Properties of bacterial luciferase/NADH : FMN oxidoreductase and firefly luciferase immobilized onto sepharose

NADH : FMN oxidoreductase and bacterial luciferase have been efficiently coimmobilized onto Sepharose 4B. This luminescent immobilized enzyme system can be used to assay NADH. The assay is rapid and sensitive with a lower limit of detection of 0.2 pmol/assay tube. The intra-assay precision was 3.5% at 2 × 10^-5 M and 5.8% at 2 × 10^-6 M NADH. Light intensity was proportional to NADH concentration from 0.2 to 1000 pmol. Added serum and certain dehydrogenases were found to be inhibitory; however, inhibition could be eliminated by a combination of heat treatment and dilution. Firefly luciferase has also been immobilized onto both Sepharose 4B and CL 6B. The detection limit for ATP using this immobilized enzyme was 0.2 pmol and the assay was linear from 0.2 to 2000 pmol. The intra-assay precision was 4.8% at 2 × 10^-4 M and 3.2% at 1 × 10^-5 M ATP. The immobilized enzymes remained fully active when rapidly frozen in the presence of glycerol and DTT. Such preparations could be stored for at least two months with no loss of activity. A variety of different compounds were used to block any remaining reactive groups on the Sepharose following immobilization of the enzymes. Glycine, 2-aminoethanol, and ethylenediamine were examined. The preparations where ethylenediamine was used as a blocking agent exhibited better activity and stability than the others.     

Recombinant firefly luciferase in Escherichia coli

The authentic recombinant luciferase, the luciferase with the structure similar to that of the native protein, was obtained using random mutagenesis, and its properties were studied in comparison with several fusion proteins. Thermoinactivation curves of the recombinant luciferases within the 10–50°C temperature interval showed that thermoinactivation involves reversible and irreversible steps. Immobilization of the recombinant Luciola mingrelica and Photinus pyralis firefly luciferases on BrCN-activated sepharose was carried out. Immobilization resulted in the preparation of enzymes with high catalytic activity. Physicochemical properties and analytical characteristics of the immobilized recombinant and native luciferases were studied. The catalytic properties of the immobilized recombinant L. mingrelica luciferase were close to those of the native luciferase but the former enzyme appeared to be significantly more stable. The immobilized recombinant luciferases can be used for ATP assay within 0.01–10000 nM range.     

Citric Acid Interferes with Adenosine Triphosphate Determination by Bioluminescence

Abstract

Adenosine triphosphate (ATP) measurement by bioluminescence is used in microbial adsorption studies for which colony-forming units (CFU) may underestimate true microbial numbers. We observed that citric acid profoundly affected ATP measurement by luciferin-luciferase assay, and we assessed the effect of citric acid on ATP detection in water and buffer systems in the pH range 4.9 to 7.4. Bioluminescence depended on the buffer system, increasing as pH increased in each buffer system and decreasing as the buffer system changed from Na-phosphate to citrate-phosphate. Citrate-phosphate buffer decreased bioluminescence by 82% relative to water at the same pH. Consequences of poor buffer selection are potentially reduced luciferase activity in the bioassay system and underestimation of microbial numbers.     

An Improved Urea Specific Enzyme Electrode

Abstract

The enzyme urease was immobilized in a layer of acrylamide polymer on the surface of a Beckman cationic electrode sensitive to ammonium ion. The substrate urea diffuses to the enzyme electrode and reacts with the immobilized enzyme to produce ammonium ion at the surface of the glass electrode. By placing a thin film of cellophane around the enzyme gel layer to prevent leaching of urease into the surrounding solution, an electrode could be used continuously for over 21 days at 25[ddot]C with no loss of activity.     

The stress-related production of the activePhotinus pyralis and luciolaMingrelica firefly luciferases inEscherichia coli

The kinetics ofPhotinus pyralis andLuciola mingrelica luciferase gene expression was studied on plasmids with the thermoinducible λP_r promoter inEscherichia coli by SDS-gel electrophoresis of cell lysates to follow luciferase protein-synthesized, enzyme immunoassay (EIA) to follow native enzyme conformer, and the luciferase activity assay.E. coli cells were cultivated at temperature schemes 28–42–21°C or 28–21°C, or at alkali pH shift. In the cases of thermoinduction and pH shift, the luciferase expressions have similar features. The 3-h thermoinduction (42°C) followed by the incubation at 21°C, for 10 h resulted in the maximal amount of the luciferase protein of 4–5% of the total cell proteins. The yield did not change further. The amount of native luciferase conformer and the luciferase activity started to grow after incubation for 10 h at 21°C and reached the maximum after 50–60 h when the synthesized luciferase protein adopted the native-like conformation. At the same time, only 50% of the latter appeared to be catalytically active. An increase in the enzymatic activity correlates with an increase in the intracellular pH and ATP content. Intracellular metabolic reactions were shown to play a role in the conformational changes of the enzyme in a postthermoinduction period, and a possible mechanism of this effect is proposed.     

