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Family GH1 glycosyl hydrolases are ubiquitous in prokaryotes and eukaryotes and are utilized in numerous industrial applications, including bioconversion of lignocelluloses. In this study, hyperacidophilic archaeon Cuniculiplasma divulgatum (S5T=JCM 30642T) was explored as a source of novel carbohydrate-active enzymes. The genome of C. divulgatum encodes three GH1 enzyme candidates, from which CIB12 and CIB13 were heterologously expressed and characterized. Phylogenetic analysis of CIB12 and CIB13 clustered them with β-glucosidases from genuinely thermophilic archaea including Thermoplasma acidophilum, Picrophilus torridus, Sulfolobus solfataricus, Pyrococcus furiosus , and Thermococcus kodakarensis. Purified enzymes showed maximal activities at pH 4.5–6.0 (CIB12) and 4.5–5.5 (CIB13) with optimal temperatures at 50°C, suggesting a high-temperature origin of Cuniculiplasma spp. ancestors. Crystal structures of both enzymes revealed a classical (α/β)8 TIM-barrel fold with the active site located inside the barrel close to the C-termini of β-strands including the catalytic residues Glu204 and Glu388 (CIB12), and Glu204 and Glu385 (CIB13). Both enzymes preferred cellobiose over lactose as substrates and were classified as cellobiohydrolases. Cellobiose addition increased the biomass yield of Cuniculiplasma cultures growing on peptides by 50%, suggesting that the cellobiohydrolases expand the carbon substrate range and hence environmental fitness of Cuniculiplasma. [ABSTRACT FROM AUTHOR]

The laccases from white-rot fungi exhibit high redox potential in treating phenolic compounds. However, their application in commercial purposes has been limited because of the relatively low productivity of the native hosts. Here, the laccase A-encoding gene lacA of Trametes sp. AH28-2 was overexpressed under the control of the strong promoter of cbh1 (P cbh1), the gene encoding the endogenous cellobiohydrolase 1 (CBH1), in the industrial workhorse fungus Trichoderma reesei. Firstly, the lacA expression cassette was randomly integrated into the T. reesei chromosome by genetic transformation. The lacA gene was successfully transcribed, but the laccase couldn't be detected in the liquid fermentation condition. Meanwhile, it was found that the endoplasmic reticulum-associated degradation (ERAD) was strongly activated, indicating that the expression of LacA probably triggered intense endoplasmic reticulum (ER) stress. Subsequently, the lacA expression cassette was added with the downstream region of cbh1 (T cbh1) to construct the new expression cassette lacA:: Δ cbh1 , which could replace the cbh1 locus in the genome via homologous recombination. After genetic transformation, the lacA gene was integrated into the cbh1 locus and transcribed. And the unfolded protein response (UPR) and ERAD were only slightly induced, for which the loss of endogenous cellulase CBH1 released the pressure of secretion. Finally, the maximum laccase activity of 168.3 U/l was obtained in the fermentation broth. These results demonstrated that the reduction of secretion pressure by deletion of endogenous protein-encoding genes would be an efficient strategy for the secretion of heterologous target proteins in industrial fungi. One-Sentence Summary The reduction of the secretion pressure by deletion of the endogenous cbh1 gene can contribute to heterologous expression of the laccase (LacA) from Trametes sp. AH28-2 in Trichoderma reesei. [ABSTRACT FROM AUTHOR]

Glycoengineering ultimately allows control over glycosylation patterns to generate new glycoprotein variants with desired properties. A common challenge is glycan heterogeneity, which may affect protein function and limit the use of key techniques such as mass spectrometry. Moreover, heterologous protein expression can introduce nonnative glycan chains that may not fulfill the requirement for therapeutic proteins. One strategy to address these challenges is partial trimming or complete removal of glycan chains, which can be obtained through selective application of exoglycosidases. Here, we demonstrate an enzymatic O -deglycosylation toolbox of a GH92 α-1,2-mannosidase from Neobacillus novalis , a GH2 β-galactofuranosidase from Amesia atrobrunnea and the jack bean α-mannosidase. The extent of enzymatic O -deglycosylation was mapped against a full glycosyl linkage analysis of the O -glycosylated linker of cellobiohydrolase I from Trichoderma reesei (Tr Cel7A). Furthermore, the influence of deglycosylation on Tr Cel7A functionality was evaluated by kinetic characterization of native and O -deglycosylated forms of Tr Cel7A. This study expands structural knowledge on fungal O -glycosylation and presents a ready-to-use enzymatic approach for controlled O -glycan engineering in glycoproteins expressed in filamentous fungi. [ABSTRACT FROM AUTHOR]

