Hydrolysis of amorphous and crystalline cellulose by heterologously produced cellulases of Melanocarpus albomyces

Three thermostable neutral cellulases from Melanocarpus albomyces, a 20-kDa endoglucanase (Cel45A), a 50-kDa endoglucanase (Cel7A), and a 50-kDa cellobiohydrolase (Cel7B) heterologously produced in a recombinant Trichoderma reesei were purified and studied in hydrolysis (50 degrees C, pH 6.0) of crystalline and amorphous cellulose. To improve their efficiency, M. albomyces cellulases naturally harboring no cellulose-binding module (CBM) were genetically modified to carry the CBM of T. reesei CBHI/Cel7A, and were studied under similar experimental conditions. Hydrolysis performance and product profiles were used to evaluate hydrolytic features of the investigated enzymes. Each cellulase proved to be active against the tested substrates; the cellobiohydrolase Cel7B had greater activity than the endoglucanases Cel45A and Cel7A against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence of synergism was observed when mixtures of the novel enzymes were applied in a constant total protein dosage. Presence of the CBM improved the hydrolytic potential of each enzyme in all experimental configurations; it had a greater effect on the endoglucanases Cel45A and Cel7A than the cellobiohydrolase Cel7B, especially against crystalline substrate. The novel cellobiohydrolase performed comparably to the major cellobiohydrolase of T. reesei (CBHI/Cel7A) under the applied experimental conditions.     

Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose

An extremely highly active cellobiohydrolase (CBH IIb or Cel6B) was isolated from Chrysosporium lucknowense UV18-25 culture filtrate. The CBH IIb demonstrated the highest ability for a deep degradation of crystalline cellulose amongst a few cellobiohydrolases tested, including C. lucknowense CBH Ia, Ib, IIa, and Trichoderma reesei CBH I and II. Using purified C. lucknowense enzymes (CBH Ia, Ib, and IIb; endoglucanases II and V; beta-glucosidase, xylanase II), artificial multienzyme mixtures were reconstituted, displaying an extremely high performance in a conversion of different cellulosic substrates (Avicel, cotton, pretreated Douglas fir wood) to glucose. These mixtures were much or notably more effective in hydrolysis of the cellulosic substrates than the crude multienzyme C. lucknowense preparation and other crude cellulase samples produced by T. reesei and Penicillium verruculosum. Highly active cellulases are a key factor in bioconversion of plant lignocellulosic biomass to ethanol as an alternative to fossil fuels.     

Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei.

There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing beta-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and beta-glucan. The endoglucanase activity on CMC and beta-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.     

Cellulases of Penicillium verruculosum

Nine major cellulolytic enzymes were isolated from a culture broth of a mutant strain of the fungus Penicillium verruculosum: five endo-1, 4-β-glucanases (EGs) having molecular masses 25, 33, 39, 52, and 70 kDa, and four cellobiohydrolases (CBHs: 50, 55, 60, and 66 kDa). Based on amino acid similarities of short sequenced fragments and peptide mass fingerprinting, the isolated enzymes were preliminary classified into different families of glycoside hydrolases: Cel5A (EG IIa, 39 kDa), Cel5B (EG IIb, 33 kDa), Cel6A (CBH II, two forms: 50 and 60 kDa), Cel7A (CBH I: 55 and 66 kDa), Cel7B (EG I: 52 and 70 kDa). The 25 kDa enzyme was identical to the previously isolated Cel12A (EG III). The family assignment was further confirmed by the studies of the substrate specificity of the purified enzymes. High-molecular-weight forms of the Cel6A, Cel7A, and Cel7B were found to possess a cellulose-binding module (CBM), while the catalytically active low-molecular-weight forms of the enzymes, as well as other cellulases, lacked the CBM. Properties of the isolated enzymes, such as substrate specificity toward different polysaccharides and synthetic glycosides, effect of pH and temperature on the enzyme activity and stability, adsorption on Avicel cellulose and kinetics of its hydrolysis, were investigated.     

