Purification and Characterization of an Alkaline Protease from Micrococcus sp.Isolated from the South China Sea  

作　　者：HOU Enling XIA Tao ZHANG Zhaohui MAO Xiangzhao 

机构地区：[1]College of Food Science and Engineering,Ocean University of China,Qingdao 266003,P.R.China

出　　处：《Journal of Ocean University of China》2017年第2期319-325,共7页中国海洋大学学报（英文版）

基　　金：supported by the Fundamental Research Funds for the Central Universities(No.201564018);Qingdao Shinan District Science and Technology Development Funds(No.2014-14-002-SW);Major Special Science and Technology Projects in Shandong Province(No.2015ZDZX05003);the National Natural Science Foundation of China-Shandong Joint Fund for Marine Science Research Centers(No.U1406402)

摘　　要：Protease is wildly used in various fields,such as food,medicine,washing,leather,cosmetics and other industrial fields.In this study,an alkaline protease secreted by Micrococcus NH54PC02 isolated from the South China Sea was purified and characterized.The growth curve and enzyme activity curve indicated that the cell reached a maximum concentration at the 30 th hour and the enzyme activity reached the maximum value at the 36 th hour.The protease was purified with 3 steps involving ammonium sulfate precipitation,ion-exchange chromatography and hydrophobic chromatography with 8.22-fold increase in specific activity and 23.68% increase in the recovery.The molecular mass of the protease was estimated to be 25 k Da by SDS-PAGE analysis.The optimum temperature and p H for the protease activity were 50℃ and pH 10.0,respectively.The protease showed a strong stability in a wide range of pH values ranging from 6.0–11.0,and maintained 90% enzyme activity in strong alkaline environment with p H 11.0.Inhibitor trials indicated that the protease might be serine protease.But it also possessed the characteristic of metalloprotease as it could be strongly inhibited by EDTA and strongly stimulated by Mn^(2+).Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS(MALDI-TOF-TOF/MS) showed that the protease might belong to the peptidase S8 family.Protease is wildly used in various fields, such as food, medicine, washing, leather, cosmetics and other industrial fields. In this study, an alkaline protease secreted by Micrococcus NH54PC02 isolated from the South China Sea was purified and characterized. The growth curve and enzyme activity curve indicated that the cell reached a maximum concentration at the 30th hour and the enzyme activity reached the maximum value at the 36th hour. The protease was purified with 3 steps involving ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic chromatography with 8.22-fold increase in specific activity and 23.68% increase in the recovery. The molecular mass of the protease was estimated to be 25 kDa by SDS-PAGE analysis. The optimum temperature and pH for the protease activity were 50°C and pH 10.0, respectively. The protease showed a strong stability in a wide range of pH values ranging from 6.0-11.0, and maintained 90% enzyme activity in strong alkaline environment with pH 11.0. Inhibitor trials indicated that the protease might be serine protease. But it also possessed the characteristic of metalloprotease as it could be strongly inhibited by EDTA and strongly stimulated by Mn²⁺. Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) showed that the protease might belong to the peptidase S8 family.

关 键 词：PURIFICATION characterization PROTEASE MICROCOCCUS sp. the SOUTH China Sea 

分 类 号：Q814.1[生物学—生物工程]