示差脉冲伏安法测定乳酸脱氢酶活性及纳米微粒生物毒性检测新方法研究

采用示差脉冲伏安法，在乳酸脱氢酶(LDH)酶促体系“丙酮酸盐 + NADH +H⁺ (?) 乳酸盐 + NAD⁺”中，通过检测NAD⁺还原峰电流的变化，测定了不同条件下(不同酶用量、缓冲液pH值以及温度)LDH的活性、酶促体系的米氏常数K_m^NADH以及最大反应速率v_max。并且在最佳实验条件下，通过检测LDH活性的改变，实验考察了3种纳米物质(ZnS，TiO₂(R)和TiO₂(A))对乳酸脱氢酶酶促体系的影响。     

Recycling of NAD+ using coimmobilized alcohol dehydrogenase andE. coli

The use of immobilized enzymes has opened the possibility of large scale utilization of NAD⁺-linked dehydrogenases, but the applications of this technique were limited by the necessity of providing the large amounts of NAD⁺ required by its stoichiometric consumption in the reaction. After immobilization of alcohol dehydrogenase and intactE. coli by glutaraldehyde in the presence of serum albumin, the respiratory chain was found to be capable of regenerating NAD⁺ from NADH. This NAD⁺ can be recycled at least 100 times, and thus the method is far more effective than any other, and, moreover, does not require NADH oxydase purification. The total NADH oxidase activity recovered was 10–30% of the initial activity. Although, NADH is unable to cross the cytoplasmic membrane, it was able to reach the active site of NADH dehydrogenase after immobilization. The best yield of NADH oxidase activity with immobilized bacteria was obtained without prior treatment of the bacteria to render them more permeable. The denaturation by heat of NADH oxidase in cells that are permeabilized was similar before and after immobilization. In contrast, the heat denaturation of soluble Β-galactosidase required either a higher temperature or a longer exposure after immobilization. The sensitivity of immobilized NADH oxidase to denaturation by methanol was decreased compared to permeabilized cells. As a result, it is clear that the system can function in the presence of methanol, which is necessary as a solvent for certain water insoluble substrates.     

尼古丁对乳酸脱氢酶活性的影响

用计时电流法成功地研究了尼古丁对乳酸脱氢酶活性的影响,测定了尼古丁存在与否对乳酸脱氢酶酶促反应的初速度V0、酶促反应最大反应初速度Vm及米氏常数Km。实验结果表明尼古丁对乳酸脱氢酶的活性有很大影响,但在高浓度的NADH存在下,尼古丁对乳酸脱氢酶活性的影响将得到大大降低。     

Control release of lactate dehydrogenase encapsulated in poly (vinyl alcohol) nanofibers via electrospinning

Sustained release of lactate dehydrogenase (LDH, EC 1.1.1.27) from electrospun poly (vinyl alcohol) (PVA) nanofibers was successfully achieved using the coaxial electrospinning technique. The presence of the encapsulated enzyme in the nanofibers was confirmed by infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Scanning electron microscopy (SEM) was used to evaluate the morphology and diameter of the nanofibers. The conversion of lactate to pyruvate by LDH coupling with the reduction of the cofactor nicotinamide adenine dinucleotide (NAD⁺) to dihydronicotinamide adenine dinucleotide (NADH) produces an increment in the ultraviolet absorption (UV) at 340 nm. This change in the UV absorbance was used to follow the release kinetic of LDH from the PVA nanofibers and also as a measure to evaluate the residual enzymatic catalytic function. Most of the encapsulated LDH enzyme was released in a sustained manner from the PVA nanofibers within a period of 1 month.     

Attempting to remove the substrate inhibition ofL-lactate dehydrogenase fromBacillus stearothermophilus by site-directed mutagenesis

L-lactate dehydrogenase (LDH) catalyzes the interconversion of an oxoacid (pyruvate) and hydroxy-acid (lactate) using the NADH/NAD⁺ pair as a redox cofactor. The enzyme has a commercial significance, as it can be used to produce chiral building blocks for the synthesis of key pharmaceuticals and agrochemicals. However, the substrate inhibition which is due to an abortive NAD⁺-pyruvate complex reducing the steady state concentration of functional LDH limits its use in industry. This substrate inhibition can be overcome by weaking the binding of NAD⁺. The conserved aspartic acid residue at position 38 was replaced by the longer basic arginine side chain (D38R) using PCR based overlap extension mutagenesis technique in the hope of weakening NAD⁺-binding. The mutant gene was overexpressed in theEscherichia coli high-expression vector pKK223-3 in JM105 cells; then, the mutant protein was produced. Comparing the effect of substrate inhibition in the arginine-38 mutant with wild-type, substrate inhibition is decreased threefold.     

