Effect of thermal and chemical denaturants on Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase stability and activity

Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase (2 degrees ADH) was optimally active near 90 degrees C displaying thermostability half-lives of 1.2 days, 1.7 h, 19 min, 9.0 min, and 1.3 min at 80 degrees C, 90 degrees C, 92 degrees C, 95 degrees C, and 99 degrees C, respectively. Enzyme activity loss upon heating (90-100 degrees C) was accompanied by precipitation, but the soluble enzyme remaining after partial inactivation retained complete activity. Enzyme thermoinactivation was modeled by a pseudo-first order rate equation suggesting that the rate determining step was unimolecular with respect to protein and thermoinactivation preceded aggregation. The apparent 2 degrees ADH melting temperature (T(m)) occurred at approximately 115 degrees C, 20 degrees C higher than the temperature for maximal activity, suggesting that it is completely folded in its active temperature range. Thermodynamic calculations indicated that the active folded structure of the 2 degrees ADH is stabilized by a relatively small Gibbs energy (triangle upG(stab.)(double dagger) = 110 kJ mol(-1)). 2 degrees ADH catalytic activities at 37 degrees C to 75 degrees C, were 2-fold enhanced by guanidine hydrochloride (GuHCl) concentrations between 120 mM and 190 mM. These results demonstrate the extreme resistance of this thermophilic 2 degrees ADH to thermal or chemical denaturation; and suggest increased temperature or GuHCl levels seem to enhance protein fixability and activity.     

Increased thermal stability of glucose dehydrogenase by cross-linking chemical modification

A hetero-oligomeric glucose dehydrogenase (GDH) from a moderate thermophilic bacterium, SM4 was cross-linked with glutaraldehyde (GA) and it now showed only one optimum temperature for reaction at around 65°C, which approximately follows the Arrhenius equation. The native enzyme had shown optima at both 45°C and 75°C. In addition to the alteration of the optimum temperature for reaction, GA cross-linked GDH retained more than 90% of its initial activity even after 30 min of incubation at 65°C.     

Chaperone-like activity of beta-casein and thermal stability of alcohol dehydrogenase

To elucidate the correlation of structural peculiarities of beta-casein and their chaperon-like activity the modified forms of the protein (with cysteinyl residues introduced in polypeptide chain) were investigated. The aggregation of native and recombinant beta-caseins was studied as well as their chaperon-like activity towards alcohol dehydrogenase thermal aggregation. It was shown that physico-chemical and chaperone-like properties ofdimeric and oligomeric forms ofbeta-casein (which formation is due to intermolecular disulfide bonds) differ significantly from monomeric forms. It was found that thermal stability of alcohol dehydrogenase depends on beta-casein concentration.     

Increasing the thermal stability of the water-soluble pyrroloquinoline quinone glucose dehydrogenase by single amino acid replacement

Based on the characterization of a PCR mutation of water-soluble glucose dehydrogenase possessing pyrroloquinoline quinone (PQQ), PQQGDH-B, Ser231Cys, we have constructed a series of Ser231 variants. The replacement of Ser231 to Cys, Met, Leu, Asp, Asn, His, or Lys resulted in an increase in thermal stability. Among these variants, Ser231Lys showed the highest level of thermal stability and also showed high catalytic activity. Considering that Ser231Lys showed more than an 8-fold increase in its half-life during the thermal inactivation at 55 degrees C compared with the wild-type enzyme, and also retained catalytic activity similar to a wild-type enzyme, the application of this mutant enzyme as a glucose sensor constituent may develop into a stable glucose sensor construction.     

Effect of dichlorophenolindophenol on glucose dehydrogenase activity of Pseudomonas fluorescens (type R)]

The glucose dehydrogenase activity of Pseudomonas fluorescens cells grown in iron-depleted synthetic media is strongly decreased (about 80%) by dichlorophenolindophenol (DIP) 2 X 10(-3) M. In those cells, DIP seems not to play the part of an ultimate electron acceptor.     

Engineering a chimeric pyrroloquinoline quinone glucose dehydrogenase: improvement of EDTA tolerance, thermal stability and substrate specificity

An engineered Escherichia coli PQQ glucose dehydrogenase (PQQGDH) with improved enzymatic characteristics was constructed by substituting and combining the gene-encoding protein regions responsible for EDTA tolerance, thermal stability and substrate specificity. The protein region responsible for complete EDTA tolerance in Acinetobacter calcoaceticus, which is recognized as the indicator of high stability in co-factor binding, was elucidated. The region is located between 32 and 59% from the N-terminus of A. calcoaceticus PQQGDH(A27 region) and also corresponds to the same position from 32 to 59% from the N-terminus in E. coli PQQGDH, though E. coli PQQGDH is EDTA sensitive. We previously reported that the C-terminal 3% region of A. calcoaceticus (A3 region) played an important role in the increase of thermal stability, and that His775Asn substitution in E. coli PQQGDH resulted in an increase in the substrate specificity of E. coli PQQGDH towards glucose. Based on these findings, chimeric and/or mutated PQQGDHs, E97A3 H775N, E32A27E41 H782N, E32A27E38A3 and E32A27E38A3 H782N were constructed to investigate the compatibility of two protein regions and one amino acid substitution. His775 substitution to Asn corresponded to His782 substitution to Asn (H782N) in chimeric enzymes harbouring the A27 region. Since all the chimeric PQQGDHs harbouring the A27 region were EDTA tolerant, the A27 region was found to be compatible with the other region and substituted amino acid responsible for the improvement of enzymatic properties. The contribution of the A3 region to thermal stability complemented the decrease in the thermal stability due to the His775 or His782 substitution to Asn. E32A27E38A3 H782N, which harbours all the above mentioned three regions, showed improved EDTA tolerance, thermal stability and substrate specificity. These results suggested a strategy for the construction of a semi-artificial enzyme by substituting and combining the gene-encoding protein regions responsible for the improvement of enzyme characteristics. The characteristics of constructed chimeric PQQGDH are discussed based on the predicted model, beta-propeller structure.     

