Functional Characterization and Structural Modelling of Peptidoglycan Degrading ß-N-acetyl-glucosaminidase from a Dental Isolate of Serratia marcescens.

INTRODUCTION: Enzymatic degradation of peptidoglycan, a structural cell wall component of Gram-positive bacteria, has attracted considerable attention being a specific target for many known antibiotics. METHODS: Peptidoglycan hydrolases are involved in bacterial lysis through peptidoglycan degradation. ß-N-acetyl-glucosaminidase, a peptidoglycan hydrolase, acts on O-glycosidic bonds formed by N-acetylglucosamine and N-acetyl muramic acid residues of peptidoglycan. Aim of present study was to study the action of ß-N-acetylglucosaminidase, on methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-negative bacteria. RESULTS: We investigated its dynamic behaviour using molecular dynamics simulation and observed that serine and alanine residues are involved in catalytic reaction in addition to aspartic acid, histidine, lysine and arginine residues. When simulated in its bound state, the RMSD values were found lesser than crystal form in the time stamp of 1000 picoseconds revealing its stability. Structure remained stably folded over 1000 picoseconds without undergoing any major change further confirming the stability of complex. CONCLUSION: It can be concluded that enzymes belonging to this category can serve as a tool in eradicating Gram-positive pathogens and associated infections.

ß-N-Acetylglucosaminidase MthNAG from Myceliophthora thermophila C1, a thermostable enzyme for production of N-acetylglucosamine from chitin.

Thermostable enzymes are a promising alternative for chemical catalysts currently used for the production of N-acetylglucosamine (GlcNAc) from chitin. In this study, a novel thermostable ß-N-acetylglucosaminidase MthNAG was cloned and purified from the thermophilic fungus Myceliophthora thermophila C1. MthNAG is a protein with a molecular weight of 71 kDa as determined with MALDI-TOF-MS. MthNAG has the highest activity at 50 °C and pH 4.5. The enzyme shows high thermostability above the optimum temperature: at 55 °C (144 h, 75% activity), 60 °C (48 h, 85% activity; half-life 82 h), and 70 °C (24 h, 33% activity; half-life 18 h). MthNAG releases GlcNAc from chitin oligosaccharides (GlcNAc)2-5, p-nitrophenol derivatives of chitin oligosaccharides (GlcNAc)1-3-pNP, and the polymeric substrates swollen chitin and soluble chitosan. The highest activity was detected towards (GlcNAc)2. MthNAG released GlcNAc from the non-reducing end of the substrate. We found that MthNAG and Chitinase Chi1 from M. thermophila C1 synergistically degraded swollen chitin and released GlcNAc in concentration of approximately 130 times higher than when only MthNAG was used. Therefore, chitinase Chi1 and MthNAG have great potential in the industrial production of GlcNAc.

Significant reduction in allergenicity of ovalbumin from chicken egg white following treatment with ascidian viscera N-acetylglucosaminidase.

Ovalbumin (OA) is the most abundant ingredient of chicken egg-white allergenic proteins. In the present study we investigated the possibility of reducing OA allergenicity by treatment with a natural protein exhibiting N-acetylglucosaminidase (NA) activity. Ascidian is cultivated as a food resource in northeast Asia. The ascidian viscera NA (AVNA) with almost no other exoglycosidases or proteolytic enzymes was isolated by applying size-exclusion chromatography to a protein precipitate of ascidian viscera. Intact OA was mixed with AVNA containing 0.2, 1.0, and 5.0 Units of NA. Anion-exchange chromatography was then used to isolate OA from AVNA-treated OA. The electrophoretic patterns and N-glycans of each isolated OA from AVNA-treated OA (iOA) were analyzed, and the terminal N-acetylglucosamines of iOA were selectively cleaved with no other degradation occurring. A competitive indirect enzyme-linked immunosorbent assay using rabbit anti-OA sera was performed to investigate the allergenicity of iOA, which was found to be significantly reduced depending on the increased NA activity compared to that of intact OA. These results indicate that OA allergenicity was reduced using a simple and mild treatment process with AVNA, and suggest that ascidian NA is an efficient natural protein for reducing the allergenicity of OA without requiring the use of harsh physical treatments or chemical conjugation.

