scholarly journals Hydrolysis of Cellulose by Soluble Clostridium Thermocellum and Acidothermus Cellulolyticus Cellulases

2018 ◽  
Vol 1 (1) ◽  
pp. 5-19
Author(s):  
Phillip Brumm ◽  
Phillip Brumm ◽  
Dan Xie ◽  
Dan Xie ◽  
Larry Allen ◽  
...  
Keyword(s):  
Filter Paper ◽  
Clostridium Thermocellum ◽  
Cellulose Degradation ◽  
Cellulase Activity ◽  
Cellulose Hydrolysis ◽  
Reducing Sugar ◽  
Crystalline Cellulose ◽  
Amorphous Cellulose ◽  
Highly Active ◽  
The Individual

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%.

scholarly journals Comparison of Family 9 Cellulases from Mesophilic and Thermophilic Bacteria

2010 ◽  
Vol 77 (4) ◽  
pp. 1436-1442 ◽  
Author(s):  
Florence Mingardon ◽  
John D. Bagert ◽  
Cyprien Maisonnier ◽  
Devin L. Trudeau ◽  
Frances H. Arnold
Keyword(s):  
High Temperatures ◽  
Catalytic Domain ◽  
Thermophilic Bacteria ◽  
Cellulose Degradation ◽  
Cellulase Activity ◽  
Crystalline Cellulose ◽  
Wild Type ◽  
Thermostable Enzymes ◽  
Highly Active ◽  
Homology Module

ABSTRACTCellulases containing a family 9 catalytic domain and a family 3c cellulose binding module (CBM3c) are important components of bacterial cellulolytic systems. We measured the temperature dependence of the activities of three homologs:Clostridium cellulolyticumCel9G,Thermobifida fuscaCel9A, andC. thermocellumCel9I. To directly compare their catalytic activities, we constructed six new versions of the enzymes in which the three GH9-CBM3c domains were fused to a dockerin both with and without aT. fuscafibronectin type 3 homology module (Fn3). We studied the activities of these enzymes on crystalline cellulose alone and in complex with a miniscaffoldin containing a cohesin and a CBM3a. The presence of Fn3 had no measurable effect on thermostability or cellulase activity. The GH9-CBM3c domains of Cel9A and Cel9I, however, were more active than the wild type when fused to a dockerin complexed to scaffoldin. The three cellulases in complex have similar activities on crystalline cellulose up to 60°C, butC. thermocellumCel9I, the most thermostable of the three, remains highly active up to 80°C, where its activity is 1.9 times higher than at 60°C. We also compared the temperature-dependent activities of different versions of Cel9I (wild type or in complex with a miniscaffoldin) and found that the thermostable CBM is necessary for activity on crystalline cellulose at high temperatures. These results illustrate the significant benefits of working with thermostable enzymes at high temperatures, as well as the importance of retaining the stability of all modules involved in cellulose degradation.

scholarly journals Effects of Pretreatment with Ionic Liquids on Cellulose Hydrolysis under Hydrothermal Conditions

Molecules ◽  
2019 ◽  
Vol 24 (19) ◽  
pp. 3572 ◽  
Author(s):  
Toshitaka Funazukuri ◽  
Shingo Ozawa
Keyword(s):  
Ionic Liquids ◽  
Filter Paper ◽  
Biomass Conversion ◽  
Batch Reactor ◽  
Cellulose Hydrolysis ◽  
Reaction Rates ◽  
Crystalline Cellulose ◽  
Hydrothermal Conditions ◽  
Imidazolium Chloride ◽  
Product Yields

Hydrothermal hydrolysis in hot pressurized liquid water (HPLW) is attractive for biomass conversion into valuable products because it achieves high reaction rates without catalysts and additives. The hydrothermal hydrolysis of high crystalline cellulose requires higher reaction temperature than polysaccharides having low crystallinity. It can be expected to increase the reaction rate or decrease temperature by decreasing the crystallinity. In the present study ashless filter paper as a fibrous pure cellulose sample was pretreated with ionic liquids (ILs) such as imidazolium chloride ILs containing alkyl side chains ranging from two to six carbons, and with an aqueous solution of bis(ethylenediamine ammonium) copper (BEDC). Herein, the pretreatment with ILs was to regenerate filter paper: dissolving in ILs at 373 K for 120 min or in an aqueous BEDC solution at room temperature, precipitating by adding water, washing the solid, and then drying. Subsequently, the pretreated filter paper samples were hydrolyzed at 533 K and 5.0 MPa in HPLW in a small semi-batch reactor, and the effects of the pretreatment with ILs or BEDC on reaction rates and product yields were examined. While the crystallinity indexes with all ILs and BEDC after the pretreatments decreased to 44 to 47 from the original sample of 87, the reaction rates and product yields were significantly affected by the IL species. At 533 K and 5.0 MPa, the dissolution rate with [AMIM][Cl] was nine times as fast as that for untreated sample.

