Š. Bauer

Extracellular β-glucanases of the yeast, Saccharomyces cerevisiae

Abstract Fractionation of proteins secreted by protoplasts and intact cells of Saccharomyces cerevisiae by DEAE-cellullose chromatography in 0.05 M phosphate buffer (pH 7) using a linear gradient of NaCl revealed the presence of at least three different fractions hydrolyzing laminarin (β-1,3-glucan). The first fraction, not retained by the column, appeared to consist of two enzymes exhibiting activity for laminarin, p- nitrophenyl -β- d -glucopyranoside , pustulan (β-1,6-glucan) and a low but detectable activity for laminarin treated with NaIO4. The second fraction, eluted from the DEAE-cellulose column at 0.23 M NaCl possessed the substrate specificity of an endo-β-1,3-glucanase. It exhibited the maximum activity at pH 5.5 in 0.1 M acetate buffer. The third fraction, eluted from the column at 0.35 M NaCl, was an unspecific exo-β-glucanase hydrolyzing terminally both laminarin and pustulan. The pH optimum of the latter enzyme was in the range 4.5–5.0 with laminarin, p- nitrophenyl -β- d -glucopyranoside or pustulan as substrates. The β-glucanases found extracellularly were compared with those occurring in protoplast lysates and cell-free extracts of intact cells.

Extracellular polysaccharide-protein complexes produced by selected strains ofCandida albicans (Robin) Berkhout

Water-soluble extracellular polysaccharide-protein complexes of a molecular weight of about 200,000 were isolated from glucose-containing nutrient media after cultivation of slightly, moderately and highly virulent strains ofCandida albicans (Robin) Berkhout. Chemical, physico-chemical and immunological properties of these compounds were studied. The complexes contain 74–86% of mannose, 21–31% of glucose, 1–1.5% of glucosamine and 11–14% of proteins built from 17 aminoacids. The polysaccharideprotein complexes are immunologically active and differ from each other by their precipitation ability if added to specific antisera prepared against moderately highly virulentCandida albicans strains.

Xylan-degrading activity in yeasts: growth on xylose, xylan and hemicelluloses.

The ability to grow in liquid media withD-xylose, xylan from deciduous trees, and hemicelluloses from conifers was tested in 95 strains of 35 genera of yeasts and yeast-like organisms. Of 54 strains thriving on xylose, only 13 (generaAureobasidium, Cryptococcus andTrichosporon) utilized xylan and hemicelluloses as growth substrates. The árowth media of these strains were found to contain xylandegrading enzymes splitting the substrate to xylose and a mixture of xylose oligosaccharides. The ability of these yeasts to utilize the wood components (hitherto unknown in the genusCryptococeus) makes them potential producers of microbial proteins from industrial wood wastes containing xylose oligosaccharides, xylan, and hemicelluloses as the major saccharide components without previous saccharification.

Metabolism of 2-deoxy-D glucose by Baker's yeast. IV. Incorporation of 2-deoxy-D-glucose into cell wall mannan.

Abstract 1. 1. Saccharomyces cerevisiae grown in the presence of 2-deoxy- d -glucose incorporate this glucose and mannose analogue into cell wall polysaccharides. Fractionation of cell walls to mannan- and glucan-containing fractions followed by analysis for glucose, mannose and deoxyglucose showed that deoxyglucose was incorported mainly, if not exclusively, into cell wall mannan. 2. 2. Mild acid hydrolysis of the mannan-containing fraction isolated from deoxyglucose-grown cells afforded besides free deoxyglucose two oligosaccharides containing both mannose and deoxyglucose. They were tentatively identified as 3-O-α, d -mannopyranosyl-2-deoxy- d -glucose and a trisaccharide containing the former disaccharide and an additional mannose residue at non reducing terminal. 3. 3. The results demonstrate direct interaction of deoxyglucose metabolites with the enzyme system of Saccharomyces cerevisiae responsible for the cell-wall mannan biosynthesis.

Preparation of mutants of Trichoderma viride with increased production of cellulase

Four mutant strains exhibiting increased production of cullulases were prepared by UV irradiation of conidia ofTrichoderma viride QM 9414. Selected mutants were tested for production of cellulases in submerged cultivations in shake flasks and in a 30-L fermentor in a synthetic medium containing 1 % microcrystaline cellulose as the carbon source. Some mutants showed considerable morphological differences when compared to the parent strain, the most noticeable being a higher degree of branching of the mutant hyphae. The branched mutants produced 2 to 3 times higher levels of β-glucosidase than the parent strain QM 9414.

