On-site cellulase production and efficient saccharification of corn stover employing cbh2 overexpressing Trichoderma reesei with novel induction system.

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Li, Yonghao; Zhang, Xiaoyue; Xiong, Liang; Mehmood, Muhammad Aamer; Zhao, Xinqing; Bai, Fengwu

2017-08-01

Although on-site cellulase production offers cost-effective saccharification of lignocellulosic biomass, low enzyme titer is still a barrier for achieving robustness. In the present study, a strain of T. reesei was developed for enhanced production of cellulase via overexpression of Cellobiohydrolase II. Furthermore, optimum enzyme production was achieved using a novel inducer mixture containing synthesized glucose-sophorose (MGD) and alkali pre-treated corn stover (APCS). Within 60h, a remarkably higher cellulase productivity and activity were achieved in the fed-batch fermentation using the optimized ratio of MGD and APCS in the inducer mixture, compared to those reported using cellulosic biomass as the sole inducer. After the enzyme production, APCS was added directly into the fermentation broth at 20% solid loading, which produced 122.5g/L glucose and 40.21g/L xylose, leading to the highest yield reported so far. The improved enzyme titers during on-site cellulase production would benefit cost-competitive saccharification of lignocellulosic biomass. Copyright © 2017 Elsevier Ltd. All rights reserved.

Enzymatic hydrolyzing performance of Acremonium cellulolyticus and Trichoderma reesei against three lignocellulosic materials

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Murakami Katsuji

2009-10-01

Full Text Available Abstract Background Bioethanol isolated from lignocellulosic biomass represents one of the most promising renewable and carbon neutral alternative liquid fuel sources. Enzymatic saccharification using cellulase has proven to be a useful method in the production of bioethanol. The filamentous fungi Acremonium cellulolyticus and Trichoderma reesei are known to be potential cellulase producers. In this study, we aimed to reveal the advantages and disadvantages of the cellulase enzymes derived from these fungi. Results We compared A. cellulolyticus and T. reesei cellulase activity against the three lignocellulosic materials: eucalyptus, Douglas fir and rice straw. Saccharification analysis using the supernatant from each culture demonstrated that the enzyme mixture derived from A. cellulolyticus exhibited 2-fold and 16-fold increases in Filter Paper enzyme and β-glucosidase specific activities, respectively, compared with that derived from T. reesei. In addition, culture supernatant from A. cellulolyticus produced glucose more rapidly from the lignocellulosic materials. Meanwhile, culture supernatant derived from T. reesei exhibited a 2-fold higher xylan-hydrolyzing activity and produced more xylose from eucalyptus (72% yield and rice straw (43% yield. Although the commercial enzymes Acremonium cellulase (derived from A. cellulolyticus, Meiji Seika Co. demonstrated a slightly lower cellulase specific activity than Accellerase 1000 (derived from T. reesei, Genencor, the glucose yield (over 65% from lignocellulosic materials by Acremonium cellulase was higher than that of Accellerase 1000 (less than 60%. In addition, the mannan-hydrolyzing activity of Acremonium cellulase was 16-fold higher than that of Accellerase 1000, and the conversion of mannan to mannobiose and mannose by Acremonium cellulase was more efficient. Conclusion We investigated the hydrolysis of lignocellulosic materials by cellulase derived from two types of filamentous fungi. We

The VELVET A Orthologue VEL1 of Trichoderma reesei Regulates Fungal Development and Is Essential for Cellulase Gene Expression

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Atanasova, Lea; Fekete, Erzsébet; Paholcsek, Melinda; Sándor, Erzsébet; Aquino, Benigno; Druzhinina, Irina S.; Karaffa, Levente; Kubicek, Christian P.

2014-01-01

Trichoderma reesei is the industrial producer of cellulases and hemicellulases for biorefinery processes. Their expression is obligatorily dependent on the function of the protein methyltransferase LAE1. The Aspergillus nidulans orthologue of LAE1 - LaeA - is part of the VELVET protein complex consisting of LaeA, VeA and VelB that regulates secondary metabolism and sexual as well as asexual reproduction. Here we have therefore investigated the function of VEL1, the T. reesei orthologue of A. nidulans VeA. Deletion of the T. reesei vel1 locus causes a complete and light-independent loss of conidiation, and impairs formation of perithecia. Deletion of vel1 also alters hyphal morphology towards hyperbranching and formation of thicker filaments, and with consequently reduced growth rates. Growth on lactose as a sole carbon source, however, is even more strongly reduced and growth on cellulose as a sole carbon source eliminated. Consistent with these findings, deletion of vel1 completely impaired the expression of cellulases, xylanases and the cellulase regulator XYR1 on lactose as a cellulase inducing carbon source, but also in resting mycelia with sophorose as inducer. Our data show that in T. reesei VEL1 controls sexual and asexual development, and this effect is independent of light. VEL1 is also essential for cellulase gene expression, which is consistent with the assumption that their regulation by LAE1 occurs by the VELVET complex. PMID:25386652

A Mitogen-Activated Protein Kinase Tmk3 Participates in High Osmolarity Resistance, Cell Wall Integrity Maintenance and Cellulase Production Regulation in Trichoderma reesei

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Wang, Mingyu; Zhao, Qiushuang; Yang, Jinghua; Jiang, Baojie; Wang, Fangzhong; Liu, Kuimei; Fang, Xu

2013-01-01

The mitogen-activated protein kinase (MAPK) pathways are important signal transduction pathways conserved in essentially all eukaryotes, but haven't been subjected to functional studies in the most important cellulase-producing filamentous fungus Trichoderma reesei. Previous reports suggested the presence of three MAPKs in T. reesei: Tmk1, Tmk2, and Tmk3. By exploring the phenotypic features of T. reesei Δtmk3, we first showed elevated NaCl sensitivity and repressed transcription of genes involved in glycerol/trehalose biosynthesis under higher osmolarity, suggesting Tmk3 participates in high osmolarity resistance via derepression of genes involved in osmotic stabilizer biosynthesis. We also showed significant downregulation of genes encoding chitin synthases and a β-1,3-glucan synthase, decreased chitin content, ‘budded’ hyphal appearance typical to cell wall defective strains, and increased sensitivity to calcofluor white/Congo red in the tmk3 deficient strain, suggesting Tmk3 is involved in cell wall integrity maintenance in T. reesei. We further observed the decrease of cellulase transcription and production in T. reesei Δtmk3 during submerged cultivation, as well as the presence of MAPK phosphorylation sites on known transcription factors involved in cellulase regulation, suggesting Tmk3 is also involved in the regulation of cellulase production. Finally, the expression of cell wall integrity related genes, the expression of cellulase coding genes, cellulase production and biomass accumulation were compared between T. reesei Δtmk3 grown in solid state media and submerged media, showing a strong restoration effect in solid state media from defects resulted from tmk3 deletion. These results showed novel physiological processes that fungal Hog1-type MAPKs are involved in, and present the first experimental investigation of MAPK signaling pathways in T. reesei. Our observations on the restoration effect during solid state cultivation suggest that T. reesei

A mitogen-activated protein kinase Tmk3 participates in high osmolarity resistance, cell wall integrity maintenance and cellulase production regulation in Trichoderma reesei.

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Mingyu Wang

Full Text Available The mitogen-activated protein kinase (MAPK pathways are important signal transduction pathways conserved in essentially all eukaryotes, but haven't been subjected to functional studies in the most important cellulase-producing filamentous fungus Trichoderma reesei. Previous reports suggested the presence of three MAPKs in T. reesei: Tmk1, Tmk2, and Tmk3. By exploring the phenotypic features of T. reesei Δtmk3, we first showed elevated NaCl sensitivity and repressed transcription of genes involved in glycerol/trehalose biosynthesis under higher osmolarity, suggesting Tmk3 participates in high osmolarity resistance via derepression of genes involved in osmotic stabilizer biosynthesis. We also showed significant downregulation of genes encoding chitin synthases and a β-1,3-glucan synthase, decreased chitin content, 'budded' hyphal appearance typical to cell wall defective strains, and increased sensitivity to calcofluor white/Congo red in the tmk3 deficient strain, suggesting Tmk3 is involved in cell wall integrity maintenance in T. reesei. We further observed the decrease of cellulase transcription and production in T. reesei Δtmk3 during submerged cultivation, as well as the presence of MAPK phosphorylation sites on known transcription factors involved in cellulase regulation, suggesting Tmk3 is also involved in the regulation of cellulase production. Finally, the expression of cell wall integrity related genes, the expression of cellulase coding genes, cellulase production and biomass accumulation were compared between T. reesei Δtmk3 grown in solid state media and submerged media, showing a strong restoration effect in solid state media from defects resulted from tmk3 deletion. These results showed novel physiological processes that fungal Hog1-type MAPKs are involved in, and present the first experimental investigation of MAPK signaling pathways in T. reesei. Our observations on the restoration effect during solid state cultivation suggest

Development of a low-cost cellulase production process using Trichoderma reesei for Brazilian biorefineries.

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Ellilä, Simo; Fonseca, Lucas; Uchima, Cristiane; Cota, Junio; Goldman, Gustavo Henrique; Saloheimo, Markku; Sacon, Vera; Siika-Aho, Matti

2017-01-01

During the past few years, the first industrial-scale cellulosic ethanol plants have been inaugurated. Although the performance of the commercial cellulase enzymes used in this process has greatly improved over the past decade, cellulases still represent a very significant operational cost. Depending on the region, transport of cellulases from a central production facility to a biorefinery may significantly add to enzyme cost. The aim of the present study was to develop a simple, cost-efficient cellulase production process that could be employed locally at a Brazilian sugarcane biorefinery. Our work focused on two main topics: growth medium formulation and strain improvement. We evaluated several Brazilian low-cost industrial residues for their potential in cellulase production. Among the solid residues evaluated, soybean hulls were found to display clearly the most desirable characteristics. We engineered a Trichoderma reesei strain to secrete cellulase in the presence of repressing sugars, enabling the use of sugarcane molasses as an additional carbon source. In addition, we added a heterologous β-glucosidase to improve the performance of the produced enzymes in hydrolysis. Finally, the addition of an invertase gene from Aspegillus niger into our strain allowed it to consume sucrose from sugarcane molasses directly. Preliminary cost analysis showed that the overall process can provide for very low-cost enzyme with good hydrolysis performance on industrially pre-treated sugarcane straw. In this study, we showed that with relatively few genetic modifications and the right growth medium it is possible to produce considerable amounts of well-performing cellulase at very low cost in Brazil using T. reesei . With further enhancements and optimization, such a system could provide a viable alternative to delivered commercial cellulases.

Nutrient control for stationary phase cellulase production in Trichoderma reesei Rut C-30.

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Callow, Nicholas V; Ray, Christopher S; Kelbly, Matthew A; Ju, Lu-Kwang

2016-01-01

This work describes the use of nutrient limitations with Trichoderma reesei Rut C-30 to obtain a prolonged stationary phase cellulase production. This period of non-growth may allow for dependable cellulase production, extended fermentation periods, and the possibility to use pellet morphology for easy product separation. Phosphorus limitation was successful in halting growth and had a corresponding specific cellulase production of 5±2 FPU/g-h. Combined with the addition of Triton X-100 for fungal pellet formation and low shear conditions, a stationary phase cellulase production period in excess of 300 h was achieved, with a constant enzyme production rate of 7±1 FPU/g-h. While nitrogen limitation was also effective as a growth limiter, it, however, also prevented cellulase production. Copyright © 2015 Elsevier Inc. All rights reserved.

Trpac1, a pH response transcription regulator, is involved in cellulase gene expression in Trichoderma reesei.

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He, Ronglin; Ma, Lijuan; Li, Chen; Jia, Wendi; Li, Demao; Zhang, Dongyuan; Chen, Shulin

2014-12-01

Fungi grow over a relatively wide pH range and adapt to extracellular pH through a genetic regulatory system mediated by a key component PacC, which is a pH transcription regulator. The cellulase production of the filamentous fungi Trichoderma reesei is sensitive to ambient pH. To investigate the connection between cellulase expression regulation and ambient pH, an ortholog of Aspergillus nidulans pacC, Trpac1, was identified and functionally characterized using a target gene deletion strategy. Deleting Trpac1 dramatically increased the cellulase production and the transcription levels of the major cellulase genes at neutral pH, which suggested Trpac1 is involved in the regulation of cellulase production. It was further observed that the expression levels of transcription factors xyr1 and ace2 also increased in the ΔTrpac1 mutant at neutral pH. In addition, the ΔTrpac1 mutant exhibited conidiation defects under neutral and alkaline pH. These results implied that Trpac1 in involved in growth and development process and cellulase gene expression in T. reesei. Copyright © 2014 Elsevier Inc. All rights reserved.

Regulation of cellulase expression, sporulation, and morphogenesis by velvet family proteins in Trichoderma reesei.

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Liu, Kuimei; Dong, Yanmei; Wang, Fangzhong; Jiang, Baojie; Wang, Mingyu; Fang, Xu

2016-01-01

Homologs of the velvet protein family are encoded by the ve1, vel2, and vel3 genes in Trichoderma reesei. To test their regulatory functions, the velvet protein-coding genes were disrupted, generating Δve1, Δvel2, and Δvel3 strains. The phenotypic features of these strains were examined to identify their functions in morphogenesis, sporulation, and cellulase expression. The three velvet-deficient strains produced more hyphal branches, indicating that velvet family proteins participate in the morphogenesis in T. reesei. Deletion of ve1 and vel3 did not affect biomass accumulation, while deletion of vel2 led to a significantly hampered growth when cellulose was used as the sole carbon source in the medium. The deletion of either ve1 or vel2 led to the sharp decrease of sporulation as well as a global downregulation of cellulase-coding genes. In contrast, although the expression of cellulase-coding genes of the ∆vel3 strain was downregulated in the dark, their expression in light condition was unaffected. Sporulation was hampered in the ∆vel3 strain. These results suggest that Ve1 and Vel2 play major roles, whereas Vel3 plays a minor role in sporulation, morphogenesis, and cellulase expression.

Ras GTPases Modulate Morphogenesis, Sporulation and Cellulase Gene Expression in the Cellulolytic Fungus Trichoderma reesei

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Zhang, Jiwei; Zhang, Yanmei; Zhong, Yaohua; Qu, Yinbo; Wang, Tianhong

2012-01-01

Background The model cellulolytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In T. reesei the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from S. cerevisiae, but their functions remain unknown. Methodology/Principal Findings Here, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of TrRas2 results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated TrRas2G16V allele exhibits increased cellulase gene transcription, the cbh1 and cbh2 expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway. Conclusions/Significance Together, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the

PEA PEEL WASTE: A LIGNOCELLULOSIC WASTE AND ITS UTILITY IN CELLULASE PRODUCTION BY Trichoderma reesei UNDER SOLID STATE CULTIVATION

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Nitin Verma

2011-03-01

Full Text Available A wide variety of waste bioresources are available on our planet for conversion into bioproducts. In the biological systems, microorganisms are used to utilize waste as an energy source for the synthesis of valuable products such as biomass proteins and enzymes. The large quantities of byproducts generated during the processing of plant food involve an economic and environmental problem due to their high volumes and elimination costs. After isolation of the main constituent, there are abundant remains which represent an inexpensive material that has been undervalued until now. Pea peel waste is one of the undervalued, unused sources of energy that can serve as a potential source for cellulase production. Batch experiments have been performed, using pea peel waste as a carbon source for cellulase production under solid state cultivation by Trichoderma reesei. It was observed that 30 oC temperature and pH 5.0 are the most favorable conditions for cellulase production by T. reesei. FPase activity significantly increases by incorporation of whey as well as wheat starch hydrolysate in the basal salt media used in the production study. The present study describes the utility of pea peel waste, whey as well as wheat starch hydrolysate in cellulase production by T. reesei. The utilization of economically cheap, pea peel waste for cellulase production could be a novel, cost effective, and valuable approach in cellulase production as well as in solid waste management.

Two Major Facilitator Superfamily Sugar Transporters from Trichoderma reesei and Their Roles in Induction of Cellulase Biosynthesis*

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

2013-01-01

Proper perception of the extracellular insoluble cellulose is key to initiating the rapid synthesis of cellulases by cellulolytic Trichoderma reesei. Uptake of soluble oligosaccharides derived from cellulose hydrolysis represents a potential point of control in the induced cascade. In this study, we identified a major facilitator superfamily sugar transporter Stp1 capable of transporting cellobiose by reconstructing a cellobiose assimilation system in Saccharomyces cerevisiae. The absence of Stp1 in T. reesei resulted in differential cellulolytic response to Avicel versus cellobiose. Transcriptional profiling revealed a different expression profile in the Δstp1 strain from that of wild-type strain in response to Avicel and demonstrated that Stp1 somehow repressed induction of the bulk of major cellulase and hemicellulose genes. Two other putative major facilitator superfamily sugar transporters were, however, up-regulated in the profiling. Deletion of one of them identified Crt1 that was required for growth and enzymatic activity on cellulose or lactose, but was not required for growth or hemicellulase activity on xylan. The essential role of Crt1 in cellulase induction did not seem to rely on its transporting activity because the overall uptake of cellobiose or sophorose by T. reesei was not compromised in the absence of Crt1. Phylogenetic analysis revealed that orthologs of Crt1 exist in the genomes of many filamentous ascomycete fungi capable of degrading cellulose. These data thus shed new light on the mechanism by which T. reesei senses and transmits the cellulose signal and offers potential strategies for strain improvement. PMID:24085297

Roles of Protein Kinase A and Adenylate Cyclase in Light-Modulated Cellulase Regulation in Trichoderma reesei

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Schuster, André; Tisch, Doris; Seidl-Seiboth, Verena; Kubicek, Christian P.

2012-01-01

The cyclic AMP (cAMP) pathway represents a central signaling cascade with crucial functions in all organisms. Previous studies of Trichoderma reesei (anamorph of Hypocrea jecorina) suggested a function of cAMP signaling in regulation of cellulase gene expression. We were therefore interested in how the crucial components of this pathway, adenylate cyclase (ACY1) and cAMP-dependent protein kinase A (PKA), would affect cellulase gene expression. We found that both ACY1 and PKA catalytic subunit 1 (PKAC1) are involved in regulation of vegetative growth but are not essential for sexual development. Interestingly, our results showed considerably increased transcript abundance of cellulase genes in darkness compared to light (light responsiveness) upon growth on lactose. This effect is strongly enhanced in mutant strains lacking PKAC1 or ACY1. Comparison to the wild type showed that ACY1 has a consistently positive effect on cellulase gene expression in light and darkness, while PKAC1 influences transcript levels of cellulase genes positively in light but negatively in darkness. A function of PKAC1 in light-modulated cellulase gene regulation is also reflected by altered complex formation within the cel6a/cbh2 promoter in light and darkness and in the absence of pkac1. Analysis of transcript levels of cellulase regulator genes indicates that the regulatory output of the cAMP pathway may be established via adjustment of XYR1 abundance. Consequently, both adenylate cyclase and protein kinase A are involved in light-modulated cellulase gene expression in T. reesei and have a dampening effect on the light responsiveness of this process. PMID:22286997

Thermal stability of Trichoderma reesei c30 cellulase and aspergillus niger; -glucosidase after ph and chemical modification

Energy Technology Data Exchange (ETDEWEB)

Woodward, J.; Whaley, K.S.; Zachry, G.S.; Wohlpart, D.L.

1981-01-01

Treatment of Trichoderma reesei C30 cellulase at pH 10.0 for 1 h at room temperature increased its pH and thermal stability. Chemical modification of the free epsilon-amino groups of cellulase at pH 10.0 resulted in no further increase in stability. Such chemical modification, however, decreased the thermal stability of the cellulose-cellulase complex. On the contrary, the chemical modification of Aspergillus niger glucosidase with glutaraldehyde at pH 8.0 increased the thermal stability of this enzyme.

Efficient Coproduction of Mannanase and Cellulase by the Transformation of a Codon-Optimized Endomannanase Gene from Aspergillus niger into Trichoderma reesei.

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Sun, Xianhua; Xue, Xianli; Li, Mengzhu; Gao, Fei; Hao, Zhenzhen; Huang, Huoqing; Luo, Huiying; Qin, Lina; Yao, Bin; Su, Xiaoyun

2017-12-20

Cellulase and mannanase are both important enzyme additives in animal feeds. Expressing the two enzymes simultaneously within one microbial host could potentially lead to cost reductions in the feeding of animals. For this purpose, we codon-optimized the Aspergillus niger Man5A gene to the codon-usage bias of Trichoderma reesei. By comparing the free energies and the local structures of the nucleotide sequences, one optimized sequence was finally selected and transformed into the T. reesei pyridine-auxotrophic strain TU-6. The codon-optimized gene was expressed to a higher level than the original one. Further expressing the codon-optimized gene in a mutated T. reesei strain through fed-batch cultivation resulted in coproduction of cellulase and mannanase up to 1376 U·mL -1 and 1204 U·mL -1 , respectively.

Revisiting overexpression of a heterologous β-glucosidase in Trichoderma reesei: fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities.

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Xue, Xianli; Wu, Yilan; Qin, Xing; Ma, Rui; Luo, Huiying; Su, Xiaoyun; Yao, Bin

2016-07-11

The filamentous fungus Trichoderma reesei has the capacity to secret large amounts of cellulase and is widely used in a variety of industries. However, the T. reesei cellulase is weak in β-glucosidase activity, which results in accumulation of cellobiose inhibiting the endo- and exo-cellulases. By expressing an exogenous β-glucosidase gene, the recombinant T. reesei cellulase is expected to degrade cellulose into glucose more efficiently. The thermophilic β-glucosidase NfBgl3A from Neosartorya fischeri is chosen for overexpression in T. reesei due to its robust activity. In vitro, the Pichia pastoris-expressed NfBgl3A aided the T. reesei cellulase in releasing much more glucose with significantly lower amounts of cellobiose from crystalline cellulose. The NfBgl3A gene was hence fused to the cbh1 structural gene and assembled between the strong cbh1 promoter and cbh1 terminator to obtain pRS-NfBgl3A by using the DNA assembler method. pRS-NfBgl3A was transformed into the T. reesei uridine auxotroph strain TU-6. Six positive transformants showed β-glucosidase activities of 2.3-69.7 U/mL (up to 175-fold higher than that of wild-type). The largely different β-glucosidase activities in the transformants may be ascribed to the gene copy numbers of NfBgl3A or its integration loci. The T. reesei-expressed NfBgl3A showed highly similar biochemical properties to that expressed in P. pastoris. As expected, overexpression of NfBgl3A enhanced the overall cellulase activity of T. reesei. The CBHI activity in all transformants increased, possibly due to the extra copies of cbh1 gene introduced, while the endoglucanase activity in three transformants also largely increased, which was not observed in any other studies overexpressing a β-glucosidase. NfBgl3A had significant transglycosylation activity, generating sophorose, a potent cellulase inducer, and other oligosaccharides from glucose and cellobiose. We report herein the successful overexpression of a thermophilic N

Systems Analysis of Lactose Metabolism in Trichoderma reesei Identifies a Lactose Permease That Is Essential for Cellulase Induction

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Ivanova, Christa; Bååth, Jenny A.; Seiboth, Bernhard; Kubicek, Christian P.

2013-01-01

Trichoderma reesei colonizes predecayed wood in nature and metabolizes cellulose and hemicellulose from the plant biomass. The respective enzymes are industrially produced for application in the biofuel and biorefinery industry. However, these enzymes are also induced in the presence of lactose (1,4-0-ß-d-galactopyranosyl-d-glucose), a waste product from cheese manufacture or whey processing industries. In fact, lactose is the only soluble carbon source that induces these enzymes in T. reesei on an industrial level but the reason for this unique phenomenon is not understood. To answer this question, we used systems analysis of the T. reesei transcriptome during utilization of lactose. We found that the respective CAZome encoded all glycosyl hydrolases necessary for cellulose degradation and particularly for the attack of monocotyledon xyloglucan, from which ß-galactosides could be released that may act as the inducers of T. reesei’s cellulases and hemicellulases. In addition, lactose also induces a high number of putative transporters of the major facilitator superfamily. Deletion of fourteen of them identified one gene that is essential for lactose utilization and lactose uptake, and for cellulase induction by lactose (but not sophorose) in pregrown mycelia of T. reesei. These data shed new light on the mechanism by which T. reesei metabolizes lactose and offers strategies for its improvement. They also illuminate the key role of ß-D-galactosides in habitat specificity of this fungus. PMID:23690947

Regulation of transcription of cellulases- and hemicellulases-encoding genes in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei)

NARCIS (Netherlands)

Stricker, A.R.; Mach, R.L.; Graaff, de L.H.

2008-01-01

The filamentous fungi Aspergillus niger and Hypocrea jecorina (Trichoderma reesei) have been the subject of many studies investigating the mechanism of transcriptional regulation of hemicellulase- and cellulase-encoding genes. The transcriptional regulator XlnR that was initially identified in A.

Application of Statistical Design for the Production of Cellulase by Trichoderma reesei Using Mango Peel

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P. Saravanan

2012-01-01

Full Text Available Optimization of the culture medium for cellulase production using Trichoderma reesei was carried out. The optimization of cellulase production using mango peel as substrate was performed with statistical methodology based on experimental designs. The screening of nine nutrients for their influence on cellulase production is achieved using Plackett-Burman design. Avicel, soybean cake flour, KH2PO4, and CoCl2·6H2O were selected based on their positive influence on cellulase production. The composition of the selected components was optimized using Response Surface Methodology (RSM. The optimum conditions are as follows: Avicel: 25.30 g/L, Soybean cake flour: 23.53 g/L, KH2PO4: 4.90 g/L, and CoCl2·6H2O: 0.95 g/L. These conditions are validated experimentally which revealed an enhanced Cellulase activity of 7.8 IU/mL.

Thermal stability of Trichoderma reesei C30 cellulase and Aspergillus niger. beta. -glucosidase after pH and chemical modification

Energy Technology Data Exchange (ETDEWEB)

Woodward, J.; Whaley, K.S.; Zachry, G.S.; Wohlpart, D.L.

1981-01-01

Treatment of Trichoderma reesei C30 cellulase at pH 10.0 for 1 h at room temperature increased its pH and thermal stability. Chemical modification of the free epsilon-amino groups of cellulase at pH 10.0 resulted in no further increase in stability. Such chemical modification, however, decreased the thermal stability of the cellulose-cellulase complex. On the contrary, the chemical modification of Aspergillus niger ..beta..-glucosidase with glutaraldehyde at pH 8.0 increased the thermal stability of this enzyme.

Comparative analysis of the Trichoderma reesei transcriptome during growth on the cellulase inducing substrates wheat straw and lactose

OpenAIRE

Bischof, Robert; Fourtis, Lukas; Limbeck, Andreas; Gamauf, Christian; Seiboth, Bernhard; Kubicek, Christian P

2013-01-01

Background Renewable lignocellulosic biomass is an advantageous resource for the production of second generation biofuels and other biorefinery products. In Middle Europe, wheat straw is one of the most abundant low-cost sources of lignocellulosic biomass. For its efficient use, an efficient mix of cellulases and hemicellulases is required. In this paper, we investigated how cellulase production by T. reesei on wheat straw compares to that on lactose, the only soluble and also cheap inducing ...

Expression of three Trichoderma reesei cellulase genes in Saccharomyces pastorianus for the development of a two-step process of hydrolysis and fermentation of cellulose.

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Fitzpatrick, J; Kricka, W; James, T C; Bond, U

2014-07-01

To compare the production of recombinant cellulase enzymes in two Saccharomyces species so as to ascertain the most suitable heterologous host for the degradation of cellulose-based biomass and its conversion into bioethanol. cDNA copies of genes representing the three major classes of cellulases (Endoglucanases, Cellobiohydrolases and β-glucosidases) from Trichoderma reesei were expressed in Saccharomyces pastorianus and Saccharomyces cerevisiae. The recombinant enzymes were secreted by the yeast hosts into the medium and were shown to act in synergy to hydrolyse cellulose. The conditions required to achieve maximum release of glucose from cellulose by the recombinant enzymes were defined and the activity of the recombinant enzymes was compared to a commercial cocktail of T. reesei cellulases. We demonstrate that significantly higher levels of cellulase activity were achieved by expression of the genes in S. pastorianus compared to S. cerevisiae. Hydrolysis of cellulose by the combined activity of the recombinant enzymes was significantly better at 50°C than at 30°C, the temperature used for mesophilic yeast fermentations, reflecting the known temperature profiles of the native enzymes. The results demonstrate that host choice is important for the heterologous production of cellulases. On the basis of the low activity of the T. reesei recombinant enzymes at fermentation temperatures, we propose a two-step process for the hydrolysis of cellulose and its fermentation into alcohol using cellulases produced in situ. © 2014 The Society for Applied Microbiology.

Genome sequencing and transcriptome analysis of Trichoderma reesei QM9978 strain reveals a distal chromosome translocation to be responsible for loss of vib1 expression and loss of cellulase induction.

Science.gov (United States)

Ivanova, Christa; Ramoni, Jonas; Aouam, Thiziri; Frischmann, Alexa; Seiboth, Bernhard; Baker, Scott E; Le Crom, Stéphane; Lemoine, Sophie; Margeot, Antoine; Bidard, Frédérique

2017-01-01

The hydrolysis of biomass to simple sugars used for the production of biofuels in biorefineries requires the action of cellulolytic enzyme mixtures. During the last 50 years, the ascomycete Trichoderma reesei , the main source of industrial cellulase and hemicellulase cocktails, has been subjected to several rounds of classical mutagenesis with the aim to obtain higher production levels. During these random genetic events, strains unable to produce cellulases were generated. Here, whole genome sequencing and transcriptomic analyses of the cellulase-negative strain QM9978 were used for the identification of mutations underlying this cellulase-negative phenotype. Sequence comparison of the cellulase-negative strain QM9978 to the reference strain QM6a identified a total of 43 mutations, of which 33 were located either close to or in coding regions. From those, we identified 23 single-nucleotide variants, nine InDels, and one translocation. The translocation occurred between chromosomes V and VII, is located upstream of the putative transcription factor vib1 , and abolishes its expression in QM9978 as detected during the transcriptomic analyses. Ectopic expression of vib1 under the control of its native promoter as well as overexpression of vib1 under the control of a strong constitutive promoter restored cellulase expression in QM9978, thus confirming that the translocation event is the reason for the cellulase-negative phenotype. Gene deletion of vib1 in the moderate producer strain QM9414 and in the high producer strain Rut-C30 reduced cellulase expression in both cases. Overexpression of vib1 in QM9414 and Rut-C30 had no effect on cellulase production, most likely because vib1 is already expressed at an optimal level under normal conditions. We were able to establish a link between a chromosomal translocation in QM9978 and the cellulase-negative phenotype of the strain. We identified the transcription factor vib1 as a key regulator of cellulases in T. reesei whose

Is an organic nitrogen source needed for cellulase production by Trichoderma reesei Rut-C30?

DEFF Research Database (Denmark)

Rodríguez Gómez, Divanery; Hobley, Timothy John

2013-01-01

The effect of organic and inorganic nitrogen sources on Trichoderma reesei Rut-C30 cellulase production was investigated in submerged cultivations. Stirred tank bioreactors and shake flasks, with and without pH control, respectively, were employed. The experimental design involved the addition...... of individual organic nitrogen sources (soy peptone, glutamate, glycine and alanine) within a basal medium containing Avicel (i.e. micro crystalline cellulose) and ammonium sulphate. It was found that in the shake flask experiments, the highest cellulase activities (~0.1 ± 0.02 FPU ml−1) were obtained...... with media containing soy peptone (3–6 g l−1) and glutamate (3.6 g l−1). However, these improvements in the cellulase titers in the presence of the organic nitrogen sources appeared to be related to smaller changes in the pH of the medium. This was confirmed using stirred tank bioreactors with pH control...

Intracellular β-Glucosidases CEL1a and CEL1b Are Essential for Cellulase Induction on Lactose in Trichoderma reesei

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Xu, Jintao; Zhao, Guolei; Kou, Yanbo; Zhang, Weixin; Zhou, Qingxin; Chen, Guanjun

2014-01-01

Lactose (1,4-O-β-d-galacto-pyranosyl-d-glucose) induces cellulolytic enzymes in Trichoderma reesei and is in fact one of the most important soluble carbon sources used to produce cellulases on an industrial level. The mechanism underlying the induction is, however, not fully understood. In this study, we investigated the cellular functions of the intracellular β-glucosidases CEL1a and CEL1b in the induction of cellulase genes by lactose in T. reesei. We demonstrated that while CEL1a and CEL1b were functionally equivalent in mediating the induction, the simultaneous absence of these intracellular β-glucosidases abolished cbh1 gene expression on lactose. d-Galactose restored the efficient cellulase gene induction in the Δcel1a strain independently of its reductive metabolism, but not in the Δcel1a Δcel1b strain. A further comparison of the transcriptional responses of the Δcel1a Δcel1b strain complemented with wild-type CEL1a or a catalytically inactive CEL1a version and the Δcel1a strain constitutively expressing CEL1a or the Kluyveromyces lactis β-galactosidase LAC4 showed that both the CEL1a protein and its glycoside hydrolytic activity were indispensable for cellulase induction by lactose. We also present evidence that intracellular β-glucosidase-mediated lactose induction is further conveyed to XYR1 to ensure the efficiently induced expression of cellulase genes. PMID:24879125

Study of a High-Yield Cellulase System Created by Heavy-Ion Irradiation-Induced Mutagenesis of Aspergillus niger and Mixed Fermentation with Trichoderma reesei

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Chen, Ji-Hong; Li, Wen-Jian; Liu, Jing; Hu, Wei; Xiao, Guo-Qing; Dong, Miao-Yin; Wang, Yu-Chen

2015-01-01

The aim of this study was to evaluate and validate the efficiency of 12C6+ irradiation of Aspergillus niger (A. niger) or mutagenesis via mixed Trichoderma viride (T. viride) culturing as well as a liquid cultivation method for cellulase production via mixed Trichoderma reesei (T. reesei) and A. niger culture fermentation. The first mutagenesis approach was employed to optimize yield from a cellulase-producing strain via heavy-ion mutagenesis and high-throughput screening, and the second was to effectively achieve enzymatic hydrolysis of cellulase from a mixed culture of mutant T. viride and A. niger. We found that 12C6+-ion irradiation induced changes in cellulase biosynthesis in A. niger but had no effect on the time course of the synthesis. It is notable that the exoglucanases (CBH) activities of A. niger strains H11-1 and H differed (6.71 U/mL vs. 6.01 U/mL) and were significantly higher than that of A. niger mutant H3-1. Compared with strain H, the filter paper assay (FPA), endoglucanase (EG) and β-glucosidase (BGL) activities of mutant strain H11-1 were increased by 250.26%, 30.26% and 34.91%, respectively. A mixed culture system was successfully optimized, and the best ratio of T. reesei to A. niger was 5:1 for 96 h with simultaneous inoculation. The BGL activity of the mixed culture increased after 72 h. At 96 h, the FPA and BGL activities of the mixed culture were 689.00 and 797.15 U/mL, respectively, significantly higher than those of monocultures, which were 408.70 and 646.98 U/mL for T. reesei and 447.29 and 658.89 U/mL for A. niger, respectively. The EG activity of the mixed culture was 2342.81 U/mL, a value that was significantly higher than that of monocultures at 2206.57 U/mL for T. reesei and 1727.62 U/mL for A. niger. In summary, cellulose production and hydrolysis yields were significantly enhanced by the proposed combination scheme. PMID:26656155

Study of a High-Yield Cellulase System Created by Heavy-Ion Irradiation-Induced Mutagenesis of Aspergillus niger and Mixed Fermentation with Trichoderma reesei.

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Shu-Yang Wang

Full Text Available The aim of this study was to evaluate and validate the efficiency of 12C6+ irradiation of Aspergillus niger (A. niger or mutagenesis via mixed Trichoderma viride (T. viride culturing as well as a liquid cultivation method for cellulase production via mixed Trichoderma reesei (T. reesei and A. niger culture fermentation. The first mutagenesis approach was employed to optimize yield from a cellulase-producing strain via heavy-ion mutagenesis and high-throughput screening, and the second was to effectively achieve enzymatic hydrolysis of cellulase from a mixed culture of mutant T. viride and A. niger. We found that 12C6+-ion irradiation induced changes in cellulase biosynthesis in A. niger but had no effect on the time course of the synthesis. It is notable that the exoglucanases (CBH activities of A. niger strains H11-1 and H differed (6.71 U/mL vs. 6.01 U/mL and were significantly higher than that of A. niger mutant H3-1. Compared with strain H, the filter paper assay (FPA, endoglucanase (EG and β-glucosidase (BGL activities of mutant strain H11-1 were increased by 250.26%, 30.26% and 34.91%, respectively. A mixed culture system was successfully optimized, and the best ratio of T. reesei to A. niger was 5:1 for 96 h with simultaneous inoculation. The BGL activity of the mixed culture increased after 72 h. At 96 h, the FPA and BGL activities of the mixed culture were 689.00 and 797.15 U/mL, respectively, significantly higher than those of monocultures, which were 408.70 and 646.98 U/mL for T. reesei and 447.29 and 658.89 U/mL for A. niger, respectively. The EG activity of the mixed culture was 2342.81 U/mL, a value that was significantly higher than that of monocultures at 2206.57 U/mL for T. reesei and 1727.62 U/mL for A. niger. In summary, cellulose production and hydrolysis yields were significantly enhanced by the proposed combination scheme.

Kinetic studies on batch cultivation of Trichoderma reesei and application to enhance cellulase production by fed-batch fermentation.

Science.gov (United States)

Ma, Lijuan; Li, Chen; Yang, Zhenhua; Jia, Wendi; Zhang, Dongyuan; Chen, Shulin

2013-07-20

Reducing the production cost of cellulase as the key enzyme for cellulose hydrolysis to fermentable sugars remains a major challenge for biofuel production. Because of the complexity of cellulase production, kinetic modeling and mass balance calculation can be used as effective tools for process design and optimization. In this study, kinetic models for cell growth, substrate consumption and cellulase production in batch fermentation were developed, and then applied in fed-batch fermentation to enhance cellulase production. Inhibition effect of substrate was considered and a modified Luedeking-Piret model was developed for cellulase production and substrate consumption according to the growth characteristics of Trichoderma reesei. The model predictions fit well with the experimental data. Simulation results showed that higher initial substrate concentration led to decrease of cellulase production rate. Mass balance and kinetic simulation results were applied to determine the feeding strategy. Cellulase production and its corresponding productivity increased by 82.13% after employing the proper feeding strategy in fed-batch fermentation. This method combining mathematics and chemometrics by kinetic modeling and mass balance can not only improve cellulase fermentation process, but also help to better understand the cellulase fermentation process. The model development can also provide insight to other similar fermentation processes. Copyright © 2013 Elsevier B.V. All rights reserved.

Dehydrogenase GRD1 Represents a Novel Component of the Cellulase Regulon in Trichoderma reesei (Hypocrea jecorina) ▿ † §

Science.gov (United States)

Schuster, André; Kubicek, Christian P.; Schmoll, Monika

2011-01-01

Trichoderma reesei (Hypocrea jecorina) is nowadays the most important industrial producer of cellulase and hemicellulase enzymes, which are used for pretreatment of cellulosic biomass for biofuel production. In this study, we introduce a novel component, GRD1 (glucose-ribitol dehydrogenase 1), which shows enzymatic activity on cellobiose and positively influences cellulase gene transcription, expression, and extracellular endo-1,4-β-d-glucanase activity. grd1 is differentially transcribed upon growth on cellulose and the induction of cellulase gene expression by sophorose. The transcription of grd1 is coregulated with that of cel7a (cbh1) under inducing conditions. GRD1 is further involved in carbon source utilization on several carbon sources, such as those involved in lactose and d-galactose catabolism, in several cases in a light-dependent manner. We conclude that GRD1 represents a novel enhancer of cellulase gene expression, which by coregulation with the major cellulase may act via optimization of inducing mechanisms. PMID:21602376

Characterization and Strain Improvement of a Hypercellulytic Variant, Trichoderma reesei SN1, by Genetic Engineering for Optimized Cellulase Production in Biomass Conversion Improvement.

Science.gov (United States)

Qian, Yuanchao; Zhong, Lixia; Hou, Yunhua; Qu, Yinbo; Zhong, Yaohua

2016-01-01

The filamentous fungus Trichoderma reesei is a widely used strain for cellulolytic enzyme production. A hypercellulolytic T. reesei variant SN1 was identified in this study and found to be different from the well-known cellulase producers QM9414 and RUT-C30. The cellulose-degrading enzymes of T. reesei SN1 show higher endoglucanase (EG) activity but lower β-glucosidase (BGL) activity than those of the others. A uracil auxotroph strain, SP4, was constructed by pyr4 deletion in SN1 to improve transformation efficiency. The BGL1-encoding gene bgl1 under the control of a modified cbh1 promoter was overexpressed in SP4. A transformant, SPB2, with four additional copies of bgl1 exhibited a 17.1-fold increase in BGL activity and a 30.0% increase in filter paper activity. Saccharification of corncob residues with crude enzyme showed that the glucose yield of SPB2 is 65.0% higher than that of SP4. These results reveal the feasibility of strain improvement through the development of an efficient genetic transformation platform to construct a balanced cellulase system for biomass conversion.

Characterization and strain improvement of a hypercellulytic variant, Trichoderma reesei SN1, by genetic engineering for optimized cellulase production in biomass conversion improvement

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Qian Yuanchao

2016-08-01

Full Text Available The filamentous fungus Trichoderma reesei is a widely used strain for cellulolytic enzyme production. A hypercellulolytic T. reesei variant SN1 was identified in this study and found to be different from the well-known cellulase producers QM9414 and RUT-C30. The cellulose-degrading enzymes of T. reesei SN1 show higher endoglucanase (EG activity but lower β-glucosidase (BGL activity than those of QM9414 and RUT-C30. A uracil auxotroph strain, SP4, was constructed by pyr4 deletion in SN1 to improve transformation efficiency. The BGL1-encoding gene bgl1 under the control of a modified cbh1 promoter was overexpressed in SP4. A transformant, SPB2, with four additional copies of bgl1 exhibited a 17.1-fold increase in BGL activity and a 30% increase in filter paper activity. Saccharification of corncob residues with crude enzyme showed that the glucose yield of SPB2 is 65% higher than that of SP4. These results reveal the feasibility of strain improvement through the development of an efficient genetic transformation platform to construct a balanced cellulase system for biomass conversion.

Optimization of the Medium for the Production of Cellulase by the Mutant Trichoderma reesei WX-112 Using Response Surface Methodology

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Xue-Cai Hao

2006-01-01

Full Text Available The mutant strain Trichoderma reesei WX-112 with high cellulase activity was isolated by a newly invented plate. The mutant’s ability to produce cellulase increased 1.95 times after the treatment with UV and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG. Also, the medium composition was optimized using response surface methodology (RSM. A fractional factorial design (26–2 was applied to elucidate the medium components that significantly affect cellulase production. The concentration of Avicel and soybean cake flour in the medium were significant factors. The steepest ascent method was used to locate the optimal domain and a central composite design was used to estimate the quadratic response surface from which the factor levels for maximum production of cellulase were determined. The composition of fermentation medium optimized with response surface methodology was (in g/L: wheat bran 30, Avicel 36.4, soybean cake flour 24.7, KH2PO4 4 and corn steep flour 5. Compared to the original medium, the cellulase activity increased from 7.2 to 10.6 IU/mL.

The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30

Science.gov (United States)

2011-01-01

Background Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI) produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P. Results We deleted the gene pgi1, encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a Δpgi1 mutant. Both Δpgi1 and cre1+Δpgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, Δpgi1 and cre1+Δpgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1. Δpgi1 mutants were also affected in the extracellular β-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δpgi1 during growth on glucose. Conclusions The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δpgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in Rut-C30 increased cellulase

Immobilization of Trichoderma reesei cells by radiation polymerization

International Nuclear Information System (INIS)

Kumakura, M.; Kaetsu, I.

1983-01-01

Trichoderma reesei cells were immobilized by radiation polymerization 2-hydroxyethyl acrylate monomer at low temperature. Cellulase production resulting from the growth of the cells in the porous polymer matrix of immobilized cell composites was confirmed by measuring the cellulase activity and pH during the culture. (orig.)

Exploring the Synergy between Cellobiose Dehydrogenase from Phanerochaete chrysosporium and Cellulase from Trichoderma reesei

OpenAIRE

Wang, Min; Lu, Xuefeng

2016-01-01

Recent demands for the production of lignocellulose biofuels boosted research on cellulase. Hydrolysis efficiency and production cost of cellulase are two bottlenecks in biomass to biofuels process. The Trichoderma cellulase mixture is one of the most commonly used enzymes for cellulosic hydrolysis. During hydrolytic process cellobiose accumulation causes feedback inhibition against most cellobiohydrolases and endoglucanases. In this study, we demonstrated the synergism effects between cellob...

The phosducin-like protein PhLP1 impacts regulation of glycoside hydrolases and light response in Trichoderma reesei

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Tisch Doris

2011-12-01

Full Text Available Abstract Background In the biotechnological workhorse Trichoderma reesei (Hypocrea jecorina transcription of cellulase genes as well as efficiency of the secreted cellulase mixture are modulated by light. Components of the heterotrimeric G-protein pathway interact with light-dependent signals, rendering this pathway a key regulator of cellulase gene expression. Results As regulators of heterotrimeric G-protein signaling, class I phosducin-like proteins, are assumed to act as co-chaperones for G-protein beta-gamma folding and exert their function in response to light in higher eukaryotes. Our results revealed light responsive transcription of the T. reesei class I phosducin-like protein gene phlp1 and indicate a light dependent function of PhLP1 also in fungi. We showed the functions of PhLP1, GNB1 and GNG1 in the same pathway, with one major output being the regulation of transcription of glycoside hydrolase genes including cellulase genes in T. reesei. We found no direct correlation between the growth rate and global regulation of glycoside hydrolases, which suggests that regulation of growth does not occur only at the level of substrate degradation efficiency. Additionally, PhLP1, GNB1 and GNG1 are all important for proper regulation of light responsiveness during long term exposure. In their absence, the amount of light regulated genes increased from 2.7% in wild type to 14% in Δphlp1. Besides from the regulation of degradative enzymes, PhLP1 was also found to impact on the transcription of genes involved in sexual development, which was in accordance with decreased efficiency of fruiting body formation in Δphlp1. The lack of GNB1 drastically diminished ascospore discharge in T. reesei. Conclusions The heterotrimeric G-protein pathway is crucial for the interconnection of nutrient signaling and light response of T. reesei, with the class I phosducin-like protein PhLP1, GNB1 and GNG1 acting as important nodes, which influence light

Compatible ionic liquid-cellulases system for hydrolysis of lignocellulosic biomass.

Science.gov (United States)

Wang, Ying; Radosevich, Mark; Hayes, Douglas; Labbé, Nicole

2011-05-01

Ionic liquids (ILs) have been increasingly recognized as novel solvents for dissolution and pretreatment of cellulose. However, cellulases are inactivated in the presence of ILs, even when present at low concentrations. To more fully exploit the benefits of ILs it is critical to develop a compatible IL-cellulases system in which the IL is able to effectively solubilize and activate the lignocellulosic biomass, and the cellulases possess high stability and activity. In this study, we investigated the stability and activity of a commercially available cellulases mixture in the presence of different concentrations of 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]). A mixture of cellulases and β-glucosidase (Celluclast1.5L, from Trichoderma reesei, and Novozyme188, from Aspergillus niger, respectively) retained 77% and 65% of its original activity after being pre-incubated in 15% and 20% (w/v) IL solutions, respectively, at 50°C for 3 h. The cellulases mixture also retained high activity in 15% [Emim][OAc] to hydrolyze Avicel, a model substrate for cellulose analysis, with conversion efficiency of approximately 91%. Notably, the presence of different amounts of yellow poplar lignin did not interfere significantly with the enzymatic hydrolysis of Avicel. Using this IL-cellulase system (15% [Emim][OAc]), the saccharification of yellow poplar biomass was also significantly improved (33%) compared to the untreated control (3%) during the first hour of enzymatic hydrolysis. Together, these findings provide compelling evidence that [Emim][OAc] was compatible with the cellulase mixture, and this compatible IL-cellulases system is promising for efficient activation and hydrolysis of native biomass to produce biofuels and co-products from the individual biomass components. Copyright © 2010 Wiley Periodicals, Inc.

Optimization of a synthetic mixture composed of major Trichoderma reesei enzymes for the hydrolysis of steam-exploded wheat straw

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Billard Hélène

2012-02-01

Full Text Available Abstract Background An efficient hydrolysis of lignocellulosic substrates to soluble sugars for biofuel production necessitates the interplay and synergistic interaction of multiple enzymes. An optimized enzyme mixture is crucial for reduced cost of the enzymatic hydrolysis step in a bioethanol production process and its composition will depend on the substrate and type of pretreatment used. In the present study, an experimental design was used to determine the optimal composition of a Trichoderma reesei enzyme mixture, comprising the main cellulase and hemicellulase activities, for the hydrolysis of steam-exploded wheat straw. Methods Six enzymes, CBH1 (Cel7a, CBH2 (Cel6a, EG1 (Cel7b, EG2 (Cel5a, as well as the xyloglucanase Cel74a and the xylanase XYN1 (Xyl11a were purified from a T. reesei culture under lactose/xylose-induced conditions. Sugar release was followed in milliliter-scale hydrolysis assays for 48 hours and the influence of the mixture on initial conversion rates and final yields is assessed. Results The developed model could show that both responses were strongly correlated. Model predictions suggest that optimal hydrolysis yields can be obtained over a wide range of CBH1 to CBH2 ratios, but necessitates a high proportion of EG1 (13% to 25% which cannot be replaced by EG2. Whereas 5% to 10% of the latter enzyme and a xylanase content above 6% are required for highest yields, these enzymes are predicted to be less important in the initial stage of hydrolysis. Conclusions The developed model could reliably predict hydrolysis yields of enzyme mixtures in the studied domain and highlighted the importance of the respective enzyme components in both the initial and the final hydrolysis phase of steam-exploded wheat straw.

The intracellular galactoglycome in Trichoderma reesei during growth on lactose

NARCIS (Netherlands)

Karaffa, L.; Coulier, L.; Fekete, E.; Overkamp, K.M.; Druzhinina, I.S.; Mikus, M.; Seiboth, B.; Novák, L.; Punt, P.J.; Kubicek, C.P.

2013-01-01

Lactose (1,4-0-β-d-galactopyranosyl-d-glucose) is used as a soluble carbon source for the production of cellulases and hemicellulases for - among other purposes - use in biofuel and biorefinery industries. The mechanism how lactose induces cellulase formation in T. reesei is enigmatic, however.

Concurrent production of cellulase and xylanase from Trichoderma reesei NCIM 1186: enhancement of production by desirability-based multi-objective method.

Science.gov (United States)

Jampala, Preethi; Tadikamalla, Satish; Preethi, M; Ramanujam, Swathy; Uppuluri, Kiran Babu

2017-05-01

Application of multiple response optimizations using desirability function in the production of microbial metabolites improves economy and efficiency. Concurrent production of cellulase and xylanase in Trichoderma reesei NCIM 1186 using an agricultural weed, Prosopis juliflora pods, was studied. The main aim of the study was to optimize significant medium nutrient parameters for maximization of cellulase and xylanase by multi-objective optimization strategy using biomass. Process parameters such as the nutrient concentrations (pods, sucrose, and yeast extract) and pH were investigated to improve cellulase and xylanase activities by one factor at a time approach, single response optimization and multi-objective optimization. At the corresponding optimized process parameters in single response optimization, the maximum cellulase activity observed was 3055.65 U/L where xylanase highest activity was 422.16 U/L. Similarly, the maximum xylanase activity, 444.94 U/L, was observed with the highest cellulase activity of 2804.40 U/L. The multi-objective optimization finds a tradeoff between the two objectives and optimal activity values in between the single-objective optima were achieved, 3033.74 and 439.13 U/L for cellulase and xylanase, respectively.

Quantitative site-specific phosphoproteomics of Trichoderma reesei signaling pathways upon induction of hydrolytic enzyme production

NARCIS (Netherlands)

Nguyen, E.V.; Imanishi, S.Y.; Haapaniemi, P.; Yadav, A.; Saloheimo, M.; Corthals, G.L.; Pakula, T.M.

2016-01-01

The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the carbon source it grows on, where the regulation

Effect of physical treatment on Trichoderma reesei cells

International Nuclear Information System (INIS)

Kumakura, M.; Kaetsu, I.

1984-01-01

The effect of physical treatment such as freezing and gamma-ray irradiation on Trichoderma reesei cells was studied. The decrease phenomena of cellulase production, which was observed in the culture of the cells using wheat bran extract, was improved by physical treatment. (author)

Influence of the carbon source on production of cellulases, hemicellulases and pectinases by Trichoderma reesei Rut C-30

DEFF Research Database (Denmark)

Olsson, Lisbeth; Christensen, T.M.I.E.; Hansen, K.P.

2003-01-01

The growth and enzyme production by Trichoderma reesei Rut C-30 using different lignocellulosic materials as carbon source were investigated. Cellulose, sugar beet pulp and alkaline extracted sugar beet pulp (resulting in partial removal of hemicellulose, lignin and pectin) or mixtures thereof were...

Overexpression of an exotic thermotolerant β-glucosidase in trichoderma reesei and its significant increase in cellulolytic activity and saccharification of barley straw

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Dashtban Mehdi

2012-05-01

Full Text Available Abstract Background Trichoderma reesei is a widely used industrial strain for cellulase production, but its low yield of β-glucosidase has prevented its industrial value. In the hydrolysis process of cellulolytic residues by T. reesei, a disaccharide known as cellobiose is produced and accumulates, which inhibits further cellulases production. This problem can be solved by adding β-glucosidase, which hydrolyzes cellobiose to glucose for fermentation. It is, therefore, of high vvalue to construct T. reesei strains which can produce sufficient β-glucosidase and other hydrolytic enzymes, especially when those enzymes are capable of tolerating extreme conditions such as high temperature and acidic or alkali pH. Results We successfully engineered a thermostable β-glucosidase gene from the fungus Periconia sp. into the genome of T. reesei QM9414 strain. The engineered T. reesei strain showed about 10.5-fold (23.9 IU/mg higher β-glucosidase activity compared to the parent strain (2.2 IU/mg after 24 h of incubation. The transformants also showed very high total cellulase activity (about 39.0 FPU/mg at 24 h of incubation whereas the parent strain almost did not show any total cellulase activity at 24 h of incubation. The recombinant β-glucosidase showed to be thermotolerant and remains fully active after two-hour incubation at temperatures as high as 60°C. Additionally, it showed to be active at a wide pH range and maintains about 88% of its maximal activity after four-hour incubation at 25°C in a pH range from 3.0 to 9.0. Enzymatic hydrolysis assay using untreated, NaOH, or Organosolv pretreated barley straw as well as microcrystalline cellulose showed that the transformed T. reesei strains released more reducing sugars compared to the parental strain. Conclusions The recombinant T. reesei overexpressing Periconia sp. β-glucosidase in this study showed higher β-glucosidase and total cellulase activities within a shorter incubation

Overexpression of an endo-1,4-β-glucanase V gene (EGV) from Trichoderma reesei leads to the accumulation of cellulase activity in transgenic rice.

Science.gov (United States)

Li, X Y; Liu, F; Hu, Y F; Xia, M; Cheng, B J; Zhu, S W; Ma, Q

2015-12-21

The ectopic expression of cellulase in biomass can reduce the cost of biofuel conversion. This trait modification technique is highly beneficial for biofuel production. In this study, we isolated an endo-1,4-beta-glucanase gene (EGV) from Trichoderma reesei and inserted this gene downstream of a fragment encoding the signal peptide Apo-SP in a modified pCAMBIA1301 vector to obtain an Apo-SP and AsRed fusion protein. Transient expression of this fusion protein in onion epidermal cells showed that the Apo-SP signal was localized to the plastids. EGV transgenic rice plants that did not carry screening marker genes were obtained through overexpression of the pDTB double T-DNA vector. Western blotting showed that EGV was expressed in the dry straw of T0 generation transgenic rice plants and in fresh leaves of the T1 generation. More importantly, our results also showed that the peptide product of EGV in the transgenic plants folded correctly and was capable of digesting the cellulase substrate CMC. Additionally, cellulase activity remained stable in the straw that had been dried at room temperature for three months. This study presents an important technical approach for the development of transgenic rice straw that has stable cellulase activity and can be used for biofuel conversion.

Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis.

Science.gov (United States)

Florencio, Camila; Cunha, Fernanda M; Badino, Alberto C; Farinas, Cristiane S; Ximenes, Eduardo; Ladisch, Michael R

2016-08-01

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5-15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme

Product inhibition of five Hypocrea jecorina cellulases

DEFF Research Database (Denmark)

Murphy, Leigh; Westh, Peter; Bohlin, Christina

2013-01-01

Product inhibition of cellulolytic enzymes has been deemed a critical factor in the industrial saccharification of cellulosic biomass. Several investigations have addressed this problem using crude enzyme preparations or commercial (mixed) cellulase products, but quantitative information...... on individual cellulases hydrolyzing insoluble cellulose remains insufficient. Such knowledge is necessary to pinpoint and quantify inhibitory weak-links in cellulose hydrolysis, but has proven challenging to come by. Here we show that product inhibition of mono-component cellulases hydrolyzing unmodified...... cellulose may be monitored by calorimetry. The key advantage of this approach is that it directly measures the rate of hydrolysis while being essentially blind to the background of added product. We investigated the five major cellulases from Hypocrea jecorina (anamorph: Tricoderma reesei), Cel7A (formerly...

A new stoichiometric miniaturization strategy for screening of industrial microbial strains: application to cellulase hyper-producing Trichoderma reesei strains

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Jourdier Etienne

2012-05-01

Full Text Available Abstract Background During bioprocess development, secondary screening is a key step at the boundary between laboratory and industrial conditions. To ensure an effective high-throughput screening, miniaturized laboratory conditions must mimic industrial conditions, especially for oxygen transfer, feeding capacity and pH stabilization. Results A feeding strategy has been applied to develop a simple screening procedure, in which a stoichiometric study is combined with a standard miniaturization procedure. Actually, the knowledge of all nutriments and base or acid requirements leads to a great simplification of pH stabilization issue of miniaturized fed-batch cultures. Applied to cellulase production by Trichoderma reesei, this strategy resulted in a stoichiometric mixed feed of carbon and nitrogen sources. While keeping the pH between shake flask and stirred bioreactor comparable, the developed shake flask protocol reproduced the strain behaviour under stirred bioreactor conditions. Compared to a an already existing miniaturized shake flasks protocol, the cellulase concentration was increased 5-fold, reaching about 10 g L-1. Applied to the secondary screening of several clones, the newly developed protocol succeeded in selecting a clone with a high industrial potential. Conclusions The understanding of a bioprocess stoichiometry contributed to define a simpler and more effective miniaturization. The suggested strategy can potentially be applied to other fed-batch processes, for the screening of either strain collections or experimental conditions.

Filamentous fungi and media for cellulase production in solid state cultures

Science.gov (United States)

Kilikian, B.V.; Afonso, L.C.; Souza, T.F.C.; Ferreira, R.G.; Pinheiro, I.R.

