流动注射分析法测定固定化酶的催化活力

本文介绍了脲酶的固定方法以及用流动注射分析法测定固定化酶的催化活力，测得脲的平均转化率为９５．０％。     

固定化脲酶的制备及应用

以壳聚糖作为载体,戊二醛作为交联剂对脲酶进行固定化。固定化的最适条件为:酶的偶联时间60min,戊二醛浓度0.5%,pH值7.0。对游离及固定化脲酶的酶学性质研究表明,酶促反应的最适pH均为7.0,最适温度分别为33℃和70℃。米氏常数分别为29.8mmol/L和13.9mmol/L。与游离酶相比,固定化酶的热稳定性和贮存稳定性更佳。应用固定化酶测定了试样中的微量组分。     

酶法测定水中的尿素含量

利用脲酶、谷氨酸脱氢酶偶联催化尿素水解的原理,通过测定还原型烟酰胺腺嘌呤二核苷酸吸光度变化率得出其酶促反应速度,对应不同的尿素浓度制得标准曲线,讨论了ｐＨ值和抑制剂对测定的影响,实测了水样中尿素的含量。     

复合纤维素固定化脲酶对铜离子的吸附作用

脲酶对重金属离子极为敏感。由复合纤维素（CC）固定化脲酸对铜离子的吸附现象得出的结论是：脲酶吸附的Cu~(2+)总量与其活性没有定量关系，而是与固酶重量成比例。从而可以认为，重金属离子不是直接作用于脲酶活性部位使其中毒的，很可能是作用于脲酶大分子其他部位，使分子构象变形后导致脲酶失活的。由于CC固酶对Cu~(2+)有吸附作用，且又不污染系统，所以可用其除去食品、药物及水中的Cu~(2+)离子。     

磁性壳聚糖微球用于酶的固定化研究Ⅱ磁性壳聚糖微球对脲酶的吸附及其酶学性质的研究

本文用磁性壳聚糖作为载体用吸附法对脲酶进行固定化研究。结果表明,磁性壳聚糖对脲酶的固载量与磁性壳聚糖微球的粒径、交联度及酶溶液的离子强度成反比;固定化脲酶和自由酶的最适温度分别为80℃和70℃,固定化脲的最适合pH值变化不大,固定化脲酶和自由酶的米氏常数km分别为0.00546mol/L和0.19mol/L。     

以伴刀豆球蛋白为固定基质的脲酶生物传感器

本文制备了一种以溶剂聚合膜pH电极作原电极,利用伴刀豆球蛋白(Con A)与糖蛋白间的特异性识别作用,将Con A和脲酶表面的麦芽糖残基结合,采用交替沉积Con A和脲酶,进行多层脲酶膜组装的脲酶生物传感器。研究了酶固定化条件的影响,优化了实验条件,测试了传感器对尿素的生物电化学响应。在6.9×10-5~1.0×10-3mol.L-1的浓度范围内传感器响应的电极电位与尿素浓度的对数成正比,检出限为4.5×10-5mol.L-1。将传感器用于牛奶样品中回收率的测定,结果满意。     

磁性壳聚糖微球用于酶的固定化研究*Ⅲ磁性壳聚糖微球的活化及其对脲酶的固定化研究

以环氧氯丙烷活化的磁性壳聚糖微球作为载体,对脲酶进行固定化研究。结果表明, 在25℃时, 活化磁性壳聚糖微球对脲酶的固定化在2h时就达到了最大值,固定化酶和自由酶的最适温度都在65℃左右,自由酶和固定化酶的米氏常数Km值分别为0.042mol/L和0.008mol/L,固定化酶的Km降低了5倍。     

磁性壳聚糖微球用于酶的固定化研究：Ⅲ磁性壳聚糖微球的活化及其对脲酶的固化研究

以环氧氯丙烷活化的磁性壳聚糖微球作为载体、对脲酶进行固定化研究,结果表明,在25℃时,活化磁性壳聚糖微球对脲酶的固定化在2h时就达到了最大值,固定化酶和自由酶的最适温度都在65℃左右,自由酶和固定化酶的米氏常数Km值分别为0．042mol/L和0．008mol/L,固定化酶的Km降低了5倍。     

