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。     

A Rigidoporus Vinctus Alcohol Dehydrogenase and Its Characterization

Alcohol dehydrogenases (ADHs; E.C. 1.1.1.1) are widely distributed enzymes found in many microorganisms. ADHs are oxidoreductases that catalyze the NAD(P)⁺‐dependent conversion of alcohols to aldehydes or ketones as well as the reverse reaction. The ADH cloned from Rigidoporus vinctus (RvADH) was 1035 bp that encodes a protein of 344 amino acid residues with calculated molecular mass of 38.39 kDa. This ADH is belonging to the medium‐chain family (medium‐chain dehydrogenase/reductase (MDR) and has the highly conserved GXXGXXG sequence found in the MDR family which found as the coenzyme‐binding pocket. To characterize the ADH protein, the coding region was subcloned into an expression vector pET‐20b(+) and transformed into E. coli Rosetta (DE3). The recombinant His6‐tagged ADH was overexpressed and purified by Ni²⁺‐nitrilotriacetic acid Sepharose. The purified enzyme showed one band on 12 % sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The Michaelis constant (K_M) value of the recombinant enzyme for ethanol was 0.79 mM. In substrates specificity analysis showed that RvADH had great oxidative activity toward primary alcohols. However, the less activtiy toward secondary alcohols and alcohol derivatives were compared with ethanol. Regarding the reductase activity showed low or even no activity to aldehydes and ketone.     

Detection of NAD+-dependent alcohol dehydrogenase activities in YR-1 strain of Mucor circinelloides, a potential bioremediator of petroleum contaminated soils

Different soluble NAD⁺-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleumcontaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD⁺-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (≈7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD⁺-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol-dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.     

计时电流法研究4-(N-亚硝基甲氨基)-1-(3-吡啶基)-1-丁酮对谷氨酸脱氢酶活性的影响

用计时电流法研究了烟草特有亚硝胺之一4 (N 亚硝基甲氨基) 1 (3 吡啶基) 1 丁酮(NNK)对谷氨酸脱氢酶(GLDH)催化活性的影响,测定了NNK存在与否时GLDH酶促反应的最大反应速率及米氏常数或表观米氏常数。实验结果表明,NNK对GLDH的催化活性有明显抑制作用,而且属于可逆竞争性抑制,测得在25℃,pH 8.0时抑制常数Ki为176μmol·L-1。     

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.     

Novel aromatic carboxamides from dehydroabietylamine as potential fungicides: Design,synthesis and antifungal evaluation

The present study was carried out to design and synthesize a number of novel aromatic carboxamide derivatives of dehydroabietylamine. The preliminary antifungal assay indicated that most of title compounds displayed moderate to good antifungal activity toward the six fungal strains in vitro. Compounds 3i, 3q, 4b and 4d showed significant antifungal activity against Sclerotinia sclerotiorum, with EC₅₀ values ranging from 0.067 ～ 0.393 mg/L. Compounds 3i, 4b and 4d also showed pronounced mycelial growth inhibition activities against B. cinerea and A. solani. Furthermore, in the in vivo assay, compound 4b exhibited brilliant protective activity against S. sclerotiorum-infected rape leaves. Meanwhile, the in vivo bioassay on tomato plants infected by B. cinerea showed that compound 3i and 4d displayed excellent protective activity at 200 mg/L, which were near to boscalid. Primary mechanistic study revealed that 4b could inhibit sclerotia formation as well as reduce the exopolysaccharide level. SEM and TEM analysis indicated that 4b possessed a strong ability to destroy the surface morphology of mycelia, cell structure and seriously interfere with the growth of the fungal pathogen. In addition, 4b exhibited good inhibitory activity (IC₅₀ = 23.3 ± 1.6 μM) toward succinate dehydrogenase (SDH). Molecular modeling study confirmed the binding modes between compound 4b and SDH. The above antifungal results and fungicidal mechanism study revealed that this class of dehydroabietylamine derivatives could be potential SDH inhibitors and lead compounds for novel fungicides development.     

