地衣芽孢杆菌B13耐高温α-淀粉酶基因的克隆和表达
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摘要
耐高温α-淀粉酶是目前最重要的工业酶制剂之一,广泛应用于发酵、制糖等工业。地衣芽孢杆菌(Bacillus licheniformis)是国内生产高温α-淀粉酶的代表菌株之一。本文主要研究地衣芽孢杆菌B13高温α-淀粉酶(BLA)编码基因(amy)的克隆、序列分析及在大肠杆菌和枯草芽孢杆菌中的表达。
用PCR方法从地衣芽孢杆菌B13的总DNA中扩增出一条约1.8kb的DNA片段,测序结果表明,该片段与B.licheniformis584中的耐高温α-淀粉酶基因的同源性高达99%。将B.licheniformis B13耐高温α-淀粉酶基因在大肠杆菌中进行表达和表达产物活性测定,结果表明在大肠杆菌中,耐高温α-淀粉酶基因能利用自身的启动子及信号肽合成有活性的目标蛋白,并能分泌到胞外。
枯草芽孢杆茵是被广泛利用的来表达外源基因的良好宿主,但其本身包含有淀粉酶基因。利用无标记同源重组载体pDGIEF,敲除B.subtilis WB800的淀粉酶基因,构建了枯草芽孢杆菌WB800D,以此做为外源淀粉酶基因表达的良好宿主。利用穿梭载体pUBC19,将含有高温α-淀粉酶自身启动子序列和信号肽序列的amy基因与其进行体外重组,并转化B.subtilis WB800D,获得了表达菌株B.subtilis WB800D(pUBC19-amy)。但是,其表达量较低,活性不高。
利用穿梭载体pP43NMK,将含有信号肽序列的amy基因片段与其进行体外重组,并转化B.subtilis WB800D,获得了表达菌株B.subtilis WB800D(pP43NMK-amy)。实验结果表明,在P43带动下,α-淀粉酶基因在枯草芽孢杆菌中得到了表达,与地衣芽孢杆菌的耐高温α-淀粉酶基因自身启动子相比,采用P43启动子,α-淀粉酶表达水平提高了8.9倍。这项研究为构建高效表达耐高温α-淀粉酶基因工程菌奠定基础。
Thermostableα-amylase,catalyzing hydrolysis of starch at high temperatures,is one of the most important enzymes,widely applied in sugar and fermentation industry.Bacillus licheniformis is one of the stains used to produce thermostableα-amylase in industry.The aim of this research was focused on cloning and expression of the gene encoding a thermostableα-amylase from Bacillus licheniformis B13.
A DNA fragment containingα-amylase gene,amy with its own promoter and signal peptide sequences was obtained by PCR from genomic DNA of B.licheniformis B13.The result of sequencing indicated thatα-amylase gene from B.licheniformis B13 had 99% similarity to that from B.licheniformis 548.The amy gene could be expressed in E.coli using its own promoter andα-amylase expressed could secreted into the culture medium.
B.subtilis WB800 was widely used as host for the expression of heterologous gene, containingα-amylase.By knocking out its amylase gene B.subtilis WB800D was constructed in order to expression heterologous amy gene.The amy gene from B.licheniformis B13 was cloned into shuttle vector pUBC19 and expressed in B.subtilis WB800D.But expression level was very low.
The amy gene and signal peptide sequences was integrated into shuttle vector pP43NMK and expressed in B.subtilis WB800D.The results showed that theα-amylase gene was highly expressed under the control of promoter P43.The amount ofα-amylase expressed under the control of promoter P43 was 8.9 more times of that expressed under the control of itself promoter ofα-amylase gene of B.lichenformis B13.It was very helpful to constructα-amylase gene engineering strain.
用PCR方法从地衣芽孢杆菌B13的总DNA中扩增出一条约1.8kb的DNA片段,测序结果表明,该片段与B.licheniformis584中的耐高温α-淀粉酶基因的同源性高达99%。将B.licheniformis B13耐高温α-淀粉酶基因在大肠杆菌中进行表达和表达产物活性测定,结果表明在大肠杆菌中,耐高温α-淀粉酶基因能利用自身的启动子及信号肽合成有活性的目标蛋白,并能分泌到胞外。
枯草芽孢杆茵是被广泛利用的来表达外源基因的良好宿主,但其本身包含有淀粉酶基因。利用无标记同源重组载体pDGIEF,敲除B.subtilis WB800的淀粉酶基因,构建了枯草芽孢杆菌WB800D,以此做为外源淀粉酶基因表达的良好宿主。利用穿梭载体pUBC19,将含有高温α-淀粉酶自身启动子序列和信号肽序列的amy基因与其进行体外重组,并转化B.subtilis WB800D,获得了表达菌株B.subtilis WB800D(pUBC19-amy)。但是,其表达量较低,活性不高。
利用穿梭载体pP43NMK,将含有信号肽序列的amy基因片段与其进行体外重组,并转化B.subtilis WB800D,获得了表达菌株B.subtilis WB800D(pP43NMK-amy)。实验结果表明,在P43带动下,α-淀粉酶基因在枯草芽孢杆菌中得到了表达,与地衣芽孢杆菌的耐高温α-淀粉酶基因自身启动子相比,采用P43启动子,α-淀粉酶表达水平提高了8.9倍。这项研究为构建高效表达耐高温α-淀粉酶基因工程菌奠定基础。
Thermostableα-amylase,catalyzing hydrolysis of starch at high temperatures,is one of the most important enzymes,widely applied in sugar and fermentation industry.Bacillus licheniformis is one of the stains used to produce thermostableα-amylase in industry.The aim of this research was focused on cloning and expression of the gene encoding a thermostableα-amylase from Bacillus licheniformis B13.
A DNA fragment containingα-amylase gene,amy with its own promoter and signal peptide sequences was obtained by PCR from genomic DNA of B.licheniformis B13.The result of sequencing indicated thatα-amylase gene from B.licheniformis B13 had 99% similarity to that from B.licheniformis 548.The amy gene could be expressed in E.coli using its own promoter andα-amylase expressed could secreted into the culture medium.
B.subtilis WB800 was widely used as host for the expression of heterologous gene, containingα-amylase.By knocking out its amylase gene B.subtilis WB800D was constructed in order to expression heterologous amy gene.The amy gene from B.licheniformis B13 was cloned into shuttle vector pUBC19 and expressed in B.subtilis WB800D.But expression level was very low.
The amy gene and signal peptide sequences was integrated into shuttle vector pP43NMK and expressed in B.subtilis WB800D.The results showed that theα-amylase gene was highly expressed under the control of promoter P43.The amount ofα-amylase expressed under the control of promoter P43 was 8.9 more times of that expressed under the control of itself promoter ofα-amylase gene of B.lichenformis B13.It was very helpful to constructα-amylase gene engineering strain.
引文
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