A novel endo-type chitinase possessing chitobiase activity derived from the chitinolytic bacterium, Chitiniphilus shinanonensis SAY3T.

One of the chitinases (ChiG) derived from the chitinolytic bacterium Chitiniphilus shinanonensis SAY3T exhibited chitobiase activity cleaving dimers of N-acetyl-D-glucosamine (GlcNAc) into monomers, which is not detected in typical endo-type chitinases. Analysis of the reaction products for GlcNAc hexamers revealed that all the five internal glycosidic bonds were cleaved at the initial stage. The overall reaction catalyzed by chitobiases toward GlcNAc dimers was similar to that catalyzed by N-acetyl-D-glucosaminidases (NAGs). SAY3 possesses two NAGs (ChiI and ChiT) that are thought to be important in chitin catabolism. Unexpectedly, a triple gene-disrupted mutant (ΔchiIΔchiTΔchiG) was still able to grow on synthetic medium containing GlcNAc dimers or powdered chitin, similar to the wild-type SAY3, although it exhibited only 3% of total cellular NAG activity compared to the wild-type. This indicates the presence of unidentified enzyme(s) capable of supporting normal bacterial growth on the chitin medium by NAG activity compensation.

Structures and functions of carbohydrate-active enzymes of chitinolytic bacteria Paenibacillus sp. str. FPU-7.

Chitin and its derivatives have valuable potential applications in various fields that include medicine, agriculture, and food industries. Paenibacillus sp. str. FPU-7 is one of the most potent chitin-degrading bacteria identified. This review introduces the chitin degradation system of P. str. FPU-7. In addition to extracellular chitinases, P. str. FPU-7 uses a unique multimodular chitinase (ChiW) to hydrolyze chitin to oligosaccharides on the cell surface. Chitin oligosaccharides are converted to N-acetyl-d-glucosamine by ß-N-acetylhexosaminidase (PsNagA) in the cytosol. The functions and structures of ChiW and PsNagA are also summarized. The genome sequence of P. str. FPU-7 provides opportunities to acquire novel enzymes. Genome mining has identified a novel alginate lyase, PsAly. The functions and structure of PsAly are reviewed. These findings will inform further improvement of the sustainable conversion of polysaccharides to functional materials.

Polymorphic variants (p.Ser141Ser and p.Arg737Gly) at the NAGLU gene are really indicative of pseudodeficiency alleles?

Filocamo et al. recently published a paper describing the presence of a pseudodeficiency allele, constituted by p.Ser141Ser and p.Arg737Gly polymorphisms at the NAGLU gene, which leads to a reduced level of the alpha-N-acetyl-D-glucosaminidase activity. Based on analysis performed in Brazilian patients, using a customized gene panel containing SGSH, NAGLU, HGSNAT and GNS we observed that p.Ser141Ser (rs659497) and p.Arg737Gly (rs86312) variants were present in homozygosis in all of our MPS IIIB patients and in the majority of MPS IIIA, IIIC and IIID patients, and there was no significant decrease of the alpha-N-acetyl-D-glucosaminidase enzyme activity in this group when compared with those without the "pseudodeficiency allele". Thus, we suggest that these two variants are not producing a pseudodeficiency allele.

Leptospira seropositivity as a risk factor for Mesoamerican Nephropathy.

BACKGROUND: Leptospirosis is postulated as a possible cause of Mesoamerican Nephropathy (MeN) in Central American workers. OBJECTIVES: Investigate job-specific Leptospira seroprevalence and its association with kidney disease biomarkers. METHODS: In 282 sugarcane workers, 47 sugarcane applicants and 160 workers in other industries, we measured anti-leptospiral antibodies, serum creatinine, and urinary injury biomarkers, including neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18), and N-acetyl-D-glucosaminidase (NAG). RESULTS: Leptospira seroprevalence differed among job categories and was highest among sugarcane cutters (59%). Seropositive sugarcane workers had higher NGAL concentrations (relative mean: 1.28; 95% CI: 0.94-1.75) compared to those who were seronegative, with similar findings among field and non-field workers. CONCLUSIONS: Leptospira seroprevalence varied by job category. There was some indication that seropositivity was associated with elevated biomarker levels, but results were inconsistent. Additional studies may help establish whether Leptospira infection plays any role in MeN among Central American workers.

