The following reagents 4-methylumbelliferyl acetate, 4-methyllumbelliferyl-α-D galactopyranoside, N-acetil-D-galactosamine, 4-methyllumbelliferyl-β-D-glucopyranoside, taurodeoxycholate hydrate, acarbose, 4-methyllumbelliferyl-α-D-glucopyranoside, 4-methylumbelliferylβ-D-N,N’,N’’triacetylchitotrioside, ethylenediamine dihydrochloride, formic acid, chondroitin sulfate sodium salt from shark cartilage, 1,9-dimethyl-methylene blue zinc chloride double salt, sodium hydroxide (NaOH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 4-methyllumbelliferyl sulfate potassium and 4-methyllumbelliferyl α-L-iduronide were purchased from Glycosynth (Warrington, UK). Sodium acetate, sodium citrate, sodium phosphate, and aminoacetic acid (glycine) were purchased from Synth (São Paulo, Brazil).
4 methylumbelliferyl acetate
Manufactured by Merck Group
Sourced in United States
4-methylumbelliferyl acetate is a fluorogenic substrate used in laboratory settings. It serves as a sensitive indicator for the detection and quantification of various enzymes, including phosphatases and esterases.
Automatically generated - may contain errors
Lab products found in correlation
α naphtyl acetate, by Merck Group (2 mentions)
β d glucose pentaacetate, by Merck Group (2 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Acarbose, by Merck Group (1 mentions)
Ethylenediamine dihydrochloride, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Chondroitin sulfate sodium salt, by Merck Group (1 mentions)
1 9 dimethyl methylene blue zinc chloride double salt, by Merck Group (1 mentions)
Acetate colorimetric assay kit, by Merck Group (1 mentions)
β d galactose pentaacetate, by Merck Group (1 mentions)
Chlorogenic acid, by Merck Group (1 mentions)
Pectin, by Merck Group (1 mentions)
Benzyl cinnamate, by Merck Group (1 mentions)
4 nitrophenyl octanoate, by Merck Group (1 mentions)
4 nitrophenyl butyrate, by Merck Group (1 mentions)
4 nitrophenyl palmitate, by Merck Group (1 mentions)
4 nitrophenyl acetate, by Merck Group (1 mentions)
4 nitrophenyl decanoate, by Merck Group (1 mentions)
Avicel, by Merck Group (1 mentions)
Ppiczαa, by Thermo Fisher Scientific (1 mentions)
5 protocols using 4 methylumbelliferyl acetate
1
Enzymatic Activity Assays for Glycosaminoglycans
2
Deacetylase Activity Quantification
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MZ0003 deacetylase activity was detected using the Acetate Colorimetric Assay Kit (Sigma-Aldrich, St Louis, MO, USA). The substrates used, purchased from Sigma-Aldrich, were: 4-methylumbelliferyl acetate and 7-aminocephalosporanic acid (dissolved in DMSO); α-naphtyl acetate, β-D-glucose pentaacetate and β-D-galactose pentaacetate (dissolved in methanol); 5-bromo-4-chloro-3-indoyl acetate (dissolved in ethanol); p-NP acetate and cellulose acetate (dissolved in acetonitrile). The reaction mixtures, each containing 10 μ g of protein and substrate, were pre-incubated in 5 mM Na-phosphate buffer pH 7.3 for 30 min at 35°C. Thereafter the assay was developed according to the manufacturer’s instructions and the absorbance was read at 450 nm. Values for each substrate were taken as the mean of three independent measurements after subtraction of a blank sample, without MZ0003, to correct for background degradation. The amount of acetate liberated was calculated in nM μ l-1 from a standard curve according to the manufacturer’s instructions.
3
Gut Metagenome Enzyme Screening
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The slug gut metagenome DNA was provided by Dr Natalie Ferry, University of Salford, Manchester, United Kingdom. The substrates—4-nitrophenyl acetate, 4-nitrophenyl butyrate, 4-nitrophenyl octanoate, 4-nitrophenyl decanoate, and 4-nitrophenyl palmitate, β-D-glucose pentaacetate, pectin, chlorogenic acid, benzyl cinnamate, and 4-methylumbelliferyl acetate—were purchased from Sigma-Aldrich (Gillingham, United Kingdom). Xylan (birchwood, partially acetylated) was purchased from Megazyme (Wicklow, Ireland). Other chemicals were of molecular grade and purchased from Sigma-Aldrich (Gillingham, United Kingdom). Primers for PCR were synthesised and sequenced by Eurofins (Konstanz, Germany). BL21 and TOP10 competent cells were purchased from ThermoFisher Scientific (Leicester, United Kingdom).
4
Constructing Recombinant Acetyl Xylan Esterases
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The plasmid pPICZαA-axe1, which has been previously constructed to express mature AXE1 [39 (link)], was used as a template for recombinant acetyl xylan esterases construction. The gene sequences encoding xylan-specific CBM4-2, CBM6, and CBM22-2 modules were originally from Rhodothermus marinus Xyn10A, Clostridium thermocellum Xyn11A, and Clostridium thermocellum Xyn10B, respectively (Additional file 1 ) [40 (link)–42 (link)]. The corresponding gene fragments were synthesized by Springen Biotech Co. (Jiangsu, China) and carried into plasmids pUC57 with modified codon according to the codon preference of Pichia pastoris. Escherichia coli DH5α was used for plasmid construction and propagation. P. pastoris KM71H and the plasmid pPICZαA from Invitrogen (Carlsbad, CA, USA) were used for heterologous expression.
The substrates 4-methylumbelliferyl acetate and Avicel were purchased from Sigma-Aldrich (St. Louis, MO). Wheat bran was obtained from Yongfang (Shandong, China) and wheat straw was obtained from Lian Yungang (Jiangsu, China). Water-insoluble wheat arabinoxylan (inAX) was purchased from Megazyme (P-WAXYI, Wicklow Ireland). TransStart FastPfu DNA polymerase and T4 DNA ligase were purchased from TransGen Biotech (Beijing, China). The restriction enzymes and endoglycosidase H were purchased from New England Biolabs (Beverly, MA, USA). All other chemicals used were analytical grade.
The substrates 4-methylumbelliferyl acetate and Avicel were purchased from Sigma-Aldrich (St. Louis, MO). Wheat bran was obtained from Yongfang (Shandong, China) and wheat straw was obtained from Lian Yungang (Jiangsu, China). Water-insoluble wheat arabinoxylan (inAX) was purchased from Megazyme (P-WAXYI, Wicklow Ireland). TransStart FastPfu DNA polymerase and T4 DNA ligase were purchased from TransGen Biotech (Beijing, China). The restriction enzymes and endoglycosidase H were purchased from New England Biolabs (Beverly, MA, USA). All other chemicals used were analytical grade.
5
Purification and Analysis of Proteins
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Isopropyl thio-β-D-galactoside (IPTG), p-Nitrophenyl esters, phenyl acetate, α-naphtyl acetate, β-naphtyl acetate, and 4-methyl umbelliferyl acetate were procured from SIGMA ALDRICH (USA). Ni-NTA resin for affinity chromatography was purchased from Qiagen, Germany. Standard proteins for size exclusion chromatography were purchased from Bio-Rad laboratories (USA). PCR primers were procured from Bio Serve, India. Other reagents and chemicals were procured from Himedia.
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