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Journal articles on the topic 'Cellulose'
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1
Deng, Yijie, and Shiao Y. Wang. "Sorption of Cellulases in Biofilm Enhances Cellulose Degradation by Bacillus subtilis." Microorganisms 10, no. 8 (July 26, 2022): 1505. http://dx.doi.org/10.3390/microorganisms10081505.
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Biofilm commonly forms on the surfaces of cellulosic biomass but its roles in cellulose degradation remain largely unexplored. We used Bacillus subtilis to study possible mechanisms and the contributions of two major biofilm components, extracellular polysaccharides (EPS) and TasA protein, to submerged biofilm formation on cellulose and its degradation. We found that biofilm produced by B. subtilis is able to absorb exogenous cellulase added to the culture medium and also retain self-produced cellulase within the biofilm matrix. The bacteria that produced more biofilm degraded more cellulose compared to strains that produced less biofilm. Knockout strains that lacked both EPS and TasA formed a smaller amount of submerged biofilm on cellulose than the wild-type strain and also degraded less cellulose. Imaging of biofilm on cellulose suggests that bacteria, cellulose, and cellulases form cellulolytic biofilm complexes that facilitate synergistic cellulose degradation. This study brings additional insight into the important functions of biofilm in cellulose degradation and could potentiate the development of biofilm-based technology to enhance biomass degradation for biofuel production.
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Kumar, Amit. "Dissolving pulp production: Cellulases and xylanases for the enhancement of cellulose accessibility and reactivity." Physical Sciences Reviews 6, no. 5 (April 30, 2021): 111–29. http://dx.doi.org/10.1515/psr-2019-0047.
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Abstract Dissolving pulps are high-grade cellulose pulps that have minimum amount of non-cellulosic impurities. Dissolving pulps are the basic source for the manufacturing of several cellulosic products such as viscose, lyocell, cellulose acetates, cellulose nitrates, carboxymethyl-cellulose, etc. Dissolving pulps are mainly manufactured by pre-hydrolysis kraft and acid sulphite pulping. A high reactivity of dissolving pulps is desirable for its eco-friendly utilization for several purposes. Several approaches including mechanical, chemical, ultrasonic, and enzymatic treatments have been employed for the improvement of pulp reactivity. This review mainly focussed on pulp reactivity improvement through enzymatic approaches. Cellulases and xylanase have been proved effective for the improvement of pulp reactivity of dissolving pulp from different sources. The different combinations of cellulase, xylanase, and mechanical refining have been tested and found more effective rather than the single one.
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Hetzler, Stephan, Daniel Bröker, and Alexander Steinbüchel. "Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains." Applied and Environmental Microbiology 79, no. 17 (June 21, 2013): 5159–66. http://dx.doi.org/10.1128/aem.01214-13.
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ABSTRACTThe noncellulolytic actinomyceteRhodococcus opacusstrain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation inR. opacusPD630, we engineered strains that episomally expressed six different cellulase genes fromCellulomonas fimiATCC 484 (cenABC,cex,cbhA) andThermobifida fuscaDSM43792 (cel6A), thereby enablingR. opacusPD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml−1, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose byR. opacus. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabledR. opacusPD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain ofR. opacusPD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step.
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Hall, J., G. W. Black, L. M. A. Ferreira, S. J. Millward-Sadler, B. R. S. Ali, G. P. Hazlewood, and H. J. Gilbert. "The non-catalytic cellulose-binding domain of a novel cellulase from Pseudomonas fluorescens subsp. cellulosa is important for the efficient hydrolysis of Avicel." Biochemical Journal 309, no. 3 (August 1, 1995): 749–56. http://dx.doi.org/10.1042/bj3090749.
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A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA, constructed in lambda ZAPII, was screened for carboxymethyl-cellulase activity. The pseudomonad insert from a recombinant phage which displayed elevated cellulase activity in comparison with other cellulase-positive clones present in the library, was excised into pBluescript SK- to generate the plasmid pC48. The nucleotide sequence of the cellulase gene, designated celE, revealed a single open reading frame of 1710 bp that encoded a polypeptide, defined as endoglucanase E (CelE), of M(r) 59663. The deduced primary structure of CelE revealed an N-terminal signal peptide followed by a 300-amino-acid sequence that exhibited significant identity with the catalytic domains of cellulases belonging to glycosyl hydrolase Family 5. Adjacent to the catalytic domain was a 40-residue region that exhibited strong sequence identity to non-catalytic domains located in two other endoglucanases and a xylanase from P. fluorescens. The C-terminal 100 residues of CelE were similar to Type-I cellulose-binding domains (CBDs). The three domains of the cellulase were joined by linker sequences rich in serine residues. Analysis of the biochemical properties of full-length and truncated derivatives of CelE confirmed that the enzyme comprised an N-terminal catalytic domain and a C-terminal CBD. Analysis of purified CelE revealed that the enzyme had an M(r) of 56000 and an experimentally determined N-terminal sequence identical to residues 40-54 of the deduced primary structure of full-length CelE. The enzyme exhibited an endo mode of action in hydrolysing a range of cellulosic substrates including Avicel and acid-swollen cellulose, but did not attack xylan or any other hemicelluloses. A truncated form of the enzyme, which lacked the C-terminal CBD, displayed the same activity as full-length CelE against soluble cellulose and acid-swollen cellulose, but exhibited substantially lower activity than the full-length cellulase against Avicel. The significance of these data in relation to the role of the CBD is discussed.
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Brumm, Phillip, Phillip Brumm, Dan Xie, Dan Xie, Larry Allen, Larry Allen, David A. Mead, and David A. Mead. "Hydrolysis of Cellulose by Soluble Clostridium Thermocellum and Acidothermus Cellulolyticus Cellulases." Journal of Enzymes 1, no. 1 (April 26, 2018): 5–19. http://dx.doi.org/10.14302/issn.2690-4829.jen-18-2025.
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The goal of this work was to clone, express, characterize and assemble a set of soluble thermostablecellulases capable of significantly degrading cellulose. We successfully cloned, expressed, and purified eleven Clostridium thermocellum (Cthe) cellulases and eight Acidothermuscellulolyticus(Acel) cellulases. The performance of the nineteen enzymes was evaluated on crystalline (filter paper) and amorphous (PASC) cellulose. Hydrolysis products generated from these two substrates were converted to glucose using beta-glucosidase and the glucose formed was determined enzymatically. Ten of the eleven Cthe enzymes were highly active on amorphous cellulose. The individual enzymes all produced <10% reducing sugar equivalents from filter paper. Combinations of Cthe cellulases gave higher conversions, with the combination of CelE, CelI, CelG, and CelK converting 34% of the crystalline cellulose. All eight Acel cellulases showed endo-cellulase activity and were highly active on PASC. Only Acel_0615 produced more than 10% reducing sugar equivalents from filter paper, and a combination of six Acel cellulases produced 32% conversion. Acel_0617, a GH48 exo-cellulase, and Acel_0619, a GH12 endo-cellulase, synergistically stimulated cellulose degradation by the combination of Cthe cellulases to almost 80%. Addition of both Acel enzymes to the Cthe enzyme mix did not further stimulate hydrolysis. Cthe CelG and CelI stimulated cellulose degradation by the combination of Acel cellulases to 66%.
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Chatterjee, Soumya, Sonika Sharma, Rajesh Kumar Prasad, Sibnarayan Datta, Dharmendra Dubey, Mukesh K. Meghvansi, Mohan G. Vairale, and Vijay Veer. "Cellulase Enzyme based Biodegradation of Cellulosic Materials: An Overview." South Asian Journal of Experimental Biology 5, no. 6 (March 11, 2016): 271–82. http://dx.doi.org/10.38150/sajeb.5(6).p271-282.
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Cellulose, a macromolecule of β -D- anhydroglucopyranose units linked by β (1,4)-glycosidic bonds, is the world’s most abundant organic polymer and is the main component of plant biomass that provides stability. Due to its sta-ble fibrous property, it has become one of the most important commercial raw materials for many industries. However, accumulation of waste cellulose due to natural and/or anthropogenic sources is a matter of concern in terms of environmental pollution. Wastes cellulosic substrates can be utilized as sources of energy through controlled hydrolysis using cellulases- a complex group of enzymes capable of degrading all types of cellulosic waste materials. A number of bacteria, fungi and insects are having the capability to degrade cellulose by production of cellulase enzymes. Further, the symbiotic insect-microbe relationships present in the insect gut microbiome for the production of cellulolytic system is of immense importance as this would lead to applications in different fields like biodegradation of cellulosic wastes, pollution reduction, biofuel production, insect/pest control etc. Cel-lulase gene can also be improved by genetic or protein engineering methods using recent technological advances. This review deals with the advances of cellulase enzymes and its utilization for different application.
