information provides new insights on the key genes encoding hydrolytic enzymes involved in biomass deconstruction employed by soil bacteria.

Growth and consumption of L-malic acid in wine-like medium by acclimated and non-acclimated cultures of Patagonian Oenococcus oeni strains.

Science.gov (United States)

Bravo-Ferrada, Bárbara Mercedes; Hollmann, Axel; Brizuela, Natalia; La Hens, Danay Valdés; Tymczyszyn, Elizabeth; Semorile, Liliana

2016-09-01

Five Oenococcus oeni strains, selected from spontaneous malolactic fermentation (MLF) of Patagonic Pinot noir wine, were assessed for their use as MLF starter cultures. After the individual evaluation of tolerance to some stress conditions, usually found in wine (pH, ethanol, SO2, and lysozyme), the behavior of the strains was analyzed in MLO broth with 14 % ethanol and pH 3.5 in order to test for the synergistic effect of high ethanol level and low pH and, finally, in a wine-like medium. Although the five strains were able to grow in MLO broth under low pH and/or high ethanol, they must be acclimated to grow in a wine-like medium. Additionally, glycosidase and tannase activities were evaluated, showing differences among the strains. The potential of the strains to ferment citrate was tested and two of the five strains showed the ability to metabolize this substrate. We did not detect the presence of genes encoding histidine, tyrosine descarboxylase, and putrescine carbamoyltransferase. All the strains tested exhibited good growth capacity and ability to consume L-malic acid in a wine-like medium after cell acclimation, and each of them showed a particular enzyme profile, which might confer different organoleptic properties to the wine.

Directing filtration to optimize enzyme immobilization in reactive membranes

DEFF Research Database (Denmark)

Luo, Jianquan; Marpani, Fauziah; Brites, Rita

2014-01-01

enzymatic reaction efficiency were evaluated in terms of enzyme loading, conversion rate and biocatalytic stability. Alcohol dehydrogenase (ADH) was selected as a model enzyme. Lower pressure, higher enzyme concentration and lower pH resulted in higher irreversible fouling resistance and lower permeate flux....... High pH during immobilization produced increased permeate flux but declines in conversion rates, likely because of the weak immobilization resulting from strong electrostatic repulsion between enzymes and membrane. The results showed that pore blocking as a fouling mechanism permitted a higher enzyme...... loading but generated more permeability loss, while cake layer formation increased enzyme stability but resulted in low loading rate. Low pH (near isoelectric point) favored hydrophobic and electrostatic adsorption of enzymes on the membrane, which reduced the enzyme stability. Neutral pH, however...

Evaluation of Oil Removal Efficiency and Enzymatic Activity in Some fungal Strains for Bioremediation of Petroleum-Polluted Soils

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Fariba Mohsenzadeh

2012-12-01

Full Text Available Background: Petroleum pollution is a global disaster and there are several soil cleaning methods including bioremediation.Methods: In a field study, fugal strains were isolated from oil-contaminated sites of Arak refinery (Iran and their growth ability was checked in potato dextrose agar (PDA media containing 0-10% v/v crude oil, the activity of three enzymes (Catalase, Peroxidase and Phenol Oxidase was evaluated in the fungal colonies and bioremediation ability of the fungi was checked in the experimental pots containing 3 kg sterilized soil and different concentrations of petroleum (0-10% w/w.Results: Four fungal strains, Acromonium sp., Alternaria sp., Aspergillus terreus and Penicillium sp., were selected asthe most resistant ones. They were able to growth in the subjected concentrations and Alternaria sp. showed thehighest growth ability in the petroleum containing media. The enzyme assay showed that the enzymatic activity was increased in the oil-contaminated media. Bioremediation results showed that the studied fungi were able to decrease petroleum pollution. The highest petroleum removing efficiency of Aspergillus terreus, Penicillium sp.,Alternaria sp. and Acromonium sp. was evaluated in the 10%, 8%, 8% and 2% petroleum pollution respectively.Conclusions: Fungi are important microorganisms in decreasing of petroleum pollution. They have bioremediation potency that is related to their enzymatic activities.

Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing.

Science.gov (United States)

Vester, Jan Kjølhede; Glaring, Mikkel Andreas; Stougaard, Peter

2014-05-20

The use of cold-active enzymes has many advantages, including reduced energy consumption and easy inactivation. The ikaite columns of SW Greenland are permanently cold (4-6°C) and alkaline (above pH 10), and the microorganisms living there and their enzymes are adapted to these conditions. Since only a small fraction of the total microbial diversity can be cultured in the laboratory, a combined approach involving functional screening of a strain collection and a metagenomic library was undertaken for discovery of novel enzymes from the ikaite columns. A strain collection with 322 cultured isolates was screened for enzymatic activities identifying a large number of enzyme producers, with a high re-discovery rate to previously characterized strains. A functional expression library established in Escherichia coli identified a number of novel cold-active enzymes. Both α-amylases and β-galactosidases were characterized in more detail with respect to temperature and pH profiles and one of the β-galactosidases, BGalI17E2, was able to hydrolyze lactose at 5°C. A metagenome sequence of the expression library indicated that the majority of enzymatic activities were not detected by functional expression. Phylogenetic analysis showed that different bacterial communities were targeted with the culture dependent and independent approaches and revealed the bias of multiple displacement amplification (MDA) of DNA isolated from complex microbial communities. Many cold- and/or alkaline-active enzymes of industrial relevance were identified in the culture based approach and the majority of the enzyme-producing isolates were closely related to previously characterized strains. The function-based metagenomic approach, on the other hand, identified several enzymes (β-galactosidases, α-amylases and a phosphatase) with low homology to known sequences that were easily expressed in the production host E. coli. The β-galactosidase BGalI17E2 was able to hydrolyze lactose at low

