E. Coli Fusion Expression Of β-mannanase And Characterizing

Hemicelluloses are a kind of regenerate energy sources which seconds only cellulose and account 1/4-1/3 of the plant dry weight. Its decomposition means a lot to the energy sources and environment problem in future. The enzymatic reaction in hemicelluloses decomposition and the enzyme system screening of hemicelluloses hydrolysis have been the focal point of research. Our research group had got a fungi Arimillariela tabescens (EJLY2098) which secreted β-1,4-D-mannanase while induced. Since it takes more than a month to ferment the natural microbe, constructing a recombinant of E.coli by gene engineering to produce the mannanase will make it much more convenient. We were to clone the target gene man to the fusion expression vector pET-32a with trx-tag His-tag S-tag at N-terminal.Digested the recombinant plasmid pPICZαA-man with EcoR I and Not I to get target gene man, ligased with pET-32a which containing the confusion protein trx-tag S-tag His-tag. Then transformed to DH5α. After identified by enzyme digestion and sequencing, the recombinant plasmid was transformed into BL21(DE3) for expression. Screened positive clones by PCR. Induced with IPTG and identified target protein by SDS-PAGE. But detected not any target protein and activity. Optimized the induced condition and changed the host but still no target protein was detected.Digested the recombinant plasmid pET-32a-man with Nco I and Not I to get man gene, ligased with pET-22b which containing the pelB signal peptide. Transformed the recombinant plasmid to TOP10F', then identified by enzyme digestion and sequencing. Transformed the recombinant plasmid into BL21(DE3) for expression. Induced the recombinants with IPTG and analyzed by SDS-PAGE. It showed activity while detected periplasmic extracts by DNS. The induced supernatant showed no activity while detected with the same method, but showed little activity at the quality analysis.The fusion expressed β -mannanase with C-terminal 6 × His-tag had been secreted to the periplasmic. The optimum temperature and pH was 60℃ and 7.0,and showed relevant activity at the range of 50℃-60℃ and pH 5-9. Temperature stability was 30℃-60℃ and pH stability was 8.0-10.0.We constructed pET-32a-man and pET-22b-man correctly and fulfilled the latter...