原生质体融合提高产谷氨酰胺转氨酶菌株产量

doi:10.7506/spkx1002-6630-201603027

食品科学

原生质体融合提高产谷氨酰胺转氨酶菌株产量

侯孝仑1，刘雅清1，郭玮婷1，高红亮1，常忠义1，步国建2，鲁伟2，解秀娟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，江苏泰兴 225411

出版日期:2016-02-15 发布日期:2016-02-26

Strain Improvement by Protoplast Fusion for Enhanced Transglutaminase Production

HOU Xiaolun1, LIU Yaqing1, GUO Weiting1, GAO Hongliang1, CHANG Zhongyi1, BU Guojian2, LU Wei2, XIE Xiujuan2, JIN Mingfei1,*

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

Online:2016-02-15 Published:2016-02-26

摘要/Abstract

摘要：

目的：研究并建立产谷氨酰胺转氨酶茂原链霉菌原生质体制备技术，通过原生质体融合技术筛选高产菌株。方法：以溶菌酶处理茂原链霉菌获得原生质体，采用原生质体融合技术，通过96 孔板高通量初筛、试管复筛、摇瓶验证选育高产菌株。结果：茂原链霉菌形成的原生质体再生出的菌落直径比较小，而菌丝生成的菌落直径比较大，两种形态的菌落96 孔板发酵后酶活力有显著性差异。不同的茂原链霉菌株制备原生质体，酶解程度、原生质体纯度、再生率有很大的差异，再生率最高可达1 804.25%，最低仅为12.76%。原生质融合后的高产菌株，选取96 孔板初筛酶活力前3%的菌株进行试管发酵，得到高产菌株比例为32.2%，酶活力比亲本高22.4%，经摇瓶验证产酶比对照提高16.28%。结论：利用基因组重排技术，可以初步对茂原链霉菌进行高产菌株选育。

关键词: 茂原链霉菌, 原生质体, 再生, 融合, 基因组重排

Abstract:

Objective: To obtain a high-yield transglutaminase (TGase)-producing strain through protoplast fusion between
mutant strains of Streptomyces mobaraensis. Methods: Protoplasts were obtained by lysozyme digestion, and after protoplast
fusion, the selected fusants were screened for TGase production by primary high-throughput screening in 96-well plates and
fermentation in test tubes and shake flasks, respectively. Results: The colonies regenerated from protoplasts had a smaller
diameter, and the diameter of colonies formed by mycelia was larger. The two different colonies had a significant difference
in TGase activity during fermentation in 96-well microplates. Different mutant strains of Streptomyces mobaraensis were
very different with respect to TGase activity, protoplast purity and protoplast regeneration rate. The maximum protoplast
regeneration rate was 1 804.25%, and the minimum was only 12.76%. After genome shuffling, we selected the top 3% of
fusant strains with high TGase activity through high-throughput screening in 96-well plates to carry out fermentation in test
tubes. Of these strains, 32.2% were high-yield strains having a 22.4% higher TGase activity than the parental strain. The
production was increased by 16.28% in shake flask fermentation. Conclusion: Genome shuffling can be used to improve
Streptomyces mobaraensis for enhanced TGase production.

Key words: Streptomyces mobaraensis, protoplasts, regeneration, fusion, genome shuffling

中图分类号:

Q939.97

侯孝仑，刘雅清，郭玮婷，高红亮，常忠义，步国建，鲁伟，解秀娟，金明飞. 原生质体融合提高产谷氨酰胺转氨酶菌株产量[J]. 食品科学, doi: 10.7506/spkx1002-6630-201603027.

HOU Xiaolun, LIU Yaqing, GUO Weiting, GAO Hongliang, CHANG Zhongyi, BU Guojian, LU Wei, XIE Xiujuan, JIN Mingfei. Strain Improvement by Protoplast Fusion for Enhanced Transglutaminase Production[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201603027.

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原生质体融合提高产谷氨酰胺转氨酶菌株产量

Strain Improvement by Protoplast Fusion for Enhanced Transglutaminase Production

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