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头孢氨苄酶催化合成过程调控及应用基础研究
范宜晓
Thesis Advisor刘庆芬
2020-07-01
Degree Grantor中国科学院大学
Degree Name博士
Degree Discipline生物化工
Keyword酶催化合成,动力学控制,头孢氨苄,过程调控,反应体系
Abstract

与传统的β-内酰胺抗生素相比,半合成β-内酰胺抗生素具有抗菌谱广和稳定性好的优点,是应用最广泛的一类抗感染药物。目前,化学合成法主导着半合成β-内酰胺抗生素的工业化生产,能耗高、效率低、排放大量难降解污染物是其难以逾越的弊端。环境友好的酶催化合成法一直受到研究者的重视,其中动力学控制下的酶催化合成路线是一种相对高效的方法。但由于对该路线的合成过程缺乏系统的研究和认识,致使其一直存在底物摩尔比例高、转化率低、反应产率低等问题。本论文以生产量较大的半合成β-内酰胺抗生素头孢氨苄(CEX)作为研究目标,以7-氨基-3-去乙酰氧基头孢烷酸(7-ADCA)为母核,固定化青霉素G酰化酶(IPGA)为生物催化剂,系统研究了其动力学控制下的酶催化合成过程,开发了悬浮液体系和增溶体系中的酶催化合成工艺。主要研究内容和结论如下:在全溶液体系中,系统研究了动力学控制下头孢氨苄的酶催化合成过程。考察了不同侧链苯甘氨酸甲酯盐酸盐(PGME)和苯甘氨酰胺(PGA)下的酶催化合成过程,在此基础上研究了pH、温度、酶量、底物浓度和底物摩尔比例对酶催化合成头孢氨苄的综合影响。与PGA作为侧链的反应相比,以PGME作为侧链的转化率和反应产率分别提高45.9%和1234.5%。得到了较优的工艺条件为:pH 6.5、温度15℃、7-ADCA 60 mmol/L、PGME/7-ADCA摩尔比例 1.5和IPGA/7-ADCA 30.46 IU/mmol。在较优反应条件下,最大转化率为98.5%,反应产率为29.5 mmol/L/h,PGME水解率为35.9%。以7-ADCA为母核,PGME为侧链,IPGA为生物催化剂,研究了酶催化合成头孢氨苄的反应动力学。推导了酶催化合成头孢氨苄的反应速率方程,通过酶催化水解PGME和酶催化水解头孢氨苄相结合的方法,求解了酶催化合成头孢氨苄的反应速率常数。反应速率常数分析表明,酶与侧链供体的结合是酶催化合成头孢氨苄的速控步骤。与头孢氨苄的水解相比,PGME的水解是限制酶催化合成头孢氨苄转化率的主要原因。酶催化合成的最佳pH为6.0-6.5,升高pH会加剧PGME和头孢氨苄的水解,不利于头孢氨苄的合成。对温度影响的动力学和活化能分析表明,与头孢氨苄的合成反应相比,升高温度更能加剧PGME水解和头孢氨苄水解,适当的低温有利于头孢氨苄的合成。酶量的过度增加会加剧固定化酶颗粒内部内扩散限制效应发生。为了提高酶催化合成头孢氨苄的反应产率,开发了悬浮液体系中头孢氨苄酶催化合成工艺。考察了PGME加入方式、pH、温度、酶量、PGME加入时间、初始7-ADCA浓度和PGME/7-ADCA摩尔比对酶催化合成的影响。与批次加入比较,PGME连续加入可使其水解率降低了12%,转化率和反应产率分别提高2.8%和2.7%,PGME连续加入更有利于头孢氨苄的酶催化合成。在pH 7.0、温度15℃、初始7-ADCA浓度659 mmol/L、PGME/7-ADCA摩尔比例1.12、IPGA/7-ADCA 22.85 IU/mmol和PGME加入时间60 min下,最大转化率为99.3%,反应产率为200 mmol/L/h,PGME水解率为11.4%。与相同条件下全溶液体系中酶催化合成比较,悬浮液体系中转化率提高5.4%,反应产率提高395%,水解率降低36.7%。为了实现高底物浓度下酶催化合成头孢氨苄,研究了PGME存在下7-ADCA在水相介质中的溶解行为。溶解度的测定表明,PGME的存在会提高7-ADCA的溶解度。在此基础上,采用pH调控的策略制备得到了7-ADCA的增溶体系,显著增强了7-ADCA在水中的溶解。在pH 6.5、温度15℃和PGME 900 mmol/L下,7-ADCA在水中的浓度达到673 mmol/L,比相同条件下7-ADCA的溶解度提高25.88倍。与PGME不存在的7-ADCA溶液相比,该PGME存在下的7-ADCA增溶体系具有更好的稳定性。紫外光谱、荧光光谱、拉曼光谱以及量化计算的结果表明,PGME和7-ADCA分子之间存在氢键相互作用,其是PGME存在下7-ADCA溶解增强的推动力。在7-ADCA溶解增强研究的基础上,进一步开展了增溶体系中酶催化合成头孢氨苄的研究。研究了底物浓度、底物摩尔比例以及晶种添加对酶催化合成的影响。在pH 6.5、温度15℃、7-ADCA 400 mmol/L、PGME/7-ADCA摩尔比例 1.12、晶种添加时间8 min和晶种添加量6.8 mmol/L下,最大转化率为99.4%,反应产率为312.3 mmol/L/h,PGME水解率为11.3%。将该合成工艺与悬浮液体系中的合成工艺进行了对比,它们的转化率均达到了比较高的水平,PGME水解率也相当,但是增溶体系的反应产率比悬浮液体系的提高56.2%,并且前者的合成工艺比后者更为简单。;Compared with traditional β-lactam antibiotics, semi-synthetic β-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 β-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 β-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% at 120 min 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% at 120 min 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 prepared 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. 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% at 80 min 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 ratio was also comparable. However, the reaction productivity at the 7-ADCA solubilization system was 56.2% higher than that of the suspension solution system.

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/49688
Collection中国科学院过程工程研究所
Recommended Citation
GB/T 7714
范宜晓. 头孢氨苄酶催化合成过程调控及应用基础研究[D]. 中国科学院大学,2020.
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