Metabolomic Comparison of Saccharomyces cerevisiae and the Cryotolerant Species S. bayanus var. uvarum and S. kudriavzevii during Wine Fermentation at Low Temperature

Temperature is one of the most important parameters affecting the length and rate of alcoholic fermentation and final wine quality. Wine produced at low temperature is often considered to have improved sensory qualities. However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase, and sluggish or stuck fermentations. To investigate the effects of temperature on commercial wine yeast, we compared its metabolome growing at 12°C and 28°C in a synthetic must. Some species of the Saccharomyces genus have shown better adaptation at low temperature than Saccharomyces cerevisiae. This is the case of the cryotolerant yeasts Saccharomyces bayanus var. uvarum and Saccharomyces kudriavzevii. In an attempt to detect inter-specific metabolic differences, we characterized the metabolome of these species growing at 12°C, which we compared with the metabolome of S. cerevisiae (not well adapted at low temperature) at the same temperature. Our results show that the main differences between the metabolic profiling of S. cerevisiae growing at 12°C and 28°C were observed in lipid metabolism and redox homeostasis. Moreover, the global metabolic comparison among the three species revealed that the main differences between the two cryotolerant species and S. cerevisiae were in carbohydrate metabolism, mainly fructose metabolism. However, these two species have developed different strategies for cold resistance. S. bayanus var. uvarum presented elevated shikimate pathway activity, while S. kudriavzevii displayed increased NAD⁺ synthesis.     

Influence of Calcium Ion on Ethanol Tolerance of Saccharomyces bayanus and Alcoholic Fermentation by Yeasts

The addition of Ca²⁺ (as CaCl₂) in optimal concentrations (0.75 to 2.0 mM) to a fermentation medium with a trace contaminating concentration of Ca²⁺ (0.025 mM) led to the rapid production of higher concentrations of ethanol by Saccharomyces cerevisiae, Saccharomyces bayanus, and Kluyveromyces marxianus. The positive effect of calcium supplementation (0.75 mM) on alcoholic fermentation by S. bayanus was explained by the increase in its ethanol tolerance. The ethanol inhibition of growth and fermentation followed the equation μ_xi = μ_oi [1 - (X/X_mi)]ⁿⁱ, where μ_oi and μ_xi are, respectively, the specific growth (i = g) and fermentation (i = f) rates in the absence or presence of a concentration (X) of added ethanol, and X_mi is the maximal concentration of ethanol which allows growth or fermentation. The toxic power is given by n_i. In Ca²⁺ - supplemented medium (0.75 mM), n_g = 0.42 for growth and n_f = 0.43 for fermentation compared with 0.52 and 0.55, respectively, in unsupplemented medium; for both media, X_mg = 10% (vol/vol) and X_mf = 13% (vol/vol). For lethal concentrations of ethanol, the specific death rates were minimal for cells that were grown and incubated with ethanol in medium with an optimal concentration of Ca²⁺, maximal for cells grown and incubated with ethanol in unsupplemented medium, and intermediate for cells grown in unsupplemented medium and incubated with ethanol in calcium-supplemented medium. The effect of Ca²⁺ on the acidification curve of energized cells in the presence of ethanol was found to be closely associated with its protective effect on growth, fermentation, and viability.     

Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae

The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC 2.7.4.2) from Saccharomyces cerevisiae, a previously unstudied enzyme. An E. coli codon-optimized version of the S. cerevisiae gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in E. coli BL21(DE3) and purified on a Ni²⁺ column. The K_M of the ATP binding site was determined to be 98.3 µM at 30°C, the optimal growth temperature for S. cerevisiae, and 74.3 µM at 37°C, the optimal growth temperature for E. coli. The K_M of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The V_max was determined to be 4.51 µmol/min/mg enzyme at 30°C and 5.33 µmol/min/mg enzyme at 37°C. PMK is Mg²⁺ dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH = 7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 µM FPP.     

