Identification And Function Characterization Of Novel Glucan Hydrolases CoMA And LamC From Corallococcus Sp.Strain EGB

The myxobacteria are a special kind of Gram-negative unicellular bacteria with rod-shaped vegetative cells.Because of their gliding movement,complicated multicellular behavior and morphogenesis process,the myxobacteria are generally considered as advanced prokaryotes,which is an important model organism and may live on the edge of eukaryotes.Another outstanding trait of the myxobacteria is that they can produce a large number of bioactive secondary metabolites with novel structures.So myxobacteria is another kind of important drug source microbial community,besides the actinomycete,with great potential for drug development.Meantime,myxobacteria can produce abundant glucoside hydrolases with unique structures functions,which can degraded macromolecules in the external environment for gaining energy.Till date the largest complete bacterial genomes Sorangium cellulosum So 0157-2?14.78Mb?have been reported,and?2.7%of total proteome were annotated with diverse combinations of CAZY domains.In this study,we try to unearth some glycoside hydrolases within the genome of Corallococcus sp.strain EGB.A novel amylase CoMA gene was cloned from the chromosomal DNA of EGB strain.The open reading frame that corresponds to CoMA consists of 1665 nucleotides and encodes a protein of 554 amino acids with a predicted molecular mass of 59,946 Daltons and a calculated pI of 6.08.The initial 26-amino acid sequence was detected as signal peptide by using SignalP 4.1.BLASTP analysis showed that CoMA showed high identity?34%?with the well-characterized a-amylase HOAMYA?PDB:1WZA?from the thermophilic halophile Halothermothrix orenii,and 30%identity with the trehalose synthase TreS?PDB:3Z09?from Mycobacterium smegmatis.However,CoMA exhibited unexpectedly low sequence identity?10-20%?with the group multispecific enzymes of GH13₂0.The gene?coMA?was expressed in E.coli BL21?DE3?.The specific activities of recombinant CoMA was 980 U/mg for soluble starch.CoMA can hydrolyze soluble starch,amylose,amylopectin,glycogen,pullulan,maltooligosaccharides??G3?and y-cyclodextrin.The optimal pH and temperature for CoMA were 7.0 and 50?.The optimum substrate of CoMA was soluble starch,with km and kcat values of 2.9 mg/mL and 3021.2 s-1.The enzyme was activated by Mn2+and inhibited by Cu2+?Zn2+?Ni2+?Cr3+?Fe3+.CoMA was inhibited strongly by SDS,but EGTA had little effect on enzyme activity.Thus CoMA was a Ca2+-independent amylase.Moreover,it hydrolyzed ?-cyclodextrin and soluble starch to maltose and hydrolyzed pullulan to panose with relative activities of 0.2,1 and 0.14,respectively.In addition,CoMA has a most remarkable feature:its ability to catalyze maltooligosaccharides?maltotriose to maltoheptaose?and starch to produce high levels of maltose without synchronous glucose production.Thus,CoMA can be classified as a maltogenic a-amylase,which produce high levels of maltose without synchronous glucose production.CoMA is a kind of a-amylase that yields a-maltose by splitting the ?,1,4-glucosidic linkages in starch through an endo-type mechanism.Degradation of 1%?w/v?starch resulted in the formation of oligosaccharides in the maltose to maltopentaose range at early time points,followed by an accumulation of maltose,maltotetraose and small amounts of glucose after 1 h,and its final end-products consisted of a high level of 58%?w/w?maltose without concomitant production of glucose.Thus,the participation of some mechanism other than simple hydrolysis such as transglycosylation or condensation was strongly suggested.In an attempt to gain some insight into the mechanism utilized by CoMA,maltotriose was used as a substrate and the end-product profile was examined.As a small quantity of maltohexaose was detected when CoMA reacted with maltotriose,transglycosylation is postulated to be the major multimolecular mechanism.Glucose was not detected in the initial stages of the hydrolysis