Azide anions inhibit GH-18 endochitinase and GH-20 Exo ß-N-acetylglucosaminidase from the marine bacterium Vibrio harveyi.

Vibrio harveyi is a bioluminescent marine bacterium that utilizes chitin as its sole source of energy. In the course of chitin degradation, the bacterium primarily secretes an endochitinase A (VhChiA) to hydrolyze chitin, generating chitooligosaccharide fragments that are readily transported into the cell and broken down to GlcNAc monomers by an exo ß-N-acetylglucosaminidase (VhGlcNAcase). Here we report that sodium salts, especially sodium azide, inhibit two classes of these chitin-degrading enzymes (VhChiA and VhGlcNAcase) with distinct modes of action. Kinetic analysis of the enzymatic hydrolysis of pNP-glycoside substrates reveals that sodium azide inhibition of VhChiA has a mixed-type mode, but that it inhibits VhGlcNAcase competitively. We propose that azide anions inhibit chitinase activity by acting as strong nucleophiles that attack Cγ of the catalytic Glu or Cß of the neighbouring Asp residues. Azide anions may bind not only to the catalytic centre, but also to the other subsites in the substrate-binding cleft of VhChiA. In contrast, azide anions may merely occupy the small-binding pocket of VhGlcNAcase, thereby blocking the accessibility of its active site by short-chain substrates.

Expression, purification, crystallization and preliminary crystallographic analysis of a GH20 ß-N-acetylglucosaminidase from the marine bacterium Vibrio harveyi.

Vibrio harveyi ß-N-acetylglucosaminidase (VhGlcNAcase) is a new member of the GH20 glycoside hydrolase family responsible for the complete degradation of chitin fragments, with N-acetylglucosamine (GlcNAc) monomers as the final products. In this study, the crystallization and preliminary crystallographic data of wild-type VhGlcNAcase and its catalytically inactive mutant D437A in the absence and the presence of substrate are reported. Crystals of wild-type VhGlcNAcase were grown in 0.1 M sodium acetate pH 4.6, 1.4 M sodium malonate, while crystals of the D437A mutant were obtained in 0.1 M bis-tris pH 7.5, 0.1 M sodium acetate, 20% PEG 3350. X-ray data from the wild-type and the mutant crystals were collected at a synchrotron-radiation light source and were complete to a resolution of 2.5 Å. All crystals were composed of the same type of dimer, with the substrate N,N'-diacetylglucosamine (GlcNAc2 or diNAG) used for soaking was cleaved by the active enzyme, leaving only a single GlcNAc molecule bound to the protein.

Antibacterial metabolites and bacteriolytic enzymes produced by Bacillus pumilus during bacteriolysis of Arthrobacter citreus.

The marine isolate Bacillus pumilus SBUG 1800 is able to lyse living cells of Arthrobacter citreus on solid media as well as pasteurized A. citreus cells in liquid mineral salt medium. The cultivation of B. pumilus in the presence of pasteurized A. citreus is accompanied by an enhanced production of 2,5-diketopiperazines (DKPs). DKPs inhibit bacterial growth, but do not seem to cause bacteriolysis. This study shows that B. pumilus also lyses living cells of A. citreus in co-culture experiments as an intraguild predator, even if the inoculum of B. pumilus is low. In order to characterize the bacteriolytic process, more precisely changes in the extracellular metabolome and proteome have been analyzed under different culture conditions. Besides the known DKPs, a number of different pumilacidins and bacteriolytic enzymes are produced. Two lipopeptides with [M + H](+) = 1008 and [M + H](+) = 1022 were detected and are proposed to be pumilacidin H and I. While the lipopeptides lyse living bacterial cells in lysis test assays, a set of extracellular enzymes degrades the dead cell material. Two of the cell wall hydrolases involved have been identified as N-acetylmuramoyl-L-alanine amidase and beta-N-acetylglucosaminidase. These findings together with electron microscopic and cell growth monitoring during co-culture experiments give a detailed view on the bacteriolytic process.