An evolutionary approach uncovers a diverse response of tRNA 2-thiolation to elevated temperatures in yeast

Chemical modifications of transfer RNA (tRNA) molecules are evolutionarily well conserved and critical for translation and tRNA structure. Little is known how these nucleoside modifications respond to physiological stress. Using mass spectrometry and complementary methods, we defined tRNA modification levels in six yeast species in response to elevated temperatures. We show that 2-thiolation of uridine at position 34 (s²U₃₄) is impaired at temperatures exceeding 30°C in the commonly used Saccharomyces cerevisiae laboratory strains S288C and W303, and in Saccharomyces bayanus. Upon stress relief, thiolation levels recover and we find no evidence that modified tRNA or s²U₃₄ nucleosides are actively removed. Our results suggest that loss of 2-thiolation follows accumulation of newly synthesized tRNA that lack s²U₃₄ modification due to temperature sensitivity of the URM1 pathway in S. cerevisiae and S. bayanus. Furthermore, our analysis of the tRNA modification pattern in selected yeast species revealed two alternative phenotypes. Most strains moderately increase their tRNA modification levels in response to heat, possibly constituting a common adaptation to high temperatures. However, an overall reduction of nucleoside modifications was observed exclusively in S288C. This surprising finding emphasizes the importance of studies that utilize the power of evolutionary biology, and highlights the need for future systematic studies on tRNA modifications in additional model organisms.     

Minimization of Glycerol Production during the High-Performance Fed-Batch Ethanolic Fermentation Process in Saccharomyces cerevisiae, Using a Metabolic Model as a Prediction Tool

On the basis of knowledge of the biological role of glycerol in the redox balance of Saccharomyces cerevisiae, a fermentation strategy was defined to reduce the surplus formation of NADH, responsible for glycerol synthesis. A metabolic model was used to predict the operating conditions that would reduce glycerol production during ethanol fermentation. Experimental validation of the simulation results was done by monitoring the inlet substrate feeding during fed-batch S. cerevisiae cultivation in order to maintain the respiratory quotient (RQ) (defined as the CO₂ production to O₂ consumption ratio) value between 4 and 5. Compared to previous fermentations without glucose monitoring, the final glycerol concentration was successfully decreased. Although RQ-controlled fermentation led to a lower maximum specific ethanol production rate, it was possible to reach a high level of ethanol production: 85 g · liter⁻¹ with 1.7 g · liter⁻¹ glycerol in 30 h. We showed here that by using a metabolic model as a tool in prediction, it was possible to reduce glycerol production in a very high-performance ethanolic fermentation process.     

Ethanol Production from Colpomenia sinuosa by an Alginate Fermentation Strain Meyerozyma guilliermondii

With the consumption of energy and the spread of COVID-19, the demand for ethanol production is increasing in the world. The industrial ethanol fermentation microbes cannot metabolize the alginate component of macro algae, which affects the ethanol yield. In this research, the ethanol production process from macro algae by an alginate fermentation yeast Meyerozyma guilliermondii, especially the pretreatment process of Colpomenia sinuosa, was studied. At the same time, the experimental design of Box-Behnken was carried out to achieve the optimum fermentation performance. The concentration of KH₂PO₄ (A: 2–6 g.L⁻¹), pH (B: 4–7), reaction time (C: 60–120 h) and temperature (D: 24–34 °C) were variable input parameters. During the ethanol production process, the algae powder was firstly mixed with water at 90 °C for 0.5 h. Later the fermentation culture medium was prepared and then it was fermented by the yeast Meyerozyma guilliermondii to produce ethanol. And the optimal fermentation parameters were as follows: fermentation temperature of 28 °C, KH₂PO₄ dosage of 4.7 g.L⁻¹, initial pH of 6, and fermentation time of 99 h. The ethanol yield reached 0.268 g.g⁻¹ (ethanol to algae), close to the predicted value of model. The generation of alginate lyase during the fermentation of algae was also examined. The highest alginate lyase activity reached 46.42 U.mL⁻¹.     

