Production of xylanase by the thermophilic fungusThielavia terrestris

Summary The effect of varying fermentation pH and temperature on extracellular xylanase production by the thermophilic fungus,Thielavia terrestris (ATCC 26917), was studied using a stirred tank bioreactor. Maximum xylanase activity (18.8 I.U/mL) was obtained when the temperature was controlled at 48°C and the initial pH set at 4.0. Under these conditions, the volumetric productivity of xylanase was 1044 l. U./L. h. which is superior to that achieved by many mesophilic xylanolytic micro-organisms.     

Production of thermostable xylanase by a thermophilic fungus,Thermoascus aurantiacus

Twenty-one strains of thermophilic fungi in the Forintek culture collection were screened for their production of xylanolytic (and cellulolytic) enzymes in both solid and aqueous media containing various hemicellulosic and cellulosic substrates. Thermoascus aurantiacus strain C436 was selected as the best producer of extracellular xylanase (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) enzymes. High xylanase activity was detected in fungal culture filtrates even when realistic lignocellulosic residues (including steam-exploded aspenwood and untreated aspenwood sawdust) were used as substrates. Maximum xylanase activity (575.9 U ml⁻¹) was detected in cultures grown in Vogel's medium containing oat-spelt xylan. The xylanase activity exhibited a temperature optimum of 75°C and pH optimum around 5.0. The half-lives of the xylanase activity at 70 and 60°C were 1.5 h and 4 days, respectively. Over 90% of the xylanase activity was retained after 12 weeks at 50°C. Crude culture filtrates concentrated by membrane ultrafiltration could effectively hydrolyse xylan and steam-exploded aspenwood hemicellulose to release near theoretical yields of low molecular weight pentose oligomers.     

Secretion of thermophilic bacterial cellobiohydrolase in Saccharomyces cerevisiae

The partial DNA sequence corresponding to the N-terminal amino acid sequence of cellobiohydrolase derived from a thermophilic anaerobe NA10 was determined. The cellobiohydrolase gene fused to the secretion signal (signal peptide and T-S region) from Saccharomyces diastaticus was expressed in an ethanologenic yeast, S. cerevisiae YIY345, under control of the glucoamylase promoter. The recombinant yeast produced cellobiohydrolase: approximately 40% of the total cellobiohydrolase activity was detected in the medium, and the remaining cellobiohydrolase was localized in the intracellular fraction. An analysis of the N-terminal amino acid sequence of the main intracellular cellobiohydrolase revealed that the signal peptide and T-S region were removed proteolytically. Alteration of the amino acid residues at the cleavage site by insertion of a Bgl II linker led to an approximately 3.5-fold increase in the total cellobiohydrolase production, but did not affect the efficiency of secretion into the medium. Cellobiohydrolase production was not repressed in the presence of glucose. The recombinant yeast hydrolyzed carboxymethyl cellulose in the medium. The results suggest the possibility of the direct bioconversion of cellulose to ethanol by the recombinant yeast.     

Mating reaction inSaccharomyces cerevisiae

The effect of proteolytic enzymes on sexual agglutinability of haploid cells of the yeastSaccharomyces cerevisiae was examined. Sexual agglutinability of cells of botha and α types was lost on treatment with alkaline protease and two kinds of neutral proteases ofBacillus subtilis, pronase and α-chymotrypsin. Agglutinability of α type cells was lost after treatment with acid protease ofRhizopus chinensis and trypsin, but that ofa type cells was not. These results indicate that the sex-specific substance responsible for the sexual agglutination (agglutination factor) ina type cells differs from that in α type cells. Agglutination factors were solubilized from cell-wall fractions of both mating types by Glusulase treatment. These crude factors specifically inhibited the agglutinability of cells of the opposite mating type with little effect on the agglutinability of cells of the same mating type.     

