Administration of immobilized firefly luciferase for quantitative estimation of ATP and enzymes synthesizing and destroying ATP]

A possibility of using immobilized Luciola mingrelica luciferase for quantitative test of ATP and ATP synthetizing and degrading enzymes activities is demonstrated. A kinetic scheme is given, and experimental conditions for using L. mingrelica luciferin-luciferase system for kinetic enzyme analysis are determined.     

Kinetic properties of highly purified luciferase from fireflies Luciola mingrelica.]

Luciferase of the fireflies Luciola mingrelica was isolated from dried lanterns of fireflies and purified by chromatography on DEAE-Sephadex. The homogeneity of the preparation was determined by polyacrylamide gel disc electrophoresis. The molecular weight of the enzyme equal to 45000 was determined by disc electrophoresis in the presence of sodium dodecyl sulfate. The kinetic properties of the enzyme (V and Km for luciferin and ATP) within the pH-range of 7,0--8,5 were studied. The kinetic curves of the pH-dependences of log V and log Km for both substrates are bell-shaped, with a slope equal to 2. At pH optimum (7,7--7,9) the Km values for luciferin and ATP are 6,6 mkM and 0,3 mM, respectively. The properties of luciferase L. m. were compared to those of luciferase from fireflies Phophinus pyralis previously described in literature.     

Random mutagenesis of Luciola mingrelica firefly luciferase. Mutant enzymes with bioluminescence spectra showing low pH sensitivity

Most firefly luciferases demonstrate a strong pH-dependence of bioluminescence spectra. Gene region encoding first 225 residues of Luciola mingrelica luciferase was subjected to random mutagenesis, and four mutants with altered pH-sensitivity of bioluminescence spectra were isolated. F16L substitution showed distinctly lower pH-dependence of bioluminescence spectra, and Y35N,H and F16L/A40S substitutions resulted in the enzymes with bioluminescence spectra virtually independent from pH in the range of 6.0-7.8. The structural explanation is proposed for the effect of mutations on pH-sensitivity of bioluminescence spectra.     

Interaction of firefly luciferase with substrates and their analogs: a study using fluorescence spectroscopy methods.

The results of the author's laboratory on the interaction of Luciola mingrelica firefly luciferase with substrates and their analogs using both steady-state and time resolved fluorescence are reviewed. The contribution of fluorescence of Trp and Tyr residues of the protein to its intrinsic fluorescence spectrum was estimated. Studies of quenching of Trp and Tyr fluorescence by luciferin and ATP allowed one to determine binding constants of the luciferase with substrates and to show that the binding of one substrate to the luciferase decreases the affinity of the enzyme for the other one. Fluorescence of oxyluciferin and its analogs (dimethyl- and monomethyloxyluciferins) was shown to be a good model of native firefly bioluminescence. A comparison of the fluorescence spectra of oxyluciferin and its analogs in aqueous solutions and in the presence of the luciferase revealed specific and nonspecific effects of the microenvironment on the equilibrium between different ionic forms of oxyluciferin. An approach based on photo-physical concepts of the correlation between luminescence spectra and structure of the emitter and its microenvironment was proposed and this approach was used to analyze bioluminescence spectra of wild-type and mutant luciferases.     

Comparison of acyl-CoA synthetic activities and enantioselectivity toward 2-arylpropanoic acids in firefly luciferases

Measurement of thioesterification activities for dodecanoic acid (C12) and ketoprofen was done using five firefly luciferases, from Pyrocoelia miyako (PmL), Photinus pyralis (PpL), Luciola cruciata (LcL), Hotaria parvura (HpL), and Luciola mingrelica (LmL). Among these, PmL, PpL, and LcL showed the expected thioesterification activities toward both substrates. All the enzymes exhibited (R)-enantioselectivity toward ketoprofen, which had same tendency as firefly luciferase from Luciola lateralis (LUC-H). HpL and LmL, however, did not accept ketoprofen, although they had thioesterification activity toward C12. These results indicate that the substrate acceptance of luciferases for the thioesterification reaction varies dramatically relying on the origin of firefly. Hence we focused primarily on PmL and investigated the effect of pH on enzymatic activity. In addition, by determining the kinetic parameters at various pH values, we verified that the k(cat) parameter contributed to the preferential enantioselectivity of this enzyme.     

