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KMID : 0043319800030010007
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Archives of Pharmacal Research
1980 Volume.3 No. 1 p.7 ~ p.12
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Ampicillin Biosynthesis by Immobilized Enzyme
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Kim YS
Ryu DY
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Abstract
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Ampicillin was synthesized from 6-aminopenicillanic acid (6-APA) and D-alpha-phenylglycine methyl ester by using ampicillin synthesizing enzyme from Pseudomonas melanogenum (IAM 1655). The whole cell enzyme was immobilized by entrapping it in the polyacrylamide gel lattices. The polymer used in the enzyme entrapment was made from 150mg per ml of acrylamide monomer and 8mg per ml of N,N’’-methylenebisacrylamide. About 200mg/whole cell enzyme was mixed in the polymer for entrapment. The maximal activity retention after immobilization was 56%. The optimal pH values for the whole cell enzyme and the immobilized whole cell enzyme were 6.0 and 5.9, respectively. The optimal temperature for the enzyme activity were the same for both type of preparations. The enzyme stabilities against pH and heat increased for immobilized whole cell enzyme. Immobilized cell was more stable especially in the acidic condition while both type were found to be very susceptible to thermal inactivation at a temperature above 40oC. The kinetic constants obtained from Lineweaver-Burk plot based on two substrate reaction mechanism showed somewhat higher value for immobilized whole cell enzyme as compared to the whole cell enzyme: the Km value for 6-APA were 7.0mM and 12.5mM while Km values for phenylglycine methyl ester were 4.5mM and 8.2mM, respectively. Using the immobilized whole cell enzyme packed i a column reactor, the productivity of ampicillin was studied by varying the flow rate of substrate solution. At the space velocity, SV, 0.14 hr-1 the conversion wis 45%. Operational stability found in terms of half life was 30 hr at SV=0.2 hr.
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