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 |
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Authors: | Bing-Qiang Yan Xiu-Lan Chen Xiao-Yan Hou Hailun He Bai-Cheng Zhou Yu-Zhong Zhang |
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Institution: | (1) The State Key Lab of Microbial Technology, Marine Biotechnology Research Center, Shandong University, 250100 Jinan, People’s Republic of China |
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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.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
X.-L. Chen and B.-Q. Yan contributed equally to this work. |
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Keywords: | MCP-03 Subtilase Cold-adapted Halophilic Pseudoalteromonas sp SM9913 PPC domain Deep sea |
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