Molecular Mechanisms of Interactions between Monolayered Transition Metal Dichalcogenides and Biological Molecules.

Single layered two-dimensional (2D) materials such as transition metal dichalcogenides (TMDs) show great potential in many microelectronic or nanoelectronic applications. For example, because of extremely high sensitivity, TMD-based biosensors have become promising candidates for next-generation label-free detection. However, very few studies have been conducted on understanding the fundamental interactions between TMDs and other molecules including biological molecules, making the rational design of TMD-based sensors (including biosensors) difficult. This study focuses on the investigations of the fundamental interactions between proteins and two widely researched single-layered TMDs, MoS2, and WS2 using a combined study with linear vibrational spectroscopy attenuated total reflectance FTIR and nonlinear vibrational spectroscopy sum frequency generation vibrational spectroscopy, supplemented by molecular dynamics simulations. It was concluded that a large surface hydrophobic region in a relatively flat location on the protein surface is required for the protein to adsorb onto a monolayered MoS2 or WS2 surface with preferred orientation. No disulfide bond formation between cysteine groups on the protein and MoS2 or WS2 was found. The conclusions are general and can be used as guiding principles to engineer proteins to attach to TMDs. The approach adopted here is also applicable to study interactions between other 2D materials and biomolecules.

Enzymatic and molecular characterization of an endoglucanase E from Clostridium cellulovorans 743B.

Endoglucanase E (EngE) is a cellulosomal enzyme of the glycoside hydrolase family 5 generated by the cellulosome-producing bacterium Clostridium cellulovorans 743B. Although its basic activities and properties have been characterized, its substrate specificity, product range, and steady-state kinetics remain unclear. The current study prepared recombinant EngE (rEngE) and analyzed its substrate specificity and product range using thin layer chromatography. When carboxymethyl cellulose (CMC) or phosphoric acid swollen cellulose was used as a substrate, disaccharides and trisaccharides were the main products. However, no product was detected with microcrystalline cellulose as the substrate. This indicated that rEngE is a cellulase that hydrolyzes low-crystallinity cellulose. Furthermore, products were detected when glucomannan, lichenan, or ß-glucan was used, but no product was obtained with xylan. These results suggested that rEngE hydrolyzes the ß-1,4 glycosidic bond between glucose residues of the substrate. In the kinetic analysis, at CMC concentrations of ≥3 mg/mL, the reaction rate decreased. Application of the above data to three substrate inhibition models generated a better fit to a model that generates products not only from the enzyme-substrate complex but also from enzyme-substrate-substrate (ESS) complexes, in which two substrates are bound to the enzymes. In addition, it was found that a carbohydrate-binding module (CBM) contained in EngE binds to cellulose. Therefore, substrate inhibition likely occurred because the binding site of CBM may correspond to one of the substrate-binding sites in the ESS complex.

Xylanase B from Clostridium cellulovorans 743B: overexpression, purification, crystallization and X-ray diffraction analysis.

Clostridium cellulovorans produces multi-enzyme complexes called cellulosomes capable of efficiently degrading cellulosic biomass. There are three xylanase genes containing a sequence corresponding to a dockerin domain that are necessary for constructing cellulosomes in the genome. Among the xylanases encoded by these genes, xylanase B (XynB) contains a catalytic domain belonging to glycoside hydrolase family 10 and a carbohydrate-binding module (CBM) at the N-terminus, making it a member of CBM family 22. In this study, XynB was cloned, overexpressed, purified and crystallized. XynB was crystallized using the hanging-drop vapour-diffusion method in the presence of 0.2 M sodium acetate trihydrate, 0.1 M Tris-HCl pH 8.5, 32%(w/v) PEG 4000 at 293 K. X-ray diffraction analysis revealed that the crystal diffracted to 1.95 Šresolution and belonged to space group P212121, with unit-cell parameters a = 74.28, b = 77.55, c = 88.20 Å, α = ß = γ = 90°. The data-evaluation statistics revealed high quality of the collected data, thereby establishing a solid basis for determination of the structure of cellulosomal xylanase from C. cellulovorans.

A pepper mottle virus-based vector enables systemic expression of endoglucanase D in non-transgenic plants.

