Cytosolic alcohol dehydrogenases from yeast Torulopsis candida]

A comparative study of cell cytosol alcohol dehydrogenase (ADH) from yeast Torulopsis candida IBFM-Y-127 grown on glucose and hexadecane which were the only source of carbon, was made. In both cases ADH had a pH optimum within the range of 7.0--10.0, when various normal primary alcohols (C2--C16) were used. The enzyme was active only in the presence of NAD, which cannot be substituted by NADP. The total activity of ADH decreased approximately 8-fold when the length of hydrocarbon radicals was changed from C2 up to C16. When the cells were grown on hexadecane, only ethyl, n-buthyl, n-amyl and n-hexyl alcohols were active as substrates. The dehydration rate of each alcohol was far lower than that for the cytosol of glucose-grown cells. In the latter case the enzyme activity also decreased with an increase in the alcohol radical from C2 to C6. In all cases studied methyl alcohol and cyclic (cinnamyl alcohol--C8) alcohol were not dehydrated at all. Disc-electrophoresis in polyacrylamide gel, involving gel colouration for the assay of enzyme activity showed that glucose--grown cell cytosol contained three forms of ADH. One of those forms was highly active when short--chain normal primary alcohols were used; this form may be probably regarded as "classical" ADH (EC 1.1.1.1). The two other forms caused intensive dehydration of long-chain alcohols (the best substrates were C7--C10 alcohols for one form and C10--C14 for the others). The two forms of ADH are probably isoenzymes of octanol dehydrogenase (EC 1.1.1.73). Cytosol of cells grown on n-alcane, had a reduced number of ADH forms. The data obtained are discussed in terms of the regulatory role of carbon and energy source (glucose or hexadecane) in the redistribution of alcohol dehydrogenases between structural components of cells (mitochondria) and cytosol.     

Isolation of a bioemulsifier from Candida lipolytica.

The yeast Candida lipolytica produced an inducible extracellular emulsification activity when it was grown with a number of water-immiscible carbon substrates. Negligible emulsification activity was produced by this yeast when it was grown with glucose as the carbon substrate. In hexadecane-supplemented cultures, emulsification activity was first detected after 36 h of growth, with maximum production after 130 h. A water-soluble emulsification activity was partially purified by repeated solvent extractions of the culture filtrate. This emulsifier, which we named liposan, was primarily composed of carbohydrate. Maximum emulsification activity was obtained when the ratio of hexadecane to liposan was 50:1. Maximum emulsification activity was obtained from pH 2 to 5. Liposan was heat stable to temperatures up to 70 degrees C, with a 60% loss in activity after heating for 1 h at 100 degrees C. Liposan effected stable oil-in-water emulsions with a variety of hydrocarbons.     

Isolation of a bioemulsifier from Candida lipolytica

The yeast Candida lipolytica produced an inducible extracellular emulsification activity when it was grown with a number of water-immiscible carbon substrates. Negligible emulsification activity was produced by this yeast when it was grown with glucose as the carbon substrate. In hexadecane-supplemented cultures, emulsification activity was first detected after 36 h of growth, with maximum production after 130 h. A water-soluble emulsification activity was partially purified by repeated solvent extractions of the culture filtrate. This emulsifier, which we named liposan, was primarily composed of carbohydrate. Maximum emulsification activity was obtained when the ratio of hexadecane to liposan was 50:1. Maximum emulsification activity was obtained from pH 2 to 5. Liposan was heat stable to temperatures up to 70 degrees C, with a 60% loss in activity after heating for 1 h at 100 degrees C. Liposan effected stable oil-in-water emulsions with a variety of hydrocarbons.     

Plasma membrane from Candida tropicalis grown on glucose or hexadecane. II. Biochemical properties and substrate-induced alterations.

Isolated plasma membranes from the yeast Candida tropicalis grown on two different carbon sources (glucose or hexadecane), had similar contents of protein (60% of total dry weight), lipid (21-24%) and carbohydrates (16-21%). Sodium dodecyl sulphate gel electrophoresis of the membrane proteins revealed 17 and 19 protein bands, respectively, for glucose and hexadecane grown cells. There were marked differences in RF values and relative peak heights between the two gels. Sterols and free fatty acids were the major components of the plasma membrane lipids. Phospholipid content was less than 2% of total plasma membrane lipids. Membrane microviscosity, as determined by fluorescence polarization, was very high (16.6 P). Fatty acid determination of membrane lipids by gas chromatography showed a significant increase of C16 fatty acids in plasma membranes of cells grown on hexadecane. Reduced-oxidized difference spectra demonstrated the presence of a b-type cytochrome in both Saccharomyces cerevisiae and C. tropicalis plasma membranes. Its concentration in C. tropicalis plasma membranes was three-fold greater in cells grown on hexadecane than in glucose grown cells.     