Long-term clinical course of a patient with mucopolysaccharidosis type IIIB.

Mucopolysaccharidosis type III (MPS III) is a rare genetic disorder caused by lysosomal storage of heparan sulfate. MPS IIIB results from a deficiency in the enzyme alpha-N-acetyl-D-glucosaminidase (NAGLU). Affected patients begin showing behavioral changes, progressive profound mental retardation, and severe disability from the age of 2 to 6 years. We report a patient with MPS IIIB with a long-term follow-up duration. He showed normal development until 3 years. Subsequently, he presented behavioral changes, sleep disturbance, and progressive motor dysfunction. He had been hospitalized owing to recurrent pneumonia and epilepsy with severe cognitive dysfunction. The patient had compound heterozygous c.1444C>T (p.R482W) and c.1675G>T (p.D559Y) variants of NAGLU. Considering that individuals with MPS IIIB have less prominent facial features and skeletal changes, evaluation of long-term clinical course is important for diagnosis. Although no effective therapies for MPS IIIB have been developed yet, early and accurate diagnosis can provide important information for family planning in families at risk of the disorder.

Production of N-Acetyl-d-glucosamine from Mycelial Waste by a Combination of Bacterial Chitinases and an Insect N-Acetyl-d-glucosaminidase.

N-Acetyl-d-glucosamine (GlcNAc) has great potential to be used as a food additive and medicine. The enzymatic degradation of chitin-containing biomass for producing GlcNAc is an eco-friendly approach but suffers from a high cost. The economical efficiency can be improved by both optimizing the member and ratio of the chitinolytic enzymes and using new inexpensive substrates. To address this, a novel combination of bacterial and insect chitinolytic enzymes was developed in this study to efficiently produce GlcNAc from the mycelia of Asperillus niger, a fermentation waste. This enzyme combination contained three bacterial chitinases (chitinase A from Serratia marcescens (SmChiA), SmChiB, SmChiC) and one insect N-acetyl-d-glucosaminidase from Ostrinia furnacalis (OfHex1) in a ratio of 39.1% of SmChiA, 26.7% of SmChiB, 32.9% of SmChiC, and 1.3% of OfHex1. A yield of 6.3 mM (1.4 mg/mL) GlcNAc with a purity of 95% can be obtained from 10 mg/mL mycelial powder in 24 h. The enzyme combination reported here exhibited 5.8-fold higher hydrolytic activity over the commercial chitinase preparation derived from Streptomyces griseus.

Long-term clinical course of a patient with mucopolysaccharidosis type IIIB / 소아과

Mucopolysaccharidosis type III (MPS III) is a rare genetic disorder caused by lysosomal storage of heparan sulfate. MPS IIIB results from a deficiency in the enzyme alpha-N-acetyl-D-glucosaminidase (NAGLU). Affected patients begin showing behavioral changes, progressive profound mental retardation, and severe disability from the age of 2 to 6 years. We report a patient with MPS IIIB with a long-term follow-up duration. He showed normal development until 3 years. Subsequently, he presented behavioral changes, sleep disturbance, and progressive motor dysfunction. He had been hospitalized owing to recurrent pneumonia and epilepsy with severe cognitive dysfunction. The patient had compound heterozygous c.1444C>T (p.R482W) and c.1675G>T (p.D559Y) variants of NAGLU. Considering that individuals with MPS IIIB have less prominent facial features and skeletal changes, evaluation of long-term clinical course is important for diagnosis. Although no effective therapies for MPS IIIB have been developed yet, early and accurate diagnosis can provide important information for family planning in families at risk of the disorder.

Symmetric dimethyl arginine and N-acetyl-ß-D-glucosaminidase lysozimuria of proximal renal tubules as a target for nephrotoxicity in patients with rheumatoid arthritis treated with disease modifying antirheumatic drugs.