scholarly journals X-Ray Crystal Structure of the Multidomain Endoglucanase Cel9G from Clostridium cellulolyticum Complexed with Natural and Synthetic Cello-Oligosaccharides

2003 ◽  
Vol 185 (14) ◽  
pp. 4127-4135 ◽  
Author(s):  
David Mandelman ◽  
Anne Belaich ◽  
J. P. Belaich ◽  
Nushin Aghajari ◽  
Hugues Driguez ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Domain ◽  
Cellulose Degradation ◽  
Cellulase Activity ◽  
Structural Basis ◽  
Crystalline Cellulose ◽  
X Ray ◽  
Clostridium Cellulolyticum ◽  
Active Site Cleft ◽  
Cellulose Binding

ABSTRACT Complete cellulose degradation is the first step in the use of biomass as a source of renewable energy. To this end, the engineering of novel cellulase activity, the activity responsible for the hydrolysis of the β-1,4-glycosidic bonds in cellulose, is a topic of great interest. The high-resolution X-ray crystal structure of a multidomain endoglucanase from Clostridium cellulolyticum has been determined at a 1.6-Å resolution. The endoglucanase, Cel9G, is comprised of a family 9 catalytic domain attached to a family IIIc cellulose-binding domain. The two domains together form a flat platform onto which crystalline cellulose is suggested to bind and be fed into the active-site cleft for endolytic hydrolysis. To further dissect the structural basis of cellulose binding and hydrolysis, the structures of Cel9G in the presence of cellobiose, cellotriose, and a DP-10 thio-oligosaccharide inhibitor were resolved at resolutions of 1.7, 1.8, and 1.9 Å, respectively.

scholarly journals Isolation of Cellulose-Degrading Bacteria and Determination of Their Cellulolytic Potential

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Pratima Gupta ◽  
Kalpana Samant ◽  
Avinash Sahu
Keyword(s):  
Filter Paper ◽  
Cellulose Degradation ◽  
Potassium Dichromate ◽  
Gravimetric Method ◽  
Cellulase Activity ◽  
Degrading Bacteria ◽  
Agar Media ◽  
Filter Paper Cellulase ◽  
Simultaneous Saccharification

Eight isolates of cellulose-degrading bacteria (CDB) were isolated from four different invertebrates (termite, snail, caterpillar, and bookworm) by enriching the basal culture medium with filter paper as substrate for cellulose degradation. To indicate the cellulase activity of the organisms, diameter of clear zone around the colony and hydrolytic value on cellulose Congo Red agar media were measured. CDB 8 and CDB 10 exhibited the maximum zone of clearance around the colony with diameter of 45 and 50 mm and with the hydrolytic value of 9 and 9.8, respectively. The enzyme assays for two enzymes, filter paper cellulase (FPC), and cellulase (endoglucanase), were examined by methods recommended by the International Union of Pure and Applied Chemistry (IUPAC). The extracellular cellulase activities ranged from 0.012 to 0.196 IU/mL for FPC and 0.162 to 0.400 IU/mL for endoglucanase assay. All the cultures were also further tested for their capacity to degrade filter paper by gravimetric method. The maximum filter paper degradation percentage was estimated to be 65.7 for CDB 8. Selected bacterial isolates CDB 2, 7, 8, and 10 were co-cultured withSaccharomyces cerevisiaefor simultaneous saccharification and fermentation. Ethanol production was positively tested after five days of incubation with acidified potassium dichromate.

scholarly journals Identification, Detection, and Spatial Resolution of Clostridium Populations Responsible for Cellulose Degradation in a Methanogenic Landfill Leachate Bioreactor

