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D-Hase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。
何然峰
,
杨宇
,
宋显炳
,
李小连
,
王自强
,
王鹏
,
王云山
. Ensifer adhaerens CAS22-03发酵工艺优化及D-p-HPG的酶法制备[J]. 过程工程学报, 2024
, 24(9)
: 1096
-1105
.
DOI: 10.12034/j.issn.1009-606X.224040
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.