Rapid Identification and Enumeration of Saccharomyces cerevisiae Cells in Wine by Real-Time PCR

Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated S. cerevisiae from its sibling species Saccharomyces bayanus, Saccharomyces pastorianus, and Saccharomyces paradoxus. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of S.cerevisiae directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify S. cerevisiae in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 × 10⁵ to 3.8 CFU/ml in sweet wine and from 5 × 10⁶ to 50CFU/ml in red wine.     

Fermentation Temperature Modulates Phosphatidylethanolamine and Phosphatidylinositol Levels in the Cell Membrane of Saccharomyces cerevisiae

During alcoholic fermentation, Saccharomyces cerevisiae is exposed to a host of environmental and physiological stresses. Extremes of fermentation temperature have previously been demonstrated to induce fermentation arrest under growth conditions that would otherwise result in complete sugar utilization at “normal” temperatures and nutrient levels. Fermentations were carried out at 15°C, 25°C, and 35°C in a defined high-sugar medium using three Saccharomyces cerevisiae strains with diverse fermentation characteristics. The lipid composition of these strains was analyzed at two fermentation stages, when ethanol levels were low early in stationary phase and in late stationary phase at high ethanol concentrations. Several lipids exhibited dramatic differences in membrane concentration in a temperature-dependent manner. Principal component analysis (PCA) was used as a tool to elucidate correlations between specific lipid species and fermentation temperature for each yeast strain. Fermentations carried out at 35°C exhibited very high concentrations of several phosphatidylinositol species, whereas at 15°C these yeast strains exhibited higher levels of phosphatidylethanolamine and phosphatidylcholine species with medium-chain fatty acids. Furthermore, membrane concentrations of ergosterol were highest in the yeast strain that experienced stuck fermentations at all three temperatures. Fluorescence anisotropy measurements of yeast cell membrane fluidity during fermentation were carried out using the lipophilic fluorophore diphenylhexatriene. These measurements demonstrate that the changes in the lipid composition of these yeast strains across the range of fermentation temperatures used in this study did not significantly affect cell membrane fluidity. However, the results from this study indicate that fermenting S. cerevisiae modulates its membrane lipid composition in a temperature-dependent manner.     

In Vivo Perturbation of Membrane-Associated Calcium by Freeze-Thaw Stress in Onion Bulb Cells : Simulation of This Perturbation in Extracellular KCl and Alleviation by Calcium

Incipient freeze-thaw stress in onion bulb scale tissue is known to cause enhanced efflux of K⁺, along with small but significant loss of cellular Ca²⁺. During the post-thaw period, irreversibly injured cells undergo a cytological aberration, namely, `protoplasmic swelling.' This cellular symptom is thought to be caused by replacement of Ca²⁺ from membrane by extracellular K⁺ and subsequent perturbation of K⁺ transport properties of plasma membrane. In the present study, onion (Allium cepa L. cv Sweet Sandwich) bulbs were slowly frozen to either −8.5°C or −11.5°C and thawed over ice. Inner epidermal peels from bulb scales were treated with fluorescein diacetate for assessing viability. In these cells, membrane-associated calcium was determined using chlorotetracycline fluorescence microscopy combined with image analysis. Increased freezing stress and tissue infiltration (visual water-soaking) were paralleled by increased ion leakage. Freezing injury (−11.5°C; irreversible) caused a specific and substantial loss of membrane-associated Ca²⁺ compared to control. Loss of membrane-associated Ca²⁺ caused by moderate stress (−8.5°C; reversible) was much less relative to −11.5°C treatment. Ion efflux and Ca²⁺-chlorotetracycline fluorescence showed a negative relationship. Extracellular KCl treatment simulated freeze-thaw stress by causing a similar loss of membrane-associated calcium. This loss was dramatically reduced by presence of extracellular CaCl₂. Our results suggest that the loss of membrane-associated Ca²⁺, in part, plays a role in initiation and progression of freezing injury.     