Cheap production of glucose is the current challenge for the production of cheap bioethanol. Ideal protein engineering approaches are required for improving the efficiency of the members of the cellulase, the enzyme complex involved in the saccharification process of cellulose. An attempt was made to improve the efficiency of the cellobiohydrolase (Cel6A), the important member of the cellulase isolated from Aspergillus fumigatus (Af Cel6A). Structure-based variants of Af Cel6A were designed. Amino acids surrounding the catalytic site and conserved residues in the cellulose-binding domain were targeted (N449V, N168G, Y50W and W24YW32Y). I mutant 3 server was used to identify the potential variants based on the free energy values (∆∆G). In silico structural analyses and molecular dynamics simulations evaluated the potentiality of the variants for increasing thermostability and catalytic activity of Cel6A. Further enzyme studies with purified protein identified the N449V is highly thermo stable (60°C) and pH tolerant (pH 5–7). Kinetic studies with Avicel determined that substrate affinity of N449V (Km =0.90 ± 0.02) is higher than the wild type (1.17 ± 0.04) and the catalytic efficiency (Kcat/Km) of N449V is ~2-fold higher than wild type. All these results suggested that our strategy for the development of recombinant enzyme is a right approach for protein engineering. [ABSTRACT FROM AUTHOR]

Fungus-derived GH-7 family cellobiohydrolase I (CBHI, EC 3.2.1.91) is one of the most important industrial enzymes for cellulosic biomass saccharification. Talaromyces cellulolyticus is well known as a mesophilic fungus producing a high amount of CBHI. Thermostability enhances the economic value of enzymes by making them more robust. However, CBHI has proven difficult to engineer, a fact that stems in part from its low expression in heterozygous hosts and its complex structure. Here, we report the successful improvement of the thermostability of CBHI from T. cellulolyticus using our homologous expression system and protein engineering method. We examined the key structures that seem to contribute to its thermostability using the 3D structural information of CBHI. Some parts of the structure of the Talaromyces emersonii CBHI were grafted into T. cellulolyticus CBHI and thermostable mutant CBHIs were constructed. The thermostability was primarily because of the improvement in the loop structures, and the positive effects of the mutations for thermostability were additive. By combing the mutations, the constructed thermophilic CBHI exhibits high hydrolytic activity toward crystalline cellulose with an optimum temperature at over 70°C. In addition, the strategy can be applied to the construction of the other thermostable CBHIs. [ABSTRACT FROM AUTHOR]

Various cellulases consist of a catalytic domain connected to a carbohydrate-binding module (CBM) by a flexible linker peptide. The linker if often strongly O-glycosylated and typically has a length of 20-50 amino acid residues. Functional roles, other than connecting the two folded domains, of the linker and its glycans, have been widely discussed, but experimental evidence remains sparse. One of the most studied cellulose degrading enzymes is the multi-domain cellobiohydrolase Cel7A from Hypocrea jecorina. Here, we designed variants of Cel7A with mutations in the linker region to elucidate the role of the linker. We found that moderate modification of the linker could result in significant changes in substrate affinity and catalytic efficacy. These changes were quite different for different linker variants. Thus, deletion of six residues near the catalytic domain had essentially no effects on enzyme function. Conversely, a substitution of four glycosylation sites near the middle of the linker reduced substrate affinity and increased maximal turnover. The observation of weaker binding provides some support of recent suggestions that linker glycans may be directly involved in substrate interactions. However, a variant with several inserted glycosylation sites near the CBM also showed lower affinity for the substrate compared to the wild-type and we suggest that substrate interactions of the glycans depend on their exact location as well as other factors such as changes in structure and dynamics of the linker peptide. [ABSTRACT FROM AUTHOR]