Plant-virus-based expression vectors have been used as an alternative to the creation of transgenic plants. Using a virus-based vector, we investigated the feasibility of producing the endoglucanase D (EngD) from Clostridium cellulovorans in Nicotiana benthamiana. This protein has endoglucanase, xylanase, and exoglucanase activities and may be of value for cellulose digestion in the generation of biofuels from plant biomass. The EngD gene was cloned between the nuclear inclusion b (NIb)- and coat protein (CP)-encoding sequences of pSP6PepMoV-Vb1. In vitro transcripts derived from the clone (pSP6PepMoV-Vb1/EngD) were infectious in N. benthamiana but caused milder symptoms than wild-type PepMoV-Vb1. RT-PCR amplification of total RNA from non-inoculated upper leaves infected with PepMoV-Vb1/EngD produced the target band for the CP, partial NIb and EngD-CP regions of PepMoV-V1/EngD, in addition to nonspecific bands. Western blot analysis showed the CP target bands of PepMoV-Vb1/EngD as well as non-target bands. EngD enzymatic activity in infected plants was detected using a glucose assay. The plant leaves showed increased senescence compared with healthy and PepMoV-Vb1-infected plants. Our study suggests the feasibility of using a viral vector for systemic infection of plants for expression of heterologous engD for the purpose of digesting a cellulose substrate in plant cells for biomass production.

Characterization of the cellulosomal scaffolding protein CbpC from Clostridium cellulovorans 743B.

Clostridium cellulovorans 743B, an anaerobic and mesophilic bacterium, produces an extracellular enzyme complex called the cellulosome on the cell surface. Recently, we have reported the whole genome sequence of C. cellulovorans, which revealed that a total of 4 cellulosomal scaffolding proteins: CbpA, HbpA, CbpB, and CbpC were encoded in the C. cellulovorans genome. In particular, cbpC encoded a 429-residue polypeptide that includes a carbohydrate-binding module (CBM), an S-layer homology module, and a cohesin. CbpC was also detected in the culture supernatant of C. cellulovorans. Genomic DNA coding for CbpC was subcloned into a pET-22b+ vector in order to express and produce the recombinant protein in Escherichia coli BL21(DE3). Measurement of CbpC adsorption to crystalline cellulose indicated a dissociation constant of 0.60 µM, which is a similar to that of CBM from CbpA. We also subcloned the region encoding xylanase B (XynB) with the dockerin from C. cellulovorans and analyzed the interaction between XynB and CbpC by GST pull-down assay. It was observed that GST-CbpC assembles with XynB to form a minimal cellulosome. The activity of XynB against rice straw tended to be increased in the presence of CbpC. These results showed a synergistic effect on rice straw as a representative cellulosic biomass through the formation of a minimal cellulosome containing XynB bound to CbpC. Thus, our findings provide a foundation for the development of cellulosic biomass saccharification using a minimal cellulosome.

Mutation of a conserved tryptophan residue in the CBM3c of a GH9 endoglucanase inhibits activity.

The presence of the family of 3c cellulose binding module (CBM3c) is important for the catalytic activity of family 9 endoglucanases such as the EngZ from Clostridium cellulovorans. To determine the role of CBM3c in catalytic activity, we made a tryptophan to alanine substitution because tryptophan can bind strongly to both substrates and other amino acids. The conserved tryptophan substitution (W483A) did not influence substrate binding, but it reduced enzyme activity to 10-14% on both amorphous and crystalline cellulose. CBM3c is directly involved in the endoglucanase reaction independent of substrate binding. EngZ W483A was also inactivated independent of substrate concentrations. Specially, EngZ W483A restored its catalytic base activity (31.6±1.2U/nM) which is similar to the wild-type (29.4±0.3U/nM) on Avicel in the presence of 50mM sodium azide which is instead of catalytic base reaction. These results suggest that CBM3c is deeply involved in the cellulolytic reaction, specifically at the catalytic base region. Moreover, EngZ W483A was also easily denatured by DTT, an outer disulfide bond breaker, compared to the wild-type. CBM3c could influence the surface stability. These features of CBM3c result from the hydrophobic interaction of tryptophan with the catalytic domain that is unrelated to substrate binding.

In vitro flow cytometry-based screening platform for cellulase engineering.

Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 10(7) events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-ß-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg).

Enhanced thermostability of mesophilic endoglucanase Z with a high catalytic activity at active temperatures.

