Role In Hydrolysis Of Cellulose Research Articles

Structural and thermostability insights into cellobiohydrolase of a thermophilic mould Myceliophthora thermophila: in-silico studies

Cellobiohydrolase (CBH) is one of the cellulases with a wide range of industrial applications; it plays a pivotal role in cellulose hydrolysis and thus in biofuel production. The structural and thermostability analysis of a CBHII of the thermophilic mold Myceliophthora thermophila (MtCel6A) had been carried out using various in-silico approaches. The validation of 3 D model by the Ramachandran plot indicated 88.5% amino acid residues in the favoured regions. Docking analysis suggested MtCel6A to display a high affinity towards cellotetraose as compared to other substrates. The enzyme exhibited a high tolerance to the end product, cellobiose. The thermostability evaluation by molecular dynamic simulations and principal component analysis confirmed its tolerance to elevated temperatures. The identified thermolabile regions could be targeted for site-directed mutagenesis in order to ameliorate thermostability further. Our experimental data published earlier confirmed the present findings of in-silico studies. The structural and functional characteristics of MtCel6A highlighted its critical features that make it a useful biocatalyst in several industrial processes. Communicated by Ramaswamy H. Sarma

English

Enzymatic cellulose hydrolysis is an important step for the production of second-generation biofuels. The filamentous fungus Trichoderma reesei is among the most important organisms for obtaining cellulolytic enzymes. The Cel6A (CBH II) cellulase from T. ressei plays an important role in cellulose hydrolysis and acts on the non-reducing end of cellulose, in contrast to Cel7B (CBH I), which acts on the reducing end of cellulose thus releasing cellobiose. Therefore, Cel6A deficiency becomes a limiting factor in cellulose saccharification. This work attempted to use codon optimization to enhance Cel6A expression in Escherichia coli. A plasmid expression vector, pUCITD04, was designed; this vector contains: the cel6a gene, regulatory regions (the promoter and terminator T7 sequences), the OmpT signal peptide that allows the secretion of proteins into the culture medium, and a 6His tail to allow purification of the protein by affinity chromatography. The protein expression experiment using a strain of E. coli transformed with pUCITD04 resulted in a 31 kDa polypeptide being secreted into the culture medium that did not possess enzymatic activity, meanwhile, the control strain transformed with the empty plasmid did not secrete any protein fragments, indicating that a truncated Cel6A was being produced by the experimental strain. This phenomenon has been reported during the production of recombinant cellulases in E. coli. In this research, we discuss probable causes of this phenomenon, as well as the drawbacks in the production of cellulases by E. coli, directing efforts to elucidate the causes of the production of truncated cellulases by this bacterial factory.   Key words: Gene construct, recombinant cellobiohydrolase, Escherichia coli.

Co-evolution of β-glucosidase activity and product tolerance for increasing cellulosic ethanol yield.

β-Glucosidase (BGL) plays a key role in cellulose hydrolysis. However, it is still a great challenge to enhance product tolerance and enzyme activity of BGL simultaneously. Here, we utilized one round error-prone PCR to engineer the Penicillium oxalicum 16 BGL (16BGL) for improving the cellulosic ethanol yield. We identified a new variant (L-6C), a triple mutant (M280T/V484L/D589E), with enhanced catalytic efficiency ([Formula: see text]) for hydrolyzing pNPG and reduced strength of inhibition ([Formula: see text]) by glucose. To be specific, L-6C achieved a [Formula: see text] of 0.35 at a glucose concentration of 20mM, which was 3.63 times lower than that attained by 16BGL. The catalytic efficiency for L-6C to hydrolyze pNPG was determined to be 983.68mM-1s-1, which was 22% higher than that for 16BGL. However, experiments showed that L-6C had reduced binding affinity (2.88mM) to pNGP compared with 16BGL (1.69mM). L-6C produced 6.15 g/L ethanol whose yield increased by about 10% than 16BGL.We performed molecular docking and molecular dynamics (MD) simulation, and binding free energy calculation using the Molecular Mechanics/Poisson Boltzmann surface area (MM/PBSA) method. MD simulation together with the MM/PBSA calculation suggested that L-6C had reduced binding free energy to pNPG, which was consistent with the experimental data.

Heterologous secretory expression of β-glucosidase from Thermoascus aurantiacus in industrial Saccharomyces cerevisiae strains.

