Department of Chemistry, Gebze Institute of Technology, 41400, Gebze, Kocaeli, Turkey.
Appl Biochem Biotechnol. 2013 Dec;171(8):2142-52. doi: 10.1007/s12010-013-0507-5. Epub 2013 Sep 13.
A novel method was developed for the immobilization of Saccharomyces cerevisiae invertase within supermacroporous polyacrylamide cryogel and was used to produce invert sugar. First, the cross-linking of invertase with soluble polyglutaraldehyde (PGA) was carried out prior to immobilization in order to increase the bulkiness of invertase and thus preventing the leakage of the cross-linked enzyme after immobilization by entrapment. And then, in situ immobilization of PGA cross-linked invertase within cryogel synthesis was achieved by free radical polymerization in semi-frozen state. The method resulted in 100 % immobilization and 74 % activity yields. The immobilized invertase retained all the initial activity for 30 days and 30 batch reactions. Immobilization had no effect on optimum temperature and it was 60 °C for both free and immobilized enzyme. However, optimum pH was affected upon immobilization. Optimum pH values for free and immobilized enzyme were 4.5 and 5.0, respectively. The immobilized enzyme was more stable than the free enzyme at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes and microorganisms.
开发了一种将酿酒酵母转化酶固定在超大孔聚丙烯酰胺水凝胶内的新方法，并用于生产转化糖。首先，在固定化之前，通过与可溶性聚戊二醛（PGA）交联，增加转化酶的体积，从而通过包埋防止交联酶在固定化后泄漏。然后，通过在半冻结状态下的自由基聚合，在水凝胶合成中实现 PGA 交联转化酶的原位固定化。该方法的固定化效率为 100%，酶活回收率为 74%。固定化酶在 30 天和 30 批反应中保留了全部初始活性。固定化对最适温度没有影响，游离酶和固定化酶的最适温度均为 60°C。然而，最适 pH 值在固定化后受到影响。游离酶和固定化酶的最适 pH 值分别为 4.5 和 5.0。固定化酶在高 pH 值和温度下比游离酶更稳定。还测定了游离和固定化转化酶的动力学参数。新开发的方法简单有效，可用于固定化其他一些酶和微生物。