2014-01-01

Cellulase production was evaluated in two reference strains (T. reesei Rut-C30 and T. reesei QM9414), two strains isolated from a sugarcane cultivation area (Trichoderma sp. IPT778 and T. harzianum rifai IPT821) and one strain isolated in a program for biodiversity preservation in São Paulo state (Myceliophthora thermophila M77). Solid state cultures were performed using sugarcane bagasse (C), wheat bran (W) and/or soybean bran (S). The highest FPA was 10.6 U/gdm for M77 in SC (10:90) at 80% moisture, which was 4.4 times higher than production in pure W. C was a strong inducer of cellulase production, given that the production level of 6.1 U/gdm in WC (40:60) was 2.5 times higher than in pure W for strain M77; T. reesei Rut-C30 did not respond as strongly with about 1.6-fold surplus production. S advantageously replaced W, as the surplus production on SC (20:80) was 2.3 times relative to WC (20:80) for M77. PMID:24948946

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility.

Science.gov (United States)

Jeoh, Tina; Ishizawa, Claudia I; Davis, Mark F; Himmel, Michael E; Adney, William S; Johnson, David K

2007-09-01

Attempts to correlate the physical and chemical properties of biomass to its susceptibility to enzyme digestion are often inconclusive or contradictory depending on variables such as the type of substrate, the pretreatment conditions and measurement techniques. In this study, we present a direct method for measuring the key factors governing cellulose digestibility in a biomass sample by directly probing cellulase binding and activity using a purified cellobiohydrolase (Cel7A) from Trichoderma reesei. Fluorescence-labeled T. reesei Cel7A was used to assay pretreated corn stover samples and pure cellulosic substrates to identify barriers to accessibility by this important component of cellulase preparations. The results showed cellulose conversion improved when T. reesei Cel7A bound in higher concentrations, indicating that the enzyme had greater access to the substrate. Factors such as the pretreatment severity, drying after pretreatment, and cellulose crystallinity were found to directly impact enzyme accessibility. This study provides direct evidence to support the notion that the best pretreatment schemes for rendering biomass more digestible to cellobiohydrolase enzymes are those that improve access to the cellulose in biomass cell walls, as well as those able to reduce the crystallinity of cell wall cellulose.

Examining the Potential of Plasma-Assisted Pretreated Wheat Straw for Enzyme Production by Trichoderma reesei

DEFF Research Database (Denmark)

Rodríguez Gómez, Divanery; Lehmann, Linda Olkjær; Schultz-Jensen, Nadja

2012-01-01

Plasma-assisted pretreated wheat straw was investigated for cellulase and xylanase production by Trichoderma reesei fermentation. Fermentations were conducted with media containing washed and unwashed plasma-assisted pretreated wheat straw as carbon source which was sterilized by autoclavation....... To account for any effects of autoclavation, a comparison was made with unsterilized media containing antibiotics. It was found that unsterilized washed plasma-assisted pretreated wheat straw (which contained antibiotics) was best suited for the production of xylanases (110 IU ml(-1)) and cellulases (0...... other nonrefined feedstocks suggests that plasma pretreated wheat straw is a promising and suitable substrate for cellulase and hemicellulase production....

Fermentative production and kinetics of cellulase protein on ...

African Journals Online (AJOL)

AJB SERVER

2006-10-16

Oct 16, 2006 ... various carbon sources on the production of cellulase using strains of T. reesei QM 9414, 97.177 and Tm3. Pretreatment of sugarcane ... of cellulose chains; endo-1,4-β-D-glucanses which cleave internal glucosidic bonds ..... production, the Leudeking piret model (Rakshit and Sahai, 1991) was developed.

Enhancing saccharification of wheat straw by mixing enzymes from genetically-modified Trichoderma reesei and Aspergillus niger

NARCIS (Netherlands)

Jiang, Yanping; Duarte, Alexandra Vivas; van Den Brink, Joost; Wiebenga, Ad; Zou, Gen; Wang, Chengshu; de Vries, Ronald P.; Zhou, Zhihua; Benoit, Isabelle

2015-01-01

Objectives: To increase the efficiency of enzymatic hydrolysis for plant biomass conversion into renewable biofuel and chemicals. Results: By overexpressing the point mutation A824 V transcriptional activator Xyr1 in Trichoderma reesei, carboxymethyl cellulase, cellobiosidase and β-d-glucosidase

Enhancing saccharification of wheat straw by mixing enzymes from genetically-modified Trichoderma reesei and Aspergillus niger

NARCIS (Netherlands)

Jiang, Yanping; Duarte, Alexandra Vivas; van den Brink, Joost; Wiebenga, Ad; Zou, Gen; Wang, Chengshu; de Vries, Ronald P; Zhou, Zhihua; Benoit, Isabelle

OBJECTIVES: To increase the efficiency of enzymatic hydrolysis for plant biomass conversion into renewable biofuel and chemicals. RESULTS: By overexpressing the point mutation A824 V transcriptional activator Xyr1 in Trichoderma reesei, carboxymethyl cellulase, cellobiosidase and β-D-glucosidase

Identification of microRNA-Like RNAs in the filamentous fungus Trichoderma reesei by solexa sequencing.

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Kang Kang

Full Text Available microRNAs (miRNAs are non-coding small RNAs (sRNAs capable of negatively regulating gene expression. Recently, microRNA-like small RNAs (milRNAs were discovered in several filamentous fungi but not yet in Trichoderma reesei, an industrial filamentous fungus that can secrete abundant hydrolases. To explore the presence of milRNA in T. reesei and evaluate their expression under induction of cellulose, two T. reesei sRNA libraries of cellulose induction (IN and non-induction (CON were generated and sequenced using Solexa sequencing technology. A total of 726 and 631 sRNAs were obtained from the IN and CON samples, respectively. Global expression analysis showed an extensively differential expression of sRNAs in T. reesei under the two conditions. Thirteen predicted milRNAs were identified in T. reesei based on the short hairpin structure analysis. The milRNA profiles obtained in deep sequencing were further validated by RT-qPCR assay. Computational analysis predicted a number of potential targets relating to many processes including regulation of enzyme expression. The presence and differential expression of T. reesei milRNAs imply that milRNA might play a role in T. reesei growth and cellulase induction. This work lays foundation for further functional study of fungal milRNAs and their industrial application.

Studies on quantitative physiology of Trichoderma reesei with two-stage continuous culture for cellulase production

Energy Technology Data Exchange (ETDEWEB)

Ryu, D; Andreotti, R; Mandels, M; Gallo, B; Reese, E T

1979-11-01

By employing a two-stage continuous-culture system, some of the more important physiological parameters involved in cellulase biosynthesis have been evaluated with an ultimate objective of designing an optimally controlled cellulase process. The two-stage continuous-culture system was run for a period of 1350 hr with Trichoderma reesei strain MCG-77. The temperature and pH were controlled at 32/sup 0/C and pH 4.5 for the first stage (growth) and 28/sup 0/C and pH 3.5 for the second stage (enzyme production). Lactose was the only carbon source for both stages. The ratio of specific uptake rate of carbon to that of nitrogen, Q(C)/Q(N), that supported good cell growth ranged from 11 to 15, and the ratio for maximum specific enzyme productivity ranged from 5 to 13. The maintenance coefficients determined for oxygen, M/sub 0/, and for carbon source, M/sub c/, are 0.85 mmol O/sub 2//g biomass/hr and 0.14 mmol hexose/g biomass/hr, respectively. The yield constants determined are: Y/sub X/O/ = 32.3 g biomass/mol O/sub 2/, Y/sub X/C/ = 1.1 g biomass/g C or Y/sub X/C/ = 0.44 g biomass/g hexose, Y/sub X/N/ = 12.5 g biomass/g nitrogen for the cell growth stage, and Y/sub X/N/ = 16.6 g biomass/g nitrogen for the enzyme production stage. Enzyme was produced only in the second stage. Volumetric and specific enzyme productivities obtained were 90 IU/liter/hrand 8 IU/g biomass/hr, respectively. The maximum specific enzyme productivity observed was 14.8 IU/g biomass/hr. The optimal dilution rate in the second stage that corresponded to the maximum enzyme productivity was 0.026 approx. 0.028 hr/sup -1/, and the specific growth rate in the second stage that supported maximum specific enzyme productivity was equal to or slightly less than zero.

Cellulases: biosynthesis and applications

Energy Technology Data Exchange (ETDEWEB)

Ryu, D D.Y.; Mandels, M

1980-04-01

Strains of Trichoderma, particularly T. reesei and its mutants, are good sources of extracellular cellulose suitable for practical saccharification. They secrete a complete cellulase complex containing endo- and exo-glucanases plus beta-glucosidase (cellobiase) which act syngergistically to degrade totally even highly resistant crystalline cellulose to soluble sugar. All strains investigated to date are inducible by cellulose, lactose, or sophorose, and all are repressible by glucose. The composition and properties of the enzyme complex are similar regardless of the strain or inducing substrate although quantities of the enzyme secreted by the mutants are higher. Enzyme yields are proportional to initial cellulose concentrations. Up to 15 filter paper cellulase units (20 mg of cellulase protein) per ml and productivities up to 80 cellulase units (130 mg cellulase protein) per litre per hour have been attained on 6% cellulose. The economics of glucose production are not yet competitive due to the low specific activity of cellulase (0.6 filter paper cellulase units/mg protein) and poor efficiency (about 15% of predicted sugar levels in 24 h hydrolyses of 10 to 25% substrate). As hydrolysis proceeds, the enzyme reaction slows due to increasing resistance of the residue, product inhibition, and enzyme inactivation. These problems are being attacked by use of pretreatments to increase the quantity of amorphous cellulose, addition of beta-glucosidase to reduce cellobiose inhibition, and studies of means to overcome instability and increase efficiency of the cellulases. Indications are that carbon compounds derived from enzymatic hydrolysis of cellulose will be used as fermentation and chemical feedstocks as soon as the process economics are favorable for such application.

Enhanced Production of Cellulase from Pineapple Waste by Response Surface Methodology

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P. Saravanan

2013-01-01

Full Text Available Optimization of the media components for cellulase production using Trichoderma reesei was carried out. The optimization of cellulase production using pineapple waste as substrate was performed with statistical methodology based on experimental designs. The screening of nutrients and their influence on the cellulase production was studied using a Plackett-Burman design. Avicel, soybean cake flour, KH2PO4, and yeast extract were found to have the positive influence for the production of cellulase. The selected components were optimized using response surface methodology. The optimum concentrations are avicel: 26.5 g/L, soybean cake flour: 22.5 g/L, KH2PO4: 4.5 g/L, and yeast extract: 12.3 g/L. A maximum cellulase activity of 8.61 IU/mL was obtained under the optimized medium in the validation experiment.

CBH1 homologs and varian CBH1 cellulase

Energy Technology Data Exchange (ETDEWEB)

Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Neefe, Paulien

2014-07-01

Disclosed are a number of homologs and variants of Hypocrea jecorina Cel7A (formerly Trichoderma reesei cellobiohydrolase I or CBH1), nucleic acids encoding the same and methods for producing the same. The homologs and variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted and/or deleted.

Recycling cellulases during the hydrolysis of steam exploded and ethanol pretreated Lodgepole pine.

Science.gov (United States)

Tu, Maobing; Chandra, Richard P; Saddler, Jack N

2007-01-01

Recycling of cellulases is one way of reducing the high cost of enzymes during the bioconversion process. The effects of surfactant addition on enzymatic hydrolysis and the potential recycling of cellulases were studied during the hydrolysis of steam exploded Lodgepole pine (SELP) and ethanol pretreated Lodgepole pine (EPLP). Three cellulase preparations (Celluclast, Spezyme CP, and MSUBC) were evaluated to determine their hydrolysis efficiencies over multiple rounds of recycling. The surfactant, Tween 80, significantly increased the yield from 63% to 86% during the hydrolysis of the SELP substrate. The addition of surfactant to the hydrolysis of the EPLP substrate increased the free enzymes in the supernatant from 71% of the initial protein to 96%. Based on the Langmuir adsorption constants, cellulases (Celluclast and Spezyme CP) from Trichoderma reesei showed a higher affinity (3.48 mL/mg and 3.17 mL/mg) for the EPLP substrate than did the Penicillium enzyme (0.62 mg/mg). The Trichoderma reesei enzyme was used in four successive rounds of enzyme recycling using surfactant addition and readsorption onto fresh substrates during the hydrolysis of EPLP. In contrast, the Penicillium-derived enzyme preparation (MSUBC) could only be recycled once. When the same recycling strategy was carried out using the SELP substrate, the hydrolysis yield declined during each enzyme recycling round. These results suggested that the higher lignin content of the SELP substrate, and the low affinity of cellulases for the SELP substrate limited enzyme recycling by readsorption onto fresh substrates.

Production of Recombinant Trichoderma reesei Cellobiohydrolase II in a New Expression System Based on Wickerhamomyces anomalus

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Dennis J. Díaz-Rincón

2017-01-01

Full Text Available Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII in a native strain of Wickerhamomyces anomalus. Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L−1. The enzyme extract showed optimum pH and temperature of 5.0–6.0 and 40°C, respectively. Enzyme kinetic parameters (KM of 2.73 mM and Vmax of 23.1 µM min−1 were between the ranges of values reported for other CBHII enzymes. Finally, the results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi. These results show the first report on the use of W. anomalus as a host to produce recombinant proteins. In addition, recombinant T. reesei CBHII enzyme could potentially be used in the degradation of lignocellulosic residues to produce bioethanol, based on its pH and temperature activity profile.

Production of Recombinant Trichoderma reesei Cellobiohydrolase II in a New Expression System Based on Wickerhamomyces anomalus

Science.gov (United States)

Díaz-Rincón, Dennis J.; Duque, Ivonne; Osorio, Erika; Rodríguez-López, Alexander; Espejo-Mojica, Angela; Parra-Giraldo, Claudia M.

2017-01-01

Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII) in a native strain of Wickerhamomyces anomalus. Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L−1. The enzyme extract showed optimum pH and temperature of 5.0–6.0 and 40°C, respectively. Enzyme kinetic parameters (KM of 2.73 mM and Vmax of 23.1 µM min−1) were between the ranges of values reported for other CBHII enzymes. Finally, the results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi. These results show the first report on the use of W. anomalus as a host to produce recombinant proteins. In addition, recombinant T. reesei CBHII enzyme could potentially be used in the degradation of lignocellulosic residues to produce bioethanol, based on its pH and temperature activity profile. PMID:28951785

Secretome data from Trichoderma reesei and Aspergillus niger cultivated in submerged and sequential fermentation methods

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Camila Florencio

2016-09-01

Full Text Available The cultivation procedure and the fungal strain applied for enzyme production may influence levels and profile of the proteins produced. The proteomic analysis data presented here provide critical information to compare proteins secreted by Trichoderma reesei and Aspergillus niger when cultivated through submerged and sequential fermentation processes, using steam-explosion sugarcane bagasse as inducer for enzyme production. The proteins were organized according to the families described in CAZy database as cellulases, hemicellulases, proteases/peptidases, cell-wall-protein, lipases, others (catalase, esterase, etc., glycoside hydrolases families, predicted and hypothetical proteins. Further detailed analysis of this data is provided in “Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation process: enzyme production for sugarcane bagasse hydrolysis” C. Florencio, F.M. Cunha, A.C Badino, C.S. Farinas, E. Ximenes, M.R. Ladisch (2016 [1]. Keywords: Tricoderma reesei, Aspergillus Niger, Enzyme Production, Secretome

Production and characterization of cellulolytic enzymes from Trichoderma reesei grown on various carbon sources

Energy Technology Data Exchange (ETDEWEB)

Warzywoda, Michel; Labre, Elisabeth; Pourquie, Jacques [Institut Francais du Petrole (IFP), 92 - Rueil-Malmaison (France)

1992-01-01

Ethanol production from lignocellulosics is considered, using a process in which biomass is first pretreated by steam explosion, yielding freely water-extractible pentoses and a cellulose-rich residue which can be further hydrolyzed by cellulases into glucose to be fermented into ethanol. Results that are reported show that both the pentose extracts and the glucose-rich hydrolyzates can be used as carbon sources for cellulase production by Trichoderma reesei. When compared with lactose as the main carbon source, pentose extracts support lower but satisfactory protein productions which are characterized by an increase in hemicellulolytic activities, which significantly improves the saccharifying potential of these enzyme preparations. (author).

Effects of different cellulases on the release of phenolic acids from rice straw during saccharification.

Science.gov (United States)

Xue, Yiyun; Wang, Xiahui; Chen, Xingxuan; Hu, Jiajun; Gao, Min-Tian; Li, Jixiang

2017-06-01

Effects of different cellulases on the release of phenolic acids from rice straw during saccharification were investigated in this study. All cellulases tested increased the contents of phenolic acids during saccharification. However, few free phenolic acids were detected, as they were present in conjugated form after saccharification when the cellulases from Trichoderma reesei, Trichoderma viride and Aspergillus niger were used. On the other hand, phenolic acids were present in free form when the Acremonium cellulolyticus cellulase was used. Assays of enzyme activity showed that, besides high cellulase activity, the A. cellulolyticus cellulase exhibited high feruloyl esterase (FAE) activity. A synergistic interaction between FAE and cellulase led to the increase in free phenolic acids, and thus an increase in antioxidative and antiradical activities of the phenolic acids. Moreover, a cost estimation demonstrated the feasibility of phenolic acids as value-added products to reduce the total production cost of ethanol. Copyright © 2017 Elsevier Ltd. All rights reserved.

Multifunctional Cellulolytic Enzymes Outperform Processive Fungal Cellulases for Coproduction of Nanocellulose and Biofuels.

Science.gov (United States)

Yarbrough, John M; Zhang, Ruoran; Mittal, Ashutosh; Vander Wall, Todd; Bomble, Yannick J; Decker, Stephen R; Himmel, Michael E; Ciesielski, Peter N

2017-03-28

Producing fuels, chemicals, and materials from renewable resources to meet societal demands remains an important step in the transition to a sustainable, clean energy economy. The use of cellulolytic enzymes for the production of nanocellulose enables the coproduction of sugars for biofuels production in a format that is largely compatible with the process design employed by modern lignocellulosic (second generation) biorefineries. However, yields of enzymatically produced nanocellulose are typically much lower than those achieved by mineral acid production methods. In this study, we compare the capacity for coproduction of nanocellulose and fermentable sugars using two vastly different cellulase systems: the classical "free enzyme" system of the saprophytic fungus, Trichoderma reesei (T. reesei) and the complexed, multifunctional enzymes produced by the hot springs resident, Caldicellulosiruptor bescii (C. bescii). We demonstrate by comparative digestions that the C. bescii system outperforms the fungal enzyme system in terms of total cellulose conversion, sugar production, and nanocellulose production. In addition, we show by multimodal imaging and dynamic light scattering that the nanocellulose produced by the C. bescii cellulase system is substantially more uniform than that produced by the T. reesei system. These disparities in the yields and characteristics of the nanocellulose produced by these disparate systems can be attributed to the dramatic differences in the mechanisms of action of the dominant enzymes in each system.

A high performance Trichoderma reesei strain that reveals the importance of xylanase III in cellulosic biomass conversion.

Science.gov (United States)

Nakazawa, Hikaru; Kawai, Tetsushi; Ida, Noriko; Shida, Yosuke; Shioya, Kouki; Kobayashi, Yoshinori; Okada, Hirofumi; Tani, Shuji; Sumitani, Jun-Ichi; Kawaguchi, Takashi; Morikawa, Yasushi; Ogasawara, Wataru

2016-01-01

The ability of the Trichoderma reesei X3AB1strain enzyme preparations to convert cellulosic biomass into fermentable sugars is enhanced by the replacement of xyn3 by Aspergillus aculeatus β-glucosidase 1 gene (aabg1), as shown in our previous study. However, subsequent experiments using T. reesei extracts supplemented with the glycoside hydrolase (GH) family 10 xylanase III (XYN III) and GH Family 11 XYN II showed increased conversion of alkaline treated cellulosic biomass, which is rich in xylan, underscoring the importance of XYN III. To attain optimal saccharifying potential in T. reesei, we constructed two new strains, C1AB1 and E1AB1, in which aabg1 was expressed heterologously by means of the cbh1 or egl1 promoters, respectively, so that the endogenous XYN III synthesis remained intact. Due to the presence of wild-type xyn3 in T. reesei E1AB1, enzymes prepared from this strain were 20-30% more effective in the saccharification of alkaline-pretreated rice straw than enzyme extracts from X3AB1, and also outperformed recent commercial cellulase preparations. Our results demonstrate the importance of XYN III in the conversion of alkaline-pretreated cellulosic biomass by T. reesei. Copyright © 2015 Elsevier Inc. All rights reserved.

Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates.

Science.gov (United States)

Várnai, Anikó; Viikari, Liisa; Marjamaa, Kaisa; Siika-aho, Matti

2011-01-01

The adsorption of purified Trichoderma reesei cellulases (TrCel7A, TrCel6A and TrCel5A) and xylanase TrXyn11 and Aspergillus niger β-glucosidase AnCel3A was studied in enzyme mixture during hydrolysis of two pretreated lignocellulosic materials, steam pretreated and catalytically delignified spruce, along with microcrystalline cellulose (Avicel). The enzyme mixture was compiled to resemble the composition of commercial cellulase preparations. The hydrolysis was carried out at 35 °C to mimic the temperature of the simultaneous saccharification and fermentation (SSF). Enzyme adsorption was followed by analyzing the activity and the protein amount of the individual free enzymes in the hydrolysis supernatant. Most enzymes adsorbed quickly at early stages of the hydrolysis and remained bound throughout the hydrolysis, although the conversion reached was fairly high. Only with the catalytically oxidized spruce samples, the bound enzymes started to be released as the hydrolysis degree reached 80%. The results based on enzyme activities and protein assay were in good accordance. Copyright © 2010 Elsevier Ltd. All rights reserved.

Cellulase production using different streams of wheat grain- and wheat straw-based ethanol processes.

Science.gov (United States)

Gyalai-Korpos, Miklós; Mangel, Réka; Alvira, Pablo; Dienes, Dóra; Ballesteros, Mercedes; Réczey, Kati

2011-07-01

Pretreatment is a necessary step in the biomass-to-ethanol conversion process. The side stream of the pretreatment step is the liquid fraction, also referred to as the hydrolyzate, which arises after the separation of the pretreated solid and is composed of valuable carbohydrates along with compounds that are potentially toxic to microbes (mainly furfural, acetic acid, and formic acid). The aim of our study was to utilize the liquid fraction from steam-exploded wheat straw as a carbon source for cellulase production by Trichoderma reesei RUT C30. Results showed that without detoxification, the fungus failed to utilize any dilution of the hydrolyzate; however, after a two-step detoxification process, it was able to grow on a fourfold dilution of the treated liquid fraction. Supplementation of the fourfold-diluted, treated liquid fraction with washed pretreated wheat straw or ground wheat grain led to enhanced cellulase (filter paper) activity. Produced enzymes were tested in hydrolysis of washed pretreated wheat straw. Supplementation with ground wheat grain provided a more efficient enzyme mixture for the hydrolysis by means of the near-doubled β-glucosidase activity obtained.

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

Science.gov (United States)

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

2015-03-01

Fluorescence-assisted carbohydrate electrophoresis (FACE) is a sensitive and simple method for the separation of oligosaccharides. It relies on labeling the reducing ends of oligosaccharides with a fluorophore, followed by PAGE. Concentration changes of oligosaccharides following hydrolysis of a carbohydrate polymer could be quantitatively measured continuously over time using the FACE method. Based on the quantitative analysis, we suggested that FACE was a relatively high-throughput, repeatable, and suitable method for the analysis of the action modes of cellulases. On account of the time courses of their hydrolytic profiles, the apparent processivity was used to show the different action modes of cellulases. Cellulases could be easily differentiated as exoglucanases, β-glucosidases, or endoglucanases. Moreover, endoglucanases from the same glycoside hydrolases family had a variety of apparent processivity, indicating the different modes of action. Endoglucanases with the same binding capacities and hydrolytic activities had similar oligosaccharide profiles, which aided in their classification. The hydrolytic profile of Trichoderma reesei Cel12A, an endoglucanases from T. reesei, contained glucose, cellobiose, and cellotriose, which revealed that it may have a new glucosidase activity, corresponding to that of EC 3.2.1.74. A hydrolysate study of a T. reesei Cel12A-N20A mutant demonstrated that the FACE method was sufficiently sensitive to detect the influence of a single-site mutation on enzymatic activity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Production of cellulase from immobilized Trichoderma reesei

International Nuclear Information System (INIS)

Kasai, Noboru; Tamada, Masao; Kumakura, Minoru

1989-05-01

This report completed the results that obtained on the study of the enzyme activity in the culture of immobilized Trichoderma reesei cells in flask scale (100ml) and bench scale (30l). In the flask scale culture, the batch and repeated batch culture were carried out, and in the bench scale culture, the batch, repeated batch and continuous culture were done by using a culture equipment that is an unit process of the bench scale test plant for saccharification of cellulosic wastes. The enzyme activity of the immobilized cells was higher than that of the intact cells in the flask scale culture and it was confirmed that the enzyme activity was not decreased on the repeated batch culture of six times even. In the bench scale culture, it was found that a optimum culture condition of the immobilized cells was not different from that of the free cells and the immobilized cells gave the enzyme solution with a high enzyme activity in the culture condition of 450rpm stirring speed and air supply of 0.1v/v/m above. The technique of the repeated batch and continuous culture for long times in bench scale without contamination was established. The enzyme activity of the immobilized cells in continuous culture became to be 85 % to that in batch culture and it was found that the enzyme solution with high enzyme activity was continuously obtained in the continuous culture for long times. (author)

Screening of highly cellulolytic fungi and the action of their cellulase enzyme systems

Energy Technology Data Exchange (ETDEWEB)

Saddler, J N

1982-11-01

Over 100 strains of wood-rotting fungi were compared for their ability to degrade wood blocks. Some of these strains were then assayed for extracellular cellulase (1,4-(1,3;1,4)-beta-D-glucan 4- glucanohydrolase, EC 3.2.1.4) activity using a variety of different solid media containing carboxymethyl cellulose or acid swollen cellulose. The diameter of clearing on these plates gave an approximate indication of the order of cellulase activities obtained from culture filtrates of these strains. Trichoderma strains grown on Vogels medium gave the highest cellulase yields. The cellulase enzyme production of T. reesei C30 and QM9414 was compared with that of eight other Trichoderma strains. Trichoderma strain E58 had comparable endoglucanase and filter paper activities with the mutant strains while the beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) activity was approximately six to nine times greater. (Refs. 26).

Overexpression of D-Xylose Reductase (xyl1 Gene and Antisense Inhibition of D-Xylulokinase (xyiH Gene Increase Xylitol Production in Trichoderma reesei

Directory of Open Access Journals (Sweden)

Yuanyuan Hong

2014-01-01

Full Text Available T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH, which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable. The copy number of the xylose reductase gene (xyl1 in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol.

Overexpression of D-Xylose Reductase (xyl1) Gene and Antisense Inhibition of D-Xylulokinase (xyiH) Gene Increase Xylitol Production in Trichoderma reesei

Science.gov (United States)

Hong, Yuanyuan; Dashtban, Mehdi; Kepka, Greg; Chen, Sanfeng; Qin, Wensheng

2014-01-01

T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH), which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable). The copy number of the xylose reductase gene (xyl1) in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol. PMID:25013760

Linking Hydrolysis Performance to Trichoderma reesei Cellulolytic Enzyme Profile

DEFF Research Database (Denmark)

Lehmann, Linda Olkjær; Petersen, Nanna; I. Jørgensen, Christian

2016-01-01

Trichoderma reesei expresses a large number of enzymes involved in lignocellulose hydrolysis and the mechanism of how these enzymes work together is too complex to study by traditional methods, e.g. by spiking with single enzymes and monitoring hydrolysis performance. In this study a multivariate...... approach, partial least squares regression, was used to see if it could help explain the correlation between enzyme profile and hydrolysis performance. Diverse enzyme mixtures were produced by Trichoderma reesei Rut-C30 by exploiting various fermentation conditions and used for hydrolysis of washed...

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

Energy Technology Data Exchange (ETDEWEB)

Medve, J.

1997-02-01

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

A Newly Isolated Penicillium oxalicum 16 Cellulase with High Efficient Synergism and High Tolerance of Monosaccharide.

Science.gov (United States)

Zhao, Xi-Hua; Wang, Wei; Tong, Bin; Zhang, Su-Ping; Wei, Dong-Zhi

2016-01-01

Compared to Trichoderma reesei RUT-C30 cellulase (Trcel), Penicillium oxalicum 16 cellulase (P16cel) from the fermentation supernatant produced a 2-fold higher glucose yield when degrading microcrystalline cellulose (MCC), possessed a 10-fold higher β-glucosidase (BGL) activity, but obtained somewhat lower other cellulase component activities. The optimal temperature and pH of β-1,4-endoglucanase, cellobiohydrolase, and filter paperase from P16cel were 50-60 °C and 4-5, respectively, but those of BGL reached 70 °C and 5. The cellulase cocktail of P16cel and Trcel had a high synergism when solubilizing MCC and generated 1.7-fold and 6.2-fold higher glucose yields than P16cel and Trcel at the same filter paperase loading, respectively. Additional low concentration of fructose enhanced the glucose yield during enzymatic hydrolysis of MCC; however, additional high concentration of monosaccharide (especially glucose) reduced cellulase activities and gave a stronger monosaccharide inhibition on Trcel. These results indicate that P16cel is a more excellent cellulase than Trcel.

Mutagenesis and evaluation of cellulase properties and cellulose hydrolysis of Talaromyces piceus.

Science.gov (United States)

He, Ronglin; Cai, Pengli; Wu, Gaihong; Zhang, Can; Zhang, Dongyuan; Chen, Shulin

2015-11-01

A fungal species with a high yield of β-glucosidase was isolated and identified as Talaromyces piceus 9-3 (anamorph: Penicillium piceum) by morphological and molecular characterization. Through dimethyl sulphate mutagenesis, the cellulase over-producing strain T. piceus H16 was obtained. The FPase activity and β-glucosidase activity of T. piceus H16 were 5.83 and 53.12 IU ml(-1) respectively--a 5.34- and 4.43-times improvement from the parent strain T. piceus 9-3. The optimum pH and temperature for enzyme activity were pH 5.0 and 50 °C for FPase activity and pH 5.0 and 55 °C for β-glucosidase activity, respectively. The cellulase were quite stable at 37 °C, only losing cellulase from T. piceus H16 with that from Trichoderma reesei RUT C30 on hydrolyzing different substrates due to the high β-glucosidase activity of T. piceus H16. These data suggest that T. piceus H16 can be used as a potential cellulase producer with good prospects.

Thermostable Cellulases: Why & How?

Energy Technology Data Exchange (ETDEWEB)

Kumar, Manoj [Royal DSM, San Francisco, CA (United States)

2010-03-24

These are a set of slides from the conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

Thermostable Cellulases: Why & How?

Energy Technology Data Exchange (ETDEWEB)

Kumar, Manoj [DSM Innovation, Incorporated, San Francisco, CA (United States)

2010-04-19

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

Engineering Cellulases for Biorefinery

Energy Technology Data Exchange (ETDEWEB)

Kumar, Manoj [Royal DSM, San Francisco, CA (United States)

2010-06-27

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

Enhancing saccharification of wheat straw by mixing enzymes from genetically-modified Trichoderma reesei and Aspergillus niger.

Science.gov (United States)

Jiang, Yanping; Duarte, Alexandra Vivas; van den Brink, Joost; Wiebenga, Ad; Zou, Gen; Wang, Chengshu; de Vries, Ronald P; Zhou, Zhihua; Benoit, Isabelle

2016-01-01

To increase the efficiency of enzymatic hydrolysis for plant biomass conversion into renewable biofuel and chemicals. By overexpressing the point mutation A824 V transcriptional activator Xyr1 in Trichoderma reesei, carboxymethyl cellulase, cellobiosidase and β-D-glucosidase activities of the best mutant were increased from 1.8 IU/ml, 0.1 IU/ml and 0.05 IU/ml to 4.8 IU/ml, 0.4 IU/ml and 0.3 IU/ml, respectively. The sugar yield of wheat straw saccharification by combining enzymes from this mutant and the Aspergillus niger genetically modified strain ΔcreA/xlnR c/araR c was improved up to 7.5 mg/ml, a 229 % increase compared to the combination of wild type strains. Mixing enzymes from T. reesei and A. niger combined with the genetic modification of transcription factors is a promising strategy to increase saccharification efficiency.

Development of cellulase-nanoconjugates with enhanced ionic liquid and thermal stability for in situ lignocellulose saccharification.

Science.gov (United States)

Grewal, Jasneet; Ahmad, Razi; Khare, S K

2017-10-01

The present work aimed to improve catalytic efficiency of Trichoderma reesei cellulase for enhanced saccharification. The cellulase was immobilized on two nanomatrices i.e. magnetic and silica nanoparticles with immobilization efficiency of 85% and 76% respectively. The nanobioconjugates exhibited increase in V max , temperature optimum, pH and thermal stability as compared with free enzyme. These could be efficiently reused for five repeated cycles and were stable in 1-ethyl-3-methylimidazoliumacetate [EMIM][Ac], an ionic liquid. Ionic liquids (IL) are used as green solvents to dissolve lignocellulosic biomass and facilitate better saccharification. The cellulase immobilized on magnetic nanoparticles was used for in situ saccharification of [EMIM][Ac] pretreated sugarcane bagasse and wheat straw for two cycles. The structural deconstruction and decrease in biomass crystallinity was confirmed by SEM, XRD and FTIR. The high hydrolysis yields (∼89%) obtained in this one-pot process coupled with IL stability and recycled use of immobilized cellulase, potentiates its usefulness in biorefineries. Copyright © 2017 Elsevier Ltd. All rights reserved.

Utilization of recombinant Trichoderma reesei expressing Aspergillus aculeatus β-glucosidase I (JN11) for a more economical production of ethanol from lignocellulosic biomass.

Science.gov (United States)

Treebupachatsakul, Treesukon; Shioya, Koki; Nakazawa, Hikaru; Kawaguchi, Takashi; Morikawa, Yasushi; Shida, Yosuke; Ogasawara, Wataru; Okada, Hirofumi

2015-12-01

The capacity of Trichoderma reesei cellulase to degrade lignocellulosic biomass has been enhanced by the construction of a recombinant T. reesei strain expressing Aspergillus aculeatus β-glucosidase I. We have confirmed highly efficient ethanol production from converge-milled Japanese cedar by recombinant T. reesei expressing A. aculeatus β-glucosidase I (JN11). We investigated the ethanol productivity of JN11 and compared it with the cocktail enzyme T. reesei PC-3-7 with reinforced cellobiase activity by the commercial Novozyme 188. Results showed that the ethanol production efficiency under enzymatic hydrolysis of JN11 was comparable to the cocktail enzyme both on simultaneous saccharification and fermentation (SSF) or separate hydrolysis and fermentation (SHF) processes. Moreover, the cocktail enzyme required more protein loading for attaining similar levels of ethanol conversion as JN11. We propose that JN11 is an intrinsically economical enzyme that can eliminate the supplementation of BGL for PC-3-7, thereby reducing the cost of industrial ethanol production from lignocellulosic biomass. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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

Energy Technology Data Exchange (ETDEWEB)

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

2010-07-15

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

Dose-dependency of radiation on enzyme production in Trichoderma reesei

International Nuclear Information System (INIS)

Kumakura, Minoru

1993-01-01

Effect of irradiation dose on the production of cellulase and amylase related enzymes in Trichoderma reesei was studied in which post-irradiation time response pattern was measured. The damage of the cells irradiated with certain irradiation doses (1.40±0.20x10 5 , 2.20±0.10x10 5 , 3.00±0.50x10 5 and 3.50±0.20x10 5 rad) was rapidly recovered. The increased enzyme production in the culture of the irradiated cells resulted from the recovery of radiation damage after irradiation. The function of cell growth was not affected by irradiation below dose of 5x10 5 rad, though the function of enzyme synthesis was drastically affected. (orig.)

The productive cellulase binding capacity of cellulosic substrates.

Science.gov (United States)

Karuna, Nardrapee; Jeoh, Tina

2017-03-01

Cellulosic biomass is the most promising feedstock for renewable biofuel production; however, the mechanisms of the heterogeneous cellulose saccharification reaction are still unsolved. As cellulases need to bind isolated molecules of cellulose at the surface of insoluble cellulose fibrils or larger aggregated cellulose structures in order to hydrolyze glycosidic bonds, the "accessibility of cellulose to cellulases" is considered to be a reaction limiting property of cellulose. We have defined the accessibility of cellulose to cellulases as the productive binding capacity of cellulose, that is, the concentration of productive binding sites on cellulose that are accessible for binding and hydrolysis by cellulases. Productive cellulase binding to cellulose results in hydrolysis and can be quantified by measuring hydrolysis rates. In this study, we measured the productive Trichoderma reesei Cel7A (TrCel7A) binding capacity of five cellulosic substrates from different sources and processing histories. Swollen filter paper and bacterial cellulose had higher productive binding capacities of ∼6 µmol/g while filter paper, microcrystalline cellulose, and algal cellulose had lower productive binding capacities of ∼3 µmol/g. Swelling and regenerating filter paper using phosphoric acid increased the initial accessibility of the reducing ends to TrCel7A from 4 to 6 µmol/g. Moreover, this increase in initial productive binding capacity accounted in large part for the difference in the overall digestibility between filter paper and swollen filter paper. We further demonstrated that an understanding of how the productive binding capacity declines over the course of the hydrolysis reaction has the potential to predict overall saccharification time courses. Biotechnol. Bioeng. 2017;114: 533-542. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Production of nanotubes in delignified porous cellulosic materials after hydrolysis with cellulase.

Science.gov (United States)

Koutinas, Αthanasios Α; Papafotopoulou-Patrinou, Evgenia; Gialleli, Angelika-Ioanna; Petsi, Theano; Bekatorou, Argyro; Kanellaki, Maria

2016-08-01

In this study, tubular cellulose (TC), a porous cellulosic material produced by delignification of sawdust, was treated with a Trichoderma reesei cellulase in order to increase the proportion of nano-tubes. The effect of enzyme concentration and treatment duration on surface characteristics was studied and the samples were analyzed with BET, SEM and XRD. Also, a composite material of gelatinized starch and TC underwent enzymatic treatment in combination with amylase (320U) and cellulase (320U) enzymes. For TC, the optimum enzyme concentration (640U) led to significant increase of TC specific surface area and pore volume along with the reduction of pore diameter. It was also shown that the enzymatic treatment did not result to a significant change of cellulose crystallinity index. The produced nano-tubular cellulose shows potential for application to drug and chemical preservative delivery systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

Highly Efficient Thermostable DSM Cellulases: Why & How?

Energy Technology Data Exchange (ETDEWEB)

Kumar, Manoj [DSM Innovation, Inc., San Francisco, CA (United States)

2011-04-26

These are the slides from this presentation. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

Effect of pH on production of xylanase by Trichoderma reesei on xylan- and cellulose-based media

Energy Technology Data Exchange (ETDEWEB)

Bailey, M.J. (VTT, Biotechnical Lab., Espoo (Finland)); Buchert, J. (VTT, Biotechnical Lab., Espoo (Finland)); Viikari, L. (VTT, Biotechnical Lab., Espoo (Finland))

1993-11-01

Trichoderma reesei VTT-D-86271 (Rut C-30) was cultivated on media based on cellulose and xylan as the main carbon source in fermentors with different pH minimum controls. Production of xylanase was favoured by a rather high pH minimum control between 6.0 and 7.0 on both cellulose- and xylan-based media. Although xylanase was produced efficiently on cellulose as well as on xylan as the carbon source, significant production of cellulase was observed only on the cellulose-based medium and best production was at lower pH (4.0 minimum). Production of xylanase at pH 7.0 was shown to be dependent on the nature of the xylan in the cultivation medium but was independent of other organic components. Best production of xylanase was observed on insoluble, unsubstituted beech xylan at pH 7.0. Similar results were obtained in laboratory and pilot (200-1) fermentors. Downstream processing of the xylanase-rich, low-cellulase culture filtrate presented no technical problems despite apparent autolysis of the fungus at the high pH. Enzyme produced in the 200-1 pilot fermentor was shown to be suitable for use in enzyme-aided bleaching of kraft pulp. Due to the high xylanase/cellulase ratio of enzyme activities in the culture filtrate, pretreatment for removal of cellulase activity prior to pulp bleaching was unnecessary. (orig.)

Enzymatic hydrolysis of alkali-pretreated rice straw by Trichoderma reesei ZM4-F3

Energy Technology Data Exchange (ETDEWEB)

Zhang, QiuZhuo [Department of Environmental Science and Engineering, Harbin Institute of Technology, Harbin 150090 (China); Cai, WeiMin [Department of Environmental Science and Engineering, Harbin Institute of Technology, Harbin 150090 (China); School of Environmental Science and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang District, Shanghai 200240 (China)

2008-12-15

To minimize the cost of cellulase production, both pretreatment of the rice straw and on-site enzyme production were realized. Rice straw was first pretreated by 2% NaOH, which could increase cellulose by 54.83%, and decreased hemicellulose by 61.07% and lignin by 36.24%, respectively. Detected by SEM, significant morphological changes were observed in the tissue. Through orthogonal experiments, temperature 35 C, initial pH value 4.5 and the rotation speed of shaking bed 180 rpm were determined to be the optimal conditions for hydrolysis of rice straw by Trichoderma reesei ZM4-F3. After hydrolysis for 96 h, the production of FPA and reducing sugars could achieve 2.231 g l{sup -1} and 12.92 U ml{sup -1}, respectively. Moreover, T. reesei ZM4-F3 can decompose 68.21% of pretreated rice straw after 120 h of hydrolysis. By GC analysis, it showed that glucose is the main component of the enzymatic hydrolysates, which made GC seem to be more effective than the DNS method for analysis of the enzymatic hydrolysates as it can detect the concentration of each kind of monosaccharide more accurately. (author)

Cellulase production through solid-state tray fermentation, and its use for bioethanol from sorghum stover.

Science.gov (United States)

Idris, Ayman Salih Omer; Pandey, Ashok; Rao, S S; Sukumaran, Rajeev K

2017-10-01

The production of cellulase by Trichoderma reesei RUT C-30 under solid-state fermentation (SSF) on wheat bran and cellulose was optimized employing a two stage statistical design of experiments. Optimization of process parameters resulted in a 3.2-fold increase in CMCase production to 959.53IU/gDS. The process was evaluated at pilot scale in tray fermenters and yielded 457IU/gDS using the lab conditions and indicating possibility for further improvement. The cellulase could effectively hydrolyze alkali pretreated sorghum stover and addition of Aspergillus niger β-glucosidase improved the hydrolytic efficiency 174%, indicating the potential to use this blend for effective saccharification of sorghum stover biomass. The enzymatic hydrolysate of sorghum stover was fermented to ethanol with ∼80% efficiency. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production

Energy Technology Data Exchange (ETDEWEB)

Sukumaran, Rajeev K.; Singhania, Reeta Rani; Mathew, Gincy Marina; Pandey, Ashok [Biotechnology Division, National Institute for Interdisciplinary Science and Technology, CSIR, Trivandrum-695 019 (India)

2009-02-15

A major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which include the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). In the present study, cellulolytic enzymes for biomass hydrolysis were produced using solid state fermentation on wheat bran as substrate. Crude cellulase and a relatively glucose tolerant BGL were produced using fungi Trichoderma reesei RUT C30 and Aspergillus niger MTCC 7956, respectively. Saccharification of three different feed stock, i.e. sugar cane bagasse, rice straw and water hyacinth biomass was studied using the enzymes. Saccharification was performed with 50 FPU of cellulase and 10 U of {beta}-glucosidase per gram of pretreated biomass. Highest yield of reducing sugars (26.3 g/L) was obtained from rice straw followed by sugar cane bagasse (17.79 g/L). The enzymatic hydrolysate of rice straw was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of ethanol was 0.093 g per gram of pretreated rice straw. (author)

Understanding the Role of the Master Regulator XYR1 in Trichoderma reesei by Global Transcriptional Analysis

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dos Santos Castro, Lilian; de Paula, Renato G.; Antoniêto, Amanda C. C.; Persinoti, Gabriela F.; Silva-Rocha, Rafael; Silva, Roberto N.

2016-01-01

We defined the role of the transcriptional factor—XYR1—in the filamentous fungus Trichoderma reesei during cellulosic material degradation. In this regard, we performed a global transcriptome analysis using RNA-Seq of the Δxyr1 mutant strain of T. reesei compared with the parental strain QM9414 grown in the presence of cellulose, sophorose, and glucose as sole carbon sources. We found that 5885 genes were expressed differentially under the three tested carbon sources. Of these, 322 genes were upregulated in the presence of cellulose, while 367 and 188 were upregulated in sophorose and glucose, respectively. With respect to genes under the direct regulation of XYR1, 30 and 33 are exclusive to cellulose and sophorose, respectively. The most modulated genes in the Δxyr1 belong to Carbohydrate-Active Enzymes (CAZymes), transcription factors, and transporters families. Moreover, we highlight the downregulation of transporters belonging to the MFS and ABC transporter families. Of these, MFS members were mostly downregulated in the presence of cellulose. In sophorose and glucose, the expression of these transporters was mainly upregulated. Our results revealed that MFS and ABC transporters could be new players in cellulose degradation and their role was shown to be carbon source-dependent. Our findings contribute to a better understanding of the regulatory mechanisms of XYR1 to control cellulase gene expression in T. reesei in the presence of cellulosic material, thereby potentially enhancing its application in several biotechnology fields. PMID:26909077

Co-fermentation using Recombinant Saccharomyces cerevisiae Yeast Strains Hyper-secreting Different Cellulases for the Production of Cellulosic Bioethanol.

Science.gov (United States)

Lee, Cho-Ryong; Sung, Bong Hyun; Lim, Kwang-Mook; Kim, Mi-Jin; Sohn, Min Jeong; Bae, Jung-Hoon; Sohn, Jung-Hoon

2017-06-30

To realize the economical production of ethanol and other bio-based chemicals from lignocellulosic biomass by consolidated bioprocessing (CBP), various cellulases from different sources were tested to improve the level of cellulase secretion in the yeast Saccharomyces cerevisiae by screening an optimal translational fusion partner (TFP) as both a secretion signal and fusion partner. Among them, four indispensable cellulases for cellulose hydrolysis, including Chaetomium thermophilum cellobiohydrolase (CtCBH1), Chrysosporium lucknowense cellobiohydrolase (ClCBH2), Trichoderma reesei endoglucanase (TrEGL2), and Saccharomycopsis fibuligera β-glucosidase (SfBGL1), were identified to be highly secreted in active form in yeast. Despite variability in the enzyme levels produced, each recombinant yeast could secrete approximately 0.6-2.0 g/L of cellulases into the fermentation broth. The synergistic effect of the mixed culture of the four strains expressing the essential cellulases with the insoluble substrate Avicel and several types of cellulosic biomass was demonstrated to be effective. Co-fermentation of these yeast strains produced approximately 14 g/L ethanol from the pre-treated rice straw containing 35 g/L glucan with 3-fold higher productivity than that of wild type yeast using a reduced amount of commercial cellulases. This process will contribute to the cost-effective production of bioenergy such as bioethanol and biochemicals from cellulosic biomass.

Bioethanol production from rice straw residues

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Elsayed B. Belal

2013-01-01

Full Text Available A rice straw -cellulose utilizing mold was isolated from rotted rice straw residues. The efficient rice straw degrading microorganism was identified as Trichoderma reesei. The results showed that different carbon sources in liquid culture such as rice straw, carboxymethyl cellulose, filter paper, sugar cane bagasse, cotton stalk and banana stalk induced T. reesei cellulase production whereas glucose or Potato Dextrose repressed the synthesis of cellulase. T. reesei cellulase was produced by the solid state culture on rice straw medium. The optimal pH and temperature for T. reesei cellulase production were 6 and 25 ºC, respectively. Rice straw exhibited different susceptibilities towards cellulase to their conversion to reducing sugars. The present study showed also that, the general trend of rice straw bioconversion with cellulase was more than the general trend by T. reesei. This enzyme effectively led to enzymatic conversion of acid, alkali and ultrasonic pretreated cellulose from rice straw into glucose, followed by fermentation into ethanol. The combined method of acid pretreatment with ultrasound and subsequent enzyme treatment resulted the highest conversion of lignocellulose in rice straw to sugar and consequently, highest ethanol concentration after 7 days fermentation with S. cerevisae yeast. The ethanol yield in this study was about 10 and 11 g.L-1.

The ACEII recombinant Trichoderma reesei QM9414 strains with enhanced xylanase production and its applications in production of xylitol from tree barks.

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Xiong, Lili; Kameshwar, Ayyappa Kumar Sista; Chen, Xi; Guo, Zhiyun; Mao, Canquan; Chen, Sanfeng; Qin, Wensheng

2016-12-28

ACEII transcription factor plays a significant role in regulating the expression of cellulase and hemicellulase encoding genes. Apart from ACEII, transcription factors such as XYR1, CRE1, HAP2/3/5 complex and ACEI function in a coordinated pattern for regulating the gene expression of cellulases and hemicellulases. Studies have demonstrated that ACEII gene deletion results in decreased total cellulase and xylanase activities with reduced transcript levels of lignocellulolytic enzymes. In this study, we have successfully transformed the ACEII transcription factor encoding gene in Trichoderma reesei to significantly improve its degrading abilities. Transformation experiments on parental strain T. reesei QM9414 has resulted in five genetically engineered strains T/Ace2-2, T/Ace2-5, T/Ace2-8, T/Ace5-4 and T/Ace10-1. Among which, T/Ace2-2 has exhibited significant increase in enzyme activity by twofolds, when compared to parental strain. The T/Ace2-2 was cultured on growth substrates containing 2% bark supplemented with (a) sugar free + MA medium (b) glucose + MA medium and (c) xylose + MA medium. The bark degradation efficiency of genetically modified T/Ace2-2 strain was assessed by analyzing the xylitol production yield using HPAEC. By 6th day, about 10.52 g/l of xylitol was produced through enzymatic conversion of bark (2% bark + MA + xylose) by the T/Ace2-2 strain and by 7th day the conversion rate was found to be 0.21 g/g. Obtained results confirmed that bark growth medium supplemented with D-xylose has profoundly increased the conversion rate of bark by T/Ace2-2 strain when compared to sugar free and glucose supplemented growth media. Results obtained from scanning electron microscopy has endorsed our current results. Bark samples inoculated with T/Ace2-2 strain has showed large number of degraded cells with clearly visible cavities and fractures, by exposing the microfibrillar interwoven complex. We propose a cost effective and ecofriendly method for

Synergetic effect of yeast cell-surface expression of cellulase and expansin-like protein on direct ethanol production from cellulose

Science.gov (United States)

2013-01-01

Background Numerous studies have examined the direct fermentation of cellulosic materials by cellulase-expressing yeast; however, ethanol productivity in these systems has not yet reached an industrial level. Certain microorganisms, such as the cellulolytic fungus Trichoderma reesei, produce expansin-like proteins, which have a cellulose-loosening effect that may increase the breakdown of cellulose. Here, to improve the direct conversion of cellulose to ethanol, yeast Saccharomyces cerevisiae co-displaying cellulase and expansin-like protein on the cell surface were constructed and examined for direct ethanol fermentation performance. Results The cellulase and expansin-like protein co-expressing strain showed 246 mU/g-wet cell of phosphoric acid swollen cellulose (PASC) degradation activity, which corresponded to 2.9-fold higher activity than that of a cellulase-expressing strain. This result clearly demonstrated that yeast cell-surface expressed cellulase and expansin-like protein act synergistically to breakdown cellulose. In fermentation experiments examining direct ethanol production from PASC, the cellulase and expansin-like protein co-expressing strain produced 3.4 g/L ethanol after 96 h of fermentation, a concentration that was 1.4-fold higher than that achieved by the cellulase-expressing strain (2.5 g/L). Conclusions The PASC degradation and fermentation ability of an engineered yeast strain was markedly improved by co-expressing cellulase and expansin-like protein on the cell surface. To our knowledge, this is the first report to demonstrate the synergetic effect of co-expressing cellulase and expansin-like protein on a yeast cell surface, which may be a promising strategy for constructing direct ethanol fermenting yeast from cellulose. PMID:23835302

Use of inedible wheat residues from the KSC-CELSS breadboard facility for production of fungal cellulase

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Strayer, R. F.; Brannon, M. A.; Garland, J. L.

1990-01-01

Cellulose and xylan (a hemicellulose) comprise 50 percent of inedible wheat residue (which is 60 percent of total wheat biomass) produced in the Kennedy Space Center Closed Ecological Life Support System (CELSS) Breadboard Biomass Production Chamber (BPC). These polysaccharides can be converted by enzymatic hydrolysis into useful monosaccharides, thus maximizing the use of BPC volume and energy, and minimizing waste material to be treated. The evaluation of CELSS-derived wheat residues for production for cellulase enzyme complex by Trichoderma reesei and supplemental beta-glucosidase by Aspergillus phoenicis is in progress. Results to date are given.

Trichoderma reesei xylanase 5 is defective in the reference strain QM6a but functional alleles are present in other wild-type strains.

Science.gov (United States)

Ramoni, Jonas; Marchetti-Deschmann, Martina; Seidl-Seiboth, Verena; Seiboth, Bernhard

2017-05-01

Trichoderma reesei is a paradigm for the regulation and industrial production of plant cell wall-degrading enzymes. Among these, five xylanases, including the glycoside hydrolase (GH) family 11 XYN1 and XYN2, the GH10 XYN3, and the GH30 XYN4 and XYN6, were described. By genome mining and transcriptome analysis, a further putative xylanase, encoded by xyn5, was identified. Analysis of xyn5 from the genome-sequenced reference strain T. reesei QM6a shows that it encodes a non-functional, truncated form of XYN5. However, non-truncated orthologues are present in other genome sequenced Trichoderma spp., and sequencing of xyn5 in other T. reesei wild-type isolates shows that they harbor a putative functional xyn5 allele. In silico analysis and 3D modeling revealed that the encoded XYN5 has significant structural similarities to xylanases of the GH11 family, including a GH-typical substrate binding groove and a carboxylate pair in the active site. The xyn5 of wild-type strain TUCIM1282 was recombinantly expressed in a T. reesei strain with a (hemi)cellulase-free background and the corresponding protein purified to apparent homogeneity. The pH and temperature optima and the kinetic parameters of the purified XYN5 were pH 4, 50 °C, and V max  = 2646 nkat/mg with a K m of 9.68 mg/ml. This functional xyn5 allele was used to replace the mutated version which led to an overall increase of the xylanolytic activity. These findings are of particular importance as GH11 xylanases are of high biotechnological relevance, and T. reesei is one of the main industrial producers of such lignocellulose-degrading enzymes.

Evolution and ecophysiology of the industrial producer Hypocrea jecorina (Anamorph Trichoderma reesei and a new sympatric agamospecies related to it.

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Irina S Druzhinina

Full Text Available BACKGROUND: Trichoderma reesei, a mitosporic green mould, was recognized during the WW II based on a single isolate from the Solomon Islands and since then used in industry for production of cellulases. It is believed to be an anamorph (asexual stage of the common pantropical ascomycete Hypocrea jecorina. METHODOLOGY/PRINCIPAL FINDINGS: We combined molecular evolutionary analysis and multiple methods of phenotype profiling in order to reveal the genetic relationship of T. reesei to H. jecorina. The resulting data show that the isolates which were previously identified as H. jecorina by means of morphophysiology and ITS1 and 2 (rRNA gene cluster barcode in fact comprise several species: i H. jecorina/T. reesei sensu stricto which contains most of the teleomorphs (sexual stages found on dead wood and the wild-type strain of T. reesei QM 6a; ii T. parareesei nom. prov., which contains all strains isolated as anamorphs from soil; iii and two other hypothetical new species for which only one or two isolates are available. In silico tests for recombination and in vitro mating experiments revealed a history of sexual reproduction for H. jecorina and confirmed clonality for T. parareesei nom. prov. Isolates of both species were consistently found worldwide in pantropical climatic zone. Ecophysiological comparison of H. jecorina and T. parareesei nom. prov. revealed striking differences in carbon source utilization, conidiation intensity, photosensitivity and mycoparasitism, thus suggesting adaptation to different ecological niches with the high opportunistic potential for T. parareesei nom. prov. CONCLUSIONS: Our data prove that T. reesei belongs to a holomorph H. jecorina and displays a history of worldwide gene flow. We also show that its nearest genetic neighbour--T. parareesei nom. prov., is a cryptic phylogenetic agamospecies which inhabits the same biogeographic zone. These two species thus provide a so far rare example of sympatric speciation

77 FR 35331 - Trichoderma reesei; Proposed Significant New Use Rule

Science.gov (United States)

2012-06-13

... Trichoderma reesei; Proposed Significant New Use Rule AGENCY: Environmental Protection Agency (EPA). ACTION... Control Act (TSCA) for the genetically modified microorganism identified generically as Trichoderma reesei...: Trichoderma reesei (MCAN J-10-2) (generic). Chemical Abstracts Service (CAS) Registry Number: Not available...

Kinetics of cellobiose hydrolysis using cellobiase composites from Trichoderma reesei and Aspergillus niger

Energy Technology Data Exchange (ETDEWEB)

Grous, W.; Converse, A.; Grethlein, H.; Lynd, L.

1985-01-01

The enzymatic hydrolysis of cellulose to glucose involves the formation of cellobiose as an intermediate. It has been found necessary to add cellobiase from Aspergillus niger (NOVO) to the cellobiase component of Trichoderma reesei mutant Rut C-30 (Natick) cellulase enzymes in order to obtain after 48 h complete conversion of the cellobiose formed in the enzymatic hydrolysis of biomass. This study of the cellobiase activity of these two enzyme sources was undertaken as a first step in the formation of a kinetic model for cellulose hydrolysis that can be used in process design. In order to cover the full range of cellobiose concentrations, it was necessary to develop separate kinetic parameters for high- and low-concentration ranges of cellobiose for the enzymes from each organism. Competitive glucose inhibition was observed with the enzymes from both organisms. Substrate inhibition was observed only with the A. niger enzymes.

A CRE1- regulated cluster is responsible for light dependent production of dihydrotrichotetronin in Trichoderma reesei.

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Alberto Alonso Monroy

Full Text Available Changing light conditions, caused by the rotation of earth resulting in day and night or growth on the surface or within a substrate, result in considerably altered physiological processes in fungi. For the biotechnological workhorse Trichoderma reesei, regulation of glycoside hydrolase gene expression, especially cellulase expression was shown to be a target of light dependent gene regulation. Analysis of regulatory targets of the carbon catabolite repressor CRE1 under cellulase inducing conditions revealed a secondary metabolite cluster to be differentially regulated in light and darkness and by photoreceptors. We found that this cluster is involved in production of trichodimerol and that the two polyketide synthases of the cluster are essential for biosynthesis of dihydrotrichotetronine (syn. bislongiquinolide or bisorbibutenolide. Additionally, an indirect influence on production of the peptaibol antibiotic paracelsin was observed. The two polyketide synthetase genes as well as the monooxygenase gene of the cluster were found to be connected at the level of transcription in a positive feedback cycle in darkness, but negative feedback in light, indicating a cellular sensing and response mechanism for the products of these enzymes. The transcription factor TR_102497/YPR2 residing within the cluster regulates the cluster genes in a light dependent manner. Additionally, an interrelationship of this cluster with regulation of cellulase gene expression was detected. Hence the regulatory connection between primary and secondary metabolism appears more widespread than previously assumed, indicating a sophisticated distribution of resources either to degradation of substrate (feed or to antagonism of competitors (fight, which is influenced by light.

