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1.
乙基纤维素微胶囊对过氧化氢酶固定化的研究 总被引:5,自引:1,他引:4
以乙基纤维素作模材,用液中干燥法使过氧化氢酶微胶囊化,研究了微胶囊化操作条件对酶活性影响。通过测定微胶囊化酶的释放曲线,证明微胶囊膜对过氧化氢酶具有较好的固定性能力,固定化酶用于催化底物过氧化氢分解,测定米氏常数为0.55mol/L。 相似文献
2.
微胶囊固定化过氧化氢酶的制取及对H2O的分解作用 总被引:5,自引:0,他引:5
以乙基纤维素为膜材,用溶液干燥法制取了微胶囊固定化过氧化氢酶,探讨了制取过程中明胶的加入对微胶囊产率及固定化过化氢酶活性的影响,同时论述了存放时间、温度以及环境PH值对微胶囊固定化过氧化氢酶稳定性的影响,深入研究了微胶囊固定化过氧化氢酶对H2O的分解作用,获得了十分有意义的结果。 相似文献
3.
微胶囊固定化过氧化氢酶的制取及对H_2O_2的分解作用 总被引:1,自引:0,他引:1
以乙基纤维素为膜材,用溶液干燥法制取了微胶囊固定化过氧化氢酶,探讨了制取过程中明胶的加入对微胶囊产率及固定化过氧化氢酶活性的影响,同时论述了存放时间、温度以及环境pH值对微胶囊固定化过氧化氢酶稳定性的影响.深入研究了微胶囊固定化过氧化氢酶对H2O2的分解作用,获得了十分有意义的结果 相似文献
4.
抗坏血酸对花生原生质体分离过程中膜损伤的保护作用 总被引:7,自引:0,他引:7
酶解处理使花生叶肉细胞原生质体膜脂质过氧化产物丙二醛(MDA)积累,添加抗坏血酸(ASA)能降低MDA的积累。未添加ASA时,酶解初期,过氧化物酶(POD)、过氧化氢酶(CAT)活性上升,说明原生质体存在各种抵御不良变化的机制,但酶解时间加长,POD活性下降。添加ASA能使超氧化物歧化酶(SOD)活性明显上升。试验结果说明:在原生质体分离过程中会导致膜损伤,细胞自身存在一个响应的防御机制,添加ASA能降低酶解过程对原生质膜的损伤。 相似文献
5.
嗜碱芽孢杆菌Bacillus sp.F26过氧化氢酶的分离纯化及性质研究 总被引:3,自引:0,他引:3
从一株低度嗜盐、兼性嗜碱芽孢杆菌Bacillus sp.F26中纯化得到一种碱性过氧化氢酶,并对该酶进行了性质研究。纯化过程经硫酸铵沉淀、阴离子交换层析、凝胶过滤层析及疏水层析四步最终获得电泳纯的目标酶(纯化58.5倍)。该过氧化氢酶的分子量为140kD,由两个大小相同的亚基组成。天然酶分子在408nm处显示特征吸收峰(Soret band)。吡啶血色素光谱显示了酶分子以原卟啉Ⅸ(protoheme Ⅸ)作为辅基。计算获得酶的表观米氏常数为32.5mmol/L。该过氧化氢酶不受连二亚硫酸钠的还原作用影响,但被氰化物、叠氮化物和3.氨基.1,2,4-三唑(单功能过氧化氢酶的专一抑制剂)强烈抑制。以邻联茴香胺、邻苯二胺和二氨基联苯胺作为电子供体测定酶活时,该酶不显示过氧化物酶活性。同时,酶的N-端序列比对结果说明,该过氧化氢酶与单功能过氧化氢酶亚群有一定的相似性,而与双功能过氧化氢酶亚群及猛过氧化氢酶亚群均没有同源性。因此,本文将纯化的碱性过氧化氢酶定性为单功能过氧化氢酶。此外,该酶具有热敏感的特点,且酶活在pH5~9的范围内不受pH影响,此后,活性随着pH的升高而升高,并在pH 11处有明显的酶活高峰。20℃、pH 11条件下的酶活半衰期达49h。在pH 11的高碱条件下表现出最高活力和一定的稳定性,这在已报道的过氧化氢酶中还未见描述。同时,该酶也显示了良好的盐碱稳定性,0.5mol/L NaCl、pH 10.5条件下的酶活半衰期达90h。另一方面,本文所研究的过氧化氢酶是第一个来源于嗜碱微生物的同源二聚体单功能过氧化氢酶,也是第一个来源于天然碱湖的单功能过氧化氢酶,它能部分地反映出细胞抗氧化体系对相应环境的适应情况。 相似文献
6.
