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PubMedDate not supplied

Effect of pH on simultaneous saccharification and isomerization by glucoamylase and glucose isomerase.

pH and temperature play critical roles in multistep enzymatic conversions. In such conversions, the optimal pH for individual steps differs greatly. In this article, we describe the production of glucoamylase (from Aspergillus oryzae MTCC152 in solid-state fermentation) and glucose isomerase (from Streptomyces griseus NCIM2020 in submerged fermentation), used in industries for producing high-fructose syrup. Optimum pH for glucoamylase was found to be 5.0. For glucose isomerase, the optimum pH ranged between 7.0 and 8.5, depending on the type of buffer used. Optimum temperature for glucoamylase and glucose isomerase was 50 and 60 degrees C, respectively. When both the enzymatic conversions were performed simultaneously at a compromised pH of 6.5, both the enzymes showed lowered activity. We also studied the kinetics at different pHs, which allows the two-step reaction to take place simultaneously. This was done by separating two steps by a thin layer of urease. Ammonia generated by the hydrolysis of urea consumed the hydrogen ions, thereby allowing optimal activity of glucose isomerase at an acidic pH of 5.0.

Aldose-Ketose Isomerases↗

PubMed1987

Clinical significance and source of raised catalytic activities of phosphohexose isomerase in the CSF in meningitis.

The value of phosphohexose isomerase (EC 5.3.1.9) determination in the CSF in the diagnosis of meningitis was tested under routine conditions. In 48 patients with untreated bacterial meningitis, enzyme activity concentrations between 40 and 2335 U/l were measured, whereas the highest phosphohexose isomerase activity concentration in 92 patients with viral meningitis was 34 U/l. Lysis of the CSF leukocytes with Triton X-100 resulted in a substantial increase of phosphohexose isomerase activity. In bacterial meningitis these values increased to 200 to 7000 U/l, but remained under 150 U/l in viral meningitis. The raised enzyme activities are derived from the leukocytes and appear to reflect their functional and metabolic state. The information provided by the leukocyte morphology can thus be appreciably extended by phosphohexose isomerase determination.

Adolescent↗

PubMedDate not supplied

[Biosynthetic features and properties of xylose isomerases from Arthrobacter nicotianae, Escherichia coli, and Erwinia carotovota subsp. atroseptica].

The characteristics of xylose isomerase biosynthesis in the bacteria Arthrobacter nicotianae BIM B-5, Erwinia carotovota subsp atroseptica jn42xylA, and Escherichia coli HB101xylA have been studied. The bacteria formed the enzyme constitutively. Out of the carbon sources studied, D-glucose and D-xylose were most favorable for the biosynthesis of xylose isomerase in E. carotovota subsp atroseptica, but the least appropriate in terms of the enzyme production efficiency in E. coli. Minimum and maximum levels of xylose isomerase formation in A. nicotianae were noted, respectively, during D-xylose and sucrose utilization. An addition to the nutrient medium of 0.1-1.5% D-glucose (together with D-xylose) did not affect the enzyme synthesis in A. nicotianae, but suppressed it in Erwinia carotovota subsp atroseptica (by 7% at the highest concentration) and Escherichia coli (by 63 and 75% at concentrations of 0.1 and 1.0%, respectively). The enzyme proteins produced by the bacteria exhibited the same substrate specificity and electrophoretic mobility (PAGE) as xylose isomerase A. nicotianae, although insignificant differences in the major physicochemical properties were noted.

Aldose-Ketose Isomerases↗

PubMed1990

[Cloning and expression of E. coli glucose isomerase gene in Streptomyces lividans].

E. coli glucose isomerase gene was cloned into Streptomyces lividans using shuttle plasmid vector pSE-3. First plasmid pXI203 was constructed in E. coli using plasmids pXI200 (containing 1.6 kb glucose isomerase gene) and pGEM-3 digested with EcoR1. Then, recombinant plasmid pSEX100 was also constructed in E. coli using plasmids pXI203 and pSE-3 digested with HindIII. When the pSEX100 was transformed into Streptomyces lividans protoplasts, recombinants were obtained on R5YE medium containing 50 micrograms/ml neomycin and 50 micrograms/ml thiostrepton. The results showed that the E. coli glucose isomerase gene cloned and expressed in Streptomyces lividans via the analysis of restriction enzyme digestion as well as the detection of the glucose isomerase activity.

Aldose-Ketose Isomerases↗

PubMed1979

Stabilization and purification of glucosamine 6-phosphate isomerase from rat kidney.