ACTIVITY OF SUPEROXIDE DISMUTASE ENZYME IN YEAST SACCHAROMYCES CEREVISIAE

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Blažena Lavová

2014-02-01

Full Text Available Reactive oxygen species (ROS with reactive nitrogen species (RNS are known to play dual role in biological systems, they can be harmful or beneficial to living systems. ROS can be important mediators of damage to cell structures, including proteins, lipids and nucleic acids termed as oxidative stress. The antioxidant enzymes protect the organism against the oxidative damage caused by active oxygen forms. The role of superoxide dismutase (SOD is to accelerate the dismutation of the toxic superoxide radical, produced during oxidative energy processes, to hydrogen peroxide and molecular oxygen. In this study, SOD activity of three yeast strains Saccharomyces cerevisiae was determined. It was found that SOD activity was the highest (23.7 U.mg-1 protein in strain 612 after 28 hours of cultivation. The lowest SOD activity from all tested strains was found after 56 hours of cultivation of strain Gyöng (0.7 U.mg-1 protein.

Extracellular Phytase Production by the Wine Yeast S. cerevisiae (Finarome Strain) during Submerged Fermentation.

Science.gov (United States)

Kłosowski, Grzegorz; Mikulski, Dawid; Jankowiak, Oliwia

2018-04-08

One of the key steps in the production of phytases of microbial origin is selection of culture parameters, followed by isolation of the enzyme and evaluation of its catalytic activity. It was found that conditions for S. cerevisiae yeast culture, strain Finarome, giving the reduction in phytic acid concentration of more than 98% within 24 h of incubation were as follows: pH 5.5, 32 °C, continuous stirring at 80 rpm, the use of mannose as a carbon source and aspartic acid as a source of nitrogen. The highest catalytic activity of the isolated phytase was observed at 37 °C, pH 4.0 and using phytate as substrate at concentration of 5.0 mM. The presence of ethanol in the medium at a concentration of 12% v / v reduces the catalytic activity to above 60%. Properties of phytase derived from S. cerevisiae yeast culture, strain Finarome, indicate the possibility of its application in the form of a cell's free crude protein isolate for the hydrolysis of phytic acid to improve the efficiency of alcoholic fermentation processes. Our results also suggest a possibility to use the strain under study to obtain a fusant derived with specialized distillery strains, capable of carrying out a highly efficient fermentation process combined with the utilization of phytates.

Performance response and egg qualities of laying birds fed enzyme ...

African Journals Online (AJOL)

Theperformance response and egg qualities o laying birds fed enzyme supplemented PKC diets asreplacement for maize was investigated wth 210, 20 week old layng pullets of Dominant Black strain at the Teaching and Research Farm of the Delta State University, Asaba Campus, Nigeria. The birds which ust come into ...

Current concepts on selected plant secondary metabolites with promising inhibitory effects against enzymes linked to Alzheimer's disease.

Science.gov (United States)

Orhan, I Erdogan

2012-01-01

Alzheimer's disease (AD) has become one of the deadliest diseases for human beings with special incidence in elderly population. It is a progressive neurodegenerative disease and the most prevalent cause of dementia. The neuropathology of AD has not been fully elucidated yet, however, cholinergic hypothesis is the most accepted theory nowadays, resulting from the cholinergic deficit emerging in the brains of AD patients. Shortage of the neurotransmitters, acetylcholine and butyrylcholine has been demonstrated, and therefore, inhibition of the enzymes; acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) that break down acetylcholine and butyrylcholine has become a standard approach for AD treatment. However, cholinesterase inhibitors are only effective in symptomatic treatment and have no ability to impede the disease. The pathogenesis of AD is highly complex and another hypothesis is the formation of amyloid plaques containing beta-amyloid peptide, which causes neurolesions in the brains of AD patients. Beta-amyloid peptide is generated after the sequential cleavage of amyloid precursor protein, especially by the beta- and gamma-secretase in the amyloidogenic pathway. The secretases involved in the processing of amyloid precursor protein are of particular interest and, consequently, the inhibition of secretase enzyme family of protease type has become another desired treatment strategy for AD. On the other hand, medicinal plants are attractive sources for drug research and development as they produce chemically-varying molecules with preferred biological activities. The aim of this article is to review the available data on selected inhibitors from plant secondary metabolites with emphasis on cholinesterase, prolyl endopeptidase, and secretase enzyme families as being the current treatments of AD.

Antagonistic activity of selected strains of Bacillus thuringiensis ...

African Journals Online (AJOL)

121, were effective in the reduction of R. solani infection. In addition, GM-23 increased the length of pepper seedlings. These results suggest that the B. thuringiensis strains studied have an excellent potential to be used as bio-control agents of R.