产耐高温谷氨酰胺转胺酶菌株的快速筛选方法  

A Rapid Method of Screening for Thermostable Transglutaminase from Streptomyces mobaraensis

作　　者：郭玮婷[1] 张慧[1] 查东风 黄汉峰 黄静[1] 高红亮[1] 常忠义[1] 金明飞[1] 鲁伟 

机构地区：[1]华东师范大学生命科学学院,上海200241 [2]河南省商丘市农业局经作中心,商丘476000 [3]泰兴市东圣食品科技有限公司,泰兴225411

出　　处：《中国生物工程杂志》2015年第8期83-89,共7页China Biotechnology

基　　金：中央高校基本科研业务费专项资金资助项目(78210203)

摘　　要：目的：采用亚硝基胍（NTG）诱变结合96孔板高通量筛选方法筛选产耐高温谷氨酰胺转胺酶（MTG）的茂原链霉菌（Streptomyces mobaraensis）。方法：通过优化96孔板高通量测定MTG活性的方法、确定筛选温度和时间,建立了产耐高温MTG菌株的快速筛选方法;通过优化NTG诱变条件建立了筛选突变库;通过96孔板高通量初筛、摇瓶复筛获得了产耐高温MTG的突变株12-82,并通过摇瓶发酵对12-82所产MTG进行热稳定性分析。结果：采用2mg/ml NTG、p H8.0、60min的诱变条件获得突变株,将突变株的发酵上清液于70℃水浴7.5min,再在37℃空气浴、反应10min的条件下测定MTG活性,从5 200株突变株中筛选出5株产耐高温MTG的突变株,其中突变株12-82在50℃水浴60min以及70℃水浴1.5min的酶活残留率均比出发株高出近20%,且80℃保温2min仍有11.9%的酶活残留率。结论：利用NTG诱变结合96孔板高通量筛选的方法筛选到5株所产MTG热稳定性相对较高的突变株,其中突变株12-82在50℃、70℃和80℃的酶活残留率均有10%~20%的提高。这为高温食品加工领域所需耐高温MTG生产菌株的高效筛选提供了可行性方案。Objective： The aims are to obtain thermostable MTG from Streptomyces mobaraensis mutants library producing by nitrosoguanidine（ NTG） radom mutagenesis and high-throughput screening. Methods： A rapid method of screening for thermostable MTG from Streptomyces mobaraensis is established by optimization of high-throughput MTG activity assay,screening temperature and time,respectively. Streptomyces mobaraensis mutants library is obtained by optimization of the concentration of NTG. A mutant 12-82 with thermostable MTG was chosen by preliminary screening and second screening from the 5200 mutants library. Results： The spores from Streptomyces mobaraensis were treated by NTG（ 2mg / ml,p H8. 0） for 60 min, then the fermented supernatant was incubated at 70℃ for 7. 5 min after fermentation in 96 well microtiters. Finally,MTG activity was determined at 37 ℃ air bath for 10 min. 5 mutants with thermostable MTG were selected from 5200 mutants.When incubated at 50℃ for 60 min and 70℃ for 1. 5min,the relative activity of 12-82 was almost 20% higher than the parent strain. When incubated at 80℃ for 2min,the relative activity of 12-82 was 11. 9%. Conclusion：5 mutants with thermostable MTG were selected by the screening method of NTG random mutagenesis combined with high-throughput screening. Among them,the relative activity of the mutant 12-82 was 10% ~ 20% higher than that of the parent strain when incubated at 50℃,70℃ and 80℃,respectively. This established rapid method to obtain thermostable MTG is practical and the increased thermostability of MTG will be advantageous for its application in food processing at high temperature.

关 键 词：茂原链霉菌 谷氨酰胺转胺酶 热稳定性 NTG诱变 96孔板高通量筛选 

分 类 号：Q819[生物学—生物工程]