Thermodynamic and kinetic aspects of the hydrolysis of ethyl(R)-2- (benzyloxycarbonylamino)-3-sulfamoylpropionate in the presence of free and immobilized alpha-chymotrypsin

The hydrolysis of ethyl (R)-2-(benzyloxycarbonylamino)-3-sulfamoylpropionate (blocked cysteic acid S-amide) by native and immobilized alpha-chymotrypsin was studied. The experiments were performed using a constant enzyme/substrate ratio of 1:8 and at a temperature of 10-40 degrees C; the immobilized enzyme was bound to a dialdehyde cellulose matrix. A kinetic equation (Eq.10) was found to be applicable which confirms that the mechanism of the enzyme reaction consists of several stages, irrespective of the enzyme state. The temperature dependence of the reaction velocity was investigated and applied using the Arrhenius equation. The constant value thus obtained for the activating energy showed that the active centres retained their character during immobilization. The differences between the velocities of the reaction with immobilized and with native enzyme corresponded to the different number of active centres during the reaction time. Based on these results a kinetic model of the mechanism of the studied reaction is presented which includes an initial balanced stage of the chemosorption type.     

The recording of ATP in the erythrocytes using luciferase injected into the cells

The luciferase preparation obtained from fireflies Luciola mingrelica has entrapped into the human erythrocytes by means of reversible osmotic lysis. The addition of luciferin to such erythrocytes leads to the appearance of luminescence, conditioned by the entrance of luciferin into the cells. Luciferin is uniformly distributed between cells and external medium. Luciferin transport through the erythrocyte membrane is a result of simple diffusion. Values of rate constant of luciferin transport through the membrane lie between 0.009-0.021 l/s 1 cells for erythrocytes of different donors. The maximum luminescence intensity increases monotonously with rise of temperature and luciferin concentration. The dependence of the maximum luminescence intensity on luciferin concentration is described by Michaelis kinetics. Obtained in different experiments, values of luciferase Michaelis constant for luciferin inside erythrocytes lie between 4.1-21.5 microM. Luminescence intensity of the luciferase containing erythrocytes depends on the intracellular ATP concentration. Under the same luciferin concentration the correlation of luminescence intensities of control erythrocytes with normal ATP level and erythrocytes depleted without glucose is near to correlation of their ATP concentrations. After the addition of glucose to the depleted erythrocytes their ATP concentration rises and luminescence intensity approaches to the level of control erythrocytes. Luciferase entrapment permit one to control rapid ATP concentration changes in the erythrocytes.     

ATP-insulin conjugates and their use for immunofactor analysis

A method resulting in ATP-insulin conjugates by covalent binding of ATP modified at C(6) amino group of the adenine residue with insulin was developed. The modified ATP was bound to insulin by means of metha-p-toluene sulfonate-N-cyclohezyl Nf [2-morpholinyl(4)ethyl]-carbodiimide. The ATP analogs and ATP-insulin conjugates possess the coenzyme activity in a reaction of luciferin oxidation by luciferase from the fireflies Luciola mingrelica. the catalytic properties of soluble and immobilize on CNBR-activated. Sepharose enzymes in reactions with native ATR, its modified derivatives and ATP--insulin conjugates were compared. The bioluminescence reaction involving ATP--insulin conjugate is inhibited by antibodies against insulin. This effect can form a basis for insulin detection in solution, which is based on competitive binding of free and antibody-labelled ATP--insulin conjugates.     

Properties of a reversible soluble-insoluble cellulase and its application to repeated hydrolysis of crystalline cellulose

Cellulase was covalently immobilized on an enteric coating polymer, Eudragit L, that is reversibly soluble and insoluble depending on the pH of the medium. The hydrolysis of solid cellulose with the immobilized enzyme can take advantage of the soluble property of the immobilized enzyme itself at the most reactive pH value; on the other hand, recovery of the enzyme can take advantage of the insoluble property of the enzyme at other pH values. It was experimentally confirmed that 100% of immobilized enzyme activity in solution can be recovered by precipitation and by dissolving it again by alternative change of pH. After a period of hydrolysis, immobilized enzyme and unreacted cellulose were precipitated together to remove the product-the soluble sugar solution-by changing pH. Following this, a new buffer solution was added to the precipitate to dissolve it and resume the reaction. This was repeated several times. The hydrolysis rate of this process increased significantly compared with that of a batch process. Utilization of the reversible soluble-insoluble carrier for immobilizing enzyme is promising, not only for cellulose-cellulase systems, but also for other heterogeneous reaction systems.     

