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      2. 檸檬烯轉化酶的提取條件優化及酶學性質研究
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        國家自然科學基金項目(31671824);國家重點研發計劃專項(2017YFD0400101)


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

          以指狀青霉(Penicillium digitatum) DSM62840為研究對象,采用高壓細胞破碎法破碎菌體,對其轉化檸檬烯生成α-松油醇過程中的相關酶的提取條件進行優化。以檸檬烯轉化酶活性為指標,在單因素試驗的基礎上結合響應面試驗法,確定最佳提取條件;同時,對檸檬烯轉化酶的酶學性質進行了初步探索。結果顯示,最佳提取條件為:破碎壓力100 MPa、破碎6次、液料比15∶1 (mL/g),在此條件下,α-松油醇的質量濃度為855.75 mg/L,酶活力達到71.31 U。檸檬烯轉化酶最佳轉化時間為4 h,在磷酸鹽緩沖液中獲得的酶活性最高;該酶可能為細胞色素P450,且不同的金屬離子對酶活性的影響不同:Fe2+對酶有輕微的抑制作用,而Ca2+、Mg2+、Mn2+、Zn2+、Co2+、Ba2+、Na+、K+、Fe3+、Ni2+則對酶有不同程度的激活作用。粗酶液SDS-PAGE電泳結果表明優化后的提取工藝效果較好。

          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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        張瑩瑩,李志,張璐璐,李曉,范剛,彭芷芊,任婧楠,潘思軼.檸檬烯轉化酶的提取條件優化及酶學性質研究[J].華中農業大學學報,2020,39(4):105-113

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        • 收稿日期:2020-02-22
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        • 在線發布日期: 2020-07-30
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