Detergent-compatible bacterial cellulases.

Cellulases, lipases, proteases, and amylases are employed in the detergent preparation to speed up the detergency process. Microbial cellulases are now commercially manufactured and are being used by various industries like detergent industry. Currently, the supplementation of detergent-compatible enzymes is a new trend followed by most of the detergent industries. The cellulases are supplemented to the detergents to improve the fabric smoothness and soil removal without damaging them. They act by passing through the textile interfibril spaces and thus the fabric quality is preserved. The process is environment friendly, and the use of cellulases and other detergent-compatible enzymes diminishes the utilization of toxic detergent constituents that are hazardous to humans. Alkaline cellulases active at ambient and low temperature are now preferred to maintain the fabric quality and use of low energy. The review reports on the production, purification, and properties studies of detergent-compatible proteases, amylases, and lipases are available. However, there is no report on detergent-compatible bacterial cellulases. In the present review, an overview on the production, purification, and characterization of detergent bacterial cellulases is presented. The stability and compatibility of the alkaline bacterial cellulases in the presence of the detergents and the detergent constituents are also discussed.

Metagenomic mining of glycoside hydrolases from the hindgut bacterial symbionts of a termite (Trinervitermes trinervoides) and the characterization of a multimodular ß-1,4-xylanase (GH11).

In recent years, there have been particular emphases worldwide on the development and optimization of bioprocesses for the utilization of biomass. An essential component of the biomass processing conduit has been the need for robust biocatalysts as high-performance tools for both the depolymerization of lignocellulosic biomass and synthesis of new high-value bio-based chemical entities. Through functional screening of the metagenome of the hindgut bacterial symbionts of a termite, Trinervitermes trinervoides, we discovered open reading frames for 25 cellulases and hemicellulases. These were classified into 14 different glycoside hydrolase (GH) families: eight GH family 5; four GH9, two GH13, and one each in GH2, GH10, GH11, GH26, GH29, GH43, GH44, GH45, GH67, and GH94 families. Of these, eight were overexpressed and partially characterized to be shown to be endocellulases (GH5C, GH5E, GH5F, and GH5G), an exocellulase (GH5D), endoxylanases (GH5H and GH11), and an α-fucosidase (GH29). The GH11 (Xyl1) was of particular interest as it was discovered to be a multimodular ß-1,4-xylanase, consisting of a catalytic domain and two carbohydrate-binding modules (CBMs). The CBM functions to selectively bind insoluble xylan and increases the rate of hydrolysis. The primary structure of GH11 showed a classical catalytic dyad of glutamic acid residues that generally forms part of the active site in GH11 enzyme family. This endoxylanase was optimal at pH 6 and 50 °C, and generated xylobiose and xylotriose from various xylan sources, including beechwood, birchwood, and wheat arabinoxylan. The catalytic ability of GH11 against natural substrate (e.g., wheat arabinoxylan) renders GH11 as a potential useful biocatalyst in the effective dismantling of complex plant biomass architecture.

Potential Role of Exoglucanase Genes (WaEXG1 and WaEXG2) in the Biocontrol Activity of Wickerhamomyces anomalus.

The use of yeasts, including Wickerhamomyces anomalus, as biocontrol agents of fungi responsible for postharvest diseases of fruits and vegetables has been investigated for the past two decades. Among a variety of mechanisms, the production of glucanases coded by the "killer genes" WaEXG1 and WaEXG2 have been reported to play a role in the ability of yeast to inhibit other fungi. The objective of the present study was to determine the expression of these genes by RT-qPCR, utilizing gene-specific primers, when W. anomalus was grown on grape berries and oranges that were either non-inoculated or inoculated with Botrytis cinerea or Penicillium digitatum, or in minimal media supplemented with cell walls of various plant pathogens and different amounts of glucose. Results indicated that WaEXG2 was more responsive than WaEXG1 to the nutritional environment (including the addition of glucose to cell wall-amended media) in vitro and appeared to play a greater role in the cellular metabolism of W. anomalus. WaEXG2 expression also appeared to be more responsive to the presence of cell walls of P. digitatum and B. cinerea than other fungal species, whereas the same level of induction was not seen in vivo when the yeast was grown in wounded/pathogen-inoculated fruits.

