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

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.