Purifying and Characterizing Bacterially Expressed Soluble Lactate Dehydrogenase from Plasmodium knowlesi for the Development of Anti-Malarial Drugs

Plasmodium lactate dehydrogenase (pLH) is one of the enzymes in glycolysis with potential target for chemotherapy. This study aimed to clone, overexpress and characterize soluble recombinant lactate dehydrogenase from Plasmodium knowlesi in a bacterial system. Synthetic P. knowlesi lactate dehydrogenase (Pk-LDH) gene was cloned into pET21a expression vector, transformed into Escherichia coli strain BL21 (DE3) expression system and then incubated for 18 h, 20 °C with the presence of 0.5 mM isopropyl β-d-thiogalactoside in Terrific broth supplemented with Magnesium sulfate, followed by protein purifications using Immobilized Metal Ion Affinity Chromatography and size exclusion chromatography (SEC). Enzymatic assay was conducted to determine the activity of the enzyme. SDS-PAGE analysis revealed that protein of 34 kDa size was present in the soluble fraction. In SEC, a single peak corresponding to the size of Pk-LDH protein was observed, indicating that the protein has been successfully purified. From MALDI-TOF analysis findings, a peptide score of 282 was established, which is significant for lactate dehydrogenase from P. knowlesi revealed via MASCOT analysis. Secondary structure analysis of CD spectra indicated 79.4% α helix and 1.37% β strand structure. Specific activity of recombinant Pk-LDH was found to be 475.6 U/mg, confirming the presence of active protein. Soluble Pk-LDH that is biologically active was produced, which can be used further in other malaria studies.     

Determination of breath alcohol using a differential-type amperometric biosensor based on alcohol dehydrogenase

A differential-type amperometric biosensor based on conventional thick-film technology has been developed for breath alcohol measurement. The amperometric breath alcohol biosensor utilizes the alcohol dehydrogenase (ADH) and nicotinamide adenine dinucleotide (NAD⁺) cofactor which produce reduced NADH as a product of the oxidation of alcohol. The biosensor was designed in a differential format consisting of a common Ag/AgCl reference electrode, an active working electrode containing the ADH, and the inactive working electrode containing only bovine serum albumin instead of the ADH. The differential signal between the active working electrode and the inactive working electrode minimized the interference from a large number of oxidizable species present in a person's breath. Prior to the amperometric measurement the biosensor was hydrated simply by dipping it into a phosphate buffer solution at pH 7.4. The NADH produced from the enzymatic reaction was oxidized at the working electrode biased at a potential of 470 mV vs. an on-board Ag/AgCl reference electrode. The biosensor can measure a person's breath alcohol over the concentration range 20–800 ppm routinely required in a test of drunken driving.     

Liquid—liquid extraction of lactate dehydrogenase from muscle using polymer-bound triazine dyes

An extract from porcine muscle containing soluble enzymes has been partitioned between the two liquid phases of an aqueous, biphasic system consisting of dextran, polyethylene glycol, and water. The influence of polymer-bound triazine dyes (Cibacron blue F3G-A and Procion yellow HE-3G) on the partition of lactate dehydrogenase and total protein was studied. The effects of pH and concentrations of polymers and buffer on this so-called affinity partitioning were also determined. The two-phase systems were used in extraction procedures for purification of lactate dehydrogenase to a specific activity of 456–494 U (7.6–8.4 μkat) per mg protein. The use of these systems for extraction of enzymes in technical scale is discussed.     

Immobilization of the restriction endonuclease PstI

PstI has been immobilized in agarose. A solution of low melting agarose containing 1,6-hexamethylenediamine and PstI formed a gel that was effective in the linearization of pBR322 DNA. The gel containing PstI could be treated with 1,5-bis(N-acetylamino-N-succinimidoxy carbonyl)pentane, a crosslinking agent, without affecting the enzyme activity. Polymerization of acrylamide in presence of PstI led to conisderably reduced enzyme activity, although EcoRI under identical conditions showed high activity. It was found that acetylation of amino groups in PstI, by reaction with hydroxysuccinimide acetate, led to total inactivation of the enzyme activity. This reaction showed the presence of reactive amino groups that were essential for the enzyme activity of PstI. Involvement of these amino groups in binding to activated Sepharose 4B, during covalent immobilization, was responsible for inactive enzyme preparations.     