Improvement of glycosylation structure by suppression of ß-N-acetylglucosaminidases in silkworm.

The baculovirus-silkworm recombinant protein expression system is an excellent method for achieving high-level expression and post-translational modifications, especially glycosylation. However, the presence of paucimannosidic-type N-glycan in glycoproteins restricts their clinical use. Paucimannosidic-type N-glycan is produced by insect-specific membrane-binding-type ß-N-acetylglucosaminidase (GlcNAcase). In the silkworm, BmGlcNAcase1, BmGlcNAcase2, and BmFDL are membrane-binding-type GlcNAcases. We investigated the localization of these GlcNAcases and found that BmFDL and BmGlcNAcase2 were mainly located in the fat body and hemolymph, respectively. The fat body is the main tissue of recombinant protein expression by baculovirus, and many glycoproteins are secreted into the hemolymph. These results suggest that inhibition of BmFDL and BmGlcNAcase2 could increase GlcNAc-type N-glycan levels. We therefore injected a GlcNAcase inhibitor into silkworms to investigate changes in the N-glycan structure of the glycoprotein expressed by baculovirus; modest levels of GlcNAc-type N-glycan were observed (0.8% of total N-glycan). Next, we generated a transgenic silkworm in which RNA interference (RNAi) reduced the BmFDL transcript level and enzyme activity to 25% and 50%, respectively, of that of the control silkworm. The proportion of GlcNAc-type N-glycan increased to 4.3% in the RNAi-transgenic silkworm. We conclude that the structure of N-glycan can be changed by inhibiting the GlcNAcases in silkworm.

A chitinolytic endochitinase and ß-N-acetylglucosaminidase-based system from Hevea latex in generating N-acetylglucosamine from chitin.

An endochitinase and ß-N-acetylglucosaminidase (NAGase) were purified and characterised from fresh rubber latex serum. These enzymes were used in a total enzyme-based system to produce pure N-acetylglucosamine (NAG) from chitin. The N-terminal amino acid sequences of both purified endochitinase (KEESRRRRHR) and NAGase (AAVDSDTLEI) lacked homology with other known chitinases, including hevamine from rubber latex lutoids. The apparent kinetic parameters, Km and Vmax, for the endochitinase using 4-MU-ß-(NAG)3 as a substrate were 99.73 µM and 29.49 pkat mg(-1), respectively. For NAGase, using 4-MU-ß-NAG as a substrate, the corresponding Km and Vmax values were 20.4 µM and 25.82 pkat mg(-1). When an enzyme incubation mixture containing a 1:1 (pkat/pkat) activity mixed ratio of endochitinase: NAGase was employed, the maximum yield of N-acetylglucosamine (NAG) obtained was 98% from ß-chitin and 20% from α-chitin. These yields were obtained after 4 days of hydrolysis of equal amounts of ß-chitin and α-chitin in the mixture. Thus, ß-chitin was the preferred substrate compared to α-chitin by a ratio of nearly five to one. Mass spectroscopic analysis, using electrospray ionisation mass spectrometry (ESI-MS), of the product obtained from ß-chitin after digestion (for 24h) depicted one distinct major molecular ion peak m/z 260.1, a small minor ion peak m/z 481.2, a potassium adduct of NAG and a potassium adduct of two NAG molecules. Furthermore, experiments to establish the commercial production of NAG using crude enzymes of Hevea latex serum are currently in progress.

Expression, characterization and homology modeling of a novel eukaryotic GH84 ß-N-acetylglucosaminidase from Penicillium chrysogenum.