2004 ◽  
Vol 70 (4) ◽  
pp. 2414-2419 ◽  
Author(s):  
P. C. Burrell ◽  
C. O'Sullivan ◽  
H. Song ◽  
W. P. Clarke ◽  
L. L. Blackall
Keyword(s):  
Landfill Leachate ◽  
Cellulose Degradation ◽  
Cellulose Hydrolysis ◽  
Crystalline Cellulose ◽  
Rrna Gene ◽  
Clone Libraries ◽  
Planktonic Phase ◽  
Group 3 ◽  
Group 5 ◽  
Clone Group

ABSTRACT An anaerobic landfill leachate bioreactor was operated with crystalline cellulose and sterile landfill leachate until a steady state was reached. Cellulose hydrolysis, acidogenesis, and methanogenesis were measured. Microorganisms attached to the cellulose surfaces were hypothesized to be the cellulose hydrolyzers. 16S rRNA gene clone libraries were prepared from this attached fraction and also from the mixed fraction (biomass associated with cellulose particles and in the planktonic phase). Both clone libraries were dominated by Firmicutes phylum sequences (100% of the attached library and 90% of the mixed library), and the majority fell into one of five lineages of the clostridia. Clone group 1 (most closely related to Clostridium stercorarium), clone group 2 (most closely related to Clostridium thermocellum), and clone group 5 (most closely related to Bacteroides cellulosolvens) comprised sequences in Clostridium group III. Clone group 3 sequences were in Clostridium group XIVa (most closely related to Clostridium sp. strain XB90). Clone group 4 sequences were affiliated with a deeply branching clostridial lineage peripherally associated with Clostridium group VI. This monophyletic group comprises a new Clostridium cluster, designated cluster VIa. Specific fluorescence in situ hybridization (FISH) probes for the five groups were designed and synthesized, and it was demonstrated in FISH experiments that bacteria targeted by the probes for clone groups 1, 2, 4, and 5 were very abundant on the surfaces of the cellulose particles and likely the key cellulolytic microorganisms in the landfill bioreactor. The FISH probe for clone group 3 targeted cells in the planktonic phase, and these organisms were hypothesized to be glucose fermenters.

scholarly journals Mutations in the Scaffoldin Gene, cipA, of Clostridium thermocellum with Impaired Cellulosome Formation and Cellulose Hydrolysis: Insertions of a New Transposable Element, IS1447, and Implications for Cellulase Synergism on Crystalline Cellulose

2008 ◽  
Vol 190 (12) ◽  
pp. 4321-4327 ◽  
Author(s):  
Vladimir V. Zverlov ◽  
Martina Klupp ◽  
Jan Krauss ◽  
Wolfgang H. Schwarz
Keyword(s):  
Clostridium Thermocellum ◽  
Culture Supernatant ◽  
Cellulose Degradation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Crystalline Cellulose ◽  
Target Sequence ◽  
Protein Patterns ◽  
Transposase Gene

ABSTRACT Mutants of Clostridium thermocellum that had lost the ability to adhere to microcrystalline cellulose were isolated. Six of them that showed diminished ability to depolymerize crystalline cellulose were selected. Size exclusion chromatography of the proteins from the culture supernatant revealed the loss of the supramolecular enzyme complex, the cellulosome. However, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulted in extracellular protein patterns comparable to those of isolated cellulosomes, except for a missing CipA band. Sequencing of the six mutant cipA genes revealed a new insertion (IS) element, IS1447, belonging to the IS3 family. It was inserted into the cipA reading frame in four different locations: cohesin module 1, two different positions in the carbohydrate binding module, and cohesin module 3. The IS sequences were identical and consisted of a transposase gene and the inverted repeats IRR and IRS. The insertion resulted in an obviously nonspecific duplication of 3 base pairs within the target sequence. This lack of specificity allows transposition without the need of a defined target DNA sequence. Eighteen copies of IS1447 were identified in the genomic sequence of C. thermocellum ATCC 27405. At least one of them can be activated for transposition. Compared to the wild type, the mutant culture supernatant, with a completely defective CipA protein, showed equal specific hydrolytic activity against soluble β-glucan but a 15-fold reduction in specific activity with crystalline cellulose. These results identify a genetic basis for the synergistic effect of complex formation on crystalline-cellulose degradation.

scholarly journals Carbohydrate-binding properties of a separately folding protein module from β-1,3-glucanase Lic16A of Clostridium thermocellum