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Pratama, Rahadian, I. Made Artika, Tetty Chaidamsari, Herti Sugiarti, and Soekarno Mismana Putra. "Isolation and Molecular Cloning of Cellulase Gene from Bovine Rumen Bacteria." Current Biochemistry 1, no. 1 (September 2, 2017): 29–36. http://dx.doi.org/10.29244/cb.1.1.29-36.
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Cellulases are the enzymes that hydrolyze cellulosic biomass and are produced by the microorganisms that grow over cellulosic matters. The objective of this research was to isolate and clone cellulase gene from cellulose-degrading bacteria of bovine rumen. Cellulose-degrading bacteria was isolated from rumen fluid using a selective medium. Total RNA was isolated from selected colony having cellulose degrading activity and was used as a template for cDNA construction using reverse transcriptase polymerase chain reaction (RT-PCR) technique. The resulted cDNA was employed as a template for PCR amplification of cellulase gene using specific primers. The cellulase gene candidate obtained was cloned into the pGEM-T-Easy vector followed by determination of its nucleotide sequence. The sequence was then aligned with sequences of cellulase genes from GenBank. Results showed that a number of isolates of rumen bacteria exhibit cellulase activity and the CR-8 isolate was selected for further analysis. The successful isolation of total RNA from CR-8 was indicated by the presence of two intense bands of ribosomal RNA (23S and 16S). The reverse transcription process was successful and the amplification of cellulase gene using the specific primers F1 and R1 resulted in a DNA fragment of 1900 bp as a candidate of cellulase gene. The fragment was successfully cloned into the pGEM-T-Easy vector, and the resulted recombinant plasmid was successfully introduced into the E. coli cells. Nucleotide sequence analysis suggested that the cloned gene is cellulase gene and shares 99% homology with the endo-1,6-beta-glucanase of T. harzianum.
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Li, Xia, Xiaoyan Geng, Lu Gao, Yanfang Wu, Yongli Wang, Alei Geng, Jianzhong Sun, and Jianxiong Jiang. "Optimized expression of a hyperthermostable endoglucanase from Pyrococcus horikoshii in Arabidopsis thaliana." BioResources 14, no. 2 (February 19, 2019): 2812–26. http://dx.doi.org/10.15376/biores.14.2.2812-2826.
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Manufacturing microbial cellulase in plants is an attractive strategy for the cost-effective production of cellulosic ethanol, especially the expression of thermostable cellulase, which causes no negative effects on plant growth and development. The beta-1,4-endogenous cellulase from Pyrococcus horikoshii (EGPh) is considered one of the most promising glycosyl hydrolase in the biofuel and textile industry for its hyperthermostability and its capability to hydrolyze crystalline celluloses, which has been researched extensively during recent years. In this study, the coding sequence of EGPh was expressed in Arabidopsis thaliana under the control of a CaMV35S promoter after codon optimization, with the addition of a eukaryotic Kozak sequence. The expression of EGPh caused no deleterious effects to the growth and development of transgenic A. thaliana. The heterologous EGPh showed relatively high activities, up to 111.69 and 13.35 U.mg-1 total soluble protein against soluble cellulose carboxymethyl cellulose (CMC) and insoluble microcrystalline cellulose (Avicel), respectively. The subcellular localization analysis showed that the EGPh protein was targeted to the plasma membrane and cell wall. Based on these results, it is proposed that EGPh is an ideal candidate for the commercial production of hyperthermostable endoglucanase using plants as biofactories.
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Mizuno, Masahiro, Shuji Kachi, Eiji Togawa, Noriko Hayashi, Kouichi Nozaki, Toshiyuki Itoh, and Yoshihiko Amano. "Structure of Regenerated Celluloses Treated with Ionic Liquids and Comparison of their Enzymatic Digestibility by Purified Cellulase Components." Australian Journal of Chemistry 65, no. 11 (2012): 1491. http://dx.doi.org/10.1071/ch12342.
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In this study, regenerated celluloses were prepared from microcrystalline cellulose (MCC) by treatment with three ionic liquids (ILs) having 1-ethyl-3-methylimidazolium (Emim) as the cation, and the IL N-(2-methoxyethyl)-N,N-diethyl-N-methylammonium alanine ([N221ME][Ala]), where the amino acid moiety is the anion. The crystal form of cellulose was transformed from cellulose I to cellulose II by dissolution with an IL and regeneration with anti-solvent. However, the crystallinity of the regenerated cellulose was different; the disordered chain region was increased in the order of [N221ME][Ala] < [Emim][OAc] < [Emim][DEP] < [Emim][Cl]. The monocomponent cellulase, especially endoglucanase, showed high hydrolyzing activity for regenerated cellulose compared with untreated cellulose. Furthermore, the degree of increase of hydrolyzing activity was almost coincident with the order of crystallinity. For the effective hydrolysis of cellulose treated with an IL, it is necessary to prepare the cellulase mixture containing an adequate ratio of each cellulase component according to crystal allomorph and the crystallinity of regenerated cellulose.
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Bu, Yingjie, Bassam Alkotaini, Bipinchandra K. Salunke, Aarti R. Deshmukh, Pathikrit Saha, and Beom Soo Kim. "Direct ethanol production from cellulose by consortium of Trichoderma reesei and Candida molischiana." Green Processing and Synthesis 8, no. 1 (January 28, 2019): 416–20. http://dx.doi.org/10.1515/gps-2019-0009.
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Abstract Industrial cellulosic ethanol production is a challenge due to the high cost of cellulases for hydrolysis when lignocellulosic materials are used as feedstock. In this study, direct ethanol production from cellulose was performed by consortium of Trichoderma reesei and Candida molischiana. Cellulose was hydrolyzed by a fully enzymatic saccharification process using Trichoderma reesei cellulases. The produced reducing sugar was further utilized by Candida molischiana for ethanol production. Because the optimal temperature for the cellulase system is approximately 50°C, the effect of temperature rise from 30°C to 50°C on cellulose hydrolysis was investigated. The results showed that the temperature rise from 30°C to 50°C after 36 h of cultivation was the best for reducing sugar and glucose production. Under these conditions, the maximum concentrations of reducing sugar and glucose produced by T. reesei were 8.0 g/L and 4.6 g/L at 60 h, respectively. The maximum production of ethanol by C. molischiana was 3.0 g/L after 120 h.
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Kashcheyeva, Ekaterina I., Yulia A. Gismatulina, Galina F. Mironova, Evgenia K. Gladysheva, Vera V. Budaeva, Ekaterina A. Skiba, Vladimir N. Zolotuhin, Nadezhda A. Shavyrkina, Aleksey N. Kortusov, and Anna A. Korchagina. "Properties and Hydrolysis Behavior of Celluloses of Different Origin." Polymers 14, no. 18 (September 18, 2022): 3899. http://dx.doi.org/10.3390/polym14183899.
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The present paper is a fundamental study on the physicochemical properties and hydrolysis behavior of cellulose samples differing in origin: bacterial, synthetic, and vegetal. Bacterial cellulose was produced by Medusomyces gisevii Sa-12 in an enzymatic hydrolyzate derived from oat-hull pulp. Synthetic cellulose was obtained from an aqueous glucose solution by electropolymerization. Plant-based cellulose was isolated by treatment of Miscanthus sacchariflorus with dilute NaOH and HNO3 solutions. We explored different properties of cellulose samples, such as chemical composition, degree of polymerization (DP), degree of crystallinity (DC), porosity, and reported infrared spectroscopy and scanning electron microscopy results. The hydrolysis behavior was most notable dependent on the origin of cellulose. For the bacterial cellulose sample (2010 DP, 90% DC, 89.4% RS yield), the major property affecting the hydrolysis behavior was its unique nanoscale reticulate structure promoting fast penetration of cellulases into the substrate structure. The study on enzymatic hydrolysis showed that the hydrolysis behavior of synthetic and Miscanthus celluloses was most influenced by the substrate properties such as DP, DC and morphological structure. The yield of reducing sugars (RS) by hydrolysis of synthetic cellulose exhibiting a 3140 DP, 80% DC, and highly depolymerization-resistant fibers was 27%. In contrast, the hydrolysis of Miscanthus-derived cellulose with a 1030 DP, 68% DC, and enzyme-accessible fibers provided the highest RS yield of 90%. The other properties examined herein (absence/presence of non-cellulosic impurities, specific surface, pore volume) had no considerable effect on the bioconversion of the cellulosic substrates.
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Lynd, Lee R., Paul J. Weimer, Willem H. van Zyl, and Isak S. Pretorius. "Microbial Cellulose Utilization: Fundamentals and Biotechnology." Microbiology and Molecular Biology Reviews 66, no. 3 (September 2002): 506–77. http://dx.doi.org/10.1128/mmbr.66.3.506-577.2002.
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SUMMARY Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for “consolidated bioprocessing” (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.
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Tokuda, Gaku, and Hirofumi Watanabe. "Hidden cellulases in termites: revision of an old hypothesis." Biology Letters 3, no. 3 (March 20, 2007): 336–39. http://dx.doi.org/10.1098/rsbl.2007.0073.
