Molecular Cloning, Purification And Characterization Of OfHex1, The β-N-acetyl-D-hexosaminidase From The Insect Ostrinia Furnacalis

β-N-acetyl-D-hexosaminidases are widely distributed in bacteria,fungi,plants and animals,catalyzing the removal ofβ-linked N-acetyl-D-glucosamine or N-acetyl-D-galactosamine residues from various glycoconjugates.Insectβ-N-acetyl-D-hexosaminidases are encoded by multiple genes and involved in several physiological processes including chitin biodegradation,N-glycan modification, glycoconjugates degradation and egg-sperm interaction.Chitin is a linear polysaccharide composed ofβ-1,4 linked N-acetyl-D-glucosamine(GlcNAc).Because chitin is indispensable for insects as structural compotent but does not exist in higher plants and animals,theβ-N-acetyl-D-hexosaminidase that is only involved in chitin degradation will be the ideal species-specific target potential for developing green pesticides.This dissertation aimed at exploiting the specific chitinolyticβ-N-acetyl-D-hexosaminidase from the pest,Ostrinia furnacalis(Guenee).The comprehensive properties of this enzyme are of research interests as well.The work in this thesis includes:â… .Sequence analysis and transcriptional level of the gene encodingβ-N-acetyl-D-hexosaminidase from Ostrinia furnacalis.(1) Based on the conserved sequences ofβ-N-acetyl-D-hexosaminidases,specific primers were synthesized and the gene encodingβ-N-acetyl-D-hexosaminidase termed OfHEXl was obtained from total cDNA of the fifth instar larvae of Ostrinia furnacalis by RT-PCR and RACE.DNA sequencing analysis indicates the full-length nucleotide sequence of the OfHEXl is 2593 bp,which encodes 594 amino acids and has been deposited in Genbank by assession number DQ887769.1.(3) Multiple amino acid sequence alignment was performed between the deduced enzyme encoded by OfHEXl and four other well-knownβ-N-acetyl-D-hexosaminidases.The residues involved in catalysis including Glu³⁵⁵,Asp²⁴⁰,His²⁹⁴,Asp³⁵⁴,Tyr⁴⁵⁰ and Trp⁴²⁴ are highly conserved.The sequence identities between OfHexl andβ-N-acetyl-D-hexosaminidases from plants,birds and mammals are all about 30%.But the sequence identities between OfHexl and other insectβ-N-acetyl-D-hexosaminidases vary from 27%to 76%.Phylogenetic analysis showed that insectβ-N-acetyl-D-hexosaminidases can be divided into four branches each with higher sequence identity,similar deduced structure as well as physiological functions. OfHEXl belongs to the branchâ…£with the identities between 48%-76%,some enzymes among which are presumed to be involved in chitin degradation.(3) The transcriptional level of OfHEXl was revealed to be dramatically upregulated during the molting,pupation and eclosion processes by both RT-PCR and Real-time PCR, indicating its physiological role is related to chitin biodegradation during insect metamorphosis.â…¡.Purification of theβ-N-acetyl-D-bexosaminidase(OfHexl) from Ostrinia furnacalisBy a combination of SOURCE30Q anion exchange,CHT hydroxyapatite,MonoQ anion exchange and Superdex200 gel filtration chromatographies,0.47 mg of OfHexl was purified from 300 insects by 1468 folds with an activity yield of 19.67%.â…¢.Identification of biochemical properties of OfHexl.(1) Molecular mass and enzyme composition of OfHexl.The molecular mass of OfHexl was determined to be 68 kDa by SDS-PAGE and 67.0±0.3 kDa by MALDI-TOF MS, respectively.Under natural condition,the molecular mass of OfHexl was determined to be 128 kDa by analytical gel filtration chromatography.OfHexl is a homodimer and the force between two subunits is non-covalent binding.One disulfide bond(Cys³⁶-Cys⁵⁵) within subunit was identified by combining partial reducing methods and mass spectrometry. According to the difference between theoretical and practical molecular mases,OfHexl was estimated to have one or two paucimannosidic N-glycans.(2) pI of OfHexl was determined to be 4.7 by isoelectric focusing electrophoresis.(3) Segments of the amino acid sequence analysis of OfHexl by combining peptide mass fingerprinting and tandem mass spectrometry matched well with the deduced amino acid sequence of OfHEXl.â…£.Characterization of the enzymatic properties of OfHexl.(1) The optimum reaction conditions for OfHexl.The optimal pH and temperature of OfHexl are 7 and 30℃.(2) Substrate spectrum of OfHexl.The natural substrate(GlcNAc)₂ is the optimal substrate for OfHexl,the k_cat/K_m value is 3069 s^-1mM^-1.OfHexl has the highest turnover number toward(GlcNAc)₂ among the reportedβ-N-acetyl-D-hexosaminidases.OfHexl can not hydrolyze the N-glycan substrate GnGn-PA.As for substrate specificity,the ratio of enzyme activity with MU-β-GlcNAc to enzyme activity with MU-β-GalNAc is 1.56 and the enzyme activity with MU-β-GlcNAc to enzyme activity with MU-β-GlcNAc-6-SO₄^- is 156.7. The OfHexl can not hydrolyze MU-β-Glc,indicating its substrate-assisted mechanism. According to HPLC analysis of hydrolsis products,OfHexl was revealed to be a typical exo-glycosidase with preference of chitooligosaccharides with low degree of polymerization. OfHexl cleaves the glycosidic bond from the non-reducing end,following retaining mechanism by which theβ-configuration of the product is retained.(3) Inhibition of OfHexl.Compared to plant and humanβ-N-acetyl-D-hexosaminidases, OfHexl is more sensitive to higher concentration of substrates.It suggests substrate inhibition by(GlcNAc)₂ is most likely caused by the second substrate binding with the K_is value of 69μM.Both family 20 glycosyl hydrolase inhibitor 2-ADN and family 18 glycosyl hydrolase inhibitor allosamidin can effectively inhibit OfHexl and the K_i values are 3.5μM and 3.2μM, respectively.It selectively inhibits OfHexl but can not inhibitβ-N-acetyl-D-hexosaminidases from higher plants and animals..To summarize,the gene encodingβ-N-acetyl-D-hexosaminidases from Ostrinia furnacalis was obtained and its transcriptional level at different developmental stage was characterized.OfHexl in the native form was obtained with high purity and its biochemical characteristics were identified.According to its cDNA sequence,mRNA transcriptional level and substrate spectrum,we deduce OfHexl is mostly possible only involved in insect chitin degradation.At the same time,the composition of the enzyme,the special substrate spectrum, as well as the unique inhibitory spectrum of allosamidin also deepened the understanding ofβ-N-acetyl-D-hexosaminidases.