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• 作者：Kamal Deep ; Abhijit Poddar ; Subrata K. Das
• 关键词：Photobacterium panuliri LBS5T ; β ; endoglucanase ; Halostable ; Solvent tolerant ; Cloning ; Overexpression
• 刊名：Applied Biochemistry and Biotechnology
• 出版年：2016
• 出版时间：February 2016
• 年：2016
• 卷：178
• 期：4
• 页码：695-709
• 全文大小：1,655 KB
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• 作者单位：Kamal Deep (1)
  Abhijit Poddar (1)
  Subrata K. Das (1)
  
  1. Department of Biotechnology, Institute of Life Sciences, Nalco Square, Bhubaneswar, 751 023, India
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Biochemistry
  
• 出版者：Humana Press Inc.
• ISSN：1559-0291

文摘

A 1329 nucleotide long endoglucanase gene was amplified from marine bacterium Photobacterium panuliri strain LBS5T.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 Ca2+, Ba2+, and Ni2+ and inhibited by Co2+, Cd2+, Zn2+, Cu2+, and Hg2+. 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.