Hanswerner Dellweg

Quantitative chromatography of homologous glucose oligomers and other saccharides using polyacrylamide gel

Abstract A rapid method for the separation and quantitation of mono- and oligosaccharides is described. The procedure is based on the use of Bio-Gel P-2, minus 400 mesh, in a properly designed column with water as eluent at 65°. For the colorimetric estimation of the carbohydrates the effluent is monitored by an automated analyzing system. Homologous oligosaccharides containing up to thirteen glucose units are separated within four to seven hours for analytical and preparative purposes. Separation of glucose from ribose and maltose from isomaltose has also been obtained. Quantitative calibration by peak height was performed in the range of 10 to 100 μg.

Xylose fermentation by yeasts. IV: Purification and kinetic studies of xylose reductase from Pichia stipitis

SummaryXylose reductase from the xylose-fermenting yeastPichia stipitis was purified to electrophoretic homogeneity via ion-exchange, gel and affinity chromatography. At physiological pH values the thermodynamic equilibrium constant was determined to be 0.575x1010 (l·mol-1). Product inhibiton studies are reported which clearly show that the kinetic mechanism of the xylose reductase is ordered-bi-bi with isomerisation of a stable enzyme form.

Xylose fermentation by yeasts

SummaryUtilization and fermentation of xylose by the yeasts Pachysolen tannophilus I fGB 0101 and Pichia stipitis 5773 to 5776 under aerobic and anaerobic conditions are investigated. Pa. tannophilus requires biotin and thiamine for growth, whereas Pi. stipitis does not, and growth of both yeasts is stimulated by yeast extract. Pi. stipitis converts xylose (30 g/l) to ethanol under anaerobic conditions with high yields of 0,40 and it produces only low amounts of xylitol. The yield coefficient is further increased at lower xylose concentrations.

Purification and properties of the NAD+-xylitol-dehydrogenase from the yeast Pichia stipitis☆

Abstract Cell-free extracts of the xylose fermenting yeast Pichia stipitis exhibited xylitol dehydrogenase activity with NAD + and NADP + . During the purification step on DEAE-sephadex A-50 a NAD + -dependent xylitol dehydrogenase could be separated from a NADP + -dependent. The NAD + -xylitol dehydrogenase was further purified to electrophoretic homogeneity via gel and affinity chromatography. The purified enzyme was most active at pH 9 and 35°C. Its molecular weight was determined to be 63,000 dalton by Sephadex G-200 column chromatography, and that of its subunit was 32,000 dalton by sodium dodecyl sulphate polyacrylamide gel electrophoresis. From the results of substrate specificity, the enzyme should be named l -iditol:NAD + -5-oxidoreductase (EC 1.1.1.14, sorbitol dehydrogenase).

Fermentation of xylose with the yeast Pachysolen tannophilus

SummaryPachysolen tannophilus IfGB 0101, is able to grow aerobically on xylose, glucose and fructose. Galactose too is assimilated after long adaption times. In the complete absence of oxygen, xylose is fermented, forming mainly xylitol, lower amounts of ethanol and CO2. According to the mass balance, it may be concluded that the pentose phosphate enzymes together with the oxidative phosphogluconate way are in action simultaneously. At semi-aerobic conditions (0.45 l air per liter, per hour) ethanol production is somewhat increased but no aeration conditions could as yet be found at which ethanol was the main fermentation product. The significance of xylitol formation seems to be mainly that of an electron sink of the phosphogluconate pathway.

A kinetic study of the NAD+-xylitol-dehydrogenase from the yeast Pichia stipitis

Abstract At physiological pH values the thermodynamic equilibrium constant was determined to be 6,9,10 −11 (mol/ l ). Product inhibition studies are reported which clearly show that the kinetic mechanism of the NAD + -xylitol dehydrogenase is “Ordered-bi-bi”. It can be seen from experimental results that a cosubstrate inhibition with a dead end EA 2 -complex occurs at elevated NAD + -concentrations. Simulations were carried out which indicate, that under intracellular conditions the NAD + -xylitol dehydrogenase is regulated by the catabolic reduction charge and not by the total concentrations of NAD + and NADH.

Xylose fermentation by yeasts. VIII: Influence of furfural on the aerobic growth of the yeast Pichia stipitis

SummaryFurfural as a product from thermic wood hydrolysis processes may be inhibitory to growth and fermentation of yeast cells. In order to determine the influence on the aerobic growth of the yeastPichiastipitis, expermiments were conducted in stirred reactors with the addition of furfural.We found that the added furfural was rapidly reduced to furfuryl alcohol. Furfural inhibited the respiration and the produced furfuryl alcohol influenced the growth rate ofPichiastipitis. As a result from enzyme studies it can be concluded that the alcohol-dehydrogenase (ADH) was mainly responsible for the reduction of furfural to furfuryl alcohol.

Gel permeation chromatography of maltooligosaccharides at different temperatures

Abstract The separation of maltodextrins (glucose to maltoheptaose) on polyacrylamide gel has been studied at various temperatures. A negative temperature dependence of elution volume was established so that the distribution coefficients decreased with increasing temperature. This peculiarity gives rise to a negative enthalpy for the interacting effects between solute and the gel matrix.

Gel chromatographic separation of oligosaccharides at elevated temperature

The separation of carbohydrates on Sephadex-gels has been described in several papers [l-4] . Preliminary studies on the chromatographic behavior of polyacrylamide gels indicate the usefulness of this support in the separation of sugars [S--7]. In our work polyacrylamide gel was found to be superibr‘ to gels based on dextran because it does not give off carbohydrates [8] and is not attacked by bacteria. A survey of the literature showed that the effect of elevated temperatures on the resolution of carbohydrates by gel chromatography has not been described. Hough, Jones and Wadman reported that the separation of sugars on paper chromatograms is markedly improved at elevated temperatures [9]. Thoma, Wright and French separated maltose homologs on a heated cellulose column and found that high temperatures increase both the resolution and mobility of saccharides [lo] . In this paper we discuss the effect of elevated temperatures on the chromatographic separation of oligosaccharides on polyacrylamide gel.

Comparison between the process performance of an UASB-reactor and an UASB-fixed film-combination with an acetic acid enrichment culture

SummaryAn Upflow Sludge Blanket-(USB)-reactor and an USB-Fixed Film-(UBFF)-combination were operated simultaneously, running as pH-auxostats with a synthetic wastewater containing acetic acid as the only carbon source. In the UBFF-reactor accelerated growth of the sludge blanket volume could be observed in addition to fast surface biofilm development and a granular bacterial sludge of high density. Hydraulic retention times (HRT) of 2.3 h, and gasproductivities of 100 v/v.d were achieved in the UBFF-reactor, compared to 3.5 h and 70 v/v.d respectively in the USB-reactor. At maximum loading rates the acetic acid conversion efficiency exeeded values of 90% in both reactors.