Search

In a previous study, we reported an alkaliphilic and thermostable endoglucanase (BsGH7-3) of glycoside hydrolase family 7 (GH7) from the hemibiotrophic plant pathogen Bipolaris sorokiniana. However, the catalytic efficiency of the enzyme was lower than for some other endoglucanases of the GH7 family reported in the literature. To engineer a more active enzyme, we identified conserved residues in the substrate-binding tunnel and on the surface of the protein that could play a role in charge-charge interaction and stabilize the structure. The mutants D257W and Q225H in the substrate-binding tunnel and Y222R and Q401N on the protein surface showed a 2-fold increase in specific activity and a 1.5-fold increase in turnover number and were active over a broader range of pH. The mutants also showed a higher tolerance to NaCl. The rational design of the BsGH7-3 mutants helped in increasing the catalytic efficiency of the thermostable enzyme and may be useful in combination with other cellulases like cellobiohydrolase and β-glucosidase towards complete saccharification of cellulose into glucose.

In this study, extended artificial scaffoldins possessing multiple cohesin modules were created in vivo by employing split-intein-mediated protein ligation. Artificial scaffoldins having one Clostridium thermocellum cohesin (Coh t ), one carbohydrate binding module (CBM) from Clostridium cellulolyticum scaffolding protein CipC, and one to five cohesins (Coh c ) derived from CipC, were assembled. These scaffoldins were used to assemble cellulosomal enzyme complexes for investigating the interplay among endoglucanase, exoglucanase, and scaffoldin-borne CBM, on the hydrolysis of a model microcrystalline cellulose substrate, Avicel. The cellulosomal complexes were assembled in vitro by incubating recombinant C. thermocellum endoglucanase (A t ) and C. cellulolyticum exoglucanase (E c ), with the various artificial scaffoldins. Under a fixed total cellulase concentration, improved hydrolysis is noted by recruiting both E c and A t on the same scaffoldin, for all scaffoldins tested, compared with free cellulases. The improvement is more profound with scaffoldins having a higher Coh c /Coh t ratio (i.e., increased E c /A t ratio). Furthermore, among scaffoldins having the same Coh c /Coh t ratio, highest rates of Avicel hydrolysis are noted when Coh t , and hence an endoglucanase, is situated next to the CBM and not flanked by Coh c . These results point to the importance of using scaffoldins with sufficiently high numbers of cohesin units to achieve an optimal exo-/endo-glucanase ratio to create efficient designer cellulosomes. Furthermore, intein-trans-splicing is proven here to be an effective method for assembling complex scaffoldins and more intricate cellulosomes.

Cellulolytic microorganisms play important roles in global carbon cycling and have evolved diverse strategies to digest cellulose. Some are 'generous,' releasing soluble sugars from cellulose extracellularly to feed both themselves and their neighbors. The gliding soil bacterium Cytophaga hutchinsonii exhibits a more 'selfish' strategy. It digests crystalline cellulose using cell-associated cellulases and releases little soluble sugar outside of the cell. The mechanism of C. hutchinsonii cellulose utilization is still poorly understood. In this review, we discuss novel aspects of the C. hutchinsonii cellulolytic system. Recently developed genetic manipulation tools allowed the identification of proteins involved in C. hutchinsonii cellulose utilization. These include periplasmic and cell-surface endoglucanases and novel cellulose-binding proteins. The recently discovered type IX secretion system is needed for cellulose utilization and appears to deliver some of the cellulolytic enzymes and other proteins to the cell surface. The requirement for periplasmic endoglucanases for cellulose utilization is unusual and suggests that cello-oligomers must be imported across the outer membrane before being further digested. Cellobiohydrolases or other predicted processive cellulases that play important roles in many other cellulolytic bacteria appear to be absent in C. hutchinsonii. Cells of C. hutchinsonii attach to and glide along cellulose fibers, which may allow them to find sites most amenable to attack. A model of C. hutchinsonii cellulose utilization summarizing recent progress is proposed.

