Transglycosyl-Amylase of Candida tropicalis

Transglucosyl-amylase was purified 96-fold and partially characterized. The K_m value with dextrin as substrate was 9.1 mg/ml. Glycerol, an acceptor of d-glucose, appeared to inhibit dextrin hydrolysis noncompetitively. The energy of activation of the enzyme was 7,920 cal/mole. Indirect determinations showed that synthesis of d-glucosyl glycerol was significantly affected by the nature of the amylaceous substrate. Glucosyl-glycerol synthesis did not increase as incubation temperature was raised from 50 to 60 C. Direct determinations by gas-liquid chromatography indicated that the synthesis of glucosyl glycerol, as a function of the concentration of either enzyme, substrate, or glycerol, traced a curvilinear path approaching 15 mg/ml as the maximum. When enzyme, substrate, and glycerol at high concentrations were varied in all possible combinations, however, conditions for producing as much as 47.5 mg/ml of glucosyl glycerol were established.     

Trnasglucosyl-amylase of Candida tropicalis

Transglucosyl-amylase was purified 96-fold and partially characterized. The K(m) value with dextrin as substrate was 9.1 mg/ml. Glycerol, an acceptor of d-glucose, appeared to inhibit dextrin hydrolysis noncompetitively. The energy of activation of the enzyme was 7,920 cal/mole. Indirect determinations showed that synthesis of d-glucosyl glycerol was significantly affected by the nature of the amylaceous substrate. Glucosyl-glycerol synthesis did not increase as incubation temperature was raised from 50 to 60 C. Direct determinations by gas-liquid chromatography indicated that the synthesis of glucosyl glycerol, as a function of the concentration of either enzyme, substrate, or glycerol, traced a curvilinear path approaching 15 mg/ml as the maximum. When enzyme, substrate, and glycerol at high concentrations were varied in all possible combinations, however, conditions for producing as much as 47.5 mg/ml of glucosyl glycerol were established.     

Acyl-CoA oxidase from Candida tropicalis

Acyl coenzyme A oxidase (acyl-CoA oxidase) has been isolated in good yield from Candida tropicalis pK 233 grown on n-alkanes. Gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measurement of flavin content suggest that the oxidase is an octamer of Mr 75 000 subunits each containing one flavin. The oxidase yields the red semiquinone form on dithionite or photochemical reduction, slowly forms an N-5 adduct with 0.16 M sulfite at pH 7.4, and is rapidly reduced by borohydride, forming the 3,4-dihydroflavin isomer. The red flavosemiquinone is only kinetically stabilized with respect to disproportionation in the free enzyme but is thermodynamically stabilized on binding enoyl-CoA derivatives. The enzyme is reduced by butyryl-, octanoyl-, and palmitoyl-CoA without formation of prominent long-wavelength bands. Acyl-CoA oxidase and the acyl-CoA dehydrogenases share many similarities in their interaction with CoA derivatives. For example, both enzymes stabilize the anionic radical on binding enoyl-CoA derivatives, both dehydrogenate 2-oxoheptadecyldethio-CoA but cannot utilize S-heptadecyl-CoA, both form long-wavelength bands with CoA persulfide species, and both enzymes are attacked by the suicide substrates 3,4-pentadienoyl-CoA and (methylene-cyclopropyl)acetyl-CoA at the flavin prosthetic group.     

Phagocyte-mediated killing of Candida tropicalis

Human peripheral monocytes (MO), neutrophils (PMN), and lymphocytes (PBL) were tested for their ability to kill Candida tropicalis. With incubation times between 30 min and 2 h, unstimulated MO and PMN, but not PBL, were efficient killers of C. tropicalis. Both leukocyte subsets were able to kill at minimum 2.5 1 effector to target ratios. Pre-incubation of MO for 24 h with interferon-gamma or tumor necrosis factor (TNF) increased their ability to kill yeast targets. TNF alone had no effect on C. tropicalis targets at concentrations up to 1000 U/ml. PBL activated for 4 d with interleukin-2 did not kill yeast targets. PMN exhibited more cytocidal efficiency per cell than MO in these assays. Direct contact of effectors and targets was required; no significant killing by PMN or MO supernatants was measured. PMN-mediated killing, but not MO killing, was inhibited by a mixture of catalase and Superoxide dismutase suggesting that oxygen-dependent killing mechanisms were partially responsible for candidacidal activity.     

Acyl-CoA oxidase from Candida tropicalis

The preparation of a highly purified acyl-CoA oxidase from the cell extract of an n-alkane-utilizing yeast, Candida tropicalis, is described. It can be crystallized from ammonium sulfate solutions without an increase in specific activity, and is homogeneous on ultracentrifuge and disc electrophoresis. The enzyme is an octamer with approximately a 600,000 molecular weight, and has an isoelectric point of 5.5. It exhibits a typical flavoprotein spectrum with absorption maxima at 277, 365 and 445 nm, and contains 8 mol of FAD per mol of enzyme. The enzyme catalyzes the stoichiometric conversion of palmitoyl-CoA and O₂ into 2-hexadecenoyl-CoA and H₂O₂. It oxidizes acyl-CoAs with carbon chain lengths of 4 to 20, and is most active toward lauroyl-CoA, but acetyl- and succinyl-CoAs are not oxidized. The enzyme is sulfhydryl dependent and is inactivated by silver and mercury compounds.     