7.
抗坏血酸对沙打旺原生质体分离和培养中膜损伤及相关酶活性的影响 总被引:5,自引:0,他引:5
抗坏血酸(ASA) 能减轻沙打旺原生质体的褐化,改善原生质体的培养状况。ASA的作用可能与它增强原生质体抗过氧化能力有关。酶解处理诱导原生质体超氧化物歧化酶(SOD) 和抗坏血酸过氧化物酶(APX)活性升高,但培养过程使APX 活性明显下降,原生质体清除过氧化物能力减弱,膜脂过氧化产物丙二醛( MDA) 积累增加,膜发生损伤。向酶溶液和培养基中添加ASA 可显著提高SOD 尤其是APX 活性,减轻膜脂过氧化,增强原生质体的存活力,促进原生质体的分裂和细胞克隆的形成。所有处理中过氧化氢酶(CAT) 活性变化不大,表明它在原生质体清除过氧化物过程中不具主要作用。 相似文献
8.
低温胁迫对巨尾桉幼苗膜脂过氧化及保护酶的影响 总被引:17,自引:0,他引:17
以木本植物巨尾桉幼苗为材料 ,研究低温胁迫对巨尾桉膜脂过氧化及保护酶的影响 ,测定了幼苗叶片的O 2(超氧阴离子 )产生速率、H2 O2 、(过氧化氢 )、MDA(丙二醛 )含量、相对电导率和SOD(超氧化物歧化酶 )、POD(过氧化物酶 )、CAT(过氧化氢酶 )、APX(抗坏血酸过氧化物酶 )活性。结果表明 :低温胁迫使叶片O 2 产生速率、H2 O2 、MDA含量和相对电导率增加 ,但抗寒锻炼植株的增幅远小于对照 ;抗寒锻炼植株的SOD、POD、CAT和APX活性均低于对照。 相似文献
9.
谷氨酸氧化酶电极的研究 总被引:2,自引:1,他引:1
用戊二醛作交联剂将谷氨酸氧化酶和牛血清白蛋白共交联,置于内层醋酸纤维素抗干扰膜和外层聚碳酸酯扩散控制膜之间,制成酶膜,将其与过氧化氢探头复合制成了扩散控制型谷氨酸氧化酶电极,并构建了采用该电极的流动注射分析芽纺。酶电极红性范围0—1000mg/L。50s响应电流达稳态值的95%。流动注射分析系统响应时间20s,检测速度60次/h,线性范围5—8 000mg,L,酶膜使用寿命两周以上。系统选择性好,仅对干扰物L-谷氨酰胺和L-天冬氨酸有微弱响应。对同一样品连续测定41次的变异系数2.8%。测量味精发酵液和瓦勃呼吸计的结果相吻合。可望应用于味精发酵及食品工业中。 相似文献
10.
幽门螺杆菌过氧化氢酶基因的克隆、序列测定及其生物信息学分析 总被引:2,自引:0,他引:2
幽门螺杆菌(Helicobacter pylorl,Hp)感染是慢性活动性胃炎和消化性溃疡的主要病因,与胃腺癌、胃粘膜相关淋巴样组织(MALT)淋巴瘤的发生亦密切相关。其有效抗原成份过氧化氢酶可刺激机体产生保护性的免疫反应。用高保真PCR扩增系统扩增出过氧化氢酶基因片段,将其定向插入载体pET-22b( ),对其全序列进行了测定。并以生物信息学软件分析其生物学特性。克隆的过氧化氢酶基因序列与报道的序列完全一致,并显示出良好的抗原性和疏水性。这一研究获得了序列正确的过氧化氢酶基因,为将来以过氧化氢酶分子作抗原的Hp疫苗研制工作打下了良好基础。 相似文献
11.
