Flow system for fish freshness determination based on double multi-enzyme reactor electrodes

A double reactor system for the determination of fish and shellfish freshness using the freshness indicator, K-value (K=[(HxR+Hx)/(ATP+ADP+AMP+IMP+HxR+Hx)]x100), was developed, where ATP, ADP, AMP, IMP, HxR and Hx are adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, inosine monophosphate, inosine and hypoxanthine, respectively. The system consisted of a pair of enzyme reactors with an oxygen electrode positioned close to the respective reactor. The enzyme reactor (I) was packed with nucleoside phosphorylase and xanthine oxidase immobilized simultaneously on chitosan beads (immobilized enzyme A). Similarly, the enzyme reactor (II) was packed with immobilized enzyme A and immobilized enzyme B (co-immobilized alkaline phosphatase and adenosine deaminase). Moreover, this reactor consisted of two layers, the enzyme A and enzyme B (1:1). A good correlation was obtained between K values, which were determination by the proposed system and by the HPLC method. One assay could be completed within 5 min. The signal for the determination of K value of fish and shellfish was reproducible within 2.3%. The long-term stability of the enzyme reactors was evaluated at 30 degrees C for 28 days.     

Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors

This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bioreactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h(-1) in the CST bioreactor and between 0.111 and 0.500 h(-1) in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The ATP was extracted from the cells using boiling tris-EDTA buffer (pH 7.75), and the quantity determined using a firefly (bioluminescence) technique. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g(-1) dry weight (dw) as dilution rate increases from 0.027 to 0.115 h(-1). At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g(-1) dw, which is assumed to be the quantity of ATP in 100% viable biomass. In the TPAL bioreactor, the ATP level increased with dilution rate in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g(-1) dw at dilution rates between 0.111 and 0.200 h(-1) to approximately 0.119 mg ATP g(-1) dw at dilution rates between 0.300 and 0.500 h(-1). This indicates that the immobilized biomass contained a viable cell fraction of around 5%. Based on these results, kinetic data for freely suspended cells should not be applied to the modeling of immobilized cell systems on the assumption that immobilized biomass is 100% viable. (c) 1993 John Wiley & Sons, Inc.     

Glycolytic pathway as an ATP generation system and its application to the production of glutathione and NADP

Efficient ATP generation is required to produce glutathione and NADP. Hence, the generation of ATP was investigated using the glycolytic pathway of yeast. Saccharomyces cerevisiae cells immobilized using polyacrylamide gel generated ATP from adenosine, consuming glucose and converting it to ethanol and carbon dioxide. Under optimal conditions, the ATP-generating activity of immobilized yeast cells was 7.0 μmol h^?1 ml^?1 gel. A column packed with these immobilized yeast cells was used for continuous ATP generation. The half-life of the column was 19 days at a space velocity of (SV) 0.3 h^?1 at 30°C. The properties of glutathione- and NADP-producing reactions coupled with the ATP-generating reaction were investigated. Escherichia coli cells with glutathione synthesizing activity and Brevibacterium ammoniagenes cells with NAD kinase activity were immobilized in a polyacrylamide gel lattice. Under optimal conditions, the immobilized E. coli cells and immobilized B. ammoniagenes cells produced glutathione and NADP at the rates of 2.1 and 0.65 μmol h^?1 ml^?1 gel, respectively, adding ATP to the reaction mixture. In order to produce glutathione and NADP economically and efficiently, the glutathione- and NADP-producing reactions were finally coupled with the ATP-generating reaction catalysed by immobilized S. cerevisiae cells. To compare the productivities of glutathione and NADP, and to compare the efficiency of ATP utilization for the production of these two compounds, the two reactor systems, co-immobilized cell system and mixed immobilized cell system, were designed. As a result, these two compounds were also found to be produced by these two kinds of reactor systems. Using the data obtained, the feasibility and properties of ATP generation by immobilized yeast cells are discussed in terms of the production of glutathione and NADP.     

