Aims: Cellulases are enzymes that convert cellulose into glucose molecules, and are produced by various microorganisms in the environment. Due to their importance to the biofuel industry, there is a need to screen for more efficient varieties of cellulases. In this study, leachate samples from a landfill site were screened for cellulolytic bacteria. Methodology and results: Leachate samples obtained from a landfill collection pond were cultured in an enriched cellulose medium. Two cellulolytic isolates, designated MAEPY1 and MAEPY2, were isolated and further characterized. Phenotypic profiles and phylogenetic analyses using sequences of 16S rRNA, gyrB and whole genome suggested that these isolates are new strains of the Paenibacillus genera. The crude enzyme extracts from both isolates have cellulose degradation activity at approximately 0.1-0.2 IU/mg under working conditions of pH 6 and 55 °C. Assays using other lignocellulosic substrates showed that the crude enzyme extracts also have high xylan degradation activity. Conclusion, significance and impact of study: Paenibacillus sp. are known to produce multiple enzymes for lignocellulolytic degradation and the present results suggest that isolates described in this study, MAEPY1 and MAEPY2, are excellent candidates deserving further study as potential producers of efficient cellulases for use in industries associated with cellulosic biomass.
Cellulases

Aims: High cost of cellulases remains the most significant barrier to the economical production of bio-ethanol from lignocellulosic biomass (LB). The present study aims at developing a local cellulolytic fungal strain through random mutagenesis coupled with the feasibility of solid-state fermentation (SSF) by utilizing agricultural wastes such as oil palm frond (OPF) as the substrate. Methodology and results: Out of 95 wild isolates tested, native fungal strain Aspergillus niger, designated DWA8 was isolated as the top enzymatic secretor. For quantitative enzyme analysis, SSF was conducted using 1x106 spore/mL inoculated onto 5 g of ground OPF, incubated at room temperature for 7 days, with 70% moisture content and an initial medium pH of 7. Random mutagenesis has always been tempting in the enhancement of enzyme production. In this work, the compounded treatment of microwave, ultraviolet (UVC) and Ethyl Methanesulfonate (EMS) have generated an Aspergillus niger MUE3.06 mutant with an overall increase of 114% in CMCase activity, approximately 70% in FPase and Xylanase activity respectively compared with the parental DWA8 strain. Thus this finding is capable to be fully developed as an established mutational scheme to create highly productive filamentous fungus in a cheap, simple and sustainable way. Conclusion, significance and impact of study: It was the first attempt to explore the combine effect of the three popular mutagens upon cellulases and xylanases. It is believed that more diversified of mutagen types induce more diversified mutation pattern (with instructive planning), which is very desirable in creating new enzymes with novel abilities.
Cellulases

This study aims to explore the resource utilization of used fungus-growing materials produced in the cultivation of Gastrodia elata. To be specific, based on the production practice, this study investigated the recycling mechanism of used fungus-growing materials of G. elata by Phallus inpudicus. To screen edible fungi with wide adaptability, this study examined the allelopathic effects of Armillaria mellea secretions on P. impudicus and 6 kinds of large edible fungi and the activities of enzymes related to degradation of the used fungus-growing materials of G. elata. The results showed that P. impudicus can effectively degrade cellulose, hemicellulose, and lignin in used fungus-growing materials of G. elata. The cellulase activity of A. mellea was significantly higher than that of P. impudicus, and the activities of lignin peroxidase, polyphenol oxidase, and xylanase of P. impudicus were significantly higher than those of A. mellea, which was the important reason why A. mellea and P. impudicus used different parts and components of the used fungus-growing materials to absorb carbon sources and develop ecological niche differences. The growth of P. impudicus was significantly inhibited on the used fungus-growing materials of G. elata. The secretions of A. mellea had allelopathic effects on P. impudicus and other edible fungi, and the allelopathic effects were related to the concentration of allelopathy substances. The screening result showed that the growth and development of L. edodes and A. auricular were not significantly affected by 30% of A. mellea liquid, indicating that they had high resistance to the allelopathy of A. mellea. The results showed that the activities of extracellular lignin peroxidase, polyphenol oxidase, and xylanase of the two edible fungi were similar to those of P. impudicus, and the cellulase activity was higher than that of P. impudicus. This experiment can be further verified by small-scale production tests.
Agaricales ; Ascomycota ; Basidiomycota ; Catechol Oxidase ; Cellulases ; Gastrodia

