基因组重排筛选高产谷氨酰胺转胺酶菌株

doi:10.13523/j.cb.20170914

中国生物工程杂志

2017, Vol. 37

Issue (9): 105-111 DOI: 10.13523/j.cb.20170914

技术与方法

基因组重排筛选高产谷氨酰胺转胺酶菌株

宋佳雯¹, 田苏¹, 张玉如², 王志珍¹, 常忠义¹, 高红亮¹, 步国建², 金明飞¹

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泰兴 225411

Genome Shuffling Enhances Transglutaminase Production of Streptomyces mobaraensis

SONG Jia-wen¹, TIAN Su¹, ZHANG Yu-ru², WANG Zhi-zhen¹, CHANG Zhong-yi¹, GAO Hong-liang¹, BU Guo-jian², JIN Ming-fei¹

1. School of Life Science, East China Normal University, Shanghai 200241, China;
2. Taixing Dongsheng Food Co. Ltd, Taixing 225411, China

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摘要： 谷氨酰胺转胺酶（TGase）的产量不足的问题一直限制其工业化生产规模，故采用基因组重排的方法，筛选高产谷氨酰胺转胺酶菌株。通过对不同制备条件下原生质体纯度和形成率的考量，获得制备原生质体的最优条件为以6mg/ml的溶菌酶浓度进行酶解，酶解时间2h。再优化融合条件，以2min紫外灭活和40min热灭活结合的方法挑选出融合子。通过两轮基因组重排，经过96孔板发酵高通量筛选和摇瓶发酵复筛验证，获得了一株产酶达7.12U/ml的茂源链霉菌，相比最初选用菌株的平均酶活提高65.5%。发酵结果显示，酶活提高的原因可能是在重组后原酶成熟更快、更彻底，且得到的菌株遗传稳定性良好。证明基因组重排能够有效提高菌株的产酶水平，同时为谷氨酰胺转胺酶产量提高提供理论依据。

关键词： 微生物谷氨酰胺转胺酶(TGase); 茂源链霉菌; 高通量筛选; 基因组重排

Abstract: Transglutaminase can catalyze Trans-phosphatidylation reaction, and is widely used in food industry. But the production of transglutaminase cannot meet the requirement of industrial production. Nowadays, random mutagenesis is the most frequent but inefficiency method to improve transglutaminase production. Transglutaminase production of Streptomyces mobaraensis is enhanced by genome shuffling. With 96-well plates high-throughput screening, the time and work are reduced. Firstly, protoplasts presentation conditions are optimized. The optimization conditions are treating mycelium by 6mg/ml lysozyme solution for 2 hours. Protoplasts are inactivated by UV illumination for 2 minutes and water-bath in 60℃ for 40min. Then fusants are picked up regenerating in regeneration medium and then screened by 96-well plates high-throughput screening and verified the high-production strains by fermentation in shaking flask. After two rounds of genome shuffling, a strain can produce TGase 65.5% higher than incipient strain is obtained and the production reaches 7.12U/ml.

Key words: Transglutaminase Streptomyces mobaraensis Genome shuffling High-throughput screening

收稿日期: 2017-02-28 出版日期: 2017-09-25

ZTFLH:

Q819

基金资助: 中央高校基本科研业务费专项基金资助项目（78210203）

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

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引用本文:

宋佳雯, 田苏, 张玉如, 王志珍, 常忠义, 高红亮, 步国建, 金明飞. 基因组重排筛选高产谷氨酰胺转胺酶菌株[J]. 中国生物工程杂志, 2017, 37(9): 105-111.

SONG Jia-wen, TIAN Su, ZHANG Yu-ru, WANG Zhi-zhen, CHANG Zhong-yi, GAO Hong-liang, BU Guo-jian, JIN Ming-fei. Genome Shuffling Enhances Transglutaminase Production of Streptomyces mobaraensis. China Biotechnology, 2017, 37(9): 105-111.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170914 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I9/105

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