响应面法优化羟化酶催化酪醇合成羟基酪醇工艺  

Optimization in Synthesis of Hydroxytyrosol from Tyrosol Catalyzed by Hydroxylase Through Response Surface Methodology

作　　者：刘英梅 郭新艳 张秀华 张小刚 刘飞 张艳艳 於怀龙 袁丹丹 孙晓康 LIU Yingmei;GUO Xinyan;ZHANG Xiuhua;ZHANG Xiaogang;LIU Fei;ZHANG Yanyan;YU Huailong;YUAN Dandan;SUN Xiaokang(Shandong Key Laboratory of Biopharmaceuticals,Shandong Engineering Laboratory of Polysaccharide Drugs,National-Local Joint Engineering Laboratory of Polysaccharide Drugs Fermentation and Refining,Shandong Academy of Pharmaceutical Sciences,Jinan 250101,China)

机构地区：[1]山东省药学科学院,山东省生物药物重点实验室,山东省多糖类药物工程实验室,多糖类药物发酵与精制国家地方联合工程实验室,山东济南250101

出　　处：《化学与生物工程》2024年第7期44-49,55,共7页Chemistry & Bioengineering

基　　金：山东省重点研发计划(科技示范工程)项目(2022SFGC0105);山东省重大科技创新工程项目(2021ZDSYS07);济南市“新高校20条”项目(2021GXRC072)。

摘　　要：以pET-22b为载体,将基因HpaB、HpaC在E.coli BL21(DE3)中表达,得到高纯度的羟化酶HpaB、HpaC,并采用单因素实验和响应面法对羟化酶催化酪醇合成羟基酪醇的反应条件进行优化。确定最佳反应条件如下:温度为32℃、缓冲液为20 mmol·L^(-1)磷酸钾缓冲液(pH值7)、酪醇浓度为17.67 mmol·L^(-1)、HpaC加量为0.78 mg·mL^(-1)、NADH浓度为9.88 mmol·L^(-1)、FAD浓度为2.88μmol·L^(-1),在此条件下,酪醇转化率为99.99%,底物酪醇几乎完全转化为羟基酪醇。该研究为酶法催化合成羟基酪醇的工业化生产提供了理论基础。The genes HpaB and HpaC were expressed in E.coli BL21(DE3) by using pET-22b as a carrier,and hydroxylase HpaB and HpaC with high-purity were obtained.Moreover,we optimized the reaction conditions for the synthesis of hydroxytyrosol from tyrosol catalyzed by hydroxylase through single-factor experiments and response surface methodologies.The optimal reaction conditions are determined as follows:the temperature is 32 ℃,the buffer is 20 mmol·L^(-1) potassium phosphate buffer(pH value of 7),the tyrosol concentration is 17.67 mmol·L^(-1),the HpaC dosage is 0.78 mg·mL^(-1),the NADH concentration is 9.88 mmol·L^(-1),and the FAD concentration is 2.88 μmol·L^(-1).Under above conditions,the conversion rate of tyrosol is 99.99%,and the substrate tyrosol is almost completely transformed into hydroxyl tyrosol.The study provides a theoretical basis for the industrial production of hydroxyl tyrosol catalyzed by enzymatic method.

关 键 词：羟基酪醇 羟化酶 响应面法 酪醇转化率 生物合成 

分 类 号：TQ243[化学工程—有机化工] Q554[生物学—生物化学]