Characterization of the Factors that Influence Sinapine Concentration in Rapeseed Meal during Fermentation

We analyzed and compared the difference in sinapine concentration in rapeseed meal between the filamentous fungus, Trametes sp 48424, and the yeast, Saccharomyces cerevisiae, in both liquid and solid-state fermentation. During liquid and solid-state fermentation by Trametes sp 48424, the sinapine concentration decreased significantly. In contrast, the liquid and solid-state fermentation process by Saccharomyces cerevisiae just slightly decreased the sinapine concentration (P ≤ 0.05). After the solid-state fermented samples were dried, the concentration of sinapine in rapeseed meal decreased significantly in Saccharomyces cerevisiae. Based on the measurement of laccase activity, we observed that laccase induced the decrease in the concentration of sinapine during fermentation with Trametes sp 48424. In order to eliminate the influence of microorganisms and the metabolites produced during fermentation, high moisture rapeseed meal and the original rapeseed meal were dried at 90°C and 105°C, respectively. During drying, the concentration of sinapine in high moisture rapeseed meal decreased rapidly and we obtained a high correlation coefficient between the concentration of sinapine and loss of moisture. Our results suggest that drying and enzymes, especially laccase that is produced during the solid-state fermentation process, may be the main factors that affect the concentration of sinapine in rapeseed meal.     

Effect of ATP on the Calcium Efflux in Dialyzed Squid Giant Axons

Dialysis perfusion technique makes it possible to control the internal composition of squid giant axons. Calcium efflux has been studied in the presence and in the virtual absence (<5 µM) of ATP. The mean calcium efflux from axons dialyzed with 0.3 µM ionized calcium, [ATP]_i > 1,000 µM, and bathed in artificial seawater (ASW) was 0.24 ± 0.02 pmol·cm^-2·s^-1 (P/CS) (n = 8) at 22°C. With [ATP]_i < 5 µM the mean efflux was 0.11 ± 0.01 P/CS (n = 15). The curve relating calcium efflux to [ATP]_i shows a constant residual calcium efflux in the range of 1–100 µM [ATP]_i. An increase of the calcium efflux is observed when [ATP]_i is >100 µM and saturates at [ATP]_i > 1,000 µM. The magnitude of the ATP-dependent fraction of the calcium efflux varies with external concentrations of Na⁺, Ca⁺⁺, and Mg⁺⁺. These results suggest that internal ATP changes the affinity of the calcium transport system for external cations.     

The Efflux of Substances from Frog Ventricles to Sucrose and to Ringer's Solutions

The frog ventricle in sucrose solution contracts for several hours at 25°C, and for as long as 24 hours at 5°G. The possibility that a fraction of the extracellular fluid remains outside of the excitable membrane was examined by measuring the efflux of tracers. The half-time for the efflux to sucrose solution at 25°C of C¹⁴ sucrose is about 1 minute, for Na²⁴ is 6.5 minutes, and for Cl⁸⁶ is 4 minutes. There is no evidence for the retention of an extracellular Na fraction. The Q₁₀ for Na and Cl efflux is about 1.3. The half-time for K⁴² efflux is about 180 minutes; the Q₁₀ is 1.7. The efflux rates of Na²⁴, Cl³⁶ and K⁴² to sucrose and to Ringer's solutions are quite similar. Ca⁴⁵ efflux is only one-fifth as fast to sucrose solution as to Ringer's; the retention of Ca⁺⁺ may be important for maintaining excitability in sucrose solution. P³² efflux is five times faster to sucrose solution than to Ringer's solution, and there is a similar increase in the rate of inosine loss to sucrose solution. The Q₁₀ for efflux to sucrose solution is 2.2 for P³²O₄ and 2.4 for inosine. We suggest that energy metabolism is abnormal in ventricles in sucrose solution and that low temperature prolongs excitability by slowing the metabolic change.     

Improved yeast tolerance to ethanol-induced death by centrifugates of dense cell suspensions incubated with ethanol

Summary Ethanol-induced death rate was higher for cells ofSaccharomyces bayanus orKluyveromyces marxianus in spanse suspensions (2×10⁴ cells/ml) compared with dense suspensions (2×10⁵–2×10⁷ cells/ml). Specific death rates of sparse suspensions decreased to values similar to dense suspensions if ethanol-induced death experiments were undertaken in the media obtained after harvesting the cells previously incubated with the same lethal concentration of ethanol.     

Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro

COVID-19 has become a global pandemic and there is an urgent call for developing drugs against the virus (SARS-CoV-2). The 3C-like protease (3CL^pro) of SARS-CoV-2 is a preferred target for broad spectrum anti-coronavirus drug discovery. We studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredients. We found that the ethanol extract of S. baicalensis and its major component, baicalein, inhibit SARS-CoV-2 3CL^pro activity in vitro with IC₅₀’s of 8.52 µg/ml and 0.39 µM, respectively. Both of them inhibit the replication of SARS-CoV-2 in Vero cells with EC₅₀’s of 0.74 µg/ml and 2.9 µM, respectively. While baicalein is mainly active at the viral post-entry stage, the ethanol extract also inhibits viral entry. We further identified four baicalein analogues from other herbs that inhibit SARS-CoV-2 3CL^pro activity at µM concentration. All the active compounds and the S. baicalensis extract also inhibit the SARS-CoV 3CL^pro, demonstrating their potential as broad-spectrum anti-coronavirus drugs.     

New Hybrids between Saccharomyces Sensu Stricto Yeast Species Found among Wine and Cider Production Strains

Two yeast isolates, a wine-making yeast first identified as a Mel⁺ strain (ex. S. uvarum) and a cider-making yeast, were characterized for their nuclear and mitochondrial genomes. Electrophoretic karyotyping analyses, restriction fragment length polymorphism maps of PCR-amplified MET2 gene fragments, and the sequence analysis of a part of the two MET2 gene alleles found support the notion that these two strains constitute hybrids between Saccharomyces cerevisiae and Saccharomyces bayanus. The two hybrid strains had completely different restriction patterns of mitochondrial DNA as well as different sequences of the OLI1 gene. The sequence of the OLI1 gene from the wine hybrid strain appeared to be the same as that of the S. cerevisiae gene, whereas the OLI1 gene of the cider hybrid strain is equally divergent from both putative parents, S. bayanus and S. cerevisiae. Some fermentative properties were also examined, and one phenotype was found to reflect the hybrid nature of these two strains. The origin and nature of such hybridization events are discussed.     

Low-resolution structure of the soluble domain GPAA1 (yGPAA170–247) of the glycosylphosphatidylinositol transamidase subunit GPAA1 from Saccharomyces cerevisiae

The GPI (glycosylphosphatidylinositol) transamidase complex catalyses the attachment of GPI anchors to eukaryotic proteins in the lumen of ER (endoplasmic reticulum). The Saccharomyces cerevisiae GPI transamidase complex consists of the subunits yPIG-K (Gpi8p), yPIG-S (Gpi17p), yPIG-T (Gpi16p), yPIG-U (CDC91/GAB1) and yGPAA1. We present the production of the two recombinant proteins yGPAA1^70–247 and yGPAA1^70–339 of the luminal domain of S. cerevisiae GPAA1, covering the amino acids 70–247 and 70–339 respectively. The secondary structural content of the stable and monodisperse yGPAA1^70–247 has been determined to be 28% α-helix and 27% β-sheet. SAXS (small-angle X-ray scattering) data showed that yGPAA1^70–247 has an R_g (radius of gyration) of 2.72±0.025 nm and D_max (maximum dimension) of 9.14 nm. These data enabled the determination of the two domain low-resolution solution structure of yGPAA1^70–247. The large elliptical shape of yGPAA1^70–247 is connected via a short stalk to the smaller hook-like domain of 0.8 nm in length and 3.5 nm in width. The topological arrangement of yGPAA1^70–247 will be discussed together with the recently determined low-resolution structures of yPIG-K^24–337 and yPIG-S^38–467 from S. cerevisiae in the GPI transamidase complex.     