To obtain basic information on the biochemical property of basidiospores of shiitake mushroom (Lentinula edodes), the ability of producing extracellular enzyme was assessed using a chromogenic plate-based assay. For the aim, amylase, avicelase, beta-glucosidase, CM-cellulase, pectinase, proteinase, and xylanase were tested against monokaryotic strains generated from forty basidiospores of two different parental dikaryotic strains of shiitake mushroom, Sanjo-101Ho and Sanjo-108Ho. These two parental strains showed different degree of extracellular enzyme activity. No identical patterns of the degree of enzyme activity were observed between monokaryotic strains and parental strains of the two shiitake cultivars. The degree of extracellular enzyme activity also varied among monokaryotic strains of the two shiitake cultivars. Our results showed that dikaryotic parental strains of shiitake mushroom produce monokaryotic basidiospores having very diverse biochemical properties.
Amylases ; beta-Glucosidase ; Cellulases ; Humans ; Parents ; Polygalacturonase ; Shiitake Mushrooms

The ability of Ganoderma to produce extracellular enzymes, including beta-glucosidase, cellulase, avicelase, pectinase, xylanase, protease, amylase, and ligninase was tested in chromogenic media. beta-glucosidase showed the highest activity, among the eight tested enzymes. In particular, Ganoderma neo-japonicum showed significantly stronger activity for beta-glucosidase than that of the other enzymes. Two Ganoderma lucidum isolates showed moderate activity for avicelase; however, Ganoderma neo-japonicum showed the strongest activity. Moderate ligninase activity was only observed in Ganoderma neo-japonicum. In contrast, pectinase, amylase, protease, and cellulase were not present in Ganoderma. The results show that the degree of activity of the tested enzymes varied depending on the Ganoderma species tested.
Amylases ; beta-Glucosidase ; Cellulase ; Cellulases ; Ganoderma ; Oxygenases ; Polygalacturonase ; Reishi

Filamentous fungi are widely used for large-scale production of cellulases. Morphological characteristics of mycelia under submerged condition are closely correlated with cellulases productivity. In order to find out the critical genes involved in the mycelial morphology development and cellulases production in liquid fermentation, 95 Neurospora crassa morphological mutants (named as SZY1-95) were screened for cellulases production. Compared with the wild type, cellulases production in four mutants SZY32, SZY35, SZY39 and SZY43 were significantly decreased, whereas mutants SZY63, SZY69, SZY87 and SZY11 produced much more cellulases than that of the wild type strain. Meanwhile, endo-beta-1,4-glucanase activity, beta-glucosidase activity, viscosity of broth and dry weight of these mutants were measured. The mycelial morphology of the mutants was also studied by microscope. Particularly, pellets were formed in mutant SZY11 and SZY43, whose viscosities were 25% and 50% of the wild type strain, respectively. Mutant SZY87 appeared long hyphae, and the viscosity of its broth was at least 2 folds of the wild type strain. These results indicate that a single gene deletion could influence the mycelial morphology in liquid fermentation, and increased the cellulases production. The low-viscosity related genes identified in our study will be the potential candidates for genetic modification of filamentous fungi.
Cellulases ; biosynthesis ; Fermentation ; Gene Deletion ; Industrial Microbiology ; Neurospora crassa ; genetics ; metabolism

There is a great diversity for cellulolytic microbes in nature and the strategies they use to digest cellulose. In addition to the cultured cellulolytic microbes, there are still a great number of microbes being not readily culturable in natural environments, which may represent great potential for identifying novel cellulases and their encoding genes. The rise of metagenomics and metaproteomics provides essential technologic tools to dig up these resources and significant progress has been made so far. This review gives an insight into some relative results that have arisen from the meta-genomic or proteomic analysis of definitive uncultured microbe communities. Their potential role in elucidating the process and mechanisms of cellulose degradation in natural environment from the point of "community system microbiology" is also discussed.
Bacteria ; enzymology ; genetics ; Cellulases ; genetics ; metabolism ; Cellulose ; metabolism ; Cloning, Molecular ; Environmental Microbiology ; Genome, Bacterial ; Metagenomics