Interspecific hybridisation between Candida albicans and Candida tropicalis

Abstract Protoplasts from auxotrophic mutants of Candida albicans and Candida tropicalis were produced by snail enzyme treatment and their fusion was induced with polyethylene glycol (PEG). During selective regeneration, nutritionally complemented interspecific hybrids were obtained. Their cells contained one nucleus, and the DNA content per cell was higher than in the parents. The isoenzymic and sugar assimilation patterns of the mutants, and those of the hybrids and the products after their haploidisation, were also analysed. The results indicated that the hybrids were partial alloploids containing the total chromosomal set of either of the parental species and one or a few chromosomes of the other.     

Parasexuality and Ploidy Change in Candida tropicalis

Candida species exhibit a variety of ploidy states and modes of sexual reproduction. Most species possess the requisite genes for sexual reproduction, recombination, and meiosis, yet only a few have been reported to undergo a complete sexual cycle including mating and sporulation. Candida albicans, the most studied Candida species and a prevalent human fungal pathogen, completes its sexual cycle via a parasexual process of concerted chromosome loss rather than a conventional meiosis. In this study, we examine ploidy changes in Candida tropicalis, a closely related species to C. albicans that was recently revealed to undergo sexual mating. C. tropicalis diploid cells mate to form tetraploid cells, and we show that these can be induced to undergo chromosome loss to regenerate diploid forms by growth on sorbose medium. The diploid products are themselves mating competent, thereby establishing a parasexual cycle in this species for the first time. Extended incubation (>120 generations) of C. tropicalis tetraploid cells under rich culture conditions also resulted in instability of the tetraploid form and a gradual reduction in ploidy back to the diploid state. The fitness levels of C. tropicalis diploid and tetraploid cells were compared, and diploid cells exhibited increased fitness relative to tetraploid cells in vitro, despite diploid and tetraploid cells having similar doubling times. Collectively, these experiments demonstrate distinct pathways by which a parasexual cycle can occur in C. tropicalis and indicate that nonmeiotic mechanisms drive ploidy changes in this prevalent human pathogen.     

Isolation of Candida tropicalis auxotrophic mutants

An enrichment scheme using nystatin was designed for the isolation of auxotrophic mutants from the diploid-alkane-utilizing yeast Candida tropicalis. A collection of 194 auxotrophs representing 7 phenotypes was isolated. One class of mutants was identified as having a defect in histidinol dehydrogenase activity and a second class of mutants was identified as having a defect in orotidine monophosphate decarboxylase activity. These strains are good candidates to be carrying mutations corresponding to the HIS4 and URA3 genes of Saccharomyces cerevisiae.     

Preparatory metabolism of p-hydroxybenzoic acid in Candida tropicalis

A technique of experimental adaptation was used to obtain mutants of Candida tropicalis which were able to utilize p-hydroxybenzoic acid as the sole source of carbon and energy. The preparatory metabolism of p-hydroxybenzoic acid involves the following stages: PHBA leads to quinol leads to hydroxyquinol leads to maleylacetic acid leads to beta-ketoadipic acid. The enzyme system which catalyzes oxidative decarboxylation of PHBA mediates also oxidative decarboxylation of protocatechuic, beta-resorcylic and gallic acids, i.e. compounds having a hydroxyl group in para position with respect to the carboxyl of hydroxyl derivatives of benzoic acid. Benzoic, salicyclic and gentisic acids are not substrates of this enzyme system. A technique is proposed for rapid indentification of beta-ketoadipic acid by thin-layer chromatography. The authors believe that methods used to study preparatory metabolism, on the basis of the Stanier theory of "simultaneous adaptation", are not quite reliable and may lead to erroneous conclusions.     

热带假丝酵母唑类耐药相关基因的研究进展

临床上热带假丝酵母(又称热带念珠菌)的分离率越来越高,唑类抗真菌药物因较低的细胞毒性且大多可口服给药,是治疗热带念珠菌感染的常用药物。我国耐唑类药物热带念珠菌的分离率较高,因此有必要了解其具体机制,为寻求新的药物作用靶点提供依据。目前认为,与热带念珠菌唑类耐药有关的主要机制有靶基因ERG11过度表达和突变、编码转录因子的upc2基因过度表达和突变、外排泵基因过度表达及其他相关基因过度表达等。本文就目前热带念珠菌唑类耐药机制的基因水平研究进展进行综述。     

The route of lysine breakdown in Candida tropicalis

Candida tropicalis was found to contain high levels of the following enzymes after growth in defined medium on L-lysine as sole nitrogen source: L-lysine N6-acetyltransferase, N6-acetyl-lysine aminotransferase, and aminotransferase activity for 5-aminovalerate and 4-aminobutyrate. Extracts were also capable of converting 5-acetamidovalerate (and 4-acetamidobutyrate) to acetate. N6-Acetyllysine however, only gave rise to acetate in the presence of 2-oxoglutarate, NAD+ and thiamine pyrophosphate. These activities were undetectable or present in much lower concentrations in cells that had been grown on ammonium sulphate as sole nitrogen source. It is concluded that L-lysine is degraded in this organism via N6-acetyllysine, 5-acetamidovalerate and 5-aminovalerate, both nitrogen atoms being removed by transamination.