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关于固定化酶的文献报导很多,但是,对于需要辅酶的酶的固定化的报导却很少。国外S.Ikeda等人用5′-磷酸吡哆醛联接琼脂糖来固定化各种含辅酶B_6(PLP)的酶或直接用琼脂糖来固定化含辅酶B_6(PLP)的酶。如图1。辅酶B_6(5′-磷酸吡哆醛;简称PLP)。以PLP为辅酶的不同酶催化各种氨基酸的反应,包括转氨作用,脱羧作用,β-羟基氨基酸的脱水作用,α-D-氨基酸的消旋作用和半胱氨酸的脱巯基作用等。在这些酶促反应中,PLP起了非常重要的作用,PLP不仅是通过吡啶氮以及5′-磷酸酯基团以离子键形式与活性中心相连,而且PLP以醛基与酶蛋白上赖氨酸的ε-氨基形成西佛碱,见图2,这些作用力造成PLP与需要PLP作为辅酶的酶之间特异亲和力。因此,固定化PLP不仅作为需要PLP作为辅因子的酶的固定化;而且可以用来提纯酶蛋白,并可进一步探讨结构-功能关系。通过固定色氨酸酶柱和乳酸脱氢酶系统的偶联作用,以NADH在340 nm处光吸收值降低来定
There are many reports on immobilized enzymes, however, there are few reports on the immobilization of enzymes requiring coenzymes. Foreign S. Ikeda et al. Immobilized various Coenzyme B_6 (PLP) -enzymes with pyridoxal 5’-phosphate and immobilized Coenzyme B_6 (PLP) -containing enzyme directly with agarose. Figure 1. Coenzyme B_6 (pyridoxal 5’-phosphate; PLP for short). Different enzymes catalyzed by PLP as coenzymes catalyze various amino acid reactions, including transamination, decarboxylation, dehydration of β-hydroxyamino acids, racemization of α-D-amino acids and desulfhydryls of cysteines. In these enzymatic reactions, PLP plays a very important role. PLP is not only linked to the active site by ionic bond between pyridinium nitrogen and 5’-phosphate group, but also PLP binds to the lysine Of the epsilon amino groups form sifuvone, see Figure 2, these forces result in a specific affinity between the PLP and the enzyme that needs PLP as a coenzyme. Therefore, the immobilized PLP is not only immobilized as an enzyme requiring PLP as a cofactor but also can be used to purify enzyme proteins, and the structure-function relationship can be further explored. By coupling the immobilized tryptophanase column with the lactate dehydrogenase system, the decrease in light absorbance at 340 nm for NADH is determined