The functional role of lipids in hydrocarbon assimilation

The yeast Candida tropicalis utilizes both glucose and hydrocarbons as sole carbon sources. When grown on hydrocarbons, the cells contain twice as much lipid as when grown on glucose. In transient continuous culture experiments, following a substrate change from glucose to hexadecane, an adaption phase occurred. During this phase the lipid concentration per cell increased greatly. It is proposed that a high cellular lipid concentration is necessary for hydrocarbon assimilation, and this is not just a reflection of the lipophilic nature of the substrate.     

Degradation of tetrahydrofurfuryl alcohol by Ralstonia eutropha is initiated by an inducible pyrroloquinoline quinone-dependent alcohol dehydrogenase.

An organism tentatively identified as Ralstonia eutropha was isolated from enrichment cultures containing tetrahydrofurfuryl alcohol (THFA) as the sole source of carbon and energy. The strain was able to tolerate up to 200 mM THFA in mineral salt medium. The degradation was initiated by an inducible ferricyanide-dependent alcohol dehydrogenase (ADH) which was detected in the soluble fraction of cell extracts. The enzyme catalyzed the oxidation of THFA to the corresponding tetrahydrofuran-2-carboxylic acid. Studies with n-pentanol as the substrate revealed that the corresponding aldehyde was released as a free intermediate. The enzyme was purified 211-fold to apparent homogeneity and could be identified as a quinohemoprotein containing one pyrroloquinoline quinone and one covalently bound heme c per monomer. It was a monomer of 73 kDa and had an isoelectric point of 9.1. A broad substrate spectrum was obtained for the enzyme, which converted different primary alcohols, starting from C2 compounds, secondary alcohols, diols, polyethylene glycol 6000, and aldehydes, including formaldehyde. A sequence identity of 65% with a quinohemoprotein ADH from Comamonas testosteroni was found by comparing 36 N-terminal amino acids. The ferricyanide-dependent ADH activity was induced during growth on different alcohols except ethanol. In addition to this activity, an NAD-dependent ADH was present depending on the alcohol used as the carbon source.     

Alcohol dehydrogenase (ADH) in yeasts. II. NAD+-and NADP+-dependent alcohol dehydrogenases in Saccharomycopsis lipolytica.

In Sm. lipolytica one NAD+-dependent and three NADP+-dependent alcohol dehydrogenases are detectable by polyacrylamide gelelectrophoresis. The NAD+-dependent ADH (ADH I), with a molecular weight of 240,000 daltons, reacts more intensively with long-chain alcohols (octanol) than with short-chain alcohols (methanol, ethanol). The ADH I is not or only minimally subject to glucose repression. Besides the ADH I band no additional inducible NAD+-dependent ADH band is gel-electrophoretically detectable during growth of yeast cells in medium containing ethanol or paraffin. The ADH I band is very probably formed by two ADH enzymes with the same electrophoretic mobility. The NADP+-dependent alcohol dehydrogenases (ADH II--IV) react with methanol, ethanol and octanol with different intensity. In polyacrylamide gradients two bands of NADP+-dependent ADH are detectable: one with a molecular weight of 70,000 daltons and the other with 120,000 daltons. The occurrence of the three NADP+-dependent alcohol dehydrogenases is regulated by the carbon source of the medium. Sm. lipolytica shows a high tolerance against allylalcohol. Resistant mutants can be isolated only at concentrations of 1 M allylalcohol in the medium. All isolates of allylalcohol-resistant mutants show identical growth in medium containing ethanol as the wild type strain.     

