利用聚丙烯酰胺凝胶电泳研究硫酸软骨素酶的性质

本文运用聚丙烯酰胺凝胶电泳（PAGE）研究了RC3菌株所产硫酸软骨素A酶（CSAase）的部分酶学性质。通过对菌株RC3进行菌种活化、扩大培养和发酵产酶培养，再通过对微生物菌体进行超声波破碎获得胞内粗酶液，将其与硫酸软骨素A等体积混合，反应后通过PAGE研究各条件对酶活力的影响。结果表明，RC3菌株所产硫酸软骨素A酶的最适反应温度为20~40 ℃，最适pH值为8.0，镁离子是该酶的激活剂，铜离子和锌离子是该酶的抑制剂，钾离子、钙离子、钠离子、锰离子和钡离子对该酶活力没有显著影响；当镁离子浓度为10 mM时对该酶表现出最强的激活作用；当铜离子和锌离子的浓度为5 mM时对该酶活力表现出强烈的抑制作用；在上述最优条件下，该酶作用4 h后获得的低分子量产物比大分子CSA具有更好的抗氧化活性。上述研究结果对该CSAase的应用奠定基础。
In this study, polyacrylamide gel electrophoresis (PAGE) was used to explore the enzymatic properties of chondroitinase A (CSAase) produced by strain RC3. A crude solution of intracellular chondroitinase was obtained by ultrasonication of the microbial cells, which was then mixed with an equal volume of chondroitin sulfate A (CSA) and incubated. The effect of various culture conditions on enzyme activity was studied using PAGE. The results showed that the optimal temperature and pH value for fermentation of CSAase were 20 ℃ to 40 ℃ and 8.0, respectively. Magnesium ion was found to activate CSAase, while copper and zinc ions were strong inhibitors. Potassium, calcium, sodium, manganese, and barium ions did not significantly affect enzyme activity. Magnesium ions at a concentration of 10 mM showed the strongest activating effect on CSAase. Copper and zinc ions showed strong inhibitory effect on the enzyme activity at a concentration of 5 mM. Under the above optimal conditions, the low-molecular-weight products that were obtained after a 4-hour hydrolysis reaction using this enzyme, showed a higher antioxidant activity than that of CSA, which is a macromolecule. This study suggests the potential applicability of CSAase.