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Cultural and metagenomic approaches for isolation of genes encoding enzymes involved in hydrolysis of cellulose

KrishiKosh

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Title Cultural and metagenomic approaches for isolation of genes encoding enzymes involved in hydrolysis of cellulose
Ph.D.
 
Creator PANDEY, SANGEETA
 
Contributor Saxena, A. K.
 
Subject enzymes, bacteria, cellulose, genes, cloning, productivity, acidity, amino acids, cellulose products, proteins
 
Description The Earth‘s most abundant renewable organic material, the lignocellulosic biomass are the credible source of bioenergy and commodity chemicals, which can be made economically viable through identification of cellulase producing bio agents having capability to accelerate the conversion of cellulose to glucose. The aim of present study was to search for efficient cellulase producing microorganisms and their enzymes from extreme environments. A set of 300 bacterial strains isolated from various extreme environments ( hot springs, saline lake, acidic soil, mangroves and desert soil) of India were screened for presence of cellulase activity. The phylogenetic analysis of positive strains, based on 16S rRNA gene sequences indicated that isolates were clustered within Firmicutes and Actinobacteria. The majority (17) of isolates was identified as Bacillus and Bacillus derived genera and rest 3were identified as Arthobacter, Rhodococcus, and Bhargavaea cecembensis. Cellulase producers from soils irrigated with paper and pulp mill effluents having have high pH and organic matter content were also isolated. Ten cellulase producers were isolated and were identified as different species of Bacillus based on sequencing of 16s rRNA gene. B. subtilis IARI-SP-1 exhibited highest β-1, 4-endoglucanase, β-1, 4-exoglucanase and β-glucosidase activity followed by B. amyloliquefaciens IARI-SP-2. The pH and temperature optima of IARI-SP-1 was 8 and 50ºC. CMC was the best carbon source for the production of endo/exoglucanase and β-glucosidase. The β-1, 4-endoglucanase gene was amplified from all isolates and their deduced amino acid sequences belonged to glycosyl hydrolase family 5. Among the domains of different isolates, the catalytic domains exhibited the highest homology of 93.7% while the regions of signal, leader, linker and carbohydrate binding domain indicated low homology (73-74%). The complete ORF of β-1, 4-endoglucanase gene of B. subtilis IARI-SP-1 was amplified and cloned in pET-28a and over expressed in E. coli BL21 (DE3). In comparison to wild strain (B. subtilis), the transformed E. coli exhibited four times increase in cellulase production. Higher enzyme activity was observed in supernatant than cell pellet suggesting more extracellular production of β-1,
4-endoglucanase. The pH and temperature optima of expressed β-1, 4-exoglucanase enzyme was identical to that of wild strain. A metagenomic library consisting of 7500 clones was also constructed to explore the uncultured microorganisms from the soil irrigated with effluents of paper and pulp mill. A cellulase positive clone H-10 was identified by function based screening. A β-1, 4- endoglucanase with an open reading frame of 1499 bp was identified from this positive clone. The deduced amino acid sequence analysis revealed that amplified β-1, 4-endoglucanase belongs to GH5 family and shared 98% similarity with B. subtilis and 95% with uncultured bacterium clone. Clone H-10 indicated higher CMCase activity of 1 IU/ml on the substrate CMC as compared to rice straw (0.5 IU/ml). The characterization of enzymes revealed its pH optima between 8.5-9 and retained 80% of activity at pH 11. The findings of current study would facilitate development of efficient and cost-effective technology for conversion of lignocellulosic biomass into biofuels and value added products.
 
Date 2016-03-10T13:53:35Z
2016-03-10T13:53:35Z
2013
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/65042
 
Language en_US
 
Format application/pdf
 
Publisher IARI, DIVISION OF MICROBIOLOGY, NEW DELHI