Hilda Kersters-Hilderson

Catalytic properties of D-xylose isomerase from Streptomyces violaceoruber

Abstract The catalytic properties and stability of d -xylose isomerase from Streptomyces violaceoruber have been studied. The enzyme was activated by Mg 2+ , Co 2+ and Mn 2+ but Ni 2+ , Ca 2+ , Zn 2+ , Cu 2+ and Hg were ineffective. Optimum catalytic conditions were obtained at 80°C in the pH range 7.5–9.5 and in the presence of 10 m m Mg 2+ . The specific activity of the enzyme increased after treatment with 10 m m EDTA (factor 2.4). A further increase of activity (factor 2.0–2.8) was observed after preincubation of the enzyme with Mg 2+ or Co 2+ , the preincubation time depending on the incubation temperature. The thermal stability of the enzyme is very high. At 60°C the enzyme retained optimum activity following 30 days of storage in the presence of 1 m m Co 2+ or 10 m m Mg 2+ . At 80°C, Co 2+ is superior as a protector against thermal denaturation. At saturating concentrations of Mg 2+ (35°C) the K m -values of the EDTA-treated enzyme with respect to d -xylose and d -glucose were 2.8 and 149 m m and the dissociation constants of the enzyme-Mg 2+ complex for xylitol and d -sorbitol were 0.455 and 4.47 m m , respectively .

[60] β-d-xylosidase from Baccilus pumilus

Publisher Summary In this chapter the purification and properties of an intracellular β- D-xylosidase (β-o-xyloside xylohydrolase), induced in the same organism by xylose as sole carbon source, is described. For purification Bacillus pumilus PRL B12 is obtained. The purification procedure involves the preparation of cell-free extract, streptomycin sulfate treatment, ammonium sulfate fractionation, hydrophobic chromatography on Phenyl-Sepharose CL-4B, gel filtration on Sephadex G-200, and affinity chromatography on Sepharose 2B coupled to p-aminobenzyl l-thio-β-D xylopyranoside. The final preparation (97% pure) represents a 185-fold enrichment of the specific activity with a total yield of 65% and appears to be homogeneous on polyacrylamide gel electrophoresis. Only a second very weak protein band with fl-D-xylosidase activity is detectable, representing the tetrameric form of the enzyme. The p-nitrophenol released from the routine substrate p-nitrophenylβ-o-xylopyranoside is determined by its absorbance at 400 nm, at pH 7.15 and 25 °. The release of phenol or xylose from other arylβ-D-xylopyranosides is followed, respectively, by the method of Asp 3 (4-aminoantipyrin) or of Winckers 4 (o-toluidine). With (l→4)-β-D-xylooligosaccharides as substrates, an enzymic assay based on a coupled reaction of D-xylose isomerase with sorbitol dehydrogenase is used.

Purification of Bacillus pumilus β-D-xylosidase by affinity chromatography

We recently described the induction of a highly specific/3-D-xylosidase 03-D-xyloside xylohydrolase, EC 3.2.1.21) in a Bacillus pumilus strain [ 1 ]. ttowever, the purification method yielded a non-homogeneous preparation, as judged by disc-gel electrophoresis. The present note deals with a very efficient purification procedure, based on affinity chromatography on p-aminobenzyl-I -thio-~3-D-xylopyranoside coupled, as inhibitor-ligand, to a matrix of Sepharose 2B.

Determination of the anomeric configuration of D-xylose with D-xylose isomerases.

Abstract The anomeric configuration of D -xylose, resulting from hydrolysis of β- D xylopyranosides by β- D -xylosidase from Bacillus pumilus , has been determined by an enzymic procedure, based on the stereospecificity of D -xylose isomerases. The initial hydrolysis product is α- D -xylose. β- D -Xylosidase from Bacillus pumilus thus acts by inversion of configuration in contrast to most other glycosidases.

Kinetic characterization of D-xylose isomerases by enzymatic assays using D-sorbitol dehydrogenase

Abstract The enzymatic and coupled d -xylose isomerase/ d -sorbitol dehydrogenase assay is a rapid and specific method, permitting accurate quantification of d -xylose isomerization and of d -xylose. The method is based on the isomerization of d -xylose to d -xylulose, followed by reduction of the latter to xylitol by commercially available d -sorbitol dehydrogenase and NADH. The application of this one-step method cannot be extended to d -glucose isomerization since the conditions for a valid coupled assay are not fulfilled. For quantification of d -glucose isomerization, the two-step procedure with d -sorbitol dehydrogenase is recommended. Kinetic parameters for d -xylose and d -glucose using d -xylose isomerase from Streptomyces violaceoruber are reported. The results are compared with the widely used colorimetric cysteine-carbazole method.

