Enzymatic Synthesis Of Ampicillin In Fully Organic Medium And Water-Solvent Mixtures

Ampicillin is one of the most widely usedβ-lactam antibiotics and suitable for a broad spectrum of bacterial infections.It is usually produced via traditional chemical procedures,which need unhydrous organic solvents,very low temperature, multi-step protection and unprotection,with heavy pollution.Enzymatic synthesis, which markedly reduces the production of waste and the dependence on organic solvents,is an efficient alternative to the chemical synthesis for the production ofβ-lactam antibiotics.This thesis focuses on the enzymatic synthesis of ampicillin in fully organic medium and the application of organic solvents in aqueous medium.We also researched the water-hexane two-phase system and one-pot enzymatic synthesis of ampicillin.Ampicillin was synthesized with D-PGM-HCl and 6-APA at appropriate pH in aqueous medium by the catalysis of immobilized penicillin G acylase IPA-750.The effects of the IPA concentration,pH,substrate concentration and ratio,as well as cosolvents on the enzymatic synthesis were investigated.The optimal result was the yield of 65.6%and S/H ratio of 1.41.The efficient enzymatic synthesis of ampicillin in fully organic medium was successfully achieved with D-PGM as the activated acyl donor.A series of organic solvents with different polarity were screened and it was shown that remarkable catalytic activity of the IPA was retained in relatively apolar organic solvents, among which ethyl acetate was the best.The influence fators including the initial substrate concentration,molar ratio of substrates,IPA concentration,temperature and water content of IPA on the enzymatic synthesis of ampicillin in pure ethyl acetate were systematically examined.As a result,high yield(92.9%)and S/H ratio (1.50)were successfully achieved with 300 mM 6-APA and 600 mM D-PGM under the optimum conditions.Furthermore,the synthetic and hydrolytic activities of IPA were both improved by the addition of hexane to ethyl acetate.However,it was better for the hydrolysis of D-PGM.The water-hexane two-phase system was studied.The hydrolysis of D-PGM was remarkably restrained in the water-hexane system(30/70,v/v).The conversion of 6-APA and D-PGM was improved,and the reaction was fast.High yield of 90.5%and S/H ratio of 2.14 were achieved with 200 mM 6-APA and 400 mM D-PGM in 2 h at 25℃.The method of one-pot enzymatic synthesis of ampicillin was developed. 6-APA was obtained from the hydrolysis of penicillin G potassium salt(PGK) catalyzed by IPA,and subsequently acylated by D-PGM with IPA.Ampicillin was directly produced from PGK without the separation and purification of 6-APA.The ampicillin yield of one-pot enzymatic synthesis in water-hexane two-phase system was much higher than that in fully aqueous medium,while the hydrolysis of D-PGM was restrained.As a result,the yield of 70.2%and S/H ratio of 1.09 were achieved.