1. Glucosamine 6-phosphate (GlcN-6-P) isomerase of rat kidney was resistant to heating at 50--55 degrees in crude extract but not after several purification steps. GlcN-6-P and N-acetylglucosamine 6-phosphate were found to stabilize the isomerase under these conditions. They also protected the enzyme from tryptic digestion, but only GlcN-6-P was effective against inactivation by p-chloromercuribenzoate. 2. When GlcN-6P isomerase was purified from fresh kidney and kidney stored at -20 degrees, separately and under GlcN-6-P, the two preparations were different in elution profile from a hydroxyapatite column. It was subsequently found that storage of crude extract at -20 degrees resulted in molecular alterations of the enzyme. Prolonged purification appeared to affect the enzyme similarly. The molecular alterations, however, were suppressed if the extract was stored at -70 degrees. 3. These findings have been utilized to develop a procedure, which enables us to purify rat kidney GlcN-6-P isomerase without any molecular alteration and in good yield.

Aldose-Ketose Isomerases↗

PubMed1985

The isozymes of glucose-phosphate isomerase (GPI-A2 and GPI-B2) from the teleost fish Fundulus heteroclitus (L.).

The fish, Fundulus heteroclitus (L.), like most advanced teleosts, possesses duplicate loci for the glycolytic enzyme, glucose-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9). The locus for the GPI-A2 (where GPI represents glucose-phosphate isomerase) isozyme is preferentially expressed in anaerobic tissues such as white skeletal muscle, while GPI-B2 predominates in aerobic tissues like liver and red muscle. We questioned whether this tissue specificity would be reflected in unique structural and functional characteristics of the respective isozymes. Consequently, an analysis of the two isozymes was undertaken. The enzymes were purified by a combination of ion-exchange chromatography and isoelectric focusing. Each isozyme was characterized as to native and subunit molecular weight, isoelectric pH, and susceptibility to thermal denaturation. Both were dimeric enzymes, with native molecular masses of 110 kDa. The isoelectric pH values for GPI-A2 and GPI-B2 were 7.9 and 6.4, respectively. Differences were apparent in thermal stability, i.e. GPI-A2 was more stable than GPI-B2. Kinetic properties were investigated as a function of both pH and temperature. The Km values for fructose 6-phosphate (Fru-6-P) differed between the isozymes at low pH, but no significant differences were observed at higher pH. The inhibition constant (Ki) for 6-phosphogluconate (6-P-gluconate) was pH dependent. GPI-A2 was slightly more sensitive to 6-P-gluconate inhibition than GPI-B2 between pH 7.0 and 8.5. The Km for Fru-6-P was temperature dependent for the GPI-B2 isozyme, but relatively temperature independent for GPI-A2 between 10 and 35 degrees C. The Ki for 6-P-gluconate was temperature dependent for both isozymes. The Ki values for GPI-A2 were consistently lower than those for GPI-B2. Energies of activation differed between the two isozymes by 4.4 kcal with GPI-A2 having the lower value. While delta G values were identical for the isozymes, their delta H and delta S values differed significantly. The structural and kinetic differences that exist between the glucose-phosphate isomerase isozymes appear to be tailored to the unique metabolic demands of the tissues in which these Gpi loci are expressed.

Animals↗

PubMedDate not supplied

[Properties of glucose isomerase from Streptomyces robeus S-606].

Optimal conditions of the glucose isomerase fixation in a cell are determined by thermal treatment of Str. robeus S-606 biomass. Under these conditions the maximal enzyme activation (by 50-55 percent) is simultaneously observed. Basic properties of glucose isomerase fixed inside the cell are studied in comparison with the enzymic cell-free extract of this enzyme. The pH-optimum for preparations coincides and is observed at pH 7.5; the temperature optimum for the soluble enzyme is 70 degrees C, and for the intracellular enzyme it is higher by 5 degrees C. Thermostability of the intracellular enzyme is also higher than that of the soluble one. The Michaelis constants are calculated for the glucose isomerase preparations in a form of producer cells and enzymic extract: they equal to 0.375 M and 0.285 M, respectively. A comparison of properties permits considering intracellular glucose isomerase as an immobilized enzymic preparation.

Aldose-Ketose Isomerases↗

PubMed1984

Xylose isomerase from Escherichia coli. Characterization of the protein and the structural gene.

The gene that codes for xylose isomerase in Escherichia coli has been cloned by complementation of a xylose isomerase-negative E. coli mutant. The structural gene is 1320 nucleotides in length and codes for a protein of 440 amino acids. An additional 209 nucleotides 5' and 82 nucleotides 3' to the structural gene were also sequenced. To verify that the cloned gene encodes E. coli xylose isomerase, the enzyme was purified to homogeneity and the sequence of the first 25 amino acid residues was determined by a semimicromanual Edman procedure. These results establish that the NH2-terminal methionine of xylose isomerase is specified by an ATG which is 7 nucleotides downstream from a Shine-Dalgarno sequence.

