【作者】 陈辉；

【导师】 杨立荣； 吴坚平； 徐刚；

【作者基本信息】 浙江大学 ， 生物化工， 2014， 博士

【摘要】 l-薄荷醇是一种重要的手性化合物,具有特别的薄荷香气和强烈的清凉作用,气味新鲜轻快,同时它还具有兴奋镇痛、杀菌止痒、促渗透、助消化等功效,因此具有很高的工业和医药应用价值。由于天然提取的l-薄荷醇的品质易受各种因素的影响且近年来市场需求越来越大,天然来源的l-薄荷醇已不再满足日益增长的需求。采用生物催化的方法获得l-薄荷醇正逐渐成为研究的热点。本文在前期筛选得到一株产碱假单胞菌(Pseudomonas alcaligenes CGMCC4405),能够产生对四对外消旋薄荷醇丙酸酯进行选择性水解的脂肪酶(PaL)。在此基础上,通过构建基因文库对PaL的基因进行了克隆和异源表达。对重组PaL进行了表达纯化测定其精确分子量为58094.3Da。通过肽指纹图谱验证了由DNA序列推测得到的氨基酸序列的准确性。远紫外区圆二色谱结果表明PaL含有26.8%的α螺旋,34.2%的β折叠,14.2%的转角和24.7%无规卷曲。PaL拆分四对外消旋薄荷醇丙酸酯具有较好的对映体选择性,其E>200。但其非对映体选择性有待于进一步提高。摇瓶发酵脂肪酶PaL的酶活为4625U/L。最适温度为35℃,最适pH为9.0。在pH6.5-10.0之间都有较好的稳定性,但热稳定性相对较差。针对PaL具有非常好的对映体选择性这一问题,我们研究了PaL的对映体识别机理。通过对酶-底物复合物结构进行分析发现：当脂肪酶PaL与d-薄荷醇丙酸酯结合时,位于R构型的手性中心C2上的异丙基的取向朝向催化组氨酸His271。异丙基的空间位阻迫使催化组氨酸His271的咪唑环偏转了约30°。这一偏转直接导致了Hε(His271)与O(alcohol)之间距离由2.2A拉大到3.7A,进而使得这两个原子之间的必须氢键无法形成。从而最终导致了d-薄荷醇丙酸酯与脂肪酶PaL形成的酶-底物复合物不够稳定,与l-薄荷醇丙酸酯相比无法正常水解。这可能是脂肪酶PaL具有很好对映体选择性的根本结构原因通过底物分子共价对接技术研究了l-薄荷醇丙酸酯和非目标底物与PaL的结合方式,并以此为指导构建了突变蛋白V180L/A272F。突变型PaL催化的水解产物中l-薄荷醇对l-新薄荷醇的非对映体比率由野生型的6.6：1提高到30.7：1。而对d-新异薄荷醇的非对映体比率则由12.0：1提高到25.5：1。分子动力学模拟表明定点突变在特定位置引入了较大的空间位阻效应,限制了底物手性中心上所连基团的空间取向,同时突变位点附近区域增大的结构刚性巩固了由空间位阻引起的限制作用从而最终强化了PaL对不同构型底物的分子识别,提高了非对映体选择性。为进一步提高非对映体选择性,我们采用了定点突变结合体外化学修饰的方法。首先构建突变体A272C,引入了化学修饰的特异性靶点。然后使用DTNB对半胱氨酸进行特异性修饰,获得了A272C-DTNB修饰脂肪酶。MALDI-TOF mass验证了DTNB的有效修饰。远紫外区和近紫外区圆二色谱结果表明DTNB的修饰没有引起PaL二级结构和三级结构的明显变化。综合拆分实验结果和分子动力学模拟结果发现：由DTNB的化学修饰引入了较大的空间位阻相应和降低了PaL修饰位点附近区域结构柔性,使得脂肪酶对非目标底物的Km值有了较大幅度的提高。这种区域结构柔性与非目标底物Km值的相关性可以解释为由DTNB的化学修饰所引起的增大的局部空间位阻效应和降低的区域结构柔性阻碍了非目标底物的诱导契合过程,从而降低了A272C-DTNB修饰脂肪酶对非目标底物的亲和性,提高了非目标底物的Km值,从而最终导致了化学修饰脂肪酶的非对映体选择性的进一步提高。为实现PaL的高效制备,我们研究了重组E.coli的高密度发酵技术。分批发酵阶段研究确定了葡萄糖对细胞干重的得率系数Yx/s=0.7,同时研究确定了20℃为最有利于重组脂肪酶诱导表达的温度。在补料发酵阶段,我们发现控制比生长速率μ=0.21h-1时对细胞的生长和积累最为有利。此时,乙酸浓度可以控制在较低水平,同时葡萄糖也不存在积累现象。所得最大细胞干重在70g/L左右。诱导时机的研究结果表明第15小时开始诱导,IPTG加入量为20μmol/g干菌体最为合适。重组脂肪酶发酵游离酶活为200000U/L左右。全细胞酶活为20000U/L。SDS-PAGE电泳结果表明重组脂肪酶以可溶性表达为主,基本没有包涵体形成。在前面实现高密度发酵的基础上,使用重组E.coli全细胞对四对外消旋薄荷醇进行拆分。研究确定了转化温度为30℃。随后研究了重组E.coli全细胞的底物和产物抑制情况。对对拆分反应有显著影响的催化剂载量、底物浓度和助溶剂加量进行了优化。确定最优催化剂载量为2U/mL酶活所对应的全细胞量,底物浓度为1000mM,添加5%助溶剂加量。在该条件下拆分四对外消旋薄荷醇制备l-薄荷醇的时空产率为1.54gL-1h-1。通过精馏得到的产物l-薄荷醇dep最高为87.4%,纯度为90.35%。实际总收率可达88.42%。更多还原

