Effect of minerals on glucose isomerase production by Streptomyces kanamyceticus

Summary A study has been made of the mineral requirements of Streptomyces kanamyceticus KCC S-0433 for production of glucose isomerase. The optimal concentrations of MgSO₄ and K₂HPO₄ for enzyme production are 0.07% and 0.05%, respectively. The elements Fe, Mn and Zn are required at levels of 10, 3 and 3 mg/l, respectively. Cu, Co and Ca have inhibitory effects on the production of the enzyme.     

Glucose diffusion in crosslinked proteins containing streptomyces

Summary Diffusion measurements of glucose in a crosslinked water-swollen protein matrix containingStreptomyces thermovulgaris strain 127, are described. Solute diffusion coefficients in the gel phase were found to be considerably lower than for free diffusion. The range of diffusion coefficients was 0.83×10⁻¹⁰ to 2.68×10⁻¹⁰m²/s at corresponding ratios to free diffusion values 0.11 to 0.38. It is shown that polymer structure does not pose any significant effect on mass transfer for the small glucose molecule.     

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.     

Preparation and properties of glucose isomerase of Streptomyces kanamyceticus cells entrapped in calcium alginate

Summary The properties of glucose isomerase in native, heat-treated and immobilized cells of Streptomyces kanamyceticus after heat and mineral treatment have been compared. The optimum pH for glucose isomerase in native cells was shifted from 8.2 to 8.6 by heat treatment and immobilization. There is no change in the optimum temperature (90°C) for activity of the enzyme by the above treatment. Heat-treated cells and immobilized cells show greater pH and thermal stability of the enzyme. The Km values of the enzyme of native cells, heat-treated cells and immobilized heat-mineral-treated cells are 208 mM, 212 mM and 166 mM respectively; Mg⁺⁺ and Co⁺⁺ enhance the activity of isomerase in all cases.     

Glucose isomerase immobolized on porous glass

Partially purified glucose isomerase from a Streptomyces species was immobilized on porous glass particles and studied for various characteristics concerning its use as an industrial catalyst. The activities were investigated in relation to the reaction parameters and the enzyme deactivation was studied systematically under various reaction conditions. The half-life of the immobilized enzyme was found to exceed 200 days at 50°C. The rate equation of the reversible glucose ? fructose reaction was derived and the kinetic constants were determined. The rate equation was found to be in good agreement with experimental data for both forward and reverse reactions. The degree of diffusional effects was experimentally measured and theoretically analyzed.     

The activity of streptomyces phage-virus in fresh water

Streptomyces (Actinomycetales) host strains and their virulent phages were obtained from a restricted locality, both fresh waters and soils being sampled. There was some evidence that one category of host strains was confined to fresh water and was susceptible to phage there. There were several instances where a particular host strain elicited phages both from fresh water and from soil and the phages from these two environments subsequently exhibited differing polyvalency ranges in the laboratory. The possible significance of this finding is that it hints at the existence of phage ecotypes, with properties and polyvalency ranges adapted to the immediate environment.     

Glucose isomerase production byStreptomyces sp. CCM 4102

The newly isolatedStreptomyces sp. CCM 4102 strain produced a high level of intracellular glucose isomerase in the media containingd-xylose as inducer of the enzyme, corn-steep liquor, yeast extract and magnesium sulfate. The enzyme synthesis was repressed byd-glucose andd-fructose. The strain did not require cobalt ions for enzyme production.     

Glucose isomerase extraction fromStreptomyces nigrificans : A comparison of methods

Summary Seventeen methods of glucose isomerase extraction fromStreptomyces nigrificans 82/20 were tested and compared. Autolysis with lysozyme, X-press disintegration, sonication, and disintegration using Novotny's procedure were the best.     