CBH1 (cellobiohydrolase) comprises the majority of secreted proteins by Trichoderma reesei. For expression of Talaromyces thermophilus lipase gene in T. reesei, a self-designed CBH1 promoter was applied to drive the lipase gene expression cassette which was bracketed by flanking sequences of cbh1 gene for homologous recombination. Protoplast and Agrobacterium-mediated plasmid transformations were performed and compared, resultantly, transformation mediated by Agrobacterium was overall proved to be more efficient. Stable integration of lipase gene into chromosomal DNA of T. reesei transformants was verified by PCR. After shaking flask fermentation, lipase activity of transformant reached 375 IU mL, whereas no cellobiohydrolase activity was detected. SDS-PAGE analysis further showed an obvious protein band about 39 kDa and no CBH1 band in fermentation broth, implying lipase gene was successfully extracellularly expressed in T. reesei via homologous recombination at cbh1 locus. This study herein would benefit genetic engineering of filamentous fungi and industrial application of thermo-alkaline lipase like in paper making and detergents addition. [ABSTRACT FROM AUTHOR]

We describe here the composition of the O-linked glycans on the Neurospora crassa cellobiohydrolase I (CBHI), which accounts for approximately 40% of the protein secreted by cells growing in the presence of cellulose. CBHI is O-glycosylated with six types of linear, and three types of branched, O-glycans containing approximately equal amounts of mannose and galactose. In addition to the classic fungal O-glycans with reducing end mannoses, we also identified reducing end galactoses which suggest the existence of a protein-O-galactosyltransferase in N. crassa. Because of the excellent genetic resources available for N. crassa, the knowledge of the CBHI O-glycans may enable the future evaluation of the role of O-glycosylation on cellulase function and the development of directed O-glycan/cellulase engineering. [ABSTRACT FROM AUTHOR]

Genomic sequencing analysis and previous studies have shown that there are eight genes in Streptomyces coelicolor A3(2) encoding putative cellulases. One of these genes, sco6548, was cloned into the Streptomyces/Escherichia coli shuttle vector pUWL201PW. The recombinant protein was successfully overexpressed in S. lividans TK24 under the control of the strong ermE promoter. Sco6548 was 1740 bp in length, and encoded a 579-amino acid-, 60.8-kDa protein with strong hydrolyzing activity toward Avicel and filter paper, yielding cellobiose as the final product. SCO6548 showed optimal activity at 50°C and pH 5. The Km values of SCO6548 toward Avicel and filter paper were 15.38 and 16.1 mg/mL, respectively. The Vmax values toward Avicel and filter paper were 0.432 and 0.084 μM/min, respectively. EDTA did not affect cellulase activity; however, several divalent cations, including Co2+, Cu2+, Ni2+ and Mn2+ (at 10 mM) had severe inhibitory effects on enzyme activity. Our analysis showed that SCO6548 is a cellulose 1,4-β-cellobiosidase that hydrolyzes cellulose into cellobiose. [ABSTRACT FROM AUTHOR]

Trichoderma reesei is the preferred organism for producing industrial cellulases. However, cellulases derived from T. reesei have their highest activity at acidic pH. When the pH value increased above 7, the enzyme activities almost disappeared, thereby limiting the application of fungal cellulases under neutral or alkaline conditions. A lot of heterologous alkaline cellulases have been successfully expressed in T. reesei to improve its cellulolytic profile. To our knowledge, there are few reports describing the co-expression of two or more heterologous cellulases in T. reesei. We designed and constructed a promoter collection for gene expression and co-expression in T. reesei. Taking alkaline cellulase as a reporter gene, we assessed our promoters with strengths ranging from 4 to 106 % as compared to the pWEF31 expression vector (Lv D, Wang W, Wei D (2012) Construction of two vectors for gene expression in Trichoderma reesei. Plasmid 67(1):67-71). The promoter collection was used in a proof-of-principle approach to achieve the co-expression of an alkaline endoglucanase and an alkaline cellobiohydrolase. We observed higher activities of both cellulose degradation and biostoning by the co-expression of an endoglucanase and a cellobiohydrolase than the activities obtained by the expression of only endoglucanase or cellobiohydrolase. This study makes the process of engineering expression of multiple genes easier in T. reesei. [ABSTRACT FROM AUTHOR]