of starch and maltotriose and only maltose was detected throughout the process of hydrolyzing 2 mM maltotriose.Thus,glucosyl-transfer,rather than maltosyl-transfer was postulated to be the major transglycosidation reaction.It also means that CoMA prefer to release maltose from the non-reducing end and precludes glucose formation by a glucosyl-transfer reaction.Due to the expression level of CoMA in E.coli is low,the full-sequence and the gene fragment without the signal peptide of CoMA have also been expressed in P.pastoris.The yield of CoMA27 was increased to 54 mg/mL.Compared with liquefying amylose AmyM,the yied of maltose was increased by 5 time from 10%?w/v?starch.Subsequently,we constructed three bifunctional eznymes fusion of liquefying amylose AmyM and maltogenic amylose CoMA with different linker peptides.Three AmyM-CoMA fusion enzymes were all expressed as the protein of ca.111.2 kDa in P.pastoris.The flexible peptide linker?GGGGS?2 resulted in the best fusion,whose expression level had been increased to 140 mg/mL and the yied of maltose was 6%.In accordance with a putative ?-?1,3?-glucanase A1?GenBank accession no.AFE08907?sequence from C.coralloides DSM 2259,the full-length ?-?1,3?-glucanase gene was PCR-amplified from Corallococcus sp.strain EGB.This specific?-?1,3?-glucanase?designated LamC,a laminarinase from Corallococcus sp.strain EGB?contains 438 amino acid residues and has a calculated pI of 5.26 and molecular mass of 47,040 Da.LamC contains several putative modules,including a predicted N-terminal signal peptide?residues 1-26?,a fascin-like module?residues 56-182?,and a?-?1,3?-glucanase catalytic module?residues 196-438?.In eukaryotes,Fascin is a 55 kDa actin-bundling protein with four homologous ?-trefoil domains.Fascin belongs to actin-binding protein family.The ability of fascin to bind and bundle filamentous actin plays a central role in the regulation of cell adhesion,migration and invasion.The fascin-like module found in the glycoside hydrolases is not common in bacteria and has not been functionally characterized.To characterize the function of the catalytic and non-catalytic modules,all LamC derivatives were Trx-and His6-tagged on the N-terminus and successfully expressed in redox-deficient E.coli Origami B?DE3?.LamC displays broad hydrolytic activity toward various polysaccharides.Analysis of the hydrolytic products revealed that LamC is an exo-acting enzyme on ?-?1,3?-and ?-?1,6?-linked glucan substrates and an endo-acting enzyme on ?-?1,4?-linked glucan and xylan substrates.Site-directed mutagenesis of conserved catalytic Glu residues?E304A and E309A?demonstrated that these activities were derived from the same active site.Excision of the fascin-like module resulted in decreased activity toward ?-?1,3?-linked glucans.The carbohydrate-binding assay showed that the fascin-like module was a novel ?-?1,3?-linked glucan-binding module.The functional characterization of the fascin-like module and catalytic module will help us better understand these enzymes and modules.The gene fragment without the signal peptide of LamC have also been expressed in P.pastoris.The recombainant LamC27 was purified from the culture supernatant of strain P.pastoris GS115?pEFaA-lamC27?.After ammonium sulfate precipitation and Ni2+-NTA purification process,LamC27 was purified by 2.8 folds,with a 48%recovery.The enzyme was activated by Mn2+and DTT,and inhibited strongly by Cu2+?Co2+?EDTA.Meantime,LamC27 can dissolved the cell wall of rice blast spore and hypha.It means LamC has potential antifungal activity toward rice blast.In summary,a novel maltogenic amylase CoMA was identified from Corallococcus sp.strain EGB.The most outstanding feature of CoMA is its ability to catalyze the conversion of maltooligosaccharides??G3?and soluble starch to maltose as the sole hydrolysate.And a novel ?-1,3-glucanase LamC gene with broad-spectrum activity was also cloned from the EGB strain and the fascin-like module was a novel ?-?1,3?-linked glucan-binding module.