Biochemical studies on yeasts : 1. Studies on glutamic acid decarboxylase in rhodotorula glutinis:2. Studies on riboflavin-exreting mutant yeast BY2

Abstract

he thesis is divided into two parts:

Part I – Studies on the Glutamic Acid Decarboxylase in Rhodotorula Part II – Studies on a Riboflavin-Excreting Mutant Yeast

Part I In the first part of the thesis are described the results of investigations carried out by the author on the enzyme glutamic acid decarboxylase in Rhodotorula. The study of this enzyme is of considerable interest on account of its close association with nitrogen metabolism in biological systems.

After a brief reference to the important contributions on amino acid decarboxylases to indicate the present state of our knowledge on the biological functions of these enzyme systems, experiments are described to demonstrate the occurrence of ?-aminobutyric acid and glutamic acid decarboxylase in Rhodotorula strains. The occurrence of this enzyme in yeast is reported for the first time. Also, the suitability of the circular paper chromatographic technique to study this reaction in detail has been indicated with experimental support.

The various environmental conditions necessary for the optimum formation of glutamic acid decarboxylase in this organism are investigated and their implications are discussed. Various methods tried for obtaining active preparations are described.

The properties of the enzyme such as coenzyme specificity, pH and temperature optima, stability at different temperatures, quantitative nature of decarboxylation, effect of substrate concentration, Michaelis constant, etc., have been studied. The significance of the results obtained therein are discussed. Attempts have been made to purify the enzyme by different methods available.

The effect of pyridoxal phosphate (commonly known inhibitor) on the enzyme is studied with a view to elucidate the nature of the enzyme and the mechanism of the inhibition. The competitive nature of the inhibitory effect of some amino acids is investigated. The inhibitory effect of lysine is studied in detail. Its great affinity for the enzyme, the purely competitive nature of inhibition, and the reversibility of inhibition by pyridoxal phosphate are established with experimental data. The importance of these results with respect to the nature of enzyme action is discussed.

Part II The usefulness of mutant organisms in the study of metabolic processes, the important contributions relating to the role of riboflavin in biological systems, and the various riboflavin-excreting microorganisms encountered are referred to. A brief review is given of the history of the riboflavin-excreting yeast and the work already done on the study of the various cultural conditions affecting riboflavin synthesis by the organism.

The effect of different carbohydrates, amino acids, purine and pyrimidine compounds on the riboflavin synthesis by the organism is studied. The possible role of some of the purines as precursors in the synthesis of riboflavin is suggested. These results are discussed in comparison with the behaviour of other riboflavin-excreting organisms reported in literature.

The influence of biotin, which was a critical growth factor for the mutant yeast (biotin deficiency acquired on mutation), on its capacity to assimilate amino acids is investigated. The parent and the mutant are compared with each other in these studies. The effect of biotin on the nitrogen content, rate of growth, and the partial replaceability of biotin with fatty acids and aspartic acid are observed.

Studies are made to elucidate the role of biotin in the deaminase systems in the mutant and the parent yeasts (aspartic acid deaminase was chosen for these studies) since the function of biotin in these systems is of current interest. The effect of cultivating the organism in varying concentrations of biotin on the deaminase activity and the effect of starvation are studied. The core salient features of these results signifying the functions of biotin in the mutant yeast vis-à-vis its abnormal activity of riboflavin excretion are discussed.

In a supplementary chapter, the application of circular paper chromatography to the study of the amino acid composition of a number of microorganisms is described. Incidentally, the identification of a degradation product of adenine and nucleic acid compounds (including nucleic acids) as 6-amino-5-imidazole carboxamide riboside was made, following the observation of purple-coloured bands on the chromatogram (sprayed with Folin’s reagent) of some bacterial hydrolysates. Further, the compound was prepared from adenine and its identity established.