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Expression and Characterization of a New L-amino Acid Oxidase AAO Producing α-ketoglutaric Acid from L-glutamic Acid

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  • Protein Engineering and Enzyme Technology
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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 multicopy 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.

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Abbreviations

AAO:

L-amino acid oxidase

α-KG:

α-aketoglutaric acid

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Acknowledgments

We acknowledge financial support of National Key R D Program of China (2017YFD0200900) Subject 2 (2017YFD0200902), Joint Open Fund of National Biopesticide Engineering Research Centre and Scientific Observation and Experimental Station of Utilization of microbial resources (Central China), Ministry of Agriculture and Rural Affairs (Grant No. JF-NBCOES-1807), Natural Science Foundation of China (31300074), Natural Science Foundation of Hubei Province (2014CFB541), Specialized Research Fund for the Doctoral Program of Higher Education (20124208120004), Key project of educational commission of Hubei province of China (D20171002).

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Authors and Affiliations

  1. National Biopesticide Engineering Research Center, Hubei Academy of Agricultural, Wuhan, Hubei Province, 430064, China

    Rao Ben, Liao Xianqing, Liu Fang, Chen Wei & Zhou Ronghua

  2. 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

    Ma Lixin & Wang YaPing

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  1. Rao Ben
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  2. Liao Xianqing
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  3. Liu Fang
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  4. Chen Wei
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  5. Zhou Ronghua
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  6. Ma Lixin
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  7. Wang YaPing
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Correspondence to Wang YaPing.

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Ben, R., Xianqing, L., Fang, L. et al. Expression and Characterization of a New L-amino Acid Oxidase AAO Producing α-ketoglutaric Acid from L-glutamic Acid. Biotechnol Bioproc E 24, 981–989 (2019). https://doi.org/10.1007/s12257-019-0182-7

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