Purification and characterization of the d-xylose isomerase gene from Escherichia coli

A DNA fragment containing both the Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene and the d-xylulokinase (ATP: d-xylulose 5-phosphotransferase, EC 2.7.1.17) gene has been cloned on an E. coli plasmid. The d-xylose isomerase gene was separated from the d-xylulokinase gene by the construction of a new deletion plasmid, pLX7. The d-xylose isomerase gene cloned on pLX7 was found still to be an intact gene. The precise location of the d-xylose isomerase gene on the plasmid pLX7 was further determined by the construction of two more plasmids, pLX8 and pLX9. This is believed to be the first d-xylose isomerase gene that has been isolated and extensively purified from any organism. d-Xylose isomerase, the enzyme product of the d-xylose isomerase gene, is responsible for the conversion of d-xylose to d-xylulose, as well as d-glucose to d-fructose. It is widely believed that yeast cannot ferment d-xylose to ethanol primarily because of the lack of d-xylose isomerase in yeast. d-Xylose isomerase (also known as d-glucose isomerase) is also used for the commercial production of high-fructose syrups. The purification of the d-xylose isomerase gene may lead to the following industrial applications: (1) cloning and expression of the gene in yeast to make the latter organism capable of directly fermenting d-xylose to ethanol, and (2) cloning of the gene on a high-copy-number plasmid in a proper host to overproduce the enzyme, which should have a profound impact on the high-fructose syrup technology.     

Effects of borate on isomerization and yeast fermentation of high xylulose solution and acid hydrolysate of hemicellulose

d-Xylose has been isomerized by immobilized d-glucose isomerase (EC nomenclature is now d-xylose isomerase, d-xylose ketol-isomerase, EC 5.3.1.5; EC 5.3.1.18 is a deleted EC entry). Temperature has a profound influence on the equilibrium concentration of d-xylulose. When 1 md-xylose was isomerized in the presence of various concentrations of borate, maximum conversion (80%) was observed at 0.2 m sodium tetraborate. Temperature (40–69°C) and pH (6.0–7.5) had an insignificant effect on the equilibrium when borate was present. d-Xylose (0.5 m) was isomerized by d-glucose isomerase in the presence of various concentrations of sodium tetraborate (0.0125–0.25 m). Based on the initial rate of ethanol production and the fraction of total sugar converted into ethanol after 24 h of yeast fermentation, an optimum tetraborate concentration of 0.05 m was determined for both isomerization and fermentation. At an acidic pH, the rate of fermentation was faster than at neutral pH when borate was included in the d-xylose—d-xylulose system. Acid hydrolysate of bagasse hemicellulose could not be fermented at a pH lower than 5. Therefore, a compromise condition, pH 6.0, was chosen for fermentation.     

Effect of oxyanions on the glucose isomerase catalysed equilibrium: 1. Complexes of d-glucose and d-fructose with germanate

The complexing parameters of d-glucose and d-fructose with germanate, derived from various forms of germanium dioxide, have been studied under the conditions pertaining to the d-glucose isomerase (d-xylose isomerase, d-xylose ketol-isomerase, EC 5.3.1.5) reaction. The interaction of germanate with d-glucose and d-fructose at various pH values has been investigated by means of optical rotation methods. The effects of temperature and concentration on the extent of complex formation are reported. The results are used to predict suitable conditions for the enhancement of d-fructose yield in the reaction of d-glucose with this enzyme.     

Production of Ethanol from d-Xylose by Using d-Xylose Isomerase and Yeasts

d-Xylulose, an intermediate of d-xylose catabolism, was observed to be fermentable to ethanol and carbon dioxide in a yield of greater than 80% by yeasts (including industrial bakers' yeast) under fermentative conditions. This conversion appears to be carried out by many yeasts known for d-glucose fermentation. In some yeasts, xylitol, in addition to ethanol, was produced from d-xylulose. Fermenting yeasts are also able to produce ethanol from d-xylose when d-xylose isomerizing enzyme is present. The results indicate that ethanol could be produced from d-xylose in a yield of greater than 80% by a two-step process. First, d-xylose is converted to d-xylulose by xylose isomerase. d-Xylulose is then fermented to ethanol by yeasts.     

