南极海洋产耐有机溶剂变性剂氧化剂蛋白酶链霉菌菌株XE-1的初步鉴定及酶学性质

Characterization of solvent，detergent and oxidizing agent stable protease from isolated Antarctic marine Streptomyces sp.XE-1

摘要:

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14 kDa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1.9 mg/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14 kDa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。

Abstract:

Abstract:［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℃ ．Km 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.