Mass production of thermostable D-hydantoinase by batch culture of recombinant Escherichia coli with a constitutive expression system

• Published in: Biotechnology and bioengineering Vol. 56; no. 4; pp. 449 - 455
• Main Authors: Lee, Dong-Cheol, Kim, Geun-Joong, Cha, Yoo-Kyong, Lee, Cheo-Young, Kim, Hak-Sung
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 20.11.1997; Wiley
• Subjects: Batch cell culture; batch cultivation; Biological and medical sciences; Biomass; Biotechnology; Catalyst activity; Coliform bacteria; constitutive expression; Enzyme engineering; Enzyme kinetics; Fermentation; Fundamental and applied biological sciences. Psychology; Genetic engineering; Glycerol; Methods. Procedures. Technologies; Production of selected enzymes; recombinant E. coli; Thermal effects; Thermodynamic stability; thermostable D-hydantoinase; Recombinant microorganism; Mass production; Stability; Enzyme; Escherichia coli; Dihydropyrimidinase; Environmental factor; Bacillaceae; Gene expression; Optimization; Operating conditions; Plasmid; Bacillales; Batchwise; Bacillus stearothermophilus; Microorganism culture; Bacteria; Hydrolases; Enterobacteriaceae
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/(SICI)1097-0290(19971120)56:4<449::AID-BIT10>3.0.CO;2-7

D‐Hydantoinase is an industrial enzyme widely used for the synthesis of optically active D‐amino acids. A gene encoding thermostable D‐hydantoinase of Bacillus stearothermophilus SD‐1 has previously been cloned and constitutively expressed by its native promoter in Escherichia coli XL1‐Blue (Lee et al., 1996b). In this work, we attempted mass production of the D‐hydantoinase by batch culture of the recombinant E. coli using glycerol as a carbon source. The plasmid content in cells increased in proportion to the culture temperature, which resulted in a two‐ or three‐fold increase of the specific D‐hydantoinase activity at 37°C compared with that at 30°C. The plasmid was stably maintained over 80 generations. When glycerol was initially added to a concentration of 100 g/L, the final biomass concentration reached about 50 g‐dry cell weight/L in a 50 L‐scale fermentation, resulting in the specific enzyme production of 3.8 × 104 unit/g‐dry cell weight in a soluble form. Glycerol‐using batch cultivation of recombinant E. coli was found to be a cost‐effective process for the mass production of industrially useful D‐hydantoinase. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 449–455, 1997.