Engineering chimeric thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae

We report here the effect of adding different types of carbohydrate-binding modules (CBM) to a single-module GH7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (TeCel7A). Both bacterial and fungal CBMs derived from families 1, 2 and 3, all reported to bind to crystalline cellulose, were used. Chimeric cellobiohydrolases with an additional S–S bridge in the catalytic module of TeCel7A were also made. All the fusion proteins were secreted in active form and in good yields by Saccharomyces cerevisiae. The purified chimeric enzymes bound to cellulose clearly better than the catalytic module alone and demonstrated high thermal stability, having unfolding temperatures (T _m) ranging from 72 °C to 77 °C. The highest activity enhancement on microcrystalline cellulose could be gained by a fusion with a bacterial CBM3 derived from Clostridium thermocellum cellulosomal-scaffolding protein CipA. The two CBM3 fusion enzymes tested were more active than the reference enzyme Trichoderma reesei Cel7A both at moderate (45 °C and 55 °C) and at high temperatures (60 °C and 65 °C), the hydrolysis yields being two- to three-fold better at 60 °C, and six- to seven-fold better at 65 °C. The best enzyme variant was also tested on a lignocellulosic feedstock hydrolysis, which demonstrated its potency in biomass hydrolysis even at 70 °C.     

嗜热真菌纤维素酶的CBD与海栖热袍菌的纤维素酶融合表达

将嗜热真菌毛壳菌纤维素酶Cel7A的纤维素结合结构域编码区与极端嗜热厌氧菌海栖热袍菌的纤维素酶CelB基因进行融合, 构建重组质粒pHsh-CBD-CelB, 并在大肠杆菌中表达。对融合蛋白进行纯化, 通过热处理和离子交换层析, 纯化到的融合蛋白SDS-PAGE 电泳图谱显示为单一条带。对融合蛋白的特性研究, 结果表明融合蛋白降解CMC的最适反应温度为90°C, 结晶纤维素吸附实验表明该融合蛋白具有结合结晶纤维素的能力, 并且融合蛋白降解CMC与结晶纤维素的能力得到提高。     

Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates

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

Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have studied the biochemical diversity of several GH 12 homologs. The H. schweinitzii Cel12A enzyme differs from the T. reesei Cel12A enzyme by only 14 amino acids (93% sequence identity), but is much less thermally stable. The bacterial Cel12A enzyme from S. sp. 11AG8 shares only 28% sequence identity to the T. reesei enzyme, and is much more thermally stable. Each of the 14 sequence differences from H. schweinitzii Cel12A were introduced in T. reesei Cel12A to determine the effect of these amino acid substitutions on enzyme stability. Several of the T. reesei Cel12A variants were found to have increased stability, and the differences in apparent midpoint of thermal denaturation (T(m)) ranged from a 2.5 degrees C increase to a 4.0 degrees C decrease. The least stable recruitment from H. schweinitzii Cel12A was A35S. Consequently, the A35V substitution was recruited from the more stable S. sp. 11AG8 Cel12A and this T. reesei Cel12A variant was found to have a T(m) 7.7 degrees C higher than wild type. Thus, the buried residue at position 35 was shown to be of critical importance for thermal stability in this structural family. There was a ninefold range in the specific activities of the Cel12 homologs on o-NPC. The most and least stable T. reesei Cel12A variants, A35V and A35S, respectively, were fully active. Because of their thermal tolerance, S. sp. 11AG8 Cel12A and T. reesei Cel12A variant A35V showed a continual increase in activity over the temperature range of 25 degrees C to 60 degrees C, whereas the less stable enzymes T. reesei Cel12A wild type and the destabilized A35S variant, and H. schweinitzii Cel12A showed a decrease in activity at the highest temperatures. The crystal structures of the H. schweinitzii, S. sp. 11AG8, and T. reesei A35V Cel12A enzymes have been determined and compared with the wild-type T. reesei Cel12A enzyme. All of the structures have similar Calpha traces, but provide detailed insight into the nature of the stability differences. These results are an example of the power of homolog recruitment as a method for identifying residues important for stability.     

