生物不对称催化中酶立体选择性的定向调控

发布时间：2018-08-23 08:36

【摘要】：生物催化法由于具有立体选择性强、反应条件温和、环境友好等优点而深受青睐，酶立体选择性的定向调控在生物催化中具有重要意义。通过对酶不对称催化选择性的定向调控，可以控制反应进行的方向与程度，获得高光学纯度的、具有特殊结构的目标产物。本文以磷脂酶B（PLB）和氧化还原酶为研究对象，探讨其立体选择性的定向调控策略，主要研究结果如下：（1）对菌株Candida albicans MYA-2876产磷脂酶B的发酵条件进行了优化，确定了最适发酵温度30℃，初始pH6.5，接种量为8%，培养时间为72h。初始纯化酶条件为30%浓度饱和硫酸铵，80%浓度沉淀目的蛋白，经过透析得到浓缩酶液，酶活可达201.36U/mg-protein。（2）考察了Candida albicans MYA-2876PLB的酶学性质，结果表明其最适反应温度是30℃，最适pH为7.8。温度与pH对酶活影响较大，0.1mM Mg2+与Ca2+对酶活有促进作用，分别提高了23.5%与18.8%，PLB对卵磷脂乳化液的底物适应性较好。（3）考察反应条件(反应温度、pH以及金属离子)、介质工程（有机溶剂、离子液体、两相体系）、化学修饰与固定化酶对磷脂酶B的催化位点选择性的影响。结果表明，离子液体对磷脂酶B的催化位点选择性的调控效果较好，在[Omim][D-HL]作用下磷脂酶B的Sn-1位点催化选择性CSS1得到较大提高（接近100%），而在[Emim]Cl作用下Sn-2位点催化选择性CSS2提高到47.08%；两相体系对磷脂酶B的催化位点选择性的调控效果一般，对产率影响较大，大部分相比水相提高3倍以上。正己烷-水（体积比1:1）两相系统CSS1提高最大至84.78%，2-庚醇-水（体积比1:1）两相系统CSS2提高最大至43.77%。（4）考察了乙醇脱氢酶ADH-A3263，ADH-A7011以及大肠杆菌重组酮还原酶E-Ketoreductase与八个底物2-辛酮、2-庚酮、苯乙酮、1-苯丙酮、1-苯乙醇、1-苯丙醇、2-庚醇和2-辛醇间的酶催化反应。结果表明，此三种酶均在30℃到35℃时酶活较高；ADH-A3263、ADH-A7011酶活最适pH偏碱性，E-Ketoreductase酶活最适pH偏中性；利用自动旋光仪测旋光率通过考察反应温度与pH，初步筛选出更合适的酶催化底物苯乙酮、1-苯丙酮、1-苯乙醇1-苯丙醇。（5）考察反应系统(反应温度、pH以及金属离子)、介质工程（有机溶剂、离子液体、两相体系）、酶的化学修饰、固定化酶以及辅酶再生对氧化还原酶的对映体选择性的影响。结果表明，本实验中有机溶剂对E-Ketoreductase催化1-苯丙酮的对映体选择性调控有较好效果，在异辛烷（含水量1%）中反应S型对映体选择率（E）高达30.885，，而在正己烷（含水量1%）中反应时却为R型选择性，E值为3.207；离子液体对E-Ketoreductase催化1-苯丙酮的对映体选择性调控有十分明显的效果，产物发生构象翻转，反应体系添加[Emim][HL]后R型E值为6.769，而添加[Bmim]PF6时却为S型选择性，E值高达到200以上；两相体系对E-Ketoreductase催化1-苯丙酮的对映体选择性调控有很好的效果，且与有机溶剂体系相比，转化率普遍都有一定提高，在异辛烷-水（体积比1:1）中反应S型对映体选择率E最大达到333.448，而在辛酸乙酯-水（体积比1:1）中反应时R型E最大达到7.890。
[Abstract]:Biocatalytic method is very popular because of its strong stereoselectivity, mild reaction conditions and friendly environment. The directional regulation of enzyme stereoselectivity is of great significance in biocatalysis. In this paper, phospholipase B (PLB) and oxidoreductase were studied to investigate the stereoselective directional regulation strategies. The main results are as follows:
(1) The optimum fermentation conditions for the production of phospholipase B by Candida albicans MYA-2876 were optimized. The optimum fermentation temperature was 30 C, initial pH 6.5, inoculation amount was 8%, culture time was 72 h. The initial purified enzyme conditions were 30% saturated ammonium sulfate, 80% precipitated the target protein, and the concentrated enzyme solution was obtained after dialysis. The enzyme activity was 201.36 U/mg-protein.
(2) The enzymatic properties of Candida albicans MYA-2876 PLB were investigated. The results showed that the optimum reaction temperature was 30 C and the optimum pH was 7.8. Temperature and pH had a great influence on the enzyme activity. 0.1 mMg2+ and Ca2+ promoted the enzyme activity by 23.5% and 18.8% respectively. PLB had a better substrate adaptability to lecithin emulsion.
(3) The effects of reaction conditions (reaction temperature, pH and metal ions), medium engineering (organic solvents, ionic liquids, two-phase system), chemical modification and immobilized enzyme on the site selectivity of phospholipase B were investigated. Catalytic selectivity of CSS1 at the Sn-1 site of enzyme B was greatly improved (nearly 100%) while the selectivity of CSS2 at the Sn-2 site was increased to 47.08% under the action of [Emim] Cl. The two-phase system had a general effect on the selectivity of catalytic site of phospholipase B, and had a greater effect on the yield, most of which was more than three times higher than that in the aqueous phase. The phase system CSS1 increased to the maximum of 84.78%, and the CSS2 of 2- heptanol water (volume ratio 1:1) two-phase system increased to the maximum of 43.77%..
(4) The enzymatic reactions of alcohol dehydrogenase ADH-A3263, ADH-A7011, recombinant ketone reductase E-Ketoreductase with eight substrates, 2-octanone, 2-heptanone, acetophenone, 1-phenylacetone, 1-phenylethanol, 1-phenylpropanol, 2-heptanol and 2-octanol, were investigated. The optimum pH of enzyme activity was alkaline, and the optimum pH of E-Ketoreductase activity was neutral. The optimum substrate for enzyme catalysis was acetophenone, 1-phenylacetone and 1-phenylethanol-1-phenylpropanol by measuring the optical rotation with automatic polarimeter.
(5) The effects of reaction system (reaction temperature, pH and metal ions), medium engineering (organic solvent, ionic liquid, two-phase system), chemical modification of enzyme, immobilized enzyme and coenzyme regeneration on the enantioselectivity of redox enzyme were investigated. The results showed that the enantioselectivity of 1-phenylacetone catalyzed by E-Ketoreductase in organic solvents was improved. The selectivity of S-type enantiomers (E) was as high as 30.885 in isooctane (water content 1%) and R-type enantiomers (E value 3.207) in n-hexane (water content 1%). Ionic liquids had a very obvious effect on the enantioselective regulation of 1-phenylacetone catalyzed by E-Ketoreductase. When [Emim] [HL] was added, the R-type E value was 6.769, but when [Bmim] PF6 was added, it was S-type selectivity, and the E value was above 200. The two-phase system had a good effect on enantioselective regulation of 1-phenylacetone catalyzed by E-Ketoreductase, and the conversion of 1-phenylacetone was generally higher than that of organic solvent system. The maximum enantioselectivity E of S-type in the medium reaction was 333.448, and that of R-type E in the ethyl octanoate-water (volume ratio 1:1) was 7.890.
【学位授予单位】：北京理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TQ032.4

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