Significantly enhanced stability of glucose dehydrogenase by directed evolution

An NaCl-independent stability-enhanced mutant of glucose dehydrogenase (GlcDH) was obtained by using in vitro directed evolution. The family shuffling method was applied for in vitro directed evolution to construct a mutant library of GlcDH genes. Three GlcDH-coding genes from Bacillus licheniformis IFO 12200, Bacillus megaterium IFO 15308 and Bacillus subtilis IFO 13719 were each cloned by direct PCR amplification into the p Trc99A expression vector and expressed in the host, Escherichia coli. In addition to these three GlcDH genes, a gene encoding a previously obtained GlcDH mutant, F20 (Q252L), derived from B. megaterium IWG3, was also subjected to directed evolution by the family shuffling method. A highly thermostable mutant, GlcDH DN-46, was isolated in the presence or absence of NaCl after the second round of family shuffling and filter-based screening of the mutant libraries. This mutant had only one novel additional amino acid residue exchange (E170K) compared to F20, even though DN-46 was obtained by family shuffling of four different GlcDH genes. The effect of temperature and pH on the stability of the GlcDH mutants F20 and DN46 was investigated with purified enzymes in the presence or absence of NaCl. In the absence of NaCl, F20 showed very poor thermostability (half-life =1.3 min at 66 degrees C), while the half-life of isolated mutant DN-46 was 540 min at 66 degrees C, i.e., 415-fold more thermostable than mutant F20. The activity of the wild-type and F20 enzymes dropped critically when the pH value was changed to the alkaline range in the absence of NaCl, but no such decrease was apparent with the DN-46 enzyme in the absence of NaCl.     

The consequence of stimulating glucose dehydrogenase activity by the addition of PQQ on metabolite production by Agrobacterium radiobacter NCIB 11883

Summary Agrobacterium radiobacter NCIB 11 883 does not produce gluconate under conditions of glucose excess in batch or continuous culture. However, the addition of micromolar concentrations of pyrrolo quinoline quinone (PQQ) to fermentation media resulted in rapid excretion of gluconate by batch and continuous cultures. This rapid dehydrogenation of glucose was found in cells grown under carbon and nitrogen limitation and is constitutive which suggests that the only reason why this activity is not normally expressed is due to the inability of the organism to synthesize the prosthetic group (PQQ) of the glucose dehydrogenase enzyme.Although the addition of PQQ to batch and continuous cultures caused a very rapid specific rate of gluconate production (0.6–1.1 g gluconate g^-1 dry wt. h^-1) the rate of exopolysaccharide production remained unaltered. Indeed, when the rates of substrate and oxygen uptake are corrected for the rate of gluconate production in the presence of PQQ there appears to be little physiological consequence as a result of this oxidation.     

Effects of water activity and aqueous solvent ordering on thermal stability of lysozyme, alpha-chymotrypsinogen A, and alcohol dehydrogenase

Effects of water activity (aW) and solvent ordering were separately analyzed on the thermal unfolding of lysozyme and alpha-chymotrypsinogen A, and also on the thermal deactivation of yeast alcohol dehydrogenase (YADH) in aqueous solutions with various additives. With the coexistence of additives, water activity was the determinant of the extent of the change in the thermal stability of proteins while solvent ordering was the determinant of the direction of the change. The parameter alpha, determined from the activity coefficient of water, representing the deviation of aW from that of the ideal solution, was useful as a quantitative index of the solvent ordering showing good correlations with the unfolding temperature and enthalpy of lysozyme and alpha-chymotrypsinogen A and also with the thermal deactivation rate constant of YADH at a constant aW. Solvent ordering seemed to affect the thermal stability of proteins mainly through its effect on the intramolecular hydrophobic interaction among amino acid residues in a protein molecule but the contribution of the electrostatic interaction including hydrogen bonding through the change in permittivity of solution was also suggested.     

Effect of estrogens on glucose phosphate dehydrogenase activity of liver and oviduct in the amphibian Bufo arenarum.