Alcohol oxidation by Candida guilliermondii yeasts grown on hexadecanol

A number of enzyme systems involved in the first steps of hexadecane oxidation can be induced by hexadecanol, an intermediate product of hexadecane degradation. It has also been found that, in Candida guilliermondii cells and in their mitochondrial fraction, an oxidase system is induced when the cells are grown on hexadecanol. This system is similar to that in cells grown on hexadecane; it oxidises higher alcohols at a high rate and is not inhibited by the inhibitors of the man phosphorylating respiration chain. The membrane-bound alcohol dehydrogenase and aldehyde dehydrogenase activities resistant to pyrazole, an inhibitor of cytosol ethanol dehydrogenase, are induced together with the oxidase activity when the cells are grown on hexadecanol as well as on hexadecane. The oxidation of higher alcohols by whole cells is entirely inhibited by azide although their oxidation by mitochondria is resistant to the action of azide; apparently, azide inhibits the transport of alcohols into the cell.     

Activity of pentose phosphate pathway enzymes in alkane-oxidizing yeast cells]

The activity of the key enzymes of the pentose phosphate pathway (glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, transketolase) was determined in cell-free homogenates of Candida lipolytica 695 and Candida tropicalis 303 growing on different carbon sources. The activity of these enzymes remained almost the same in the course of growth of both cultures. The activity of the enzymes differed only slightly in the cells metabolizing hexadecane and glucose. The activity of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the cell-free homogenates of C. tropicalis 303 was twice as high as in the cells of C. lipolytica 695. The activity of transketolase was the same in both cultures. The main role of the pentose phosphate pathway is presumed to consist not in catabolism of the carbon source, but in biosynthesis of pentoses and other important intermediates.     

Production of long-chain alcohols by yeasts

Fourteen yeast strains from six genera were analysed for the presence of long-chain alcohols. Six strains from three genera contained long-chain alcohols, highest levels being found in Candida albicans. The alcohols were identified and determined by TLC, GLC and GLC-MS. The major long-chain alcohols synthesized by these organisms were saturated, primary alcohols with C14, C16 or C18 chain length. Unsaturated long-chain alcohols were not detected. In all strains that produced long-chain alcohols, the relative proportions were C16 greater than C18 greater than C14. Long-chain alcohol contents were higher in organisms from anaerobically, as compared with aerobically, grown cultures reaching about 650 micrograms (g dry wt organisms)-1 in stationary-phase cultures of C. albicans. In cultures of C. albicans, synthesis of long-chain alcohols occurred only after the end of exponential growth. The alcohols were predominantly present as free alcohols. The fatty-acyl chain-length profile of the triacylglycerol and to a lesser extent the sterol/wax ester fractions from C. albicans reflected that of the long-chain alcohols produced by this yeast.     

Characterization of alcohol dehydrogenase 3 of the thermotolerant methylotrophic yeast Hansenula polymorpha

In this study, we identified and characterized mitochondrial alcohol dehydrogenase 3 from the thermotolerant methylotrophic yeast Hansenula polymorpha (HpADH3). The amino acid sequence of HpADH3 shares over 70% of its identity with the alcohol dehydrogenases of other yeasts and exhibits the highest similarity of 91% with the alcohol dehydrogenase 1 of H. polymorpha. However, unlike the cytosolic HpADH1, HpADH3 appears to be a mitochondrial enzyme, as a mitochondrial targeting extension exists at its N terminus. The recombinant HpADH3 overexpressed in Escherichia coli showed similar catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The HpADH3 displayed substrate specificities with clear preferences for medium chain length primary alcohols and acetaldehyde for an oxidation reaction and a reduction reaction, respectively. Although the H. polymorpha ADH3 gene was induced by ethanol in the culture medium, both an ADH isozyme pattern analysis and an ADH activity assay indicated that HpADH3 is not the major ADH in H. polymorpha DL-1. Moreover, HpADH3 deletion did not affect the cell growth on different carbon sources. However, when the HpADH3 mutant was complemented by an HpADH3 expression cassette fused to a strong constitutive promoter, the resulting strain produced a significantly increased amount of ethanol compared to the wild-type strain in a glucose medium. In contrast, in a xylose medium, the ethanol production was dramatically reduced in an HpADH3 overproduction strain compared to that in the wild-type strain. Taken together, our results suggest that the expression of HpADH3 would be an ideal engineering target to develop H. polymorpha as a substrate specific bioethanol production strain.     