Glucose isomerase activity in suspension of Arthrobacter nicotianae cells and adsorption immobilization of the microorganisms on inorganic carriers

Kinetics of monosaccharide isomerization has been studied in suspensions of intact, non-growing Arthrobacter nicotianae cells. Under the conditions of the study, glucose and fructose were isomerized at the same maximum rate of 700 micromol/min per 1 g dried cells, which increased with temperature (the dependence was linear at 60-80 degrees C). The proposed means of adsorption immobilization of A. nicotianae cells involve inorganic carriers differing in macrostructure, chemical nature, and surface characteristics. Biocatalysts obtained by adsorbing the cells of A. nicotianae on carbon-containing foam ceramics in the coarse of submerged cultivation were relatively stable and retained original activity (catalysis of monosaccharide isomerization) throughout 14 h of use at 70 degrees C. Maximum glucose isomerase activity (2 micromol/min per 1 g) was observed with biocatalysts prepared by adsorption of non-growing A. nicotianae cells to the macroporous carbon-mineral carrier Sapropel and subsequent drying of the cell suspension together with the carrier.     

Anhydrotetracycline oxygenase activity and biosynthesis of tetracyclines in streptomyces aureofaciens

Summary A new method for the assay of the enzyme converting anhydrotetracycline into dehydrotetracycline is described. The enzyme activity increases sharply during the growth phase and reaches its maximum at the beginning of the production phase; it is depressed when the concentration of inorganic phosphate in the cultivation medium is increased.     

Glucose isomerase production on a xylan-based medium by Bacillus thermoantarcticus

Effects of medium components on intracellular glucose isomerase (GI) production were investigated by Bacillus thermoantarcticus. The highest GI activity was obtained as 1630 U dm^?3 in the medium containing (g dm^?3): 10.6, birchwood-xylan; 5.6, yeast extract; 5.9 (NH₄)₂SO₄ at T = 55 °C in 33 cm^?3 shake-flasks. When birchwood-xylan was replaced with oat spelt- or beechwood-xylan, GI activity decreased to 1372 and 1308 U dm^?3, respectively. Effects of pH at uncontrolled-pH (pH_UC = 6.0) and controlled-pH (pH_C = 6.0) operations, and oxygen transfer at the air inlet rate of 0.5 vvm and agitation rates of 300, 500 and 700 min^?1, were investigated in 3.0 dm³ bioreactor system with 1.65 dm³ working volume in the designed medium. The highest GI activity was attained at 500 min^?1, 0.5 vvm, pH_UC = 6 as 1840 U dm^?3 where cell concentration was 2.3 g dm^?3. The use of agricultural waste xylan, as the carbon source resulted in concomitant production of xylanase and GI. The highest xylanase activity was attained as 9300 U dm^?3 at 500 min^?1 and 0.5 vvm. K_La varied between 0.008–0.033 s^?1 whereas the highest oxygen uptake rate was 0.002 mmol dm^?3 s^?1. Initially biochemical reaction limitations were effective; thereafter, mass transfer resistances became more effective.     

Glucose isomerase: insights into protein engineering for increased thermostability

Thermostable glucose isomerases are desirable for production of 55% fructose syrups at >90 degrees C. Current commercial enzymes operate only at 60 degrees C to produce 45% fructose syrups. Protein engineering to construct more stable enzymes has so far been relatively unsuccessful, so this review focuses on elucidation of the thermal inactivation pathway as a future guide. The primary and tertiary structures of 11 Class 1 and 20 Class 2 enzymes are compared. Within each class the structures are almost identical and sequence differences are few. Structural differences between Class 1 and Class 2 are less than previously surmised. The thermostabilities of Class 1 enzymes are essentially identical, in contrast to previous reports, but in Class 2 they vary widely. In each class, thermal inactivation proceeds via the tetrameric apoenzyme, so metal ion affinity dominates thermostability. In Class 1 enzymes, subunit dissociation is not involved, but there is an irreversible conformational change in the apoenzyme leading to a more thermostable inactive tetramer. This may be linked to reversible conformational changes in the apoenzyme at alkaline pH arising from electrostatic repulsions in the active site, which break a buried Arg-30-Asp-299 salt bridge and bring Arg-30 to the surface. There is a different salt bridge in Class 2 enzymes, which might explain their varying thermostability. Previous protein engineering results are reviewed in light of these insights.     