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The intestinal flagellates of termites produce cellulases that contribute to cellulose digestion of their host termites. However, 75% of all termite species do not harbour the cellulolytic flagellates; the endogenous cellulase secreted from the midgut tissue has been considered a sole source of cellulases in these termites. Using the xylophagous flagellate-free termites Nasutitermes takasagoensis and Nasutitermes walkeri , we successfully solubilized cellulases present in the hindgut pellets. Zymograms showed that the hindguts of these termites possessed several cellulases and contained up to 59% cellulase activity against crystalline cellulose when compared with the midgut. Antibiotic treatment administered to N. takasagoensis significantly reduced cellulase activity in the hindgut, suggesting that these cellulases were produced by symbiotic bacteria.
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Poomai, Nutt, Wilailak Siripornadulsil, and Surasak Siripornadulsil. "Cellulase Enzyme Production from Agricultural Waste by Acinetobacter sp. KKU44." Advanced Materials Research 931-932 (May 2014): 1106–10. http://dx.doi.org/10.4028/www.scientific.net/amr.931-932.1106.
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Due to a high ethanol demand, the approach for effective ethanol production is important and has been developed rapidly worldwide. Several agricultural wastes are highly abundant in celluloses and the effective cellulase enzymes do exist widely among microorganisms. Accordingly, the cellulose degradation using microbial cellulase to produce a low-cost substrate for ethanol production has attracted more attention. In this study, the cellulase producing bacterial strain has been isolated from rich straw and identified by 16S rDNA sequence analysis as Acinetobacter sp. KKU44. This strain is able to grow and exhibit the cellulase activity. The optimal temperature for its growth and cellulase production is 37 °C. The optimal temperature of bacterial cellulase activity is 60 °C. The cellulase enzyme from Acinetobacter sp. KKU44 is heat-tolerant enzyme. The bacterial culture of 36 h. showed highest cellulase activity at 120 U/mL when grown in LB medium containing 2% (w/v). The capability of Acinetobacter sp. KKU44 to grow in cellulosic agricultural wastes as a sole carbon source and exhibiting the high cellulase activity at high temperature suggested that this strain could be potentially developed further as a cellulose degrading strain for a production of low-cost substrate used in ethanol production.
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Liu, Jun, and Huiren Hu. "The role of cellulose binding domains in the adsorption of cellulases onto fibers and its effect on the enzymatic beating of bleached kraft pulp." BioResources 7, no. 1 (January 11, 2012): 878–92. http://dx.doi.org/10.15376/biores.7.1.878-892.
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The adsorption of cellulases onto fibers may be one of the most important factors affecting the enzymatic reaction between cellulases and fibers. This study investigated the adsorption kinetics involved, using isothermal adsorption equations. Cellulose binding domains (CBDs) were isolated from a commercial cellulase, and their role in the adsorption and enzymatic reaction was evaluated. Approximately 13% to 24% of the refining energy was saved after northern bleached softwood kraft pulp samples were pretreated with full cellulase, CBDs, or cellulase lacking CBDs under optimal conditions. The absence of CBDs in cellulase resulted in less effective enzyme adsorption and hydrolysis of the fibers. These data suggest that pretreatment of northern bleached softwood kraft pulp with CBDs may not only improve the beating degree of the pulp and reduce refining energy consumption but also improve the tensile index of the handsheet. Analysis of the degree of cellulose crystallinity and fiber surface morphology by X-ray diffraction and scanning electron microscopy revealed that the CBDs in cellulase help modify the crystalline area and facilitate the enzymatic degradation of cellulose. The adsorption parameters of the cellulases calculated from isothermal adsorption experiments confirmed the role of CBDs in the adsorption of cellulases onto fibers.
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Liu, Wenjin, Xiao-Zhou Zhang, Zuoming Zhang, and Y. H. Percival Zhang. "Engineering of Clostridium phytofermentans Endoglucanase Cel5A for Improved Thermostability." Applied and Environmental Microbiology 76, no. 14 (May 28, 2010): 4914–17. http://dx.doi.org/10.1128/aem.00958-10.
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ABSTRACT A family 5 glycoside hydrolase from Clostridium phytofermentans was cloned and engineered through a cellulase cell surface display system in Escherichia coli. The presence of cell surface anchoring, a cellulose binding module, or a His tag greatly influenced the activities of wild-type and mutant enzymes on soluble and solid cellulosic substrates, suggesting the high complexity of cellulase engineering. The best mutant had 92%, 36%, and 46% longer half-lives at 60°C on carboxymethyl cellulose, regenerated amorphous cellulose, and Avicel, respectively.
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Wang, Hongliang, Fabio Squina, Fernando Segato, Andrew Mort, David Lee, Kirk Pappan, and Rolf Prade. "High-Temperature Enzymatic Breakdown of Cellulose." Applied and Environmental Microbiology 77, no. 15 (June 17, 2011): 5199–206. http://dx.doi.org/10.1128/aem.00199-11.
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ABSTRACTCellulose is an abundant and renewable biopolymer that can be used for biofuel generation; however, structural entrapment with other cell wall components hinders enzyme-substrate interactions, a key bottleneck for ethanol production. Biomass is routinely subjected to treatments that facilitate cellulase-cellulose contacts. Cellulases and glucosidases act by hydrolyzing glycosidic bonds of linear glucose β-1,4-linked polymers, producing glucose. Here we describe eight high-temperature-operating cellulases (TCel enzymes) identified from a survey of thermobacterial and archaeal genomes. Three TCel enzymes preferentially hydrolyzed soluble cellulose, while two preferred insoluble cellulose such as cotton linters and filter paper. TCel enzymes had temperature optima ranging from 85°C to 102°C. TCel enzymes were stable, retaining 80% of initial activity after 120 h at 85°C. Two modes of cellulose breakdown, i.e., with endo- and exo-acting glucanases, were detected, and with two-enzyme combinations at 85°C, synergistic cellulase activity was observed for some enzyme combinations.
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Ilić, Nevena, Marija Milić, Sunčica Beluhan, and Suzana Dimitrijević-Branković. "Cellulases: From Lignocellulosic Biomass to Improved Production." Energies 16, no. 8 (April 21, 2023): 3598. http://dx.doi.org/10.3390/en16083598.
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Cellulases are enzymes that are attracting worldwide attention because of their ability to degrade cellulose in the lignocellulosic biomass and transform it into highly demanded bioethanol. The enzymatic hydrolysis of cellulose by cellulases into fermentable sugars is a crucial step in biofuel production, given the complex structure of lignocellulose. Due to cellulases’ unique ability to hydrolyze the very recaltricant nature of lignocellulosic biomass, the cellulase market demand is rapidly growing. Although cellulases have been used in industrial applications for decades, constant effort is being made in the field of enzyme innovation to develop cellulase mixtures/cocktails with improved performance. Given that the main producers of cellulases are of microbial origin, there is a constant need to isolate new microorganisms as potential producers of enzymes important for biofuel production. This review provides insight into current research on improving microbial cellulase production as well as the outlook for the cellulase market with commercial cellulase preparation involved in industrial bioethanol production.
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KIPPER, Kalle, Priit VÄLJAMÄE, and Gunnar JOHANSSON. "Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as ‘burst’ kinetics on fluorescent polymeric model substrates." Biochemical Journal 385, no. 2 (January 7, 2005): 527–35. http://dx.doi.org/10.1042/bj20041144.
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Reaction conditions for the reducing-end-specific derivatization of cellulose substrates with the fluorogenic compound, anthranilic acid, have been established. Hydrolysis of fluorescence-labelled celluloses by cellobiohydrolase Cel7A from Trichoderma reesei was consistent with the active-site titration kinetics (burst kinetics), which allowed the quantification of the processivity of the enzyme. The processivity values of 88±10, 42±10 and 34±2.0 cellobiose units were found for Cel7A acting on labelled bacterial cellulose, bacterial microcrystalline cellulose and endoglucanase-pretreated bacterial cellulose respectively. The anthranilic acid derivatization also provides an alternative means for estimating the average degree of polymerization of cellulose and, furthermore, allows the quantitative monitoring of the production of reducing end groups on solid cellulose on hydrolysis by cellulases. Hydrolysis of bacterial cellulose by cellulases from T. reesei revealed that, by contrast with endoglucanase Cel5A, neither cellobiohydrolases Cel7A nor Cel6A produced detectable amounts of new reducing end groups on residual cellulose.
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Ma, Yuan Yuan, Xin Wang, Han Ze Wang, Kun Zhang, and Min Hua Zhang. "The Expression In Vitro and Application on Cellulose Degradation of LeEXP2." Advanced Materials Research 183-185 (January 2011): 790–94. http://dx.doi.org/10.4028/www.scientific.net/amr.183-185.790.
