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11.
A flow system for substrate recycling of NAD+/NADH was set up with an enzyme reactor containing coimmobilized glycerol dehydrogenase (GDH) and diaphorase. The product from the diaphorase catalysis, hexacyanoferrate(II), aws detected amperometrically at a glassy carbon electrode. The amplification factor was 150 for a reactor volume of 100 μ l at a flow-rate of 0.5 ml/min. With a stopped flow of four minutes, the signal increased another 88 times, resulting in a signal amplification of 13 300 times. Equations are derived for the amplification factor and used for a discussion of the optimization of amplification systems. The Km for GDH with glycerol as a substrate was found to be 5 × 10−3 M at pH 8.0. GDH from Cellulomonas sp. was purified on a gel filtration column and the purified enzyme showed a specificity toward NAD+, compared to NADP+, that was higher than 99.9%. Due to the NAD+ specificity of the purified GDH, the enzyme amplification system reported here could be used in detection systems for enzyme immunoassays when using alkaline phosphatase as a label and NADP+ as a substrate. The stability of immobilized GDH and diaphorase is several orders of magnitude better than that of alcohol dehydrogenase, which is the enzyme commonly used for NAD+-specific detection in these applications. 相似文献
12.
《Electroanalysis》2004,16(12):1051-1058
The voltammetric behavior of α‐ketoglutarate (α‐KG) at the hanging mercury drop electrode (HMDE) has been investigated in acetate buffer solution. Under the optimum experimental conditions (pH 4.5, 0.2 M NaAc‐HAc buffer solution), a sensitive reductive wave of α‐KG was obtained by linear scan voltammetry (LSV) and the peak potential was ?1.18 V (vs. SCE), which was an irreversible adsorption wave. The kinetic parameters of the electrode process were α=0.3 and ks=0.72 1/s. There was a linear relationship between peak current ip, α‐KG and α‐KG concentration in the range of 2×10?6–8×10?4 M α‐KG. The detection limit was 8×10?7 M and the relative standard deviation was 2.0% (Cα‐KG=8×10?4 M, n=10). Applications of the reductive wave of α‐KG for practical analysis were addressed as follows: (1) It can be used for the quantitative analysis of α‐KG in biological samples and the results agree well with those obtained from the established ultraviolet spectrophotometric method. (2) Utilizing the complexing effect between α‐KG and aluminum, a linear relationship holds between the decrease of peak current of α‐KG Δip and the added Al concentration Cequation/tex2gif-inf-5.gif in the range of 5.0×10?6–2.5×10?4 M. The detection limit was 2.2×10?6 M and the relative standard deviation was 3.1% (Cequation/tex2gif-inf-6.gif=4×10?5 M, n=10). It was successfully applied to the detection of aluminum in water and synthetic biological samples with satisfactory results, which were consistent with those of ICP‐AES. (3) It was also applied to study the effect of AlIII on the glutamate dehydrogenase (GDH) activity in the catalytically reaction of α‐KG+NH +NADH?L ‐glutamate+NAD++H2O by differential pulse polarography (DPP) technique. By monitoring DPP reductive currents of NAD+ and α‐KG, an elementary important result was found that Al could greatly affect the activity of GDH. This study could be attributed to intrinsic understanding of the aluminum's toxicity in enzyme reaction processes. 相似文献
13.
