摘要
镰孢霉菌内酯水解酶是一种高效的生物催化剂,主要功能是催化内酯水解生成羟基酸。它对底物专一性强且立体选择性优越,因而广泛应用于工业化生产D-泛内酯。近二十年来,随着对镰孢霉菌内酯水解酶研究的深入,关于它的筛选、催化、纯化、克隆和表达方面的报道不断涌现。然而这些研究都围绕着一个产品D-泛内酯而展开,虽然偶有关于镰孢霉菌内酯水解酶催化水解其它底物的研究报道,但是不够系统和深入。
本实验室已经从土壤微生物中筛选得到一株具有较高活性和选择性的真菌,镰孢霉菌Fusarium proliferatum (Matsushima) Nirenberg ECU2002,它能不对称水解手性2-羟基-4-丁内酯。在这样的背景下,本文进一步研究了镰孢霉菌ECU2002内酯水解酶对各种内酯的催化效果,致力于探索其在不对称合成尤其是不对称催化中的新应用。
本文主要分为三个部分,第一部分是镰孢霉菌ECU2002内酯水解酶的克隆、表达、纯化和催化性能研究。重组后的镰孢霉菌内酯水解酶(recombinant Fusarium proliferatum lactonase,简写reFPL)在大肠杆菌体内表达,从而进一步提高了reFPL的产酶效率,使其成为一种方便易得的生物催化剂。人为地在酶分子的N-端添加组氨酸标签并建立了方便快捷的纯化方法。通过广泛尝试reFPL对一系列内酯化合物的水解活性,本文发现2-羟基-4-取代-4-丁内酯也是reFPL较为合适的底物。
第二部分详细研究了重组后的镰孢霉菌ECU2002内酯水解酶(reFPL)对2-羟基-4-取代-4-丁内酯这一类化合物的水解性能。首先通过经典的克莱森缩合反应合成了2-羟基-4-取代-4-丁内酯,并通过硅胶柱层析分离了内酯的顺、反两种非对映异构体。然后分别以顺式和反式内酯为底物,研究了酶促拆分的效果。结果表明,2-羟基-4-取代-4-丁内酯的四种立体异构体中的三种都能通过reFPL以光学纯的形式制备出来。而且,reFPL的底物谱比较广、能耐受较高的底物浓度(200 mM)并可以回收重复利用(至少3次)。最后,对水解产物的构型从多个角度进行了表征。通过核磁共振技术(1H-NMR和NOE),对内酯的相对构型进行了鉴定,并经过X-射线衍射数据进行了验证。对于绝对构型,本文通过X-射线衍射(XRD)技术、圆二色光谱(CD)技术、化学相关法和比对旋光等手段从多个角度进行了鉴定。
第三部分的主要内容,是在酶促水解2-羟基-4-苯基-4-丁内酯的基础上,建立新路线合成2-羟基-4-苯基丁酸：一种合成血管紧张肽转化酶抑制剂(Angiotensin Converting Enzyme inhibitor)类药物的合成前体。本文的研究结果表明,reFPL对顺／反两种内酯的对映选择性水解可以在相同的时间内完成。因此通过酶促水解顺／反-2-羟基-4-苯基-4-丁内酯的混合物,再将水解产物直接氢化(Pd／C催化)还原的方式,最终制备获得了光学纯2-羟基-4-苯基丁酸的两种对映异构体。
Fusarium lactonase is a kind of efficient biocatalyst and its most important capacity is the catalytic hydrolysis of lactones into hydroxyl acids. Due to its great specificity and enantioselectivity, Fusarium lactonase has now been used widely in the industrialized production of D-pantolactone. In past twenty years, reports have been emerging regarding its screening, catalysis, purification, cloning and expression. However, all of these researches focused on the production of D-pantolactone, and only isolated reports are on Fusarium lactonase-mediated hydrolysis of other substrates.
One fungus, Fusarium proliferatum (Matsushima) Nirenberg ECU2002, has been isolated from soil samples. It shows great activity and stereoselectivity in hydrolyzing 2-hydroxy-4-butyrolactones. In this paper, we carry out further researches on the Fusarium lactonase-catalyzed hydrolysis of chiral lactones, focusing on new application in asymmetric synthesis, especially in asymmetric catalysis.
This paper is consisted of three parts. In the first part, we describe the cloning and expression of the lactonase gene from Fusarium proliferatum ECU2002 in E. coli JM109 (DE3) (reFPL). By optimizing expression conditions, the lactonase production was significantly enhanced making reFPL an easy-to-make biocatalyst. The reFPL was then purified with the aid of His tags before the N-terminal of mutual enzyme. Furthermore, biocatalytic properties of reFPL were also investigated, particularly its hydrolytic activity towards lactones, and we found that 4-substituted 2-hydroxy-4-butyrolactone was also suitable substrate for reFPL.
The second part of this paper focused on the reFPL-catalyzed hydrolysis of 4-substituted 2-hydroxy-4-butyrolactones.4-Substituted 2-hydroxy-4-butyrolactones was synthesized via classical Claisen condensation, and in most cases cis-and trans-lactones were separable via chromatography on silica gel. Enzymatic resolution was then carried out on these cis-and trans-racemic mixtures. Three out of four possible stereoisomers of 4-substituted 2-hydroxy-4-butyrolactones could be prepared in good optical purity. ReFPL shows hydrolytic activity towards a broad substrate spectrum against high substrate concentration (up to 200 mM), and it is also recyclable (at least three times). In the end, the configuration of the enzymatic hydrolyzed product was also assigned using different measures. The relative configuration was assigned according to the1H NMR and Nuclear Overhauser Effect (NOE) experiments and confirmed by X-ray diffraction. As for the absolute structure, it was assigned by different methods including X-ray diffraction and circular dichroism.
In the third part, a new route to chiral 2-hydroxy-4-phenylbutyrates, precursors to the angiotensin converting enzyme inhibitors, was developed on the basis of reFPL-catalyzed hydrolysis of 2-hydroxy-4-phenyl-4-butyrolactone. The enantioselective hydrolysis of cis-and trans-lactones could be completed within the same time. Hydrogenation of this mixture over Pd/C affords chiral 2-hydroxy-4-phenylbutyrates. This route has great potential for industrial application.
引文
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