Process development for the production of optically pure D-p-hydroxyphenyl glycine using thermostable D-hydantoinase내열성 D-hydantoinase 를 이용하여 광학적으로 순수한 D-p-hydroxyphenylglycine을 제조하는 공정 개발

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D-Hydantoinase is an industrial enzyme widely used for the synthesis of optically active D-amino acids. In this study, we developed the practical processes for the production of N-carbamoyl-D-p-hydroxyphenylglycine, which is easily hydrolyzed to D-p-hydroxyphenylglycine under acidic conditions, from 5-(4-hydroxyphenyl)hydantoin using thermostable D-hydantoinase. 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. 1996. Ann. N. Y. Acad. Sci. 799: 401-405). At first, 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 degree of Celsius compared with that at 30 degree of Celsius. 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 $3.8\times 10^{4}$ 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. Secondly, we optimized the process for the production of N-carbamoyl-D-p-hydroxyphenylglycine, which is readily hydrolyzed to D-p-hydroxyphenyl glycine under acidic conditions, from 5-(4-hydroxyphenyl)hydantoin using the mass-produced D-hydantoinase. The enzyme reaction was carried out in a heterogeneous system consisting of a high substrate concentration up to 300 g/L. In this reaction system, most of substrate is present in suspended particles. Optimal temperature and pH were determined to be 45 degree of Celsius and...
Kim, Hak-Sungresearcher김학성researcher
한국과학기술원 : 생물과학과,
Issue Date
144210/325007 / 000945280

학위논문(박사) - 한국과학기술원 : 생물과학과, 1998.2, [ ix, 102 p. ]


Heterogeneous; Glycerol; Hydroxyphenylglycine; Hydantoinase; Modeling; 모델링; 비균질; 글리세롤; 히드록시페닐글리신; 히단토인

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