聚乙烯醇固定化胆固醇氧化酶的研究  

Study on cholesterol oxidase immobilized by polyvinyl alcohol(PVA)

作　　者：李杰民[1,2] 王双双[1] 刘倩倩[1] 赵芳[3] 王发合 周河治[1] 杨辉[1] 

机构地区：[1]广西大学生命科学与技术学院,南宁530005 [2]广西农业科学院农产品加工研究所,南宁530007 [3]邯郸职业技术学院,河北邯郸056001 [4]青岛赛特香料有限公司,山东青岛266423

出　　处：《南方农业学报》2013年第4期657-661,共5页Journal of Southern Agriculture

基　　金：广西自然科学基金项目(桂科自0728017)

摘　　要：【目的】探讨类芽孢杆菌胆固醇氧化酶(COD)的固定化,为COD的应用研究提供参考依据。【方法】以不同浓度的聚乙烯醇、硼酸为主要材料制备固定化酶,并通过试验确定制备固定化酶的最适浓度、最佳反应温度、最适pH、热稳定性、酸稳定性、米氏常数及操作稳定性。【结果】选用12%聚乙烯醇、5%硼酸制备的固定化酶形状好、强度适中、易操作、固定化率高,其最适反应温度60℃,最适反应pH7.0,热、酸稳定性比游离酶好,且稳定范围较游离酶宽,表观米氏常数Km(2.55×10-5mol/L)相对游离酶变小,重复使用5次后酶活力仍能保持在67.8%以上,半衰期为8.91次。【结论】聚乙烯醇制备的固定化COD稳定性好,与底物亲和力较强,可重复回收利用,具有良好的应用前景。[Objective]The immobilization of cholesterol oxidase （COD） was investigated to provide technical support for COD application. [Method]The immobilized enzyme was prepared by polyvinyl alcohol and boric acid. The optimum concentration, reaction temperature, heat stability, pH value, acid stability, Miehaelis constant and operation stability were deter- mined through experiments. [Result]In the optimal conditions for the immobilized enzyme preparation （12% PVA, 5% boric acid, and pH 7.0 at the reaction temperature of 60 ~C）, the immobilized enzyme was easily produced with good shape, mod- erate intensity and high immobilization rate. The optimum reaction pH was 7.0. The thermal and acidic stabilities were better than those of the free enzyme. Its Km was 2.55/10-5 mol/L. After five times of repeated reaction, the relative activity of the immobilized enzyme remained above 67.8% with the half-life period of 8.91 times. [Conclusion]The cholesterol oxidase immobilized by polyvinyl alcohol exhibited good stability and strong affinity to cholesterol. It possesses good application prospects due to its easy recovery and application.

关 键 词：聚乙烯醇 胆固醇氧化酶 固定化 酶活性 稳定性 米氏常数 

分 类 号：Q814.2[生物学—生物工程]