A combined theoretical and experimental study to improve the thermal stability of recombinant D-lactate dehydrogenase immobilized on a novel superparamagnetic Fe3O4NPs@metal–organic framework

Lactate dehydrogenase (LDH) is an enzyme that catalyzes the reduction of nicotinamide adenine dinucleotide (NADH) and pyruvate to nicotinamide adenine dinucleotide (NAD⁺) and D-lactate in the final step of anaerobic glycolysis. This enzyme belongs to the family of oxidoreductases. Humans possess two isoforms of LDH enzyme: NAD-dependent L-lactate dehydrogenase (L-LDH) and NAD-dependent D-lactate dehydrogenase (D-LDH). D-LDH is released during tissue damage, and is a sign of diseases such as kidney stones, heart failure, and some types of cancers and appendicitis. Accordingly, the design and construction of biosensors for the determination of lactate levels are important. The thermal sensitivity of D-LDH and low protein production in the host bacteria limit the use of this protein in certain applications. To solve these problems, two solutions were used in this study. First, the codon-optimized 1008 bp D-LDH gene fused with a histidine tag was cloned at the NcoI/XhoI sites and expressed in E. coli BL21. Second, a new metal–organic framework (Fe₃O₄NPs@Ni-MOF) was synthesized and used for immobilization and stabilization of D-LDH. Fe₃O₄NPs@Ni-MOF core-shell nanocomposites were characterized by Fourier transform infrared spectroscopy, vibrating sample magnetometer, scanning electron microscopy, X-ray diffraction, and the Brunauer–Emmett–Teller method. In comparison with the free enzyme, the immobilized enzyme presented better stability at high temperatures. The immobilization of the enzyme could be useful because most reactions happen at high temperatures in industry. To examine the effect of Fe₃O₄NPs@Ni-MOF on the adsorption and conformation of D-LDH at the atomistic level, a molecular dynamics simulation was carried out. Our study showed that the interaction between Fe₃O₄NPs@Ni-MOF and D-LDH involved van der Waals interactions, hydrophobic interaction energies, cation–π interaction between the Ni ions of the MOF with the enzyme residues and also, the hydrogen bond interactions between enzyme and heteroatoms in the MOF. Root mean square fluctuation and secondary structure analysis showed that Fe₃O₄NPs@Ni-MOF protected the conformation of the enzyme.     

Effect of methyltin trichloride on the activity of lactate dehydrogenase

The effect of MeSnCl₃, which is a highly toxic compound, on the activity of L-lactate:NAD oxidoreductase (lactate dehydrogenase) in the extract from the liver of Russian sturgeon (Asipenser gueldenstaedtiB.). Noncompetitive inhibition of the enzymatic reaction was discovered. This can be due to a change in the enzyme conformation caused by the action on the thiol groups, important for enzyme activity.     

Polyelectrolyte complex capsules as a material for enzyme immobilization

The polyelectrolyte complex (PEC) membrane formed by cellulose sulfate and poly(dimethyldiallylammonium chloride) was used to encapsulate lactate dehydrogenase. The exclusion limit of the membrane was found to be low enough to secure irreversible entrapping of the enzyme. The obtained capsules were checked for their functionality in a stirred-batch reactor by following the kinetics of NADH oxidation. The data were fitted with an isotropic kinetic model including competitive product-inhibition phenomenon. The results of mathematical modeling demonstrated that the anisotropic system, like PEC capsules, could be satisfactorily described by the isotropic model.     

Acrolein detection based on alcohol dehydrogenase inhibition

This paper presents the effect of acrolein on three dehydrogenases and proposes a fast spectrometric method for acrolein analysis. We have found that alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AlDH) are inhibited by low acrolein concentrations (0.2?mM) while inhibition of glutamate dehydrogenase (GDH) is not observed even at higher acrolein concentrations (1?mM). Acrolein is a suicide substrate for AlDH and ADH inhibition by acrolein is competitive. Cysteine (L-Cys) and glutathione (GSH) react with acrolein and thus reduce its expected inhibitory effect. ADH was chosen to develop a spectrophotometric method for acrolein analysis based on enzyme inhibition. The calibration curve is linear between 0.2 and 1.0?mM acrolein.     

