Paenibacillus sp.E18双功能木聚糖酶基因克隆及性质研究
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摘要
木聚糖酶是可将木聚糖降解成低聚糖和木糖的一类酶的总称,是半纤维素酶类中最具代表性的组分,主要存在于微生物、植物和低等动物中。在造纸、食品、饲料、制药、能源以及环境保护等多方面都有着重要的应用价值。微生物是木聚糖酶最主要的来源,具有产量高、成本低、生产过程易控制等优点。
本研究通过模拟菌株生长自然环境和功能性平板筛选,从玉米青贮中分离得到一株具有木聚糖降解能力的菌株,经16S rDNA鉴定为类芽胞杆菌,命名为Paenibacillus sp. E18。
利用生物信息学方法,对微生物来源糖苷水解酶第十家族木聚糖酶氨基酸序列进行同源比对,设计保守区简并引物。利用Touchdown-PCR技术,克隆得到一个木聚糖酶基因片段,并通过两步Tail-PCR法获得该片段上下游侧翼序列。经序列比对、ORF分析,确定该段序列为一个基因簇,目前已得到三个完整的基因,分别命名为xynB-E18、abf43A-E18、axeE18。这三个基因同已发表的基因序列最高相似性分别为85%、56%和70%。其中xynB-E18是一个木聚糖酶编码基因,具有十家族保守氨基酸位点:Glu129和Glu236。abf43A-E18属于四十三家族,其表达产物可能具有木聚糖酶或阿拉伯呋喃糖苷酶活性,axeE18与一个假设的乙酰酯酶基因具有最高相似性。后两者所编码蛋白可能作为辅酶在木聚糖降解过程中发挥作用。由于目前得到的序列中未预测到有启动子存在,推测该基因簇可能包含更多相关基因。
xynB-E18插入pET22b(+)构建表达载体,在大肠杆菌BL21(DE3)中得到了表达。通过盐析和离子交换层析纯化得到电泳纯重组蛋白XynB-E18。该酶的最适pH和最适温度分别为7.0和50℃;有较好的pH稳定性,在pH 6.0~10.5范围内酶活性维持在80%以上;但热稳定性较差。
其最适底物为桦木木聚糖,同时对大麦葡聚糖、地衣多糖、昆布多糖也有一定水解能力,活性相对于桦木木聚糖在20~40%。经基因序列分析和混合底物条件下动力学研究,确定XynB-E18为单结构域双功能木聚糖酶,即在同一催化位点实现对木聚糖和葡聚糖的水解作用。
本实验进一步确立了从环境菌群中快速分离产酶菌株并应用简并引物获得基因片段的方法,克隆得到同一基因簇中三个基因,并对其中木聚糖酶基因作了表达及性质研究,为多功能木聚糖酶的深入研究提供了参考和经验。
Xylanases are enzymes to catalyze the hydrolysis of xylan into xylose and oligosaccharide. They are crucial hemicellulases and produced mainly by microorganisms, plants and lower animals. Xylanases have been widely used in many industry processes, such as pulp and paper, foodstuff, animal feed, pharmacy, energy and environmental protection. Microbial xylanases are more promising due to high production, easy to control, low cost and so on.
In this study, a strain, E18, showing xylan-degrading activity was isolated by simulation of the natural environment and using selective screening plates, and was identified to be Paenibacillus sp. based on 16S rDNA sequence.
By using bioinformatical methods, the amino acid sequences of microbial xylanases from glycoside hydrolase (GH) family 10 were aligned, and a pair of degenerate primers was designed based on the conserved regions. A xyalanse gene fragment was cloned by a Touchdown-PCR, and the flanking regions were obtained by using two step TAIL-PCR. Based on sequence comparison and open reading frame (ORF) analysis, the fragment assembly contained a gene cluster including three genes, xynB-E18, abf43A-E18 and axeE18. The highest homologies of these three genes to known sequences were 85%, 56% and 70%, respectively. xynB-E18 was a xylanase-coding gene containing two highly conserved glutamate, Glu129 and Glu236, of GH 10 members. The deduced amino acid sequence of abf43A-E18 encoded a protein belonging to GH 43, which recombinant protein had both xylanase and arabinofuranohydrolase activities. The sequence of axeE18 exhibited high identity with a putative esterase. The encoded proteins of abf43A-E18 and axeE18 might play a role in xylan degradation as coenzymes. No promoter has been identified yet, suggesting that the xylanolytic gene cluster had more than three members.