Comparative Analysis of Secretome Profiles of Manganese(II)-Oxidizing Ascomycete Fungi.

Fungal secretomes contain a wide range of hydrolytic and oxidative enzymes, including cellulases, hemicellulases, pectinases, and lignin-degrading accessory enzymes, that synergistically drive litter decomposition in the environment. While secretome studies of model organisms such as Phanerochaete chrysosporium and Aspergillus species have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates or conducted side-by-side comparisons of diverse species. Thus, the mechanisms of carbon degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of LC-MS/MS, genomic, and bioinformatic analyses to characterize and compare the protein composition of the secretomes of four recently isolated, cosmopolitan, Mn(II)-oxidizing Ascomycetes (Alternaria alternata SRC1lrK2f, Stagonospora sp. SRC1lsM3a, Pyrenochaeta sp. DS3sAY3a, and Paraconiothyrium sporulosum AP3s5-JAC2a). We demonstrate that the organisms produce a rich yet functionally similar suite of extracellular enzymes, with species-specific differences in secretome composition arising from unique amino acid sequences rather than overall protein function. Furthermore, we identify not only a wide range of carbohydrate-active enzymes that can directly oxidize recalcitrant carbon, but also an impressive suite of redox-active accessory enzymes that suggests a role for Fenton-based hydroxyl radical formation in indirect, non-specific lignocellulose attack. Our findings highlight the diverse oxidative capacity of these environmental isolates and enhance our understanding of the role of filamentous Ascomycetes in carbon turnover in the environment.

Cellulases: Classification, Methods of Determination and Industrial Applications.

Microbial cellulases have been receiving worldwide attention, as they have enormous potential to process the most abundant cellulosic biomass on this planet and transform it into sustainable biofuels and other value added products. The synergistic action of endoglucanases, exoglucanases, and ß-glucosidases is required for the depolymerization of cellulose to fermentable sugars for transformation in to useful products using suitable microorganisms. The lack of a better understanding of the mechanisms of individual cellulases and their synergistic actions is the major hurdles yet to be overcome for large-scale commercial applications of cellulases. We have reviewed various microbial cellulases with a focus on their classification with mechanistic aspects of cellulase hydrolytic action, insights into novel approaches for determining cellulase activity, and potential industrial applications of cellulases.

Medium initial pH and carbon source stimulate differential alkaline cellulase time course production in Stachybotrys microspora.

The production profile of cellulases of the mutant strain A19 from the filamentous fungus Stachybotrys microspora was studied in the presence of various carbon sources (glucose, lactose, cellulose, carboxymethylcellulose (CMC), and wheat bran) and a range of medium initial pH (5, 7, and 8). Two extracellular cellulases from the Stachybotrys strain (endoglucanases and ß-glucosidases) were monitored by enzymatic assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and zymogram analysis. Glucose and lactose repressed CMCase time course production while they permitted a strong ß-glucosidase one. On Avicel cellulose, CMC, and wheat bran, both activities were highly produced. Wheat bran (WB) is the best carbon source with an optimum of production at days 5 and 6. The production kinetics of both activities were shown to depend on the medium initial pH, with a preference for neutral or alkaline pH in the majority of conditions. The exception concerned the ß-glucosidase which was much more produced at acidic pH, on glucose and cellulose. Most interestingly, a constitutive and conditional expression of an alkaline endoglucanase was revealed on the glucose-based medium at an initial pH of 8 units. The zymogram analysis confirmed such conclusions and highlighted that carbon sources and the pH of the culture medium directed a differential induction of various endoglucanases and ß-glucosidases.

Metagenomic insights into the carbohydrate-active enzymes carried by the microorganisms adhering to solid digesta in the rumen of cows.