This is the first study for therrmostable mutants of mesophilic endoglucanase EngZ from Clostridium cellulovorans using by site-directed mutagenesis. K94R, S365P and their double mutant K94R/S365P had a wide range of active temperatures (30-60 °C). In addition, the optimal temperature of K94R/S365P was increased by 7.5 °C. K94R/S365P retained 78.3% relative activity at 70 °C, while the wild type retained only 5.8%. Especially, K94R/S365P remained 45.1-fold higher activity than the wild type at 70 °C. In addition, K94R/S365P was 3.1-fold higher activity than the wild type at 42.5 °C, which is the optimal temperature of the wild type. K94R/S365P showed also stimulated in 2.5-fold lower concentration of CaCl2 and delayed aggregation temperature in the presence of CaCl2 compared to the wild type. In pH stability, K94R/S365P was not influenced, but the optimum pH was transferred from pH 7 to pH 6. In long-term hydrolysis, K94R/S365P reduced the newly released reducing sugar yields after 12h reaction; however, the yields consistently increased until 72h. Finally, the total reducing sugar of K94R/S365P was 5.0-fold higher than the wild type at 50 °C, pH6. EngZ (K94R/S365P) can support information to develop thermostability of GH9 endoglucanase with a high catalytic efficiency as the potential industrial bioprocess candidate.

Restriction modification system analysis and development of in vivo methylation for the transformation of Clostridium cellulovorans.

Clostridium cellulovorans, a cellulolytic bacterium producing butyric and acetic acids as main fermentation products, is a promising host for biofuel production from cellulose. However, the transformation method of C. cellulovorans was not available, hindering its genetic engineering. To overcome this problem, its restriction modification (RM) systems were analyzed and a novel in vivo methylation was established for its successful transformation in the present study. Specifically, two RM systems, Cce743I and Cce743II, were determined. R. Cce743I has the same specificity as LlaJI, recognizing 5'-GACGC-3' and 5'-GCGTC-3', while M. Cce743I methylates the external cytosine in the strand (5'-GACG(m)C-3'). R. Cce743II, has the same specificity as LlaI, recognizing 5'-CCAGG-3' and 5'-CCTGG-3', while M. Cce743II methylates the external cytosine of both strands. An in vivo methylation system, expressing M. Cce743I and M. Cce743II from C. cellulovorans in Escherichia coli, was then established to protect plasmids used in electrotransformation. Transformants expressing an aldehyde/alcohol dehydrogenase (adhE2), which converted butyryl-CoA to n-butanol and acetyl-CoA to ethanol, were obtained. For the first time, an effective transformation method was developed for metabolic engineering of C. cellulovorans for biofuel production directly from cellulose.

Structures of exoglucanase from Clostridium cellulovorans: cellotetraose binding and cleavage.

Exoglucanase/cellobiohydrolase (EC 3.2.1.176) hydrolyzes a ß-1,4-glycosidic bond from the reducing end of cellulose and releases cellobiose as the major product. Three complex crystal structures of the glycosyl hydrolase 48 (GH48) cellobiohydrolase S (ExgS) from Clostridium cellulovorans with cellobiose, cellotetraose and triethylene glycol molecules were solved. The product cellobiose occupies subsites +1 and +2 in the open active-site cleft of the enzyme-cellotetraose complex structure, indicating an enzymatic hydrolysis function. Moreover, three triethylene glycol molecules and one pentaethylene glycol molecule are located at active-site subsites -2 to -6 in the structure of the ExgS-triethylene glycol complex shown here. Modelling of glucose into subsite -1 in the active site of the ExgS-cellobiose structure revealed that Glu50 acts as a proton donor and Asp222 plays a nucleophilic role.

Short-time dynamics of pH-dependent conformation and substrate binding in the active site of beta-glucosidases: A computational study.

