| Glycans and glycoconjugates play an important physiological role in all kinds of lives,As is known,these important physiological functions always depend on the glycan sturctures.Thus,structural analysis of different glycans is becoming one of the main tasks in glycomics research.Exo-hydrolases treatment combining with different analytic instruments is commonly considerd to be an efficient and green approach for structural analysis.Among all the exo-glycosidases,β-N-acetylhexosaminidases is necessary due to its related sugar motie,GlcNAc,exists in most of the natural glycans.Currently,the commercially available β-N-acetylhexosaminidases are mainly from jack bean,S.plicatus and S.pneumonia.Among them,Jack bean sourced enzyme is usually isolated from plant tissue and unable to be obtained recombinantly.Even though the latter two could be produced recombinantly,one is not able to hydrolyze bisecting GlcNAc,the other one doesn’t catalyze the hydrolysis of β 1,2 linked GlcNAc residue,which limits their application in N-glycan analysis.Therefore,we explored β-N-acetylhexosaminidases with different properties and specificities from different bacteria and aimed to develop them into alternative enzymatic tools for glycan analysis recombinantly.It can not only provide us efficient enzymes in large scale but also enlarge the current pool of β-N-acetylhexosaminidase.In this study,we attempted to clone some unstudied genes encoding β-N-acetylhexosaminidases from different bacteria strains.Finally,three active recombinant β-N-acetylhexosaminidases were obtained from Akkermansia muciniphila and Stackerbrandtia nassaunsis and then termed as AmBHexN2301,AmBHexN2446 and SnBHexN384.They showed their own properties in the characterization and specificity assays.As SnBHexN384 exhibited relatively broad spcifity and better acitivty,it was chosen for applications in glycan structural analysis,in which SnBHexN384 exhibited some advantages over jack bean hexosaminidase in degradation of bisecting structrues.Details of the results were described below:1.Gene cloning,heterologous expression and activity detection of three recombinantβ-N-actetylhexosaminidases,AmBHexN2301,AmBHexN2446 and SnBHexN384Three genes encoding for β-N-acetylhexosaminidases were found from genome of A.muciniphila and S.nassauensis by searching the gene database.They were amplified and over-expressed in E.coli successfully via a series of molecular techniques.The molecular sizes of these three recombinant enzymes were revealed to be consistent with their theoretical size after a series of protein analysis.Activity detection was performed with a panel of pNP-glycosides as substrates,it turned that all three enzymes were selective to GlcNAc and GalNAc glycons with β configuration among the synthetic substrates.Kinetic parameters suggested that Km values of AmBHexN2301,AmBHexN2446 and SnBHexN384 were 0.52±0.02 mM,1.36±0.09 mM,2.47±0.05 mM to pNP-β-GlcNAc and 0.11±0.01 mM,0.38±0.03 mM,3.29±0.32 mM to pNP-β-GalNAc.2.Biochemical characterization and catalytic site verification of three recombinant β-N-acetylhexosaminidases,AmBHexN2301,AmBHexN2446 and SnBHexN384The optimum pH of AmBHexN2301,AmBHexN2446 and SnBHexN384 were 5.0,6.5,and 6.0 respectively.AmBHexN2301 was more sensitive to small pH changes and its activity of AmBHexN2301 decreased dramatically when pH was over 5.0.The optimum temperature of both AmBHexN2301 and AmBHexN2446 were 37℃,and they were stable at their optimum temperature.Optimum temperature of SnBHexN384 was determined to be 60℃.SnBHexN384 was stable up to 50℃ after a 24h-incubation but couldnot keep stable at its optimum temperature.Among all the measured metal ions,Cu2+reduced the activity of AmBHexN2301 the most,and Fe3+reduced activity of AmBHexN2446 the most.However,recombinant SnBHexN384 showed good tolerance to all the