Using $\underline{Acetobacter} \underline{turbidans}$ enzyme, enzymatic synthesis of cephalexin from D-α-phenylglycine methylester(PGM) and 7-animo-3-deacetoxycephalosporanic acid(7-ADCA) was attempted. This enzyme was partially purified through ammonium sulfate fractionation, DEAE, CM, and S-200 gel filtration. This enzyme showed its maximal synthetic activity at pH 6.0 and 40 C.
An unique kinetic pattern that the maximal conversion achieved was greatly affected by molar ratio of two substrates and the amount of enzyme loaded, was observed. In order to elucidate this phenomenon, the reaction mixture of all steps involved in enzymatic synthesis of cephalexin was analyzed by TLC and HPLC techniques.
From this result, it was confirmed that this enzyme could catalyze simultaneously three reactions, which are the hydrolysis of PGM, transfer of phenylglycyl group to 7-ADCA to form cephalexin, and hydrolysis of cephalexin synthesized. It was also revealed that the hydrolysis of PGM and cephalexin, and synthetic reaction of cephalexin were not fully reversible. The optimal pH for hydrolysis was slightly higher than the other two reactions and the hydrolysis rate of PGM was decreased greatly by increasing 7-ADCA concentration.
Considering all reaction steps, it was proposed that reaction mechanism of this enzyme was nearly identical with that of $\underline{X. citri}$. From this proposed reaction mechanism, the rate equations derived in case of $\underline{X. citri}$ were used to evaluated the kinetic parameters. The unwanted product, phenylglycin, was found to act as a competitive inhibitor in the hydrolyzing reactions of PGM and cephalexin, and as a non-competitive inhibitor in the synthetic reaction. Using the rate equations and kinetic parameters determined, the reaction progress was simulated by computer and it was found that simulated result fitted well the experimental one.
Acetobacter turbidans에서 분리한 효소를 이용하여 피.지.엠. (PGM)과 7-에.디.시.에. (7-ADCA)로부터 세팔랙신의 합성에 관련된 여러 인자들을 연구하였다.
이 균주에서 분리한 효소는 비활성도가 165.5였으며 산도 6.0과 섭씨 40 도에서 최적 합성 조건을 가졌다.
세팔랙신 합성의 전환율은 기질 농도의 비에 크게 좌우되며 또한 효소의 양을 증가시킬 경우 최대 증가율이 약간 감소되는 경향을 보였다. 이 반응계를 박층 크로마도그래피와 고속 액체 크로마토그래피를 이용하여 분석한 결과 3 가지 반응이 동시에 일어남을 알 수 있었다. 이 3 가지 반응은 피.지.엠.의 가수분해, 페닐글라이신 기의 7-에.디.시.에.로의 전이반응, 그리고 생성된 세팔랙신의 가수분해 반응이다. 피.지.엠.의 가수분해 반응과 세팔랙신의 분해 반응은 비가역적으로 일어남이 실험 결과 밝혀졌다. 위의 모든 반응 현상이 Xanthomonas citri와 유사한 사실을 근거로 해서 반응 모형을 확인하였다.
Xanthomoas citri의 반응식을 이용해 과련된 모든 속도변수의 값을 구했다. 생성되는 페닐글라이신은 분해와 합성반응에서 유사한 저해 현상을 보였다. 이렇게 구한 속도변수와 반응식을 이용하여 컴퓨터 시뮬레이션 한 결과 실험치와 비교적 잘 일치함을 알 수 있었다.
