Production and characterization of tyrosinase activity in Pycnoporus sanguineus CCT-4518 crude extract

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.

Development and validation of spectrophotometric and spectrofluorimetric methods for simultaneous determination of Tofisopam.

Two simple, rapid and accurate spectrophotometric and spectrofluorimetric methods developed for the determination of Tofisopam (TF) in pure form and in pharmaceutical formulation. The spectrophotometric method (A) is based on the reduction of ferric into ferrous in presence of 1, 10-phenanthroline to give an orange –red colored ferroin complex measured at 510 nm. Method (B) spectrofluorimetric method is based on the oxidative coupling reaction of TF with 3-methylbenzothiazolin-2-one hydrazone (MBTH) hydrochloride in presence of cerium (IV) ammonium sulfate in an acidic medium. The quenching effect of TF on the fluorescence of excess cerous ions is measured at the emission λem 345 nm with excitation λex at 296 nm. The factors affecting the reactions were carefully studied and optimized. Beer,s law is obeyed in the range of 2-12 μg ml-1 for both methods with the mean percentages recovery of 100.04 ± 0.445 and 99.29 ± 0.563 for method (A) and (B), respectively. The two proposed methods were successfully applied for the determination of TF in Nodeprine tablets. Statistical comparison between the results obtained by these methods and that obtained by the official method for the determination of the drug was done, and it was found that there was no significant differences between them.