Glucose- 6phosphate dehydrogenase (G-6PDH) stimulation by estradiol- 17beta has been studied in oviduct and liver of Bufa arenarum. OviducalG-6PDH has been found to be stimulated by a single dose of estradiol- 17beta (100 mug/100 g body weight), the stimulation being dependent on season. Hepatic G-6PDH of females is susceptible to hormonal stimulation, without seasonal variation, while in males the enzymatic activity is not modified under the same conditions. The stimulating effect of estrogen on oviducal and hepatic G- 6PDH was inhibited by Actynomicin D. The susceptibility of G- 6PDH to estrogenic action would assure NADPH production, indispensable for the biosynthesis of lipids which are required for cell growth and for hepatic vitellogenesis.     

Enzymatic activity and stability of D-fructose dehydrogenase and sarcosine dehydrogenase immobilized onto giant vesicles

Stable vesicles with diameters between about 1 and 10 mum were prepared by a particular emulsification technology that involved the use of the surfactants Span 80 and Tween 80 and the phospholipid lecithin (phosphatidylcholine from soybeans). Two membrane enzymes, d-fructose dehydrogenase from Gluconobacter sp. (FDH) and sarcosine dehydrogenase from Pseudomonas putida (SDH), were for the first time immobilized onto the bilayer membranes of these type of vesicles; and the catalytic activity and enzymatic stability were measured and compared with the enzymes in a vesicle-free solution. The enzyme activity as well as stability considerably increased upon immobilization. In particular, immobilized FDH at 25 degrees C was stable for at least 20 days, while the activity of the free enzyme dropped to about 20% of its initial value during the same period of time.In contrast to FDH and SDH, immobilization of sorbitol dehydrogenase from Gluconobacter suboxydans (SODH) was not successful, as no improved activity or stability could be obtained.     

Effect of compatible and noncompatible osmolytes on the enzymatic activity and thermal stability of bovine liver catalase

Catalase is an important antioxidant enzyme that catalyzes the disproportionation of H₂O₂ into harmless water and molecular oxygen. Due to various applications of the enzyme in different sectors of industry as well as medicine, the enhancement of stability of the enzyme is important. Effect of various classes of compatible as well as noncompatible osmolytes on the enzymatic activity, disaggregation, and thermal stability of bovine liver catalase have been investigated. Compatible osmolytes, proline, xylitol, and valine destabilize the denatured form of the enzyme and, therefore, increase its disaggregation and thermal stability. The increase in the thermal stability is accompanied with a slight increase of activity in comparison to the native enzyme at 25?°C. On the other hand, histidine, a noncompatible osmolyte stabilizes the denatured form of the protein and hence causes an overall decrease in the thermal stability and enzymatic activity of the enzyme. Chemometric results have confirmed the experimental results and have provided insight into the distribution and number of mole fraction components for the intermediates. The increase in melting temperature (T_m) and enzymatic rate could be further amplified by the intrinsic effect of temperature enhancement on the enzymatic activity for the industrial purposes.     

Temporal relationship between glucose 6-phosphate dehydrogenase activity and DNA-synthesis

Summary Wounds have been inflicted in the skin of guinea-pigs. Measurements were made on the basal cells 39–54 h later, at different times of the day. It has been shown that there can be two peaks of mitosis, one about mid-day and the other about 22 h. Synthesis of DNA, measured by Feulgen microdensitometry, preceded mitotic activity. Marked changes were found in glucose 6-phosphate dehydrogenase activity, measured by quantitative cytochemistry and microdensitometry. The greatest activity preceded DNA-synthesis, indicating that pentose-shunt metabolic activity is involved in biosynthetic processes required for cell proliferation.     

Effects of Ca2+ on the activity and stability of methanol dehydrogenase

The effects of exogenously added Ca²⁺ on the enzymatic activity and structural stability of methanol dehydrogenase were studied for various Ca²⁺ concentrations. Methanol dehydrogenase activity increased significantly with increasing concentration of Ca²⁺, approaching saturation at 200 mM Ca²⁺. The effect of Ca²⁺ on the activation of MDH was time dependent and Ca²⁺ specific and was due to binding of the metal ions to the enzyme. Addition of increasing concentration of Ca²⁺ caused a decrease of the intrinsic tryptophan fluorescence intensity in a concentration-dependent manner to a minimum at 200 mM, but with no change in the fluorescence emission maximum wavelength or the CD spectra. The results revealed that the activation of methanol dehydrogenase by Ca²⁺ occurred concurrently with the conformational change. In addition, exogenously bound Ca²⁺ destabilized MDH. The potential biological significance of these results is discussed.     

The histochemical localization of glucose dehydrogenase activity in sheep liver

Synopsis A method is described for the histochemical localization of glucose dehydrogenase activity in sheep liver. Fresh-frozen sections were fixed in acetone at –20°C and incubated in a medium at pH 8.0 containing glucose, NAD or NADP, Nitro BT and EDTA.p-Hydroxymercuribenzoate was added to suppress the nothing dehydrogenase effect. Activity was mainly confined to hepatocyte cytoplasm and, with NAD, was strongest in periportal areas. Activity was less with NADP and was strongest in centrilobular areas. The specificity of the enzyme for different substrates and inhibitors was compatible with the known properties of glucose dehydrogenase.