Ampicillin was synthesized from 6-aminopenicillanic acid(6-APA) and D- α-phenylglycine methyl ester(PGM) by $\mbox{\underline{Pseudomonas}}$ $\mbox{\underline{melannogenum}}$ IAM 1655 containing the acylating enzyme. The specific activity of the enzyme was 2.7 unit per mg after 24 hour to 30 hour cultivation.
The whole cell enzyme was immobilized into polyacrylamide gel lattices. The enzyme activity was highest when 200 mg per ml of cell (wet weight) was entrapped into the polymer which was made from 150 mg per ml of acrylamide monomer and 8 mg per ml of N,N'-methylenebisacrylamide.
The maximal activity retention after immobilization was 56%.
The optimum pH values for intact cells and immobilized cells were 6.0 and 5.9, respectively, and they were practically the same. The optimum temperatures for the enzyme activity were the same for both type of preparations. The enzyme stabilities against pH and thermal changes increased for immobilized whole cell enzyme. Immobilized whole cells were especially more stable in the acidic condition while both found to be very susceptible to thermal deactivation at a temperature above 40 ℃. The activation energy was estimated as 6.15 kcal/mole for the immobilized whole cell enzyme and that for the intact whole cell enzyme was 8,43 kcal/mole.
The kinetic constants obtained from Lineweaver-Burk polt based on two substrate mechanism showed somewhat higher values for immobilized whole cell enzyme : Km values for 6-APA were 7.0 mM and 12.5 mM while for PGM were 4.5 mM and 8.2 mM, respectively. Substrate inhibition by PGM was observed ; the inhibition constant, Kis, for intact whole cell was 98 mM and that for immobilized whole cell enzyme was estimated as about 100 mM. From the experimental result of the product inhibition study the reaction seemed to obey the ordered mechanism. Namely, the leading substrate and product were PGM and methanol, respectively.
The use of some organic solvents and detergents, especially, benzene, chloroform, xylene and cetyltrimethyl ammonium bromide increased the enzyme activity by about 1.5 fold.
By increasing the amount of cell loading the time necessary to reach the maximal conversion (about 60%) was reduced but the final equilibrium level was about same.
When the optimal molar ratio of 6-APA to PGM was 1:3, ampicillin productivity was very good.
In the continuous production of ampicillin by immobilwhole cell enzyme packed in a column reactor, the conversion was examined by varying the flow rate of substrate solution. At $SV = 0.14 \mbox{hour}^{-1}$ the conversion was 45%. Operational stability was expressed in halflife and its value obtained as 30 hour at $SV = 0.2 hour^{-1}$.
Pseudomonas melanogenum IAM 1655 의 균체를 polyacrylamide gel에 고정화 시켜서 ampicillin 합성에 이용할때 여러가지 반응 조건을 조사 했으며 일반적인 효소의 성질도 찾았다.
최적의 pH 는 고정화 균체의 경우 5.9 이었고 온도는 35 ℃ 이었다. 이 결과는 미생물 균체에 비해 pH 는 약간의 이동 (6.0 → 5.9) 이 있었지만 최적 온도는 같았다. 기질 농도의 최적 조건은 6-APA 에 대한 PGM의 비가 1 : 3 일때 가장 좋았다. 특히 고정화 시켰을때 pH 와 온도의 변화에 대한 안정성이 증가되어서 공업적으로 이용시 이점이 될수 있었다.
이 반응에 있어서 고정화 균체에 대한 $K_m$ 값은 PGM 의 경우 12.5 mM 로 나타났고, 6-APA 에 있어서는 8.2 mM 로 추정되었다. 이 값들은 미생물 균체의 값보다 증가됨을 알수 있었고, PGM 에 의한 기질 저해 현상도 조사 되어졌다. 반응기전은 순차적으로 반응 양식이며, 우선적으로 반응되는 기질은 PGM 이고 생성물은 methanol 로 사료되었다. 고정화 균체를 column 에 채워 연속적으로 ampicillin 을 생성할때 $SV = 0.14 hr^{-1}$ 일때 생성물은 45% 정도의 수율로 얻어졌고 $0.2 hr^{-1}$ 에서 연속 가동 시켰을 경우 반감기는 30 시간 정도로 관찰 되어졌다.
