柠檬烯转化酶的提取条件优化及酶学性质研究  

Optimization of extraction conditions of limonene converting-enzyme and study on enzymatic properties

作　　者：张莹莹 李志 张璐璐 李晓 范刚 彭芷芊 任婧楠 潘思轶 ZHANG Yingying;LI Zhi;ZHANG Lulu;LI Xiao;FAN Gang;PENG Zhiqian;REN Jingnan;PAN Siyi(College of Food Science and Technology,Huazhong Agricultural University/Key Laboratory of Environment Correlative Dietology,Ministry of Education,Wuhan 430070,China)

机构地区：[1]华中农业大学食品科学技术学院/环境食品学教育部重点实验室,武汉430070

出　　处：《华中农业大学学报》2020年第4期105-113,共9页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(31671824);国家重点研发计划专项(2017YFD0400101)。

摘　　要：以指状青霉(Penicillium digitatum)DSM62840为研究对象,采用高压细胞破碎法破碎菌体,对其转化柠檬烯生成α-松油醇过程中的相关酶的提取条件进行优化。以柠檬烯转化酶活性为指标,在单因素试验的基础上结合响应面试验法,确定最佳提取条件;同时,对柠檬烯转化酶的酶学性质进行了初步探索。结果显示,最佳提取条件为:破碎压力100 MPa、破碎6次、液料比15∶1(mL/g),在此条件下,α-松油醇的质量浓度为855.75 mg/L,酶活力达到71.31 U。柠檬烯转化酶最佳转化时间为4 h,在磷酸盐缓冲液中获得的酶活性最高;该酶可能为细胞色素P450,且不同的金属离子对酶活性的影响不同:Fe^2+对酶有轻微的抑制作用,而Ca^2+、Mg^2+、Mn^2+、Zn^2+、Co^2+、Ba^2+、Na^+、K^+、Fe^3+、Ni^2+则对酶有不同程度的激活作用。粗酶液SDS-PAGE电泳结果表明优化后的提取工艺效果较好。With Penicillium digitatum DSM62840 as the object of research,the extraction conditions of related enzymes in the process of converting limonene toα-terpenol were optimized to obtain high activity.And limonene converting-enzyme was extracted by high pressure cell crushing method.Taking the limonene converting-enzyme activity as evaluation indicator,the response surface methodology was used to optimize the extraction technology based on single factor test.At the same time,the properties of limonene converting-enzyme were preliminarily studied.The results showed that the optimum conditions for extracting were as follows:disruption pressure of 100 MPa,crushing times of 6,and liquid-to material ratio of 15∶1(mL/g).Under the conditions,the yield ofα-terpenol was 855.75 mg/L,and the enzyme activity was 71.31 U.The optimum conversion time of limonene converting-enzyme was 4 h,and the highest enzyme activity was obtained in phosphate buffer.Limonene converting-enzyme might be cytochrome P450,and different metal ions have different effects on the enzyme:Fe^2+had slight inhibition on the enzyme,while Ca^2+,Mg^2+,Mn^2+,Zn^2+,Co^2+,Ba^2+,Na^+,K^+,Fe^3+and Ni^2+had different activation effects on the enzyme.SDS-PAGE analysis of the crude enzyme solution showed that the extraction effect could be better under the optimum conditions.

关 键 词：指状青霉 柠檬烯 提取条件 生物转化 柠檬烯转化酶 响应面优化 酶活性 

分 类 号：TS201.25[轻工技术与工程—食品科学]