Increase in the Thermal Stability of Phytase from Citrobacter freundii by Site-Directed Saturation Mutagenesis


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Abstract

Phytases are widely used to improve the properties of mixed fodder. Due to its high specific activity, PhyА-Cf phytase from Citrobacter freundii is of special interest for agrobiotechnology. In order to decrease the thermal sensitivity of the enzyme during fodder granulation at 60–80°C, the thermal stability of phytase was enhanced by site-directed saturation mutagenesis. The mutant genes were cloned in a pP10 vector under the control of the GAP promoter and expressed in Pichia pastoris yeast cells. A mutant variant of the enzyme, K46M/K138E, showed a 35% increase in the thermal stability (10 min at 80°C) as compared to phytase from the wild-type strain. Analysis of the 3D enzyme model showed that the substitution of K138E located on the enzyme surface in the region of a loop with a highly irregular structure (a so-called coil) exerted a significant effect on the change in the protein thermal stability. It was shown that the introduced mutations did not noticeably affect the industrially valuable enzyme characteristics, including the specific activity, temperature, and pH profile.

About the authors

T. L. Gordeeva

State Research Institute for Genetics and Selection of Industrial Microorganisms, Kurchatov Institute National Research Center (GOSNIIGENETIKA, Kurchatov Institute NRC)

Author for correspondence.
Email: tatiana.gordeeva@mail.ru
Russian Federation, Moscow, 117545

L. N. Borshchevskaya

State Research Institute for Genetics and Selection of Industrial Microorganisms, Kurchatov Institute National Research Center (GOSNIIGENETIKA, Kurchatov Institute NRC)

Email: tatiana.gordeeva@mail.ru
Russian Federation, Moscow, 117545

A. N. Kalinina

State Research Institute for Genetics and Selection of Industrial Microorganisms, Kurchatov Institute National Research Center (GOSNIIGENETIKA, Kurchatov Institute NRC)

Email: tatiana.gordeeva@mail.ru
Russian Federation, Moscow, 117545

S. P. Sineoky

State Research Institute for Genetics and Selection of Industrial Microorganisms, Kurchatov Institute National Research Center (GOSNIIGENETIKA, Kurchatov Institute NRC)

Email: tatiana.gordeeva@mail.ru
Russian Federation, Moscow, 117545

M. D. Kashirskaya

BIOAMIDE Joint-Stock Venture

Email: tatiana.gordeeva@mail.ru
Russian Federation, Saratov, 410033

S. P. Voronin

BIOAMIDE Joint-Stock Venture

Email: tatiana.gordeeva@mail.ru
Russian Federation, Saratov, 410033

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