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食品与发酵工业  2020, Vol. 46 Issue (2): 1-6    DOI: 10.13995/j.cnki.11-1802/ts.022306
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)来源耐热β-半乳糖苷酶BgaB转糖苷催化活性改造
董艺凝1, 陈卫2, 陈海琴2*, 赵建新2, 陈永泉2, 张灏2
1 (滁州学院 生物与食品工程学院,安徽 滁州,239000)
2 (江南大学 食品学院,食品科学与技术国家重点实验室,江苏 无锡,214122)
Enhances transglycosylation activity of thermostable β-galactosidase BgaB from Geobacillus stearothermophilus
DONG Yining1, CHEN Wei2, CHEN Haiqin2*, ZHAO Jianxin2, CHEN Yongquan2, ZHANG Hao2
1 (Faculty of Biological Science and Food Engineering, Chuzhou University, Chuzhou 239000, China)
2 (State Key Laboratoryof Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China)
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摘要 该研究以GH42家族嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)来源耐热β-半乳糖苷酶BgaB为研究对象,针对其转糖苷活性弱的问题,采用定点突变与化学修饰相结合的方法,对其预测亲核催化位点Glu303进行了功能研究与分子改造。所得突变体Ox-E303C与野生型酶相比,可将低聚半乳糖合成量由0%提高到11.5%。研究结果表明对耐热β-半乳糖苷酶BgaB亲核催化位点进行半胱氨酸亚磺酸(—SOO-)替换,能够提高其转糖苷催化活性。该研究对GH42家族β-半乳糖苷酶转糖苷催化功能的分子改造具有广泛的参考价值。
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关键词:  β-半乳糖苷酶  低聚半乳糖  转糖苷活性  嗜热脂肪芽孢杆菌  定点突变  化学修饰    
Abstract: Weak transglycosylation is a common problem with β-galactosidases from the glycoside hydrolase family 42 (GH42). The β-galactosidase BgaB from Geobacillus stearothermophilus, a typical thermostable enzyme of the GH42 family was investigated. Glu303 was predicted to be the catalytic nucleophile of BgaB. To improve the transglycoside activity of BgaB, functional studies and molecular modifications were carried out on the Glu303. Using site-directed mutagenesis and chemical modification to replace the carboxyl group of the Glu303 with a cysteine sulfinate (—SOO-), the Ox-E303C mutant was generated. Compared with the wild-type enzyme, the Ox-E303C mutant was found to increase galactooligosaccharides (GOS) synthesis from 0% to 11.5%. The result shows that introduction of a—SOO- group on to the Glu303 could improve transglycoside activity, and the catalytic nucleophile was involved in the transglycosylation regulation of BgaB. The results presented here have significant implications for the molecular modification of the transglycoside activity of GH42 β-galactosidases.
Key words:  β-galactosidases    galactooligosaccharides (GOS)    Geobacillus stearothermophilus    transglycoside activity    site-directed mutation    chemical modification
收稿日期:  2019-09-18                出版日期:  2020-01-25      发布日期:  2020-03-13      期的出版日期:  2020-01-25
基金资助: 国家自然科学基金项目(31301523;31171636);安徽省自然科学基金面上项目(1708085MC72);“十二五”国家“863”计划(2011AA100905);校级科技创新团队支持计划(00001702);滁州市第六批“221”产业创新团队项目(特色农产品开发与利用)
作者简介:  博士,副教授(陈海琴教授为通讯作者,。
董艺凝,陈卫,陈海琴,等. 嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)来源耐热β-半乳糖苷酶BgaB转糖苷催化活性改造[J]. 食品与发酵工业, 2020, 46(2): 1-6.
DONG Yining,CHEN Wei,CHEN Haiqin,et al. Enhances transglycosylation activity of thermostable β-galactosidase BgaB from Geobacillus stearothermophilus[J]. Food and Fermentation Industries, 2020, 46(2): 1-6.
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