Enzymatic synthesis of cephalexin directly from D-α-phenylglycine methyl ester (PGM) and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) was attempted by using $\underline{Xanthomonas}$ $\underline{citri}$ enzyme. This enzyme showed its maximal reaction rate at pH 6.0 and $37^\circ{C}$. An unique kinetic pattern that the maximal conversion achieved was decreased by increasing the amount of enzyme loaded or phenylglycine methyl ester was observed. In order to elucidate this phenomena, all the reaction steps involved in enzymatic synthesis of cephalexin were carefully analyzed by thin layer chrmatographic technique and high performance liquid chromatographic technique. It was confirmed that this enzyme could catalyze the three reactions at least, which are the hydrolysis of phenylglycine methyl ester, transfer of phenylglycyl group to 7-amino-3-deacetoxycephalosporanic acid to form cephalexin, and hydrolysis of cephalexin produced. It was also revealed that the hydrolyzing reactions of phenylglycine methyl ester and cephalexin were not fully reversible. An interesting finding was that the addition of 7-amino-3-deacetoxycephalosporanic acid in hydrolyzing system of phenylglycine methyl ester caused to decrease this hydrolytic rate and to increase the synthetic reaction rate of cephalexin. It was also found that the optimal molar ratio of two substrates in cephalexin synthesis was 3 for its maximal reaction rate. The optimal pHs for three reactions mentioned above were nearly identical at pH 6.0. Based on these findings, a new reaction model for, cephalexin synthesis was proposed having acyl enzyme intermediate. However, the acylated site in this enzyme was not yet detected by chemical modification method. Form the proposed reaction model, the reaction rate equations for three reactions catalyzed by this enzyme were derived by quasi-steady state method. The kinetic parameters were evaluated with varying the concentration of substrates by Lineweaver-Burk plot. The unwanted product, phenylglycine, was found to act as a competitive inhibitor in the hydrolyzing reactions of phenylglycine methyl ester and cephalexin, and to act as a noncompetitive inhibitor in cephalexin synthetic reaction.
Using the developed rate equations and kinetic parameters, the reaction progress was simulated by computer. A relative good agreement of simulated results with experimental data was obtained. Using the same enzyme obtained from $\underline{X.}$ ${underline{citri}$, the synthesis of cephaloglycin directly from D-α-phenylglycine methyl ester and 7-amino-cephalosporanic acid (7-ACA) was also attempted. The similar kinetic pattern was observed even in this case, and some supporting evidences for a proposed reaction model for this enzyme were also obtained. It was found that the simulated results by computer using the developed rate equations and kinetic parameters obtained from Lineweaver-Burk plot had a good agreement with experimental results.
So as to expand the applicability of this proposed reaction model to another system, enzymatic synthesis of phenoxymethylpenicillin (PNV) from phenoxyacetic acid methyl ester (POM) and 6-aminopenicillanic acid (6-APA) was investigated using $\underline{Erwinia}$ $\underline{aroideae}$ enzyme. Among the substrates tested, phenoxyacetic acid methyl ester was the most suitable for a acyl donor to 6-aminopenicillanic acid to form phenoxymethylpenicillin. In this system, it was also confirmed that three reactions including the hydrolysis of phenoxyacetic acid methyl ester, transfer of phenoxyacetyl group to 6-aminopenicillanic acid, and the hydrolysis of phenoxymethylpenicillin occurred simultaneously. Some experimental results showed the participitation of common enzyme intermediate in these reactions catalyzed by $\underline{E.}$ $\underline{aroideae}$ enzyme. However the reaction mechanism of $\underline{Erwinia}$ enzyme was somewhat different from that of $\underline{Xanthomonas}$ enzyme. $\underline{Erwinia}$ enzyme had a similar catalytic properties of metal-containing exopeptidase such as aminopeptidase and carboxypepetidase. The optimal pHs for three reactions occurred by $\underline{Erwinia}$ enzyme were quite different from each other ; esterase activity in alkaline region and peptidase activity in acidic region.
On the ground of above findings, a new name for this group of enzymes concerning the biotransformation of acyl group attached on β-lactam nuclei was proposed. It is "α-Acylamino-β-lactam acylhydrolase"(ALAH). This group of enzymes was classified into 4 types such as α-acylamino-β-lactam acylhydrolase I, II, III, and IV according to their preferential substrate specificities in place of using penicillin V acylase, penicillin V acylase, and ampicillin acylase.
