纳米磁性壳聚糖微球固定化Alcalase碱性蛋白酶的制备及酶学性质研究  

Study on preparation and enzymatic properties of immobilized alcalase alkaline protease magnetic chitosan microspheres

作　　者：梁玉杰[1,2] 芦鑫[2] 黄纪念[2] 谢新华[1] 

机构地区：[1]河南农业大学食品科学技术学院,河南郑州450002 [2]河南省农科院农副产品加工研究所,河南郑州450002

出　　处：《食品与发酵工业》2014年第8期66-71,共6页Food and Fermentation Industries

基　　金：河南省重大科技攻关计划项目(122101110500)

摘　　要：以自制磁性壳聚糖微球作固定化酶载体,考察给酶量、pH、戊二醛浓度和交联时间对固定酶酶活和酶活回收率的影响,并研究固定化酶的酶学性质及其微观结构。结果表明:给酶量112 000 u/g载体,pH 8.5,戊二醛体积分数8%,交联时间11 h条件下酶活达最高(86 779±119.26)u/g,酶活回收率达(77.48±0.11)%。固定化酶和游离酶最适pH分别为11和10.5,最适温度皆为60℃,且固定酶pH和温度稳定性明显高于游离酶;重复使用5次固定酶酶活保持(80.89±0.20)%;由米氏常数可知固定酶具有更强的底物亲和力;电镜显示Fe3O4磁核和磁性壳聚糖微球皆为表面光滑球形的纳米粒子,高比表面积能提供更多酶结合位点;红外光谱证明Fe3O4已被壳聚糖包埋,振动样品磁强计检测固定化酶具有良好磁响应性。In this paper, taking the magnetic chitosan microspheres （M-CS） as the carrier, the effects of en- zyme-to-carrier ratio, pH, glutaral concentration, reaction time on the activity and activity recovery of immobilized A1- calase were investigated. The characterizations and microstructure of immobilized enzyme were also studied. The re- suits showed that the maximum activity of immobilized Alcalase reached （86779± 119.26） u/g and the activity re- covery of immobilized Alcalase was （77.48 ± 0.11 ）% under the conditions of enzyme-to-carrier ratio 112000 u/g, pH 8.5, glutaral concentration 8% （V/V） , and reaction time 11 h. The optimal reaction pH values for free and im- mobilized Alcalase were 11 and 10.5 respectively, both The optimal reaction temperatures for free and immobilized Alcalase were 60 ℃. The pH and temperature stabilities of immobilized Alcalase were more excellent than those of free Alcalase. The activity of immobilized Alcalase reused 5 times remained 80.89% （ ± 0.20% ）. According to Km value, immobilized Alcalase showed stronger affinity than free Alcalase. Transmission electron microscope showed that Fe304 and chitosan microspheres were all spherical nanopaticles with smooth surface, so more enzyme could be bound due to high specific area. FT-IR revealed that Fe304 was well wrapped up in M-CS, Vibrating sample magnetometer indicated that immobilized Alcalase had a good magnetic response.

关 键 词：磁性壳聚糖微球 固定化 ALCALASE碱性蛋白酶 酶学性质 微观形态 

分 类 号：TQ925.2[轻工技术与工程—发酵工程]