生物素化ATP硫酸化酶的表达、固定化与应用

现代大规模焦测序技术的产生是DNA测序技术的一次革命,其关键技术之一是得到高活性的、固定于磁性微球表面的ATP硫酸化酶．生物素化的ATP硫酸化酶可以通过生物素与亲和素之间的特异结合特性固定在包被亲和素的磁性微球表面,但是利用化学修饰法将ATP硫酸化酶进行生物素化修饰很可能会影响酶的活性．利用融合表达策略,将大肠杆菌生物素酰基载体蛋白C端87个氨基酸肽段(BCCP87)与ATP硫酸化酶在大肠杆菌内融合表达,经SDS-PAGE和Western blot分析,表达的融合蛋白分子质量约为64 ku,并且能够在大肠杆菌内被生物素化．生物素化的ATP硫酸化酶能够与亲和素包被的磁珠结合,固定后的ATP硫酸化酶具有活性,并且能够用于定量检测焦磷酸盐(PPi)和焦测序,为今后建立高通量大规模焦测序系统提供了一个有效的工具酶．     

Immobilized polyphosphate kinase: preparation, properties, and potential for use in adenosine 5'-triphosphate regeneration

Polyphosphate kinase (ATP:polyphosphate phosphotransferase; EC 2.7.4.1), partially purified from Escherichia coli, has been immobilized on glutaraldehyde-activated aminoethyl cellulose with a 10% retention of enzymatic activity. The immobilized enzyme can carry out the synthesis of ATP from ADP, using long-chain inorganic polyphosphate as a phosphoryl donor. Chromatographic analyses of the product mixture produced from ADP and [32P]polyphosphate demonstrated that 98% of the 32P was incorporated into ATP, indicating that the immobilized polyphosphate kinase is substantially free from contaminating polyphosphate phosphohydrolase (EC 3.6.1.11), adenosine triphosphatase (EC 3.6.1.4), and adenylate kinase (EC 2.7.4.3). Immobilized polyphosphate kinase loses no activity when stored in an aqueous suspension for 2 months at 5 degrees C or for 1-2 weeks at 25 degrees C. It may be stored indefinitely as a lyophilized powder at -10 degrees C. Michaelis constants for ADP and polyphosphate were determined to be 160 and 120 microM, respectively, for the immobilized enzyme. A small-batch reactor was found to produce ATP linearly with time up to 65% conversion of polyphosphate into ATP and to attain greater than 85% conversion to ATP at equilibrium. The ease of purification and immobilization of E. coli polyphosphate kinase, its storage stability, the purity and yield of its ATP product, and the low values of the Michaelis constants for its substrates make it a highly promising enzyme for ATP regeneration.     

Reciprocating-flow ATP amplification system for increasing the number of amplification cycles

We constructed a novel ATP amplification reactor using a reciprocating-flow system to increase the number of ATP amplification cycles without an increase in backpressure. We previously reported a continuous-flow ATP amplification system that effectively and quantitatively amplified ATP and increased the sensitivity of a quantitative bioluminescence assay. However, it was difficult to increase the number of amplification cycles due to backpressure in the system. Because addition of immobilized adenylate kinase (ADK) and pyruvate kinase (PK) columns increased backpressure, the maximum number of ATP amplification cycles within column durability was only 4. In this study, ATP amplification was performed using a reciprocating-flow system, and 10 cycles of ATP amplification could be achieved without an increase in backpressure. As a result, ATP was amplified more than 100-fold after 10 cycles of reciprocating flow. The gradient of ATP amplification was approximately 1.76^N. The backpressure on the columns was 0.03 MPa in 1–10 ATP amplification cycles, and no increases in backpressure were observed.     

