高活性谷氨酰胺酶豆豉芽孢杆菌的筛选及glsA基因的克隆  

Screening and glsA Cloning of Do chi Bacillus with High Glutaminase Activity

作　　者：詹寿年[1] 吴拥军[1] 郭倩倩[1] 卢彪[1] 

机构地区：[1]贵州大学生命科学学院贵州省农业生物工程重点实验室,贵州贵阳550025

出　　处：《贵州农业科学》2011年第6期119-122,I0001,共5页Guizhou Agricultural Sciences

基　　金：贵阳市工业攻关项目"Red重组系统改良豆豉芽孢杆菌GA酶的关键技术研究及工业应用"[筑科工合同字(2010)第1-68号];贵阳市人才基金"应用Red重组系统改良豆豉芽孢杆菌glsA基因"[筑人才办合同字(2010)第23号];贵州大学人才引进项目"高活性谷氨酰胺酶基因(glsA)的克隆与表达"[贵大人基合字(2010)第022号]

摘　　要：为筛选获得高活性谷氨酰胺酶豆豉芽孢杆菌菌株,提高谷氨酸的含量,试验通过康维皿弥散法对525株芽孢杆菌进行酶活检测,获得一株产谷氨酰胺酶活力为68.407 mg NH3/(g曲.h.37℃)的B.subtilisGA317菌株,其酶活力较B.subtilisBJ3-2高5.59倍。对2个菌株谷氨酰胺酶编码基因glsA进行克隆测序,结果表明:2个菌株glsA基因核酸序列和蛋白序列同源性分别为63%和47%,表现出较大的差异。B.subtilisGA317与B.subtilisBJ3-2蛋白序列中都含有α3,α6,β8 3个酶活性位点,分别位于蛋白序列中67-80、117-125、260-267的3个区域,B.subtilisG317的α3区域有7个氨基酸发生改变,在α6和β8区域各有1个氨基酸发生改变。研究为进一步利用基因工程技术改良菌种产谷氨酰胺酶活力奠定了一定基础。Enzyme activities of 525 Bacillus strains were detected by Conway diffusion method to screen out Do chi Bacillus stain with high glutaminase activity and enhance glutamic acid content.The results showed that B.subtilis GA317,a Do chi Bacillus stain with glutaminase activity 68.407mg NH3/（g yeasy·h·37℃） which is 5.59 times higher than that of B.subtilis BJ3-2,was obtained.The glsA gene were cloned and sequenced with these two strains.According to sequence alignment on nucleic acid and protein,their homologies were 63% and 47% respectively,which showed great differences.Protein sequences of B.subtilis GA317 and B.subtilis BJ3-2 all contained three active sites of enzyme α3,α6 and β8.These active sites separately located in three regions ranged of 67-80,117-125 and 260-267 respectively.There were 7 amino acids changed in α3 region of B.subtilis G317,while there was only one amino acid changed in region α6 and β8.This research established foundation for further studies on improvement of glutamine activity by genetic engineering technology.

关 键 词：谷氨酰胺酶 酶活中心 枯草芽孢杆菌 glsA基因 

分 类 号：S182[农业科学—农业基础科学] Q939.97[生物学—微生物学]