产耐高温谷氨酰胺转胺酶菌株的快速筛选方法

doi:10.13523/j.cb.20150812

中国生物工程杂志

2015, Vol. 35

Issue (8): 83-89 DOI: 10.13523/j.cb.20150812

技术与方法

产耐高温谷氨酰胺转胺酶菌株的快速筛选方法

郭玮婷¹, 张慧¹, 查东风², 黄汉峰², 黄静¹, 高红亮¹, 常忠义¹, 金明飞¹, 鲁伟³

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

A Rapid Method of Screening for Thermostable Transglutaminase from Streptomyces mobaraensis

GUO Wei-ting¹, ZHANG Hui¹, ZHA Dong-feng², HUANG Han-feng², HUANG Jing¹, GAO Hong-liang¹, CHANG Zhong-yi¹, JIN Ming-fei¹, LU Wei³

1. School of Life Science, East China Normal University, Shanghai 200241, China;
2. Economic Crops Center Henan Shangqiu Agriculture Bureau, Shangqiu 476000, China;
3. Taixing Dongsheng Food and Technology Limited Company, Taixing 225411, China

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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、pH8.0、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。

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

Abstract:

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, pH8.0) 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 60min 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.

Key words: Streptomyces mobaraensis Transglutaminase Thermostablility NTG mutagenesis High-throughput screening in 96 well microtiters

收稿日期: 2015-04-29 出版日期: 2015-08-25

ZTFLH:

Q819

基金资助:

中央高校基本科研业务费专项资金资助项目(78210203)

通讯作者: 金明飞 E-mail: mfjin@bio.ecnu.edu.cn

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引用本文:

郭玮婷, 张慧, 查东风, 黄汉峰, 黄静, 高红亮, 常忠义, 金明飞, 鲁伟. 产耐高温谷氨酰胺转胺酶菌株的快速筛选方法[J]. 中国生物工程杂志, 2015, 35(8): 83-89.

GUO Wei-ting, ZHANG Hui, ZHA Dong-feng, HUANG Han-feng, HUANG Jing, GAO Hong-liang, CHANG Zhong-yi, JIN Ming-fei, LU Wei . A Rapid Method of Screening for Thermostable Transglutaminase from Streptomyces mobaraensis. China Biotechnology, 2015, 35(8): 83-89.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150812 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I8/83

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