효소를 이용한 반합성 베타 락탐 항생제의 생산을 시도하기 위하여 $underline{잰토모나스 시트리} 균의 효소와 \underline{어위니아 애로이디}$ 균의 효소를 이용하였다. $\underline{잰토모나스}$ 효소를 이용하여 디-페닐글라이신 메틸 에스터(피.지.엠) 와 7-아미노-3-데아세톡시세팔로스포란산(7-에이.디.시.에이)으로부터 세팔렉신의 생산을 시도하였다. 이 효소는 산도 6.0 과 섭씨 37도에서 최적조건을 가졌다. 이 효소합성계는 효소의 양을 증가시키면 최대전환율이 감소하는 특이한 현상을 나타내었다. 이 반응계를 박층 크로마토그래피와 고속 액체 크로마토그래피를 이용하여 조사해 본 결과 적어도 3가지 반응이 동시에 일어남을 알 수 있었다. 이 3가지 반응은 피.지.엠의 가수분해, 페닐글라이실 기의 7-에이.디.시.에이 로의 전이반응, 그리고 생성된 세팔렉신의 가수분해이며, 피.지.엠과 세팔렉신의 가수분해반응은 비가역임이 밝혀졌다. 피.지.엠만의 가수분해반응계에서 7-에이.디.시.에이를 첨가하면 이의 가수분해속도가 크게 저하되었다. 또한 세팔렉신 합성계에서는 기질인 피.지.엠과 7-에이.디.시.에이의 몰 비가 3일 때 가장 좋은 합성율을 나타내었다. 위의 3가지 반응에 대한 최적 산도를 조사해 본 결과 모두 산도 6.0 부근임을 알 수 있었다. 이러한 실험결과를 토대로 하여 새로 반응모형을 설계하여 제안하였다. 이 모형은 아실 효소 중간체를 갖는 것으로 가정하였으나 원 효소의 아실화되는 부위에 대한 검출은 실패하였다. 이 모형으로부터 반응식을 구하였고 이 반응식을 이용하여 속도변수를 구하였다. 이렇게 구한 반응식과 속도변수를 이용하여 컴퓨터 시뮬레이션 한 결과 실험치와 비교적 잘 일치하는 결과를 얻을 수 있었다.
이와 동일한 방법으로 피.지.엠과 7-아미노세팔로스포란 산 (7-에이. 시.에이)으로부터 세팔로글라이신의 합성도 $\underline{잰토모나스}$ 효소를 이용하여 시도되었다. 이러한 세팔로글라이신의 합성계에서도 대체적으로 세팔렉신의 경우와 비슷한 결과를 나타내었다.
제안된 모형의 응용을 확대하기 위하여 $\underline{어위니아 에로이디}$의 효소를 이용하여 페니실린 뷔(페녹시메틸 페니실린)의 합성도 시도하였다. 이러한 페니실린 뷔의 합성을 위한 6-아미노페니실란 산에 대한 아실 공여 체로는 페녹시 초산 메틸 에스터가 페녹시아세틸글라이신 보다 훨씬 좋았다. 이 효소계에서도 $\underline{잰토모나스}$의 효소와 같이 페니실린 뷔의 합성계에서 3가지 반응이 촉매되고 있음을 확인할 수 있었다. 그러나 $\underline{어위니아} 효소는 \underline{잰토모나스}$ 효소와는 촉매기전이 상이함을 알 수 있었다. 특히 에스터레이즈의 역가는 산도 9.0 에서, 펩티데이즈의 역가(아미데이즈의 역가)는 산도 6.0 에서 잘 나타내었다. 또한 효소변형실험으로부터 금속 이온과 타이로신의 페놀 기가 $\underline{어위니아}$ 효소의 반응계에 관여함을 보여주었는데, 이러한 결과들은 이 효소가 카복시펩티데이즈 나 아미노펩티데이즈 와 같은 금속을 함유한 엑소펩티데이즈 와 성질이 유사함을 나타내주고 있다.
이러한 실험결과로 부터 반합성 베타 락탐 항생제의 합성과 가수분해에 관여하는 효소들을 공히 "알파-아실아미노-베타-락탐 아실하이드롤레이즈"라고 명명하는 것이 타당하다고 사료된다. 또한 이러한 효소군들은 그 기질특이성에 따라 알파-아실아미노-베타-락탐 아실하이드롤레이즈 I, II, III, IV로 분류하는 것이 보다 더 타당하리라고 생각한다.
