H. J. Phaff

Studies on polygalacturonase of certain yeasts

Abstract 1. 1. Among a considerable number of yeast species, representing nearly all yeast genera, only six were found which were capable of causing certain changes in pectin, when grown in its presence. These six cultures could all be identified with Saccharomyces fragilis (and a variety of this species) and its imperfect form Candida pseudotropicalis , as well as certain varieties of the latter. (See Discussion section.) These organisms cannot use pectin as a source of carbon, but require an added sugar for development. 2. 2. S. fragilis var. No. 351 was used for most of the studies reported herein. When it was grown in the presence of pectin, clarification of the liquid culture media took place with the formation of an amorphous precipitate consisting of about 1% of the pectin added. The jellying power of the pectin is destroyed, but the amount of calcium pectate and alcohol precipitate, obtainable from a clarified solution, is practically unchanged as compared to control solutions. 3. 3. The action was found to be due to an exocellular, nonadaptive, polygalacturonase-like enzyme, free of pectinesterase. The action of this yeast polygalacturonase is characteristic in that pectic acid, when used as a substrate, is only partially hydrolyzed. In contrast to fungus polygalacturonase, the enzyme cannot hydrolyze pectic acid completely to monogalacturonic acid. The velocity constant of the (initially) first-order reaction was found to be 0.196 at 22 °C. and at a pH of 3.40. The optimum pH was approximately 3.5–4.0, and the maximum rate was in the region of 55–60 °C.

Properties of yeast polygalacturonase

Abstract Various properties were studied of the exocellular polygalacturonase of the yeast Saccharomyces fragilis (strain #351). Yeast polygalacturonase (YPG) was partially purified, until the activity was 2.03 (PGu)mgTN. YPG is not accompanied by pectinesterase. The initial rate of pectic acid hydrolysis was about 158 times as fast as that of pectin (N.F.). Up to about 0.00147 PGu/ml. of pectic acid reaction mixture, the initial velocity of hydrolysis was proportional to the enzyme concentration. The extent of hydrolysis of pectinic acids of increasing methoxyl content (prepared by alkali-demethoxylation of pectin) was an inverse linear function of the methoxyl content. By addition of purified orange pectinesterase to YPG in the presence of Mg ++ , the initial rate of pectin hydrolysis approached that of pectic acid hydrolysis. The maximum extent of hydrolysis of pectic acid was found to be about 48% (based on polyuronide content). The optimum pH was found to be 4.4. YPG was found to be less contaminated with other hydrolases than fungal PG preparations, and on the basis of experience with a limited number of substrates was found to be specific for pectic acid. Of a number of enzyme inhibitors, only nitrous acid showed significant inhibitory action on YPG.

THE ASSOCIATION OF YEASTS WITH CERTAIN BARK BEETLES

There is now a vast literature describing various aspects of the rela? tionship between insects and microorganisms (see e.g. Steinhaus, 1946). Taxonomic studies on yeasts associated with bark beetles began when Beck (1922) described a new species of yeast, Endomyces bisporus, which she isolated repeatedly from the galleries of Ips typographus attacking spruce in Austria. Siemaszko (1939) also noted this yeast associated with Ips typographus in Picea excelsa in Poland. StellingDekker (1931) renamed this yeast Endomycopsis bispora, because it forms blastospores besides true mycelium. It was finally renamed Hansenula beckii (Beck) by Wickerham (1951) since it utilizes nitrate-ion as a single source of nitrogen, a typical characteristic for species of Hansenula. Lodder and van Rij (1952), however, feel that the yeast should be maintained in Endomycopsis in spite of the fact that it is nitrate positive, because of its abundant production of true mycelium. It may be well to point out that all studies on this yeast were done with the original isolate of Beck. The only other report in the literature, which describes the occurrence of this yeast in Ips typographus, is a paper by Grosmann (1930). Her insect material was collected from two well separated areas in central Germany. Beside E. bisporus, she found various wood staining fungi and another yeast producing hatshaped spores (2-4 per ascus) which dehisce early. This yeast did not produce mycelium or pseudomycelium. The possibility was considered that this yeast might belong to Hansenula. A third type did not pro? duce spores. Grosmann also found yeasts in the intestinal tract of adults and larvae and in fresh excreta. The view was expressed that the yeasts are not essential for the growth of the insects, but merely live in association with them as commensals. Rumbold (1931) and Leach, Orr and Christensen (1934) also noted the regular occurrence of yeasts and yeast-like organisms in association with bark beetles and bluestaining fungi. Somewhat later Holst (1936) showed that a new spe? cies of yeast, which was named Zygosaccharomyces pini, could be iso? lated regularly from phloem galleries and the bark beetles Dendroctonus and Ips from various parts of the United States. This yeast, lacking

The preparation of tetragalacturonic acid

Abstract Pectic acid was subjected to partial hydrolysis by yeast polygalacturonase which was halted when the oligouronides larger than tetragalacturonic acid had disappeared, as shown paper-chromatographically. From this hydrolyzate, tetragalacturonic acid was isolated in 12.7% yield by fractional precipitation with cupric ions. Various properties of the acid were determined.

