Enzyme Inhibition Rate Method for Rapid Detection of Organophosphorus and Carbamate Pesticides in Cowpea

[Objectives ] The paper was to explore enzyme inhibition rate method for rapid detection of organophosphorus and carbamate pesticides in cowpea. [ Methods ] Acetylcholinesterase （ACHE） was added to cowpea extract, to determine the inhibition rate of extract against enzyme. The influences of different sampiing methods and sampling parts on detection results were compared. [ Results] The positive rate of standard sampling was 18.18% higher than that of non-stand- ard sampling, and the positive rate of samples collected from cowpea tail was 16.67% higher than that collected from other parts. [ Condmions] Enzyme inhibi- tion rate method is suitable for rapid detection of organophosphorus and carbamate pesticides in cowpea.

HOMOGENEOUS ENZYME IMMUNOASSAY OF SERUM AFP BY FLUORIMETRIC KINETIC METHOD

The activity of the Ab-bound enzyme(HRP)changed after immunochemical reaction,and can be indicated by a sensitive fluorimetric kinetic method.The finding set the stage for developing sensitive homogeneous immunoassay.AFP was measured as an example.

A NOVEL POLYMER ENZYME LINKED IMMUNOASSAY METHOD AND ITS APPLICATIONS

During the course of study, we found that both poly (N-isopropylacrylamide ) (PNIP) and PNIP-Ab (enzyme-labelled antibody)could be adhere tightly to a cellulose acetale-nitrate membrane, and that the retention of PNIP-Ab on the membrane increased over 30-fold when compared with the unconjugated Ab.Thus we used this characteristic to develop a novel immunoassay method-polymer enzyme linked immunoassay method: homogeneous antigen-antibody reaction and heterogeneous separation process. When applied for detection of human serum HBsAg, this immunoassay system can detect as little as 1 ng/ml of human serum HBsAg.

Studies on Reduced Extent Method for Inhibited Thermokinetics Of Enzyme－Catalyzed Reaction──Inhibition of the laccase－catalyzed oxidation of o－dihydroxybenzene by m－dihydroxybenzene

The thermokinetic reduced extent equations of reversible inhibitions for Michaiels-Menten enzymatic reaction were deduced, and then the criteria for distingushing inhibition type was given and the methods for calculating kinetic parameters, KM,Ki and Urn were suggested. This theory was applied to inverstigate the inhibited thermokinetics of laccase-catalyzed oxidation of o-dihydroxybenzene by m-dihydroxybenzene. The experimental results show the inhibition belongs to reversible competitive type, KM=6.224×10-3 mol L-1, Ki=2. 363 × 10-2 mol. L-1.

Study on Process Conditions of Preparation of Microporous Potatoes Starch by Complex Enzyme Method

[Objective] The technology of using α-amylase and glucoamylase to prepare microporous potato starch was studied.[Method] Taking potato starch as raw materials,starch hydrolysis rate and the oil absorption as a measure of index,the influences of the reaction temperature,two enzymes proportion,the quantity of enzyme,the chroma of substrate,buffer solution pH and reaction time on microporous potato starch were investigated.[Result] The experimental results showed that the best technological conditions were reaction temperature 45 ℃,enzyme ratio(glucoamylase /α-amylase) 6,the quantity of enzyme(amount of enzyme and starch quality than) is 1.0%,the substrate quantity chroma of 0.14 g/ml,buffer solution pH 4,the reaction time 8 h.In such process condition,the oil adsorption rate of hydrolyzed potato starch was as high as 70.2%,starch hydrolytic ratio was 34.16%.[Conclusion] The study provided a basis for the development and utilization of microporous starch.

Enzyme extraction by ultrasound from sludge flocs

Enzymes play essential roles in the biological processes of sludge treatment. In this article, the ultrasound method to extract enzymes from sludge flocs was presented. Results showed that using ultrasound method at 20 kHz could extract more types of enzymes than that at 40 kHz and ethylenediamine tetraacetic acid （EDTA） methods. The optimum parameters of ultrasound extraction at 20 kHz were duration of 10 min and intensity of 552 W/g TSS. Under the optimum condition, ultrasound could break the cells and extract both the extracellular and a small part of intercellular enzymes. Ultrasound intensity was apparently more susceptive to enzyme extraction than duration, suggesting that the control of intensity during ultrasound extraction was more important than that of duration. The Pearson correlation analysis between enzyme activities and cation contents revealed that the different types of enzymes had distinct cation binding characteristics.

Analytical Solutions of System of Non-Linear Differential Equations in the Single-Enzyme, Single-Substrate Reaction with Non-Mechanism-Based Enzyme Inactivation

A closed form of an analytical expression of concentration in the single-enzyme, single-substrate system for the full range of enzyme activities has been derived. The time dependent analytical solution for substrate, enzyme-substrate complex and product concentrations are presented by solving system of non-linear differential equation. We employ He’s Homotopy perturbation method to solve the coupled non-linear differential equations containing a non-linear term related to basic enzymatic reaction. The time dependent simple analytical expressions for substrate, enzyme-substrate and free enzyme concentrations have been derived in terms of dimensionless reaction diffusion parameters ε, λ1, λ2 and λ3 using perturbation method. The numerical solution of the problem is also reported using SCILAB software program. The analytical results are compared with our numerical results. An excellent agreement with simulation data is noted. The obtained results are valid for the whole solution domain.