Identification and purification of the main components of cellulases from a mutant strain of Trichoderma viride T 100-14

A new mutant strain of fungus Trichoderma viride T 100-14 was cultivated on 1% microcrystalline cellulose (Avicel) for 120h and the resulting culture filtrate was prepared for protein identification and purification. To identify the predominant catalytic components, cellulases were separated by an adapted two-dimensional electrophoresis technique. The apparent major spots were identified by high performance liquid chromatography electrospray ionization mass (HPLC-ESI-MS). Seven of the components were previously known, i.e., the endoglucanases Cel7B (EG I), Cel12A (EG III), Cel61A (EG IV), the cellobiohydrolases Cel7A (CBH I), Cel6A (CBH II), Cel6B (CBH IIb) and the beta-glucosidase. The seven major components in the fermentation broth of T. viride T 100-14 probably constitute the essential enzymes for crystalline cellulose hydrolysis and they were further purified to electrophoretic homogeneity by a series of chromatography column. Hydrolysis studies of the purified elements revealed that three of the cellulases were classified as cellobiohydrolases due to their main activities on p-nitrophenyl-beta-d-cellobioside (pNPC). Three of the cellulases, with the abilities of hydrolyzing both carboxymethyl-cellulose (CMC) and Avicel indicate their endoglucanase activities. It deserved noting that the beta-glucosidase from the T 100-14 displayed an extremely high activity on p-nitrophenyl-beta-D-glycopyranoside (pNPG), which suggested it was a good candidate for the conversion of cellobiose to glucose.     

Dynamic interaction of Trichoderma reesei cellobiohydrolases Cel6A and Cel7A and cellulose at equilibrium and during hydrolysis

The binding of cellobiohydrolases to cellulose is a crucial initial step in cellulose hydrolysis. In the search for a detailed understanding of the function of cellobiohydrolases, much information concerning how the enzymes and their constituent catalytic and cellulose-binding domains interact with cellulose and with each other and how binding changes during hydrolysis is still needed. In this study we used tritium labeling by reductive methylation to monitor binding of the two Trichoderma reesei cellobiohydrolases, Cel6A and Cel7A (formerly CBHII and CBHI), and their catalytic domains. Measuring hydrolysis by high-performance liquid chromatography and measuring binding by scintillation counting allowed us to correlate activity and binding as a function of the extent of degradation. These experiments showed that the density of bound protein increased with both Cel6A and Cel7A as hydrolysis proceeded, in such a way that the adsorption points moved off the initial binding isotherms. We also compared the affinities of the cellulose-binding domains and the catalytic domains to the affinities of the intact proteins and found that in each case the affinity of the enzyme was determined by the linkage between the catalytic and cellulose-binding domains. Desorption of Cel6A by dilution of the sample showed hysteresis (60 to 70% reversible); in contrast, desorption of Cel7A did not show hysteresis and was more than 90% reversible. These findings showed that the two enzymes differ with respect to the reversibility of binding.     

Cel9M, a new family 9 cellulase of the Clostridium cellulolyticum cellulosome

A new cellulosomal protein from Clostridium cellulolyticum Cel9M was characterized. The protein contains a catalytic domain belonging to family 9 and a dockerin domain. Cel9M is active on carboxymethyl cellulose, and the hydrolysis of this substrate is accompanied by a decrease in viscosity. Cel9M has a slight, albeit significant, activity on both Avicel and bacterial microcrystalline cellulose, and the main soluble sugar released is cellotetraose. Saccharification of bacterial microcrystalline cellulose by Cel9M in association with two other family 9 enzymes from C. cellulolyticum, namely, Cel9E and Cel9G, was measured, and it was found that Cel9M acts synergistically with Cel9E. Complexation of Cel9M with the mini-CipC1 containing the cellulose binding domain, the X2 domain, and the first cohesin domain of the scaffoldin CipC of the bacterium did not significantly increase the hydrolysis of Avicel and bacterial microcrystalline cellulose.     

Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B

Two different types of approach were taken to improve the hydrolytic activity towards crystalline cellulose at elevated temperatures of Melanocarpus albomyces Cel7B (Ma Cel7B), a single-module GH-7 family cellobiohydrolase. Structure-guided protein engineering was used to introduce an additional tenth disulphide bridge to the Ma Cel7B catalytic module. In addition, a fusion protein was constructed by linking a cellulose-binding module (CBM) and a linker from the Trichoderma reesei Cel7A to the C terminus of Ma Cel7B. Both approaches proved successful. The disulphide bridge mutation G4C/M70C located near the N terminus, close to the entrance of the active site tunnel of Ma Cel7B, led to improved thermostability (ΔT _m = 2.5°C). By adding the earlier found thermostability-increasing mutation S290T (ΔT _m = 1.5°C) together with the disulphide bridge mutation, the unfolding temperature was increased by 4°C (mutant G4C/M70C/S290T) compared to that of the wild-type enzyme, thus showing an additive effect on thermostability. Both disulphide mutants had increased activity towards microcrystalline cellulose (Avicel) at 75°C, apparently solely because of their improved thermostability. The addition of a CBM also improved the thermostability (ΔT _m = 2.5°C) and caused a clear (sevenfold) increase in the hydrolysis activity of Ma Cel7B towards Avicel at 70°C.     

A cellulose-binding module of the Trichoderma reesei beta-mannanase Man5A increases the mannan-hydrolysis of complex substrates

Endo-beta-1,4-D-mannanases (beta-mannanase; EC 3.2.1.78) are endohydrolases that participate in the degradation of hemicellulose, which is closely associated with cellulose in plant cell walls. The beta-mannanase from Trichoderma reesei (Man5A) is composed of an N-terminal catalytic module and a C-terminal carbohydrate-binding module (CBM). In order to study the properties of the CBM, a construct encoding a mutant of Man5A lacking the part encoding the CBM (Man5ADeltaCBM), was expressed in T. reesei under the regulation of the Aspergillus nidulans gpdA promoter. The wild-type enzyme was expressed in the same way and both proteins were purified to electrophoretic homogeneity using ion-exchange chromatography. Both enzymes hydrolysed mannopentaose, soluble locust bean gum galactomannan and insoluble ivory nut mannan with similar rates. With a mannan/cellulose complex, however, the deletion mutant lacking the CBM showed a significant decrease in hydrolysis. Binding experiments using activity detection of Man5A and Man5ADeltaCBM suggests that the CBM binds to cellulose but not to mannan. Moreover, the binding of Man5A to cellulose was compared with that of an endoglucanase (Cel7B) from T. reesei.     

Molecular and Biochemical Analyses of CbCel9A/Cel48A,a Highly Secreted Multi-Modular Cellulase by Caldicellulosiruptor bescii during Growth on Crystalline Cellulose

During growth on crystalline cellulose, the thermophilic bacterium Caldicellulosiruptor bescii secretes several cellulose-degrading enzymes. Among these enzymes is CelA (CbCel9A/Cel48A), which is reported as the most highly secreted cellulolytic enzyme in this bacterium. CbCel9A/Cel48A is a large multi-modular polypeptide, composed of an N-terminal catalytic glycoside hydrolase family 9 (GH9) module and a C-terminal GH48 catalytic module that are separated by a family 3c carbohydrate-binding module (CBM3c) and two identical CBM3bs. The wild-type CbCel9A/Cel48A and its truncational mutants were expressed in Bacillus megaterium and Escherichia coli, respectively. The wild-type polypeptide released twice the amount of glucose equivalents from Avicel than its truncational mutant that lacks the GH48 catalytic module. The truncational mutant harboring the GH9 module and the CBM3c was more thermostable than the wild-type protein, likely due to its compact structure. The main hydrolytic activity was present in the GH9 catalytic module, while the truncational mutant containing the GH48 module and the three CBMs was ineffective in degradation of either crystalline or amorphous cellulose. Interestingly, the GH9 and/or GH48 catalytic modules containing the CBM3bs form low-density particles during hydrolysis of crystalline cellulose. Moreover, TM3 (GH9/CBM3c) and TM2 (GH48 with three CBM3 modules) synergistically hydrolyze crystalline cellulose. Deletion of the CBM3bs or mutations that compromised their binding activity suggested that these CBMs are important during hydrolysis of crystalline cellulose. In agreement with this observation, seven of nine genes in a C. bescii gene cluster predicted to encode cellulose-degrading enzymes harbor CBM3bs. Based on our results, we hypothesize that C. bescii uses the GH48 module and the CBM3bs in CbCel9A/Cel48A to destabilize certain regions of crystalline cellulose for attack by the highly active GH9 module and other endoglucanases produced by this hyperthermophilic bacterium.     