Aims Nitrogen (N) addition could affect the rate of forest litter and soil organic matter decomposition by regulating extracellular enzyme activity (EEA). The impact of N addition on EEA may differ across different age stands with different organic matter quality. We were interested in whether the impact of N addition on EEA in litter and mineral soil during the growing season was dependent on stand age of a larch plantation in North China. Methods We added three levels of N (0, 20 and 50kg N ha−1 year−1) in three age stands (11, 20 and 45 years old) of Larix principis-rupprechtii plantation in North China. We measured potential activities of β-1,4-glucosidase (BG), cellobiohydrolase (CB), β-1,4-N-acetyl-glucosaminidase (NAG) and phenol oxidase (PO) in litter (organic horizon) and mineral soil (0–10cm) during the second growing season after N amendment. We also measured C and N concentrations, microbial biomass C and N, and KCl-extractable ammonium and nitrate in both litter and mineral soil. Important Findings We observed unimodal patterns of EEA during the growing season in all three stands, consistent with the seasonal variations of soil temperature. Stand age had a strong effect on EEA in both litter and mineral soil, and this effect differed between litter and mineral soil as well as between different enzymes. N addition did not significantly affect the activities of BG or CB but significantly suppressed the activity of NAG in litter. We also found stand age-specific responses of PO activity to N addition in both litter and mineral soil. N addition suppressed PO activity of the high C:N ratio litters in 20- and 45-year-old stands but had no significant effect on PO activity of the low C:N ratio litter in 11-year-old stand. Moreover, N addition inhibited PO activity of the high C:N ratio soil in 20-year-old stand but had no significant impact on PO activity of the low C:N ratio soils in 11- and 45-year-old stands. Overall, stand age had a greater effect on EEA in litter and mineral soil compared to 2 years of N addition. Moreover, the effect of N addition on PO activity is stand age dependent, which may affect the long-term soil carbon storage in this forest. [ABSTRACT FROM AUTHOR]

Endocellulases are one kind of the important biodegrading cellulose enzymes. Experimental results show that a rotated and distorted preactivated structure exists before the substrate entering the transition state. The molecular dynamic simulation of endocellulase Cel6A models revealed a correlation between the rotation and distortion of pyranoside ring in −1 glycosyl unit of the substrate. The two key residues, Tyr73 and Ser189, in Cal6A cooperate to rotate and distort the pyranoside ring in the cellulose hydrolysis. [ABSTRACT FROM AUTHOR]

A starch-inducible homologous expression system in Acremonium cellulolyticus was constructed to successfully produce recombinant cellulolytic enzymes. A. cellulolyticus Y-94 produced amylolytic enzymes and cellulolytic enzymes as major proteins in the culture supernatant when grown with soluble starch (SS) and Solka-Flock cellulose (SF), respectively. To isolate a strong starch-inducible promoter, glucoamylase (GlaA), which belongs to glycoside hydrolase family 15, was purified from the SS culture of Y-94, and its gene was identified in the genome sequence. The 1.4-kb promoter and 0.4-kb terminator regions of glaA were amplified by polymerase chain reaction (PCR) and used in the construction of a plasmid that drives the expression of the cellobiohydrolase I (Cel7A) gene from A. cellulolyticus. The resultant expression plasmid, containing pyrF as a selection marker, was randomly integrated into the genome of the A. cellulolyticus Y-94 uracil auxotroph. The prototrophic transformant, Y203, produced recombinant Cel7A as an extracellular protein under control of the glaA promoter in the SS culture. Recombinant and wild-type Cel7A were purified from the SS culture of Y203 and the SF culture of A. cellulolyticus CF-2612, respectively. Both enzymes were found to have the same apparent molecular weight (60 kDa), thermostability ( T 67.0 °C), and optimum pH (pH 4.5), and showed similar catalytic properties for soluble and insoluble substrates. These results suggest that the A. cellulolyticus starch-inducible expression system will be helpful for characterization and improvement of fungal cellulolytic enzymes. [ABSTRACT FROM AUTHOR]

