Rolf K. Hommel

Production of water-soluble surface-active exolipids by Torulopsis apicola

SummarySeveral Torulopsis yeasts were screened for production of extracellular surface-active compounds. One strain, Torulopsis apicola IMET 43747, was studied in greater detail. Both on nalkanes and on carbohydrates it produced a mixture of water-soluble biosurfactants with remarkable interfacial activities and surface-tension values around 30 mN m-1 and interfacial tension below 1 mN m-1. Most of the biosurfactants are produced in the late exponential and in the early stationary growth phase. Production was increased by using hydrophobic compounds as the carbon source. The yields on n-alkanes were influenced by the concentrations of both the carbon source and the yeast extract. The effects of one purified biosurfactant on microbial growth on nalkanes and its antibacterial and antiphagal activities reveal new physiological aspects of biosurfactant generation by T. apicola.

Crotonobetaine reductase fromEscherichia coli — a new inducible enzyme of anaerobic metabolization of L(-)-carnitine

Crotonobetaine reductase fromEscherichia coli 044 K74 is an inducible enzyme detectable only in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine as inducers. Enzyme activity was not detected in cells cultivated in the presence of inducer plus glucose, nitrate, γ-butyrobetaine or oxygen, respectively. Fumarate caused an additional stimulation of growth and an increased expression of crotonobetaine reductase. The reaction product, γ-butyrobetaine, was identified by autoradiography. Crotonobetaine reductase is localized in the cytoplasm, and has been characterized with respect to pH (pH 7.8) and temperature optimum (40–45 °C). The Km value for crotonobetaine was determined to be 1.1×10−2M. γ-Butyrobetaine,D(+)-carnitine and choline are inhibitors of crotonobetaine reduction. For γ-butyrobetaine (Ki=3×10−5M) a competitive inhibition type was determined. Various properties suggest that crotonobetaine reductase is different from other reductases of anaerobic respiration.

Elucidation of the structure of an unusual cyclic glycolipid from Torulopsis apicola

Abstract The yeast Torulopsis apicola produces a microcrystalline mixture of glycolipids, the major component of which has been identified as the novel 16-(2′- O -β- d -glucopyranosyl-β- d -glucopyranosyloxy)hexadecanoic acid 1′,4″-lactone 6′,6″-diacetate ( 2 ) by spectroscopic methods.

Oxygenation of Hexadecane in the Biosynthesis of Cyclic Glycolipids in Torulopsis Apicola

Biotransformation of [1-13C] labelled hexadecane, hexadecanol and hexadecanoic acid have been investigated using the yeast Torulopsis apicola. The yeast produces a microcrystalline mixture of two glycolipids, the lipophilic moiety of which consists of ω- or (ω-l)-hydroxylated hexadecanoic acid. Biosynthesis of these glycolipids takes place via hydroxylation of hexadecane, oxidation to hexadecanoic acid and ω or (ω-l)-hydroxylation of hexadecanoic acid. Feeding the cell cultures with a mixture of hexadecane and [1-13C] labelled hexadecane derivatives one observes 13C enrichment ratios which indicate that neither of the biohydroxylation or oxidation steps are rate limiting in the formation of the glycolipids, furthermore, two different monooxygenase systems appear to be involved in hydroxylation of hexadecane and hexadecanoic acid.

Effect of ammonium ions on glycolipid production byCandida (Torulopsis)apicola

The effect of ammonium on growth ofCandida apicola and on production of sophorose lipid was studied. Sophorose lipid production increased with increasing initial ammonium sulphate concentration. Both growth and product formation were strongly reduced at 73.6mm ammonium. With 58.9mm ammonium a microcrystalline sophorose lipid was formed. The ratio of the two isomers of the sophorose lipid, harbouring either ω- or ω-1 hydroxy fatty acid, was influenced by the initial concentration of ammonium. Both production kinetics, yields and profiles of the total cellular fatty acids express alterations with enlarged ammonium concentrations. These results suggest regulatory effects of ammonium onC. apicola and its sophorose lipid synthesis.

The inducible microsomal fatty alcohol oxidase of Candida (Torulopsis) apicola

In the transition phase of Candida apicola IMET 43747 from logarithmic to stationary growth a pyridine-nucleotide-independent alcohol oxidase was induced coinciding with the beginning of sophorose lipid production. This enzyme was not repressed by glucose and was measurable in stationary cells grown on glucose or on a mixture of n-hexadecane and glucose. An NAD+-dependent aldehyde dehydrogenase behaved in the same way. Both enzymes were localized in the microsomal fraction. The alcohol oxidase accepted long-chain (fatty) aliphatic alcohols (C8 to at least C16) and diols starting from decanediol. Trace activities were found with ω-hydroxy fatty acids. Aromatic, secondary and tertiary alcohols were not oxidized. In the stationary growth phase the substrate specificity of the alcohol oxidase tends to be changed to more hydrophobic substrates. The physiological role of both enzymes, the alcohol oxidase and aldehyde dehydrogenase, is discussed including their possible involvement in the synthesis of sophorose lipid.

Cytochrome P-450 in the sophorose-lipid-producing yeast Candida (Torulopsis) apicola

The appearance of cytochrome P-450 and of cytochrome oxidase aa3 were determined in the sophorose lipid producing yeast Candida (Torulopsis) apicola IMET 43 747 grown on a mixture of glucose and n-hexadecane. Cytochrome P-450, detectable in both the logarithmic and the stationary growth phase was not repressed by glucose. At the end of the logarithmic growth phase the content of cytochrome P-450 was three- to fivefold increased, which was connected with initiation of sophorose lipid biosynthesis. After that it dropped to the basal level, which remained constant during sophorose lipid biosynthesis. Cytochrome P-450 from logarithmic cells was cross-reactive with an antibody derived against cytochrome P-450alk from C. tropicalis. With microsomal proteins of stationary cells no cross-reactivity was obtained. The microsomal hydroxylase system of stationary cells seem to be regulated by the carbohydrate used as carbon source.

Oxidation of long-chain alkanes by Acetobacter rancens

SummaryThe degradation of hexadecane and tetradecane by Acetobacter rancens CCM 1774 was investigated. It was found that this strain is able to grow to a limited extent on hexadecane as a carbon source. The occurrence of n-alkanoic acids and alcohols among the reaction products of growing as well as resting cells indicates a monoterminal degradation of long-chain alkanes. Both alkane-grown and glucose-grown resting cells exhibited alkane oxidizing activities which were not influenced by chloramphenicol. This suggested a constitutive nature of the appropriate enzymes.

Kinetics of enzymatic lysis, formation and regeneration of protoplasts ofCandida (Torulopsis) apicola.

The optimal conditions for protoplast formation ofCandida apicola were by using an enzyme fromArthrobacter sp. in combination with 2-mercaptoethanol. The kinetic data support the two-layered structure model of cell wall for this yeast but the structure of the cell wall depended on the age of cells and culture conditions. To regenerate the protoplasts, the type of osmotic stabilizer was important: sorbitol gave 16 to 30% regeneration. Electron microscopy revealed the presence of vesicles in the sections of protoplasts and whole cells ofCandida apicola grown in production medium and producing glycolipids. In sections of whole cells, vesicle-like structures are located in the periplasmic space and in protoplasts they can either be attached to, or released from, the cell surface. These vesicles are thought to be involved in the transport of the surface-active glycolipids and in the protection of the cell against denaturing effects.