Study on Gene Cloning and Expression of β-mannanase from Enterobacter aerogenes B19 and Characterization of the Recombinant Enzyme

The Enterobacter aerogenes B19 β-mannanase gene(ManE)cloned and full length gene of β-mannanase(ManE)were obtained successfully. The ManE gene was connected with pET-28a(+)expression vector,then the recombinant plasmid was transformed into Escherichia coli BL21(DE3). The recombinant strain BL21(DE3)-pET-28a(+)-ManE was constructed and the recombinant β-mannanase was expressed successfully. The results showed that the ManE gene(2196 bp)was cloned and encoded 731 amino acid residues. The crude enzyme was purified by HisTrap HP Ni-affinity chromatography and the molecular mass of the purified recombinant β-mannanase was detected about 82.5 ku by SDS-PAGE protein electrophoresis. The enzymatic properties analysis showed that the optimal reaction temperature and pH of this recombinant enzyme were 55℃ and 6.5,respectively. The recombinant enzyme exhibited high thermal stability when the temperature ranged from 50 to 55℃,and the pH value ranged from 4.0 to 7.0. The recombinant β-mannanase activity was activated in different degree using Co²⁺,Mn²⁺,Zn²⁺,Ba²⁺ and Ca²⁺ with low concentration. But it was inhibited by K⁺,Mg²⁺ and Cu²⁺. In addition,Cu²⁺ was inhibitor of enzyme activity,had the most obvious inhibitory effect on the recombinant enzyme and the relative enzymatic activity only retained 65.3%. The heterrologous expression of β-mannanase was achieved,and the results provided an important theoretical basis for further industrial application.