克雷伯杆菌O852产反式二氢香芹酮关键酶转化条件及超声提取工艺优化

李志; 张莹莹; 张璐璐; 李晓; 范刚; 彭芷芊; 任婧楠; 潘思轶

doi:10.13386/j.issn1002-0306.2020030411

克雷伯杆菌O852产反式二氢香芹酮关键酶转化条件及超声提取工艺优化

Optimization of Ultrasonic Extraction of Key Enzymes for Trans-dihydrocarvone Transformation in Klebsiella sp. O852

摘要

摘要: 本研究以Klebsiella sp. O852为研究对象,使用超声提取的方法对其进行细胞破碎,对其转化柠檬烯生成反式二氢香芹酮的关键酶所在位置及转化条件进行了研究。通过单因素实验研究了超声时间、料液比、振幅对细胞破碎的效果的影响。最终设计Box-Behnken中心组合试验确定了最优提取条件。结果表明,该酶是一种胞内酶。最佳培养时间为8 h,此时为细胞生长的对数中期。转化时间为4 h时,在磷酸盐缓冲液中获得的酶活最高。通过单因素及响应面试验,获得提取该酶的最优条件为,超声时间21 min,料液比1:13,振幅36%,在此条件下,得到的酶活为(40.97±2.02) U/g。本研究为进一步研究该酶的纯化及酶学性质的探究提供了参考。

Abstract: In this study, the cells of Klebsiella sp. O852 were disrupted by ultrasonic extraction. The location and the conversion conditions of the key enzymes for the conversion of limonene to trans-dihydrocarvone were investigated. Then, the effects of ultrasonic time, material-liquid ratio, and amplitude on cell disruption were studied using the single factor experiments. Additionally, the Box-Behnken central combination test was designed to determine the optimal extraction conditions. The results showed that the enzyme was an intracellular enzyme. The optimal cultivation time was 8 h, which was the mid-logarithmic phase of cell growth. The optimum conversion time was 4 h, and the highest enzyme activity was obtained in phosphate buffer solution. The single factor experiments and response surface test showed that the optimal ultrasonic time was 21 min, material-liquid ratio was 1:13, and amplitude was 36%. And the content of the enzyme activity was (40.97±2.02) U/g under these optimal conditions. This study would provide a reference for further study on the purification and enzymatic properties of this enzyme.