Cloning And Expression Of Endoglucanase Gene From Aspergillus Oryzae GiF-10 And Characterization Of Recombinant Enzyme

Endoglucanase is one of the most important cellulose enzymes, which cooperates with cellobihydrolase andβ-1,4-glucosidase to translate cellulose to glucose completely. Cellulase produced by natural microorganism has low activity which make the production cost very high. By means of genetic engineering to improve the activity of cellulase is an effective way to reduce the cost of cellulase production.According to the sequence of endoglucanase CelB gene (GenBank Accession No.: D83732.1) from Aspergillus oryzae, a pair of primers was designed and synthesized, sequence analysis revealed that the gene has no intron, and a high-fidelity polymerase was used for amplifying endoglucanase eg gene by using Aspergillus oryzae giF-10 genomic DNA as template, PCR amplified a specific band about 1200bp, then connected it to pMD19-T simple vector.Sequencing showed that the length of eg gene was 1251bp, OFR was 1251bp, encoding 417 amino acids,5'end had a signal peptide sequence coding 17 amino acid, The full gene had an approximate molecular mass of 44.46KDa. Sequence alignment of endoglucanase gene with GenBank database showed it shared a 100% sequence identity with the CelB gene from Aspergillus oryzae submitted in GenBank. Then submitted endoglucanase eg gene sequence in GenBank, accession number was HQ739052.The eg gene was cloned into the E.coli expression vector pET32a, then obtained recombinant plasmid pET32a-eg, expressed in Escherichia coli BL21 (DE3). Hydrolysis cycle tests showed that the expression was successful.The eg gene was cloned into the yeast expression vector pPICZaA,then the pPICZaA-eg plasmid was linearized and electroporated into yeast competent cells of X33 host strain and get a lot of positive transformants, hydrolysis cycle tests showed that the expression was successful. and the SDS-PAGE showed that the molecular weight was about 65 KDa In the condition of shake flask culture, fermentation conditions of yeast expression engineering strain were optimized, the results showed that the optimal concentration of methanol induction was 0.75%, five-day induction culture with 1L flask reached the highest activity 120 U/mL.Comparing original enzyme with recombinant of the enzymatic properties found that the original enzyme activity was 250 U/mL, the recombinant enzyme unit activity of was 120 U/mL, the original enzyme's optimum pH and temperature were 4.0 and 55℃, respectively.In the range of 45℃-65℃and pH3.4-4.4 it could maintain above 80% of the highest endoglucanase activity. Recombinant enzyme optimal pH and temperature were 4.0 and 45℃,respectively. In the range of 35℃-45℃and pH3.4-4.4 it could maintain above 80% of the highest endoglucanase activity.