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过程工程学报 ›› 2024, Vol. 24 ›› Issue (9): 1096-1105.DOI: 10.12034/j.issn.1009-606X.224040CSTR: 32067.14.jproeng.224040

• 研究论文 • 上一篇    下一篇

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

何然峰1,2,3， 杨宇1,2,3， 宋显炳2,3,4， 李小连2,3， 王自强2,3*， 王鹏1*， 王云山2,3   

  1. 1. 河北科技大学化学与制药工程学院，河北 石家庄 050018 2. 中国科学院过程工程研究所，生化工程国家重点实验室，北京 100190 3. 中国科学院过程工程研究所，生物药制备与递送重点实验室(中国科学院)，北京 100190 4. 河北大学生命科学学院，河北 保定 071002
  • 收稿日期:2024-01-29 修回日期:2024-03-21 出版日期:2024-09-28 发布日期:2024-09-23
  • 通讯作者: 王自强 zqwang@ipe.ac.cn

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

Ranfeng HE1,2,3,  Yu YANG1,2,3,  Xianbing SONG2,3,4,  Xiaolian LI2,3,  Ziqiang WANG2,3*, Peng WANG1*,  Yunshan WANG2,3   

  1. 1. School of Chemical and Pharmaceutical Engineering, Hebei University of Science and Technology, Shijiazhuang, Hebei 050018, China 2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China 3. Key Laboratory of Biopharmaceutical Preparation and Delivery (Chinese Academy of Sciences), Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China 4. College of Life Sciences, Hebei University, Baoding, Hebei 071002, China
  • Received:2024-01-29 Revised:2024-03-21 Online:2024-09-28 Published:2024-09-23
  • Contact: Zi QiangWANG zqwang@ipe.ac.cn

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摘要： 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。

关键词: Ensifer adhaerens CAS22-03, D-海因酶, N-氨甲酰水解酶, 发酵工艺优化, 全细胞催化

Abstract: 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.

Key words: Ensifer adhaerens CAS22-03, D-hydantoinase, N-carbamoyl hydrolase, fermentation process optimization, whole-cell catalysis