ABSTRACT
A D-hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity from Bacillus circulans. Purification of two hundred forty-three-fold was achieved with an overall yield of 12%. The relative molecular mass of the native enzyme is 212,000 and that of the subunit is 53,000. This enzyme is an acidic protein with an isoelectric point of 4.55. The enzyme is sensitive to thiol reagent and requires metal ions for its activity. The optimal conditions for the hydantoinase activity are pH 8.0-10.0 and a temperature of 75 degrees C. The enzyme is the most stable in a pH range of 8.5-9.5 and up to 60 degrees C. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturant SDS. These remarkable properties are used for the purification procedure.
Subject(s)
Amidohydrolases/isolation & purification , Bacillus/enzymology , Amidohydrolases/chemistry , Enzyme Inhibitors/chemistry , Enzyme Stability , Hydrogen-Ion Concentration , Metals/chemistry , Molecular Weight , Peptide Fragments/chemistry , Stereoisomerism , Substrate Specificity , TemperatureABSTRACT
The first and second derivatives of progress curves are obtained from the cubic spline function. The new approach is based on a development of the splining quality test which was used for estimating the precision of the splining. The proposed method is used on a computer with a FORTRAN 77 program. The method may also be applied for an approximate estimation of experimental error.
Subject(s)
Computer Simulation , Mathematical Computing , Models, Biological , Enzymes/metabolism , Kinetics , Programming Languages , Reaction Time , Reproducibility of ResultsABSTRACT
The present communication describes a novel method for estimating initial velocities (v) of enzyme-catalysed reactions. It is based on an approximation of experimental data obtained by the cubic spline function. The initial velocity of a reaction is calculated as a derivative of the approximating function at a time value equal to zero. The proposed method is usable on a computer with a FORTRAN IV program. The method can be successfully used in such cases as substantial extents of substrate conversion, the inactivation of an enzyme in the course of a reaction, the existence of large experimental error or when the reaction mechanism is unknown.