利用基因工程菌HC01固定化细胞转化生产D-对羟基苯甘氨酸

Bioconversion of D-HPG Using Immobilized Genetic Engineered Strain HC01

摘要:

对一菌两酶工程菌HC01转化底物DL-对羟基苯海因(DL-HPH)的最适条件及其细胞固定化进行了研究, HC01游离细胞转化DL-HPH的最适条件为40°C, pH 7.5。通过对固定化细胞酶活力测定, 确定细胞固定化的最优条件为海藻酸钠浓度2.5%, 细胞浓度为0.029 g/mL, 钙离子浓度为3%。固定化HC01的热稳定性比游离细胞高5°C, 二价金属离子Mn2+、Mg2+、Cu2+、Co2+和Ni2+在浓度为0.1 mmol/L时对固定化细胞中D-海因酶(HYD)和N-氨甲酰-D-氨基酸酰胺水解酶(CAB)两酶的活力无显著影响, Mn2+和Mg2+可分别使游离细胞中CAB活力提高至原来的2.1和2.7倍。在氮气保护下, 当初始pH为9.0、转化温度为40°C、转速为80 r/min、利用固定化HC01转化30 g/L的DL-HPH时, 36 h后转化率可达97%左右, 产物D-HPG经纯化后光学纯度达到99.7%, 得率可达85%。

Abstract:

The cells of engineered strain HC01 were immobilized in the form of Ca2+-alginate beads. The conditions for immobilization were investigated. The optimal gel concentration and cell concentration were found to be 2.5% and 0.029 g/mL in the presence of 3% CaCl2. The thermo-stability of immobilized cells was 5°C higher than that of free cells in the same condition. Divalent metal ions, such as Mn2+, Mg2+, Cu2+, Co2+ and Ni2+ did not affect significantly the enzymatic activity of D-hydantoinase (HYD) and N-carbamoylase (CAB) in immobilized cells. By contrast, Mn2+ and Ni2+ could independently enhance the activity of CAB up to 210% and 270% in free cells. The present data showed that the optimal reaction condition of immobilized cells was at pH 7.5 and 40°C as same as that of free cells. The immobilized cells were applied to produce D-p-Hydroxyphenylglycine (D-HPG) directly from the substrate DL-Hydroxyphenyl Hydantoin (DL-HPH) in a batch reactor. Conversion of about 97% was reached after 36-h reaction when the substrate concentration was 30 g/L with the initial pH of 9.0 under the protection of nitrogen gas. The overall yield of D-HPG was 85% with the optical purity of 99.7% after purification.