【作者】 于丽娟；

【导师】 徐岩；

【作者基本信息】 江南大学 ， 发酵工程， 2008， 博士

【摘要】 天然薄荷醇由于受天气和地域等的影响,产量和质量波动很大,近年来,利用生物法拆分dl-薄荷醇制备l-薄荷醇己经成为l-薄荷醇生产的研究热点。本文利用透明圈平板初筛与转化反应气相检测复筛相结合的方法,筛选出了一株具有高度不对称水解dl-乙酸薄荷酯生成l-薄荷醇的菌株洋葱布克氏菌（Burkholderia cepacia）;以此菌株为材料,对其培养条件,整细胞体系的转化条件进行了详细研究;并对该菌株细胞中立体选择性酯酶同工酶进行了分离纯化,研究了其酶学性质,为其进一步克隆表达和相关应用提供实验依据。主要工作包括:1)以提高单位发酵液中的酶活为目标,选择菌体生长和产酶为指标,利用响应面法对B. cepacia的培养条件和营养条件进行了优化,确定摇瓶发酵产酶的最优培养基组成为:葡萄糖2.27%,牛肉膏1.58%,蛋白胨1.83%,K₂HPO₄ 0.42%,MgSO₄·7H₂O 0.02%,CaCl₂ 0.01%,NaCl 0.5%;较优环境条件为:接种量6%,培养温度30℃,装液量15%,初始pH值为7.0⁷.5,培养时间为48 h;在此最优条件下进行摇瓶发酵产酶,产酶量比未优化时提高了2.3倍。2)采用硫铵盐析、疏水层析、离子交换层析和凝胶过滤对B. cepacia的胞内酯酶进行了分离纯化。同工酶Est1的比酶活提高了近3倍,为0.77 U/mg,分子量为66 kDa;同工酶Est2的比酶活提高了近23倍,为5.91 U/mg,分子量约为37 kDa。Est1和Est2的最适pH都为7.0,最适反应温度为30℃,活性中心都存在丝氨酸残基,且都不属于金属酶类。立体选择性受温度影响不明显,但受pH影响严重,pH过高或过低都会引起立体选择性的明显下降,添加大部分表面活性剂或抑制剂均使酶的立体选择性下降。Est1没有明显位置特异性,而Est2具有2-位特异性,它们都优先水解短链脂肪酸酯。除甲酸薄荷酯外,均优选短链l-型脂肪酸薄荷酯进行水解。因此,两目的酶都属于短链酯酶。Est1的N-末端氨基酸组成为:MHITTT,Est2的N-末端氨基酸组成为:MGARTDA,与已知Burkholderia sp.水解酶（酯酶/脂肪酶）均没有明显的同源性,说明Est1和Est2都可能是一种新酶,都属于羧酸酯酶大类（EC3.1.1.1）。3)对B. cepacia整细胞催化的dl-乙酸薄荷酯不对称水解制备l-薄荷醇的生物催化过程进行了研究。发现添加助溶剂可改变底物与酶的接触面积和整细胞的通透性,从而对催化反应转化率和产物ee值（对映体过剩量）有一定的影响,添加15%的二甲亚砜对反应速率和产物ee值都有提高作用,最佳反应条件为:pH 7.0,0.067 mol/L的Na₂HPO₄-KH₂PO₄缓冲体系,温度30℃,15%二甲亚砜,1%底物,15 g/L干细胞量,反应24 h。在此最优条件下得到的l-薄荷醇的光学纯度达95%以上。通过反应产物的分离纯化,纯化得率为85%,l-薄荷醇单次循环总收率为66%,说明了先对dl-薄荷醇进行化学酯化再通过B. cepacia整细胞催化的不对称拆分制备l-薄荷醇是完全可行的。更多还原

【Abstract】 The yield and quality of natural menthol are affected seriously by weather and region, etc., synthetic menthol can overcome all these drawbacks. Therefore, there is a great interest in separation of dl-menthol to prepare l-menthol by microbial methods.A strain named Burkholderia cepacia ATCC 25416 with high enantioselectivity and hydrolysis activity toward dl-menthyl acetate was screened through combined screening strategy of hydrolysis ability with stereoselectivity. The cultivation conditions and the enantioselective hydrolysis conditions of B. cepacia whole cells were studied in this work. In order to extend the usage and clone the enzyme in a large scale, the key enzymes were also purified and characterized. The main research contents were shown as follows:（1） To increase the enzyme activity, the cultivation conditions of B. cepacia were optimized using response surface methodology （RSM）. The optimum culture medium components were 2.27% glucose, 1.58% beef extract, 1.83% peptone, 0.42% K₂HPO₄, 0.02% MgSO₄·7H₂O, 0.01% CaCl₂, 0.5% NaCl. The optimum environment conditions were 6% inoculation size, 15% medium volume, initial pH 7.0⁷.5, 30℃and 48 h. Under these conditions, the enzyme activity was increased by 2.3 times.（2） The key enzymes were purified from B. cepacia by a four-step purification, i.e., ammonium sulfate precipitation, butyl-Sepharose, DEAE-Sepharose, and Superdex G-75 column chromatography. Est1 was obtained with activity of 0.77 U/mg and molecular weight of 66 kDa. Est2 was obtained with activity of 5.91 U/mg and molecular weight of 37 kDa. The optimum pH and the optimum temperature of Est1 and Est2 were pH 7.0 and 30℃, respectively. These two enzymes belonged to the class of serine hydrolases, but not metalloenzyme. The enantioselectivities of the two enzymes were not temperature depended, but pH depended, and decreased sharply under harsh pH conditions. The enantioselectivities were also droped when different detergents or inhibitors used in the reaction systems. There was not position specificity about Est1, while Est2 had a 2-position specificity. The two enzymes showed higher hydrolytic activity toward short acyl chain p-nitrophenol esters and short acyl chain menthol esters. Except menthyl formate, they all preferentially hydrolyzed short-chain fatty acid esters of menthol with high stereospecificity, which indicated that the two purified enzymes were short-chain fatty acid esters hydrolase. The N-terminal amiano acid sequence of Est1 had been determined as MHITTT, and the N-terminal sequence of Est2 was MGARTDA. There were no homologies of the two sequences with other Burkholderia sp. hydrolase （esterase/lipase）. Considering the N-terminal sequences and their high stereospecificity for short chain menthyl esters, these two esterases were concluded as new enzymes belonging to the carboxylesterase group （EC 3.1.1.1） of esterase family.（3） The catalytic reaction process about enzntioselective hydrolysis of dl-menthyl acetate to l-menthol catalyzed by B. cepacia whole cells was studied. The results showed that the permeability of the whole cells and the interface area between substrate and enzyme were changed after addition of co-solvents, which influenced the hydrolysis conversion and ee-value （enantiomeric excess） of product. High hydrolysis rate and ee-value of product were achieved when 15% （v/v） dimethyl sulfoxide （DMSO） was added. The optimum hydrolysis conditions catalyzed by B. cepacia whole cells were 1 mL Na₂HPO₄-KH₂PO₄ （pH 7.0, 0.067 mol/L）, 15% dimethyl sulfoxide （DMSO）, 1% dl-menthyl acetate, 15 g/L dry cells, 30℃and 24 h. Under these conditions, l-menthol could be obtained with the optical purity higher than 95%. The product l-menthol was separated with a recovery of 85%, and the total yield of l-menthol was 66% in each cycle. These results proved that it is reasonable to split dl-menthyl acetate in preparation l-menthol using B. cepacia whole cells as biocatalyst after chemical esterification of dl-menthol with acetic anhydride.更多还原