Cylindrocarpon destructans is an ascomycete soil-borne pathogen that causes ginseng root rot. To identify effective biocontrol agents, we isolated several bacteria from ginseng cultivation soil and evaluated their antifungal activity. Among the isolated bacteria, one isolate (named JH7) was selected for its high antibiotic activity and was further examined for antagonism against fungal pathogens. Strain JH7 was identified as a Chromobacterium sp. using phylogenetic analysis based on 16S rRNA gene sequences. This strain was shown to produce antimicrobial molecules, including chitinases and proteases, but not cellulases. Additionally, the ability of JH7 to produce siderophore and solubilize insoluble phosphate supports its antagonistic and beneficial traits for plant growth. The JH7 strain suppressed the conidiation, conidial germination, and chlamydospore formation of C. destructans. Furthermore, the JH7 strain inhibited other plant pathogenic fungi. Thus, it provides a basis for developing a biocontrol agent for ginseng cultivation.
Ascomycota ; Bacteria ; Cellulases ; Chromobacterium* ; Fungi ; Genes, rRNA ; Germination ; Panax* ; Peptide Hydrolases ; Plants ; Soil

A beta-glucosidase from Penicillium italicum was purified with a specific activity of 61.8 U/mg, using a chromatography system. The native form of the enzyme was an 88.5-kDa tetramer with a molecular mass of 354 kDa. Optimum activity was observed at pH 4.5 and 60degrees C, and the half-lives were 1,737, 330, 34, and 1 hr at 50, 55, 60, and 65degrees C, respectively. Its activity was inhibited by 47% by 5 mM Ni2+. The enzyme exhibited hydrolytic activity for p-nitrophenyl-beta-D-glucopyranoside (pNP-Glu), p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-xyloside, and cellobiose, however, no activity was observed for p-nitrophenyl-beta-D-lactopyranoside, p-nitrophenyl-beta-D-galactopyranoside, carboxymetyl cellulose, xylan, and cellulose, indicating that the enzyme was a beta-glucosidase. The kcat/Km (s-1 mM-1) values for pNP-Glu and cellobiose were 15,770.4 mM and 6,361.4 mM, respectively. These values were the highest reported for beta-glucosidases. Non-competitive inhibition of the enzyme by both glucose (Ki = 8.9 mM) and glucono-delta-lactone (Ki = 11.3 mM) was observed when pNP-Glu was used as the substrate. This is the first report of non-competitive inhibition of beta-glucosidase by glucose and glucono-delta-lactone.
beta-Glucosidase ; Cellobiose ; Cellulases ; Cellulose ; Chromatography ; Citrus ; Fungi ; Glucose ; Glucosides ; Hydrogen-Ion Concentration ; Penicillium

To promote the selection of promising monokaryotic strains of button mushroom (Agaricus bisporus) during breeding, 61 progeny strains derived from basidiospores of two different lines of dikaryotic parental strains, ASI1038 and ASI1346, were analyzed by nucleotide sequencing of the intergenic spacer I (IGS I) region in their rDNA and by extracellular enzyme assays. Nineteen different sizes of IGS I, which ranged from 1,301 to 1,348 bp, were present among twenty ASI1346-derived progeny strains, while 15 different sizes of IGS I, which ranged from 700 to 1,347 bp, were present among twenty ASI1038-derived progeny strains. Phylogenetic analysis of the IGS sequences revealed that different clades were present in both the ASI10388- and ASI1346-derived progeny strains. Plating assays of seven kinds of extracellular enzymes (beta-glucosidase, avicelase, CM-cellulase, amylase, pectinase, xylanase, and protease) also revealed apparent variation in the ability to produce extracellular enzymes among the 40 tested progeny strains from both parental A. bisporus strains. Overall, this study demonstrates that characterization of IGS I regions and extracellular enzymes is useful for the assessment of the substrate-degrading ability and heterogenicity of A. bisporus monokaryotic strains.
Agaricales* ; Amylases ; Breeding ; Cellulases ; DNA, Ribosomal ; Enzyme Assays ; Humans ; Parents ; Polygalacturonase