一株产蛋白酶嗜碱菌株的分离、鉴定及酶学特性  

Isolation,identification and enzyme profiling of an alkaliphilic protease producing bacterium

作　　者：郝建国[1,2] 薛燕芬[1] 马延和[1] 

机构地区：[1]中国科学院微生物研究所,北京100101 [2]中国科学院研究生院,北京100049

出　　处：《微生物学报》2010年第1期54-59,共6页Acta Microbiologica Sinica

基　　金：国家"863计划"(2007AA021306; 2006AA020202)~~

摘　　要：【目的】筛选产蛋白酶嗜碱菌并对其进行鉴定和特性分析。【方法】利用碱性脱脂牛奶培养基分离纯化产蛋白酶嗜碱菌,通过形态特征、生理生化、16S rRNA基因序列分析以及DNA-DNA杂交实验确定菌株的分类地位,利用酪蛋白水解法分析所产蛋白酶的pH和温度作用范围、稳定性和耐氧化剂能力。【结果】从我国西藏盐碱湖样品中分离到一株产碱性蛋白酶的菌株ZL223,该菌株为革兰氏阳性菌,最适生长温度为37℃,最适生长pH9.0,16S rRNA基因序列分析显示,菌株ZL223与假强芽孢杆菌Bacillus pseudo firmus OF4亲缘关系最近,16S rRNA基因序列相似性为98.6%,DNA-DNA杂交结果显示与B.pseudofirmus OF4同源性为86%。菌株ZL223产生的蛋白酶作用的最适pH为12.0,最适温度为40℃。【结论】结合生理生化指标测定的结果,鉴定菌株为假强芽孢杆菌ZL223(B.pseudofirmus ZL223)。该菌株产生的碱性蛋白酶具有较高的pH适应性,值得进一步研究。[ Objective ] To isolate and characterize alkaliphilic bacteria with protease activity. [ Methods] Proteaseproducing alkaliphiles were isolated by skim milk agar method. The morphological, biochemical and physiological characteristics, 16S rRNA gene sequence and DNA-DNA hybridization were determined to identify the taxonomic position of strain ZL223. Meanwhile, the enzyme characterization of the protease including optimal temperature and pH range, stability and oxidation resistance were tested by using casein hydrolysis test. [ Results] A new strain named ZL223 was isolated from Bange Lake in Tibet, China. Cells were Gram-positive, spore-forming, motile rods. Growth pH range was from 7.0 to 10. 0, optimum at pH 9.0. Growth temperature range was from 15℃ to 45℃ , optimum at 37℃. The G ＋ C content of DNA was 40.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain belonged to the genus Bacillus and most closely related to Bacillus pseudofirmus OF4 with 98.6 % sequence similarity. DNA-DNA hybridization analysis confirmed the close relationship of strain ZL223 with Bacillus pseudofirmu OF4 （86% relatedness）. Strain ZL223 produced extracellular alkaline protease and its maximal enzyme activity was observed at 40℃ and pH 12.0. [ Conclusion] Base on polyphasic taxonomy, strain ZL223 was a new member of Bacillus pseudofirmus. It produced alkaline protease which was worth further research.

关 键 词：嗜碱微生物 假强芽孢杆菌 16S RRNA基因 DNA-DNA杂交 

分 类 号：Q936[生物学—微生物学]