Transglycosylic reactions of nucleotides of 2-deoxy-sugars II. 2-deoxyglucose incorporation into glycogen☆

Abstract • The kinetics of 2-deoxy- d -glucose (dGlc) incorporation into glycogen was investigated in a reaction catalyzed with yeast glycogen synthetase (UDPG-glycogen glucosyl-transferase, EC 2.4.I.II). The Michaelis constant Km found for UDPdGlc as a substrate was 5.6 mM, whereas the Km for UDPG was 1.2 mM. Maximal velocity νmax for dGlc incorporation into glycogen was 26% of the value for glucose incorporation. • UDPdGlc acts as a competitive inhibitor in the reaction of glycogen biosynthesis from UDPG. The apparent inhibition constant Ki for UDPdGlc was found to be 1.1 mM. • Glycogen containing incorporated dGlc is a substrate for β-amylase. Products of β-amylolysis were isolated and identified as maltose, monodeoxymaltose (2-deoxy- d -glucopyranosyl-α(1→4)- d -glucopytanose) and dideoxymaltose (2-deoxy- d -glucopyranosyl)-α(1→4)-2-deoxy- d -glucopyranose). The formation of both dGlc containing disaccharides in the course of β-amylolysis confirmed that more than one dGlc residue is transferred successively per glycogen chain in the reaction with glycogen synthetase.

Biosynthesis of yeast mannan. Diversity of mannosyltransferases in the mannan-synthesizing enzyme system from yeast.

1. A microsomal enzyme preparation from the yeast Saccharomyces cerevisiae catalyzes the transfer of mannosyl units from GDPmannose to mannose and a number of mannose-containing oligosaccharides and glycosides whereby different glycosidic bonds are formed. 2. Of the compounds tested besides mannose, only those containing an alpha-linked mannosyl unit at the nonreducing position of their molecule were effective as acceptors. Monodeoxyanalogues of mannose as well as alpha-mannose phosphates did not serve as acceptors in the above reaction. 3. The structure of the product formed with mannose as acceptor was determined to be O-alpha-D-mannosyl-(1 leads to 2)-mannose; with alphaMan (1 leads to 6)mannose as the acceptor, the product was alphaMan(1 leads to 6)mannose and with alphaMan-(1 leads to 2)mannose the product was tentatively characterized as a mixture of alphaMan-(1 leads to 3)alphaMan(1 leads to 2)mannose and alphaMan(1 leads to 2)alphaMan(1 leads to 2)mannose. 4. The enzymes catalyzing the formation of different types of glycosidic bonds differed in their acceptor specificity, pH-activity curves and rates of heat denaturation. 5. Radioactive disaccharides were unable to enter the mannan protein molecule in the cell-free system while free radioactive mannose did incorporate into polysaccharide to a minor extent under the same conditions.

Characterization of cellulolytic enzyme complexes obtained from mutants of Trichoderma reesei with enhanced cellulase production

SummaryThe cellulolytic enzyme complexes secreted by the fungus Trichoderma reesei QM 9414 and its mutants M 5, M 6, MHC 15, and MHC 22 were characterized by determining their specific filter-paper (FP)-, carboxymethylcellulase (Cx)-and β-glucosidase (βG)-activities. They were characterised further by measuring their Cx and βG profiles after separation on an isoelectrofocusing column over the pH range 3–10. While the overall FP-activity was roughly equal in all preparations, the specific β-glucosidase activity was highest in mutants MHC 15 and MHC 22 which are distingiushed morphologically from the parent strain, QM 9414, by a higher degree of branching of their hyphae. Two peaks of β-glucosidase activity were detected by isoelectric focusing in preparations from QM 9414 and M 6, none in the enzyme from the mutant M 5 while 3 and 4 peaks respectively were found in preparations from morphological mutants MHC 15 and MHC 22. The higher β-glucosidase activity in these last two preparations was also reflected in the higher glucose to cellobiose ratio in the initial stages of cellulose hydrolysis by the individual enzyme preparations.

Metabolism of 2-deoxy-d-glucose in baker's yeast V. Formation of 2-deoxy-α,α′-trehalose☆

Abstract A non-reducing disaccharide containing only 2-deoxy- d -glucose (dGlc) was isolated and purified from yeast incubated with dGlc. The disaccharide is alkali-stable, extremenly acid-labile and exhibits an [α] d + 153.8 to 158.7° and [M] d + 477.3 to 492.4°. Based on these properties the disaccharide was identified as 2,2′-dideoxy-α,α′-trehalose ( O-α-2- deoxy- d -glucopyranosyl-( i → i )-O-α-2-deoxy- d -glucopyranoside ). The formation of this disaccharide was about 15 times greater under aerobic than under anaerobic conditions. A stimulatory effect of dGlc on degradation of glycogen stores was observed in yeast incubated aerobically with dGlc. A scheme surveying the metabolism of dGlc in yeast is presented.

Transglycosylic reactions of nucleotides of 2-deoxy-sugars: I. Biosynthesis of 2-deoxysucrose

Abstract Uridine 5′-diphosphate 2-deoxy- d -glucose (UDP-dGlc) has been shown to serve as a 2-deoxyglucosyl donor in the reaction catalyzed by UDPglucose-fructose glucosyltransferase. The product of the reaction was identified as 2-deoxysucrose. The Michaelis constant for UDP-dGlc as a substrate in the reaction of 2-deoxysucrose synthesis was found to be 5.5·10−3M. The reaction exhibited reduced initial and maximal velocities compared with those in which UDPglucose was used as a substrate.