Effect of gelatin on properties of <Emphasis Type="Italic">Luciola mingrelica</Emphasis> firefly luciferase

An ATP-reagent containing thermostable mutant of Luciola mingrelica firefly luciferase, MgSO₄, components of buffer solution, and stabilizers, which is widely used to detect nano- and picomolar concentrations of adenosine-5′-triphosphate (ATP) in various biological samples, is been object of this study. The activity, stability, and analytical characteristics of the ATP-reagent have been assessed in the presence and absence of 5% gelatin and in gelatin gel. The solution of ATP-reagent was obtained at 30°C and a gelatin concentration of 5%, while gel formation occurred at 22°C. The gelatin addition decreased the activity and stability of luciferase slightly. The sensitivity of ATP detection (above 0.96) did not depend on the gelatin presence and the aggregate state of the disperse system. Limits of detection were 2 × 10⁻¹², 7 × 10⁻¹³, and 7 × 10⁻¹⁴ M ATP, when the ATP-reagent was used in gelatin films and in the solution in the presence of 5% gelatin and in the absence, respectively. It was shown that the storage of ATP-reagent in gelatin gel not only preserved enzymatic activity, but protected the enzyme from bacterial contamination, which was the cause of the enzyme activity loss.     

Bioluminescent determination of reduced nicotinamide adenine dinucleotide with immobilized bacterial luciferase and flavin mononucleotide oxidoreductase on collagen film

Bacterial luciferase flavin mononucleotide oxidoreductase were co-immobilized on collagen strips. Reduced nicotinamide adenine dinucletodie was determined in the range 1 X 10^?9?2 X 10^?5 M, with a precision of 5%. The immobilized system retained 70% of its initial activity after two weeks.     

Use of a special Brazilian red-light emitting railroad worm Luciferase in bioassays of NEK7 protein Kinase and Creatine Kinase

Background

Luciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP.

Methods

Here, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP.

Results

In this work we used, after several optimization reactions, creatine kinase isoforms as well as ?NEK7 protein kinase in the absence or presence of ATP analogous inhibitors  to validate this new luminescence method.

Conclusion

With this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme.

     

Efficient Immobilization of Porcine Pancreatic α-Amylase on Amino-Functionalized Magnetite Nanoparticles: Characterization and Stability Evaluation of the Immobilized Enzyme

The potential of the modified magnetic nanoparticles for covalent immobilization of porcine pancreatic α-amylase has been investigated. The synthesis and immobilization processes were simple and fast. The co-precipitation method was used for synthesis of magnetic iron oxide (Fe₃O₄) nanoparticles (NPs) which were subsequently coated with silica through sol–gel reaction. The amino-functionalized NPs were prepared by treating silica-coated NPs with 3-aminopropyltriethoxysilane followed by covalent immobilization of α-amylase by glutaraldehyde. The optimum enzyme concentration and incubation time for immobilization reaction were 150 mg and 4 h, respectively. Upon this immobilization, the α-amylase retained more than 50 % of its initial specific activity. The optimum pH for maximal catalytic activity of the immobilized enzyme was 6.5 at 45 °C. The kinetic studies on the immobilized enzyme and its free counterpart revealed an acceptable change of K_m and V_max. The Km values were found as 4 and 2.5 mM for free and immobilized enzymes, respectively. The V_max values for the free and immobilized enzymes were calculated as 1.75 and 1.03 μmol mg^?1 min^?1, in order, when starch was used as the substrate. A quick separation of immobilized amylase from reaction mixture was achieved when a magnetically active support was applied. In comparison to the free enzyme, the immobilized enzyme was thermally stable and was reusable for 9 cycles while retaining 68 % of its initial activity.     

Surface functionalization via in situ interaction of plasma-generated free radicals with stable precursor-molecules on cellulose

The surface functionalization process was accomplished in a consecutive 3 step process including: (1) Argon- and oxygen-plasma enhanced generation of free radical sites on cellophane surfaces; (2) “In situ” gas phase derivatization in the absence of plasma using hydrazine, ethylene diamine, or propylene diamine; (3) Second “in situ”, gas phase derivatization in the absence of plasma using oxallyl chloride or “ex situ” derivatization in the presence of glutaraldehyde. The presence of free radical sites on the plasma exposed cellophane surfaces was demonstrated using “in situ” sulfur dioxide and nitric oxide labeling techniques. It was shown that the free radical sites readily react under “in situ” conditions with the stable chain-precursor components and generate the desired spacer-chain molecules. ESCA, ATR-FTIR analysis and dying techniques were used to monitor the cellophane surface changes. A factorial design was used for selecting the optimal plasma parameters. Functionalized cellophane substrates were used for immobilization of α-chymotrypsin in the presence of spacer-chain molecules. The activity of the immobilized α-chymotrypsin was found to be lower in comparison to the activity of the free enzyme and the presence of virgin cellophane in the free enzyme solution also reduced significantly the activity of the enzyme. It is suggested that the swollen state of the cellophane plays a significant role in the decrease of the immobilized enzyme activity.     

