An automated Cobas Fara method was developed determining the activity of recombinant M. thermophila laccase (rMtL). The chromogenic substrate used was syringaldazine. Under aerobic conditions, rMtL catalyses the oxidation of syringaldazine forming tetrametoxy-azo bis methylene quinone. The developed violet colour was measured kinetically at 530 nm as an expression of the enzyme activity, rMtL is a very sensitive oxidoreductase, therefore many factors had to be carefully controlled in order to get a robust analytical assay. In order to stabilize rMtL, PEG 6000 was added to the enzyme dilution medium. Furthermore, Triton X-I00 was included in the enzyme incubation solution. The analytical as well as technical conditions have been optimized, resulting in a method with good precision, sensitivity and speed of analysis. The Michaelis-Menten constant, Km, was determined to be 22μM syringaldazine. LOQ was determined to be 0.010 Uml-1, LOD to be 0.0002 Uml-1 The analytical range of the enzyme dilution curve was from 0.01 to 0.044 Uml-1 The repeatability was 1.9%, the reproducibility 3.1%. Testing the robustness of the method showed that the most sensible factors in the rMtL analysis in decreasing range were: incubation temperature, concentration of Triton X-I00, molarity and pH of the incubation buffer, and finally the concentration of syringaldazine.

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An automated flow injection method has been developed for the determination of microbial peroxidase activity. The substrate used was hydrogen peroxide and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) was used as the electron donor. In the presence of hydrogen peroxide, peroxidase catalyses the dehydrogenation of ABTS, resulting in the formation of a resonance-stabilized radical cation of ABTS. The green-blue colour formed, recorded at 418 nm, is taken as a measure of the peroxidase activity. The general technical conditions and the general enzymic kinetics have been optimized. Conditions for activation and stabilization of the enzyme were found, e.g., ammonium sulfate acts as a peroxidase activator. The resulting method has a good precision, sensitivity and speed.

An automated method for the determination of acid proteinase activity has been developed. The enzyme is incubated with a haemoglobin substrate after which the reaction solution is dialysed. The concentration of the liberated amino acids and peptides is determined by reaction with a trinitrobenzenesulphonic acid reagent. The technical parameters of the process and the general analytical conditions have been optimised. The method offers great advantages in its flexibility of selecting substrate and buffer independently of the conditions for the colour reaction. The final result is an automated method with high sensitivity, precision and speed. The sensitivity of the colour reaction is 1 mg 1-1 of nitrogen from leucine.

An automated, simplified, ultramicro method for the continuous determination of penicillins V and G, based on Pan's fundamental work, is described. Penicillin is hydrolyzed enzymatically by penicillinase into the corresponding penicilloic acid, which, in the presence of mercuric chloride, has a reducing effect on molybdoarsenic acid. The intensity of the molybdenum blue color which develops is measured. The simplification consists of an omission of the very time-consuming shaking procedure which Pan's method involved, and in the use of penicillinase conversion of penicillin into penicilloic acid. The reagent concentrations were modified in automating the procedure. The automation results in an analysis with a high degree of specificity, increased accuracy and precision (M = 8764, S% = 0.9), as well as a greater speed of analysis when compared with the manual method described in the text. The sensitivity is about 1 pg of penicillin V per ml.ONE OF THE FOLLOWING three methods is generally used for the determination of penicillin: the iodine method, described by Goodall and Davies (I), which has a low specificity and is per-