【摘要】 D 对羟基苯甘氨酸 (D p HPG)是制备羟氨苄青霉素、羟氨苄头孢菌素和羟氨唑头孢菌素等β 内酰胺类抗生素的重要中间体 ,同时它也用于多种多肽类激素及农药的合成 .在D 对羟基苯甘氨酸的多种制备方法中 ,生物酶转化法具有原料易得、工艺简单、耗能少、产率高、成本低、光学纯度好、三废污染少等优势 .为利用生物酶转化法制备D 对羟基苯甘氨酸 ,首先用尿素、乙醛酸和苯酚合成底物D ,L 对羟基苯海因 ,然后利用D 海因酶基因工程菌E .coliBL2 1 pMD T7 dht细胞作酶源 ,进行底物D ,L 对羟基苯海因到中间体N 氨基甲酰 D 对羟基苯甘氨酸的酶法转化 ,最后用化学法将N 氨基甲酰 D 对羟基苯甘氨酸进一步转化为D 对羟基苯甘氨酸 .结果表明 ,底物D ,L 对羟基苯海因的收率为 60 % .8L体积的发酵小试实验表明 ,发酵 12h ,工程菌E .coliBL2 1 pMD T7 dht的海因酶活力为 30 0 0U L ,SDS 聚丙烯酰胺凝胶电泳薄层扫描结果显示海因酶表达量约占菌体总可溶性蛋白质的 60 % ,菌体收率为 6% .8L体积的海因酶转化实验表明 ,在 4 %底物D ,L 对羟基苯海因和 1%菌体 (湿重 )pH 9 0情况下 ,反应 5h ,D ,L 对羟基苯海因的转化率可达 96% .在酸性条件下 ,用NaNO2 将N 氨基甲酰 D 对羟基苯甘氨酸转化为D 对羟基苯甘氨酸 ,反应 2h ,转?更多还原

【Abstract】 For the industrial enzymatic production of D-p-hydroxyphenylglycine (D-p-HPG), substrate D,L-p-hydroxyphenylhydantoin (D,L-p-HPH) was synthesized using glyoxylic acid-urea-phenol, the enzymatic conversion of D,L-p-HPH to intermediate N-carbamoyl-D-p-hydroxyphenylglycine (CpHPG) was carried out by D-hydantoinase genetic engineering strain E. coli BL21/pMD-T 7-dht resting cells and the chemical conversion of CpHPG to D-p-HPG was performed using sodium nitrite under acidic conditions. The results showed that chemical yield of D, L-p-HPH was 60%. The fermentation was carried out for 12 hours in a 10 L fermentor containing 8 L medium. The D-hydantoinase activity of engineering strain E. coli BL21/pMD-T 7-dht was 3000 U/L and the amount of the recombinant D-hydantoinase was about 60% of the total bacterial soluble proteins by SDS-polyacrylamide gel electrophoresis thin layer scanning. The yield of bacterial cell was 6%. Biotransformation was carried out at 37℃ in 8 L of reaction mixture containing 4% (W/V) D, L-p-HPH, 1% intact cell (wet weight) and 0.1 mol/L sodium phosphate (pH 9.0), with a molar yield of 96% within 5 hours. The pure product CpHPG was further chemically converted to D-p-HPG with an 89% yield. Thus, the overall conversion efficiency of D, L-p-HPH to D-p-HPG was 84%. The chemical purity and optical purity of D-p-HPG was 99% and 99.5%, respectively. The melting point (mp), optical rotation and infrared (IR) absorption spectrum of the product were identical with the control product of D-p-HPG.更多还原