Xylitol dehydrogenase from Pachysolen tannophilus

Abstract NAD-xylitol-dehydrogenase (EC 1.1.1.9) from Pachysolen tannophilus was investigated in relation to xylitol byproduction during xylose fermentation by this yeast. For this purpose the enzyme was partially purified by a combination of affinity chromatography and fast liquid protein chromatography. The enzyme catalyzes an equilibrium reaction which at physiological pH values favours the accumulation of xylitol. The kinetics of the enzyme were shown to be Michaelis-Menten type with respect to both reaction directions. The activity of the enzyme was shown to be under the influence of the 'catabolic reduction charge' (NADH/NAD + NADH) and ATP. The apparent equilibrium constant of the enzyme may explain the considerable byproduction of xylitol during xylose fermentation by P. tannophilus .     

Microaerophilic metabolism of Pachysolen tannophilus at different pH values

Microaerophilic production of xylitol by Pachysolen tannophilus from detoxified hemicellulose hydrolyzate was optimal between pH values 6.0 to 7.5 when about 90% of xylose was utilized for xylitol production, the rest being fermented to ethanol. At pH values of 3.0 and 12.0, respiration became important, consuming up to 30% of available xylose. A graphic procedure suggests that histamine and cysteine are at the active site of xylose reductase in this yeast.     

Assignment of cloned genes to electrophoretically separated chromosomes of the yeast Pachysolen tannophilus

An electrophoretic karyotype of Pachysolen tannophilus has been obtained using Pulsed Field Gel Electrophoresis. Seven chromosomal bands were separated with one of the bands migrating probably as a doublet. The sizes of the chromosomes were estimated to be between 1 and 3.1 megabase pairs. Eleven loci have been assigned to chromosomal bands, including four involved in the metabolism of D-xylose.     

Reversible inactivation of d-xylose utilization by d-glucose in the pentose-fermenting yeast Pachysolen tannophilus

Abstract A major problem in fermenting pentoses using lignocellulosic substrates is the presence of d -glucose which inhibits d -xylose utilization. We previously showed that d -glucose represses the induction of xylose reductase and xylitol dehydrogenase activities, thereby inhibiting d -xylose utilization in Pachysolen tannophilus . The question arose whether d -glucose can also inactivate d -xylose fermentation. P. tannophilus cells were grown on a defined d -xylose-containing liquid medium. At about 40 h, d -glucose was added to a final concentration of 3% (w/v). This led to a rapid cessation of d -xylose utilization, which resumed after 10–12 h before d -glucose was completely consumed. This suggests that d -glucose inactivated existing d -xylose catabolic enzymes and that inactivation was reversed at low d -glucose concentrations. This reversible inactivation was distinct from d -glucose repression. Addition of cycloheximide did not block the resumption of d -xylose consumption, suggesting that reactivation was independent of protein synthesis.     

Mutants of Pachysolen tannophilus showing enhanced rates of growth and ethanol formation from d-xylose

Mutants of Pachysolen tannophilus NRRL Y-2460 have been sought that show enhanced rates of d-xylose fermentation. Mutagenesis followed by enrichment in urea-xylitol broth generally resulted in a lower frequency of good ethanol producers than enrichment in nitrate-xylitol broth. Under aerobic conditions, the best xylose-fermenting strains (which were obtained from nitrate-xylitol broth) produced ethanol from xylose twice as fast and in 32% better yield than the parent strain. Under anaerobic conditions, these strains produced ethanol from xylose 50% faster than (but in the same yield as) the parent strain. These findings show that enrichment in nitrate-xylitol broth is a promising method for obtaining mutants of Pachysolen having enhanced fermentation rates.     

