固定化的β-半乳糖苷酶合成低聚半乳糖的研究
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摘要
β-半乳糖苷酶可以利用乳糖合成低聚半乳糖(GOS)，用作功能性食品配料。β-半乳糖苷酶固定化是工业化处理乳清，使其乳糖合成GOS的必要步骤。本论文研究β-半乳糖苷酶的固定化方法，以期获得性能良好的固定化酶，将其用于处理乳清合成GOS。
将乳清粉用蒸馏水溶解复原成一定乳糖浓度的溶液作为实验样品。GOS得率使用TLC法检测，TLC法的准确性用HPLC法进行确证，证明TLC法是检测GOS的有效方法。
选择壳聚糖作载体，戊二醛交联的方法固定化β-半乳糖苷酶，研究了固定化的最适条件：确定出先固化后交联的顺序较好，4℃固定化6h，pH6.0，1％戊二醛室温交联30min，加酶量为0.4mg／g干重壳聚糖珠，活力回收率达到26.43％，该结果不理想。
实验制备了海藻酸钙-明胶凝胶包埋法固定化酶，其最优条件为：高粘度海藻酸钠2.54％，明胶0.88％，低粘度海藻酸钠0.24％时，凝胶获得最大的稳定性，重复使用次数可以达到8次。经过戊二醛交联防止酶泄漏，并且提高了固定化酶的操作稳定性，加酶量0.06％(w/v)，活力回收率最高可达80％，即固定化酶的比活力为15.1u/g凝胶。海藻酸钙凝胶包埋制备的固定化酶最适温度为55℃，较游离酶高5℃；最适pH升高0.5个单位，耐热性和pH耐受性变宽，而且具有向碱偏移的趋势，这些特性有利于固定化酶的直接使用。
利用海藻酸钙-明胶凝胶包埋法固定化酶，以复原乳清为底物合成GOS，经单因素试验和响应面法(RSA)优化分析，当反应时间为2.0h，乳糖含量37.4％，温度为58.2℃，pH为5.0～6.0，加酶量为0.14％时，GOS得率最高，理论值为28.9％，经验证实验可以达到29.1％。1升的放大实验中GOS得率为28.3％，与小试相差无几。
本论文证实海藻酸钙—明胶凝胶包埋法可以制备活力回收率较高的固定化乳糖酶，并且具有较好的操作稳定性，该酶可以用于处理高浓度乳清，合成GOS，并具有进一步扩大试验的价值。
β-galactosidase can synthesizes galactooligosacchrides (GOS) at high level of lactose. The immobilization of β -galactosidase was investigated in order to make full use of lactose from whey industrially. The good method of immobilization of β -galactosidase was studied, then the immobilized β-galactosidase was applied to synthesize GOS.
Whey powder was reconstituted to the solution with determined concentration of lactose for the working samples. The yield of GOS was monitored with TLC corrected with HPLC.
Aspergillus-oryza-β-galactosidase was adsorbed on chitosan and cross-linked with glutaraldehyde. The optimal parameters included: cross-linking followed with immobilization, under 4 C, immobilization for 6h, glutaraldehyde pH6.0, 1.0%; under the room temperature for 30min. The enzyme concentration was 0.4mg/g dry chitosan beads. The activity yield reached 26.43%, which was not ideal for enzymatic preparation of GOS.
Consequently, calcium alginate gel was used to entrap β-galactosidase. The optimums as follows: 2.54% high-viscosity-sodium alginate, 0.88%gelatin, 0.24%low- viscosity-sodium alginate, 2.0%CaCl2, under which condition the gel got the best stability that can be used 8 times on end and the activity yield was 80%. Then immobilized enzyme entrapped in gel was characterized. The immobilized enzyme entrapped with alginate was showed that its optimal temperature rises 5 C to 55 C, optimal pH raised 0.5, endurance to temperature and pH were both better than soluble enzyme which showed that this kind of immobilized enzyme can be used directly and conveniently.
The immobilized enzyme entrapped in alginate-gelatin gel was used to produce GOS from reconstituted whey. Through single factor experiments and SAS analysis, optimal conditions included lactose concentration, 37.4%; reaction time, 2.0h; temperature, 58.2 C; pH 5.0-6.0, enzyme concentration, 0.14%. The maximum GOS concentration was estimated as 28.9%, which was proved as 29.1 % in the trials with 1L reconstituted whey.