Application of kinetic-based biospecific affinity chromatographic systems to ATP-dependent enzymes: studies with yeast hexokinase

This study is concerned with the development of kinetic-based bioaffinity chromatographic systems for purification of ATP-dependent kinases, with a particular focus on the allosteric yeast hexokinase enzyme (EC 2.7.1.1). Synthesis and characterization of highly substituted N(6)-linked and S(6)-linked immobilized ATP derivatives are described using a rapid solid-phase modular approach. Evaluation of the new immobilized ATP derivatives has been carried out using model chromatographic studies with yeast hexokinase, employing specific substrate analogues (N-acetyl-D-glucosamine and suramin) to promote biospecific adsorption, in the presence and absence of citrate (a so-called allosteric activator of hexokinase activity). In this paper, successful bioaffinity chromatography systems were developed for yeast hexokinase and, as a result, interesting binding and catalytic properties of the enzyme were highlighted and explored. The overall results confirm the potential for extrapolation of the kinetic locking-on tactic, a general kinetic-based bioaffinity approach already developed for the NAD(P)(+)-dependent dehydrogenases, to ATP/ADP-dependent enzymes. However, in view of the enhancement of the intrinsic ATPase activity of hexokinase with glucosamine derivatives, and the coincidental hydrolysis of immobilized ATP to immobilized ADP, future developments necessary to support adaptation of the approach to ATP-dependent enzymes are discussed.     

ATP regeneration by thermostable ATP synthase

We investigated the possibility of using thermostable ATP synthase (TF(0)F(1)) for a new ATP regeneration method. TF(0)F(1) was purified from a thermophilic bacterium, PS3, and reconstituted into liposomes. ATP synthesis experiments showed that TF(0)F(1) liposomes could synthesize ATP in micromole concentrations by acid-base change. The acid-base change was repeated six times over an 11-day period with no detectable loss of activity at the reaction temperature (45 degrees C). Given these encouraging results, we conceptualized and modeled a system to synthesize ATP using ATP synthase with energy supplied by acid-base change. In this system, liposomes containing ATP synthase are immobilized on small glass spheres that facilitate separation of buffers from the liposomes after the acid-base change. Compared to an alternate system that uses membranes to separate the buffers from the liposomes, the glass spheres reduce inefficient mixing of acidic and basic buffers during the acid-base change. To increase the ATP synthesis yield, this system uses electrodialysis to regenerate a potassium gradient after the acid-base change. It also employs water-splitting electrodialysis to regenerate KOH and HCl required to adjust the pH of acidic and basic buffers. All reagents are recycled, so electrical energy is the only required input.     

Comparative studies on soluble and immobilized yeast hexokinase

Comparative studies have been carried out on soluble and immobilized yeast hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1). The enzyme was immobilized by covalent attachment to a polyacrylamide type support containing carboxylic functional groups. The effects of immobilization on the catalytic properties and stability of hexokinase were studied. As a result of immobilization, the pH optimum for catalytic activity was shifted in the alkaline direction to ～pH 9.7. The apparent optimum temperature of the immobilized enzyme was higher than that of the soluble enzyme. The apparent K_m value with D-glucose as substrate increased, while that with ATP as substrate decreased, compared with the data for the soluble enzyme. Differences were found in the thermal inactivation processes and stabilities of the soluble and immobilized enzymes. The resistance to urea of the soluble enzyme was higher at alkaline pH values, while that for the immobilized enzyme was greatest at ～pH 6.0.     

固定化细胞法在5'-三磷酸腺苷合成中的应用

从生物催化活性细胞、细胞的固定化形式、固定化细胞反应器3个方面对固定化细胞法合成5’-三磷酸腺苷(ATP)的研究情况作了扼要综述。分析了我国ATP生产的工业化现状,并对今后的研究和开发提出了建议。     

Preparation of N6-[N-(6-aminohexyl) carbamoyl]-adenine nucleotides and their application to coenzymically active immobilized ADP and ATP, and affinity adsorbents.

