【作者】 毕洪书；

【导师】 胡永红； 周华； 沈树宝；

【作者基本信息】 南京工业大学 ， 生物化工， 2003， 硕士

【摘要】 海因酶（Hydantoinase）是一类能够可逆催化海因或5-取代海因及其衍生物开环水解的酶，在生产光学活性氨基酸方面具有广泛的工业应用前景。海因酶法生产药物前体——手性氨基酸是一种经济有效的方法，将来这些药物进入市场后会大大刺激对手性氨基酸的需求，进而增加了海因酶的重要性。 本文在产海因酶菌种的选育、菌种的鉴定、菌株B.cereus BP-6的产酶条件优化及酶转化条件等几个方面进行了系统的研究，最终得到以下结果： 1．建立了微生物L-海因酶简便而有效的筛选模型。采用富集法、添加苯酚红指示剂的平板快速显色法对30多份土样进行大量初筛，得到具有海因酶活力的菌株62株。采用摇瓶发酵测定酶活和茚三酮显色法定量测定L-Phe相结合的方法进行复筛，以复筛最高酶活的40％为基准得到11株具有较高海因酶活力的菌株。然后进一步复筛，利用高效毛细管电泳手性拆分法检测终产物L-Phe，并计算对映体过量值（ee）。经验证实验，最终筛选得到一株海因酶活力和ee值都较高的菌株BP-6，酶活为1.98U／g．wet cell，ee值=0.73。 2．菌株BP-6进一步分离纯化后，对菌株的形态特征、生理生化特征、生长特性等项目进行鉴定。依据《伯杰氏细菌鉴定手册》第8版第15部分，将该菌株鉴定为真细菌目、芽孢杆菌科（Bacillaceae）、芽孢杆菌属（Bacillus）、蜡样芽孢杆菌（B.cereus），暂命名为B.cereus BP-6。 3．以所筛得的菌株B.cereus BP-6为出发菌株，首先采用单因素试验对最佳产酶培养基组分进行筛选。实验结果显示该菌株的最佳碳源是葡萄糖，最佳氮源是以蛋白胨和牛肉膏组成的复合氮源，金属离子中以Ca²⁺对酶活的影响最大，最适诱导物为苄基海因。然后利用响应面回归分析法对产酶培养基进行优化，得出最佳培养基组成为：葡萄糖2.0％，牛肉膏0.7％，蛋白胨1.4％，苄基海因0.2％，NaCl 0.3％，K₂HPO₄．3H₂O 0.2％，CaCl₂ 0.02％。进一步进行最佳发酵条件的考察，实验结果表明该菌株最适产酶温度为30～32℃，pH 7.0～7.2，装液量为100ml／500ml，发酵时间16-18h。在以上优化条件下进行液体发酵产酶，酶活为2.49U／g，是最初产酶量的1.26倍。 4．最后，对酶转化过程的主要影响因素如温度、pH、酶用量等进行研究，确定了转化的最佳条件，即酶转化最适温度为38～40℃，酶转化最适pH8.0～ 8．5，加入有机溶剂对转化率没有明显提高，最适底物浓度为 10 g／L，菌体浓度 为底物浓度的 4倍，转化时间 48小时，在此条件下转化率可达对％。更多还原

【Abstract】 Hydantoinases are valuable enzymes for the production of optically pure D- and L-amino acids. The hydantoinase process is an economically attractive method for the production of many unnatural amino acids, which are components of potential Pharmaceuticals. As soon as these pharmaceuticals enter the commercial market there will be an augmented demand for these amino acids, which will in turn increase the importance of the hydantoinase process in the future.The screening and deteminant of microbial hydantoinase-producing, the fementationconditions of the B.cereus BP-6 were studied systematically, the following results were:Based on studies and comparisons among methods of screening microbial hydantoinase, a rapid, simple and efficient Screening Modle for microbial hydantoinase-producing was developed. More than thirty soil samples from all kinds of abundant nitrogen. An efficient and rapid plating method for determinant of microbial hydantoinase activity by observing the color-changing on the culture was adopted. This was preliminary screening and 62 strains obtained. After culturing in a triangle bottle on the shake and ninhydrin reaction is used to determine their hydantoinase activity and concentration of L-phenylalanine, and investigated the stereospecificity of obtained strains. This was secondary screening and 11 fine strains were selected, also detecting the enantiom excess of production using capillary electrophoresis. The highest activity of strain B.cereus BP-6 was 1.98U/g,ee was 0.73.Isolation and purification the strain BP-6, and through detection of its appearance, physiological and biochemical characteristics, growth characters, it was identified Bacillaceae, Bacillus, B.cereus according to Bergeysmanual of Determiutue Bacteriology(8th), and named B.cereus BP-6.Using B.cereus BP-6 abtained in lab, which can produce hydantoinase, the optimum composition of the fermentation medium was screened in individual factor.Experiment results showed that the best carbon source was glucose, the best nitrogenis peptone combined with beef extract. Ca2+ was the positive ion which had the best effect on enzyme synthesis. Benzylhydantoin was the best inducer. Next the fermentation medium was optimized using response surface analysis (RSA). The optimal medium was: glucose 2.0%, peptone 1.4%, beef extract 0.7%, NaCl 0.3%, KH2PO4 0.2%, CaCl2 0.02%, benzylhydantoin 0.2%. The optimum fermentation conditions as following: the optimum fermentation temperature was 32 , the optimum fermentation time was 16-18h, the optimum fermentation shaking frequency was 150rpm, the optimum fermentation pH was pH7.2. In doing so B.cereus BP-6 hydantoinase activity was 2.49U/g, enlarging 1.26 times.Finally the influence factors including temperature, pH, the quantity of hydantoinase of the conversion were studied in detail. The results showed that the optimal temperature of B.cereus BP-6 hydantoinase reaction was 38~40 , pH8.0~ 8. 5, adding organic solvent had no evident influence, the optimal concentration of substrate was 10g/L, the quantity of hydantoinase was 4 times than that of substrate, keeping reaction for 48h 31% benzylhydantoin was transformed to phenylalanine.更多还原