Family 7 cellobiohydrolases from Phanerochaete chrysosporium: crystal structure of the catalytic module of Cel7D (CBH58) at 1.32 A resolution and homology models of the isozymes.

Cellobiohydrolase 58 (Cel7D) is the major cellulase produced by the white-rot fungus Phanerochaete chrysosporium, constituting approximately 10 % of the total secreted protein in liquid culture on cellulose. The enzyme is classified into family 7 of the glycosyl hydrolases, together with cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) from Trichoderma reesei. Like those enzymes, it catalyses cellulose hydrolysis with net retention of the anomeric carbon configuration.The structure of the catalytic module (431 residues) of Cel7D was determined at 3.0 A resolution using the structure of Cel7A from T. reesei as a search model in molecular replacement, and ultimately refined at 1.32 A resolution. The core structure is a beta-sandwich composed of two large and mainly antiparallel beta-sheets packed onto each other. A long cellulose-binding groove is formed by loops on one face of the sandwich. The catalytic residues are conserved and the mechanism is expected to be the same as for other family members. The Phanerochaete Cel7D binding site is more open than that of the T. reesei cellobiohydrolase, as a result of deletions and other changes in the loop regions, which may explain observed differences in catalytic properties. The binding site is not, however, as open as the groove of the corresponding endoglucanase. A tyrosine residue at the entrance of the tunnel may be part of an additional subsite not present in the T. reesei cellobiohydrolase.The Cel7D structure was used to model the products of the five other family 7 genes found in P. chrysosporium. The results suggest that at least two of these will have differences in specificity and possibly catalytic mechanism, thus offering some explanation for the presence of Cel7 isozymes in this species, which are differentially expressed in response to various growth conditions.     

Degradation of microcrystalline cellulose: synergism between different endoglucanases of Cellulomonas sp. ATCC 21399

Three immunologically and enzymatically distinct endoglucanases of Cellulomonas sp. ATCC 21399 were purified previously. Endoglucanase A and endoglucanase B acted synergistically on microcrystalline cellulose (Avicel), whereas no synergistic action was observed between endoglucanase B or endoglucanase C. Only endoglucanase A was capable of hydrolyzing Avicel when acting alone and this enzyme resulted in "short fiber formation" when acting on Avicel. The end product of hydrolysis of acid swollen Avicel produced by the three endoglucanases was in all cases dominated by cellobiose and showed lower content of glucose and cellotriose. Higher cellodextrins appeared as transient end products. The results indicate that the function of endoglucanase A in the cellulase system of Cellulomonas might be very similar to the function of the cellobiohydrolases of Trichoderma reesei.     

Exo-mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP-23. A unique type of cellulase among Bacillales.