Cloning of Xanthomonas DNA that expresses d-xylose catabolic enzymes

A gene bank of the d-xylose utilizing, cellulolytic Xanthomonas (XA1-1) DNA, inserted into the Hind III site of pKT230, was screened for clones which encoded d-xylose isomerase. One clone (pND70) was identified which complemented d-xylose isomerase negative mutants of Escherichia coli and this clone contained an insert of XA1-1 DNA of approximately 15 kb. Enzyme assays showed that pND70 appeared to encode d-xylose permease, and xylulose kinase in addition to d-xylose isomerase. Specific activities of all 3 enzymes from E. coli JA200 (pND70) grown in d-xylose were double those detected in XA1-1 when also grown on d-xylose.     

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

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.     

Effect of oxyanions on the d-glucose isomerase catalysed equilibrium: 2. Effect of germanate on the equilibrium of d-glucose and d-fructose with immobilized d-glucose isomerase

The addition of germanate anions to high d-glucose feed syrups, which are passed through an immobilized d-glucose isomerase [xylose isomerase, d-xylose ketol-isomerase, EC 5.3.1.5] column, displaces a ca. 50/50 d-glucose/d-fructose mixture (produced in the absence of germanate) in favour of d-fructose. A maximum conversion of 94% from a d-glucose feed (40% w/v) is obtained with no detrimental effect on the enzyme. This is related to the germanate: sugar ratio. Optimization of the d-fructose yield from d-glucose germanate substrate has been carried out. The effects due to temperature, pH and concentration were taken into consideration. Confirmation of the quantitative identification of the d-fructose was obtained by isotope dilution analysis. The theory behind the displacement is also discussed, and shows close agreement with practical results.     

Ethanol production from pentoses by immobilized microorganisms

The capabilities of immobilized Fusarium oxysporum f. sp. lini, Mucor sp., and Saccharomyces cerevisiae in fermenting pentose to ethanol have been compared. S. cerevisiae was found to have the best fermentation rate on d-xylulose of 0.3 g l^?1 h^?1. By using a separate isomerase column for converting d-xylose to d-xylulose and a yeast column for converting d-xylulose to ethanol, an ethanol concentration of 32 g l^?1 was obtained from 10% d-xylose. The ethanol yield was calculated to be 64% of the theoretical yield.     

l-Xylose and l-lyxose production from xylitol using Alcaligenes 701B strain and immobilized l-rhamnose isomerase enzyme

Xylitol was used as a raw material for production of l-xylose and l-lyxose using Alcaligenes 701B strain and immobilized l-rhamnose isomerase enzyme. Alcaligenes 701B converted xylitol to l-xylulose with a yield of 34% in the bioreactor. l-Xylulose was converted to l-xylose and l-lyxose using immobilized l-rhamnose isomerase enzyme. The final equilibrium between l-xylulose, l-xylose and l-lyxose was 53:26:21. The enzyme assays indicated that Alcaligenes 701B strain has an NAD-dependent xylitol dehydrogenase enzyme responsible for l-xylulose production. Furthermore, NAD(P)H-dependent l-xylulose reductase enzyme was active during conversion of xylitol to l-xylulose. The highest l-xylulose production rate corresponded with the highest growth rate. The Alcaligenes 701B strain used d-xylose for biomass growth, but xylitol was used only for l-xylulose production during conversion phase.     

Studies on sugar isomerases of Lactobacillus sp.: Whole cell immobilization of d-glucose isomerase

A new soil isolate of Lactobacillus sp. grown in Yamanaka medium under submerged conditions showed the presence of d-glucose, d-xylose and d-ribose isomerases in washed cell suspension and cell free extracts. d-Xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) and d-ribose isomerase (d-ribose ketol-isomerase, EC 5.3.1.20) activities reached a maximum in 48 h of growth and then declined. d-Glucose isomerase (d-glucose 6-phosphate isomerase, d-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) activity was maximum after 72 h and remained constant for ～120 h of growth. d-Glucose isomerase activity increased with the increase in number of generations of culture and reached a maximum in 5–6 generations, whereas d-xylose and d-ribose isomerase activities decreased. The washed and starved whole cells could be heat treated and immobilized on the rough surface of glass rods or glass slides using acetone treatment. The heat treated immobilized cells showed only the presence of d-glucose isomerase activity and showed no d-xylose and d-ribose isomerase activities. d-Glucose isomerase activity of heat treated immobilized cells was inhibited less by sorbitol, mannitol, sodium arsenate, cysteine and calcium ions than the free d-glucose isomerase activity in fresh untreated washed whole cells and cell free extracts. EDTA inhibition had the same effect for both forms. Ca²⁺inhibition could be reversed by adding Mg²⁺ions.     

