Endophytic fungi in plant tissues produce a wide range of secondary metabolites and enzymes, which exhibit a variety of biological activities. In the present study, litter endophytic fungi were isolated from a fire-prone forest and screened for thermostable cellulases. Among nine endophytic fungi tested, two isolates, Bartalinia pondoensis and Phoma sp., showed the maximum cellulase activity. Bartalinia pondoensis was further selected for its cellulase production and characterization. Among the carbon and nitrogen sources tested, maximum cellulase production was observed with maltose and yeast extract, and the eucalyptus leaves and rice bran served as the best natural substrates. The cellulase activity increased with increasing temperature, with maximum activity recorded at 100 °C. The maximum CMCase activity was observed between pH 6.0 and 7.0 and retained 80% of its activity in the pH range of 8-10. Partially purified cellulase of B. pondoensis retained 50% of its activity after 2 h of incubation at 60 °C, 80 °C and 100 °C. These results suggest that litter endophytic fungus B. pondoensis is a potential source for the production of thermostable and alkali-tolerant cellulase.
- Klíčová slova
- Agni fungi, Bartalinia pondoensis, Cellulase, Litter endophytic fungi, Thermal stability,
- MeSH
- alkálie MeSH
- Ascomycota * metabolismus MeSH
- celulasa * chemie MeSH
- celulasy * MeSH
- endofyty metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- alkálie MeSH
- celulasa * MeSH
- celulasy * MeSH
This study aimed to isolate and characterize a novel cellulolytic enzyme from black goat rumen by using a culture-independent approach. A metagenomic fosmid library was constructed from black goat rumen contents and screened for a novel cellulase. The KG37 gene encoding a protein of 858 amino acid residues (92.7 kDa) was isolated. The deduced protein contained a glycosyl hydrolase family 74 (GH74) domain and showed 77% sequence identity to two endo-1,4-β-glucanases from Fibrobacter succinogenes. The novel GH74 cellulase gene was overexpressed in Escherichia coli, and its protein product was functionally characterized. The recombinant GH74 cellulase showed a broad substrate spectrum. The enzyme exhibited its optimum activity at pH 5.0 and temperature range of 20-50 °C. The enzyme was thermally stable at pH 5.0 and at a temperature of 20-40 °C. The novel GH74 cellulase can be practically exploited to convert lignocellulosic biomass to value-added products in various industrial applications in future.
- MeSH
- bachor mikrobiologie MeSH
- celulasa chemie genetika izolace a purifikace MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- Fibrobacter enzymologie genetika MeSH
- genetické testování MeSH
- genová knihovna MeSH
- klonování DNA MeSH
- koncentrace vodíkových iontů MeSH
- kozy mikrobiologie MeSH
- metagenom * MeSH
- metagenomika MeSH
- molekulová hmotnost MeSH
- rekombinantní proteiny genetika izolace a purifikace metabolismus MeSH
- sekvenční homologie MeSH
- stabilita enzymů MeSH
- substrátová specifita MeSH
- teplota MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- celulasa MeSH
- rekombinantní proteiny MeSH
Cellulose is the main polymeric component of the plant cell wall, the most abundant polysaccharide on Earth, and an important renewable resource. Basidiomycetous fungi belong to its most potent degraders because many species grow on dead wood or litter, in environment rich in cellulose. Fungal cellulolytic systems differ from the complex cellulolytic systems of bacteria. For the degradation of cellulose, basidiomycetes utilize a set of hydrolytic enzymes typically composed of endoglucanase, cellobiohydrolase and beta-glucosidase. In some species, the absence of cellobiohydrolase is substituted by the production of processive endoglucanases combining the properties of both of these enzymes. In addition, systems producing hydroxyl radicals based on cellobiose dehydrogenase, quinone redox cycling or glycopeptide-based Fenton reaction are involved in the degradation of several plant cell wall components, including cellulose. The complete cellulolytic complex used by a single fungal species is typically composed of more than one of the above mechanisms that contribute to the utilization of cellulose as a source of carbon or energy or degrade it to ensure fast substrate colonization. The efficiency and regulation of cellulose degradation differs among wood-rotting, litter-decomposing, mycorrhizal or plant pathogenic fungi and yeasts due to the different roles of cellulose degradation in the physiology and ecology of the individual groups.
- MeSH
- Basidiomycota chemie enzymologie genetika metabolismus MeSH
- benzochinony metabolismus MeSH
- biodegradace MeSH
- celulasa chemie genetika metabolismus MeSH
- celulosa metabolismus MeSH
- hydroxylový radikál metabolismus MeSH
- nemoci rostlin mikrobiologie MeSH
- rostliny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- benzochinony MeSH
- celulasa MeSH
- celulosa MeSH
- hydroxylový radikál MeSH
- quinone MeSH Prohlížeč
Three cellulase components (FP-ase, CMC-ase and cellobiase) were purified by affinity binding on Avicel followed by Sephadex G-25, DEAE-Sepharose, DEAE-cellulose and Sephadex G-100 chromatography from the culture filtrate of the newly isolated strain Penicillium camemberti. The isolated enzymes had the properties of cellobiohydrolase, endo-1,4-beta-D-glucanase and cellobiase and their respective molar masses were 99, 87 and 61 kDa as determined by molecular sieve chromatography on Sephadex G-100. The amino acid composition of each fraction was also determined.
- MeSH
- aminokyseliny analýza MeSH
- celulasa chemie izolace a purifikace metabolismus MeSH
- celulosa metabolismus MeSH
- chromatografie iontoměničová MeSH
- metabolismus sacharidů MeSH
- Penicillium enzymologie MeSH
- spektrofotometrie infračervená MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aminokyseliny MeSH
- celulasa MeSH
- celulosa MeSH
The reaction mechanism of the non-specific endo-1,4-beta-glucanase from Trichoderma reesei QM 9414 (endoglucanase I) was investigated using both reducing-end3H-labelled and universally 14C-labelled cellooligosaccharides, as well as reducing-end3H-labelled xylooligosaccharides. The bond cleavage frequencies of cellooligosaccharides proved to be dependent upon the substrate concentration, especially in the case of cellotriose. In addition to simple hydrolytic cleavage, the enzyme catalyzes reactions along alternative pathways, including transglycosylations leading to products larger than the substrate. Some of these pathways were shown to be reversible. During cellotriose or cellopentaose degradation, substrate resynthesis was demonstrated by incorporation of added radioactive D-glucose or cellobiose. The endoglucanase I is active on xylan and xylooligosaccharides, but less than on soluble cellulose derivatives (e.g. hydroxyethylcellulose) and cellooligosaccharides. The fact that for these different types of substrates the same active site is operative is proven by the ability of the enzyme to utilize cellooligosaccharides and xylooligosaccharides as both glycosyl donors and acceptors. The mixed substrate reactions lead to products composed of D-glucosyl and D-xylosyl residues. The kinetic parameters for cellooligosaccharide degradation can be used for the description of an extended substrate binding site. Of the four putative glycosyl subsites, -II and +II show the highest affinities, 16.7 kJ.mol-1 and 7.1 kJ.mol-1, respectively.
- MeSH
- celulasa chemie metabolismus MeSH
- endo-1,4-beta-xylanasy MeSH
- glukosa metabolismus MeSH
- glykosidhydrolasy metabolismus MeSH
- oligosacharidy metabolismus MeSH
- substrátová specifita MeSH
- Trichoderma enzymologie MeSH
- xylosa metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- celulasa MeSH
- endo-1,4-beta-xylanasy MeSH
- glukosa MeSH
- glykosidhydrolasy MeSH
- oligosacharidy MeSH
- xylosa MeSH