Cel6D from Paenibacillus barcinonensis is a modular cellobiohydrolase with a novel molecular architecture among glycosyl hydrolases of family 6. It contains an N-terminal catalytic domain (family 6 of glycosyl hydrolases (GH6)), followed by a fibronectin III-like domain repeat (Fn3 1,2 ) and a C-terminal family 3b cellulose-binding domain (CBM3b). The enzyme has been identified and purified showing catalytic activity on cellulosic substrates and cellodextrins, with a marked preference for phosphoric acid swollen cellulose (PASC). Analysis of mode of action of Cel6D shows that it releases cellobiose as the only hydrolysis product from cellulose. Kinetic parameters were determined on PASC showing a K m of 68.73 mg/ml and a V max of 1.73 U/mg. A series of truncated derivatives of Cel6D have been constructed and characterized. Deletion of CBM3b caused a notable reduction in hydrolytic activity, while deletion of the Fn3 domain abolished activity, as the isolated GH6 domain was not active on any of the substrates tested. Mutant enzymes Cel6D-D146A and Cel6D-D97A were constructed in the residues corresponding to the putative acid catalyst and to the network for the nucleophilic attack. The lack of activity of the mutant enzymes indicates the important role of these residues in catalysis. Analysis of cooperative activity of Cel6D with cellulases from the same producing P. barcinonensis strain reveals high synergistic activity with processive endoglucanase Cel9B on hydrolysis of crystalline substrates. The characterized cellobiohydrolase can be a good contribution for depolymerization of cellulosic substrates and for the deconstruction of native cellulose.

We recently discovered a novel glycoside hydrolase family 6 (GH6) cellobiohydrolase from Paenibacillus curdlanolyticus B-6 (PcCel6A), which is rarely found in bacteria. This enzyme is a true exo-type cellobiohydrolase which exhibits high substrate specificity on amorphous cellulose and low substrate specificity on crystalline cellulose, while this showed no activity on substitution substrates, carboxymethyl cellulose and xylan, distinct from all other known GH6 cellobiohydrolases. Product profiles, HPLC analysis of the hydrolysis products and a schematic drawing of the substrate-binding subsites catalysing cellooligosaccharides can explain the new mode of action of this enzyme which prefers to hydrolyse cellopentaose. PcCel6A was not inhibited by glucose or cellobiose at concentrations up to 300 and 100 mM, respectively. A good synergistic effect for glucose production was found when PcCel6A acted together with processive endoglucanase Cel9R from Clostridium thermocellum and β-glucosidase CglT from Thermoanaerobacter brockii. These properties of PcCel6A make it a suitable candidate for industrial application in the cellulose degradation process.

Despite multiple research efforts, the current strategies for exploitation of lignocellulosic plant matter are still far from optimal, being hampered mostly by the difficulty of degrading the recalcitrant parts. An interesting approach is to use lignocellulose-degrading microbial communities by using different environmental sources of microbial inocula. However, it remains unclear whether the inoculum source matters for the degradation process. Here, we addressed this question by verifying the lignocellulose degradation potential of wheat (Triticum aestivum) straw by microbial consortia generated from three different microbial inoculum sources, i.e., forest soil, canal sediment and decaying wood. We selected these consortia through ten sequential-batch enrichments by dilution-to-stimulation using wheat straw as the sole carbon source. We monitored the changes in microbial composition and abundance, as well as their associated degradation capacity and enzymatic activities. Overall, the microbial consortia developed well on the substrate, with progressively-decreasing net average generation times. Each final consortium encompassed bacterial/fungal communities that were distinct in composition but functionally similar, as they all revealed high substrate degradation activities. However, we did find significant differences in the metabolic diversities per consortium: in wood-derived consortia cellobiohydrolases prevailed, in soil-derived ones β-glucosidases, and in sediment-derived ones several activities. Isolates recovered from the consortia showed considerable metabolic diversities across the consortia. This confirmed that, although the overall lignocellulose degradation was similar, each consortium had a unique enzyme activity pattern. Clearly, inoculum source was the key determinant of the composition of the final microbial degrader consortia, yet with varying enzyme activities. Hence, in accord with Beyerinck's, "everything is everywhere, the environment selects" the source determines consortium composition.

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.

Cellulases can be engineered with enhanced properties for broad use in scientific and industrial applications. In this study, the wild-type cbh2 gene of the thermophilic fungus Chaetomium thermophilum encoding cellobiohydrolase II (CBHII) was mutagenized through in vitro directed evolution. The resulting Pichia pastoris yeast library was screened, and two transformants were selected for enhanced CBHII activities that were not attributed to increased gene copy numbers. The optimum fermentation times of the two mutant transformants were shortened to 4-5 days after methanol induction compared to 6 days for the wild-type. The optimum reaction temperature (60 °C) and pH level (5 or 6) of the mutant CBHII proteins, designated CBHIIX16 and CBHIIX305, were higher than those of wild-type CBHII (50 °C and pH 4). Kept at 80 °C for 1 h, CBHIIX16 and CBHIIX305 retained >50% of their activities, while the wild-type CBHII lost all activity. Sequence analysis of CBHIIX16 and CBHIIX305 revealed that they contained five and six mutated amino acids, respectively. Structural modeling confirmed the presence of carbohydrate binding type-1 and catalytic domains, where the hydrogen bond numbers between the 227th and 203rd amino acids were increased, which perhaps contributed to the elevated enzyme stability. Therefore, the two CBHII mutants selected for increased enzymatic activities also demonstrated elevated optimum reaction temperature and pH levels and enhanced thermal stability. These properties may be beneficial in practical applications for CBHII.