Screening and isolation of potential lactate dehydrogenase inhibitors from five Chinese medicinal herbs: Soybean,Radix pueraria,Flos pueraria,Rhizoma belamcandae,and Radix astragali

Stroke is among the leading causes of death and severe disability worldwide. Flavonoids have been extensively used in the treatment of ischemic stroke by reducing lactate dehydrogenase levels and thereby enhancing blood perfusion to the ischemic region. Here, we used ultrafiltration high‐performance liquid chromatography coupled with diode array detection and mass spectrometry for the rapid screening and identification of flavonoids from five Chinese medicinal herbs: soybean, Radix pueraria, Flos pueraria, Rhizoma belamcandae, and Radix astragali. Using PC12 cells as a suitable in vitro model of toxicity, cell viability was quantitated using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. The results showed that the extracts of soybean and the six major components, namely, acetyldaidzin, malonylgenistin, daidiain, glycitin, genistin, and acetylcitin; the extract of R. pueraria and its main component daidzein; the extract of F. pueraria and its three major components, tectorigenin, tectoridin, and tectorigenin‐7‐O‐xylosylglucosid; and the extract of R. belamcandae and its main component, tectoridin, were strong lactate dehydrogenase inhibitors. Also, the components of R. astragali showed no bioactivity. These findings indicate that the ultrafltration high‐performance liquid chromatography coupled with diode array detection and mass spectrometry method could be utilized in rapid screening and separation of bioactive compounds from a complex matrix.     

含离子液体［bmim］Cl的反应介质中马肝醇脱氢酶的催化特性

 研究了马肝醇脱氢酶(HLADH)在含离子液体1-丁基-3-甲基咪唑氯酸盐(［bmim］Cl)的反应介质中的催化特性. 以乙醇为底物时,该酶在［bmim］Cl含量≤0.15 g/ml的体系中的活力高于在不含离子液体的体系中的活力; 离子液体浓度过高(＞0.15 g/ml)对酶活性有明显的抑制作用. 反应温度和pH对含离子液体的反应介质中酶活力的影响规律与不含离子液体时的规律相似. 与不含离子液体的反应介质相比, HLADH在含0.05 g/ml ［bmim］Cl的体系中催化乙醇氧化的活化能下降,酶反应的Vmax和Km均升高. 反应体系中低浓度(≤0.1 g/ml)的离子液体能提高酶的热稳定性,但高浓度(＞0.1 g/ml)的离子液体可降低酶的热稳定性. 紫外二阶导数光谱显示,在含不同浓度离子液体的反应介质中酶分子构象的变化有较大的差异.     

An HPLC method for the determination of the free cortisol/cortisone ratio in human urine

The measurement of the urinary free cortisol-cortisone ratio has been reported to be a sensitive indicator of renal 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD 2) activity. This converts biologically active cortisol to inactive cortisone. A decrease in its activity (e.g. through disease or inhibition caused by a therapeutic agent or a foodstuff) may increase cortisol levels and susceptibility towards hypertension. The method presented here uses a simple isocratic tandem column HPLC system. The method has been validated and found to be robust and reproducible. The lower limit of quantification (LLOQ) was found to be 10 ng/mL for both cortisol and cortisone. Samples of urine (n = 99) from patients, most of whom were on complex combinations of drugs, were analyzed and 92% of samples were found to give successful results with this method (cortisol and cortisone above LLOQ). The ratio ranged from 0.07 to 5.61. No interferences were noted from the drugs that the patients were taking. It was also found that a morning spot urine sample gave comparable results to 24 h collection samples, thus making sample collection much easier.     

Oxidase/peroxidase bilayer-modified electrodes as sensors for lactate,pyruvate, cholesterol and uric acid

Enzyme heterobilayer-modified electrodes were fabricated by successively covalently binding to the surface of a tin(IV) oxide plate horseradish peroxidase (HRP), then an oxidase (lactate, pyruvate or cholesterol oxidase or uricase), which liberates hydrogen peroxide by reaction with the respective substrate. The cooperative action of oxidase-HRP leads to an efficient amperometric sensor system with the minimum amount of enzyme immobilized on an electrode.