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当前位置: 首页 > 文章 > 新型GH5家族β-甘露聚糖酶的基因挖掘及表达鉴定 食品科学 2016,37 (11) 90-96
Position: Home > Articles > Gene Mining, Expression and Characterization of Novel GH5 Family β-Mannanases FOOD SCIENCE 2016,37 (11) 90-96

新型GH5家族β-甘露聚糖酶的基因挖掘及表达鉴定

作  者：
唐存多;史红玲;蔚晓华;张梅;唐青海;岳超;姚伦广;夏敏;阚云超
单  位：
南阳师范学院昆虫生物反应器河南省工程实验室;南阳师范学院生命科学与技术学院;蓬莱市市场监督管理局
关键词：
β-甘露聚糖酶;基因挖掘;生物信息学分析;米曲霉;蛋白表达
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p PIC9KM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Ao Man5A含有20个氨基酸残基的信号肽,而Ao Man5B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Ao Man5A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,re Ao Man5A和re Ao Man5B上清液的酶活力分别为2.9、12.5 IU/m L;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℃,p H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re Ao Man5A和re Ao Man5B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。
译  名：
Gene Mining, Expression and Characterization of Novel GH5 Family β-Mannanases
作  者：
TANG Cunduo;SHI Hongling;YU Xiaohua;ZHANG Mei;TANG Qinghai;YUE Chao;YAO Lunguang;XIA Min;KAN Yunchao;Henan Provincial Engineering Laboratory of Insect Bio-reactor, Nanyang Normal University;School of Life Science and Technology, Nanyang Normal University;Penglai Market Supervisory Authority;
关键词：
β-mannanase;;genome mining;;bioinformatic analysis;;Aspergillus oryzae;;protein expression
摘  要：
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 Aoman5 A and Aoman5 B, 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 p PIC9 KM, and then the expressed products were purified and identified. The results of bioinformatic analysis showed that Ao Man5 A contained a signal peptide with 20 amino acid residues, while Ao Man5 B 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 Ao Man5 A 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 re Ao Man5 A and re Ao Man5 B were 2.9 and 12.5 IU/m L, 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 re Ao Man5 A and re Ao Man5 B 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.

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