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Improving catalytic efficiency and maximum activity at low pH of Aspergillus neoniger phytase using rational design

文献类型： 外文期刊

作者： Zhou, Sijia 1 ; Liu, Zhemin 1 ; Xie, Wancui 2 ; Yu, Yuan 3 ; Ning, Chen 1 ; Yuan, Mingxue 1 ; Mou, Haijin 1 ;

作者机构： 1.Ocean Univ China, Coll Food Sci & Engn, Qingdao 266003, Shandong, Peoples R China

2.Qingdao Univ Sci & Technol, Coll Marine Sci & Biol Engn, Qingdao 266042, Shandong, Peoples R China

3.Chinese Acad Fishery Sci, Yellow Sea Fisheries Res Inst, Qingdao 266071, Shandong, Peoples R China

关键词： Phytase; Rational design; Surface charges; Flexibility

期刊名称：INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES （ 影响因子：6.953； 五年影响因子：6.737 ）

ISSN： 0141-8130

年卷期： 2019 年 131 卷

页码：

收录情况： SCI

摘要： A preferable phytase for use in animal feeds for industrial applications should have high, optimal activity at low pH in the monogastric gut environment and high thermostability. To obtain enzymes with enhanced catalytic efficiency (pH 5.5) and excellent activity in acidic pH range, we performed structure-based rational design of a thermostable phytase (PhyAn). For this, six mutants based on different rational design strategies were constructed and heterologously expressed in Pichia pastoris. Particularly, the extracellular enzymatic activity was assessed to ensure that the produced enzymes met requirements of further analyses. Several positive mutants with enhanced catalytic efficiency or pH-profile shifts were carefully examined. Biochemical and kinetic investigations of purified mutants revealed that E79K, E80K, E79K + E80K and D68K had higher catalytic efficiency than the parent enzyme by approximately 49%, 67%, 86% and 15%, respectively. Moreover, the optimum pH of mutant Y65H was shifted from 5.0 to 3.0, and the peak of D68K shifted to pH 5.5. Analysis of the structural-functional relationships revealed that changes in amino acid charges, structural flexibility and space hindrance could significantly influence certain enzyme characteristics. Our results illustrate the feasibility and present a structural foundation for enhancing the phytase-catalytic efficiency and acid resistance by assembling mutations derived using rational design. (C) 2019 Published by Elsevier B.V.

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