本文選題：酯酶　切入點：海洋微生物　出處：《催化學(xué)報》2016年08期 　論文類型：期刊論文

【摘要】：手性藥物不同對映體往往表現(xiàn)出截然不同的生理活性和毒性,為了減少有毒副作用的對映體,并降低其生物活性,光學(xué)純手性藥物的合成一直是制藥行業(yè)的研究熱點.由于手性藥物中間體是合成手性藥物的重要構(gòu)建模塊,因此手性藥物中間體的合成至關(guān)重要.手性乳酸及其酯是合成各類藥物、農(nóng)藥和聚合物的重要中間體,在制藥工業(yè)和材料工業(yè)中手性乳酸及其酯的制備非常重要.手性乳酸及其酯可以通過傳統(tǒng)的有機化學(xué)合成和生物酶催化合成,通過有機化學(xué)合成法往往很難得到光學(xué)純度較高的手性乳酸及其酯,而生物酶催化法可以得到光學(xué)純的手性乳酸及其酯,同時避免了有機化學(xué)合成所導(dǎo)致的金屬殘留和環(huán)境污染等問題.生物酶法合成光學(xué)純的乳酸及其酯可以通過脫氫酶不對稱還原酮的前體得到,然而生物催化使用脫氫酶法需要價格昂貴的輔助因子,如NADH和NADPH.而另外一種生物催化方法是通過利用酯酶或者脂肪酶不對稱水解外消旋的酯,從而得到光學(xué)純度較高的手性中間體.目前市場上的L-乳酸甲酯價格不太昂貴,因為L-乳酸甲酯可以直接通過大發(fā)酵的方法取代有機化學(xué)法和酶法直接得到.然而D-乳酸甲酯不能使用廉價的發(fā)酵法直接得到,因而其價格昂貴.生物酶催化法可能會成為制備D-乳酸甲酯的主要方法,因為利用生物酶法可以得到光學(xué)純度較高的D-乳酸甲酯.本文從西太平洋深海來源的微生物Pseudomonas oryzihabitans HUP022中克隆并異源表達(dá)了一種新穎酯酶PHE14.通過對酯酶PHE14的酶學(xué)性質(zhì)鑒定表明,酯酶PHE14的最適反應(yīng)底物為對硝基苯酚乙酸酯(C2),最適pH為9.0,最適溫度為60°C.PHE14催化最適反應(yīng)底物C2的活性達(dá)到293.07U/mg,Vmax和Km分別為200μM/(mg·min)和0.24mmol/L.酯酶PHE14對多種有機溶劑、表面活性劑和金屬離子都具有非常好的耐受性.深海微生物酯酶PHE14對高濃度NaCl具有很好的耐受性,在4mol/LNaCl存在下,相對酶活力為71.4%.同時,酯酶PHE14能夠催化消旋乳酸甲酯的不對稱水解反應(yīng)制備重要的手性化工產(chǎn)品—D-乳酸甲酯.與先前的一些酯酶拆分的報道不同,有機溶劑和表面活性劑對酯酶PHE14催化的動力學(xué)水解反應(yīng)沒有促進(jìn)作用.而且,本研究是首次通過酶動力學(xué)水解拆分反應(yīng)制備光學(xué)純的D-乳酸甲酯.經(jīng)過實驗優(yōu)化,在pH9.0和30°C的條件下,反應(yīng)產(chǎn)物D-乳酸甲酯的對映體過量值和產(chǎn)率分別為99%和88.7%.深海微生物酯酶PHE14作為一種綠色生物催化劑,在多種工業(yè)的不對稱合成中都具有非常好的應(yīng)用潛力.
[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 擄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 擄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.
【作者單位】： 中國科學(xué)院南海海洋研究所中國科學(xué)院熱帶海洋生物資源與生態(tài)重點實驗室;中國科學(xué)院大學(xué);中國科學(xué)院南海海洋研究所廣東省海洋藥物重點實驗室;南海生物資源開發(fā)和利用協(xié)同創(chuàng)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)~~
【分類號】：TQ460.1;O629.8

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