耐热β-半乳糖苷酶(Pyrococcus furiosus DSM 3638)活性位点N415S点突变对其酶活性的影响

doi:10.6040/j.issn.1671-9352.0.2016.399

山东大学学报（理学版） ›› 2016, Vol. 51 ›› Issue (11): 74-78.doi: 10.6040/j.issn.1671-9352.0.2016.399

耐热β-半乳糖苷酶(Pyrococcus furiosus DSM 3638)活性位点N415S点突变对其酶活性的影响

尹蕾^1,2,王原媛³,辛志豪⁴,王向东^1*

1.山东大学医学院细胞生物学研究所, 山东济南 250012;2.潍坊生物园发展有限公司, 山东潍坊 261205;3.山东东兴汇智有限公司, 山东潍坊 261205;4.潍坊睿真生物科技有限公司, 山东潍坊 261205

收稿日期:2016-08-24 出版日期:2016-11-20 发布日期:2016-11-22
通讯作者: 王向东(1962— ),男,教授,研究方向为蛋白、酶与代谢. E-mail:xdongw@sdu.edu.cn E-mail:yinleijnu@163.com
作者简介:尹蕾(1986— ),女,硕士研究生,研究方向为蛋白、酶与代谢. E-mail:yinleijnu@163.com

The effect of point mutation N415S in Pyrococcus furiosus DSM 3638 (a thermo-stable β-galactosidase)on its enzymatic activities

YIN Lei^1,2, WANG Yuan-yuan³, XIN Zhi-hao⁴, WANG Xiang-dong^1*

1. Department of Cell Biology, Shandong University School of Medicine, Jinan 250012, Shandong, China;
2. Biomedical Science and Technology Industrial Park of Weifang Hi-tech Zone, Weifang 261205, Shandong, China;
3. Shandong Dongxinghuizhi Biotech Ltd. Weifang 261205, Shandong, China;
4. Weifang Ruizhen Biotech Ltd. Weifang 261205, Shandong, China

Received:2016-08-24 Online:2016-11-20 Published:2016-11-22

摘要/Abstract

摘要： β-半乳糖苷酶具有将乳糖分解为半乳糖和葡萄糖的能力,也具有把半乳糖聚合成低聚半乳糖的能力。这两种能力在工业生产中具有不同的意义。通过点突变使β-galactosidase［Pyrococcus furiosus DSM 3638］415位天冬酰胺(Asn or N)突变为丝氨酸(Ser or S),突变体构建于毕赤酵母表达质粒载体,并筛选以毕赤酵母为宿主细胞的工程菌种,以此制备β-半乳糖苷酶的突变体酶蛋白。研究发现,该突变体的β-半乳糖苷酶活性在95 ℃、pH5.5时达到最高酶活,为耐高温乳糖酶,且该突变体水解牛奶的能力较好,可用于低乳糖牛奶及其相关制品加工中。而突变后β-半乳糖苷酶的另一个活性,即产生低聚半乳糖的能力的活性也有所提高,但不够明显。因此该位点突变可用于乳制品的加工,但不能期盼产生更多的半乳糖寡聚体。

关键词: 酶活力, 点突变, 低聚半乳糖, β-半乳糖苷酶

Abstract: β-galactosidase has both activities to hydrolyze lactose into galactose /glucose and to polymerize a couple of galactoses into oligo-galactose. These two β-galactosidase activities have application potential in manufactory industries. By exploiting site mutation technique, a mutant N415S(Asn was replaced by Ser)of thermo-stable β-galactosidase(Pyrococcus furiosus DSM 3638)was obtained. The mutant cDNA was cloned into yeast expression vector, and a yeast expression cell strain was selected to express the mutant enzyme protein. By fermentation, we obtained a product of this mutant enzyme. We further characterized the mutant enzyme, and found that the optimum pH is 5.5, the optimum temperature is 95 ℃. The enzyme activity of this mutant to hydrolyze lactose is much better than that of the wild type(WT); while the enzyme activity to polymerize galactose into oligomer is better that that of the wild type, but not so good as expected for industry application. This mutant will be useful for milk treatment to largely reduce the content of lactose.

Key words: β-galactosidase, galactooligosaccharides, point mutation, enzyme activity

中图分类号:

Q939.97

尹蕾,王原媛,辛志豪,王向东. 耐热β-半乳糖苷酶(Pyrococcus furiosus DSM 3638)活性位点N415S点突变对其酶活性的影响[J]. 山东大学学报（理学版）, 2016, 51(11): 74-78.

YIN Lei, WANG Yuan-yuan, XIN Zhi-hao, WANG Xiang-dong. The effect of point mutation N415S in Pyrococcus furiosus DSM 3638 (a thermo-stable β-galactosidase)on its enzymatic activities[J]. JOURNAL OF SHANDONG UNIVERSITY(NATURAL SCIENCE), 2016, 51(11): 74-78.

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耐热β-半乳糖苷酶(Pyrococcus furiosus DSM 3638)活性位点N415S点突变对其酶活性的影响

The effect of point mutation N415S in Pyrococcus furiosus DSM 3638 (a thermo-stable β-galactosidase)on its enzymatic activities

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