Microbiology ◽  
2009 ◽  
Vol 155 (7) ◽  
pp. 2442-2449 ◽  
Author(s):  
Igor A. Dvortsov ◽  
Natalia A. Lunina ◽  
Ludmila A. Chekanovskaya ◽  
Wolfgang H. Schwarz ◽  
Vladimir V. Zverlov ◽  
...  
Keyword(s):  
Cell Wall ◽  
Yeast Cell ◽  
Clostridium Thermocellum ◽  
Specific Activity ◽  
Yeast Cell Wall ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Binding Properties ◽  
Highly Active ◽  
Catalytic Module

The multi-modular non-cellulosomal endo-1,3(4)-β-glucanase Lic16A from Clostridium thermocellum contains a so-called X module (denoted as CBMX) near the N terminus of the catalytic module (191–426 aa). Melting of X-module-containing recombinant proteins revealed an independent folding of the module. CBMX was isolated and studied as a separate fragment. It was shown to bind to various insoluble polysaccharides, including xylan, pustulan, chitin, chitosan, yeast cell wall glucan, Avicel and bacterial crystalline cellulose. CBMX thus contains a hitherto unknown carbohydrate-binding module (CBM54). It did not bind soluble polysaccharides on which Lic16A is highly active. Ca2+ ions had effects on the binding, e.g. stimulated complex formation with chitosan, which was observed only in the presence of Ca2+. The highest affinity to CBMX was shown for xylan (binding constant K=3.1×104 M−1), yeast cell wall glucan (K=1.4×105 M−1) and chitin (K=3.3.105 M−1 in the presence of Ca2+). Lic16A deletion derivatives lacking CBMX had lower affinity to lichenan and laminarin and a slight decrease in optimum temperature and thermostability. However, the specific activity was not significantly affected.

The Expression In Vitro and Application on Cellulose Degradation of LeEXP2

2011 ◽  
Vol 183-185 ◽  
pp. 790-794 ◽  
Author(s):  
Yuan Yuan Ma ◽  
Xin Wang ◽  
Han Ze Wang ◽  
Kun Zhang ◽  
Min Hua Zhang
Keyword(s):  
Filter Paper ◽  
Plant Cell Wall ◽  
Cellulose Degradation ◽  
Biomass Conversion ◽  
Cellulose Hydrolysis ◽  
Cellulosic Biomass ◽  
Functional Study ◽  
Extracellular Medium ◽  
Yeast Strains

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.

scholarly journals Cellodextrin and Laminaribiose ABC Transporters in Clostridium thermocellum

2008 ◽  
Vol 191 (1) ◽  
pp. 203-209 ◽  
Author(s):  
Yakir Nataf ◽  
Sima Yaron ◽  
Frank Stahl ◽  
Raphael Lamed ◽  
Edward A. Bayer ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Binding Proteins ◽  
Cell Attachment ◽  
Binding Protein ◽  
Cellulose Hydrolysis ◽  
Thermophilic Bacterium ◽  
Cellulosic Biomass ◽  
Titration Calorimetry ◽  
Sugar Binding ◽  
Highly Active

ABSTRACT Clostridium thermocellum is an anaerobic thermophilic bacterium that grows efficiently on cellulosic biomass. This bacterium produces and secretes a highly active multienzyme complex, the cellulosome, that mediates the cell attachment to and hydrolysis of the crystalline cellulosic substrate. C. thermocellum can efficiently utilize only β-1,3 and β-1,4 glucans and prefers long cellodextrins. Since the bacterium can also produce ethanol, it is considered an attractive candidate for a consolidated fermentation process in which cellulose hydrolysis and ethanol fermentation occur in a single process. In this study, we have identified and characterized five sugar ABC transporter systems in C. thermocellum. The putative transporters were identified by sequence homology of the putative solute-binding lipoprotein to known sugar-binding proteins. Each of these systems is transcribed from a gene cluster, which includes an extracellular solute-binding protein, one or two integral membrane proteins, and, in most cases, an ATP-binding protein. The genes of the five solute-binding proteins were cloned, fused to His tags, overexpressed, and purified, and their abilities to interact with different sugars was examined by isothermal titration calorimetry. Three of the sugar-binding lipoproteins (CbpB to -D) interacted with different lengths of cellodextrins (G2 to G5), with disassociation constants in the micromolar range. One protein, CbpA, binds only cellotriose (G3), while another protein, Lbp (laminaribiose-binding protein) interacts with laminaribiose. The sugar specificity of the different binding lipoproteins is consistent with the observed substrate preference of C. thermocellum, in which cellodextrins (G3 to G5) are assimilated faster than cellobiose.

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