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Cellulosic ethanol has become a hotspot in recent years. However, its crystal structure makes the efficiency of cellulosic degradation by cellulase very low. Traditional ways to disrupt of connection between microfiber consumes a deal of energy and would pollute the environment as well. Plant expansin is known to loosen the plant cell wall, and might provide a synergistic effect on the activities of cellulase. Whereas, the expression level of expansin in plants has been a limit to the functional study and application in cellulose degradation. Thus, it is essential to screen expansin proteins for biomass deconstruction and express them effectively in vitro. Therefore, we cloned expansin gene LeEXP2 from tomato leaves and obtained recombinant Pichia yeast strains integrated with LeEXP2 gene. When incubated in the same culture condition, recombinant strains can secrete the LeEXP2 protein to extracellular medium, while wild-type strain cannot. Preliminary cellulose degradation experiment confirmed that the secreted protein had synergistic the effect of cellulose hydrolysis by cellulase. The experiments of extension strength of filter-paper strips shows that LeEXP2 has a texture-loosening effect on the filter paper, which might make cellulase prone to access cellulose. Above data suggests that LeEXP2 could be expressed effectively in vitro and might become a kind of potential biochemical agent applied in cellulosic biomass conversion for bioenergy production.
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Wu, Bin, Yue Zhao, and Pei Ji Gao. "A new approach to measurement of saccharifying capacities of crude cellulase." BioResources 1, no. 2 (October 3, 2006): 189–200. http://dx.doi.org/10.15376/biores.1.2.189-200.
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A practical, quantitative approach has been designed, which makes it possible to accurately estimate the saccharifying activities of crude cellulase preparations for insoluble cellulosics. The challenge in activity determination imposed by changes in hydrolysis time and concentration of cellulase and cellulosics on the assay could be overcome by selection of the specific conversion percentage of cellulose as a function of cellulase concentration, that is, the hydrolysis percentage of filter paper by unit cellulase per minute, as the objective function with respect to different concentrations of crude cellulase. A rational and governing equation for crude cellulase assay was derived , and reliable results for quantitatively estimating the saccharifying activities of crude cellulases during the progress of hydrolysis of several cellulosic substrates were obtained.
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Liang, Youyun, Tong Si, Ee Lui Ang, and Huimin Zhao. "Engineered Pentafunctional Minicellulosome for Simultaneous Saccharification and Ethanol Fermentation in Saccharomyces cerevisiae." Applied and Environmental Microbiology 80, no. 21 (August 22, 2014): 6677–84. http://dx.doi.org/10.1128/aem.02070-14.
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ABSTRACTSeveral yeast strains have been engineered to express different cellulases to achieve simultaneous saccharification and fermentation of lignocellulosic materials. However, successes in these endeavors were modest, as demonstrated by the relatively low ethanol titers and the limited ability of the engineered yeast strains to grow using cellulosic materials as the sole carbon source. Recently, substantial enhancements to the breakdown of cellulosic substrates have been observed when lytic polysaccharide monooxygenases (LPMOs) were added to traditional cellulase cocktails. LPMOs are reported to cleave cellulose oxidatively in the presence of enzymatic electron donors such as cellobiose dehydrogenases. In this study, we coexpressed LPMOs and cellobiose dehydrogenases with cellobiohydrolases, endoglucanases, and β-glucosidases inSaccharomyces cerevisiae. These enzymes were secreted and docked onto surface-displayed miniscaffoldins through cohesin-dockerin interaction to generate pentafunctional minicellulosomes. The enzymes on the miniscaffoldins acted synergistically to boost the degradation of phosphoric acid swollen cellulose and increased the ethanol titers from our previously achieved levels of 1.8 to 2.7 g/liter. In addition, the newly developed recombinant yeast strain was also able to grow using phosphoric acid swollen cellulose as the sole carbon source. The results demonstrate the promise of the pentafunctional minicellulosomes for consolidated bioprocessing by yeast.
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Zhou, Qingxin, Jintao Xu, Yanbo Kou, Xinxing Lv, Xi Zhang, Guolei Zhao, Weixin Zhang, Guanjun Chen, and Weifeng Liu. "Differential Involvement of β-Glucosidases from Hypocrea jecorina in Rapid Induction of Cellulase Genes by Cellulose and Cellobiose." Eukaryotic Cell 11, no. 11 (September 21, 2012): 1371–81. http://dx.doi.org/10.1128/ec.00170-12.
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ABSTRACTAppropriate perception of cellulose outside the cell by transforming it into an intracellular signal ensures the rapid production of cellulases by cellulolyticHypocrea 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 inH. jecorina. Here we demonstrated that CEL1b, like CEL1a, was an intracellular β-glucosidase displayingin vitrotransglycosylation 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 ofcel1aandcel1bsignificantly 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 inH. jecorina. They may otherwise contribute to the accumulation of cellobiose from cellulose as inducing signals.
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Kudanga, T., and E. Mwenje. "Extracellular cellulase production by tropical isolates of Aureobasidium pullulans." Canadian Journal of Microbiology 51, no. 9 (September 1, 2005): 773–76. http://dx.doi.org/10.1139/w05-053.
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Cellulase production by Aureobasidium pullulans from the temperate regions has remained speculative, with most studies reporting no activity at all. In the current study, tropical isolates from diverse sources were screened for cellulase production. Isolates were grown on a synthetic medium containing cell walls of Msasa tree (Brachystegia sp.) as the sole carbon source, and their cellulolytic activities were measured using carboxymethyl cellulose and α-cellulose as substrates. All isolates studied produced carboxymethyl cellulase (endoglucanase) and alpha-cellulase (exoglucanase) activity. Endoglucanase-specific activities of ten selected isolates ranged from 2.375 to 12.884 µmol glucose·(mg protein)–1·h–1, while activities on α-cellulose (exoglucanase activity) ranged from 0.293 to 22.442 µmol glucose·(mg protein)–1·day–1. Carboxymethyl cellulose induced the highest cellulase activity in the selected isolates, while the isolates showed variable responses to nitrogen sources. The current study indicates that some isolates of A. pullulans of tropical origin produce significant extracellular cellulolytic activity and that crude cell walls may be good inducers of cellulolytic activity in A. pullulans.Key words: Aureobasidium pullulans, plant cell wall, cellulases, endoglucanase, exoglucanase.
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Nicomrat, Duongruitai, and Jirasak Tharajak. "Synergistic Effects of Cellulase-Producing Microorganisms for Future Bioconversion of Lignocellulosic Biomass." Applied Mechanics and Materials 804 (October 2015): 255–58. http://dx.doi.org/10.4028/www.scientific.net/amm.804.255.
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Lignocellulosic biomass can nowadays be bioconverted to value-added biofuels by numerous cellulases purified from diverse microbes. In nature, complex microbial communities produce multifunctional cellulase systems with broader substrate utilization and act sequentially in the synergistic action by subsequently converting cellulose into an utilizable energy source and glucose. This research was to apply cellulase producing isolates based on their possible synergistic action to degrade complex cellulose containing biomass. In the study, the microorganism species, isolated species from durian peels after macerated for 3 days and shown for their high biodegradation activity, Bacillus spp. (B12, B13, and B16) and Pseudomonas spp., (B23 and B55), could express high cellulase activity on carboxymethylcellulose (CMC) and filter paper (FP). Bacillus spp. B13 and B16 showed high cellulase activity on soluble cellulose of CMC while B12 and B55 displayed high cellulase activity on crystalline insoluble cellulose of FP. To observe the synergistic effect of the cellulase-producing consortia, co-cultures of B12,B23 and F23 were grew well on both CMC and FP. Therefore, these findings of synergistic effects of microbial consortia could bring us a future work to develop high efficient cellulase producing systems for further industry application.
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Igarashi, Kiyohiko, Takayuki Uchihashi, Anu Koivula, Masahisa Wada, Satoshi Kimura, Tetsuaki Okamoto, Merja Penttilä, Toshio Ando, and Masahiro Samejima. "Traffic Jams Reduce Hydrolytic Efficiency of Cellulase on Cellulose Surface." Science 333, no. 6047 (September 1, 2011): 1279–82. http://dx.doi.org/10.1126/science.1208386.
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A deeper mechanistic understanding of the saccharification of cellulosic biomass could enhance the efficiency of biofuels development. We report here the real-time visualization of crystalline cellulose degradation by individual cellulase enzymes through use of an advanced version of high-speed atomic force microscopy. Trichoderma reesei cellobiohydrolase I (TrCel7A) molecules were observed to slide unidirectionally along the crystalline cellulose surface but at one point exhibited collective halting analogous to a traffic jam. Changing the crystalline polymorphic form of cellulose by means of an ammonia treatment increased the apparent number of accessible lanes on the crystalline surface and consequently the number of moving cellulase molecules. Treatment of this bulky crystalline cellulose simultaneously or separately with T. reesei cellobiohydrolase II (TrCel6A) resulted in a remarkable increase in the proportion of mobile enzyme molecules on the surface. Cellulose was completely degraded by the synergistic action between the two enzymes.
