食品科学

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新型GH5家族β-甘露聚糖酶的基因挖掘及表达鉴定

唐存多1,史红玲1,蔚晓华2,张 梅3,唐青海1,岳 超1,姚伦广1,夏 敏2,阚云超1,*   

  1. 1.南阳师范学院 昆虫生物反应器河南省工程实验室,河南 南阳 473061;
    2.南阳师范学院生命科学与技术学院,河南 南阳 473061;3.蓬莱市市场监督管理局,山东 蓬莱 265600
  • 出版日期:2016-06-15 发布日期:2016-06-27

Gene Mining, Expression and Characterization of Novel GH5 Family β-Mannanases

TANG Cunduo1, SHI Hongling1, YU Xiaohua2, ZHANG Mei3, TANG Qinghai1, YUE Chao1, YAO Lunguang1, XIA Min2, KAN Yunchao1,*   

  1. 1. Henan Provincial Engineering Laboratory of Insect Bio-reactor, Nanyang Normal University, Nanyang 473061, China;
    2. School of Life Science and Technology, Nanyang Normal University, Nanyang 473061, China;
    3. Penglai Market Supervisory Authority, Penglai 265600, China
  • Online:2016-06-15 Published:2016-06-27

摘要:

为了获得性能优良的新型β-甘露聚糖酶,本研究采用基因挖掘技术从米曲霉(Aspergillus oryzae)RIB40基因组中挖掘到了2 个假定的新型GH5家族β-甘露聚糖酶基因,分别命名为Aoman5A和Aoman5B。对这两条序列做了相关的生物信息学分析,同时借助pPIC9KM质粒将两个酶的成熟肽编码基因在Pichia pastoris GS115中实现了表达,并对表达产物进行了纯化和鉴定。生物信息学分析的结果表明AoMan5A含有20 个氨基酸残基的信号肽,而AoMan5B含有21 个氨基酸残基的信号肽和12 个氨基酸残基的前导肽;序列比对的结果显示两个酶与目前已报道的序列最高的同源性分别为68%和79%,且AoMan5A的N端还携带有一个1家族的CBM;三维结构预测的结果显示,两者均符合(α/β)8的TIM-桶状结构。表达鉴定的结果表明,在同样表达条件下,reAoMan5A和reAoMan5B上清液的酶活力分别为2.9、12.5 IU/mL;纯化后它们的酶比活力分别为8.3、104.2 IU/mg,前者的最适温度为35 ℃,而后者的最适温度为50 ℃,pH值特性的测定结果表明这两种酶均为酸性酶。硫酸-苯酚法测得reAoMan5A和reAoMan5B的糖含量分别为25.4%和12.6%,表明这两种重组酶均经过了糖基化修饰。本研究获得了两种新型的β-甘露聚糖酶,比较分析了它们的序列特征,并实现了它们的异源表达,为β-甘露聚糖酶的进一步研究及应用奠定了坚实的基础,也为其他新型酶的基因挖掘提供了可资借鉴的经验。

关键词: &beta, -甘露聚糖酶, 基因挖掘, 生物信息学分析, 米曲霉, 蛋白表达

Abstract:

In order to obtain novel β-mannanases with excellent performance, two putative novel GH5 family β-mannanase
genes were excavated from the Aspergillus oryzae RIB40 genome by genome mining, named as Aoman5A and Aoman5B,
respectively. The bioinformatic analysis of the two gene sequences was conducted using corresponding softwares or web
server, and the genes encoding two mature peptides were expressed in Pichia pastoris GS115 with the aid of plasmid
pPIC9KM, and then the expressed products were purified and identified. The results of bioinformatic analysis showed
that AoMan5A contained a signal peptide with 20 amino acid residues, while AoMan5B contained a signal peptide with
21 amino acid residues and a propeptide with 12 amino acid residues. The results of sequence alignment displayed that
the sequences of two enzymes had the highest similarity of 68% and 79% with the reported sequences, and the N-terminal
of AoMan5A also carried a GH1 family CBM. The results of 3-D structure prediction showed that both of them were
in accordance with the (α/β)8 TIM-barrel structure. Under the same expression condition, the enzymes activities in
supernatants from reAoMan5A and reAoMan5B were 2.9 and 12.5 IU/mL, respectively, with specific activity of 8.3
and 104.2 IU/mg, respectively, after purification. The optimum temperature for the former was 35 ℃, while that for
the later was 50 ℃. It turned out that both of them were acidic enzymes. The carbohydrate contents of reAoMan5A
and reAoMan5B were determined to be 25.4% and 12.6% using the phenol sulfuric acid method indicating both to
be glycosylated. This study will lay a solid foundation for further research and application of β-mannanases, and also
provide a referential guidance for the gene mining of other novel enzymes.

Key words: β-mannanase, genome mining, bioinformatic analysis, Aspergillus oryzae, protein expression

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