Cloning, Overexpression, and Characterization of Halostable, Solvent-Tolerant Novel β-Endoglucanase from a Marine Bacterium Photobacterium panuliri LBS5(T) (DSM 27646(T)).
A 1329 nucleotide long endoglucanase gene was amplified from marine bacterium Photobacterium panuliri strain LBS5(T).The enzyme sequence was novel as protein-based similarity search revealed that it shared maximum similarity of 99% with hypothetical protein of P. aquae and 40% with endoglucanase of P. marinum AK15. The gene was cloned, overexpressed in Escherichia coli BL21 (DE3), and purified up to homogeneity using Ni-NTA affinity chromatography. The purified enzyme, designated as Cel8, was monomeric and has a molecular mass of 53 kDa. The enzyme was halostable and exhibited optimal carboxymethyl cellulase (CMCase) activity and stability at 2 M NaCl. Optimal activity was obtained at 40 °C and at pH 4. The enzyme exhibited remarkable stability in different organic solvents (50%, v/v), and activity increased nearly 1.5-fold in presence of butanol, isopropanol, petroleum ether, benzene, acetone, and n-hexane. It was active in Ca(2+), Ba(2+), and Ni(2+) and inhibited by Co(2+), Cd(2+), Zn(2+), Cu(2+), and Hg(2+). Under normal physiological conditions, the enzyme has 25% helix, 30% sheets, and 56% irregularities, whereas salt leads to helix to sheet transition in enzyme. Three-dimensional reconstruction analysis revealed that the enzyme has (α/β)8 structure and a TIM barrel fold-like structure at the central groove of enzyme. This is the first evidenced report on halostable, organic solvent tolerant cellulase in the marine bacterial genus Photobacterium.
Department of Biotechnology, Institute of Life Sciences, Nalco Square, Bhubaneswar, 751 023, India.
Kamal
K
Deep
Department of Biotechnology, Institute of Life Sciences, Nalco Square, Bhubaneswar, 751 023, India.
Abhijit
A
Poddar
Department of Biotechnology, Institute of Life Sciences, Nalco Square, Bhubaneswar, 751 023, India. subratkdas@hotmail.com.
Subrata K
SK
Das
D000595:Amino Acid Sequence; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007700:Kinetics; D008386:Marine Biology; D008969:Molecular Sequence Data; D010776:Photobacterium / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012997:Solvents / Q000737:chemistry
D011994:Recombinant Proteins; D012997:Solvents; D002480:Cellulase
Cloning; Halostable; Overexpression; Photobacterium panuliri LBS5T; Solvent tolerant; β-endoglucanase