食品科学 ›› 2021, Vol. 42 ›› Issue (14): 129-136.doi: 10.7506/spkx1002-6630-20200229-329

• 生物工程 • 上一篇    下一篇

产黄青霉抗真菌几丁质酶的纯化及特性分析

高兆建,丁飞鸿,陈欢,耿云龙,赵宜峰   

  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)
  • 发布日期:2021-07-27
  • 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 Characterization of Antifungal Chitinase from Penicillium chrysogenum

GAO Zhaojian, DING Feihong, CHEN Huan, GENG Yunlong, ZHAO Yifeng   

  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)
  • Published:2021-07-27

摘要: 为实现抗真菌性能的耐热新型几丁质酶在食品防腐及生物防治方面的应用,从产黄青霉Xch23中分离纯化几丁质酶(Chi-Pc76)并研究其应用特性。通过硫酸铵沉淀、DEAE-Cellulose A52离子交换层析和Sephadex G-100凝胶过滤层析纯化得到均一的Chi-Pc76。最终得到Chi-Pc76纯化倍数17.1 倍,回收率21.6%,比活力为584.8 U/mg。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测得Chi-Pc76分子质量61 kDa。纯化后的Chi-Pc76最佳反应条件为pH 6.0、温度55 ℃。Chi-Pc76在宽泛的pH 4.0~10.0之间稳定性强,其显著的热稳定性可达到70 ℃。Chi-Pc76对底物胶体几丁质具有高的底物特异性,对乙二醇几丁质、α-几丁质、β-几丁质有中等活性,而对其他受试底物无活性或痕量活性。以胶体几丁质为底物Km和Vmax值分别为0.25 mg/mL和20 μmol/(min·mg)。金属离子Ca2+、Ba2+、K+、Na+对酶有明显激活作用;十二烷基硫酸钠、吐温80、苯甲基磺酰氟和尿素对酶活力基本无影响,但Mn2+、Co2+、Fe2+、Ag+、Cu2+、Zn2+、Pb2+、Hg2+和β-巯基乙醇、二硫苏糖醇均对酶活力有抑制作用。Chi-Pc76对黑曲霉、黄曲霉、尖孢镰刀菌和橘青霉均有显著的抗真菌活性,对比文献为产黄青霉抗真菌几丁质酶。鉴于Chi-Pc76纯化简便、热稳定性好、宽广的pH值稳定性、高效降解几丁质和抗真菌等特点,产黄青霉Xch23几丁质酶在几丁质废料综合利用、生物转化药理活性产品、食品保鲜以及植物病原性真菌和昆虫幼虫生物防治等方面具有潜在价值。

关键词: 几丁质酶;产黄青霉;分离纯化;酶学特性;抗真菌

Abstract: A chitinase (named Chi-Pc76) was isolated and purified from the fermentation broth of Penicillium chrysogenum Xch23 and its application potential was evaluated. The enzyme was purified to homogeneity by consecutive ammonium sulfate precipitation, DEAE-Cellulose A52 ion exchange chromatography and Sephadex G-100 gel filtration chromatography, yielding a 17.1-fold purification with 21.6% recovery and an specific activity of 584.8 U/mg. The molecular mass of Chi-Pc76 was estimated to be 61 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the purified Chi-Pc76 was 6.0 and 55 ℃, respectively. The enzyme showed high stability in the broad pH range of 4.0–10.0 and temperature stability up to 70 ℃. Chi-Pc76 exhibited high activity toward colloidal chitin and moderate activity toward glycol chitin, α-chitin, β-chitin, while it showed no or trace activity toward other tested substrates. Its Km and Vmax values for colloidal chitin were 0.25 mg/mL and 20 μmol/(min·mg), respectively. The activity of the enzyme was stimulated by Ca2+, Ba2+, Mg2+, K+, Na+ ions, but not affected by SDS, Tween 80, Triton X100, PMSF and urea. Mn2+, Co2+, Fe2+, Ag+, Cu2+, Zn2+, Pb2+ and Hg2+ and β-mercaptoethanol, as well as DTT inhibited the enzyme activity. Chi-Pc76 showed antifungal activity toward Aspergillus niger, Aspergillus flavus, Fusarium oxysporum, and P. citrinum. To the best of our knowledge, this is the first report of the antifungal chitinase from P. chrysogenum. In view of its advantages such as simple purification, high thermostability, broad-pH range stability, high-efficiency chitin-degrading ability, and antifungal activity, Chi-Pc76 will have potential applications in the comprehensive utilization of chitin waste, the bioconversion of chitin to pharmacologically active products, food preservation, and the biological control of phytopathogenic fungi and insect larvae.

Key words: chitinase; Penicillium chrysogenum; purification; enzymatic characteristics; antifungal activity

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