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Ndukwe, Nelly A., J. Boitumelo M. Sibiya, and J. Pieter H. Van Wyk. "Saccharification of Sawdust with Aspergillus Niger Cellulase." Journal of Solid Waste Technology and Management 46, no. 3 (August 1, 2020): 321–27. http://dx.doi.org/10.5276/jswtm/2020.321.
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Accumulated sawdust is a major waste product produced by numerous active sawmills around the Lagos Lagoon in Nigeria. The potential of this wood waste as a resource for bio-product development through the hydrolysis of its cellulose component into glucose, a fermentable sugar is not yet appreciated. Not only is the environment exposed to this organic pollutant but the health of humans is also at risk. The cellulose content of wood sawdust from five different tropical tree species dumped along the Lagos lagoon has been saccharified with cellulase from Aspergillus niger. In order to increase the amount of fermentable sugars released from the cellulose content the various sawdust samples have been delignified with the Kraft process and hydrogen peroxide treatment prior to A. niger cellulase catalyzed degradation at incubation temperatures of 30 0C, 400C, 500C and 600C, respectively. The delignification process was successful by triggering an increase in sugar formation during cellulase catalyze hydrolyses of all waste cellulose materials with different amounts of sugar produced from the various celluloses.
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Poole, D. M., A. J. Durrant, G. P. Hazlewood, and H. J. Gilbert. "Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains." Biochemical Journal 279, no. 3 (November 1, 1991): 787–92. http://dx.doi.org/10.1042/bj2790787.
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The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp. cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum. A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P. fluorescens subsp. cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus. The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained. The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively. Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively. The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD. The putative role of the P. fluorescens subsp. cellulosa CBD in cellulase action is discussed.
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Yadav, Vikas, Bruce J. Paniliatis, Hai Shi, Kyongbum Lee, Peggy Cebe, and David L. Kaplan. "Novel In Vivo-Degradable Cellulose-Chitin Copolymer from Metabolically Engineered Gluconacetobacter xylinus." Applied and Environmental Microbiology 76, no. 18 (July 23, 2010): 6257–65. http://dx.doi.org/10.1128/aem.00698-10.
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ABSTRACT Despite excellent biocompatibility and mechanical properties, the poor in vitro and in vivo degradability of cellulose has limited its biomedical and biomass conversion applications. To address this issue, we report a metabolic engineering-based approach to the rational redesign of cellular metabolites to introduce N-acetylglucosamine (GlcNAc) residues into cellulosic biopolymers during de novo synthesis from Gluconacetobacter xylinus. The cellulose produced from these engineered cells (modified bacterial cellulose [MBC]) was evaluated and compared with cellulose produced from normal cells (bacterial cellulose [BC]). High GlcNAc content and lower crystallinity in MBC compared to BC make this a multifunctional bioengineered polymer susceptible to lysozyme, an enzyme widespread in the human body, and to rapid hydrolysis by cellulase, an enzyme commonly used in biomass conversion. Degradability in vivo was demonstrated in subcutaneous implants in mice, where modified cellulose was completely degraded within 20 days. We provide a new route toward the production of a family of tailorable modified cellulosic biopolymers that overcome the longstanding limitation associated with the poor degradability of cellulose for a wide range of potential applications.
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Bae, Jungu, Kouichi Kuroda, and Mitsuyoshi Ueda. "Proximity Effect among Cellulose-Degrading Enzymes Displayed on the Saccharomyces cerevisiae Cell Surface." Applied and Environmental Microbiology 81, no. 1 (October 10, 2014): 59–66. http://dx.doi.org/10.1128/aem.02864-14.
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ABSTRACTProximity effect is a form of synergistic effect exhibited when cellulases work within a short distance from each other, and this effect can be a key factor in enhancing saccharification efficiency. In this study, we evaluated the proximity effect between 3 cellulose-degrading enzymes displayed on theSaccharomyces cerevisiaecell surface, that is, endoglucanase, cellobiohydrolase, and β-glucosidase. We constructed 2 kinds of arming yeasts through genome integration: ALL-yeast, which simultaneously displayed the 3 cellulases (thus, the different cellulases were near each other), and MIX-yeast, a mixture of 3 kinds of single-cellulase-displaying yeasts (the cellulases were far apart). The cellulases were tagged with a fluorescence protein or polypeptide to visualize and quantify their display. To evaluate the proximity effect, we compared the activities of ALL-yeast and MIX-yeast with respect to degrading phosphoric acid-swollen cellulose after adjusting for the cellulase amounts. ALL-yeast exhibited 1.25-fold or 2.22-fold higher activity than MIX-yeast did at a yeast concentration equal to the yeast cell number in 1 ml of yeast suspension with an optical density (OD) at 600 nm of 10 (OD10) or OD0.1. At OD0.1, the distance between the 3 cellulases was greater than that at OD10 in MIX-yeast, but the distance remained the same in ALL-yeast; thus, the difference between the cellulose-degrading activities of ALL-yeast and MIX-yeast increased (to 2.22-fold) at OD0.1, which strongly supports the proximity effect between the displayed cellulases. A proximity effect was also observed for crystalline cellulose (Avicel). We expect the proximity effect to further increase when enzyme display efficiency is enhanced, which would further increase cellulose-degrading activity. This arming yeast technology can also be applied to examine proximity effects in other diverse fields.
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Touijer, Hanane, Najoua Benchemsi, Mohamed Ettayebi, Abdellatif Janati Idrissi, Bouchra Chaouni, and Hicham Bekkari. "Thermostable Cellulases from the Yeast Trichosporon sp." Enzyme Research 2019 (April 17, 2019): 1–6. http://dx.doi.org/10.1155/2019/2790414.
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Objectives. Identification of cellulolytic microorganisms is of great interest to the hydrolysis of cellulosic biomass. This study focuses on the identification of cellulolytic yeasts and the optimization of cellulase activities produced by the best performing isolate. Results. 30 cellulolytic yeast isolates were selected. Enzymes produced by an isolate from the Trichosporon genus showed the property to hydrolyze different substrates: carboxymethyl cellulose (CMC), cellulose fiber, and filter paper (FP). The optimum measured temperature was 55°C for CMCase and 60°C for FPase. The optimal pH was 5 for CMCase and 4 to 6 for FPase. The effect of the substrates concentration showed that the best activities were obtained at 100 mg/mL CMC or FP. The highest activities were 0.52 for the CMCase and 0.56 for the cellulase fiber at 10 min incubation, 0.44 IU/mL at 15 min incubation, and 24 h FPase preincubation. Conclusion. Cellulases produced by the studied yeast are capable of hydrolyzing soluble and insoluble substrates at elevated temperatures and at a wide pH range. They are considerable interest in the production of fermentable sugars from lignocellulosic substrates.
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Wang, Na, Zhihua Yan, Na Liu, Xiaorong Zhang, and Chenggang Xu. "Synergy of Cellulase Systems between Acetivibrio thermocellus and Thermoclostridium stercorarium in Consolidated-Bioprocessing for Cellulosic Ethanol." Microorganisms 10, no. 3 (February 24, 2022): 502. http://dx.doi.org/10.3390/microorganisms10030502.
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Anaerobes harbor some of the most efficient biological machinery for cellulose degradation, especially thermophilic bacteria, such as Acetivibrio thermocellus and Thermoclostridium stercorarium, which play a fundamental role in transferring lignocellulose into ethanol through consolidated bioprocessing (CBP). In this study, we compared activities of two cellulase systems under varying kinds of hemicellulose and cellulose. A. thermocellus was identified to contribute specifically to cellulose hydrolysis, whereas T. stercorarium contributes to hemicellulose hydrolysis. The two systems were assayed in various combinations to assess their synergistic effects using cellulose and corn stover as the substrates. Their maximum synergy degrees on cellulose and corn stover were, respectively, 1.26 and 1.87 at the ratio of 3:2. Furthermore, co-culture of these anaerobes on the mixture of cellulose and xylan increased ethanol concentration from 21.0 to 40.4 mM with a high cellulose/xylan-to-ethanol conversion rate of up to 20.7%, while the conversion rates of T. stercorarium and A. thermocellus monocultures were 19.3% and 15.2%. The reason is that A. thermocellus had the ability to rapidly degrade cellulose while T. stercorarium co-utilized both pentose and hexose, the metabolites of cellulose degradation, to produce ethanol. The synergistic effect of cellulase systems and metabolic pathways in A. thermocellus and T. stercorarium provides a novel strategy for the design, selection, and optimization of ethanol production from cellulosic biomass through CBP.
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Ahmed, Mohamed, Soad El-Zayat, and Magdi El-Sayed. "Cellulolytic activity of cellulose-decomposing fungi isolated from Aswan hot desert soil, Egypt." Journal of Biological Studies 1, no. 2 (June 2, 2018): 35–48. http://dx.doi.org/10.62400/jbs.v1i2.9.
