Purification and properties of d-xylulose reductase from Pachysolen tannophilus

d-Xylulose reductase (EC 1.1.1.9) from Pachysolen tannophilus IFO 1007 was purified by Sephadex G-100 gel chromatography with three columns and DEAE cellulose chromatography. The purified enzyme was entirely homogeneous on disc gel electrophoresis. It was most active at pH 9.1–10.0 and 55°C, and stable at pH 7–9 and below 25 °C. Its activity was stimulated by NH₄Cl,NaCl,MgCl₂,KCl, glutathione, cysteine and glycine, and inhibited remarkably by SH inhibitor such as lead acetate, HgCl₂ and AgNO₃. It oxidized xylitol, sorbitol, ribitol and glycerine but not mannitol, inositol, arabitol and erythritol. Its K_m values of enzyme against xylitol, sorbitol and ribitol were 1.1 × 10⁻² M, 3.0 × 10⁻² M and 5.0 × 10⁻² M, respectively. Its molecular weight was determined to be 120,000 by Sephadex G-200 column chromatography, and that of its subunit was 40,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Comparison of the catalytic and inhibitory properties of Pachysolen tannophilus xylose reductase to rat lens aldose reductase

Summary The catalytic and inhibitory profiles of xylose reductase isolated from the yeast Pachysolen tannophilus (PTXR) are compared to those of aldose reductase (AR) obtained form rat lens. While both PTXR and rat lens AR are NADPH-specific enzymes and have an affinity for a variety of substrates such as d-xylose, d,l-glyceraldehyde, and 4-nitrobenzaldehyde, the enzymes differ in their substrate affinity profiles. Also, PTXR is not inhibited by standard inhibitors of AR thus supporting a hypothesis that this enzyme may not possess the inhibitor binding site found in rat lens AR. Offprint requests to: J. DeRuiter     

Demonstration of D-xylose reductase and D-xylitol dehydrogenase in Pachysolen tannophilus

Summary The yeast, Pachysolen tannophilus, can utilize the pentose D-xylose with accumulation of significant quantities of ethanol. Cell extracts of the organism contain NADPH-linked D-xylose reductase (aldose reductase EC 1.1.1.21) and NAD-dependent D-xylitol dehydrogenase (D-xylulose reductase EC 1.1.1.9). D-Xylose was required for induction of both the D-xylitol dehydrogenase and the D-xylose reductase. Neither enzyme was found in glucose grown cell-free extracts.     

Multiple forms of xylose reductase in Pachysolen tannophilus CBS4044

Abstract Cell-free extracts of xylose-grown Pachysolen tannophilus exhibited xylose reductase activity with both NADPH and NADH. The ratio of the NADPH- and NADH-dependent activities varied with growth conditions. Affinity chromatography of cell-free extracts resulted in a separation of two xylose reductases. One was active with both NADPH and NADH, the other was specific for NADPH. Apart from this coenzyme specificity, the two enzymes also differed in their affinities for xylose and NADPH. The role of the two enzymes in xylose metabolism is discussed in relation to attempts to use P. tannophilus for the alcoholic fermentation of wood sugars.     

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 .     

Purification and properties of aldose reductase and aldehyde reductase II from human erythrocyte

Aldose reductase (EC 1.1.1.21) and aldehyde reductase II (L-hexonate dehydrogenase, EC 1.1.1.2) have been purified to homogeneity from human erythrocytes by using ion-exchange chromatography, chromatofocusing, affinity chromatography, and Sephadex gel filtration. Both enzymes are monomeric, Mr 32,500, by the criteria of the Sephadex gel filtration and polyacrylamide slab gel electrophoresis under denaturing conditions. The isoelectric pH's for aldose reductase and aldehyde reductase II were determined to be 5.47 and 5.06, respectively. Substrate specificity studies showed that aldose reductase, besides catalyzing the reduction of various aldehydes such as propionaldehyde, pyridine-3-aldehyde and glyceraldehyde, utilizes aldo-sugars such as glucose and galactose. Aldehyde reductase II, however, did not use aldo-sugars as substrate. Aldose reductase activity is expressed with either NADH or NADPH as cofactors, whereas aldehyde reductase II can utilize only NADPH. The pH optima for aldose reductase and aldehyde reductase II are 6.2 and 7.0, respectively. Both enzymes are susceptible to the inhibition by p-hydroxymercuribenzoate and N-ethylmaleimide. They are also inhibited to varying degrees by aldose reductase inhibitors such as sorbinil, alrestatin, quercetrin, tetramethylene glutaric acid, and sodium phenobarbital. The presence of 0.4 M lithium sulfate in the assay mixture is essential for the full expression of aldose reductase activity whereas it completely inhibits aldehyde reductase II. Amino acid compositions and immunological studies further show that erythrocyte aldose reductase is similar to human and bovine lens aldose reductase, and that aldehyde reductase II is similar to human liver and brain aldehyde reductase II.     

