文献类型： 外文期刊

作者： Rao Ben ¹ ; Liao Xianqing ¹ ; Liu Fang ¹ ; Chen Wei ¹ ; Zhou Ronghua ¹ ; Ma Lixin ² ; Wang YaPing ² ;

作者机构： 1.Hubei Acad Agr, Natl Biopesticide Engn Res Ctr, Wuhan 430064, Hubei, Peoples R China

2.Hubei Univ, Hubei Collaborat Innovat Ctr Green Transformat Bi, State Key Lab Biocatalysis & Enzyme Engn, Hubei Key Lab Ind Biotechnol,Biol Fac, Wuhan 430062, Hubei, Peoples R China

关键词： alpha-Ketoglutaric acid; high density fermentation; L-amino acid oxidase

期刊名称：BIOTECHNOLOGY AND BIOPROCESS ENGINEERING （ 影响因子：2.836； 五年影响因子：2.281 ）

ISSN： 1226-8372

年卷期： 2019 年 24 卷 6 期

页码：

收录情况： SCI

摘要： L-amino acid oxidase (AAO) was reported to be capable of converting L-glutamic acid to alpha-aketoglutaric acid (alpha-KG). The sequence of AAO from Kitasatospora cheerisanensis was synthesized based on Pichia pastoris codon-usage preferences. AAO gene was cloned into plasmid pPICZ alpha 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 pPICZ alpha. 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 degrees C and 6, respectively. After optimization of the conversion conditions, the average production rate of L-glutamic acid to alpha-KG was 3.46 g/L/h and the highest alpha-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.