利用融合标签纯化和表达β-甘露聚糖酶
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摘要
甘露聚糖酶(β-mannanase; endo-1,4-β-D-mannan mannohydrolase; EC3.2.1.78)是一种非常重要的半纤维素酶,尤其是在麻类生物脱胶领域。本研究以甘露聚糖酶产生菌地衣芽孢杆菌(Bacillus licheniform)HDYM04的基因组DNA为模板,采用PCR扩增的方法扩增得到其甘露聚糖酶基因,将基因片段与融合表达载体pET28a连接后转化进入大肠杆菌BL21(DE3),进而对重组酶的酶学性质进行了研究。
克隆所得甘露聚糖酶基因片段长度为1282bp。对所获得片段进行相应生物信息学分析,结果表明该酶与芽孢杆菌属的甘露聚糖酶同源性最高,为酸性甘露聚糖酶,分子量约为38kD,无信号肽和跨膜区,具有MANB和糖苷水解酶两个结构域。构建甘露聚糖酶的异源重组菌株后对重组菌株进行诱导表达,筛选出酶活性最高的HDEM8菌株进行诱导条件和酶学性质的研究,菌株HDEM8的初始酶活力为38.60U/mL。经以IPTG终浓度、诱导温度和时间为因子的正交试验优化后,确定最优诱导条件为IPTG终浓度1.0mmol/L、30℃、诱导培养7h。酶活最高达到45.57U/mL。将诱导后的HDEM8菌株细胞裂解物经His·Bind Columns纯化后进行酶学性质分析,结果表明:适合反应温度为50℃-60℃,最适温度为55℃;适合反应pH为pH4.5-pH6.0,pH酸性时稳定性,中性和偏碱性时稳定性差。该酶表现出较好的耐高温活性,但是pH稳定性区域较窄。
利用融合标签表达和纯化甘露聚糖酶,为HDYM04及其所产甘露聚糖酶在生物脱胶和其他领域中的应用提供了理论依据,同时也为本实验室建立在大肠杆菌BL21(DE3)融合表达外源基因的平台。
Mannanase is a very important enzyme for hemicellulose degradation,especially in biological-degumming field. In this research, the genome DNA of Bacillus licheniformis HDYM-04, the mannanase-producing strain, was used as the template of PCR, after the gain of mannanase gene by PCR, the fragment of mannanase gene was inserted into the fusion expression vector pET28a, and the recombinant DNA was transferred into Escherichia coli BL21(DE3) , and then the enzymological properties of the fusion enzyme was analyzed.
The length of mannanase gene, named as man3, gained by PCR was 1282bp. The sequence of man3 was analyzed by bioinformatics methods. The results showed that the enzyme was homologous with Bacillus sp., acidic, MW 38kD, without signal peptide and transmembrane region, two structural region of MANB and Glycosyl hydrolase. The original enzyme activity of Escherichia coli BL21(DE3) recombinant train HDEM8 of mannanase gene was determined as 38.60U/mL, and after optimized by orthogonal experiment, it was 45.57U/mL with the induced condition IPTG 1.0mmol/L, 30℃, 7h. After the purification by His·Bind Columns, the results of enzymological analysis showed that the optimum temperature and pH was 55℃and 5.5, the relevant activity at the range 50℃-60℃and 4.5-6.0, temperature stability was 30℃-60℃and pH stability was 3.0-6.0.
The gain and analysis of fusion expressed mannanase made a foundation for using HDYM04 and it’s mannanase in the biological-degumming and the other applications. In this research, the system of an allogenetic gene fusion expressed in Escherichia coli BL21(DE3) was established for our laboratory.
克隆所得甘露聚糖酶基因片段长度为1282bp。对所获得片段进行相应生物信息学分析,结果表明该酶与芽孢杆菌属的甘露聚糖酶同源性最高,为酸性甘露聚糖酶,分子量约为38kD,无信号肽和跨膜区,具有MANB和糖苷水解酶两个结构域。构建甘露聚糖酶的异源重组菌株后对重组菌株进行诱导表达,筛选出酶活性最高的HDEM8菌株进行诱导条件和酶学性质的研究,菌株HDEM8的初始酶活力为38.60U/mL。经以IPTG终浓度、诱导温度和时间为因子的正交试验优化后,确定最优诱导条件为IPTG终浓度1.0mmol/L、30℃、诱导培养7h。酶活最高达到45.57U/mL。将诱导后的HDEM8菌株细胞裂解物经His·Bind Columns纯化后进行酶学性质分析,结果表明:适合反应温度为50℃-60℃,最适温度为55℃;适合反应pH为pH4.5-pH6.0,pH酸性时稳定性,中性和偏碱性时稳定性差。该酶表现出较好的耐高温活性,但是pH稳定性区域较窄。
利用融合标签表达和纯化甘露聚糖酶,为HDYM04及其所产甘露聚糖酶在生物脱胶和其他领域中的应用提供了理论依据,同时也为本实验室建立在大肠杆菌BL21(DE3)融合表达外源基因的平台。
Mannanase is a very important enzyme for hemicellulose degradation,especially in biological-degumming field. In this research, the genome DNA of Bacillus licheniformis HDYM-04, the mannanase-producing strain, was used as the template of PCR, after the gain of mannanase gene by PCR, the fragment of mannanase gene was inserted into the fusion expression vector pET28a, and the recombinant DNA was transferred into Escherichia coli BL21(DE3) , and then the enzymological properties of the fusion enzyme was analyzed.
The length of mannanase gene, named as man3, gained by PCR was 1282bp. The sequence of man3 was analyzed by bioinformatics methods. The results showed that the enzyme was homologous with Bacillus sp., acidic, MW 38kD, without signal peptide and transmembrane region, two structural region of MANB and Glycosyl hydrolase. The original enzyme activity of Escherichia coli BL21(DE3) recombinant train HDEM8 of mannanase gene was determined as 38.60U/mL, and after optimized by orthogonal experiment, it was 45.57U/mL with the induced condition IPTG 1.0mmol/L, 30℃, 7h. After the purification by His·Bind Columns, the results of enzymological analysis showed that the optimum temperature and pH was 55℃and 5.5, the relevant activity at the range 50℃-60℃and 4.5-6.0, temperature stability was 30℃-60℃and pH stability was 3.0-6.0.
The gain and analysis of fusion expressed mannanase made a foundation for using HDYM04 and it’s mannanase in the biological-degumming and the other applications. In this research, the system of an allogenetic gene fusion expressed in Escherichia coli BL21(DE3) was established for our laboratory.
引文
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