Intrinsic isomerase activity of medium-chain acyl-CoA dehydrogenase

Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We found that the enzyme has intrinsic isomerase activity, which was confirmed using incubation followed with HPLC analysis. The isomerase activity of the enzyme was thoroughly characterized through studies of kinetics, substrate specificity, pH dependence, and enzyme inhibition. E376 mutants were constructed, and mutant enzymes were purified and characterized. It was shown that E376 is the catalytic residue for both dehydrogenase and isomerase activities of the enzyme. The isomerase activity of medium-chain acyl-CoA dehydrogenase is probably a spontaneous process driven by thermodynamic equilibrium with the formation of a conjugated structure after deprotonation of substrate alpha proton. The energy level of the transition state may be lowered by a stable dienolate intermediate, which gains further stabilization via charge transfer with the electron-deficient FAD cofactor of the enzyme. This raises the question as to whether the dehydrogenase might function as an isomerase in vivo in conditions in which the activity of the isomerase is decreased.     

Identification of protein-disulfide isomerase activity in fibronectin

Assembly and degradation of fibronectin-containing extracellular matrices are dynamic processes that are up-regulated during wound healing, embryogenesis, and metastasis. Although several of the early steps leading to fibronectin deposition have been identified, the mechanisms leading to the accumulation of fibronectin in disulfide-stabilized multimers are largely unknown. Disulfide-stabilized fibronectin multimers are thought to arise through intra- or intermolecular disulfide exchange. Several proteins involved in disulfide exchange reactions contain the sequence Cys-X-X-Cys in their active sites, including thioredoxin and protein-disulfide isomerase. The twelfth type I module of fibronectin (I12) contains a Cys-X-X-Cys motif, suggesting that fibronectin may have the intrinsic ability to catalyze disulfide bond rearrangement. Using an established protein refolding assay, we demonstrate here that fibronectin has protein-disulfide isomerase activity and that this activity is localized to the carboxyl-terminal type I module I12. I12 was as active on an equal molar basis as intact fibronectin, indicating that most of the protein-disulfide isomerase activity of fibronectin is localized to I12. Moreover, the protein-disulfide isomerase activity of fibronectin appears to be partially cryptic since limited proteolysis of I10-I12 increased its isomerase activity and dramatically enhanced the rate of RNase refolding. This is the first demonstration that fibronectin contains protein-disulfide isomerase activity and suggests that cross-linking of fibronectin in the extracellular matrix may be catalyzed by a disulfide isomerase activity contained within the fibronectin molecule.     

Glucose phosphate isomerase and 6-phosphogluconate dehydrogenase polymorphism in Malaysian leaf monkeys

Glucose phosphate isomerase and 6-phosphogluconate dehydrogenase were found to be polymorphic in Malaysian leaf monkeys. Two glucose phosphate isomerase electrophoretic phenotypes were presumed to be homozygous. Three 6-phosphogluconate alleles and four electrophoretic phenotypes were present. The allele frequencies inPresbytis obscura werePgd ^A=0.64,Pgd ^B=0.27 andPgd ^C=0.09. The frequencies of the 6-PGD phenotypes inP. obscura were not in accord with Hardy-Weinberg expectations. All the biochemical markers examined show identical electrophoretic patterns in the Malaysian leaf monkeys.     