Reaction of ADP with hexamethylene diisocyanate in hexamethylphosphoramide followed by treatment in an acidic medium afforded three new adenine nucleotide analogues, N6-[N-(6-aminohexyl)carbamoyl]-ADP, N6-[N-(6-aminohexyl)carbamoyl]-ATP, and N6-[N-(6-aminohexyl)carbamoyl]-AMP in yields of 13%, 12% and 17%, respectively. The occurrence of the ATP analogue may be interpreted in terms of the equilibrium, 2ADP = ATP + AMP. Coenzymic activities of the ADP analogue against acetate kinase and pyruvate kinase were 82% and 20%, respectively, relative to ADP and those of the ATP analogue against hexokinase and glycerokinase were 63% and 87%, respectively, relative to ATP. These analogues were bound to CNBr-activated soluble dextran through their terminal amino group to give an immobilized ADP and an immobilized ATP, each of which was recycled in a system comprising acetate kinase and hexokinase, and when placed in a membrane reactor together with the enzymes, functioned as an immobilized coenzyme continuously yielding glucose 6-phosphate. A series of chemically defined affinity adsorbents were obtained by coupling these analogues to CNBr-activated Sepharose, and were used to separate the enzymes in a mixture of hexokinase, pyruvate kinase, phosphoglycerate kinase, lactate dehydrogenase, and alcohol dehydrogenase.     

Identification of l-Baikiain from Calcareous Red Algae Serraticardia maxima (Yendo) Silva and Its Distribution in Some Rhodophyta

The conjugated enzyme system of NAD kinase (NK) and acetate kinase (AK) was investigated to produce NADP continuously by the dynamic recycling of ATP. The enzymes were physically entrapped in an ultrafiltration hollow fiber tube and the cosubstrate, ATP, as well as the substrates were fed to the reactor continuously. The ATP turnover increased with a decrease in the concentration of ATP fed and with an increase in the concentration of immobilized enzymes. The maximal productivity of the reactor was 0.228 μmol/hr/ml-reactor with an ATP turnover of 8.35. The experimental results were approximately predicted using a theoretical model in which the equilibrium random mechanism was assumed for the AK reaction and neglecting the backward reaction for NK. When the concentration of ATP fed was much lower than the Michaelis constant of NK for ATP, the ATP turnover took a constant value irrespective of the ATP feed concentration, and this value was proportional to the concentration of NK. A rate limiting step was proved to exist in the reaction of NK. The half-life of the enzyme system was over 10 days in a continuous operation.     

Immobilization of pig muscle 3-phosphoglycerate kinase

3-Phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 88 units g^?1 xerogel. The activity versus pH profile showed a sharper maximum at pH 6.5 in the case of the immobilized enzyme. The immobilized enzyme had a broad apparent optimum temperature range between 40 and 50°C. The apparent K_m values of the immobilized 3-phosphoglycerate kinase were lower for both 3-phosphoglycerate and ATP than those of the soluble enzyme. In the case of the immobilized enzyme stabilities were enhanced.     

Continuous monitoring of adenosine 5'-triphosphate in the microenvironment of immobilized enzymes by firefly luciferase

The study of enzymes sequestered in artificial or biological systems is generally conducted by indirect methodology with macroscopic measurements of reactants in the bulk medium. This paper describes a new approach with firefly luciferase to monitor ATP concentration directly in the microenvironment of enzymes producing or consuming ATP. Upon addition of ATP to immobilized firefly luciferase, the onset of light production is slower than that observed with the soluble enzyme, due to a slower diffusion of ATP to the immobilized enzyme. With immobilized pyruvate kinase, a relative accumulation of ATP inside the beads is demonstrated, as measured with coimmobilized firefly luciferase. The accumulation of product (ATP) is enhanced when the bead suspension is not stirred. This ATP in the beads is relatively inaccessible to soluble hexokinase added to the bulk medium. Similarly, a rapid ATP depletion in the microenvironment of immobilized hexokinase is demonstrated. This microscopic event is kinetically distinguishable from the slower macroscopic depletion of substrate in the bulk medium. The rate of depletion in the microenvironment depends on the local activity of the immobilized enzyme but not on the total amount of enzyme in suspension, as does the macroscopic phenomenon. The theoretical principles for the interaction of diffusion and catalysis in these systems are briefly summarized and discussed. These results are relevant to various molecular mechanisms proposed for membrane-bound enzyme action and regulation, derived from macroscopic kinetic measurements assuming a negligible diffusion control.     