Aldose-Ketose Isomerases↗

PubMed1995

Molecular cloning and characterization of the xylose isomerase gene from a thermophilic Bacillus species.

The gene (xylA) encoding a thermostable xylose isomerase has been isolated and characterized from a thermophilic Bacillus species for the first time. The xylA open reading frame of 1323 bp encoded a protein containing 441 amino acids with a calculated molecular weight of 50,176. The amino acid sequence of this protein showed 76% homology to xylose isomerase isolated from Bacillus subtilis and contained all the important catalytic domains of the enzyme. The gene complemented the xyl-5 mutation and produced a functional enzyme constitutively in Escherichia coli. The crude cell-free extract of E. coli recombinants exhibited xylose isomerase activity over a wide range of temperatures from 60 to 100 degrees C with an optimal enzyme activity of 10.4 Units/mg protein at 85 degrees C. This optimal temperature was one of the highest reported so far for thermostable xylose isomerases. The recombinant enzyme was found to be a tetramer with each subunit having molecular weight of 50,000.

Aldose-Ketose Isomerases↗

PubMed1993

Beta-oxidation of unsaturated fatty acids in humans. Isoforms of delta 3, delta 2-enoyl-CoA isomerase.

This investigation was undertaken in order to elucidate the human enzymes which participate in metabolism of the double bonds of unsaturated fatty acids during beta-oxidation. The results indicate that the human monofunctional delta 3, delta 2-enoyl-CoA isomerase (EC 5.3.3.8) with the native M(r) of 70,000 differed significantly from its rat counterpart [Palosaari et al. (1990) J. Biol. Chem. 265, 3347-3353]; the isoelectric point of the human isoform was over three pH-units more acidic, it showed different chromatographic behaviour, the human enzyme did not show any clear-cut substrate chain-length specificity and only a weak immunological cross-reactivity was detected with the antibody to rat liver mitochondrial short-chain enzyme. This explains the failure of attempts to apply the rat data directly to human beings. Another isomerase activity from human liver was found to be a part of the isomerase-hydratase-dehydrogenase polypeptide showing immunological cross-reactivity with the previously characterized peroxisomal multifunctional enzyme (MFE) from rat liver.

Animals↗

PubMed1990

Purification and characterization of bovine liver 3-cis-2-trans-enoyl-CoA isomerase.

The purification to homogeneity of 3-cis-2-trans-enoyl-CoA isomerase from bovine liver is described. This procedure has also been successfully applied to the isolation of the enzyme from rat liver mitochondria. Its molecular mass was determined to be 30 kDa by several methods. Km of the isomerase for 3-cis-dodecanoyl-CoA, the physiological intermediate in the beta-oxidation of oleic acid and of the 3-trans-isomer was determined to be 3.2 x 10(-5) M. The velocity of isomerization of the 3-trans-substrate is reduced 10-15 times. Inhibition experiments and pH-dependency of the reaction kinetics suggest the participation of a histidine residue in the isomerization. Protein analytical studies revealed that the N-terminus is blocked (acetylated). Cyanogen bromide cleavage of the purified enzyme resulted in five fragments. The N-terminus of one fragment is blocked. Partial amino-terminal sequences of two of the fragments were obtained by Edman degradation, a prerequisite for further studies on the structure of this isomerase on the DNA level.

Amino Acid Sequence↗

PubMed1991

Differential inhibition of dehydrogenase and 5-ene----4-ene isomerase activities of purified 3 beta-hydroxysteroid dehydrogenase. Evidence for two distinct sites.

The success in synthesis of [3H]5-androstene-3,17-dione, the intermediate product in the transformation of DHEA to 4-androstenedione by 3 beta-hydroxysteroid dehydrogenase/5-ene----4-ene isomerase (3 beta-HSD) offers the opportunity to determine whether or not the two activities reside in one active site or in two closely related active sites. The finding that N,N-dimethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) inhibits competitively and specifically the dehydrogenase activity whereas a non-competitive inhibition type with a Ki value 1000 fold higher was observed for the isomerase activity, indicated that dehydrogenase and isomerase activities belong to separate sites. Using 5 alpha-dihydro-testosterone and 5 alpha-androstane-3 beta, 17 beta-diol, exclusive substrates for dehydrogenase activity, it was shown that dehydrogenase is reversible and strongly inhibited by 4-MA and that thus the irreversible step in the transformation of DHEA to 4-androstenedione is due to the isomerase activity.

