一种新颖海洋微生物酯酶的功能鉴定及其在D-乳酸甲酯制备中的应用（英文）

发布时间：2018-03-16 20:14

本文选题：酯酶　切入点：海洋微生物　出处：《催化学报》2016年08期 　论文类型：期刊论文

【摘要】：手性药物不同对映体往往表现出截然不同的生理活性和毒性,为了减少有毒副作用的对映体,并降低其生物活性,光学纯手性药物的合成一直是制药行业的研究热点.由于手性药物中间体是合成手性药物的重要构建模块,因此手性药物中间体的合成至关重要.手性乳酸及其酯是合成各类药物、农药和聚合物的重要中间体,在制药工业和材料工业中手性乳酸及其酯的制备非常重要.手性乳酸及其酯可以通过传统的有机化学合成和生物酶催化合成,通过有机化学合成法往往很难得到光学纯度较高的手性乳酸及其酯,而生物酶催化法可以得到光学纯的手性乳酸及其酯,同时避免了有机化学合成所导致的金属残留和环境污染等问题.生物酶法合成光学纯的乳酸及其酯可以通过脱氢酶不对称还原酮的前体得到,然而生物催化使用脱氢酶法需要价格昂贵的辅助因子,如NADH和NADPH.而另外一种生物催化方法是通过利用酯酶或者脂肪酶不对称水解外消旋的酯,从而得到光学纯度较高的手性中间体.目前市场上的L-乳酸甲酯价格不太昂贵,因为L-乳酸甲酯可以直接通过大发酵的方法取代有机化学法和酶法直接得到.然而D-乳酸甲酯不能使用廉价的发酵法直接得到,因而其价格昂贵.生物酶催化法可能会成为制备D-乳酸甲酯的主要方法,因为利用生物酶法可以得到光学纯度较高的D-乳酸甲酯.本文从西太平洋深海来源的微生物Pseudomonas oryzihabitans HUP022中克隆并异源表达了一种新颖酯酶PHE14.通过对酯酶PHE14的酶学性质鉴定表明,酯酶PHE14的最适反应底物为对硝基苯酚乙酸酯(C2),最适pH为9.0,最适温度为60°C.PHE14催化最适反应底物C2的活性达到293.07U/mg,Vmax和Km分别为200μM/(mg·min)和0.24mmol/L.酯酶PHE14对多种有机溶剂、表面活性剂和金属离子都具有非常好的耐受性.深海微生物酯酶PHE14对高浓度NaCl具有很好的耐受性,在4mol/LNaCl存在下,相对酶活力为71.4%.同时,酯酶PHE14能够催化消旋乳酸甲酯的不对称水解反应制备重要的手性化工产品—D-乳酸甲酯.与先前的一些酯酶拆分的报道不同,有机溶剂和表面活性剂对酯酶PHE14催化的动力学水解反应没有促进作用.而且,本研究是首次通过酶动力学水解拆分反应制备光学纯的D-乳酸甲酯.经过实验优化,在pH9.0和30°C的条件下,反应产物D-乳酸甲酯的对映体过量值和产率分别为99%和88.7%.深海微生物酯酶PHE14作为一种绿色生物催化剂,在多种工业的不对称合成中都具有非常好的应用潜力.
[Abstract]:Different enantiomers of chiral drugs often exhibit distinct physiological activities and toxicity, in order to reduce the enantiomers with toxic side effects and their biological activities. The synthesis of optical pure chiral drugs has been a hot topic in the pharmaceutical industry. Because chiral intermediates are important building blocks for the synthesis of chiral drugs, Therefore, the synthesis of chiral pharmaceutical intermediates is very important. Chiral lactic acid and its esters are important intermediates for the synthesis of various drugs, pesticides and polymers. The preparation of chiral lactic acid and its esters is very important in the pharmaceutical and material industries. Chiral lactic acid and its esters can be synthesized by traditional organic chemistry and catalyzed by biological enzymes. Chiral lactic acid and its esters with high optical purity are often difficult to be obtained by organic chemical synthesis, while chiral lactic acid and its esters with optical purity can be obtained by bioenzyme catalysis. At the same time, the problems of metal residue and environmental pollution caused by organic chemical synthesis are avoided. The synthesis of optical pure lactic acid and its esters by biological enzyme method can be obtained by the precursor of dehydrogenase asymmetric reductor. However, the use of dehydrogenase in biocatalysis requires expensive cofactors, such as NADH and NADPH.Another biocatalysis involves asymmetric hydrolysis of racemic esters using esterase or lipase. Thus the chiral intermediates with high optical purity can be obtained. At present, the price of L-lactate methyl ester on the market is not too high. Because methyl L-lactic acid can be obtained directly by large fermentation instead of organic and enzymatic methods, but D- methyl lactate can't be obtained directly by cheap fermentation. So it's expensive. Bioenzyme catalysis may be the main method for preparing methyl Dlactate. In this paper, a novel esterase PHE14 was cloned from Pseudomonas oryzihabitans HUP022 from the deep-sea microorganism of the western Pacific Ocean. The enzymatic properties of the esterase PHE14 were identified. The optimum substrate of esterase PHE14 is p-nitrophenol acetate C2O, the optimum pH is 9.0, the optimum temperature is 60 掳C. PHE14, the activity of substrate C2 is 293.07UmgGN Vmax and km is 200 渭 Mr / mg 路min and 0.24mmol / L respectively. Both surfactants and metal ions have very good tolerance. Deep sea microbial esterase PHE14 has good tolerance to high concentration of NaCl. In the presence of 4mol / L NaCl, the relative enzyme activity is 71.4. Esterase PHE14 can catalyze asymmetric hydrolysis of racemic methyl lactate to produce important chiral chemical product D- methyl lactate. Organic solvents and surfactants did not promote the kinetic hydrolysis of esterase PHE14. Moreover, this study was the first time to prepare optically pure methyl Dlactate by enzymatic hydrolysis and resolution. Under the conditions of pH9.0 and 30 掳C, the enantiomeric excess value and yield of D- methyl lactate were 99% and 88.7, respectively. Deep-sea microbial esterase PHE14 was used as a green biocatalyst. It has good application potential in asymmetric synthesis of many industries.
【作者单位】：中国科学院南海海洋研究所中国科学院热带海洋生物资源与生态重点实验室;中国科学院大学;中国科学院南海海洋研究所广东省海洋药物重点实验室;南海生物资源开发和利用协同创新中心;
【基金】：supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA11030404);Key Project from the Chinese Academy of Sciences (KGZD-EW-606) the National Natural Science Foundation of China (21302199) Guangzhou Science and Technology Plan Projects (201510010012)~~
【分类号】：TQ460.1;O629.8

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