Glucose phosphate isomerase enzyme-activity mutants inMus musculus: Genetical and biochemical characterization

Two glucose-6-phosphate isomerase (GPI) mutants with approximately 60% residual activity in blood compared to wild type have been independently detected in offspring derived from 1-ethyl-1-nitrosourea-treated male mice. Homozygous mutants with about 20% residual activity were recovered in progeny of inter se matings of heterozygotes. However, in both mutant lines the number of homozygous mutants was less than expected suggesting an increased lethality of these animals. Results of linkage studies and of investigations of physicochemical properties of the mutant enzymes indicate point mutations at theGpi-1s structural locus on chromosome 7. Based on these findings the two new alleles were designatedGpi-1s ^b-m1Neu andGpi-1s ^b-m2Neu, respectively. The b-m1Neu allele codes for an erythrocyte enzyme which, in the homodimeric form, exhibits a decreased stability toward heat and urea, an altered isoelectric point, normalpH dependence, an increasedK _m for fructose-6-phosphate, and increasedK _i's for 6-phosphogluconate and 2,3-diphosphoglycerate (2,3-DPG) compared to the wild-type enzyme. The GPI-1s^b-m2Neu homodimer, in contrast, is characterized by an even stronger instability, slightly alteredpH dependence, an increasedK _i for 2,3-DPG, normal other kinetics, and normal isoelectric point. The different degree of stability of the mutant homodimersin vitro seems to be reflected in a different degree of stabilityin vivo, since GPI deficiency in general is more strongly expressed in the tissues of the homozygousGpi-1s ^b-m2Neu mutant compared to the homozygousGpi-1s ^b-m1Neu mutant. The similarity of the mutant enzymes to the allozymes found in human GPI deficiencies indicates the GPI deficient mouse mutants to be excellent models for the human disease.This research was supported in part by Contract BI6-156-D from the Commission of the European Communities.     

Marmoset glutathione transferases with ketosteroid isomerase activity

The common marmoset Callithrix jacchus encodes two glutathione transferase (GST) enzymes with ketosteroid double-bond isomerase activity. The most active enzyme is CjaGST A3-3 showing a specific activity with 5-androsten-3,17-dione (Δ⁵-AD) of 62.1 ± 1.8 μmol min^-1 mg^-1, and a k_cat value of 261 ± 49 s^-1. The second ketosteroid isomerase CjaGST A1-1 has a 30-fold lower specific activity with Δ⁵-AD and a 37-fold lower k_cat value. Thus, the marmoset CjaGST A3-3 would be the main contributor to the biosynthesis of the steroid hormones testosterone and progesterone, like the human ortholog HsaGST A3-3. Two residues differ in the H-site of the 91.4% sequence identical CjaGST A1-1 and CjaGST A3-3, and modeling of the structures suggests that the bulky phenyl ring of Phe111 in CjaGST A1-1 causes steric hindrance in the binding of the steroid substrate. Tributyltin acetate (IC₅₀=0.16 ± 0.004 μM) and ethacrynic acid (IC₅₀=3.3 ± 0.2 μM) were found to be potent inhibitors of CjaGST A3-3, as previously demonstrated with the human and equine orthologs.     

Preparation and characteristics of protoplasts of Streptomyces kanamyceticus

To prepare actively regenerating protoplasts of S. kanamyceticus, the influence of the conditions of the mycelium cultivation, the culture age, lytic conditions, composition of the regeneration medium, the procedure of the culture inoculation to the regeneration medium and other parameters were studied. The study resulted in development of optimal conditions for preparation of S. kanamyceticus protoplasts in a number of 1.10(9) protoplasts per ml. The cultivation on the ST medium with 10 to 15% sucrose and addition of glycine up to 1% for 30 hours (the stationary growth phase) followed by treatment of the culture with lysozyme in an amount of 2 mg/ml for 1 hour at 32 degrees C provided preparation of up to 100% of actively regenerating protoplasts free of mycelium fragments. The size of the protoplasts increased up to 1.5 micron against the usually observed size of 0.7 to 1.0 micron with using modified lyzing buffer with 20% of sucrose according to the method recommended for S. erythreus. However, 50 to 70% of the protoplasts had point of linear regions in the cell walls, which suggested that spheroplasts were mainly forming and the phenomenon was associated with the characteristic properties of the strain cell wall structure.