Isomerization of glucose to fructose: production and some properties of glucose isomerase from Streptomyces sp. strain C7

Nine strains of actinomycetes isolated from Iraqi soils were investigated for glucose isomerase production. Only one strain, Streptomyces sp., C₇, was active. The maximum conversion ratio of the enzyme for the cells grown in d-xylose medium after 24 h incubation at 70°C and pH 6.9, was 64 and 48% for crude extract and cell-bound enzyme, respectively. The optimum pH value and temperatures for both enzymes were 8.0 and 70°C.     

Emerging technology for fermenting d-xylose

In the past four years, numerous yeasts which convert d-xylose to ethanol have been reported. The conversion occurs most readily under aerobic conditions. Various aspects of this conversion have provided new insight into the mechanisms and metabolic regulation of ethanol fermentation in yeasts. Although specific fermentation rates, product yields and product concentrations are significantly lower with d-xylose than with d-glucose, technology is emerging which may prove to be feasible for commercial fermentation of d-xylose-containing waste streams.     

Overproduction of d-xylose isomerase in Escherichia coli by cloning the d-xylose isomerase gene

The Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene, xylA, has been cloned on various E. coli plasmids. However, it has been found that high levels of overproduction of the d-xylose isomerase, the protein product of the xylA gene, cannot be accomplished by cloning the intact gene on high copy-number plasmids alone. This is believed to be due to the fact that the expression of the gene through its natural promoter is highly regulated in E. coli. In order to overcome this, the xylA structural gene has been fused with other strong promoters such as tac and lac, resulting in the construction of a number of fused genes. Analysis of the E. coli transformants containing the fused genes, cloned on high copy-number plasmids, indicated that a 20-fold overproduction of the enzyme can now be obtained. It is expected that overproduction of the enzyme in E. coli can still be substantially improved through additional manipulation with recombinant DNA techniques.     

Overproduction of d-xylose isomerase in Escherichia coli by cloning the d-xylose isomerase gene

The Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene, xylA, has been cloned on various E. coli plasmids. However, it has been found that high levels of overproduction of the d-xylose isomerase, the protein product of the xylA gene, cannot be accomplished by cloning the intact gene on high copy-number plasmids alone. This is believed to be due to the fact that the expression of the gene through its natural promoter is highly regulated in E. coli. In order to overcome this, the xylA structural gene has been fused with other strong promoters such as tac and lac, resulting in the construction of a number of fused genes. Analysis of the E. coli transformants containing the fused genes, cloned on high copy-number plasmids, indicated that a 20-fold overproduction of the enzyme can now be obtained. It is expected that overproduction of the enzyme in E. coli can still be substantially improved through additional manipulation with recombinant DNA techniques.     

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.     

Selective isolation of acidophilic streptomyces strains for glucose isomerase production

Approximately 260 Streptomyces strains were isolated from neutral pH farmland soil and evaluated for their ability to produce glucose isomerase. The number of acidophilic Streptomyces organisms growing at pH 4.0 was low, i.e., 10 organisms per g of soil. All of the isolates showed glucose isomerase activity when they were grown in a medium containing d-xylose, an inducer for glucose isomerase. More than half of the strains tested developed heavy growth in 24 h, and many produced high titers of glucose isomerase after 24 h of growth in a medium buffered at pH 5.0.     

