一株产甘露聚糖酶菌株的分离鉴定及酶的纯化与性质  

Purification and characterization of mannanase from an alkaliphilic mannanase producing bacterium HMTS15

作　　者：廖婷婷[1,2] 翟磊[1,2] 高成华[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》2011年第11期1520-1526,共7页Acta Microbiologica Sinica

基　　金：中国科学院创新项目(KSCXZ-EW-G-3)~~

摘　　要：【目的】筛选性能良好的产碱性甘露聚糖酶的菌株,对菌株进行多项分类鉴定,分离纯化所产甘露聚糖酶并进行性质研究。【方法】利用碱性魔芋粉培养基分离纯化产甘露聚糖酶的嗜碱菌,通过形态特征观察、生理生化测定、16S rRNA序列分析等实验确定菌株的分类地位。利用硫酸铵沉淀、阴离子交换层析和分子筛层析得到电泳纯的酶,分析了酶的最适温度、最适pH、温度和pH稳定性、NaCl以及金属离子等的耐受性。【结果】从我国内蒙古碱湖样品中分离得到一株产碱性甘露聚糖酶的菌株HMTS15,经过多项分类鉴定显示其是与Bacillus agaradhaerens DSM 8721不同的新菌株。菌株HMTS15所产的甘露聚糖酶反应的最适pH为10.0,最适温度75℃。【结论】多项分类结果鉴定菌株为Bacillus agaradhaerens HMTS15。该菌株产生的碱性甘露聚糖酶与同类其他来源的酶相比具有更好的热稳定性和pH适应性,有进一步的研究价值。[Objective] To isolate and identify an alkaliphilic mannanase producing bacterium,purify and characterize mannanase thereof.[Methods] Mannanase-producing alkaliphilic bacterium HMTS15 was isolated by alkaline agar with konjak from water sample of Hamatai Lake in Inner Mongolia,China.The morphological,biochemical and physiological characteristics and 16S rRNA gene were analyzed to identify the taxonomic position of strain HMTS15.Mannanase produced by strain HMTS15 was purified by four steps including（NH4）2SO4 precipitation,cellulose DEAE-sepharose,twice Superdex 200.The enzyme properties including optimal temperature,optimal pH,thermal stability,pH stability,NaCl tolerance,metal ion tolerance,EDTA and SDS tolerance were tested.[Results] Strain HMTS15 was Gram-positive rod.Its growth pH ranged from 7.0 to 11.0 and growth temperature ranged from 10 ℃ to 45 ℃.The G＋C content of the DNA was 40 mol%.Phylogenetic analvses based on 16S rRNA gene sequence comparisons indicated that strain HMTS15 was a member of Bacillus.The extracellular mannanase from strain HMTS15 was purified as a single band with molecular weight of about 45 kD on SDS-PAGE.The optimal catalytic activity was showed at 75 ℃ and pH 10.The mananase was stable up to 60 ℃ and retained about 60% residual activity at 65 ℃ for 30 min.The ions Fe2＋,Mn2＋,Co2＋,Zn2＋,Ag＋,Hg2＋ and EDTA inhibited the acitivity of the mannanase.[Conclusion] Polyphasic taxonomy revealed that strain HTMS15 was a new member of Bacillus agaradhaerens.The alkaline mannanase produced by strain HMTS15 hold the valuable property in stability at high temperature and broad range of pH.

关 键 词：嗜碱芽胞杆菌 碱性甘露聚糖酶 多项分类 纯化 性质 

分 类 号：TQ925[轻工技术与工程—发酵工程]