Elizabeth Nunes Fernandes Mariele Nair de Campos Moura Jos Luis F. Costa Lima Boaventura F. Reis 《Microchemical Journal》2004,77(2):107-112
An automatic flow procedure for the determination of glycerol in wines by employing a flow system based on multicommutation and enzymatic reaction is described. Glycerol dehydrogenase was immobilized on aminopropyl glass beads and packed into a column that was coupled to the flow system. The NADH produced by the enzymatic reaction was monitored by spectrophotometry at 340 nm and its radiation absorption presented a relationship with glycerol concentration. The system manifold comprised a set of three-way solenoid valves controlled by a microcomputer, which was furnished with electronic interfaces and runs a software that was designed to carry out on-line sample dilution, reagent addition, and data acquisition. The procedure allows the determination of glycerol in wine samples without any prior pretreatment. The procedure presented as profitable features a linear response range between 2.0 and 10.0 g l−1 glycerol (R=0.998), a detection limit of 0.006 g l−1 glycerol, a relative standard deviation of 1.8% (n=14) for a typical wine sample presenting 5.3 g l−1 glycerol, a sampling throughput of 33 determinations per hour, and a NAD+ consumption of 0.8 mg per determination. The results were compared with those obtained using a reference method and no significant difference at 90% confidence level was observed. 相似文献
14.
基于可见吸收信号的乳酸脱氢酶光纤传感器 总被引:1,自引:0,他引:1
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。 相似文献
15.
介体型乳酸脱氢酶生物传感器的研制及应用 总被引:3,自引:0,他引:3
本报道了能对丙酮酸产生良好响应的电流型乳酸脱氢酶生物传感器。该传感器以耐尔兰A修饰浸石墨电极为基体电极,将酶直接固化在蚕丝蛋白膜上。在PH7.4的NaH2PO4-NaOH介质中,当2.4×10^-4mol/L的辅酶I(NADH)存在下,该传感器的响应电流与丙酮酸浓度在3.2×10^-5mol/L范围内有良好的线性关系。响应时间为60s.本讨论了影响传感器响应的各种因素,用此传感器测定了人血清中 相似文献
16.
Nils Siegbahn Mats-Olle Maånsson Klaus Mosbach 《Applied biochemistry and biotechnology》1987,12(2):91-105
A soluble, bifunctional enzyme complex has been prepared by crosslinking lactate dehydrogenase and alcohol dehydrogenase with
glutaraldehyde. The crosslinking was performed on a solid phase while the active sites of alcohol dehydrogenase and lactate
dehydrogenase were held adjacent to one another with the aid of a bis-NAD analog. Subsequently, the enzyme complex was released
from the solid phase. The soluble enzyme complex was then purified by using NAD-Sepharose as an affinity adsorbent. Based
on gel filtration experiments, the complex was estimated to consist of one of each dehydrogenase.
By using a third enzyme, lipoamide dehydrogenase, which competes with lactate dehydrogenase for NADH produced by alcohol dehydrogenase,
the effect of site-to-site orientation was studied. It was found that about 83% of the NADH produced by alcohol dehydrogenase
was oxidized by site-to-site oriented lactate dehydrogenase compared to a figure of only about 61% obtained in an identical
system of separate enzymes. This indicates that given two alternative routes, the preference for the one to lactate dehydrogenase
over the one to lipoamide dehydrogenase is enhanced when lactate dehydrogenase and alcohol dehydrogenase are site-to-site
oriented. 相似文献
17.
Fumihiko Hasumi Yasumitsu Miyamoto Ichiro Okura 《Applied biochemistry and biotechnology》1995,55(1):1-4
Glutamate synthesis by reductive amination of 2-oxoglutarate was performed by the combination of NADH regeneration system
and glutamate dehydrogenase (GluDH). The conversion of 2-oxoglutamate to glutamate was 98% after 3 h, and the turnover number
of NAD+was 17. 相似文献
18.
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20.
介体型乙醇生物传感器的研制及应用 总被引:1,自引:0,他引:1
本文报道了电流型乙醇生物传感器的研制与应用。该传感器以健合型耐尔蓝修饰浸蜡石墨电极为基体电极,将乙醇脱氢酶及NAD^+固定在人造丝网上,成为一种无试剂的乙醇生物传感器。在pH8.8的Tris/HCl介质中,该传感器的响应电流与乙醇浓度在0.10-1.0mmol/l范围内有良好的线性关系。 相似文献