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.
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