To explore the potential of Ashbya gossypii as a host for the expression of recombinant proteins and to assess whether protein secretion would be more similar to the closely related Saccharomyces cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were successfully expressed in A. gossypii from plasmids containing the two micron sequences from S. cerevisiae, under the S. cerevisiae PGK1 promoter. The native signal sequences of EGI and CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. gossypii. Although CBHI activity was not detected using 4-methylumbelliferyl-beta-D: -lactoside as substrate, the protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable, the specific activity being comparable to that produced by a similar EGI producing S. cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared with the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae.

Family 48 glycoside hydrolases (cellobiohydrolases) are among the most important cellulase components for crystalline cellulose hydrolysis mediated by cellulolytic bacteria. Open reading frame (Cphy_3368) of Clostridium phytofermentans ISDg encodes a putative family 48 glycoside hydrolase (CpCel48) with a family 3 cellulose-binding module. CpCel48 was successfully expressed as two soluble intracellular forms with or without a C-terminal His-tag in Escherichia coli and as a secretory active form in Bacillus subtilis. It was found that calcium ion enhanced activity and thermostability of the enzyme. CpCel48 had high activities of 15.1 U micromol(-1) on Avicel and 35.9 U micromol(-1) on regenerated amorphous cellulose (RAC) with cellobiose as a main product and cellotriose and cellotetraose as by-products. By contrast, it had very weak activities on soluble cellulose derivatives (e.g., carboxymethyl cellulose (CMC)) and did not significantly decrease the viscosity of the CMC solution. Cellotetraose was the smallest oligosaccharide substrate for CpCel48. Since processivity is a key characteristic for cellobiohydrolases, the new initial false/right attack model was developed for estimation of processivity by considering the enzyme's substrate specificity, the crystalline structure of homologous Cel48 enzymes, and the configuration of cellulose chains. The processivities of CpCel48 on Avicel and RAC were estimated to be approximately 3.5 and 6.0, respectively. Heterologous expression of secretory active cellobiohydrolase in B. subtilis is an important step for developing recombinant cellulolytic B. subtilis strains for low-cost production of advanced biofuels from cellulosic materials in a single step.

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 (DeltaT (m) = 2.5 degrees C). By adding the earlier found thermostability-increasing mutation S290T (DeltaT (m) = 1.5 degrees C) together with the disulphide bridge mutation, the unfolding temperature was increased by 4 degrees 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 degrees C, apparently solely because of their improved thermostability. The addition of a CBM also improved the thermostability (DeltaT (m) = 2.5 degrees C) and caused a clear (sevenfold) increase in the hydrolysis activity of Ma Cel7B towards Avicel at 70 degrees C.

Production of cellulolytic enzymes, such as cellobiohydrolases (CBH) and cellobiose dehydrogenase (CDH), by the basidiomycete Phanerochaete chrysosporium is significantly repressed in glucose-containing media; this is known as carbon catabolite repression. We have analyzed the glucose concentration dependence of transcript numbers of the cellulolytic genes (cel6A, cel7D, and cdh) and beta-glucosidase gene (bgl3A) by means of real-time quantitative reverse transcriptase polymerase chain reaction to investigate the roll of carbon catabolite derepression in these gene expression. When the mycelium of P. chrysosporium grown in glucose culture was transferred to media containing various concentrations of glucose (0-5,000 microM), the expression levels of cel6A, cel7D, and cdh were drastically influenced by glucose, whereas no significant change was observed in bgl3A. The numbers of transcripts of cel6A, cel7D, and cdh increased exponentially during incubation for 6 h in the culture without glucose, and the rates of increase were 2.1 times per hour for cel6A transcripts and 2.7 times per hour for cel7D transcripts. Moreover, derepression of cel6A and cel7D was delayed (by 1.6 and 0.6 h, respectively) when the culture contained 50 microM glucose compared with that in the absence of glucose, suggesting that the promoter activities of cel7D and cel6A are distinct under conditions of carbon catabolite derepression.

