Studies on Chrysanthemic Acid

Ribose-5-phosphate ketol-isomerase, an enzyme isomerizing ribose-5-phosphate to ribulose-5-phosphate, is isolated from Candida utilis which is grown in a medium containing xylose. The enzyme is also purified by means of fractionation with ammonium sulfate, acetone, and by DEAE-cellulose column chromatography.

The enzyme has its optimum pH at 7.5 and optimum temperature at 50°C.

Michaelis-Menten constant for d-ribose-5-phosphate is 7.38 × 10^?4 M and activation energy of the enzyme reaction is 10,525 calories.

The enzyme activity is inhibited by p-CMB, EDTA and sodium pyrophosphate, and activated by the addition of magnesium ion.

Extract of Candida utilis contains polyol: NAD oxidoreductase which catalyzes the conversion of polyols to the corresponding ketoses.

By fractionation with ammonium sulfate and on DEAE-cellulose column chromatography, the purity of enzyme has been increased about 14-fold.

The relatively high activity with both xylitol and sorbitol suggests that they may be the natural substances for the enzyme.

Evidence suggests that this enzyme relates to the metabolism of d-xylose in Candida utilis.     

Inhibition ofCandida utilis by cycloheximide and acetoxycycloheximide

Cycloheximide and acetoxycyloheximide were similar in their effect on the synthesis of RNA and protein by exponentially growing cells ofCandida utilis. Both antibiotics inhibited the synthesis of protein more than that of RNA. During inhibition there was preferential synthesis of ribosomal protein and some completed ribosomes were formed. However, the synthesis of ribosomal RNA was reduced more than that of transfer RNA. The actions of these drugs onCandida utilis are compared with the effects of other antibiotics whose primary effect on bacteria is to inhibit protein synthesis.     

Influence of specific growth limitation and dilution rate on the phosphorylation efficiency and cytochrome content of mitochondria of Candida utilis NCYC 321

With Candida utilis cells that had been removed directly from a 61 chemostat culture, in steady state, well-coupled mitochondria generally could be isolated. This required a modified snail-gut enzyme procedure that allowed the total processing time to be decreased to 3 h, or less. Examination of these mitochondria in an oxygraph showed the presence of 3 sites of energy conservation when the cells were grown at various dilution rates between 0.1 and 0.45 h^-1 in environments that were, successively, glucose-, ammonia-, magnesium-, phosphate- and sulphate-limited. Potassium-limited cells also apparently possessed 3 sites of oxidative phosphorylation when growing at dilution rates greater than 0.2 h^-1, but only 2 sites when growing at lower dilution rates. Analysis of cytochrome spectra obtained with these intact mitochondria revealed large quantitative (but not qualitative) differences, depending on the environmental conditions under which the yeast had been cultured. In particular, comparison of the ratio of cytochrome b to cytochrome a showed a pattern of change with dilution rate in mitochondria from potassium-limited cells that was distinctly different from those evident in mitochondria from cells that had been limited in their growth by the availability of other nutrients.     

Isolation of aCandida utilis variant by using a nitrogen-limited continuous culture

During continuous culture ofCandida utilis the appearance of a morphologic variant yeast was detected. The new microorganism developed systematically whenever it was changed from normal to stressed propagation conditions. A simple system was used for the isolation of the yeast variant, which was defective in cellular division and showed improved kinetic parameters and oxygen uptake rate. An asynchronic nitrogen-limited continuous culture ofCandida utilis allowed us to enrich the population in the chemostat with the modified yeast and isolate it in a defined medium. Assimilation and fermentation tests indicated it to be a variant ofCandida utilis that showed stable morphologic and physiologic differences with the parental yeast.Candida utilis growing in this nitrogen-limited continuous culture also showed a high mutation rate.     

