Isolation and properties of cytoplasmic L(+)-lactatoxydase of Candida lipolytica yeasts]

The enzyme preparation of L(+)-lactatoxydase (K.F. 1.1.3.2) with molecular weight of 230 000 has been isolated from the soluble fraction of the C. lipolytica cells and purified similar 360 times. The enzyme oxydizes L(+)-lactate, the optimum activity of the enzyme being observed at pH 8.0. Oxydation of the substrate is followed by accumulation of H2O2. Silver ions, p-chloromercurybenzoate and dicumarol inhibit the activity of L(+)-lactatoxydase. Iron complexones, cyanide and L-malate do not inhibit oxydation of the substrate. Pyruvate and its fluorine derivative practically do not produce any inhibiting effects either. The enzyme preparation contains 0.6 moles of flavin and 2 moles of nonhaem iron per a mole of the enzyme. Km value for the substrate is equal to 4-10(-4) M, Vmax--4.5 mkatom O/min/mg. Acidation of incubation medium leads to a decrease both of Km and Vmax. Km value for oxygen is equal to 3.1 mkM O2. Beside oxygen, ferricyanide, 2.6-dichlorphenolindophenol, phenazine methosulphate and cytochrome C may also serve as acceptors of L(+)-lactatoxydase electrons. The oxydized enzyme preparation is characterized by a spectrum absorption maximum at 410 nm. Upon L(+)-lactatoxydase reduction the maximum is shifted up to 420 nm.     

Reverse electron transfer from the cytochrome c level in cyanide-resistant mitochondria of the yeast, Candida lipolytica]

The ability of cyanide-resistant mitochondria of yeast Candida lipolytica to perform reverse electron transfer from cytochrome c to alternative oxidase was studied. It was shown that the energy for such a transfer can be provided by high energy intermediates or membrane potential but not by ATP. Reverse electron transfer from cytochrome c is impossible due to energy of NADH and alpha-glycerophosphate oxidation via alternative pathway in the presence of cyanide. These results prove once again that electron transfer via alternative pathway is not connected with the energy accumulation.     

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.     

Sterols from Candida lipolytica yeast grown on n-alkanes

The sterols of Candida lipolytica grown on n-alkanes were isolated by reverse phase HPLC and found to be mainly ergosterol, with small quantities of ergost-7-en-3β-ol, ergosta-7,22-dien-3β-ol, ergosta-7,24(28)-dien-3β-ol and ergosta-5,7,9(11),22-tetraen-3β-ol.     

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.     

The preparation and some properties of yeast mitochondria

Summary A comparison of the structural and functional integrity of yeast mitochondria prepared by a mechanical disintegration method with that reported for mitochondria prepared by enzyme methods has been made.The mitochondria prepared by mechanical disruption show at least as great a degree of functional integrity as that reported for enzymically prepared mitochondria. This was judged by the substrates oxidised, the rates of these oxidations, the respiratory control and ADP:0 ratios and difference spectra.The respiration of citrate and of ethanol by the mechanically prepared mitochondria has been shown to be stimulated by exogenous NAD; in the case of the latter substrate a degree of respiratory coupling became apparent as a consequence of this stimulation.The penetration of NADH₂, and of citrate or of ethanol in the presence of exogenous NAD, into the mitochondria has been examined.The results are consistent with the reported existence of a NADH₂ dehydrogenase system located in the outer area of the inner mitochondrial membrane. The NAD stimulation of ethanol respiration also suggests the occurrence of an external alcohol dehydrogenase in the mitochondrial preparation; similar results with citrate suggest external forms of aconitic hydratase and isocitric dehydrogenase to be present.Unlike animal mitochondria the rate of penetration of oxidisable substrates in uninhibited suspensions appeared to exceed respiration and/or outward diffusion rates in this preparation.Evidence of a malate stimulated citrate and isocitrate transport system similar to that reported for animal mitochondria is presented, but the specificity of the system for tricarboxylate anion appears to differ, at least in respect of propane 1.2.3 tricarboxylate (tricarbalyllate).     

Detection, isolation and some properties of membrane proteinases from yeast mitochondria

The complete amino-acid sequences of two isocytochromes c (larval- and adult-type cytochromes c) purified from the housefly Musca domestica L. were determined. Their sequences differed at six positions from each other. More than 90% of the total cytochrome c was larval-type during larval stages. The amount of adult-type cytochrome c increased rapidly from 1 day before adult emergence, making the total cytochrome c content increase to approximately 2.5-times as much cytochrome c content as in larvae. Although the major cytochrome c species in flight muscles was adult-type, imaginal disks contained mainly larval-type cytochrome c.     

