Identification Of A 1,4-β-D-Glucan Glucohydrolase Belonging To Glycoside Hydrolase Family 9 From Cytophaga Hutchinsonii

Cytophaga hutchinsonii is a common aerobic cellulose decomposing bacterium in soil,which can rapidly utilize crystalline cellulose.The genome of C.hutchinsonii encodes 22 glycoside hydrolases(GHs)related to cellulose degradation,but it does not contain genes encode exocellulase and cellulosome that responsible for crystalline cellulose degradation.Therefore,the predicted strategy of C.hutchinsonii to degrade cellulose is different from other known cellulose decomposing bacteria and fungi.The purpose of this study was to identify the enzymological characteristics of Cel9G,which belonging to GH9 family of C.hutchinsonii and its role in degradation of cellulose in the strain.Cel9G is a predicted β-glycosidase of uncertain specificity,which is composed of1452 amino acids and contains several domains,including secreted signal peptide(amino acids 1-20),Cel D＿N domain(amino acids 180-266),GH9 catalytic domain(amino acids 277-719),PKD domain(amino acids 762-844)and Cterminal sorting domain(CTD,amino acids 1379-1450).In order to understand the role of each domain in the degradation of cellulose by Cel9G,six Cel9G derivatives were constructed in this work,including Cel9G＿A(unknown functional domain,amino acids 21-179),Cel9G＿B(Cel D＿N+GH9 catalytic domain,amino acids 160-761),Cel9G＿C(unknown domain+Cel D＿N+GH9catalytic domain,amino acids 21-761),Cel9G＿D(Cel D＿N+GH9 catalytic domain+PKD,amino acids 160-864),Cel9G＿E(GH9 catalytic domain,amino acids 267-761),Cel9G＿F(CTD-truncated,amino acids 21-1378).We successfully expressed and purified five recombinant proteins,including Cel9G＿A,Cel9G＿B,Cel9G＿C,Cel9G＿D and Cel9G＿E.Among the four derivatives contained catalytic domain,only Cel9G＿E had no enzyme activity,while the other three enzymes Cel9G＿B,Cel9G＿C and Cel9G＿D had similar substrate specificity and temperature stability,so Cel9G＿B was selected as the representative to identify the detailed enzymatic characteristics of Cel9G.The results of substrate specificity showed that Cel9G＿B had the highest activity against barley glucan(β-1,3/1,4 glycosidic linkage),followed by lichenan,and lower activity on other polysaccharides(mannan,Avicel,PASC,CMC,etc).In addition,Cel9G＿B also showed very weak activity to laminarin(β-1,3/1,6glycosidic linkage),which may be a side effect of Cel9G＿B against β-1,3glycosidic bonds.Interestingly,Cel9G＿B showed similar activities against pnitrophenyl-β-D-cellobioside(p-NPC)and p-nitrophenyl-β-D-glucopyranoside(p-NPG),suggesting that Cel9G＿B had β-glucosidase activity.The final products of hydrolysis of cellooligosaccharides and barley glucan by Cel9G＿B was glucose.Since the Blast P search showed that Cel9G had high homology with chitobiases from genus Vibrio,we tested the hydrolysis of chitooligosaccharides by Cel9G＿B.The results showed that Cel9G＿B can degrade the deacetyl-chitobiose into glucosamine,but it can’t degrade diacetyl-chitobiose.The time course experiment of p-NPC hydrolyzsis by Cel9G＿B showed that it hydrolyzed single glucose from the non reducing end,and the time course experiment of Cel9G＿B against cellopentose showed that Cel9G＿B only cut one glucose from the end of saccharide and then left the substrate,indicating that glucose was released from the end of cellulose by Cel9G＿B with an the exo-type and non-processive mode.Cel9G＿B also released glucosamine from the end of chitooligosaccharides in a non-processive mode.The catalytic efficiency(kcat /km)of Cel9G＿B against cellooligosaccharides increases with the degree of polymerization,and its kcat/Km value for cellohexaose was 750 times higher than that of cellobiose.The above-mentioned results showed that Cel9G was a β-1,4-D glucan glucohydrolase,which was completely different from the cellulose hydrolysis mode of the known typical GH9 cellulase,and the final product of cellulose hydrolysis of the latter is generally cellotetraose or cellobiose.We constructed the phylogenetic tree of the catalytic domain of GH9 enzymes,and found that the enzymes of the GH9 were divided into four groups,while Cel9G and its homologs formed a new group(Group C).Multiple sequence alignment of the catalytic domains of the GH9 enzymes showed that there were two main differences between Group C and other GH9 enzymes.One was that enzymes from Group C has an additional residue(S/T,T321 in Cel9G),another difference was the insertion sequence of 11 residues(N338-S348 in Cel9G).However,the two known catalytic amino acids in typical GH9 enzymes were conserved in group C enzymes(D323 and E734 in Cel9G,respectively).In order to verify whether D323 and E734 were the real catalytic residues of Cel9G,twopoint mutants D323 A and E734 A were constructed in Cel9G＿B,respectively.It was found that the two mutants were completely inactivated or retained very weak activity to barley glucan,cellotetraose,and p-NPG,indicating that D323 and E734 were probably the catalytic residues of Cel9G.In order to explain the unique characteristics of Cel9G compared to other typical GH9 cellulases,two deletion mutants ΔT321 and ΔN338–S348 were constructed respectively,and point mutants T321 A and T321 S were also constructed for T321.The results of enzyme activity showed that the deletion mutant ΔT321 had no activity againt barley glucan,the activity ofΔT321 against cellotetraose and p-NPG was less than 4% compared with the wild-type activity;the mutant ΔN338–S348 had no activity for cellotetraose,but low activity for p-NPG and barley glucan.When the T321 was replaced by alanine or serine,the activity of the mutants T321 A and T321 S against cellotetrose was only decreased slightly,but their activity against p-NPG and cellobiose decreased significantly,which indicated that T321 played an important role in cellobiose hydrolysis.Based on the fact that Cel9G can hydrolyze cellooligosaccharide to glucose,and it may be localized on the cell surface,we speculate that Cel9G can cooperate with the endocellulase on cell surfaces to convert amorphous cellulose to glucose.Glucose is a necessary nutrient for the early growth stage of C.hutchinsonii.We speculate that the biological function of Cel9G is to provide nutrient for the proliferation of bacterial in the early growth stages of C.hutchinsonii.