解淀粉芽孢杆菌I型耐热普鲁兰酶的纯化及酶特性分析

Purification and Enzymatic Characterization of Thermolabile Type I Pullulanase from Bacillus amyloliquefaciens

对来源于菌株解淀粉芽孢杆菌HxP-21的普鲁兰酶(命名为PulBa)分离纯化,研究其酶学特性,为普鲁兰酶在淀粉加工中的应用提供理论基础。通过硫酸铵沉淀、阴离子交换层析和葡聚糖凝胶过滤层析从菌株HxP-21发酵液中分离纯化出一种新型的普鲁兰酶。酶的纯化倍数20.8,回收率53.2%,比活力176.5 U/mg。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测得PulBa达到电泳纯,分子质量51.2 kDa。PulBa在45~70℃和pH 3~6范围内具有较高酶活力,最适反应温度55℃、pH 4.5。PulBa有良好的pH值稳定性和热稳定性,40~70℃孵育120 min保留最初活性的80%以上;pH 3~7范围内具有很高的稳定性,孵育6 h后仍保留60 U/mL以上活性。PulBa对各种金属离子和化学试剂表现出不同的敏感性,Mg^(2+)和Ca^(2+)能够显著增强酶活力。PulBa最适作用底物为普鲁兰糖,对马铃薯支链淀粉、玉米支链淀粉、可溶性淀粉和糖原也有一定水解活性,但对α-环糊精和β-环糊精和直链淀粉无活性。以普鲁兰糖为底物PulBa的K_(m)和V_(max)值分别为1.34 mg/mL和24.6μmol/(min·mg)。研究表明PulBa是典型的I型普鲁兰酶。薄层层析进一步证明,PulBa专一性水解支链淀粉α-1,6-糖苷键,产生麦芽三糖。本研究确定了一种新型的普鲁兰酶,该酶在高热稳定性和酸性环境下具有高活性,在淀粉加工等生物技术产业中有较好的应用潜力。

A pullulanase(named PulBa)from Bacillus amyloliquefaciens HxP-21 was isolated and purified,and its enzymatic properties were studied to provide a theoretical basis for the application of pullulanase in starch processing.The pullulanase was isolated and purified from the fermentation broth of strain HxP-21 by sequential ammonium sulfate precipitation,anion exchange chromatography and dextran gel filtration chromatography.The yield of PulBa was 53.2%,and the procedure resulted in a 20.8-fold purification and a specific enzyme activity of 176.5 U/mg.Sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE)measured PulBa to be electrophoretically pure,with a molecular mass of 51.2 kDa.PulBa had high enzyme activity in the range of 45–70℃ and pH 3–6,with optimum temperature of 55℃ and optimum pH of 4.5.PulBa also exhibited good pH stability and thermostability.More than 80%of its initial activity was retained after being incubated at 40–70℃ for 120 min.PulBa was highly stable over an acidic pH range of 3-7,and more than 60 U/mL of its activity was retained after 6 h incubation under these pH conditions.PulBa showed different sensitivities to various metal ions and chemical reagents,and Mg^(2+) and Ca^(2+) were able to significantly enhance its enzyme activity.The most suitable substrate for PulBa was pullulan,and it also showed hydrolytic activity on potato amylopectin,corn amylopectin,soluble starch and glycogen,but no effect on α-cyclodextrin and β-cyclodextrin and amylose.When pullulan was used as a substrate,its K_(m) and V_(max) were 1.34 mg/mL and 24.6μmol/(min·mg),respectively.Results indicated that PulBa was a typical type I pullulanase.Thin layer chromatography(TLC)further demonstrated that PulBa specifically cleave the α-1,6-glycosidic bonds of amylopectin to produce maltotriose.Therefore,PulBa had high thermostability and acidic pH tolerance,making it a promising candidate for application in biotechnological industries such as starch processing.