食品科学 ›› 2017, Vol. 38 ›› Issue (12): 104-111.doi: 10.7506/spkx1002-6630-201712016

• 生物工程 • 上一篇    下一篇

巴氏葡萄球菌TS-82类胡萝卜素裂解酶的分离纯化及其酶学特性

朱明明,樊明涛,何鸿举,马汉军   

  1. 1.河南科技学院食品学院,河南?新乡 453003;2.西北农林科技大学食品科学与工程学院,陕西?杨凌 712100
  • 出版日期:2017-06-25 发布日期:2017-06-26
  • 基金资助:
    河南科技学院高层次人才科研启动项目(2015015;2016020);国家自然科学基金面上项目(31171728); 河南省高校科技创新团队支持计划项目(13IRTSTHN006)

Isolation, Purification and Characterization of a Novel Carotenoid-Cleaving Enzyme from Staphylococcus pasteuri TS-82

ZHU Mingming, FAN Mingtao, HE Hongju, MA Hanjun   

  1. 1. School of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, China; 2. College of Food Science and Engineering, Northwest A&F University, Yangling 712100, China
  • Online:2017-06-25 Published:2017-06-26

摘要: 巴氏葡萄球菌TS-82类胡萝卜素裂解酶经强阴离子柱、高效制备液相色谱和多肽分子筛纯化得到液相级纯酶(95.6%)。该酶比活力为125?U/g,纯化倍数为446,回收率为2.39%。纯化后的类胡萝卜素裂解酶经液相色谱-质谱联用测定,得其分子质量为655.093?D。关于酶学特性,研究发现该酶对C40类胡萝卜素底物的最适温度为60?℃,而作用于β-阿朴-8?ˉ-胡萝卜醛的最适温度是50?℃,该酶的稳定温度为50?℃以下;该酶对所测定底物的最适pH值为3.0;该酶与5?种底物亲和力排列为:玉米黄质>虾青素>β-胡萝卜素>角黄质>β-阿朴-8?ˉ-胡萝卜醛;Al3+和Fe3+是该酶的强效催化剂,Fe2+是该酶的强效抑制剂;H2O2在低浓度范围内(0~16?mmol/L)可促进酶活性;低体积分数乙醇(4%~16%)的添加对酶活性无明显抑制作用。结果表明该酶具有很好的耐酸性和热稳定性,能够适应果酒环境,为其工业化应用提供依据。

关键词: 巴氏葡萄球菌TS-82, 类胡萝卜素裂解酶, 类胡萝卜素, 分离纯化, 酶学特性

Abstract: A carotenoid cleavage enzyme with HPLC grade purity of 95.6% was obtained from Staphylococcus pasteuri (S. pasteuri) TS-82 by using anion-exchange chromatography, preparative high performance liquid chromatography (PHPLC), and superdex peptide chromatography together. The purified enzyme had a specific activity of 125 U/g with 466-fold purification and 2.39% recovery. Its molecular weight was 655.093 D as identified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI/MS). Enzymatic characterization indicated that the purified enzyme had optimal temperature of 60 ℃ for degrading C40 carotenoids and 50 ℃ for degrading β-apo-8?ˉ-carotenal, and it maintained stable activity at 50 ℃. The optimum pH value was 3.0 for degrading all investigated substrates. The Km and vmax values indicated that the enzyme showed the highest affinity towards zeaxanthin, followed by astaxanthin, β-carotene, canthaxanthin, and β-apo-8?ˉ-carotenal in a decreasing order. The metal ions Al3+ and Fe3+ were identified as potent activators for the purified enzyme, whereas Fe2+ as a potent inhibitor. H2O2 was able to increase the enzyme activity in degrading β-carotene at levels of 0-16 mmol/L. Alcohol at low concentration (4%–16%) did not inhibit the activity of the enzyme. In conclusion, the enzyme showed great acid resistance and thermostability, which makes it stable in wine environment and provides a basis for industrial application.

Key words: Staphylococcus pasteuri TS-82, carotenoid cleavage enzyme, carotenoids, purification, characterization

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