Evidence of peroxisomes and peroxisomal enzyme activities in the oleaginous yeast Apiotrichum curvatum

The presence of peroxisomes and peroxisomal enzyme activities were investigated in the oleaginous yeast Apiotrichum curvatum ATCC 20509 (formerly Candida curvata D.) Catalase, a marker enzyme for peroxisomes, was measured in cell-free extracts prepared by sonication. The nature of the carbon and nitrogen sources in the growth medium greatly affected catalase activity. Cells grown on corn oil had high specific activity of catalase, but those grown on glucose, sucrose, or maltose had low specific activity. High specific activity of catalase was measured in cultures grown on media that supported poor growth (with soluble starch as carbon source or with methylamine, urea, or asparagine as nitrogen source). Peroxisomes from cells grown on corn oil were separated from other subcellular fractions in a discontinuous sucrose gradient. Major peaks of activity of fatty acid beta-oxidation and of two key enzymes in the glyoxylate cycle were found in fractions containing peroxisomes, but not in fractions corresponding to the mitochondria. Peroxisomal beta-oxidation showed equivalent activity with palmitoyl CoA or n-octanoyl CoA as substrate. Mitochondria did not seem to contain NAD-linked glutamate dehydrogenase. Peroxisomes with a homogeneous matrix and core surrounded by a single-layer membrane were observed with an electron microscope in cells grown on corn oil, but not in those grown on glucose. Staining with 3,3'-diaminobenzidine revealed that catalase activity was located in peroxisomes. Peroxisomes in this oleaginous yeast play important roles in lipid metabolism.     

Cell surface properties of five polycyclic aromatic compound-degrading yeast strains

To investigate the effects of physiological properties on polycyclic aromatic compound (PAH) degradation, the surface tension and emulsification activities, and cell surface hydrophobicity of five PAH-degrading yeast isolates were compared to Saccharomyces cerevisiae from cultures grown with glucose, hexadecane, or naphthalene as carbon sources. The cell surface hydrophobicity values for the five yeast strains were significantly higher than for S. cerevisiae for all culture conditions, although these were highest with hexadecane and naphthalene. Strains with higher hydrophobicity showed higher rates of naphthalene and phenanthrene degradation, indicating that increased cell hydrophobicity might be an important strategy in PAH degradation for the five strains. Emulsification activities increased for all five yeast strains with naphthalene culturing, although no relationship existed between emulsification activity and PAH degradation rate. Surface tensions were not markedly reduced with naphthalene culturing.     

The activity of yeast ADH I and ADH II with long-chain alcohols and diols

The activities of yeast ADH I and ADH II towards long chain alcohols and diols were studied using rather unusual conditions (1.0 M Tris pH 8.75, approximately 0.3 mg/ml enzyme and [S]相似文献   

Determination of carbohydrates, sugar alcohols, and glycols in cell cultures and fermentation broths using high-performance anion-exchange chromatography with pulsed amperometric detection

Cell cultures and fermentation broths are complex mixtures of organic and inorganic compounds. Many of these compounds are synthesized or metabolized by microorganisms, and their concentrations can impact the yields of desired products. Carbohydrates serve as carbon sources for many microorganisms, while sugar alcohols (alditols), glycols (glycerol), and alcohols (methanol and ethanol) are metabolic products. We used high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) to simultaneously analyze for carbohydrates, alditols, and glycerol in growing yeast (Saccharomyces cerevisiae) cultures and their final fermentation broths. Both cultures were grown on complex undefined media, aliquots centrifuged to remove particulates, and the supernatants diluted and directly injected for analysis. Pulsed amperometry allowed a direct detection of the carbohydrates, alditols, and glycols present in the cultures and fermentation broths with very little interference from other matrix components. The broad linear range of three to four orders of magnitude allowed samples to be analyzed without multiple dilutions. Peak area RSDs were 2-7% for 2, 3-butanediol, ethanol, glycerol, erythritol, rhamnose, arabitol, sorbitol, galactitol, mannitol, arabinose, glucose, galactose, lactose, ribose, raffinose, and maltose spiked into a heat-inactivated yeast culture broth supernatant that was analyzed repetitively for 48 h. This method is useful for directly monitoring culture changes during fermentation. The carbohydrates in yeast cultures were monitored over 1 day. A yeast culture with medium consisting primarily of glucose and trace levels of trehalose and arabinose showed a drop in sugar concentration over time and an increase in glycerol. Yeast growing on a modified culture medium consisting of multiple carbohydrates and alditols showed preference for specific carbon sources and showed the ability to regulate pathways leading to catalysis of alternative carbon sources.     

