产谷氨酰胺转胺酶菌株的高通量筛选  

Novel high-throughput screening of Streptoverticillium mobaraense with high microbial transglutaminase yield

作　　者：刘颖[1] 田沛霖[1] 陈佳[1] 高红亮[1] 鲁伟 鲁心安[1] 常忠义[1] 金明飞[1] 

机构地区：[1]华东师范大学生命科学学院,上海200241 [2]泰兴市东圣食品科技有限公司,江苏泰兴225441

出　　处：《西北农林科技大学学报（自然科学版）》2013年第6期167-172,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目"IL-17A调节REG3A促进伤口愈合的作用机理"(81072422)

摘　　要：【目的】用醋酸纤维素膜和96孔板2种方法高通量筛选高产谷氨酰胺转胺酶(MTG)的茂原轮枝链霉菌菌株。【方法】利用紫外诱变获得茂原轮枝链霉菌突变体库后,分别利用醋酸纤维素膜和96孔板2种方法高通量初筛产MTG菌株,然后依次用试管和摇瓶发酵法复筛,并对96孔板高通量筛选方法的条件进行了优化。【结果】利用醋酸纤维素膜显色法从3 000株紫外诱变的菌株中筛选出1株高产MTG茂原轮枝链霉菌,其MTG活性为4.9U/mL,较出发株(MTG活性2.7U/mL)提高了80%。优化的96孔板高通量筛选方法为:直径为3mm左右的单菌落接种至150μL发酵培养基中,30℃200r/min培养72h;利用该法从3 415株紫外诱变的菌株中筛选获得了2株MTG高产株,其MTG活性为5.4U/mL,较出发株(MTG活性为3.0U/mL)提高了80%。【结论】利用基于醋酸纤维素膜和96孔板的2种高通量筛选方法分别筛选到1株和2株高产MTG的茂原轮枝链霉菌;相较传统的选育方法,这2种高通量筛选方法都大大降低了经济成本,提高了筛选效率,缩短了选育周期。【Objective】 This study aimed to screen Streptoveticillium mobaraense mutants with high-yield of microbial transglutaminase（MTG）using cellulose acetate film（CAF）and 96-well microtiter plate based high-throughput screening（HTS）methods.【Method】 CAF and 96-well microtiter plate were applied in high-throughput primary screening of UV treated S.mobaraense.High-yield candidates were further confirmed by stout tube fermentation and shaking flask method.Conditions of 96-well microtiter plate cultivation were optimized.【Result】 1 strain with high-yield of MTG（MTG activity as 4.9 U/mL）was selected by CAF assay from a library of over 3 000 mutants with the enzyme activity improved by 80% compared to the parent strain（MTG activity as 2.7 U/mL）.Optimized conditions of 96-well microtiter plate fermentation were as follows：colonies with a diameter of 3 mm were inoculated to 150 μL/well fermentation medium before being cultivated at 30 ℃ 200 r/min for 72 h for enzyme assay.2 strains were screened from 3 415 mutants with MTG activities of 5.4 U/mL,80% higher than and starting strain with MTG activity of 3.0 U/mL.【Conclusion】 1 and 2 high-yield strains were selected using CAF and 96-well microtiter plate,respectively.Both methods developed in this research provided efficient HTS approaches at a low cost in short period.

关 键 词：茂原轮枝链霉菌 微生物谷氨酰胺转胺酶 高通量筛选 醋酸纤维素膜 96孔板 

分 类 号：Q939.97[生物学—微生物学]