The neutral endo-β-glucanase gene cel5A from Humicola insolens was cloned and connected with the cellobiohydrolase 1 promoter from Trichoderma reesei to construct a recombinant plasmid pCB-hEG with the hygromycin B resistance marker. The plasmid was introduced into conidia of T. reesei using the Agrobacterium tumefaciens mediated transformation method. Eight transformants were obtained on screening plates with sodium carboxymethyl cellulose as the sole carbon source. Stable integration of the cel5A gene into the chromosomal DNA of T. reesei was confirmed by PCR. An obvious protein band (approximately 52 kDa) was detected by SDS-PAGE from fermentation broth, which showed that the cel5A gene in recombinant T. reesei successfully fulfilled efficient expression and extracellular secretion. After 96 h shaking-flask fermentation, the endo-β-glucanase activity at pH 6.5 from recombinant T. reesei reached 3,068 U/ml, which was 11 times higher than that of the host strain. In a 2 m fermenter, the endo-β-glucanase activity could be further increased to 8,012 U/ml after 96 h fermentation. The results showed a good prospect for application of neutral endo-β-glucanase in the textile industry. [ABSTRACT FROM AUTHOR]

Fungi are considered the primary decomposers of dead plant biomass in terrestrial ecosystems. However, current knowledge regarding the successive changes in fungal communities during litter decomposition is limited. Here we explored the development of the fungal community over 24 months of litter decomposition in a temperate forest with dominant Quercus petraea using 454-pyrosequencing of the fungal internal transcribed spacer (ITS) region and cellobiohydrolase I (cbhI) genes, which encode exocellulases, to specifically address cellulose decomposers. To quantify the involvement of phyllosphere fungi in litter decomposition, the fungal communities in live leaves and leaves immediately before abscission were also analysed. The results showed rapid succession of fungi with dramatic changes in the composition of the fungal community. Furthermore, most of the abundant taxa only temporarily dominated in the substrate. Fungal diversity was lowest at leaf senescence, increased until month 4 and did not significantly change during subsequent decomposition. Highly diverse community of phyllosphere fungi inhabits live oak leaves 2 months before abscission, and these phyllosphere taxa comprise a significant share of the fungal community during early decomposition up to the fourth month. Sequences assigned to the Ascomycota showed highest relative abundances in live leaves and during the early stages of decomposition. In contrast, the relative abundance of sequences assigned to the Basidiomycota phylum, particularly basidiomycetous yeasts, increased with time. Although cellulose was available in the litter during all stages of decomposition, the community of cellulolytic fungi changed substantially over time. The results indicate that litter decomposition is a highly complex process mediated by various fungal taxa. [ABSTRACT FROM AUTHOR]

Building on our previous efforts to generate thermostable chimeric fungal cellobiohydrolase I (CBH I, also known as Cel7A) cellulases by structure-guided recombination, we used FoldX and a ‘consensus’ sequence approach to identify individual mutations present in the five homologous parent CBH I enzymes which further stabilize the chimeras. Using the FoldX force field, we calculated the effect on ΔGFolding of each candidate mutation in a number of CBH I structures and chose those predicted to be stabilizing in multiple structures. With an alignment of 41 CBH I sequences, we also used amino acid frequencies at each candidate position to calculate predicted effects on ΔGFolding. A combination of mutations chosen using these methods increased the T50 of the most thermostable chimera by an additional 4.7°C, to yield a CBH I with T50 of 72.1°C, which is 9.2°C higher than that of the most stable native CBH I, from Talaromyces emersonii. This increased stability resulted in a 10°C increase in the optimal temperature for activity, to 65°C, and a 50% increase in total sugar production from crystalline cellulose at the optimal temperature, compared with native T.emersonii CBH I. [ABSTRACT FROM PUBLISHER]