To utilize glucoamylase-displaying yeast cells for enzymatic desizing of starched cotton cloth, we constructed yeast strains that codisplayed Rhizopus oryzae glucoamylase and two kinds of Trichoderma reesei cellulose-binding domains (CBD1, CBD of cellobiohydrolase I (CBHI); and CBD2, CBD of cellobiohydrolase II (CBHII)). In this study, we aimed to obtain a high efficiency of enzymatic desizing of starched cotton cloth. Yeast cells that codisplayed glucoamylase and CBD had higher activity on starched cotton cloth than yeast cells that displayed only glucoamylase. Glucoamylase and double CBDs (CBD1 and CBD2) codisplaying yeast cells exhibited the highest activity ratio (4.36-fold), and glucoamylase and single CBD (CBD1 or CBD2) codisplaying yeast cells had higher relative activity ratios (2.78- and 2.99-fold, respectively) than glucoamylase single-displaying cells. These results indicate that the glucoamylase activity of glucoamylase-displaying cells would be affected by the binding ability of CBD codisplayed on the cell surface to starched cotton cloth. These novel strains might play useful roles in the enzymatic desizing of starched cotton cloth in the textile industry.

In the current studies, we sequenced and characterized the genomic and complementary deoxyribonucleic acid clones encoding the cellobiohydrolase encoding genes cel1 and cel2 of Polyporus arcularius. The predicted amino acid sequences of Cel1 and Cel2 are similar to glycosyl hydrolase family 7 and 6 proteins, respectively. The expression of cel1 and cel2 was induced by microcrystalline cellulose (Avicel) and cellopentaose but repressed by glucose, cellobiose, cellotriose, and cellotetraose. There was a very low level of cel1 and cel2 transcription regardless of the carbon source. These results suggest that P. arcularius cells constitutively express a very low level of cellulase that can degrade insoluble crystalline cellulose and that the transcription of cel1 and cel2 in the cells is induced by products produced by these endoglucanases such as cellooligosaccharides.

A cellobiohydrolase-encoding cDNA, Tvcel7a, from Trametes versicolor has been cloned and expressed in Aspergillus niger. The deduced amino acid sequence shows that Tvcel7a encodes a 456-amino acid polypeptide belonging to glycosyl hydrolase family 7. TvCel7a possesses a 19-amino acid secretion signal but does not possess a linker region nor a carbohydrate-binding domain. Two peaks of activity were obtained after TvCel7a was purified to apparent homogeneity by gel-filtration followed by anion-exchange chromatography. Mass spectrometry performed on the purified proteins confirmed that both peaks corresponded to the predicted sequence of the T. versicolor cellulase. The biochemical properties of the purified TvCel7a obtained from both peaks were studied in detail. The pH and temperature optima were 5.0 and 40 degrees C, respectively. The enzyme was stable over a pH range extending from pH 3.0 to 9.0 and at temperatures lower than 50 degrees C. The kinetic parameters with the substrate p-nitrophenyl beta-D: -cellobioside (pNPC) were 0.58 mM and 1.0 micromol/min/mg protein for the purified TvCel7a found in both peaks 1 and 2. TvCel7a catalyzes the hydrolysis of pNPC, filter paper, beta-glucan, and avicel to varying extents, but no detectable hydrolysis was observed when using the substrates carboxymethylcellulose, laminarin and pNPG.

Cellulose was treated with different extracellular microbial enzymes. The treatment of cellulose with the enzymes can improve alkaline solubility. Both endoglucanase and crude cellulase decreased the average degrees of polymerization ([see text]) and improved the alkaline solubility of cellulose most efficiently. The composition of the enzyme, the type of cellulosic materials, pretreatment, and the treatment conditions are the key factors for its effective processing, using the enzymes to improve on alkaline solubility of cellulose. The improvement in the alkaline solubility is caused by the decrease in [see text] and hydrogen bond because of enzymatic hydrolysis.

A gene encoding a cellobiohydrolase (CBH) was isolated from Thermoascus aurantiacus IFO 9748 and designated as cbh1. The deduced amino acid sequence encoded by cbh1 showed high homology with the sequence of glycoside hydrolase family 7. To confirm the sequence of the gene encoding the CBH, the cloned gene was expressed in the yeast Saccharomyces cerevisiae, in which no cellulase activity was found, and the gene product was purified and subjected to enzymatic characterization. The recombinant enzyme was confirmed as a CBH by analysis of the reaction product and designated as CBHI. Recombinant CBHI retained more than 80% of its initial activity after 1 h of incubation at 65 degrees C and was stable in the pH range 3.0-9.0. The optimal temperature for enzyme activity was about 65 degrees C and the optimal pH was about 6.0. The recombinant enzyme was found to be highly glycosylated and this glycosylation was shown to contribute to the thermostability of the enzyme. CBHI expression was shown to be induced at higher temperature in T. aurantiacus.