3-Hydroxysteroid Dehydrogenases↗

PubMed1974

Intracellular distribution of hydroperoxide isomerase.

Differential centrifugation of several plant extracts indicates that the majority of the hydroperoxide isomerase activity is present in the cytoplasm of the cell. However, lesser amounts of isomerase activity were found in the mitochondrial and microsomal fractions of sunflower seedlings. Sucrose density gradient centrifugation of extracts from sunflower, watermelon, and flax seedlings and from cauliflower buds showed that isomerase activity was associated with the mitochondria. There was no evidence for presence of hydroperoxide isomerase activity in the microbodies.

Journal Article↗

PubMed1981

Metabolism of linoleic Acid by barley lipoxygenase and hydroperoxide isomerase.

The oxidation of linoleic acid in incubation mixtures containing extracts of barley lipoxygenase and hydroperoxide isomerase, and the production of these enzymes in quiescent and germinated barley, were investigated. The ratio of 9-hydroperoxylinoleic acid to 13-hydroperoxylinoleic acid was higher for incubation mixtures containing extracts of quiescent barley than for mixtures containing extracts of germinated barley; production of 13-hydroperoxylinoleic acid from germinated barley exceeded that of quiescent barley. Hydroperoxy metabolites of linoleic acid were converted to 9-hydroxy-10-oxo-cis-12-octadecenoic acid, 13-hydroxy-10-oxo-trans-11-octadecenoic acid, and small amounts of 11-hydroxy-12,13-epoxy-cis-9-octadecenoic acid and 11-hydroxy-9,10-epoxy-cis-13-octadecenoic acid whether quiescent or germinated barley was the enzyme source; a fifth product, 13-hydroxy-12-oxo-cis-9-octadecenoic acid was formed only when germinated barley was the enzyme source.Lipoxygenase was readily extracted by buffer, but hydroperoxide isomerase was bound in a catalytically active state to the insoluble barley grist and was efficiently extracted only when Triton X-100 was included in the extraction buffer. Hydroperoxide isomerase was localized in the embryo of quiescent barley, but it was present in the embryo, acrospire, and in small but concentrated amounts in the rootlet of germinating barley. The levels of both lipoxygenase and hydroperoxide isomerase increased through the thirteenth day of germination.

Journal Article↗

PubMed2004

Chalcone isomerase family and fold: no longer unique to plants.

Chalcone isomerase, an enzyme in the isoflavonoid pathway in plants, catalyzes the cyclization of chalcone into (2S)-naringenin. Chalcone isomerase sequence family and three-dimensional fold appeared to be unique to plants and has been proposed as a plant-specific gene marker. Using sensitive methods of sequence comparison and fold recognition, we have identified genes homologous to chalcone isomerase in all completely sequenced fungi, in slime molds, and in many gammaproteobacteria. The residues directly involved in the enzyme's catalytic function are among the best conserved across species, indicating that the newly discovered homologs are enzymatically active. At the same time, fungal and bacterial species that have chalcone isomerase-like genes tend to lack the orthologs of the upstream enzyme chalcone synthase, suggesting a novel variation of the pathway in these species.

Amino Acid Sequence↗

PubMed1984

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.

Journal Article↗

PubMed1994

Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp.

An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50 degrees C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and beta-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85 degrees C for 20 min. Divalent metal ions Mg, Co, and Mn were required for maximum activity of the enzyme. The K(m) values for D-xylose and D-glucose at 80 degrees C and pH 7.5 were 6.66 and 142 mM, respectively, while K(cat) values were 2.3 x 10 s and 0.5 x 10 s, respectively.

Journal Article↗

PubMed1975

D-Glucose isomerase: constitutive and catabolite repression-resistant mutants of Streptomyces phaeochromogenes.

As in other Streptomyces species, the enzymatic conversion of D-glucose to D-fructose is carried out in Streptomyces phaeochromogenes NRRL B-3559 by the inducible enzyme, D-xylose keto isomerase (EC 5.3.1.5). Mutants of this microorganism were selected for their ability to grow on D-lyxose (2-epimer of D-xlose). As a result of the mutational event, the microorganism constitutively produced D-xylose isomerase. As in the parent strain, the constitutive formation of the isomerase was repressed by D-glucose. The fact that this mutant was unable to grow in low D-xylose concentrations in the presence of the D-glucose analogue, 3-O-methylglucose, permitted the isolation of D-xylose isomerase constitutive mutants which were insensitive to D-glucose repression.

Carbohydrate Epimerases↗