Sequence analysis of a Paenibacillus sp. BP-23 recombinant clone coding for a previously described endoglucanase revealed the presence of an additional truncated ORF with homology to family 48 glycosyl hydrolases. The corresponding 3509-bp DNA fragment was isolated after gene walking and cloned in Escherichia coli Xl1-Blue for expression and purification. The encoded enzyme, a cellulase of 1091 amino acids with a deduced molecular mass of 118 kDa and a pI of 4.85, displayed a multidomain organization bearing a canonical family 48 catalytic domain, a bacterial type 3a cellulose-binding module, and a putative fibronectin-III domain. The cloned cellulase, unique among Bacillales and designated Cel48C, was purified through affinity chromatography using its ability to bind Avicel. Maximum activity was achieved at 45 degrees C and pH 6.0 on acid-swollen cellulose, bacterial microcrystalline cellulose, Avicel and cellodextrins, whereas no activity was found on carboxy methyl cellulose, cellobiose, cellotriose, pNP-glycosides or 4-methylumbeliferyl alpha-d-glucoside. Cellobiose was the major product of cellulose hydrolysis, identifying Cel48C as a processive cellobiohydrolase. Although no chromogenic activity was detected from pNP-glycosides, TLC analysis revealed the release of p-nitrophenyl-glycosides and cellodextrins from these substrates, suggesting that Cel48C acts from the reducing ends of the sugar chain. Presence of such a cellobiohydrolase in Paenibacillus sp. BP-23 would contribute to widen up its range of action on natural cellulosic substrates.     

The Humicola grisea Cel12A enzyme structure at 1.2 A resolution and the impact of its free cysteine residues on thermal stability

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have extended our previous work on the structural and biochemical diversity of GH 12 homologs to include the most stable fungal GH 12 found, Humicola grisea Cel12A. The H. grisea enzyme was much more stable to irreversible thermal denaturation than the Trichoderma reesei enzyme. It had an apparent denaturation midpoint (T(m)) of 68.7 degrees C, 14.3 degrees C higher than the T. reesei enzyme. There are an additional three cysteines found in the H. grisea Cel12A enzyme. To determine their importance for thermal stability, we constructed three H. grisea Cel12A single mutants in which these cysteines were exchanged with the corresponding residues in the T. reesei enzyme. We also introduced these cysteine residues into the T. reesei enzyme. The thermal stability of these variants was determined. Substitutions at any of the three positions affected stability, with the largest effect seen in H. grisea C206P, which has a T(m) 9.1 degrees C lower than that of the wild type. The T. reesei cysteine variant that gave the largest increase in stability, with a T(m) 3.9 degrees C higher than wild type, was the P201C mutation, the converse of the destabilizing C206P mutation in H. grisea. To help rationalize the results, we have determined the crystal structure of the H. grisea enzyme and of the most stable T. reesei cysteine variant, P201C. The three cysteines in H. grisea Cel12A play an important role in the thermal stability of this protein, although they are not involved in a disulfide bond.     

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility

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

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

This study examined the cellulytic effects on steam-pretreated barley straw of cellulose-degrading enzyme systems from the five thermophilic fungi Chaetomium thermophilum, Thielavia terrestris, Thermoascus aurantiacus, Corynascus thermophilus, and Myceliophthora thermophila and from the mesophile Penicillum funiculosum. The catalytic glucose release was compared after treatments with each of the crude enzyme systems when added to a benchmark blend of a commercial cellulase product, Celluclast, derived from Trichoderma reesei and a beta-glucosidase, Novozym 188, from Aspergillus niger. The enzymatic treatments were evaluated in an experimental design template comprising a span of pH (3.5-6.5) and temperature (35-65 degrees C) reaction combinations. The addition to Celluclast + Novozym 188 of low dosages of the crude enzyme systems, corresponding to 10 wt % of the total enzyme protein load, increased the catalytic glucose yields significantly as compared to those obtained with the benchmark Celluclast + Novozyme 188 blend. A comparison of glucose yields obtained on steam-pretreated barley straw and microcrystalline cellulose, Avicel, indicated that the yield improvements were mainly due to the presence of highly active endoglucanase activity/activities in the experimental enzyme preparations. The data demonstrated the feasibility of boosting the widely studied T. reeseicellulase enzyme system with additional enzymatic activity to achieve faster lignocellulose degradation. We conclude that this supplementation strategy appears feasible as a first step in identifying truly promising fungal enzyme sources for fast development of improved, commercially viable, enzyme preparations for lignocellulose degradation.