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H J Phaff

Publications and source records attributed to H J Phaff.

At least 37 records · Page 2Linked to original sources
PubMed1979

Identification of Candida lusitaniae as an opportunistic yeast in humans.

Four yeast strains, causally associated with infection in a patient with acute myelogenous leukemia, were identified by standard methods currently used in yeast taxonomy as representatives of Candida lusitania van Uden et do Carmo-Sousa. Because this species has not been recognized previously as an opportunistic yeast in humans, molecular taxonomic methods were applied to confirm its identity. The nuclear deoxyribonucleic acid (DNA) base composition of two clinical isolates was shown to be 45.1 mol% guanine plus cytosine as compared to 44.7 mol% guanine plus cytosine for the type strain of this species. DNA/DNA reassociation experiments revealed more than 95% complementarity between the DNAs from the clinical isolates and that of the type strain of C. lusitaniae, thus confirming their classification by conventional taxonomy. A key is provided to differentiate C. lusitaniae from two phenotypically similar Candida species.

Blood↗
PubMed1978

Simple method for the isolation of astaxanthin from the basidiomycetous yeast Phaffia rhodozyma.

A method is described for the quantitative and, possibly, large-scale extraction of astaxanthin from the yeast Phaffia rhodozyma. The method utilizes extracellular enzymes produced by the bacterium Bacillus circulans WL-12, which partially digests the yeast cell wall and renders the carotenoid pigments extractable by acetone or ethanol. Complete recovery of astaxanthin from heat-killed P. rhodozyma cells was obtained after growing B. circulans WL-12 on these yeast cells for 26 h and then extracting the yeast-bacterium mixture with acetone. A bacteria-free lytic system, which gave quantitative extraction of astaxanthin from P. rhodozyma, was obtained by concentrating the culture broth from the growth of B. circulans WL-12 on P. rhodozyma cells. Hydrolytic enzyme activities detected in this concentrate included beta-(1 leads to 3)-glucanase, beta-(1 leads to 6)-glucanase, alpha-(1 leads to 3)-glucanase, xylanase, and chitinase. The lytic system was found to work most efficiently at pH 6.5 and with low concentrations of yeast.

Bacillus↗
PubMed1977

Purification and partial characterization of an exo-beta-glucanase from the yeast Kluyveromyces aestaurii.

The intracellular-periplasmic exo-1,3-beta-glucanase (EC 3.2.1.58) has been extracted from the yeast Kluyveromyces aestuarii and purified to immunoelectrophoretic homogeneity by ion-exchange and gel-exclusion chromatography. The kinetic constants and activation energies for laminarin, p-nitrophenyl-beta-D-glucoside, and pustulan have been determined, along with the effect of pH. Evidence is presented indicating that the enzyme is composed of a single polypeptide chain, about 24% carbohydrates, and its molecular weight was estimated to be 43 000.

Ascomycota↗
PubMed1976

Lysis of yeast cell walls. Lytic beta-(1 leads to 6)-glucanase from Bacillus circulans WL-12.

When grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, Bacillus circulans WL-12 produces wall-lytic enzymes in addition to non-lytic beta-(1 leads to 3) and beta-(1 leads to 6)-glucananases. The lytic enzymes were isolated from the culture liquid by adsorption on insoluble yeast glucan in batch operation. After digestion of the glucan, the mixture of enzymes was chromatographed on hydroxylapatite on which the lytic activity could be resolved into one lytic beta-(1 leads to 6)glucanase and two lytic beta-(1 leads to 3)-glucanase was further purified by chromatography over diethylamino-ehtyl-agarose and carboxymethyl cellulose. Its specific activity on pustulan was 6.2 units per mg of protein. The enzyme moved as a single protein with a molecular weight of 54000 during sodium dodecylsulphate electrophoresis in slab gels. Hydrolysis of pustulan went thorugh a series of oligosaccharides, leading to a mixture of gentiotriose, gentiobiose and glucose. The enzyme also produced small amounts of gentiobiose from laminarin and pachyman and on this basis its lytic activity on yeast cell walls,was attribut beta-(1 leads to 3)-linked oligosaccharides were not detected. The lytic beta-(1 leads to 6)-glucanase has an optimum pH of 6.0. Pustulan hydrolysis followed Michaelis-Menten kinetics. A Km of 0.29 mg pustulan per ml and a V of 9.1 micro-equivalents of glucose released/min per mg of enzyme were calculated. The enzyme has no metal ion requirement. The lytic beta-(1 leads to 6)-glucanase differs in essence from the non-lytic beta-(1 leads to 6)-glucanase of the same organism by its positive action on yeast cell walls and yeast glucan and its much lower specific activity on soluble pustulan.

Bacillus↗
PubMed1976

Lysis of yeast cell walls. Lytic beta-(1 leads to 3)-glucanases from Bacillus circulans WL-12.

