STABILIZATION STUDY OF TETRAMERIC Kluyveromyces lactis β-GALACTOSIDASE BY IMMOBILIZATION ON IMMOBEAD: THERMAL, PHYSICO-CHEMICAL, TEXTURAL AND CATALYTIC PROPERTIES

Abstract We investigated the immobilization of a tetrameric Kluyveromyces lactis β-galactosidase (EC: 3.2.1.23) (KL-Gal) on Immobead 150 using different support modification strategies. Immobead support was modified using an acid solution of H2SO4:HNO3 (3:1) (Immobead-Ac) or 5 % (v/v) glutaraldehyde (Immobead-Glu). Its unmodified form (Immobead) was also tested. Immobilization yields and efficiencies were evaluated by testing protein loads from 10 to 200 mg.g-1 support. The thermal, physico-chemical, textural and catalytic properties of the supports (modified and unmodified) and their derivatives (Immobead-KL-Gal, Immobead-Ac-KL-Gal and Immobead-Glu-KL-Gal) were analyzed. The highest immobilization yields and efficiencies were achieved with a protein load of 100 mg.g-1 support. Surface and pore areas of the Immobead support were greatly decreased after modification. Michaelis constant of the immobilized β-galactosidase increased in the derivatives. Maximum velocity decreased approximately 2.8 times for Immobead-KL-Gal and Immobead-Glu-KL-Gal, and approximately 1.4 times for Immobead-Ac-KL-Gal. In batch processes, the three derivatives could be reused successfully at least 15 times, maintaining high residual enzymatic activity during the lactose hydrolysis (in both cheese whey and milk). The tetrameric K. lactis β-galactosidase immobilized on Immobead supports via the tested treatments was stabilized and is an alternative tool for lactose hydrolysis in the dairy industry.

摘要 本研究探究了将四聚体乳酸克鲁维酵母（Kluyveromyces lactis）β-半乳糖苷酶（β-galactosidase，EC: 3.2.1.23，简称KL-Gal）通过不同载体修饰策略固定于固定化载体Immobead 150上的方法。分别采用浓硫酸-浓硝酸（体积比3:1）溶液（处理后载体记为Immobead-Ac）、5%（体积分数）戊二醛（glutaraldehyde，处理后载体记为Immobead-Glu）对原始Immobead载体进行修饰，并设置未修饰的原始载体（Immobead）作为对照。通过设置10~200 mg·g⁻¹载体的蛋白负载量梯度，评估了各体系的固定化得率与固定化效率。对修饰及未修饰的载体，以及所制备的三类固定化酶衍生物（Immobead-KL-Gal、Immobead-Ac-KL-Gal与Immobead-Glu-KL-Gal）的热学、物理化学、织构及催化性能进行了表征分析。结果显示，当蛋白负载量为100 mg·g⁻¹载体时，可获得最高的固定化得率与固定化效率。载体经修饰后，其表面与孔道面积均出现显著下降。固定化β-半乳糖苷酶的米氏常数（Michaelis constant）在各衍生物中均有所升高；而最大反应速率（maximum velocity）方面，Immobead-KL-Gal与Immobead-Glu-KL-Gal约下降2.8倍，Immobead-Ac-KL-Gal则约下降1.4倍。在批次反应体系中，三类固定化酶衍生物均可至少重复使用15次，且在乳清（cheese whey）与牛乳体系的乳糖水解（lactose hydrolysis）反应中均能维持较高的残余酶活。经上述三种修饰方式固定于Immobead载体的四聚体乳酸克鲁维酵母β-半乳糖苷酶稳定性得到提升，可作为乳制品工业（dairy industry）中乳糖水解的备选工具。