Transport of glucose and galactose in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25° was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S]_i) and extracellular ([S]_o) glucose concentrations was increased by 0·4mm-phlorrhizin and 0·3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S]_i/[S]_o fell below 1·0 only at [S]_o higher than 0·5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K_m 1·16mm; V_max. 4·5μmoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0·1mm-2,4-dinitrophenol, 0·4mm-phlorrhizin and by the absence of external Na⁺. 6. The kinetic parameters of galactose entry into the cells were: K_m 1·5mm; V_max 10μmoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0°, was inhibited by 0·4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na⁺-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na⁺-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

Punica granatum waste to ethanol valorisation employing optimized levels of saccharification and fermentation

Pomegranate peels (PPW) as municipal waste is inexpensive biomass that could be a renewable source of sugars particularly rich in hemicellulosic contents. The subsequent conversion of available sugars in PPW can provide prospective strategy for cost-effective bioenergy production. In this study, an experimental setup based on CCD was implemented with the aim of bioconversion of biomass into bioethanol. The factors considered were Hydrochloric acid concentration (X₁), the hydrolysis temperature (X₂) and time (X₃) for optimization with dilute Hydrochloric acid (HCl) saccharification. The present study investigates the optimised level of bioethanol synthesis from acid pre-treated PPW explained by RSM. Subsequently, three yeasts viz. Saccharomyces cerevisiae K7, Metschnikowia sp. Y31 and M. cibodasensis Y34 were utilized for fermentation of acid hydrolysed and detoxified feed stocks. Optimum values of reducing sugars 48.02 ± 0.02 (gL^?1) and total carbohydrates 205.88 ± 0.13 (gL^?1) were found when PPW was hydrolyzed with 1% HCl concentration at 100?C of temperature for 30 min. Later on, fermentation of PPWH after detoxification with 2.5% activated charcoal. The significant ethanol (g ethanol/g of reducing sugars) yields after fermentation with Metschnikowia sp. Y31 and M. cibodasensis Y34 found to be 0.40 ± 0.03 on day 5 and 0.41 ± 0.02 on last day of experiment correspondingly. Saccharomyces cerevisiae K7 also produce maximum ethanol 0.40 ± 0.00 on last day of incubation utilizing the PPWH. The bioconversion of commonly available PPW into bioethanol as emphasize in this study could be a hopeful expectation and also cost-effective to meet today energy crisis.     

Molecular and Biochemical Characterization of a β-Fructofuranosidase from Xanthophyllomyces dendrorhous

An extracellular β-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70°C) and thermostability (with a T₅₀ in the range 66 to 71°C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-β-(2→1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the k_cat/K_m ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial β-fructofuranosidases, the enzyme from X. dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Using a 41% (wt/vol) sucrose solution, the maximum fructooligosaccharide concentration reached was 65.9 g liter⁻¹. In addition, we isolated and sequenced the X. dendrorhous β-fructofuranosidase gene (Xd-INV), showing that it encodes a putative mature polypeptide of 595 amino acids and that it shares significant identity with other fungal, yeast, and plant β-fructofuranosidases, all members of family 32 of the glycosyl-hydrolases. We demonstrate that the Xd-INV could functionally complement the suc2 mutation of Saccharomyces cerevisiae and, finally, a structural model of the new enzyme based on the homologous invertase from Arabidopsis thaliana has also been obtained.     

Effects of Temperature on Methanogenesis in a Thermophilic (58°C) Anaerobic Digestor

The short-term effects of temperature on methanogenesis from acetate or CO₂ in a thermophilic (58°C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with ¹⁴C-labeled methane precursors (¹⁴CH₃COO⁻ or ¹⁴CO₂). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60°C and was completely inhibited at 65°C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58°C and did not grow or produce methane at 65°C. An accidental shift of digestor temperature from 58 to 64°C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from ¹⁴CH₃COO⁻ was optimal at 65°C and completely inhibited at 75°C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70°C. Methanogenesis from ¹⁴CO₂ in the sludge was optimal at 65°C and still proceeded at 75°C. A CO₂-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75°C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65°C produced more methane than sludge incubated at 60°C, and no acetate accumulated at 65°C. Methanogenesis was severely inhibited in sludge incubated at 70°C, but since neither acetate nor H₂ accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.