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 $3.8\times10^4$ 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 8.5, respectively, by taking into account the reaction rate and conversion yield. When the free enzyme was employed as a biocatalyst, enzyme loading higher than 300 unit/g-substrate was required to achieve maximum conversion. Use of whole cell enzyme resulted in the maximum conversion even at lower enzyme loadings than the free enzyme, showing 96% conversion yield at 300 g/L substrate. The heterogeneous reaction system used in this work might be applied to the enzymatic production of other valuable compound from a rarely water-soluble substrate.
Finally, we developed a kinetic model which describes the heterogeneous reaction system consisting of solid substrate particles for the production of D-amino acid by using the mass-produced D-hydantoinase. The heterogeneous reaction system involves dissolution of solid substrate, enzymatic conversion of dissolved D-form substrate, spontaneous racemization of L-form substrate to D-form, and deactivation of the enzyme. In the case of using whole cell enzyme, transfer of dissolved substrate and product through the cell membrane was considered. The kinetic parameters were determined from experiments, literature data, and Marquardt's method of non-linear regression analysis. The model was simulated using the kinetic parameters, and compared with experimental data. As a result, good agreement was observed between the experimental results and the simulated ones. Factors affecting the kinetics of the heterogeneous reaction system was analyzed based on the kinetic model. Optimal biocatalyst loading and the efficiencies of the free and whole cell reaction systems were also compared. The model might be useful for more convenient and time-saving scale-up of the bioconversion process.
Productivity of the bioconversion process developed here is enough to produce annual 300 tons of D-p-hydroxyphenylglycine using 30 kL of the culture broth with the D-hydantoinase. The media cost is less than 1% of the total production cost.
광학적으로 순수한 D-p-hydroxyphenylglycine을 생산하기 위하여, 내열성 D-hydantoinase를 이용하여 5-(4-hydroxyphenyl)hydantoin 으로부터 N-carbamoyl-D-p-hydroxyphenylglycine을 대량 생산하는 생물 전환 공정을 개발하였다.
산업적으로 유용한 효소를 실제로 이용하기 위해서는, 우선 경제적인 대량 생산법이 필히 요구된다. 내열성 D-hydantoinase를 대량 생산하는 데는, 고농도의 glycerol을 carbon source로 하여 재조합 대장균을 회분식으로 고농도 배양하는 방법이 적합한 것으로 확인되었다. 초기 glycerol 농도 100 g/L를 사용하여 50 L fermentor에서 배양한 결과, 최종 건조 균체 농도는 약 50 g-DCW/L를 얻을 수 있었으며, 배양액 당 D-hydantoinase의 생산성은 $2\times10^6$ unit/L 수준이었다. 재조합 plasmid 는 매우 안정하여, 배지에 선택력으로서 항생제의 투여는 필요치 않았다.
생산된 D-hydantoinase를 이용하여 기질인 5-(4-hydroxyphenyl)hydantoin 으로부터 N-carbamoyl-D-p-hydroxyphenylglycine을 대량 생산하는 효소 반응 공정을 최적화 하였다. 배양된 대장균 세포를 그대로 biocatalyst로 이용하는 전세포 전환 반응법이 적합하였으며 생산성도 충분한 것으로 확인되었다. 기질은 300 g/L의 고농도로 투여하여, 반응액 중의 기질 대부분이 고체 분말 형태로 존재하는 비균질 형태의 효소 반응을 수행하였다. 최적의 반응 조건은 전환 수율과 반응 속도를 고려하여 45도, pH 8.5로 결정되었으며, 여기서 50000 unit/L의 전세포 효소를 시용하여 300 g/L의 기질을 반응시키면, 약 96%의 수율로 N-carbamoyl-D-p-hydroxyphenylglycine을 생산할 수 있었다.
본 D-Hydantoinase 효소 반응 공정에 대하여 개발된 수학적 모델은, 실제의 실험 결과들을 잘 예측할 수 있었다. 유리 효소를 이용할 때와 전 세포를 이용할 때에 대해, 각각 구축된 모델을 이용하여 효소 반응을 동적으로 해석할 수 있었으며, 반응의 효율을 비교 분석할 수 있었다. 또한, 재조합 대장균을 배양할 때마다 전세포의 비 효소 활성이 편차를 나타내더라도, 계획된 생산 schedule에 따라 정해진 반응 시간 내에 효소 반응이 완수 될 수 있도록, 최적의 전세포 효소 투여량 역시 모델을 이용하여 결정할 수 있었다.
개발된 생물 전환 공법에 의하여 연간 300 ton 규모의 D-p-hydroxyphenylglycine을 생산하기 위해서는, 재조합 대장균을 연간 약 30 kL 정도 배양하면 효소 생산은 충분할 것으로 추산 되었다. 또한, D-p-Hydroxyphenylglycine을 생산하는 전 공정에서, 본 생물 전환 단계의 비용은 매우 낮은 비율을 차지하여, 기질의 합성 단계와 decarbamoylation 단계가 총 비용을 주로 결정하게 되었다. 생물 전환 공정의 전 단계를 모두 포함한 본 연구의 결과는, 실제 산업적으로 D-p-hydroxyphenylglycine 생산하는 공정 개발에 바탕이 될 것이다.