ß-N-acetylglucosaminidases from the family 84 of glycoside hydrolases form a small group of glycosidases in eukaryotes responsible for the modification of nuclear and cytosolic proteins with O-GlcNAc, thus they are involved in a number of important cell processes. Here, the first fungal ß-N-acetylglucosaminidase from Penicillium chrysogenum was expressed in Pichia pastoris and secreted into the media, purified and characterized. Moreover, homology modeling and substrate and inhibitor docking were performed to obtain structural information on this new member of the GH84 family. Surprisingly, we found that this fungal ß-N-acetylglucosaminidase with its sequence and structure perfectly fitting to the GH84 family displays biochemical properties rather resembling the ß-N-acetylhexosaminidases from the family 20 of glycoside hydrolases. This work helped to increase the knowledge on the scarcely studied glycosidase family and revealed a new type of eukaryotic ß-N-acetylglucosaminidase.

Evidence for lytic transglycosylase and ß-N-acetylglucosaminidase activities located at the polyhydroxyalkanoates (PHAs) granules of Thermus thermophilus HB8.

The thermophilic bacterium Thermus thermophilus HB8 accumulates polyhydroxyalkanoates (PHAs) as intracellular granules used by cells as carbon and energy storage compounds. PHAs granules were isolated from cells grown in sodium gluconate (1.5 % w/v) as carbon source. Lytic activities are strongly associated and act to the PHAs granules proved with various methods. Specialized lytic trasglycosylases (LTGs) are muramidases capable of locally degrading the peptidoglycan (PG) meshwork of Gram negative bacteria. These enzymes cleave the ß-1,4-glycosidic linkages between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues of PG. Lysozyme-like activity/-ies were detected using lysoplate assay. Chitinolytic activity/-ies, were detected as N-acetyl glucosaminidases (NAG) (E.C.3.2.1.5.52) hydrolyzing the synthetic substrate p-nitrophenyl-N-acetyl-ß-D-glucosaminide (pNP-GlcNAc) releasing pNP and GlcNAc. Using zymogram analysis two abundant LTGs were revealed hydrolyzing cell wall of Micrococcus lysodeikticus or purified PG incorporated as natural substrates, in SDS-PAGE and then renaturation. These proteins corresponded in a SDS-PAGE and Coomassie-stained gel in molecular mass of 110 and 32 kDa respectively, were analyzed by MALDI-MS (Matrix-assisted laser desorption/ionization-Mass Spectrometry). The 110 kDa protein was identified as an S-layer domain-containing protein [gi|336233805], while the 32 kDa similar to the hypothetical protein VDG1235_2196 (gi/254443957). Overall, the localization of PG hydrolases in PHAs granules appears to be involved to their biogenesis from membranes, and probably promoting septal PG splitting and daughter cell separation.

Enzymatic characterizations and activity regulations of N-acetyl-ß-D-glucosaminidase from the spermary of Nile tilapia (Oreochromis niloticus).

N-Acetyl-ß-D-glucosaminidase (NAGase) is proved to be correlated with reproduction of male animals. In this study, enzymatic characterizations of NAGase from spermary of Nile tilapia (Oreochromis niloticus) were investigated in order to further study its reproductive function in fish. Tilapia NAGase was purified to be PAGE homogeneous by the following techniques: (NH4)2SO4 fractionation (40-55%), DEAE-cellulose (DE-32) ion exchange chromatography, Sephadex G-200 gel filtration and DEAE-Sephadex (A-50). The specific activity of the purified enzyme was 4100 U/mg. The enzyme molecular weight was estimated as 118.0 kD. Kinetic studies showed that the hydrolysis of p-nitrophenyl-N-acetyl-ß-D-glucosaminide (pNP-NAG) by the enzyme followed Michaelis-Menten kinetics. The Michaelis-Menten constant (Km) and maximum velocity (Vm) were determined to be 0.67 mM and 23.26 µM/min, respectively. The optimum pH and optimum temperature of the enzyme for hydrolysis of pNP-NAG was to be at pH 5.7 and 55°C, respectively. The enzyme was stable in a pH range from 3.3 to 8.1 at 37°C, and inactive at temperature above 45°C. The enzyme activity was regulated by the following ions in decreasing order: Hg(2+) > Zn(2+) > Cu(2+) > Pb(2+) > Mn(2+). The IC50 of Cu(2+), Zn(2+) and Hg(2+) was 1.23, 0.28, and 0.0027 mM, respectively. However, the ions Li(+), Na(+), K(+), Mg(2+) and Ca(2+) had almost no influence on enzyme activity. In conclusion, the enzymatic characterizations of NAGase from tilapia were special to the other animals, which were correlated with its living habit; besides, CuSO4 and ZnSO4 should used very carefully as insecticides in tilapia cultivation since they both had strong regulations on the enzyme.