Direct electrochemistry and electrocatalytic activity of catalase immobilized onto electrodeposited nano-scale islands of nickel oxide 总被引:1,自引:0,他引:1
Cyclic voltammetry was used for simultaneous formation and immobilization of nickel oxide nano-scale islands and catalase on glassy carbon electrode. Electrodeposited nickel oxide may be a promising material for enzyme immobilization owing to its high biocompatibility and large surface. The catalase films assembled on nickel oxide exhibited a pair of well defined, stable and nearly reversible CV peaks at about -0.05 V vs. SCE at pH 7, characteristic of the heme Fe (III)/Fe (II) redox couple. The formal potential of catalase in nickel oxide film were linearly varied in the range 1-12 with slope of 58.426 mV/pH, indicating that the electron transfer is accompanied by single proton transportation. The electron transfer between catalase and electrode surface, (k(s)) of 3.7(+/-0.1) s(-1) was greatly facilitated in the microenvironment of nickel oxide film. The electrocatalytic reduction of hydrogen peroxide at glassy carbon electrode modified with nickel oxide nano-scale islands and catalase enzyme has been studied. The embedded catalase in NiO nanoparticles showed excellent electrocatalytic activity toward hydrogen peroxide reduction. Also the modified rotating disk electrode shows good analytical performance for amperometric determination of hydrogen peroxide. The resultant catalase/nickel oxide modified glassy carbon electrodes exhibited fast amperometric response (within 2 s) to hydrogen peroxide reduction (with a linear range from 1 microM to 1 mM), excellent stability, long term life and good reproducibility. The apparent Michaelis-Menten constant is calculated to be 0.96(+/-0.05)mM, which shows a large catalytic activity of catalase in the nickel oxide film toward hydrogen peroxide. The excellent electrochemical reversibility of redox couple, high stability, technical simplicity, lake of need for mediators and short preparations times are advantages of this electrode. Finally the activity of biosensor for nitrite reduction was also investigated. 相似文献
12.
The predominant physiological activity of myeloperoxidase is to convert hydrogen peroxide and chloride to hypochlorous acid. However, this neutrophil enzyme also degrades hydrogen peroxide to oxygen and water. We have undertaken a kinetic analysis of this reaction to clarify its mechanism. When myeloperoxidase was added to hydrogen peroxide in the absence of reducing substrates, there was an initial burst phase of hydrogen peroxide consumption followed by a slow steady state loss. The kinetics of hydrogen peroxide loss were precisely mirrored by the kinetics of oxygen production. Two mols of hydrogen peroxide gave rise to 1 mol of oxygen. With 100 microM hydrogen peroxide and 6 mM chloride, half of the hydrogen peroxide was converted to hypochlorous acid and the remainder to oxygen. Superoxide and tyrosine enhanced the steady-state loss of hydrogen peroxide in the absence of chloride. We propose that hydrogen peroxide reacts with the ferric enzyme to form compound I, which in turn reacts with another molecule of hydrogen peroxide to regenerate the native enzyme and liberate oxygen. The rate constant for the two-electron reduction of compound I by hydrogen peroxide was determined to be 2 x 10(6) M(-1) s(-1). The burst phase occurs because hydrogen peroxide and endogenous donors are able to slowly reduce compound I to compound II, which accumulates and retards the loss of hydrogen peroxide. Superoxide and tyrosine drive the catalase activity because they reduce compound II back to the native enzyme. The two-electron oxidation of hydrogen peroxide by compound I should be considered when interpreting mechanistic studies of myeloperoxidase and may influence the physiological activity of the enzyme. 相似文献
13.