Immobilized β-galactosidase can be made by entrapment in alginate and gelatin with high activity yield and good practical stability, having the industrial prospect to synthesize GOS.
将乳清粉用蒸馏水溶解复原成一定乳糖浓度的溶液作为实验样品。GOS得率使用TLC法检测，TLC法的准确性用HPLC法进行确证，证明TLC法是检测GOS的有效方法。
选择壳聚糖作载体，戊二醛交联的方法固定化β-半乳糖苷酶，研究了固定化的最适条件：确定出先固化后交联的顺序较好，4℃固定化6h，pH6.0，1％戊二醛室温交联30min，加酶量为0.4mg／g干重壳聚糖珠，活力回收率达到26.43％，该结果不理想。
实验制备了海藻酸钙-明胶凝胶包埋法固定化酶，其最优条件为：高粘度海藻酸钠2.54％，明胶0.88％，低粘度海藻酸钠0.24％时，凝胶获得最大的稳定性，重复使用次数可以达到8次。经过戊二醛交联防止酶泄漏，并且提高了固定化酶的操作稳定性，加酶量0.06％(w/v)，活力回收率最高可达80％，即固定化酶的比活力为15.1u/g凝胶。海藻酸钙凝胶包埋制备的固定化酶最适温度为55℃，较游离酶高5℃；最适pH升高0.5个单位，耐热性和pH耐受性变宽，而且具有向碱偏移的趋势，这些特性有利于固定化酶的直接使用。
利用海藻酸钙-明胶凝胶包埋法固定化酶，以复原乳清为底物合成GOS，经单因素试验和响应面法(RSA)优化分析，当反应时间为2.0h，乳糖含量37.4％，温度为58.2℃，pH为5.0～6.0，加酶量为0.14％时，GOS得率最高，理论值为28.9％，经验证实验可以达到29.1％。1升的放大实验中GOS得率为28.3％，与小试相差无几。
本论文证实海藻酸钙—明胶凝胶包埋法可以制备活力回收率较高的固定化乳糖酶，并且具有较好的操作稳定性，该酶可以用于处理高浓度乳清，合成GOS，并具有进一步扩大试验的价值。
β-galactosidase can synthesizes galactooligosacchrides (GOS) at high level of lactose. The immobilization of β -galactosidase was investigated in order to make full use of lactose from whey industrially. The good method of immobilization of β -galactosidase was studied, then the immobilized β-galactosidase was applied to synthesize GOS.
Whey powder was reconstituted to the solution with determined concentration of lactose for the working samples. The yield of GOS was monitored with TLC corrected with HPLC.
Aspergillus-oryza-β-galactosidase was adsorbed on chitosan and cross-linked with glutaraldehyde. The optimal parameters included: cross-linking followed with immobilization, under 4 C, immobilization for 6h, glutaraldehyde pH6.0, 1.0%; under the room temperature for 30min. The enzyme concentration was 0.4mg/g dry chitosan beads. The activity yield reached 26.43%, which was not ideal for enzymatic preparation of GOS.
Consequently, calcium alginate gel was used to entrap β-galactosidase. The optimums as follows: 2.54% high-viscosity-sodium alginate, 0.88%gelatin, 0.24%low- viscosity-sodium alginate, 2.0%CaCl2, under which condition the gel got the best stability that can be used 8 times on end and the activity yield was 80%. Then immobilized enzyme entrapped in gel was characterized. The immobilized enzyme entrapped with alginate was showed that its optimal temperature rises 5 C to 55 C, optimal pH raised 0.5, endurance to temperature and pH were both better than soluble enzyme which showed that this kind of immobilized enzyme can be used directly and conveniently.
The immobilized enzyme entrapped in alginate-gelatin gel was used to produce GOS from reconstituted whey. Through single factor experiments and SAS analysis, optimal conditions included lactose concentration, 37.4%; reaction time, 2.0h; temperature, 58.2 C; pH 5.0-6.0, enzyme concentration, 0.14%. The maximum GOS concentration was estimated as 28.9%, which was proved as 29.1 % in the trials with 1L reconstituted whey.
Immobilized β-galactosidase can be made by entrapment in alginate and gelatin with high activity yield and good practical stability, having the industrial prospect to synthesize GOS.
引文
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