The complete degradation of cellulose to glucose is essential to carbon turnover in terrestrial ecosystems and to engineered biofuel production. A rate-limiting step in this pathway is catalyzed by beta-glucosidase (BG) enzymes, which convert cellulobiose into two glucose molecules. The activity of these enzymes has been shown to vary with solution pH. However, it is not well understood how pH influences the enzyme conformation required for catalytic action on the substrate. A structural understanding of this pH effect is important for predicting shifts in BG activity in bioreactors and environmental matrices, in addition to informing targeted protein engineering. Here we applied molecular dynamics simulations to explore conformational and substrate binding dynamics in two well-characterized BGs of bacterial (Clostridium cellulovorans) and fungal (Trichoderma reesei) origins as a function of pH. The enzymes were simulated in an explicit solvated environment, with NaCl as electrolytes, at their prominent ionization states obtained at pH 5, 6, 7, and 7.5. Our findings indicated that pH-dependent changes in the ionization states of non-catalytic residues localized outside of the immediate active site led to pH-dependent disruption of the active site conformation. This disruption interferes with favorable H-bonding interactions with catalytic residues required to initiate catalysis on the substrate. We also identified specific non-catalytic residues that are involved in stabilizing the substrate at the optimal pH for enzyme activity. The simulations further revealed the dynamics of water-bridging interactions both outside and inside the substrate binding cleft during structural changes in the enzyme-substrate complex. These findings provide new structural insights into the pH-dependent substrate binding specificity in BGs.

ß-mannanase (Man26A) and α-galactosidase (Aga27A) synergism - a key factor for the hydrolysis of galactomannan substrates.

This study investigated the behavior of mannan-degrading enzymes, specifically focusing on differences with respect to their substrate specificities and their synergistic associations with enzymes from different glycoside hydrolase (GH) families. Galactosidases from Cyamopsis tetragonolobus seeds (Aga27A, GH27) and Aspergillus niger (AglC, GH36) were evaluated for their abilities to synergistically interact with mannanases from Clostridium cellulovorans (ManA, GH5) and A. niger (Man26A, GH26) in hydrolysis of guar gum and locust bean gum. Among the mannanases, Man26A was more efficient at hydrolyzing both galactomannan substrates, while among the galactosidases; Aga27A was the most effective at removing galactose substituents on both galactomannan substrates and galactose-containing oligosaccharides. An optimal protein mass ratio of glycoside hydrolases required to maximize the release of both reducing sugar and galactose residues was determined. Clear synergistic enhancement of locust bean gum hydrolysis with respect to reducing sugar release was observed when both mannanases at 75% enzyme dosage were supplemented with 25% enzyme protein dosage of Aga27A. At a protein ratio of 75% Man26A to 25% Aga27A, the presence of Man26A significantly enhanced galactose release by 25% Aga27A (2.36 fold) with locust bean gum, compared to when Aga27A was used alone at 100% enzyme protein dosage. A dosage of Aga27A at 75% and ManA at 25% protein content liberated the highest reducing sugar release on guar gum hydrolysis. A dosage of Man26A and Aga27A at 75-25% protein content, respectively, liberated reducing sugar release equivalent to that when Man26A was used alone at 100% protein content. From the findings obtained in this study, it was observed that the GH family classification of an enzyme affects its substrate specificity and synergistic interactions with other glycoside hydrolases from different families (more so than its EC classification). The GH26 Man26A and GH27 Aga27A enzymes appeared to be more promising for applications that involve the hydrolysis of galactomannan containing biomass. This method of screening for maximal compatibility between various GH families can ultimately lead to a more rational development of tailored enzyme cocktails for lignocellulose hydrolysis.

A noncellulosomal mannanase26E contains a CBM59 in Clostridium cellulovorans.

A multicomponent enzyme-complex prevents efficient degradation of the plant cell wall for biorefinery. In this study, the method of identifying glycoside hydrolases (GHs) to degrade hemicelluloses was demonstrated. The competence of C. cellulovorans, which changes to be suitable for degradation of each carbon source, was used for the method. C. cellulovorans was cultivated into locust bean gum (LBG) that is composed of galactomannan. The proteins produced by C. cellulovorans were separated into either fractions binding to crystalline cellulose or not. Proteins obtained from each fraction were further separated by SDS-PAGE and were stained with Coomassie Brilliant Blue and were detected for mannanase activity. The proteins having the enzymatic activity for LBG were cut out and were identified by mass spectrometry. As a result, four protein bands were classified into glycosyl hydrolase family 26 (GH26) mannanases. One of the identified mannanases, Man26E, contains a carbohydrate-binding module (CBM) family 59, which binds to xylan, mannan, and Avicel. Although mannose and galactose are the same as a hexose, the expression patterns of the proteins from C. cellulovorans were quite different. More interestingly, zymogram for mannanase activity showed that Man26E was detected in only LBG medium.

Unique contribution of the cell wall-binding endoglucanase G to the cellulolytic complex in Clostridium cellulovorans.