Some Biochemical Properties of Mitochondria Isolated from Euglena gracilis*

SYNOPSIS. Mitochondria were isolated from Euglena gracilis strain Z by pressure-breakage of the cells and sucrose-cushion centrifugation. Multiple peaks (2-4) were observed in the rate of phosphorylation with Mg-ADP-phosphate concentration curves. The phosphorylative and oxidative activities were highest with NADH as the substrate, moderate with succinate, and lowest with glutamate. Inhibition of phosphorylation with 2,4-dinitrophenol and carbonyl cyanide, m-chlorophenylhydrazone gave sigmoidal concentration curves, with the extent of inhibition by DNP depending on the substrate used. Inhibition of phosphorylation by valinomycin, atractyloside, or carboxyatractyloside was only ～ 60%. Oligomycin inhibited phosphorylation in 2 phases at low and high concentrations; it inhibited Mg-ATPase in a sigmoidal fashion. Both phosphorylation and oxidation had discontinuities in Arrhenius plots at 34 C and 18 C. The relative Mg²⁺-dependent nucleoside triphosphatase activity was: 1 for ATP and GTP, 0.6 for ITP, 0.15 for CTP and and UTP; with Ca²⁺ in place of Mg²⁺ this activity was 0.35. Both DNP and CCCP stimulated the Mg-ATPase 50-200%. The optimal pH for the stimulation was ～ 7 regardless of the uncoupler used, and ～ 8 without the uncouplers. The few differences observed between mitochondria from Euglena and those from other sources are probably due to the fragmentation of the reticular mitochondrial structure during isolation and not to unique characteristics of these mitochondria.     

Process for the Stereoselective Biotransformation of Acetoacetic Acid Esters Using Yeasts

A process for the stereospecific reduction of acetoacetic acid esters to the 3-(S)-hydroxy-butanoic acid esters by the yeasts Saccharomyces cerevisiae and Candida utilis grown on glucose and ethanol media was developed. A continuous single stage steady state production system was found to be superior to pulse-, batch- and fed-batch systems in terms of optical product purity, biomass concentration and production rates.

Optical purity of 3-(S)-hydroxybutanoic acid esters produced with Saccharomyces cerevisiae and Candida utilis was dependent on pH. A maximal optical purity was obtained at pH2.2 from S. cerevisiae growing on ethanol medium. The specific product formation rate of the chemostat cultures was 0.02…0.05 gg^?1 h^?1. C. utilis was more productive than S. cerevisiae but it reconsumed the product under carbon limited growth conditions.     

Effect of Sclerin on Incorporation of Oleic Acid into Phospholipids and Activity of Phospholipase in Mitochondria

Sclerin (SCL) stimulated the oxidation and the incorporation into the phospholipids of Na-[1-¹⁴C]-oleate in mitochondria isolated from rat liver, preventing the depression of the phosphorylating functions and protecting 2,4-dinitrophenol (DNP)-activated ATPase in mitochondria during incubation with oleate. Also, SCL markedly enhanced the activity of phospholipase to hydrolyze endogenous substrates in mitochondria. The increase in the activity was due to reconstruction of phospholipids through esterification of oleate in mitochondrial membrane, but not to the de novo enzyme synthesis. It was concluded that the level of endogenous phospholipase in mitochondria during incubation reflects the energy- dependent reacylation of the lysophospholipids produced by the action of phospholipase in mitochondrial membrane.     

Molecular approaches of a mitochondrial mutation inCandida utilis

Analysis of the cytochrome spectra of a mitochondrial mutant ofCandida utilis showed complete absence of apocytochromeb; this suggests a certain degree of damage, probably a small deletion in themit genes of mitochondrial DNA. Oxygen uptake measurements with and without cyanide of the respiratory-competentCandida utilis parent strain and its derivative mitochondrial mutant P_1,2 indicated the absence of the cyanide-sensitive or normal respiratory chain and a lowered rate of cyanide-insensitive or alternate respiration. Mitochondrial profiles and distribution of parental and mutant cells account for an altered mitochondrial DNA which affects mitochondria in the latter cell shape and function. The mutant cells ofCandida utilis were considered asmit ^– mutants from the observations reported here.     

Effect of 2,4-dinitrophenol on the rat liver respiratory activity and ATP content after hypothermic storage and following reperfusion

The influence of oxidative phosphorylation uncoupler 2,4-dinitrophenol (DNP) presence in preserving solution on the rat liver respiratory activity and ATP content after 18 h of hypothermic storage (HS) and following normothermic reperfusion (NR) was investigated. DNP presence on the HS stage led to decrease of ATP level as compared with the control. After DNP removal during NR the gradual recovery of oxidative phosphorylation coupling occurred. This fact resulted in improvement of mitochondrial functional state (V4 respiration rate decrease, respiratory control and ATP level increase).     