Bacillus circulans WL-12 when grown in a mineral medium with yeast cell walls or yeast glucan as the soli carbon source, produced five beta-glucanases. Two beta-(1 leads to 3)-glucanases (I and II), which are lytic to yeast cell walls, were isolated from the culture liquid by batch adsorption on yeast glucan, and separated by chromatography on hydroxylapatite. Lytic beta-(1 leads to 3)-glucanase I was further purified by carboxymethylcellulose chromatography. The specific activity of lytic beta-(1 leads to 3)-glucanase I on laminarin was 4.1 U per mg of protein. The enzyme moved as a single protein with a molecular weight of 40000 during sodium dodecylsulfate electrophoresis in slab gels. It was specific for the beta-(1 leads to 3)-glucosidic bond but the enzyme did not hydrolyze laminaribiose. Hydrolysis of laminarin went through a series of oligosaccharides, and laminaribiose and glucose accumulated till the end of the reaction. A small amount of gentibiose was also produced from laminarin. Products from yeast cell walls and yeast glucan included laminaripentaose, laminaritriose, laminaribiose, glucose and gentiobiose, but no laminaritetraose was detected. This glucanase has an optimum pH of 5.5.

Bacillus↗
PubMed1976

Dehiscence and active spore release in pathogenic strains of the yeast Metschnikowia bicuspidata var. australis: possible predatory implication.

Strains of Metschnikowia bicuspidata var. australis, pathogenic to brine shrimp (Artemia salina), were observed to form asci which, upon reaching maturity, forcibly expelled their needle-shaped spores. The mechanical force responsible apparently originates from the formation of an ectoplasmic mucilage capable of exerting pressure over all of the ascus contents; when the apex of the peduncle ruptures, the ascospores are violently released. Cytochemical analyses indicated that the gel is a substance highly resistant to chemical and enzymatic hydrolysis. Its chemical nature is not known as yet. The morphogenetic events of this process are described, and its ecological implication, the possibility of active mechanical predation in yeast, is discussed.

Aldehyde-Lyases↗
PubMed1975

Glucanases in Schizosaccharomyces. Isolation and properties of an exo-beta-glucanase from the cell extracts and culture fluid of Schizosaccharomyces japonicus var. versatilis.

(11 Cell extracts and extracellular culture fluids of species of the yeast genus Schizosaccharomyces exhibited exo-beta-(1 leads to 3)- and exo-beta-(1 leads to 6)-glucanase (EC 3.2.1.-) activities. (2) Using a combination of Sephadex G-100 and DEAE-cellulose chromatography, the exo-beta-(1 leads to 3)-glucanases from the cell extracts and culture fluid of Schizosaccharomyces japonicus var. versatilis were purified extensively. The enzymes from either location exhibited similar purification and other properties. (3) The purified enzymes hydrolysed the beta-(1 leads to 6)-glucosidic linkage in addition to the beta-(1 leads to 3) linkage. Heat denaturation, inhibition and electrophoretic studies indicated that both hydrolytic activities were properties of a single protein. Laminarin and pustulan hydrolysis followed Michaelis-Menten kinetics. The Km and V for laminarin hydrolysis were 6.25 mg/ml and 350 mumol of glucose released/min/mg protein, and for pustulan they were 166 mg/ml and 52 mumol of glucose released/min/mg protein. (4) The exo-beta-glucanase was assigned a molecular weight of 43 000. (5) the purified enzyme failed to hydrolyse isolated cell walls from either baker's yeast or Schizosaccharomyces pombe or to induce protoplast formation from intact cells of S. japonicus var. versatilis or Saccharomyces cerevisiae.

Ascomycota↗
PubMed1974

Demonstration of a fibrillar component in the cell wall of the yeast Saccharomyces cerevisiae and its chemical nature.

The ultrastructure of isolated cell walls of Saccharomyces cerevisiae from the log and stationary phases of growth was studied after treatment with the following enzymes: purified endo-beta-(1 --> 3)-glucanase and endo-beta-(1 --> 6)-glucanase produced by Bacillus circulans; purified exo-beta-glucanase and endo-beta-(1 --> 3)-glucanase produced by Schizosaccharomyces versatilis; commercial Pronase. While exo-beta-glucanase from S. versatilis had no electron microscopically detectable effect on the walls, Pronase removed part of the external amorphous wall material disclosing an amorphous wall layer in which fibrils were indistinctly visible. Amorphous wall material was completely removed by the effect of either endo-beta-(1 --> 3)- or endo-beta-(1 --> 6)-glucanase of B. circulans or by a mixture of the two enzymes. As a result of these treatments a continuous fibrillar component appeared, composed of densely interwoven microfibrils resisting further action by both of the B. circulans enzymes. The fibrillar wall component was also demonstrated in untreated cell walls by electron microscopy after negative staining. Because of the complete disappearance of the fibrils following treatment with the S. versatilis endo-beta-(1 --> 3)-glucanase it can be concluded that this fibrillar component is composed of beta-(1 --> 3)-linked glucan. Bud scars were the only wall structures resistant to the effect of the latter enzyme.

