A method of quantitative determination of alcohol oxidase and catalase in yeast colonies

A rapid, simple and highly reproducible method is developed for qualitative determination of alcohol oxidase and catalase in yeast colonies using digitonin as a permeabilizing agent. The method permits finding differences in regulation of catalase by ethanol in methylotrophic and nonmethylotrophic yeasts.     

A new CSLM-based method for determination of the phase behaviour of aqueous mixtures of biopolymers

On mixing different types of high molecular weight (bio)polymers in an aqueous solution, phase separation often occurs. In some cases, the occurrence of phase separation may be readily observed, because due to density differences the heavier of the two phases is accumulated at the bottom of the vessel in which the mixture is contained. By using classical techniques, the composition of the two phases may then be determined. In the case where the density differences are not so large, and the viscosity of the system is high, the two phases remain intimately mixed. An alternative route to determine the phase behaviour of these systems might be a microscopic technique (Confocal Scanning Laser Microscopy, CSLM), using the fluorescence intensity of labelled biopolymers to quantify their concentration and phase volume in the system. Experiments were performed with several mixtures of sodium alginate, labelled with fluorescein, and sodium caseinate, fluorescently labelled with Texas Red. The viscosity of the mixtures studied was low enough to allow bulk phase separation of the phases by using an ultracentrifuge. Results of the phase volumes, and the composition of the phases, obtained independently by applying the two different methods (CSLM, or analysis of the separate phases after centrifugation) were compared and found to be in reasonable agreement.     

A method for separate quantitative determination of 2-keto-3-deoxy-octonate and 3,6-dideoxyhexoses in mixtures.

A method for analysing ³²P in small aqueous samples by measuring the ?erenkov radiation is described. Centering problems with small sample volumes are eliminated by placing the sample in a polystyrene tube in the scintillation vial. The detection efficiency is high, 54.5 ± 0.6% (2 SD) at a sample volume of 25 μl. The reproducibility is good and independent of the sample volume. The detection efficiency of ³²P in polyacrylamide gel is shown to be as good as in water.     

A rapid method for quantitative determination of L-tryptophan

Summary A modified colorimetric technique automatized by an Alpkem micro-continuous flow analyser was described for estimating the concentration of L-tryptophan in fermentation broth. This approach provided a convenient alternative to HPLC for L-tryptophan estimation and may help to avoid the time-consuming and laborious screening work encountered in the strain improvement programme.     

A simple method for the quantitative determination of muramic acid

A simple method for microdetermination of muramic acid is elaborated. The method is based on the degradation of muramic acid to lactic acid, followed by degradation of the latter to acetaldehyde which can be determined colorimetrically with p-hydroxydiphenyl (PHD). A linear relationship exists between the concentration of muramic acid (up to 20 μg), and absorbance at 560 nm. Substances usually present in the hydrolysates of bacterial cell wall peptidoglycan do not interfere in the determination.     

Occurrence of cytochromes in R and S yeast mutants of the genusSaccharomyces

Visible region of an absorption spectrum was followed in cells of original strains and of rough mutants ofSaccharomyces cerevisiae andS. cerevisiae var.ellipsoideus. It was found that there are no substantial differences in relative content of cytochromesb andc in aerobically grown rough and smooth yeast forms, in spite of the fact that both forms differ substantially in the metabolic oxygen quotient. If the cytochromes present were not reduced in washed cells by dithionite or by substrate addition, the rough forms exhibited a lower cytochrome b:c ratio than the smooth forms. Under anaerobic conditions of cultivation, the rough forms retained a typical aerobic spectrum, lacking, however, the cytochromea and a₃ band; the ratio of cytochromesb andc was changed in favour of cytochromeb (from the original 1.7: 1 up to 3.4: 1). The inability of the rough mutants to produce anaerobic cytochrome spectrum represented by cytochrome b₁ was connected with their inability to reproduce under anaerobic conditions.     

A rapid method to differentiate between five species of the genusSaccharomyces

Summary Long-chain fatty acids of 8 yeast strains representing 5 species of the genusSaccharomyces, were extracted from yeast cells by saponification and analyzed as methyl esters by gasliquid chromatography (GLC). When these species were grown under standard conditions, each produced a distinctive mean fatty-acid “fingerprint” characterized by certain fatty acid compositions. With this method, it was possible to distinguish between the 5 species within 4 h after they were obtained from 48 h cultures as compared with 7 to 10 days for more conventional methods. Factors such as speed and sensitivity of this method make it an attractive alternative to conventional laboratory tests for distinguishing between species ofSaccharomyces.     

A method for the quantitative determination of neutral glycosphingolipids in urine sediment

A method is described for the isolation and quantitation of six neutral glycosyl ceramides from human urinary sediment. Total lipids were extracted from sediments of 24-hr urine collections, and the glycosyl ceramides were isolated by silicic acid column chromatography followed by thin-layer chromatography. Methanolysis of the individual glycosyl ceramides yielded methyl glycosides which were quantitated as the trimethylsilyl ethers by gas-liquid chromatography. By this technique, the submicromolar concentrations of six glycosyl ceramides in normal subjects and in individuals with Fabry's disease, an hereditary glycosphingolipid storage disease, were determined. Trihexosyl ceramide (galactosyl-galactosylglucosyl ceramide) and a digalactosyl ceramide accumulated in the urinary sediment of patients with Fabry's disease.     

A quantitative method for the determination of the activities of mushroom tyrosinase

A sensitive, rapid, quantitative method for the determination of the activities of the bifunctional enzyme, mushroom tyrosinase (o-diphenol: O₂ oxido-reductose, EC 1.10.3.1) has been developed. The spectrophotometric method utilizes p-cresol and 4-methyl catechol as substrates at pH 4.8. By maintaining this low pH value, the rates of the nonenzymic reactions are negligible during the course of the assay. Preliminary analysis of the rates of enzyme-catalyzed reactions gave typical results for both substrates: Lineweaver-Burk plots yielded straight lines and the initial velocities for the reactions were proportional to enzyme concentration. Tyrosinase preparations judged to be as pure as those previously reported could be assayed to enzyme concentrations as low as 1 mg/liter with p-cresol while catechol allowed lower concentrations to be assayed (0.3 mg/liter). The precise specific activities towards p-cresol and 4-methyl catechol were found to vary between enzyme solutions and were used to characterize enzyme preparations.