极地研究

极地研究 ›› 2011, Vol. 22 ›› Issue (1-English): 49-54.DOI: 10.3724/SP.J.1085.2011.00049

• 研究论文 • 上一篇    下一篇

Cold-adaptive alkaline protease from the psychrophilic Planomicrobium sp. 547: enzyme characterization and gene cloning

YANG XiangSheng1, CHEN XingLin2, XU XianZhong2 &  ZENG RunYing2*   

  1. 1 School of Life Sciences, Xiamen University, 361005 Xiamen, China;

    2 Third Institute of Oceanography, State Oceanic Administration, 361005 Xiamen, China

  • 收稿日期:2010-10-27 修回日期:2010-11-08 出版日期:1961-03-30 发布日期:1961-03-30
  • 通讯作者: ZENG RunYing

Cold-adaptive alkaline protease from the psychrophilic Planomicrobium sp. 547: enzyme characterization and gene cloning

YANG XiangSheng1, CHEN XingLin2, XU XianZhong2 &  ZENG RunYing2*   

  1. 1 School of Life Sciences, Xiamen University, 361005 Xiamen, China;

    2 Third Institute of Oceanography, State Oceanic Administration, 361005 Xiamen, China

  • Received:2010-10-27 Revised:2010-11-08 Online:1961-03-30 Published:1961-03-30
  • Contact: Run-Ying ZENG

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摘要：

A psychrophilic bacterium strain 547 producing cold-adaptive alkaline protease was isolated from the deep sea sediment of Prydz Bay, Antarctica. The organism was identified as a Planomicrobium species by 16S rRNA analysis. The optimal and highest growth temperatures for strain 547 were 15℃ and 30℃, respectively. The extracellular protease was purified by ammonium sulfate precipitation and DEAE cellulose-52 chromatography. The optimal temperature and pH for the activity of the purified enzyme were 35℃ and pH 9.0, respectively. The enzyme retained approximately 40% of its activity after 2 h of incubation at 50℃. The enzymatic activity was inhibited by 1 mmol/L phenylmethyl sulfonylfluoride (PMSF) and hydrochloride 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), indicating that it was a serine protease. The presence of Ca2+ and Mn2+ increased the activity of the enzyme. The protease gene with a size of 1 269 bp was cloned from Planomicrobium sp. 547 using primers designed based on the conserved sequences of proteases in GenBank. The Planomicrobium sp. 547 protease contained a domain belonging to the peptidase S8 family, which has a length of 309 amino acid (AA) residues. The alignment and phylogenetic analysis of the AA sequence indicated that the protease belonged to the subtilisin family.

关键词: cold-adaptive protease, Planomicrobium, Antarctic, subtilisin

Abstract:

A psychrophilic bacterium strain 547 producing cold-adaptive alkaline protease was isolated from the deep sea sediment of Prydz Bay, Antarctica. The organism was identified as a Planomicrobium species by 16S rRNA analysis. The optimal and highest growth temperatures for strain 547 were 15℃ and 30℃, respectively. The extracellular protease was purified by ammonium sulfate precipitation and DEAE cellulose-52 chromatography. The optimal temperature and pH for the activity of the purified enzyme were 35℃ and pH 9.0, respectively. The enzyme retained approximately 40% of its activity after 2 h of incubation at 50℃. The enzymatic activity was inhibited by 1 mmol/L phenylmethyl sulfonylfluoride (PMSF) and hydrochloride 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), indicating that it was a serine protease. The presence of Ca2+ and Mn2+ increased the activity of the enzyme. The protease gene with a size of 1 269 bp was cloned from Planomicrobium sp. 547 using primers designed based on the conserved sequences of proteases in GenBank. The Planomicrobium sp. 547 protease contained a domain belonging to the peptidase S8 family, which has a length of 309 amino acid (AA) residues. The alignment and phylogenetic analysis of the AA sequence indicated that the protease belonged to the subtilisin family.

Key words: cold-adaptive protease, Planomicrobium, Antarctic, subtilisin

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