吸附交联法和包埋法固定化混合酶研究

对从自然界分离得到的Microbacterium sp．OU01进行发酵，制得粗酶液（壳聚糖酶和伊肛氨基葡萄糖苷酶混合液），采用吸附交联法及包埋法对其进行固定，研究其固定化的最优条件和固定化酶的酶学性质。结果表明，游离混合酶液的最佳PH为5．6，最适反应温度为50℃。吸附交联法固定化酶的最佳条件为：戊二醛体积分数5．0％，加酶量15mL（49．51U／mL），固定化时间6h。该条件下制得的固定化酶的最适pH为4．5，最适反应温度为60℃。包埋法固定化酶的最佳条件为：海藻酸钠质量浓度10g／L，CaCl。质量浓度15g／L，加酶量为1mL（50．35U／mL）／5mL质量浓度10g／L海藻酸钠，固定化时间20min。该条件下制得的固定化酶的最适PH为5．0，最适反应温度为50℃。固定化酶酶解壳聚糖所得产物中含D-氨基葡萄糖，说明粗酶液中的壳聚糖酶和外切-β-D-氨基葡萄糖苷酶被同时固定。通过对游离酶和固定化酶稳定性的研究，表明吸附交联固定化酶更适于工业化应用生产D-氨基葡萄糖。

Comparative Study of Crude Enzyme Immobilization by Cross-linking and Entrapment Methods

The crude enzyme （including chitosanase and β-D-glucosaminidase） was got by the fer- mentation of Microbacterium sp. OU01 that was separated. Adsorption cross-linking method and entrapment method by sodium alginate were adopted to immobilize crude enzyme. The effect of reaction conditions on the relative activity of immobilized crude enzyme by two kinds of immobi- lization methods was tested. The results show that the best efficiency of immobilization by ad- sorption cross-linking method was obtained when glutaraldehyde concentration was 5.0%, crude enzyme dosage was 15 mL （49.51 U/mL） and immobilized time was 6 h. The best efficiency of immobilization by entrapment method was obtained when sodium alginate concentration was 10 g/ L, crude enzyme dosage was 1 mL （50.35 U/mL）/5 mL 10 g/L sodium alginate, CaC12 concen-tration was 15 g/L and immobilized time was 20 rain. The optimal pH and temperature of the crude enzyme were 5.6 and 50 ℃. But the optimal pH and temperature of the immobilized enzyme by adsorption cross-linking method were 4.5 and 60 ℃. The optimal pH and temperature of the immobilized enzyme by entrapment method were 5.0 and 50 ℃. The chitosan was hydrolyzed into D-glucosame by two kinds of immobilized enzyme, which showed that the chitosanase and β-D- glueosaminidase in the crude enzyme was simultaneously immobilized. The study of stability in the free enzyme and immobilized enzyme shows that the immobilized enzyme by adsorption cross-linking method might have a prospective industrial application.