Plant succinic semialdehyde dehydrogenase: dissection of nucleotide binding by surface plasmon resonance and fluorescence spectroscopy

Recent kinetic studies revealed distinct modes of inhibition of mitochondrial Arabidopsis thaliana succinic semialdehyde dehydrogenase (At-SSADH1) by AMP and ATP. Inhibition of SSADH by ATP may represent an important mechanism of feedback regulation of the GABA shunt by the respiratory chain. Here we used two approaches to investigate the interaction of ATP with At-SSADH1. Cofactor displacement studies based on the reduced fluorescence intensity of free NADH versus that of enzyme-bound NADH revealed that both AMP and ATP decreased NADH-At-SSADH1 complex formation. The competitive inhibitor AMP displaced all bound NADH, while ATP, a noncompetitive inhibitor, could not, even in great excess, release all NADH from its binding site. To assess the effect of ATP on NAD-At-SSADH, we employed surface plasmon resonance to monitor nucleotide binding to immobilized At-SSADH1. For this, we used a Strep-tag II modified derivative of At-SSADH1 (designated ST-At-SSADH1). The tagged enzyme was tightly and reversibly captured by StrepTactin, which was covalently immobilized on a CM5 chip. The binding constants for NAD(+) and ATP were determined from titration curves and were in good agreement with the constants obtained from enzyme kinetics. Surface plasmon resonance measurements confirmed that ATP binds to a site different from the binding site for NAD(+). GTP competed with ATP. However, only ATP increased the dissociation constant of NAD(+) from SSADH. This explains the reduced affinity of NAD(+)/NADH to At-SSADH1 in the presence of ATP, as revealed by enzymatic kinetics, and supports our model of feedback regulation of SSADH and the GABA shunt by ATP.     

固定化尿苷-胞苷激酶和聚磷酸激酶偶联催化制备5′-胞苷酸

尿苷-胞苷激酶作为生物体核苷酸代谢补偿途径中的重要催化剂,可以催化胞苷的磷酸化反应合成5′-胞苷酸(简称胞苷酸),但需要NTP作为磷酸供体。为了提高胞苷酸的生产效率,文中首先使用大肠杆菌分别异源表达来源于嗜热栖热菌Thermus thermophiles HB8的尿苷-胞苷激酶和来源于类球红细菌Rhodobacter sphaeroides的聚磷酸激酶,其中尿苷-胞苷激酶用于催化胞苷和ATP形成胞苷酸,聚磷酸激酶则用于ATP的循环再生。然后,使用D403金属螯合树脂吸附Ni²⁺形成固定化载体,再利用固定化载体特异性吸附重组酶形成固定化酶。最后,单因素优化实验确定固定化酶的催化反应条件,在30℃、pH 8.0的条件下,以60 mmol/L胞苷和0.5 mmol/L ATP为底物,可实现5批次的高效连续催化反应,胞苷酸平均摩尔得率达到91.2%。上述制备方法反应成本低,产物得率高,酶利用率高,在工业生产中具有较好的应用潜力。     

Two weeks of muscle immobilization impairs functional sympatholysis but increases exercise hyperemia and the vasodilatory responsiveness to infused ATP

