Biotechnology and Bioprocess Engineering 2019; 24(6): 981-989  
Expression and Characterization of a New L-amino Acid Oxidase AAO Producing α-ketoglutaric Acid from L-glutamic Acid
Rao Ben1, Liao Xianqing1, Liu Fang1, Chen Wei1, Zhou Ronghua1, Ma Lixin2, and Wang YaPing2,*
1National Biopesticide Engineering Research Center, Hubei Academy of Agricultural, Wuhan, Hubei Province 430064, China
2State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio- Resources, Hubei university, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan, Hubei Province 430062, China
Correspondence to: Wang YaPing*
State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio- Resources, Hubei university, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan, Hubei Province 430062, China
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Received: May 14, 2019; Revised: July 7, 2019; Accepted: July 24, 2019; Published online: December 31, 2019.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
L-amino acid oxidase (AAO) was reported to be capable of converting L-glutamic acid to α-aketoglutaric acid (α-KG). The sequence of AAO from Kitasatospora cheerisanensis was synthesized based on Pichia pastoris codon-usage preferences. AAO gene was cloned into plasmid pPICZα which was transformed into P. pastoris. Next, multi-copy expression plasmids were constructed by using plasmid pHBM905BDM. High-density fermentation was performed and the recombinant enzyme was characterized. The conversion conditions were optimized. By using Escherichia coli expression system, no soluble or active AAO was obtained from two strains after fermentation and induction. We can't obtain high-level expression of recombinant strains by using plasmid pPICZα. Therefore, we constructed multi-copy expression plasmids using plasmid pHBM905BDM. By using this plasmid, multi-copy strains were constructed and named as PAAO1, PAAO2, PAAO3, PAAO4, and PAAO5, respectively. The following results showed that expression of AAO in multi-copy strains increased as designed and strain PAAO5 was chosen for high-density fermentation and enzyme activity experiments. After high-density fermentation, we achieved an AAO-expression yield of 120.8 U/mL. After temperature and pH optimization, the highest AAO activity was observed at a temperature and pH of 20°C and 6, respectively. After optimization of the conversion conditions, the average production rate of L-glutamic acid to α-KG was 3.46 g/L/h and the highest α-KG titer (103 g/L) was converted from 120 g/L L-glutamic acid. In this study, AAO was abundantly expressed by using P. pastoris expression system. The following experiments indicated that AAO is suitable for use in industrial applications.
Keywords: α-Ketoglutaric acid, high density fermentation, L-amino acid oxidase


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