基于可见吸收信号的乳酸脱氢酶光纤传感器

报道一种测定乳酸脱氢酶活力的基于可见吸收信号的光纤生物传感器，在该传感体系中，通过辅酶Ｉ的氧化还原对（ＮＡＤ＾＋／ＮＡＤＨ）将乳酸脱氢酶和心肌黄酶催化的两个脱氢过阳以耦合，第一个脱氢过程对分析对象进行了化学识别，第二个脱氢过程引起可见吸收信号的变化，该传感器对０～４００Ｕ／Ｌ的乳酸脱氢酶有线性响应关系，检测下限为４８ＵＬ，该传感器已用于人体血清中乳酸脱氢酶活力的测定。     

Amperometric determination of serum lactate dehydrogenase activity using an ADP-modified graphite electrode

Graphite electrodes modified with a drop-coated layer of polyethyleneimine (PEI) and adenosine diphosphate (ADP) displayed an electrocatalytic response to NADH after the adenine moiety of ADP was electrochemically oxidised. NADH can be detected amperometrically in alkaline solution (pH 9.0) at low applied potentials (+50 mV (Ag/AgCl)). Using a stationary electrode arrangement, linear response for NADH concentrations between 1.0×10⁻⁸ and 1.0×10⁻⁴ M was found, with a response time of 12 s and a detection limit of 8×10⁻⁹ M. The electrode was applied to the amperometric monitoring of the reaction between lactate and NAD⁺ catalysed by lactate dehydrogenase (LDH). A flow injection-amperometric method for the determination of LDH activity in human serum was developed. The method allows a fast and accurate discrimination between pathological and normal LDH activity levels, with a sampling rate of 40 h⁻¹. Quantitative results for a random set of human serum samples were found to be in good agreement with the standard spectrophotometric method.     

Extraction and separation of lactate dehydrogenase inhibitors from Poria cocos (Schw.) Wolf based on a hyphenated technique and in vitro methods

Stroke is one of the most common diseases worldwide. Lactate dehydrogenase inhibitors are widely used in the treatment of ischemic stroke, with natural products considered a promising source of lactate dehydrogenase inhibitors. In this study, ultrafiltration liquid chromatography coupled with mass spectrometry was used for the screening and identification of lactate dehydrogenase inhibitors from Poria cocos . Five lactate dehydrogenase inhibitors were selected: dehydropachymic acid, pachymic acid, dehydrotrametenolic acid, trametenolic acid, and eburicoic acid. The inhibitors were extracted and isolated with purities of 96.75, 98.15, 97.25, 95.46, and 94.88%, respectively, by using a new “hyphenated” strategy of microwave‐assisted extraction coupled with counter‐current chromatography and centrifugal partition chromatography by a two‐phase solvent system of n‐hexane/ethyl acetate/ethanol/water at the volume ratio 0.965:1.000:0.936:0.826 v/v/v/v. The bioactivity of the isolated compounds was assessed using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay in PC12 cells. The results also showed that the hyphenated technique of microwave‐assisted extraction coupled with counter‐current chromatography and centrifugal partition chromatography was an efficient method for the continuous extraction and online isolation of chemical constituents from medicinal herbs. Furthermore, the research route based on the activity screening, extraction, separation, and activity verification of the compounds offered advantages of efficiency, orientation, and objectivity.     

Capillary electrophoresis-integrated immobilized enzyme microreactor with graphene oxide as support: Immobilization of negatively charged L-lactate dehydrogenase via hydrophobic interactions

We report the first application of hydrophobic interaction between graphene oxide (GO) and negatively charged enzymes to fabricate CE-integrated immobilized enzyme microreactors (IMERs) by a simple and reliable immobilization procedure based on layer by layer assembly. L -lactate dehydrogenase (L -LDH), which is negatively charged during the enzymatic reaction, is selected as the model enzyme. Various spectroscopic techniques, including SEM, FTIR, and UV-vis are used to characterize the fabricated CE-IMERs, demonstrating the successful immobilization of enzymes on the negatively charged GO layer in the capillary surface. The IMER exhibits excellent repeatability with RSDs of inter-day and batch-to-batch less than 3.49 and 6.37%, respectively, and the activity of immobilized enzymes remains about 90% after five-day usage. The measured K_m values of pyruvate and NADH of the immobilized L -LDH are in good agreement with those obtained by free enzymes. The results demonstrate that the hydrophobic interactions and/or π-π stacking is significant between the GO backbone and the aromatic residues of L -LDH and favorable to fabrication of CE-integrated IMERs. Finally, the method is successfully applied to the determination of pyruvate in beer samples.     

