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.
YANG XiangShengCHEN XingLinXU XianZhongZENG RunYing
