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.
YANG XiangShengCHEN XingLinXU XianZhongZENG RunYing