ß-hexosaminidase from Xenopus laevis eggs and oocytes: from gene to immunochemical characterization.

Glycosidases are present both in sperm and eggs in vertebrates and have been associated with different fertilization steps as gamete binding, egg coat penetration, and polyspermy prevention. In this manuscript, we have analyzed the activity of different glycosidases of Xenopus laevis eggs. The main activity corresponded to N-acetyl-ß-D-glucosaminidase (Hex), which was reported to participate both in gamete binding and polyspermy prevention among phylogenetically distant animals. We have raised homologous antibodies against a recombinant N-terminal fragment of a X. laevis Hex, and characterized egg's Hex both by Western blot and immunohistochemical assays. Noteworthy, Hex was mainly localized to the cortex of animal hemisphere of full-grown oocytes and oviposited eggs, and remained unaltered after fertilization. Hex is constituted by different pair arrangements of two subunits (α and ß), giving rise to three possible Hex isoforms: A (αß), B (ßß), and S (αα). However, no information was available regarding molecular identity of Hex in amphibians. We present for the first time the primary sequences of two isoforms of X. laevis Hex. Interestingly, our results suggest that α- and ß-like subunits that constitute Hex isoforms could be synthesized from a same gene in Xenopus, by alternative exon use. This finding denotes an evolutionary divergence with mammals, where α and ß Hex subunits are synthesized from different genes on different chromosomes.

The Listeria monocytogenes serotype 4b autolysin IspC has N-acetylglucosaminidase activity.

IspC is a novel peptidoglycan (PG) hydrolase that is conserved in Listeria monocytogenes serotype 4b strains and is involved in virulence. The aim of this study was to establish the hydrolytic bond specificity of IspC. Purified L. monocytogenes peptidoglycan was digested by recombinant IspC and the resulting muropeptides were separated by reverse phase high-performance liquid chromatography. The structure of each muropeptide was determined using matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry in combination with MALDI-post-source decay mass spectrometry. The structure of muropeptides resulting from IspC-mediated hydrolysis indicated that IspC has N-acetylglucosaminidase activity. These muropeptides also had a high proportion of N-acetylated glucosamine residues. To determine whether IspC is more effective at hydrolysing N-acetylated peptidoglycan than de-N-acetylated peptidoglycan, a peptidoglycan deacetylase (PgdA) in-frame deletion mutant was created. This mutant was shown to have fully N-acetylated peptidoglycan and was more susceptible to hydrolysis by IspC when compared with the partially de-N-acetylated wild-type peptidoglycan. This indicates that IspC is more efficient when hydrolysing a fully N-acetylated peptidoglycan substrate. The finding that IspC acts as an N-acetylglucosaminidase is consistent with its categorization, based on amino acid sequence, as a member of the GH73 family. As with other members of this family, de-N-acetylation seems to be an important mechanism for regulating the activity of IspC.

Purification and characterization of an exo-type ß-N-acetylglucosaminidase from Pseudomonas fluorescens JK-0412.

AIMS: To purify and characterize an exo-acting chitinolytic enzyme produced from a Gram-negative bacterium Pseudomonas fluorescens JK-0412. METHODS AND RESULTS: A chitinolytic bacterial strain that showed confluent growth on a minimal medium containing powder chitin as the sole carbon source was isolated and identified based on a 16S ribosomal DNA sequence analysis and named Ps. fluorescens JK-0412. From the culture filtrates of this strain, a chito-oligosaccharides-degrading enzyme was purified to apparent homogeneity with a molecular mass of 50 kDa on SDS-PAGE gels. The kinetics, optimum pH and temperature, and substrate specificity of the purified enzyme (named as NagA) were determined. CONCLUSIONS: An extracellular chitinolytic enzyme was purified from the Ps. fluorescens JK-0412 and shown to be an exo-type ß-N-acetylglucosaminidase yielding GlcNAc as the final product from the natural chito-oligosaccharides, (GlcNAc)(n) , n = 2-5. SIGNIFICANCE AND IMPACT OF THE STUDY: As NagA is secreted extracellularly in the presence of colloidal chitin, Ps. fluorescens JK-0412 can be recognized as a potent producer for industry-level and cost-effective production of chitinolytic enzyme. This enzyme appears to have potential applications as an efficient tool for the degradation of chitinous materials and industry-level production of GlcNAc. To the best of our knowledge, this is the first report on an exo-type chitinolytic enzyme of Pseudomonas species.

