D407A/D430A双位点突变对嗜热酸性生淀粉α-淀粉酶GTamy酶学性质的影响

曾静; 郭建军; 袁林

doi:10.13386/j.issn1002-0306.2018.14.019

D407A/D430A双位点突变对嗜热酸性生淀粉α-淀粉酶GTamy酶学性质的影响

江西省科学院微生物研究所, 江西南昌 330096

基金项目:

国家自然科学基金青年科学基金项目(31501422)

江西省青年科学基金项目(20171BAB214003)

江西省科学院项目(2018-JCQN-02、2018-YCXY-02)。

详细信息

作者简介:
曾静(1986-),女,博士,助理研究员,研究方向:极端嗜热酶的开发与应用,E-mail:zengjingwhu@126.com。

通讯作者:
袁林(1980-),男,博士,副研究员,研究方向:工业微生物的应用研究,E-mail:yuanlincn2003@aliyun.com。

中图分类号: TS201.2⁺5
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出版历程
- 收稿日期: 2017-11-23
- 网络出版日期: 2020-11-26
- 刊出日期: 2018-07-14

Effects of Double Point Mutation D407A/D430A on the Enzymatic Properties of Thermoacidiphilic Raw Starch Degrading α-amylase GTamy

Institute of Microbiology, Jiangxi Academy of Sciences, Nanchang 330096, China

摘要

摘要: 旨在研究位于嗜热酸性生淀粉α-淀粉酶GTamy的结构域A与生淀粉结合域RSBD间的Ca²⁺结合位点对其酶学性质的影响,构建了GTamy的Ca²⁺结合位点突变体GTamyD407A/D430A,并比较了GTamy与突变体GTamyD407A/D430A的酶学性质。结果表明,与GTamy相比,突变体的高温活性和热稳定性明显降低。突变体于80℃的比活力由1 756.75 U/mg降低至1 484.48 U/mg;80℃的半衰期由3 h降低至2.5 h。以可溶性淀粉为底物时,突变体的底物结合能力不变,反应速率为GTamy的79%;以玉米淀粉为底物时,突变体的底物吸附率为GTamy的67.9%,底物降解率为GTamy的59.3%。该Ca²⁺结合位点可能通过改变RSBD和催化活性中心的分子结构来影响GTamy的高温活性和热稳定性。本研究表明该Ca²⁺结合位点有利于GTamy维持高温活性和热稳定性。
- 嗜热酸性生淀粉α-淀粉酶 /
- 生淀粉结合域 /
- Ca²⁺结合位点 /
- 定点突变 /
- 酶学性质
Abstract: This work aims to investigate the effect of Ca²⁺-binding site which locates between the domain A and the raw starch binding domain of thermoacidiphilic raw starch degrading α-amylase GTamy on the enzymatic properties of GTamy. A Ca²⁺-binding site mutant GTamyD407A/D430A was constructed based on the strucutre analysis of GTamy. Results showed that:Compared to GTamy,the mutant exhibited significant decrease in terms of thermal activity and stability. The specific activity of the mutant at 80℃ decreased from 1 756.75 U/mg to 1 484.48 U/mg,and the half-life of the mutant at 80℃ decreased from 3 h to 2.5 h. The mutant showed similar substrate binding capacity but 79% of the catalytic efficiency of GTamy on the soluble starch. And the mutant exhibited 67.9% of the substrate binding rate and 59.3% of the hydrolysis rate of GTamy towards corn starch. The Ca²⁺-binding site might influence the thermal activity and stability of GTamy by changing the molecular structure of the raw starch binding domain and the catalytic active center. This result indicated that the Ca²⁺-binding site was important to the thermal activity and stability of GTamy.
- thermoacidiphilic raw starch degrading α-amylase /
- raw starch binding domain /
- Ca²⁺-binding site /
- site-directed mutagenesis /
- enzymatic properties