Nitrogen-source-induced activation of neutral trehalase in Schizosaccharomyces pombe and Pachysolen tannophilus: role of cAMP as second messenger

Abstract Resting cells of the fission yeast Schizosaccharomyces pombe , suspended in buffer with glucose, responded to the addition of asparagine by increasing trehalase activity. This response was preceded by a peak in cAMP concentration. The addition of the nitrogen source to resting cells, devoid of the catalytic subunit of cAMP-dependent protein kinase, produced the transient increase in cAMP but did not promote any change in trehalase activity. In the budding yeast Pachysolen iannophilus , the activation of trehalase by nitrogen source was also accompanied by a sharp peak in cAMP. These results suggest that in the two yeasts cAMP acts as a second messenger in the transduction of the nitrogen-source-induced signal causing the activation of trehalase.     

Fermentation of acid hydrolysates from olive-tree pruning debris by Pachysolen tannophilus

The influence of the type and concentration of acid in the hydrolysis process and its effect on the subsequent fermentation by Pachysolen tannophilus (ATCC 32691) to produce ethanol and xylitol was studied. The hydrolysis experiments were performed using hydrochloric, sulphuric and trifluoroacetic acids in concentrations ranging from 0.1 to 1.0 N, a temperature of 90 degrees C, and a time of 240 min. The fermentation experiments were conducted on a laboratory scale in a batch-culture reactor at pH 4.5 and 30 degrees C. The hydrolysis with the highest acid concentration produced the complete solubilization of hemicellulose to monosaccharides. The highest values for the specific rate of ethanol production were registered in cultures hydrolyzed with trifluoroacetic acid, and values were found to decrease as the acid concentration increased. The highest values of overall ethanol yields ( [Formula: see text] = 0.37 kg kg(-1)) were also found in the fermentation of the hydrolysates of trifluoroacetic acid.     

Acetone stimulation of ethanol production from d-xylose by Pachysolen tannophilus

Summary Pachysolen tannophilus, a homothallic yeast, converts xylose to ethanol at a yield of 0.3 (g/g xylose). Concomitant with ethanol production, xylitol accumulates in the culture medium at similar yields (0.3 g/g xylose). The addition of the hydrogen-accepting compound, acetone, increases the amount of ethanol produced by this organism by 50–70%. The increase in ethanol is directly correlated with a decrease in xylitol secreted. The results indicate that conversion of acetone to 2-propanol by the cells provides the NAD⁺ used as a cofactor by xylitol dehydrogenase, the enzyme responsible for converting xylitol to xylulose.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U. S. Department of Agriculture over other firms or similar products not mentioned.     

Characterization of Ethanol Production from Xylose and Xylitol by a Cell-Free Pachysolen tannophilus System

Whole cells and a cell extract of Pachysolen tannophilus converted xylose to xylitol, ethanol, and CO₂. The whole-cell system converted xylitol slowly to CO₂ and little ethanol was produced, whereas the cell-free system converted xylitol quantitatively to ethanol (1.64 mol of ethanol per mol of xylitol) and CO₂. The supernatant solution from high-speed centrifugation (100,000 × g) of the extract converted xylose to ethanol, but did not metabolize xylitol unless a membrane fraction and oxygen were also present. Fractionation of the crude cell extract by gel filtration resulted in an inactive fraction in which ethanol production from xylitol was fully restored by the addition of NAD⁺ and ADP. The continued conversion of xylose to xylitol in the presence of fluorocitrate, which inhibited aconitase, demonstrated that the tricarboxylic acid cycle was not the source of the electrons for the production of xylitol from xylose. Therefore, the source of the electrons is indirectly identified as an oxidative pentose-hexose cycle.     

Enzymatic production of glycerol acetate from glycerol

In this study, we report the enzymatic production of glycerol acetate from glycerol and methyl acetate. Lipases are essential for the catalysis of this reaction. To find the optimum conditions for glycerol acetate production, sequential experiments were designed. Type of lipase, lipase concentration, molar ratio of reactants, reaction temperature and solvents were investigated for the optimum conversion of glycerol to glycerol acetate. As the result of lipase screening, Novozym 435 (Immobilized Candida antarctica lipase B) was turned out to be the optimal lipase for the reaction. Under the optimal conditions (2.5 g/L of Novozym 435, 1:40 molar ratio of glycerol to methyl acetate, 40 °C and tert-butanol as the solvent), glycerol acetate production was achieved in 95.00% conversion.     

