Release of galactosyl oligosaccharides by endo-β-N-acetylglucosaminidase D

Endo-β-N-acetylglucosaminidase D from Diplococcus pneumoniae released galactosyl oligosaccharides from IgG glycopeptides treated with β-N-acetylglucosaminidase. The structure of the major oligosaccharide was proposed to be as follows.

Since α-mannosidase digestion of the β-N-acetylglucosaminidase-treated glycopeptides made them again resistant to the endoglycosidase, we concluded that an unsubstituted α-mannosyl residue was required for the enzymic action.     

Purification and properties of Acinetobacter sp. endo-β-N-acetylglucosaminidase acting on complex oligosaccharides of glycoproteins

A novel endo-β-N-acetylglucosaminidase capable of acting on complex type sugar chains of glycoproteins was found in the culture broth of a bacterium which was isolated from soil and identified as Acinetobacter sp. The enzyme was purified to homogeneity on polyacrylamide gel electrophoresis by successive purification procedures involving ammonium sulfate fractionation and chromatographies on DEAE-cellulose, hydroxylapatite and Sephadex G-150. Its molecular weight was about 35,000 on gel filtration. The optimum pH was 3.0–3.5, and the enzyme was stable in the pH range from 6–8. The enzyme had high activity on dansyl ovalbumin glycopeptide, and also could hydrolyze dansyl asialotransferrin glycopeptide and dansyl transferrin glycopeptide containing complex type sugar chains. The K_m value for dansyl asialotransferrin glycopeptide as the substrate of enzyme assay was 0.68 mM. The enzyme could release complex type sugar chains from intact asialotransferrin without the addition of any detergent.     

The release of oligosaccharides from glycoproteins by endo-β-N-acetylglucosaminidase of Flavobacterium sp.

Endo-β-N-acetylglucosaminidase, purified to homogenicity from the cultural filtrate of Flavobacterium sp., liberated oligosaccharides from various glycoproteins. The enzyme could liberate the carbohydrate chain from native ovalbumin. The release of oligosaccharides from ribonuclease B, yeast carboxypeptidase and a Ricinus lectin was also observed. These glycoproteins contain neutral oligosaccharides that are attached to the protein through glycosyl asparagine bonds. The treatment of glycoprotein with SDS and boiling was more effective for removal of oligosaccharides by the enzyme. The enzyme hydrolyzed all five heterogeneous ovalbumin glycopeptides, although the rate of hydrolysis decreased as the size of the sugar moiety increased. Removal of the neutral oligosaccharides did not appear to effect the enzymatic properties of the hemagglutination ability of these glycoproteins.     

Subcellular localization of endo-β-N-acetylglucosaminidase and high-mannose type free N-glycans in plant cell

Subcellular distribution of plant endo-β-N-acetylglucosaminidase (endo-β-GlcNAc-ase) and high-mannose type free N-glycans produced by the endoglycosidase has been analyzed using cotyledons of pumpkin seedlings as the model plant cells. Each organelle in the cotyledons was fractionated by ultracentrifugation with the sucrose density gradient system and the endo-β-GlcNAc-ase activity in each fraction was assayed with fluorescence labeled N-glycans as substrates. The endoglycosidase activity was exclusively recovered in the soluble fraction (cytosol fraction) but not in other specific organellar fractions, suggesting that the endoglycosidase would reside predominantly in the cytosol. The quantitative analysis of high-mannose type free N-glycans occurring in each fraction showed that more than 70% of the free N-glycans was recovered from the soluble fraction, suggesting the endoglycosidase would work in the cytosol and the resulting free N-glycans would accumulate in the same fraction. The pumpkin endo-β-GlcNAc-ase (endo-CM) partially purified from the cotyledons showed optimum activity around pH 6.5, supporting this enzyme would reside in the cytosol. Furthermore, the detailed analysis of substrate specificity of endo-CM using various high-mannose type N-glycans showed that the pumpkin enzyme, as well as other plant endo-β-N-acetylglucosaminidases, were highly active toward the high-mannose type glycans bearing the Manα1-2Manα1-3Manβ1-structural unit.     

