一种内切-1,3-岩藻聚糖酶的乳酸菌表达及性质

Expression and properties of an endo-1,3-fucoidanase in Lactobacillus

岩藻聚糖是一种具有良好应用潜力的多糖,具备多种生理活性。内切-1,3-岩藻聚糖酶是岩藻寡糖制备的关键工具。然而,作用方式清晰的食品工业用岩藻聚糖酶的缺乏阻碍了该类酶的应用。本研究旨在获得来源于GH174家族的食品工业用内切-1,3-岩藻聚糖酶Fun174Sb,并探究其作用方式。利用乳酸菌表达系统实现了一种内切-1,3-岩藻聚糖酶Fun174Sb的异源表达。结果显示,Fun174Sb对于来源于土耳其刺参的岩藻聚糖(Ht-FUC)具有降解能力,酶活性为1.90 U/mg。该酶在35~50°C、pH 7.5~8.5条件下显示出较高的活性,且具有良好的温度稳定性及pH稳定性。通过超高效凝胶排阻色谱串联质谱以及核磁共振技术阐明了Fun174Sb的酶解产物中主要寡糖的结构,从而推断出Fun174Sb切割Ht-FUC的Fucp2(OSO_(3)^(−))与Fucp2,4(OSO_(3)^(−))之间的α-1,3-糖苷键。研究表明,乳酸菌表达的Fun174Sb具有良好的生化性质以及清晰的作用方式。本研究为作用方式清晰的食品级岩藻聚糖的应用奠定基础。

Fucoidan is a marine polysaccharide with diverse physiological activities.Endo-1,3-fucoidanase is a favorable tool for the preparation of fucooligosaccharides.Nevertheless,the lack of food-grade fucoidanase with clear cleavage sites has hindered its application.This study aimed to obtain a food-grade endo-1,3-fucoidanase from GH174 family(Fun174Sb)and to investigate its cleavage site.The study demonstrated that the food-grade expression of Fun174Sb could be achieved through the Lactobacillus NICE system.Fun174Sb exhibits significant endo-acting hydrolysis activity towards fucoidan from Holothuria tubulosa(Ht-FUC),with an enzymatic activity of 1.90 U/mL.Fun174Sb showed significant activity within a temperature range of 35-50°C and a pH range of 7.5-8.5,and it possessed good temperature and pH stability.Furthermore,the structure of the dominant oligosaccharide in the enzymatic product was analyzed using ultra performance size exclusion chromatography-mass spectrum(UPSEC-MS)and nuclear magnetic resonance(NMR).It could be inferred that Fun174Sb cleaves theα-1,3-glycosidic bond between Fucp2(OSO_(3)^(−))and Fucp2,4(OSO_(3)^(−))in Ht-FUC.The study showed that Fun174Sb expressed in Lactobacillus exhibited favorable biochemical properties and a clear mode of action.It laid the foundation for the application of food-grade fucoidanase.