Clutivation of yeast on gas oil

The results achieved by the cultivation of the yeast. Candida lipolytica on gas oil are referred. By using a distillation fraction of gas oil distilling between 180–400°C, containing 10–20% of n-alkanes, the optimal condition for biomass production and deparaffination were estimated for various dilution rates and various amounts of gas oil in the medium. The main factor, which influences the yield coefficient by hydrocarbon fermentation is the polyauxie of the hydrocarbon substrate. The penetration of dispersed hydrocarbons into the yeast cell is demonstrated on electron micrographs and the velocity and reversibility of this process is estimated by using tritium-traced hexadecane.     

Production of extracellular higher fatty acids by yeasts grown on hexadecanoic acid

Production of exocellular higher fatty acids by Candida yeasts was studied during their growth in a mineral medium with hexadecane. The qualitative and quantitative composition of exocellular higher fatty acids was investigated during cultivation of Candida lipolytica VCM Y 2378(695) under the conditions of different aeration (5 and 80% saturation of the medium with oxygen). Palmitic (C16:0), palmitooleic (C16:1), oleic (C18:1) and linoleic (C18:2) acids predominated among other higher fatty acids. The overall amount of fatty acids increased and the content of unsaturated fatty acids decreased when the yeast growth was limited with oxygen.     

Effect of glucose and long-chain fatty acids on synthesis of long-chain alcohols by Candida albicans

Candida albicans, grown aerobically in glucose-containing media, produced C14, C16 and C18 saturated long-chain alcohols only after the end of exponential growth. Contents of C14 alcohols were always lowest, and C16 and C18 alcohol contents about equal. Contents of all three classes of alcohol increased as the concentration of glucose in aerobic cultures harvested after 168 h incubation was raised from 1.0 to 30.0% (w/v). However, in 168 h anaerobic cultures, greatest long-chain alcohol contents in organisms were obtained using media containing 10% (w/v) glucose. Substituting glucose (10%, w/v) with the same concentration of galactose in aerobic cultures greatly decreased contents of long-chain alcohols, while inclusion of 10% (w/v) glycerol virtually abolished their synthesis. Supplementing anaerobic cultures with odd-chain fatty acids induced synthesis of odd-chain alcohols. Maximum conversion of fatty acid to the corresponding long-chain alcohol was observed with heptadecanoic acid. The effect of glucose on production of heptadecanol from exogenously provided heptadecanoic acid was similar to that observed on synthesis of the three major even-chain alcohols in media lacking a fatty-acid supplement. Cell-free extracts of organisms catalysed in vitro conversion of palmitoyl-CoA to 1-hexadecanol.     

Degradation of Hydrocarbons by Members of the Genus Candida III. Oxidative Intermediates from 1-Hexadecene and 1-Heptadecene by Candida lipolytica

Candida lipolytica (Phaff) was grown in a mineral-salts medium amended with either 1-hexadecene or 1-heptadecene as substrate. Intermediates of the same chain length as the substrate were isolated and identified by various analytical procedures. The following intermediates of 16 and 17 carbon atoms were identified: omega-unsaturated acids, omega-unsaturated primary and secondary alcohols, 1,2-epoxides, 1,2-diols, and 2-hydroxy acids. Based on the chemical structure of these compounds, three oxidative mechanisms are proposed for the degradation of long-chain 1-alkenes by this yeast: (i) methyl-group oxidation, (ii) double-bond oxidation, and (iii) subterminal oxidation.     

Effect of aeration and carbon dioxide on cell morphology of Candida utilis

The effect of CO2 availability on cell size, shape, and aggregation in continuous cultures of Candida utilis was studied in minimal medium with glucose or ethanol as the sole carbon and energy source. Enrichment with CO2 was achieved (i) by using the substrate with more C atoms, (ii) by using pure oxygen and thus decreasing aeration intensity at the same dissolved-oxygen concentration, or (iii) by adding CO2 to the aeration gas. The cells were always of yeast shape, and no filaments were formed. In cultures with a biomass concentration above 6 g (dry weight) per liter, no cell aggregates were observed. In cultures with a lower biomass, the daughter cells failed to separate from the parent cells and formed aggregates with thickened walls. The average cell number per aggregate was found to be higher, and the average protoplast volume lower, under conditions of probable CO2 limitation. Simultaneously, the ratio of total dry weight to wet weight of protoplasts was considerably higher, indicating an increased share of wall or extracellular material. The possible effect of the observed morphological changes for maintaining a suitable concentration gradient of CO2 around the cell is discussed.