Fluorogenic probe for measuring high-mannose type glycan-specific endo-β-N-acetylglucosaminidase H activity

We synthesized a fluorogenic probe with a high-mannose type heptasaccharide structure to detect the hydrolytic activity of endo-β-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H). The heptasaccharide derivative (1) was labeled with an N-methylanthraniloyl group as a reporter dye at the branching point of the β-mannoside residue and 2,4-dinitrophenyl group as a quencher molecule at the reducing end, which was hydrolyzed by Endo-H, resulting in increased fluorescence intensity. Thus, Endo-H activities could be evaluated easily and quantitatively by measuring the fluorescence signal. Using both this probe (1) and a previously synthesized pentasaccharide probe, the hydrolysis activity of Endo-H and Endo-M were investigated. The results clearly showed a correlation with the substrate specificity of each enzyme.     

Activation of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae by various sugars

Endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae was activated by the addition of glucose, mannose, N-acetylglucosamine, and β-allose. While the enzyme did not appear to be significantly affected by the addition of galactose or N-acetylgalactosamine. These results indicate that the C-4 and C-6 positions of the monosaccharide are the most important for enzyme activation. Moreover, the enzyme was activated by the addition of disaccharides such as cellobiose, gentiobiose, and di-N-acetylchitobiose, but not by polysaccharides such as starch and yeast mannan. In the presence of N-acetylglucosamine, the enzyme activation occurred well over pH 4.0 and the K_m value of the enzyme for (Man)₆(GlcNAc)₂-Asn-dansyl changes from 1.2 mM to 3.2 mM.     

Transfer of Man9GlcNAc tol-fucose by endo-β-N-acetylglucosaminidase fromArthrobacter protophormiae

We have reported that transglycosylation activity of endo--N-acetylglucosaminidase fromArthrobacter protophormiae (endo-A) can be enhanced to near completion using GlcNAc as an acceptor in a medium containing 30% acetone (Fan J-Q, Takegawa K, Iwahara S, Kondo A, Kato I, Abeygunawardana C, Lee YC (1995)J Biol Chem 270: 17723–29). In this paper, we found that the endo-A can also transfer an oligosaccharide, Man₉GlcNAc, tol-Fuc using Man₉GlcNAc₂Asn as donor substrate in a medium containing 35% acetone. The transglycosylation yield was greater than 25% when 0.2m l-Fuc was used as acceptor. The transglycosylation product was purified by high performance liquid chromatography on a graphitized carbon column and the presence ofl-Fuc was confirmed by sugar composition analysis and electrospray mass spectrometry. Sequential exo-glycosidase digestion of pyridyl-2-aminated transglycosylation product, Man₉GlcNAc-l-Fuc-PA, revealed that a -anomeric configuration linkage was formed between GlcNAc andl-Fuc. The GlcNAc was found to be 1,2-linked tol-Fuc by two methods; i) collision-induced decomposition on electrospray mass spectrometry after periodate oxidation, reduction and permethylation of Man₉GlcNAc-l-Fuc; and ii) preparation of Man₉GlcNAc-l-Fuc-PA, its periodate oxidation and reduction, followed by hydrolysis and HPLC analysis. Thus, the structure of the oligosaccharide synthesized by endo-A transglycosylation was determined to be Man₉GlcNAc(1,2)-l-Fuc. Methyl -l-fucopyranoside,l-Gal are also acceptors for the enzymic transglycosylation. However, transglycosylation failed when methyl -l-fucopyranoside,d-Fuc andd-Gal were used. These results indicate that the endo-A requires not only 3-OH and 4-OH to be equatorial but also a⁴C₁-conformation or equivalent conformation of the acceptor to perform transglycosylation.Abbreviations endo-A endo--N-acetylglucosaminidase fromArthrobacter protophormiae - PA pyridyl-2-amino- - AP aminopyridine - GlcNAc N-acetyl-d-glucosamine - Man mannose - Gal galactose - Fuc fucose - Glc glucose - PA-C₂ PA-glycolaldehyde - PA-C₃ PA-l-glyceraldehyde - PA-C₄ PA-d-threose - HPAEC-PAD high performance anion exchange chromatography with pulsed amperometric detector - HPLC high performance liquid chromatography - ODS octadecylsilyl - ES-MS electrospray mass spectrometry - CID collision-induced decomposition     

