Pichia Pinus Research Articles

Use of formate gradients for improving biomass yield of Pichia pinus growing continuously on methanol

Investigations were made into the improvement of growth yield (Y) of Pichia pinus MH 4 growing continuously on methanol by feeding formate so as to create an increasing concentration gradient (transient state). Under particular formate supply conditions, Y could be increased from 0.37 g·g-1 on methanol alone to 0.55 and 0.47 g·g-1 in the presence of formate at dilution rates (D) of 0.045 and 0.075 h-1, respectively. These differences could be explained as being due to a limiting formate consumption rate of 50–60 nmol·min-1·g-1 dry wt., coupled to a net-energy generation independent of D. Any further formate oxidation proceeded without energy gain. Deviations from optimum conditions of biomass increase are discussed in terms of different formate oxidizing systems and uncoupling properties of formate itself. These results are compared to and confirmed by steady-state considerations.

Threonine dehydratase activity from several yeast species is activated and affected by phosphate

The threonine dehydratase activity of several yeast species was examined. Of 14 species tested, Candida maltosa, Pichia guilliermondii, Pichia pinus and Yarrowia lipolytica were found to produce an enzyme whose activity was increased about 20-fold in the presence of phosphate ions. It is suggested that phosphate affects the allosteric properties of the enzyme and leads, thereby, to an alteration of the binding of threonine (substrate), isoleucine (negative effector), and valine (positive effector) as well as to an alteration of the effects of temperature on the enzyme including changes in temperature optimum, energy of activation, and heat inactivation.

Threonine dehydratase activity from several yeast species is activated and affected by phosphate

The threonine dehydratase activity of several yeast species was examined. Of 14 species tested, Candida maltosa, Pichia guilliermondii, Pichia pinus and Yarrowia lipolytica were found to produce an enzyme whose activity was increased about 20-fold in the presence of phosphate ions. It is suggested that phosphate affects the allosteric properties of the enzyme and leads, thereby, to an alteration of the binding of threonine (substrate), isoleucine (negative effector), and valine (positive effector) as well as to an alteration of the effects of temperature on the enzyme including changes in temperature optimum, energy of activation, and heat inactivation.

Quantification of different yeasts associated with the bark beetle,Ips typographus, during its attack on a spruce tree

There were different amounts and types of yeasts associated with individuals ofIps typographus spruce bark beetles during different phases of their attack on a healthy spruce tree. The yeasts were isolated on Sabouraud agar medium in order to identify them and estimate their numbers.Hansenula holstii andCandida diddensii type yeasts were most frequently isolated. The increase in number of these two yeast types probably accounted for most of the total yeast increase found during the later attack phases of the bark beetles. Lesser amounts ofHansenula capsulata, Pichia pinus, Candida nitratophila, and twoCryptococcus type yeasts were also found.

Serological differences between the multiple amine oxidases of yeasts and comparison of the specificities of the purified enzymes from Candida utilis and Pichia pastoris.

1. Antiserum to purified methylamine oxidase of Candida boidinii formed precipitin lines (with spurs) in double-diffusion tests with crude extracts of methylamine-grown cells of the following yeast species: Candida nagoyaensis, Candida nemodendra, Hansenula minuta, Hansenula polymorpha and Pichia pinus. No cross-reaction was observed with extracts of Candida lipolytica, Candida steatolytica, Candida tropicalis, Candida utilis, Pichia pastoris, Sporobolomyces albo-rubescens, Sporopachydermia cereana or Trigonopsis variabilis. Quantitative enzyme assays enabled the relative titre of antiserum against the various methylamine oxidases to be determined. 2. The amine oxidases from two non-cross-reacting species, C. utilis and P. pastoris, were purified to near homogeneity. 3. The methylamine oxidases, despite their serological non-similarity, showed very similar catalytic properties to methylamine oxidase from C. boidinii. Their heat-stability, pH optima, molecular weights, substrate specificities and sensitivity to inhibitors are reported. 4. The benzylamine oxidases of C. utilis and P. pastoris both oxidized putrescine, and the latter enzyme failed to show any cross-reaction with antibody to C. boidinii methylamine oxidase. Benzylamine oxidase from C. boidinii itself also did not cross-react with antibody to methylamine oxidase. The heat-stability, molecular weights, substrate specificities and sensitivity to inhibitors of the benzylamine/putrescine oxidases are reported. 5. The benzylamine/putrescine oxidase of C. utilis differed only slightly from that of C. boidinii. 6. Benzylamine/putrescine oxidase from P. pastoris differed from the Candida enzymes in heat-stability, subunit molecular weight and substrate specificity. In particular it catalysed the oxidation of the primary amino groups of spermine, spermidine, lysine, ornithine and 1,2-diaminoethane, which are not substrates for either of the Candida benzylamine oxidases that have been purified. 7. Spermine and spermidine were oxidized at both primary amino groups; in the case of spermidine this is a different specificity from that of plasma amine oxidase. 8. Under appropriate conditions, P. pastoris benzylamine/putrescine oxidase (which is very easy to purify) can be a useful analytical tool in measuring polyamines.

