Penicillin amidase, an enzyme that hydrolyzed benzylpenicillin to yield 6-APA, was produced by culture of $\underline{Bacillus}$ $\underline{megaterium}$ (ATCC 14945) and purified 70 fold by means of adsorption on Celite followed by dialysis or ammonium sulfate precipitation.
Little attempts have been made to utilize synthetic nylon fiber and natural cotton for enzyme immobilization. New enzymatic process for the preparation of 6-APA was developed by employing the enzyme immobilized onto these fibers as carriers. The feasibility of utilizing these materials as enzyme carriers was studied by evaluating four different immobilization methods.
Cotton was activated with CNBr or $TiCl_4$, followed by coupling of enzyme. The optimal pH for activation was found in the range of 11.5 to 12, and the amount of protein coupled to cotton was as high as 163 mg/g of cotton matrix, showing a good possibility of employing natural cellulosic fiber as an enzyme carrier. Activation with $TiCl_4$ was not recommended because of degradation of fiber structure by strongly acidic $TiCl_4$ solution.
Native nylon polymer has few end groups, and it must be pretreated in order to generate potentially reactive groups that can be covalently bound with enzyme molecule. Therefore, the nylon fiber was treated with N,N-dimethylaminopropylamine of HCl to generate reactive amino groups on it, and the enzyme was coupled to it by crosslinking with glutaraldehyde.
Only 11% of enzyme activity was retained by employing N, N-dimethylaminopropylamine, so the partial hydrolysis with HCl was further investigated. The optimal hydrolysis time was found about 5 to 10 minutes at 45℃ when 3.75N-HCl solution was used.
Under these conditions, the amount of enzyme coupled onto the nylon was up to 63mg/g nylon matrix, and the activity retention was as high as 72%.
From the experiments on the effect of enzyme loading, two interesting features were found.
First, the maximum coupling efficiency was 77%, and Second, the activity of immobilized enzyme gradually decreased when the amount of enzyme loading was above a certain level. It results from the blocking of reactive groups on nylon fiber by the impurities which exist in partially purified enzyme preparation.
This fact strongly indicates that the enzyme loading factor should be considered as a criterion by which the efficiency of immobilization procedure is evaluated, especially when impure enzyme is used.
In comparison with soluble enzyme, the optimal pH was not changed, and the overall rate profile became broader with respect to pH range. The temperature effect on enzyme activity was same as that of soluble enzyme below 40℃, and showed somewhat increased stability at slightly higher temperature.
The kinetic constants obtained by Lineweaver-Burk plot showed somewhat higher values for immobilized enzyme, the $K_m$ value; 4.3 mM and 4.8mM, Kia value; 40mM and 45 mM, Kip value; 22mM and 50mM for the soluble and immobilized enzyme, respectively. Thermal deactivation of immobilized enzyme conformed approximately the first order decay, and the half-life was about 7.8 days.
From the information available, a rate equation was derived to predict the performances of penicillin amidase reactors. The progress of batch reaction agreed well with the result of computer simulation for both the immobilized and soluble enzymes, confirming the reliability of the rate equation and the values of kinetic constants.
The performance of a single-stage CSTR type enzyme reactor system was simulated by solving the steady-state equation using Newton-Raphson method.
The immobilized enzyme reactor system gave higher conversion values than the soluble enzyme reactor for all feed concentrations tested. This observation could be explained by higher values of Kia and Kip for the immobilized enzyme indicating that the inhibitory effect of the products on the immobilized enzyme were less than on the soluble enzyme at the same product concentration.
We found that the reactor system with immobilized enzyme gave higher productivity than the one with soluble enzyme, at a given space time or for a level of conversion desired.
It showed that 70 to 80% greater productivity can be achieved using the immobilized enzyme as compared with the soluble enzyme while maintaining 90% to 95% conversion of penicillin to 6-APA.
화학적 처리 방법 대신 새로운 물리적 방법을 개발하여 나일론 fiber의 표면적을 증가시키고, 이것에 산처리에 의한 활성 amino기를 도입시키는 최적조건을 찾으므로써 나일론 fiber 를 penicillin amidase 효소의 고정화에 사용할수 있는 가능성을 제시하였다. Penicillin amidase 를 나일론 fiber 에 glutaraldehyde로서 공유 결합시키므로써 고정화하여 그 특성을 조사하여 가용성 효소와 비교 검토 하였다.
고정화 효소와 가용성 효소의 단일 공정 반응은 computer 에 의한 simulation 결과와 잘 일치하므로써 효소의 반응속도식과 속도상수 값들의 정확성을 확인하였다.
가용성 효소 대신에 고정화 효소를 사용하므로써 얻어질수 있는 장점은 :
1) 고정화 효소의 최적 pH 의 범위가 넓어지므로써 pH 7.5-8.0에서 반응을 진행 시킬수 있으므로 강 알칼리 조건하에서의 페니실린의 불활성화를 감소 시킬수 있다.
2) 고정화 효소의 반응 생성물에 의한 inhibition 상수들이 증가 하므로 말미암아 연속 반응조를 사용하여 90-95% 까지 반응을 진행시킬때 가용성 효소 대신에 고정화 효소를 사용하므로써 70-80% 이상의 productivity를 증가시킬수 있다. 이와같은 실험결과 아직도 경비 절약에 대한 여러 관점에서의 연구가 더 요구되나, 6-APA 를 산업적으로 생산하는데 있어서 나일론 fiber 를 이용한 고정화 효소의 실제 응용이 가능함을 충분히 시사하고 있다.
