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~C and 30~C, 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~C 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 Cae＋ and Mnz＋ 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.
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~C and 30~C, 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~C 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 Cae＋ and Mnz＋ 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.
基金
supported by the National High Technology Research and Development Program of China (Grant no. 2007AA091407)
the Project of China Ocean Mineral Resources Research and Development Association (COMRA, Grant no. DYXM-115-02-2-04)