BACKGROUND: The aim of this study was to determine the effect of initial therapy with some disease modifying antirheumatic drugs (DMARDs) (Methotrexate and Ketoprofen) on glomerular and tubular integrity in patients with Rheumatoid arthritis (RA). OBJECTIVES: To determine whether there is a change in clinical and laboratory indicators of renal function in course of the follow up of treatment and whether that change correlates with the dynamics of the quantity of enzymes excreted in urine and reactants of the acute phase. MATERIALS AND METHODS: Using colorimetric method for determination of NAG, samples of 70 participants were examined (35 RA patients treated with Ketoprofen only, 35 RA patients treated with combined use of Methotrexate and Ketoprofen). The follow up was 5 time-intervals in the course of 24 weeks. RESULTS: There was moderate correlation between NAG and microalbuminuria (r=0,34) in the group of patients treated with Ketoprofen only, while statistically significant correlation (r=0,21) was seen in group of patients with combined use of Methotrexate and Ketoprofen. NAG enzymuria in size, number of patients registered, and time of appearance were greater and appears earlier in the group with the combined use of Methotrexate and Ketoprofen compared with the mono-therapy with Ketoprofen. Mean urinary NAG induction was increasing with the concomitant use of Methotrexate and Ketoprofen. CONCLUSIONS: Methotrexate is more potent NAG inductor than Ketoprofen and provokes greater tubular enzymuria than Ketoprofen.

Clinical Value of Renal Dynamic Imaging and Urinary N-Acetyl-?-D-Glucosaminidase,Apoptosis DNA Fragment Detection in Evaluating Damage Degree of Hydronephrotic Kidneys in Children with Hydronephrosis / 实用儿科临床杂志

Objective To explore the clinical value of renal dynamic imaging and urinary N-acetyl-?-D-glucosaminidase(NAG),apoptosis DNA fragment(ADF) in evaluating the damage degree of hydronephrotic kidneys(HnK) in children with hydronephrosis.Methods Level of glomerular filtration rate(GFR) was detected in 41 children with congenital hydronephrosis by renal dynamic imaging,and urine NAG,ADF in pelvis in HnK and healthy kidneys (as controls) were detected by enzyme-linked immuno-sorbent assay(ELISA).Patholo-gic changes of HnK in 41 children were graded intoⅠ~Ⅴ according to Elder standard. And GFR,urinary NAG and ADF of HnK were divi-ded into subgroup according to pathologic changes ,at the same time statistical analysis was performed within each groups. And the correlations of pathologic grades with GFR,urinary NAG and ADF of HnK were analyzed.Results 1.Kindneys GFR in healthy kidneys and Hnk were (174.33?20.43)?10-3 L/min,(143.86?17.51)?10-3 L/min respectinely,and there was significant difference between healthy kidneys and Hnk (P0.05).3.There was significant negative correlation between GFR levels of HnK and pathologic grades(r=-0.814 P0.05).Conclusions For hydronephrotic kidneys,urinary NAG can eva-luate impaired nephric tubule whereas renal dynamic imaging may evaluate the damage level of glomeruli;urine ADF may not indicate the damage level of diseased kidneys in children with congenital hydronephrosis.

Inhibition Kinetics of Acetic Anhydride on Enzyme Activity of β-N-Acetyl-D-glucosaminidase from Pacific White Shrimp ( Penaeus vannamei ) / 中国生物化学与分子生物学报

β- N-Acetyl- D-glucosaminidase ( NAGase, EC3.2.1.52) is a composition of the chitinases and cooperates with endo-chitinase and exo-chitinase to disintegrate chitin into N-acetylglucosamine. Pacific White Shrimp (P. vannamei) NAGase is involved in digestion and molting processes. Some pollutants in seawater affect the enzyme activity causing loss of the biological function of the enzyme, which affects the exuviating shell and threatens the survival of the animal. The effects of acetic anhydride on the enzyme activity for the hydrolysis of pNP-NAG have been studied. The results show that acetic anhydride can lead to reversible non-competitive inhibition at appropriate concentrations, and the IC50 is estimated to be 9.0 mmol/L. The equilibrium constants have been determined for acetic anhydride binding with the enzyme and/or the enzymesubstrate complexes. Inhibition kinetics of acetic anhydride on the enzyme has been studied using the kinetic method of the substrate reaction. The results suggest that at pH 6.2, the action of acetic anhydride on the enzyme is first quick equilibrium binding and then slow inhibition. The microscopic rate constants have been determined for inhibition and reactivation. The results show that k + 0 is much larger than k - 0, indicating the enzyme is completely inactivated at sufficiently large modificator concentration.