外源镧对茶园紫色土酶活性的影响

通过盆栽实验研究了外源稀土元素La对茶园紫色土脲酶、蔗糖酶、纤维素酶及脱氢酶活性的影响。结果表明:低浓度(≤300 mg·kg-1)La3+对茶园紫色土脲酶活性、蔗糖酶活性、纤维素酶活性具有显著促进作用(p0.05),而高浓度(≥800 mg·kg-1)则呈现显著的抑制作用(p0.05)。脲酶、蔗糖酶和纤维素酶最优促进浓度分别为100,300和300 mg·kg-1,抑制浓度分别为800,800和600 mg·kg-1。随培养时间延长,La3+对茶园紫色土脲酶、蔗糖酶、纤维素酶刺激作用有减缓趋势。各浓度La3+处理对茶园紫色土脱氢酶均呈显著抑制作用(p0.05),随浓度升高抑制作用逐步增强,随着培养时间延长,抑制作用有减缓的趋势。相关分析结果表明:脲酶分别与蔗糖酶和纤维素酶呈现极显著正相关(p0.01),蔗糖酶与纤维素酶亦呈现极显著正相关(p0.01),脱氢酶与脲酶、蔗糖酶、纤维素酶相关性不显著。脱氢酶活性可以作为评价稀土元素污染茶园紫色土土壤环境的敏感指标。     

聚乙烯醇高含水胶固定化脲酶的研究

冷冻-部分脱水法制成的聚乙烯醇高含水胶固定化脲酶活力收率及对脲素的分解能力明显高于聚丙烯酰胺囱定化脲酶,稳定性相近;用低浓度的戊二醛后处理,提高了固定化酶的稳定性。     

The Structure of the Elusive Urease–Urea Complex Unveils the Mechanism of a Paradigmatic Nickel‐Dependent Enzyme

Urease, the most efficient enzyme known, contains an essential dinuclear Ni^II cluster in the active site. It catalyzes the hydrolysis of urea, inducing a rapid pH increase that has negative effects on human health and agriculture. Thus, the control of urease activity is of utmost importance in medical, pharmaceutical, and agro‐environmental applications. All known urease inhibitors are either toxic or inefficient. The development of new and efficient chemicals able to inhibit urease relies on the knowledge of all steps of the catalytic mechanism. The short (microseconds) lifetime of the urease–urea complex has hampered the determination of its structure. The present study uses fluoride to substitute the hydroxide acting as the co‐substrate in the reaction, preventing the occurrence of the catalytic steps that follow substrate binding. The 1.42 Å crystal structure of the urease–urea complex, reported here, resolves the enduring debate on the mechanism of this metalloenzyme.     

Electrical Control of Urease Activity Immobilized to the Conducting Polymer on the Carbon Felt Electrode

The activity of urease varies by its redox reaction. Active urease has an SH group that is essential to exhibit its activity, however, oxidation agents such as quinone compounds can oxidize the SH group in urease and a S–S bond is produced, resulting in the loss of enzyme activity. The reduction potential of cystine was almost the same as that of the recovery of urease activity. In this work, it has been found that the SH group of urease can be oxidized by not only chemical reaction but also by the direct electrode oxidation of urease and the produced S–S bond can be reduced to SH group by chemical and electrode reactions, and the original enzyme activity is recovered. This research shows that the regulation of urease activity is easily possible by changing the electrode potential of the porous carbon felt immobilized urease. The variation of urease activity was monitored by ammonia or carbon dioxide electrode equipped with the urease immobilized carbon felt, and the ammonia or carbon oxide generated from urea can transfer through the carbon felt to reach the each gas permeable membrane. The combination of gas electrode with porous conducting material such as carbon can supply the novel device for the electrochemical investigation of enzyme activity.     

Effect of thermosensitive matrix-phase transition on urease-catalyzed urea hydrolysis

Temperature dependencies of kinetic and equilibrium parameters of urea hydrolysis catalyzed by native urease and the urease immobilized in a thermosensitive poly-N-isopropylacrylamide gel have been studied. The swelling ratio of the collapsed urease-containing gel is shown to increase in the presence of urea. Below a lower critical solution temperature (LCST) of the polymer, the immobilized u reaseactually has thesame catalytic properties as the native enzyme. At temperatures above LCST, the observed catalytic activity of the immobilized enzyme depends chiefly not only on the thermoreversible matrix state, but also on gel water content.     

pH-Sensitive Oscillatory Motion of a Urease Motor on the Urea Aqueous Phase

A self-propelled object coupled with an enzyme reaction between urease and urea was investigated at the air/aqueous interface. A plastic object that was fixed to a urease-immobilized filter paper was used as a self-propelled object, termed a urease motor, placed on an aqueous urea solution. The driving force of the urease motor is the difference in the surface tension around the object. Oscillatory motion or no motion was triggered depending on the initial pH of the urea solution. Both the frequency and maximum speed of the oscillatory motion varied depending on the initial pH of the water phase. The mechanisms underlying the oscillatory motion and no motion were discussed in relation to the bell-shaped enzyme activity of urease in the enzyme reaction and the surface tension around the urease motor.     