The Activity of Cellulase from Thermophilic Fungi Isolated from CaneBagasses

International Nuclear Information System (INIS)

Aris-Toharisman; Akyunul-Jannah

2000-01-01

The activity of cellulase from thermophilic fungi isolated from canebagasses has been measured. This wild strain, named fungal strain PJ-2,secreted a large amount of cellulolytic enzyme components consisting of 0.46units of avicelase, 0.8 units of carboxymethyl cellulose hydrolizing enzyme(CMCase) and 0.5 units of β-glucosidase per ml of culture broth oncultivation in Mandels Reese medium for 7 days at 500 o C. These cellulasesproduction was lower than that of Trichoderma reesei NRRL 3653 producing 0.5units/ml avicelase, 1.6 units/ml CMCase and 0.4 units/ml ofβ-glucosidase cultivated in the same medium at 30 o C. However,thermophilic fungi may be potential to be exploited in lignocellulosedegradation at the tropical areas as the process usually needs temperature ofabove 50 o C. (author)

Adsorption and mechanism of cellulase enzymes onto lignin isolated from corn stover pretreated with liquid hot water

OpenAIRE

Lu, Xianqin; Zheng, Xiaoju; Li, Xuezhi; Zhao, Jian

2016-01-01

Background In the bioconversion of lignocellulosic substrates, the adsorption behavior of cellulase onto lignin has a negative effect on enzymatic hydrolysis of cellulose, decreasing glucose production during enzymatic hydrolysis, thus decreasing the yield of fermentation and the production of useful products. Understanding the interaction between lignin and cellulase is necessary to optimize the components of cellulase mixture, genetically engineer high-efficiency cellulase, and reduce cost ...

Importance of cellulase cocktails favoring hydrolysis of cellulose.

Science.gov (United States)

Victoria, Juliet; Odaneth, Annamma; Lali, Arvind

2017-07-03

Depolymerization of lignocellulosic biomass is catalyzed by groups of enzymes whose action is influenced by substrate features and the composition of cellulase preparation. Cellulases contain a mixture of variety of enzymes, whose proportions dictate the saccharification of biomass. In the current study, four cellulase preparation varying in their composition were used to hydrolyze two types of alkali-treated biomass (aqueous ammonia-treated rice straw and sodium hydroxide-treated rice straw) to study the effect on catalytic rate, saccharification yields, and sugar release profile. We found that substrate features affected the extent of saccharification but had minimal effect on the sugar release pattern. In addition, complete hydrolysis to glucose was observed with enzyme preparation having at least a cellobiase units (CBU)/carboxymethyl cellulose (CMC) ratio (>0.15), while a modified enzyme ratio can be used for oligosaccharide synthesis. Thus, cellulase preparation with defined ratios of the three main enzymes can improve the saccharification which is of utmost importance in defining the success of lignocellulose-based economies.

Improvement on sugar cane bagasse hydrolysis using enzymatic mixture designed cocktail.

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Bussamra, Bianca Consorti; Freitas, Sindelia; Costa, Aline Carvalho da

2015-01-01

The aim of this work was to study cocktail supplementation for sugar cane bagasse hydrolysis, where the enzymes were provided from both commercial source and microorganism cultivation (Trichoderma reesei and genetically modified Escherichia coli), followed by purification. Experimental simplex lattice mixture design was performed to optimize the enzymatic proportion. The response was evaluated through hydrolysis microassays validated here. The optimized enzyme mixture, comprised of T. reesei fraction (80%), endoglucanase (10%) and β-glucosidase (10%), converted, theoretically, 72% of cellulose present in hydrothermally pretreated bagasse, whereas commercial Celluclast 1.5L converts 49.11%±0.49. Thus, a rational enzyme mixture designed by using synergism concept and statistical analysis was capable of improving biomass saccharification. Copyright © 2015 Elsevier Ltd. All rights reserved.

Sugar-binding sites on the surface of the carbohydrate-binding module of CBH I from Trichoderma reesei.

Science.gov (United States)

Tavagnacco, Letizia; Mason, Philip E; Schnupf, Udo; Pitici, Felicia; Zhong, Linghao; Himmel, Michael E; Crowley, Michael; Cesàro, Attilio; Brady, John W

2011-05-01

Molecular dynamics simulations were carried out for a system consisting of the carbohydrate-binding module (CBM) of the cellulase CBH I from Trichoderma reesei (Hypocrea jecorina) in a concentrated solution of β-D-glucopyranose, to determine whether there is any tendency for the sugar molecules to bind to the CBM. In spite of the general tendency of glucose to behave as an osmolyte, a marked tendency for the sugar molecules to bind to the protein was observed. However, the glucose molecules tended to bind only to specific sites on the protein. As expected, the hydrophobic face of the sugar molecules, comprising the axial H1, H3, and H5 aliphatic protons, tended to adhere to the flat faces of the three tyrosine side chains on the planar binding surface of the CBM. However, a significant tendency to bind to a groove-like feature on the upper surface of the CBM was also observed. These results would not be inconsistent with a model of the mechanism for this globular domain in which the cellodextrin chain being removed from the surface of crystalline cellulose passes over the upper surface of the CBM, presumably then available for hydrolysis in the active site tunnel of this processive cellulase. Copyright © 2011 Elsevier Ltd. All rights reserved.

Computer Simulations Reveal Multiple Functions for Aromatic Residues in Cellulase Enzymes (Fact Sheet)

Energy Technology Data Exchange (ETDEWEB)

2012-07-01

NREL researchers use high-performance computing to demonstrate fundamental roles of aromatic residues in cellulase enzyme tunnels. National Renewable Energy Laboratory (NREL) computer simulations of a key industrial enzyme, the Trichoderma reesei Family 6 cellulase (Cel6A), predict that aromatic residues near the enzyme's active site and at the entrance and exit tunnel perform different functions in substrate binding and catalysis, depending on their location in the enzyme. These results suggest that nature employs aromatic-carbohydrate interactions with a wide variety of binding affinities for diverse functions. Outcomes also suggest that protein engineering strategies in which mutations are made around the binding sites may require tailoring specific to the enzyme family. Cellulase enzymes ubiquitously exhibit tunnels or clefts lined with aromatic residues for processing carbohydrate polymers to monomers, but the molecular-level role of these aromatic residues remains unknown. In silico mutation of the aromatic residues near the catalytic site of Cel6A has little impact on the binding affinity, but simulation suggests that these residues play a major role in the glucopyranose ring distortion necessary for cleaving glycosidic bonds to produce fermentable sugars. Removal of aromatic residues at the entrance and exit of the cellulase tunnel, however, dramatically impacts the binding affinity. This suggests that these residues play a role in acquiring cellulose chains from the cellulose crystal and stabilizing the reaction product, respectively. These results illustrate that the role of aromatic-carbohydrate interactions varies dramatically depending on the position in the enzyme tunnel. As aromatic-carbohydrate interactions are present in all carbohydrate-active enzymes, the results have implications for understanding protein structure-function relationships in carbohydrate metabolism and recognition, carbon turnover in nature, and protein engineering

Deactivation of Cellulase at the Air-Liquid Interface Is the Main Cause of Incomplete Cellulose Conversion at Low Enzyme Loadings.

Science.gov (United States)

Bhagia, Samarthya; Dhir, Rachna; Kumar, Rajeev; Wyman, Charles E

2018-01-22

Amphiphilic additives such as bovine serum albumin (BSA) and Tween have been used to improve cellulose hydrolysis by cellulases. However, there has been a lack of clarity to explain their mechanism of action in enzymatic hydrolysis of pure or low-lignin cellulosic substrates. In this work, a commercial Trichoderma reesei enzyme preparation and the amphiphilic additives BSA and Tween 20 were applied for hydrolysis of pure Avicel cellulose. The results showed that these additives only had large effects on cellulose conversion at low enzyme to substrate ratios when the reaction flasks were shaken. Furthermore, changes in the air-liquid interfacial area profoundly affected cellulose conversion, but surfactants reduced or prevented cellulase deactivation at the air-liquid interface. Not shaking the flasks or adding low amounts of surfactant resulted in near theoretical cellulose conversion at low enzyme loadings given enough reaction time. At low enzyme loadings, hydrolysis of cellulose in lignocellulosic biomass with low lignin content suffered from enhanced enzyme deactivation at the air-liquid interface.

fA cellular automaton model of crystalline cellulose hydrolysis by cellulases

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Little Bryce A

2011-10-01

Full Text Available Abstract Background Cellulose from plant biomass is an abundant, renewable material which could be a major feedstock for low emissions transport fuels such as cellulosic ethanol. Cellulase enzymes that break down cellulose into fermentable sugars are composed of different types - cellobiohydrolases I and II, endoglucanase and β-glucosidase - with separate functions. They form a complex interacting network between themselves, soluble hydrolysis product molecules, solution and solid phase substrates and inhibitors. There have been many models proposed for enzymatic saccharification however none have yet employed a cellular automaton approach, which allows important phenomena, such as enzyme crowding on the surface of solid substrates, denaturation and substrate inhibition, to be considered in the model. Results The Cellulase 4D model was developed de novo taking into account the size and composition of the substrate and surface-acting enzymes were ascribed behaviors based on their movements, catalytic activities and rates, affinity for, and potential for crowding of, the cellulose surface, substrates and inhibitors, and denaturation rates. A basic case modeled on literature-derived parameters obtained from Trichoderma reesei cellulases resulted in cellulose hydrolysis curves that closely matched curves obtained from published experimental data. Scenarios were tested in the model, which included variation of enzyme loadings, adsorption strengths of surface acting enzymes and reaction periods, and the effect on saccharide production over time was assessed. The model simulations indicated an optimal enzyme loading of between 0.5 and 2 of the base case concentrations where a balance was obtained between enzyme crowding on the cellulose crystal, and that the affinities of enzymes for the cellulose surface had a large effect on cellulose hydrolysis. In addition, improvements to the cellobiohydrolase I activity period substantially improved overall

Sequential and simultaneous strategies for biorefining of wheat straw using room temperature ionic liquids, xylanases and cellulases.

Science.gov (United States)

Husson, Eric; Auxenfans, Thomas; Herbaut, Mickael; Baralle, Manon; Lambertyn, Virginie; Rakotoarivonina, Harivoni; Rémond, Caroline; Sarazin, Catherine

2018-03-01

Sequential and simultaneous strategies for fractioning wheat straw were developed in combining 1-ethyl-3-methyl imidazolium acetate [C2mim][OAc], endo-xylanases from Thermobacillus xylanilyticus and commercial cellulases. After [C2mim][OAc]-pretreatment, hydrolysis catalyzed by endo-xylanases of wheat straw led to efficient xylose production with very competitive yield (97.6 ± 1.3%). Subsequent enzymatic saccharification allowed achieving a total degradation of cellulosic fraction (>99%). These high performances revealed an interesting complementarity of [C2mim][OAc]- and xylanase-pretreatments for increasing enzymatic digestibility of cellulosic fraction in agreement with the structural and morphological changes of wheat straw induced by each of these pretreatment steps. In addition a higher tolerance of endo-xylanases from T. xylaniliticus to [C2mim][AcO] until 30% v/v than cellulases from T. reesei was observed. Based on this property, a simultaneous strategy combining [C2mim][OAc]- and endo-xylanases as pretreatment in a one-batch produced xylose with similar yield than those obtained by the sequential strategy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Immobilization of cellulase mixtures on magnetic particles for hydrolysis of lignocellulose and ease of recycling

DEFF Research Database (Denmark)

Alftrén, Johan; Hobley, Timothy John

2014-01-01

In the present study whole cellulase mixtures were covalently immobilized on non-porous magnetic particles to enable enzyme reuse. It was shown that CellicCTec2 immobilized on magnetic particles activated with cyanuric chloride gave the highest bead activity measured by mass of reducing sugar...... serum albumin (BSA)) on hydrolysis yield was studied for free and immobilized CellicCTec2. It was observed that for both free and immobilized CellicCTec2 the hydrolysis yield was increased when Tween 80, PEG 6000 or BSA was included. Interaction between magnetic particles (containing immobilized Cellic......CTec2) and lignin was examined and it was demonstrated that addition of BSA completely inhibited interaction while Tween 80 and PEG 6000 had no effect on decreasing magnetic particle-lignin interaction. Hydrolysis of pretreated wheat straw biomass was performed in two consecutive cycles using...

Efficiency of new fungal cellulase systems in boosting enzymatic degradation of barley straw lignocellulose

DEFF Research Database (Denmark)

Rosgaard, L.; Pedersen, S.; Meyer, Anne Boye Strunge

2006-01-01

This study examined the cellulytic effects on steam-pretreated barley straw of cellulose-degrading enzyme systems from the five thermophilic fungi Chaetomium thermophilum, Thielavia terrestris, Thermoascus aurantiacus, Corynascus thermophilus, and Myceliophthora thermophila and from the mesophile...... Penicillum funiculosum. The catalytic glucose release was compared after treatments with each of the crude enzyme systems when added to a benchmark blend of a commercial cellulase product, Celluclast, derived from Trichoderma reesei and a P-glucosidase, Novozym 188, from Aspergillus niger. The enzymatic...... treatments were evaluated in an experimental design template comprising a span of pH (3.5-6.5) and temperature (35-65 degrees C) reaction combinations. The addition to Celluclast + Novozym 188 of low dosages of the crude enzyme systems, corresponding to 10 wt % of the total enzyme protein load, increased...

Recycling cellulases for cellulosic ethanol production at industrial relevant conditions

DEFF Research Database (Denmark)

Lindedam, Jane; Haven, Mai Østergaard; Chylenski, Piotr

2013-01-01

Different versions of two commercial cellulases were tested for their recyclability of enzymatic activity at high dry matter processes (12% or 25% DM). Recyclability was assessed by measuring remaining enzyme activity in fermentation broth and the ability of enzymes to hydrolyse fresh, pretreated...... to preserve enzymatic activity. Best results for enzyme recycling at 25% DM was 59% and 41% of original enzyme load for a Celluclast:Novozyme188 mixture and a modern cellulase preparation, respectively. However, issues with stability of enzymes and their strong adsorption to residual solids still pose...

Separation and quantification of cellulases and hemicellulases by capillary electrophoresis

DEFF Research Database (Denmark)

Jørgensen, Henning; Kutter, Jörg Peter; Olsson, Lisbeth

2003-01-01

Cellulases and hemicellulases are two classes of enzymes produced by filamentous fungi and secreted into the cultivation medium. Both classes of enzymes consist of a subset of classes of which the fungi produce several enzymes with varying molecular mass and pI but similar enzymatic activities....... Current methods are limited in their ability to quantify all of these enzymes when all are present simultaneously in a mixture. Five different cellulases (two cellobiohydrolases and three endoglucanases) and one hemicellulase (endoxylanase) were separated using capillary electrophoresis (CE) in a fused...

Development of Specific Substrates for Hypocrea jecorina Cellulases

DEFF Research Database (Denmark)

Rasmussen, Tina Secher

  During the last decades a considerable amount of interest has focused on transformation of cellulosic biomass to renewable energy sources such as ethanol.1,2 Cellulases, secreted by different microorganisms, are key enzymes in this process. However, the degradation of cellulose is a difficult......, a commonly encountered problem during this process is the "dying off" of enzymes over time,4 possibly caused by one component in the mixture becoming rate-limiting. Currently, no methodologies exists that can accurately profile, identify and quantify active enzymes in a complex mixture and such a methodology...... of the three-dimensional (X-ray) structures of different cellulases indicated that modifications at other positions would occlude binding, while, typically some space is available around the 4' and 6' position. The substituents were chosen so that further modifications would be possible either by click...

Cellulases for biomass degradation: comparing recombinant cellulase expression platforms.

Science.gov (United States)

Garvey, Megan; Klose, Holger; Fischer, Rainer; Lambertz, Camilla; Commandeur, Ulrich

2013-10-01

Improvement of cellulase expression has the potential to change the nature of the biofuel industry. Increasing the economic feasibility of cellulase systems would significantly broaden the range of practicable biomass conversion, lowering the environmental impact of our civilisations' fuel needs. Cellulases are derived from certain fungi and bacteria, which are often difficult to culture on an industrial scale. Accordingly, methods to recombinantly express important cellulases and other glycosyl hydrolase (GH) enzymes are under serious investigation. Herein, we examine the latest developments in bacterial, yeast, plant, and fungal expression systems. We discuss current strategies for producing cellulases, and evaluate the benefits and drawbacks in yield, stability, and activity of enzymes from each system, and the overall progress in the field. Copyright © 2013 Elsevier Ltd. All rights reserved.

Study of cellulase enzymes self-assembly in aqueous-acetonitrile solvent: Viscosity measurements

Energy Technology Data Exchange (ETDEWEB)

Ghaouar, N., E-mail: naoufel-ghaouar@lycos.co [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 2092 (Tunisia); Institut National des Sciences Appliquees et de Technologie, INSAT, Centre Urbain Nord, BP. 676, Tunis (Tunisia); Aschi, A. [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 2092 (Tunisia); Belbahri, L. [School of Engineering of Lullier, University of Applied Sciences of Western Switzerland, 150, Route de Presinge, 1254 Jussy (Switzerland); Trabelsi, S.; Gharbi, A. [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 2092 (Tunisia)

2009-11-15

The present study extends the viscosity measurements performed by Ghaouar et al. [Physica B, submitted for publication.] to study the conformational change of the cellulase enzymes in aqueous-acetonitrile mixture. We aim to investigate: (i) the denaturation process by measuring the specific viscosity for temperatures varying between 25 and 65 deg. C and acetonitrile concentrations between 0% and 50%, (ii) the enzyme-enzyme interaction by calculating the Huggins coefficient and (iii) the enzyme sizes by following the hydrodynamic radius for various temperatures. The precipitation of cellulases versus acetonitrile concentration is also considered. We show from all physical quantities measured in this work that the precipitation and the denaturation processes of cellulase enzymes exist together.

Study of cellulase enzymes self-assembly in aqueous-acetonitrile solvent: Viscosity measurements

International Nuclear Information System (INIS)

Ghaouar, N.; Aschi, A.; Belbahri, L.; Trabelsi, S.; Gharbi, A.

2009-01-01

The present study extends the viscosity measurements performed by Ghaouar et al. [Physica B, submitted for publication.] to study the conformational change of the cellulase enzymes in aqueous-acetonitrile mixture. We aim to investigate: (i) the denaturation process by measuring the specific viscosity for temperatures varying between 25 and 65 deg. C and acetonitrile concentrations between 0% and 50%, (ii) the enzyme-enzyme interaction by calculating the Huggins coefficient and (iii) the enzyme sizes by following the hydrodynamic radius for various temperatures. The precipitation of cellulases versus acetonitrile concentration is also considered. We show from all physical quantities measured in this work that the precipitation and the denaturation processes of cellulase enzymes exist together.

Accessory enzymes influence cellulase hydrolysis of the model substrate and the realistic lignocellulosic biomass.

Science.gov (United States)

Sun, Fubao Fuebiol; Hong, Jiapeng; Hu, Jinguang; Saddler, Jack N; Fang, Xu; Zhang, Zhenyu; Shen, Song

2015-11-01

The potential of cellulase enzymes in the developing and ongoing "biorefinery" industry has provided a great motivation to develop an efficient cellulase mixture. Recent work has shown how important the role that the so-called accessory enzymes can play in an effective enzymatic hydrolysis. In this study, three newest Novozymes Cellic CTec cellulase preparations (CTec 1/2/3) were compared to hydrolyze steam pretreated lignocellulosic substrates and model substances at an identical FPA loading. These cellulase preparations were found to display significantly different hydrolytic performances irrelevant with the FPA. And this difference was even observed on the filter paper itself when the FPA based assay was revisited. The analysis of specific enzyme activity in cellulase preparations demonstrated that different accessory enzymes were mainly responsible for the discrepancy of enzymatic hydrolysis between diversified substrates and various cellulases. Such the active role of accessory enzymes present in cellulase preparations was finally verified by supplementation with β-glucosidase, xylanase and lytic polysaccharide monooxygenases AA9. This paper provides new insights into the role of accessory enzymes, which can further provide a useful reference for the rational customization of cellulase cocktails in order to realize an efficient conversion of natural lignocellulosic substrates. Copyright © 2015 Elsevier Inc. All rights reserved.

Immobilization of cellulases on magnetic particles to enable enzyme recycling during hydrolysis of lignocellulose

DEFF Research Database (Denmark)

Alftrén, Johan

feedstocks containing insolubles. This could potentially be overcome by immobilizing the cellulases on magnetically susceptible particles. Consequently, the immobilized cellulases could be magnetically recovered and recycled for a new cycle of enzymatic hydrolysis of cellulose. The main objective...... of this thesis was to examine the possibility of immobilizing cellulases on magnetic particles in order to enable enzyme re-use. Studies at lab and pilot scale (20 L) were conducted using model and real substrates. In paper I and III beta-glucosidase or a whole cellulase mixture was covalently immobilized...... on commercial, but expensive, magnetic particles activated with different chemistries. It was observed that the highest immobilized enzyme activities were obtained using magnetic particles activated with cyanuric chloride. In paper II biotinylated recombinant beta-glucosidase was produced and immobilized...

Use of a new Trichoderma harzianum strain isolated from the Amazon rainforest with pretreated sugar cane bagasse for on-site cellulase production.

Science.gov (United States)

Delabona, Priscila da Silva; Farinas, Cristiane Sanchez; da Silva, Mateus Ribeiro; Azzoni, Sindelia Freitas; Pradella, José Geraldo da Cruz

2012-03-01

The on-site production of cellulases is an important strategy for the development of sustainable second-generation ethanol production processes. This study concerns the use of a specific cellulolytic enzyme complex for hydrolysis of pretreated sugar cane bagasse. Glycosyl hydrolases (FPase, xylanase, and β-glucosidase) were produced using a new strain of Trichoderma harzianum, isolated from the Amazon rainforest and cultivated under different conditions. The influence of the carbon source was first investigated using shake-flask cultures. Selected carbon sources were then further studied under different pH conditions using a stirred tank bioreactor. Enzymatic activities up to 121 FPU/g, 8000 IU/g, and 1730 IU/g of delignified steam-exploded bagasse+sucrose were achieved for cellulase, xylanase and β-glucosidase, respectively. This enzymatic complex was used to hydrolyze pretreated sugar cane bagasse. A comparative evaluation, using an enzymatic extract from Trichoderma reesei RUTC30, indicated similar performance of the T. harzianum enzyme complex, being a potential candidate for on-site production of enzymes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Biotransformation of Geniposide into Genipin by Immobilized Trichoderma reesei and Conformational Study of Genipin

Directory of Open Access Journals (Sweden)

Yishun Yang

2018-01-01

Full Text Available Trichoderma reesei QM9414, Trichoderma viride 3.316, Aspergillus niger M85, and Aspergillus niger M92 were screened for hydrolyzing geniposide into genipin. T. reesei was selected according to the β-glucosidase activity of the fermentation broths using geniposide as a substrate. T. reesei was immobilized by embedding method using sodium alginate as the carrier. Geniposide was hydrolyzed by immobilized T. reesei at 28°C (200 rpm for 34 h, and the yield of genipin was 89%. The product was purified and identified by UV, IR, EIMS, and 1H-NMR. Since there were two sets of signals in 1H-NMR spectra, a series of experiments were performed and verified that the existence of two conformations was the main reason. Generally, biotransformation of geniposide into genipin by immobilized T. reesei provides a promising solution to the genipin production.

LPMOs in cellulase mixtures affect fermentation strategies for lactic acid production from lignocellulosic biomass.

Science.gov (United States)

Müller, Gerdt; Kalyani, Dayanand Chandrahas; Horn, Svein Jarle

2017-03-01

Enzymatic catalysis plays a key role in the conversion of lignocellulosic biomass to fuels and chemicals such as lactic acid. In the last decade, the efficiency of commercial cellulase cocktails has increased significantly, in part due to the inclusion of lytic polysaccharide monooxygenases (LPMOs). However, the LPMOs' need for molecular oxygen to break down cellulose demands reinvestigations of process conditions. In this study, we evaluate the efficiency of lactic acid production from steam-exploded birch using an LPMO-containing cellulase cocktail in combination with lactic acid bacteria, investigating both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). While the SSF set up generally has been considered to be more efficient because it avoids sugar accumulation which may inhibit the cellulases, the SHF set up in our study yielded 26-32% more lactic acid than the SSF. This was mainly due to competition for oxygen between LPMOs and the fermenting organisms in the SSF process, which resulted in reduced LPMO activity and thus less efficient saccharification of the lignocellulosic substrate. By means of aeration it was possible to activate the LPMOs in the SSF, but less lactic acid was produced due to a shift in metabolic pathways toward production of acetic acid. Overall, this study shows that lactic acid can be produced efficiently from lignocellulosic biomass, but that the use of LPMO-containing cellulase cocktails in fermentation processes demands re-thinking of traditional process set ups due to the requirement of oxygen in the saccharification step. Biotechnol. Bioeng. 2017;114: 552-559. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Purification and characterization of five cellulases and one xylanase from Penicillium brasilianum IBT 20888

DEFF Research Database (Denmark)

Jørgensen, Henning; Eriksson, T.; Borjesson, J.

2003-01-01

The filamentous fungus Penicillium brasilianum IBT 20888 was cultivated on a mixture of 30 g l(-1) cellulose and 10 g l(-1) xylan for 111 h and the resulting culture filtrate was used for protein purification. From the cultivation broth, five cellulases and one xylanase were purified. Hydrolysis...... studies revealed that two of the cellulases were acting as cellobiohydrolases by being active on only microcrystalline cellulose (Avicel). Three of the cellulases were active on both Avicel and carboxymethyl cellulose indicating endoglucanase activity. Two of these showed furthermore mannanase activity...... the cellulose-binding domain or an essential part of it. The basic xylanase (pI > 9) was only active towards xylan. Two of the purified cellulases with endoglucanase activity were partly sequenced and based on sequence homology with known enzymes they were classified as belonging to families 5 and 12...

Cellulase production by Trichoderma harzianum in static and mixed solid-state fermentation reactors under nonaseptic conditions

Energy Technology Data Exchange (ETDEWEB)

Deschamps, F.; Giuliano, C.; Asther, M.; Huet, M.C.; Roussos, S.

1985-09-01

Cellulase production from lignocellulosic materials was studied in solid-state cultivation by both static and mixed techniques under nonaseptic conditions. The effects of fermentation conditions, such as moisture content, pH, temperature, and aeration, on cellulase production by Trichoderma harzianum using a mixture of wheat straw (80%) and bran (20%) were investigated. With a moisture content of 74% and a pH of 5.8, 18 IU filter paper activity and 198 IU endoglucanase activity/g initial substrate content were obtained in 66 hours. The extension from static column cultivation to stirred tank reactor of 65 l capacity gave similar yields of cellulase.

Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins

Energy Technology Data Exchange (ETDEWEB)

Yarbrough, John M.; Mittal, Ashutosh; Katahira, Rui; Mansfield, Elisabeth; Taylor, Larry E.; Decker, Stephen R.; Himmel, Michael E.; Vinzant, Todd

2017-04-24

Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. To evaluate lignin binding affinities of different enzyme activities in various commercial cellulase formulations in order to determine if enzyme losses due to lignin binding can be modulated by using different enzymes of the same activity We used water:dioxane (1:9) to extract lignin from pretreated corn stover. Commercial cellulases were incubated with lignin and the unbound supernatants were evaluated for individual enzyme loss by SDS=PAGE and these were correlated with activity loss using various pNP-sugar substrates. Colorimetric assays for general glycosyl hydrolase activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native systems demonstrated low binding of endo- and exo-cellulases, high binding of xylanase, and moderate ..beta..-glucosidase binding. Engineered cellulase mixtures exhibited low binding of exo-cellulases, very strong binding of endocellulases and ..beta..- glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of ..beta..-glucosidase activities. Bound and unbound activities were correlated with general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated with binding of ..beta..-glucosidase activity. While ..beta..-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated with xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between the three classes of cellulases preparations indicate that it is certainly possible to alter the binding of specific

Radiation pretreatment of cellulosic wastes and immobilization of cells producing cellulase for their conversion to glucose

International Nuclear Information System (INIS)

Kumakura, Minoru; Kaetsu, Isao

1988-01-01

Radiation pretreatment of cellulosic wastes such as saw dust and chaff was studied by using electron beam accelerator, in which irradiation effect was increased by increasing irradiation dose and dose rate, by after heating irradiated materials at 100∼140deg C, and by irradiation in the addition of alkaline solution. Trichoderma reesei cells producing cellulase were immobilized by using fibrous porous carrier obtained from radiation polymerization. The filter paper, cellobiose, and CMC activities in the immobilized growing cells were higher than those in free cells. The activity in the immobilized cells obtained with hydrophobic carrier was higher than that obtained with hydrophilic one. Durability of the immobilized cells was examined by repeated batch culture. It was found that the enzyme solution produced in the culture of the immobilized cells can hydrolyze effectively saw dust pretreated by radiation. (author)

Biosynthesis of the enzymes of the cellulase system by T. Reesei QM 9414 in the presence of sophorose

Science.gov (United States)

Gritzali, M.

1982-12-01

As conventional, nonrenewable energy sources are rapidly depleted and it was necessary to search for alternative sources of energy. It was increasingly apparent that biomass and waste are alternatives well worth exploring. The sources of biomass and wastes that considered for conversion to useful products are quite diverse, but the most abundant constituent of almost every type is cellulose. Cellulose is cleanly converted to soluble fermentable sugars enzymatically, and cellulose enzymes were isolated from a number of microbial sources. It is generally agreed that the most effective system of enzymes for the conversion of cellulose to glucose is produced by species of the imperfect fungus Trichoderma. The mutant organism Trichoderma reesei QM 9414 is among the best producers of high levels of enzymes; these are extracellular and have carbonhydrate covalently bound to the peptide. Trichoderma produces three types of enzymes which, in a sequential and cooperative manner, convert cellulose to soluble oligosaccharides and glucose.

Radiation pre-treating straw hydrolyzed by cellulase resulted from immobilized Trichoderma reesei growing cells

International Nuclear Information System (INIS)

Lu Zhaoxin; Minoru Kumakura

1992-01-01

Wheat and rice straw was irradiated by electron beam with different dose at the presence of 4% NaOH or without 4% NaOH. The powder fraction above 200 meshes in pretreated rice straw increased with increasing doses and it was more at presence of 4% NaOH than that without 4% NaOH. The pretreated straw was hydrolyzed with 1% cellulase at 40 degree C for 48 h. The glucose yield (%) was given a rise with the increase of irradiation dose and it was 70% and 80% over that of un-pretreated rice and wheat straw, respectively. At the presence of 4% NaOH, the glucose yield increased as the irradiation dose increased from 0 to 5 x 10 5 Gy, reaching a maximum, 35% for wheat straw and 36.6% for rice straw, which increased by about 2.5 times in comparison with un-pretreated straw, then decrease with increasing the irradiation dose to 10 x 10 5 Gy. The glucose yield reached 19% and 22% for rice and wheat straw in 6 days of hydrolysis, respectively

A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina)

DEFF Research Database (Denmark)

Jørgensen, Mikael Skaanning; Skovlund, Dominique Aubert; Johannesen, Pia Francke

2014-01-01

ABSTRACT: BACKGROUND: The industrially applied filamentous fungus Trichoderma reesei has received substantial interest due to its highly efficient synthesis apparatus of cellulytic enzymes. However, the production of heterologous enzymes in T. reesei still remains low mainly due to lack of tools...

Peran Trichoderma reesei E. G Simmons pada Pengendalian Damping-Off Semai Cendana (Santalum Album Linn.

Directory of Open Access Journals (Sweden)

S. M. Widyastuti

2006-12-01

Full Text Available The sandalwood seedling has been planted in the nursery of Balitbang Kehutanan NTT to experience the destruction is about 20%, seedling is attacked by  damping-off. Biological control has been developed as alternate method against soil born diseases to eliminate  damage  on the environment. One of  the biological  agents  having high  antagonistic potential against soil born pathogen is Trichoderma spp. The direction of experiment  to search the cause of lodoh on the sandalwood  seedling and the application influences T. reesei on the control lodoh in the sandalwood seedling. Methods  of  the experiment was (1 isolation the sandalwood seedling of  painful and soil example  from NTT, (2 Postulat Koch test on the sandalwood seedling, (3 antagonistic test of T.  reesei  in vitro and (4 effectiveness  test of T. reesei against developing patogen in vivo. The result indicated that lodoh on the sandalwood seedling caused by Fusarium sp. T. reesei in vitro inhibited Fusarium sp. at average of 100% accordingly. The application of T. reesei as biological control agent against Fusarium sp. showed high  effectivity. The attact percentage of lodoh on sandalwood seedlings showed that the lowest one was growth on compost formulated with T. reesei (5%, whereas seedlings growth on compost without T. reesei,  unsteril  sands and soil samples  from NTT were 30%, 35% and 65%.

Effect of multiple short highly energetic X-ray pulses on the synthesis of endoglucanase by a mutant strain of Trichoderma reesei-M7

International Nuclear Information System (INIS)

Gemishev, Orlin; Markova, Maya; Savov, Valentin; Zapryanov, Stanislav; Blagoev, Alexander

2014-01-01

Bioconversion of cellulose-containing substrate to glucose represents an important area of modern biotechnology. Enzymes for the degradation of the polysaccharide part of biomass have been produced, mostly by fungi belonging to genus Trichoderma. Studies were carried out with the mutant strain Trichoderma reesei-M7, a cellulase producer. Spores of the enzyme producer were irradiated with different doses of characteristic X-ray radiation from metallic tungsten (mainly the W Ka1 and Ka2 lines) with a high dose rate. The latter is a specific property of the dense plasma focus (DPF) device, which has pulsed operation and thus gives short and highly energetic pulses of multiple types of rays and particles. In this case, we focused our study on the influence of hard X-rays. The doses of X-rays absorbed by the spores varied in the range of approximately 5-11,000 mSv measured with thermoluminescent dosimeters (TLD). The influence of the applied doses in combination with exceptionally high dose rates (in the order of tens of millisieverts per microsecond) on the activity of the produced endoglucanase, amount of biomass and extra-cellular protein, was studied in batch cultivation conditions. In the dose range of 200-1200 mSv, some enhancement of endoglucanase activity was obtained: around 18%-32%, despite the drop of the biomass amount, compared with the untreated material. Keywords: endoglucanase; X-ray pulses; thermoluminescent dosimeters (TLD); dense plasma focus (DPF); Trichoderma reesei

Insight into the process of product expulsion in cellobiohydrolase Cel6A from Trichoderma reesei by computational modeling.

Science.gov (United States)

Huang, Houhou; Han, Fei; Guan, Shanshan; Qian, Mengdan; Wan, Yongfeng; Shan, Yaming; Zhang, Hao; Wang, Song

2018-03-24

Glycoside hydrolase cellulase family 6 from Trichoderma reesei (TrCel6A) is an important cellobiohydrolase to hydrolyze cellooligosaccharide into cellobiose. The knowledge of enzymatic mechanisms is critical for improving the conversion efficiency of cellulose into ethanol or other chemicals. However, the process of product expulsion, a key component of enzymatic depolymerization, from TrCel6A has not yet been described in detail. Here, conventional molecular dynamics and steered molecular dynamics (SMD) were applied to study product expulsion from TrCel6A. Tyr103 may be a crucial residue in product expulsion given that it exhibits two different posthydrolytic conformations. In one conformation, Tyr103 rotates to open the -3 subsite. However, Tyr103 does not rotate in the other conformation. Three different routes for product expulsion were proposed on the basis of the two different conformations. The total energy barriers of the three routes were calculated through SMD simulations. The total energy barrier of product expulsion through Route 1, in which Tyr103 does not rotate, was 22.2 kcal·mol -1 . The total energy barriers of product expulsion through Routes 2 and 3, in which Tyr103 rotates to open the -3 subsite, were 10.3 and 14.4 kcal·mol -1 , respectively. Therefore, Routes 2 and 3 have lower energy barriers than Route 1, and Route 2 is the thermodynamically optimal route for product expulsion. Consequently, the rotation of Tyr103 may be crucial for product release from TrCel6A. Results of this work have potential applications in cellulase engineering.

Chimeric enzymes with improved cellulase activities

Science.gov (United States)

Xu, Qi; Baker, John O; Himmel, Michael E

2015-03-31

Nucleic acid molecules encoding chimeric cellulase polypeptides that exhibit improved cellulase activities are disclosed herein. The chimeric cellulase polypeptides encoded by these nucleic acids and methods to produce the cellulases are also described, along with methods of using chimeric cellulases for the conversion of cellulose to sugars such as glucose.

Ionic liquid-tolerant cellulase enzymes

Science.gov (United States)

Gladden, John; Park, Joshua; Singer, Steven; Simmons, Blake; Sale, Ken

2017-10-31

The present invention provides ionic liquid-tolerant cellulases and method of producing and using such cellulases. The cellulases of the invention are useful in saccharification reactions using ionic liquid treated biomass.

Immobilization of cellulase by radiation polymerization

International Nuclear Information System (INIS)

Kumakura, M.; Kaetsu, I.

1983-01-01

Immobilization of cellulase by radiation polymerization at low temperatures was studied. The enzymatic activity of immobilized cellulase pellets varied with the monomer, enzyme concentration, and the thickness of immobilized cellulase pellets. The optimum monomer concentration in the immobilization of cellulase was 30-50% at the pellet thickness of 1.0 mm, in which the enzymatic activity was 50%. The enzymatic activity of immobilized cellulase pellets was examined using various substrates such as cellobiose, carboxymethylcellulose, and paper pretreated by radiation. It was found that irradiated paper can be hydrolyzed by immobilized cellulase pellets. (author)

Exo-endo cellulase fusion protein

Science.gov (United States)

Bower, Benjamin S [Palo Alto, CA; Larenas, Edmund A [Palo Alto, CA; Mitchinson, Colin [Palo Alto, CA

2012-01-17

The present invention relates to a heterologous exo-endo cellulase fusion construct, which encodes a fusion protein having cellulolytic activity comprising a catalytic domain derived from a fungal exo-cellobiohydrolase and a catalytic domain derived from an endoglucanase. The invention also relates to vectors and fungal host cells comprising the heterologous exo-endo cellulase fusion construct as well as methods for producing a cellulase fusion protein and enzymatic cellulase compositions.

Unravelling the molecular basis for light modulated cellulase gene expression - the role of photoreceptors in Neurospora crassa

Science.gov (United States)

2012-01-01

Background Light represents an important environmental cue, which exerts considerable influence on the metabolism of fungi. Studies with the biotechnological fungal workhorse Trichoderma reesei (Hypocrea jecorina) have revealed an interconnection between transcriptional regulation of cellulolytic enzymes and the light response. Neurospora crassa has been used as a model organism to study light and circadian rhythm biology. We therefore investigated whether light also regulates transcriptional regulation of cellulolytic enzymes in N. crassa. Results We show that the N. crassa photoreceptor genes wc-1, wc-2 and vvd are involved in regulation of cellulase gene expression, indicating that this phenomenon is conserved among filamentous fungi. The negative effect of VVD on production of cellulolytic enzymes is thereby accomplished by its role in photoadaptation and hence its function in White collar complex (WCC) formation. In contrast, the induction of vvd expression by the WCC does not seem to be crucial in this process. Additionally, we found that WC-1 and WC-2 not only act as a complex, but also have individual functions upon growth on cellulose. Conclusions Genome wide transcriptome analysis of photoreceptor mutants and evaluation of results by analysis of mutant strains identified several candidate genes likely to play a role in light modulated cellulase gene expression. Genes with functions in amino acid metabolism, glycogen metabolism, energy supply and protein folding are enriched among genes with decreased expression levels in the wc-1 and wc-2 mutants. The ability to properly respond to amino acid starvation, i. e. up-regulation of the cross pathway control protein cpc-1, was found to be beneficial for cellulase gene expression. Our results further suggest a contribution of oxidative depolymerization of cellulose to plant cell wall degradation in N. crassa. PMID:22462823

Bioprospecting thermophiles for cellulase production: a review.

Science.gov (United States)

Acharya, Somen; Chaudhary, Anita

2012-07-01

Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of lignocellulosic biomass hydrolysis is through enzymatic saccharification using cellulases. Significant information has also been gained about the physiology of thermophilic cellulases producers and process development for enzyme production and biomass saccharification. The review discusses the current knowledge on cellulase producing thermophilic microorganisms, their physiological adaptations and control of cellulase gene expression. It discusses the industrial applications of thermophilic cellulases, their cost of production and challenges in cellulase research especially in the area of improving process economics of enzyme production.

Bioprospecting thermophiles for cellulase production: a review

Directory of Open Access Journals (Sweden)

Somen Acharya

2012-09-01

Full Text Available Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of lignocellulosic biomass hydrolysis is through enzymatic saccharification using cellulases. Significant information has also been gained about the physiology of thermophilic cellulases producers and process development for enzyme production and biomass saccharification. The review discusses the current knowledge on cellulase producing thermophilic microorganisms, their physiological adaptations and control of cellulase gene expression. It discusses the industrial applications of thermophilic cellulases, their cost of production and challenges in cellulase research especially in the area of improving process economics of enzyme production.

Formation and release of cellulolytic enzymes during growth of Trichoderma reesei on cellobiose and glycerol

Energy Technology Data Exchange (ETDEWEB)

Vaheri, M.P.; Vaheri, M.E.O.; Kaupinen, V.S.

1979-01-01

Production and release of cellulolytic enzymes by T. reesei QM 9414 were studied under induced and non-induced conditions and glycerol, respectively, as the only C source. There was a base level of cell debris-bound hydrolytic activity against filter paper and p-nitrophenyl glycoside even in T. reesei grown non-induced on glycerol. T. reesei grown on cellobiose was induced to produce large amounts of extracellular filter paper- and CMC-hydrolyzing enzymes, which were actively released even in the early stages of cultivation. Beta-Glucosidase was mainly detected in the cell debris and was not released unless the cells were autolyzing.

Process relevant screening of cellulolytic organisms for consolidated bioprocessing.

Science.gov (United States)

Antonov, Elena; Schlembach, Ivan; Regestein, Lars; Rosenbaum, Miriam A; Büchs, Jochen

2017-01-01

Although the biocatalytic conversion of cellulosic biomass could replace fossil oil for the production of various compounds, it is often not economically viable due to the high costs of cellulolytic enzymes. One possibility to reduce costs is consolidated bioprocessing (CBP), integrating cellulase production, hydrolysis of cellulose, and the fermentation of the released sugars to the desired product into one process step. To establish such a process, the most suitable cellulase-producing organism has to be identified. Thereby, it is crucial to evaluate the candidates under target process conditions. In this work, the chosen model process was the conversion of cellulose to the platform chemical itaconic acid by a mixed culture of a cellulolytic fungus with Aspergillus terreus as itaconic acid producer. Various cellulase producers were analyzed by the introduced freeze assay that measures the initial carbon release rate, quantifying initial cellulase activity under target process conditions. Promising candidates were then characterized online by monitoring their respiration activity metabolizing cellulose to assess the growth and enzyme production dynamics. The screening of five different cellulase producers with the freeze assay identified Trichoderma   reesei and Penicillium   verruculosum as most promising. The measurement of the respiration activity revealed a retarded induction of cellulase production for P.   verruculosum but a similar cellulase production rate afterwards, compared to T.   reesei . The freeze assay measurement depicted that P.   verruculosum reaches the highest initial carbon release rate among all investigated cellulase producers. After a modification of the cultivation procedure, these results were confirmed by the respiration activity measurement. To compare both methods, a correlation between the measured respiration activity and the initial carbon release rate of the freeze assay was introduced. The analysis revealed that the

Immobilization of Trichoderma reesei by radiation polymerization

International Nuclear Information System (INIS)

Zhou Ruimin; Ma Zueteh; Kaetus, Isao; Kumakura, Minoro

1993-01-01

Immobilization of Trichoderma reesei was carried out by radiation polymerization. It was found that the activity of fixed cells increased with increasing surface area of the carrier and was affected by the concentration of monomer tetraethylenglycol dimethacrylate and the shape of the substrate composition and structure of cotton textile fabrics. (author)

Ultrasonic hyperactivation of cellulase immobilized on magnetic nanoparticles.

Science.gov (United States)

Ladole, Mayur Ramrao; Mevada, Jayesh Sevantilal; Pandit, Aniruddha Bhalchandra

2017-09-01

In the present work, effect of low power, low frequency ultrasound on cellulase immobilized magnetic nanoparticles (cellulase@MNPs) was studied. To gain maximum activity recovery in cellulase@MNPs various parameters viz. ratio of MNPs:cellulase, concentration of glutaraldehyde and cross-linking time were optimized. The influence of ultrasonic power on cellulase@MNPs was studied. Under ultrasonic conditions at 24kHz, 6W power, and 6min of incubation time there was almost 3.6 fold increased in the catalytic activity of immobilized cellulase over the control. Results also indicated that there was improvement in pH and temperature stability of cellulase@MNPs. Furthermore, thermal deactivation energy required was more in cellulase@MNPs than that of the free cellulase. Secondary structural analysis revealed that there were conformational changes in free cellulase and cellulase@MNPs before and after sonication which might be responsible for enhanced activity after ultrasonication. Finally, the influence of ultrasound and cellulase@MNPs for biomass hydrolysis was studied. Copyright © 2017 Elsevier Ltd. All rights reserved.

An Integrated Process of Ionic Liquid Pretreatment and Enzymatic Hydrolysis of Lignocellulosic Biomass with Immobilised Cellulase

Directory of Open Access Journals (Sweden)

Mihaela Ungurean

2014-08-01

Full Text Available An integrated process of lignocellulosic biomass conversion was set up involving pretreatment by an ionic liquid (IL and hydrolysis of cellulose using cellulase immobilised by the sol-gel method, with recovery and reuse of both the IL and biocatalyst. As all investigated ILs, regardless of the nature of the anion and the cation, led to the loss of at least 50% of the hydrolytic activity of cellulase, the preferred solution involved reprecipitation of cellulose and lignin after the pretreatment, instead of performing the enzymatic hydrolysis in the same reaction system. The cellulose recovered after pretreatment with 1-ethyl-3-methylimidazolium acetate ([Emim][Ac] and dimethylsulfoxide (DMSO (1:1 ratio, v/v was hydrolysed with almost double yield after 8 h of reaction time with the immobilised cellulase, compared to the reference microcrystalline cellulose. The dissolution capacity of the pretreatment mixture was maintained at satisfactory level during five reuse cycles. The immobilised cellulase was recycled in nine reaction cycles, preserving about 30% of the initial activity.

[The isolation and characterization of beta-glucosidase gene and beta-glucosidase of Trichoderma viride]: Progress report

International Nuclear Information System (INIS)

Stafford, D.W.

1983-01-01

Our project was to isolate and characterize the enzyme β-glucosidase and to clone and characterize the β-glucosidase gene; our goal is to clone and characterize each of the cellulase genes from Trichoderma. The induction of the Trichoderma reesei cellulase complex by cellulose and by the soluble inducer, sophorose, has been demonstrated. Although the induction of the cellulase complex has previously been well documented, the induction of β-glucosidase had been questioned. 49 refs., 6 figs., 2 tabs

Evaluation of bacterial expansin EXLX1 as a cellulase synergist for the saccharification of lignocellulosic Agro-industrial wastes.

Directory of Open Access Journals (Sweden)

Hui Lin

Full Text Available Various types of lignocellulosic wastes extensively used in biofuel production were provided to assess the potential of EXLX1 as a cellulase synergist. Enzymatic hydrolysis of natural wheat straw showed that all the treatments using mixtures of cellulase and an optimized amount of EXLX1, released greater quantities of sugars than those using cellulase alone, regardless of cellulase dosage and incubation time. EXLX1 exhibited different synergism and binding characteristics for different wastes, but this can be related to their lignocellulosic components. The cellulose proportion could be one of the important factors. However, when the cellulose proportion of different biomass samples exhibited no remarkable differences, a higher synergism of EXLX1 is prone to occur on these materials, with a high proportion of hemicellulose and a low proportion of lignin. The information could be favorable to assess whether EXLX1 is effective as a cellulase synergist for the hydrolysis of the used materials. Binding assay experiments further suggested that EXLX1 bound preferentially to alkali pretreated materials, as opposed to acid pretreated materials under the assay condition and the binding preference would be affected by incubation temperature.

Evaluation of Bacterial Expansin EXLX1 as a Cellulase Synergist for the Saccharification of Lignocellulosic Agro-Industrial Wastes

Science.gov (United States)

Lin, Hui; Shen, Qi; Zhan, Ju-Mei; Wang, Qun; Zhao, Yu-Hua

2013-01-01

Various types of lignocellulosic wastes extensively used in biofuel production were provided to assess the potential of EXLX1 as a cellulase synergist. Enzymatic hydrolysis of natural wheat straw showed that all the treatments using mixtures of cellulase and an optimized amount of EXLX1, released greater quantities of sugars than those using cellulase alone, regardless of cellulase dosage and incubation time. EXLX1 exhibited different synergism and binding characteristics for different wastes, but this can be related to their lignocellulosic components. The cellulose proportion could be one of the important factors. However, when the cellulose proportion of different biomass samples exhibited no remarkable differences, a higher synergism of EXLX1 is prone to occur on these materials, with a high proportion of hemicellulose and a low proportion of lignin. The information could be favorable to assess whether EXLX1 is effective as a cellulase synergist for the hydrolysis of the used materials. Binding assay experiments further suggested that EXLX1 bound preferentially to alkali pretreated materials, as opposed to acid pretreated materials under the assay condition and the binding preference would be affected by incubation temperature. PMID:24086425

Production of cellulase from Pellicularia filamentosa

Energy Technology Data Exchange (ETDEWEB)

Mizukoshi, S; Sugi, H; Mori, H; Ichihashi, M

1977-01-01

In the screening test for cellulase producers from 237 strains of microorganisms, 3 strains of Fusarium sp., 2 strains of Trichoderma sp., and 4 strains of Pellicularia sp. were found to produce a lot of cellulase in their culture filtrates. Of these cellulase producing fungi, Pellicularia filamentosa isolated from diseased cucumber seedlings showed high cellulase activity comparable to that of Trichoderma viride. This preparation was active toward filter paper, avicel, carboxymethyl cellulose, soluble starch, insulin, and p-nitrophenyl ..beta..-D-glucopyranoside. The optimum temperature and pH for the filter paper degrading activity of this crude cellulase were 45/sup 0/C and 5.0, respectively. The filter paper degrading activity was inhibited by Cu/sup + +/, Fe/sup + + +/, N-bromosuccinimide, and sodium dodecyl sulfate.

Prospecting Agro-waste Cocktail: Supplementation for Cellulase Production by a Newly Isolated Thermophilic B. licheniformis 2D55.

Science.gov (United States)

Kazeem, Muinat Olanike; Shah, Umi Kalsom Md; Baharuddin, Azhari Samsu; AbdulRahman, Nor' Aini

2017-08-01

Bacteria isolated from thermophilic environment that can produce cellulase as well as utilise agro-waste biomass have a high potential for developing thermostable cellulase required in the biofuel industry. The cost for cellulase represents a significant challenge in converting lignocellulose to fermentable sugars for biofuel production. Among three potential bacteria examined, Bacillus licheniformis 2D55 (accession no. KT799651) was found to produce the highest cellulolytic activity (CMCase 0.33 U/mL and FPase 0.09 U/mL) at 18-24 h fermentation when grown on microcrystalline cellulose (MCC) as a carbon source in shake flask at 50 °C. Cellulase production process was further conducted on the untreated and NaOH pretreated rice straw (RS), rice husk (RH), sugarcane bagasse (BAG) and empty fruit bunch (EFB). Untreated BAG produced the highest FPase (0.160 U/mL), while the highest CMCase (0.150 U/mL) was supported on the pretreated RH. The mixture of untreated BAG and pretreated RH as agro-waste cocktail has remarkably improved CMCase (3.7- and 1.4-fold) and FPase (2.5- and 11.5-fold) compared to the untreated BAG and pretreated RH, respectively. The mechanism of cellulase production explored through SEM analysis and the location of cellulase enzymes of the isolate was also presented. Agro-waste cocktail supplementation provides an alternative method for an efficient production of cellulase.

Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

Science.gov (United States)

Iwakuma, Hidekazu; Koyama, Yoshiyuki; Miyachi, Ayako; Nasukawa, Masashi; Matsumoto, Hitoshi; Yano, Shuntaro; Ogihara, Jun; Kasumi, Takafumi

2016-01-01

We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

Thermostable cellulase from a thermomonospora gene

Science.gov (United States)

Wilson, D.B.; Walker, L.P.; Zhang, S.

1997-10-14

The invention relates to a gene isolated from Thermomonospora fusca, wherein the gene encodes a thermostable cellulase. Disclosed is the nucleotide sequence of the T. fusca gene; and nucleic acid molecules comprising the gene, or a fragment of the gene, that can be used to recombinantly express the cellulase or a catalytically active polypeptide thereof, respectively. The isolated and purified recombinant cellulase or catalytically active polypeptide may be used to hydrolyze substrate either by itself; or in combination with other cellulases, with the resultant combination having unexpected hydrolytic activity. 3 figs.

Utilization of radiation technique on the saccharification and fermentation of biomass

International Nuclear Information System (INIS)

Kaetsu, I.; Kumakura, M.; Fujimura, T.; Yoshii, F.; Kojima, T.; Tamada, M.

1981-01-01

The application of irradiation technique to the process of saccharification and subsequent fermentation of cellulosic wastes such as chaff and rice straw to obtain ethanol, was investigated. It was found that when waste raw materials were irradiated by γ-ray or electron beam, they became accessible to the subsequent enzymatic saccharification reaction. Irradiation of 10 7 to 10 8 Rad was enough for this effect. Some kind of additives reduced necessary dosage for this pretreatment. Cellulase, Trichoderma reesei which produce cellulase, and yeast were immobilized as biocatalysts for biomass conversion by radiation-induced polymerization of glass-forming monomer at low temperature. The immobilized cellulase showed almost the same activity of glucose production as the native cellulase. Continuous saccharification reaction was carried out by using the immobilized cellulase. The immobilized Trichoderma reesei and the immobilized yeast showed almost the same activity as the intact biocatalysts. It was concluded that the continuous saccharification and subsequent fermentation could be carried out effectively by using the immobilized biocatalysts. Spinach chloroplasts were immobilized by the same method as the first step for the conversion of water into hydrogen gas using solar energy. The immobilized chloroplasts kept the O 2 evolution activity in storage more than 30 days at 4 0 C. (author)

Utilization of radiation technique on the saccharification and fermentation of biomass

Energy Technology Data Exchange (ETDEWEB)

Kaetsu, I.; Kumakura, M.; Fujimura, T.; Yoshii, F.; Kojima, T.; Tamada, M. (Japan Atomic Energy Research Inst., Takasaki, Gunma. Takasaki Radiation Chemistry Research Establishment)

1981-01-01

The application of irradiation technique to the process of saccharification and subsequent fermentation of cellulosic wastes such as chaff and rice straw to obtain ethanol, was investigated. It was found that when waste raw materials were irradiated by ..gamma..-ray or electron beam, they became accessible to the subsequent enzymatic saccharification reaction. Irradiation of 10/sup 7/ to 10/sup 8/ Rad was enough for this effect. Some kind of additives reduced necessary dosage for this pretreatment. Cellulase, Trichoderma reesei which produce cellulase, and yeast were immobilized as biocatalysts for biomass conversion by radiation-induced polymerization of glass-forming monomer at low temperature. The immobilized cellulase showed almost the same activity of glucose production as the native cellulase. Continuous saccharification reaction was carried out by using the immobilized cellulase. The immobilized Trichoderma reesei and the immobilized yeast showed almost the same activity as the intact biocatalysts. It was concluded that the continuous saccharification and subsequent fermentation could be carried out effectively by using the immobilized biocatalysts. Spinach chloroplasts were immobilized by the same method as the first step for the conversion of water into hydrogen gas using solar energy. The immobilized chloroplasts kept the O/sub 2/ evolution activity in storage more than 30 days at 4/sup 0/C.

Utilization of radiation technique on the saccharification and fermentation of biomass

Science.gov (United States)

Kaetsu, I.; Kumakura, M.; Fujimura, T.; Yoshii, F.; Kojima, T.; Tamada, M.

The application of irradiation technique to the process of saccharification and subsequent fermentation of cellulosic wastes such as chaff and rice straw to obtain ethanol, was investigated. It was found that when waste raw materials were irradiated by ?-ray or electron beam, they became accessible to the subsequent enzymatic saccharification reaction. Irradiation of 10 7-10 8 Rad was enough for this effect. Some kind of additives reduced necessary dosage for this pretreatment. Cellulase, Trichoderma reesei which produce cellulase, and yeast were immobilized as biocatalysts for biomass conversion by radiation-induced polymerization of glass-forming monomer at low temperature. The immobilized cellulase showed almost same activity of glucose production as the native cellulase. Continuous saccharification reaction was carried out by using the immobilized cellulase. The immobilized Trichoderma reesei and the immobilized yeast showed almost same activity as the intact biocatalysts. It was concluded that the continuous saccharification and subsequent fermentation could be carried out effectively by using the immobilized biocatalysts. Spinach chloroplasts were immobilized by the same method as the first step for the conversion of water into hydrogen gas using solar energy. The immobilized chloroplasts kept the O 2 evolution activity in storage more than 30 days at 4°C. Thermostatility of chloroplasts was also improved greatly by the immobilization.

Investigating commercial cellulase performances toward specific biomass recalcitrance factors using reference substrates.

Science.gov (United States)

Ju, Xiaohui; Bowden, Mark; Engelhard, Mark; Zhang, Xiao

2014-05-01

Three commercial cellulase preparations, Novozymes Cellic(®) Ctec2, Dupont Accellerase(®) 1500, and DSM Cytolase CL, were evaluated for their hydrolytic activity using a set of reference biomass substrates with controlled substrate characteristics. It was found that lignin remains a significant recalcitrance factor to all the preparations, although different enzyme preparations respond to the inhibitory effect of lignin differently. Also, different types of biomass lignin can inhibit cellulase enzymes in different manners. Enhancing enzyme activity toward biomass fiber swelling is an area significantly contributing to potential improvement in cellulase performance. While the degree of polymerization of cellulose in the reference substrates did not present a major recalcitrance factor to Novozymes Cellic(®) Ctec2, cellulose crystallite has been shown to have a significant lower reactivity toward all enzyme mixtures. The presence of polysaccharide monooxygenases (PMOs) in Novozymes Ctec2 appears to enhance enzyme activity toward decrystallization of cellulose. This study demonstrated that reference substrates with controlled chemical and physical characteristics of structural features can be applied as an effective and practical strategy to identify cellulosic enzyme activities toward specific biomass recalcitrance factor(s) and provide specific targets for enzyme improvement.

Overexpressing key component genes of the secretion pathway for enhanced secretion of an Aspergillus niger glucose oxidase in Trichoderma reesei.

Science.gov (United States)

Wu, Yilan; Sun, Xianhua; Xue, Xianli; Luo, Huiying; Yao, Bin; Xie, Xiangming; Su, Xiaoyun

2017-11-01

Vast interest exists in developing T. reesei for production of heterologous proteins. Although rich genomic and transcriptomic information has been uncovered for the T. reesei secretion pathway, little is known about whether engineering its key components could enhance expression of a heterologous gene. In this study, snc1, a v-SNARE gene, was first selected for overexpression in T. reesei. In engineered T. reesei with additional copies of snc1, the Aspergillus niger glucose oxidase (AnGOD) was produced to a significantly higher level (2.2-fold of the parental strain). hac1 and bip1, two more component genes in the secretion pathway, were further tested for overexpression and found to be also beneficial for AnGOD secretion. The overexpression of one component gene more or less affected the expression of the other two genes, suggesting a complex regulating mechanism. Our study demonstrates the potential of engineering the secretion pathway for enhancing heterologous gene production in T. reesei. Copyright © 2017 Elsevier Inc. All rights reserved.

