Improved thermostability and enzyme activity of a recombinant phyA mutant phytase from Aspergillus niger N25 by directed evolution and site-directed mutagenesis.

Abstract:

:We previously constructed three recombinant phyA mutant strains (PP-NPm-8, PP-NPep-6A and I44E/T252R-PhyA), showing improved catalytic efficiency or thermostability of Aspergillus niger N25 phytase, by error-prone PCR or site-directed mutagenesis. In this study, directed evolution and site-directed mutagenesis were further applied to improve the modified phytase properties. After one-round error-prone PCR for phytase gene of PP-NPep-6A, a single transformant, T195L/Q368E/F376Y, was obtained with the significant improvements in catalytic efficiency and thermostability. The phytase gene of T195L/Q368E/F376Y, combined with the previous mutant phytase genes of PP-NPep-6A, PP-NPm-8 and I44E/T252R-PhyA, was then sequentially modified by DNA shuffling. Three genetically engineered strains with desirable properties were then obtained, namedQ172R, Q172R/K432R andQ368E/K432R. Among them, Q172R/K432R showed the highest thermostability with the longest half-life and the greatest remaining phytase activity after heat treatment, while Q368E/K432R showed the highest catalytic activity. Five substitutions (Q172R, T195L, Q368E, F376Y, K432R) identified from random mutagenesis were added sequentially to the phytase gene of PP-NPep-6A to investigate how the mutant sites influence the properties of phytase. Characterization and structural analysis demonstrated that these mutations could produce cumulative or synergistic improvements in thermostability or catalytic efficiency of phytase.

journal_name

Enzyme Microb Technol

authors

Tang Z,Jin W,Sun R,Liao Y,Zhen T,Chen H,Wu Q,Gou L,Li C

doi

10.1016/j.enzmictec.2017.09.010

subject

Has Abstract

pub_date

2018-01-01 00:00:00

pages

74-81

eissn

0141-0229

issn

1879-0909

pii

S0141-0229(17)30183-7

journal_volume

108

pub_type

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