Extremophiles

, Volume 13, Issue 4, pp 725–733

Molecular analysis of the gene encoding a cold-adapted halophilic subtilase from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913: cloning, expression, characterization and function analysis of the C-terminal PPC domains

  • Bing-Qiang Yan
  • Xiu-Lan Chen
  • Xiao-Yan Hou
  • Hailun He
  • Bai-Cheng Zhou
  • Yu-Zhong Zhang
Original Paper

DOI: 10.1007/s00792-009-0263-1

Cite this article as:
Yan, BQ., Chen, XL., Hou, XY. et al. Extremophiles (2009) 13: 725. doi:10.1007/s00792-009-0263-1

Abstract

Only a few cold-adapted halophilic proteases have been reported. Here, the gene mcp03 encoding a cold-adapted halophilic protease MCP-03 was cloned from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913, which contains a 2,130-bp ORF encoding a novel subtilase precursor. The recombinant MCP-03, expressed in Escherichia coli BL21 and purified from fermented broth, is a multi-domain protein with a catalytic domain and two PPC domains. Compared to mesophilic subtilisin Carlsberg, MCP-03 had characteristics of a typical cold-adapted enzyme (e.g., higher activity at low temperatures, lower optimum temperature and higher thermolability). MCP-03 also exhibited good halophilic ability with maximal activity at 3 M NaCl/KCl and good stability in 3 M NaCl. Deletion mutagenesis showed that the C-terminal PPC domains were unnecessary for enzyme secretion but had an inhibitory effect on MCP-03 catalytic efficiency and were essential for keeping MCP-03 thermostable.

Keywords

MCP-03 Subtilase Cold-adapted Halophilic Pseudoalteromonas sp. SM9913 PPC domain Deep sea 

Abbreviations

C-t

C-terminal

N-t

N-terminal

PPC domain

Bacterial pre-peptidase C-terminal domain

PKD domain

Polycystic kidney disease domain

FAAF

N-Succinyl-Phe-Ala-Ala-Phe-p-nitroanilide

AAPF

N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide

AAPR

N-Succinyl-Ala-Ala-Pro-Arg-p-nitroanilide

AAPL

N-Succinyl-Ala-Ala-Pro-Leu-p-nitroanilide

FVR

N-Benzoyl-Phe-Val-Arg-p-nitroanilide

AAPK

N-Succinyl-Ala-Ala-Pro-Lys-p-nitroanilide

AAA

N-Succinyl-Ala-Ala-Ala-p-nitroanilide

AAV

N-Succinyl-Ala-Ala-Val-p-nitroanilide

GGG

N-Succinyl-Gly-Gly-Gly-p-nitroanilide

GGF

N-Succinyl-Gly-Gly-Phe-p-nitroanilide

Supplementary material

792_2009_263_MOESM1_ESM.doc (422 kb)
Supplementary material 1 (DOC 422 kb)

Copyright information

© Springer 2009

Authors and Affiliations

  • Bing-Qiang Yan
    • 1
  • Xiu-Lan Chen
    • 1
  • Xiao-Yan Hou
    • 1
  • Hailun He
    • 1
  • Bai-Cheng Zhou
    • 1
  • Yu-Zhong Zhang
    • 1
  1. 1.The State Key Lab of Microbial Technology, Marine Biotechnology Research CenterShandong UniversityJinanPeople’s Republic of China

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