The ruminal microbial community is a unique source of enzymes that underpin the conversion of cellulosic biomass. In this study, the microbial consortia adherent on solid digesta in the rumen of Jersey cattle were subjected to an activity-based metagenomic study to explore the genetic diversity of carbohydrolytic enzymes in Jersey cows, with a particular focus on cellulases and xylanases. Pyrosequencing and bioinformatic analyses of 120 carbohydrate-active fosmids identified genes encoding 575 putative Carbohydrate-Active Enzymes (CAZymes) and proteins putatively related to transcriptional regulation, transporters, and signal transduction coupled with polysaccharide degradation and metabolism. Most of these genes shared little similarity to sequences archived in databases. Genes that were predicted to encode glycoside hydrolases (GH) involved in xylan and cellulose hydrolysis (e.g., GH3, 5, 9, 10, 39 and 43) were well represented. A new subfamily (S-8) of GH5 was identified from contigs assigned to Firmicutes. These subfamilies of GH5 proteins also showed significant phylum-dependent distribution. A number of polysaccharide utilization loci (PULs) were found, and two of them contained genes encoding Sus-like proteins and cellulases that have not been reported in previous metagenomic studies of samples from the rumens of cows or other herbivores. Comparison with the large metagenomic datasets previously reported of other ruminant species (or cattle breeds) and wallabies showed that the rumen microbiome of Jersey cows might contain differing CAZymes. Future studies are needed to further explore how host genetics and diets affect the diversity and distribution of CAZymes and utilization of plant cell wall materials.

ß -Glucosidases from the fungus trichoderma: an efficient cellulase machinery in biotechnological applications.

ß-glucosidases catalyze the selective cleavage of glucosidic linkages and are an important class of enzymes having significant prospects in industrial biotechnology. These are classified in family 1 and family 3 of glycosyl hydrolase family. ß-glucosidases, particularly from the fungus Trichoderma, are widely recognized and used for the saccharification of cellulosic biomass for biofuel production. With the rising trends in energy crisis and depletion of fossil fuels, alternative strategies for renewable energy sources need to be developed. However, the major limitation accounts for low production of ß-glucosidases by the hyper secretory strains of Trichoderma. In accordance with the increasing significance of ß-glucosidases in commercial applications, the present review provides a detailed insight of the enzyme family, their classification, structural parameters, properties, and studies at the genomics and proteomics levels. Furthermore, the paper discusses the enhancement strategies employed for their utilization in biofuel generation. Therefore, ß-glucosidases are prospective toolbox in bioethanol production, and in the near future, it might be successful in meeting the requirements of alternative renewable sources of energy.

Proteomic analysis of temperature dependent extracellular proteins from Aspergillus fumigatus grown under solid-state culture condition.

Fungal species of the genus Aspergillus are filamentous ubiquitous saprophytes that play a major role in lignocellulosic biomass recycling and also are considered as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. Analysis of extracellular secreted biomass degrading enzymes using complex lignocellulosic biomass as a substrate by solid-state fermentation could be a more practical approach to evaluate application of the enzymes for lignocellulosic biorefinery. This study isolated a fungal strain from compost, identified as Aspergillus fumigatus, and further analyzed it for lignocellulolytic enzymes at different temperatures using label free quantitative proteomics. The profile of secretome composition discovered cellulases, hemicellulases, lignin degrading proteins, peptidases and proteases, and transport and hypothetical proteins; while protein abundances and further their hierarchical clustering analysis revealed temperature dependent expression of these enzymes during solid-state fermentation of sawdust. The enzyme activities and protein abundances as determined by exponentially modified protein abundance index (emPAI) indicated the maximum activities at the range of 40-50 °C, demonstrating the thermophilic nature of the isolate A. fumigatus LF9. Characterization of the thermostability of secretome suggested the potential of the isolated fungal strain in the production of thermophilic biomass degrading enzymes for industrial application.

Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production.

