β-Lactamase activity was detected in cell-free preparations obtained from ultrasonic treatment of Neisseria gonorrhoeae after growth in liquid medium. Crude preparations of β-lactamase were subjected to affinity chromatography, using several β-lactam antibiotics as ligands bound to agarose supports. Affinity gels produced by coupling 7-aminocephalosporanic acid or 6-aminopenicillanic acid by their amino groups to carboxyl-terminal agarose via a five- to eight-carbon spacer arm proved to be effective chromatography media. β-Lactamase preparations subjected to chromatography using these gels were purified 200-fold, with approximately 80% recovery of active material. Purified preparations were judged homogeneous by their behavior during electrophoresis on polyacrylamide in both the presence and absence of sodium dodecyl sulfate. Characterization of the purified enzyme established a molecular weight of approximately 25,000 and an isoelectric point of 5.4. Analyses of substrate specificity, effect of inhibitors, pH, and kinetic parameters were performed. The evidence suggests that the β-lactamase produced in N. gonorrhoeae closely resembles the character of class IIIa (TEM-type) β-lactamases.

Both beta-N-acetylglucosaminidase nad beta-N-acetylgalactosaminidase activities were detected in the culture fluids of Paecilomyces persicinus P-10 after growth in a soybean meal-corn meal medium. The active material was purified by means of protamine sulfate fractionation and ultrafiltration, followed by ion exchange and gel chromatography. The ratio of the two activities remained constant throughout the purification, and the final product was shown to migrate as a single band by using gel isoelectric focusing, disc electrophoresis, and detergent gel electrophoresis. Temperature, pH, inhibition, and kinetic studies were performed to characterize both activities. The molecular weight of the enzyme was estimated to be about 100,000 by high-resolution gel chromatography. Based on the data obtained, it is suggested that both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities reside in the same protein.

Small intracellular peptides containing a-aminoadipic acid, cysteine, and a valine moiety were obtained from mycelia of Paecilomyces persicinus P-10 by ethanol or trichloroacetic acid extraction. After performic acid oxidation and ionexchange chromatography, analysis of the peptide fractions by two-dimensional thin-layer electrophoresis and chromatography revealed the presence of three related peptides, as sulfonic acid derivatives, each containing a-aminoadipic acid. Each peptide was isolated in chromatographically pure form by semipreparative thin-layer electrophoresis and chromatography. The purified peptides were subjected to differential hydrolysis, dansylation, and combined dansylation-phenylisothiocyanate sequence analysis. Based on these studies, the structures of the isolated peptides were determined to be (i) glycl-8-(a-aminoadipyl)-cysteinyl-fihydroxyvaline, (ii) glycyl-$-(a-aminoadipyl)-cysteinylvaline, and (iii) 8-(a-aminoadipyl)-cysteinylvaline. The peptides isolated from Paecilomyces are similar to the a-aminoadipic acid-cysteine-valine moiety complex peptides isolated from Cephalosporium.

A qualitative micromethod (IDS Rapid NH system) employing conventional and single-substrate enzyme tests was developed for the biochemical characterization of Neisseria spp., Haemophilus spp., and other gram-negative species. A total of over 140 dehydrated, miniaturized biochemical tests were investigated for their ability to distinguish species. Computer-assisted test selection and pair separation analysis of the data allowed the selection of 11 4-h tests that would identify Haemophilus and Neisseria spp. implicated as etiological agents as well as differentiate them from other Neisseria spp., Moraxella spp., Branhamella catarrhalis, Centers for Disease Control M groups, and Kingella spp. The final test configuration included modified glucose, sucrose, galactosidase, nitrate, phosphatase, resazurin reduction, and two arylamidase tests. In addition, indole, urea, and ornithine decarboxylase tests were included to biochemically type strains of Haemophilus influenzae and Haemophilus parainfluenzae.

An enzyme-linked immumosorbent assay (ELISA), based upon the detection of streptolysin 0 antibodies in human sera, was developed. Disposable polystyrene tubes, sensitized with streptolysin 0 antigen, were used as the test vehicles. Corresponding antibodies, present in test sera, were detected by binding of the antibodies to goat anti-human immunoglobulin G conjugated to horseradish peroxidase. Demonstration of bound conjugate was accomplished by monitoring peroxidase activity spectrophotometrically at 450 nm, using 5-aminosalicylic acid as the indicator. A total of 97 human sera, previously analyzed by means of the anti-streptolysin 0 titration technique, were evaluated with the ELISA procedure. A direct quantitative relationship, found to be statistically significant, was demonstrated between Todd units and absorbance values obtained with ELISA.