Biofilms of K. pneumoniae prepared as described in Section 2.2.1 using a volume of 200 μL were grown over night (~20 h) on 96-well flat-bottomed (Nunc) cell culture plates (polystyrene). Wells were vigorously washed three times with sterile distilled water to remove non-adherent bacteria, filled with 100 μg/mL DNaseI (5-Prime/Termofisher, Hilden, Germany) in PBS, 100 μg/mL proteinase K (Applichem, Darmstadt, Germany) or 1.2 units β-N-acetylglucosaminidase (Sigma, St. Louis, MO, USA) in acetate buffer pH 5.0 and further incubated at 37 °C for 1 h (DNaseI and proteinase K) or 2 h (β-N-acetylglucosaminidase) as previously described [12 (link),13 (link)]. Biofilms incubated with PBS or acetate buffer pH 5.0 were used as controls. After incubation wells were washed once with sterile distilled water and stained for 15 min with 100 μL 1.4% crystal violet at room temperature, washed with distilled water three times to remove excess dye and allowed to dry at room temperature. The crystal violet was dissolved in 100 μL of 95% ethanol (Merck, Darmstadt, Germany) and the optical density at 570 nm was read using a SpectraMax 340PC (Molecular Devices, Sunnyvale, CA, USA).
β n acetylglucosaminidase
Manufactured by Merck Group
Sourced in Japan, Germany, United States, Italy
β-N-acetylglucosaminidase is an enzyme that catalyzes the hydrolysis of the N-acetyl-beta-D-glucosaminide linkage in N-acetyl-beta-D-glucosaminides. It is a key component in the degradation of glycosaminoglycans and glycoproteins.
Automatically generated - may contain errors
Lab products found in correlation
Microtof mass spectrometer, by Bruker (1 mentions)
P 2200 polarimeter, by Jasco (1 mentions)
α l fucosidase, by Merck Group (1 mentions)
Dnasei, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by AppliChem (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Spectramax 340pc, by Molecular Devices (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
96 well tc treated microplate, by Corning (1 mentions)
6 protocols using β n acetylglucosaminidase
1
Degradation of K. pneumoniae Biofilms
2
Purification and Characterization of Pochonicine
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Pochonicine (1 ) was purified using the method previously reported.1 (link)) β-N-acetylglucosaminidase (GlcNAcase) from Spodoptera litura was prepared as described in our previous papers.6 ,7 ) β-N-acetylglucosaminidases from the jack bean (Canavalia ensiformis) and human placenta were purchased from Sigma-Aldrich (Sigma-Aldrich, Tokyo, Japan). These enzymes were used for enzyme inhibition assay without further purification. Cation-exchange column chromatography was performed on Amberlite CG-50 (H+ form), which was purchased from the Organo Co. (Organo Co., Tokyo, Japan). HPLC separation was conducted on an Asahipak ES502C (7.5×100 mm, Showa Denko KK, Tokyo, Japan) with a detection wavelength of 210 nm. The NMR spectra were obtained using a Varian Inova AS600 spectrometer (Varian, Palo Alto, CA, USA) in CD3OD; the spectra were referenced according to the solvent peaks (δH 3.35 or δC 49.0). High-resolution ESI mass spectra were recorded on a micrOTOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Optical rotation was measured on a P-2200 polarimeter (JASCO, Tokyo, Japan).
3
Glycan Structure Elucidation via Exoglycosidase Digestion
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Selected glycan pools were treated with specific exoglycosidases to support glycan structure assignments. Each enzyme reaction mixture contained a 1 μl aliquot of labeled glycans, 6 μl 100 mm sodium phosphate buffer pH 5 and 5 μl of either one of the following enzymes: β-N-acetylglucosaminidase from jack bean (NAG; 312.5 mU)(Sigma-Aldrich), α-l -fucosidase from bovine kidney (BKF; 33.3 mU)(Sigma-Aldrich), α(1–3,4)-fucosidase from Xanthomonas manihotis (XMF; 2.5 mU)(Sigma-Aldrich). For treatment with β(1–4,6)-galactosidase from jack bean (JBG) (227.3 mU) (Prozyme, Hayward, CA) 250 mm sodium citrate pH 4.0 was used. All exoglycosidase digestions were performed at 37 °C for 24 h.
4
Measuring Lysosomal Enzyme Activities
5
Phosphorylation Sites Mapping of Native OBP Isoforms
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Phosphorylation sites of native OBP isoforms were mapped by mild Beta-Elimination, followed by Michael Addition of DTT (BEMAD) and then by MALDI-TOF MS analysis (20 (link)). Prior to mild β-elimination and Michael addition of DTT, isoforms were treated with β-N-acetylglucosaminidase (Sigma-Aldrich, 1 unit per 5 μg of protein) to remove GlcNAc groups. Proteins were not reduced and alkylated to avoid addition of DTT on cysteines. They were then digested with either trypsin (T) or chymotrypsin (CT) or both (T + CT) (Sigma-Aldrich). After a step of enrichment of DTT-modified peptides by using thiol columns [fully described in Nagnan-Le Meillour et al. (7 (link))], peptides were eluted directly into the matrix (α-cyano-4-hydroxycinnamic acid) with 12.5, 25, and 50% acetonitrile (ACN). Eluates were not pooled in order to obtain higher percentages of peptide recovery in mass spectrometry. The theoretical masses of DTT-modified peptides were calculated from the OBP peptide map (GenBank accession number NP_998961 ) by using the Peptide Mass software at https://web.expasy.org/peptide_mass/ .
6
Biodegradation of Dual-Functioning Scaffolds
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The biodegradation of dual-functioning scaffolds was investigated after sample soaking in three different solvents: (i) PBS (phosphate buffer solution, without Ca2+ and Mg2+, Euroclone, Milan, Italy); (ii) H2O2 1.25 mM in PBS; and (iii) β-N-Acetylglucosaminidase (from Canavalia ensiformis (Jack bean), NAGase, Sigma-Aldrich, USA) 5 U/L in PBS.
In details, weighted circular portions of each scaffold (0.32 cm2) were sterilized through UV irradiation for 24 h and, then, placed in a 96-well plate (Corning® 96 Well TC-Treated MicroPlates, New York, NY, USA). The scaffolds were soaked in 200 μL of solvent (PBS, H2O2 or NAGase) and maintained in incubator (Shellab® Sheldon® Manufacturing Inc., Cornelius, OR, USA) (95% air and 5% CO2 atmosphere) at 37 °C for 7 and 14 days; in order to alter nor the pH neither the enzymatic unit, the solvent was changed every 24 h. After both 7 and 14 days, sample degradation was evaluated in terms of morphological alterations and weight loss. Each condition was investigated in triplicate.
In details, weighted circular portions of each scaffold (0.32 cm2) were sterilized through UV irradiation for 24 h and, then, placed in a 96-well plate (Corning® 96 Well TC-Treated MicroPlates, New York, NY, USA). The scaffolds were soaked in 200 μL of solvent (PBS, H2O2 or NAGase) and maintained in incubator (Shellab® Sheldon® Manufacturing Inc., Cornelius, OR, USA) (95% air and 5% CO2 atmosphere) at 37 °C for 7 and 14 days; in order to alter nor the pH neither the enzymatic unit, the solvent was changed every 24 h. After both 7 and 14 days, sample degradation was evaluated in terms of morphological alterations and weight loss. Each condition was investigated in triplicate.
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