一种高耐热乳糖酶的异源表达、固定化及酶学性质研究  

Heterologous expression and enzymatic characterization of a highlythermostable lactase and its immobilized enzyme

作　　者：徐惠东 尤扬 游颖欣 王周平[1] 夏雨[1] XU Huidong;YOU Yang;YOU Yingxin;WANG Zhouping;XIA Yu(School of Food Science and Technology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学食品学院,江苏无锡214122

出　　处：《食品与发酵工业》2024年第21期1-8,共8页Food and Fermentation Industries

基　　金：国家重点研发计划项目“政府间国际科技创新合作”重点专项(2021YFE0101800);江苏省重点研发计划(现代农业)项目(BE2022324)。

摘　　要：乳糖酶在乳制品生产中具有重要作用,但是目前商用的乳糖酶耐热性低,可重复利用率差。为了解决以上问题,该实验用枯草芽孢杆菌WB800异源表达了激烈热球菌(Pyrococcus furiosus)来源的β-半乳糖苷酶(β-galactosidase,BgaS)。该酶最适温度为90℃,最适pH值为7,具有良好的应用潜力。为了提高该乳糖酶的重复利用率,该实验用壳聚糖微球为载体,采用交联法实现了BgaS的固定化并对交联条件进行优化,确定最适交联条件为:戊二醛26.50 g/L,交联温度35℃,交联时间2.5 h,酶添加量5.94 U/mL,优化后酶活力回收率达55.11%。固定化酶相较游离酶,最适pH与最适温度相同,且在非最适pH条件下,固定化酶的相对酶活力较游离酶普遍提升了18.23%以上,并且固定化酶在95℃条件下孵育90 min仍保有80%以上的相对活性,较游离酶提升了11.31%。并且固定化酶回收利用率高,操作稳定性好,最适条件下处理3 h即可使牛乳中乳糖含量降低至0.5%以下,达到无乳糖水平,为工业化应用提供了技术支撑。Lactase plays a significant role in the dairy industry.However,currently commercial lactase have low thermal stability and poor reusability.To convert these drawbacks,β-galactosidase(BgaS)from Pyrococcus furiosus was selected and heterologously expressed in Bacillus subtilis WB800.The optimum temperature and pH of BgaS were at 90℃and 7.These enzymic properties showed a promising potential for application in lactose-free product processing.To enhance the reusability of the BgaS,chitosan microspheres were employed as carriers for enzyme immobilization using a cross-linking method,and the cross-linking conditions were optimized.The optimal conditions were as follows:26.50 g/L of glutaraldehyde,35℃of cross-linking temperature,2.5 h of cross-linking time,and 5.94 U/mL of enzyme dosage.After optimization,the enzyme activity recovery reached 55.11%.Compared to free enzymes,immobilized enzymes had the same optimal pH and temperature.Moreover,under non-optimal pH conditions,the relative enzyme activity of immobilized enzymes generally increased by more than 18.23%compared to free enzymes.In terms of thermostability,immobilized enzymes retained over 80%relative activity after being incubated at 95℃for 90 min,which was an improvement of 11.31%compared to free enzymes.Meanwhile,immobilized enzymes had a high recovery rate and good operational stability,and could reduce lactose in milk to below 0.5%in just 3 h under optimal conditions,achieving lactose-free levels.This provides technical support for potential industrial applications.

关 键 词：耐热乳糖酶 壳聚糖 固定化 脱乳糖产品 枯草芽孢杆菌 

分 类 号：TS252.1[轻工技术与工程—农产品加工及贮藏工程]