measured metal ions.SDS could impire the activity of AmBHexN2301 and AmBHexN2446.N-ethylmaleimide could decrease the acitivy of AmBHexN2301 by amount while it didn’t inhibit the acitivy of AmBHexN2446 too much.SDS,urea and N-ethylmaleimide could inhibit activity of SnBHexN384 partially,the higher concentration of these compounds the stronger inhibition there was.Triton X-100,Iodoacetamide and 2ME didn’t affect activity of SnBHexN384 that much.IC50 of β-N-acetylhexosaminidase specific inhibitor,PUGNAc,were 0.5 μM to AmBHexN2301 and AmBHexN2446 both,and was 0.2 μM to SnBHexN384.Base on the aminio acids sequences of already known β-N-acetylhexosaminidases from differnent GH families,phylogenetic tree was made and it indicated that all these three recombinant β-N-acetylhexosaminidases could be classified into GH20 family.A further multiple alignmengt with already known GH20 β-N-acetylhexosaminidases showed that the conserved sequence moties existed in these three recombinant hexosaminidases.Moreover,two amino acids,Asp and Glu,which was estimated to be active sites in GH20 β-N-acetylhexosaminidases also conserved in three β-N-acetylhexosaminidases reported here.SnBHexN384 were selected for homology modelling,the resultant structure formed a(α/β)8 catalytic domain which was typically conserved in most of the GH20 hydrolases.Site-directed mutagensis was performed to the supposed catalytic sites of SnBHexN384 showing that any changes of these two amino acids lead to a great loss to the activity.3.Substrate specificity of recombinant β-N-acetylhexosaminidases,AmBHexN2301,AmBHexN2446 and SnBHexN384All of AmBHexN2301,AmBHexN2446 and SnBHexN384 could cleave β1,2/3/4/6 linked GlcNAc from the none-recucing end of simple unbranched glycooligosaccharides,but they showed different substrate selectivity to N-glycan structures.AmBHexN2301 and AmBHexN2446 could only hydrolyze the single attached GlcNAc in α 1,3 or 1,6 antennary of N-glycan core structure,M3.No GlcNAc residue could be released by AmBHexN2301 and AmBHexN2446 if there was a bisecting GlcNAc in the N-glycan structures.Recombinant SnBHexN384 was able to efficiently hydrolyze terminal GlcNAc from common N-glycan structures,such as A2,A3 and A4.When it comes to bisecting structures,concentrated recombinant SnBHexN384 could hydrolyze both of the two GlcNAc residues in hybrid N-glycan structure,M5AlB,whereas it could not cleave the besceting GlcNAc from complex N-glycan structures like FA2B and FA2G2B.Due to the existence of bisecting GlcNAc,SnBHexN384 could only catalyse the hydrolysis of one GlcNAc in one of the anntenaris in FA2B structure.5.Applications of recombinant SnBHexN384 in glycan structural analysis and GlcNAc preparationRecombiant SnBHexN384 was applied to natural N-glycomes prepared from chicken egg white and bovine milk taking jack bean sourced β-N-acetylhexosaminidase as a positive control.Hydrolysis activity was detected by UPLC.Though both of the hexosaminidases could not able to hydrolyze the bisecting GlcNAc,SnBHexN384 could generate more products in smaller structures as AlB than Jack bean hexosaminidase when applied to albumen N-glycans.Therefore,it was concluded that the huge peak in the chromatography corresponding to AlB could be a sign of large-amount of besceting structures in the sample when SnBHexN384 was used for N-glycan structure analysis.When it comes to bovine milk N-glycan structural analysis,recombinant SnBHexN384 was as good as jack bean hexosaminidae.It could be developed to be an alternative enzymatic tool for structural analysis.More AlB was produced by SnBHexN384 when comparing with jack bean hexosaminidase.Moreover,recombinant SnBHexN384 was used to prepare GlcNAc from insoluble chitin in small-scale experiments,miligrams of GlcNAc could be obtained finally. |
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