Ethanol from wood. Cellulase enzyme production

Energy Technology Data Exchange (ETDEWEB)

Szengyel, Zsolt

2000-03-01

Conversion of biomass to liquid fuels, such as ethanol, has been investigated during the past decades. First due to the oil crisis of the 1970s and lately because of concerns about greenhouse effect, ethanol has been found to be a suitable substitute for gasoline in transportation. Although ethanol is produced in large quantities from corn starch, the conversion of lignocellulosic biomass to ethanol is rather problematic. However, cellulosic raw materials are important as they are available in large quantities from agriculture and forestry. One of the most extensively investigated processes is the enzymatic process, in which fungal cellulolytic enzymes are used to convert the cellulose content of the biomass to glucose, which is then fermented to ethanol. In order to make the raw material accessible to biological attack, it has to be pretreated first. The most successful method, which has been evaluated for various lignocellulosic materials, is the steam pretreatment. In this thesis the utilization of steam pretreated willow (hardwood) and spruce (softwood) was examined for enzyme production using a filamentous fungus T. reesei RUT C30. Various carbon sources originating from the steam pretreated materials have been investigated. The replacement of the solid carbon source with a liquid carbon source, as well as the effect of pH, was studied. The effect of toxic compounds generated during pretreatment was also examined. Comparative study of softwood and hardwood showed that steam pretreated hardwood is a better carbon source than softwood. The hydrolytic potential of enzyme solutions produced on wood derived carbon sources was better compared to commercial cellulases. Also enzyme solutions produced on steam pretreated spruce showed less sensitivity towards toxic compounds formed during steam pretreatment.

Bioprospecting thermophiles for cellulase production: a review

OpenAIRE

Acharya,Somen; Chaudhary,Anita

2012-01-01

Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of...

Space mutagenic effect of Trichoderma reesei

International Nuclear Information System (INIS)

Tian Xingshan; Zhou Fengzheng; Huang Xiaoguang; Kuang Zheshi; Pan Mushui; Li Guoli; Guo Yong

2005-01-01

The slant mycelia cultured with or without mutagenic agent diethyl sulfate (DS) and spores of Trichoderma reesei were loaded in the 18th returning satellite. Systematical screening showed that the life cycle and morphology of some strains had changed after space flight. After selection and domestication, 3 mutant strains with high cellulose enzyme activity were isolated. The cellulose enzyme productivities of the mutants were significantly increased more than 50%, and the mutant were generically stable. (authors)

Cellulase hydrolysis of unsorted MSW

DEFF Research Database (Denmark)

Jensen, Jacob Wagner; Felby, Claus; Jørgensen, Henning

2011-01-01

A recent development in waste management and engineering has shown that the cellulase can be used for the liquefaction of organic fractions in household waste. The focus of this study was to optimize the enzyme hydrolysis of thermally treated municipal solid waste (MSW) by the addition of surfact......A recent development in waste management and engineering has shown that the cellulase can be used for the liquefaction of organic fractions in household waste. The focus of this study was to optimize the enzyme hydrolysis of thermally treated municipal solid waste (MSW) by the addition...... of calcium, potassium, sodium, chloride and others that may affect cellulolytic enzymes. Cellulase performance showed no effect of adding the metal ion-chelating agent EDTA to the solution. The cellulases were stable, tolerated and functioned in the presence of several contaminants....

Construction of a novel selection system for endoglucanases exhibiting carbohydrate-binding modules optimized for biomass using yeast cell-surface engineering.

Science.gov (United States)

Nakanishi, Akihito; Bae, Jungu; Kuroda, Kouichi; Ueda, Mitsuyoshi

2012-10-23

To permit direct cellulose degradation and ethanol fermentation, Saccharomyces cerevisiae BY4741 (Δsed1) codisplaying 3 cellulases (Trichoderma reesei endoglucanase II [EG], T. reesei cellobiohydrolase II [CBH], and Aspergillus aculeatus β-glucosidase I [BG]) was constructed by yeast cell-surface engineering. The EG used in this study consists of a family 1 carbohydrate-binding module (CBM) and a catalytic module. A comparison with family 1 CBMs revealed conserved amino acid residues and flexible amino acid residues. The flexible amino acid residues were at positions 18, 23, 26, and 27, through which the degrading activity for various cellulose structures in each biomass may have been optimized. To select the optimal combination of CBMs of EGs, a yeast mixture with comprehensively mutated CBM was constructed. The mixture consisted of yeasts codisplaying EG with mutated CBMs, in which 4 flexible residues were comprehensively mutated, CBH, and BG. The yeast mixture was inoculated in selection medium with newspaper as the sole carbon source. The surviving yeast consisted of RTSH yeast (the mutant sequence of CBM: N18R, S23T, S26S, and T27H) and wild-type yeast (CBM was the original) in a ratio of 1:46. The mixture (1 RTSH yeast and 46 wild-type yeasts) had a fermentation activity that was 1.5-fold higher than that of wild-type yeast alone in the early phase of saccharification and fermentation, which indicates that the yeast mixture with comprehensively mutated CBM could be used to select the optimal combination of CBMs suitable for the cellulose of each biomass.

Interrelationships of VEL1 and ENV1 in light response and development in Trichoderma reesei.

Directory of Open Access Journals (Sweden)

Hoda Bazafkan

Full Text Available Sexual development is regulated by a complex regulatory mechanism in fungi. For Trichoderma reesei, the light response pathway was shown to impact sexual development, particularly through the photoreceptor ENVOY. Moreover, T. reesei communicates chemically with a potential mating partner in its vicinity, a response which is mediated by the velvet family protein VEL1 and its impact on secondary metabolism. We therefore studied the regulatory interactions of ENV1 and VEL1 with a focus on sexual development. Although individual mutants in both genes are female sterile under standard crossing conditions (light-dark cycles, an altered light regime enabled sexual development, which we found to be due to conditional female sterility of Δenv1, but not Δvel1. Phenotypes of growth and asexual sporulation as well as regulation of the peptide pheromone precursors of double mutants suggested that ENV1 and VEL1 balance positive and negative regulators of these functions. Additionally, VEL1 contributed to the strong deregulation of the pheromone system observed in env1 mutants. Female sterility of Δvel1 was rescued by deletion of env1 in darkness in MAT1-1, indicating a block of sexual development by ENV1 in darkness that is balanced by VEL1 in the wild-type. We conclude that ENV1 and VEL1 exert complementing functions in development of T. reesei. Our results further showed that the different developmental phenotypes of vel1/veA mutants in T. reesei and Aspergillus nidulans are not due to the presence or function of ENV1 in the VELVET regulatory pathway in T. reesei.

Cellulase Production by Bacteria: A Review

OpenAIRE

Sadhu Sangrila; Maiti Tushar Kanti

2013-01-01

Cellulose is an abundant natural biopolymer on earth and most dominating Agricultural waste. This cellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. It can be degraded by cellulase produced by cellulolytic bacteria. This enzyme has various industrial applications and now considered as major group of industrial enzyme. The review discusses application of cellulase, classification of cellulase, quantification...

Immobilization of cellulase on functionalized cobalt ferrite nanoparticles

International Nuclear Information System (INIS)

Bohara, Raghvendra Ashok; Thorat, Nanasaheb Devappa; Pawar, Shivaji Hariba

2016-01-01

Amine functionalized cobalt ferrite (AF-CoFe 2 O 4 ) magnetic nanoparticles (MNPs) were used for immobilization of cellulase enzyme via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDS) and N-hydroxysuccinimide (NHS) coupling reaction. The structural, morphological and magnetic properties of AF-CoFe 2 O 4 were determined. TEM micrograph revealed a mean diameter of -8 nm and showed that the AF-CoFe 2 O 4 remain distinct with no significant change in size after binding with cellulase. Fourier transform infrared (FT-IR) spectroscopy confirmed the binding of cellulase to AF-CoFe 2 O 4 . The properties of immobilized cellulase were investigated by optimizing binding efficiency, pH, temperature and reusability. The results showed that the immobilized cellulase has higher thermal stability than free cellulase, which might be due to covalent interaction between cellulase and AF-CoFe 2 O 4 surface. The immobilized cellulase also showed good reusability after recovery. Therefore, AF-CoFe 2 O 4 MNPs can be considered as promising candidate for enzyme immobilization.

Reaction mechanism of dicofol removal by cellulase.

Science.gov (United States)

Wang, Ziyuan; Yang, Ting; Zhai, Zihan; Zhang, Boya; Zhang, Jianbo

2015-10-01

It remains unclear whether dicofol should be defined as a persistent organic pollutant. Its environmental persistence has gained attention. This study focused on its degradation by cellulase. Cellulase was separated using a gel chromatogram, and its degradation activity towards dicofol involved its endoglucanase activity. By analyzing the kinetic parameters of cellulase reacting with mixed substrates, it was shown that cellulase reacted on dicofol and carboxyl methyl cellulose through two different active centers. Thus, the degradation of dicofol was shown to be an oxidative process by cellulase. Next, by comparing the impacts of tert-butyl alcohol (a typical OH free-radical inhibitor) on the removal efficiencies of dicofol under both cellulase and Fenton reagent systems, it was shown that the removal of dicofol was initiated by OH free radicals produced by cellulase. Finally, 4,4'-dichloro-dibenzophenone and chloride were detected using gas chromatography mass spectrometry and ion chromatography analysis, which supported our hypothesis. The reaction mechanism was analyzed and involved an attack by OH free radicals at the orthocarbon of dicofol, resulting in the degradation product 4,4'-dichloro-dibenzophenone. Copyright © 2015. Published by Elsevier B.V.

Undefined cellulase formulations hinder scientific reproducibility.

Science.gov (United States)

Himmel, Michael E; Abbas, Charles A; Baker, John O; Bayer, Edward A; Bomble, Yannick J; Brunecky, Roman; Chen, Xiaowen; Felby, Claus; Jeoh, Tina; Kumar, Rajeev; McCleary, Barry V; Pletschke, Brett I; Tucker, Melvin P; Wyman, Charles E; Decker, Stephen R

2017-01-01

In the shadow of a burgeoning biomass-to-fuels industry, biological conversion of lignocellulose to fermentable sugars in a cost-effective manner is key to the success of second-generation and advanced biofuel production. For the effective comparison of one cellulase preparation to another, cellulase assays are typically carried out with one or more engineered cellulase formulations or natural exoproteomes of known performance serving as positive controls. When these formulations have unknown composition, as is the case with several widely used commercial products, it becomes impossible to compare or reproduce work done today to work done in the future, where, for example, such preparations may not be available. Therefore, being a critical tenet of science publishing, experimental reproducibility is endangered by the continued use of these undisclosed products. We propose the introduction of standard procedures and materials to produce specific and reproducible cellulase formulations. These formulations are to serve as yardsticks to measure improvements and performance of new cellulase formulations.

Immobilization of cellulase on functionalized cobalt ferrite nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Bohara, Raghvendra Ashok; Thorat, Nanasaheb Devappa; Pawar, Shivaji Hariba [Center for Interdisciplinary Research, D. Y. Patil University, Kolhapur (India)

2016-01-15

Amine functionalized cobalt ferrite (AF-CoFe{sub 2}O{sub 4}) magnetic nanoparticles (MNPs) were used for immobilization of cellulase enzyme via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDS) and N-hydroxysuccinimide (NHS) coupling reaction. The structural, morphological and magnetic properties of AF-CoFe{sub 2}O{sub 4} were determined. TEM micrograph revealed a mean diameter of -8 nm and showed that the AF-CoFe{sub 2}O{sub 4} remain distinct with no significant change in size after binding with cellulase. Fourier transform infrared (FT-IR) spectroscopy confirmed the binding of cellulase to AF-CoFe{sub 2}O{sub 4}. The properties of immobilized cellulase were investigated by optimizing binding efficiency, pH, temperature and reusability. The results showed that the immobilized cellulase has higher thermal stability than free cellulase, which might be due to covalent interaction between cellulase and AF-CoFe{sub 2}O{sub 4} surface. The immobilized cellulase also showed good reusability after recovery. Therefore, AF-CoFe{sub 2}O{sub 4} MNPs can be considered as promising candidate for enzyme immobilization.

Enhanced Cellulose Degradation Using Cellulase-Nanosphere Complexes

Science.gov (United States)

Blanchette, Craig; Lacayo, Catherine I.; Fischer, Nicholas O.; Hwang, Mona; Thelen, Michael P.

2012-01-01

Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS) and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC); however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production. PMID:22870287

Enhanced cellulose degradation using cellulase-nanosphere complexes.

Directory of Open Access Journals (Sweden)

Craig Blanchette

Full Text Available Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC; however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production.

Enhanced cellulose degradation using cellulase-nanosphere complexes.

Science.gov (United States)

Blanchette, Craig; Lacayo, Catherine I; Fischer, Nicholas O; Hwang, Mona; Thelen, Michael P

2012-01-01

Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS) and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC); however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production.

Alteration of white-rot basidiomycetes cellulase and xylanase activities in the submerged co-cultivation and optimization of enzyme production by Irpex lacteus and Schizophyllum commune.

Science.gov (United States)

Metreveli, Eka; Kachlishvili, Eva; Singer, Steven W; Elisashvili, Vladimir

2017-10-01

Mono and dual cultures of four white-rot basidiomycete species were evaluated for cellulase and xylanase activity under submerged fermentation conditions. Co-cultivation of Pycnoporus coccineus or Trametes hirsuta with Schizophyllum commune displayed antagonistic interactions resulting in the decrease of endoglucanase and total cellulase activities. In contrast, increases in cellulase and xylanase activity were revealed through the compatible interactions of Irpex lacteus with S. commune. Co-cultivation conditions were optimized for maximum enzyme production by I. lacteus and S. commune, the best producers of cellulase/xylanase and β-glucosidase, respectively. An optimized medium for the target enzyme production by the mixed culture was established in a laboratory fermenter yielding 7U/mL total cellulase, 142U/mL endoglucanase, 104U/mL xylanase, and 5.2U/mL β-glucosidase. The dual culture approach resulted in an enzymatic mixture with 11% improved lignocellulose saccharification potential compared to enzymes from a monoculture of I. lacteus. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cellulase activities in biomass conversion: measurement methods and comparison.

Science.gov (United States)

Dashtban, Mehdi; Maki, Miranda; Leung, Kam Tin; Mao, Canquan; Qin, Wensheng

2010-12-01

Cellulose, the major constituent of all plant materials and the most abundant organic molecule on the Earth, is a linear biopolymer of glucose molecules, connected by β-1,4-glycosidic bonds. Enzymatic hydrolysis of cellulose requires mixtures of hydrolytic enzymes including endoglucanases, exoglucanases (cellobiohydrolases), and β-glucosidases acting in a synergistic manner. In biopolymer hydrolysis studies, enzyme assay is an indispensable part. The most commonly used assays for the individual enzymes as well as total cellulase activity measurements, including their advantages and limitations, are summarized in this review article. In addition, some novel approaches recently used for enzyme assays are summarized.

Effects of Lytic Polysaccharide Monooxygenase Oxidation on Cellulose Structure and Binding of Oxidized Cellulose Oligomers to Cellulases

Energy Technology Data Exchange (ETDEWEB)

Vermaas, Josh V.; Crowley, Michael F.; Beckham, Gregg T.; Payne, Christina M.

2015-05-21

with different affinities relative to cellobiose itself, which potentially affects hydrolytic turnover through product inhibition. To examine the effect of oxidation on cello-oligomer binding, we use thermodynamic integration to compute the relative change in binding free energy between the hydrolyzed and oxidized products in the active site of Family 7 and Family 6 processive glycoside hydrolases, Trichoderma reesei Cel7A and Cel6A, which are key industrial cellulases and commonly used model systems for fungal cellulases. Our results suggest that the equilibrium between the two reducing end oxidized products, favoring the linear aldonic acid, may increase product inhibition, which would in turn reduce processive substrate turnover. In the case of LMPO action at the nonreducing end, oxidation appears to lower affinity with the nonreducing end specific cellulase, reducing product inhibition and potentially promoting processive cellulose turnover. Overall, this suggests that oxidation of recalcitrant polysaccharides by LPMOs accelerates degradation not only by increasing the concentration of chain termini but also by reducing decrystallization work, and that product inhibition may be somewhat reduced as a result.

Carboxymethyl-cellulase from Erwina chrysanthemi. I. Production and regulation of extracellular carboxymethyl-cellulase

Energy Technology Data Exchange (ETDEWEB)

Boyer, M.H.; Chambost, J.P.; Magnan, M.; Cattaneo, J.

1984-01-01

Erwinia chrysanthemi strain 3665 growing aerobically in a mineral salts medium containing various carbon sources constitutively secreted low levels of carboxymethyl-cellulase activity. Increased production of this activity was triggered by conditions which reduced the growth rate. The results obtained with continuous culture suggested that this production was controlled by a mechanism similar to catabolite repression. However, other factors might be implicated in the regulation of cellulase production.

Increase in stability of cellulase immobilized on functionalized magnetic nanospheres

Energy Technology Data Exchange (ETDEWEB)

Zhang, Wenjuan [Department of Machine Intelligence and Systems Engineering, Faculty of Systems Engineering, Akita Prefectural University, Akita 015-0055 (Japan); Qiu, Jianhui, E-mail: qiu@akita-pu.ac.jp [Department of Machine Intelligence and Systems Engineering, Faculty of Systems Engineering, Akita Prefectural University, Akita 015-0055 (Japan); Feng, Huixia [College of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou 730050 (China); Zang, Limin; Sakai, Eiichi [Department of Machine Intelligence and Systems Engineering, Faculty of Systems Engineering, Akita Prefectural University, Akita 015-0055 (Japan)

2015-02-01

Functionalized magnetic nanospheres were prepared by co-condensation of tetraethylorthosilicate with three different amino-silanes: 3-(2-aminoethylamino propyl)-triethoxysilane (AEAPTES), 3-(2-aminoethylamino propyl)-trimethoxysilane (AEAPTMES) and 3-aminopropyltriethoxysilane (APTES). Then three functionalized magnetic nanospheres were used as supports for immobilization of cellulase. The three functionalized magnetic nanospheres with core–shell morphologies exhibited higher capacity for cellulase immobilization than unfunctionalized magnetic nanospheres. The increasing of surface charge of functionalized magnetic nanospheres leads to an enhancement of the capacity of cellulase immobilization. Particularly, AEAPTMES with methoxy groups was favored to be hydrolyzed and grafted on unfunctionalized magnetic nanospheres than the others. AEAPTMES functionalized magnetic nanospheres with the highest zeta potential (29 mV) exhibited 87% activity recovery and the maximum amount of immobilized cellulase was 112 mg/g support at concentration of initial cellulase of 8 mg/mL. Immobilized cellulase on AEAPTMES functionalized magnetic nanospheres had higher temperature stability and broader pH stability than other immobilized cellulases and free cellulase. In particular, it can be used in about 40 °C, demonstrating the potential of biofuel production using this immobilized cellulase. - Highlights: • Three Amino-silane modified magnetic nanospheres were prepared. • Cellulase immobilized AEAPTMES functionalized magnetic nanospheres had higher temperature stability and broader pH stability than free cellulase. • The potential of biofuel production using this immobilized cellulase.

Increase in stability of cellulase immobilized on functionalized magnetic nanospheres

International Nuclear Information System (INIS)

Zhang, Wenjuan; Qiu, Jianhui; Feng, Huixia; Zang, Limin; Sakai, Eiichi

2015-01-01

Functionalized magnetic nanospheres were prepared by co-condensation of tetraethylorthosilicate with three different amino-silanes: 3-(2-aminoethylamino propyl)-triethoxysilane (AEAPTES), 3-(2-aminoethylamino propyl)-trimethoxysilane (AEAPTMES) and 3-aminopropyltriethoxysilane (APTES). Then three functionalized magnetic nanospheres were used as supports for immobilization of cellulase. The three functionalized magnetic nanospheres with core–shell morphologies exhibited higher capacity for cellulase immobilization than unfunctionalized magnetic nanospheres. The increasing of surface charge of functionalized magnetic nanospheres leads to an enhancement of the capacity of cellulase immobilization. Particularly, AEAPTMES with methoxy groups was favored to be hydrolyzed and grafted on unfunctionalized magnetic nanospheres than the others. AEAPTMES functionalized magnetic nanospheres with the highest zeta potential (29 mV) exhibited 87% activity recovery and the maximum amount of immobilized cellulase was 112 mg/g support at concentration of initial cellulase of 8 mg/mL. Immobilized cellulase on AEAPTMES functionalized magnetic nanospheres had higher temperature stability and broader pH stability than other immobilized cellulases and free cellulase. In particular, it can be used in about 40 °C, demonstrating the potential of biofuel production using this immobilized cellulase. - Highlights: • Three Amino-silane modified magnetic nanospheres were prepared. • Cellulase immobilized AEAPTMES functionalized magnetic nanospheres had higher temperature stability and broader pH stability than free cellulase. • The potential of biofuel production using this immobilized cellulase

Hydrolysis of cellulose in a cellulase-bead fluidized bed reactor

Energy Technology Data Exchange (ETDEWEB)

Karube, I; Tanaka, S; Shirai, T; Suzuki, S

1977-08-01

Cellulase was immobilized in a collagen fibril matrix, and no leakage of cellulase from the collagen fibril matrix was observed. The immobilized cellulase was more stable than the native cellulase. The substrate cellulose was hydrolyzed quantitatively with immobilized cellulase. The final reaction product was identified as glucose. Immobilized cellulase was used in a fluidized bed reactor where the pressure drop of the fluidized bed reactor was low and constant. Cellulose was hydrolyzed to glucose by the cellulase-bead fluidized bed reactor. The minimum flow velocity (U/sub mf/) was 0.5 cm/sec and the optimum flow velocity of the cellulose hydrolysis was 1 cm/sec.

Enhanced cellulase production by Penicillium oxalicum for bio-ethanol application.

Science.gov (United States)

Saini, Reetu; Saini, Jitendra Kumar; Adsul, Mukund; Patel, Anil Kumar; Mathur, Anshu; Tuli, Deepak; Singhania, Reeta Rani

2015-01-01

Present study was focused on cellulase production from an indigenously isolated filamentous fungal strain, identified as Penicillium oxalicum. Initially, cellulase production under submerged fermentation in shake flasks resulted in cellulase activity of 0.7 FPU/mL. Optimization of process parameters enhanced cellulase production by 1.7-fold and resulted in maximum cellulase activity of 1.2 FPU/mL in 8 days. Cellulase production was successfully scaled-up to 7 L fermenter under controlled conditions and incubation time was reduced from 8 days to 4 days for achieving similar cellulase titer. Optimum pH and temperature for activity of the crude enzyme were pH 5 and 50 °C, respectively. At 50 °C the produced cellulase retained approximately 50% and 26% of its activity at 48 h and 72 h, respectively. Hydrolytic efficiency of P. oxalicum was comparable to commercial cellulase preparations which indicate its great potential for application in the lignocellulose hydrolysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

cellulase and pectinase production potentials of aspergillus niger

African Journals Online (AJOL)

Prof Oyeleke

Production of pectinase and cellulase by Aspergillus niger from corn cob was examined. ... organism was screened for enzymatic activity using Carboxyl Methyl ... preparation of denim fabrics in textile industries, ... exploitation of cellulase is its high cost of production ... catabolite repression influence economics of cellulase.

Characterization of a thermophilic cellulase from Geobacillus sp. HTA426, an efficient cellulase-producer on alkali pretreated of lignocellulosic biomass.

Science.gov (United States)

Potprommanee, Laddawan; Wang, Xiao-Qin; Han, Ye-Ju; Nyobe, Didonc; Peng, Yen-Ping; Huang, Qing; Liu, Jing-Yong; Liao, Yu-Ling; Chang, Ken-Lin

2017-01-01

A themophilic cellulase-producing bacterium was isolated from a hot spring district and identified as Geobacillus sp. HTA426. The cellulase enzyme produced by the Geobacillus sp. HTA426 was purified through ammonium sulfate precipitation and ion exchange chromatography, with the recovery yield and fold purification of 10.14% and 5.12, respectively. The purified cellulase has a molecular weight of 40 kDa. The optimum temperature and pH for carboxymethyl cellulase (CMCase) activity of the purified cellulase were 60°C and pH 7.0, respectively. The enzyme was also stable over a wide temperature range of 50°C to 70°C after 5 h of incubation. Moreover, the strain HTA426 was able to grow and produce cellulase on alkali-treated sugarcane bagasse, rice straw and water hyacinth as carbon sources. Enzymatic hydrolysis of sugarcane bagasse, which was regarded as the most effective carbon source for cellulase production (CMCase activity = 103.67 U/mL), followed by rice straw (74.70 U/mL) and water hyacinth (51.10 U/mL). This strain producing an efficient thermostable cellulose is a potential candidate for developing a more efficient and cost-effective process for converting lignocellulosic biomass into biofuel and other industrial process.

Using an Inducible Promoter of a Gene Encoding Penicillium verruculosum Glucoamylase for Production of Enzyme Preparations with Enhanced Cellulase Performance.

Directory of Open Access Journals (Sweden)

Alexander G Bulakhov

Full Text Available Penicillium verruculosum is an efficient producer of highly active cellulase multienzyme system. One of the approaches for enhancing cellulase performance in hydrolysis of cellulosic substrates is to enrich the reaction system with β -glucosidase and/or accessory enzymes, such as lytic polysaccharide monooxygenases (LPMO displaying a synergism with cellulases.Genes bglI, encoding β-glucosidase from Aspergillus niger (AnBGL, and eglIV, encoding LPMO (formerly endoglucanase IV from Trichoderma reesei (TrLPMO, were cloned and expressed by P. verruculosum B1-537 strain under the control of the inducible gla1 gene promoter. Content of the heterologous AnBGL in the secreted multienzyme cocktails (hBGL1, hBGL2 and hBGL3 varied from 4 to 10% of the total protein, while the content of TrLPMO in the hLPMO sample was ~3%. The glucose yields in 48-h hydrolysis of Avicel and milled aspen wood by the hBGL1, hBGL2 and hBGL3 preparations increased by up to 99 and 80%, respectively, relative to control enzyme preparations without the heterologous AnBGL (at protein loading 5 mg/g substrate for all enzyme samples. The heterologous TrLPMO in the hLPMO preparation boosted the conversion of the lignocellulosic substrate by 10-43%; however, in hydrolysis of Avicel the hLPMO sample was less effective than the control preparations. The highest product yield in hydrolysis of aspen wood was obtained when the hBGL2 and hLPMO preparations were used at the ratio 1:1.The enzyme preparations produced by recombinant P. verruculosum strains, expressing the heterologous AnBGL or TrLPMO under the control of the gla1 gene promoter in a starch-containing medium, proved to be more effective in hydrolysis of a lignocellulosic substrate than control enzyme preparations without the heterologous enzymes. The enzyme composition containing both AnBGL and TrLPMO demonstrated the highest performance in lignocellulose hydrolysis, providing a background for developing a fungal strain capable

Activity-based protein profiling of secreted cellulolytic enzyme activity dynamics in Trichoderma reesei QM6a, NG14, and RUT-C30

Energy Technology Data Exchange (ETDEWEB)

Anderson, Lindsey N.; Culley, David E.; Hofstad, Beth A.; Chauvigne-Hines, Lacie M.; Zink, Erika M.; Purvine, Samuel O.; Smith, Richard D.; Callister, Stephen J.; Magnuson, Jon M.; Wright, Aaron T.

2013-12-01

Development of alternative, non-petroleum based sources of bioenergy that can be applied in the short-term find great promise in the use of highly abundant and renewable lignocellulosic plant biomass.1 This material obtained from different feedstocks, such as forest litter or agricultural residues, can yield liquid fuels and other chemical products through biorefinery processes.2 Biofuels are obtained from lignocellulosic materials by chemical pretreatment of the biomass, followed by enzymatic decomposition of cellulosic and hemicellulosic compounds into soluble sugars that are converted to desired chemical products via microbial metabolism and fermentation.3, 4 To release soluble sugars from polymeric cellulose multiple enzymes are required, including endoglucanase, exoglucanase, and β-glucosidase.5, 6 However, the enzymatic hydrolysis of cellulose into soluble sugars remains a significant limiting factor to the efficient and economically viable utilization of lignocellulosic biomass for transport fuels.7, 8 The primary industrial source of cellulose and hemicellulases is the mesophilic soft-rot fungus Trichoderma reesei,9 having widespread applications in food, feed, textile, pulp, and paper industries.10 The genome encodes 200 glycoside hydrolases, including 10 cellulolytic and 16 hemicellulolytic enzymes.11 The hypercellulolytic catabolite derepressed strain RUT-C30 was obtained through a three-step UV and chemical mutagenesis of the original T. reesei strain QM6a,12, 13 in which strains M7 and NG14 were intermediate, having higher cellulolytic activity than the parent strain but less activity and higher catabolite repression than RUT-C30.14 Numerous methods have been employed to optimize the secreted enzyme cocktail of T. reesei including cultivation conditions, operational parameters, and mutagenesis.3 However, creating an optimal and economical enzyme mixture for production-scale biofuels synthesis may take thousands of experiments to identify.

Product inhibition of enzymatic hydrolysis of cellulose: are we running the reactions all wrong?

DEFF Research Database (Denmark)

Meyer, Anne S.

2012-01-01

cellobiose and glucose. The reported KI for glucose on the T. reesei cellulases and -glucosidase varies from 0.04 to 5 g/L. The type of inhibition is debated, and probably varies for different -glucosidases, but with a required goal of sufficient glucose concentration to support ethanol concentrations....... This is because the currently used Trichoderma reesei derived cellulases, i.e. exoglucanases (mainly the cellobiohydrolases Cel7A and Cel6A), endo-1,4--glucanases, and now boosted with -glucosidase and other enzymes, now considered the “industry standard” enzymes, are significantly inhibited by the products...... of minimum ∼5–6% v/v, the glucose product concentrations exceed the critical limit for product inhibition. Hence, regardless of the recent progress in enzyme development for cellulose hydrolysis, the glucose product inhibition remains an issue, which is exacerbated as the reaction progresses, especially...

Cytochemical localization of cellulases in decayed and nondecayed wood

Energy Technology Data Exchange (ETDEWEB)

Murmanis, L.; Highley, T.L.; Palmer, J.G.

1987-01-01

Sawdust from undecayed western hemlock wood and from wood previously decayed by the brown-rot fungus Poria placenta or by the white-rot fungus Ganoderma applanatum was incubated with commercial cellulase from Trichoderma viride. Samples were treated cytochemically to locate cellulase activity and examined by TEM. Results showed that cellulase degraded undecayed wood extensively, with the attack starting on the outer border of a cell wall and progressing inside. Wood decayed by P. placenta, with or without cellulase incubation, and treated by the cytochemical test showed uniform distribution of electron dense particles throughout the cell walls. In wood decayed by G. applanatum, cellulase degradation was similar to that in undecayed wood. From measurements of particle diameter it is suggested that electron dense particles are cellulase. It is concluded that brown-rot and white-rot fungi have different effects on the microstructure of wood. The brown-rot fungus appears to open the wood microstructure so that cellulase can diffuse throughout the degraded tracheid wall.

Increase in stability of cellulase immobilized on functionalized magnetic nanospheres

Science.gov (United States)

Zhang, Wenjuan; Qiu, Jianhui; Feng, Huixia; Zang, Limin; Sakai, Eiichi

2015-02-01

Functionalized magnetic nanospheres were prepared by co-condensation of tetraethylorthosilicate with three different amino-silanes: 3-(2-aminoethylamino propyl)-triethoxysilane (AEAPTES), 3-(2-aminoethylamino propyl)-trimethoxysilane (AEAPTMES) and 3-aminopropyltriethoxysilane (APTES). Then three functionalized magnetic nanospheres were used as supports for immobilization of cellulase. The three functionalized magnetic nanospheres with core-shell morphologies exhibited higher capacity for cellulase immobilization than unfunctionalized magnetic nanospheres. The increasing of surface charge of functionalized magnetic nanospheres leads to an enhancement of the capacity of cellulase immobilization. Particularly, AEAPTMES with methoxy groups was favored to be hydrolyzed and grafted on unfunctionalized magnetic nanospheres than the others. AEAPTMES functionalized magnetic nanospheres with the highest zeta potential (29 mV) exhibited 87% activity recovery and the maximum amount of immobilized cellulase was 112 mg/g support at concentration of initial cellulase of 8 mg/mL. Immobilized cellulase on AEAPTMES functionalized magnetic nanospheres had higher temperature stability and broader pH stability than other immobilized cellulases and free cellulase. In particular, it can be used in about 40 °C, demonstrating the potential of biofuel production using this immobilized cellulase.

Genetic modification: a tool for enhancing cellulase secretion

Directory of Open Access Journals (Sweden)

Anusuiya Singh

2017-06-01

Full Text Available Lignocellulosic (LC biomass is abundantly available as a low-cost resource on the Earth. LC conversion into energy carriers is the most accepted alternative energy production policy because it is non-competitor to food or feed. LC ethanol has brought cellulases to the forefront which was otherwise lost in oblivion during last decades. LC biomass can be converted into value added products or into sugars by various routes, e.g., thermo-chemical, chemical, or biological methods. Biological route via enzymes is one of the most eco-friendly and feasible method. Both fungi and bacteria are known to degrade biomass. Fungi have been greatly exploited for cellulase production due to their inherent properties of secreting extracellular cellulase. These microorganisms are known as cellulase producers for many decades, however, to bring the enzymatic biomass conversion to an economically feasible status, extensive research efforts have been made in last decade to enhance cellulase titers. Mutations and genetic interventions along with bioprocess development have played a very important role for enhancing cellulase production. This review will present a critical overview of the on-going research towards improving cellulase production for biofuel industry via genetic modification, which will include mutation and genetic engineering employed to exert changes at genetic level in microorganisms.

Comparison of Different Methods of Denim Stone Washing by Pumice Stone, Acid Cellulases and Neutral Cellulases

Directory of Open Access Journals (Sweden)

M. Montazer

2007-10-01

Full Text Available Denim is a casual garment normally used by young people and extremely influential in shaping the fashion industry. Among various garments, these garments are subjected to innovations. This work is an attempt to compare the different methods of stone washing using pumice stones, acid cellulases and neutral cellulases or in combination of these methods. The effects of different processing conditions on the garment are compared and reported. Color differences of samples are probed by reflective colorimeter on the front side as well as the backside and also the white pocket of the garment.The abrasion resistance, tensile strength and crease recovery angle of samples are also reported. The XRD spectra are used to calculate the crystalline degrees of the selected samples. Moreover, fiber surfaces of some treated samples have been observed by SEM. The results indicate that treatment of denim with pumice stone with equal weight of garment causes a small color differences. The addition of cellulases to the washing, however, accelerates the color fading. Also, lower staining observed on the white pocket when the garment was treated with cellulases. However, the neutralcellulases increase the garment fading and decrease the staining on the white pocket. It is also observed that pumice stone with cellulases damages the fabric surface, although it is of a minimal damage.

Purification and Characterization of β-1,3-Glucanase from the Antagonistic Fungus Trichoderma reesei

Directory of Open Access Journals (Sweden)

SRI WAHYUNI BUDIARTI

2009-09-01

Full Text Available Trichoderma enzymes that inhibit fungal cell walls have been suggested to play an important role in mycoparasitic action against fungal root rot pathogen Ganoderma philippii. This experiment was aimed to purify and characterize the β-1,3-glucanase of T. reesei. Extracellular β-1,3-glucanase was produced by growing mycoparasite T. reesei isolate T13 in colloidal chitin and sucrose as carbon sources. The enzyme was then purified to its homogeneity by precipitation with ammonium sulfate, followed by gel filtration chromatography and chromatofocusing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE 12% was used to confirm the purity of enzyme at each stage of preparation and to characterize purified protein. The results showed that T. reesei produced at least three extracellular β-1,3-glucanases. Estimation of molecular weight based on SDS-PAGE 12% have three isoform of β-1,3-glucanase were 90 kDa for β-1,3-glucanase-I, 75 kDa for β-1,3-glucanase-II, and 64 kDa for β-1,3-glucanase-III. Their optimum pH and temperature were 5 and 50 oC, respectively.

High-throughput selection for cellulase catalysts using chemical complementation.

Science.gov (United States)

Peralta-Yahya, Pamela; Carter, Brian T; Lin, Hening; Tao, Haiyan; Cornish, Virginia W

2008-12-24

Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases, however, is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Because of the large number of enzyme variants that selections can now test as compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity.

Pulsed laser deposition and characterization of cellulase thin films

Science.gov (United States)

Cicco, N.; Morone, A.; Verrastro, M.; Viggiano, V.

2013-08-01

Thin films of cellulase were obtained by pulsed laser deposition (PLD) on an appropriate substrate. Glycoside hydrolase cellulase has received our attention because it emerges among the antifouling enzymes (enzymes being able to remove and prevent the formation of micro-organism biofilms) used in industry and medicine field. Pressed cellulase pellets, used as target material, were ablated with pulses of a Nd-YAG laser working at wavelength of 532 nm. In this work, we evaluated the impact of PLD technique both on molecular structure and hydrolytic activity of cellulase. Characteristic chemical bonds and morphology of deposited layers were investigated by FTIR spectroscopy and SEM respectively. The hydrolytic activity of cellulase thin films was detected by a colorimetric assay.

Exogenous cellulases of thermophilic micromycetes. Pt. 2. Thermostability of enzyme preparations

Energy Technology Data Exchange (ETDEWEB)

Kvesitadze, G; Gogilashvili, L; Svanidze, R; Buachidze, T; Chirgadze, L; Nizharadze, D

1986-01-01

The ability of a large number of higher fungi to form extracellular cellulases is investigated. Some representatives of these fungi grow at 40-50/sup 0/C, and form extracellular cellulases exceeding cellulases of mesophilic fungi in thermostability. It is shown that cellulases of higher thermophilic fungi differ by their thermostability. The temperature optimum of cellulase action of higher fungi occurs within 60-62/sup 0/C.

A steady state theory for processive cellulases

DEFF Research Database (Denmark)

Cruys-Bagger, Nicolaj; Olsen, Jens Elmerdahl; Præstgaard, Eigil

2013-01-01

coefficient’, which represents the probability of the enzyme dissociating from the substrate strand before completing n sequential catalytic steps, where n is the mean processivity number measured experimentally. Typical processive cellulases have high substrate affinity, and therefore this probability is low....... This has significant kinetic implications, for example the maximal specific rate (Vmax/E0) for processive cellulases is much lower than the catalytic rate constant (kcat). We discuss how relationships based on this theory may be used in both comparative and mechanistic analyses of cellulases....

Trichoderma reesei FS10-C enhances phytoremediation of Cd-contaminated soil by Sedum plumbizincicola and associated soil microbial activities

Science.gov (United States)

Teng, Ying; Luo, Yang; Ma, Wenting; Zhu, Lingjia; Ren, Wenjie; Luo, Yongming; Christie, Peter; Li, Zhengao

2015-01-01

This study aimed to explore the effects of Trichoderma reesei FS10-C on the phytoremediation of Cd-contaminated soil by the hyperaccumulator Sedum plumbizincicola and on soil fertility. The Cd tolerance of T. reesei FS10-C was characterized and then a pot experiment was conducted to investigate the growth and Cd uptake of S. plumbizincicola with the addition of inoculation agents in the presence and absence of T. reesei FS10-C. The results indicated that FS10-C possessed high Cd resistance (up to 300 mg L-1). All inoculation agents investigated enhanced plant shoot biomass by 6–53% of fresh weight and 16–61% of dry weight and Cd uptake by the shoots by 10–53% compared with the control. All inoculation agents also played critical roles in increasing soil microbial biomass and microbial activities (such as biomass C, dehydrogenase activity and fluorescein diacetate hydrolysis activity). Two inoculation agents accompanied by FS10-C were also superior to the inoculation agents, indicating that T. reesei FS10-C was effective in enhancing both Cd phytoremediation by S. plumbizincicola and soil fertility. Furthermore, solid fermentation powder of FS10-C showed the greatest capacity to enhance plant growth, Cd uptake, nutrient release, microbial biomass and activities, as indicated by its superior ability to promote colonization by Trichoderma. The solid fermentation powder of FS10-C might serve as a suitable inoculation agent for T. reesei FS10-C to enhance both the phytoremediation efficiency of Cd-contaminated soil and soil fertility. PMID:26113858

Cellulase variants with improved expression, activity and stability, and use thereof

Science.gov (United States)

Aehle, Wolfgang; Bott, Richard R; Bower, Benjamin; Caspi, Jonathan; Estell, David A; Goedegebuur, Frits; Hommes, Ronaldus W.J.; Kaper, Thijs; Kelemen, Bradley; Kralj, Slavko; Van Lieshout, Johan; Nikolaev, Igor; Van Stigt Thans, Sander; Wallace, Louise; Vogtentanz, Gudrun; Sandgren, Mats

2014-03-25

The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having improved expression, activity and/or stability. Also described are nucleic acids encoding the cellulase variants, compositions comprising the cellulase variants, and methods of use thereof.

Cellulase variants with improved expression, activity and stability, and use thereof

Energy Technology Data Exchange (ETDEWEB)

Aehle, Wolfgang; Bott, Richard R.; Bower, Benjamin S.; Caspi, Jonathan; Goedegebuur, Frits; Hommes, Ronaldus Wilhelmus Joannes; Kaper, Thijs; Kelemen, Bradley R.; Kralj, Slavko; Van Lieshout, Johannes Franciscus Thomas; Nikolaev, Igor; Wallace, Louise; Van Stigt Thans, Sander; Vogtentanz, Gudrun; Sandgren, Mats

2016-12-20

The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having improved expression, activity and/or stability. Also described are nucleic acids encoding the cellulase variants, compositions comprising the cellulase variants, and methods of use thereof.

Approaches for improving thermostability characteristics in cellulases.

Science.gov (United States)

Anbar, Michael; Bayer, Edward A

2012-01-01

Many efforts have been invested to reduce the cost of biofuel production to substitute renewable sources of energy for fossil-based fuels. At the forefront of these efforts are the initiatives to convert plant-derived cellulosic material to biofuels. Although significant improvements have been achieved recently in cellulase engineering in both efficiency and cost reduction, complete degradation of lignocellulosic material still requires very long periods of time and high enzyme loads. Thermostable cellulases offer many advantages in the bioconversion process, which include increase in specific activity, higher levels of stability, inhibition of microbial growth, increase in mass transfer rate due to lower fluid viscosity, and greater flexibility in the bioprocess. Besides rational design methods, which require deep understanding of protein structure-function relationship, two of the major methods for improvement in specific cellulase properties are directed evolution and knowledge-based library design based on multiple sequence alignments. In this chapter, we provide protocols for constructing and screening of improved thermostable cellulases. Modifications of these protocols may also be used for screening for other improved properties of cellulases such as pH tolerance, high salt, and more. Copyright © 2012 Elsevier Inc. All rights reserved.

Behaviour of the activity of cellulase irradiated under various conditions

International Nuclear Information System (INIS)

Kumakura, M.; Kaetsu, I.

1988-01-01

The activity of cellulase irradiated at various conditions has been studied. The activity of cellulase irradiated at low temperature (-78 0 C) increased by heating at 40 0 C, but that of cellulase irradiated at high temperature above 0 0 C decreased. The activity of cellulase irradiated in the dry state at room temperature increased with irradiation dose. The effect of adding biological substances such as amino acids, enzymes, and agar on the irradiation of cellulase was studied. It was shown that EDTA and p-benzoquinone have a protective ability against radiation-induced inactivation of the enzyme. (author)

Behaviour of the activity of cellulase irradiated under various conditions

Energy Technology Data Exchange (ETDEWEB)

Kumakura, M; Kaetsu, I

1988-04-18

The activity of cellulase irradiated at various conditions has been studied. The activity of cellulase irradiated at low temperature (-78 /sup 0/C) increased by heating at 40 /sup 0/C, but that of cellulase irradiated at high temperature above 0 /sup 0/C decreased. The activity of cellulase irradiated in the dry state at room temperature increased with irradiation dose. The effect of adding biological substances such as amino acids, enzymes, and agar on the irradiation of cellulase was studied. It was shown that EDTA and p-benzoquinone have a protective ability against radiation-induced inactivation of the enzyme.

Purification and properties of the cellulases from the thermophilic fungus Thermoascus aurantiacus

Energy Technology Data Exchange (ETDEWEB)

Tong, C C; Cole, A L; Shepherd, M G

1980-10-01

Three cellulases and a beta-glucosidase were purified from the culture filtrate of the thermophilic fungus Thermoascus aurantiacus. The isolated enzymes were all homogenous on polyacrylamide-disc-gel electrophoresis. Data from chromatography on Bio-Gel P-60 and solium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated molecular weights of 87000 (beta-glucosidase), 78000 (cellulase I), 49000 (cellulase II) and 34000 (cellulase III); the carbohydrate contents of the enzymes were 33.0 5.5, 2.6 and 1.8% (w/w) respectively. Although the three purified cellulases were active toward filter paper, only cellulases I and III were active towards CM (Carboxymethyl)-cellulose. Cellulase I was also active towards yeast glucan. The Km and catalytic-centre-activity values for the enzymes were as follows; 0.52 mu mol/ml and 6.5 by 10 to the power of 4 for beta-glucosidase on p-nitrophenyl beta-d-glucoside, 3.9 mg/ml and 6.3 for cellulase I on CM-cellulose, 1.2 mg/ml and 1.1 for cellulase I on yeast glucan, 34.4 mg/ml and 0.34 for cellulase II on filter paper, and 1.9 mg/ml and 33 for cellulase III on CM-cellulose.

Studies on the enzymology of cellulose degradation by the anaerobic bacterium Clostridium thermocellum and the anaerobic fungus Neocallimastix frontalis

Energy Technology Data Exchange (ETDEWEB)

Bhat, K.M.; Gow, L.A.; Wilson, C.A.; Wood, T.W. (Rowett Research Inst., Aberdeen (UK))

1990-01-01

The extracellular cellulases from the anaerobic bacterium Clostridium thermocellum and the anaerobic rumen fungus Neocallimastix frontalis are very active on crystalline cellulose. In both cases the activity resides in a high molecular weight complex. The complex from C. thermocellum (termed the cellulosome) was found to be readily dissociated at pH 5.0 and at room temperature by a mixture of SDS, EDTA and DTT. Virtually all the activity of the unfractionated cellulosome was recovered when the dissociated enzyme components were reassociated by dialysis. Thus, the route is now established for the first time for a meaningful study of the mechanism of cellulase action of this commercially important enzyme system. Nearly all of the activity to crystalline cellulose shown by the cellulase of N. frontalis was associated with a fraction which comprised only 2% of the extracellular protein, 3% of the endoglucanase and 3% of the {beta}-glucosidase. This fraction, which could be isolated by affinity chromatography on cellulose, was produced in greater quantity when the fungus was grown in co-culture with the methanogen, Methanobrevibacter smithii. The specific activity of the partially purified enzyme for degradation of crystalline cellulose was several-fold greater than that produced by the aerobic fungus T. reesei, which is being developed world-wide for its commercial potential for converting cellulose to fermentable soluble sugars. The cellulase of N. frontalis clearly has great commercial potential. 39 refs., 19 figs., 22 tabs.

Measurement and characterization of cellulase activity in sclerophyllous forest litter.

Science.gov (United States)

Criquet, Stéven

2002-07-01

Cellulases are enzymatic proteins which hydrolyze cellulose polymers to smaller oligosaccharides, cellobiose and glucose. They consist in three major types of enzymes: endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91) and beta-glucosidases (EC 3.2.1.21) which play an essential role in carbon turnover of forest ecosystem. The aim of this study was firstly to determine the parameters (i.e. buffer type, pH, temperature, quantity of litter, incubation time and reagent type) which affect the measurement of cellulase activity in a sclerophyllous forest litter, and secondly to compare two methods for measuring cellulase activity: a direct method and an extraction method. In the direct method, the litter was directly incubated with a buffered solution containing the enzyme substrate, whereas in the extraction method, the cellulases were firstly extracted before measuring their activity. The results were compared with other studies about soil cellulase activity, and it appeared that several parameters (buffer type, pH, temperature and sample quantity) which influence the measurement of cellulase activity differ according to whether a soil or a litter is considered. Concerning the procedure used for the measurement of cellulase activity, results showed that the activity values were higher when using an extraction procedure than when using a direct procedure. The extraction procedure, combined with a concentration stage of the extract, also allowed electrophoretic analysis (PAGE) of the cellulases extracted from the litter. The electrophoretic pattern revealed two cellulase isoenzymes which may be related to the occurrence of two pH-activity peaks of these enzymes when citrate buffer was used for the measurement of cellulase activity in the litter.

A rapid, one step molecular identification of Trichoderma citrinoviride and Trichoderma reesei.

Science.gov (United States)

Saroj, Dina B; Dengeti, Shrinivas N; Aher, Supriya; Gupta, Anil K

2015-06-01

Trichoderma species are widely used as production hosts for industrial enzymes. Identification of Trichoderma species requires a complex molecular biology based identification involving amplification and sequencing of multiple genes. Industrial laboratories are required to run identification tests repeatedly in cell banking procedures and also to prove absence of production host in the product. Such demands can be fulfilled by a brief method which enables confirmation of strain identity. This communication describes one step identification method for two common Trichoderma species; T. citrinoviride and T. reesei, based on identification of polymorphic region in the nucleotide sequence of translation elongation factor 1 alpha. A unique forward primer and common reverse primer resulted in 153 and 139 bp amplicon for T. citrinoviride and T. reesei, respectively. Simplification was further introduced by using mycelium as template for PCR amplification. Method described in this communication allows rapid, one step identification of two Trichoderma species.

A High-throughput Selection for Cellulase Catalysts Using Chemical Complementation

Science.gov (United States)

Peralta-Yahya, Pamela; Carter, Brian T.; Lin, Hening; Tao, Haiyan; Cornish, Virginia W.

2010-01-01

Efficient enzymatic hydrolysis of lignocellulosic material remains one of the major bottlenecks to cost-effective conversion of biomass to ethanol. Improvement of glycosylhydrolases however is limited by existing medium-throughput screening technologies. Here, we report the first high-throughput selection for cellulase catalysts. This selection was developed by adapting chemical complementation to provide a growth assay for bond cleavage reactions. First, a URA3 counter selection was adapted to link chemical dimerizer activated gene transcription to cell death. Next, the URA3 counter selection was shown to detect cellulase activity based on cleavage of a tetrasaccharide chemical dimerizer substrate and decrease in expression of the toxic URA3 reporter. Finally, the utility of the cellulase selection was assessed by isolating cellulases with improved activity from a cellulase library created by family DNA shuffling. This application provides further evidence that chemical complementation can be readily adapted to detect different enzymatic activities for important chemical transformations for which no natural selection exists. Due to the large number of enzyme variants selections can test compared to existing medium-throughput screens for cellulases, this assay has the potential to impact the discovery of improved cellulases and other glycosylhydrolases for biomass conversion from libraries of cellulases created by mutagenesis or obtained from natural biodiversity. PMID:19053460

Cellulases from Thermophilic Fungi: Recent Insights and Biotechnological Potential

Directory of Open Access Journals (Sweden)

Duo-Chuan Li

2011-01-01

Full Text Available Thermophilic fungal cellulases are promising enzymes in protein engineering efforts aimed at optimizing industrial processes, such as biomass degradation and biofuel production. The cloning and expression in recent years of new cellulase genes from thermophilic fungi have led to a better understanding of cellulose degradation in these species. Moreover, crystal structures of thermophilic fungal cellulases are now available, providing insights into their function and stability. The present paper is focused on recent progress in cloning, expression, regulation, and structure of thermophilic fungal cellulases and the current research efforts to improve their properties for better use in biotechnological applications.

Exogenous cellulases of thermophilic micromycetes. Pt. 1. Selection of producers

Energy Technology Data Exchange (ETDEWEB)

Kvesitadze, G; Kvachadze, L; Aleksidze, T; Chartishvili, D K

1986-01-01

More than 600 micromycetes - representatives of different genera have been investigated for their ability to produce exogenous cellulases. Most of the investigated cultures were found to produce these enzymes, 24 cultures being thermophilic, and 18 thermotolerant. Cellulase or its derivatives proved to be the most favourable carbon source for cellulase secretion. None of the thermophilic cultures studied manifested the ability of exogenous exoglucanase biosynthesis. Using UV-rays as mutagen, a mutant strain A. terreus T-49 has been obtained being characterized by an increased endo-glucanase and cellobiase activity, as compared to the initial strains. The cellulase preparations of thermophilic micromycetes contain one cellulasic component: endo-glucanase, or two: endo-glucanase and cellobiase.

Improved biomass degradation using fungal glucuronoyl-esterases-hydrolysis of natural corn fiber substrate

DEFF Research Database (Denmark)

d'Errico, Clotilde; Börjesson, Johan; Ding, Hanshu

2016-01-01

of improved degradation of lignocellulosic biomass by the use of GEs. Improved C5 sugar, glucose and glucuronic acid release was observed when heat pretreated corn fiber was incubated in the presence of GEs from Cerrena unicolor and Trichoderma reesei on top of different commercial cellulase...

cellulase and pectinase production potentials of aspergillus niger

African Journals Online (AJOL)

Prof Oyeleke

preparation of denim fabrics in textile industries, maceration of protoplasts ... exploitation of cellulase is its high cost of production ... catabolite repression influence economics of cellulase ... of enzyme production is to replace pure cellulose by.

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.

Intercalation of cellulase enzyme into a hydrotalcite layer structure

Science.gov (United States)

Zou, N.; Plank, J.

2015-01-01

A new inorganic-organic hybrid material whereby cellulase enzyme is incorporated into a hydrotalcite type layered double hydroxide (LDH) structure is reported. The Mg2Al-cellulase-LDH was synthesized via co-precipitation from Mg/Al nitrate at pH=9.6. Characterization was performed using X-ray powder diffraction (XRD), small angle X-ray scattering (SAXS), elemental analysis, infrared spectroscopy (IR) and thermogravimetry (TG). From XRD and SAXS measurements, a d-value of ~5.0 nm was identified for the basal spacing of the Mg2Al-cellulase-LDH. Consequently, the cellulase enzyme (hydrodynamic diameter ~6.6 nm) attains a slightly compressed conformation when intercalated. Formation of the LDH hybrid was also confirmed via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Mg2Al-cellulase-LDH phases appear as ~20 nm thin foils which are intergrown to flower-like aggregates. Activity of the enzyme was retained after deintercalation from the Mg2Al-LDH framework using anion exchange. Accordingly, cellulase is not denatured during the intercalation process, and LDH presents a suitable host structure for time-controlled release of the biomolecule.

Cellulase producing microorganism ATCC 55702

Science.gov (United States)

Dees, H. Craig

1997-01-01

Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

Expression of a thermotolerant laccase from Pycnoporus sanguineus in Trichoderma reesei and its application in the degradation of bisphenol A.

Science.gov (United States)

Zhao, Jie; Zeng, Shengquan; Xia, Ying; Xia, Liming

2018-04-01

The laccase gene from Pycnoporus sanguineus was cloned and inserted between the strong Pcbh1 promoter and the Tcbh1 terminator from Trichoderma reesei to form the recombinant plasmid pCH-lac. Using Agrobacterium-mediated technique, the pCH-lac was integrated into the chromosomes of T. reesei. Twenty positive transformants were obtained by employing hygromycin B as a selective agent. PCR was used to confirm that the laccase gene was integrated into the chromosomal DNA of T. reesei. Laccase production by recombinant transformants was performed in shaking flasks, and the activity of laccase reached 8.8 IU/mL after 96-h fermentation under a batch process, and 17.7 IU/mL after 144-h fermentation using a fed-batch process. SDS-PAGE analysis of the fermentation broth showed that the molecular mass of the protein was about 68 kDa, almost the same as that of the laccase produced by P. sanguineus, which indicated that laccase was successfully expressed in T. reesei and secreted out of the cells. The laccase produced by the recombinant T. reesei showed good thermal stability, and could degrade the toxic phenolic material bisphenol A efficiently, after 1-h reaction with 0.06 IU/mL laccase and 0.5 mmol/L ABTS as the mediator at 60 °C and pH 4.5, the degradation rate reached 95%, which demonstrated that it had great potential value in treating the household garbage and wastewater containing the bisphenol A. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Enhanced production and application of acidothermophilic Streptomyces cellulase.

Science.gov (United States)

Budihal, Saikumar R; Agsar, Dayanand; Patil, Sarvamangala R

2016-01-01

An efficient cellulolytic and acidothermophilic actinobacterium was isolated from soil, adhered to decomposing tree bark and was identified as Streptomyces DSK59. Screening of synthetic media and the media components identified that, a medium based on starch casein minerals containing carboxy methyl cellulose (CMC) and beef extract (BE) could support enhanced cellulase production by the organism. CMC, BE, NaCl, temperature and pH were accounted as significant for cellulase production and these were optimized using a response surface central composite design (CCD). Optimization of cellulase production resulted in an enhancement of endoglucanase activity to 27IUml(-1). Acidothermophillic Streptomyces cellulase was found to be efficient for hydrolysis of pretreated sorghum stover and liberated 0.413gg(-1) of total reducing sugars which was higher than previously reported sugar yields obtained using fungal enzymes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparison of the thermostability of cellulases from various thermophilic fungi

Energy Technology Data Exchange (ETDEWEB)

Wojtczak, G; Breuil, C; Yamada, J; Saddler, J N

1987-10-01

The cellulase activities of six thermophilic fungi were compared. Although the thermophilic fungi grew at relatively high temperatures (> 45/sup 0/C) the optimum temperatures for assaying the various cellulase activities were only slightly higher than the optimum temperatures for the mesophilic fungi, Trichoderma harzianum. Over prolonged incubation (> 24 h) the thermophilic strains demonstrated a higher hydrolytic potential as a result of the greater thermostability of the cellulase components. Although the extracellular cellulase activities had similar pH and temperature optima, in some cases the thermostability of the extracellular components were considerably lower.

Banana peel: A novel substrate for cellulase production under solid ...

African Journals Online (AJOL)

These results indicated that banana peel provided necessary nutrients for cell growth and cellulase synthesis. It can be used as a potential substrate for cellulase production by T. viride GIM 3.0010 under solid-state fermentation. To the best of our knowledge, this is the first report on cellulase production using banana peel.

Secretion of clostridium cellulase by E. coli

Science.gov (United States)

Yu, Ida Kuo

1998-01-01

A gene, encoding an endocellulase from a newly isolated mesophilic Clostridium strain IY-2 which can digest bamboo fibers, cellulose, rice straw, and sawdust, was isolated by shotgun cloning in an E. coli expression plasmid pLC2833. E. coli positive clones were selected based on their ability to hydrolyze milled bamboo fibers and cellulose present in agar plates. One clone contained a 2.8 kb DNA fragment that was responsible for cellulase activity. Western blot analyses indicated that the positive clone produced a secreted cellulase with a mass of about 58,000 daltons that was identical in size to the subunit of one of the three major Clostridium cellulases. The products of cellulose digestion by this cloned cellulase were cellotetraose and soluble higher polymers. The cloned DNA contained signal sequences capable of directing the secretion of heterologous proteins from an E. coli host. The invention describes a bioprocess for the treatment of cellulosic plant materials to produce cellular growth substrates and fermentation end products suitable for production of liquid fuels, solvents, and acids.

