title: Cloning and expression of β-1, 4-endoglucanase gene from Bacillus subtilis isolated from soil long term irrigated with effluents of paper and pulp mill.
abstract: A strain of Bacillus subtilis IARI-SP-1 isolated from soil long term irrigated with effluents of paper and pulp mill showed high β-1, 4-endoglucanase (2.5 IU/ml) but low activity of β-1, 4-exoglucanase (0.8 IU/ml) and β-glucosidase (0.084 IU/ml). The β-1, 4-endoglucanase gene of IARI-SP-1 was amplified using degenerate primers designed based on sequences already available in NCBI GenBank. A full length gene of β-1, 4-endonuclease consisting of 1499 nucleotides was identified through sequence analysis of the amplified product. The ORF encoded for a protein of 500 amino acids with a predicted molecular weight of 55 kDa. The gene was cloned in pET-28a and over expressed in Escherichia 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 (8.2 IU/ml) than cell pellet (2.8 IU/ml) suggesting more extracellular production of β-1, 4-endoglucanase. SDS-PAGE and CMC plate assay also confirmed the overproduction by the transformed E. coli. The pH and temperature optima of expressed β-1, 4-endoglucanase enzyme was identical to that of wild strain and was 8 and 50-60 °C, respectively.
authors.affiliation: Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, India.
authors.forename: Sangeeta
authors.initials: S
authors.lastname: Pandey
authors.affiliation: Division of Nematology, Indian Agricultural Research Institute, New Delhi 110012, India.
authors.forename: Jyoti
authors.initials: J
authors.lastname: Kushwah
authors.affiliation: Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, India.
authors.forename: Rameshwar
authors.initials: R
authors.lastname: Tiwari
authors.affiliation: Division of Nematology, Indian Agricultural Research Institute, New Delhi 110012, India.
authors.forename: Ram
authors.initials: R
authors.lastname: Kumar
authors.affiliation: Division of Nematology, Indian Agricultural Research Institute, New Delhi 110012, India.
authors.forename: Vishal Singh
authors.initials: VS
authors.lastname: Somvanshi
authors.affiliation: Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, India.
authors.forename: Lata
authors.initials: L
authors.lastname: Nain
authors.affiliation: Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, India. Electronic address: saxena461@yahoo.com.
authors.forename: Anil Kumar
authors.initials: AK
authors.lastname: Saxena
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004787:Environmental Pollution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007220:Industrial Waste; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D013696:Temperature
chemical_list: D003470:Culture Media; D007220:Industrial Waste; D011994:Recombinant Proteins; D002480:Cellulase
keywords: Cellulase; Cloning; Overexpression; β-1, 4-Endoglucanase