Purification and characterization of the -xylose isomerase gene from Escherichia coli

A DNA fragment containing both the Escherichia coli -xylose isomerase ( -xylose ketol-isomerase, EC 5.3.1.5) gene and the -xylulokinase (ATP: -xylulose 5-phosphotransferase, EC 2.7.1.17) gene has been cloned on an E. coli plasmid. The -xylose isomerase gene was separated from the -xylulokinase gene by the construction of a new deletion plasmid, pLX7. The -xylose isomerase gene cloned on pLX7 was found still to be an intact gene. The precise location of the -xylose isomerase gene on the plasmid pLX7 was further determined by the construction of two more plasmids, pLX8 and pLX9. This is believed to be the first -xylose isomerase gene that has been isolated and extensively purified from any organism. -Xylose isomerase, the enzyme product of the -xylose isomerase gene, is responsible for the conversion of -xylose to -xylulose, as well as -glucose to -fructose. It is widely believed that yeast cannot ferment -xylose to ethanol primarily because of the lack of -xylose isomerase in yeast. -Xylose isomerase (also known as -glucose isomerase) is also used for the commercial production of high-fructose syrups. The purification of the -xylose isomerase gene may lead to the following industrial applications: (1) cloning and expression of the gene in yeast to make the latter organism capable of directly fermenting -xylose to ethanol, and (2) cloning of the gene on a high-copy-number plasmid in a proper host to overproduce the enzyme, which should have a profound impact on the high-fructose syrup technology.     

Purification and characterization of chalcone isomerase from soybeans

Chalcone isomerase from soybean has been purified 11,000-fold over the crude extract. The purification procedure features pseudo-affinity chromatography on an Amicon Matrex Orange A column with selective elution by a product of the enzymatic reaction. The purified enzyme is greater than 99.5% pure and possesses a specificity activity of 340 IU/mg, which is 520-fold greater than previously reported. The apparent molecular weight of the chalcone isomerase is 24,000 as determined from sodium dodecyl sulfate-polyacrylamide gels and from size exclusion chromatography under native conditions on Sephacryl S-200. The enzyme exists as a monomer that migrates on isoelectric focusing gels with a pI of 5.7. Amino acid analysis indicates that almost 50% of the residues are hydrophobic and yields a partial specific volume of 0.750 ml/g. Chalcone isomerase contains no carbohydrate moieties and has a blocked N terminus. The purified enzyme catalyzes the conversion of 2', 4',4-trihydroxychalcone (I) to (2S)-4',7-dihydroxyflavanone (II) at pH 7.6 with a second order rate constant, kcat/Km, of 1.1 X 10(9) M-1 min-1 and an apparent equilibrium constant, [II]/[I], of 7.6. The rate constant for the conversion of enzyme-bound substrate to the (2S)-flavanone, kcat = 11,000 min-1, exceeds the spontaneous conversion by 36 million-fold. The enzyme catalyzes the formation of (2S)-flavanone over 100,000-fold faster than to the (2R)-flavanone, indicating that the enzyme is highly stereoselective, yielding over 99.999% of the (2S)-flavanone.     

Optimum temperature operation mode for glucose isomerase reactor operating at constant glucose conversion

The optimum temperature operation mode required to achieve constant outlet glucose conversion is determined for immobilized glucose isomerase continuous packed bed reactor. The reactor design equation assumes reversible Michaelis-Menten kinetics with both enzyme deactivation and substrate protection. An increasing temperature profiles are determined for different operating periods, residence times and glucose conversions. The temperature increase with time is very small at low degree of glucose conversion and at relatively long residence time. The temperature rise with time increases at high degree of conversion and at relatively short residence time.     

Kinetics of glucose isomerization to fructose by immobilized glucose isomerase: anomeric reactivity of D-glucose in kinetic model

The substrate specificity of immobilized D-glucose isomerase (EC 5.3. 1.5) is investigated with an immobilized enzyme-packed reactor. A series of isomerization experiments with alpha-, beta-, and equilibrated D-glucose solutions indicates that beta anomer as well as alpha anomer is a substrate of the glucose isomerase at pH 7.5 and 60 degrees C. For substrate concentration of 0.028 mol l(-1) (1% w/v), the initial conversion rate of alpha-D-glucose was 43% higher than that with equilibrated glucose at the same concentration and 113% higher than beta-D-glucose conversion rate. This anomeric reactivity of glucose isomerase is mathematically described with a set of kinetic equations based on the reaction steps complying with Briggs-Haldane mechanism and the experimentally determined kinetic constants. The proposed reaction mechanism includes the mutarotation and the isomerization reactions of alpha- and beta-D-glucose with different rate constants.