Production Of Cellulase In Plastids Of Transgenic Plants

Science.gov (United States)

Lamppa, Gayle

2002-08-06

A genetic construct encoding a fusion protein including endogluconase E1 and a transit peptide is used to transform plants. The plants produce cellulase by expressing the genetic construct. The cellulase is targeted to plastids and can be collected and purified.

Strong cellulase inhibition by Mannan polysaccharides in cellulose conversion to sugars.

Science.gov (United States)

Kumar, Rajeev; Wyman, Charles E

2014-07-01

Cellulase enzymes contribute a major fraction of the total cost for biological conversion of lignocellulosic biomass to fuels and chemicals. Although a several fold reduction in cellulase production costs and enhancement of cellulase activity and stability have been reported in recent years, sugar yields are still lower at low enzyme doses than desired commercially. We recently reported that hemicellulose xylan and its oligomers strongly inhibit cellulase and that supplementation of cellulase with xylanase and β-xylosidase would significantly reduce such inhibition. In this study, mannan polysaccharides and their enzymatically prepared hydrolyzates were discovered to be strongly inhibitory to fungal cellulase in cellulose conversion (>50% drop in % relative conversion), even at a small concentration of 0.1 g/L, and inhibition was much greater than experienced by other known inhibitors such as cellobiose, xylooligomers, and furfural. Furthermore, cellulase inhibition dramatically increased with heteromannan loading and mannan substitution with galactose side units. In general, enzymatically prepared hydrolyzates were less inhibitory than their respective mannan polysaccharides except highly substituted ones. Supplementation of cellulase with commercial accessory enzymes such as xylanase, pectinase, and β-glucosidase was effective in greatly relieving inhibition but only for less substituted heteromannans. However, cellulase supplementation with purified heteromannan specific enzymes relieved inhibition by these more substituted heteromannans as well, suggesting that commercial preparations need to have higher amounts of such activities to realize high sugar yields at the low enzyme protein loadings needed for low cost fuels production. © 2014 Wiley Periodicals, Inc.

Cellulase immobilization on superparamagnetic nanoparticles for reuse in cellulosic biomass conversion

Directory of Open Access Journals (Sweden)

Fernando Segato

2016-07-01

Full Text Available Current cellulosic biomass hydrolysis is based on the one-time use of cellulases. Cellulases immobilized on magnetic nanocarriers offer the advantages of magnetic separation and repeated use for continuous hydrolysis. Most immobilization methods focus on only one type of cellulase. Here, we report co-immobilization of two types of cellulases, β-glucosidase A (BglA and cellobiohydrolase D (CelD, on sub-20 nm superparamagnetic nanoparticles. The nanoparticles demonstrated 100% immobilization efficiency for both BglA and CelD. The total enzyme activities of immobilized BglA and CelD were up to 67.1% and 41.5% of that of the free cellulases, respectively. The immobilized BglA and CelD each retained about 85% and 43% of the initial immobilized enzyme activities after being recycled 3 and 10 times, respectively. The effects of pH and temperature on the immobilized cellulases were also investigated. Co-immobilization of BglA and CelD on MNPs is a promising strategy to promote synergistic action of cellulases while lowering enzyme consumption.

Properties of cellulase as template molecule on chitosan—methyl methacrylate membrane

Science.gov (United States)

Lian, Qi; Zheng, Xuefang; Wu, Haixia; Song, Shitao; Wang, Dongjun

2015-12-01

In this study, a novel molecular imprinting membrane made of chitosan and methyl methacrylate (MMA) was fabricated with cellulase as template molecule and the thermal response to cellulase was characterized. The film was characterized by infrared spectroscopy (IR), X-ray diffraction (XRD), scanning electron microscopy (SEM) and the permeation experiment. The results showed that the space structure of the film was as similar as the cellulase. Moreover, the membrane had advanced molecular imprinting capability to cellulase comparing to pepsin and pectinase at any temperature and the film had excellent ability to identify specific template molecule (cellulase) at the synthesis temperature compared to other temperatures.

Kinetic Studies on Trichoderna Viride Cellulase

International Nuclear Information System (INIS)

Saw Aung; Oo Aung; Aung Myint

2002-02-01

Studies on cellulase enzyme (EC 3.2.1.4), which catalyzes the hydrolysis of. cellulose to yield glucose, were made. Cellulase from a fungus source, Trichoderma viride was cultivated on Czapek's agar medium and enzyme production broth medium was employed for parameter tests. The microscopic examination and cellulase hydrolysis test on subcultured fungi were applied to confirm the T. viride species. A calibration curve for standard glucose was plotted by using visible spectroscopy. Dinitrosalicylic acid was used as enzyme reaction inhibitor and the colour intensity was measured in a UV-visible spectrophotometer at a λ max of 570 nm. The parameters such as optimum pH, optimum temperature, effect of substrate concentration, effect, of enzyme concentration, enzyme unit (EU), reaction order (n), maximum velocity (V max ), Michaelis-Menten constant (K m ) using various substrates, viz., carboxy methylcellulose, cotton fibre and filter paper determined. (author)

Induction of Cellulase by Gentiobiose and Its Sulfur-Containing Analog in Penicillium purpurogenum

OpenAIRE

Kurasawa, Takashi; Yachi, Makoto; Suto, Manabu; Kamagata, Yoichi; Takao, Shoichi; Tomita, Fusao

1992-01-01

Cellulase induction by β-glucodisaccharides was investigated by using non-cellulase-induced mycelia of Penicillium purpurogenum P-26, a highly-cellulase-producing fungus. Gentiobiose induced significant amounts of cellulase compared with cellobiose when nojirimycin was added to the induction medium to inhibit extracellular β-glucosidase activity. Thiogentiobiose (6-S-β-d-glucopyranosyl-6-thio-d-glucose), a sulfur-containing analog of gentiobiose, was more effective for cellulase induction tha...

Exploration of indigenous agrowastes for cellulase production by ...

African Journals Online (AJOL)

admin

2012-05-10

May 10, 2012 ... However, high lignin content of B. campestris made the cellulose inaccessible and resulted in poor yield of enzyme. Therefore, S. spontaneum has a great potential to serve as a cheaper, easily available and reasonable substrate for cellulase production. Key words: Agrowastes, cellulase, indigenous, ...

Cationic polyacrylamide enhancing cellulase treatment efficiency of hardwood kraft-based dissolving pulp.

Science.gov (United States)

Wang, Qiang; Liu, Shanshan; Yang, Guihua; Chen, Jiachuan; Ni, Yonghao

2015-05-01

Cellulase treatment for decreasing viscosity and increasing Fock reactivity of dissolving pulp is a promising approach to reduce the use of toxic chemicals, such as hypochlorite in the dissolving pulp manufacturing process in the industry. Improving the cellulase treatment efficiency during the process is of practical interest. In the present study, the concept of using cationic polyacrylamide (CPAM) to enhance the cellulase treatment efficiency was demonstrated. This was mainly attributed to the increased cellulase adsorption onto cellulose fibers based on the patching/bridging mechanism. Results showed that the cellulase adsorption was increased by about 20% with the addition of 250 ppm of CPAM under the same conditions as those of the control. It was found that the viscosity decrease and Fock reactivity increase for the cellulase treatment was enhanced from using CPAM. The CPAM-assisted cellulase treatment concept may provide a practical alternative to the present hypochlorite-based technology for viscosity control in the industry. Copyright © 2015 Elsevier Ltd. All rights reserved.

Safety evaluation of β-glucanase derived from Trichoderma reesei: Summary of toxicological data

NARCIS (Netherlands)

Coenen, T.M.M.; Schoenmakers, A.C.M.; Verhagen, H.

1995-01-01

Barlican, a β-glucanase enzyme obtained from Trichoderma reesei, was produced by a fermentation process and subjected to a series of toxicological tests to document its safety for use as a feed additive. The enzyme product was examined for general oral toxicity, inhalation toxicity, irritation to

Adsorption of cellulase on cellulolytic enzyme lignin from lodgepole pine.

Science.gov (United States)

Tu, Maobing; Pan, Xuejun; Saddler, Jack N

2009-09-09

Enzymatic hydrolysis of lignocellulosic materials is significantly affected by cellulase adsorption onto the lignocellulosic substrates and lignin. The presence of lignin plays an important role in lignocellulosic hydrolysis and enzyme recycling. Three cellulase preparations (Celluclast, Spezyme CP, and MSUBC) were evaluated to determine their adsorption onto cellulolytic enzyme lignin (CEL) from steam-exploded Lodgepole pine (SELP) and ethanol (organosolv)-pretreated Lodgepole pine (EPLP). The adsorption affinity of cellulase (Celluclast) onto isolated lignin (CEL-EPLP and CEL-SELP) was slightly higher than that from corresponding EPLP and SELP substrates on the basis of the Langmuir constants. Effects of temperature, ionic strength, and surfactant on cellulase adsorption onto isolated lignin were also explored in this study. Thermodynamic analysis of enzyme adsorption onto isolated lignin (Gibbs free energy change DeltaG(0) approximately -30 kJ/mol) indicated this adsorption was a spontaneous process. The addition of surfactant (0.2% w/v) could reduce the adsorption of cellulase onto CEL-SELP by 60%. Two types of adsorption isotherm were compared for cellulase adsorption onto isolated lignin. A Langmuir adsorption isotherm showed better fit for the experimental data than a Freundlich adsorption isotherm.

Xyloglucan oligosaccharides promote growth and activate cellulase: Evidence for a role of cellulase in cell expansion

International Nuclear Information System (INIS)

McDougall, G.J.; Fry, S.C.

1990-01-01

Oligosaccharides produced by the action of fungal cellulase on xyloglucans promoted the elongation of etiolated pea (Pisum sativum L.) stem segments in a straight-growth bioassay designed for the determination of auxins. The oligosaccharides were most active at about 1 micromolar. We tested the relative growth-promoting activities of four HPLC-purified oligosaccharides which shared a common glucose 4 ·xylose 3 (XG7) core. The substituted oligosaccharides XG8 (glucose 4 ·xylose 3 ·galactose) and XG9n (glucose 4 ·xylose 3 ·galactose 2 ) were more effective than XG7 itself and XG9 (glucose 4 ·xylose 3 ·galactose·fucose). The same oligosaccharides also promoted the degradation, assayed viscometrically, of xyloglucan by an acidic cellulase from bean (Phaseolus vulgaris L.) leaves. The oligosaccharides were highly active at 10 -4 molar, causing up to a fourfold increase in activity, but the effect was still detectable at 1 micromolar. Those oligosaccharides (XG8 and XG9n) which best promoted growth, stimulated cellulase activity to the greatest extent. The oligosaccharides did not stimulate the action of the cellulase in an assay based on the conversion of [ 3 H]xyloglucan to ethanol-soluble fragments. This suggests that the oligosaccharides enhanced the midchain hydrolysis of xyloglucan molecules (which would rapidly reduce the viscosity of the solution), at the expense of cleavage near the termini (which would yield ethanol-soluble products)

Compositions and methods comprising cellulase variants with reduced affinity to non-cellulosic materials

Energy Technology Data Exchange (ETDEWEB)

Cascao-Pereira, Luis; Kaper, Thijs; Kelemen, Bradley R.; Liu, Amy D.

2017-07-04

The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having reduced binding to non-cellulosic materials. Also described are nucleic acids encoding the cellulase, compositions comprising said cellulase, methods of identifying cellulose variants and methods of using the compositions.

Compositions and methods comprising cellulase variants with reduced affinity to non-cellulosic materials

Science.gov (United States)

Cascao-Pereira, Luis G.; Kaper, Thijs; Kelemen, Bradley R; Liu, Amy D.

2012-08-07

The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having reduced binding to non-cellulosic materials. Also described are nucleic acids encoding the cellulase, compositions comprising said cellulase, methods of identifying cellulose variants and methods of using the compositions.

Compositions and methods comprising cellulase variants with reduced affinity to non-cellulosic materials

Energy Technology Data Exchange (ETDEWEB)

Cascao-Pereira, Luis G; Kaper, Thijs; Kelemen, Bradley R; Liu, Amy D

2015-04-07

The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having reduced binding to non-cellulosic materials. Also described are nucleic acids encoding the cellulase, compositions comprising said cellulase, methods of identifying cellulose variants and methods of using the compositions.

Xylanase XYN IV from Trichoderma reesei showing exo- and endo-xylanase activity

Science.gov (United States)

A novel xylanase from Trichoderma reesei Rut C30, named XYN IV, was purified from the cellulolytic system of the fungus. The enzyme was discovered on its ability to attack aldotetraohexenuronic acid (HexA-2Xyl-4Xyl-4Xyl, HexA3Xyl3), releasing the reducing-end xylose residue. XYN IV exhibited catalyt...

Corn stover-enhanced cellulase production by Aspergillus niger ...

African Journals Online (AJOL)

The production of extracellular cellulases by Aspergilus niger NRRL 567 on corn stover was studied in liquid state fermentation. In this study, three cellulases, exoglucanase (EXG), endoglucanase (EG) and β-glucosidase (BGL) were produced by A. niger NRRL 567. The optimal pH, temperature and incubation time for ...

Functional diversity for biomass deconstruction in family 5 subfamily 5 (GH5_5) of fungal endo-β1,4-glucanases.

Science.gov (United States)

Li, Bingyao; Walton, Jonathan D

2017-05-01

Endo-β1,4-glucanases in glycosyl hydrolase family 5 (GH5) are ubiquitous enzymes in the multicellular fungi and are common components of enzyme cocktails for biomass conversion. We recently showed that an endo-glucanase of subfamily 5 of GH5 (GH5_5) from Sporotrichum thermophile (StCel5A) was more effective at releasing glucose from pretreated corn stover, when part of an eight-component synthetic enzyme mixture, compared to its closely related counterpart from Trichoderma reesei, TrCel5A. StCel5A and TrCel5A belong to different clades of GH5_5 (GH5_5_1 and GH5_5_2, respectively). To test whether the superior activity of StCel5A was a general property of all enzymes in the GH5_5_2 clade, StCel5A, TrCel5A, and two additional members of each subfamily were expressed in a common host that had been engineered to suppress its native cellulases (T. reesei Δxyr1) and compared against each other alone on pure substrates, in synthetic mixtures on pure substrates, and against each other in synthetic mixtures on real biomass. The results indicated that superiority is a unique property of StCel5A and not of GH5_5_2 generally. The six Cel5A enzymes had significant differences in relative activities on different substrates, in specific activities, and in sensitivities to mannan inhibition. Importantly, the behavior of the six endo-glucanases on pure cellulose substrates did not predict their behavior in combination with other cellulolytic enzymes on a real lignocellulosic biomass substrate.

Thermostable cellulases, and mutants thereof, capable of hydrolyzing cellulose in ionic liquid

Science.gov (United States)

Sapra, Rajat; Datta, Supratim; Chen, Zhiwei; Holmes, Bradley M.; Simmons, Blake A.; Blanch, Harvey W.

2016-04-26

The present invention provides for a composition comprising an ionic liquid and a thermostable cellulose, and a method of hydrolyzing a cellulose, comprising: (a) providing a composition comprising a solution comprising an ionic liquid and a cellulose, and (b) introducing a thermostable cellulase to the solution, such that the cellulose is hydrolyzed by the cellulase. The present invention also provides for a Thermatoga maritima thermostable cellulase mutant with increased cellulase activity.

Enhanced processive cellulases

Science.gov (United States)

Adney, William S.; Beckham, Gregg T.; Jarvis, Eric; Himmel, Michael E.; Decker, Stephen R.; Linger, Jeffrey G.; Podkaminer, Kara; Baker, John O.; Taylor, II, Larry; Xu, Qi; Singh, Arjun

2017-06-20

Nucleic acid sequences encoding chimeric polypeptides that exhibit enhanced cellulase activities are disclosed herein. These nucleic acids may be expressed in hosts such as fungi, which in turn may be cultured to produce chimeric polypeptides. Also disclosed are chimeric polypeptides and their use in the degradation of cellulosic materials.

Factors affecting endoglucanase production by Trichoderma reesei ...

African Journals Online (AJOL)

Jane

2011-08-22

Aug 22, 2011 ... from the ANOVA analysis have a significant value of Pmodel>F= 0.0008 and R2 .... there are various environmental and nutritional factors ... reported to affect cellulase production from wheat straw ... many factors affecting simultaneously the fermentation ..... and control its stability (Kalra and Sandhu, 1986).

Expression of Acidothermus cellulolyticus thermostable cellulases in tobacco and rice plants

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Xiran Jiang

2017-01-01

Full Text Available The production of cellulases in plants is an economical method for the conversion of lignocellulosic biomass into fuels. Herein we report the expressions of two thermostable Acidothermus cellulolyticus cellulases, endo-1,4-β-D-glucanase (E1 and exoglucanase (Gux1, in tobacco and rice. To evaluate the expression of these recombinant cellulases, we expressed the full-length E1, the catalytic domains of E1 (E1cd and Gux1 (Gux1cd, as well as an E1–Gux1cd fusion enzyme in various subcellular compartments. In the case of tobacco, transgenic plants that expressed apoplast-localized E1 showed the highest level of activity, about three times higher than those that expressed the cytosolic E1. In the case of rice, the level of cellulase-specific activity in the transgenic plants ranged from 11 to 20 nmol 4-methylumbelliferone min−1 mg−1 total soluble protein. The recombinant cellulases exhibited good thermostability below 70 °C. Furthermore, transgenic rice leaves that were stored at room temperature for a month lost about 20% of the initial cellulase activity. Taken together, the results suggested that heterologous expression of thermostable cellulases in plants may be a viable option for biomass conversion.

Investigation and Isolation of Cellulase-Producing microorganisms in the Red Sea

KAUST Repository

Fatani, Siham

2016-05-01

Cellulolytic microorganisms are considered to be key players in biorefinery, especially for the utilization of plant biomass. These organisms have been isolated from various environments. The Red Sea is one of the seas with high biodiversity and a unique environment, characterized by high water temperature and high salinity . However, there is little information regarding cellulases in Red Sea environments. The aim of the present study is to evaluate the Red Sea as a gene resource for microbial cellulase. I first surveyed microbial cellulases in the Red Sea using a method called metagenomes, and then investigated their abundance and diversity. My survey revealed that the Red Sea biome has a substantial abundance and a wide range of cellulase enzymes with substantial abundance, when compared with those in other environments. Next, I tried to isolate cellulase-active microorganisms from the Red Sea and I successfully obtained seven strains of four different taxonomic groups. These strains showed a similarity of 99% identity to Aspergillus ustus, 99% to Staphylococcus pasteuri, 99% to Bacillus aerius and 99% to Bacillus subtilis. The enzyme assay I conducted, revealed that these strains actually secreted active cellulases. These results suggest that the Red Sea environment can be, indeed, an excellent gene resource of microbial cellulases.

Production and Partial Characterization of Cellulases from ...

African Journals Online (AJOL)

The optimum temperature of CCI was 55°C whereas that of CCO was 50°C. The stability of enzymes from 35°C to 70°C was studied. At 70°C, CCO cellulase has lost 45.88% of its original activity while CCI cellulase lost 58.14%. The results show that corn cob could serve as a cheap carbon source for the production of fungi ...

Direct ethanol production from lignocellulosic sugars and sugarcane bagasse by a recombinant Trichoderma reesei strain HJ48.

Science.gov (United States)

Huang, Jun; Chen, Dong; Wei, Yutuo; Wang, Qingyan; Li, Zhenchong; Chen, Ying; Huang, Ribo

2014-01-01

Trichoderma reesei can be considered as a candidate for consolidated bioprocessing (CBP) microorganism. However, its ethanol yield needs to be improved significantly. Here the ethanol production of T. reesei CICC 40360 was improved by genome shuffling while simultaneously enhancing the ethanol resistance. The initial mutant population was generated by nitrosoguanidine treatment of the spores, and an improved population producing more than fivefold ethanol than wild type was obtained by genome shuffling. The results show that the shuffled strain HJ48 can efficiently convert lignocellulosic sugars to ethanol under aerobic conditions. Furthermore, it was able to produce ethanol directly from sugarcane bagasse, demonstrating that the shuffled strain HJ48 is a suitable microorganism for consolidated bioprocessing.

CELLULASES FROM THE BASIDIO - MYCETES CULTURAL LIQUID

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К. G. Dreval

2013-04-01

Full Text Available Adsorption of cellulases on substrate taking place during the cultivation process was determined. Adsorbed enzymes can be eluted by buffer solution with high ionic strength, but for determine their activity they should be transferred into the aqueous solution. On the basis of the results a method for obtaining of cellulases preparations from cultural liquids of basidiomycetes was developed. This method is the elution of cellulases from the cultivation substrate of basidiomycetes. It was found that using of the last allows to obtain enzymatic preparations with a high degree of purification in 3 stages (salting out of proteins — dialysis — gelchromatography. Cellulase preparations received original products of basidiomycetes strains К-1, А-Дон-02, Д-1 Irpex lacteus and AnSc-1 Daedaleopsis confragosa f. confragosa were obtained. They contained different proteins, enzymes with specific peaks out of column and their activity. However, common to them was a distinct maximum of outing from the column by endoglucanases or cellobiases, which may indicate that the studied cellulolytic complexes of basidiomycetes do not contain multiple forms of cellulases with different molecular mass. This method allowed to obtain preparations with different degree of purification in comparing with the original culture filtrate 7,3 for endoglucanase and 33,3 for cellobiase of strain А-Дон-02 I. lacteus; 13,1 for endoglucanase and 25,5 for cellobiase of strain Д-1 I. lacteus; 29,9 for endoglucanase and 90,1 for cellobiase of strain К-1 I. lacteus; 2,1 for endoglucanase and 30,6 for cellobiase of strain AnSc-1 D. confragosa f. confragosa.

Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain.

Science.gov (United States)

Okuda, Naoyuki; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Hoshino, Tamotsu

2016-10-01

We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression.

TESTING OF THE EFFICIENCY OF SOME ENZYMATIC MIXTURES, CONCERNING THE CONVERSION OF SEVERAL LIGNOCELLULOSIC BIOMASS’ SOURSES, TO REDUCING SUGARS

Directory of Open Access Journals (Sweden)

RADIANA TAMBA-BEREHOIU

2008-05-01

Full Text Available Three lignocelluloses substrates have been used, as following: Mischantus, Maize stalk and Wheat bran, in order to obtain fermentescible sugars, which will be transformed into bioethanol. The substrates were hydrolysed using commercial enzymes: MethaPlus (b-glucanase, xylanase, cellulase, Veron 191 (xylanase, Hep C (cellulase. The hydrolysis was performed at 550 C, for 20 h, at pH = 5,5. The best results were obtained by using MethaPlus enzyme. The efficiency of hydrolysis was 110.80 % for Miscanthus, 126.15 % for maize stalk and 118.76 % for wheat bran, reported to the control. The most enhanced quantities of reducing sugars were obtained in maize stalk, namely: 126.15 % using MethaPlus, 112.07 % using Veron 191 and 113.52 % using Hep C. The wheat bran was hydrolysed with enzymatic mixtures, for emphasizing the reducing sugars’ grow, coming from residual starch’s content (flour. In comparison to the control, the efficiency of hydrolysis was of: 181.004 % for MethaPlus-Veron M4 mixture, 168.83 % for MethaPlus-Veron 393 mixture, 205.86 % for MethaPlus-BG a-malt mixture and of 176.57 % for MethaPlus-Veron MX mixture. The enzymatic mixture which contains BG a-malt was the most productive, the hydrolysis efficiency being superior to all other variants.

Inhibitory effect of vanillin on cellulase activity in hydrolysis of cellulosic biomass.

Science.gov (United States)

Li, Yun; Qi, Benkun; Wan, Yinhua

2014-09-01

Pretreatment of lignocellulosic material produces a wide variety of inhibitory compounds, which strongly inhibit the following enzymatic hydrolysis of cellulosic biomass. Vanillin is a kind of phenolics derived from degradation of lignin. The effect of vanillin on cellulase activity for the hydrolysis of cellulose was investigated in detail. The results clearly showed that vanillin can reversibly and non-competitively inhibit the cellulase activity at appropriate concentrations and the value of IC50 was estimated to be 30 g/L. The inhibition kinetics of cellulase by vanillin was studied using HCH-1 model and inhibition constants were determined. Moreover, investigation of three compounds with similar structure of vanillin on cellulase activity demonstrated that aldehyde group and phenolic hydroxyl groups of vanillin had inhibitory effect on cellulase. These results provide valuable and detailed information for understanding the inhibition of lignin derived phenolics on cellulase. Copyright © 2014 Elsevier Ltd. All rights reserved.

Engineering ionic liquid-tolerant cellulases for biofuels production.

Science.gov (United States)

Wolski, Paul W; Dana, Craig M; Clark, Douglas S; Blanch, Harvey W

2016-04-01

Dissolution of lignocellulosic biomass in certain ionic liquids (ILs) can provide an effective pretreatment prior to enzymatic saccharification of cellulose for biofuels production. Toward the goal of combining pretreatment and enzymatic hydrolysis, we evolved enzyme variants of Talaromyces emersonii Cel7A to be more active and stable than wild-type T. emersonii Cel7A or Trichoderma reesei Cel7A in aqueous-IL solutions (up to 43% (w/w) 1,3-dimethylimdazolium dimethylphosphate and 20% (w/w) 1-ethyl-3-methylimidazolium acetate). In general, greater enzyme stability in buffer at elevated temperature corresponded to greater stability in aqueous-ILs. Post-translational modification of the N-terminal glutamine residue to pyroglutamate via glutaminyl cyclase enhanced the stability of T. emersonii Cel7A and variants. Differential scanning calorimetry revealed an increase in melting temperature of 1.9-3.9°C for the variant 1M10 over the wild-type T. emersonii Cel7A in aqueous buffer and in an IL-aqueous mixture. We observed this increase both with and without glutaminyl cyclase treatment of the enzymes. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Mutant strain screening and its enzyme production conditions of cellulase

International Nuclear Information System (INIS)

Dong Zhiyang; Zhu Lingxiang; Yu Wei

2001-01-01

Trichoderma koeningii T-801, which can produce relatively high cellulase, was isolated. The ability of producing cellulase of mutant T-801 had increased 1.77 times after treated with nitrous guanide and γ-ray and was higher than that of Trichoderma QM9414. The medium with straw powder as carbon source and peptone as nitrogen source is optimal and the maximum cellulase activity is reached at 30 degree C and pH 5.0 when cultured for 5 days

Comparative production of cellulases by mutants of Trichoderma parceramosume PTCC5140

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Hoda Nouri

2017-06-01

Discussion and conclusion: Evaluation of cellulase production in mutant strains of Trichoderma parceramosume PTCC 5140 showed that use of chemical mutagenesis with 2 to 11 fold increasing in enzyme activity is a potent method to improve cellulase complex activity. In the current study, obtained mutant strains could be introduced as a potent cellulase producer for further studies in bioconversion processes.

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

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Elijah Johnson

2006-01-01

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

Processive and nonprocessive cellulases for biofuel production. Lessons from bacterial genomes and structural analysis

Energy Technology Data Exchange (ETDEWEB)

Wilson, David B. [Cornell Univ. Ithaca, New York, NY (United States). Dept. of Molecular Biology and Genetics

2012-01-15

Cellulases are key enzymes used in many processes for producing liquid fuels from biomass. Currently there many efforts to reduce the cost of cellulases using both structural approaches to improve the properties of individual cellulases and genomic approaches to identify new cellulases as well as other proteins that increase the activity of cellulases in degrading pretreated biomass materials. Fungal GH-61 proteins are important new enzymes that increase the activity of current commercial cellulases leading to lower total protein loading and thus lower cost. Recent work has greatly increased our knowledge of these novel enzymes that appear to be oxido-reductases that target crystalline cellulose and increase its accessibility to cellulases. They appear to carry out the C1 activity originally proposed by Dr Reese. Cellobiose dehydrogenase appears to interact with GH-61 proteins in this function, providing a role for this puzzling enzyme. Cellulase research is making considerable progress and appears to be poised for even greater advances. (orig.)

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)

Light-dependent roles of the G-protein α subunit GNA1 of Hypocrea jecorina (anamorph Trichoderma reesei

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Kubicek Christian P

2009-09-01

Full Text Available Abstract Background The filamentous ascomycete Hypocrea jecorina (anamorph Trichoderma reesei is primarily known for its efficient enzymatic machinery that it utilizes to decompose cellulosic substrates. Nevertheless, the nature and transmission of the signals initiating and modulating this machinery are largely unknown. Heterotrimeric G-protein signaling represents one of the best studied signal transduction pathways in fungi. Results Analysis of the regulatory targets of the G-protein α subunit GNA1 in H. jecorina revealed a carbon source and light-dependent role in signal transduction. Deletion of gna1 led to significantly decreased biomass formation in darkness in submersed culture but had only minor effects on morphology and hyphal apical extension rates on solid medium. Cellulase gene transcription was abolished in Δgna1 on cellulose in light and enhanced in darkness. However, analysis of strains expressing a constitutively activated GNA1 revealed that GNA1 does not transmit the essential inducing signal. Instead, it relates a modulating signal with light-dependent significance, since induction still required the presence of an inducer. We show that regulation of transcription and activity of GNA1 involves a carbon source-dependent feedback cycle. Additionally we found a function of GNA1 in hydrophobin regulation as well as effects on conidiation and tolerance of osmotic and oxidative stress. Conclusion We conclude that GNA1 transmits a signal the physiological relevance of which is dependent on both the carbon source as well as the light status. The widespread consequences of mutations in GNA1 indicate a broad function of this Gα subunit in appropriation of intracellular resources to environmental (especially nutritional conditions.

Production and properties of fungal cellulase from native isolates ...

African Journals Online (AJOL)

Orange bagasse was employed as the sole carbon source for production of cellulase having adapted the organisms on cellulose and on orange bagasse. The extracellular cellulase produced by Aspergillus niger and Rhizopus species were partially purified by ammonium sulphate precipitation in a single step and dialyzed ...

Optimization for cellulase production by Aspergillus niger using saw ...

African Journals Online (AJOL)

Cellulases are a group of hydrolytic enzymes and are capable of degrading lignocellulosic materials. Cellulases have wide range of applications. This work focuses on factors relevant for improvement of enzymatic hydrolysis of saw dust by using Aspergillus niger. Different cultural conditions were examined to assess their ...

Cellulase immobilization on magnetic nanoparticles encapsulated in polymer nanospheres.

Science.gov (United States)

Lima, Janaina S; Araújo, Pedro H H; Sayer, Claudia; Souza, Antonio A U; Viegas, Alexandre C; de Oliveira, Débora

2017-04-01

Immobilization of cellulases on magnetic nanoparticles, especially magnetite nanoparticles, has been the main approach studied to make this enzyme, economically and industrially, more attractive. However, magnetite nanoparticles tend to agglomerate, are very reactive and easily oxidized in air, which has strong impact on their useful life. Thus, it is very important to provide proper surface coating to avoid the mentioned problems. This study aimed to investigate the immobilization of cellulase on magnetic nanoparticles encapsulated in polymeric nanospheres. The support was characterized in terms of morphology, average diameter, magnetic behavior and thermal decomposition analyses. The polymer nanospheres containing encapsulated magnetic nanoparticles showed superparamagnetic behavior and intensity average diameter about 150 nm. Immobilized cellulase exhibited broader temperature stability than in the free form and great reusability capacity, 69% of the initial enzyme activity was maintained after eight cycles of use. The magnetic support showed potential for cellulase immobilization and allowed fast and easy biocatalyst recovery through a single magnet.

Recycling cellulases for cellulosic ethanol production at industrial relevant conditions: potential and temperature dependency at high solid processes.

Science.gov (United States)

Lindedam, Jane; Haven, Mai Østergaard; Chylenski, Piotr; Jørgensen, Henning; Felby, Claus

2013-11-01

Different versions of two commercial cellulases were tested for their recyclability of enzymatic activity at high dry matter processes (12% or 25% DM). Recyclability was assessed by measuring remaining enzyme activity in fermentation broth and the ability of enzymes to hydrolyse fresh, pretreated wheat straw. Industrial conditions were used to study the impact of hydrolysis temperature (40 or 50°C) and residence time on recyclability. Enzyme recycling at 12% DM indicated that hydrolysis at 50°C, though ideal for ethanol yield, should be kept short or carried out at lower temperature to preserve enzymatic activity. Best results for enzyme recycling at 25% DM was 59% and 41% of original enzyme load for a Celluclast:Novozyme188 mixture and a modern cellulase preparation, respectively. However, issues with stability of enzymes and their strong adsorption to residual solids still pose a challenge for applicable methods in enzyme recycling. Copyright © 2013 Elsevier Ltd. All rights reserved.

Production of cellulases by fungal cultures isolated from forest litter soil

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A. Sri Lakshmi

2012-06-01

Full Text Available The aims of this study were the isolation and screening of fungal cultures from forest litter soil for cellulases production. In the present study, four fungal cultures were isolated and identified. Among these fungal cultures, three belonged to the genus Aspergillus and one belonged to the genus Pencillium. These fungal cultures were tested to find their ability to produce cellulases, that catalyze the degradation of cellulose, which is a linear polymer made of glucose subunits linked by beta-1, 4 glycosidic bonds. The fungal isolate 3 (Aspergillus sp. was noticed to show maximum zone of hydrolysis of carboxy-methyl cellulose and produce higher titers of cellulases including exoglucanase, endoglucanase and beta -D-glucosidase. The activities of the cellulases were determined by Filter paper assay (FPA, Carboxy-methly cellulase assay (CMCase and beta -D-glucosidase assay respectively. The total soluble sugar and extracellular protein contents of the fungal filtrates were also determined.

High consistency cellulase treatment of hardwood prehydrolysis kraft based dissolving pulp.

Science.gov (United States)

Wang, Qiang; Liu, Shanshan; Yang, Guihua; Chen, Jiachuan; Ni, Yonghao

2015-01-01

For enzymatic treatment of dissolving pulp, there is a need to improve the process to facilitate its commercialization. For this purpose, the high consistency cellulase treatment was conducted based on the hypothesis that a high cellulose concentration would favor the interactions of cellulase and cellulose, thus improves the cellulase efficiency while decreasing the water usage. The results showed that compared with a low consistency of 3%, the high consistency of 20% led to 24% increases of cellulase adsorption ratio. As a result, the viscosity decrease and Fock reactivity increase at consistency of 20% were enhanced from 510 mL/g and 70.3% to 471 mL/g and 77.6%, respectively, compared with low consistency of 3% at 24h. The results on other properties such as alpha cellulose, alkali solubility and molecular weight distribution also supported the conclusion that a high consistency of cellulase treatment was more effective than a low pulp consistency process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Heterologous expression of two Aspergillus niger feruloyl esterases in Trichoderma reesei for the production of ferulic acid from wheat bran.

Science.gov (United States)

Long, Liangkun; Zhao, Haoyuan; Ding, Dafan; Xu, Meijuan; Ding, Shaojun

2018-05-01

Feruloyl esterase (FAE)-encoding genes AnfaeA and AnfaeB were isolated from Aspergillus niger 0913. For overexpression of the two genes in Trichoderma reesei, constitutive and inductive expression plasmids were constructed based on parental plasmid pAg1-H3. The constructed plasmids contained AnfaeA or AnfaeB gene under the control of glyceraldehyde-3-phosphate dehydrogenase A gene (gpdA) promoter (from A. nidulans) or cellobiohydrolases I (cbh I) gene promoter (from T. reesei), and cbh I terminator from T. reesei. The target plasmids were transferred into T. reesei D-86271 (Rut-C30) by Agrobacterium tumefaciens-mediated transformation (ATMT), respectively. A high level of feruloyl esterase was produced by the recombinant fungal strains under solid-state fermentation, and the cbh I promoter was more efficient than the gpdA promoter in the expression of AnfaeA. The optimum temperatures and pH values were 50 °C and 5.0 for AnFAEA, and 35 °C and 6.0 for AnFAEB. The maximum production levels were 20.69 U/gsd for AnFAEA and 15.08 U/gsd for AnFAEB. The recombinant fungal enzyme systems could release 62.9% (for AnFAEA) and 52.2% (for AnFAEB) of total ferulic acids from de-starched wheat bran, which was higher than the 46.3% releasing efficiency of A. niger 0913. The supplement of xylanase from T. longibrachiatum in the enzymatic hydrolysis led to a small increment of the ferulic acids release.

Recycling cellulase towards industrial application of enzyme treatment on hardwood kraft-based dissolving pulp.

Science.gov (United States)

Wang, Qiang; Liu, Shanshan; Yang, Guihua; Chen, Jiachuan; Ji, Xingxiang; Ni, Yonghao

2016-07-01

Cost-effectiveness is vital for enzymatic treatment of dissolving pulp towards industrial application. The strategy of cellulase recycling with fresh cellulase addition was demonstrated in this work to activate the dissolving pulp, i.e. decreasing viscosity and increasing Fock reactivity. Results showed that 48.8-35.1% of cellulase activity can be recovered from the filtered liquor in five recycle rounds, which can be reused for enzymatic treatment of dissolving pulp. As a result, the recycling cellulase with addition fresh cellulase of 1mg/g led to the pulp of viscosity 470mL/g and Fock reactivity 80%, which is comparable with cellulase charge of 2mg/g. Other pulp properties such as alpha-cellulose, alkaline solubility and molecular weight distribution were also determined. Additionally, a zero-release of recycling cellulase treatment was proposed to integrate into the dissolving pulp production process. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synergistic effect of cellulase and xylanase during hydrolysis of natural lignocellulosic substrates.

Science.gov (United States)

Song, Hui-Ting; Gao, Yuan; Yang, Yi-Min; Xiao, Wen-Jing; Liu, Shi-Hui; Xia, Wu-Cheng; Liu, Zi-Lu; Yi, Li; Jiang, Zheng-Bing

2016-11-01

Synergistic combination of cellulase and xylanase has been performed on pre-treated substrates in many previous studies, while few on natural substrates. In this study, three unpretreated lignocellulosic substrates were studied, including corncob, corn stover, and rice straw. The results indicated that when the mixed cellulase and xylanase were applied, reducing sugar concentrations were calculated as 19.53, 15.56, and 17.35mg/ml, respectively, based on the 3,5 dinitrosalicylic acid (DNS) method. Compared to the treatment with only cellulose, the hydrolysis yields caused by mixed cellulase and xylanase were improved by 133%, 164%, and 545%, respectively. In addition, the conversion yield of corncob, corn stover, and rice straw by cellulase-xylanase co-treatment reached 43.9%, 48.5%, and 40.2%, respectively, based on HPLC analysis, which confirmed the synergistic effect of cellulase-xylanase that was much higher than either of the single enzyme treatment. The substrate morphology was also evaluated to explore the synergistic mechanism of cellulase-xylanase. Copyright © 2016 Elsevier Ltd. All rights reserved.

Engineering Cellulase Enzymes for Bioenergy

Science.gov (United States)

Atreya, Meera Elizabeth

Sustainable energy sources, such as biofuels, offer increasingly important alternatives to fossil fuels that contribute less to global climate change. The energy contained within cellulosic biofuels derives from sunlight energy stored in the form of carbon-carbon bonds comprising sugars such as glucose. Second-generation biofuels are produced from lignocellulosic biomass feedstocks, including agricultural waste products and non-food crops like Miscanthus, that contain lignin and the polysaccharides hemicellulose and cellulose. Cellulose is the most abundant biological material on Earth; it is a polymer of glucose and a structural component of plant cell walls. Accessing the sugar is challenging, as the crystalline structure of cellulose resists degradation; biochemical and thermochemical means can be used to depolymerize cellulose. Cellulase enzymes catalyze the biochemical depolymerization of cellulose into glucose. Glucose can be used as a carbon source for growth of a biofuel-producing microorganism. When it converts glucose to a hydrocarbon fuel, this microbe completes the biofuels process of transforming sunlight energy into accessible, chemical energy capable of replacing non-renewable transportation fuels. Due to strong intermolecular interactions between polymer chains, cellulose is significantly more challenging to depolymerize than starch, a more accessible polymer of glucose utilized in first-generation biofuels processes (often derived from corn). While most mammals cannot digest cellulose (dietary fiber), certain fungi and bacteria produce cellulase enzymes capable of hydrolyzing it. These organisms secrete a wide variety of glycoside hydrolase and other classes of enzymes that work in concert. Because cellulase enzymes are slow-acting and expensive to produce, my aim has been to improve the properties of these enzymes as a means to make a cellulosic biofuels process possible that is more efficient and, consequently, more economical than current

Enzymatic hydrolysis of oil palm empty fruits bunch fiber using Celluclast® and Accellerase® BG for sugar production

Science.gov (United States)

Salleh, Noor Shafryna; Murad, Abdul Munir Abdul

2016-11-01

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

Bioconversion potential of Trichoderma viride HN1 cellulase for a lignocellulosic biomass Saccharum spontaneum.

Science.gov (United States)

Iqtedar, Mehwish; Nadeem, Mohammad; Naeem, Hira; Abdullah, Roheena; Naz, Shagufta; Qurat ul Ain Syed; Kaleem, Afshan

2015-01-01

The industrialisation of lignocellulose conversion is impeded by expensive cellulase enzymes required for saccharification in bioethanol production. Current research undertakes cellulase production from pretreated Saccharum spontaneum through Trichoderma viride HN1 under submerged fermentation conditions. Pretreatment of substrate with 2% NaOH resulted in 88% delignification. Maximum cellulase production (2603 ± 16.39 U/mL/min carboxymethyl cellulase and 1393 ± 25.55 U/mL/min FPase) was achieved at 6% substrate at pH 5.0, with 5% inoculum, incubated at 35°C for 120 h of fermentation period. Addition of surfactant, Tween 80 and metal ion Mn(+2), significantly enhanced cellulase yield. This study accounts proficient cellulase yield through process optimisation by exploiting cheaper substrate to escalate their commercial endeavour.

Carboxymethyl cellulase and cellobiase production by Clostridium acetobutylicum in an industrial fermentation medium.

OpenAIRE

Allcock, E R; Woods, D R

1981-01-01

The production of a carboxymethyl cellulase and a cellobiase by Clostridium acetobutylicum was demonstrated. In liquid medium the carboxymethyl cellulase was induced by molasses, and it was not repressed by glucose. Optimum carboxymethyl cellulase activity occurred at pH 4.6 and 37 degrees C.

Cellulase production by white-rot basidiomycetous fungi: solid-state versus submerged cultivation

DEFF Research Database (Denmark)

Bentil, Joseph A.; Thygesen, Anders; Mensah, Moses

2018-01-01

fungi for improved enzyme expression, as well as on-site approaches for production of enzyme blends for industrial biomass conversion. The quantitative comparisons made have implications for selection of the most appropriate cultivation method for WRB fungi for attaining maximal cellulase production....... on different biomass residues in SSC or SmC systems. Although some variation in cellulase production yields have been reported for certain substrates, the analysis convincingly shows that SmC is generally more efficient than SSC for obtaining high cellulase production yields and high cellulase production rates......) or submerged cultivation (SmC) systems. In this review, we quantitatively assess the data available in the literature on cellulase production yields by WRB fungi cultivated by SSC or SmC. The review also assesses cellulolytic enzyme production rates and enzyme recovery when WRB fungi are cultivated...

Cellulase Recycling after High-Solids Simultaneous Saccharification and Fermentation of Combined Pretreated Corncob

Energy Technology Data Exchange (ETDEWEB)

Du, Ruoyu [State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin (China); Su, Rongxin, E-mail: surx@tju.edu.cn [State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin (China); Collaborative Innovation Center of Chemical Science and Engineering, Tianjin (China); Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin (China); Zhang, Mingjia [State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin (China); Qi, Wei [State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin (China); Collaborative Innovation Center of Chemical Science and Engineering, Tianjin (China); Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin (China); He, Zhimin [State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin (China)

2014-06-26

Despite the advantageous prospect of second-generation bioethanol, its final commercialization must overcome the primary cost impediment due to enzyme assumption. To solve this problem, this work achieves high-concentration ethanol fermentation and multi-round cellulase recycling through process integration. The optimal time and temperature of the re-adsorption process were determined by monitoring the adsorption kinetics of cellulases. Both glucose and cellobiose inhibited cellulase adsorption. After 96 h of ethanol fermentation, 40% of the initial cellulase remained in the broth, from which 62.5% of the cellulase can be recycled and reused in fresh substrate re-adsorption for 90 min. Under optimum conditions, i.e., pH 5.0, dry matter loading of 15 wt%, cellulase loading of 45 FPU/g glucan, two cycles of fermentation and re-adsorption can yield twofold increased ethanol outputs and reduce enzyme costs by over 50%. The ethanol concentration in each cycle can be achieved at levels >40 g/L.

Cellulase recycling after high-solids simultaneous saccharification and fermentation of combined pretreated corncob

Directory of Open Access Journals (Sweden)

Ruoyu eDu

2014-06-01

Full Text Available Despite the advantageous prospect of second-generation bioethanol, its final commercialization must overcome the primary cost impediment due to enzyme assumption. To solve this problem, this work achieves high-concentration ethanol fermentation and multi-round cellulase recycling through process integration. The optimal time and temperature of the re-adsorption process were determined by monitoring the adsorption kinetics of cellulases. Both glucose and cellobiose inhibited cellulase adsorption. After 96 h of ethanol fermentation, 40% of the initial cellulase remained in the broth, from which 62.5% of the cellulase can be recycled and reused in fresh substrate re-adsorption for 90 min. Under optimum conditions, i.e., pH 5.0, dry matter loading of 15 wt%, cellulase loading of 45 FPU/g glucan, two cycles of fermentation and re-adsorption can yield two-fold increased ethanol outputs and reduce enzyme costs by over 50%. The ethanol concentration in each cycle can be achieved at levels greater than 40 g/L.

Cellulase Recycling after High-Solids Simultaneous Saccharification and Fermentation of Combined Pretreated Corncob

International Nuclear Information System (INIS)

Du, Ruoyu; Su, Rongxin; Zhang, Mingjia; Qi, Wei; He, Zhimin

2014-01-01

Despite the advantageous prospect of second-generation bioethanol, its final commercialization must overcome the primary cost impediment due to enzyme assumption. To solve this problem, this work achieves high-concentration ethanol fermentation and multi-round cellulase recycling through process integration. The optimal time and temperature of the re-adsorption process were determined by monitoring the adsorption kinetics of cellulases. Both glucose and cellobiose inhibited cellulase adsorption. After 96 h of ethanol fermentation, 40% of the initial cellulase remained in the broth, from which 62.5% of the cellulase can be recycled and reused in fresh substrate re-adsorption for 90 min. Under optimum conditions, i.e., pH 5.0, dry matter loading of 15 wt%, cellulase loading of 45 FPU/g glucan, two cycles of fermentation and re-adsorption can yield twofold increased ethanol outputs and reduce enzyme costs by over 50%. The ethanol concentration in each cycle can be achieved at levels >40 g/L.

ARA1 regulates not only l-arabinose but also d-galactose catabolism in Trichoderma reesei

NARCIS (Netherlands)

Benocci, Tiziano; Aguilar-Pontes, Maria Victoria; Kun, Roland Sándor; Seiboth, Bernhard; de Vries, Ronald P; Daly, Paul

2017-01-01

Trichoderma reesei is used to produce saccharifying enzyme cocktails for biofuels. There is limited understanding of the transcription factors (TFs) that regulate genes involved in release and catabolism of l-arabinose and d-galactose, as the main TF XYR1 is only partially involved. Here, the T.

The identification of and relief from Fe3+ inhibition for both cellulose and cellulase in cellulose saccharification catalyzed by cellulases from Penicillium decumbens.

Science.gov (United States)

Wang, Mingyu; Mu, Ziming; Wang, Junli; Hou, Shaoli; Han, Lijuan; Dong, Yanmei; Xiao, Lin; Xia, Ruirui; Fang, Xu

2013-04-01

Lignocellulosic biomass is an underutilized, renewable resource that can be converted to biofuels. The key step in this conversion is cellulose saccharification catalyzed by cellulase. In this work, the effect of metal ions on cellulose hydrolysis by cellulases from Penicillium decumbens was reported for the first time. Fe(3+) and Cu(2+) were shown to be inhibitory. Further studies on Fe(3+) inhibition showed the inhibition takes place on both enzyme and substrate levels. Fe(3+) treatment damages cellulases' capability to degrade cellulose and inhibits all major cellulase activities. Fe(3+) treatment also reduces the digestibility of cellulose, due to its oxidation. Treatment of Fe(3+)-treated cellulose with DTT and supplementation of EDTA to saccharification systems partially relieved Fe(3+) inhibition. It was concluded that Fe(3+) inhibition in cellulose degradation is a complicated process in which multiple inhibition events occur, and that relief from Fe(3+) inhibition can be achieved by the supplementation of reducing or chelating agents. Copyright © 2013 Elsevier Ltd. All rights reserved.

Trichoderma Reesei single cell protein production from rice straw pulp in solid state fermentation

Science.gov (United States)

Zaki, M.; Said, S. D.

2018-04-01

The dependency on fish meal as a major protein source for animal feed can lead toit priceinstability in line with the increasing in meat production and consumption in Indonesia. In order todeal with this problem, an effort to produce an alternative protein sources production is needed. This scenario is possible due to the abundantavailability of agricultural residues such as rice straw whichcould be utilized as substrate for production of single cell proteins as an alternative proteinsource. This work investigated the potential utilization of rice straw pulp and urea mixture as substrate for the production of local Trichoderma reesei single cell protein in solid state fermentation system. Some parameters have been analyzed to evaluate the effect of ratio of rice straw pulp to urea on mixed single cell protein biomass (mixed SCP biomass) composition, such as total crude protein (analyzed by kjedhal method) and lignin content (TAPPI method).The results showed that crude protein content in mixed SCP biomassincreases with the increasing in fermentation time, otherwise it decreases with the increasing insubstrate carbon to nitrogen (C/N) ratio. Residual lignin content in mixed SCP biomass decreases from 7% to 0.63% during fermentationproceeded of 21 days. The highest crude protein content in mixed SCP biomasswas obtained at substrate C/N ratio 20:1 of 25%.

Preparation of cellulase concoction using differential adsorption phenomenon.

Science.gov (United States)

Birhade, Sachinkumar; Pednekar, Mukesh; Sagwal, Shilpa; Odaneth, Annamma; Lali, Arvind

2017-05-28

Controlled depolymerization of cellulose is essential for the production of valuable cellooligosaccharides and cellobiose from lignocellulosic biomass. However, enzymatic cellulose hydrolysis involves multiple synergistically acting enzymes, making difficult to control the depolymerization process and generate desired product. This work exploits the varying adsorption properties of the cellulase components to the cellulosic substrate and aims to control the enzyme activity. Cellulase adsorption was favored on pretreated cellulosic biomass as compared to synthetic cellulose. Preferential adsorption of exocellulases was observed over endocellulase, while β-glucosidases remained unadsorbed. Adsorbed enzyme fraction with bound exocellulases when used for hydrolysis generated cellobiose predominantly, while the unadsorbed enzymes in the liquid fraction produced cellooligosaccharides majorly, owing to its high endocellulases activity. Thus, the differential adsorption phenomenon of the cellulase components can be used for the controlling cellulose hydrolysis for the production of an array of sugars.

Glycosylation Helps Cellulase Enzymes Bind to Plant Cell Walls (Fact Sheet)

Energy Technology Data Exchange (ETDEWEB)

2012-06-01

Computer simulations suggest a new strategy to design enhanced enzymes for biofuels production. Large-scale computer simulations predict that the addition of glycosylation on carbohydrate-binding modules can dramatically improve the binding affinity of these protein domains over amino acid mutations alone. These simulations suggest that glycosylation can be used as a protein engineering tool to enhance the activity of cellulase enzymes, which are a key component in the conversion of cellulose to soluble sugars in the production of biofuels. Glycosylation is the covalent attachment of carbohydrate molecules to protein side chains, and is present in many proteins across all kingdoms of life. Moreover, glycosylation is known to serve a wide variety of functions in biological recognition, cell signaling, and metabolism. Cellulase enzymes, which are responsible for deconstructing cellulose found in plant cell walls to glucose, contain glycosylation that when modified can affect enzymatic activity-often in an unpredictable manner. To gain insight into the role of glycosylation on cellulase activity, scientists at the National Renewable Energy Laboratory (NREL) used computer simulation to predict that adding glycosylation on the carbohydrate-binding module of a cellulase enzyme dramatically boosts the binding affinity to cellulose-more than standard protein engineering approaches in which amino acids are mutated. Because it is known that higher binding affinity in cellulases leads to higher activity, this work suggests a new route to designing enhanced enzymes for biofuels production. More generally, this work suggests that tuning glycosylation in cellulase enzymes is a key factor to consider when engineering biochemical conversion processes, and that more work is needed to understand how glycosylation affects cellulase activity at the molecular level.

Daily dynamics of cellulase activity in arable soils depending on management practices

Science.gov (United States)

Lavrent'eva, E. V.; Semenov, A. M.; Zelenev, V. V.; Chzhun, Yu.; Semenova, E. V.; Semenov, V. M.; Namsaraev, B. B.; van Bruggen, A. H. C.

2009-08-01

The daily dynamics of cellulase activity was studied during 27 days by the cellophane membrane method on soils managed using the conventional high-input farming system (application of mineral fertilizers and pesticides) and the biological conservation farming system (application of organic fertilizers alone) in a microfield experiment. The regular oscillatory dynamics of the cellulase activity were revealed and confirmed by the harmonic (Fourier) analysis. The oscillatory dynamics of the cellulase activity had a self-oscillatory nature and was not directly caused by the disturbing impacts of both the uncontrolled (natural) changes in the temperature and moisture (rainfall) and the controlled ones (the application of different fertilizers). The disturbing impacts affected the oscillation amplitude of the cellulase activity but not the frequency (periods) of the oscillations. The periodic oscillations of the cellulase activity were more significant in the soil under the high-input management compared to the soil under the biological farming system.

Variants of cellobiohydrolases

Energy Technology Data Exchange (ETDEWEB)

Bott, Richard R.; Foukaraki, Maria; Hommes, Ronaldus Wilhelmus; Kaper, Thijs; Kelemen, Bradley R.; Kralj, Slavko; Nikolaev, Igor; Sandgren, Mats; Van Lieshout, Johannes Franciscus Thomas; Van Stigt Thans, Sander

2018-04-10

Disclosed are a number of homologs and variants of Hypocrea jecorina Ce17A (formerly Trichoderma reesei cellobiohydrolase I or CBH1), nucleic acids encoding the same and methods for producing the same. The homologs and variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted and/or deleted.

Fractionation of Aspergillus niger cellulases by combined ion exchange affinity chromatography

Energy Technology Data Exchange (ETDEWEB)

Boyer, R.F.; Allen, T.L.; Dykema, P.A.

1987-02-05

Eight chemically modified cellulose supports were tested for their ability to adsorb components of the Aspergillus niger cellulase system. At least two of the most effective adsorbents, aminoethyl cellulose and carboxymethyl cellulose, were shown to be useful for the fractionation of cellulases. These supports apparently owe their resolving capacity to both ion exchange and biospecific binding effects; however, the relative importance of each effect is unknown. These observations form the basis for a new cellulase fractionation technique, combined ion exchange-affinity chromatography. 22 references.

Exogenous Cellulase Contributes to Mycoherbicidal Activity of Fusarium arthrosporioides on Orobanche aegyptiaca

Directory of Open Access Journals (Sweden)

Olubukola O. Babalola

2010-01-01

Full Text Available This paper investigates an association between the tubercle size of Orobanche aegyptiaca, tubercle death, and days to tubercle death in relation to cellulase-assisted mycoherbicide. Fusarium arthrosporioides killed 56% of tubercles when applied with cellulase compared to 35% when no cellulase was added. Death was inversely correlated with days over the two fungal treatment types. O. aegyptiaca tubercle size significantly correlated with the two other infection parameters studied. For F. arthrosporioides, only 9% (2 of the variation in days to death was explained by variation in tubercle size, whereas with cellulase it reaches 14%. In this study, mycelia of F. arthrosporioides did not show apparent damage to the tomato roots.

Immobilization of microbial cell and yeast cell and its application to biomass conversion using radiation techniques

International Nuclear Information System (INIS)

Kaetsu, Isao; Kumakura, Minoru; Fujimura, Takashi; Kasai, Noboru; Tamada, Masao

1987-01-01

The recent results of immobilization of cellulase-producing cells and ethanol-fermentation yeast by radiation were reported. The enzyme of cellulase produced by immobilized cells was used for saccharification of lignocellulosic wastes and immobilized yeast cells were used for fermentation reaction from glucose to ethanol. The wastes such as chaff and bagasse were treated by γ-ray or electron-beam irradiation in the presence of alkali and subsequent mechanical crushing, to form a fine powder less than 50 μm in diameter. On the other hand, Trichoderma reesei as a cellulase-producing microbial cell was immobilized on a fibrous carrier having a specific porous structure and cultured to produce cellulase. The enzymatic saccharification of the pretreated waste was carried out using the produced cellulase. The enhanced fermentation process to produce ethanol from glucose with the immobilized yeast by radiation was also studied. The ethanol productivity of immobilized growing yeast cells thus obtained was thirteen times that of free yeast cells in a 1:1 volume of liquid medium to immobilized yeast cells. (author)

Effects of gas periodic stimulation on key enzyme activity in gas double-dynamic solid state fermentation (GDD-SSF).

Science.gov (United States)

Chen, Hongzhang; Shao, Meixue; Li, Hongqiang

2014-03-05

The heat and mass transfer have been proved to be the important factors in air pressure pulsation for cellulase production. However, as process of enzyme secretion, the cellulase formation has not been studied in the view of microorganism metabolism and metabolic key enzyme activity under air pressure pulsation condition. Two fermentation methods in ATPase activity, cellulase productivity, weight lose rate and membrane permeability were systematically compared. Results indicated that gas double-dynamic solid state fermentation had no obviously effect on cell membrane permeability. However, the relation between ATPase activity and weight loss rate was linearly dependent with r=0.9784. Meanwhile, the results also implied that gas periodic stimulation had apparently strengthened microbial metabolism through increasing ATPase activity during gas double-dynamic solid state fermentation, resulting in motivating the production of cellulase by Trichoderma reesei YG3. Therefore, the increase of ATPase activity would be another crucial factor to strengthen fermentation process for cellulase production under gas double-dynamic solid state fermentation. Copyright © 2013 Elsevier Inc. All rights reserved.

Immobilization of microbial cell and yeast cell and its application to biomass conversion using radiation techniques

Science.gov (United States)

Kaetsu, Isao; Kumakura, Minoru; Fujimura, Takashi; Kasai, Noboru; Tamada, Masao

The recent results of immobilization of cellulase-producing cells and ethanol-fermentation yeast by radiation were reported. The enzyme of cellulase produced by immobilized cells was used for saccharification of lignocellulosic wastes and immobilized yeast cells were used for fermentation reaction from glucose to ethanol. The wastes such as chaff and bagasse were treated by γ-ray or electron-beam irradiation in the presence of alkali and subsequent mechanical crushing, to form a fine powder less than 50 μm in diameter. On the other hand, Trichoderma reesei as a cellulase-producing microbial cell was immobilized on a fibrous carrier having a specific porous structure and cultured to produce cellulase. The enzymatic saccharification of the pretreated waste was carried out using the produced cellulase. The enhanced fermentation process to produce ethanol from glucose with the immobilized yeast by radiation was also studied. The ethanol productivity of immobilized growing yeast cells thus obtained was thirteen times that of free yeast cells in a 1:1 volume of liquid medium to immobilized yeast cells.

