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High-level expression of L-glutamate oxidase in Pichia pastoris using multi-copy expression strains and high cell density cultivation

文献类型： 外文期刊

作者： Wang YaPing 1 ; Ben, Rao 2 ; Hong, Yan 1 ; Rui, Han 1 ; Li, Li 1 ; Liao Ping'an 1 ; Ma Lixin 1 ;

作者机构： 1.Hubei Univ, Fac Biol, Hubei Key Lab Ind Biotechnol, Hubei Collaborat Innovat Ctr Green Transformat Bi, Wuhan 430062, Hubei Province, Peoples R China

2.Hubei Acad Agr, Natl Biopesticide Engn Res Ctr, Wuhan 430064, Hubei Province, Peoples R China

关键词： L-glutamate oxidase;Pichia pastoris;Exponential feeding;Copy number;Fermentation

期刊名称：PROTEIN EXPRESSION AND PURIFICATION （ 影响因子：1.65； 五年影响因子：1.548 ）

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收录情况： SCI

摘要： L-glutamate oxidase (CLOD), encoded by the gox gene, catalyses the transformation of L-glutamic acid into alpha-ketoglutaric acid (a-KG). In the present study, Pichia pastoris was used for heterologous production of CLOD following optimization of the gox coding sequence for expression in the yeast host. A series of constructs based on the pHBM905BDM plasmid were engineered and transformed into P. pastoris to increase the gox copy number. The results indicated that CLOD protein levels and enzyme activity increased with increasing gox copy number. Strain PGLOD4, which contained four copies of the target gene, was chosen for subsequent fermentation experiments, and a fermentation strategy involving two exponential feeding phases was developed. During the preinduction phase, glycerol was fed exponentially at mu(G) = 0.15/h. When the cell density reached 300 g/l, methanol was fed exponentially at mu(M) = 0.03/h to induce CLOD production. After 84 h of cultivation, the final cell density and total enzyme activity reached 420 g/L, and 247.8 U/mL, respectively. The recombinant enzyme displayed an optimum temperature of 40 degrees C, which was higher than recombinant enzyme expressed in E. coll. This is important because increasing the temperature could accelerate enzymatic transformation of L-glutamic acid to a KG. Experiments also demonstrated superior thermo-stability for the enzyme produced in yeast, which further enhances its potential for industrial applications. (C) 2016 Published by Elsevier Inc.

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