【作者】 李远颂；

【导师】 刘四新； 李从发；

【作者基本信息】 华南热带农业大学 ， 农产品加工及贮藏工程， 2006， 硕士

【摘要】 3-苯基乳酸是一种重要的化学合成前体,广泛应用于医药、化工、生物合成等领域。它有两种对映体,单一对映体的3-苯基乳酸对生物体的作用及其在医药、化工方面的应用不同。因此,采用简便快速的方法合成手性3-苯基乳酸具有非常重要的意义。生物合成法能满足上述要求。本研究利用高效毛细管电泳建立了3-苯基乳酸的手性毛细管电泳拆分方法:以0.1mol/L pH 5.5的磷酸盐缓冲液为毛细管电泳缓冲液,0.03mol/L羟丙基-β-环糊精作为手性选择剂,在分离电压26KV、分离温度25℃的条件下,采用未涂层石英毛细管(60cm/75cm×50μm i.d.)和毛细管区带电泳法可使3-苯基乳酸对映体达到基线分离。以此为基础建立了3-苯基乳酸的定性和定量分析方法。分析了溶血葡萄球菌(Staphylococcus haemolyticus)T01转化苯丙酮酸生成R-3-苯基乳酸的可行性。研究表明:菌株T01能将底物苯丙酮酸立体选择性地还原成对映体过量(e.e.)值为100%的R-3-苯基乳酸。优化了菌株T01的发酵产酶条件:产酶培养基的最佳组成为D-果糖2%、酵母膏0.5%、氯化钠0.5%、FeSO4?7H2O 0.01%、初始pH为7.0。将20 mL产酶培养基装入250 mL三角瓶中,30℃,180 r/min条件下摇床培养30h后,酶比活力达到最高值。优化了菌株T01转化苯丙酮酸的条件:底物苯丙酮酸的浓度为3.0 g/L,细胞量与苯丙酮酸的比率为2.5时,选用磷酸盐缓冲液作为转化缓冲液,在pH为8.0的转化反应体系中,加入0.03 g/L的表面活性剂溴化十六烷基吡啶和5%的能量化合物葡萄糖,30℃,180 r/min条件下摇床转化16h,底物苯丙酮酸的转化率可达到46.38%。研究了菌株T01所产酶的酶学性质,试验结果表明:菌株T01所产的氧化还原酶催化还原羰基所需的辅酶为NADH,催化还原苯丙酮酸反应的最适pH值为9.0,最适温度为35℃。该酶在pH8.5～9.5范围内较为稳定,当温度达40℃以上时酶活力损失较大;添加金属离子对酶活会产生一定的影响。本文还探讨了3-苯基乳酸的定性和定量分析方法、溶血葡萄球菌的致病性及其改造等,并展望了手性3-苯基乳酸的生物合成。更多还原

【Abstract】 3-phenyllactic acid is an important precursor for chemical synthesis, and it was widely used in many fields, such as medicine, chemical industry, biosynthesis and so on. However, the two enantiomers of 3-phenyllactic acid show different biological activities and different applications in pharmaceutical and chemical industry. As a consequence, synthesis of chiral 3-phenyllactic acid is very significant. These could be met by biosynthesis.The method was established to resolve chiral 3-phenyllactic acids by capillary electrophoresis. Using 0.1mol/L phosphate solution (pH5.5) as running buffer and 0.03mol/L hydroxypropyl-β-cyclodextrin as chiral selector, the baseline seperation of 3-phenyllactic acid enantiomers was achieved with resolution of 1.51 by quartz capillary without coating(60cm/75cm×50μm i.d.)and capillary zone electrophoresis under seperation voltage 26kv at 25℃. Based on these results, the methods for qualitative and quantitative analysis of 3-phenyllactic acid were established.The feasibility was analyzed to transforme phenylpyruvic acid to R-3-phenyllactic acid by Staphylococcus haemolyticus T01. S. haemolyticus T01 could reduce asymmetrically phenylpyruvic acid to R-3-phenyllactic acid of 100% enantiomeric excess.The fermentation conditions of S. haemolyticus T01 were studied. The optimal medium was as follows: 2% D-fructose, 0.5% yeast paste, 0.5% NaCl, 0.01%FeSO4?7H2O, and initial pH was adjusted to 7.0. The strain showed highest activity after incubation at 30℃,180r/min for 30h. The specific activity of the enzyme was 0.6265mU/g.The optimal conditions were investigated for bioreduction of phenylpyruvic acid to R-3-phenyllactic acid. With phenylpyruvic acid concentration being 3.0g/L, cell / phenylpyruvic acid ratio being 2.5, and 0.03g/L cetyl pyridinium bromide and 5% glucose added, the conversion rate of phenylpyruvic acid to R-3-phenyllactic acid could reach 46.38% after 16h at 30℃,180r/min in pH 8.0 reaction system(0.05mol/L phosphate solution as buffer).更多还原