M.H. Gil J.P. Sardinha M.T. Vieira M. Vivan D. Costa C. Rodrigues F.-M. Matysik A.M. Oliveira Brett 《Biotechnology Techniques》1999,13(9):595-599
An electrochemical biosensor is described consisting of a thin-layer gold film electrode prepared by cathodic sputtering using a poly(vinyl chloride) sheet as substrate, with voltammetric behaviour comparable to that of conventional polycrystalline gold electrodes, coated with the hydrolysed copolymer hydroxyethyl methacrylate-co-methyl methacrylate onto which glucose oxidase was immobilized. The mechanical properties of the plastic foil substrate permit easy construction of circular-shaped electrodes which were employed as working electrodes for batch injection analysis. The electrochemical biosensor fabrication is inexpensive and can be used as disposable enzyme sensor for the detection of hydrogen peroxide. The biosensor was tested for the determination of glucose in serum and a good correlation was obtained with the measurement using the electrochemical and the spectrophotometric methods. 相似文献
14.
Erol Akyilmaz Gizem Oyman Ezgi Cınar Gorkem Odabas 《Preparative biochemistry & biotechnology》2017,47(1):86-93
A new biosensor based on catalase enzyme immobilized on electrochemically constructed polyaniline (PANI) film modified with glutaraldehyde has been developed for the determination of hydrogen peroxide (H2O2) in milk samples. Assembly processes of polyaniline and immobilization of the enzyme were monitored with the help of electrochemical impedance spectroscopy. Amperometric measurements have been performed at cathodic peak (?0.3?V vs. Ag/AgCI) which was attributed to reduction of PANI. Hydrogen peroxide was determined by using amperometric method at ?0.3?V. The biosensor responses were correlated linearly with the hydrogen peroxide concentrations between 5.0?×?10?6 and 1.0?×?10?4?M by amperometric method. Detection limit of the biosensor is 2.18?×?10?6?M for H2O2. In the optimization studies of the biosensor, some parameters such as optimum pH, temperature, concentration of aniline, amount of enzyme, and number of scans during electropolymerization were investigated. 相似文献
15.
1. The presence of xanthine was required for the inhibition of bovine milk xanthine oxidase by o-iodosobenzoate, iodoacetamide, hydrogen peroxide or p-chloromercuribenzoate. 2. Inactivation by p-chloromercuribenzoate was very rapid, was reversed by cysteine and was less in the presence of FAD. Lineweaver-Burk plots showed that the inactivation by p-chloromercuribenzoate was competitive with substrate. 3. Inactivation by o-iodosobenzoate, iodoacetamide or hydrogen peroxide could not be reversed by cysteine or xanthine. However, the presence of xanthine during the incubation with inhibitor protected the enzyme against o-iodosobenzoate but not against iodoacetamide or hydrogen peroxide. 4. p-Chloromercuribenzoate protected the enzyme against inactivation by hydrogen peroxide. 相似文献
16.
Gulay Bayramoglu M. Yakup Arica Aysenur Genc V. Cengiz Ozalp Ahmet Ince Niyazi Bicak 《Bioprocess and biosystems engineering》2016,39(6):871-881
A novel method was developed for facile immobilization of enzymes on silica surfaces. Herein, we describe a single-step strategy for generating of reactive double bonds capable of Michael addition on the surfaces of silica particles. This method was based on reactive thin film generation on the surfaces by heating of impregnated self-curable polymer, alpha-morpholine substituted poly(vinyl methyl ketone) p(VMK). The generated double bonds were demonstrated to be an efficient way for rapid incorporation of enzymes via Michael addition. Catalase was used as model enzyme in order to test the effect of immobilization methodology by the reactive film surface through Michael addition reaction. Finally, a plug flow type immobilized enzyme reactor was employed to estimate decomposition rate of hydrogen peroxide. The highly stable enzyme reactor could operate continuously for 120 h at 30 °C with only a loss of about 36 % of its initial activity. 相似文献
17.