Bacteriolytic enzymes from Staphylococcus aureus. Specificity of action of endo-β-N-acetylglucosaminidase

The bacteriolytic enzyme with an isoelectric point of 9.5 that is produced by all strains of Staphylococcus aureus investigated was purified from strain M18 (Wadström & Hisatsune, 1970). This enzyme released reducing groups from cell walls of Micrococcus lysodeikticus and was thus shown to be a bacteriolytic hexosaminidase. Although dinitrophenylation and acid hydrolysis of cell walls hydrolysed by a partially purified enzyme gave DNP-alanine and DNP-glycine from staphylococcal peptidoglycan, which indicated the presence of a peptidase and probably also an N-acetylmuramyl-l-alanine amidase, hydrolysis of cell walls by the extensively purified enzyme did not give any DNP-amino acids. The enzyme digest was purified by Amberlite CG-120 and Sephadex G-10 chromatography. Reduction by sodium borohydride of the disaccharide obtained was followed by acid hydrolysis and paper chromatography. Glucosamine completely disappeared after this treatment and a new spot identical with glucosaminitol appeared. The muramic acid spot remained unchanged. The purified enzyme was found to be devoid of exo-β-N-acetylglucosaminidase activity. These results are compatible with the action of a bacteriolytic endo-β-N-acetylglucosaminidase. It is also proposed that this enzyme is probably identical with the staphylococcal lysozyme. The mode of action of this has not previously been investigated.     

Bacteriolytic enzymes from Staphylococcus aureus. Properties of the endo-β-N-acetylglucosaminidase

An extracellular bacteriolytic endo-β-N-acetylglucosaminidase has been purified and its specificity of action has been investigated (Wadström & Hisatsune, 1970a,b). Some enzymic properties, such as optimum pH for enzyme activity on whole cells and cell walls of Micrococcus lysodeikticus and Staphylococcus aureus and optimum pH for stability, have been studied. The activity was maximum in 0.05m-tris–hydrochloric acid buffer, pH7.0. A higher ionic strength inhibited cell-wall hydrolysis. Since the crude and purified enzymes were found to be unstable on storage, the stabilizing and inhibiting effects of several compounds were investigated. Several heavy metal ions inactivated the enzyme at very low concentrations. Thiol compounds stabilized and thiol-reacting compounds partly inhibited the activity. Crude and purified glucosaminidase was found to be heat-stable at acidic pH and unstable at alkaline pH as previously found for several lysozymes (endo-β-N-acetylmuramidases). Other properties of the staphylococcal enzyme and hen''s-egg-white lysozyme have been compared, since the modes of action of the two are quite similar (Wadström & Hisatsune, 1970b).     

Bacteriolytic enzymes from Staphylococcus aureus. Purification of an endo-β-N-acetylglucosaminidase

On cultivation of Staphylococcus aureus in a complex liquid medium, bacteriolytic activity is found extracellularly. The maximal amount was found at the end of the exponential growth phase in batch culture, but in continuous culture run under similar conditions the yield was doubled. Isoelectric focusing of dialysed crude culture supernatants showed that the bacteriolytic activity of all four strains studied (M18, 524, Wood 46 and Duncan) was heterogeneous. The most alkaline peak of activity (isoelectric point 9.5±0.1) was assayed against Micrococcus lysodeikticus turbidimetrically. This bacteriolytic activity was purified more than 70-fold after continuous dialysis by adsorption on CM-Sephadex, precipitation with ethanol, heat purification, isoelectric focusing and Sephadex G-100 chromatography. The purified enzyme (isoelectric point 9.6±0.1) was found to give a single band on polyacrylamide-gel and cellulose acetate electrophoresis and was devoid of all 14 staphylococcal enzymes and toxins assayed for. The molecular weight is 70000±5000 as estimated by Sephadex G-100 and G-200 chromatography. The marked instability of the partially and highly purified enzyme was investigated. The mode of action and some properties of this enzyme are given in the following papers (Wadström & Hisatsune, 1970; Wadström, 1970). These results indicate that this extracellular enzyme which is produced by several strains of S. aureus is not a `lysozyme' (endo-β-N-acetylmuramidase) as previously suggested, but an endo-β-N-acetylglucosaminidase.     

