Mateo César, Monti Rubens, Pessela Benevides C C, Fuentes Manuel, Torres Rodrigo, Guisán José Manuel, Fernández-Lafuente Roberto
Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Biotechnol Prog. 2004 Jul-Aug;20(4):1259-62. doi: 10.1021/bp049957m.
The kinetic constants (Km, Vmax, and inhibition constants for the different products) of soluble and different immobilized preparations of beta-galactosidase from Kluyveromyces lactis were determined. For the soluble enzyme, the Km was 3.6 mM, while the competitive inhibition constant by galactose was 45 mM and the noncompetitive one by glucose was 758 mM. The immobilized preparations conserved similar values of Km and competitive inhibition, but in some instances much higher values for the noncompetitive inhibition constants were obtained. Thus, when glyoxyl or glutaraldehyde supports were used to immobilize the enzyme, the noncompetitive inhibition was greatly reduced (Ki approximately 15,000 and >40,000 mM, respectively), whereas when using sugar chains to immobilize the enzyme the behavior had an effect very similar to the soluble enzyme. These results presented a great practical relevance. While using the soluble enzyme or the enzyme immobilized via the sugar chain as biocatalysts in the hydrolysis of lactose in milk only around 90% of the substrate was hydrolyzed, by using of these the enzyme immobilized via the glyoxyl or the glutaraldehyde groups, more than 99% of the lactose in milk was hydrolyzed.
测定了乳酸克鲁维酵母β-半乳糖苷酶的可溶性制剂和不同固定化制剂的动力学常数（Km、Vmax以及不同产物的抑制常数）。对于可溶性酶，Km为3.6 mM，而半乳糖的竞争性抑制常数为45 mM，葡萄糖的非竞争性抑制常数为758 mM。固定化制剂的Km值和竞争性抑制作用相近，但在某些情况下，非竞争性抑制常数的值要高得多。因此，当使用乙二醛或戊二醛载体固定酶时，非竞争性抑制作用大大降低（Ki分别约为15,000和>40,000 mM），而使用糖链固定酶时，其行为与可溶性酶非常相似。这些结果具有很大的实际意义。在使用可溶性酶或通过糖链固定的酶作为生物催化剂水解牛奶中的乳糖时，只有约90%的底物被水解，而使用通过乙二醛或戊二醛基团固定的酶时，牛奶中超过99%的乳糖被水解。