Characterization of reactions catalyzed by manganese peroxidase from Phanerochaete chrysosporium 总被引:2,自引:0,他引:2
Manganese peroxidase (MnP) is one of two extracellular peroxidases believed to be involved in lignin biodegradation by the white-rot basidiomycete Phanerochaete chrysosporium. The enzyme oxidizes Mn(II) to Mn(III), which accumulates in the presence of Mn(III) stabilizing ligands. The Mn(III) complex in turn can oxidize a variety of organic substrates. The stoichiometry of Mn(III) complex formed per hydrogen peroxide consumed approaches 2:1 as enzyme concentration increases at a fixed concentration of peroxide or as peroxide concentration decreases at a fixed enzyme concentration. Reduced stoichiometry below 2:1 is shown to be due to Mn(III) complex decomposition by hydrogen peroxide. Reaction of Mn(III) with peroxide is catalyzed by Cu(II), which explains an apparent inhibition of MnP by Cu(II). The net decomposition of hydrogen peroxide to form molecular oxygen also appears to be the only observable reaction in buffers that do not serve as Mn(III) stabilizing ligands. The nonproductive decomposition of both Mn(III) and peroxide is an important finding with implications for proposed in vitro uses of the enzyme and for its role in lignin degradation. Steady-state kinetics of Mn(III) tartrate and Mn(III) malate formation by the enzyme are also described in this paper, with results largely corroborating earlier findings by others. Based on a comparison of pH effects on the kinetics of enzymatic Mn(III) tartrate and Mn(III) malate formation, it appears that pH effects are not due to ionizations of the Mn(III) complexing ligand. 相似文献
18.
Alcohol oxidase, an enzyme which exhibits relatively weak substrate specificity among short chain alcohols, forms the corresponding aldehyde and hydrogen peroxide as coproduct. The ability of alcohol oxidase from Pichia pastoris yeast to convert ethanol to acetaldehyde and hydrogen peroxide was examined in an oxygen pressure reactor under conditions, such that oxygen availability was sufficient to permit rapid catalysis. Hydrogen peroxide levels of approximately 1.8/M (6% w/w) were attained in 2-3 h with 2.8 muM enzyme, corresponding to a productivity of approximately 30 g peroxide/g enzyme. Optimal conditions (within equipment limitations) were 900 psi oxygen, 2.6M ethanol, at 4 degrees C. Similar levels of products were reached in the reactor using enzyme immobilized covalently on controlled pore glass and noncovalently on an anion exchange support. Recycle of covalently immobilized enzyme was not possible as a result of enzyme inactivation after a single run. Limited recycle of noncovalently immobilized enzyme was accomplished with substantial decreases in levels of product attainable on each cycle. 相似文献
19.
Detection of the common electrochemical interferents, ascorbic acid and hydrogen peroxide, using a SIRE (Sensors based on Injection of the Recognition Element) technology based biosensor in reverse mode operation is reported. The differential measuring principle employed in the SIRE biosensor during operation in reverse mode is such that the sample is measured first in the presence of enzyme (yielding matrix signal only), and then measured again in the absence of enzyme (yielding signal from matrix+analyte). Subtraction of the signal obtained in the presence of enzyme from the signal obtained in the absence of enzyme gives a specific signal for the analyte only and correlates directly to its concentration in solution. The linear range for the determination of ascorbic acid and hydrogen peroxide was 0-3 mM and 0-2 mM, respectively, with an enzyme concentration of 25 U ascorbate oxidase/ml and 1000 U catalase/ml. The reproducibility was 5% for ascorbic acid (R.S.D. n=15) and 10% for hydrogen peroxide (R.S.D. n=18). The cost per measurement was 0.28 USD for ascorbic acid analysis and 0.0008 USD for hydrogen peroxide analysis. The degradation of ascorbic acid in cereal was followed in real-time, as was the stabilization of low pH on the degradation process. 相似文献
20.
M Santimone 《Biochimie》1975,57(1):91-96
The oxidation of ferrocytochrome c catalysed by highly purified horse-radish isoperoxidase P2 was studied kinetically. To take into account the low turnover number of the enzyme and the tendency to autocatalytic oxidation of ferrocytochrome c, experimental conditions were used which prevented us from using the steady-state treatment. According to kinetic results reported by several authors, a kinetic scheme involving a ternary complex between the enzyme and the substrates was postulated and simulated on a hybrid computer. By assuming that the interaction of peroxidase with hydrogen peroxide is much faster than the interaction with ferrocytochrome c, one can verify that this scheme explains the fact that initial velocity does not vary in relation to the hydrogen peroxide concentration and that a sudden change of slope occurs in the kinetic curve for an initial hydrogen peroxide/ferrocytochrome c ratio lower than 0.5. 相似文献