β-d-xylosidase from Bacillus pumilus: Molecular properties and oligomeric structure

Bacillus pumilus beta-D-xylosidase, purified by affinity chromatography, seems to be homogeneous, as judged by disc electrophoresis and gel filtration. The absorption coefficient at 280 nm, Ao, 1% 1cm, determined by the dry weight method, is 1.78. The complete amino acid composition is determined. Sedimentation velocity studies show the presence of two components with S20, W values of 10.0 S and 6.6 S. After glutaraldehyde cross-linking two, enzymically active, components, with apparent molecular weights 126 000 and 243 000, can be isolated by preparative sucrose gradient ultracentrifugation. These values are confirmed by analytical disc electrophoresis at different acrylamide concentrations. The subunit molecular weight is 60 000. L-Methionine is the only N-terminal amino acid detectable. The possible presence of both dimeric and tetrameric forms of the beta-D-xylosidase in solution has to be envisaged.

D-xylose isomerase from Streptomyces violaceoruber: structural and catalytic roles of bivalent metal ions

Abstract d -Xylose isomerase from Streptomyces violaceoruber has two nonequivalent bivalent metal ion sites. The activity and stability of the enzyme-[M(II),—] and the enzyme-[M(II),M(II)]-complexes under various denaturing conditions have been studied and indicate that (i) denaturation is always accompanied by irreversible dissociation of the tetrameric structure to monomers; (ii) treatment with 0.1% sodium dodecyl sulfate causes reversible dissociation into active dimers; the equilibrium tetramer dimers is unaffected by Co(II) or Mg(II); (iii) addition of 1 eq Co(II) per monomer or binding of Co(II) at the conformational high-affinity site markedly enhances the structural stability of d -xylose isomerase against elevated temperature, urea, guanidinium hydrochloride, ethanol, and acetone; (iv) the stabilizing effect of Mg(II) at the conformational site is less evident; (v) binding of bivalent metal ions at the lower-affinity site is required for initiation of catalytic activity; (vi) the activating effect of Mg(II) is superior to that of Co(II).

β-d-xylosidase from Bacillus pumilus prl b12: hydrolysis of aryl β-d-xylopyranosides

Abstract The influence of substituents on the binding and hydrolysis of several substituted β- d -xylopyranosides by β- d -xylosidase Bacillus pumilus PRL B12 has been investigated. From a comparison of the inhibition constants of 1-thio-β- d -xylopyranosides with the apparent Michaelis-Menten constants of the substrates, it followed that the latter constants are good approximations of the true equilibrium constants. The influence of the substituent on the rate and activation parameters is small. The results are in agreement with, but do not prove, a one-step mechanism without the formation of a glycosyl-enzyme intermediate.

Quantitative determination of d-xylose by a coupled reaction of d-xylose isomerase with d-glucitol dehydrogenase

Abstract An enzymie assay for the quantitative determination of d -xylose has been developed. The methods is based on a coupled reaction of d -xylose isomerase with d -glucitol dehydrogenase. Perfect agreement between the results, obtained with the o -toluidine-acetic acid method and with the coupled enzymic assay, was found. The method has also been successfully used in a kinetic study of β- d -xylosidase using xylobiose as substrate. Accurate and reproducible results were obtained throughout all experiments.

Kinetic α-deuterium isotope effects for enzymatic and acid hydrolysis of aryl-β-d-glycopyranosides

Abstract The kinetic α-deuterium isotope effect for the enzymatic- and acid-catalyzed hydrolysis of a series of aryl-β- d -[1- 2 H]glycopyranosides has been measured. The magnitude of the effect indicates a considerable steric hindrance of the anomeric CH (CD) bond in an early transition state for both kinds of reactions. Better leaving aryl groups decrease the isotope effect for the acid-catalyzed hydrolysis, as predicted by the predominant A-1 character of the reaction. In contrast, the α-deuterium effect for the enzymatic-catalyzed reactions is increased by better leaving aglycon groups, suggesting a mechanism with considerable S N 2 characteristics. The isotope effect for the acid hydrolysis of 4-methylumbelliferyl- and 4-methylfenyl-β- d -glucopyranoside has been measured over the temperature range 40–85°C. The results indicate a different temperature dependence of the effect for both β- d -glucopyranosides.