The secretome of Penicillium funiculosum contains two family GH7 enzymes, one of which (designated XynA) has been described as a xylanase. This is unusual because it is the only xylanase in family GH7, which is mainly composed of cellobiohydrolases and endoglucanases, and also because XynA is highly similar to the cellobiohydrolase I from Talaromyces emersonii and Trichoderma reesei (72 and 65 % identity, respectively). To probe this enigma, we investigated the biochemical properties of XynA, notably its activity on xylans and β- d-glucans. A highly pure sample of XynA was obtained and used to perform hydrolysis tests on polysaccharides. These revealed that XynA is 100-fold more active on β-1,4-glucan than on xylan. Likewise, XynA was active on both 4-nitrophenyl-β- d-lactopyranoside ( pNP-β- d-Lac) and 4-nitrophenyl-β- d-cellobioside ( pNP-cellobiose), which shows that XynA is principally an exo-acting type 1 cellobiohydrolase enzyme that displays 5.2-fold higher performance on pNP-cellobiose than on pNP-β- d-Lac. Finally, analyses performed using cellodextrins as substrate revealed that XynA mainly produced cellobiose (C2) from substrates containing three or more glucosyl subunits, and that C2 inhibits XynA at high concentrations (IC = 17.7 μM). Overall, this study revealed that XynA displays typical cellobiohydrolase 1 activity and confirms that the description of this enzyme in public databases should be definitively amended. Moreover, the data provided here complete the information provided by a previous proteomics investigation and reveal that P. funiculosum secretes a complete set of cellulose-degrading enzymes. [ABSTRACT FROM AUTHOR]

The endoglucanase (Cel5B) from the filamentous fungus Gloeophyllum trabeum was cloned and expressed without a signal peptide, and alanine residue 22 converted to glutamine in Pichia pastoris GS115. The DNA sequence of Cel5B had an open reading frame of 1,077 bp, encoding a protein of 359 amino acid residues with a molecular weight of 47 kDa. On the basis of sequence similarity, Cel5B displayed active site residues at Glu-175 and Glu-287. Both residues lost full hydrolytic activity when replaced with alanine through point mutation. The purified recombinant Cel5B showed very high specific activity, about 80- to 1,000-fold and 13- to 70-fold in comparison with other endoglucanases and cellobiohydrolase, on carboxymethylcellulose and filter paper, respectively, at pH 3.5 and 55°C. Cel5B displayed bifunctional characteristics under acidic conditions. The kinetic properties of the enzyme determined using a Lineweaver-Burk plot indicated that Cel5B is a catalytically efficient cellulolytic enzyme. These results suggest that Cel5B has high bifunctional endo- and exoglucanase activity under acidic conditions and is a good candidate for bioconversion of lignocellulose. [ABSTRACT FROM AUTHOR]

Organic matter decomposition in the globally widespread coniferous forests has an important role in the carbon cycle, and cellulose decomposition is especially important in this respect because cellulose is the most abundant polysaccharide in plant litter. Cellulose decomposition was 10 times faster in the fungi-dominated litter of Picea abies forest than in the bacteria-dominated soil. In the soil, the added 13 C-labelled cellulose was the main source of microbial respiration and was preferentially accumulated in the fungal biomass and cellulose induced fungal proliferation. In contrast, in the litter, bacterial biomass showed higher labelling after 13 C-cellulose addition and bacterial biomass increased. While 80% of the total community was represented by 104-106 bacterial and 33-59 fungal operational taxonomic units ( OTUs), 80% of the cellulolytic communities of bacteria and fungi were only composed of 8-18 highly abundant OTUs. Both the total and 13 C-labelled communities differed substantially between the litter and soil. Cellulolytic bacteria in the acidic topsoil included Betaproteobacteria, Bacteroidetes and Acidobacteria, whereas these typically found in neutral soils were absent. Most fungal cellulose decomposers belonged to Ascomycota; cellulolytic Basidiomycota were mainly represented by the yeasts Trichosporon and Cryptococcus. Several bacteria and fungi demonstrated here to derive their carbon from cellulose were previously not recognized as cellulolytic. [ABSTRACT FROM AUTHOR]