Interactive forces between lignin and cellulase as determined by atomic force microscopy

OpenAIRE

Qin, Chengrong; Clarke, Kimberley; Li, Kecheng

2014-01-01

Background Lignin is a complex polymer which inhibits the enzymatic conversion of cellulose to glucose in lignocellulose biomass for biofuel production. Cellulase enzymes irreversibly bind to lignin, deactivating the enzyme and lowering the overall activity of the hydrolyzing reaction solution. Within this study, atomic force microscopy (AFM) is used to compare the adhesion forces between cellulase and lignin with the forces between cellulase and cellulose, and to study the moiety groups invo...

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...

The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass.

Science.gov (United States)

Maki, Miranda; Leung, Kam Tin; Qin, Wensheng

2009-07-29

Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology.

Biotechnological applications of bacterial cellulases

Czech Academy of Sciences Publication Activity Database

Menéndez, E.; García-Fraile, Paula; Rivas, R.

2015-01-01

Roč. 2, č. 3 (2015), s. 163-182 ISSN 2306-5354 R&D Projects: GA MŠk(CZ) EE2.3.30.0003 Institutional support: RVO:61388971 Keywords : Biotechnological applications * Bacterial cellulases * Cellulose degradation Subject RIV: EE - Microbiology, Virology

21 CFR 173.120 - Carbohydrase and cellulase derived from Aspergillus niger.

Science.gov (United States)

2010-04-01

... cellulase derived from Aspergillus niger. Carbohydrase and cellulase enzyme preparation derived from Aspergillus niger may be safely used in food in accordance with the following prescribed conditions: (a) Aspergillus niger is classified as follows: Class, Deuteromycetes; order, Moniliales; family, Moniliaceae...

Exploring the natural fungal biodiversity of tropical and temperate forests toward improvement of biomass conversion.

Science.gov (United States)

Berrin, Jean-Guy; Navarro, David; Couturier, Marie; Olivé, Caroline; Grisel, Sacha; Haon, Mireille; Taussac, Sabine; Lechat, Christian; Courtecuisse, Régis; Favel, Anne; Coutinho, Pedro M; Lesage-Meessen, Laurence

2012-09-01

In this study, natural fungal diversity in wood-decaying species was explored for biomass deconstruction. In 2007 and 2008, fungal isolates were collected in temperate forests mainly from metropolitan France and in tropical forests mainly from French Guiana. We recovered and identified 74 monomorph cultures using morphological and molecular identification tools. Following production of fungal secretomes under inductive conditions, we evaluated the capacity of these fungal strains to potentiate a commercial Trichoderma reesei cellulase cocktail for the release of soluble sugars from biomass. The secretome of 19 isolates led to an improvement in biomass conversion of at least 23%. Of the isolates, the Trametes gibbosa BRFM 952 (Banque de Ressources Fongiques de Marseille) secretome performed best, with 60% improved conversion, a feature that was not universal to the Trametes and related genera. Enzymatic characterization of the T. gibbosa BRFM 952 secretome revealed an unexpected high activity on crystalline cellulose, higher than that of the T. reesei cellulase cocktail. This report highlights the interest in a systematic high-throughput assessment of collected fungal biodiversity to improve the enzymatic conversion of lignocellulosic biomass. It enabled the unbiased identification of new fungal strains issued from biodiversity with high biotechnological potential.

The Effects of Bioprocess Parameters on Cellulase Production with Trichoderma viride CMIT35

Directory of Open Access Journals (Sweden)

Teodor Vintila

2010-05-01

Full Text Available Fungal cellulases are well-studied, and have various applications in industry, health or agriculture. Species of Trichoderma can produce substantial amounts of endoglucanase, exoglucanase (saccharifying cellulases, and some strains are able to produce important quantities of β-glucosidase. A number of fungi were isolated abroad and screened for cellulolytic potential. In this study, the kinetics of cellulase production from an indigenous strain of T. viride CMIT35 is reported. Product formation parameters of different types of cellulases indicate that the studied strain of T. viride is capable of producing important levels of cellulases when grown on Mandels medium with wheat bran as carbon source. Furthermore, it was observed that production of endoglucanase reaches its maximum during exponential phase of growth, while exoglucanase during the stationary phase. Enzyme production by solid-state fermentation was also investigated and found to be more efficient than liquid state fermentation. High production of cellulase was noted at the following parameters for liquid cultures: 4% wheat bran, 5% inoculum, 180 r.p.m. agitation, pH 5; and 60% humidity in the case of solid state fermentation.

Assimilating Text-Mining & Bio-Informatics Tools to Analyze Cellulase structures

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Satyasree, K. P. N. V., Dr; Lalitha Kumari, B., Dr; Jyotsna Devi, K. S. N. V.; Choudri, S. M. Roy; Pratap Joshi, K.

2017-08-01

Text-mining is one of the best potential way of automatically extracting information from the huge biological literature. To exploit its prospective, the knowledge encrypted in the text should be converted to some semantic representation such as entities and relations, which could be analyzed by machines. But large-scale practical systems for this purpose are rare. But text mining could be helpful for generating or validating predictions. Cellulases have abundant applications in various industries. Cellulose degrading enzymes are cellulases and the same producing bacteria - Bacillus subtilis & fungus Pseudomonas putida were isolated from top soil of Guntur Dt. A.P. India. Absolute cultures were conserved on potato dextrose agar medium for molecular studies. In this paper, we presented how well the text mining concepts can be used to analyze cellulase producing bacteria and fungi, their comparative structures are also studied with the aid of well-establised, high quality standard bioinformatic tools such as Bioedit, Swissport, Protparam, EMBOSSwin with which a complete data on Cellulases like structure, constituents of the enzyme has been obtained.

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

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Fourage L.

2013-08-01

Full Text Available Enzymatic hydrolysis of lignocellulose is one of the major bottlenecks in the development of biological conversion of lignocellulosic biomass to biofuels. One of the most efficient organisms for the production of cellulolytic enzymes is the fungus Trichoderma reesei, mainly thanks to its high secretion capacity. The conversion of cellulose to glucose involves three types of cellulases working in synergy: endoglucanases (EC 3.2.1.4 randomly cleave 13-1,4 glycosidic linkages of cellulose, cellobiohydrolases (EC 3.2.1.91 attack cellulose chain ends to produce cellobiose dimers which are converted into glucose by the 13-glucosidases (EC 3.2.1 21. Unexpectedly, the amount of l3-glucosidase (BGLI from T. reesei hyperproducing strains represents a very low percentage of the total secreted proteins. A suboptimal content of this enzyme limits the performance of commercial cellulase preparations as cellobiose represents the main inhibitor of the cellulolysis reaction by cellobiohydrolases. This bottleneck can be alleviated either by overexpressing the f3-glucosidase in T. reesei or optimized its specific activity. After giving a brief overview of the main available technologies, this example will be used to illustrate the potential of directed evolution technologies to devolop enzymes tailored to fit industrial needs. We describe the L-ShuffiingTM strategy implemented with three parental genes originating from microbial biodiversity leading to identification of an efficient 13-glucosidase showing a 242 fold increase in specific activity for the pNPGIc substrate compared to WT (Wild Type Cel3a beta-glucosidase of T. reesei. After expression of the best improved 13-glucosidase in T. reesei and secretion of a new enzymatic cocktail, improvement of the glucosidase activity allows a 4-fold decrease of cellulase loading for the saccharification of an industrial pretreated biomass compared to the parental cocktail. L’hydrolyse enzymatique de la lignocellulose

Effects of metal ions on the catalytic degradation of dicofol by cellulase.

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Zhai, Zihan; Yang, Ting; Zhang, Boya; Zhang, Jianbo

2015-07-01

A new technique whereby cellulase immobilized on aminated silica was applied to catalyze the degradation of dicofol, an organochlorine pesticide. In order to evaluate the performance of free and immobilized cellulase, experiments were carried out to measure the degradation efficiency. The Michaelis constant, Km, of the reaction catalyzed by immobilized cellulase was 9.16 mg/L, and the maximum reaction rate, Vmax, was 0.40 mg/L/min, while that of free cellulase was Km=8.18 mg/L, and Vmax=0.79 mg/L/min, respectively. The kinetic constants of catalytic degradation were calculated to estimate substrate affinity. Considering that metal ions may affect enzyme activity, the effects of different metal ions on the catalytic degradation efficiency were explored. The results showed that the substrate affinity decreased after immobilization. Monovalent metal ions had no effect on the reaction, while divalent metal ions had either positive or inhibitory effects, including activation by Mn2+, reversible competition with Cd2+, and irreversible inhibition by Pb2+. Ca2+ promoted the catalytic degradation of dicofol at low concentrations, but inhibited it at high concentrations. Compared with free cellulase, immobilized cellulase was affected less by metal ions. This work provided a basis for further studies on the co-occurrence of endocrine-disrupting chemicals and heavy metal ions in the environment. Copyright © 2015. Published by Elsevier B.V.

Nucleotide sequences of two cellulase genes from alkalophilic Bacillus sp. strain N-4 and their strong homology.

OpenAIRE

Fukumori, F; Sashihara, N; Kudo, T; Horikoshi, K

1986-01-01

Two genes for cellulases of alkalophilic Bacillus sp. strain N-4 (ATCC 21833) have been sequenced. From the DNA sequences the cellulases encoded in the plasmids pNK1 and pNK2 consist of 488 and 409 amino acids, respectively. The DNA and protein sequences of the pNK1-encoded cellulase are related to those of the pNK2-encoded cellulase. The pNK2-encoded cellulase lacks the direct repeat sequence of a stretch of 60 amino acids near the C-terminal end of the pNK1-encoded cellulase. The duplicatio...

Cellulase assisted synthesis of nano-silver and gold: Application as immobilization matrix for biocatalysis.

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Mishra, Abhijeet; Sardar, Meryam

2015-01-01

In the present study, we report in vitro synthesis of silver and gold nanoparticles (NPs) using cellulase enzyme in a single step reaction. Synthesized nanoparticles were characterized by UV-VIS spectroscopy, Dynamic Light Spectroscopy (DLS), Transmission Electron Microscopy (TEM), Energy-dispersive X-ray Spectroscopy (EDX), X-ray Diffraction (XRD), Circular Dichroism (CD) and Fourier Transform Infrared Spectroscopy (FTIR). UV-visible studies shows absorption band at 415nm and 520nm for silver and gold NPs respectively due to surface plasmon resonance. Sizes of NPs as shown by TEM are 5-25nm for silver and 5-20nm for gold. XRD peaks confirmed about phase purity and crystallinity of silver and gold NPs. FTIR data shows presence of amide I peak on both the NPs. The cellulase assisted synthesized NPs were further exploited as immobilization matrix for cellulase enzyme. Thermal stability analysis reveals that the immobilized cellulase on synthesized NPs retained 77-80% activity as compared to free enzyme. While reusability data suggests immobilized cellulase can be efficiently used up to sixth cycles with minimum loss of enzyme activity. The secondary structural analysis of cellulase enzyme during the synthesis of NPs and also after immobilization of cellulase on these NPs was carried out by CD spectroscopy. Copyright © 2015 Elsevier B.V. All rights reserved.

Metagenome Analysis of Protein Domain Collocation within Cellulase Genes of Goat Rumen Microbes

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SooYeon Lim

2013-08-01

Full Text Available In this study, protein domains with cellulase activity in goat rumen microbes were investigated using metagenomic and bioinformatic analyses. After the complete genome of goat rumen microbes was obtained using a shotgun sequencing method, 217,892,109 pair reads were filtered, including only those with 70% identity, 100-bp matches, and thresholds below E−10 using METAIDBA. These filtered contigs were assembled and annotated using blastN against the NCBI nucleotide database. As a result, a microbial community structure with 1431 species was analyzed, among which Prevotella ruminicola 23 bacteria and Butyrivibrio proteoclasticus B316 were the dominant groups. In parallel, 201 sequences related with cellulase activities (EC.3.2.1.4 were obtained through blast searches using the enzyme.dat file provided by the NCBI database. After translating the nucleotide sequence into a protein sequence using Interproscan, 28 protein domains with cellulase activity were identified using the HMMER package with threshold E values below 10−5. Cellulase activity protein domain profiling showed that the major protein domains such as lipase GDSL, cellulase, and Glyco hydro 10 were present in bacterial species with strong cellulase activities. Furthermore, correlation plots clearly displayed the strong positive correlation between some protein domain groups, which was indicative of microbial adaption in the goat rumen based on feeding habits. This is the first metagenomic analysis of cellulase activity protein domains using bioinformatics from the goat rumen.

Lignin-based polyoxyethylene ether enhanced enzymatic hydrolysis of lignocelluloses by dispersing cellulase aggregates.

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Lin, Xuliang; Qiu, Xueqing; Yuan, Long; Li, Zihao; Lou, Hongming; Zhou, Mingsong; Yang, Dongjie

2015-06-01

Water-soluble lignin-based polyoxyethylene ether (EHL-PEG), prepared from enzymatic hydrolysis lignin (EHL) and polyethylene glycol (PEG1000), was used to improve enzymatic hydrolysis efficiency of corn stover. The glucose yield of corn stover at 72h was increased from 16.7% to 70.1% by EHL-PEG, while increase in yield with PEG4600 alone was 52.3%. With the increase of lignin content, EHL-PEG improved enzymatic hydrolysis of microcrystalline cellulose more obvious than PEG4600. EHL-PEG could reduce at least 88% of the adsorption of cellulase on the lignin film measured by quartz crystal microbalance with dissipation monitoring (QCM-D), while reduction with PEG4600 was 43%. Cellulase aggregated at 1220nm in acetate buffer analyzed by dynamic light scattering. EHL-PEG dispersed cellulase aggregates and formed smaller aggregates with cellulase, thereby, reduced significantly nonproductive adsorption of cellulase on lignin and enhanced enzymatic hydrolysis of lignocelluloses. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cellulase and Xylanase Production from Three Isolates of Indigenous Endophytic Fungi

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Yopi; Tasia, W.; Melliawati, R.

2017-12-01

Cellulases and hemicellulases have good potential to be used in energy production, in pulp, paper, textile industries, as well as in animal feed industries. Moreover, its utilization in food industries also cannot be ignored, among others, cellulase and xylanase roles in bakery, wine, and fruit and vegetables juice production. One of the potential enzyme source is endophytic fungi. Object of this study is to explore the potency of endophytic fungi isolated from medicinal plants as source of cellulolytic and xylanolytic enzymes. HL.47F.216 is endophytic fungi isolated from traditional medicinal plants ironwood tree was determined as xylanase producer. HL.51F.235 from pin-flower tree is cellulase producer, while CBN.6F.29 which produces both xylanase and cellulase is originated from Madagascar periwinkle. HL.47F.216 showed 2.5 cm in clear zone diameter and its xylanase activity was 0.262 U/mL with optimum condition pH 7 at 50°C. HL.51F.235 showed 2.4 cm clear zone diameter and 0.239 U/mL of cellulase activity at pH 5 and 70°C. CBN.6F.29 showed 2.8 cm and 0.394 U/mL (pH 5, 40°C) for its cellulase activity, while 2.3 cm and 0.439 U/mL (pH 8, 70°C) for its xylanase activity. Xylanase from HL.47F.216 and CBN.6F.29 showed low molecular masses of 20 kDa and 37-50 kDa, respectively. Molecular masses for cellulases from HL.51F.235 and CBN.6F.29 were 25 and 50 kDa for HL.51F.235 and 100 kDa for CBN.6F.29. Based on macroscopic and microscopic identification, fungal isolate CBN.6F.29 is a member of Class Coelomycetes, while HL.47F.216 was Acremonium sp. and HL.51F.235 was Aspergillus nigri.

Reliable simultaneous zymographic method of characterization of cellulolytic enzymes from fungal cellulase complex.

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Dojnov, Biljana; Grujić, Marica; Vujčić, Zoran

2015-08-01

A method for zymographic detection of specific cellulases in a complex (endocellulase, exocellulase, and cellobiase) from crude fermentation extracts, after a single electrophoretic separation, is described in this paper. Cellulases were printed onto a membrane and, subsequently, substrate gel. Cellobiase isoforms were detected on the membrane using esculine as substrate, endocellulase isoforms on substrate gel with copolymerized carboxymethyl cellulose (CMC), while exocellulase isoforms were detected in electrophoresis gel with 4-methylumbelliferyl-β-d-cellobioside (MUC). This can be a useful additional tool for monitoring and control of fungal cellulase production in industrial processes and fundamental research, screening for particular cellulase producers, or testing of new lignocellulose substrates. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose

DEFF Research Database (Denmark)

Murphy, Leigh; Cruys-Bagger, Nicolaj; Baumann, Martin J.

2012-01-01

by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B...

Synergistic and Dose-Controlled Regulation of Cellulase Gene Expression in Penicillium oxalicum.

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Li, Zhonghai; Yao, Guangshan; Wu, Ruimei; Gao, Liwei; Kan, Qinbiao; Liu, Meng; Yang, Piao; Liu, Guodong; Qin, Yuqi; Song, Xin; Zhong, Yaohua; Fang, Xu; Qu, Yinbo

2015-09-01

Filamentous fungus Penicillium oxalicum produces diverse lignocellulolytic enzymes, which are regulated by the combinations of many transcription factors. Here, a single-gene disruptant library for 470 transcription factors was constructed and systematically screened for cellulase production. Twenty transcription factors (including ClrB, CreA, XlnR, Ace1, AmyR, and 15 unknown proteins) were identified to play putative roles in the activation or repression of cellulase synthesis. Most of these regulators have not been characterized in any fungi before. We identified the ClrB, CreA, XlnR, and AmyR transcription factors as critical dose-dependent regulators of cellulase expression, the core regulons of which were identified by analyzing several transcriptomes and/or secretomes. Synergistic and additive modes of combinatorial control of each cellulase gene by these regulatory factors were achieved, and cellulase expression was fine-tuned in a proper and controlled manner. With one of these targets, the expression of the major intracellular β-glucosidase Bgl2 was found to be dependent on ClrB. The Bgl2-deficient background resulted in a substantial gene activation by ClrB and proved to be closely correlated with the relief of repression mediated by CreA and AmyR during cellulase induction. Our results also signify that probing the synergistic and dose-controlled regulation mechanisms of cellulolytic regulators and using it for reconstruction of expression regulation network (RERN) may be a promising strategy for cellulolytic fungi to develop enzyme hyper-producers. Based on our data, ClrB was identified as focal point for the synergistic activation regulation of cellulase expression by integrating cellulolytic regulators and their target genes, which refined our understanding of transcriptional-regulatory network as a "seesaw model" in which the coordinated regulation of cellulolytic genes is established by counteracting activators and repressors.

Cellulase retention and sugar removal by membrane ultrafiltration during lignocellulosic biomass hydrolysis.

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Knutsen, Jeffrey S; Davis, Robert H

2004-01-01

Technologies suitable for the separation and reuse of cellulase enzymes during the enzymatic saccharification of pretreated corn stover are investigated to examine the economic and technical viability of processes that promote cellulase reuse while removing inhibitory reaction products such as glucose and cellobiose. The simplest and most suitable separation is a filter with relatively large pores on the order of 20-25 mm that retains residual corn stover solids while passing reaction products such as glucose and cellobiose to form a sugar stream for a variety of end uses. Such a simple separation is effective because cellulase remains bound to the residual solids. Ultrafiltration using 50-kDa polyethersulfone membranes to recover cellulase enzymes in solution was shown not to enhance further the saccharification rate or overall conversion. Instead, it appears that the necessary cellulase enzymes, including beta-glucosidase, are tightly bound to the substrate; when fresh corn stover is contacted with highly washed residual solids, without the addition of fresh enzymes, glucose is generated at a high rate. When filtration was applied multiple times, the concentration of inhibitory reaction products such as glucose and cellobiose was reduced from 70 to 10 g/L. However, an enhanced saccharification performance was not observed, most likely because the concentration of the inhibitory products remained too high. Further reduction in the product concentration was not investigated, because it would make the reaction unnecessarily complex and result in a product stream that is much too dilute to be useful. Finally, an economic analysis shows that reuse of cellulase can reduce glucose production costs, especially when the enzyme price is high. The most economic performance is shown to occur when the cellulase enzyme is reused and a small amount of fresh enzyme is added after each separation step to replace lost or deactivated enzyme.

Effects of lignin and surfactant on adsorption and hydrolysis of cellulases on cellulose

OpenAIRE

Li, Yanfei; Sun, Zongping; Ge, Xiaoyan; Zhang, Junhua

2016-01-01

Background Considerable works have been reported concerning the obstruction of enzymatic hydrolysis efficiency by lignin. However, there is a lack of information about the influence of lignin on the adsorption of cellulases on cellulose, along with the hydrolytic activity of the cellulases adsorbed on lignin. In addition, limited discovery has been reported about the influence of additives on cellulase desorption from lignin and lignocellulosic materials. In this work, the effects of lignin o...

Cellulase biosynthesis by trichoderma viride on soluble substrates

Energy Technology Data Exchange (ETDEWEB)

Shin, S B; Kitagawa, Y; Suga, K; Ichikawa, K

1978-01-01

Batch and continuous cultures of Trichoderma viride QM 6a were carried out using either glucose or cellobiose as the sole carbon source. From the data obtained in the continuous culture with glucose as substrate, growth parameters of this fungus ..mu../sub m/, K/sub s/, m and Y were identified. In the case of glucose as substrate, there were extremely low levels of cellobiase and no detectable cellulase activity in both batch and continuous cultures. The inducible cellobiase was an intracellular enzyme, produced in association with cell growth in batch culture on cellobiose as substrate. A kinetic model for cellobiose degradation and cell growth is proposed. A significant increase in the extracellular cellulase productivity was obtained in the range of low dilution rates from 0.025 h/sup -1/ to 0.2 h/sup -1/ in the continuous culture on cellobiose. From the results of these experiments, it was concluded that in continuous culture on cellobiose as substrate the cellulase activity was determined by the balance between induction and catabolite repression.

Effect of cellulase producing fungi on plant residues degradation used as organic fertilizer

International Nuclear Information System (INIS)

Ibrahim, R.M.M

2009-01-01

Series of laboratory and field experiments were conducted at Soil microbiology Unit and Farm of soil and Water research department, Nuclear Research Center, Atomic Energy Authority, Egypt. Laboratory experiments revealed that between nine fungal strain, A. niger was the most potent cellulolytic fungus able to degrade many cellulosic sources (CP, CMC, and FP). Study the effect of cellulolytic fungi on degradation of plant residues used as organic fertilizer in addition to nitrogen fixing bacteria (symbiotically) on lupine growth, yield and nutrients uptake (Field experiment) had been carried out. This objective aims to recycling different plant residues in soil which is consistent with (sustainable development) and utilization of these organic residues as a single carbon source for cellulolytic fungi.Application of 15 N- tracer technique gave us the chance and opportunity to quantify the exact amounts of N derived from the different sources of nitrogen available to lupine plant under the effect of cellulolytic fungi on different plant residues.The obtained results could be summarized as following:I.Laboratory Technique Selection of the most potent cellulolytic fungi 1-Nine fungal strains of Aspergillus niger; Penicillium oxalicum; Trichoderma longibranchiatum; Aspergillus terreus; Aspergillus flavus; Alterrnaria sp.; Trichderma harzianum ; Rhizopus sp. and Syncephalastrum sp. obtained from different sources and tested for their cellulolytic activity. 2-Aspergillus niger and Pencillium oxalicum exhibited the highest cellulase productivity followed by Trichoderma longibranchiatum and Aspergillus terreus.3- fungal mixtures of the most potent four genera Aspergillus niger; Penicillium oxalicum; Trichoderma longibranchiatum and Aspergillus terreus found to have a lower cellulolytic activities for all substrates compared with single inoculation with A. niger.4-Highest FPase activities were exhibited by A. niger when filter paper (FP) used as a carbon source.5-A. niger is

The putative cellodextrin transporter-like protein CLP1 is involved in cellulase induction in Neurospora crassa.

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

2015-01-09

Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, cellodextrin transporter-like protein 1 (CLP1; NCU05853), a putative cellodextrin transporter-like protein that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain Δ3βG formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with Δ3βG. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Putative Cellodextrin Transporter-like Protein CLP1 Is Involved in Cellulase Induction in Neurospora crassa*

Science.gov (United States)

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

2015-01-01

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

Accelerating effects of cellulase in the removal of denture adhesives from acrylic denture bases.

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Harada-Hada, Kae; Mimura, Sumiyo; Hong, Guang; Hashida, Tatsumi; Abekura, Hitoshi; Murata, Hiroshi; Nishimura, Masahiro; Nikawa, Hiroki

2017-04-01

Studies of effective methods for the easy removal of denture adhesives from a denture base are not well represented in the literature. We previously assessed the removability of denture adhesives by immersing within denture cleaners, showing that some cleaners have a weak effect, insufficiently effective in daily use. In this study, we prepared a cellulase, as a potential component for denture adhesive removers, and we examined whether the addition of cellulase to denture cleaners is effective in the removal of cream denture adhesives. We prepared the cellulase Meicelase as one component for the liquefaction of denture adhesives. We used two denture cleaners and two cream adhesives. After the immersion of plates in sample solutions, we evaluated the area of the sample plate still covered with adhesives. Biofilm removal assay was also performed using denture cleaners containing cellulase. The addition of cellulase accelerated the removal of cream adhesives in immersion experiments to a rate faster than that of water and denture cleaners. However, it did not influence the removability of Candida albicans biofilms from acrylic resin specimens. Cellulase hastened the liquefaction of cream adhesives. Copyright © 2016 Japan Prosthodontic Society. Published by Elsevier Ltd. All rights reserved.

Novel Magnetic Cross-Linked Cellulase Aggregates with a Potential Application in Lignocellulosic Biomass Bioconversion

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Junqi Jia

2017-02-01

Full Text Available The utilization of renewable biomass resources to produce high-value chemicals by enzymatic processes is beneficial for alternative energy production, due to the accelerating depletion of fossil fuels. As immobilization techniques can improve enzyme stability and reusability, a novel magnetic cross-linked cellulase aggregate has been developed and applied for biomass bioconversion. The crosslinked aggregates could purify and immobilize enzymes in a single operation, and could then be combined with magnetic nanoparticles (MNPs, which provides easy separation of the materials. The immobilized cellulase showed a better activity at a wider temperature range and pH values than that of the free cellulase. After six cycles of consecutive reuse, the immobilized cellulase performed successful magnetic separation and retained 74% of its initial activity when carboxylmethyl cellulose (CMC was used as the model substrate. Furthermore, the structure and morphology of the immobilized cellulase were studied by Fourier transform infrared spectroscopy (FTIR and scanning electron microscopy (SEM. Moreover, the immobilized cellulase was shown to hydrolyze bamboo biomass with a yield of 21%, and was re-used in biomass conversion up to four cycles with 38% activity retention, which indicated that the immobilized enzyme has good potential for biomass applications.

Novel Magnetic Cross-Linked Cellulase Aggregates with a Potential Application in Lignocellulosic Biomass Bioconversion.

Science.gov (United States)

Jia, Junqi; Zhang, Weiwei; Yang, Zengjie; Yang, Xianling; Wang, Na; Yu, Xiaoqi

2017-02-10

The utilization of renewable biomass resources to produce high-value chemicals by enzymatic processes is beneficial for alternative energy production, due to the accelerating depletion of fossil fuels. As immobilization techniques can improve enzyme stability and reusability, a novel magnetic cross-linked cellulase aggregate has been developed and applied for biomass bioconversion. The crosslinked aggregates could purify and immobilize enzymes in a single operation, and could then be combined with magnetic nanoparticles (MNPs), which provides easy separation of the materials. The immobilized cellulase showed a better activity at a wider temperature range and pH values than that of the free cellulase. After six cycles of consecutive reuse, the immobilized cellulase performed successful magnetic separation and retained 74% of its initial activity when carboxylmethyl cellulose (CMC) was used as the model substrate. Furthermore, the structure and morphology of the immobilized cellulase were studied by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Moreover, the immobilized cellulase was shown to hydrolyze bamboo biomass with a yield of 21%, and was re-used in biomass conversion up to four cycles with 38% activity retention, which indicated that the immobilized enzyme has good potential for biomass applications.

Recovery and reuse of cellulase catalyst in an enzymatic cellulose hydrolysis process

Science.gov (United States)

Woodward, J.

1987-09-18

A process for recovering cellulase from the hydrolysis of cellulose, and reusing it in subsequent hydrolyois procedures. The process utilizes a commercial adsorbent that efficiently removes cellulase from reaction products which can be easily removed by simple decantation. 1 fig., 4 tabs.

A liquid crystal-based sensor for the simple and sensitive detection of cellulase and cysteine.

Science.gov (United States)

Wang, Yi; Hu, Qiongzheng; Tian, Tongtong; Gao, Yan'an; Yu, Li

2016-11-01

A liquid crystal (LC)-based sensor, which is capable of monitoring enzymatic activity at the aqueous/LC interface and detecting cellulase and cysteine (Cys), was herein reported. When functionalized with a surfactant, dodecyl β-d-glucopyranoside, the 4-cyano-4'-pentylbiphenyl (5CB) displays a dark-to-bright transition in the optical appearance for cellulase. We attribute this change to the orientational transition of LCs, as a result of enzymatic hydrolysis between cellulase and surfactant. Furthermore, by adding cellulase and Cu(2+), our surfactant-LCs system performs an interesting ability to detect Cys, even though Cys could not interact with surfactant or LC directly. Alternatively, through the strong binding between Cys and Cu(2+), cellulase was able to hydrolyze surfactant in the presence of Cu(2+), leading to the transition of LCs from dark to bright. The detection limit of the LC sensor was around 1×10(-5)mg/mL and 82.5μM for cellulase and Cys, respectively. The LC-based sensor may contribute to the development of low-cost, expedient, and label-free detection for cellulase and Cys and the design strategy may also provide a novel way for detecting multiple analytes. Copyright © 2016 Elsevier B.V. All rights reserved.

Purification and Characterization of Thermostable Cellulase from ...

African Journals Online (AJOL)

Available online at http://www.tjpr.org ... Methods: Molecular community structure of the newly selected thermophilic bacterial ... Keywords: Thermostable cellulase, Sugarcane bagasse, Purification, Characterization, Hot spring ... Currently, one.

Plant carbohydrate binding module enhances activity of hybrid microbial cellulase enzyme

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Caitlin Siobhan Byrt

2012-11-01

Full Text Available A synthetic, highly active cellulase enzyme suitable for in planta production may be a valuable tool for biotechnological approaches to develop transgenic biofuel crops with improved digestibility. Here, we demonstrate that the addition of a plant derived carbohydrate binding module (CBM to a synthetic glycosyl hydrolase (GH improved the activity of the hydrolase in releasing sugar from plant biomass. A CEL-HYB1-CBM enzyme was generated by fusing a hybrid microbial cellulase, CEL-HYB1, with the carbohydrate-binding module (CBM of the tomato (Solanum lycopersicum SlCel9C1 cellulase. CEL-HYB1 and CEL-HYB1-CBM enzymes were produced in vitro using Pichia pastoris and the activity of these enzymes was tested using CMC, MUC and native crystalline cellulose assays. The presence of the CBM substantially improved the endo-glucanase activity of CEL-HYB1, especially against the native crystalline cellulose encountered in Sorghum plant cell walls. These results indicate that addition of an endogenous plant derived CBM to cellulase enzymes may enhance hydrolytic activity.

Cellulase Activity in Solid State Fermentation of Palm Kernel Cake with Trichoderma sp.

Directory of Open Access Journals (Sweden)

Massaud, M. B. N.

2012-01-01

Full Text Available Aims: The effect of different types of fungal inocula to the cellulase activity measured on palm kernel cake (PKC was studied. Methodology and Results: Isolate Pro-A1 which was identified as Trichoderma sp. was selected as a potential producer of cellulase via solid state fermentation technique (SSF. Two types of PKCs were used; raw PKC (containing residual oil and defatted PKC. The PKCs were inoculated with different concentrations of conidia and varying amounts (g of solid mycelia plugs (SMP for SSF. The effect of ultrafiltered crude fungal filtrate (CFF as inocula was also being tested. The highest cellulase activity of 2.454 FPU/mL was detected with 60% (wt/wt SMP applied to the raw PKC. Conversely, 2.059 FPU/mL of cellulase activity was measured when 80% (wt/wt of SMP was applied to the defatted PKC which is 62.3% higher than the untreated defatted PKC; and more than 100% increase in enzymatic activity compared to raw PKC. The cellulase activity in the SSF inoculated with 8 x 106 conidia /mL and 12 x 106 conidia /mL were 1.704 FPU/mL for raw PKC and 1.856 FPU/mL for defatted PKC, an enhancement of about 46% from uninoculated batch. Inoculation with CFF bears corresponding maximum improvement of the cellulase activity on both PKCs of 13.58% (raw and 2.86% (defatted. Conclusion, significance and impact of study: The current study proves that Trichoderma sp. in the form of SMP can enhance the cellulase activity on PKCs effectively with more than 100% increment. Fungal conidia are also a better choice in enhancing cellulase activity of Trichoderma sp. permitted that the PKC used is devoid of oil. From this study, Trichoderma sp. holds the potential of converting lignocellulosic materials into products of commercial and industrial values such as glucose and other biofuels.

Mechanism of interaction between cellulase action and applied shear force, an hypothesis

NARCIS (Netherlands)

Lenting, H.B.M.; Lenting, H.B.M.; Warmoeskerken, Marinus

2001-01-01

An overview is given of what is known in literature concerning the structure of both cellulose and cellulase enzymes and the enzymatic degradation of cellulose. Based on this knowledge, a hypothesis is formulated about the relation between cellulase performance and required applied shear force on

Improvement of ethanol production from crystalline cellulose via optimizing cellulase ratios in cellulolytic Saccharomyces cerevisiae.

Science.gov (United States)

Liu, Zhuo; Inokuma, Kentaro; Ho, Shih-Hsin; den Haan, Riaan; van Zyl, Willem H; Hasunuma, Tomohisa; Kondo, Akihiko

2017-06-01

Crystalline cellulose is one of the major contributors to the recalcitrance of lignocellulose to degradation, necessitating high dosages of cellulase to digest, thereby impeding the economic feasibility of cellulosic biofuels. Several recombinant cellulolytic yeast strains have been developed to reduce the cost of enzyme addition, but few of these strains are able to efficiently degrade crystalline cellulose due to their low cellulolytic activities. Here, by combining the cellulase ratio optimization with a novel screening strategy, we successfully improved the cellulolytic activity of a Saccharomyces cerevisiae strain displaying four different synergistic cellulases on the cell surface. The optimized strain exhibited an ethanol yield from Avicel of 57% of the theoretical maximum, and a 60% increase of ethanol titer from rice straw. To our knowledge, this work is the first optimization of the degradation of crystalline cellulose by tuning the cellulase ratio in a cellulase cell-surface display system. This work provides key insights in engineering the cellulase cocktail in a consolidated bioprocessing yeast strain. Biotechnol. Bioeng. 2017;114: 1201-1207. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Characterization of the enzymes present in the cellulase system of Thielavia terrestris 255B

Energy Technology Data Exchange (ETDEWEB)

Gilbert, Michel; Breuil, Colette; Saddler, J N [Forintek Canada Corp., Ottawa, ON (CA). Dept. of Biotechnology and Chemistry

1992-01-01

The authors initiated a study of the cellulases from the thermophilic fungus Thielavia terrestris 255B to see how they compared with enzymes derived from mesophilic fungi such as Trichoderma. To try to obtain maximum production of a complete cellulase system, the fungus was first grown on a variety of soluble and insoluble substrates. As well as assaying the culture filtrates for cellulase activity and protein concentration, the enzyme profiles were compared using non-denaturing electrophoretic techniques (IEF and native-PAGE). The separation by native-PAGE and IEF was followed by activity staining methods to detect endoglucanase and xylanase activities. Native-PAGE could not be used to determine accurately the M{sub r} of the cellulases because of possible differences in mass/charge ratios. Bands with apparent M{sub r} values above 200000 were reproducibly detected. This suggested that the various cellulase components may be organized into high molecular weight complexes. (author).

A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities

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Gupta Munishwar

2007-06-01

Full Text Available Abstract Background The use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex™ Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities. Results Complete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase were completely retained after cross-linking. The Vmax/Km values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50°C, 60°C and 70°C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity. Conclusion A single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1 hydrolysis of pectin, 2 hydrolysis of xylan and 3 hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes.

Preparation, characterisation and use for antioxidant oligosaccharides of a cellulase from abalone (Haliotis discus hannai) viscera.

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Tao, Zhi-Peng; Sun, Le-Chang; Qiu, Xu-Jian; Cai, Qiu-Feng; Liu, Guang-Ming; Su, Wen-Jin; Cao, Min-Jie

2016-07-01

In China, abalone (Haliotis discus hannai) production is growing annually. During industrial processing, the viscera, which are abundant of cellulase, are usually discarded or processed into low-value feedstuff. Thus, it is of interest to obtain cellulase from abalone viscera and investigate its application for preparation of functional oligosaccharides. A cellulase was purified from the hepatopancreas of abalone by ammonium sulfate precipitation and two-steps column chromatography. The molecular weight of the cellulase was 45 kDa on SDS-PAGE. Peptide mass fingerprinting analysis yielded 103 amino acid residues, which were identical to cellulases from other species of abalone. Substrate specificity analysis indicated that the cellulase is an endo-1,4-β-glucanase. Hydrolysis of seaweed Porphyra haitanensis polysaccharides by the enzyme produced oligosaccharides with degree of polymerisation of two to four, whose monosaccharide composition was 58% galactose, 4% glucose and 38% xylose. The oligosaccharides revealed 2,2'-diphenyl-1-picrylhydrazyl free radical as well as hydrogen peroxide scavenging activity. It is feasible and meaningful to utilise cellulase from the viscera of abalone for preparation of functional oligosaccharides. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Comparative studies on production of cellulases from three strains of aspergillus niger

International Nuclear Information System (INIS)

Sohail, M.; Ahmad, A.; Khan, S.

2014-01-01

Three strains of Aspergillus niger were retrieved from culture collection of the Department of Microbiology, University of Karachi, Pakistan and were studied for their ability to produce cellulases. Cultivation at different temperatures and in presence of various carbon sources revealed that all the three strains produced more amounts of endoglucanase, glucosidase and filter-paperase activities at 35 degree C; carboxymethyl cellulose promotes the production of filter paperase and endoglucanase activities whereas salicin induced glucosidase activity. Experiments on growth and enzyme production kinetics showed that generation time and hence volumetric rate of biomass production is influenced by the carbon source used in the medium; simple carbon source, such as glucose favored the growth of all the strains. Cellulases from all the strains showed optimum activity at temperature >50 degree C and under acidic range of pH, while melting temperature was 64-65 degree C. These findings affirm that cellulases from A. niger are potential candidates as alternative to Trichoderma cellulases. (author)

Adsorption, immobilization and activity of cellulase in soil: the impacts of maize straw and its humification

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Ali Akbar Safari Sinegani

2013-12-01

Full Text Available The present work aimed to study some aspects of sorption and immobilization of cellulase molecules on soil components by the analysis of the reactions of cellulase in a soil treated with different levels of maize residue and incubated for 90 days. The analysis of variance showed that the effects of the treatments of maize straw, incubation time and their interaction on cellulase adsorption, desorption and immobilization were statistically significant. The adsorption and immobilization capacities of soil by application of maize straw increased significantly. However they decreased with decreasing the soil organic matter (SOM after 45 days of incubation. The desorption of adsorbed cellulase molecules from the soil by washing with distilled water depended on the SOM contents and its humification. The binding strength of cellulase molecule with fresh miaze straw was significantly stronger than that with humified maize straw. The immobilized cellulase activity, particularly its specific activity increased significantly by increasing the OC contents in the soil treated with maize straw.

Cellulase activity of a thermophilic Aspergillus fumigatus (fresenius) strain

Energy Technology Data Exchange (ETDEWEB)

Vandamme, E J; Logghe, J M; Geeraerts, A M

1982-10-01

A thermophilic fungus, isolated from horse manure on Whatman-cellulose CF-11 as sole carbon source was identified as Aspergillus fumigatus. It grew optimally at 45 degrees C and displayed highest cellulase activity at 55 degrees C and pH 5.0 towards a range of soluble and crude insolulble cellulosic substrates. Germination and outgrowth of the spore inoculum in carboxymethylcellulose (CMCellulose) medium was accompanied by high endoglucanase (E.C. 3.2.1.4) activity. The cellulose complex e.g. exo-beta-1,4-glucanase (E.C.3.2.1.-); endo-beta-1,4glucanase (E.C.3.2.1.4.) and beta-glucosidase (E.C.3.2.1.21.) displayed quite different properties depending on whether it was formed on low or highly substituted CMCellulose. Extracellular cellulase formation followed biomass accumulation. Upon prolonged incubation cell lysis occurred which resulted in a further increase in cellulase activity. Ball-milled crude cellulosics, such as newsprint, de-inked newsprint, glossy writing paper, toilet paper and beech sawdust were substantially solubilized with 110 hours of growth. The type of crude cellulosic material greatly influenced the pattern of enzyme production. The enzyme complex formed when A. fumigatus was grown on soluble CMC-4M6F, displayed a different activity spectrum towards crude cellulosics compared with that formed on ball-milled glossy writing paper. The extracellular cellulase of A. fumigatus looks promising for a rapid and substantial solubilisation and saccharification of crude cellulosics. (Refs. 32).

Design and characterization of synthetic fungal-bacterial consortia for direct production of isobutanol from cellulosic biomass.

Science.gov (United States)

Minty, Jeremy J; Singer, Marc E; Scholz, Scott A; Bae, Chang-Hoon; Ahn, Jung-Ho; Foster, Clifton E; Liao, James C; Lin, Xiaoxia Nina

2013-09-03

Synergistic microbial communities are ubiquitous in nature and exhibit appealing features, such as sophisticated metabolic capabilities and robustness. This has inspired fast-growing interest in engineering synthetic microbial consortia for biotechnology development. However, there are relatively few reports of their use in real-world applications, and achieving population stability and regulation has proven to be challenging. In this work, we bridge ecology theory with engineering principles to develop robust synthetic fungal-bacterial consortia for efficient biosynthesis of valuable products from lignocellulosic feedstocks. The required biological functions are divided between two specialists: the fungus Trichoderma reesei, which secretes cellulase enzymes to hydrolyze lignocellulosic biomass into soluble saccharides, and the bacterium Escherichia coli, which metabolizes soluble saccharides into desired products. We developed and experimentally validated a comprehensive mathematical model for T. reesei/E. coli consortia, providing insights on key determinants of the system's performance. To illustrate the bioprocessing potential of this consortium, we demonstrate direct conversion of microcrystalline cellulose and pretreated corn stover to isobutanol. Without costly nutrient supplementation, we achieved titers up to 1.88 g/L and yields up to 62% of theoretical maximum. In addition, we show that cooperator-cheater dynamics within T. reesei/E. coli consortia lead to stable population equilibria and provide a mechanism for tuning composition. Although we offer isobutanol production as a proof-of-concept application, our modular system could be readily adapted for production of many other valuable biochemicals.

Abscisic acid and ethephon regulation of cellulase in the endosperm cap and radicle during lettuce seed germination.

Science.gov (United States)

Chen, Bingxian; Ma, Jun; Xu, Zhenjiang; Wang, Xiaofeng

2016-10-01

The purpose of this study was to investigate the role of cellulase in endosperm cap weakening and radicle elongation during lettuce (Lactuca sativa L.) seed germination. The application of abscisic acid (ABA) or ethephon inhibits or promotes germination, respectively, by affecting endosperm cap weakening and radicle elongation. Cellulase activities, and related protein and transcript abundances of two lettuce cellulase genes, LsCEL1 and LsCEL2, increase in the endosperm cap and radicle prior to radicle protrusion following imbibition in water. ABA or ethephon reduce or elevate, respectively, cellulase activity, and related protein and transcript abundances in the endosperm cap. Taken together, these observations suggest that cellulase plays a role in endosperm cap weakening and radicle elongation during lettuce seed germination, and that the regulation of cellulase in the endosperm cap by ABA and ethephon play a role in endosperm cap weakening. However, the influence of ABA and ethephon on radicle elongation may not be through their effects on cellulase. © 2016 Institute of Botany, Chinese Academy of Sciences.

Using temperature-responsive zwitterionic surfactant to enhance the enzymatic hydrolysis of lignocelluloses and recover cellulase by cooling.

Science.gov (United States)

Cai, Cheng; Pang, Yuxia; Zhan, Xuejuan; Zeng, Meijun; Lou, Hongming; Qian, Yong; Yang, Dongjie; Qiu, Xueqing

2017-11-01

Some zwitterionic surfactants exhibit upper critical solution temperature (UCST) in aqueous solutions. For the zwitterionic surfactant solution mixed with cellulase, when its temperature is below UCST, the cellulase can be recovered by coprecipitation with zwitterionic surfactant. In this work, 3-(Hexadecyldimethylammonio) propanesulfonate (SB3-16) was selected to enhance the enzymatic hydrolysis of lignocelluloses and recover the cellulase. After adding 2mmol/L of SB3-16, the enzymatic digestibility of eucalyptus pretreated by dilute acid (Eu-DA) and by sulfite (Eu-SPORL) increased from 27.9% and 35.1% to 72.6% and 89.7%, respectively. The results showed that SB3-16 could reduce the non-productive adsorption of cellulase on hydrophobic interface, while it did not significantly inhibit the activity of cellulase. For the solution contained 1wt% SB3-16 and 200mg protein/L CTec2 cellulase, 55.2% of protein could be recovered by cooling. The filter paper activity of the recovered cellulase was 1.93FPU/mg protein, which was 95.8% of its initial activity. Copyright © 2017. Published by Elsevier Ltd.

Effects of different dietary cation-anion balance and cellulase on blood biochemical indexes in sheep

International Nuclear Information System (INIS)

Niu Xuejia; Sun Yongqiang; Zheng Guoping; Li Mangxue; Liu Dasen

2007-01-01

Five sheep with average live weight of 38 kg were selected and fitted with permanent ruminal fistulae for the study of the influence of DCAB and cellulase on blood biochemical indexes in sheep. With the design of 5 x 5 Latin square, the goats were undergone four groups of treatments. As sheep in control group were only fed with basic feed, other treatments were fed with basic feed and additives. Those additives were: treatment 1 with cellulase (0.2% of dietary), treatment 2 with DCAB1 (20 mEq/100g DM), treatment 3 with cellulase (0.2% of dietary) and DCAB1 (20 mEq/100g DM) and treatment 4 with cellulase (0.2% of dietary) and DCAB2 (40 mEq/100 g DM). The results show that different DCAB and cellulase had no apparent influence on the concentrations of T 3 and T 4 (P>0.05), but they had significant influence on the concentration of insulin (P 0.05). The study indicated that addition of 0.2% cellulase to dietary while adjusting DCAB to 0.2-40 mEq/100 g DM respectively did not destroy tissues and organs of sheep. (authors)

Electrotransformation and expression of cellulase genes in wild-type Lactobacillus reuteri.

Science.gov (United States)

Li, Wang; Yang, Ming-Ming; Zhang, Guang-Qin; He, Wan-Ling; Li, Yuan-Xiao; Chen, Yu-Lin

2012-01-01

Two cellulase genes, Cel15 and Cel73, were amplified from Bacillus subtilis genome DNA in a previous study. Two integrative vectors, pLEM4153 and pLEM4154, containing the genes Cel15 and Cel73, respectively, were constructed and successfully electroporated into the wild-type Lactobacillus reuteri which was isolated from chick guts through an optimized procedure. Two recombinant L. reuteri were selected from a Man, Rogosa, and Sharp (MRS) plate with 10 µg/ml erythromycin, and named L. reuteri XNY-Cel15 and L. reuteri XNY-Cel73, respectively. To verify the transcription and expression of the two cellulase genes in the recombinant L. reuteri strains, the mRNA relative quantity (RQ) and the cellulase activity were determined. The mRNA RQ of Cel15 in L. reuteri XNY-Cel15 is 1,8849.5, and that of Cel73 in L. reuteri XNY-Cel73 is 1,388, and the cellulase activity of the modified MRS broth cultured with L. reuteri XNY-Cel15 was 0.158 U/ml, whereas that with L. reuteri XNY-Cel73 was 0.15 U/ml. Copyright © 2012 S. Karger AG, Basel.

Enzymatic activity of the cellulolytic complex produced by Trichoderma reesei. Enzymatic hydrolysis of cellulose; Actividad enzimatica del complejo celulolitico producido por Trichoderma reesei. Hidrolisis enzimatica de la celulosa

Energy Technology Data Exchange (ETDEWEB)

Alfonsel, M; Negro, M J; Saez, R; Martin, C

1986-07-01

The enzymatic activity characterization of the cellulolytic complex obtained from Trichoderma reesei QM 9414 and the influence of the enzymatic hydrolysis conditions on the hydrolysis yield are studied. Pure cellulose and native or alkali pretreated biomass Onopordum nervosum have been used as substrates. The values of pH, temperature, substrate concentration and enzyme-substrate ratio for the optimum activity of that complex, evaluated as glucose and reducing sugars production, have been selected. Previous studies on enzymatic hydrolysis of 0. nervosum have shown a remarkable effect of the alkaline pretreatments on the final hydrolysis yield. (Author) 10 refs.

Improving cellulase productivity of Penicillium oxalicum RE-10 by repeated fed-batch fermentation strategy.

Science.gov (United States)

Han, Xiaolong; Song, Wenxia; Liu, Guodong; Li, Zhonghai; Yang, Piao; Qu, Yinbo

2017-03-01

Medium optimization and repeated fed-batch fermentation were performed to improve the cellulase productivity by P. oxalicum RE-10 in submerged fermentation. First, Plackett-Burman design (PBD) and central composite design (CCD) were used to optimize the medium for cellulase production. PBD demonstrated wheat bran and NaNO 3 had significant influences on cellulase production. The CCD results showed the maximum filter paper activity (FPA) production of 8.61U/mL could be achieved in Erlenmeyer flasks. The maximal FPA reached 12.69U/mL by submerged batch fermentation in a 7.5-L stirred tank, 1.76-fold higher than that on the original medium. Then, the repeated fed-batch fermentation strategy was performed successfully for increasing the cellulase productivity from 105.75U/L/h in batch fermentation to 158.38U/L/h. The cellulase activity and the glucan conversion of delignined corn cob residue hydrolysis had no significant difference between the enzymes sampled from different cycles of the repeated fed-batch fermentation and that from batch culture. Copyright © 2016 Elsevier Ltd. All rights reserved.

Heterologous expression, purification, crystallization and preliminary X-ray analysis of Trichoderma reesei xylanase II and four variants

International Nuclear Information System (INIS)

Wan, Qun; Kovalevsky, Andrey; Zhang, Qiu; Hamilton-Brehm, Scott; Upton, Rosalynd; Weiss, Kevin L.; Mustyakimov, Marat; Graham, David; Coates, Leighton; Langan, Paul

2013-01-01

The wild-type protein and four active-site mutants of xylanase II from Trichoderma reesei that catalyzes the hydrolysis of glycosidic bonds in xylan have successfully been crystallized. The crystallization of several structures including ligand-free and protein ligand complexes containing the substrate (xylohexaose) or product (xylotriose) are detailed. Xylanase II from Trichoderma reesei catalyzes the hydrolysis of glycosidic bonds in xylan. Crystallographic studies of this commercially important enzyme have been initiated to investigate its reaction mechanism, substrate binding and dependence on basic pH conditions. The wild-type protein was heterologously expressed in an Escherichia coli host using the defined medium and four active-site amino acids were replaced to abolish its activity (E177Q and E86Q) or to change its pH optimum (N44D and N44H). Cation-exchange and size-exclusion chromatography were used to obtain >90% protein purity. The ligand-free proteins and variant complexes containing substrate (xylohexaose) or product (xylotriose) were crystallized in several different space groups and diffracted to high resolutions (from 1.07 to 1.55 Å)

Biochemical characterization of thermostable cellulase enzyme from ...

African Journals Online (AJOL)

user

2012-05-29

May 29, 2012 ... tested for their ability to produce cellulase complex enzyme by growing on a defined substrates as well ... In the current industrial processes, cellulolytic enzymes ... energy sources such as glucose, ethanol, hydrogen and.

Production of cellulases by a thermophilic fungus, Thermoascus aurantiacus A-131

Energy Technology Data Exchange (ETDEWEB)

Kawamori, M; Takayama, K; Takasawa, S

1987-01-01

A thermophilic fungus, strain A-131, isolated from a soil sample produced cellulases in the culture fluid. The fungus (strain A-131) was identified as Thermoascus aurantiacus Miehe from its taxonomical characteristics. The cellulases of T. aurantiacus A-131 were produced constitutively without cellulase inducers. Moreover, their production was induced markedly by amorphous polysaccharides containing beta-1, 4 linkages such as alkali-treated bagasse and xylan rather than crystalline cellulose. The cultivation of T. aurantiacus A-131 at 45 degrees C with 4% alkali-treated bagasse led to the production of about 70 U/ml of CMCase after four days. The thermostability of the cellulolytic enzymes of T. aurantiacus A-131 was excellent and virtually no decreases in their activities were seen after preincubation at 60 degrees C for 24 hours. (Refs. 21).

Central carbon metabolism influences cellulase production in Bacillus licheniformis.

Science.gov (United States)

Wang, J; Liu, S; Li, Y; Wang, H; Xiao, S; Li, C; Liu, B

2018-01-01

Bacillus licheniformis that can produce cellulase including endo glucanase and glucosidase is an important industrial microbe for cellulose degradation. The purpose of this research was to assess the effect of endo glucanase gene bglC and glucosidase gene bglH on the central metabolic flux in B. licheniformis. bglC and bglH were knocked out using homologous recombination method, respectively, and the corresponding knockout strains were obtained for 13 C metabolic flux analysis. A significant change was observed in metabolic fluxes after 13 C metabolic flux ratio analysis. In both of the knockout strains, the increased fluxes of the pentose phosphate pathway and malic enzyme reaction enabled an elevated supply of NADPH which provided enough reducing power for the in vivo synthesis reactions. The fluxes through tricarboxylic acid cycle and anaplerotic reactions increased fast in the two knockout strains, which meant more energy generated. The changed fluxes in central carbon metabolism provided a holistic view of the physiological status in B. licheniformis and possible targets for further strain engineering. Cellulase is very important in the field of agriculture and bioenergy because of its degrading effect on cellulosic biomass. This study presented the effect of central carbon metabolism on cellulase production in Bacillus licheniformis. The study also provided a holistic view of the physiological status in B. licheniformis. The shifted metabolism provided a quantitative evaluation of the biosynthesis of cellulase and a priority ranked target list for further strain engineering. © 2017 The Society for Applied Microbiology.

Characterization of thermostable cellulase produced by Bacillus strains isolated from solid waste of carrageenan

Science.gov (United States)

Listyaningrum, N. P.; Sutrisno, A.; Wardani, A. K.

2018-03-01

Cellulase-producing bacteria was isolated from solid waste of carrageenan and identified as Bacillus licheniformis C55 by 16S rRNA sequencing. The optimum condition for cellulase production was obtained at pH and temperature of 8.0 and 50°C, respectively in a medium containing glucose as carbon source and 1.0% carboxymethyl cellulose (CMC) to stimulate the cellulase production. Most remarkably, the enzyme retained its relative activity over 50% after incubation at 50°C for 90 minutes. Substrate specificity suggested that the enzyme is an endoglucanase. The molecular mass of Bacillus licheniformis C55 crude cellulase was found about 18 kDa by SDS-PAGE analysis. This thermostable enzyme would facilitate development of more efficient and cost-effective forms of the process to convert lignocellulosic biomass into high-value products.

Expression of Trichoderma reesei β-mannanase in tobacco chloroplasts and its utilization in lignocellulosic woody biomass hydrolysis.

Directory of Open Access Journals (Sweden)

Pankaj Agrawal

Full Text Available Lignocellulosic ethanol offers a promising alternative to conventional fossil fuels. One among the major limitations in the lignocellulosic biomass hydrolysis is unavailability of efficient and environmentally biomass degrading technologies. Plant-based production of these enzymes on large scale offers a cost-effective solution. Cellulases, hemicellulases including mannanases and other accessory enzymes are required for conversion of lignocellulosic biomass into fermentable sugars. β-mannanase catalyzes endo-hydrolysis of the mannan backbone, a major constituent of woody biomass. In this study, the man1 gene encoding β-mannanase was isolated from Trichoderma reesei and expressed via the chloroplast genome. PCR and Southern hybridization analysis confirmed site-specific transgene integration into the tobacco chloroplast genomes and homoplasmy. Transplastomic plants were fertile and set viable seeds. Germination of seeds in the selection medium showed inheritance of transgenes into the progeny without any Mendelian segregation. Expression of endo-β-mannanase for the first time in plants facilitated its characterization for use in enhanced lignocellulosic biomass hydrolysis. Gel diffusion assay for endo-β-mannanase showed the zone of clearance confirming functionality of chloroplast-derived mannanase. Endo-β-mannanase expression levels reached up to 25 units per gram of leaf (fresh weight. Chloroplast-derived mannanase had higher temperature stability (40 °C to 70 °C and wider pH optima (pH 3.0 to 7.0 than E.coli enzyme extracts. Plant crude extracts showed 6-7 fold higher enzyme activity than E.coli extracts due to the formation of disulfide bonds in chloroplasts, thereby facilitating their direct utilization in enzyme cocktails without any purification. Chloroplast-derived mannanase when added to the enzyme cocktail containing a combination of different plant-derived enzymes yielded 20% more glucose equivalents from pinewood than the

SIMULTANEOUS PRETREATMENT OF LIGNOCELLULOSE AND HYDROLYSIS OF STARCH IN MIXTURES TO SUGARS

OpenAIRE

Hamzeh Hoseinpour; Keikhosro Karimi; Hamid Zilouei; Mohammad J. Taherzadeh

2010-01-01

Mixtures of starch and lignocelluloses are available in many industrial, agricultural, and municipal wastes and residuals. In this work, dilute sulfuric acid was used for simultaneous pretreatment of lignocellulose and hydrolysis of starch, to obtain a maximum amount of fermentable sugar after enzymatic hydrolysis with cellulase and β-glucosidase. The acid treatment was carried out at 70-150°C with 0-1% (v/v) acid concentration and 5-15% (w/v) solids concentration for 0-40 minutes. Under the ...

Improvement of halophilic cellulase production from locally isolated fungal strain.

Science.gov (United States)

Gunny, Ahmad Anas Nagoor; Arbain, Dachyar; Jamal, Parveen; Gumba, Rizo Edwin

2015-07-01

Halophilic cellulases from the newly isolated fungus, Aspergillus terreus UniMAP AA-6 were found to be useful for in situ saccharification of ionic liquids treated lignocelluloses. Efforts have been taken to improve the enzyme production through statistical optimization approach namely Plackett-Burman design and the Face Centered Central Composite Design (FCCCD). Plackett-Burman experimental design was used to screen the medium components and process conditions. It was found that carboxymethylcellulose (CMC), FeSO4·7H2O, NaCl, MgSO4·7H2O, peptone, agitation speed and inoculum size significantly influence the production of halophilic cellulase. On the other hand, KH2PO4, KOH, yeast extract and temperature had a negative effect on enzyme production. Further optimization through FCCCD revealed that the optimization approach improved halophilic cellulase production from 0.029 U/ml to 0.0625 U/ml, which was approximately 2.2-times greater than before optimization.

