Cloning, Expression And Characterization Of An Endo-β-1,4-mannanase From Penicillium Freii F63

β-Mannanases are produced by various organisms, such as plant, animal andmicroorganism. Microorganisms are the ideal source of β-mannanases due to theirhigh enzyme productivity and easy cultivation. Till now, a few β-mannannase genesfrom Penicillium spp. have been cloned and expressed. In this study, a novel GH5alkali-tolerant β-mannanase gene from Penicillium freii F63was cloned by geneticengineering technology. The recombinant enzyme was produced in Pichia pastorisGS115with a high yield and productivity to near apparent homogeneity, whereasehad higher catalytic activity in the acidic to neutral pH region. It showed betterstability from acidic to alkaline conditions.By TD-PCR with degenerate primers GH5F and GH5R, A176bp fragment wasamplified from the genomic DNA of Penicillium freii F63, and was partial sequenceof a glycosyl hydrolase family5endo-β-mannanase gene. The5′and3′flankingregions were obtained by TAIL–PCR with designing of two pairs specificity primersand assembled with the core region to generate a DNA sequence of1367bp，namedman5F63. The full-length β-mannanase gene with1260bp including the signalpeptide and mature protein coding sequences was obtained from the cDNA ofPenicillium freii F63, using the specific primers GH56F and GH56R. Two introns,55bp and52bp, respectively, interrupted the coding sequence. SignalP analysisindicated the presence of a putative signal peptide at residues1-18aa. The matureβ-mannanase （Man5F63） with the typical （β/α）8-barrel fold had a polypeptide of401amino acid residues. The calculated molecular mass and Isoelectric point of themature β-mannanase were43.1kDa and5.52, respectively. The conserved catalyticresidues, Glu246and Glu355, were estimated. The nucleotide sequence for theβ-mannanase gene （man5F63） was deposited in GenBank under accession numberJN882026.The gene fragment encoding the mature protein without the signal peptidesequence was amplified from Penicillium freii F63with primers Man5F63F andMan5F63R, cloned into the pPIC9vector, and then expressed in Pichia pastorisGS115. The positive transformant exhibiting the highest β-mannanase activity wasselected for high-cell-density fermentation in1L flasks. The recombinant enzyme（rMan5F63） was secreted into the culture supernatant with a high yield （1.1g/L）. Itsapparent molecular weight was approximately72.0kDa, and it was near toelectrophoretic homogeneity based on SDS-PAGE analysis. This result indicated thatsome post-translational modifications occurred in rMan5F63during expression in P.pastoris. There were11peptides from LC-ESI-MS/MS analysis, and they completely corresponded to the predicted sequence of Man5F63. Thus the tested sample wasproved to be the recombinant enzyme （rMan5F63） indeed.The recombinant enzyme （rMan5F63） was shown optimal activity at60°C and pH4.5, and exhibited alkaline tolerant stability. The activity of rMan5F63wassignificantly enhanced in presence of MnSO₄, CoCl₂, CuSO₄and β-mercaptoethanol,whereas it was strongly inhibited by HgCl₂and SDS. It hydrolysed heteromannanscontaining high content of manose, and then specificly hydrolased β-1,4-D-glycosidiclinkages formed with manose. The specific activity, Kmand Vmaxvalues were47.5U/mg,7.8mg/mL and70.4μmol/（min·mg）, respectively, for locust bean gum, and40.3U/mg,2.3mg/mL and61.7μmol/（min·mg）, respectively, for konjac flour.