| The cellulose-degrading bacterium Cytophaga hutchinsonii can rapidly degrade crystalline cellulose,and direct contact between bacteria and cellulose is necessary for cellulose degradation.The genome of C.hucthinsonii does not encode known carbohydrate binding modules(CBMs)that bind crystalline cellulose,therefore the mechanism of how C.hutchinosonii binds substrates is currently unclear.In the previous work of this study,a group of cellulose binding proteins(CBPs)were identified from the outer membrane proteins of C.hutchinsonii using Avicel as the substrate.In this work,one of the CBPs(CHU＿0173)was selected for further study.CHU＿0173 contains 531 amino acids with a theoretical molecular weight of 56.5 k Da,and does not show any homology with the known CBMs.According to the predicted domain,the N-terminus and C-terminus of CHU＿0173 each contain a Gld M domain,which belonging to a sliding protein.At present,it is not found that CHU＿0173 was related to the binding to crystalline cellulose.In this work,the coding sequence of CHU＿0173(excluding the signal peptide)was ligated to the expression vector p ET-30a(+),and protein expression was induced in the expression strain Transetta(DE3)and the expression product rCHU＿0173 was purified.The results of the substrate binding profile of rCHU＿0173 showed that rCHU＿0173 had no binding capacity to soluble polysaccharides such as barley glucan,soluble mannan,soluble starch,Methylcellulose and 2-Hydroxyethyl-cellulose;it had a strong binding capacity for insoluble polysaccharides such as bagasse,insoluble xylan,lichenan,and insoluble mannan,but it had weak binding capacity for filter paper,Avicel,PASC,and chitin.Since CHU＿0173 had no homology with the known CBMs,which indicated that CHU＿0173 contained a new CBM.Because the sizes of CBMs are generally between 50-250 amino acid residues.We further located the region of CHU＿0173 that bound to the substrates.We constructed a total of 8 recombinant plasmids encoding different lengths of rCHU＿0173 with N-terminal or C-terminal deletions,and these deleted polypeptides were expressed and purified.Six deleted polypeptides were successfully obtained,including CHU＿0173-1(amino acid residues 72-531),CHU＿0173-4(amino acid residues 222-531),CHU＿0173-5(amino acid residues222-481),CHU＿0173-6(amino acid residues 222-431),CHU＿0173-7(amino acid residues 222-381),CHU＿0173-8(amino acid residues 222-331).Expression products were not obtained from recombinant plasmids CHU＿0173-2(amino acid residues 122-531)and CHU＿0173-3(amino acid residues 172-531).The substrate binding profile of the deleted polypeptides was determined,when 221 amino acids at the N-terminus were deleted,the ability of CHU＿0173-4(222-531)to bind insoluble xylan decreased significantly,but the ability to bind other polysaccharides did not change.Since the binding ability of CHU＿0173-1(72-531)to xylan had not changed,which indicated that amino acids 72-222 are responsible for the binding to xylan.We constructed the recombinant protein CHU＿0173-X1、CHU＿0173-X2 containing the 72-242、72-292 amino acid of CHU＿0173,respectively.Binding experiments showed that CHU＿0173-X1 、CHU＿0173-X2 only had a weak binding capacity for insoluble xylan,indicating that amino acids 72-292 did not contain a complete xylan binding module.The partial sequence before amino acid 72 of CHU＿0173 was also necessary for binding to xylan.When 50 amino acids at the C-terminus were deleted,the binding capacity of the deletion mutant CHU＿0173-5(222-481)toward filter paper,Avicel,PASC,and chitin was significantly increased.The binding capacity of CHU＿0173-6 and CHU＿0173-7(222-381)was similar to that of CHU＿0173-5.When 200 amino acids at the C-terminus were deleted,CHU＿0173-8(222-331)significantly decreased the binding capacity against all polysaccharides.Finally the region bound to crystalline cellulose was located at amino acids 222-381 of CHU＿0173.In general,there are 2-4 conserved aromatic residues in each CBM family that play a key role in the process of binding substrates.We searched and selected the homologous peptides of CHU＿0173-7 in the NCBI database for multiple sequence alignments and found 7 conserved aromatic amino acids,including F232,Y252,Y294,Y325,Y343,Y361,and F365.The seven conservative aromatic residues of CHU＿0173-6 were mutated to alanine through site-directed mutagenesis,and the binding ability of each site-directed mutant polypeptide to various insoluble polysaccharide substrates was tested.The results showed that the mutants Y294 A,Y325A,Y343 A,and Y361 A decreased the binding capacity to chitin,indicating that they may be the key amino acids in the binding site of CHU＿0173,which needs to be confirmed by multiple site mutation.The significance of this work is that CHU＿0173 contained a new CBM that can bind to crystalline cellulose,and preliminarily identified the key aromatic residues in its binding site.Because it has no homology with the known CBMs,CHU＿0173-7 is a founding member of the new CBM family.In addition,the binding of C.hutchinsonii to cellulose and sliding of the bacteria on the cellulose surface are contradictory and have not been studied clearly.Studying on CHU＿0173 may provide a new insight for explaining this contradiction and provide a basis for explaining how C.hutchinsonii binds and utilizes crystalline cellulose. |
