Cloning, Expression And Characterization Of Endoglucanase And Mannanase From The Marine Bacterium Paenibacillus Sp. BME-14

Nowadays resource on the earth is in shortage,which makes people endeavor to explore reproducible resource.As a reproducible resource existing all over the world, cellulosic materials are attracting large attention.How to degradate it to available saccharide and use it efficiently has become a hot point in research.Exploiture of cellulase and hemicellulase used to hydrolyze cellulose is the main research direction today.In this study,we screened for marine bacteria which producted cellulase and mannanase,and studied a serial properties of an endoglucanase and a mannanase cloned from Paenibacillus sp.BME- 14.A marine bacterium Paenibacillus sp.BME-14 producing cellulase and mannanase was isolated from the sea water using Conge red staining and analyzed by 16S rDNA.By constructing a genomic library,a positive clone pUC-BC was obtained by Conge red staining and the insert contained an endoglucanase gene,a truncated mannanase gene and a truncated mannosidase gene.The gene of cel9P had an open reading frame of 1629 bp,encoding a peptide of 542-amino acid residue with a calculated molecular mass of 60,314 Da.The enzyme showed the highest amino acid identity of 52%with other known endoglucanases and had a C-terminal catalytic domain belonging to the glycosyl hydrolases family 9.The optimum pH and temperature for enzymatic activity was pH 6.5 and 35℃.The metal ions of Ca²⁺,Mg²⁺ and Mn²⁺ had a positive effect on the activity while Hg²⁺,Cu²⁺ and EDTA had a negative effect.Cel9P exhibited higher activity on barley glucan and CMC-Na,and the Vm,Km and k_cat toward CMC-Na and barley glucan were 36.82μmol min^-1 mg^-1 protein,13.24 mg ml^-1,37.01/s,and 18.11μmol min^-1 mg^-1 protein,1.65 mg ml^-1,18.21/s.TLC analysis of the soluble sugars released from CMC by Cel9P indicated that the main product was cellobiose with small amounts of cellotriose and cellotetraose. Notable,Cel9P had 65%of the maximal activity at 5℃.Based on the special characteristic of Cel9P,it had a potential significance for study of cold-active mechanism and industry applications.Based on the known fragment of pUC-BC,we obtained the complete mannanase gene by inverse PCR.The gene of man26B had an open reading frame of 1428 bp,encoding a peptide of 475-amino acid residue with a calculated molecular mass of 53 kDa.Man26B possessed two domains,a carbohydrate binding module（CBM） of family 6 and a family 26 catalytic domain（CD） of glycosyl hydrolases.The CD showed the highest homologies to Cel44C of P.polymyxa（65%identity）.The optimum pH and temperature for enzymatic activity was pH 4.5 and 60℃.The activity of Man26B was not affected by Mg²⁺ and Co²⁺,but inhibited by Hg²⁺,Ca²⁺,Cu²⁺,Mn²⁺,K⁺,Na⁺ andβ-Mercaptoethanol and slightly enhanced by Pb²⁺ and Zn²⁺.EDTA did not affect the activity of Man26B, which indicated that divalent ions were not necessary for it.Man26B showed the high specific activity on LBG and konjac glucomannan.The values of K_m,V_max and k_cat were respectively 3.80 mg ml^-1,91.70μmol min^-1 mg^-1 protein and 77.08/s with the LBG as the substrate.In addition,after deletion of the CBM6 domain,it became more stable and retained 80%and 60%of the initial activity after treatment at 80℃and 90℃for 30min, respectively.This study provided the new endoglucanase gene and mannanase gene,and both of them will be valuable in the industrial applications and mechanism research because of their special properties.