Specificity towards oligomannoside and hybrid type glycans of the endo-β-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo--N-acetylglucosaminidase (EC 3.2.1.96) named Endo B, isolated from culture filtrates of the basidiomyceteSporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of theN-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43–49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)₁(GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₄(GlcNAc)₂ were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)₂(Man)₅(GlcNAc)₂ glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme.The oligomannoside-and theN-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule.Endo B represents a powerful tool for removing oligomannoside-andN-acetyllactosamine-type glycans fromN-glycopeptides andN-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.Abbreviations Gal d-galactose - Man d-mannose - GlcNAc N-acetyl-d-glucosamine - Con A concanavalin A - Asn asparagine - GLC gas liquid chromatography - TLC thin layer chromatography - Endo endo--N-acetylglucosaminidase - Endo B endo--N-acetylglucosaminidase isolated fromSporotrichum dimorphosporum - PBE polybuffer exchanger - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Kinetic characterization of a novel endo-β-N-acetylglucosaminidase on concentrated bovine colostrum whey to release bioactive glycans

EndoBI-1 is a recently isolated endo-β-N-acetylglucosaminidase, which cleaves the N-N′-diacetyl chitobiose moiety found in the N-glycan core of high mannose, hybrid and complex N-glycans. These N-glycans have selective prebiotic activity for a key infant gut microbe, Bifidobacterium longum subsp. infantis. The broad specificity of EndoBI-1 suggests the enzyme may be useful for many applications, particularly for deglycosylating milk glycoproteins in dairy processing. To facilitate its commercial use, we determined kinetic parameters for EndoBI-1 on the model substrates ribonuclease B and bovine lactoferrin, as well as on concentrated bovine colostrum whey. K_m values ranging from 0.25 to 0.49, 0.43 to 1.00 and 0.90 to 3.18 mg/mL and V_max values ranging from 3.5 × 10⁻³ to 5.09 × 10⁻³, 4.5 × 10⁻³ to 7.75 × 10⁻³ and 1.9 × 10⁻²to 5.2 × 10⁻² mg/mL × min were determined for ribonuclease B, lactoferrin and whey, respectively. In general, EndoBI-1 showed the highest apparent affinity for ribonuclease B, while the maximum reaction rate was the highest for concentrated whey. EndoBI-1-released N-glycans were quantified by a phenol-sulphuric total carbohydrate assay and the resultant N-glycan structures monitored by nano-LC-Chip-Q–TOF MS. The kinetic parameters and structural characterization of glycans released suggest EndoBI-1 can facilitate large-scale release of complex, bioactive glycans from a variety of glycoprotein substrates. Moreover, these results suggest that whey, often considered as a waste product, can be used effectively as a source of prebiotic N-glycans.     

The use of endo-β-N-acetylglucosaminidase H in characterizing the structure and function of glycoproteins

Endo-β-N-acetylglucosaminidase H from $\text{Streptomyces plicatus}$ can be useful in determining both the molecular weight of the protein moiety of glycoproteins and their inherent number of oligosaccharide chains. In the case of carboxypeptidase Y the molecular mass of the carbohydrate free protein was confirmed as 51,000 daltons. The native enzyme was shown to contain 4 oligosaccharide chains each averaging about 14 mannose residues. On treatment of mung bean nuclease I with the endoglycosidase, the molecular mass decreased from 39,000 to 31,000 daltons. The peptides produced on reduction of this enzyme with thiol were 18,700 and 12,500 daltons, indicating that carbohydrate had been present on both. Penicillium nuclease P₁ was decreased in size from 40,000 to 30,000 daltons by the endoglycosidase. Although most of the carbohydrate was removed from each of the native enzymes by the endoglycosidase, denaturation of the glycoproteins was necessary to effect complete removal. Enzyme activitywas not affected by carbohydrate depletion of these glycoproteins, a result consistent with similar studies on other oligosaccharide-containing enzymes.     

