Identification And Biological Function Of Glucoside Hydrolase From Corallococcus Sp.EGB

Myxobacteria are gram-negative bacteria with rod-shaped vegetative cells that exhibit unique group behavior including cell growth?predatory member?motility and development.There are two main research directions involve in myxobacteria including development and secondary metabolites.As a group of bacteria that possess complex social behaviour during their life cycle,myxobacteria has aroused wide interest from more and more investigators and devoted numerous research studies in the last decades,especially the fruiting body development by several sequential cell-cell interactions.Meantime,myxobacteria can produce abundant secondary metabolites and glucoside hydrolases with unique structures and functions,which have great potential in many industrial applications.Based on the morphologies and the 16S rRNA gene sequences,the strain EGB was assigned as Corallococcus sp..The strain showed good activity for inhibiting various pathogenic fungus like Ustilaginoidea virens?Magnaporthe.oryzae?Fusarium oxysporum?Verticillium dahliae and Phytophthora capsici.The pot experiment revealed good biological control of cucumber Fusarium wilt with Corallococcus sp.EGB.Further research showed that the supernatant enzyme of strain EGB can effectively reduce the infection of M.oryzae.The results showed that the mechanism involved in biological control of cucumber Fusarium wilt with Corallococcus sp.EGB is different from the reports.We deduced that there was a antifungal protein existed in the supernatant of strain EGB.According to the substrate spectrum analysis and hydrolysis component analysis of yeast cell wall,we found that a?-1,6-glucanase involved in the antifungal process.The(3-1,6-glucanase GluM was purified from the supernatant of strain EGB with the formation of substrate-protein complex,the specific activity of GluM was estimated as 24000 U·mg-1.Spectral analysis showed GluM specificity hydrolyze?-1,6-linked glucan,especially for short-chain?-1,6-glucan.GluM has a broad temperature adaptability,the optimum temperature and pH is 50?and 7.0.The purified GluM showed effective enzymatic hydrolysis towards hyphae and spore of Magnaporthe grisea,the treated hyphae and spore revealed distortional and damaged structure.Based on the peptide mass fingerprint and complete genome sequence of Corallococcus corallococcus DSM 2259,the intact ORF of gluM gene was estimated as 3222 bp,encoding 1073 amino acids.gluM encodes a secretory?-1,6-glucanase with a predicted signal peptide 26 amino acid residues.Regional expression showed that the active site of GluM was mainly located at N-terminal with 500 amino acid residues.GluM shared 93%identity with the TonB-dependent receptor of C.coralloides DSM 225 and 80%identity with the Oar of Myxococcus xanthus DK 1622 from the NCBI database,and shared no significant similarity with the reported glucoside hydrolases(10%identity),indicating that GluM is a novel?-1,6-glucanase,which was defined as a new glycoside hydrolase family GH136.The recombinant protein GluM was purified by the method of substrate adsorption.The spore germination was uppressed effectively with the rGluM,which showed the same effect with the purified GluM from the supernatant of Corallococcus sp.EGB.Synthesize the above results,we deduced that there was a?-1,6-glucanase existed in the supernatant of strain EGB showed antifungal activity.Sequence analysis of GluM revealed that GluM amino acid sequence shared 80%identity with the Oar of Myxococcus xanthus DK 1622.The function of Oar in the development showed the Oar has been participated in the process of the fruitbody body formation and predatory behavior.Further research showed that Oar was a kind of outer membrane channel protein with sugar transport capacity.The component analysis of the supernatant from DK1622 revealed that there was an active ingredient with the capacity of restoring the fruiting body formation of the oar mutant strain.With preliminary extraction and purification,the active ingredient in the supernatant was speculated to be oligosaccharide with molecular weight of less than 10 kDa.The oligosaccharide was speculated as a signaling molecule involved in the progress of cell interaction,there by affected the entire process of development.A novel?-amylase,AmyM,was purified from the culture supernatant of Corallococcus sp.strain EGB.AmyM is a maltohexaose-forming exoamylase with an apparent molecular mass of 55.46 kDa.Based on the results of peptide mass fingerprinting of AmyM and by comparison to the genome sequence of C.coralloides DSM 2259,the AmyM gene was identified and cloned into Escherichia coli.amyM encodes a secretory amylase with a predicted signal peptide of 23 amino acid residues,which showed no significant identity with known and functionally verified amylases.Recombinant AmyM(rAmyM)was purified by NTA affinity chromatography,with a specific activity of up to 14 000 U/mg.rAmyM was optimally active at 50? in Tris-HCl buffer(50 mM;pH 7.0)and stable at temperatures of<50?.rAmyM was stable over a wide range of pH values(from pH 5.0 to 10.0)and highly tolerant to high concentrations of salts,detergents,and various organic solvents.Its activity toward starch was independent of calcium ions.Compared with the commercial amylase Novo BAN800,the amylase AmyM showed high hydrolysis efficiency in a short time.Compared with the complicated hydrolysis products of BAN800,the hydrolysis products of AmyM were relatively unitary.