The gene xynB-E18 was cloned into vector pET22b(+) and then expressed in Escherichia coli. The recombinant protein was purified to electrophoretic homogeneity by ammonium sulfate precipitation and hydrophobic chromatography. The optimum temperature and pH for activity of the recombinant protein XynB-E18 was 50oC and pH 7.0, respectively. The enzyme was pH stable, retaining more than 80% of the initial activity at pH 6.0
本研究通过模拟菌株生长自然环境和功能性平板筛选,从玉米青贮中分离得到一株具有木聚糖降解能力的菌株,经16S rDNA鉴定为类芽胞杆菌,命名为Paenibacillus sp. E18。
利用生物信息学方法,对微生物来源糖苷水解酶第十家族木聚糖酶氨基酸序列进行同源比对,设计保守区简并引物。利用Touchdown-PCR技术,克隆得到一个木聚糖酶基因片段,并通过两步Tail-PCR法获得该片段上下游侧翼序列。经序列比对、ORF分析,确定该段序列为一个基因簇,目前已得到三个完整的基因,分别命名为xynB-E18、abf43A-E18、axeE18。这三个基因同已发表的基因序列最高相似性分别为85%、56%和70%。其中xynB-E18是一个木聚糖酶编码基因,具有十家族保守氨基酸位点:Glu129和Glu236。abf43A-E18属于四十三家族,其表达产物可能具有木聚糖酶或阿拉伯呋喃糖苷酶活性,axeE18与一个假设的乙酰酯酶基因具有最高相似性。后两者所编码蛋白可能作为辅酶在木聚糖降解过程中发挥作用。由于目前得到的序列中未预测到有启动子存在,推测该基因簇可能包含更多相关基因。
xynB-E18插入pET22b(+)构建表达载体,在大肠杆菌BL21(DE3)中得到了表达。通过盐析和离子交换层析纯化得到电泳纯重组蛋白XynB-E18。该酶的最适pH和最适温度分别为7.0和50℃;有较好的pH稳定性,在pH 6.0~10.5范围内酶活性维持在80%以上;但热稳定性较差。
其最适底物为桦木木聚糖,同时对大麦葡聚糖、地衣多糖、昆布多糖也有一定水解能力,活性相对于桦木木聚糖在20~40%。经基因序列分析和混合底物条件下动力学研究,确定XynB-E18为单结构域双功能木聚糖酶,即在同一催化位点实现对木聚糖和葡聚糖的水解作用。
本实验进一步确立了从环境菌群中快速分离产酶菌株并应用简并引物获得基因片段的方法,克隆得到同一基因簇中三个基因,并对其中木聚糖酶基因作了表达及性质研究,为多功能木聚糖酶的深入研究提供了参考和经验。
Xylanases are enzymes to catalyze the hydrolysis of xylan into xylose and oligosaccharide. They are crucial hemicellulases and produced mainly by microorganisms, plants and lower animals. Xylanases have been widely used in many industry processes, such as pulp and paper, foodstuff, animal feed, pharmacy, energy and environmental protection. Microbial xylanases are more promising due to high production, easy to control, low cost and so on.
In this study, a strain, E18, showing xylan-degrading activity was isolated by simulation of the natural environment and using selective screening plates, and was identified to be Paenibacillus sp. based on 16S rDNA sequence.
By using bioinformatical methods, the amino acid sequences of microbial xylanases from glycoside hydrolase (GH) family 10 were aligned, and a pair of degenerate primers was designed based on the conserved regions. A xyalanse gene fragment was cloned by a Touchdown-PCR, and the flanking regions were obtained by using two step TAIL-PCR. Based on sequence comparison and open reading frame (ORF) analysis, the fragment assembly contained a gene cluster including three genes, xynB-E18, abf43A-E18 and axeE18. The highest homologies of these three genes to known sequences were 85%, 56% and 70%, respectively. xynB-E18 was a xylanase-coding gene containing two highly conserved glutamate, Glu129 and Glu236, of GH 10 members. The deduced amino acid sequence of abf43A-E18 encoded a protein belonging to GH 43, which recombinant protein had both xylanase and arabinofuranohydrolase activities. The sequence of axeE18 exhibited high identity with a putative esterase. The encoded proteins of abf43A-E18 and axeE18 might play a role in xylan degradation as coenzymes. No promoter has been identified yet, suggesting that the xylanolytic gene cluster had more than three members.
The gene xynB-E18 was cloned into vector pET22b(+) and then expressed in Escherichia coli. The recombinant protein was purified to electrophoretic homogeneity by ammonium sulfate precipitation and hydrophobic chromatography. The optimum temperature and pH for activity of the recombinant protein XynB-E18 was 50oC and pH 7.0, respectively. The enzyme was pH stable, retaining more than 80% of the initial activity at pH 6.0
引文
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