The asparagine-linked sugar chains of the glycoproteins in rat erythrocyte plasma membrane—Structural studies of the acidic oligosaccharides

Among the four acidic oligosaccharide fractions obtained by paper electrophoresis of the hydrazinolysate of the plasma membrane glycoproteins of rat erythrocytes, one was further separated into two by prolonged paper electrophoresis using 120-cm paper. Three fractions were mixtures of monosialyl oligosaccharides and two of disialyl oligosaccharides. After desialylation, their neutral portions were fractionated by Bio-Gel P-4 column chromatography and by affinity chromatography using a Con A-Sepharose column. Structural studies of the neutral oligosaccharides, thus obtained, indicated that at least 26 different complex-type oligosaccharides are present as a neutral portion of the acid oligosaccharides. Structurally they can be classified into bi-, tri-, and tetraantennary oligosaccharides with Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(±Fucα1 → 6)GlcNAc_OT as their common cores. Galβ1 → 3Galβ1 → 4GlcNAc, Siaα2 → 3Galβ1 → 4GlcNAc, Siaα2 → 6Galβ1 → 4GlcNAc, and a series of Siaα2 → (Galβ1 → 4GlcNAcβ1 → 3)_n · Galβ1 → 4GlcNAc were found as their outer chains. Their structures together with the structures of neutral oligosaccharides reported in the preceding paper indicated that the outer chain moieties of the asparagine-linked sugar chains of rat erythrocyte membrane glycoproteins are formed not by random concerted action of glycosyl transferases in Golgi membrane but by the mechanism in which the formation of one outer chain will regulate the elongation of others.     

The asparagine-linked sugar chains of the glycoproteins in rat erythrocyte plasma membrane—Fractionation of oligosaccharides liberated by hydrazinolysis and structural studies of the neutral oligosaccharides

The asparagine-linked sugar chains of the plasma membrane glycoproteins of rat erythrocytes were released as oligosaccharides by hydrazinolysis and labeled by NaB³H₄ reduction. The radioactive oligosaccharides were separated into a neutral and at least four acidic fractions by paper electrophoresis. The neutral oligosaccharide fraction was separated into at least 11 peaks upon Bio-Gel P-4 column chromatography. Structural studies of them by sequential exoglycosidase digestion in combination with methylation analysis revealed that they were a mixture of three high mannose-type oligosaccharides and at least 11 complex type oligosaccharides with Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(±Fucα1 → 6)GlcNAc as their cores and Galβ1 → 4GlcNAc, Galβ1 → 3Galβ1 → 4GlcNAc, and various lengths of Galβ1 → 4GlcNAc repeating chains in their outer chain moieties. Most of the complex-type Oligosaccharides were biantennary, and the tri- and tetraantennary Oligosaccharides contain only the Galβ1 → 3Galβ1 → 4GlcNAc group in their outer chain moieties.     

Remarkable transglycosylation activity of glycosynthase mutants of endo-D, an endo-β-N-acetylglucosaminidase from Streptococcus pneumoniae

Endo-β-N-acetylglucosaminidase from Streptococcus pneumoniae (Endo-D) is an endoglycosidase capable of hydrolyzing the Fc N-glycan of intact IgG antibodies after sequential removal of the sialic acid, galactose, and internal GlcNAc residues in the N-glycan. Endo-D also possesses transglycosylation activity with sugar oxazoline as the donor substrate, but the transglycosylation yield is low due to enzymatic hydrolysis of the donor substrate and the product. We report here our study on the hydrolytic and transglycosylation activity of recombinant Endo-D and its selected mutants. We found that Endo-D preferred core-fucosylated N-glycan for hydrolysis but favored nonfucosylated GlcNAc acceptor for transglycosylation. Several mutants showed significantly enhanced transglycosylation efficiency over the wild type enzyme. Two mutants (N322Q and N322A) were identified as typical glycosynthases that demonstrated remarkable transglycosylation activity with only marginal or no product hydrolysis activity. Kinetic studies revealed that the N322Q [corrected]and N322A glycosynthases had much higher catalytic efficiency for glycosylating the nonfucosylated GlcNAc acceptor. In comparison, the N322Q was much more efficient than N322A for transglycosylation. However, N322Q and N322A [corrected] could not take more complex N-glycan oxazoline as substrate for transglycosylation, indicating their strict substrate specificity. The usefulness of the N322Q glycosynthase was exemplified by its application for efficient glycosylation remodeling of IgG-Fc domain.     

