食品科学 ›› 2019, Vol. 40 ›› Issue (22): 81-87.doi: 10.7506/spkx1002-6630-20181109-112

• 生物工程 • 上一篇    下一篇

嗜热甘露聚糖酶毕赤酵母工程菌的表达及该酶在果汁澄清中的应用

陈伟,谷新晰,黄蕾,李晨,田洪涛,卢海强   

  1. (河北农业大学食品科技学院,河北 保定 071000)
  • 出版日期:2019-11-25 发布日期:2019-12-02
  • 基金资助:
    国家自然科学基金青年科学基金项目(31501417);河北省食品科学与工程学科“双一流”建设资金项目(2016SPGCA18)

Expression in Pichia pastoris and Characterization of a Thermophilic Mannanase and Its Application in Juice Clarification

CHEN Wei, GU Xinxi, HUANG Lei, LI Chen, TIAN Hongtao, LU Haiqiang   

  1. (College of Food Science and Technology, Hebei Agricultural University, Baoding 071000, China)
  • Online:2019-11-25 Published:2019-12-02

摘要: 探究嗜热真菌Neosartorya sp. HBFH9中甘露聚糖酶NsMan5B的酶学性质及其在果汁加工中的应用潜力。本研究从菌株HBFH9中获得nsMan5B基因,该基因全长1 491 bp(2 个内含子),cDNA全长1 371 bp,编码456 个氨基酸和1 个终止密码子。NsMan5B酶分子由1 个信号肽序列、碳水化合物结合序列、linker序列和催化区组成。重组NsMan5B的最适反应温度为60 ℃,最适反应pH 4.0,在pH 2.0~10.0和50 ℃条件下稳定。NsMan5B成熟蛋白分子质量约为49 kDa,不存在糖基化修饰。金属离子和化学试剂对该酶活性的影响差异较大,其中K+、Mg2+和Cu2+等离子对酶活性促进较明显,分别使酶活力提高了271%、123%和100%。随着醇类分子碳链的延长和浓度的增加,醇类物质对重组酶NsMan5B活性的抑制作用增强,其中在正丁醇体积分数为2.5%条件下,使该酶活性降低了86%。重组NsMan5B酶对柿子汁、苹果汁、桃子汁、葡萄汁和橘子汁澄清实验结果显示,除橘子汁外,重组酶对其他果汁的澄清度都有不同程度的提高,其中柿子汁澄清度提高了31.8%,其他3 种果汁的澄清度分别提高7%、4%和4%。综上所述,甘露聚糖酶NsMan5B在果汁加工中具有较好的应用前景。

关键词: 甘露聚糖酶, 果汁澄清, 异源表达, 食品酶, 嗜热酶

Abstract: A β-mannanase gene, nsMan5B, was cloned from the thermophilic fungus Neosartorya sp. HBFH9 and successfully expressed in Pichia pastoris. Sequence analysis indicated that the complete DNA sequence of nsMan5B was 1 491 bp in length (containing 2 Introns). The cDNA sequence of nsMan5B was 1 371 bp in length, which could encode a protein of 456 amino acids with a molecular mass of 49 kDa. Structural analysis indicated the presence of a putative signal peptide, a carbohydrate-binding module of family 1 (CBM 1), a linker region and a catalytic domain of GH5 in NsMan5B. The recombinant NsMan5B was optimally active at pH 4.0 and 60 ℃, and had good stability over the wide range of pH 2.0–10.0 and at 50 ℃ or below. The effects of metal ions and chemical reagents on its enzymatic activity were different. The enzymatic activity was increased by 271%, 123% and 100% in the presence of K+, Mg2+ and Cu2+, respectively. With increasing carbon chain length and concentration of alcohol, the inhibitory effect on NsMan5B activity was markedly enhanced. Its enzymatic activity was reduced by 86% by n-butanol (2.5%, V/V). It was observed that NsMan5B was effective in the clarification of orange juice as well as persimmon, apple, peach and grape juices, whose clarity was increased by 31.8%, 7%, 4% and 4%, respectively. Consequently, it was concluded that the NsMan5B could be successfully used in the fruit juice industry.

Key words: mannanase, juice clarification, expression, food enzymes, thermophilic enzyme

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