The Cloning And Expression Of β-mannanase Gene And Its Recombined Enzymatic Properties

Paenibacillus sp. CH-3which had a high level β-mannanase activity was an endophyte strain isolatedfrom locust seed in our laboratory, the β-mannanase encoding gene（manA） was isolated from it bydegenerate PCR and TAIL-PCR. Sequence analysis showed that the gene with an ORF of984bp encodinga protein of327amino acids, with a calculated molecular weight（MW）of35.270kDa and a isoelectricpoint （pI）value of7.01. And the1-32amino acid residues of N-terminal was its signal peptide. Accordingto the homology research, the deduced amino acid sequence of MANA from Paenibacillus sp. CH-3had thehighest similarity with MANA1from Paenibacillus sp. A1（81.35%）, so the enzyme should belong to theglycosyl hydrolase family5（GH5）. Enzymatic three-dimensional structure analysis showed that the secondstructure of the enzyme included14α-helix and15β-sheets, and the catalytic domain of it adopted acertain folding way to form a TIM barrel structure.The manA gene from Paenibacillus sp. CH-3were cloned into the plasmid pET32a（+） or pET28a（+）which had double restriction site of BamH I and Sal I, and then the manA in pET32a（+） or pET28a（+） wastransformed into the host E.coli BL21（the engineered bacteria of β-mannanase）. After recovered theexpression products from the engineered bacteria, we found β-mannanase activity（90.34U/ml） and obtaineda specific band approximately50.4kDa（or39.4KDa） by using SDS-PAGE electrophoresis analysising andcoomassie brilliant blue staining.The effects of fermentation temperature, inducer（IPTG） concentration and induction time onrecombinant β-manmanase production of the gene engineered bacteria were investigated by single factorexperiments, on the base of these experiments the orthogonal array experiment was designed. Theresults revealed that the fermentation conditions optimization of the gene engineered bacteria were asfollow: fermentation temperature28℃, IPTG concentration0.05mM and induction time12h, and in thisfermentation condition, the enzyme activity achieved1054.17U/ml, increased about10.7times comparedwith the initial enzyme activity. The effects of the four factors on β-manmanase production were thatinducer（IPTG） concentration>induction time>the interaction of temperature and inducer（IPTG）concentration> fermentation temperature. The gene engineered bacteria fermentation was carried out inthe50L fermentor at28℃,0.05mM of IPTG concentration. Results indicated that the recombinant β-manmanase activity had reached a high level after adding inducer within2h, and achieved its highestpeak at8h（1917.96U/ml）, increased about20.2times compared with the initial enzyme activity.Subsequently the enzyme activity declined slightly, yet remained stable overall.Enzymic properties of the gene engineered bacteria revealed that the optimal temprature and pHfor producing enzyme were45℃and7.0, respectively, the enzyme activity was stable when theenzyme was treated at3545℃, when it was treated at50℃for30min, the enzyme activity stillremained74.23%, and when the temperature rised to55℃the enzyme activity virtually disappeared;more than87.06%enzyme activity was remained after15min treatment at pH4.07.0. In addition, theenzyme activity could be activated by Ca²⁺、Ba²⁺、Mg²⁺、Mn²⁺and Co²⁺, among them Ca²⁺had the mostsignificant effect on the enzyme activity, and61%of activity was improved when the ion appeared inthe enzyme solution. However, Cu²⁺、Zn²⁺、EDTA and Fe²⁺could inhibit its activity.Bacillus subtilis HD-1which also had a high level β-mannanase activity was another endophyte strainisolated from soybean seed in the laboratory, the β-mannanase encoding gene（manB） was isolated from itby PCR. Sequence analysis showed that the gene was1089bp length, the1-27amino acid residues ofN-terminal was its signal peptide. The amino acid sequence of this enzyme shared the highest up to98.34%homology with other β-mannanases from Bacillus subtilis, and the conserved motifs in other β-mannanasesof ManB were also recognized in the sequence, so the enzyme should belong to the glycosyl hydrolasefamily26. Enzymatic three-dimensional structure analysis showed that the catalytic domain of the enzymeadopted a certain folding way to form a TIM barrel structure. There was a disulfide bond between Cys92and Cys112, and the domain between them formed the redox-active center of the enzyme. Enzymaticthree-dimensional structure analysis provided a basis for the improvement of the enzymatic catalyticactivity and the directed transformation of the enzymatic function.