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℃, 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℃. However, when considering the stabilities of whole cell hydantoinase and substrate, pH 8.0 and 30℃ 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 acrylamide solution. The activity of retained whole cell hydantoinase showed about recovery of 40-50\% for total activity. The hydrolysis of D, L-p-hydroxyphenylhydantion reached 95\% in 5 h when volume of acrylamide-immobilized whole cell hydantoinase was 50\%. As a result, the volume of acrylamide entrappted whole cell hydantoinase for enzyme reaction at bioreactor was fixed at 40\% of reaction volume. When yeast extract was added to 0.1\%, the half life of immobilized whole cell hydantoinase was increased to 22 days, about 11 times comparing to control. In the repeated batch operation, time required to obtain the conversion yield of 99\% was lengthened with increasing number of batch operation when batch operayion was conducted four times successively. In continuous stirred tank reactor operation, the maximum productivities of 5\% dimethylsulfoxide and non-solvent for N-carbamoyl-D-hydroxyphenylglycine were found to be 2.3 and 1.1 g/L/h, respectively, at a dilution rate of 3.03 and 3.69 1/h. The hydantoinase loading for immobilization on Silicate was fixed at 35 mg protein pergram wet adsorbent. The recovered activity after immobilization on Silicate was calculated to be about 15-30\%. The immobilized hydantoinase showed a maximum activity at pH 9.0, while the maximum activity of free-hydantoinase was observed at around pH 8.5. Hydantoinase showed a narrow range of pH for enzyme activity, while the immobilized hydantoinase showed its broad activity within the range of pH. The immobilized hydantoinase was stable for 30 days without any loss of activity in a continuous stirred tank reactor. Relative activity of immobilized hydantoinase slightly was dropped when repeated batch operation was conducted more than fourteen times successively, and this seems to be due to the abrasion of immobilized adsorbent during reaction. It seems to be due to the abrasion of adsorbent as stated earlier, and it could be overcome by using repeated packed bed reactor system.
KBEL101 이 토양에서 분리되어 균체효소와 조효소원으로 사용되었다. 선별된 KBEL101은 공시균주인 Pseudomonas striata IFO 12996 보다 약 3배정도 높으며, 배양조건은 배지의 초기 pH 5.5, 온도 30℃로 나타났다. Hydantoinase 를 효과적으로 생산하기 위해 여러가지 질소원을 첨가하여 본 결과 yeast extract 가 가장 좋은 질소원 이었고, yeast extract 의 농도가 0.3% 에서 1%까지 증가될 때 최대의 상대활성과 총활성을 보여주었다. 또한 탄소원으로서 glucose 는 총활이 가장 높았으며, 모든 탄소원의 상대활성은 그다지 증가하지않았다. 앞서에서 얻은 배양 및 배지조건으로 배양한 결과 약 17시간 배양후 균체를 수확하는 것이 가장 효과적인 것임을 알 수 있었다.
한편, KBEL101 에서 생성된 hydantoinase 의 특성을 살펴본 결과, constitutive enzyme 으로 판단되며, 반응산물인 N-carbamoyl amino acid 에 의해 활성저해가 일어났으며, 효소반응 조건은 pH 8.0, 30℃로 나타났다.
기질인 D,L-p-hydroxylphenylhydantoin 은 수난용성이므로 기질의 용해도를 증가 시킴으로써 효소반응 속도를 증가시키고, 효율적인 생물반응기를 구성할 수 있기때문에 이 목적을 달성하기위해 유기용매를 사용하였다. 그 결과 5% dimethylsulfoxide 를 사용하였을 때 약 2.3배의 높은 반응속도와 control과 유사한 효소 안정성을 보여주었다.
선별된 hydantoinase 의 효소 안정성이 낮기때문에 균체와 조효소의 고정화를 통하여 효소의 안정성을 높이고 생물반응기에 응용하고자 하였다. 이를 위해 polyacrylamide 에 균체를 고정화하여 연속반응기 (CSTR) 에서 운전한 결과 효소안정성은 free cell에 비해 약간 증가할 뿐 이었다. 그리하여, 기질에 탄소원과 질소원을 첨가하여 효소의 조업 안정성을 증가시키고자 하였다. 그 결과 0.1% yeast extract 에서 약 25일 동안 효소가 안정성을 유지하는 것으로 나타났다.
균체 고정화시 실질적인 효소 안정성의 증가가 없었고, 질소원 첨가시 반응 산물의 분리 정제를 어렵게 하기때문에 효소 고정화를 하였다. 먼저 조효소의 순도를 높이기 위해 PEG 6000 으로 2단계 분리 정제한 후 여러가지 흡착제에 효소를 고정화 하였다. 그 결과 Silicate에서 가장 높은 단백질 흡착 용량과 효소 활성회복을 나타내었다. 이렇게 선별된 고정화 효소의 효소반응 특성을 조사한 결과 최적 반응 조건은 효소의 안정성을 고려할 때 pH 8.0, 온도30℃ 로 판단된다. 또한 dimethylsulfoxide 의 농도에 따라 고정화 효소의 조업 안정성을 살펴 본 결과 5% 에서 약 30일의 뛰어난 효소 안정성을 보여주었다.