Soils of coniferous forest ecosystems are important for the global carbon cycle, and the identification of active microbial decomposers is essential for understanding organic matter transformation in these ecosystems. By the independent analysis of DNA and RNA, whole communities of bacteria and fungi and its active members were compared in topsoil of a Picea abies forest during a period of organic matter decomposition. Fungi quantitatively dominate the microbial community in the litter horizon, while the organic horizon shows comparable amount of fungal and bacterial biomasses. Active microbial populations obtained by RNA analysis exhibit similar diversity as DNA-derived populations, but significantly differ in the composition of microbial taxa. Several highly active taxa, especially fungal ones, show low abundance or even absence in the DNA pool. Bacteria and especially fungi are often distinctly associated with a particular soil horizon. Fungal communities are less even than bacterial ones and show higher relative abundances of dominant species. While dominant bacterial species are distributed across the studied ecosystem, distribution of dominant fungi is often spatially restricted as they are only recovered at some locations. The sequences of cbhI gene encoding for cellobiohydrolase (exocellulase), an essential enzyme for cellulose decomposition, were compared in soil metagenome and metatranscriptome and assigned to their producers. Litter horizon exhibits higher diversity and higher proportion of expressed sequences than organic horizon. Cellulose decomposition is mediated by highly diverse fungal populations largely distinct between soil horizons. The results indicate that low-abundance species make an important contribution to decomposition processes in soils. [ABSTRACT FROM AUTHOR]

The article presents a study on the analysis of cellulase hyperproducing mutants derived from the Trichoderma reesei QM9414 fungus. The mutants showed higher filter-paper degrading activity and a lower growth rate than the wild-type QM9414 strain. It suggests that cellulase hyperproduction by the mutants was regulated at the transcriptional levels.

Aims: A new cellobiohydrolase (CBH) gene ( cbh3) from Chaetomium thermophilum was cloned, sequenced and expressed in Pichia pastoris. Methods and Results: Using RACE-PCR, a new thermostable CBH gene ( cbh3) was cloned from C. thermophilum. The cDNA of the CBH was 1607 bp and contained a 1356 bp open reading frame encoding a protein CBH precursor of 451 amino acid residues. The mature protein structure of C. thermophilum CBH3 only comprises a catalytic domain and lacks cellulose-binding domain and a hinge region. The gene was expressed in P. pastoris. The recombinant CBH purified was a glycoprotein with a size of about 48·0 kDa, and exhibited optimum catalytic activity at pH 5·0 and 60 °C. The enzyme was more resistant to high temperature. The CBH could hydrolyse microcrystalline cellulose and filter paper. Conclusions: A new thermostable CBH gene of C. thermophilum was cloned, sequenced and overexpressed in P. pastoris. Significance and Impact of the Study: This CBH offers an interesting potential in saccharification steps in both cellulose enzymatic conversion and alcohol production. [ABSTRACT FROM AUTHOR]

A combined (enzymatic and chemical) process using a Bacillus pumilus strain (DKS1), isolated from the soil, was used to degum ramie bast fibres. After 24 h of incubation with the isolated pectinolytic strain using a low-cost medium, the weight loss of the ramie fibre was found to be 25% under small scale. High activity of pectate lyase was detected in the culture supernatants; 400 kg of ramie fibres was degummed with 24% weight loss in large-scale degumming under field conditions. No cellulase activity was found. Microbial intervention followed by mild (0.1%) alkali treatment showed high percentage of weight loss from the ramie fibre. Bacterial degumming followed by chemical treatment resulted in an increase of single fibre tenacity (cN/tex) by more than 20.81% as compared to non-degummed (decorticated) fibre samples. Scanning electron micrographs (SEM) and fluorescence microscope showed that after Bacillus pumilus DKS1 treatment the surface of the decorticated ramie fibre becomes very smooth. These results indicate the process provides an economical and eco-friendly method for the small scale as well as large-scale degumming of decorticated ramie fibre. This study has great relevance to the textile as well as paper industry. [ABSTRACT FROM AUTHOR]

The article highlights the study of the genetic coding for cellobiohydrolase II from the white rot basidiomycete, Irpex lacteus MC-2 and its expression in pichia pastoris. The derived amino acid sequences exposed a significant structure comprised of protein binding domain owning to glycosyl hydrolases family. The primary objective of the study is to help developing effective cellulosic biomass degradation usiing with rot fungi enzymes.