The effect of leachate recirculation with enzyme cellulase addition on waste stability in landfill bioreactor

Science.gov (United States)

Saffira, N.; Kristanto, G. A.

2018-01-01

Landfill bioreactor with leachate recirculation is known to enhance waste stabilization. However, the composition of waste in Indonesia is comprised by organic waste which is lignocellulosic materials that considered take a long time to degrade under anaerobic condition. To accelerate the degradation process, enzyme addition is ought to do. Cellulase is an enzyme that can catalyse cellulose and other polysaccharide decomposition processes. Therefore, operation of waste degradation using leachate recirculation with a cellulase addition to enhance waste stabilization was investigated using anaerobic bioreactor landfill. The experiment was performed on 2 conditions; leachate recirculation with cellulase addition and recirculation only as a control. The addition of cellulase is reported to be significant in decreasing organic content, represented by volatile solid parameter. The volatile solid reduction in the cellulase augmented reactor and control reactor was 17.86% and 7.90%, respectively. Cellulase addition also resulted in the highest cellulose reduction. Settlement of the landfill in a bioreactor with enzyme addition (32.67%) was reported to be higher than the control (19.33%). Stabilization of landfill review by the decreasing rate constant of the cellulose and lignin ratio parameter was more rapidly achieved by the enzyme addition (0.014 day-1) compared to control (0.002 day-1).

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance.

Science.gov (United States)

Kern, Marcelo; McGeehan, John E; Streeter, Simon D; Martin, Richard N A; Besser, Katrin; Elias, Luisa; Eborall, Will; Malyon, Graham P; Payne, Christina M; Himmel, Michael E; Schnorr, Kirk; Beckham, Gregg T; Cragg, Simon M; Bruce, Neil C; McQueen-Mason, Simon J

2013-06-18

Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.

Assessment of methods to determine minimal cellulase concentrations for efficient hydrolysis of cellulose

Energy Technology Data Exchange (ETDEWEB)

Hogan, C.M.; Mes-Hartree, M.; Saddler, J.N. (Forintek Canada Corp., Ottawa, ON (Canada). Biotechnology and Chemistry Dept.); Kushner, D.J. (Toronto Univ., Ontario (Canada). Dept. of Microbiology)

1990-02-01

The enzyme loading needed to achieve substrate saturation appeared to be the most economical enzyme concentration to use for hydrolysis, based on percentage hydrolysis. Saturation was reached at 25 filter paper units per gram substrate on Solka Floc BW300, as determined by studying (a) initial adsorption of the cellulase preparation onto the substrate, (b) an actual hydrolysis or (c) a combined hydrolysis and fermentation (CHF) process. Initial adsorption of the cellulases onto the substrate can be used to determine the minimal cellulase requirements for efficient hydrolysis since enzymes initially adsorbed to the substrate have a strong role in governing the overall reaction. Trichoderma harzianum E58 produces high levels of {beta}-glucosidase and is able to cause high conversion of Solka Floc BW300 to glucose without the need for exogenous {beta}-glucosidase. End-product inhibition of the cellulase and {beta}-glucosidase can be more effectively reduced by employing a CHF process than by supplemental {beta}-glucosidase. (orig.).

BIOPROCESS DEVELOPMENTS FOR CELLULASE PRODUCTION BY Aspergillus oryzae CULTIVATED UNDER SOLID-STATE FERMENTATION

Directory of Open Access Journals (Sweden)

R. D. P. B. Pirota

Full Text Available Abstract Bioprocess development studies concerning the production of cellulases are of crucial importance due to the significant impact of these enzymes on the economics of biomass conversion into fuels and chemicals. This work evaluates the effects of solid-state fermentation (SSF operational conditions on cellulase production by a novel strain of Aspergillus oryzae using an instrumented lab-scale bioreactor equipped with an on-line automated monitoring and control system. The use of SSF cultivation under controlled conditions substantially improved cellulase production. Highest production of FPase (0.40 IU g-1, endoglucanase (123.64 IU g-1, and β-glucosidase (18.32 IU g-1 was achieved at 28 °C, using an initial substrate moisture content of 70%, with an inlet air humidity of 80% and an airflow rate of 20 mL min-1. Further studies of kinetic profiles and respirometric analyses were performed. The results showed that these data could be very useful for bioprocess development of cellulase production and scale-up.

Thermostable, haloalkaline cellulase from Bacillus halodurans CAS 1 by conversion of lignocellulosic wastes.

Science.gov (United States)

Annamalai, Neelamegam; Rajeswari, Mayavan Veeramuthu; Elayaraja, Sivaramasamy; Balasubramanian, Thangavel

2013-04-15

An extracellular thermostable, haloalkaline cellulase by bioconversion of lignocellulosic wastes from Bacillus halodurans CAS 1 was purified to homogeneity with recovery of 12.54% and purity fold 7.96 with the molecular weight of 44 kDa. The optimum temperature, pH and NaCl for enzyme activity was determined as 60°C, 9.0 and 30% and it retained 80% of activity even at 80°C, 12 and 35% respectively. The activity was greatly inhibited by EDTA, indicating that it was a metalloenzyme and significant inhibition by PMSF revealed that serine residue was essential for catalytic activity. The purified cellulase hydrolyzed CMC, cellobiose and xylan, but not avicel, cellulose and PNPG. Furthermore, the cellulase was highly stable in the presence of detergents and organic solvents such as acetone, n-hexane and acetonitrile. Thus, the purified cellulase from B. halodurans utilizing lignocellulosic biomass could be greatly useful to develop industrial processes. Published by Elsevier Ltd.

An ethanolamine kinase Eki1 affects radial growth and cell wall integrity in Trichoderma reesei.

Science.gov (United States)

He, Ronglin; Guo, Wei; Zhang, Dongyuan

2015-09-01

Ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1.82) catalyzes the committed step of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. The functions of eki genes that encode ethanolamine kinase have been intensively studied in mammalian cells, fruit flies and yeast. However, the role of the eki gene has not yet been characterized in filamentous fungi. In this study, Treki1, an ortholog of Saccharomyces cerevisiae EKI1, was identified and functionally characterized using a target gene deletion strategy in Trichoderma reesei. A Treki deletion mutant was less sensitive to cell wall stressors calcofluor white and Congo red and released fewer protoplasts during cell wall digestion than the parent strain QM9414. Further transcription analysis showed that the expression levels of five genes that encode chitin synthases were drastically increased in the ΔTreki1 mutant. The chitin content was also increased in the null mutant of Treki1 comparing to the parent strain. In addition, the ΔTreki1 mutant exhibited defects in radial growth, conidiation and the accumulation of ethanolamine. The results indicate that Treki1 plays a key role in growth and development and in the maintenance of cell wall integrity in T. reesei. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Expression of naturally ionic liquid-tolerant thermophilic cellulases in Aspergillus niger.

Science.gov (United States)

Amaike Campen, Saori; Lynn, Jed; Sibert, Stephanie J; Srikrishnan, Sneha; Phatale, Pallavi; Feldman, Taya; Guenther, Joel M; Hiras, Jennifer; Tran, Yvette Thuy An; Singer, Steven W; Adams, Paul D; Sale, Kenneth L; Simmons, Blake A; Baker, Scott E; Magnuson, Jon K; Gladden, John M

2017-01-01

Efficient deconstruction of plant biomass is a major barrier to the development of viable lignocellulosic biofuels. Pretreatment with ionic liquids reduces lignocellulose recalcitrance to enzymatic hydrolysis, increasing yields of sugars for conversion into biofuels. However, commercial cellulases are not compatible with many ionic liquids, necessitating extensive water washing of pretreated biomass prior to hydrolysis. To circumvent this issue, previous research has demonstrated that several thermophilic bacterial cellulases can efficiently deconstruct lignocellulose in the presence of the ionic liquid, 1-ethyl-3-methylimadizolium acetate. As promising as these enzymes are, they would need to be produced at high titer in an industrial enzyme production host before they could be considered a viable alternative to current commercial cellulases. Aspergillus niger has been used to produce high titers of secreted enzymes in industry and therefore, we assessed the potential of this organism to be used as an expression host for these ionic liquid-tolerant cellulases. We demonstrated that 29 of these cellulases were expressed at detectable levels in a wild-type strain of A. niger, indicating a basic level of compatibility and potential to be produced at high levels in a host engineered to produce high titers of enzymes. We then profiled one of these enzymes in detail, the β-glucosidase A5IL97, and compared versions expressed in both A. niger and Escherichia coli. This comparison revealed the enzymatic activity of A5IL97 purified from E. coli and A. niger is equivalent, suggesting that A. niger could be an excellent enzyme production host for enzymes originally characterized in E. coli, facilitating the transition from the laboratory to industry.

Expression of naturally ionic liquid-tolerant thermophilic cellulases in Aspergillus niger

Energy Technology Data Exchange (ETDEWEB)

Amaike Campen, Saori; Lynn, Jed; Sibert, Stephanie J.; Srikrishnan, Sneha; Phatale, Pallavi; Feldman, Taya; Guenther, Joel M.; Hiras, Jennifer; Tran, Yvette Thuy An; Singer, Steven W.; Adams, Paul D.; Sale, Kenneth L.; Simmons, Blake A.; Baker, Scott E.; Magnuson, Jon K.; Gladden, John M.; Croft, Anna Kristina

2017-12-27

Efficient deconstruction of plant biomass is a major barrier to the development of viable lignocellulosic biofuels. Pretreatment with ionic liquids reduces lignocellulose recalcitrance to enzymatic hydrolysis, increasing yields of sugars for conversion into biofuels. However, commercial cellulases are not compatible with many ionic liquids, necessitating extensive water washing of pretreated biomass prior to hydrolysis. To circumvent this issue, previous research has demonstrated that several thermophilic bacterial cellulases can efficiently deconstruct lignocellulose in the presence of the ionic liquid, 1-ethyl-3-methylimadizolium acetate. As promising as these enzymes are, they would need to be produced at high titer in an industrial enzyme production host before they could be considered a viable alternative to current commercial cellulases. Aspergillus niger has been used to produce high titers of secreted enzymes in industry and therefore, we assessed the potential of this organism to be used as an expression host for these ionic liquid-tolerant cellulases. We demonstrated that 29 of these cellulases were expressed at detectable levels in a wild-type strain of A. niger, indicating a basic level of compatibility and potential to be produced at high levels in a host engineered to produce high titers of enzymes. We then profiled one of these enzymes in detail, the β-glucosidase A5IL97, and compared versions expressed in both A. niger and Escherichia coli. This comparison revealed the enzymatic activity of A5IL97 purified from E. coli and A. niger is equivalent, suggesting that A. niger could be an excellent enzyme production host for enzymes originally characterized in E. coli, facilitating the transition from the laboratory to industry.

Expression of naturally ionic liquid-tolerant thermophilic cellulases in Aspergillus niger

Science.gov (United States)

Lynn, Jed; Sibert, Stephanie J.; Srikrishnan, Sneha; Phatale, Pallavi; Feldman, Taya; Guenther, Joel M.; Hiras, Jennifer; Tran, Yvette Thuy An; Singer, Steven W.; Adams, Paul D.; Sale, Kenneth L.; Simmons, Blake A.; Baker, Scott E.; Magnuson, Jon K.; Gladden, John M.

2017-01-01

Efficient deconstruction of plant biomass is a major barrier to the development of viable lignocellulosic biofuels. Pretreatment with ionic liquids reduces lignocellulose recalcitrance to enzymatic hydrolysis, increasing yields of sugars for conversion into biofuels. However, commercial cellulases are not compatible with many ionic liquids, necessitating extensive water washing of pretreated biomass prior to hydrolysis. To circumvent this issue, previous research has demonstrated that several thermophilic bacterial cellulases can efficiently deconstruct lignocellulose in the presence of the ionic liquid, 1-ethyl-3-methylimadizolium acetate. As promising as these enzymes are, they would need to be produced at high titer in an industrial enzyme production host before they could be considered a viable alternative to current commercial cellulases. Aspergillus niger has been used to produce high titers of secreted enzymes in industry and therefore, we assessed the potential of this organism to be used as an expression host for these ionic liquid-tolerant cellulases. We demonstrated that 29 of these cellulases were expressed at detectable levels in a wild-type strain of A. niger, indicating a basic level of compatibility and potential to be produced at high levels in a host engineered to produce high titers of enzymes. We then profiled one of these enzymes in detail, the β-glucosidase A5IL97, and compared versions expressed in both A. niger and Escherichia coli. This comparison revealed the enzymatic activity of A5IL97 purified from E. coli and A. niger is equivalent, suggesting that A. niger could be an excellent enzyme production host for enzymes originally characterized in E. coli, facilitating the transition from the laboratory to industry. PMID:29281693

Expression of naturally ionic liquid-tolerant thermophilic cellulases in Aspergillus niger.

Directory of Open Access Journals (Sweden)

Saori Amaike Campen

Full Text Available Efficient deconstruction of plant biomass is a major barrier to the development of viable lignocellulosic biofuels. Pretreatment with ionic liquids reduces lignocellulose recalcitrance to enzymatic hydrolysis, increasing yields of sugars for conversion into biofuels. However, commercial cellulases are not compatible with many ionic liquids, necessitating extensive water washing of pretreated biomass prior to hydrolysis. To circumvent this issue, previous research has demonstrated that several thermophilic bacterial cellulases can efficiently deconstruct lignocellulose in the presence of the ionic liquid, 1-ethyl-3-methylimadizolium acetate. As promising as these enzymes are, they would need to be produced at high titer in an industrial enzyme production host before they could be considered a viable alternative to current commercial cellulases. Aspergillus niger has been used to produce high titers of secreted enzymes in industry and therefore, we assessed the potential of this organism to be used as an expression host for these ionic liquid-tolerant cellulases. We demonstrated that 29 of these cellulases were expressed at detectable levels in a wild-type strain of A. niger, indicating a basic level of compatibility and potential to be produced at high levels in a host engineered to produce high titers of enzymes. We then profiled one of these enzymes in detail, the β-glucosidase A5IL97, and compared versions expressed in both A. niger and Escherichia coli. This comparison revealed the enzymatic activity of A5IL97 purified from E. coli and A. niger is equivalent, suggesting that A. niger could be an excellent enzyme production host for enzymes originally characterized in E. coli, facilitating the transition from the laboratory to industry.

Enhancement of Cellulase Production by Cellulomonas Fimi and Bacillus Subtilis

International Nuclear Information System (INIS)

Omer, A.M.

2012-01-01

Two bacterial strains identified as Cellulomonas fimi and Baciliius subtilus are cosidered as highly active cellulytic bacteria. Trials for maximizing the cellulolytic activites of the two strains were conducted. A maximum cellulase production was achieved at 1 and 1.5%carboxy methyl cellulose as carbon source, sodium nitrate and yeast as nitrogen source for Cellulomonas fimi and Bacillus subtilis, respectively. Incubation temprature at 30 and 45 degree C, ph at 6 and 7 achieved the highest activity of cellulase for Cellulomonas fimi and bacillus subtilis, respectively

Characterization and pulp refining activity of a Paenibacillus campinasensis cellulase expressed in Escherichia coli.

Science.gov (United States)

Ko, Chun-Han; Tsai, Chung-Hung; Lin, Po-Heng; Chang, Ko-Cheng; Tu, Jenn; Wang, Ya-Nang; Yang, Chien-Ying

2010-10-01

The Cel-BL11 gene from Paenibacillus campinasensis BL11 was cloned and expressed in Escherichia coli as a His-tag fusion protein. Zymographic analysis of the recombinant protein revealed cellulase activity corresponding to a protein with a 38-kDa molecular weight. The optimum temperature and pH for purified cellulase were 60 °C and pH 7.0, respectively. The enzyme retained more than 80% activity after 8h at 60 °C at pH 6 and 7. The cellulase has a Km of 11.25 mg/ml and a Vmax of 1250 μmol/min/mg with carboxylmethyl cellulose (CMC). Then enzyme was active on Avicel, swollen Avicel, CMC, barley β-glucan, laminarin in the presence of 100 mM acetate buffer. It was inhibited by Hg²⁺, Cu²⁺ and Zn²⁺. Significant kraft pulp refining energy saving, 10%, was exhibited by the pretreatment of this cellulase applied at 2 IU per gram of oven-dried pulp. Broad pH and temperature stability render this cellulase a convenient applicability toward current mainstream biomass conversion and other industrial processes. Copyright © 2010 Elsevier Ltd. All rights reserved.

Antimicrobial and Antioxidant Activity of Chitosan/Hydroxypropyl Methylcellulose Film-Forming Hydrosols Hydrolyzed by Cellulase

Directory of Open Access Journals (Sweden)

Anna Zimoch-Korzycka

2016-09-01

Full Text Available The aim of this study was to evaluate the impact of cellulase (C on the biological activity of chitosan/hydroxypropyl methylcellulose (CH/HPMC film-forming hydrosols. The hydrolytic activity of cellulase in two concentrations (0.05% and 0.1% was verified by determination of the progress of polysaccharide hydrolysis, based on viscosity measurement and reducing sugar-ends assay. The 2,2-diphenyl-1-picrylhydrazyl (DPPH free radical scavenging effect, the ferric reducing antioxidant power (FRAP, and microbial reduction of Pseudomonas fluorescens, Yersinia enterocolitica, Bacillus cereus, and Staphylococcus aureus were studied. During the first 3 h of reaction, relative reducing sugar concentration increased progressively, and viscosity decreased rapidly. With increasing amount of enzyme from 0.05% to 0.1%, the reducing sugar concentration increased, and the viscosity decreased significantly. The scavenging effect of film-forming solutions was improved from 7.6% at time 0 and without enzyme to 52.1% for 0.1% cellulase after 20 h of reaction. A significant effect of cellulase addition and reaction time on antioxidant power of the tested film-forming solutions was also reported. Film-forming hydrosols with cellulase exhibited a bacteriostatic effect on all tested bacteria, causing a total reduction.

Analysis of cellulase and polyphenol oxidase production by southern pine beetle associated fungi

Science.gov (United States)

Abduvali Valiev; Zumrut B. Ogel; Dier D. Klepzig

2009-01-01

In this study, the production of extracellular enzymes by fungi associated with southern pine beetle was investigated for the first time. Cellulase and polyphenol oxidase production were analyzed for three beetle associated fungi. Only the mutualistic symbiont Entomocorticium sp. A was found to produce cellulases and polyphenol oxidase....

Cellulase-assisted extraction and antioxidant activity of the polysaccharides from garlic.

Science.gov (United States)

Pan, Saikun; Wu, Shengjun

2014-10-13

In the present study, the polysaccharides were prepared from garlic by using a cellulase-assisted extraction method and the antioxidant activity of garlic polysaccharides (GPs) was evaluated. To improve the yield of GPs, the influences of the several factors such as extraction time, temperature, pH, and cellulase amount on the extraction efficiency were studied. The optimal conditions for extraction of GPs were determined as follows: time, 80 min; temperature, 45 °C; pH, 5; cellulase amount, 8000 U/g. Under the optimised extraction conditions, the yield of GPS reached up to 35.34%. The GPs product exhibited strong antioxidant activity including hydroxyl radical scavenging activity, 2,2-diphenyl-β-picrylhydrazyl radical scavenging activity, and reducing power. The results suggest that the GPs could be used as potential antioxidants. Copyright © 2014 Elsevier Ltd. All rights reserved.

Silage fermentation and ruminal degradation of stylo prepared with lactic acid bacteria and cellulase.

Science.gov (United States)

Li, Mao; Zhou, Hanlin; Zi, Xuejuan; Cai, Yimin

2017-10-01

In order to improve the silage fermentation of stylo (Stylosanthes guianensis) in tropical areas, stylo silages were prepared with commercial additives Lactobacillus plantarum Chikuso-1 (CH1), L. rhamnasus Snow Lact L (SN), Acremonium cellulase (CE) and their combination as SN+CE or CH1 + CE, and the fermentation quality, chemical composition and ruminal degradation of these silages were studied. Stylo silages treated with lactic acid bacteria (LAB) or cellulase, the pH value and NH 3 -N ⁄ total-N were significantly (P fermentation and ruminal degradation than SN+CE treatment. The results confirmed that LAB or LAB plus cellulase treatment could improve the fermentation quality, chemical composition and ruminal degradation of stylo silage. Moreover, the combined treatment with LAB and cellulase may have beneficial synergistic effects on ruminal degradation. © 2017 Japanese Society of Animal Science.

The preparation and application of crude cellulase for cellulose-hydrogen production by anaerobic fermentation

Energy Technology Data Exchange (ETDEWEB)

Guo, Yi-Ping; Fan, Yao-Ting; Pan, Chun-Mei; Hou, Hong-Wei [Department of Chemistry, Zhengzhou University, Zhengzhou, Henan 450052 (China); Fan, Shao-Qun [Department of Chemistry, Zhengzhou University, Zhengzhou, Henan 450052 (China); Beijing Alcatel-Lucent R and D Center, Beijing, 100102 (China)

2010-01-15

Strategies were adopted to cost-efficiently produce cellulose-hydrogen by anaerobic fermentation in this paper. First, cellulase used for hydrolyzing cellulose was prepared by solid-state fermentation (SSF) on cheap biomass from Trichoderma viride. Several cultural conditions for cellulase production on cheap biomass such as moisture content, inoculum size and culture time were studied. And the components of solid-state medium were optimized using statistical methods to further improve cellulase capability. Second, the crude cellulase was applied to cellulose-hydrogen process directly. The maximal hydrogen yield of 122 ml/g-TVS was obtained at the substrate concentration of 20 g/L and cultured time of 53 h. The value was about 45-fold than that of raw corn stalk wastes. The hydrogen content in the biogas was 44-57%(v/v) and there was no significant methane gas observed. (author)

Cloning of cellulase genes using pUC18 and lambda 2001 vectors

International Nuclear Information System (INIS)

Bashir, A.; Ashfaq, S.R.; Rajoka, M.I.; Malik, K.A.; Batt, C.A.

1991-01-01

Chromosomal DNA from cellulomonas biazotea NIAB 442 was used for isolation and cloning of cellulase genes. For this purpose plasmid pUC18 was used for cloning fragments in the range of 109 Kb and phase vector lambda 2001 for cloning fragments in the range of 15-20 Kb respectively. Three restriction enzymes BamHI, Sau3AI and SaII were used for partial restriction of chromosomal DNA to obtain fragment size in the range of 0.5 - 20 Kb. BamHI and SaII were used to linearize pUC18 to obtain compatible ends against the three enzymes used in chromosomal DNA restriction. Linearized pUC18 was then ligated to respective compatible chromosomal DNA fragments and transformed to JM109 competent cells. A total of 6781 recombinants were tested for the production of B-glucosidase and carboxy methyl cellulase (CMC-ase) production. Only one of the recombinants was found to be positive for B-glucosidase production in solid culture. One of the recombinants was found positive for CMC-ase production in solid culture and is being verified and characterized. Larger DNA fragments in the range of 15-20 Kilobase were obtained by partial restriction of chromosomal DNA with BamHI, SaII and Xhol. Lambda 2001 was double digested with BamHI/EcoRI and Xhol/EcoRI for removal of stuffer fragment. Ligation of respective compatible ends was performed between Lambda DNA and chromosomal DNA. Ligation mixture was used for packaging and infection of P2 lysogen. No plaques could be obtained on P2 lysogen due to inefficient packaging. (author)

Production of immobilized cellulase enzyme by some microorganisms from the rice straw agro-waste using γ-irradiation

International Nuclear Information System (INIS)

Mohamed, M.A.Z.

2014-01-01

Studies were carried out using 14 fungal cultures screened for their ability to produce cellulase enzymes. A .hortai was selected for the present research as a potent cellulase producer. Cultural and nutritional factors affecting cellulase production were also investigated in order to optimize the fermentation conditions for the maximization of production. The obtained results revealed that, the maximum cellulase production (0.23 U/ml) was achieved after 96 h in a liquid medium (Ph 7.0) inoculated with 10% v/v inoculum size, at temperature 37 ºC, containing (gL -1 ) CMC, 5.0; yeast extract, 0.1; (NH 4 ) 2 SO 4 , 0.5; KH 2 PO 4 , 10.0; MgSO 4 .7H 2 O, 0.1 and NaCl, 0.2. The activity remained almost stable between ph 6.0 and 7.0. The highest cellulase activity (1.18 U/ml) was obtained at a lactose concentration of (5.0 gL -1 ). Partial purification of the crude cellulase by ammonium sulphate 70% saturation showed the highest specific enzyme activity and purification fold (2.3 U/mg protein and 2.12 fold, respectively). Different carriers and methods were used to select the suitable one for cellulase immobilization. Poly (acrylamide-co-acrylic acid) prepared by diazotization method increase S.E.A and the amount of immobilized enzyme to be (2.3 U/mg protein and 2.8 mg), respectively. The immobilized cellulase shows better operational stability, including wider ph and thermal ranges. The immobilized cellulase remained fully active up to 60°C. The kinetic parameters Km and Vmax were determined. The increase of the apparent Km after immobilization clearly indicates an apparent lower affinity of the immobilized enzyme for its substrate than the free enzyme. The resulting immobilized cellulase exhibited good reusability on degradation of rice straw agricultural wastes and also show good storage stability, that it lost only 17 % of its initial activity after 6 weeks.

Cellulase enzyme: Homology modeling, binding site identification and molecular docking

Science.gov (United States)

Selvam, K.; Senbagam, D.; Selvankumar, T.; Sudhakar, C.; Kamala-Kannan, S.; Senthilkumar, B.; Govarthanan, M.

2017-12-01

Cellulase is an enzyme that degrades the linear polysaccharide like cellulose into glucose by breaking the β-1,4- glycosidic bonds. These enzymes are the third largest enzymes with a great potential towards the ethanol production and play a vital role in degrading the biomass. The production of ethanol depends upon the ability of the cellulose to utilize the wide range of substrates. In this study, the 3D structure of cellulase from Acinetobacter sp. was modeled by using Modeler 9v9 and validated by Ramachandran plot. The accuracy of the predicted 3D structure was checked using Ramachandran plot analysis showed that 81.1% in the favored region, compatibility of an atomic model (3D) with amino acid sequence (1D) for the model was observed as 78.21% and 49.395% for Verify 3D and ERRAT at SAVES server. As the binding efficacy with the substrate might suggests the choice of the substrate as carbon and nitrogen sources, the cellobiose, cellotetraose, cellotetriose and laminaribiose were employed in the docking studies. The docking of cellobiose, cellotetraose, cellotetriose and laminaribiose with cellulase exhibited the binding energy of -6.1523 kJ/mol, -7.8759 kJ/mol,-6.1590 kJ/mol and -6.7185 kJ/mol, respectively. These docking studies revealed that cellulase has the greater potential towards the cellotetraose as a substrate for the high yield of ethanol.

Effects of non-ionic surfactants on the interactions between cellulases and tannic acid

DEFF Research Database (Denmark)

Olsen, Søren Nymand; Bohlin, Christina Helena; Murphy, Leigh

2011-01-01

of cellulases to lignin. In the current work we address this hypothesis using tannic acid (TAN) as a general poly-phenolic model compound (for lignin and soluble phenolics) and measure the mutual interactions of cellulases (CBHI, CBHII, EGI, EGII and BG), TAN and NIS (Triton X-100) using isothermal titration...

Enhancing Cellulase Commercial Performance for the Lignocellulosic Biomass Industry

Energy Technology Data Exchange (ETDEWEB)

Jarnigan, Alisha [Danisco, US Inc., Copenhagen (Denmark)

2016-06-07

Cellulase enzyme loading (Bt-G) for the economic conversion of lignocellulosic biomass to ethanol is on of the key challenges identified in the Biomass Program of DOE/EERE. The goal of Danisco’s project which ran from 2008 to 2012, was to address the technical challenge by creating more efficient enzyme that could be used at lower doses, thus reducing the enzymes’ cost contribution to the conversio process. We took the approach of protein engineering of cellulase enzymes to overcome the enzymati limitations in the system of cellulosic-hydrolyzing enzymes to improve performance in conversion o biomass, thereby creating a more effective enzyme mix.

Optimization of a natural medium for cellulase by a marine Aspergillus niger using response surface methodology.

Science.gov (United States)

Xue, Dong-Sheng; Chen, Hui-Yin; Lin, Dong-Qiang; Guan, Yi-Xin; Yao, Shan-Jing

2012-08-01

The components of a natural medium were optimized to produce cellulase from a marine Aspergillus niger under solid state fermentation conditions by response surface methodology. Eichhornia crassipes and natural seawater were used as a major substrate and a source of mineral salts, respectively. Mineral salts of natural seawater could increase cellulase production. Raw corn cob and raw rice straw showed a significant positive effect on cellulase production. The optimum natural medium consisted of 76.9 % E. crassipes (w/w), 8.9 % raw corn cob (w/w), 3.5 % raw rice straw (w/w), 10.7 % raw wheat bran (w/w), and natural seawater (2.33 times the weight of the dry substrates). Incubation for 96 h in the natural medium increased the biomass to the maximum. The cellulase production was 17.80 U/g the dry weight of substrates after incubation for 144 h. The natural medium avoided supplying chemicals and pretreating substrates. It is promising for future practical fermentation of environment-friendly producing cellulase.

Cellulase Inhibition by High Concentrations of Monosaccharides

DEFF Research Database (Denmark)

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

2014-01-01

Biological degradation of biomass on an industrial scale culminates in high concentrations of end products. It is known that the accumulation of glucose and cellobiose, end products of hydrolysis, inhibit cellulases and decrease glucose yields. Aside from these end products, however, other monosa...

The Effect of Cellulose Crystal Structure and Solid-State Morphology on the Activity of Cellulases

Energy Technology Data Exchange (ETDEWEB)

Stipanovic, Arthur J [SUNY College of Environmental Science and Forestry

2014-11-17

Consistent with the US-DOE and USDA “Roadmap” objective of producing ethanol and chemicals from cellulosic feedstocks more efficiently, a three year research project entitled “The Effect of Cellulose Crystal Structure and Solid-State Morphology on the Activity of Cellulases” was initiated in early 2003 under DOE sponsorship (Project Number DE-FG02-02ER15356). A three year continuation was awarded in June 2005 for the period September 15, 2005 through September 14, 2008. The original goal of this project was to determine the effect of cellulose crystal structure, including allomorphic crystalline form (Cellulose I, II, III, IV and sub-allomorphs), relative degree of crystallinity and crystallite size, on the activity of different types of genetically engineered cellulase enzymes to provide insight into the mechanism and kinetics of cellulose digestion by “pure” enzymes rather than complex mixtures. We expected that such information would ultimately help enhance the accessibility of cellulose to enzymatic conversion processes thereby creating a more cost-effective commercial process yielding sugars for fermentation into ethanol and other chemical products. Perhaps the most significant finding of the initial project phase was that conversion of native bacterial cellulose (Cellulose I; BC-I) to the Cellulose II (BC-II) crystal form by aqueous NaOH “pretreatment” provided an increase in cellulase conversion rate approaching 2-4 fold depending on enzyme concentration and temperature, even when initial % crystallinity values were similar for both allomorphs.

Oxidative cleavage and hydrolytic boosting of cellulose in soybean spent flakes by Trichoderma reesei Cel61A lytic polysaccharide monooxygenase

DEFF Research Database (Denmark)

Pierce, Brian; Wittrup Agger, Jane; Wichmann, Jesper

2017-01-01

The auxiliary activity family 9 (AA9) copper-dependent lytic polysaccharide monooxygenase (LPMO) from Trichoderma reesei (EG4; TrCel61A) was investigated for its ability to oxidize the complex polysaccharides from soybean. The substrate specificity of the enzyme was assessed against a variety of ...

Neurospora crassa tox-1 Gene Encodes a pH- and Temperature-Tolerant Mini-Cellulase.

Science.gov (United States)

Xiao, Yue; Zhang, Qiongsi; Luo, Yiquan; Zhang, Ying; Luo, Xi; Wang, Yuchuan; Cao, Weiguo; Pinto, Vito De; Liu, Qiuyun; Li, Gang

2016-06-15

Cellulases that endure extreme conditions are essential in various industrial sectors. This study reports a mini-cellulase gene tox-1 from Neurospora crassa. The gene tox-1 was cloned in Escherichia coli after chimerization with the YebF gene and substitutions of certain isoleucine and valine with leucine residues. The yeast transformants could grow on rice straw-agar medium. The 44-amino acid peptide and its two mutant variants displayed potent cellulase activities in Congo Red assay and enzymatic assays. Conservative replacements with leucine have substantially increased the stabilities and half-lives of the peptides at alkaline pH and low and high temperatures and also the tolerance to organic solvents and surfactants, on the basis of activities toward cellose. The small size of the mini-cellulase would allow for commercially viable automatic chemical peptide synthesis. This work suggests that conservative leucine replacements may serve as a general strategy in the engineering of more robust enzymes with special features with little loss of activities.

Xylanase, CM-cellulase and avicelase production by the thermophilic fungus Sporotrichum thermophile

Energy Technology Data Exchange (ETDEWEB)

Margaritis, A; Merchant, R; Yaguchi, M

1983-01-01

When wheat straw was used as C source, S. thermophile produced large amounts of xylanase extracellularly in addition to CM-cellulase and Avicelase. These enzymes were isolated by alcohol precipitation, desalting, and column chromatography. The molecular weights were estimated to be 25,0065,000 and 84,000 for xylanase, CM-cellulase, and Avicelase, respectively. Serine and threonine were the most abundant amino acids and these enzymes are very acidic proteins.

The role of subsite +2 of the Trichoderma reesei beta-mannanase TrMan5A in hydrolysis and transglycosylation

DEFF Research Database (Denmark)

Rosengren, Anna; Hägglund, Per; Anderson, Lars Steen

2012-01-01

The N-terminal catalytic module of beta-mannanase TrMan5A from the filamentous fungus Trichoderma reesei is classified into family 5 of glycoside hydrolases. It is further classified in clan A with a (beta/alpha)(8) barrel configuration and has two catalytic glutamates (E169 and E276). It has at ...

Strategies to increase cellulase production with submerged fermentation using fungi isolated from the Brazilian biome

Directory of Open Access Journals (Sweden)

Genilton da Silva Faheina Junior

2015-03-01

Full Text Available Studies on new microbial sources of cellulase and accurate assessment of the steps that increase cellulase production are essential strategies to reduce costs of various processes using such enzymes. This study aimed at the selection of cellulase-producing filamentous fungi, and at the research of parameters involving cellulase production by submerged fermentation. The first test consisted of selecting the best cellulase-producing microorganisms (FPase in Erlenmeyer flasks containing 200 mL of specific growth medium. The next test was designed to further investigate the enzyme production in fermentation with four types of soluble sugars: glucose, lactose, sucrose and xylose. In bioreactor tests, three different inoculation strategies were analyzed. The best FPase activity was presented by the strain Trichoderma sp. CMIAT 041 (49.9 FPU L-1 and CMCase by the fungus Lasiodiplodia theobromae CMIAT 096 (350.0 U L-1. Sucrose proved to be the best option among the soluble sugars tested, with higher rates of FPase activity (49.9 FPU L-1 and CMCase (119.7 U L-1. The best inoculation strategy for the bioreactor was a spore suspension obtained from a semi-solid state fermentation of wheat bran for 72h.

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

Science.gov (United States)

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

Biohydrolysis of Saccharum spontaneum for cellulase production by ...

African Journals Online (AJOL)

admin

2012-03-13

Mar 13, 2012 ... fermentation (SSF) for economic production of cellulase. ... Lignocellulosic biomass has been regarded as a ... concentration in the pretreated substrate. It can be fairly ... wheat straw, 54% and sorghum straw, 61%) for ethanol.

Cellulase linkers are optimized based on domain type and function: insights from sequence analysis, biophysical measurements, and molecular simulation.

Directory of Open Access Journals (Sweden)

Deanne W Sammond

Full Text Available Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs to carbohydrate-binding modules (CBMs. Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function. Sequence analysis suggests that the linker lengths between structured domains are optimized based on the GH domain and CBM type, such that linker length may be important for activity. Longer linkers are observed in eukaryotic GH Family 6 cellulases compared to GH Family 7 cellulases. Bacterial GH Family 6 cellulases are found with structured domains in either N to C terminal order, and similar linker lengths suggest there is no effect of domain order on length. O-glycosylation is uniformly distributed across linkers, suggesting that glycans are required along entire linker lengths for proteolysis protection and, as suggested by simulation, for extension. Sequence comparisons show that proline content for bacterial linkers is more than double that observed in eukaryotic linkers, but with fewer putative O-glycan sites, suggesting alternative methods for extension. Conversely, near linker termini where linkers connect to structured domains, O-glycosylation sites are observed less frequently, whereas glycines are more prevalent, suggesting the need for flexibility to achieve proper domain orientations. Putative N-glycosylation sites are quite rare in cellulase linkers, while an N-P motif, which strongly disfavors the attachment of N-glycans, is commonly observed. These results suggest that linkers exhibit features that are likely tailored for optimal function, despite possessing low sequence identity. This study suggests

Laser mutagenesis and producing cellulase condition optimization of Trichoderma virid protoplast

International Nuclear Information System (INIS)

Chen Shuli; Zhang Qin; Han Jingjing; Lv Jiangtao; Wang Shilong; Yao Side

2009-01-01

The protoplast of Trichoderma virid CICC13038 was mutated using Nd:YAG laser of 266 nm light. And a high-cellulase producing strain JG13 was bred by screening with cellulose microcrystalline. Under the condition of 28 degree C, 180 rpm and 72 h of fermentation time, optimal conditions for the celluase ferment by orthogonal experiment were: 2% bran as the carbon source, 1% (NH 4 ) 2 SO 4 as the nitrogen source, 0.5% Tween-80 as a enzyme-promoting agent,and 25 mL of medium volume in a 250 mL bottle. The cellulase activity of the mutant reached 35.68 U/mL, 25.76% higher than that of the original strain under the same conditions. The mutant JG13 has a great potential in industrial production. And it also can be used as the original strain for further mutagenesis to get the strain of higher cellulase activity. (authors)

Characterization of a GHF45 cellulase, AkEG21, from the common sea hare Aplysia kurodai

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Mohammad Matiur Rahman

2014-08-01

Full Text Available The common sea hare Aplysia kurodai is known to be a good source for the enzymes degrading seaweed polysaccharides. Recently four cellulases, i.e., 95 kDa, 66 kDa, 45 kDa and 21 kDa enzymes, were isolated from A. kurodai (Tsuji et al., PLoS ONE, 8, e65418, 2013. The former three cellulases were regarded as glycosyl-hydrolase-family 9 (GHF9 enzymes, while the 21 kDa cellulase was suggested to be a GHF45 enzyme. The 21 kDa cellulase was significantly heat stable, and appeared to be advantageous in performing heterogeneous expression and protein-engineering study. In the present study, we determined some enzymatic properties of the 21 kDa cellulase and cloned its cDNA to provide the basis for the protein engineering study of this cellulase. The purified 21 kDa enzyme, termed AkEG21 in the present study, hydrolyzed carboxymethyl cellulose with an optimal pH and temperature at 4.5 and 40oC, respectively. AkEG21 was considerably heat-stable, i.e., it was not inactivated by the incubation at 55oC for 30 min. AkEG21 degraded phosphoric-acid-swollen cellulose producing cellotriose and cellobiose as major end products but hardly degraded oligosaccharides smaller than tetrasaccharide. This indicated that AkEG21 is an endolytic -1,4-glucanase (EC 3.2.1.4. A cDNA of 1,013 bp encoding AkEG21 was amplified by PCR and the amino-acid sequence of 197 residues was deduced. The sequence comprised the initiation Met, the putative signal peptide of 16 residues for secretion and the catalytic domain of 180 residues, which lined from the N-terminus in this order. The sequence of the catalytic domain showed 47-62% amino-acid identities to those of GHF45 cellulases reported in other mollusks. Both the catalytic residues and the N-glycosylation residues known in other GHF45 cellulases were conserved in AkEG21. Phylogenetic analysis for the amino-acid sequences suggested the close relation between AkEG21 and fungal GHF45 cellulases.

Characterization of a GHF45 cellulase, AkEG21, from the common sea hare Aplysia kurodai

Science.gov (United States)

Rahman, Mohammad; Inoue, Akira; Ojima, Takao

2014-08-01

The common sea hare Aplysia kurodai is known to be a good source for the enzymes degrading seaweed polysaccharides. Recently four cellulases, i.e., 95 kDa, 66 kDa, 45 kDa and 21 kDa enzymes, were isolated from A. kurodai (Tsuji et al., PLoS ONE, 8, e65418, 2013). The former three cellulases were regarded as glycosyl-hydrolase-family 9 (GHF9) enzymes, while the 21 kDa cellulase was suggested to be a GHF45 enzyme. The 21 kDa cellulase was significantly heat stable, and appeared to be advantageous in performing heterogeneous expression and protein-engineering study. In the present study, we determined some enzymatic properties of the 21 kDa cellulase and cloned its cDNA to provide the basis for the protein engineering study of this cellulase. The purified 21 kDa enzyme, termed AkEG21 in the present study, hydrolyzed carboxymethyl cellulose with an optimal pH and temperature at 4.5 and 40oC, respectively. AkEG21 was considerably heat-stable, i.e., it was not inactivated by the incubation at 55oC for 30 min. AkEG21 degraded phosphoric-acid-swollen cellulose producing cellotriose and cellobiose as major end products but hardly degraded oligosaccharides smaller than tetrasaccharide. This indicated that AkEG21 is an endolytic ?-1,4-glucanase (EC 3.2.1.4). A cDNA of 1,013 bp encoding AkEG21 was amplified by PCR and the amino-acid sequence of 197 residues was deduced. The sequence comprised the initiation Met, the putative signal peptide of 16 residues for secretion and the catalytic domain of 180 residues, which lined from the N-terminus in this order. The sequence of the catalytic domain showed 47-62% amino-acid identities to those of GHF45 cellulases reported in other mollusks. Both the catalytic residues and the N-glycosylation residues known in other GHF45 cellulases were conserved in AkEG21. Phylogenetic analysis for the amino-acid sequences suggested the close relation between AkEG21 and fungal GHF45 cellulases.

Production and Partial Characterization of Cellulases from ...

African Journals Online (AJOL)

Prof. Ogunji

fermentation, the organism produced cellulase. Activity ... become of considerable economic interest to develop processes for effective treatment and ... industries such as textile, laundry, pulp and paper and fruit juice extraction. ..... choices for costly enzymes that lose appreciable part of their activity even when stored at low.

Identification of a haloalkaliphilic and thermostable cellulase with improved ionic liquid tolerance

Energy Technology Data Exchange (ETDEWEB)

Zhang, Tao; Datta, Supratim; Eichler, Jerry; Ivanova, Natalia; Axen, Seth D.; Kerfeld, Cheryl A.; Chen, Feng; Kyrpides, Nikos; Hugenholtz, Philip; Cheng, Jan-Fang; Sale, Kenneth L.; Simmons, Blake; Rubin, Eddy

2011-02-17

Some ionic liquids (ILs) have been shown to be very effective solvents for biomass pretreatment. It is known that some ILs can have a strong inhibitory effect on fungal cellulases, making the digestion of cellulose inefficient in the presence of ILs. The identification of IL-tolerant enzymes that could be produced as a cellulase cocktail would reduce the costs and water use requirements of the IL pretreatment process. Due to their adaptation to high salinity environments, halophilic enzymes are hypothesized to be good candidates for screening and identifying IL-resistant cellulases. Using a genome-based approach, we have identified and characterized a halophilic cellulase (Hu-CBH1) from the halophilic archaeon, Halorhabdus utahensis. Hu-CBH1 is present in a gene cluster containing multiple putative cellulolytic enzymes. Sequence and theoretical structure analysis indicate that Hu-CBH1 is highly enriched with negatively charged acidic amino acids on the surface, which may form a solvation shell that may stabilize the enzyme, through interaction with salt ions and/or water molecules. Hu-CBH1 is a heat tolerant haloalkaliphilic cellulase and is active in salt concentrations up to 5 M NaCl. In high salt buffer, Hu-CBH1 can tolerate alkali (pH 11.5) conditions and, more importantly, is tolerant to high levels (20percent w/w) of ILs, including 1-allyl-3-methylimidazolium chloride ([Amim]Cl). Interestingly, the tolerances to heat, alkali and ILs are found to be salt-dependent, suggesting that the enzyme is stabilized by the presence of salt. Our results indicate that halophilic enzymes are good candidates for the screening of IL-tolerant cellulolytic enzymes.

Characterization of Cellulase Enzyme Inhibitors Formed During the Chemical Pretreatments of Rice Straw

Science.gov (United States)

Rajan, Kalavathy

Production of fuels and chemicals from a renewable and inexpensive resource such as lignocellulosic biomass is a lucrative and sustainable option for the advanced biofuel and bio-based chemical platform. Agricultural residues constitute the bulk of potential feedstock available for cellulosic fuel production. On a global scale, rice straw is the largest source of agricultural residues and is therefore an ideal crop model for biomass deconstruction studies. Lignocellulosic biofuel production involves the processes of biomass conditioning, enzymatic saccharification, microbial fermentation and ethanol distillation, and one of the major factors affecting its techno-economic feasibility is the biomass recalcitrance to enzymatic saccharification. Preconditioning of lignocellulosic biomass, using chemical, physico-chemical, mechanical and biological pretreatments, is often practiced such that biomass becomes available to downstream processing. Pretreatments, such as dilute acid and hot water, are effective means of biomass conversion. However, despite their processing importance, preconditioning biomass also results in the production of carbohydrate and lignin degradation products that are inhibitory to downstream saccharification enzymes. The saccharification enzyme cocktail is made up of endo-cellulase, exo-cellulase and beta-glucosidase enzymes, whose role is to cleave cellulose polymers into glucose monomers. Specifically, endo-cellulase and exo-cellulase enzymes cleave cellulose chains in the middle and at the end, resulting in cellobiose molecules, which are hydrolyzed into glucose by beta-glucosidase. Unfortunately, degradation compounds generated during pretreatment inhibit the saccharification enzyme cocktail. Various research groups have identified specific classes of inhibitors formed during biomass pretreatment and have studied their inhibitory effect on the saccharification cocktail. These various research groups prepared surrogate solutions in an attempt to

A new earthworm cellulase and its possible role in the innate immunity.

Science.gov (United States)

Park, In Yong; Cha, Ju Roung; Ok, Suk-Mi; Shin, Chuog; Kim, Jin-Se; Kwak, Hee-Jin; Yu, Yun-Sang; Kim, Yu-Kyung; Medina, Brenda; Cho, Sung-Jin; Park, Soon Cheol

2017-02-01

A new endogenous cellulase (Ean-EG) from the earthworm, Eisenia andrei and its expression pattern are demonstrated. Based on a deduced amino acid sequence, the open reading frame (ORF) of Ean-EG consisted of 1368 bps corresponding to a polypeptide of 456 amino acid residues in which is contained the conserved region specific to GHF9 that has the essential amino acid residues for enzyme activity. In multiple alignments and phylogenetic analysis, the deduced amino acid sequence of Ean- EG showed the highest sequence similarity (about 79%) to that of an annelid (Pheretima hilgendorfi) and could be clustered together with other GHF9 cellulases, indicating that Ean-EG could be categorized as a member of the GHF9 to which most animal cellulases belong. The histological expression pattern of Ean-EG mRNA using in situ hybridization revealed that the most distinct expression was observed in epithelial cells with positive hybridization signal in epidermis, chloragogen tissue cells, coelomic cell-aggregate, and even blood vessel, which could strongly support the fact that at least in the earthworm, Eisenia andrei, cellulase function must not be limited to digestive process but be possibly extended to the innate immunity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enhancement in ionic liquid tolerance of cellulase immobilized on PEGylated graphene oxide nanosheets: Application in saccharification of lignocellulose.

Science.gov (United States)

Xu, Jiaxing; Sheng, Zhenhuan; Wang, Xinfeng; Liu, Xiaoyan; Xia, Jun; Xiong, Peng; He, Bingfang

2016-01-01

The objective of the present work was to improve ionic liquid (IL) tolerance of cellulase based on the exploration of functional nanoscale carriers for potential application in lignocellulosic biorefinery. PEGylated graphene oxide (GO) composite was successfully fabricated by chemical binding of 4-arm-PEG-NH2 and GO and applied to the immobilization of cellulase. The PEGylated GO-Cellulase retained 61% of the initial activity in 25% (w/v) 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) while free cellulase only retained 2%. The IL stability was enhanced more than 30 times. The relatively minor change in Km value (from 2.7 to 3.2mgmL(-1)) after the immobilization suggested that PEGylated GO-Cellulase was capable of closely mimicking the performance of free enzyme. After treating rice straw with [Bmim][Cl] and dilution to a final IL concentration of 15% (w/v), the slurry was directly hydrolyzed using PEGylated GO-Cellulase without IL removing and a high hydrolysis rate of 87% was achieved. Copyright © 2015 Elsevier Ltd. All rights reserved.

Addressing the Recalcitrance of Cellulose Degradation through Cellulase Discovery, Nano-scale Elucidation of Molecular Mechanisms, and Kinetic Modeling

Energy Technology Data Exchange (ETDEWEB)

Walker, Larry P., Bergstrom, Gary; Corgie, Stephane; Craighead, Harold; Gibson, Donna; Wilson, David

2011-06-13

This research project was designed to play a vital role in the development of low cost sugars from cellulosic biomass and contributing to the national effort to displace fossil fuel usage in the USA transportation sector. The goal was to expand the portfolio of cell wall degrading enzymes through innovative research at the nano-scale level, prospecting for novel cellulases and building a kinetic framework for the development of more effective enzymatic conversion processes. More precisely, the goal was to elucidate the molecular mechanisms for some cellulases that are very familiar to members of our research team and to investigate what we hope are novel cellulases or new enzyme combinations from the world of plant pathogenic fungi and bacteria. Hydrolytic activities of various cellulases and cellulase cocktails were monitored at the nanoscale of cellulose fibrils and the microscale of pretreated cellulose particles, and we integrated this insight into a heterogeneous reaction framework. The over-riding approach for this research program was the application of innovative and cutting edge optical and high-throughput screening and analysis techniques for observing how cellulases hydrolyze real substrates.

Enzyme production in immobilized Trichoderma reesei cells with hydrophobic polymers prepared by radiation polymerization method

International Nuclear Information System (INIS)

Luzhao Xin; Kumakura, Minoru; Kaetsu, Isao

1993-01-01

Trichoderma reesei cells were immobilized on paper covered with hydrophobic monomer, trimethylpropane triacrylate by radiation polymerization. The effect of immobilization condition on enzyme productivity was studied by measuring filter paper and cellobiose activity. The cells were adhered and grew on the surface of the carrier with the polymer giving high enzyme productivity in the immobilized cells in comparison with the free cells. Optimum concentration and volume of the coating monomer for the preparation of the immobilized cells were obtained. (author)

Fractionation and cellulase treatment for enhancing the properties of kraft-based dissolving pulp.

Science.gov (United States)

Duan, Chao; Wang, Xinqi; Zhang, YanLing; Xu, Yongjian; Ni, Yonghao

2017-01-01

The aim of this study was to investigate a combined process involving pulp fractionation and cellulase treatment of each fraction for improving the molecular weight distribution (MWD) and reactivity of a kraft-based dissolving pulp. Three pulp fractions, namely long-fiber, mid-fiber and short-fiber fractions (LF, MF and SF, respectively), were used as the substrates. The results showed that the SF had the highest accessibility, lowest viscosity, and highest cellulase adsorption capacity, while the opposite was true for the LF. At a given viscosity, the combined process led to a lower polydispersity index (3.71 vs 4.98) and a higher Fock reactivity (85.6% vs 76.3%), in comparison to the conventional single-stage cellulase treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Catalysis of Rice Straw Hydrolysis by the Combination of Immobilized Cellulase from Aspergillus niger on β-Cyclodextrin-Fe3O4 Nanoparticles and Ionic Liquid

Science.gov (United States)

Huang, Po-Jung; Chang, Ken-Lin; Chen, Shui-Tein

2015-01-01

Cellulase from Aspergillus niger was immobilized onto β-cyclodextrin-conjugated magnetic particles by silanization and reductive amidation. The immobilized cellulase gained supermagnetism due to the magnetic nanoparticles. Ninety percent of cellulase was immobilized, but the activity of immobilized cellulase decreased by 10%. In this study, ionic liquid (1-butyl-3-methylimidazolium chloride) was introduced into the hydrolytic process because the original reaction was a solid-solid reaction. The activity of immobilized cellulase was improved from 54.87 to 59.11 U g immobilized cellulase−1 at an ionic liquid concentration of 200 mM. Using immobilized cellulase and ionic liquid in the hydrolysis of rice straw, the initial reaction rate was increased from 1.629 to 2.739 g h−1 L−1. One of the advantages of immobilized cellulase is high reusability—it was usable for a total of 16 times in this study. Compared with free cellulase, magnetized cellulase can be recycled by magnetic field and the activity of immobilized cellulase was shown to remain at 85% of free cellulase without denaturation under a high concentration of glucose (15 g L−1). Therefore, immobilized cellulase can hydrolyze rice straw continuously compared with free cellulase. The amount of harvested glucose can be up to twentyfold higher than that from the hydrolysis by free cellulase. PMID:25874210

Catalysis of Rice Straw Hydrolysis by the Combination of Immobilized Cellulase from Aspergillus niger on β-Cyclodextrin-Fe3O4 Nanoparticles and Ionic Liquid

Directory of Open Access Journals (Sweden)

Po-Jung Huang

2015-01-01

Full Text Available Cellulase from Aspergillus niger was immobilized onto β-cyclodextrin-conjugated magnetic particles by silanization and reductive amidation. The immobilized cellulase gained supermagnetism due to the magnetic nanoparticles. Ninety percent of cellulase was immobilized, but the activity of immobilized cellulase decreased by 10%. In this study, ionic liquid (1-butyl-3-methylimidazolium chloride was introduced into the hydrolytic process because the original reaction was a solid-solid reaction. The activity of immobilized cellulase was improved from 54.87 to 59.11 U g immobilized cellulase−1 at an ionic liquid concentration of 200 mM. Using immobilized cellulase and ionic liquid in the hydrolysis of rice straw, the initial reaction rate was increased from 1.629 to 2.739 g h−1 L−1. One of the advantages of immobilized cellulase is high reusability—it was usable for a total of 16 times in this study. Compared with free cellulase, magnetized cellulase can be recycled by magnetic field and the activity of immobilized cellulase was shown to remain at 85% of free cellulase without denaturation under a high concentration of glucose (15 g L−1. Therefore, immobilized cellulase can hydrolyze rice straw continuously compared with free cellulase. The amount of harvested glucose can be up to twentyfold higher than that from the hydrolysis by free cellulase.

Use of Sunflower Meal with Enzyme Mixture Substituted for Soybean Meal in Broiler Diets

OpenAIRE

KIRKPINAR, Figen; BASMACIOĞLU, Hatice

2001-01-01

The study was carried out to investigate the effects of sunflower meal with an enzyme mixture containing a-amylase, ß-glucanase, cellulase, lipase and protease substituted for soybean meal in broiler diets on performance, intestinal weight, viscosity and pH, as well as the frequency of sticky droppings and abdominal fat. In the trial, a total of 960 one-day-old Avian broiler chicks were used. The trial lasted 6 weeks. A total of 12 experimental diets were supplied ad libitum. Dietary treatme...

Cellulase Production Potentials of the Microbial Profile of Some Sugarcane Bagasse Dumping Sites in Ilorin, Nigeria

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Kamoldeen Abiodun AJIJOLAKEWU

2013-11-01

Full Text Available This research work investigated cellulase production potentials of the microbial profile of three sugarcane bagasse dumping sites at Zango area, Ilorin, Nigeria. The microbial isolates were screened for cellulase production with a view to select the best organism for eventual cellulase production. Pour Plate method was used for the isolation and a total of thirteen (13 different organisms including both fungal and bacterial species were isolated and screened. Six (6 fungal isolates identified as Mucor racemosus, Aspergillus niger, Aspergillus flavus, Neurospora sitophilus, Penicillium oxalicum and Penicillium citrinum were isolated, while seven (7 different bacterial species isolated include Clostridium cellobioparum, Clostridium thermocellum,Bacillus subtilis, Bacillus pumillus, Lactobacillus spp, Pseudomonas flavescens and Serratia spp. Generally, bacterial isolates were more in abundance than fungal species. However; fungal isolates were constant and were isolated through the experimental period of three weeks. All the isolates showed cellulase production potential in varying degrees as reflected in the clearance zone around their colonies. Fungal isolates produced more cellulase than the bacterial isolates. Mucor racemosus had the highest clearance zone (75.0 mm among the fungal isolates while Clostridium cellobioparum (35.0 mm were the best producer among bacterial isolates. The least producer among fungal isolates, Penicillium citrinum (40.0 mm, is a little more than the bacterial cellulase producer (35.0 mm and is far greater than the least bacterium Serratia spp (14.0 mm.

Bioprospecting of functional cellulases from metagenome for second generation biofuel production: a review.

Science.gov (United States)

Tiwari, Rameshwar; Nain, Lata; Labrou, Nikolaos E; Shukla, Pratyoosh

2018-03-01

Second generation biofuel production has been appeared as a sustainable and alternative energy option. The ultimate aim is the development of an industrially feasible and economic conversion process of lignocellulosic biomass into biofuel molecules. Since, cellulose is the most abundant biopolymer and also represented as the photosynthetically fixed form of carbon, the efficient hydrolysis of cellulose is the most important step towards the development of a sustainable biofuel production process. The enzymatic hydrolysis of cellulose by suites of hydrolytic enzymes underlines the importance of cellulase enzyme system in whole hydrolysis process. However, the selection of the suitable cellulolytic enzymes with enhanced activities remains a challenge for the biorefinery industry to obtain efficient enzymatic hydrolysis of biomass. The present review focuses on deciphering the novel and effective cellulases from different environmental niches by unculturable metagenomic approaches. Furthermore, a comprehensive functional aspect of cellulases is also presented and evaluated by assessing the structural and catalytic properties as well as sequence identities and expression patterns. This review summarizes the recent development in metagenomics based approaches for identifying and exploring novel cellulases which open new avenues for their successful application in biorefineries.