Mutants of Pachysolen tannophilus with Improved Production of Ethanol from d-Xylose

The conversion of d-xylose to ethanol by the yeast Pachysolen tannophilus is relatively inefficient in batch culture. The inefficiency has been attributed in part to concurrent utilization of ethanol in the presence of appreciable concentrations of d-xylose and to the formation of xylitol and other by-products. To increase the concentration of ethanol accumulated in batch cultures, UV-induced mutants of P. tannophilus were selected on the basis of diminished growth on ethanol. Eleven independent mutant loci that conferred the ethanol-defective phenotype were identified. Three led to a greater yield and volumetric rate of production of ethanol than the wild type. One also produced less xylitol and was characterized by a deficiency in activity for malate dehydrogenase.     

Oxygen requirements for d-xylose fermentation to ethanol and polyols by Pachysolen tannophilus

The oxygen requirements for ethanol production from d-xylose (10 or 20 g l^?1) by Pachysolen tannophilus have been determined by controlling the availability of oxygen to shake flasks. Under anaerobic conditions no ethanol was produced whereas under aerobic conditions mainly biomass was formed. Semi-anaerobic conditions resulted in maximum ethanol production. By varying the stirring speed of a fermenter and supplying air to the liquid surface at various rates, the oxygen transfer rate (OTR) was controlled under semi-anaerobic conditions. By increasing the OTR from 0.05 to 16.04 mmol l^?1 h^?1, the ethanol yield coefficient decreased from 0.28 to 0.18 while the cell yield coefficient increased from 0.14 to 0.22. The accumulation of polyols decreased from 0.88 to 0.56 g l^?1 with increasing OTR. At OTRs between 0.09 and 1.18 mmol l^?1 h^?1, specific ethanol productivity attained a maximum value of 0.07 h^?1 and decreased with either increasing or decreasing OTR. The results indicate that the OTR must be carefully controlled for efficient ethanol production from d-xylose by P. tannophilus.     

Factors in acid treated bagasse inhibiting ethanol production from d-xylose by Pachysolen tannophilus

The fermentation of d-xylose, the major sugar-cane bagasse hemicellulose component, to ethanol by Pachysolen tannophilus is inhibited by various factors produced or released during the acid hydrolysis of the bagasse or during the fermentation process. These include ethanol, iron, chromium, copper, nickel, acetic acid and furfural. Ethanol production by P. tannophilus is inhibited by ethanol fconcentrations >24 g l^?1. Furfural and acetic acid concentrations as low as 0.3 and 7 g l^?1, respectively, and iron, chromium, nickel and copper at concentrations of 0.07, 0.01, 0.01 and 0.004 g l^?1, respectively. Similar concentrations may be found in acid-hydrolysed bagasse. The removal of these factors by treatment with ion-exchange resin resulted in the fermentation of the sugars to ethanol. The d-glucose was used rapidly and completely whereas d-xylose utilization was slow and incomplete. An ethanol concentration of 4.1 g l^?1 was produced and an ethanol yield of 0.32 was obtained. Xylitol in significant amounts was produced.     

外加肌醇对嗜鞣管囊酵母发酵的酒精产量及其耐酒精能力的影响

嗜鞣管囊酵母(Pachysolen tannophilus)是可以同时发酵葡萄糖和木糖为酒精的菌种,在其生长和发酵培养基中分别添加不同浓度((0～200mg/L)的肌醇以及不同起始浓度的酒精,以考察外加肌醇对嗜鞣管囊酵母生长、产酒精能力和耐酒精能力的影响.结果 表明,添加肌醇前后,嗜鞣管囊酵母的生物量及发酵的酒精产量均有所增加.外加肌醇对嗜鞣管囊酵母生长有轻微的刺激作用,酵母生长最适肌醇浓度为150mg/L;而对酵母生长的耐酒精能力却有明显的影响, 并且,菌种在YEPD培养基中的耐酒精能力高于在YEPX培养基中的耐酒精能力.经实验测定,肌醇对嗜鞣管囊酵母产酒精能力及发酵的耐酒精能力均有显著的影响.发酵培养基中未添加起始浓度的酒精时,菌种发酵的最适肌醇浓度为100mg/L,此时生成的酒精产量为45.20g/L.当分别添加起始酒精浓度为10%和12%时,随着肌醇浓度的增加,菌种发酵生成的酒精浓度均呈上升趋势;肌醇浓度为200mg/L时,两种起始酒精浓度下,酒精的净生成量均达到最大,分别为17.18g/L和16.68g/L.     