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily cellobiose, because it is the major soluble by-product of cellulose and acts as a strong inhibitor, especially for cellobiohydrolase, which plays a key role in cellulose hydrolysis. Commonly used ethanologenic yeast Saccharomyces cerevisiae is unable to utilize cellobiose; accordingly, genetic engineering efforts have been made to transfer β-glucosidase genes enabling cellobiose utilization. Nonetheless, laboratory yeast strains have been employed for most of this research, and such strains may be difficult to use in industrial processes because of their generally weaker resistance to stressors and worse fermenting abilities. The purpose of this study was to engineer industrial yeast strains to ferment cellobiose after stable integration of tabgl1 gene that encodes a β-glucosidase from Thermoascus aurantiacus (TaBgl1). The recombinant S. cerevisiae strains obtained in this study secrete TaBgl1, which can hydrolyze cellobiose and produce ethanol. This study clearly indicates that the extent of glycosylation of secreted TaBgl1 depends from the yeast strains used and is greatly influenced by carbon sources (cellobiose or glucose). The recombinant yeast strains showed high osmotolerance and resistance to various concentrations of ethanol and furfural and to high temperatures. Therefore, these yeast strains are suitable for ethanol production processes with saccharified lignocellulose.

High-level production of a fungal β-glucosidase with application potentials in the cost-effective production of Trichoderma reesei cellulase

Abstract β-glucosidase (BGL), an important component in cellulase complex, plays a crucial role in cellulose hydrolysis. However, its activity in cellulase system of Trichoderma reesei, the conventional cellulase producer, is extremely low. In this study, β-glucosidase gene (bgl) from Aspergillus niger was cloned and achieved secreted expression in Pichia pastoris. High-level production of BGL (129 IU/mL) was achieved in a 1 m3 fermenter by using an optimized mixed-feed strategy, in which glycerol/methanol at the ratio of 1:5 and 30 g/L corn steep powder were fed to maintain the methanol concentration of 0.7% (v/v). The obtained BGL was then used to catalyze the synthesis of sophorose, a strong inducer for cellulase production, from glucose via a transglycosylation reaction. When used the resultant glucose-sophorose mixture (GSM) containing 7.08% sophorose as both carbon source and inducer, the cellulase yield produced by Trichoderma reesei was high up to 95.23 IU/mL (filter paper activity), which was 2.1-fold of that achieved by lactose. These results indicated that the BGL is potential for application in the cost-effective and highly efficient production of Trichoderma reesei cellulase.

Optimization of Culture Conditions and Medium Components for Carboxymethyl Cellulase (CMCase) Production by a Novel Basidiomycete Strain Peniophora sp. NDVN01

Background: Cellulytic enzymes including carboxymethyl cellulases play the key role in hydrolysis of cellulose, a huge organic carbon reservoir on earth, into monomeric sugars and their eventual conversion into valuable chemicals and energy sources. Objectives: In this study, we described the identification of a basidiomycete isolate NDVN01 and optimization of culture conditions and medium components for CMCase production by this strain under liquid state fermentation. The CMCase was estimated as 32 - 33 kDa on a native Polyacrylamide gel electrophoresis (PAGE). Materials and Methods: We used 5 basidiomycetes for screening CMCase production, internal transcribed spacer (ITS) sequence analysis in combination with morphology for strain identification, and liquid state fermentation for optimization of CMCase production. Results: The maximum CMCase production by Peniophora sp. NDVN01 was obtained at 28°C, with the initial medium pH of 7 and within 120 hours of cultivation in the optimum medium containing 80% (v/v) of potato infusion, 0.6% (w/v) straw rice as additional carbon source and 0.2% (w/v) ammonium hydrogen phosphate as an additional nitrogen source, and 0.5% (w/v) pulp as inducer. Conclusions: Under optimal conditions, Peniophora sp. NDVN01 produced 24.65 ± 0.37 units of CMCase per mL of culture supernatant, which was 8.6 times higher than the amount (2.87 ± 0.28 U.mL -1) before optimization.

Molecular simulation evidence for processive motion of Trichoderma reesei Cel7A during cellulose depolymerization

We present free energy calculations for the Trichoderma reesei Cel7A (cellobiohydrolase I) linker peptide from molecular dynamics simulations directed towards understanding the linker role in cellulose hydrolysis. The calculations predict an energy storage mechanism of the linker under stretching/compression that is consistent with processive depolymerization. The linker exhibits two stable states at lengths of 2.5 nm and 5.5 nm during extension/compression, with a free energy difference of 10.5 kcal/mol between the two states separated by an energy barrier. The switching between stable states supports the hypothesis that the linker peptide has the capacity to store energy in a manner similar to a spring.