During exercise, contracting muscles can override sympathetic vasoconstrictor activity (functional sympatholysis). ATP and adenosine have been proposed to play a role in skeletal muscle blood flow regulation. However, little is known about the role of muscle training status on functional sympatholysis and ATP- and adenosine-induced vasodilation. Eight male subjects (22 ± 2 yr, Vo(2max): 49 ± 2 ml O(2)·min(-1)·kg(-1)) were studied before and after 5 wk of one-legged knee-extensor training (3-4 times/wk) and 2 wk of immobilization of the other leg. Leg hemodynamics were measured at rest, during exercise (24 ± 4 watts), and during arterial ATP (0.94 ± 0.03 μmol/min) and adenosine (5.61 ± 0.03 μmol/min) infusion with and without coinfusion of tyramine (11.11 μmol/min). During exercise, leg blood flow (LBF) was lower in the trained leg (2.5 ± 0.1 l/min) compared with the control leg (2.6 ± 0.2 l/min; P < 0.05), and it was higher in the immobilized leg (2.9 ± 0.2 l/min; P < 0.05). Tyramine infusion lowers LBF similarly at rest, but, when tyramine was infused during exercise, LBF was blunted in the immobilized leg (2.5 ± 0.2 l/min; P < 0.05), whereas it was unchanged in the control and trained leg. Mean arterial pressure was lower during exercise with the trained leg compared with the immobilized leg (P < 0.05), and leg vascular conductance was similar. During ATP infusion, the LBF response was higher after immobilization (3.9 ± 0.3 and 4.5 ± 0.6 l/min in the control and immobilized leg, respectively; P < 0.05), whereas it did not change after training. When tyramine was coinfused with ATP, LBF was reduced in the immobilized leg (P < 0.05) but remained similar in the control and trained leg. Training increased skeletal muscle P2Y2 receptor content (P < 0.05), whereas it did not change with immobilization. These results suggest that muscle inactivity impairs functional sympatholysis and that the magnitude of hyperemia and blood pressure response to exercise is dependent on the training status of the muscle. Immobilization also increases the vasodilatory response to infused ATP.     

Interaction of ox heart mitochondrial F1-ATPase with immobilized ADP and ATP.

The reaction of mitochondrial F1-ATPase with immobilized substrate was studied by using columns of agarose-hexane-ATP. Mg2+ was required for binding of the enzyme to the column matrix. The column-bound enzyme could be eluted fully by ATP and other nucleoside triphosphates. Nucleoside di- and mono-phosphates were less effective. At a fixed concentration of nucleotide the effectiveness of elution was proportional to the charge on the eluting molecule. The ATP of the column matrix was hydrolysed by the bound F1-ATPase to release phosphate, probably by a uni-site reaction mechanism. Thus the F1-ATPase was bound to the immobilized ATP by a catalytic site. Treatment of the bound F1-ATPase with 4-chloro-7-nitrobenzofurazan prevented complete release of the enzyme by ATP. Only one-third of the bound enzyme was now eluted by the nucleotide. The inhibition of release could be due either to the inhibitor blocking co-operative interactions between sites or to its increasing the tightness of binding of immobilized ADP at the catalytic site.     

Continuous-flow bioluminescent dtermination of ATP in platelets using firefly luciferase immobilized on epoxy methacrylate

Firefly luciferase was immobilized on epoxy methacrylate beads and used for a continuous-flow assay of ATP extracted from platelets. The immobilized luciferase had a half-life of 3 days at 25°C; there was a 25% recovery of luciferase activity upon immobilization, and ca 50 reactors were made from 1 mg of commercial enzyme. The sensitivity of the assay was 0.3 pmol of ATP, and the response was linear between 1 and 500 pmol of ATP. The content of platelets obtained with the present method correlated well with those obtained using soluble luciferase.     

Immobilization of acetate kinase and phosphotransacetylase on derivatized glass beads

The enzymes acetate kinase (ATP: acetate phosphotransferase, EC 2.7.2.1) and phosphotransacetylase (acetyl coenzyme A: orthophosphate acetyltransferase, EC 2.3.1.8) were separately immobilized onto controlled pore glass CPG and silica beads (pore size 50 nm). Different coupling techniques were screened and immobilized enzymes were subjected to storage stability tests. The selected method, the CPG γ-aminopropyl glutaraldehyde succinate dihydrazide, was further optimized to improve the activity of the enzyme-loaded glass beads.