Structural studies on IgG oligosaccharides of patients with primary Sjögren's syndrome

Sjögren's syndrome (SS) is an autoimmune disease, and some patients have been found to have SS complicated with rheumatoid arthritis (RA), in which IgG is known to carry abnormal N-linked oligosaccharides. In order to investigate the relationship between SS and RA, the structures of N-linked oligosaccharides of IgG from 12 primary SS patients without RA, 9 RA patients, and 8 healthy individuals were analyzed using reversed-phase high-performance liquid chromatography, in combination with sequential exoglycosidase treatment and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. All of the IgG samples obtained from primary SS patients, RA patients, and healthy individuals contained the same series of biantennary complex-type oligosaccharides, but the ratio of each oligosaccharide differed among these 3 groups. The incidence of galactose-lacking N-linked oligosaccharides obtained from the IgG of RA patients was significantly higher than that from healthy individuals, but that from the serum IgG of primary SS patients varied among individuals. The patients with primary SS were classified into two groups based on the galactosylation levels of IgG oligosaccharides; one group exhibits galactosylation levels as low as those of RA patients and another exhibits levels similar to those of healthy individuals. Measurement of levels of rheumatoid factor (RF) revealed that primary SS patients with a high incidence of RF belonged to the low galactosylation group, as did RA patients. These results suggest that appearance of IgG carrying abnormal N-linked oligosaccharides in primary SS may be related to future complication with RA.     

Evidence for vital role of endo-β-N-acetylglucosaminidase in the resistance of Arthrobacter protophormiae RKJ100 towards elevated concentrations of o-nitrobenzoate

Arthrobacter protophormiae RKJ100 was previously characterized for its ability to tolerate extremely high concentrations of o-nitrobenzoate (ONB), a toxic xenobiotic environmental pollutant. The physiological responses of strain RKJ100 to ≥30 mM ONB indicated towards a resistance mechanism manifested via alteration of cell morphology and cell wall structure. In this study, we aim to characterize gene(s) involved in the resistance of strain RKJ100 towards extreme concentrations (i.e. 150 mM) of ONB. Transposon mutagenesis was carried out to generate a mutant library of strain RKJ100, which was then screened for ONB-sensitive mutants. A sensitive mutant was defined and selected as one that could not tolerate ≥30 mM ONB. Molecular and biochemical characterization of this mutant showed that the disruption of endo-β-N-acetylglucosaminidase (ENGase) gene caused the sensitivity. ENGase is an important enzyme for oligosaccharide processing and cell wall recycling in bacteria, fungi, plants and animals. Previous reports have already indicated several possible roles of this enzyme in cellular homeostasis. Results presented here provide the first evidence for its involvement in bacterial resistance towards extreme concentrations of a toxic xenobiotic compound and also suggest that strain RKJ100 employs ENGase as an important component in osmotic shock response for resisting extreme concentrations of ONB.     

Double-knockout of putative endo-β-N-acetylglucosaminidase (ENGase) genes in Arabidopsis thaliana: loss of ENGase activity induced accumulation of high-mannose type free N-glycans bearing N,N'-acetylchitobiosyl unit

Endo-β-N-acetylglucosaminidase (ENGase) is involved in the production of high-mannose type free N-glycans during plant development and fruit maturation. In a previous study (K. Nakamura et al. Biosci. Biotechnol. Biochem., 73, 461-464 (2009)), we identified the tomato ENGase gene and found that gene expression remained relatively constant. In the present study, we constructed an Arabidopsis thaliana mutant in which the expression of two putative ENGase genes was suppressed. The mutant showed no ENGase activity, but produced high-mannose type free N-glycans carrying the N,N'-acetylchitobiosyl unit that is produced by peptide:N-glycanase, indicating that both these genes encode Arabidopsis ENGase.