【Abstract】 l-menthol is a kind of important chiral chemical. It has been used extensively in industrial and pharmaceutical field due to its unique flavor, cooling and refreshing effects. The yield and quality of natural menthol are affected seriously by weather and region. Moreover, due to the rapidly increasing demand for l-menthol among the global market in recent years, the l-menthol from natural sources has no longer satisfied the demand. How to apply biocatalysis method to produce l-menthol has became the research focus gradually.A strain named Pseudomonas alcalignens CGMCC4405producing lipase with high selectivity and hydrolysis activity toward the mixture of8diastereomers of racemic menthol to produce l-menthol was isolated from soil. The Pseudomonas alcaligenes lipase (PaL) gene was cloned and expressed in E.coli by the construction of genomic library. After PaL purification, the result of MALDI-TOF mass indicated that the precise molecular weight of PaL was58094.3Da and the peptide mass fingerprinting verified the amino acid sequence deduced from DNA sequence. The result of far-UV CD showed that PaL had26.8%a-helix,34.2%p-sheet,14.2%turn and24.7%random coli. The PaL had high enantioselectivity (E>200) in the process of resolution. However, the diastereoselectivity was not very ideal. The enzyme activity in shake flask was4625U/L. The optimal temperature and pH of PaL was35℃and9.0respectively. The PaL had good pH stability. Conversely, the thermal stability was not ideal.Contrary to the property of excellent enantioselectivity, we investigated the enantiomers recognition mechanism of PaL. Based on the analysis of the structure of PaL-substrate complex, the orientation of isopropyl connected with C2stereo center of d-menthyl propionate towards the imidazole ring of catalytic residue His271was identified. The steric exclusion effect of isopropyl forced the imidazole ring to deflect30°. The deflection caused the distance between Hs(His271) and O(alcohol) increased from2.2A to3.7A, which was unable to form the essential hydrogen bond. The lack of the essential hydrogen bond caused the PaL-d-menthyl propionate became not stable enough. Compared to l-menthyl propionate, d-menthyl propionate couldn’t be hydrolyzed normally. This may be the structure basis of excellent enantioselectivity of PaL.To resolve the problem of low diastereopreference, the interaction modes between PaL and substrate were investigated by molecular modeling and covalent docking. Under the guidance of the result of covalent docking, the double sites mutation V180L/A272F was constructed. The diastereomeric ratio of l-menthol hydrolyzed by variant PaL towards l-neomenthol increased from6.6:1of WT PaL to30.7:1. For the diastereomeric ratio of d-isoneomenthol, it increased from12.0:1to25.5:1. The result of molecular dynamic simulation indicated that stronger steric exclusion effect was introduced to certain location of PaL by site-directed mutagenesis. The increased steric exclusion effect restricted the orientation of group connected with stereo centers. The increased the structure rigidity of the region around the mutation sites consolidated the restriction effect of steric exclusion. Finally recognition specificity of substrates with different configurations and the diastereoselectivity were enhanced.The combined method of site-directed mutagenesis and chemical modification was employed to further improve diastereoselectivity of PaL. The strategy involved the introduction of a single cysteine residue into the hot spot via site-directed mutagenesis to constructe the variant A272C. This was then covalently linked with DTNB to give chemically modified mutant lipase. The result of MALDI-TOF mass verified the effectiveness of DTNB modification. The far and near-UV CD demonstrated the secondary and tertiary structure of PaL did not changed obviously after DTNB modification. The analysis of kinetic properties and molecular dynamic simulation showed that when bound with l-menthyl propionate, WT and A272C-DTNB modified PaL exhibited similar Km values. In contrast, the binding of non-target substrates produced increased Km values due to decreased flexibility and increased steric exclusion. The significant correlation of PaL flexibility with the Km value of the non-target substrate can be explained that the increased steric exclusion introduced by DTNB-modification of Cyc272and encountered by non-target isomers prevents their fitting into the PaL’s active site by restricting the orientation of the isopropyl group of the substrate.In order to achieve the effective preparation of PaL, we investigated the high-density fermentation of E. coli. The yield coefficient of glucose towards dry cell weight (Yx/s) is0.7. In the fed-batch fermentation stage, exponential feeding was used to control the specific growth rate at0.21h-1. The IPTG (20μmol/g dry cell weight) was added to fermentation broth in the15th hour at20℃. In the end of fermentation, the maximum dry cell weight is70g/L. The maximum enzyme activity of PaL is200000U/L. The result of SDS-PAGE gel electrophoresis showed that the PaL expressed effectively as soluble protein. Almost no inclusion body formed.On the basis of high-density fermentation, the recombinant E.coli whole cells were used to catalyze the hydrolysis of recemic menthyl propionate to produce/-menthol. Some key parameters of the biocatalytic process, including substrate and product inhibition, reaction temperature, catalyst loading, substrate loading and amount of cosolvent were optimized. The optimal biocatalytic process parameters were:reaction temperature30℃, catalyst loading2U/mL, substrate loading1000mM (21.2%, w/v) and cosolvent concentration5%. Under this optimal condition, the productivity of1.54gL-1h-1was achieved. The dep of target product/-menthol obtained by distillation was87.4%and the purity is90.45%. The total yield of l-menthol achieved88.24%。 These results indicate that the biocatalytic method has great industrial application prospect in the manufacturing of l-menthol.更多还原