Transport ofl-tryptophan inSaccharomyces cerevisiae

In addition to the general amino acid transport system (GAP) ofS. cerevisiae l-tryptophan is transported by another system with approximately 25% capacity of GAP, with aK _T of 0.41±0.08 mmol/L and with a similar specificity as GAP (lower inhibition by Met, Pro, Ser, Thr and 2-aminoisobutyric acid; greater inhibition by Glu and His). The pH optimum of this system is at 5.0–5.5, activation energy above the transition point (20°C) was 20 kJ/mol, below the transition point 55 kJ/mol. The transport by this system was virtually unidirectional, efflux amounting to at most 10% into a tryptophan-free medium. The transport itself was blocked by 2,4-dinitrophenol, antimycin A and uranyl nitrate. The system was synthesized de novo during preincubation with glucose=fructose>trehalose >ethanol within 30 min, and was degraded with a half-time of 15 min in the absence of further synthesis. The accumulation ratios ofl-tryptophan ingap1 mutants were concentration-dependent (200∶1 at 1 μmoll-Trp/L, 4∶1 at 2.5 mmoll-Trp/L) and decreased with increasing suspension density from 200∶1 to 5∶1 (for 10 μmoll-Trp/L). The involvement of hydrogen ions in the uptake was clearly demonstrated by the effect of D₂O even if it could not be established by either shifts of pH_out or membrane depolarization.     

The chitin-glucan complex inSaccharomyces cerevisiae

The content of glucosamine in the walls of daughter (without bud scars) and mother (multiscar) cells ofSaccharomyces cerevisiae was examined in a control and after treatment with dilute alkali, acid and buffer. The occurrence of chitin in the bud and birth scars is discussed. The results of IR and X-ray analysis of cell-wall fractions indicate the presence of α-chitin which is a part of the chitin-glucan complex. The size of the crystallite of α-chitin in this complex is about 60 Å.     

Transport of acyclic polyols inSaccharomyces cerevisiae

Acyclic polyols (erythritol, xylitol, ribitol, D-arabinitol, mannitol, sorbitol and galactitol) are not metabolized by Saccharomyces cerevisiae. They are taken up by a fast non-active process, reaching 40-70% distribution referred to total cell water. The uptake is insensitive to temperature, pH (between 4 and 8), 2,4-dinitrophenol and uranyl ions. Its initial rate rises linearly with concentration from 10(-5)M to 1M. The process resembles simple diffusion through large pores or the trapping of the whole solution on the surface. Protoplasts behave like whole cells in this respect. Only erythritol shows a second type of uptake which is inhibitor-insensitive but temperature-dependent.     

Turnover of canavanine-containing proteins inSaccharomyces cerevisiae

Saccharomyces cerevisiae grown for 2 h in the presence of 0.5 mmol/L canavanine in a synthetic medium with ethanol as the sole carbon source (OEC) exhibited a slowing down of protein synthesis for 3–4 h after a shift to fresh ethanolbased medium containing 1.0 mmol/L arginine (OEA) in comparison with untreated cells grown on OEA. The change of carbon source from ethanol to glucose (OGA) after growth in the OEC medium resulted in an even deeper decline of protein synthesis. The degradation of canavanine-containing proteins in cells pregrown and labelled in an OEC medium after transfer to OEA was more rapid than in the OGA medium. The initial rate of protein degradation during the first hour in the OGA medium was less than 1%/h whereas in the OEA medium it reached almost 10%/h. The fraction of proteins with high turnover (half-life 0.46 h) constituted 8.3% on OEA, while during subsequent growth on OGA it was only 0.75% with a half-life of 0.12 h.     

Cloning of a gene for S-adenosylmethionine synthesis inSaccharomyces cerevisiae

Summary The gene for ethionine resistance was isolated, and its phenotypic characteristics were investigated. The cells transformed with this gene showed strong resistance to DL-ethionine, and S-adenosylmethionine (SAM) was remarkably accumulated within the cells. Judging from the restriction map of this gene, it suggests that the gene is not the gene SAM1 but SAM2.     

