摘要
利用大孔吸附树脂DA-201为载体对海洋脂肪酶固定化,并探寻添加剂对固定化过程的影响。分别以NH_4Cl、甘露糖和甘氨酸为添加剂,采用单因素和正交实验相结合的方法优化条件。结果显示,以NH_4Cl为添加剂的最优条件:柠檬酸-柠檬酸钠缓冲液pH 6. 0,固定化温度30℃,载体投放量0. 5g,NH_4Cl浓度25mmol/L,固定化时间3. 0h,酶活力达到115. 27U/g;比不含有添加剂的固定化酶固定化效率提高47. 42%。以甘露糖为添加剂最优条件:磷酸二氢钾-氢氧化钠缓冲液pH7. 0,固定化温度35℃,载体投放量0. 5g,甘露糖浓度10mmol/L,固定化时间4. 5h;酶活力达到122. 75U/g,比不含有添加剂的固定化酶固定化效率提高6. 50%。以甘氨酸为添加剂的最优条件:磷酸二氢钾-氢氧化钠缓冲液pH7. 0,固定化温度20℃,载体投放量0. 5g,甘氨酸浓度为25mmol/L,固定化时间7. 5h;酶活力达到141. 69U/g,比不含有添加剂的固定化酶固定化效率提高26. 12%。采用不同添加剂对大孔吸附树脂DA-201的吸附固定化过程有较大影响,可以极大地提高吸附效率;同时发现缓冲液类型、pH、温度、添加剂浓度和固定化时间等对DA-201树脂吸附脂肪酶有很大影响,对后续吸附固定化工业酶研究有较好的参考价值。
Macroporous absorbent resin DA-201 was adopted as a carrier to immobilize marine-derived lipase,and the influence of additives on the immobilization process was explored. NH_4Cl,mannose and glycine were used as the additives,and the conditions were optimized by a combination of single factor and orthogonal experiments. The results showed that the optimal conditions with additive NH_4Cl were: citric acid-sodium citrate buffer pH6. 0,immobilization temperature 30℃,carrier quantity 0. 5 g, NH_4Cl concentration 25 mmol/L,immobilized time 3. 0 h; enzyme activity reached 115. 27 U/g,which was 47. 42% higher than that of the immobilized enzyme without additive. The optimal conditions with additive mannose were: potassium dihydrogen phosphate-sodium hydroxide buffer pH7. 0,immobilized temperature 35℃,carrier quantity 0. 5 g,mannose concentration 10 mmol/L,the immobilized time 4. 5 h; the enzyme activity reached 122. 75 U/g,which was6. 50% higher than that of the immobilized enzyme without additive. The optimal conditions with glycine additive: potassium dihydrogen phosphate-sodium hydroxide buffer pH7. 0,immobilized temperature 20℃,carrier quantity 0. 5 g,the glycine concentration 25 mmol/L,the immobilized time 7. 5 h; the enzyme activity reached 141. 69 U/g,which was 26. 12% higher than that of the immobilized enzyme without additive. The addition of different additives exhibited great effects on the immobilization through absorbtion by macroporous absorbent resin DA-201 and could greatly improve the adsorption efficiency. Additionally,buffer type,pH,temperature,additive concentration and immobilization time were found to have great influence on the adsorption of lipase by resin DA-201,which provides good reference for subsequent research of the immobilization of industrial enzymes.
引文
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