A Study on the Efficient Preparation of α-Ketoglutarate with L-Glutamate Oxidase

• Published in: Molecules (Basel, Switzerland) Switzerland), 2024-04, Vol.29 (8), p.1861
• Main Authors: Niu, Shuhui, Liu, Fang, Wang, Yaping, Rao, Ben, Wang, Yueying
• Format: Article
• Language: English
• Subjects: alpha-ketoglutaric acid (α-KG); Amino Acid Oxidoreductases - chemistry; Amino Acid Oxidoreductases - metabolism; Amino acids; Backup software; catalase; Catalase - chemistry; Catalase - metabolism; Catalysis; Cloning; E coli; Efficiency; Enzymes; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Escherichia coli; Escherichia coli - enzymology; Experiments; Glutamate; Ketoglutaric Acids - chemistry; Ketoglutaric Acids - metabolism; L-glutamate oxidase; Plasmids; Proteins; Streptomyces - enzymology; whole-cell catalysis
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules29081861

Alpha-ketoglutaric acid (α-KG), as an intermediate product of the tricarboxylic acid cycle, plays a crucial role in peptide and amino acid synthesis. In order to reduce costs and improve efficiency in the oxidative production of α-ketoglutaric acid, this study successfully synthesized and expressed L-glutamate oxidase (LGOX ) from R111 and catalase (KatG ) from H736. Two immobilization methods and the conditions for one-step whole-cell catalysis of α-ketoglutaric acid were investigated. α-Ketoglutaric acid has broad applications in the pharmaceutical, food, and chemical industries. The specific research results are as follows: (1) By fusing the sfGFP tag, L-glutamate oxidase (LGOX r) and catalase (KatG ) were successfully anchored to the outer membrane of cells, achieving one-step whole-cell catalysis of α-ketoglutaric acid with a conversion efficiency of up to 75%. (2) Through the co-immobilization of LGOX and KatG , optimization of the preparation parameters of immobilized cells, and exploration of the immobilization method using @ZIF-8, immobilized cells with conversion rates of over 60% were obtained even after 10 cycles of reuse. Under the optimal conditions, the production rate of α-ketoglutaric acid reached 96.7% in a 12 h reaction, which is 1.1 times that of @SA and 1.29 times that of free cells.