The cellulosomes produced by Clostridium cellulovorans are organized by the specific interactions between the cohesins in the scaffolding proteins and the dockerins of the catalytic components. Using a cohesin biomarker, we identified a cellulosomal enzyme which belongs to the glycosyl hydrolase family 5 and has a domain of unknown function 291 (DUF291) with functions similar to those of the surface layer homology domain in C. cellulovorans. The purified endoglucanase G (EngG) had the highest synergistic degree with exoglucanase (ExgS) in the hydrolysis of crystalline cellulose (EngG/ExgS ratio = 3:1; 1.71-fold). To measure the binding affinity of the dockerins in EngG for the cohesins of the main scaffolding protein, a competitive enzyme-linked interaction assay was performed. Competitors, such as ExgS, reduced the percentage of EngG that were bound to the cohesins to less than 20%; the results demonstrated that the cohesins prefer to bind to the common cellulosomal enzymes rather than to EngG. Additionally, in surface plasmon resonance analysis, the dockerin in EngG had a relatively weak affinity (30- to 123-fold) for cohesins compared with the other cellulosomal enzymes. In the cell wall affinity assay, EngG anchored to the cell surfaces of C. cellulovorans using its DUF291 domain. Immunofluorescence microscopy confirmed the cell surface display of the EngG complex. These results indicated that in C. cellulovorans, EngG assemble into both the cellulolytic complex and the cell wall complex to aid in the hydrolysis of cellulose substrates.

Display of Clostridium cellulovorans xylose isomerase on the cell surface of Saccharomyces cerevisiae and its direct application to xylose fermentation.

Xylose isomerase (XI) is a key enzyme in the conversion of D-xylose, which is a major component of lignocellulosic biomass, to D-xylulose. Genomic analysis of the bacterium Clostridium cellulovorans revealed the presence of XI-related genes. In this study, XI derived from C. cellulovorans was produced and displayed using the yeast cell-surface display system, and the xylose assimilation and fermentation properties of this XI-displaying yeast were examined. XI-displaying yeast grew well in medium containing xylose as the sole carbon source and directly produced ethanol from xylose under anaerobic conditions.

The processive endoglucanase EngZ is active in crystalline cellulose degradation as a cellulosomal subunit of Clostridium cellulovorans.

Clostridium cellulovorans produces an efficient enzyme complex for the degradation of lignocellulosic biomass. In our previous study, we detected and identified protein spots that interacted with a fluorescently labeled cohesin biomarker via two-dimensional gel electrophoresis. One novel, putative cellulosomal protein (referred to as endoglucanase Z) contains a catalytic module from the glycosyl hydrolase family (GH9) and demonstrated higher levels of expression than other cellulosomal cellulases in Avicel-containing cultures. Purified EngZ had optimal activity at pH 7.0, 40°C, and the major hydrolysis product from the cellooligosaccharides was cellobiose. EngZ's specific activity toward crystalline cellulose (Avicel and acid-swollen cellulose) was 10-20-fold higher than other cellulosomal cellulase activities. A large percentage of the reducing ends that were produced by this enzyme from acid-swollen cellulose were released as soluble sugar. EngZ has the capability of reducing the viscosity of Avicel at an intermediate-level between exo- and endo-typing cellulases, suggesting that it is a processive endoglucanase. In conclusion, EngZ was highly expressed in cellulolytic systems and demonstrated processive endoglucanase activity, suggesting that it plays a major role in the hydrolysis of crystalline cellulose and acts as a cellulosomal enzyme in C. cellulovorans.

Production of minicellulosomes for the enhanced hydrolysis of cellulosic substrates by recombinant Corynebacterium glutamicum.

Although cellulosic materials of plant origin are the most abundant utilizable biomass resource, the amino acid-producing organism Corynebacterium glutamicum can not utilize these materials. Here we report the engineering of a C. glutamicum strain expressing functional minicellulosomes containing chimeric endoglucanase E bound to miniCbpA from Clostridium cellulovorans that can hydrolyze cellulosic materials. The chimeric endoglucanase E consists of the endoglucanase E catalytic backbone of Clostridium thermocellum fused with the endoglucanase B dockerin domain of C. cellulovorans. The resulting strain degraded cellulose efficiently by substrate targeting via the carbohydrate binding module. The assembly of minicellulosomes increased the activity against carboxymethyl cellulose approximately 2.8-fold compared with that for the corresponding enzymes alone. This is the first report of the formation of Clostridium minicellulosomes by C. glutamicum. The development of C. glutamicum strain that is capable of more effective cellulose hydrolysis brings about a realization of consolidated bioprocessing for the utilization of cellulosic biomass.