The article discusses the results of a study characterizing the cellobiose dehydrogenase (CDH) enzyme from the white-rot basidiomycete Trametes hirsuta. The molecular weight of the enzyme was 92kDa. The potential of using CDH for the biological treatment of wood chips without negative effects on paper strength is explored.

Investigates cloning, sequencing and heterologous expression of a cellobiohydrolase cDNA from the basidiomycete Corticium rolfsii. Cloning and sequencing of the cellulase gene; Expression of the C. rolfsii CBH1 gene.

The identification of 12 Fusarium strains isolated from diseased hops (Humulus lupulus, L.) was achieved by a strategy based on cellobiohydrolase-C: cleaved amplified polymorphic sequence analysis targeting the gene and the use of an antibody directed against a peptide of the Fusarium graminearum enzyme. This strategy is shown to be rapid and reliable for all the Fusarium of our collection: F. avenaceum, F. graminearum, F. sambucinum, F. sporotrichioides, F. tricinctum and F. venenatum. [Copyright &y& Elsevier]

An exoglucanase gene was cloned from a cellulolytic fungus, Humicola grisea. DNA sequencing of this gene, designated as exol, revealed that it contained four introns in the coding region. The deduced amino acid sequence of EXO1 was 451 amino acids in length and showed 57.7% identity with that of H. grisea cellobiohydrolase 1 (CBH1), but lacked the typical domain structures of a cellulose-binding domain and a hinge region. Transcriptional analysis of the exo1 and cbh1 genes showed that the expression of these genes was induced by Avicel, and repressed in the presence of glucose. The exo1 gene was expressed in Aspergillus oryzae, and the recombinant EXO1 protein was purified. EXO1 and CBH1 produced by A. oryzae showed relatively higher activity toward Avicel, but showed much lower activity toward carboxymethyl cellulose (CMC) and p-nitrophenyl-β-D-cellobioside (PNPC), than H. grisea endoglucanase 1 (EGL1). The addition of a cellulose-binding domain and a hinge region to EXO1 caused decreases in its enzymatic activities as well as the deletion of the cellulose-binding domain from CBH1. EXO1 showed relatively weak or no synergistic activity toward Avicel with H. grisea endoglucanases, but showed a significant level of apparent synergism with H. grisea CBH1 and Trichoderma reesei EGLI. CBH1 showed a significant level of apparent endo-exo synergism with H. grisea and T. reesei endoglucanases. H. grisea has at least two different types of major exoglucanase components and shows strong cellulolytic activity through synergism with cellulase components including EXO1 and CBH1. [ABSTRACT FROM AUTHOR]

Aims: To develop a novel PCR-based method able to detect potential cellulolytic filamentous fungi and to classify them exploiting the amplification of the cellobiohydrolase gene ( cbh-I) and its polymorphism. Methods and Results: A mixed approach including the combination of (i) fungal cultivation and isolation, (ii) classification of fungal isolates through the amplification of the cbh gene using a fluorescently labelled primer (f-CBH-PCR) and (iii) final fungal identification based on amplification and sequencing of the ITS1-5.8S rDNA-ITS2 region of the selected fungal strains was developed. By this approach, it was possible to screen 77 fungal strains belonging to 14 genera and 26 species. Conclusions: The f-CBH-PCR permitted the discrimination of fungal species, producing typical f-CBH profiles. Significance and Impact of the Study: In this study, the cbh gene was used as a preliminary classification tool able to differentiate among themselves the fungal members isolated from indoor museum items and surrounding environment. Such mixed approach consented the fast identification of all isolated fungal strains. The f-CBH-PCR method demonstrated its discrimination power, and it can be considered as a new molecular system suitable for the classification of fungal strains isolated from different environments. [ABSTRACT FROM AUTHOR]