Yeast mannans inhibit binding and phagocytosis of zymosan by mouse peritoneal macrophages.

We have examined the effects of various mannans, glycoproteins, oligosaccharides, monosaccharides, and sugar phosphates on the binding and phagocytosis of yeast cell walls (zymosan) by mouse peritoneal macrophages. A phosphonomannan (PO(4):mannose ratio = 1:8:6) from kloeckera brevis was the most potent inhibitor tested; it inhibited binding and phagocytosis by 50 percent at concentrations of approximately 3-5 mug/ml and 10 mug/ml, respectively. Removal of the phosphate from this mannan by mild acid and alkaline phosphatase treatment did not appreciably reduce its capacity to inhibit zymosan phagocytosis. The mannan from saccharomyces cerevisiae mutant LB301 inhibits phagocytosis by 50 percent at 0.3 mg/ml, and a neutral exocellular glucomannan from pichia pinus inhibited phagocytosis by 50 percent at 1 mg/ml. Cell wall mannans from wild type S. cervisiae X2180, its mnn2 mutant which contains mannan with predominantly 1(arrow)6- linked mannose residues, yeast exocellular mannans and O-phosphonomannans were less efficient inhibitors requiring concentrations of 1-5 mg/ml to achieve 50 percent reduction in phagocytosis. Horseradish peroxidase, which contains high-mannose type oligosaccharides, was also inhibitory. Mannan is a specific inhibitor of zymosan binding and phagocytosis. The binding and ingestion of zymosan but not of IgG- or complement-coated erythrocytes can be obliterated by plating macrophages on substrates coated with poly-L-lysin (PLL)-mannan. Zymosan uptake was completely abolished by trypsin treatment of the macrophages and reduced by 50-60 percent in the presence of 10 mM EGTA. Pretreatment of the macrophages with chloroquine inhibited zymosan binding and ingestion. These results support the proposal that the macrophage mannose/N-acetylglucosamine receptor (P. Stahl, J.S. Rodman, M.J. Miller, and P.H. Schlesinger, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:1399-1403, mediates the phagocytosis of zymosan particles.

Genetic control of cell type and complex organization of the mating type locus in the yeast Pichia pinus

Genetic mechanisms of switching the mating type locus MAT1 in the homothallic yeast Pichia pinus were studied. By analysis of mutations affecting MAT1 complex structural and functional organization of this locus was shown. The existence of two functional regions in MAT1 is postulated. Region I controls mating ability of haploid cells, determines the neutrality of heterozygous cells and regulates the work of Region II. Region II controls meiosis and/or sporulation in the cultures heterozygous for Region I as well as controls switching MAT1 ⇋ MAT1α in haploid cells.

DISTRIBUTION OF UREASE AND EXTRACELLULAR DNASE IN YEAST SPECIES

Urease and extracellular DNase activities were tested in 292 strains belonging to 125 species of 33 genera. Basidiomycetous yeasts and related species had both urease and extracellular DNase. Species belonging to the genera Rhodotorula and Cryptococcus, and four species of Candida (Candida curvata, Candida humicola, Candida bogoriensis, and Candida diffluens) which are considered to be closely related to basidiomycetous yeasts, showed both activities. In general, ascosporogenous yeasts and their imperfect forms did not have either urease or extracellular DNase. Hansenula bimundalis and Saccharomycopsis lipolytica showed both enzymes, but the activities were weaker than those of basidiomycetous yeasts. Schizosaccharomyces pombe, Schizosaccharomyces octosporus, Citeromyces matritensis, Lipomyces starkeyi, and Nadsonia elongata had urease but not extracellular DNase. Saccharomycopsis fibuligera, Hansenula jadinii, Hansenula minuta, Pichia pinus, Pichia rhodanensis, and Candida pseudotropicalis showed extracellular DNase but not urease. Taxonomic significance of urease and extracellular DNase is discussed.