Ascomycota↗
PubMed1974

Deoxyribonucleic acid base composition and hybridization studies on the human pathogen Sporothrix schenckii and Ceratocystis species.

Deoxyribonucleic acid (DNA) base composition, as measured by guanine plus cytosine (G + C) content, was determined in 17 strains of Sporothrix schenckii and Ceratocystis species. The average G + C content for S. schenckii was 54.7 +/- 0.2 mol%. No strain of Ceratocystis, with the exception of C. minor, gave values for G + C content similar to those obtained for S. schenckii. All DNA samples from strains of S. schenckii cross-hybridized with high percentages of relative binding. DNA samples from C. stenoceras and C. pilifera showed low degrees of homology with S. schenckii DNA. The DNA from C. minor hybridized with 75% binding with that from a strain of S. schenckii, as compared with the homologous reaction.

Ascomycota↗
PubMed1974

Lysis of yeast cell walls: glucanases from Bacillus circulans WL-12.

Endo-beta-(1 --> 3)- and endo-beta-(1 --> 6)-glucanases are produced in high concentration in the culture fluid of Bacillus circulans WL-12 when grown in a mineral medium with bakers' yeast cell walls as the sole carbon source. Much lower enzyme levels were found when laminarin, pustulan, or mannitol was the substrate. The two enzyme activities were well separated during Sephadex G-100 chromatography. The endo-beta-(1 --> 3)-glucanase was further purified by diethylaminoethyl-cellulose and hydroxyapatite chromatography, whereas the endo-beta-(1 --> 6)-glucanase could be purified further by diethylamino-ethyl-cellulose and carboxymethyl cellulose chromatography. The endo-beta-(1 --> 3)-glucanase was specific for the beta-(1 --> 3)-glucosidic bond, but it did not hydrolyze laminaribiose; laminaritriose was split very slowly. beta-(1 --> 4)-Bonds in oat glucan in which the glucosyl moiety is substituted in the 3-position were also cleaved. The kinetics of laminarin hydrolysis (optimum pH 5.0) were complex but appeared to follow Michaelis-Menten theory, especially at the lower substrate concentrations. Glucono-delta-lactone was a noncompetitive inhibitor and Hg(2+) inhibited strongly. The enzyme has no metal ion requirements or essential sulfhydryl groups. The purified beta-(1 --> 6)-glucanase has an optimum pH of 5.5, and its properties were studied in less detail. In contrast to the crude culture fluid, the two purified beta-glucanases have only a very limited hydrolytic action on cell wall of either bakers' yeast or of Schizosaccharomyces pombe. Although our previous work had assumed that the two glucanases studied here are responsible for cell wall lysis, it now appears that the culture fluid contains in addition a specific lytic enzyme which is eliminated during the extensive purification process.

Ascomycota↗
PubMed1972

Deoxyribonucleic acid base composition of species in the yeast genus Kluyveromyces van der Walt emend. van der Walt.

The deoxyribonucleic acid base composition (percent guanine + cytosine [GC]) was determined for 29 strains, representing 18 species of the genus Kluyveromyces. It was concluded that on the basis of GC content (47.4%) and other properties K. veronae occupies an uncertain position in the genus Kluyveromyces. The GC content of the remaining 17 species ranged from 35.3 to 43.4%, and three groups of species were recognized. The GC content of the first ranged from 35.3 to 38.0%; that of the second group from 39.5 to 41.7%; that of the third group from 42.4 to 43.4%. Several species revealed a nearly identical GC content. The GC contents do not correspond in all instances with the five groups of species proposed by van der Walt.

Acetone↗
PubMed1970

Rhodotorulic acid from species of Leucosporidium, Rhodosporidium, Rhodotorula, Sporidiobolus, and Sporobolomyces, and a new alanine-containing ferrichrome from Cryptococcus melibiosum.

An examination of 142 strains within 19 genera of yeasts and yeastlike organisms for formation of hydroxamic acids in low-iron culture showed production of hydroxamates by two unclassified strains and by 52 strains among the genera Aessosporon (3 of 3 strains), Cryptococcus (1 of 43), Leucosporidium (3 of 11), Rhodosporidium (4 of 4), Rhodotorula (27 of 39), Sporidiobolus (2 of 2), and Sporobolomyces (12 of 13). Crystalline rhodotorulic acid was isolated in amounts sufficient to account for most or all of the measured hydroxamate in culture supernatants of 16 strains representative of the five last-mentioned hydroxamate-producing genera. A new alanine-containing ferrichrome was isolated from one strain of Cryptococcus melibiosum. Rhodotorulic acid was a major metabolic product of many of the positive strains when grown in low-iron media, and iron was shown to repress its synthesis and excretion into the culture medium. The taxonomic significance of production of hydroxamic acids is described in connection with the position of these yeast species in the subclass Heterobasidiomycetidae.

Alanine↗