【作者】 乔越美；

【导师】 武波；

【作者基本信息】 广西大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 L-丝氨酸是氨基酸输液的重要成分，化妆品的添加剂，酶法生产色氨酸的前体物质。由于L-丝氨酸处于其他几种氨基酸、核苷酸、和磷脂的合成代谢的中间位置，较其他氨基酸来说，它的体内代谢运转速度极快。因此，直接发酵生产L-丝氨酸是很困难的。酶法合成L-丝氨酸由于利用甘氨酸和甲醛特异的合成L-丝氨酸，具有原料来源方便，原料成本低和可合成高浓度产品等优点，因此是最有前景的L-丝氨酸生产方法。丝氨酸的酶法合成系利用丝氨酸羟甲基转移酶(serinehydromethyl-transferase，SHMT)催化甲醛和甘氨酸(Gly)可逆地合成L-Serine，反应过程中SHMT需PLP和四氢叶酸(THFA)作为其辅因子。丝氨酸羟甲基转移酶在生物体内由glyA基因编码，且自然界中的一般的菌体内都含有glyA基因。 本课题的工作是采用PCR技术，以大肠杆菌K12菌株和假单孢菌T46的染色体DNA为模板，扩增得到约1.3kb的glyA基因片段，将PCR产物插入到表达质粒pETblue中，以重组质粒转化E. coliTuner(DE3)placI，筛选得到含glyA基因并高表达SHMT的基因工程菌PBK，并优化了其逆向酶活测定的反应条件。更多还原

【Abstract】 L-Serine is used as a component of amino acids mfusion,an additive of cosmetics,and as a starting material for the enzymatic production of tryptophan. The fermentative production of L-serine directly from carbohydrates has not been established because the metaboic turnover of this amino acid is much quicker than that of other amino acids: L-serine is a key intermediate for the synthesis of several other amino acids, nucleic acids and phospholipids. Because of its advantages of cheao raw material, available raw material and high concentration of L-serine in the reaction solution,the enzymatic method becomes the most promising one for manufacturing L-serine.With the chromosome DNA of Escherichia coli K12 and Pseudomonas aeruginosaT46 as templates, PCR products containing glyA gene encoding SHMT was obtained with the length of about 1.3kb. The PCR products were insertsed into pETblue .Then the two recombinant plasmids were transformed into E.coliTuner (DE3)placI. Among the transformants, recombinant bacteria over-expressing SHMT were screened out. Then the conditions for converse enzymatic reaction were optimized.更多还原