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.
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.
Yan
Bing-Qiang
BQ
Marine Biotechnology Research Center, Shandong University, Jinan, People's Republic of China.
Chen
Xiu-Lan
XL
Hou
Xiao-Yan
XY
He
Hailun
H
Zhou
Bai-Cheng
BC
Zhang
Yu-Zhong
YZ
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D005285:Fermentation; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D017434:Protein Structure, Tertiary; D031161:Pseudoalteromonas / Q000201:enzymology* / Q000235:genetics*; D012623:Seawater; D020860:Subtilisin / Q000235:genetics*
D020860:Subtilisin