Ethanol production from d-galactose and glycerol by Pachysolen tannophilus

Pachysolen tannophilus has recently been shown to be able to convert d-xylose, a pentose, to ethanol. Previously, d-xylose had been considered to be nonfermentable by yeasts. The present study shows that the organism can be used to obtain ethanol from other carbohydrates previously considered as nonfermentable, either by P. tannophilus in particular, d-galactose, or by yeasts in general, glycerol. Such identification for d-galactose allows P. tannophilus to be considered for fermentation of four of the five major plant monosaccharides: d-glucose, d-mannose, d-galactose and d-xylose. The ability to ferment glycerol is of potential use, in part, for the conversion of glycerol derived from algae into ethanol.     

Purification and characterization of aldose reductase and aldehyde reductase from human kidney.

Aldose reductase and aldehyde reductases have been purified to homogeneity from human kidney and have molecular weights of 32,000 and 40,000 and isoelectric pH 5.8 and 5.3, respectively. Aldose reductase, beside catalyzing the reduction of various aldehydes, reduces aldo-sugars, whereas aldehyde reductase, does not reduce aldo-sugars. Aldose reductase activity is expressed with either NADH or NADPH as cofactor, whereas aldehyde reductase utilizes only NADPH. Both enzymes are inhibited to varying degrees by aldose reductase inhibitors. Antibodies against bovine lens aldose reductase precipitated aldose reductase but not aldehyde reductase. The sequence of addition of the substrates to aldehyde reductase is ordered and to aldose reductase is random, whereas for both the enzymes the release of product is ordered with NADP released last.     

Purification and characterization of human testis aldose and aldehyde reductase

1. Aldose reductase and aldehyde reductase were purified to homogeneity from human testis. 2. The molecular weight of aldose reductase and aldehyde reductase were estimated to be 36,000 and 38,000 by SDS-PAGE, and the pI values of these enzymes were found to be 5.9 and 5.1 by chromatofocusing, respectively. 3. Aldose reductase had activity for aldo-sugars, whereas aldehyde reductase was virtually inactive for aldo-sugars. The Km values of aldose reductase for D-glucose, D-galactose and D-xylose were 57, 49 and 6.2 mM, respectively. Aldose reductase utilized both NADPH and NADH as coenzymes, whereas aldehyde reductase only NADPH. 4. Sulfate ion caused 3-fold activation of aldose reductase, but little for that of aldehyde reductase. 5. Sodium valproate inhibited significantly aldehyde reductase, but not aldose reductase. Aldose reductase was inhibited strongly by aldose reductase inhibitors being in clinical trials at concentrations of the order of 10(-7)-10(-9) M. Aldehyde reductase was also inhibited by these inhibitors, but its susceptibility was less than aldose reductase. 6. Reaction of aldose reductase with pyridoxal 5'-phosphate (PLP) resulted ca 2.5-fold activation, but aldehyde reductase did not cause the activation. PLP-treated aldose reductase has lost the susceptibility to aldose reductase inhibitor.     

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.     

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.     

Hybrid formation and copulation activity in the yeast Pachysolen tannophilus

The copulation activity and hybrid formation efficiency have been studied in the xylose-assimilating yeast Pachysolen tannophilus. It was shown that the presence of 2% D-glucose, 0.5% yeast extract, and 2% agarose in the growth medium provided for the highest frequencies of hybrid formation. Atypical hybrid cultures similar in morphophysiological characteristics to native haploid strains of P. tannophilus were revealed in the course of hybridization. The genesis mechanism of such cultures and the reasons for the restricted applicability of hybridological analysis to genetic studies of P. tannophilus are discussed.