Cloning And Expression Of Aspergillus Niger β-glucosidase Gene In Escherichia Coli

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.