Bacterial Cellulose Hydrolysis in Anaerobic Environmental Subsystems—Clostridium thermocellumandClostridium stercorarium, Thermophilic Plant‐fiber Degraders

Cellulose degradation is a rare trait in bacteria. However, the truly cellulolytic bacteria are extremely efficient hydrolyzers of plant cell wall polysaccharides, especially those in thermophilic anaerobic ecosystems. Clostridium stercorarium, a thermophilic ubiquitous soil dweller, has a simple cellulose hydrolyzing enzyme system of only two cellulases. However, it seems to be better suited for the hydrolysis of a wide range of hemicelluloses. Clostridium thermocellum, an ubiquitous thermophilic gram-type positive bacterium, is one of the most successful cellulose degraders known. Its extracellular enzyme complex, the cellulosome, was prepared from C. thermocellum cultures grown on cellulose, cellobiose, barley beta-1,3-1,4-glucan, or a mixture of xylan and cellulose. The single proteins were identified by peptide chromatography and MALDI-TOF-TOF. Eight cellulosomal proteins could be found in all eight preparations, 32 proteins occur in at least one preparation. A number of enzymatic components had not been identified previously. The proportion of components changes if C. thermocellum is grown on different substrates. Mutants of C. thermocellum, devoid of scaffoldin CipA, that now allow new types of experiments with in vitro cellulosome reassembly and a role in cellulose hydrolysis are described. The characteristics of these mutants provide strong evidence of the positive effect of complex (cellulosome) formation on hydrolysis of crystalline cellulose.

Detection of novel Fibrobacter populations in landfill sites and determination of their relative abundance via quantitative PCR

Members of the bacterial genus Fibrobacter have long been considered important components of the anaerobic cellulolytic community in the herbivore gut, but their presence and activity in other environments is largely unknown. In this study, a specific polymerase chain reaction (PCR) primer set, targeting the 16S rRNA gene of Fibrobacter spp., was applied to community DNA from five landfill sites followed by temporal thermal gel electrophoresis (TTGE) analysis of cloned amplification products. Phylogenetic analysis of clone sequences indicated the presence of novel clusters closely related to the genus Fibrobacter. There are two named species, Fibrobacter succinogenes and F. intestinalis, and only two of the 58 sequenced clones were identified with them, and both were F. succinogenes. The clone sequences from landfill were recovered in five distinct clusters within the Fibrobacter lineage, and four of these were novel. Quantitative PCR (qPCR) assays of reverse-transcribed community RNA from landfill leachates and rumen fluid samples indicated that the abundance of Fibrobacter spp. relative to total bacteria varied from 0.2% to 40% in landfill, and 21% to 32% in the rumen, and these data demonstrate that fibrobacters can be a significant component of the microbial community in landfill ecosystems. This is the first evidence for Fibrobacter spp. outside the gut ecosystem, and as the only cultivated representatives of this group are actively cellulolytic, their diversity and abundance points to a possible role in cellulose hydrolysis in landfill, and perhaps other anaerobic environments also.

Cellulose digestion and cellulase regulation and distribution in Fibrobacter succinogenes subsp. succinogenes S85

Fibrobacter succinogenes subsp. succinogenes S85 initiated growth on microcrystalline cellulose without a lag whether inoculated from a glucose, cellobiose, or cellulose culture. During growth on cellulose, there was no accumulation of soluble carbohydrate. When the growth medium contained either glucose or cellobiose in combination with microcrystalline cellulose, there was a lag in cellulose digestion until all of the soluble sugar had been utilized, suggesting an end product feedback mechanism that affects cellulose digestion. Cl-stimulated cellobiosidase and periplasmic cellodextrinase were produced under all growth conditions tested, indicating constitutive synthesis. Both cellobiosidases were cell associated until the stationary phase of growth, whereas proteins antigenically related to the Cl-stimulated cellobiosidase and a proportion of the endoglucanase were released into the extracellular culture fluid during growth, irrespective of the substrate. Immunoelectron microscopy of cells with a polyclonal antibody to Cl-stimulated cellobiosidase as the primary antibody and 10-nm-diameter gold particles conjugated to goat anti-rabbit antibodies as the second antibody revealed protrusions of the outer surface which were selectively labeled with gold, suggesting that Cl-stimulated cellobiosidase was located on the protrusions. These data support the contention that the protrusions have a role in cellulose hydrolysis; however, this interpretation is complicated by reactivity of the antibodies with a large number of other proteins that possess related antigenic epitopes.