Comparison of Nitrogen Depletion and Repletion on Lipid Production in Yeast and Fungal Species

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Shihui Yang

2016-08-01

Full Text Available Although it is well known that low nitrogen stimulates lipid accumulation, especially for algae and some oleaginous yeast, few studies have been conducted in fungal species, especially on the impact of different nitrogen deficiency strategies. In this study, we use two promising consolidated bioprocessing (CBP candidates to examine the impact of two nitrogen deficiency strategies on lipid production, which are the extensively investigated oleaginous yeast Yarrowia lipolytica, and the commercial cellulase producer Trichoderma reesei. We first utilized bioinformatics approaches to reconstruct the fatty acid metabolic pathway and demonstrated the presence of a triacylglycerol (TAG biosynthesis pathway in Trichoderma reesei. We then examined the lipid production of Trichoderma reesei and Y. lipomyces in different media using two nitrogen deficiency strategies of nitrogen natural repletion and nitrogen depletion through centrifugation. Our results demonstrated that nitrogen depletion was better than nitrogen repletion with about 30% lipid increase for Trichoderma reesei and Y. lipomyces, and could be an option to improve lipid production in both oleaginous yeast and filamentous fungal species. The resulting distinctive lipid composition profiles indicated that the impacts of nitrogen depletion on yeast were different from those for fungal species. Under three types of C/N ratio conditions, C16 and C18 fatty acids were the predominant forms of lipids for both Trichoderma reesei and Y. lipolytica. While the overall fatty acid methyl ester (FAME profiles of Trichoderma reesei were similar, the overall FAME profiles of Y. lipolytica observed a shift. The fatty acid metabolic pathway reconstructed in this work supports previous reports of lipid production in T. reesei, and provides a pathway for future omics studies and metabolic engineering efforts. Further investigation to identify the genetic targets responsible for the effect of nitrogen depletion on

Differential Involvement of β-Glucosidases from Hypocrea jecorina in Rapid Induction of Cellulase Genes by Cellulose and Cellobiose

Science.gov (United States)

Zhou, Qingxin; Xu, Jintao; Kou, Yanbo; Lv, Xinxing; Zhang, Xi; Zhao, Guolei; Zhang, Weixin; Chen, Guanjun

2012-01-01

Appropriate perception of cellulose outside the cell by transforming it into an intracellular signal ensures the rapid production of cellulases by cellulolytic Hypocrea jecorina. The major extracellular β-glucosidase BglI (CEL3a) has been shown to contribute to the efficient induction of cellulase genes. Multiple β-glucosidases belonging to glycosyl hydrolase (GH) family 3 and 1, however, exist in H. jecorina. Here we demonstrated that CEL1b, like CEL1a, was an intracellular β-glucosidase displaying in vitro transglycosylation activity. We then found evidence that these two major intracellular β-glucosidases were involved in the rapid induction of cellulase genes by insoluble cellulose. Deletion of cel1a and cel1b significantly compromised the efficient gene expression of the major cellulase gene, cbh1. Simultaneous absence of BglI, CEL1a, and CEL1b caused the induction of the cellulase gene by cellulose to further deteriorate. The induction defect, however, was not observed with cellobiose. The absence of the three β-glucosidases, rather, facilitated the induced synthesis of cellulase on cellobiose. Furthermore, addition of cellobiose restored the productive induction on cellulose in the deletion strains. The results indicate that the three β-glucosidases may not participate in transforming cellobiose beyond hydrolysis to provoke cellulase formation in H. jecorina. They may otherwise contribute to the accumulation of cellobiose from cellulose as inducing signals. PMID:23002106

Production, Optimization, and Characterization of Organic Solvent Tolerant Cellulases from a Lignocellulosic Waste-Degrading Actinobacterium, Promicromonospora sp. VP111.

Science.gov (United States)

Thomas, Lebin; Ram, Hari; Kumar, Alok; Singh, Ved Pal

2016-07-01

High costs of natural cellulose utilization and cellulase production are an industrial challenge. In view of this, an isolated soil actinobacterium identified as Promicromonospora sp. VP111 showed potential for production of major cellulases (CMCase, FPase, and β-glucosidase) utilizing untreated agricultural lignocellulosic wastes. Extensive disintegration of microcrystalline cellulose and adherence on it during fermentation divulged true cellulolytic efficiency of the strain. Conventional optimization resulted in increased cellulase yield in a cost-effective medium, and the central composite design (CCD) analysis revealed cellulase production to be limited by cellulose and ammonium sulfate. Cellulase activities were enhanced by Co(+2) (1 mM) and retained up to 60 °C and pH 9.0, indicating thermo-alkaline tolerance. Cellulases showed stability in organic solvents (25 % v/v) with log P ow  ≥ 1.24. Untreated wheat straw during submerged fermentation was particularly degraded and yielded about twofold higher levels of cellulases than with commercial cellulose (Na-CMC and avicel) which is especially economical. Thus, this is the first detailed report on cellulases from an efficient strain of Promicromonospora that was non-hemolytic, alkali-halotolerant, antibiotic (erythromycin, kanamycin, rifampicin, cefaclor, ceftazidime) resistant, multiple heavy metal (Mo(+6) = W(+6) > Pb(+2) > Mn(+2) > Cr(+3) > Sn(+2)), and organic solvent (n-hexane, isooctane) tolerant, which is industrially and environmentally valuable.

In vitro flow cytometry-based screening platform for cellulase engineering

Science.gov (United States)

Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

2016-01-01

Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 107 events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

Molecular cloning of cellulase genes from indigenous bacterial isolates

International Nuclear Information System (INIS)

Jong Bor Chyan; Pauline Liew Woan Ying; Mat Rasol Awang

2006-01-01

Indigenous cellulolytic bacterial isolates having high activities in degrading carboxymethyl cellulose (CMC) were isolated from local environments. Identification of these isolates were performed by molecular techniques. By using polymerase chain reaction (PCR) techniques, PCR products encoding cellulase gene were amplified from the total genomic DNAs. Purified PCR product was successfully cloned and expressed in Escherichia coli host system. The complete nucleotide sequences of the cellulase genes determined. The analysis of amino acid sequences deduced from the genes indicated that the cloned DNA fragments show high homology to those of endoglucanase genes of family GH5. All cloned genes consist of an N-terminal signal peptide, a catalytic domain of family 5 glycosyl hydrolase and a cellulose-binding domain of family III. (Author)

Biosynthesis and isolation of C1 and Cx cellulases

Energy Technology Data Exchange (ETDEWEB)

Panaiotov, Kh; Cholakov, G

1981-01-01

Aspergillus usamii, Aspergillus niger, and Trichoderma viridae were grown on media containing lactose, lignin, (NH4)2SO4, urea, KH2PO4, CaCl2, MgSO4, and yeast extract. Maximum activities of cellulase C1 and Cx in Aspergillus usamii were observed after 76 and 90 h to be approximately 6 and approximately 24 units/mug protein, respectively. Maximum production by Aspergillus niger was 5 units C1/mug at 90h and 44 units Cx/mug at 34 h and Trichoderma produced 32.5 units C1 at 34 h and 16.5 units Cx at 58 h. Thus, Trichoderma viride produces cellulases C1 and Cx in a more balanced ratio than the Aspergillus strains.

Enhanced cellulase recovery without β-glucosidase supplementation for cellulosic ethanol production using an engineered strain and surfactant.

Science.gov (United States)

Huang, Renliang; Guo, Hong; Su, Rongxin; Qi, Wei; He, Zhimin

2017-03-01

Recycling cellulases by substrate adsorption is a promising strategy for reducing the enzyme cost of cellulosic ethanol production. However, β-glucosidase has no carbohydrate-binding module (CBM). Thus, additional enzymes are required in each cycle to achieve a high ethanol yield. In this study, we report a new method of recycling cellulases without β-glucosidase supplementation using lignocellulosic substrate, an engineered strain expressing β-glucosidase and Tween 80. The cellulases and Tween 80 were added to an aqueous suspension of diluted sulfuric acid/ammonia-treated corncobs in a simultaneous saccharification and fermentation (SSF) process for ethanol production. Subsequently, the addition of fresh pretreated corncobs to the fermentation liquor and remaining solid residue provided substrates with absorbed cellulases for the next SSF cycle. This method provided excellent ethanol production in three successive SSF cycles without requiring the addition of new cellulases. For a 10% (w/v) solid loading, a cellulase dosage of 30 filter paper units (FPU)/g cellulose, 0.5% Tween 80, and 2 g/L of the engineered strain, approximately 90% of the initial ethanol concentration from the first SSF process was obtained in the next two SSF processes, with a total ethanol production of 306.27 g/kg corncobs and an enzyme productivity of 0.044 g/FPU. Tween 80 played an important role in enhancing cellulase recovery. This new enzyme recycling method is more efficient and practical than other reported methods. Biotechnol. Bioeng. 2017;114: 543-551. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Endogenous cellulases in stylet secretions of cyst nematodes

NARCIS (Netherlands)

Smant, G.

1998-01-01

This thesis describes the identification ofβ-1,4-endoglucanases (cellulases) in stylet secretions of the two cyst nematodes species, Globodera rostochiensis and Heterodera glycines . A novel method was developed to raise monoclonal antibodies that were

Ecofriendly application of cellulase and xylanase producing marine ...

African Journals Online (AJOL)

windows

2012-06-05

Jun 5, 2012 ... producing marine Streptomyces clavuligerus as enhancer in ... pretreatment of cellulase, xylanase and the combination of enzymes. ... Energy from biomass holds a promising scope under ... investment, simplification of the fermentation media, ... biodegradation of lignocellulosic residues and enhanced ...

Cellulase Production by Trichoderma koningii AS3.4262 in Solid-State Fermentation Using Lignocellulosic Waste from the Vinegar Industry

Directory of Open Access Journals (Sweden)

Jian Liu

2007-01-01

Full Text Available Cellulase production was carried out in solid-state fermentation using the waste from the vinegar industry as the substrate for Trichoderma koningii AS3.4262. This waste is porous and easy to degrade by cellulolytic fungi. The effects of water content, initial pH value in solid substrate and culture temperature on cellulase synthesis were observed for optimal production in flask fermentors. An orthogonal layout was employed in the statistical process and better cellulase activity was obtained in the fermentation batch. The optimal filter paper cellulase (FPAse activity of 6.90 IU/g of substrate dry matter (SDM, and carboxylmethyl cellulase (CMCase activity of 23.76 IU/g SDM were obtained after 84 h of incubation with media containing vinegar waste, with optimal moisture content of 50 %, pH=5.0, incubation temperature of 30 °C, and additional nutrients of inorganic salts in a certain amount. To produce cellulase on a larger scale, a deep trough fermentor with forced aeration was used, so that FPAse activity of 5.87 IU/g SDM and CMCase activity of 12.98 IU/g SDM were reached after 84 hours of solid-state fermentation. Results indicate the excellent scope of utilizing vinegar waste as solid substrate for commercial production of cellulase employing fungi.

Application of Molecular Imprinted Magnetic Fe3O4@SiO2 Nanoparticles for Selective Immobilization of Cellulase.

Science.gov (United States)

Tao, Qing-Lan; Li, Yue; Shi, Ying; Liu, Rui-Jiang; Zhang, Ye-Wang; Guo, Jianyong

2016-06-01

Magnetic Fe3O4@SiO2 nanoparticles were prepared with molecular imprinting method using cellulase as the template. And the surface of the nanoparticles was chemically modified with arginine. The prepared nanoparticles were used as support for specific immobilization of cellulase. SDS-PAGE results indicated that the adsorption of cellulase onto the modified imprinted nanoparticles was selective. The immobilization yield and efficiency were obtained more than 70% after the optimization. Characterization of the immobilized cellulase revealed that the immobilization didn't change the optimal pH and temperature. The half-life of the immobilized cellulase was 2-fold higher than that of the free enzyme at 50 degrees C. After 7 cycles reusing, the immobilized enzyme still retained 77% of the original activity. These results suggest that the prepared imprinted nanoparticles have the potential industrial applications for the purification or immobilization of enzymes.

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

Science.gov (United States)

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

2012-09-01

A thermophile cellulase-producing bacterium was isolated and identified as closely related to Geobacillus subterraneus. The strain, named Geobacillus sp. T1, was able to grow and produce cellulase on cellobiose, microcrystalline cellulose, carboxymethylcellulose (CMC), barley straw, wheat straw and Whatman No. 1 filter paper. However, barley and wheat straws were significantly better substrates for cellulase production. When Geobacillus sp. T1 was cultivated in the presence of 0.5% barley straw, 0.1% Tween 80 and pH 6.5 at 50°C, the maximum level of free cellulase up to 143.50 U/mL was produced after 24h. This cellulase (≈ 54 kDa) was most active at pH 6.5 and 70°C. The enzyme in citrate phosphate buffer (10mM) was stable at 60°C for at least 1h. Geobacillus sp. T1 with efficient growth and cellulase production on straws seems a potential candidate for conversion of agricultural biomass to fuels. Copyright © 2012 Elsevier Ltd. All rights reserved.

The role of pheromone receptors for communication and mating in Hypocrea jecorina (Trichoderma reesei)

Science.gov (United States)

Seibel, Christian; Tisch, Doris; Kubicek, Christian P.; Schmoll, Monika

2012-01-01

Discovery of sexual development in the ascomycete Trichoderma reesei (Hypocrea jecorina) as well as detection of a novel class of peptide pheromone precursors in this fungus indicates promising insights into its physiology and lifestyle. Here we investigated the role of the two pheromone receptors HPR1 and HPR2 in the H. jecorina pheromone-system. We found that these pheromone receptors show an unexpectedly high genetic variability among H. jecorina strains. HPR1 and HPR2 confer female fertility in their cognate mating types (MAT1-1 or MAT1-2, respectively) and mediate induction of fruiting body development. One compatible pheromone precursor–pheromone receptor pair (hpr1–hpp1 or hpr2–ppg1) in mating partners was sufficient for sexual development. Additionally, pheromone receptors were essential for ascospore development, hence indicating their involvement in post-fertilisation events. Neither pheromone precursor genes nor pheromone receptor genes of H. jecorina were transcribed in a strictly mating type dependent manner, but showed enhanced expression levels in the cognate mating type. In the presence of a mating partner under conditions favoring sexual development, transcript levels of pheromone precursors were significantly increased, while those of pheromone receptor genes do not show this trend. In the female sterile T. reesei strain QM6a, transcriptional responses of pheromone precursor and pheromone receptor genes to a mating partner were clearly altered compared to the female fertile wild-type strain CBS999.97. Consequently, a delayed and inappropriate response to the mating partner may be one aspect causing female sterility in QM6a. PMID:22884620

Produção de celulases por Aspergillus niger e cinética da desativação celulásica=Cellulases production by Aspergillus niger and cellulase deactivation kinetic

Directory of Open Access Journals (Sweden)

Caroline Mariana de Aguiar

2011-10-01

Full Text Available O presente trabalho teve como objetivo a avaliação da cinética de produção de enzimas celulases pelo fungo Aspergillus niger e da cinética de desativação das celulases. Foi utilizado bagaço de cana-de-açúcar pré-tratado como fonte de carbono na fermentação para a produção do complexo celulásico e também como substrato da hidrólise enzimática. A. niger foi cultivado em três bateladas, cada uma contendo 10, 50 e 100 g L-1 de bagaço pré-tratado com NaOH 4% (m v-1. A cinética da produção das celulases foi obtida determinando-se a atividade enzimática das amostras coletadas ao longo do tempo. As variações do pH também foram determinadas. A deativação enzimática foi avaliada determinando-se periodicamente a atividade das amostras armazenadas nas condições de resfriamento (4°C e de congelamento (-18ºC. Conclui-se que o A. niger produz celulases quando cultivado em meio de cultivo contendo bagaço de cana-de-açúcar pré-tratado e que o tempo ideal para coleta do caldo enzimático foi de aproximadamente sete dias, com produtividade máxima de 0,0013 U mL-1∙h para a batelada com 10 g L-1 e 0,0018 U mL-1∙h para as bateladas com 50 e 100 g L-1. O complexo celulásico não sofre desativação se armazenado à temperatura de -18°C por 43 dias, mas perde cerca de 40% da sua atividade após 48h se armazenado a 4°C.This work aimed to evaluate the kinetic for the cellulase production by Aspergillus niger and the deactivation kinetic of the cellulase enzymes. Cellulase were produced in three different batches using NaOH 4% (w v-1 pre-treated sugarcane bagasse as the carbon source in the fermentation broth. The amount of the bagasse in each batch was 10, 50 and 100 g L-1. The kinetic of the cellulase production was accomplished by periodically determining the cellulasic activity of the fermentation broth using pre-treated bagasse as the hydrolysis substrate. Changes in the pH also were determined. The cellulase

Enzymatic activity of the cellulolytic complex produced by Trichoderma reesei. Enzymatic hydrolysis of cellulose

International Nuclear Information System (INIS)

Alfonsel J, M.; Negro A, M. J.; Saez A, R.; Martin M, C.

1986-01-01

The enzymatic activity characterization of the cellulolytic complex obtained from Trichoderma reesei QM 9414 and the influence of the enzymatic hydrolysis conditions on the hydrolysis yield are studied. Pure cellulose and native or alkali pretreated biomass Onopordum nervosum have been used as substrates. The values of pH, temperature, substrate concentration and enzyme-substrate ratio for the optimum activity of that complex, evaluated as glucose and reducing sugars production, have been selected. Previous studies on enzymatic hydrolysis of 0. nervosum have shown a remarkable effect of the alkaline pretreatments on the final hydrolysis yield. (Author) 10 refs

Comparative Studies on the Amylase and Cellulase Production of ...

African Journals Online (AJOL)

Michael Horsfall

from different sources and examined for their ability to produce cellulase and amylase. Ten fungal ... microorganisms for enzymes and, eventually, also secondary metabolites with anti-microbial or ... polymer i.e. cellulose to smaller sugar.

Optimization of cellulase production by Penicillium sp.

Science.gov (United States)

Prasanna, H N; Ramanjaneyulu, G; Rajasekhar Reddy, B

2016-12-01

The production of cellulolytic enzymes (β-exoglucanase, β-endoglucanase and β-glucosidase) by Penicillium sp. on three different media in liquid shake culture conditions was compared. The organism exhibited relatively highest activity of endoglucanase among three enzymes measured at 7-day interval during the course of its growth on Czapek-Dox medium supplemented with 0.5 % (w/v) cellulose. Cellulose at 0.5 %, lactose at 0.5 %, sawdust at 0.5 %, yeast extract at 0.2 % as a nitrogen source, pH 5.0 and 30 °C temperature were found to be optimal for growth and cellulase production by Penicillium sp. Yields of Fpase, CMCase and β-glucosidase, attained on optimized medium with Penicillium sp. were 8.7, 25 and 9.52 U/ml, respectively with increment of 9.2, 5.9 and 43.8-folds over titers of the respective enzyme on unoptimised medium. Cellulase of the fungal culture with the ratio of β-glucosidase to Fpase greater than one will hold potential for biotechnological applications.

Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass.

Directory of Open Access Journals (Sweden)

Gustavo Pagotto Borin

Full Text Available Our dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the world's second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses, must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant.Analyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and β-glucan (that compose the most external

Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass

Science.gov (United States)

Borin, Gustavo Pagotto; Sanchez, Camila Cristina; de Souza, Amanda Pereira; de Santana, Eliane Silva; de Souza, Aline Tieppo; Leme, Adriana Franco Paes; Squina, Fabio Marcio; Buckeridge, Marcos; Goldman, Gustavo Henrique; Oliveira, Juliana Velasco de Castro

2015-01-01

Background Our dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the world's second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G) bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses), must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant. Results Analyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and β-glucan (that compose the most external

Cleanability Improvement of Cotton Fabrics Through Their Topographical Changes Due to the Conditioning with Cellulase Enzyme

NARCIS (Netherlands)

Calvimontes, A.; Lant, N.J.; Dutschk, Victoria

2012-01-01

In this study, topographical changes of woven cotton fabrics conditioned with a cellulase enzyme during several wash–dry cycles are systematically studied. A recent study of cellulase enzyme effect on cellulose films has proven that this substance selectively attacks amorphous regions of cellulose,

Pseudo-affinity chromatography of rumen microbial cellulase on ...

African Journals Online (AJOL)

Pseudo-affinity chromatography of rumen microbial cellulase on Sepharose- Cibacron Blue F3GA. ... African Journal of Biotechnology ... Pseudo affinity adsorption of bioproducts on Sepharose-cibacron blue F3-GA was subjected to rumen microbial enzyme evaluation through batch binding and column chromatography of ...

Biohydrolysis of Saccharum spontaneum for cellulase production by ...

African Journals Online (AJOL)

Saccharum spontaneum, a wasteland weed, is utilized for cellulase production by Aspergillus terreus in solid state fermentation. S. spontaneum served as good carbon source and solid support. Various process parameters including optimal nitrogen source, initial moisture level, incubation time, initial pH, incubation ...

Optimisation of synergistic biomass-degrading enzyme systems for efficient rice straw hydrolysis using an experimental mixture design.

Science.gov (United States)

Suwannarangsee, Surisa; Bunterngsook, Benjarat; Arnthong, Jantima; Paemanee, Atchara; Thamchaipenet, Arinthip; Eurwilaichitr, Lily; Laosiripojana, Navadol; Champreda, Verawat

2012-09-01

Synergistic enzyme system for the hydrolysis of alkali-pretreated rice straw was optimised based on the synergy of crude fungal enzyme extracts with a commercial cellulase (Celluclast™). Among 13 enzyme extracts, the enzyme preparation from Aspergillus aculeatus BCC 199 exhibited the highest level of synergy with Celluclast™. This synergy was based on the complementary cellulolytic and hemicellulolytic activities of the BCC 199 enzyme extract. A mixture design was used to optimise the ternary enzyme complex based on the synergistic enzyme mixture with Bacillus subtilis expansin. Using the full cubic model, the optimal formulation of the enzyme mixture was predicted to the percentage of Celluclast™: BCC 199: expansin=41.4:37.0:21.6, which produced 769 mg reducing sugar/g biomass using 2.82 FPU/g enzymes. This work demonstrated the use of a systematic approach for the design and optimisation of a synergistic enzyme mixture of fungal enzymes and expansin for lignocellulosic degradation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Characterization of cellulase production by carbon sources in two ...

African Journals Online (AJOL)

user7

2013-11-27

Nov 27, 2013 ... 7State Key Laboratory of Urban Water Resource and Environment, Harbin ... China. Accepted 28 March, 2012. The induction of cellulase production in two .... prevent the contamination with bacteria, 0.02% sodium azide was.

Production of xylanases and cellulases by aspergillus fumigatus ms16 using crude lignocellulosic substrates

International Nuclear Information System (INIS)

Naseeb, S.; Sohai, M.; Ahmad, A.; Khan, S.A.

2015-01-01

Xylanolytic and cellulolytic potential of a soil isolate, Aspergillus fumigatus (MS16) was studied by growing it on a variety of lignocellulosics, purified cellulose and xylan supplemented media. It was noted that carboxymethyl cellulose, salicin and xylan induce the -glucosidase and xylanase, respectively production of endoglucanase. The study revealed that Aspergillus fumigatus (MS16) co-secretes xylanase and cellulase in the presence of xylan; the ratio of the two enzymes was influenced by the initial pH of the medium. The maximum titers of xylanase and cellulase were noted at initial pH of 5.0. Relatively higher titers of both the enzymes were obtained when the fungus was cultivated at 35 degree C. Whereas, cellulase production was not detected when the fungus was cultivated at 40 degree C. The volumetric productivity (Qp) of xylanase was much higher than cellulases. The organism produced 2-3 folds higher titers of xylanase when grown on lignocellulosic materials in submerged cultivation than under solid-state cultivation, suggesting a different pattern of enzyme production in presence and in absence of free water. The partial characterization of enzymes showed that xylanase from this organism has -glucosidase. The higher melting temperature than endoglucanase and optimum temperature for activity was higher for xylanases than cellulases, whereas the optimum pH differed slightly i.e. in the range of 4.0-5.0. Enzyme preparation from this organism was loaded on some crude substrates and it showed that the enzyme preparation can be used to hydrolyze a variety of vegetable and agricultural waste materials. (author)

Exploration of indigenous agrowastes for cellulase production by ...

African Journals Online (AJOL)

Regional agrowastes such as Vigna mungo, Saccharum spontaneum and Brassica campestris were collected and biohydrolysis of these substrates for cellulase production were carried out by Aspergillus niger. Proximate composition of each agrowastes was analyzed based on dry weight, to have an insight view of their ...

Optimization for cellulase production by Aspergillus niger using saw ...

African Journals Online (AJOL)

STORAGESEVER

2008-11-19

Nov 19, 2008 ... Several other parameters like inoculum size, time duration, nitrogen source and its ... β-1,4-D-glycosidic bond (Gielkens et al., 1999; Han et al.,. 1995). Cellulase is a ... management, medical/pharmaceutical industry, proto-.

A graphene screen-printed carbon electrode for real-time measurements of unoccupied active sites in a cellulase

DEFF Research Database (Denmark)

Cruys-Bagger, Nicolaj; Tatsumi, Hirosuke; Borch, Kim

2014-01-01

Cellulases hydrolyze cellulose to soluble sugars and this process is utilized in sustainable industries based on lignocellulosic feedstock. Better analytical tools will be necessary to understand basic cellulase mechanisms, and hence deliver rational improvements of the industrial process...

Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System.

Directory of Open Access Journals (Sweden)

Yoichiro Ito

Full Text Available Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering.

Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System

Science.gov (United States)

Ito, Yoichiro; Yamanishi, Mamoru; Ikeuchi, Akinori; Imamura, Chie; Matsuyama, Takashi

2015-01-01

Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering. PMID:26692026

Covalent Immobilization of Cellulase Using Magnetic Poly(ionic liquid) Support: Improvement of the Enzyme Activity and Stability.

Science.gov (United States)

Hosseini, Seyed Hassan; Hosseini, Seyedeh Ameneh; Zohreh, Nasrin; Yaghoubi, Mahshid; Pourjavadi, Ali

2018-01-31

A magnetic nanocomposite was prepared by entrapment of Fe 3 O 4 nanoparticles into the cross-linked ionic liquid/epoxy type polymer. The resulting support was used for covalent immobilization of cellulase through the reaction with epoxy groups. The ionic surface of the support improved the adsorption of enzyme, and a large amount of enzyme (106.1 mg/g) was loaded onto the support surface. The effect of the presence of ionic monomer and covalent binding of enzyme was also investigated. The structure of support was characterized by various instruments such as FT-IR, TGA, VSM, XRD, TEM, SEM, and DLS. The activity and stability of immobilized cellulase were investigated in the prepared support. The results showed that the ionic surface and covalent binding of enzyme onto the support improved the activity, thermal stability, and reusability of cellulase compared to free cellulase.

Cellulose hydrolysis by fungi. 2. Cellulase production by Trichoderma harzianum in liquid medium fermentation

Energy Technology Data Exchange (ETDEWEB)

Roussos, S.; Raimbault, M. (Laboratoire de Microbiologie ORSTOM, Centre de Recherche IRCHA, 91 - Vert-le-Petit (France))

Microcrystalline cellulose (cellulose Avicel, Merck) supported growth of Trichoderma harzianum and induced production of cellulases in liquid cultures. After 50 h growth, the total cellulasic activities present in both the supernatant and the mycelium were 3,000 IU/l of carboxymethyl cellulose, 400 IU/l of filter paper activity, and 4 IU/l of cotton activity corresponding to 1.7 g/l of proteins. Cellulase production could be increased by a preliminary treatment of cellulose, and pH regulation during growth. The influence of inoculum concentration was studied and an optimum of 3 X 10/sup 7/ conidia/g dry weight of substrate was demonstrated. Using a synthetic culture medium, a soluble factor of germination was demonstrated which could be leached out by 3 successive washings of conidia.

Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels.

Science.gov (United States)

Peng, Huan; Rübsam, Kristin; Jakob, Felix; Schwaneberg, Ulrich; Pich, Andrij

2016-11-14

This paper reports a facile approach for encapsulation of enzymes in nanogels. Our approach is based on the use of reactive copolymers able to get conjugated with enzyme and build 3D colloidal networks or biohybrid nanogels. In a systematic study, we address the following question: how the chemical structure of nanogel network influences the biocatalytic activity of entrapped enzyme? The developed method allows precise control of the enzyme activity and improvement of enzyme resistance against harsh store conditions, chaotropic agents, and organic solvents. The nanogels were constructed via direct chemical cross-linking of water-soluble reactive copolymers poly(N-vinylpyrrolidone-co-N-methacryloxysuccinimide) with proteins such as enhanced green fluorescent protein (EGFP) and cellulase in water-in-oil emulsion. The water-soluble reactive copolymers with controlled amount of reactive succinimide groups and narrow dispersity were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Poly(ethylene glycol) bis(3-aminopropyl) and branched polyethylenimine were utilized as model cross-linkers to optimize synthesis of nanogels with different architectures in the preliminary experiments. Biofluorescent nanogels with different loading amount of EGFP and varying cross-linking densities were obtained. We demonstrate that the biocatalytic activity of cellulase-conjugated nanogels (CNG) can be elegantly tuned by control of their cross-linking degrees. Circular dichroism (CD) spectra demonstrated that the secondary structures of the immobilized cellulase were changed in the aspect of α-helix contents. The secondary structures of cellulase in highly cross-linked nanogels were strongly altered compared with loosely cross-linked nanogels. The fluorescence resonance energy transfer (FRET) based study further revealed that nanogels with lower cross-linking degree enable higher substrate transport rate, providing easier access to the active site of

Response to Comment on "Revealing nature's cellulase diversity: the digestion mechanism of Caldicellulosiruptor bescii CelA".

Science.gov (United States)

Brunecky, Roman; Alahuhta, Markus; Xu, Qi; Donohoe, Bryon S; Crowley, Michael F; Kataeva, Irina A; Yang, Sung-Jae; Resch, Michael G; Adams, Michael W W; Lunin, Vladimir V; Himmel, Michael E; Bomble, Yannick J

2014-05-09

Gusakov critiques our methodology for comparing the cellulolytic activity of the bacterial cellulase CelA with the fungal cellulase Cel7A. We address his concerns by clarifying some misconceptions, carefully referencing the literature, and justifying our approach to point out that the results from our study still stand.

Study of thermal and chemical effects on cellulase enzymes: Viscosity measurements

International Nuclear Information System (INIS)

Ghaouar, N.; Aschi, A.; Belbahri, L.; Trabelsi, S.; Gharbi, A.

2009-01-01

The behaviour of cellulase enzymes in phosphate saline buffer has been studied over a wide range of temperatures and enzyme concentrations by using viscosity measurements. To characterize the conformation change of cellulase versus temperature and chemical denaturants, such as guanidinium chloride (GdmCl) and urea, the information about the intrinsic viscosity and the hydrodynamic radius are necessary. The dependence of the intrinsic viscosity and the hydrodynamic radius in its random coil conformation on temperature and denaturant concentration were studied. Our results and discussions are limited to the dilute regime of concentration because of abnormalities in conformation observed in the very dilute regime due to the presence of capillary absorption effects.

Study of thermal and chemical effects on cellulase enzymes: Viscosity measurements

Energy Technology Data Exchange (ETDEWEB)

Ghaouar, N., E-mail: naoufel-ghaouar@lycos.co [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 2092 (Tunisia); Institut National des Sciences Appliquees et de Technologie, INSAT, Centre Urbain Nord, BP. 676, Tunis (Tunisia); Aschi, A. [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 2092 (Tunisia); Belbahri, L. [Agronomy Department, School of Engineering of Lullier, University of Applied Sciences of Western Switzerland, 150, Route de Presinge, 1254 Jussy (Switzerland); Trabelsi, S.; Gharbi, A. [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 2092 (Tunisia)

2009-11-15

The behaviour of cellulase enzymes in phosphate saline buffer has been studied over a wide range of temperatures and enzyme concentrations by using viscosity measurements. To characterize the conformation change of cellulase versus temperature and chemical denaturants, such as guanidinium chloride (GdmCl) and urea, the information about the intrinsic viscosity and the hydrodynamic radius are necessary. The dependence of the intrinsic viscosity and the hydrodynamic radius in its random coil conformation on temperature and denaturant concentration were studied. Our results and discussions are limited to the dilute regime of concentration because of abnormalities in conformation observed in the very dilute regime due to the presence of capillary absorption effects.

Isolation and Screening of Thermo-Stable Cellulase Enzyme Fungal Producer at Different Temperature

International Nuclear Information System (INIS)

Noor Ashiqin Jamroo; Noor Azrimi Umor; Kamsani

2015-01-01

Thermo stable cellulase from fungi has high potential for industrial application. In this study, wild -type of fungal were isolate from different sources such as hot spring water, sea water, soft wood, rice straw and cow dung. The isolates were characterized by cultural and morphological observation. Based on morphological characteristics, the genera of all fungal cultures were identified namely Aspergillus fumigatus. The screening for thermo stable cellulase were done using 2 % carboxymethyl cellulose and congo red as an indicator at temperature 30, 37, 45 and 50 degree Celsius respectively. Out of 26 fungal isolates, only eight isolates were selected for further screening and showed the abilities to secrete cellulases by forming distinct halo zones on selective agar plate. The maximum halo zone ranging from 32 mm to 35 mm were obtained after 72 hour incubation at 50 degree Celsius by H2, SW1 and C1 isolates. As contrary other isolates showed halo zone range from 22 mm to 29 mm at same temperature. All the isolates showed the abilities to secrete cellulase enzyme at other temperature but lower when compared to 50 degree Celsius referred to the halo zone obtained. The SW1 isolates showed highest cellulolytic index which was 2.93 measured at 37 degree Celsius and 2.67 at 50 degree Celsius respectively. (author)

Self-induction system for cellulase production by cellobiose produced from glucose in Rhizopus stolonifer

OpenAIRE

Zhang, Yingying; Tang, Bin; Du, Guocheng

2017-01-01

Cellulolytic fungi have evolved a sophisticated genetic regulatory network of cellulase synthesis to adapt to the natural environment. Even in the absence of lignocellulose, it still secretes low levels of ?constitutive? cellulase for standby application. However, the mechanisms of this constitutive expression remain incompletely understood. Here we identified a cellobiose synthetase (CBS) from Rhizopus stolonifer, which has the capacity to catalyse the synthesis of cellobiose from uridine di...

Evaluation of the efficiency of alternative enzyme production technologies

DEFF Research Database (Denmark)

Albæk, Mads Orla

Enzymes are used in an increasing number of industries. The application of enzymes is extending into the production of lignocellulosic ethanol in processes that economically can compete with fossil fuels. Since lignocellulosic ethanol is based on renewable resources it will have a positive impact...... production of cellulases and hemi-cellulases. The aim of the thesiswas to use modeling tools to identify alternative technologies that have higher energy or raw material efficiency than the current technology. The enzyme production by T. reesei was conducted as an aerobic fed-batch fermentation. The process...... of the uncertainty and sensitivity of the model indicated the biological parameters to be responsible for most of the model uncertainty. A number of alternative fermentation technologies for enzyme production were identified in the open literature. Their mass transfer capabilities and their energy efficiencies were...

The composition of accessory enzymes of Penicillium chrysogenum P33 revealed by secretome and synergistic effects with commercial cellulase on lignocellulose hydrolysis.

Science.gov (United States)

Yang, Yi; Yang, Jinshui; Liu, Jiawen; Wang, Ruonan; Liu, Liang; Wang, Fengqin; Yuan, Hongli

2018-06-01

Herein, we report the secretome of Penicillium chrysogenum P33 under induction of lignocellulose for the first time. A total of 356 proteins were identified, including complete cellulases and numerous hemicellulases. Supplementing a commercial cellulase with increasing dosage of P33 enzyme cocktail from 1 to 5 mg/g substrate increased the release of reducing sugars from delignified corn stover by 21.4% to 106.8%. When 50% cellulase was replaced by P33 enzyme cocktail, release of reducing sugars was 78.6% higher than with cellulase alone. Meanwhile, glucan and xylan conversion was increased by 37% and 106%, respectively. P33 enzyme cocktail also enhanced commercial cellulase hydrolysis against four different delignified lignocellulosic biomass. These findings demonstrate that mixing appropriate amount of P33 cocktail with cellulase improves polysaccharide hydrolysis, suggesting P33 enzymes have great potential for industrial applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

Designing novel cellulase systems through agent-based modeling and global sensitivity analysis

Science.gov (United States)

Apte, Advait A; Senger, Ryan S; Fong, Stephen S

2014-01-01

Experimental techniques allow engineering of biological systems to modify functionality; however, there still remains a need to develop tools to prioritize targets for modification. In this study, agent-based modeling (ABM) was used to build stochastic models of complexed and non-complexed cellulose hydrolysis, including enzymatic mechanisms for endoglucanase, exoglucanase, and β-glucosidase activity. Modeling results were consistent with experimental observations of higher efficiency in complexed systems than non-complexed systems and established relationships between specific cellulolytic mechanisms and overall efficiency. Global sensitivity analysis (GSA) of model results identified key parameters for improving overall cellulose hydrolysis efficiency including: (1) the cellulase half-life, (2) the exoglucanase activity, and (3) the cellulase composition. Overall, the following parameters were found to significantly influence cellulose consumption in a consolidated bioprocess (CBP): (1) the glucose uptake rate of the culture, (2) the bacterial cell concentration, and (3) the nature of the cellulase enzyme system (complexed or non-complexed). Broadly, these results demonstrate the utility of combining modeling and sensitivity analysis to identify key parameters and/or targets for experimental improvement. PMID:24830736

Conversion of woody biomass into fermentable sugars by cellulase from Agaricus arvensis.

Science.gov (United States)

Jeya, Marimuthu; Nguyen, Ngoc-Phuong-Thao; Moon, Hee-Jung; Kim, Sang-Hwan; Lee, Jung-Kul

2010-11-01

Agaricus arvensis, a newly isolated basidiomycetous fungus, was found to secrete efficient cellulases. The strain produced the highest endoglucanase (EG), cellobiohydrolase (CBH) and beta-glucosidase (BGL) activities of 0.3, 3.2 and 8U/mg-protein, respectively, with rice straw as the carbon source. Saccharification of the woody biomass with A. arvensis cellulase as the enzyme source released a high level of fermentable sugars. Enzymatic hydrolysis of the poplar biomass was optimized using the response surface methodology in order to study the influence of the variables (pH, temperature, cellulases concentration and substrate concentration). The enzyme and substrate concentrations were identified as the limiting factors for the saccharification of poplar wood biomass. A total reducing sugar level of 29g/L (293mg/g-substrate) was obtained at an enzyme concentration of 65FPU/g-substrate after optimization of the hydrolysis parameters. The model validation showed a good agreement between the experimental results and the predicted responses. A. arvensis could be a good candidate for the production of reducing sugars from a cellulosic biomass.

Designing novel cellulase systems through agent-based modeling and global sensitivity analysis.

Science.gov (United States)

Apte, Advait A; Senger, Ryan S; Fong, Stephen S

2014-01-01

Experimental techniques allow engineering of biological systems to modify functionality; however, there still remains a need to develop tools to prioritize targets for modification. In this study, agent-based modeling (ABM) was used to build stochastic models of complexed and non-complexed cellulose hydrolysis, including enzymatic mechanisms for endoglucanase, exoglucanase, and β-glucosidase activity. Modeling results were consistent with experimental observations of higher efficiency in complexed systems than non-complexed systems and established relationships between specific cellulolytic mechanisms and overall efficiency. Global sensitivity analysis (GSA) of model results identified key parameters for improving overall cellulose hydrolysis efficiency including: (1) the cellulase half-life, (2) the exoglucanase activity, and (3) the cellulase composition. Overall, the following parameters were found to significantly influence cellulose consumption in a consolidated bioprocess (CBP): (1) the glucose uptake rate of the culture, (2) the bacterial cell concentration, and (3) the nature of the cellulase enzyme system (complexed or non-complexed). Broadly, these results demonstrate the utility of combining modeling and sensitivity analysis to identify key parameters and/or targets for experimental improvement.

Practical screening of purified cellobiohydrolases and endoglucanases with α-cellulose and specification of hydrodynamics

Directory of Open Access Journals (Sweden)

Jäger Gernot

2010-08-01

Full Text Available Abstract Background It is important to generate biofuels and society must be weaned from its dependency on fossil fuels. In order to produce biofuels, lignocellulose is pretreated and the resulting cellulose is hydrolyzed by cellulases such as cellobiohydrolases (CBH and endoglucanases (EG. Until now, the biofuel industry has usually applied impractical celluloses to screen for cellulases capable of degrading naturally occurring, insoluble cellulose. This study investigates how these cellulases adsorb and hydrolyze insoluble α-cellulose − considered to be a more practical substrate which mimics the alkaline-pretreated biomass used in biorefineries. Moreover, this study investigates how hydrodynamics affects cellulase adsorption and activity onto α-cellulose. Results First, the cellulases CBH I, CBH II, EG I and EG II were purified from Trichoderma reesei and CBH I and EG I were utilized in order to study and model the adsorption isotherms (Langmuir and kinetics (pseudo-first-order. Second, the adsorption kinetics and cellulase activities were studied under different hydrodynamic conditions, including liquid mixing and particle suspension. Third, in order to compare α-cellulose with three typically used celluloses, the exact cellulase activities towards all four substrates were measured. It was found that, using α-cellulose, the adsorption models fitted to the experimental data and yielded parameters comparable to those for filter paper. Moreover, it was determined that higher shaking frequencies clearly improved the adsorption of cellulases onto α-cellulose and thus bolstered their activity. Complete suspension of α-cellulose particles was the optimal operating condition in order to ensure efficient cellulase adsorption and activity. Finally, all four purified cellulases displayed comparable activities only on insoluble α-cellulose. Conclusions α-Cellulose is an excellent substrate to screen for CBHs and EGs. This current investigation

Purification and characterization of a thermostable alkaline cellulase produced by Bacillus licheniformis 380 isolated from compost

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ÉVILIN G. DE MARCO

2017-10-01

Full Text Available ABSTRACT During composting processes, the degradation of organic waste is accomplished and driven by a succession of microbial populations exhibiting a broad range of functional competencies. A total of 183 bacteria, isolated from a composting process, were evaluated for cellulase activity at different temperatures (37, 50, 60, and 70°C and pH values. Out of the 22 isolates that showed activity, isolate 380 showed the highest cellulase activity. Its ability to produce cellulase was evaluated in culture medium supplemented with carboxymethyl cellulose, microcrystalline cellulose, wheat straw, and rice husk. The culture medium supplemented with carboxymethyl cellulose induced higher enzyme activity after 6 hours of incubation (0.12 UEA mL-1 min-1. For wheat straw and rice husk, the results were 0.08 UEA mL-1 min-1 for both, while for microcrystalline cellulose, 0.04 UEA mL-1 min-1 were observed. The highest carboxymethyl cellulase activity was observed at 60°C (0.14 UEA mL-1 min-1 for both crude and partially purified enzyme after 30 and 120 min of incubation, respectively. Alkalinization of the medium was observed during cultivation in all substrates. The cellulase had a molecular mass of 20 kDa determined by SDS-Page. Isolate 380 was identified as Bacillus licheniformis. This work provides a basis for further studies on composting optimization.

Adsorption of cellulases onto sugar beet shreds and modeling of the experimental data

Directory of Open Access Journals (Sweden)

Ivetić Darjana Ž.

2014-01-01

Full Text Available This study investigated the adsorption of cellulases onto sugar beet shreds. The experiments were carried out using untreated, as well as dried and not dried dilute acid and steam pretreated sugar beet shreds at different initial enzyme loads. Both dilute acid and steam pretreatment were beneficial in respect of cellulases adsorption providing 8 and 9 times higher amounts of adsorbed proteins, respectively, in comparison to the results obtained with the untreated substrate. Although the use of higher solids load enabled by drying of pretreated substrates, could be beneficial for process productivity, at the same time it decreases the adsorption of enzymes. The obtained experimental data were fitted to five adsorption models, and the Langmuir model having the lowest residual sum of squares was used for the determination of adsorption parameters which were used to calculate the strength of cellulases binding to the substrates.[Projekat Ministarstva nauke Republike Srbije, br. TR 31002

Implications of cellobiohydrolase glycosylation for use in biomass conversion

Directory of Open Access Journals (Sweden)

Decker Stephen R

2008-05-01

Full Text Available Abstract The cellulase producing ascomycete, Trichoderma reesei (Hypocrea jecorina, is known to secrete a range of enzymes important for ethanol production from lignocellulosic biomass. It is also widely used for the commercial scale production of industrial enzymes because of its ability to produce high titers of heterologous proteins. During the secretion process, a number of post-translational events can occur, however, that impact protein function and stability. Another ascomycete, Aspergillus niger var. awamori, is also known to produce large quantities of heterologous proteins for industry. In this study, T. reesei Cel7A, a cellobiohydrolase, was expressed in A. niger var. awamori and subjected to detailed biophysical characterization. The purified recombinant enzyme contains six times the amount of N-linked glycan than the enzyme purified from a commercial T. reesei enzyme preparation. The activities of the two enzyme forms were compared using bacterial (microcrystalline and phosphoric acid swollen (amorphous cellulose as substrates. This comparison suggested that the increased level of N-glycosylation of the recombinant Cel7A (rCel7A resulted in reduced activity and increased non-productive binding on cellulose. When treated with the N-glycosidase PNGaseF, the molecular weight of the recombinant enzyme approached that of the commercial enzyme and the activity on cellulose was improved.

Hydrolysis of cellulose-containing materials by cellulase of the Trichoderma lignorum OM 534 fungus

Energy Technology Data Exchange (ETDEWEB)

Romanov, S L; Lobanok, A G

1977-01-01

Of the cellulose containing materials, hydrocellulose was most easily degraded while lignocellulose was hardest to break down with cellulase from T. lignorum grown on lactose or cellulose. Grinding and heat treatment (at 200/sup 0/) of lignocellulose enhanced its enzymic degradability. Hydrolysis was highest by cellulase from lactose-cultured Trichoderma. The hydrolysis products contained glucose, galactose, xylose, and mannose. Filtrates from T. lignorum grown on a lignocellulose were enzymically active after purification.

Kinetic and thermodynamic properties of alginate lyase and cellulase co-produced by Exiguobacterium species Alg-S5.

Science.gov (United States)

Mohapatra, Bidyut R

2017-05-01

In an effort to screen out the alginolytic and cellulolytic bacteria from the putrefying invasive seaweed Sargassum species accumulated off Barbados' coast, a potent bacterial strain was isolated. This bacterium, which simultaneously produced alginate lyase and cellulase, was identified as Exiguobacterium sp. Alg-S5 via the phylogenetic approach targeting the 16S rRNA gene. The co-produced alginate lyase and cellulase exhibited maximal enzymatic activity at pH 7.5 and at 40°C and 45°C, respectively. The K m and V max values recorded as 0.91mg/mL and 21.8U/mg-protein, respectively, for alginate lyase, and 10.9mg/mL and 74.6U/mg-protein, respectively, for cellulase. First order kinetic analysis of the thermal denaturation of the co-produced alginate lyase and cellulase in the temperature range from 40°C to 55°C revealed that both the enzymes were thermodynamically efficient by displaying higher activation energy and enthalpy of denaturation. These enzymatic properties indicate the potential industrial importance of this bacterium in algal biomass conversion. This appears to be the first report on assessing the efficacy of a bacterium for the co-production of alginate lyase and cellulase. Copyright © 2017 Elsevier B.V. All rights reserved.

Kinetic study of Escherichia coli BPPTCC-EgRK2 to produce recombinant cellulase for ethanol production from oil palm empty fruit bunch

Science.gov (United States)

Limoes, S.; Rahman, S. F.; Setyahadi, S.; Gozan, M.

2018-03-01

Oil Palm Empty Fruit Bunch (OPEFB) is an abundant biomass resource in Indonesia, which contains 46,77% (w/w) of cellulose. The high cellulose content of OPEFB can be used as a substrate for bacteria cultivation to produce cellulase. By using OPEFB as an alternative substrate, the production cost of cellulase in industrial scale can be suppressed. However, currently there are no available research that simulate a cellulase production plant design. Prior to simulating the cellulase plant design, kinetic studies of bacteria used in cultivation are needed to create an accurate simulation. In this research, kinetic studies of E. coli BPPTCC-EgRK2 growth were examined with the Monod approach to get the Monod constant (Ks) and maximum specific growth rate (μmax). This study found that E. coli BPPTCC-EgRK2 have μmax and Ks of 1.581 and 0.0709 respectively. BPPTCC-EgRK2 produced intracellular cellulase, thus gave linear correlation between cell concentration and cellulase production.

Improving cellulase production by Aspergillus niger using adaptive evolution

NARCIS (Netherlands)

Patyshakuliyeva, Aleksandrina; Arentshorst, Mark; Allijn, Iris E; Ram, Arthur F J; de Vries, Ronald P; Gelber, Isabelle Benoit

OBJECTIVES: To evaluate the potential of adaptive evolution as a tool in generating strains with an improved production of plant biomass degrading enzymes. RESULTS: An Aspergillus niger cellulase mutant was obtained by adaptive evolution. Physiological properties of this mutant revealed a five times

Construction of cellulose-utilizing Escherichia coli based on a secretable cellulase.

Science.gov (United States)

Gao, Dongfang; Luan, Yaqi; Wang, Qian; Liang, Quanfeng; Qi, Qingsheng

2015-10-09

The microbial conversion of plant biomass into value added products is an attractive option to address the impacts of petroleum dependency. The Gram-negative bacterium Escherichia coli is commonly used as host for the industrial production of various chemical products with a variety of sugars as carbon sources. However, this strain neither produces endogenous cellulose degradation enzymes nor secrets heterologous cellulases for its poor secretory capacity. Thus, a cellulolytic E. coli strain capable of growth on plant biomass would be the first step towards producing chemicals and fuels. We previously identified the catalytic domain of a cellulase (Cel-CD) and its N-terminal sequence (N20) that can serve as carriers for the efficient extracellular production of target enzymes. This finding suggested that cellulose-utilizing E. coli can be engineered with minimal heterologous enzymes. In this study, a β-glucosidase (Tfu0937) was fused to Cel-CD and its N-terminal sequence respectively to obtain E. coli strains that were able to hydrolyze the cellulose. Recombinant strains were confirmed to use the amorphous cellulose as well as cellobiose as the sole carbon source for growth. Furthermore, both strains were engineered with poly (3-hydroxybutyrate) (PHB) synthesis pathway to demonstrate the production of biodegradable polyesters directly from cellulose materials without exogenously added cellulases. The yield of PHB reached 2.57-8.23 wt% content of cell dry weight directly from amorphous cellulose/cellobiose. Moreover, we found the Cel-CD and N20 secretion system can also be used for the extracellular production of other hydrolytic enzymes. This study suggested that a cellulose-utilizing E. coli was created based on a heterologous cellulase secretion system and can be used to produce biofuels and biochemicals directly from cellulose. This system also offers a platform for conversion of other abundant renewable biomass to biofuels and biorefinery products.

Cellulase enzyme production during continuous culture growth of Sporotrichum (Chrysosporium) thermophile

Energy Technology Data Exchange (ETDEWEB)

Cossar, D; Canevascini, G

1986-07-01

The cellulolytic fungus Sporotrichum (Chrysosporium) thermophile produces an extracellular cellobiose dehydrogenase during batch culture on cellulose or cellobiose. In chemostat culture at pH 5.6 on cellobiose this enzyme was produced in parallel with endo-cellulase. At pH 5.0 in continuous or fed-batch culture such a pattern was not evident. At constant growth rate in a chemostat with varying pH, activity of these enzymes was found to be poorly correlated. Thus the induction of cellobiose dehydrogenase shows a dependence on pH and cellobiose concentration which is different to that for endo-cellulase. The natural inducer of these enzymes and the role of cellubiose dehydrogenase remain to be elucidated.

Chitinase and cellulase activity from Bacillus thuringiensis strains - doi: 10.5102/ucs.v7i1.974

Directory of Open Access Journals (Sweden)

Vinícius Fiúza Dumas

2009-12-01

Full Text Available The present study aimed to analyze the production of chitinase and cellulase enzymes by strains of Bacillus thuringiensis toxic to Spodoptera frugiperda and Anthonomus grandis larvae. In order to evaluate the relationship between cellular growth and the chitinase and cellulase production, in vitro assays were carried through with bacteria cultures grown for 16h, 24h, 48h and 72h. Chitinase and cellulase activity was determined by a colorimetric method. The amount of N-acetylglucosamine (GlcNAc or its equivalent was measured by development of color in acid medium. All strains presented enzymatic production after 16h of cellular growth until 72h. However, a Kruskal-Wallis test detected no significant differences among the chitinase and cellulase activity during the cellular growth. According to these results, was not possible to associate chitinase and cellulose activity with the different level of toxicity of Bt strains against S. frugiperda and A. grandis larvae.

Chimeric cellulase matrix for investigating intramolecular synergism between non-hydrolytic disruptive functions of carbohydrate-binding modules and catalytic hydrolysis.

Science.gov (United States)

Wang, Yuguo; Tang, Rentao; Tao, Jin; Wang, Xiaonan; Zheng, Baisong; Feng, Yan

2012-08-24

The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose.

Chimeric Cellulase Matrix for Investigating Intramolecular Synergism between Non-hydrolytic Disruptive Functions of Carbohydrate-binding Modules and Catalytic Hydrolysis*

Science.gov (United States)

Wang, Yuguo; Tang, Rentao; Tao, Jin; Wang, Xiaonan; Zheng, Baisong; Feng, Yan

2012-01-01

The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose. PMID:22778256

Inhibition of lignin-derived phenolic compounds to cellulase.

Science.gov (United States)

Qin, Lei; Li, Wen-Chao; Liu, Li; Zhu, Jia-Qing; Li, Xia; Li, Bing-Zhi; Yuan, Ying-Jin

2016-01-01

Lignin-derived phenolic compounds are universal in the hydrolysate of pretreated lignocellulosic biomass. The phenolics reduce the efficiency of enzymatic hydrolysis and increase the cost of ethanol production. We investigated inhibition of phenolics on cellulase during enzymatic hydrolysis using vanillin as one of the typical lignin-derived phenolics and Avicel as cellulose substrate. As vanillin concentration increased from 0 to 10 mg/mL, cellulose conversion after 72-h enzymatic hydrolysis decreased from 53 to 26 %. Enzyme deactivation and precipitation were detected with the vanillin addition. The enzyme concentration and activity consecutively decreased during hydrolysis, but the inhibition degree, expressed as the ratio of the cellulose conversion without vanillin to the conversion with vanillin (A 0 /A), was almost independent on hydrolysis time. Inhibition can be mitigated by increasing cellulose loading or cellulase concentration. The inhibition degree showed linear relationship with the vanillin concentration and exponential relationship with the cellulose loading and the cellulase concentration. The addition of calcium chloride, BSA, and Tween 80 did not release the inhibition of vanillin significantly. pH and temperature for hydrolysis also showed no significant impact on inhibition degree. The presence of hydroxyl group, carbonyl group, and methoxy group in phenolics affected the inhibition degree. Besides phenolics concentration, other factors such as cellulose loading, enzyme concentration, and phenolic structure also affect the inhibition of cellulose conversion. Lignin-blocking agents have little effect on the inhibition effect of soluble phenolics, indicating that the inhibition mechanism of phenolics to enzyme is likely different from insoluble lignin. The inhibition of soluble phenolics can hardly be entirely removed by increasing enzyme concentration or adding blocking proteins due to the dispersity and multiple binding sites of phenolics

Effects of some added carbohydrates on cellulases and ligninase and decomposition of whole bagasse

Energy Technology Data Exchange (ETDEWEB)

Nigam, P; Prabhu, K A

1986-01-01

Two basidiomycetes mould isolates were studied for the production of extracellular cellulases and ligninase in submerged shake culture using whole bagasse as substrate. The effects of some added carbohydrates on enzyme production, substrate decomposition and biomass formation were examined. In both cultures the addition of lactose stimulated biomass production and increased the decomposition of total carbohydrate and lignin contents of bagasse. Lactose was the only sugar which stimulated cellulase and ligninase in both cultures. 19 references.

Fabrication of graphene oxide decorated with Fe3O4@SiO2 for immobilization of cellulase

Science.gov (United States)

Li, Yue; Wang, Xiang-Yu; Jiang, Xiao-Ping; Ye, Jing-Jing; Zhang, Ye-Wang; Zhang, Xiao-Yun

2015-01-01

Fe3O4@SiO2-graphene oxide (GO) composites were successfully fabricated by chemical binding of functional Fe3O4@SiO2 and GO and applied to immobilization of cellulase via covalent attachment. The prepared composites were further characterized by transmission electron microscopy and Fourier transform infrared spectroscopy. Fe3O4 nanoparticles (NPs) were monodisperse spheres with a mean diameter of 17 ± 0.2 nm. The thickness of SiO2 layer was calculated as being 6.5 ± 0.2 nm. The size of Fe3O4@SiO2 NPs was 24 ± 0.3 nm, similar to that of Fe3O4@SiO2-NH2. Fe3O4@SiO2-GO composites were synthesized by linking of Fe3O4@SiO2-NH2 NPs to GO with the catalysis of EDC and NHS. The prepared composites were used for immobilization of cellulase. A high immobilization yield and efficiency of above 90 % were obtained after the optimization. The half-life of immobilized cellulase (722 min) was 3.34-fold higher than that of free enzyme (216 min) at 50 °C. Compared with the free cellulase, the optimal temperature of the immobilized enzyme was not changed; but the optimal pH was shifted from 5.0 to 4.0, and the thermal stability was enhanced. The immobilized cellulase could be easily separated and reused under magnetic field. These results strongly indicate that the cellulase immobilized onto the Fe3O4@SiO2-GO composite has potential applications in the production of bioethanol.

In situ, rapid, and temporally resolved measurements of cellulase adsorption onto lignocellulosic substrates by UV-vis spectrophotometry

Science.gov (United States)

Hao Liu; J. Y. Zhu; X. S. Chai

2011-01-01

This study demonstrated two in situ UV-vis spectrophotometric methods for rapid and temporally resolved measurements of cellulase adsorption onto cellulosic and lignocellulosic substrates during enzymatic hydrolysis. The cellulase protein absorption peak at 280 nm was used for quantification. The spectral interferences from light scattering by small fibers (fines) and...

Induction and optimization of cellulases using various agro-wastes ...

African Journals Online (AJOL)

SAM

2014-08-13

Aug 13, 2014 ... This study presents optimization of various lignocellulosics and alkali pretreatment for maximum cellulase production by Trichoderma virdii sp. Maximum endoglucanase (642 IU/L) and exoglucanase. (187IU/L) activity was achieved with maize straw at 5% concentration. Oat hay was the most suitable.

Evaluation of Potential Fungal Species for the in situ Simultaneous Saccharification and Fermentation (SSF of Cellulosic Material

Directory of Open Access Journals (Sweden)

Leeuwen, J.

2011-01-01

Full Text Available Three fungal species were evaluated for their abilities to saccharify pure cellulose. The three species chosen represented three major wood-rot molds; brown rot (Gloeophyllum trabeum, white rot (Phanerochaete chrysosporium and soft rot (Trichoderma reesei. After solid state fermentation of the fungi on the filter paper for four days, the saccharified cellulose was then fermented to ethanol by using Saccharomyces cerevisiae. The efficiency of the fungal species in saccharifying the filter paper was compared against a low dose (25 FPU/g cellulose of a commercial cellulase. Total sugar, cellobiose and glucose were monitored during the fermentation period, along with ethanol, acetic acid and lactic acid. Results indicated that the most efficient fungal species in saccharifying the filter paper was T. reesei with 5.13 g/100 g filter paper of ethanol being produced at days 5, followed by P. chrysosporium at 1.79 g/100 g filter paper. No ethanol was detected for the filter paper treated with G. trabeum throughout the five day fermentation stage. Acetic acid was only produced in the sample treated with T. reesei and the commercial enzyme, with concentration 0.95 and 2.57 g/100 g filter paper, respectively at day 5. Lactic acid production was not detected for all the fungal treated filter paper after day 5. Our study indicated that there is potential in utilizing in situ enzymatic saccharification of biomass by using T. reesei and P. chrysosporium that may lead to an economical simultaneous saccharification and fermentation process for the production of fuel ethanol.

Comparative Studies of Oleaginous Fungal Strains (Mucor circinelloides and Trichoderma reesei) for Effective Wastewater Treatment and Bio-Oil Production

OpenAIRE

Bhanja, Anshuman; Minde, Gauri; Magdum, Sandip; Kalyanraman, V.