Clone and Expression of High Yield Recombinant β-mannanase in Pichia pastoris

Objective:This study aimed to construct Pichia pastoris which can highly expressed β-mannanase,and to study the enzyme-producing leve1.Method:We identified a mannanase-producing strain isolated from konjac soil.The mannanase-producing strain were grown on selective plates(LB;0.5% konjac glucomannan;0.1% Congo Red;2.0% agar).Sequence analysis of the 16S rDNA fragment of the strain was conducted,and the strain was identified as Bacillus sp,named Bacillus sp.QYW-1(GenBank Accession Number JX524224).Using degenerate polymerase chain reaction a new β-mannanase gene,denoted as manW(GenBank Accession Number JX869490),was obtained from Bacillus sp.QYW-1 genome.An open reading frame(ORF) of 1 084 bp encoded a protein of 360 amino acids including a putative 25-residue signal peptide and belonging to Glycosyl hydrolase family 26.The expressed enzyme had a molecular mass of approximately 40 kD determined by SDS-PAGE.A codon optimized β-mannanase gene from Bacillus sp.QYW-1 cleaved by EcoRⅠ and NotⅠ was inserted into the expression vector pPIC9K,downstream of an α-factor signal peptide sequence,and transformed into Escherichia coli DH5α.The resulting plasmid linearized with Bgl Ⅰ was transformed into Pichia pastoris GS115.Result:After extensive screening,the recombinant strain manW1 that expresses the secretory protein at high level was obtained.Fermentation conditions and enzymatic characteristics were studied preliminarily.Optimum pH and temperature for the recombinant mannanase was 6.5 and 37°C,respectively.The recombinant enzyme was stable at pH 5.0~7.4 and maintained over 40%of original activity after incubation at 70℃ for 20min.β-mannanase activity was assayed using 3,5-dinitrosalicylic acid(DNS) method.The activity of the recombinant mannanase reached 1 180 U/mL.Conclusion:The recombinant β-mannanase has a better prospect.