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A multiplex PCR for the simultaneous detection of Staphylococcus aureus 23S rRNA, the coagulase and thermonuclease genes as well as the enterotoxin genes sea, sec, sed, seg, seh, sei, sej, sel was developed. The method was used to determine the presence of enterotoxigenic types for 93 S. aureus strains isolated from milk and dairy products. The data obtained by mPCR resulted comparable to those obtained by immunoassay methods. In addition, the mPCR assays also amplified some se genes, whose toxins are undetectable by immunoassay. Multiplex amplification can be obtained starting from 1 pg of DNA, showing the excellent specificity and high sensitivity of the assay.

NAD glycohydrolase (NADase, EC 3.2.2.5) from Neurospora crassa conidia shows marked hydrophobic properties which are related to the self inhibition of the enzyme. Both aliphatic amines and carboxylic acids are able to inhibit noncompetitively the catalytic activity of the enzyme and the inhibition depends on the non-polar moiety of the substances. Also dioxane is an inhibitor of NAD glycohydrolase even though it apparently increases the specific activity of the enzyme. This effect can be explained by the fact that NADase is present as a dimer when the enzyme is concentrated or at high temperature, and dioxane binds the enzyme breaking the hydrophobic bonds in the dimeric enzyme and yielding the most active monomeric form which is only slightly inhibited by the organic solvent.

NAD glycohydrolase from Neurospora crassa conidia was purified by affinity chromatography on a column of polyclonal antibodies bound to an agarose matrix. The procedure was easy, non-denaturating and suitable for repetitive use of the gel. The enzyme obtained appeared homogeneous by sodiumdodecyl sulphate-polyacrylamide gel electrophoresis.

Enzyme activity can be easily measured by HPLC using traces of the product itself as an internal standard. Our procedure involved the development of an equation using the experimental data obtained in the kinetic assay. The entire procedure can thus be automated and a computer program is presented here for facilitating the assay and saving time. The determination of the activity of NAD kinase is reported as an example.

Enzymes can be assayed by HPLC by calculating the amount of substrate(s) left over, or product formed, through the peak area ratios with a suitable internal standard. However, sometimes the substrates used are contaminated with small amounts of products and this can lead to errors in the determination of the enzyme activity. A method for a HPLC test of such enzymes, which prevents eventual errors, uses the ratio substrate/product at time zero as internal standard and the kinetics can be followed with the aid of a simple mathematical equation. This approach was applied to the determination of the activities of papain, urokinase, NAD glycohydrolase, and pyruvate kinase samples and it was compared with the data obtained by the internal standard method, giving reproducible results in all cases.

NAD glycohydrolase from Neurospora crassa conidia has been immobilized by hydrophobic interaction on Sepharose 4B beads coated with propyl residues through CNBr activation. The bond resulted stable under a wide range of conditions (ionic strength, temperature, pH). As a result of immobilization the pH optimum for catalytic activity shifted by about 0.2 pH unit in the acidic direction, to lie between 7.5 and 7.3. The stability of the enzymatic activity was largely enhanced by effect of immobilization but the Km value towards NAD+ was increased compared with that of the free enzyme (1 X 10(-3) and 2 X 10(-4) M respectively).