食品科学 ›› 2021, Vol. 42 ›› Issue (12): 130-137.doi: 10.7506/spkx1002-6630-20200219-194

• 生物工程 • 上一篇    下一篇

解淀粉芽孢杆菌I型耐热普鲁兰酶的纯化及酶特性分析

高兆建,胡鑫强,宋玉林,丁飞鸿,赵宜峰,陈腾   

  1. (1.徐州工程学院食品与生物工程学院,江苏 徐州 221018;2.长江桂柳食品睢宁有限公司,江苏 徐州 221000)
  • 出版日期:2021-06-25 发布日期:2021-06-29
  • 基金资助:
    江苏省高等学校自然科学研究重大项目(20KJA180008); 江苏省苏北科技计划项目(XZ-SZ201819;BC2013417;BN2015021);徐州市科技计划项目(KC17083)

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

GAO Zhaojian, HU Xinqiang, SONG Yulin, DING Feihong, ZHAO Yifeng, CHEN Teng   

  1. (1. School of Food and Biological Engineering, Xuzhou University of Technology, Xuzhou 221018, China; 2. Yangtze River Guiliu Food Suining Co. Ltd., Xuzhou 221000, China)
  • Online:2021-06-25 Published:2021-06-29

摘要: 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,Mg2+和Ca2+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Km和Vmax值分别为1.34 mg/mL和24.6 μmol/(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。

关键词: 普鲁兰酶;解淀粉芽孢杆菌;分离纯化;酶学性质

Abstract: 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 sulfate-polyacrylamide 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 Mg2+ and Ca2+ 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 Km and Vmax were 1.34 mg/mL and 24.6 μmol/(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.

Key words: pullulanase; Bacillus amyloliquefaciens; purification; enzymatic characterization

中图分类号: