马克斯克鲁维酵母β-半乳糖苷酶合成条件的优化及其酶学性质表征

Optimization of conditions for production of Kluyveromyces marxianusβ-galactosidase and characterization of its enzymatic properties

β-半乳糖苷酶是广泛使用的食品添加剂,主要用于降解乳制品中的乳糖,缓解乳糖不耐受症状。该研究对实验室保藏菌株马克斯克鲁维酵母的发酵条件进行了优化。在20 g/L半乳糖、20 g/L玉米浆干粉、40℃、初始pH 6.5、150 r/min的条件下,粗酶液的酶活力为26.3 U/mL,表明该菌株具有利用廉价碳氮源高效生产β-半乳糖苷酶的潜力。通过DEAE阴离子交换层析进一步纯化得到比活力为124.09 U/mg的纯化酶。纯化后酶的最适温度40℃,最适pH 6,Km为5.28 mmol/L,kcat为4.74 s^(-1)。此外,发现该酶受Mg^(2+)的促进,在20~40℃表现出优异的热稳定性,40℃下30 min仍能保持95.6%的活力。因此,马克斯克鲁维酵母β-半乳糖苷酶比乳酸克鲁维酵母的热稳定性更好,具有潜在的工业应用潜力。

β-Galactosidase is a widely used food additive,primarily used to degrade lactose in dairy products and alleviate symptoms of lactose intolerance.In this study,we optimized the fermentation conditions of Kluyveromyces marxianus,a strain preserved in our laboratory.Under the conditions of 20 g/L galactose,20 g/L corn steep powder,40℃,initial pH 6.5,and 150 r/min,the crude enzyme displayed an enzyme activity of 26.3 U/mL,indicating that the strain had the potential to efficiently produceβ-galactosidase using cheap carbon and nitrogen sources.Further purification via DEAE anion exchange chromatography resulted in a purified enzyme with a specific activity of 124.09 U/mg.The purified enzyme exhibited an optimal temperature of 40℃,an optimal pH of 6,a Michaelis constant of 5.28 mmol/L,and a catalytic constant of 4.74 s^(-1).Additionally,the enzyme was found to be promoted by Mg^(2+)and exhibited excellent thermal stability between 20-40℃,retaining 95.6%activity at 40℃for 30 min.As K.marxianus,which belongs to the same edible strain as Kluyveromyces lactis,itsβ-galactosidase has better thermal stability than common K.lactis,and has potential industrial application potential.