Efficient Surface Display of L-glutamate Oxidase and L-amino Acid Oxidase on Pichia pastoris Using Multi-copy Expression
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pISSN 1226-8372 eISSN 1976-3816
RESEARCH PAPER
Efficient Surface Display of L-glutamate Oxidase and L-amino Acid Oxidase on Pichia pastoris Using Multi-copy Expression Strains Rao Ben, Zhou Ronghua, Dong Qing, Liao Xianqing, Liu Fang, Chen Wei, Liu Xiaoyan, Min Yong, and Wang YaPing
Received: 30 September 2019 / Revised: 27 February 2020 / Accepted: 27 February 2020 © The Korean Society for Biotechnology and Bioengineering and Springer 2020
Abstract L-glutamate oxidase (GLOD) and L-amino acid oxidase (AAO) were reported to be capable of convert L-glutamic acid to α-aketoglutaric acid (α-KG). These two enzymes gene have been successfully expressed by using pHBM905BDM in Pichia pastoris to produce α-aketoglutaric acid from L-glutamic acid in our previous studies. Here these two enzymes were displayed on P. pastoris to achieve the conversion. We constructed multi-copy expression plasmids using plasmid pHBM905BDM. By using this plasmid, multi-copy strains were constructed and named as PGLOD(1-3)-AGα1 and PAAO(1-3)-AGα1, respectively. The following results showed that expression of GLOD(1-3)AGα1 and AAO(1-3)-AGα1 in multi-copy strains increased as designed and strain PGLOD3-AGα1 and PAAO3-AGα1 was chosen for high-density fermentation and enzyme activity experiments. By using a multi-copy expression approach and high-density fermentation, we achieved a GLOD expression yield of 688.5 U/g dry cell weight and AAO expression yield of 626.7 U/g dry cell weight. By
Rao Ben†, Zhou Ronghua†, Dong Qing, Liao Xianqing, Liu Fang, Chen Wei, Liu Xiaoyan, Min Yong National Biopesticide Engineering Technology Research Center, Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, Biopesticide Branch of Hubei Innovation Centre of Agricultural Science and Technology, Wuhan, China Wang YaPing* State Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of BioResources, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan, Hubei Province 430062, China Tel/Fax: +86-27-88661237 E-mail: [email protected] †These authors contributed equally to this work.
using displayed GLOD, the average production rate of L-glutamic acid to α-KG was 6.22 g/L/h and the highest α-KG titer (124.5 g/L) was converted from 135 g/L L-glutamic acid. By using displayed AAO, the average production rate of L-glutamic acid to α-KG was 5.78 g/L/h and the highest α-KG titer (115.6 g/L) was converted from 135 g/L L-glutamic acid. It showed that displaying enzymes on P. pastoris are suitable for use in industrial applications. Keywords: α-ketoglutaric acid, displayed enzyme, multicopy expression, Pichia pastoris
1. Introduction Alpha-ketoglutaric acid, an important intermediate of the dicarboxylic and tricarboxylic acid cycles, plays a very important role in the synthesis of amino acids and peptides, as well as in the pharmaceutical, food, organic synthesis, and nutritional supplement industries. This compound is wide
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