PREPARATION PROCESS OF N-CARBAMOYL-D-AMINO ACID WITH FIXED ENZYME

본 발명은 다공성 마그네슘 알루미늄 실리케이트에 효소를 흡착시키는 방식의 고정화 효소를 제조하는 방법 및 이에 따라 제조된 고정화 효소를 사용하여 N-카르바모일-D-아미노산을 제조하는 방법에 관한 것이다. 본 발명에 따라 제조된 고정화 효소는 매우 높은 조업 안정성을 나타내며, pH와 온도에 대한 특성에 있어서도 유리 효소의 경우보다 더 넓은 범위에서 높은 효소 활성을 나타낸다

Hydantoin 유도체로부터 D-amino acid를 생산하기 위한 효소적 공정의 개발

학위논문(박사) - 한국과학기술원 : 생물공학과, 1993.8, [ xi, 129 p. ]KBEL101 was isolated from soil and used to obtain the whole cell and crude dihydropyrimidinase (E.C.3.5.2.2., hydantoinase). Hydantoinase activity of screened KBEL101 were exhibited about 3 times higher hydantoinase acivity than pseudomonas striata IFO 12996. Cultivation conditions of KBEL 101 are initial pH 5.5, temperature 30$^\circ$C, and culture time 18 hr. In the growth conditions for production of hydantoinase activity, yeaxt extract is best nitrogen source and the formation of enzyme increased with increasing concentrations up to 1\%. Glucose as carbon source increased the total activity, while the specific activity was not increased. Hydantoinase activity was observed regardless of the presence of a hydantion dervative during cultivation. Hydantoinase was severely inhibited by products, N-carbamoylhydroxyphenlyglycine and N-carbamoyl-glycine, etc.. The optimal conditions of KBEL101 for whole cell hydantoinase are pH 9.0 and 40$^\circ$C. However, when considering the stabilities of whole cell hydantoinase and substrate, pH 8.0 and 30$^\circ$C are the best actual reaction condition. Various water-miscible organic solvents were tested with respect to both the activity and the stability of whole cell enzyme. Among the vorious organic solvents, 5\% dimethylfomamide showed increase about 2.7 times than control and 5\% dimethylsulfoxide was chosen to be a proper solvent : hydantoinase acitivity increased two fold in the presence of 5\% dimethylsulfoxide and stability in the presence of 5\% dimethylsulfoxide was like that of control. From the practical stand point, enhancement of substrate solubility is advantageous to obtain highest conversion yield by increase of reaction rate. Enzymatic synthesis fo N-carbamoyl-D-p-hydroxyphenylglycine was carried out in repeated batch operation in the presence of 5\% dimethylsulfoxide. The amount of entrapted whole cell linearly increased with increasing loading of whole cell hydantoinase up to 50 mg dry cell per mL acrylam...한국과학기술원 : 생물공학과