Table 6 Conditions for enzymatic assays, including buffer solutions, substrate concentrations, detection methods, and products. Corresponding products, chromogenic or fluorescent, were respectively measured in spectramax 190 (absorbance) or GeminiXPS (fluorescence - corresponding excitation (Exc)/emission (Ems) wavelengths).
Enzymatic Asssays Conditions | |||||
|---|---|---|---|---|---|
Enzyme | Buffer | Substrate | Detection | Standard Curve Product | Reference |
Chitinase | Citrate-phosphate 0.1 M—pH 5.25 | MU-N-triacetyl-beta-D-N-N-N-triacetylchitotriose (1.7 µM) | Exc360nm/Ems460nm | 4-Methylumbelliferone | Baker and Woo 50 |
Hexosaminidase | Citrate-phosphate 0.1 M—pH 5.25 | MU-N-acetyl-beta-D-glucosamine (10 µM) | Exc360nm/Ems460nm | 4-Methylumbelliferone | Baker and Woo 50 |
α-Mannosidase | Citrate-phosphate 0.1 M—pH 4.0 | MU α-D-mannopyranoside (2 µM) | Exc360nm/Ems460nm | 4-Methylumbelliferone | Baker and Woo 50 |
α-L-Fucosidase | Citrate-phosphate 0.1 M—pH 5.25 | MU α-L-fucopyranoside (10 µM) | Exc360nm/Ems460nm | 4-Methylumbelliferone | Baker and Woo 50 |
α-Amylase | Citrate-phosphate 0.1 M—pH 5.5 | Potato starch (62 mM) | Abs550nm | Glucose | |
Cysteine Peptidase (Cathepsin L) | Citrate-phosphate 0.1 M—pH 5.5 | Z-Phe-Arg—MCA (10 µM) | Exc330nm/Ems430nm | 7-Methylcoumarin | Alves et al.51 |
Metallopeptidase (Astacin) | Tris–HCl 0.5 M—pH 8.5 | Casein-FiTC (0.2%) | Exc365nm/Ems525nm | * | Twining 53 |
Carboxypeptidase | Tris–HCl 0.25 M—pH 8.0 | Z-Gly-Phe (20 mM) | Abs-420 nm | Phenylalanine | Nicholson and Kim 54 |
Aminopeptidase | Tris—HCl 0.1 M—pH 7.0 | L-Leucine-p-nitroanilide (1 mM) | Abs410nm | p-Nitroaniline | Erlanger et al. 55 |
Lipase | Tris–HCl 0.1 M—pH 8.5 | Dimercapto-1-Propanol Tributyrate (0.22 mM) | Abs405nm | Cysteine | Choi et al. 56 |
Serine Peptidase (Trypsin) | Tris—HCl 0.1 M—pH 7.0 | Z-Phe-Arg—MCA (10 µM) | Exc330nm/Ems430nm | 7-Methylcoumarin | Alves et al. 51 |