Enzyme and inhibition assay of urease by continuous monitoring of the ammonium formation based on capillary electrophoresis

We present here an easy‐to‐operate and efficient method for enzyme and inhibition assays of urease, which is a widely distributed and important enzyme that catalyzes the hydrolysis of urea to ammonia and CO₂. The assay was achieved by integrating CE technique and rapid on‐line derivatization method, allowing us to continuously drive the sample to the capillary, thus to measure the amount of the product ammonia from the beginning to the end of the reaction. The method exhibits excellent repeatability with RSD as low as 2.5% for the initial reaction rate (n = 5), with the LOD of ammonia of 20 μM (S/N = 5). The enzyme activity as well as the inhibition of urease by Cu²⁺ were investigated using the present method. The results show that Cu²⁺ is a noncompetitive inhibitor on urease, in accordance with the result published in the literature. The enzyme activity and inhibition kinetic constants were obtained and were found to be consistent with the results of traditional off‐line enzyme assays. Our study indicates that the present approach is a reliable and convenient method for analysis of the urease activity and inhibition kinetics by continuous on‐line monitoring of the ammonium formation based on CE.     

Availability and development of an enzyme immunomicrosensor based on an ISFET for human immunoglobulins

A critical evaluation of the potentiometric response of an enzyme immuno-ISFET sensor has demonstrated that it is an effective, simple sensor for human immunoglobulin (IgG). The sensor was constructed using an immobilized human IgG membrane and an ISFET. The assay procedure involves the competitive immunochemical reaction of ureuse-labelled anti-human IgG with human IgG in samples and membrane-bound IgG and the electrochemical determination of membrane-bound urease activity. A linear relationship was obtained between the initial rate of response and the logarithm of IgG concentration from 0.1 to 2.0 mg ml^?1.     

血红蛋白过氧化物模拟酶胶束催化显色体系

在胶束Tween80介质中，研究了以血红蛋白（hemoglobin,Hb)作为过氧化物模拟酶、隐性亮绿（recessive brilliant green,RGB)为氢供体底物、溶解氧为受氢体的酶催化反应特性。在pH5.64(NH4)2HPO4-KH2PO4缓冲溶液中，利用模拟酶对溶解氧作为受氢体催化RBG氧化生成亮绿（brilliant green,BG)而拟定了测定Hb含量的新方法。讨论了胶束介质对酶体系催化反应的影响及酶催化反应的可能机理。     

Determination of urea in urine using a conductivity cell with surface acoustic wave resonator-based measurement circuit

A conductivity cell employing a 61 MHz surface acoustic wave resonator-based measurement circuit was applied to the detection of the urea/urease reaction. The kinetic enzymatic parameters of the urease were estimated from the frequency shifts. The effects of pH, temperature and inhibitor on the response of the enzyme conductivity measurement system were investigated. The system was applied to rapid determination of urea in small urine samples. The lowest detection limit of urea was 30 ng/ml.     

血红蛋白作为过氧化物模拟酶催化显色体系的研究与应用

研究了以血红蛋白（Ｈｅｍｏｇｌｏｂｉｎ,Ｈｂ）作为过氧化物模拟酶对过氧化氢－４－氨基安替比林（４－Ａｍｉｎｏａｎｔｉｐｙｒｉｎｅ,４－ＡＡＰ）氯取代苯酚衍生物显色体系的催化反应性能,探讨了不同氯取代苯酚类衍生物作为酶催化反应氢供体底物的构效关系及酶催化反应的可能机理。拟定了Ｈｂ催化Ｈ２Ｏ２氧化４－ＡＡＰ－２,３,个三氯苯酚（２,３,４－Ｔｒｉｃｈｌｏｒｏｐｈｅｎｏｌ,ＴＣＰ）显色体系用于Ｈ２Ｏ２的测定方法。该方法测定Ｈ２Ｏ２灵敏度高,表观摩尔吸光系数为 ２．２１×１０４ Ｌ·ｍｏｌ－１·ｃｍ－１。将拟定方法与葡萄糖氧化酶催化反应偶联,用于人血清样品中葡萄糖含量的测定,得到满意的结果。     

Study on the Properties and Applications of the Silver-catalyzed Oxidation Reaction of Guaiacol

Guaiacol has been used to determine the activity of peroxide enzyme and uranium. In this paper, we lucubrate the reaction between guaiacol and ammonium persulfate and the reaction mechanism in the presence of silver's catalysis and the activator's behaviour. Based on these, we establish a new catalytic kinetic spectrophotometric method for silver determination and the results are satisfactory when applying to determine silver in the offscum of plumbum-zinc mine after extracting zinc.