Fluorescence of tryptophan residues in firefly luciferases and enzyme--substrate complexes

Quenching of tryptophan fluorescence of Luciola mingrelica (single tryptophan residue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417 and Trp-426) luciferases with different quenchers (I-, Cs+, acrylamide) was studied. The conserved Trp-417(419) residue was shown to be not accessible to charged particles, and positively and negatively charged amino acid residues are located in close vicinity to it. We found previously unreported effective energy transfer from this tryptophan to luciferin during the quenching of the tryptophan fluorescence. The distance between the luciferin molecule and Trp-417(419) was calculated: 11-15 and 12-17 A for P. pyralis and L. mingrelica luciferases, respectively. The role of the conserved Trp residue in the catalysis is discussed. ATP and AMP are also quenchers of the tryptophan fluorescence of the luciferases. In this case, an allosteric mechanism of the interaction of Trp-417(419) with an excess of ATP (AMP) is proposed.     

Activity studies of immobilized subtilisin on functionalized pure cellulose-based membranes

The activity of immobilized subtilisin BPN' on pure cellulose-based membrane support was investigated using site-directed and random immobilization approaches. The catalytic activity of site-directed immobilized subtilisin on pure cellulose fiber-based materials was found to be 81% of that in homogeneous solution, while that of randomly immobilized subtilisin was 27%. Pure cellulose membrane supports provided large surface areas for high enzyme loading without diffusional limitations. The activity of immobilized subtilisin on pure cellulose support was more than twice that on a modified polyether sulfone (MPS) membrane, which was attributed to the higher hydrophilicity of cellulose. Immobilized subtilisin maintained its initial activity for 14 days at 4 degrees C and 7 days at 24 degrees C. The immobilized enzyme could resist higher temperature and operate over a wider range of pH without loss of activity. This study showed that pure cellulose fiber-based membranes are well suited for enzyme immobilization and biocatalysis.     

A Simple Method for Beta-glucosidase Immobilization and Its Application in Soybean Isoflavone Glycosides Hydrolysis

In this study, a simple, inexpensive and fast β-glucosidase immobilization system was constructed and evaluated in isoflavone glycosides hydrolysis. A β-glucosidase gene from Thermoascus aurantiacus IFO9748 was recombinantly expressed in Pichia pastoris KM71H and immobilized on regenerated amorphous cellulose (RAC) by fused cellulose binding module 3. Through simple mixing cellulose and crude enzyme for 15 min under room temperature, 96.04% β-glucosidase was immobilized onto RAC. The optimum temperature for β-glucosidase activity was increased by 5ºC after immobilization. The half-life (t½) of heat inactivation of immobilized enzyme at 60oC was improved over 8 folds. After 30 rounds recycled at 40oC, 96.9% daidzin and 98.9% genistin could still be hydrolyzed. A continuous hydrolysis system was also constructed, and at the flow rate of 0.2 mL/min after 30 h hydrolysis, 95.6% genistin and 90.2% daidzin can still be hydrolyzed. Combined the simple and high efficient enzyme immobilization procedure and inexpensive cellulose, this scalable and practical system may have broad prospects for industrial utilization.     