Management of forest residues for rapid decay

Forest residues of two age classes (less than 1 year old and between 1 and 2 years old) were chipped and piled (1.0- to 1.5-m piles) on the cutting site. Chip piles were inoculated with decay fungi [Coriolus versicolor (L. ex Fr.) Quél., Hirschioporus abietinus (Dicks ex Fr.) Donk, or Poria placenta (Fr.) Cke.] alone and in combination with a liquid suspension of yeasts [Saccharomyces bailii Linder var. bailii and Pichia pinus (Holst) Phaff] and nitrogen-fixing bacteria (Enterobaeter spp.). Temperatures were recorded at the surface, middle, and bottom of each chip pile. Samples taken 16 months after inoculation revealed successful colonization and proliferation of the fungal inocula. Postinoculation compression tests showed that significantly more decomposition occurred in wood chips inoculated with wood-destroying fungi than those receiving no inoculation. Even greater losses of wood integrity occurred when the fungi were inoculated in combination with yeasts and bacteria. Morphological examination of wood chips using scanning electron microscopy confirmed successful colonization and glucosamine assays confirmed proliferation of the fungal inoculum in the chip pile.

S-formylglutathione: The substrate for formate dehydrogenase in methanol-utilizing yeasts

Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The Km values of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathione-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a Km of 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (Km of 40 mM) but a relatively high affinity for S-formylglutathione (Km of 1.1 mM). The Km values of formate dehydrogenase in cell-free extracts of methanol-grown Candida biodinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts.

Some physiological observations on the uptake of D-glucose and 2-deoxy-D-glucose by starving and exponentially-growing yeasts.

Some methods for measuring the uptake of sugars by yeasts were investigated critically. A study was made of the effects of starvation of Pichia pinus, Candida utilis, Saccharomyces cerevisiae and Rhodosporidium toruloides on their uptake of D-glucose and 2-deoxy-D-glucose. Marked changes in the rates of uptake of these sugars occured during 10 h of starvation, including (a) an immediate increase of up to 75% above that for growing cells and (b) a continuous decline to as little as 4%. Each yeast behaved differently. The rates did not remain constant during the periods of starvation often used for studies on the transport of sugars into yeasts. For Pichia pinus, there were striking differences, associated with starvation, between the transport of 2-deoxy-D-glucose and D-glucose, despite evidence that the two sugars enter this yeast by means of the same carrier. Some physiological explanations for these findings are discussed.

The entry of D-ribose into some yeasts of the genus Pichia.

The utilization of D-ribose by yeasts of the genus Pichia was examined with respect to aerobic growth, respiration and entry of ribose into the cells. Pichia etchellsii (CBS2011) could respire D-ribose, but not use it for aerobic growth. Pichia fermentans (CBS187) neither respired nor grew on D-ribose, though it entered the cells of this yeast either by simple diffusion, or possibly, by the D-glucose carrier, this having a very low affinity for D-ribose. Pichia pinus (CBS5097) respired and grew on D-ribose; kinetic evidence is given for this yeast having two ribose carriers, one inducible and the other constitutive.

Carbohydrate binding specificity of the lectin from the PEA (Pisum sativum)

Hapten inhibition measurements on the precipitin reaction beteeen Pisum sativum lectin and Pichia pinus phosphomannan showed the lectin to bind d-mannose, d-glucose, d-fructose and l-sorbose. Unmodified hydroxyl groups at the C-4 and the C-6 positions of the d-glucopyranose ring were essential for binding to the protein. Modification of the C-2 hydroxyl group was allowed in the d-glucopyranose ring, but not in the d-mannopyranose configuration. Substitution of the hydroxyl hydrogen atom at the C-3 position of d-glucose increased the binding efficiency. With the exception of gentiobiose, the β-linked glycobioses tested were not bound to the lectin, whereas the α-linked glycobioses were potent inhibitors.In general, the P. sativum lectin was found to be less sensitive to structural variation of inhibiting carbohydrates than concanavalin A, the lectin from Canavalia ensiformis.