One of the major challenges in the bioconversion of lignocellulosic biomass into liquid biofuels includes the search for a glucose tolerant beta-gulucosidase. Beta-glucosidase is the key enzyme component present in cellulase and completes the final step during cellulose hydrolysis by converting the cellobiose to glucose. This reaction is always under control as it gets inhibited by its product glucose. It is a major bottleneck in the efficient biomass conversion by cellulase. To circumvent this problem several strategies have been adopted which we have discussed in the article along with its production strategies and general properties. It plays a very significant role in bioethanol production from biomass through enzymatic route. Hence several amendments took place in the commercial preparation of cellulase for biomass hydrolysis, which contains higher and improved beta-glucosidase for efficient biomass conversion. This article presents beta-glucosidase as the key component for bioethanol from biomass through enzymatic route.

Re-annotation of the CAZy genes of Trichoderma reesei and transcription in the presence of lignocellulosic substrates.

BACKGROUND: Trichoderma reesei is a soft rot Ascomycota fungus utilised for industrial production of secreted enzymes, especially lignocellulose degrading enzymes. About 30 carbohydrate active enzymes (CAZymes) of T. reesei have been biochemically characterised. Genome sequencing has revealed a large number of novel candidates for CAZymes, thus increasing the potential for identification of enzymes with novel activities and properties. Plenty of data exists on the carbon source dependent regulation of the characterised hydrolytic genes. However, information on the expression of the novel CAZyme genes, especially on complex biomass material, is very limited. RESULTS: In this study, the CAZyme gene content of the T. reesei genome was updated and the annotations of the genes refined using both computational and manual approaches. Phylogenetic analysis was done to assist the annotation and to identify functionally diversified CAZymes. The analyses identified 201 glycoside hydrolase genes, 22 carbohydrate esterase genes and five polysaccharide lyase genes. Updated or novel functional predictions were assigned to 44 genes, and the phylogenetic analysis indicated further functional diversification within enzyme families or groups of enzymes. GH3 ß-glucosidases, GH27 α-galactosidases and GH18 chitinases were especially functionally diverse. The expression of the lignocellulose degrading enzyme system of T. reesei was studied by cultivating the fungus in the presence of different inducing substrates and by subjecting the cultures to transcriptional profiling. The substrates included both defined and complex lignocellulose related materials, such as pretreated bagasse, wheat straw, spruce, xylan, Avicel cellulose and sophorose. The analysis revealed co-regulated groups of CAZyme genes, such as genes induced in all the conditions studied and also genes induced preferentially by a certain set of substrates. CONCLUSIONS: In this study, the CAZyme content of the T. reesei genome was updated, the discrepancies between the different genome versions and published literature were removed and the annotation of many of the genes was refined. Expression analysis of the genes gave information on the enzyme activities potentially induced by the presence of the different substrates. Comparison of the expression profiles of the CAZyme genes under the different conditions identified co-regulated groups of genes, suggesting common regulatory mechanisms for the gene groups.

Induction of lignocellulose-degrading enzymes in Neurospora crassa by cellodextrins.

Neurospora crassa colonizes burnt grasslands in the wild and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source such as sucrose to cellulose, N. crassa dramatically upregulates expression and secretion of a wide variety of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Here, we show that an N. crassa mutant carrying deletions of two genes encoding extracellular ß-glucosidase enzymes and one intracellular ß-glucosidase lacks ß-glucosidase activity, but efficiently induces cellulase gene expression in the presence of cellobiose, cellotriose, or cellotetraose as a sole carbon source. These data indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression in N. crassa. Furthermore, the inclusion of a deletion of the catabolite repressor gene, cre-1, in the triple ß-glucosidase mutant resulted in a strain that produces higher concentrations of secreted active cellulases on cellobiose. Thus, the ability to induce cellulase gene expression using a common and soluble carbon source simplifies enzyme production and characterization, which could be applied to other cellulolytic filamentous fungi.

Toward plant cell wall degradation under thermophilic condition: a unique microbial community developed originally from swine waste.

A unique thermophilic microbial community developed initially from swine waste was investigated in this study. Cellulase activities were observed when this community was inoculated to media containing either cellulose or carboxymethylcellulose at 57 degrees C. Through constructing a clone library for the 16S ribosomal DNA, it was revealed that this community was mainly composed of three genera: Thermobacillus, Brevibacillus, and Anoxybacillus. New findings regarding the thermo- and pH stability of crude cellulases secreted by Brevibacillus sp. JXL were presented. Recent study on the growth characteristics of Anoxybacillus sp. 527 was discussed.