Quenching of the fluorescence of Tyr and Trp residues of firefly luciferase from <Emphasis Type="Italic">Luciola mingrelica</Emphasis> by the substrates

Luciferase of the firefly Luciola mingrelica is characterized by fluorescence of not only the unique Trp residue (lambda(em) = 340 nm), but also that of Tyr residues (lambda(em) = 308 nm). Quenching of the intrinsic fluorescence of the luciferase by its substrates luciferin and ATP (AMP) has been studied. Luciferin (LH2) quenches Trp fluorescence more efficiently than the fluorescence of Tyr residues. Two centers of quenching of Tyr fluorescence by ATP have been found corresponding apparently to the allosteric and active sites of the luciferase with K(s(ATP)) = 20 and 110 microM, respectively. The influence of one substrate on the affinity of luciferase to the second was investigated using fluorescence. ATP (AMP) binding to the allosteric sites of the luciferase significantly affects the affinity of luciferase to LH2. Formation of the complex between the luciferase and LH2 affects the affinity of both allosteric and active sites of the luciferase to ATP (AMP). The observed effects are probably connected with conformational changes in the luciferase molecule upon its interaction with the substrates.     

Mutant luciferase enzymes from fireflies with increased resistance to benzalkonium chloride

Benzalkonium chloride (BAC), used to extract intracellular ATP, interferes with subsequent firefly luciferase-luciferin assays. There was a significant difference among wild-type luciferases with respect to BAC resistance. Luciola lateralis luciferase (LlL) was the most tolerant, followed by Luciola cruciata luciferase (LcL) and Photinus pyralis luciferase. Random mutagenesis of thermostable mutants of LcL showed that the Glu490Lys mutation contributes to improved resistance to BAC. The corresponding Glu490Lys mutation was introduced into thermostable mutants of LlL by site-directed mutagenesis. Kinetic analysis demonstrated that the resultant LlL-217L490K mutant, having both an Ala217Leu and a Glu490Lys mutation, showed the highest resistance to BAC, with an initial remaining bioluminescence intensity of 87.4% and a decay rate per minute of 29.6% in the presence of 0.1% BAC. The Glu490Lys mutation was responsible for increased resistance to inactivation but not inhibition by BAC. The LlL-217L490K had identical thermostability and pH stability to the parental thermostable mutant. From these results, it was concluded that the LlL-217L490K enzyme is advantageous for hygiene monitoring and biomass assays based on the ATP-bioluminescence methodology. This is the first report demonstrating improved resistance to BAC of the firefly luciferase enzyme.     

Covalent immobilization of a glucoamylase to bagasse dialdehyde cellulose

Cellulose fibres from bagasse were oxidized by sodium periodate in sulphuric acid media at positions 2 and 3 of the anhydroglucose unit to produce dialdehyde cellulose. The aldehyde groups of the dialdehyde cellulose were able to react with amino groups of a glucoamylase to form covalent bonds and result in a dialdehyde cellulose immobilized enzyme. The optimum pH of this immobilized enzyme and free enzyme were in the range of 3.0–5.0 and 3.5–5.0, respectively. The optimum temperature for both the free and immobilized enzymes was 60–65 °C. The relative remaining activity of the immobilized enzyme was 36% and its stability was very good, since it could be reused for over 30 cycles. Its activity decreased from the first to the seventh reuse cycles, due to the slow detachment of non-covalently bound enzyme. However, activity tended to stabilize after the seventh cycle of reuse, indicating very stable covalent binding between the enzyme and dialdehyde cellulose.     

Interaction of dimethyl-and monomethyloxyluciferin with recombinant wild-type and mutant firefly luciferases

Dissociation constants (Ks) in the pH range 6.5-9.0 for complexes of luciferin, dimethyloxyluciferin (DMOL), and monomethylluciferin (MMOL) with recombinant wild-type and mutant (His433Tyr) luciferases from the Luciola mingrelica firefly were determined by fluorescent titration. The protonated effectors were bound by the wild-type and mutant luciferases better than the nonprotonated ones. The affinity of DMOL for the mutant luciferase was higher than for the wild-type luciferase at alkaline pH, whereas the affinity of MMOL was higher at all pH values studied. The fluorescence emission and excitation spectra of DMOL and MMOL in buffer solution (pH 7.8) were obtained in the absence and presence of luciferase. The fluorescence maxima of DMOL and MMOL complexes with luciferase were 20 and 100 nm, respectively, shifted to shorter wavelengths as compared to the values in buffer solution. This was explained by nonspecific and specific influence of the protein microenvironment on the fluorescence spectra of DMOL and its specific influence on the MMOL fluorescence spectra.