Regulation of sterol biosynthesis inSaccharomyces cerevisiae

Sterol synthesis inSaccharomyces cerevisiae was primarily controlled by the growth rate. At low specific growth rates the intermediates of ergosterol biosynthesis prevailed in cells. At the same time, the total sterol content reached about 6% of dry matter whereas the content of ergosterol was only 2–2.5%, which seems to be the maximum value forS. cerevisiae. After esterification with fatty acids these sterol intermediates are stored in lipid globules together with reserve triacylglycerols. The sporulatingS. cerevisiae cells contained 3.5% sterols and 1.5% ergosterol of dry matter.     

Patterns of wall synthesis inSaccharomyces cerevisiae

Wall formation inSaccharomyces cerevisiae seems to be the result of two main patterns of wall material deposition: (i) around the whole periphery of the cell in nonbudding ones, and (ii) mainly at the tip of the daughter cell or at the cross wall that separates dividing cells. This interpretation has been obtained following experiments in which RNA or protein synthesis has been inhibited. Under these conditions, glucan formation takes place, and wall thickening is probably due to the accumulation of this polysaccharide. Furthermore, once a pattern of wall deposition has been established, it is not modified by inhibition of RNA or protein synthesis.     

Intracellular control of proton extrusion inSaccharomyces cerevisiae

Wild-type and mutant (glucosephosphate isomerase, pyruvate kinase and respiratory deficientrho) strains were used to determine the kinetics of substrate-induced H⁺ efflux in dilute suspensions, glucose-induced production of titratable acidity in intact cells and cell-free extracts, and kinetics of extracellular titratable acidity production (pH-stat). The results indicate that (1) initial phases of H⁺ efflux proceed at the expense of preexisting cell acidity reserves while subsequent efflux is supported by de novo formed acidity, (2) apart from regulation by pH_out the H⁺ efflux is subject to intracellular control, (3) intracellular acidity level is controlled separately from H⁺ efflux. Tentative scheme is proposed for the regulation of H⁺ fluxes inS. cerevisiae.     

Effects of low X-ray doses inSaccharomyces cerevisiae

Summary Three strains ofSaccharomyces cerevisiae with different capacities for repair of radiation damage (RAD, rad18, and rad52) have been tested for their colony forming ability (CFA) and growth rates after application of small X-ray doses from 3.8 mGy to 40 Gy. There was no reproducible increase in CFA observable after application of doses between 3.8 mGy and 4.7 Gy. X-ray doses of 40 Gy causing an inactivation of CFA from 90% to 50%, depending on the repair capacity of the strains used, caused a reduced increase in optical density during 2 h buffer treatment in comparison to unirradiated cells. This reduction however, is reversible as soon as the cells are transferred into nutrient medium. One hour after transfer into growth medium the portions of cells with large buds (G2 and M phase) and cells with small buds (S phase) are drastically different in irradiated cells from those obtained in unirradiated cells. The time necessary for separation of mother and daughter cells is prolonged by X-ray irradiation and the formation of new buds is retarded.     

Rapid kinetics of glucose uptake inSaccharomyces cerevisiae

In a multiple deletion mutanthxt1Δhxt2Δhxt3Δ hxt4Δsnf3Δ ofSaccharomyces cerevisiae growing on 2 % glucose, high-affinity glucose-uptake (lowK _m) was exhibited throughout growth on glucose in contrast to the wild-type, which exhibited the usual low-affinity to high-affinity transition as the glucose in the medium was consumed. elevated levels of invertase activity throughout growth on glucose, in this mutant as compared to the wild-type, indicate that glucose repression may be impaired. Howver, in a mutant containing only theHXT2 gene (hxt1Δhxt3Δhxt4Δ snf3Δ), invertase levels were similar to those in the wild-type. It is likely, therefore, that some of these putative glucose transporters, such asHXT2, also have regulatory roles in cellular metabolism. In triple hexose-kinase mutants, rapid (200-ms) measurements of initial glucose-uptake revealed high-affinity glucose uptake (K _m approx. 2 mmol/L) while measurements on the slower 5-s scale clearly demonstrate that uptake is not linear over this longer period. These results suggest that this high-affinity component does not require a functional hexose-kinase.