Enzymatic Synthesis Of Cephalexin:Process Control And Application Fundamental

Compared with traditional ?-lactam antibiotics,semi-synthetic ?-lactam antibiotics have the advantages of broad antibacterial spectrum and good stability,and are the most widely used class of anti-infective drugs.The chemical synthesis method is dominating the industrial production of semi-synthetic ?-lactam antibiotics.High energy consumption,low efficiency,and discharge of a large number of hard-degrable pollutants are its insurmountable disadvantages.The environmental friendly enzymatic synthesis has been valued by researchers,of which the enzymatic synthesis route under kinetic control is a relatively efficient method.However,due to the lack of systematic research and understanding of the synthetic process of this route,it has the problems such as high substrate molar ratio,low conversion ratio,and low reaction productivity.In this dissertation,cephalexin(CEX),one of the semi-synthetic?-lactam antibiotics with a large production capacity,is taken as the research target,and its enzymatic synthesis under the kinetic control was systematically studied with 7-amino-3-deacetoxycephalosporanic acid(7-ADCA)as mother nucleus and immobilized penicillin G acylase(IPGA)as biocatalyst.The enzymatic synthesis processes in suspension solution systems and 7-ADCA solubilization system were developed,respectively.The main research contents and results are shown as follows:The enzymatic synthesis of CEX under kinetic control was studied in fully aqueous solution system using IPGA as biocatalyst.First,the effect of different side chain,phenylglycine methyl ester(PGME)or phenylglycine amide(PGA),on the enzymatic synthesis of CEX was investigated and compared.Based on this,the effect of reaction conditions,including pH,temperature,enzyme amount,substrate concentration,and substrate molar ratio,on the conversion ratio,reaction productivity and hydrolysis ratio of enzymatic synthesis were studied.Compared with the enzymatic synthesis using PGA as side chain,the conversion ratio and productivity using PGME as side chain was increased by 45.9%and 1234.5%,respectively.The optimized conditions for enzymatic synthesis of CEX was obtained as follow:pH 6.5,temperature 15?,7-ADCA 60 mmol/L,PGME/7-ADCA molar ratio 1.5 and IPGA/7-ADCA 30.46 IU/mmol.Under the optimized conditions,the maximum conversion ratio was obtained as 98.5%with the productivity of 29.5 mmo/L/h and hydrolysis ratio of 35.9%.The reaction kinetics of the enzyamtic synthesis of CEX was studied using PGME and 7-ADCA as substrates and IPGA as biocatalyst.The reaction rate equations of enzymatic synthesis of CEX were firstly deduced.By combining the enzymatic hydrolysis of PGME and the enzymatic hydrolysis of CEX,the reaction rate constants of the enzymatic synthesis of CEX were calculated.Analysis of the reaction rate constants indicated that the binding of the enzyme to the side chain donor was a rate-limiting step for the enzymatic synthesis of CEX.Compared with the hydrolysis of CEX,the hydrolysis of PGME in the enzymatic synthesis of CEX was the main reason that limited the conversion ratio of the enzymatic synthesis of CEX.According to the kinetic analysis,the optimal pH of the enzymatic synthesis of CEX was 6.0-6.5,and an increase in pH would promote PGME hydrolysis and CEX hydrolysis,which was not conducive to the synthesis reaction.The results of activation energy showed that high temperature could aggravate the hydrolysis of PGME and the hydrolysis of CEX,and the low temperature was beneficial to the synthesis of CEX.The increase of amount of enzyme would aggravate the occurrence of internal diffusional restrictions in the immobilized enzyme particles.In order to improve the reaction productivity of the enzymatic synthesis of CEX,the suspension solution system was developed.The effects of PGME addition method,pH,temperature,enzyme amount,PGME feeding time,initial 7-ADCA concentration and PGME/7-ADCA molar ratio on the enzymatic synthesis were investigated.Results showed that continuous addition of PGME was more efficient for the synthesis of CEX than batch addition,the PGME hydrolysis ratio decreased by 12%as well as the conversion ratio and productivity increased by 2.8%and 2.7%,respectively.Under pH 7.0,temperature 15?,7-ADCA concentration 659 mmol/L,PGME/7-ADCA molar ratio 1.12,IPGA/7-ADCA 22.85 IU/mmol and PGME feeding time 60 min,the maximum conversion ratio was obtained as 99.3%with the productivity of 200 mmol/L/h and hydrolysis ratio of 11.4%.Compared with the enzymatic synthesis in fully aqueous solution system,the conversion ratio and productivity increased by 5.4%and 395%while the hydrolysis ratio decreased by 36.7%.In order to achieve the enzymatic synthesis of CEX at very high substrate concentrations,the dissolution behavior of 7-ADCA in the aqueous solution was studied in the presence of PGME.It was found that the presence of PGME could increase the solubility of 7-ADCA.Furthermore,a 7-ADCA solubilization system was obtained using a pH shift strategy in the presence of PGME.Under pH 6.5,temperature 15? and PGME 900 mmol/L,the concentration of 7-ADCA in aqueous solution was increased to be 673 mmol/L,which was 25.88 times higher than the solubility of 7-ADCA in pure water.Based on the pH shift strategy,the 7-ADCA solubilization system with PGME had better stability than the 7-ADCA solution without PGME.The results of ultraviolet spectrum,fluorescence spectrum,Raman spectrum,and density functional theory(DFT)calculations showed that there was hydrogen bonding interaction between PGME and 7-ADCA molecules,which lead to the increase of the dissolution of 7-ADCA in aqueous solution.The enzymatic synthesis of CEX at the 7-ADCA solubilization system was performed.The effects of substrate concentrations,molar ratios of substrates and seed addition on enzymatic synthesis were investigated.Under pH 6.5,tempearure 15?,7-ADCA 400 mmol/L,PGME/7-ADCA molar ratio 1.12,seeding time 8 min and seed amount 6.8 mmol/L,the maximum conversion ratio was obtained as 99.4%with the productivity of 312.3 mmol/L/h and hydrolysis ratio of 11.3%.Finally,the 7-ADCA solubilization system and suspension solution system were compared for the enzymatic synthesis of CEX,and their conversion ratios both reached relatively high levels,and the PGME hydrolysis ratios were also comparable.However,the reaction productivity at the 7-ADCA solubilization system was 56.2%higher than that of the suspension solution system.