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Fungi are well known agents for decomposition of organic matter in common and of cellulosic material, in particular; therefore they considered as the main cellulose producing microorganisms. The present study was aimed to isolate and screen the ability of cellulolytic fungal strains from desert soil living under environmental stress to produce cellulolytic enzymes. Forty-three fungal strains in addition to two varieties were isolated from different sites at Aswan University campus and were able to degrade the cellulose with variable extents. Fungal species were grouped as high, moderate and low cellulolytic activity. The cellulase activity was assayed by carboxymethyl-cellulose "CMase" assay (endoglucanases).The highest cellulase producing isolates were Fusarium dimerum and Rhizopus oryzae. The optimum parameters for high activity of cellulase enzyme are pH 5, 35C at 9th, 11 thdays for Fusarium dimerum and Rhizopus oryzae, respectively.
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Singh, Nivisti, Bishop Bruce Sithole, and Roshini Govinden. "Optimisation of β-Glucosidase Production in a Crude Aspergillus japonicus VIT-SB1 Cellulase Cocktail Using One Variable at a Time and Statistical Methods and its Application in Cellulose Hydrolysis." International Journal of Molecular Sciences 24, no. 12 (June 9, 2023): 9928. http://dx.doi.org/10.3390/ijms24129928.
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Pulp and paper mill sludge (PPMS) is currently disposed of into landfills which are reaching their maximum capacity. Valorisation of PPMS by enzymatic hydrolysis using cellulases is an alternative strategy. Existing commercial cellulases are expensive and contain low titres of β-glucosidases. In this study, β-glucosidase production was optimised by Aspergillus japonicus VIT-SB1 to obtain higher β-glucosidase titres using the One Variable at a Time (OVAT), Plackett Burman (PBD), and Box Behnken design (BBD)of experiments and the efficiency of the optimised cellulase cocktail to hydrolyse cellulose was tested. β-Glucosidase production was enhanced from 0.4 to 10.13 U/mL, representing a 25.3-fold increase in production levels after optimisation. The optimal BBD production conditions were 6 days of fermentation at 20 °C, 125 rpm, 1.75% soy peptone, and 1.25% wheat bran in (pH 6.0) buffer. The optimal pH for β-glucosidase activity in the crude cellulase cocktail was (pH 5.0) at 50 °C. Optimal cellulose hydrolysis using the crude cellulase cocktail occurred at longer incubation times, and higher substrate loads and enzyme doses. Cellulose hydrolysis with the A. japonicus VIT-SB1 cellulase cocktail and commercial cellulase cocktails resulted in glucose yields of 15.12 and 12.33 µmol/mL glucose, respectively. Supplementation of the commercial cellulase cocktail with 0.25 U/mg of β-glucosidase resulted in a 19.8% increase in glucose yield.
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Feng, Yue, Hui-Qin Liu, Run-Cang Sun, and Jian-Xin Jiang. "Enzymatic hydrolysis of cellulose from steam-pretreated Lespedeza stalk (Lespedeza crytobotrya) with four Trichoderma cellulases." BioResources 6, no. 3 (June 7, 2011): 2776–89. http://dx.doi.org/10.15376/biores.6.3.2776-2789.
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The hydrolytic potential of cellulases produced by Trichoderma viride, Trichoderma pseudokoningii, Trichoderma koningii, and Trichoderma reesei with addition of exogenous β-glucosidase was evaluated on cellulose of steam-pretreated Lespedeza. The T. viride enzyme achieved the highest glucose conversion (90.09%), while T. pseudokoningii cellulase achieved the highest ratio of cellobiose to glucose (4.94%) at the end of hydrolysis. Enzymatic adsorption on the substrate was evaluated on filter paper activity and β-glucosidase activity in the corresponding digest with the obtained T. cellulases. T. viride cellulase possessed an efficient adsorption-desorption on the substrate and reached the highest FPA difference (0.72 U/mL) among enzyme activities, indicating to its excellent hydrolysis capability. However, β-glucosidase in T. viride cellulase system showed close bonding on the substrate, suggesting that efficiencies of adsorption-desorption on the cellulose are different between the entire cellulase system and β-glucosidase. T. viride cellulase, with active endogenous β-glucosidase (1.60 U/mL), has compatible synergism with the additional exogenous β-glucosidase.
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Caspi, Jonathan, Yoav Barak, Rachel Haimovitz, Diana Irwin, Raphael Lamed, David B. Wilson, and Edward A. Bayer. "Effect of Linker Length and Dockerin Position on Conversion of a Thermobifida fusca Endoglucanase to the Cellulosomal Mode." Applied and Environmental Microbiology 75, no. 23 (October 9, 2009): 7335–42. http://dx.doi.org/10.1128/aem.01241-09.
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ABSTRACT We have been developing the cellulases of Thermobifida fusca as a model to explore the conversion from a free cellulase system to the cellulosomal mode. Three of the six T. fusca cellulases (endoglucanase Cel6A and exoglucanases Cel6B and Cel48A) have been converted in previous work by replacing their cellulose-binding modules (CBMs) with a dockerin, and the resultant recombinant “cellulosomized” enzymes were incorporated into chimeric scaffolding proteins that contained cohesin(s) together with a CBM. The activities of the resultant designer cellulosomes were compared with an equivalent mixture of wild-type enzymes. In the present work, a fourth T. fusca cellulase, Cel5A, was equipped with a dockerin and intervening linker segments of different lengths to assess their contribution to the overall activity of simple one- and two-enzyme designer cellulosome complexes. The results demonstrated that cellulose binding played a major role in the degradation of crystalline cellulosic substrates. The combination of the converted Cel5A endoglucanase with the converted Cel48A exoglucanase also exhibited a measurable proximity effect for the most recalcitrant cellulosic substrate (Avicel). The length of the linker between the catalytic module and the dockerin had little, if any, effect on the activity. However, positioning of the dockerin on the opposite (C-terminal) side of the enzyme, consistent with the usual position of dockerins on most cellulosomal enzymes, resulted in an enhanced synergistic response. These results promote the development of more complex multienzyme designer cellulosomes, which may eventually be applied for improved degradation of plant cell wall biomass.
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Mingardon, Florence, Ang�lique Chanal, Ana M. L�pez-Contreras, Cyril Dray, Edward A. Bayer, and Henri-Pierre Fierobe. "Incorporation of Fungal Cellulases in Bacterial Minicellulosomes Yields Viable, Synergistically Acting Cellulolytic Complexes." Applied and Environmental Microbiology 73, no. 12 (April 27, 2007): 3822–32. http://dx.doi.org/10.1128/aem.00398-07.
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ABSTRACT Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.
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Mamat Razali, Nur Amira, Noriean Azraaie, Nurul Aimi Mohd Zainul Abidin, Nur Ain Ibrahim, Fauziah Abdul Aziz, and Saadah Abdul Rahman. "Effect of Chemical Treatment on Crystalline Cellulose: Changes in Crystallinity and Functional Groups of Cellulose." Advanced Materials Research 1087 (February 2015): 35–39. http://dx.doi.org/10.4028/www.scientific.net/amr.1087.35.
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Cellulosic material derived from Merbau (Intsia bijuga) was extracted at atmospheric pressure. In the initial stage the sample was delignified using peroxyacetic acid to remove the amorphous. In the second stage the samples were double bleached using hydrogen peroxide (H2O2) and sodium Hydroxide (NaOH). From the X-Ray Diffraction (XRD) data it is evident that both acid and alkali bleached celluloses have rich cellulose I structure. The results are supported by FTIR study in which all samples shown typical spectra of cellulose.
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Metreveli, Eka, Tamar Khardziani, and Vladimir Elisashvili. "The Carbon Source Controls the Secretion and Yield of Polysaccharide-Hydrolyzing Enzymes of Basidiomycetes." Biomolecules 11, no. 9 (September 10, 2021): 1341. http://dx.doi.org/10.3390/biom11091341.
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In the present study, the polysaccharide-hydrolyzing secretomes of Irpex lacteus (Fr.) Fr. (1828) BCC104, Pycnoporus coccineus (Fr.) Bondartsev and Singer (1941) BCC310, and Schizophyllum commune Fr. (1815) BCC632 were analyzed in submerged fermentation conditions to elucidate the effect of chemically and structurally different carbon sources on the expression of cellulases and xylanase. Among polymeric substrates, crystalline cellulose appeared to be the best carbon source providing the highest endoglucanase, total cellulase, and xylanase activities. Mandarin pomace as a growth substrate for S. commune allowed to achieve comparatively high volumetric activities of all target enzymes while wheat straw induced a significant secretion of cellulase and xylanase activities of I. lacteus and P. coccineus. An additive effect on the secretion of cellulases and xylanases by the tested fungi was observed when crystalline cellulose was combined with mandarin pomace. In I. lacteus the cellulase and xylanase production is inducible in the presence of cellulose-rich substrates but is suppressed in the presence of an excess of easily metabolizable carbon source. These enzymes are expressed in a coordinated manner under all conditions studied. It was shown that the substitution of glucose in the inoculum medium with Avicel provides accelerated enzyme production by I. lacteus and higher cellulase and xylanase activities of the fungus. These results add new knowledge to the physiology of basidiomycetes to improve cellulase production.