Pachysolen tannophilus: Properties and process considerations for ethanol production from d-xylose

d-Xylose comprises nearly one-third of the reducing sugars obtained from lignocellulose hydrolysis. Despite its relative abundance in crop and forest residues, xylose has been found unfermentable by most yeasts. A process for efficient xylose fermentation is expected to have significant impact on the future economics of converting lignocellulose to ethanol and may also provide additional profit for existing wood processing industries releasing xylose-rich waste streams, i.e. paper mills producing sulphite liquor. Pachysolen tannophilus was the first yeast discovered capable of significant ethanol production from xylose and has served as a model for studies of other yeasts mediating this conversion. Current knowledge about biochemical pathways involved in xylose utilization by this yeast is reviewed. Factors involved in regulating carbon flow to products are discussed in conjunction with process considerations for optimizing ethanol accumulation. Finally, the prospect of more efficient ethanol production through genetic strain improvement is considered.     

Sodium azide enhancement and inhibition of ethanol production from d-xylose Pachysolen tannophilus

The conversion of d-xylose to ethanol by the yeast Pachysolen tannophilus Y-2461 has been conducted in the presence of the respiratory inhibitor sodium azide. Conversion efficiencies improved for azide concentrations up to 0.2 mM. Concentrations above this value inhibited both ethanol production and cell growth. The work suggests that attempts to manipulate pentose conversion using extracellular factors, in this case azide, is of limited value in obtaining higher yield coefficients and better substrate conversion efficiencies.     

Ethanol production from whey permeate by immobilized yeast cells

The ability of two yeast strains to utilize the lactose in whey permeate has been studied. Kluyveromyces marxianus NCYC 179 completely utilized the lactose (9.8%), whereas Saccharomyces cerevisiae NCYC 240 displayed an inability to metabolize whey lactose for ethanol production. Of the two gel matrices tested for immobilizing K. marxianus NCYC 179 cells, sodium alginate at 2% (w/v) concentration proved to be the optimum gel for entrapping the yeast cells effectively. The data on optimization of physiological conditions of fermentation (temperature, pH, ethanol concentration and substrate concentration) showed similar effects on immobilized and free cell suspensions of K. marxianus NCYC 179, in batch fermentation. A maximum yield of 42.6 g ethanol l^?1 (82% of theoretical) was obtained from 98 g lactose l^?1 when fermentation was carried at pH 5.5 and 30°C using 120 g dry weight l^?1 cell load of yeast cells. These results suggest that whey lactose can be metabolized effectively for ethanol production using immobilized K. marxianus NCYC 179 cells.     

Concurrent Production and Consumption of Ethanol by Cultures of Pachysolen tannophilus Growing on d-Xylose

Growing cultures of Pachysolen tannophilus concurrently consumed and produced ethanol in the presence of substantial concentrations of d-xylose. Ethanol was also assimilated in the presence of other sugars, the amount depending on the sugar. Less ethanol assimilation occurred with d-glucose than with d-xylose. The rate of ethanol consumption decreased as the concentration of glucose was increased, but some consumption still occurred when 2% glucose was present. The rate increased with the amount of oxygen available to the culture when d-xylose or ethanol was the carbon source. In most instances, estimates of consumption were based on the extent of incorporation of C from [1-C]ethanol into trichloroacetic acid-insoluble material. The results are pertinent to the use of P. tannophilus for the production of ethanol from d-xylose.