Extracellular complex of chitinolytic enzymes of Clostridium paraputrificum strain J4 separated by membrane ultrafiltration.

Membrane diafiltration was used for separation of the extracellular complex of chitinolytic enzymes of C. paraputrificum J4 free from contaminants with molar mass higher than 100 kDa and lower than 30 kDa. The enzyme complex containing beta-N-acetylglucosaminidase (NAGase) and six endochitinases was concentrated on a membrane with cut-off 30 kDa. In this retentate, the NAGase/endochitinase specific activity was 13.5/6.5-times higher than in the initial culture filtrate. The proportion (in%) of endochitinases: 23 (90 kDa), 42 (86 kDa), 8 (72 kDa), 16 (68 kDa) and 8 (60 kDa) was calculated from their peak areas (determined by densitometry) in images of zymograms. NAGase (38 kDa) was less active and stable at pH lower than 4 and higher than 8 but it was more temperature-stable than endochitinases, especially at 40-60 degrees C. In contrast to endochitinases, the pH optimum of NAGase activity was shifted by ca. 0.7 pH units to the alkaline region. Extracellular NAGase together with six endochitinases secreted by C. paraputrificum J4 were separated by membrane diafiltration and characterized by molar mass, stability and activity in dependence on pH and temperature. The knowledge of composition of chitinolytic enzymes, their pH and temperature stability is useful for optimization of the separation process.

[Distribution of O-glycosylases in marine fungi of the Sea of Japan and the Sea of Okhotsk: characterization of exocellular N-acetyl-beta-D-glucosaminidase of the marine fungus Penicillium canescens].

The capacity to produce exocellular enmzymes was studied for 92 samples of fungi from various marine habitats in the Sea of Okhotsk (78 strains) and the Sea of Japan (14 strains). Strains producing highly active glycanases and glycosidases were found. Synthesis of O-glycosylhydrolases was stimulated by addition of laminaran to the nutrient medium. Highly purified N-acetyl-beta-D-glucosaminidase was isolated from the marine fungus Penicillium canescens. The molecular weight of the enzyme determined by SDS-Na-electrophoresis was 68 kDa. The enzyme displayed maximum activity at pH 4.5 and temperature 45 degrees C. Inactivation half-time of the enzyme at 50 degrees C was 25 min. N-acetyl-beta-D-glucosaminidase hydrolyzed both beta-glucosaminide and beta-galactosaminide bonds and possessed a high transglycosylazing activity.

Paracoccin from Paracoccidioides brasiliensis; purification through affinity with chitin and identification of N-acetyl-beta-D-glucosaminidase activity.

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most frequent systemic mycosis in Latin America. Our group has been working with paracoccin, a P. brasiliensis lectin with MM 70 kDa, which is purified by affinity with immobilized N-acetylglucosamine (GlcNAc). Paracoccin has been described to play a role in fungal adhesion to extracellular matrix components and to induce high and persistent levels of TNFalpha and nitric oxide production by macrophages. In the cell wall, paracoccin colocalizes with the beta-1,4-homopolymer of GlcNAc into the budding sites of the P. brasiliensis yeast cell. In this paper we present a protocol for the chitin-affinity purification of paracoccin. This procedure provided higher yields than those achieved by means of the technique based on the affinity of this lectin with GlcNAc and had an impact on downstream assays. SDS-PAGE and Western blot analysis revealed similarities between the N-acetylglucosamine- and chitin-bound fractions, confirmed by MALDI-TOF-MS of trypsinic peptides. Western blot of two-dimensional gel electrophoresis of the yeast extract showed a major spot with M(r) 70,000 and pI approximately 5.63. Moreover, an N-acetyl-beta-D-glucosaminidase activity was reported for paracoccin, thereby providing new insights into the mechanisms that lead to cell wall remodelling and opening new perspectives for its structural characterization.

