【作者】 张艳；

【导师】 赵凤生；

【作者基本信息】 上海交通大学 ， 微生物与生化药学， 2010， 硕士

【摘要】 本课题研究在亲水有机溶剂/无机盐/水两相体系和疏水有机溶剂单相体系中的生物转化反应。亲水有机溶剂(如甲醇、乙醇、丙酮、乙腈)和无机盐(如NaCl、NaH2PO4、K2HPO4、Na2SO4)在水中可以形成亲水有机溶剂/无机盐/水两相体系。这种体系的上相主要含有机溶剂,下相主要含无机盐和水,具有分相快、成本低、毒性低等诸多优点,已用于萃取分离金属和生物物质,得到较好的效果,但是用于生物转化的研究还未见报道。本课题研究在这种新型两相体系中的生物转化,用木瓜蛋白酶在正丙醇/NaCl/水两相体系中催化水解酪蛋白为氨基酸。在这种两相体系中,酶和底物主要分配在下相,产物主要分配在上相,有利于反应的进行,其转化率比亲水有机溶剂单相体系、水单相体系、疏水有机溶剂和水组成的两相体系、双水相体系的转化率都高。考察了温度、pH值、正丙醇浓度、NaCl浓度、酶浓度、底物浓度对转化率的影响。适宜的反应条件为: 44%正丙醇、60g/L NaCl的两相体系中,含0.15 g/L木瓜蛋白酶、13 g/L酪蛋白,55℃,pH 5.6。反应5 h后,转化率达到99.5%。说明这种两相体系适合于木瓜蛋白酶的催化反应。还研究了在亲水有机溶剂/无机盐/水两相体系中,假单胞菌ATCC 11250转化L-苯丙酮酸为L-苯丙氨酸的反应,大肠杆菌ATCC 9637和固定化青霉素酰化酶转化青霉素G为6-氨基青霉烷酸(6-APA)的反应,发现亲水有机溶剂和无机盐都会抑制酶的催化活性。研究了猪胰脂肪酶在亲水有机溶剂/无机盐/水两相体系中催化正己酸和无水乙醇合成己酸乙酯的反应,发现水和亲水有机溶剂对脂肪酶的活性有抑制作用。因而这种两相体系不适合上述生物转化反应。在亲水有机溶剂/无机盐/水两相体系中的生物转化只适用于某些特殊的酶。有机溶剂单相体系是另一种常用的反应介质,此体系中不含或者只含有微量的水。研究了猪胰脂肪酶在疏水有机溶剂单相中催化己酸和无水乙醇合成己酸乙酯的反应,考察了反应介质、底物浓度、底物摩尔比、加酶量、反应时间对转化率的影响。发现在高底物浓度和低底物浓度的条件下,有机溶剂介质对反应的影响是不同的。采用中等底物浓度,lg P值适中的溶剂最合适。适宜的反应条件为:以环己烷为反应介质,正己酸浓度0.6 mol/L,正己酸摩尔数:无水乙醇摩尔数=1:1.25,加酶量15 g/L,反应温度37℃,反应24 h后,转化率达到90.5%。与文献报道的结果相比较,转化率相近,但底物浓度较高,为文献报道的1.5-3.0倍。更多还原

【Abstract】 The project is about the bioconversion in hydrophilic organic solvent/salt/water two-phase system and hydrophobic organic solvent single-phase system.Hydrophilic organic solvent (such as methanol, ethanol, acetone and acetonitrile) and inorganic salt (such as NaCl, NaH2PO4, K2HPO4 and Na2SO4) dissolved in water can form hydrophilic organic solvent/salt/water two-phase system. Its top phase mainly contains organic solvent and its bottom phase mainly contains salt and water. The two-phase system has some advantages, such as easy phase separation, cheap and low-toxic raw material, which has been applied in sample enrichment and separation of metal and biomolecules, and has got some good results. But up to now, there are no any reports about the application of this two-phase system in bioconversion.Enzymatic hydrolysis of casein catalyzed by papain to produce amino acids was performed in the novel two-phase system, which was composed of n-propanol, NaCl and water. Papain and casein were mainly distributed in bottom phase, and aromatic amino acids produced by enzymatic hydrolysis were mainly in top phase. It was beneficial to the bioconversion. n-Propanol/NaCl/water two-phase system got a higher bioconversion than n-propanol/water single-phase system, aqueous single-phase system, n-hexane/water two-phase system and PEG/phosphate aqueous two-phase system. It was investigated that the effects of temperature, pH, n-propanol concentration, NaCl concentration, enzyme concentration and substrate concentration on bioconversion yield. When the two-phase system consisted of 44% n-propanol, 60 g/l NaCl, 0.15 g/l papain and 13 g/l casein at 55℃and pH 5.6, the transformation yield was 99.5% after 5 h reaction. The result showed that the two-phase system is suitable for the bioconversion by papain.The research also included the biotransformation of L-phenylpyruvic acid to L-phenylalanine by ATCC 11250 and the biotransformation of penicillin G to 6-aminopenicillanic acid (6-APA) by ATCC 9637 and immobilized penicillin acylase in hydrophilic organic solvent/salt/water two-phase systems. It was found that hydrophilic organic solvents and inorganic salts could inhibit the enzymatic activities. The synthesis of ethyl caproate from caproic acid and anhydrous ethanol catalyzed by pocine pancreas lipase in hydrophilic organic solvent/salt/water two-phase systems was investigated. It was found that water and hydrophilic organic solvents could inhibit the activity of pocine pancreas lipase. So the novel two-phase systems were not suitable to above bioconversion. The hydrophilic organic solvent/salt/water two-phase systems are only applicable for some special enzymes.Hydrophobic organic solvents are other common media for bioconversion. In the system, there is no water or little water. It was investigated that synthesis of ethyl caproate from caproic acid and anhydrous ethanol catalyzed by pocine pancreas lipase in hydrophobic organic solvent. The effects of reaction media, substrate concentration, mole ratio of substrates, enzyme amount and reaction time on bioconversion yield were studied. It was found that the influences of organic solvent on enzymatic reaction with low concentration of substrate were different from those with high concentration of substrate. Middle concentration of substrate and moderate lg P value of solvent were suitable. The appropriate conditions for biocatalysis were: 0.6 M caproic acid in cyclohexane, mole ratio of caproic acid and anhydrous ethanol with 1:1.25, 15 g/l lipase, 37℃. After 24 h reaction, the conversion yield was 90.5%. When the substrate concentration was 1.5-3.0 times as high as reports from the references, the similar conversion yield was obtained.更多还原