Identification of Antioxidant and Anti-α-amylase Components in Lotus (Nelumbo nucifera,Gaertn.) Seed Epicarp

Nano-sized Fe₃O₄ was synthesized by chemical co-precipitation and subsequently modified with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde to introduce aldehyde group on its surface. With the help of “interface activation” by adding sucrose esters-11 as surfactant, lipase from Rhizopus oryzae was successfully immobilized onto the carrier with great enhancement of activity. The hydrolysis activity of immobilized enzyme were 9.16 times and 31.6 times of free enzyme when p-nitrophenol butyrate and p-nitrophenol palmitate were used as substrates. The thermo-stability of immobilized enzyme was also enhanced compared to free enzyme. The immobilized enzyme was successfully applied in synthesis of 1,3-diacyglycerols (1,3-DAG). The specific esterification activity of immobilized enzyme was about 1.5 times of the free enzyme. The immobilized enzyme showed good region-selectivity towards 1,3-diacyglycerols and retained nearly 80% of its activity after reused for 60 times, revealing a good industrial application prospect.

     

Larval Tenebrio molitor (Coleoptera: Tenebrionidae) Fat Body Extracts Catalyze Firefly D-Luciferin- and ATP-Dependent Chemiluminescence: A Luciferase-like Enzyme*

Ultraweak light emission was detected upon injection of firefly luciferin into live Tenebrio larvae. A chemilumi-nescent enzymatic activity dependent on molecular oxygen, D-luciferin and MgATP was then isolated from larval fat body extracts by precipitation with 70% ammonium sulfate. D-Luciferin and ATP can be replaced by luciferyl-adenylate. Pyrophosphate is a main product from the chemiluminescent reaction. The in vitro chemiluminescence intensity was not affected by peroxidase inhibitors such as N₃^?_- (0.5 mM) and CN^? (1 mM), attesting to its nonperoxidatic nature but was strongly inhibited by AMP (1 mM), luciferin 6′-ethyl ether (1 mM) and sodium pyrophosphate (2 mM), well-known firefly lucifer-ase inhibitors. Some physical-chemical properties of this enzymatic activity were similar to those of firefly lucif-erase (K_MATP = 195 μM; K_0.5 luciferin - 0.8 mM; optimum pH 8.5; δ_max= 610 nm at pH 8.5; firefly lucifer-ase: δ_max= 565 nm at pH 8.0 and 619 mm at pH 6.0), but the chemiluminescence was not affected by addition of polyclonal antibodies raised against Photinus pyralis luciferase. These data suggest that this chemiluminescence results from a ligase with luciferase activity.     

Immobilization of Xylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Strain MK001 and its Application in Production of Xylo-oligosaccharides

Xylanase from Bacillus pumilus strain MK001 was immobilized on different matrices following varied immobilization methods. Entrapment using gelatin (GE) (40.0%), physical adsorption on chitin (CH) (35.0%), ionic binding with Q-sepharose (Q-S) (45.0%), and covalent binding with HP-20 beads (42.0%) showed the maximum xylanase immobilization efficiency. The optimum pH of immobilized xylanase shifted up to 1.0 unit (pH 7.0) as compared to free enzyme (pH 6.0). The immobilized xylanase exhibited higher pH stability (up to 28.0%) in the alkaline pH range (7.0–10.0) as compared to free enzyme. Optimum temperature of immobilized xylanase was observed to be 8 °C higher (68.0 °C) than free enzyme (60.0 °C). The free xylanase retained 50.0% activity, whereas xylanase immobilized on HP-20, Q-S, CH, and GE retained 68.0, 64.0, 58.0, and 57.0% residual activity, respectively, after 3 h of incubation at 80.0 °C. The immobilized xylanase registered marginal increase and decrease in K _m and V _max values, respectively, as compared to free enzyme. The immobilized xylanase retained up to 70.0% of its initial hydrolysis activity after seven enzyme reaction cycles. The immobilized xylanase was found to produce higher levels of high-quality xylo-oligosaccharides from birchwood xylan, indicating its potential in the nutraceutical industry.     

Litcrosomal Nadph Oxidase and Hybrid Electrodes

Abstract

The NADPH oxidizing activity of rat liver microsomes was investigated and found to be mainly due to the cytochrome P-450 system. The XADPH oxidase was utilized for the development of several organelle electrodes. Gelatin membrane immobilized microsomes were combined with an O₂ membrane sensor for bioelectrochemical measurement of NADPH. The dependence of the current on substrate concentration was linear up to 1 mmol·1^?1 To assemble hybrid electrodes for determination of glucose-6-phoaphate, ATP and isocitrate pure enzymes were coimmobilized with the microsomal fraction.     