Carotenoids in asporogenous yeasts

Summary1.The carotenoid pigments of a number of species ofRhodotorula andCryptococcus have been investigated.2.In both genera, so-called yellow yeasts are found which contain β-carotene as the principal pigment.3.The β-carotene content of species ofCryptococcus varies from nearly imperceptible amounts to 10 mg per 100 g dry yeast.4.β-carotene is the main pigment ofR. peneaus. The concentration of this pigment is greatly decreased when this yeast is grown at 5°C.5.The type species ofR. rubra was found to contain torularhodin, torulin, and β-and γ-carotene as the principal pigments. The composition of the pigments was quantitatively and qualitatively nearly the same, whether the yeast was cultured at 5° or at 25°C.6.Certain strains of the redR. glutinis appear yellow when grown at 5°C. Under these conditions, the proportion of carotene increased appreciably, and torularhodin and torulin content fell correspondingly.7.The taxonomic significance of carotenoid pigments for genus differentiation in the asporogenous yeasts is questioned.

End products and mechanism of hydrolysis of pectin and pectic acid by yeast polygalacturonase (YPG)

Abstract 1. 1. The change in molecular weight of pectin (N.F.) due to the action of YPG has been studied. The weight-average molecular weight decreased from 56,200 to 15,100, and the number-average molecular weight dropped from 26,000 to 11,000. Good agreement was found between the extent of breakdown based on increase in reducing value and decrease in weightaverage molecular weight. 2. 2. At pH 5.0, pectic acid was found to be randomly hydrolyzed to a mixture of mono-, di-, and trigalacturonic acids. A procedure is described for the isolation of the last two acids in pure form. 3. 3. The naphthoresorcinol reagent is largely specific for monogalacturonic acid. In samples containing 50 μg., digalacturonic acid produces 7% of the color produced by monogalacturonic acid, trigalacturonic acid 3.5%, whereas tetragalacturonic acid and pectic acid give the same color as the blank. In mixtures with monogalacturonic acid, the interference appears to be somewhat less. 4. 4. During the initial very rapid rate of hydrolysis of pectic acid, in which about 25% of the glycosidic bonds are hydrolyzed, monogalacturonic acid does not appear in significant quantities as an end product. During the second slow phase in which the per cent hydrolysis increases to about 48, monogalacturonic acid formation roughly parallels the splitting of glycosidic bonds.

The fermentation of trehalose by yeasts and its taxonomic implications.

SUMMARY: In the classification of yeasts it is customary to use an infusion of bakers’ yeast as the basal medium for fermentation tests. This extract frequently contains variable amounts of trehalose. A number of yeasts were observed to ferment yeast extract and trehalose. The fermentation of yeast extract is serious from a taxonomic point of view, since it gives the impression of positive fermentation of a sugar which actually may not be fermentable. Dilute yeast autolysate should be used as the basal fermentation medium since during autolysis trehalose is destroyed. The fermentation of yeast extract (without added sugar) is easily observed in Durham tubes by the collection of gas in the inserts, but when Einhorn fermentation tubes are used gas production is seldom apparent. One culture (N-18) isolated from spoiled apricots, and identified as Candida tropicalis showyed adaptive trehalose fermentation. The ability of various yeasts to ferment trehalose was investigated, using 133 cultures, representing twenty genera and seventy-three species. Sixteen species representing seven genera fermented yeast extract and trehalose. The fermentation of trehalose is worthy of consideration as a character for use in differentiating certain species of Candida, and perhaps other yeasts.

On the cell wall composition of the apiculate yeasts

SummarySonic oscillation was used for the purpose of obtaining clean, chemically intact cell walls. The rate of disruption was determined for cells ofHanseniaspora uvarum andSaccharomyces cerevisiae.The carbohydrate fractions of cell walls ofHanseniaspora uvarum, H. valbyensis, Kloeckera apiculata, Saccharomycodes ludwigii andSaccharmyces cerevisiae were shown to be similar. Chromatography of cell wall hydrolysates of all these species demonstrated that glucose and mannose were the only sugars present (in about equal amounts) besides traces of glucosamine.The cell walls ofH. uvarum contained 78.1 per cent carbohydrates, 7 per cent protein and approximately 0.05 per cent of chitin. Fractionation of the polysaccharides lead to a recovery of 83.3 per cent of the carbohydrates present (30.4 per cent glucan and 34.9 per cent mannan). Saccharomyces cerevisiae cell walls were found to have a carbohydrate content of 82.8 per cent, 6.5 per cent protein and a trace of chitin (0.04 per cent). Nadsonia elongata contained a relatively large amount of chitin (ca. 5 per cent) and lacked mannan in its cell walls.It was concluded thatHanseniaspora andSaccharomycodes are closely related to theSaccharomyceteae but they have little in common with species ofNadsonia.

Characteristics of trehalase inCandida tropicalis

SummaryThe trehalase content of different yeasts varies widely. A strain ofCandida tropicalis was found to be the best source of this enzyme among the yeasts tested. The trehalase activity in this yeast could be increased 8.5 times by growing it on trehalose rather than glucose. Thus trehalase is an adaptive enzyme inC. tropicalis.It was found that the amount of trehalase which could be solubilized increased with increasing pH during autolysis of the cells, none being released from the cell debris at pH 4.5 and most at pH 6.3. Some evidence was obtained to show that the solubilization was caused by an enzyme. The stability of trehalase under various conditions was studied. A partial purification was achieved by precipitation with 40% ethanol at a temperature of −18°C. The maximum temperature of the enzyme was 48°C., and the optimum pH ranged from 4.1 to 5.3