【作者】 陈琦；

【导师】 金志华；

【作者基本信息】 浙江大学 ， 化学工程（专业学位）， 2019， 硕士

【摘要】 他汀类药物是目前最为经典和有效的降脂类药物,其结构中的双手性侧链是他汀类药物发挥药效的重要活性部位,也是合成他汀类药物过程中必不可少的中间体。该手性侧链的化学合成方法存在工艺复杂、立体选择性较差、环境污染大等明显缺陷,而生物酶催化方法具有明显的条件温和、立体选择性高、环境友好等优势。本文选取2-脱氧核糖-5-磷酸醛缩酶(2-deoxyribose-5-phosphate aldolase,DERA)作为生物催化剂,催化乙醛和氯乙醛进行不对称羟醛缩合反应生成他汀类药物侧链中间体(4R,6S)-6-氯甲基-2H-吡喃-2,4-二醇。本文选取DERA原始序列上的关键位点,通过PCR技术对野生型DERA进行分子改造,寻找到多个DERA突变体。以氯乙醛醛基浓度和吸光度的线性方程y=0.2833x+0.3204为基础,通过2,4-二硝基苯肼颜色法检测并最终筛选出10株催化活力优于野生型的突变株,其中酶活提高最多的是cs-2,对应的突变位点和氨基酸是第233位的丝氨酸(Ser,S)突变为谷氨酸(Glu,E)。探索重组工程菌cs-2的最佳发酵条件,最终确定最佳的发酵条件为:培养基pH为7.0,加诱导剂的时机为发酵液OD600=0.7,加入诱导剂终浓度为1mM,诱导温度为30℃,诱导时间为12h。在最佳发酵条件下,在5L发酵罐中进行高密度发酵突变株cs-2,终止发酵时发酵液的OD600=103.5936。对重组工程菌cs-2催化乙醛和氯乙醛进行不对称羟醛缩合反应的条件进行优化,得到该反应进行的最适pH为6.0,最适反应温度为30℃,反应最适酶底比约为1.8:1。根据优化后的反应条件,按照以下条件进行催化反应:以200mM磷酸-咪唑溶液作为缓冲液,在30℃下按照酶底比1.8:1进行羟醛缩合反应,制备得到浅黄色油状液体共64.48g,底物转化率为93%,收率为73%。更多还原

【Abstract】 Nowadays statins are the most classical and effective lipid-lowering drugs.The bis chiral side-chain in the structure of statins is the important active sites for statins to exert their pharmacodynamic effects,and also is the essential intermediate in the synthesis of statins.The chemical synthesis of chiral side-chain has obvious defects,such as complex process,poor stereoselectivity and environmental pollution,while enzymatic catalysis has obvious advantages such as mild conditions,high stereoselectivity and environmental friendliness.In this paper,2-deoxyribose-5phosphate aldolase(DERA)was selected as the biocatalyst to catalyze the asymmetric aldol condensation of acetaldehyde and chloracetaldehyde to produce statin side-chain intermediate(4R,6S)-6-chloromethyl-2H-pyran-2,4-diol.In this paper,several DERA mutant strains were found by molecular modification of wild-type DERA by PCR using key sites on the original DERA sequence.Based on the linear equation y=0.2833x+0.3204 of chloracetaldehyde aldehyde concentration and absorbance,10 mutants with better catalytic activity than wild type were screened by 2,4-dinitrophenylhydrazine color method.Among them,mutant cs-2 was the most active mutant,and the corresponding mutation site and amino acid were 233-position serine(Ser,S)mutated to glutamic acid(Glu,E).The optimum fermentation conditions of recombinant engineering bacteria cs-2 were explored,and the optimum fermentation conditions were determined as follows:the pH of the medium was 7.0,the time of adding inducer was OD600=0.7,the final concentration of inducer was 1 mM,the induction temperature was 30 centigrade,and the induction time was 12 h.Under the optimum fermentation conditions,the highdensity fermentation of the mutant strain cs-2 was in progress in 5 L fermenter,and the OD600 of fermentation broth was 103.5936 at the end of fermentation.The conditions of aldol condensation of acetaldehyde and chloracetaldehyde catalyzed by recombinant engineering bacteria cs-2 were optimized.The optimum pH,temperature and enzyme-to-substrate ratio were 6.0,30 centigrade and 1.8:1.According to the optimized reaction conditions,catalytic reaction was carried out under the following conditions:using 200 mM phosphoric acid-imidazole solution as buffer solution,aldol condensation was carried out at 30 centigrade according to the enzymeto-substrate ratio of 1.8:1.A total of 64.48g of light yellow oily liquid was prepared,the conversion of substrate was 93%,and the final yield was 73%.更多还原