【作者】 周晓明；

【导师】 宫衡；

【作者基本信息】 华东理工大学 ， 微生物学， 2010， 硕士

【摘要】 β-葡萄糖苷酶(EC3.2.1.21)属于糖苷水解酶家族3,它能够水解非还原性末端的β-D葡萄糖苷键,释放出游离的葡萄糖及相应的配基。β-葡萄糖苷酶是纤维素降解中的关键酶,对于可再生资源纤维素的利用具有十分重要的意义,但其在野生型宿主菌中的低含量及低酶活成为纤维素酶解的限制因素,因此通过基因工程手段实现外源高效表达β-葡萄糖苷酶已经成为各国学者研究的热点。黑曲霉是常见的安全菌株之一,同时也是普遍公认的含有β-葡萄糖苷酶较多的菌株,实现黑曲霉β-葡萄糖苷酶基因外源高效表达具有十分重要的意义。本论文以总RNA为模板,通过逆转录PCR (RT-PCR)方法成功扩增了黑曲霉β-葡萄糖苷酶基因全长bgl序列,同时以基因组DNA为模板分别扩增出外显子5和6的序列E5,E6,PCR产物大小表明与预期一致。并成功构建三个重组表达质粒pET32a(+)-bgl, pET32a(+)-E5及pET28a(+)-E5。将bgl及E5片段进行测序,结果表明E5大小为1192bp,bgl为2583bp,将DNA序列的E5片段与cDNA外显子5进行比对,发现其同源性达100%,表明所获得的bglcDNA序列是黑曲霉β-葡萄糖苷酶正确的开放阅读框,是由转录过程中DNA模板上的外显子拼接而成。6gl与5个来源于黑曲霉的β-葡萄糖苷酶基因同源性达99%,推测的氨基酸序列同源性达99%,蛋白质功能结构域预测结果显示其属于糖苷水解酶家族3。将重组质粒转化大肠杆菌BL21(DE3)诱导表达,优化诱导条件之后,E5蛋白仍以包涵体形式存在于沉淀中。BGL可溶性表达略有增加,最佳表达条件为20℃,0.5mmol/L IPTG诱导12h,目的蛋白约占总蛋白的32.14%,可溶性部分约为15%,酶活29.8U／mg,约为野生型的30%。更多还原

【Abstract】 β-glucosidase(EC3.2.1.21) belongs to glycoside hydrolases superfamily 3, it hydrolyses P-D glucosidic bond and releases glucose and corresponding aglucone.β-glucosidase is the key enzyme of cellulose degradation, and plays an important role in the utilization of renewable resources such as cellulose. However, the low concentration and low enzyme activity ofβ-glucosidase in wild-type strains are becoming the limiting factors during cellulose hydrolysis. Now more and more researchers are contributing to the cloning and expression ofβ-glucosidase in foreign expression systems with gene engineering strategy. Aspergillus niger is known as one of the common safe strains, as well as a strain producing relative highβ-glucosidase, so it is very significant to express Aspergillus nigerβ-glucosidase gene with high performance.In this study, Aspergillus niger P-glucosidase gene bgl was amplified by reverse transcription PCR (RT-PCR) using the total RNA as template. Exon 5 and 6 sequences, E5 and E6 were amplified using the genome DNA as template.The PCR product of E5 and E6 were corresponded to the theoretical value. Three recombinant expression plasmids were then constructed:pET32a(+)-bgl, pET32a(+)-E5 and pET32a(+)-E5.The sequencing results showed E5 was 1192bp, bgl was 2583bp, the sequence alignment showed that E5 sequence was 100% identical to the exon 5 region in cDNA sequence, which demonstrated the bgl gene was the right ORF forβ-glucosidase and it was formed by exons splicing. The bgl gene was 99% homologous to five Aspergillus niger P-glucosidase genes reported and the deduced amino acid sequence was 99%. Protein function region prediction showed BGL belonged to glycoside hydrolase superfamily 3.Three recombinant expression plasmids were transformed into E.coli BL21(DE3) and induced by IPTG to expression. After optimization, E5 was still existed in the precipitation as inclusion bodies. BGL concentration was increased slightly in the supernatant. The optimized conditions for BGL expression were as follows:20℃,0.5mmol/L IPTG,12h. The whole target protein was 32.14% of the total and soluble protein was 15%.The recombinant enzyme activity was 29.8U/mg, which was 30% of the wild enzyme.更多还原