ABSTRACT
Sensitivity to phenylthiocarbamide (PTC) has a bimodal distribution pattern and the genotype of the TAS2R38 gene, which is composed of combinations of three coding single nucleotide polymorphisms (SNPs), p.A49P (c.145G>C), p.V262A (c.785T>C) and p.I296 V (c.886A>G), determines the ability or inability to taste PTC. In this study, we developed a tool for genotyping of these SNPs in the TAS2R38 gene using SNaPshot minisequencing and investigated the accuracy of the tool in 100 subjects who were genotyped by Sanger sequencing. The minor allele frequencies of the three SNPs were 0.39, and these genotypes corresponded to those determined by direct sequencing. In conclusion, we successfully developed a precise and rapid genetic tool for analysis of PTC genotype associated with bitter taste perception.
Subject(s)
Phenylthiourea/isolation & purification , Receptors, G-Protein-Coupled/genetics , Taste Perception/genetics , Taste/genetics , Genotype , Humans , Polymorphism, Single Nucleotide , Sequence Analysis, DNAABSTRACT
Tyrosinase is an enzyme of industrial interest. The production and characterization of tyrosinase from P. sanguineus CCT-4518 were investigated. The selection of inductors, luminosity influence, inoculum size and type of culture medium on the production of tyrosinase and the effect of inhibitors on enzyme activity were performed. Optimum conditions for intracellular tyrosinase production was observed after 2 days using 0.15% L-tyrosine as inducer, in the presence of light, with inoculum size of 10 mycelium discs, using 2% malt extract broth medium, incubated at 30°C, and constant agitation of 150 rpm. Tyrosinase activity was completely inhibited by the addition of 6 mM salicylhydroxamic acid or phenylthiourea, however an inhibition of 4.15% was recorded by the addition of 0.1 mM sodium azide. No inhibition could be detected in case of 0.1 mM phenyl methanesulfonyl fluoride addition. Optimal conditions for intracellular tyrosinase activity using L-dopa as substrate were observed at pH 6.6 and 45°C. Thermal stability studies indicated that the enzyme is stable at 45°C for 15 minutes. Higher temperatures decreased tyrosinase activity. Enzyme production was confirmed by non-denaturing polyacrylamide gel electrophoresis and the protein profile was investigated by denaturing polyacrylamide gel electrophoresis.
Subject(s)
Phenylthiourea/analysis , Phenylthiourea/isolation & purification , Enzyme Inhibitors/analysis , Monophenol Monooxygenase/analysis , Monophenol Monooxygenase/isolation & purification , Electrophoresis , Enzyme ActivationABSTRACT
Micellar capillary electrophoresis is optimized for separation of phenylthiohydantoin (PTH) amino acids produced in manual Edman degradation reaction for protein sequencing. There are also two major side-products produced by the Edman degradation reaction: diphenylthiourea and dimethylphenylthiourea. We report the complete separation of 19 PTH amino acids plus the two major side-reaction products in 10 min. Capillary electrophoresis is used to identify the five residues generated by manual Edman degradation sequencing of a pentapeptide.
Subject(s)
Electrophoresis/methods , Peptides/chemistry , Amino Acid Sequence , Amino Acids/isolation & purification , Amino Acids/standards , Hydrogen-Ion Concentration , Micelles , Molecular Sequence Data , Peptide Fragments/chemistry , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/isolation & purification , Phenylthiohydantoin/standards , Phenylthiourea/analogs & derivatives , Phenylthiourea/isolation & purification , Reference Standards , Thiourea/analogs & derivatives , Thiourea/isolation & purification , Thymopoietins/chemistryABSTRACT
An analytical procedure is described for the determination of 1-(2-aminoethyl)-3-(2,6-dichlorophenyl) thiourea in dietary admix. Because of the complex nature of the carrier matrix, sample cleanup was required prior to HPLC separation. Sample cleanup was achieved using a cation-exchange microcolumn which yield highly reproducible results. HPLC separation was accomplished using a 10 mu C18 column, monitoring the column effluent in the ultraviolet region at 254 nm. After sample cleanup, 1-(2-aminoethyl)-3-(2,6-dichlorophenyl) thiourea could be detected at levels as low as 3 ppm in dietary admix.
