柠檬烯转化酶的提取条件优化及酶学性质研究
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国家自然科学基金项目(31671824);国家重点研发计划专项(2017YFD0400101)


Optimization of extraction conditions of limonene converting-enzyme and study on enzymatic properties
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    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:Fe2+对酶有轻微的抑制作用,而Ca2+、Mg2+、Mn2+、Zn2+、Co2+、Ba2+、Na+、K+、Fe3+、Ni2+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。

    Abstract:

    With Penicillium digitatum DSM62840 as the object of research,the extraction conditions of related enzymes in the process of converting limonene to α-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 of α-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: Fe2+ had slight inhibition on the enzyme,while Ca2+,Mg2+,Mn2+,Zn2+,Co2+,Ba2+,Na+,K+,Fe3+ and Ni2+ 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.

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张莹莹,李志,张璐璐,李晓,范刚,彭芷芊,任婧楠,潘思轶.柠檬烯转化酶的提取条件优化及酶学性质研究[J].华中农业大学学报,2020,39(4):105-113

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  • 收稿日期:2020-02-22
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  • 在线发布日期: 2020-07-30
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