Cloning and Characterization of a novel Mannanase from Paenibacillus sp.BME-14

【摘要】：正A mannanase gene(man26B) was obtained from the sea bacterium,Paenibacillus sp.BME-14, through the constructed genomic library and inverse PCR.The gene of man26B had an open reading frame of 1428 bp that encoded a peptide of 475-amino acid residues with a calculated molecular mass of 53 kDa.Man26B possessed two domains,a carbohydrate binding module(CBM) belonging to family 6 and a family 26 catalytic domain(CD) of glycosyl hydrolases,which showed the highest homology to Cel44C of P.polymyxa(60%identity).The optimum pH and temperature for enzymatic activity of man26B were 4.5 and 60℃,respectively.The activity of Man26B was not affected by Mg~(2+) and Co~(2+),but was inhibited by Hg~(2+),Ca~(2+),Cu~(2+),Mn~(2+),K~+,Na~+ and β-Mercaptoethanol,and slightly enhanced by Pb~(2+) and Zn~(2+).EDTA did not affect the activity of Man26B,which indicates that it does not require divalent ions to function.Man26B showed a high specific activity for LBG and konjac glucomannan,with K_m,V_(max) and k_(cat),values were 3.80 mg/ml,91.70μmol/min/mg protein and 77.08/s, respectively,being observed when LBG was the substrate.Furthermore,deletion of the CBM6 domain increased the enzymes stability while enabling it to retain 80%and 60%of its initial activity after treatment at 80℃and 90℃for 30min,respectively.This finding will be useful in industrial application of Man26B because of the harsh circumstances associated with such processes.