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Uchiyama, Taku, Takayuki Uchihashi, Akihiko Nakamura, Hiroki Watanabe, Satoshi Kaneko, Masahiro Samejima, and Kiyohiko Igarashi. "Convergent evolution of processivity in bacterial and fungal cellulases." Proceedings of the National Academy of Sciences 117, no. 33 (August 3, 2020): 19896–903. http://dx.doi.org/10.1073/pnas.2011366117.
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Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteriaCellulomonas fimion various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B andCfCel48A) slide on crystalline cellulose. The direction of processive movement ofCfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungusTrichoderma reesei(TrCel7A), whose movement was first observed by this technique, whileCfCel48A moves in the same direction asTrCel7A. WhenCfCel6B andTrCel7A were mixed on the same substrate, “traffic accidents” were observed, in which the two cellulases blocked each other’s progress. The processivity ofCfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.
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Samejima, Masahiro, Takeshi Ohkubo, Kiyohiko Igarashi, Akira Isogai, Shigenori Kuga, Junji Sugiyama, and Karl‐Erik L. Eriksson. "The behaviour of Phanerochaete chrysosporium cellobiose dehydrogenase on adsorption to crystallineand amorphous celluloses." Biotechnology and Applied Biochemistry 25, no. 2 (April 1997): 135–41. http://dx.doi.org/10.1111/j.1470-8744.1997.tb00425.x.
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The adsorption of cellobiose dehydrogenase (CDH) to cellulose has been previously reported. However, the structural type of cellulose on to which CDH is adsorbed has not been investigated. In the present study we have compared the behaviour of CDH when it adsorbs on the highly crystalline cellulose microfibrils from Valonia macrophysa and on the completely amorphous cellulose prepared from a solution of Avicel in the SO2/amine system. The isotherms of CDH adsorption to both Valonia and amorphous celluloses fit well with the Langmuir adsorption theory. However, the maximum adsorption of CDH to the amorphous cellulose was much higher than that to Valonia cellulose. The location of CDH adsorbed on cellulose was revealed with colloidal‐gold‐tagged antibodies by transmission electron microscopy. By using this technique it was demonstrated that CDH adsorption to Valonia cellulose was limited to the amorphous regions attached to the crystalline microfibrils, whereas no CDH was adsorbed to the surface of the highly crystalline microfibrils. Furthermore, continuous monitoring of cellulase activity showed that limited amounts of structures susceptible to enzymic hydrolysis exist even on the surface of Valonia cellulose. From these observations, we conclude that amorphous regions of cellulose seem to the preferred sites of CDH adsorption, whereas CDH is not adsorbed to the surface of highly crystalline microfibrils.
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Hildebrand, Amanda, Edyta Szewczyk, Hui Lin, Takao Kasuga, and Zhiliang Fan. "Engineering Neurospora crassa for Improved Cellobiose and Cellobionate Production." Applied and Environmental Microbiology 81, no. 2 (November 7, 2014): 597–603. http://dx.doi.org/10.1128/aem.02885-14.
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ABSTRACTWe report engineeringNeurospora crassato improve the yield of cellobiose and cellobionate from cellulose. A previously engineered strain ofN. crassa(F5) with six of seven β-glucosidase (bgl) genes knocked out was shown to produce cellobiose and cellobionate directly from cellulose without the addition of exogenous cellulases. In this study, the F5 strain was further modified to improve the yield of cellobiose and cellobionate from cellulose by increasing cellulase production and decreasing product consumption. The effects of two catabolite repression genes,cre-1andace-1, on cellulase production were investigated. The F5 Δace-1mutant showed no improvement over the wild type. The F5 Δcre-1and F5 Δace-1Δcre-1strains showed improved cellobiose dehydrogenase and exoglucanase expression. However, this improvement in cellulase expression did not lead to an improvement in cellobiose or cellobionate production. The cellobionate phosphorylase gene (ndvB) was deleted from the genome of F5 Δace-1Δcre-1to prevent the consumption of cellobiose and cellobionate. Despite a slightly reduced hydrolysis rate, the F5 Δace-1Δcre-1ΔndvBstrain converted 75% of the cellulose consumed to the desired products, cellobiose and cellobionate, compared to 18% converted by the strain F5 Δace-1Δcre-1.
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Lucia, Arianna, Markus Bacher, Hendrikus W. G. van Herwijnen, and Thomas Rosenau. "A Direct Silanization Protocol for Dialdehyde Cellulose." Molecules 25, no. 10 (May 25, 2020): 2458. http://dx.doi.org/10.3390/molecules25102458.
Full textAbstract:
Cellulose derivatives have many potential applications in the field of biomaterials and composites, in addition to several ways of modification leading to them. Silanization in aqueous media is one of the most promising routes to create multipurpose and organic–inorganic hybrid materials. Silanization has been widely used for cellulosic and nano-structured celluloses, but was a problem so far if to be applied to the common cellulose derivative “dialdehyde cellulose” (DAC), i.e., highly periodate-oxidized celluloses. In this work, a straightforward silanization protocol for dialdehyde cellulose is proposed, which can be readily modified with (3-aminopropyl)triethoxysilane. After thermal treatment and freeze-drying, the resulting product showed condensation and cross-linking, which was studied with infrared spectroscopy and 13C and 29Si solid-state nuclear magnetic resonance (NMR) spectroscopy. The cross-linking involves both links of the hydroxyl group of the oxidized cellulose with the silanol groups (Si-O-C) and imine-type bonds between the amino group and keto functions of the DAC (-HC=N-). The modification was achieved in aqueous medium under mild reaction conditions. Different treatments cause different levels of hydrolysis of the organosilane compound, which resulted in diverse condensed silica networks in the modified dialdehyde cellulose structure.
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Krauss, Jan, Vladimir V. Zverlov, and Wolfgang H. Schwarz. "In VitroReconstitution of the Complete Clostridium thermocellum Cellulosome and Synergistic Activity on Crystalline Cellulose." Applied and Environmental Microbiology 78, no. 12 (April 20, 2012): 4301–7. http://dx.doi.org/10.1128/aem.07959-11.
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ABSTRACTArtificial cellulase complexes active on crystalline cellulose were reconstitutedin vitrofrom a native mix of cellulosomal enzymes and CipA scaffoldin. Enzymes containing dockerin modules for binding to the corresponding cohesin modules were prepared from culture supernatants of aC. thermocellum cipAmutant. They were reassociated to cellulosomes via dockerin-cohesin interaction. Recombinantly produced mini-CipA proteins with one to three cohesins either with or without the carbohydrate-binding module (CBM) and the complete CipA protein were used as the cellulosomal backbone. The binding between cohesins and dockerins occurred spontaneously. The hydrolytic activity against soluble and crystalline cellulosic compounds showed that the composition of the complex does not seem to be dependent on which CipA-derived cohesin was used for reconstitution. Binding did not seem to have an obvious local preference (equal binding to Coh1 and Coh6). The synergism on crystalline cellulose increased with an increasing number of cohesins in the scaffoldin. Thein vitro-formed complex showed a 12-fold synergism on the crystalline substrate (compared to the uncomplexed components). The activity of reconstituted cellulosomes with full-size CipA reached 80% of that of native cellulosomes. Complexation on the surface of nanoparticles retained the activity of protein complexes and enhanced their stability. Partial supplementation of the native cellulosome components with three selected recombinant cellulases enhanced the activity on crystalline cellulose and reached that of the native cellulosome. This opens possibilities forin vitrocomplex reconstitution, which is an important step toward the creation of highly efficient engineered cellulases.
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Ramalingam, Subramanian, and Dhanashekar Revathi. "De-Escalation of Saccharification Costs through Enforcement of Immobilization of Cellulase Synthesized by Wild Trichoderma viride." Catalysts 12, no. 6 (June 15, 2022): 659. http://dx.doi.org/10.3390/catal12060659.
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The economic uncertainty associated with cellulosic bioethanol can be overcome through the inclusion of cheap substrates and methodologies that can extend the shelf life of cellulolytic enzymes. In this study, wild Trichoderma viride was used to produce cellulases, media formulation studies were conducted to enhance the cellulase production further and immobilization strategies were tested for stable cellulase–iron oxide magnetic nanoparticle coupling. Out of the seven different production media designed, media containing glucose, wheat bran, cellulose and corn steep liquor supported the highest biomass growth (60 Packed cell volume) and cellulase formation (7.4 U/mL), and thus was chosen for the fiscal analysis at a larger scale (1000 m3). The profitability of the cellulase production process was assessed to be 20.86%, considering both the capital expenditure and operating expenses. Further, the effect of cost of different carbon sources, nitrogen sources and cellulase yields on the annual operating costs was explored, which led to the choice of delignified sugarcane bagasse, corn steep liquor and productivity levels to be respective decisive factors of the overall cost of the cellulase production. Likewise, the break-even period of such a large-scale operation was gauged given the market price of cellulases at USD 17 for 105 U of cellulases. Moreover, enzyme immobilization led to enhanced cellulase shelf life and ultimately contributed toward saccharification cost reduction.
