氨基甲酸乙酯降解酶的固定化研究

Study on Immobilization of Urethanase

为了制备固定化酶,以体积分数5%的戊二醛溶液与壳聚糖交联8 h制备的载体对氨基甲酸乙酯(EC)降解酶粗酶液进行固定化,并对固定化EC降解酶的酶学性质展开分析。结果表明:固定化EC降解酶的最适温度和最适pH分别是42℃和3.6;该酶在乙醇体积分数7%~25%的范围内和使用次数在8次以内的酶活均保持在50%以上,说明该固定化酶的乙醇耐受范围广,催化活性高;而且分别在4℃和20℃下贮藏6周后的酶活损失率分别不足30%和40%。基于以上特性,为了考察实际应用效果,采用双水相萃取结合气相色谱-质谱联用(GC-MS)法和顶空固相微萃取结合气相色谱-质谱联用(HS-SPME-GC/MS)法分析了游离酶和固定酶对“美乐”葡萄酒中EC和挥发性风味物质的影响。结果表明,固定化EC降解酶的EC去除率达30.90%,是游离酶的81.53%;EC降解酶的处理方式对葡萄酒中挥发性物质的相对含量没有显著影响,只是引起了香气物质种类的减少和香气成分的改变,但固定化酶对挥发性风味物质的不利影响远低于游离酶。

In order to prepare immobilized enzyme,the crude urethanase was immobilized using the carrier cross-linked by 5%glutaraldehyde solution with chitosan for 8 h and the enzymatic properties of immobilized urethanase were analyzed.The results showed that the optimal temperature and pH of immobilized urethanase were 42℃and pH 3.6,respectively.The enzyme activity could be kept above 50%within the range of 7%~25%ethanol or reused within 8 times,which indicated that the immobilized enzyme had wide ethanol tolerance range and high catalytic activity.The enzyme activity loss rate was less than 30%or 40%when stored at 4℃or 20℃for 6 weeks.Based on the above characteristics,the effect of free and immobilized enzyme on ethyl carbamate(EC)and volatile flavor substances in‘Merlot’wine were analyzed by aqueous two-phase extraction combined with gas chromatography-mass spectrometry method and headspace solid-phase microextraction combined with gas chromatography-mass spectrometry(HS-SPME-GC/MS)method in order to investigate the practical application effect.The results showed that the EC removal rate of immobilized urethanase reached 30.90%,which was 81.53%of free enzyme.The treatment of urethanase had insignificant effect on volatile substances content in wine,and only caused the reduction of aroma species and changes of aroma components.However,the adverse effects of immobilized enzymes on volatile flavor substances were much less than those of free enzyme.