Relationship between Malondialdehyde in Renal Cortex and Urinary NAG Activity of Rats exposed to Cadmium

BACKGROUND: To investigate the relationship between malondialdehyde in renal cortex and Urinary NAG activity of rats exposed to cadmium. METHODS: Rats were treated with a single intraperitoneal injection of cadmium (as CdCl2, 1 mg/kg) for cadmium-treated group and 24-hour urine were obtained prior to sacrifice on days 1, 2, 4, 8 and 16 (N=10 per each group), respectively. The concentration of malondialdehyde by thiobarbituric acid reaction and cadmium were measured in the homogenates of renal cortex. Nephrotoxocity indices such as N-acetyl--D-glucosaminidase (NAG) activity, total protein, and 24 hours urine volume, and cadmium concentration were measured in the urine. RESULT: The cadmium injection caused significant increase of cadmium concentration in the renal cortex on days 1 and 2, and in urine on days 1, 2 and 4. NAG activity and total protein concentration in urine were significantly increased on days 1, 2 and 4, and on days 1, 4 and 8, respectively. The peak values of NAG activity and total protein in urine were observed on days 1 and 4, respectively. Significant decrease of 24 hours urinary volume was induced on day 1. These results indicated that cadmium induced acute nephrotoxicity in the rats. Urinary NAG activity was changed earlier and at a higher rate than urinary total protein, which suggest that NAG activity is a more sensitive biological index in terms of early diagnosis of cadmium-induced nephrotoxicity. Renal MDA concentration was significantly increased on day 2 and on day 4, and on day 8, MDA concentration and nephrotoxicity indices except urinary total protein were returned to control level. CONCLUSION: Based on the results obtained as above, it was concluded that the malondialdehyde in renal cortex, product of lipid peroxidation was related with the changes of urinary NAG activity indicating nephrotoxic injury.

Degradation of Derivatives of N-Acetyl-D-glucosamine by Rhodococcus rhodochrous IFO 15564: Substrate Specificity and Its Application to the Synthesis of Allyl α-N-Acetyl-D-glucosaminide.

The substrate specificity was studied for the metabolic degradation of N-acetyl-D-glucosamine (GlcNAc) derivatives by Rhodococcus rhodochrous IFO 15564 which possesses N-acetyl-D-glucosamine deacetylase as a key-step enzyme. This microorganism degraded a wide range of substrates with modified N-acyl groups. The metabolizing activity of this strain became low to the substrates substituted at 1,3,4,6-positions of GlcNAc, and GlcNAc itself was suggested to be metabolized via an open-chain aldehyde form. Based on these results, a simplified procedure for the isolation of allyl α-N-acetyl-D-glucosaminide from an α, ß-anomeric mixture was developed by selectively hydrolyzing the ß-anomer with Jackbean ß-N-acetyl-D-glucosaminidase and subsequently degrading the resulting N-acetyl-D-glucosamine in the reaction mixture with this microorganism.

Combined Effects of Lead and Cadmium on NAG of Rats' Renal Tubular Epithelial Cells WANG / 环境与健康杂志

Objective To explore the combined effects of lead and cadmium on N-acetyl-?-D-glucosaminidase (NAG) of rats' renal tubular epithelial cells. Methods After extraction, purification, identification and culturing, renal tubular epithelial cells of rats were treated with lead acetate of 0, 0.02, 0. 1, 0.5 mmol/L, cadmium chloride of 0, 0.001, 0.004, 0.02 mmol/ L, and lead-cadmium of 2x2 pattern. NAG leaking out of cells was assessed. Results When cells were solely treated with lead acetate of 0.02 and 0. 1 mmol/ L, NAG activities in experimental groups showed no significant difference compared with that in the control group, but showed significant difference with a dosage of 0.5 mmol/L. When cells were solely treated with cadmium chloride of 0.001 and 0. 004mmol/L, NAG also showed no significant difference compared with that in the control group, while showing a significant difference with a dosage of 0.02 mmol/L. When cells were treated jointly with lead-cadmium of different dosages, NAG was significantly higher than that in the experimental groups treated only with lead or cadmium. Conclusion The combined effects of lead and cadmium led to more NAG leaking out of renal tubular epithelial cells of rats.