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Wang, Lunji, Yishen Zhao, Siqiao Chen, Xian Wen, Wilfred Mabeche Anjago, Tianchi Tian, Yajuan Chen, et al. "Growth, Enzymatic, and Transcriptomic Analysis of xyr1 Deletion Reveals a Major Regulator of Plant Biomass-Degrading Enzymes in Trichoderma harzianum." Biomolecules 14, no. 2 (January 24, 2024): 148. http://dx.doi.org/10.3390/biom14020148.
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The regulation of plant biomass degradation by fungi is critical to the carbon cycle, and applications in bioproducts and biocontrol. Trichoderma harzianum is an important plant biomass degrader, enzyme producer, and biocontrol agent, but few putative major transcriptional regulators have been deleted in this species. The T. harzianum ortholog of the transcriptional activator XYR1/XlnR/XLR-1 was deleted, and the mutant strains were analyzed through growth profiling, enzymatic activities, and transcriptomics on cellulose. From plate cultures, the Δxyr1 mutant had reduced growth on D-xylose, xylan, and cellulose, and from shake-flask cultures with cellulose, the Δxyr1 mutant had ~90% lower β-glucosidase activity, and no detectable β-xylosidase or cellulase activity. The comparison of the transcriptomes from 18 h shake-flask cultures on D-fructose, without a carbon source, and cellulose, showed major effects of XYR1 deletion whereby the Δxyr1 mutant on cellulose was transcriptionally most similar to the cultures without a carbon source. The cellulose induced 43 plant biomass-degrading CAZymes including xylanases as well as cellulases, and most of these had massively lower expression in the Δxyr1 mutant. The expression of a subset of carbon catabolic enzymes, other transcription factors, and sugar transporters was also lower in the Δxyr1 mutant on cellulose. In summary, T. harzianum XYR1 is the master regulator of cellulases and xylanases, as well as regulating carbon catabolic enzymes.
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Legodi, L. M., D. La Grange, E. L. Jansen van Rensburg, and I. Ncube. "Isolation of Cellulose Degrading Fungi from Decaying Banana Pseudostem and Strelitzia alba." Enzyme Research 2019 (July 25, 2019): 1–10. http://dx.doi.org/10.1155/2019/1390890.
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Cellulases are a group of hydrolytic enzymes that break down cellulose to glucose units. These enzymes are used in the food, beverage, textile, pulp, and paper and the biofuel industries. The aim of this study was to isolate fungi from natural compost and produce cellulases in submerged fermentation (SmF). Initial selection was based on the ability of the fungi to grow on agar containing Avicel followed by cellulase activity determination in the form of endoglucanase and total cellulase activity. Ten fungal isolates obtained from the screening process showed good endoglucanase activity on carboxymethyl cellulose-Congo Red agar plates. Six of the fungal isolates were selected based on high total cellulase activity and identified as belonging to the genera Trichoderma and Aspergillus. In SmF of synthetic media with an initial pH of 6.5 at 30°C Trichoderma longibrachiatum LMLSAUL 14-1 produced total cellulase activity of 8 FPU/mL and endoglucanase activity of 23 U/mL whilst Trichoderma harzianum LMLBP07 13-5 produced 6 FPU/mL and endoglucanase activity of 16 U/mL. The produced levels of both cellulases and endoglucanase by Trichoderma species were higher than the levels for the Aspergillus fumigatus strains. Aspergillus fumigatus LMLPS 13-4 produced higher β-glucosidase 38 U/mL activity than Trichoderma species.
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Malik, Muhammad Saqib, Abdul Rehman, Irfan Ullah Khan, Taj Ali Khan, Muhammad Jamil, Eui Shik Rha, and Muhammad Anees. "Thermo-neutrophilic cellulases and chitinases characterized from a novel putative antifungal biocontrol agent: Bacillus subtilis TD11." PLOS ONE 18, no. 1 (January 27, 2023): e0281102. http://dx.doi.org/10.1371/journal.pone.0281102.
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Cellulose and chitin are the most abundant naturally occurring biopolymers synthesized in plants and animals and are used for synthesis of different organic compounds and acids in the industry. Therefore, cellulases and chitinases are important for their multiple uses in industry and biotechnology. Moreover, chitinases have a role in the biological control of phytopathogens. A bacterial strain Bacillus subtilis TD11 was previously isolated and characterized as a putative biocontrol agent owing to its significant antifungal potential. In this study, cellulase and chitinase produced by the strain B. subtilis TD11 were purified and characterized. The activity of the cellulases and chitinases were optimized at different pH (2 to 10) and temperatures (20 to 90°C). The substrate specificity of cellulases was evaluated using different substances including carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), and crystalline substrates. The cellulase produced by B. subtilis TD11 had a molecular mass of 45 kDa while that of chitinase was 55 kDa. The optimal activities of the enzymes were found at neutral pH (6.0 to 7.0). The optimum temperature for the purified cellulases was in the range of 50 to 70°C while, purified chitinases were optimally active at 50°C. The highest substrate specificity of the purified cellulase was found for CMC (100%) followed by HEC (>50% activity) while no hydrolysis was observed against the crystalline substrates. Moreover, it was observed that the purified chitinase was inhibitory against the fungi containing chitin in their hyphal walls i.e., Rhizoctonia, Colletotrichum, Aspergillus and Fusarium having a dose-effect relationship.
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Cunha, Eva S., Christine L. Hatem, and Doug Barrick. "Insertion of Endocellulase Catalytic Domains into Thermostable Consensus Ankyrin Scaffolds: Effects on Stability and Cellulolytic Activity." Applied and Environmental Microbiology 79, no. 21 (August 23, 2013): 6684–96. http://dx.doi.org/10.1128/aem.02121-13.
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ABSTRACTDegradation of cellulose for biofuels production holds promise in solving important environmental and economic problems. However, the low activities (and thus high enzyme-to-substrate ratios needed) of hydrolytic cellulase enzymes, which convert cellulose into simple sugars, remain a major barrier. As a potential strategy to stabilize cellulases and enhance their activities, we have embedded cellulases of extremophiles into hyperstable α-helical consensus ankyrin domain scaffolds. We found the catalytic domains CelA (CA, GH8;Clostridium thermocellum) and Cel12A (C12A, GH12;Thermotoga maritima) to be stable in the context of the ankyrin scaffold and to be active against both soluble and insoluble substrates. The ankyrin repeats in each fusion are folded, although it appears that for the C12Acatalyticdomain (CD; where the N and C termini are distant in the crystal structure), the two flanking ankyrin domains are independent, whereas for CA (where termini are close), the flanking ankyrin domains stabilize each other. Although the activity of CA is unchanged in the context of the ankyrin scaffold, the activity of C12A is increased between 2- and 6-fold (for regenerated amorphous cellulose and carboxymethyl cellulose substrates) at high temperatures. For C12A, activity increases with the number of flanking ankyrin repeats. These results showed ankyrin arrays to be a promising scaffold for constructing designer cellulosomes, preserving or enhancing enzymatic activity and retaining thermostability. This modular architecture will make it possible to arrange multiple cellulase domains at a precise spacing within a single polypeptide, allowing us to search for spacings that may optimize reactivity toward the repetitive cellulose lattice.
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Aro, Nina, Marja Ilmén, Anu Saloheimo, and Merja Penttilä. "ACEI of Trichoderma reesei Is a Repressor of Cellulase and Xylanase Expression." Applied and Environmental Microbiology 69, no. 1 (January 2003): 56–65. http://dx.doi.org/10.1128/aem.69.1.56-65.2003.
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ABSTRACT We characterized the effect of deletion of the Trichoderma reesei (Hypocrea jecorina) ace1 gene encoding the novel cellulase regulator ACEI that was isolated based on its ability to bind to and activate in vivo in Saccharomyces cerevisiae the promoter of the main cellulase gene, cbh1. Deletion of ace1 resulted in an increase in the expression of all the main cellulase genes and two xylanase genes in sophorose- and cellulose-induced cultures, indicating that ACEI acts as a repressor of cellulase and xylanase expression. Growth of the strain with a deletion of the ace1 gene on different carbon sources was analyzed. On cellulose-based medium, on which cellulases are needed for growth, the Δace1 strain grew better than the host strain due to the increased cellulase production. On culture media containing sorbitol as the sole carbon source, the growth of the strain with a deletion of the ace1 gene was severely impaired, suggesting that ACEI regulates expression of other genes in addition to cellulase and xylanase genes. A strain with a deletion of the ace1 gene and with a deletion of the ace2 gene coding for the cellulase and xylanase activator ACEII expressed cellulases and xylanases similar to the Δace1 strain, indicating that yet another activator regulating cellulase and xylanase promoters was present.