Immobilisation of lactate dehydrogenase on poly(aniline)-poly(acrylate) and poly(aniline)-poly(vinyl sulphonate) films for use in a lactate biosensor

The immobilisation of enzymes on an electrode surface, in such a manner that they retain both substrate specificity and high levels of catalytic activity, is of great importance in bioelectrochemistry. This includes areas such as the development of enzyme-catalysed fuel cell electrodes, biosensors and other biotechnological applications. We have investigated the catalytic activity of hexahistidine tagged variants of lactate dehydrogenase (EC 1.1.1.27) from the thermophile Bacillus stearothermophilus both in solution and when immobilised on poly(aniline)-poly(acrylate) (PANi-PAA) or poly(aniline)-poly(vinyl sulphonate) (PANi-PVS) composite films. Both the C- and N-terminally tagged enzymes are readily immobilised on the modified electrode and catalyse the conversion of lactate and NAD⁺ to pyruvate and NADH. The NADH that is generated can be readily oxidised at the PANi-modified electrode surface.In solution, the activity of the C-tagged enzyme (LDH-CHis) was some 30% less that of the wild-type under comparable conditions, whereas the N-tagged enzyme was found to possess essentially the same activity as the wild-type. However, when the enzymes were immobilised on PANi-PAA and PANi-PVS the C-tagged enzyme films showed a higher NADH-dependent current than the wild-type LDH whilst the N-tagged enzyme had the highest of the three. In addition, the C-tagged enzyme film appeared more stable than the wild-type LDH-PANi film. A novel immobilisation chemistry of the enzyme is proposed to account for these observations.     

Coupled hydroperoxide lyase and alcohol dehydrogenase for selective synthesis of aldehyde or alcohol

The main objective of this work was to improve the selective synthesis of a volatile compound: aldehyde or alcohol using a coupled-enzyme system. A novel method of synthesis of C₆-aldehyde or alcohol was carried out in the presence of hydroperoxide lyase (HPLS) activity coupled to alcohol dehydrogenase (ADH) activity. After cleavage of the initial substrate, hydroperoxy fatty acid catalyzed by HPLS, the second enzyme, ADH, can catalyze the reduction of the aldehyde to the corresponding alcohol, or the oxidation of contaminating alcohol into aldehyde, depending on the cofactor present in the medium (oxidized or reduced form). We succeeded in improving the synthesis of one of the products. When coupling HPLS to NADP, the selectivity of hexanal production from 13-hydroperoxy linoleic acid was improved, and hexanol production was reduced 5 to 10 times after 15 min of reaction at 15 °C and pH 7.0. In another experiment, HPLS was coupled to ADH in the presence of NADH. The production of alcohol (hexenols) was then favored especially when using 13-hydroperoxy linolenic acid as substrate at concentrations >15 mM, reaching 95% of the products. Coupling of the enzymatic reactions (cleavage reduction) not only reduced the number of steps but also allowed us to increase the conversion rate of the initial substrate (hydroperoxy fatty acid). Structures of the compounds produced in this work were confirmed using gas chromatography-mass spectroscopy analysis. Each of these products has its own delicately different fresh odor that can be used in various applications.     

Determination of Glycerol and Serum Triglycerides with Collagen-Immobilized Glycerol Dehydrogenase and Amperometric Detection

Abstract

An amperometric procedure is described for the determination of glycerol and triglycerides in aqueous samples and in serum using glycerol dehydrogenase immobilized on a collagen membrane. Glycerol is determined by measurement of the steady-state oxidation currents generated at a platinum electrode by NADH produced in the enzyme-catalyzed reaction. The triglycerides were first hydrolyzed by the enzyme lipase in solution and the resulting glycerol determined similarly. Olive oil, determined to contain 78 % triolein, was used as the source of triglycerides in this study. For both glycerol and triglycerides the calibration plots are linear in the range from 0 to 12 μM, with detection limits of 0.2 and 0.7 μM, respectively. The immobilized glycerol dehydrogenase retained high operational activity for a period longer than 30 days.