Production of Candida lipolytica protoplasts]

Optimal conditions were found for the production and isolation of the protoplasts of Candida lipolytica. The maximum amount of the protoplasts was produced after 90 minutes of the incubation with a crude preparation of the enzyme from Helix pomatia (100 mg/g wet biomass). Longer incubation results in lysis of the protoplasts and structural damages of the intracellular components. The yield of the protoplasts does not depend on the nature of stabilizing agent. A decrease in the stabilizer concentration increases the yield of the protoplasts four times. Preliminary treatment of the yeast cells with a 0.1 M solution of SH compounds (cysteine, beta-mercaptoethanol) does not increase the yield of the protoplasts; and 0.2 M solution of these compounds decreased the yield of the protoplasts.     

Isolation and some properties of N-acetyl-beta-D-hexosaminidase from Sarcoscipha coccinea]

N-Acetyl-beta-D-hexosaminidase was isolated from the extract of the discomycet Sarcoscipha coccinea and purified 510--550-fold by gel filtration on Sephadex G-200 and by ion-exchange chromatography on KM-Sephadex C-50 and DEAE-Sephadex A-50 or by a combination of hydrophobic and affinity chromatographies. Gel electrophoresis confirmed the homogeneity of the enzyme in both cases. Some properties of purified N-acetyl-beta-D-hexosaminidase (e. g. pH optimum, thermal stability, molecular weight, etc.) were studied. The Michaelis constants and maximal cleavage rates for some substrates were determined. The tissue extract of S. coccinea was found to contain two molecular forms of N-acetyl-beta-D-hexosaminidase. At concentrations of N-acetyl-p-nitrophenyl-beta-D-glucosaminide and D-galactosaminide higher than 0,5 mM the enzyme is inhibited by an excess of the substrate.     

Puridication and properties of an intracellular ribonuclease from Candida lipolytica.

1. A ribonuclease (RNAase CL) (EC 3.1.4.23, ribonucleate 3'-oligonucleotide hydrolase) was extracted by EDTA/acetate buffer, pH 5.6 from acetonedried cells of Candida lipolytica and purified 1350-fold by acetone and (NH4)2SO4 fractionation, DEAE-cellulose and DEAE-Sephadex chromatography. 2. RNAase CL is an acidic protein having an isoelectric point of 4.2, and an approximate molecular weight of 32 000. 3. Optimal pH and temperature for the enzyme were 6.0 and 60 degrees C, respectively. It is stable at neutral pH up to 50 degrees C. At 64 degrees C for 30 min, 95, 49 and 64% inactivation of the enzyme occurred at pH values 4.2, 6.6 and 10.0, respectively. 4. RNAase CL inhibited by Zn2+ and Cu2+, sulfhydryl reactants and by high concentration of salts, but not by chelating agents. 5. RNAase CL degraded ribosomal RNA, transfer RNA, polyadenylic acid, polycytidylic acid and polyuridylic acid into acid-soluble nucleotides. Among the synthetic homopolymers, polycytidylic acid was most rapidly degraded. Polyguanylic acid and duplexes of synthetic homopolymers were less sensitive. DNA was not attacked. Specificity studies showed that RNAase CL preferentially cleaves pC-purine bonds. 6. Digestion of poly (C) by RNAase CL resulted in the liberation of cyclic 2',3'-CPM from the start of the reaction with no observable formation of intermediate oligonucleotides. This suggests that the enzyme depolymerizes by an exonucleolytic mechanism.     

Isolation and some properties of protein kinase from pigeon breast muscle]

Protein kinase was isolated from pigeon breast muscle. The preparation obtained was chromatographically homogeneous. The apparent Km varlue for histone H1 and ATP were 3,5-10(-5) M and 1,6-10(-5) M respectively. The purified enzyme displays high specificity for the lysine-rich histones (H1, H2b, H2a). The protein kinase activity is stimulated, 1,6-fold by cyclic AMP.     

The isolation, purification, and some properties of NAD-dependent isocitrate dehydrogenase from the organic acid-producing yeast Yarrowia lipolytica

The NAD+-dependent isocitrate dehydrogenase of the organic acid-producing yeast Yarrowia lipolytica was isolated, purified, and partially characterized. The purification procedure included four steps: ammonium sulfate precipitation, acid precipitation, hydrophobic chromatography, and gel-filtration chromatography. The enzyme was purified 129-fold with a yield of 31% and had a specific activity of 22 U/mg protein. The molecular mass of the enzyme was found to be 412 kDa. The enzyme consists of eight identical subunits with a molecular mass of about 52 kDa. The Km for NAD+ is 136 microM, and that for isocitrate is 581 microM. The effect of some intermediates of the citric acid cycle and nucleotides on the enzyme activity was studied. The role of isocitrate dehydrogenase (NAD+) in the overproduction of citric and keto acids is discussed.