A celluloytic complex from Clostridium cellulovorans consisting of mannanase B and endoglucanase E has synergistic effects on galactomannan degradation.

In our previous study using a fluorescently labeled cohesin biomarker, we detected and identified a putative cellulosomal mannanase belonging to the glycosyl hydrolase family 26 from Clostridium cellulovorans in xylan-containing cultures. In this study, a mannanase gene, manB from C. cellulovorans, was expressed in Escherichia coli. The optimal pH of a purified enzyme was around pH 7.0 and the optimal temperature was 40 °C. The purified mannanase B (ManB) showed high hydrolytic activity toward galactomannan. An assembly of ManB with mini-CbpA, which contains a carbohydrate-binding module that provides proximity to insoluble substrates, increased the activity toward galactomannan [locust bean gum (LBG) and guar gum] 1.7- and 2.0-fold over those without mini-CbpA. We tested the synergistic effects on galactomannan (LBG and guar gum) degradation using cellulosomal mannanase ManB with cellulosomal endoglucanase E, which was predicted to have mannanase activity in C. cellulovorans as a cellulolytic complex. When assembled with the mini-CbpA, the mixture of endoglucanase E (EngE) and ManB at a molar ratio of 1:2 showed the highest synergistic effect (2.4-fold) on LBG. The mixture at a ratio of 1:3 showed the highest synergistic effect (2.8-fold) on guar gum. These synergistic actions indicated that ManB assembled with mini-CbpA hydrolyzed insoluble galactomannan, which in turn promoted soluble galactomannan degradation by EngE.

Comparison of the mesophilic cellulosome-producing Clostridium cellulovorans genome with other cellulosome-related clostridial genomes.

Clostridium cellulovorans, an anaerobic and mesophilic bacterium, degrades native substrates in soft biomass such as corn fibre and rice straw efficiently by producing an extracellular enzyme complex called the cellulosome. Recently, we have reported the whole-genome sequence of C. cellulovorans comprising 4220 predicted genes in 5.10 Mbp [Y. Tamaru et al., (2010) J. Bacteriol., 192: 901­902]. As a result, the genome size of C. cellulovorans was about 1 Mbp larger than that of other cellulosome-producing clostridia, mesophilic C. cellulolyticum and thermophilic C. thermocellum. A total of 57 cellulosomal genes were found in the C. cellulovorans genome, and they coded for not only carbohydrate-degrading enzymes but also a lipase, peptidases and proteinase inhibitors. Interestingly, two novel genes encoding scaffolding proteins were found in the genome. According to KEGG metabolic pathways and their comparison with 11 Clostridial genomes, gene expansion in the C. cellulovorans genome indicated mainly non-cellulosomal genes encoding hemicellulases and pectin-degrading enzymes. Thus, by examining genome sequences from multiple Clostridium species, comparative genomics offers new insight into genome evolution and the way natural selection moulds functional DNA sequence evolution. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced cellulosome-producing Clostridium strains for industrial applications such as biofuel production.

Construction and characterization of different fusion proteins between cellulases and ß-glucosidase to improve glucose production and thermostability.

A ß-glucosidase from Clostridium cellulovorans (CcBG) was fused with one of three different types of cellulases from Clostridium thermocellum, including a cellulosomal endoglucanase CelD (CtCD), a cellulosomal exoglucanase CBHA (CtCA) and a non-cellulosomal endoglucanase Cel9I (CtC9I). Six bifunctional enzymes were constructed with either ß-glucosidase or cellulase in the upstream. CtCD-CcBG showed the favorable specific activities on phosphoric acid swollen cellulose (PASC), an amorphous cellulose, with more glucose production (2 folds) and less cellobiose accumulation (3 folds) when compared with mixture of the single enzymes. Moreover, CtCD-CcBG had significantly improved thermal stability with a melting temperature (T(m)) of 10.9°C higher than that of CcBG (54.5°C) based on the CD unfolding experiments. This bifunctional enzyme is thus useful in industrial application to convert cellulose to glucose.