摘要
采用离交和凝胶层析对“中国冰川1号”耐冷菌BacilluscereusSYPA23所产的蛋白酶进行分离纯化,并进行酶学性质研究.与中温酶相比,该蛋白酶具有较高的低温催化活力,其最适催化温度为42℃,适宜pH为7.0～8.5,SDSPAGE测定的分子量(Mr)为34.2×103.金属离子Mn2+、Ca2+对该酶有激活作用,Cu2+、Hg2+、Pb2+、Co2+对其活性有一定抑制作用.该酶属金属蛋白酶,其活性受EDTA强烈抑制,不受PMSF抑制.该蛋白酶具有低温酶典型的热不稳定性,0℃下半衰期24h,但Ca2+和一些低分子醇类物质能提高其稳定性.动力学数据分析表明,该蛋白酶在低温下对底物亲和能力高,在较宽温度范围内(25～45℃)均保持着较高的催化效能.
A cold-adapted protease was purified and characterized from the culture supernatant of psychrotroph Bacillus cereus SYP-A2-3, which was isolated from Chinese No. 1 Glacier in Xinjiang, China. The procedural steps of protease purification ineluded uhrafihration, ammonium sulfate precipitation, ion-exchange chromatography （ DEAE Sephadex） and gel filtration（ Superdex G-75）. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） analysis of the purified protein indicated an estimated molecular mass （Mr） of 34.2 ×10^3, The protease had the characteristics of cold-adapted enzyme, i. e. , it was more active from 25 ℃to 35 ℃, and it was most at 42 ℃ with pH 7.0 - 8.5. Characterization of the protease showed that it required certain cations such as Mn^2＋ and Ca^2＋ for maximal activity and was partly inhibited by Cu^2＋, Hg^2＋. and Co^2＋ . The protease might belong to metalloprotease, for its activity was strongly inhibited by EDTA, but not by phenylmethylsulfonyl fluoride（PMSF）. The protease also showed more thermolability, for its half-life was only 24 h at 0 ℃, but Ca^2＋ and some organic solvents such as methanol, ethanol and isopropy could improve its stability. The kinetic data under different temperatures were determined, and the enzyme affinity and catalytic efficiency were discussed in detail. Fig 7, Tab 3, Ref 17
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2006年第1期72-75,共4页
Chinese Journal of Applied and Environmental Biology
基金
国家"863"资助项目(No.2001AA214101)~~
关键词
冷适微生物
金属蛋白酶
蜡状芽孢杆菌
分离纯化
cold-adapted microorganisim
metalloprotease
Bacillus cereus
purification
