Ensifer adhaerens CAS22-03发酵工艺优化及D-p-HPG的酶法制备

Ensifer adhaerens CAS22-03 fermentation process optimization and enzymatic preparation of D-p-HPG

为了研究Ensifer adhaerens CAS22-03来源的D-海因酶(D-Hase)与N-氨甲酰水解酶(D-Case)表达活性的影响因素,提高Ensifer adhaerens CAS22-03全细胞催化制备D-对羟基苯甘氨酸(D-p-HPG)的效率,本工作采用单因素试验和正交试验优化了Ensifer adhaerens CAS22-03发酵培养基的碳源、氮源,建立了Ensifer adhaerens CAS22-03的分批补料发酵工艺,分析了DHase和D-Case的酶学特性,开发了基于Ensifer adhaerens CAS22-03全细胞催化的D-p-HPG酶促生产工艺。结果表明,Ensifer adhaerens CAS22-03的最适碳源和氮源为蔗糖和酵母浸粉,分批补料发酵过程中,D-Hase和D-Case的活性高达243.6和55.8 U/g,分别提高了56.9%和46.4%。D-Hase和D-Case的最适反应温度均为45℃,最适反应pH分别为9和8。Ensifer adhaerens CAS22-03全细胞催化制备D-p-HPG的过程中,当底物浓度为40 g/L、酶用量为底物:菌体=5:1时,40℃,200 r/min条件下反应10 h,底物转化率达到95%以上,本工作的研究结果为D-p-HPG的工业酶法生产奠定了基础。

In order to study the influence factors of the expression activities of D-Hydantoinase(D-Hase)and N-Carbamoyl hydrolase(D-Case)from Ensifer adhaerens CAS22-03,and to improve the catalytic efficiency of the whole-cell catalyzed preparation of D-p-Hydroxyphenylglycine(D-p-HPG)by Ensifer adhaerens CAS22-03,the single factor test and orthogonal test were adopted to optimize the carbon and nitrogen sources,and the fed-batch fermentation process of Ensifer adhaerens CAS22-03 was established.The enzymatic properties of D-Hase and D-Case were analyzed,and the D-p-HPG enzymatic production process based on the whole cell catalysis of Ensifer adhaerens CAS22-03 was developed.The results showed that the optimal carbon and nitrogen sources for Ensifer adhaerens CAS22-03 fermentation were sucrose and yeast extract.In the fed-batch fermentation process,the activities of D-Hase and D-Case were up to 243.6 and 55.8 U/g,which were increased by 56.9%and 46.4%,respectively.The optimal reaction temperature of D-Hase and D-Case is 45℃,and the optimal reaction pH is 9 and 8 respectively.In the process of the whole-cell catalytic preparation of D-p-HPG by Ensifer adhaerens CAS22-03,when the substrate concentration was 40 g/L and the enzyme dosage was substrate:bacterium=5:1,the substrate conversion reached more than 95%with the condition of 40℃and 200 r/min for 10 h,laying the foundation for the industrial enzymatic production of D-p-HPG.