Identification of genes encoding N-glycan processing beta-N-acetylglucosaminidases in Trichoplusia ni and Bombyx mori: Implications for glycoengineering of baculovirus expression systems.

Glycoproteins produced by non-engineered insects or insect cell lines characteristically bear truncated, paucimannose N-glycans in place of the complex N-glycans produced by mammalian cells. A key reason for this difference is the presence of a highly specific N-glycan processing beta-N-acetylglucosaminidase in insect, but not in mammalian systems. Thus, reducing or abolishing this enzyme could enhance the ability of glycoengineered insects or insect cell lines to produce complex N-glycans. Of the three insect species routinely used for recombinant glycoprotein production, the processing beta-N-acetylglucosaminidase gene has been isolated only from Spodoptera frugiperda. Thus, the purpose of this study was to isolate and characterize the genes encoding this important processing enzyme from the other two species, Bombyx mori and Trichoplusia ni. Bioinformatic analyses of putative processing beta-N-acetylglucosaminidase genes isolated from these two species indicated that each encoded a product that was, indeed, more similar to processing beta-N-acetylglucosaminidases than degradative or chitinolytic beta-N-acetylglucosaminidases. In addition, over-expression of each of these genes induced an enzyme activity with the substrate specificity characteristic of processing, but not degradative or chitinolytic enzymes. Together, these results demonstrated that the processing beta-N-acetylglucosaminidase genes had been successfully isolated from Trichoplusia ni and Bombyx mori. The identification of these genes has the potential to facilitate further glycoengineering of baculovirus-insect cell expression systems for the production of glycosylated proteins.

Inhibitory kinetics of beta-N-acetyl-D-glucosaminidase from prawn (Litopenaeus vannamei) by zinc ion.

Prawn (Litopenaeus vannamei) beta-N-acetyl-D-glucosaminidase (NAGase, EC 3.2.1.52) is involved in the digestion and molting processes. Zinc is one of the most important metals often found in the pollutant. In this article, the effects of Zn(2+) on prawn NAGase activity for the hydrolysis of pNP-NAG have been investigated. The results showed that Zn(2+) could reversibly and noncompetitively inhibit the enzyme activity at appropriate concentrations and its IC(50) value was estimated to be 6.00 +/- 0.25 mM. The inhibition model was set up, and the inhibition kinetics of the enzyme by Zn(2+) has been studied using the kinetic method of the substrate reaction. The inhibition constant was determined to be 11.96 mM and the microscopic rate constants were also determined for inactivation and reactivation. The rate constant of the inactivation (k(+0)) is much larger than that of the reactivation (k(-0)). Therefore, when the Zn(2+) concentration is sufficiently large, the enzyme is completely inactivated. On increasing the concentration of Zn(2+), the fluorescence emission peak and the UV absorbance peak are not position shifted, but the intensity decreased, indicating that the conformation of Zn(2+)-bound inactive NAGase is stable and different from that of native NAGase. We presumed that Zn(2+) made changes in the activity and conformation of prawn NAGase by binding with the histidine or cysteine residues of the enzyme.

The Enterococcus hirae Mur-2 enzyme displays N-acetylglucosaminidase activity.

Enterococcus hirae produces two autolytic enzymes named Mur-1 and Mur-2, both previously described as N-acetylmuramidases. We used tandem mass spectrometry to show that Mur-2 in fact displays N-acetylglucosaminidase activity. This result reveals that Mur-2 and its counterparts studied to date, which are members of glycosyl hydrolase family 73 from the CAZy (Carbohydrate-Active enZyme) database, display the same catalytic activity.