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

• Main Authors: Xiangsheng, Yang, Xinglin, Chen, Xianzhong, Xu, Runying, Zheng
• Format: Article in Journal/Newspaper
• Language: English
• Published: Polar Research Institute of China - PRIC 2011
• Subjects: Fauna; Prydz Bay; Advances in Polar Science; Antarc*; Antarctica; Polar Science
• Online Access: http://library.arcticportal.org/2412/; http://library.arcticportal.org/2412/1/A20110107.pdf

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.