摘要
【目的】从南极海泥土壤中分离得到一株高产蛋白酶的放线菌(XE-1),对所产蛋白酶进行纯化以得到具有特殊性质的酶产物。【方法】通过对XE-1的生理生化和16S rRNA基因序列分析,初步将其确定为灰红链霉菌的变种。利用乙醇沉淀、离子交换、凝胶等方法纯化后,利用冻干浓缩获得其胞外蛋白酶,用SDSPAGE测定分子量并进行性质表征。【结果】得到一种对高浓度的有机溶剂、变性剂、氧化剂耐受的,表观分子量为14kDa的碱性丝氨酸蛋白酶,进一步分析,其最适pH 8.2,温度为55℃,米氏常数Km和Vmax分别为1.9mg/mL和973 u/mL。此蛋白酶活性于25%的有机溶剂为50%-180%的初始活性;在不同浓度的氧化剂中为110%-140%的初始活性,在0.2 mol/L到4 mol/L的变性剂中表现为100%-150%的初始活性。【结论】从南极海洋链霉菌XE-1中分离得到一种耐有机溶剂、变性剂、氧化剂,大小约为14kDa碱性蛋白酶,其特殊的酶学性质可用于工业生产或药学等领域。
[ Objective] A polar marine actinobacterium （XE-1） was selected and used to produce a protease with special characteristics. [ Methods] The XE-1 was identified as Streptomyces based on morphological, biochemical and molecular characterizations （ 16S rRNA gene sequence analysis）. The protease was purified by 3 purification steps, including ethanol precipitation, ion exchange and gel chromatography. Its apparent molecular mass was estimated by SDS-PAGE. [ Results] A solvent, detergent and oxidizing agent stable alkaline serine protease （with a low weight molecular, 14 kDa by SDS-PAGE）, secreted by strain XE-1, was purified and characterized. The protease was stable in the pH range between 5 and 10, with optimal pH 8.2 and optimal temperature 55℃. K and Vmax towards casein activity were 1.9 mg/mL and 973 U/mL, respectively. The protease was more active and stable in various hydrophilic organic solvents （such as dimethylformamid and toluene）. Moreover, it was also active and stable in bleaching agents （such as hydrogen peroxide）; and stable in denaturant agents （such as urea and guanidine hydrochloride） at the concentration from 0. 2 mol/L to 4 mol/L, which were the new characteristics. [ Conclusion] These biochemical characteristics suggest this enzyme has the potential value in numerous industrial applications.
出处
《微生物学报》
CAS
CSCD
北大核心
2015年第3期311-320,共10页
Acta Microbiologica Sinica
基金
Supported by the Fundamental Research Funds for the Central Universities(JKZD2013017)~~
关键词
极地海洋放线菌
低分子量蛋白酶
有机溶剂耐受
变性剂和氧化剂
polar marine actinobacterium, low-molecular weight protease, solvent tolerant, detergent and oxidizing agent