Effect of crotonaldehyde on the metabolism ofCandida utilis during the production of single cell protein from ethanol

The effect of 20 low-boiling compounds on the yeastCandida utilis 49 was assessed by a screening test on agar medium. The highest toxicity was exhibited by crotonaldehyde, allyl alcohol and acrolein. Oxidation and assimilation experiments in a slightly aerobic environment showed that an increase in the level of crotonaldehyde in the medium in the range of 10–200 mg 1⁻¹ brings about a lowering of intensity of metabolic processes inCandida utilis, suppression of ethanol utilization and aerobic oxidation rate, a drop in biomass yield, prolongation of cultivation time,etc. The inhibitory effect of crotonaldehyde depends strongly on the manner of its dosage into the medium (single or continuous) and other cultivation conditions (intensity of medium aeration, physiological state of the culture,etc). Crotonaldehyde is lost from the medium partially by volatilization and partially due to chemical and biochemical transformation.     

Effect of Sclerin on Amino Acid Incorporation into Mitochondria Isolated from Rat Liver

Though sclerin (SCL) stimulated amino acid incorporation into the protein fraction of post mitochondrial supernatant of rat liver homogenate, it had no effect on the incorporation into the isolated mitochondria at pH 7.2, despite of its stimulating effect on mitochondrial oxidative phosphorylation. SCL stimulated amino acid incorporation into the mitochondria at pH 6.1, and to some extent maintained the activity on that in mitochondria during aging in hypotonic Tris-HCl buffer (pH 7.2). Since SCL prevented leakage of amino acids from the mitochondria into these buffers, it was suggested that SCL may protect a structure of mitochondrial membrane which appeared to have a significance on transport of amino acids. In liver slices, SCL stimulated amino acid incorporation only into the extra-mitochondrial fraction for the first 3 min, but gradually turned to stimulate incorporation into mitochondria within 30 min.     

Preparation of some pyrazoline derivatives and evaluation of their antifungal activities

The synthesis of a new series of 1-[(benzazole-2-yl)thioacetyl]-3,5-diaryl-2-pyrazoline derivatives was obtained by reacting 1-(chloroacetyl)-3,5-diaryl-pyrazolines with 2-mercaptobenzimidazole/benzoxazole/benzothiazole. The chemical structures of the compounds were elucidated by ¹H-NMR, ¹³C-NMR, and FAB⁺-MS spectral data. Their antifungal activities against Candida albicans, Candida glabrata, Candida utilis, Candida tropicalis, Candida krusei, and Candida parapsilosis were investigated. A significant level of activity was observed.     

Studies on hydrazone derivatives as antifungal agents

The increasing clinical importance of drug-resistant fungal pathogens has urged additional need to fungal research and new antifungal compound development. For this purpose, some N-(1-benzyl-2-phenylethylidene)-N′-[4-(aryl)thiazol-2-yl]hydrazone (1a-e) and N-(1-phenylbutylidene)-N′-[4-(aryl)thiazol-2-yl]hydrazone (2a-e) derivatives were synthesised and evaluated for antifungal activity. Their antifungal activities against standard and clinical strands of Candida albicans, Candida glabrata, Candida utilis, Candida tropicalis, Candida krusei, Candida zeylanoides, and Candida parapsilosis were investigated. A significant level of activity was observed.     

α-l-Rhamnosidase Activity in Culture Filtrate of Corticium rolfsii Enzymic Activity at Low pH

A phosphorylation system for formation of ATP from AMP by Zymolyase-treated cells of Candida boidinii (Kloeckera sp.) No. 2201 was developed as an ATP production process. This system was shown to be an energy conversion system, from a reduced C₁ -compound to ATP through reduction of NAD⁺ and oxidative phosphorylation but not substrate level phosphorylation, together with phosphorylation of AMP to ADP.

Reaction conditions for the ATP production were optimized in respect of substrate and coenzyme concentrations, pH and temperature, osmotic pressure, and oxygen supply. Under the optimal conditions, 26 mM (13 g/liter) and 8.5 dim (4g/liter) of ATP were produced with methanol and formate as C₁ -substrate, respectively.     