Characterization of a novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39.

AIMS: The aims of this study were to identify and characterize the novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39. METHODS AND RESULTS: Strain B39 was closely related to Paenibacillus cookii in 16S rRNA gene sequence. Nonetheless, this isolate can be identified as a novel Paenibacillus sp. with respect to its physiological characteristics, biochemical reactions, and profiles of fatty acid compositions. A cellulase with both CMCase and avicelase activities was secreted from strain B39 and purified by ion-exchange chromatography. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the molecular weight of B39 cellulase was determined as 148 kDa, which was much higher than other cellulases currently reported from Paenibacillus species. The enzyme showed a maximum CMCase activity at 60 degrees C and pH 6.5. Addition of 1 mmol l(-1) of Ca(2+) markedly enhanced both CMCase and avicelase activities of the enzyme. CONCLUSIONS: We have identified and characterized a novel thermophilic Paenibacillus sp. strain B39 which produced a high-molecular weight cellulase with both CMCase and avicelase activities. SIGNIFICANCE AND IMPACT OF THE STUDY: Based on the ability to hydrolyse CMC and avicel, the cellulase produced by Paenibacillus sp. strain B39 would have potential applications in cellulose biodegradation.

Random exchanges of non-conserved amino acid residues among four parental termite cellulases by family shuffling improved thermostability.

There have been two major problems preventing applications of termite cellulases; one was difficulty for their hetelologous overexpression, and another is their low thermostability. We previously achieved adaptation of termite cellulase genes to an overexpression system of Escherichia coli by family shuffling of four orthologous cDNAs (Biosci. Biotechnol. Biochem., 2005; 69: 1711-1720). Using the adapted mutant cDNAs as parental genes combined with native-form cDNAs, we performed further family shuffling and obtained mutant cDNAs, which gave enzymes with improved thermostability. The best-evolved clone (PA68) was improved by 10 degrees C in maximum stability (retaining 90% original activity for 30 min incubation) from the parental enzymes, and kept 54% of its original activity for 150 min at 50 degrees C, whereas the most thermostable enzyme amongst the parents (A18) retained 30% of its original activity. PA68 showed 889 (micromoles of reducing sugars/min/mg of protein) in V(max) and 560 (micromoles of reducing sugars/min/mg of protein) in the specific activity against carboxymethylcellulose, which corresponds to 9.8 and 13.1 times of those of one of the ancestral enzymes rRsEG. In summary, we improved thermostability of the termite cellulase and increased the V(max) value and specific activity by combining only cDNAs encoding enzymes adapted for normal temperatures.

Cellulases from two Penicillium sp. strains isolated from subtropical forest soil: production and characterization.

AIMS: To isolate new fungal strains from subtropical soils and to identify those that produce high cellulase activity. To select microbial strains producing thermostable cellulases with potential application in industry. METHODS AND RESULTS: The new strains Penicillium sp. CR-316 and Penicillium sp. CR-313 have been identified and selected because they secreted a high level of cellulase in media supplemented with rice straw. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focussing and zymography showed that the studied strains secreted multiple enzymes that hydrolyse cellulose. Cellulase activity of Penicillium sp. CR-316, the strain showing higher production, was analysed. Optimum temperature and pH of carboxymethyl cellulase activity were 65 degrees C and pH 4.5, respectively. Activity remained stable after incubation at 60 degrees C and pH 4.5 for 3 h. CONCLUSIONS: Fungal strains that secrete high levels of cellulase activity have been characterized and selected from soil. The isolated strains have complex sets of enzymes for cellulose degradation. Crude cellulase produced by Penicillium sp. CR-316 showed activity and stability at high temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: Two fungal strains with biotechnological potential have been isolated. The strains secrete high levels of cellulase, and one of them, Penicillium sp. CR-316, produces a thermostable cellulase, that makes it a good candidate for industrial applications.