Production of 2-O-α-glucopyranosyl l-ascorbic acid from ascorbic acid and β-cyclodextrin using immobilized cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) isolated and purified from Paenibacillus sp. A11 was immobilized on various carriers by covalent linkage using bifunctional agent glutaraldehyde. Among tested carriers, alumina proved to be the best carrier for immobilization. The effects of several parameters on the activation of the support and on the immobilization of enzyme were optimized. The best preparation of immobilized CGTase retained 31.2% of its original activity. After immobilization, the enzymatic properties were investigated and compared with those of the free enzyme. The optimum pH of the immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60°C). Free and immobilized CGTase showed similar pH stability profile but the thermal stability of the immobilized CGTase was 20% higher. Kinetic data (K _M and V _max) for the free and immobilized enzymes were determined from the rate of β-CD formation and it was found that the immobilized form had higher K _M and lower V _max. The immobilized CGTase also exhibited higher stability when stored at both 4°C and 25°C for 2 months. The enzyme immobilized on alumina was further used in a batch production of 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin. The yield of AA-2G was 2.92% and the immobilized CGTase retained its activity up to 74.4% of the initial catalytic activity after being used for 3 cycles. The immobilized CGTase would have a promising application in the production of various transglycosylated compounds and in the production of cyclodextrin by the hydrolysis of starch.     

Production of Mixed-Linkage Beta-Oligosaccharides from Lichenan Using Immobilized Bacillus licheniformis UEB CF Lichenase

Lichenase from Bacillus licheniformis UEB CF was immobilized on Amberlite IR120 H. The immobilization yield and lichenase activity were 87 and 92.81?% of initial activity, respectively. The immobilized enzyme exhibited a shift in the optimal pH from 5.0 to 3.0, but the activity optimal temperature was not affected. The immobilized enzyme showed a residual activity of 50?% after five uses. It also exhibited high storage stability and retained 50?% of its initial activity after 120?days at 4?°C. The main hydrolysis products yielded from lichenan were trisaccharide and tetrasaccharide. The resulting mixed-linkage beta-oligosaccharides could be used as a special nutriment for lactic bacteria.     

A Novel Streptavidin–luciferase Fusion Protein: Preparation,Properties and Application in Hybridization Analysis of DNA

A streptavidin–luciferase fusion protein comprising the thermostable mutant form of firefly luciferase Luciola mingrelica and minimal core streptavidin was constructed. The streptavidin–luciferase fusion was mainly produced in a tetrameric form with high luciferase and biotin‐binding activities. It was shown that fusion has the same K_m values for ATP and luciferin and the bioluminescence spectra as initial luciferase. The linear dependence of the bioluminescence signal on the content of the fusion was observed within the range of 10^?18–10^?13 mol per well. Successful application of obtained fusion in a biospecific bioluminescence assay based on biotin–streptavidin interactions was demonstrated by the example of a specific DNA hybridization analysis. A DNA hybridization analysis for Escherichia coli cells identification was developed using unique for these cells gadB fragment encoding glutamate decarboxylase. The amplified biotinylated GadB fragments were hybridized with the immobilized oligonucleotide probes; then, the biotin in the DNA duplexes was detected using the streptavidin–luciferase fusion protein. To reach the high sensitivity of the assay, we optimized the conditions of the assay. It was shown that the use of Pluronic for plate modification resulted in a significant reduction in the DNA detection limit which finally was 0.4 ng per well.     

Inulin Hydrolysis by Immobilized Inulinase on Functionalized Magnetic Nanoparticles Using Soy Protein Isolate and Bovine Serum Albumin

Inulin hydrolysis was performed by inulinase from Aspergillus niger covalently immobilized on magnetite nanoparticles (Fe₃O₄) covered with soy protein isolate (Fe₃O₄/SPI) functionalized by bovine serum albumin (Fe₃O₄/SPI/BSA) nanoparticles as a new bio‐functional carrier. The specific activity and protein content of the immobilized enzyme were 25.99 U/mg and 3.52 mg/mL, respectively, with 80% enzyme loading. The immobilized inulinase showed maximum activity at 45 °C, which is 5 °C higher than the optimum temperature of the free enzyme. Also, the optimum pH of the immobilized enzyme shifted from 6 to 5.5, which is more acidic compared to that of the free enzyme. The K_m value of immobilized inulinase decreased to 2.03 mg/mL. Thermal stability increased considerably at 65 and 75 °C, and a 5.13‐fold rise was detected in the enzyme half‐life at 75 °C after immobilization. Moreover, 80% of initial activity of immobilized inulinase remained after 10 cycles of hydrolysis.