Russian Article pp. 255–266

The growth of the yeast Candida utilis VKM-Y-2332 was investigated during the cultivation on a mineral medium with addition of ethanol in the regime of the chemostat and the pH-auxostat. The composition of the macroelements of the biomass produced by Candida utilis VKM-Y-2332 was studied. Stoichiometric equations of the growth process of the examined yeast strain on ethanol were presented. The growth of Candida utilis on ethanol and glucose was also characterized by the way of comparision.     

The effect of changes in the respiratory metabolism upon genome activity in Drosophila

Inhibition of hydrogen transfer between NADH and Co Q by rotenone or amytal in salivary gland cells of Drosophila hydei maintained in vitro, results in the activation of a particular group of four loci in the polytene chromosomes (puff formation). The response of these loci to the same treatment is enhanced if Na-malonate is present in the incubation medium. — Three of the loci become active if the glands are kept in a medium supplied with antimycin A or 2-heptyl-4-hydroxyquinoline-N-oxide (H QNO), specific inhibitors of the electron transfer between cytochromes b and c. — It was established that a temperature treatment and DNP raise oxygen consumption of the cells to a certain level. Following the same treatments of glands supplied with Na-malate and Na-succinate the raise in oxygen consumption attains a significantly higher level. Under these conditions no response is observed at the genome level. — Whereas DNP, which uncouples oxidative phosphorylation and enhances the respiratory chain reactions, does induce the initiation of puff formation, oligomycin, which inhibits oxidative phosphorylation and suppresses the respiratory chain reactions, is ineffective in initiating puff formation at the specific loci. However, if oligomycin is supplied to the medium in combination with KCN which inhibits the cytochrome oxidase activity, three of the four loci become active. — The presence in the medium of substances which may act as hydrogen acceptors, e.g. menadione or methylene blue, can also result in activation of the chromosome loci. — These results are interpreted as indications for the existence of a regulatory mechanism between mitochondrial respiratory metabolism and the activity of a particular group of genome loci.     

Effect of iron and EDTA on ethyl acetate accumulation in Candida utilis

Summary Production of economically-recoverable products from dilute sugar or ethanol is of practical importance. Conversion of glucose to ethyl acetate by Candida utilis was inhibited by FeCl₃ supplementation as low as 10 uM. EDTA added at the onset of growth on glucose relieved such an inhibition and also caused faster and greater amounts of accumulation of the ester. Addition of EDTA during conversion of ethanol to ethyl acetate showed little effect. EDTA may affect cell permeability and/or oxidative metabolism. For continual ethyl acetate production iron limitation must be maintained during as well as before ethanol utilization.EDTA = Ethylenediaminetetraacetic acid.     

Functional Purification of the Monocarboxylate Transporter of the Yeast Candida utilis

Plasma membranes of the yeast, Candida utilis, were solubilized with octyl-β-d-glucopyranoside and a fraction enriched in the lactate carrier was obtained with DEAE-Sepharose anion-exchange chromatography, after elution with 0.4 M NaCl. The uptake of lactic acid into proteoliposomes, containing the purified protein fraction and cytochrome c oxidase, was dependent on a proton-motive force and the transport specificity was consistent with the one of C. utilis intact cells. Overall, we have obtained a plasma membrane fraction enriched in the lactate carrier of C. utilis in which the transport properties were preserved. Given the similarities between the lactate transport of C. utilis and the one of mammalian cells, this purified system could be further explored to screen for specific lactate inhibitors, with potential therapeutic applications.     

Flavin-linked mitochondrial α-glycerophosphate dehydrogenase of Candida utilis

The 150-fold purification of the l-α-glycerophosphate dehydrogenase of Candida utilis electron-transport particles by very mild procedures is described. The active enzyme contains FAD, iron and copper. The function of the metals, if any, is not clear. Its molecular weight is about 5·10⁵. The subunit composition is complex and remains unresolved because the enzyme is contaminated with protease(s). The activity of this enzyme is very low in Saccharomyces cerevisiae unless the cells are grown in glycerol. The NAD-dependent cytoplasmic α-glycerophosphate dehydrogenase is present in C. utilis but could not be demonstrated in glucose-grown S. cerevisiae.