Analytical Expression for the Concentration of Substrate and Product in Immobilized Enzyme System in Biofuel/Biosensor

In this paper, an approximate analytical method to solve the non-linear differential equations in an immobilized enzyme film is presented. Analytical expressions for concentrations of substrate and product have been derived for all values of dimensionless parameter. Dimensionless numbers that can be used to study the effects of enzyme loading, enzymatic gel thickness, and oxidation/ reduction kinetics at the electrode in biosensor/biofuel cell performance were identified. Using the dimensionless numbers identified in this paper, and the plots representing the effects of these dimensionless numbers on concentrations and current in biosensor/biofuel cell are discussed. Analytical results are compared with simulation results and satisfactory agreement is noted.

Iterative approximate solutions of kinetic equations for reversible enzyme reactions

We study kinetic models of reversible enzyme reactions and compare two techniques for analytic approximate solutions of the model. Analytic approximate solutions of non-linear reaction equations for reversible enzyme reactions are calculated using the Homotopy Perturbation Method (HPM) and the Simple Iteration Method (SIM). The results of the approximations are similar. The Matlab programs are included in appendices.

Analytical expressions of the concentrations of substrate and product in enzyme inhibition process

The initial and boundary value problem in enzyme reactions mechanism for inhabitation process is discussed. Approximate analytical expressions for the concentrations of substrate and product are presented. Approximate analytical solutions of non-linear reaction equations containing non-linear terms related to enzymatic reaction mechanism are solved using Homotopy perturbation method. The relevant analytical expression for the substrate and product concentration profiles is discussed in terms of dimensionless reaction diffusion parameters α, β, γE, and γS. Numerical solution is also obtained using Matlab program. Our analytical expression compared with numerical estimation and good agreement is noted.

Theoretical Analysis of Immobilized Oxidase Enzyme Electrode in the Presence of Two Oxidants

In this paper, mathematical model of Martens and Hall (Analytical chemistry 66, 2763-2770 (1994)) for an immobilized oxidase enzyme electrode is discussed. The model involves the system of non-linear reaction diffusion equations under the steady state conditions. A simple and closed-form of approximate analytical expressions for the concentrations of the immobilization of three enzyme substrates has been derived by solving the system of non-linear reaction diffusion equations using new approach of homotopy perturbation method. Approximate polynomial expression of concentration of substrate, oxygen and oxidized mediator and current was obtained in terms of the Thiele moduli and the small values of parameters Bs, Bo and Bm (normalized surface concentration of substrate, oxygen and oxidized mediator). Furthermore, in this work the numerical simulation of the problem is also reported using Matlab program. An agreement between analytical expressions and numerical results is noted.

单酶法合成低分子量右旋糖酐的研究进展

低分子量右旋糖酐(Dextran)是以蔗糖为底物,由右旋糖酐蔗糖酶(Dextransucrase, DSR)催化合成葡萄糖聚合物后,再经酸水解/酶水解形成的化合物,其分子量为10 000-20 000 Da,可广泛应用于医药、食品等行业。本文综述了单酶法合成右旋糖酐的研究进展,包括微生物法/酶法生产右旋糖酐的优缺点,DSR的高效表达,单酶法合成右旋糖酐的过程调控,DSR的固定化技术、结构和催化机理,以及单酶法合成低分子量右旋糖酐的研究现状,并在此基础上提出DSR单酶一步法直接合成低分子量右旋糖酐的思路。借助人工智能技术可获得更详细的DSR结构信息,在进一步解析DSR的结构、功能和催化机理的基础上,对其进行合理的设计和改进,优化酶学性质,实现单酶一步法直接合成低分子量右旋糖酐,可为生产低分子量右旋糖酐提供了新的理论支持。

一株耐高温β-胡萝卜素降解菌的筛选及发酵工艺优化

以提升烟叶香韵为目的,对4株产香酵母菌进行发酵培养,通过产香感官评价、酶活分析、烟叶发酵成分分析(GC-MS法),筛选得到1株β-胡萝卜素降解产香酿酒酵母(Saccharomyces cerevisiae)SC35。方法:①4株酵母菌发酵产香效果感官评价;②CS35菌株β-胡萝卜素降解效果分析、样品萃取及GC-MS靶向测试分析;③酶活测定、酶促反应体系研究;④菌株SC35发酵工艺优化及响应面优化。SC35在70℃高温环境下能有效加速β-胡萝卜素的降解,降解产物为1,3-环己二酮、氧化异佛尔酮、β-紫罗兰酮等,通过单因素试验和响应面法优化菌株SC35的最佳培养基配方中各组分添加量依次为葡萄糖2.01%,NaNO20.20%,K2HPO40.10%,MgSO40.05%,FeSO4·7H2O 0.001%,KCl 0.05%;最佳发酵条件为发酵温度27.65℃,发酵时间72 h,接种量0.99%,摇床转速140 r/min,装液量100 mL/250 mL,pH值5.5(自然)。采用优化后发酵工艺SC35粗酶液降解效率可达86%以上。

荧光方法在酶抑制农药残留快速检测中的研究进展

农药残留污染是农产品质量安全的首要关注因子,基于大型仪器的实验室检测模式难以匹配检测时效性需求。基于乙酰胆碱酯酶活性抑制的农药残留快速检测方法(酶抑制法)具有操作便携、检测速度快等优势,已成为农产品质量安全的常用筛查手段。囿于常规可见光比色法性能,传统的酶抑制法在检测灵敏度方面受到制约,这限制了其进一步的发展。荧光方法利用特征的激发/发射光谱,可以有效降低非特异性干扰,因此将荧光信号作为数据采集技术是改善酶抑制法灵敏度缺陷的有效手段。本文重点介绍了荧光方法在酶抑制农药残留快速检测中的信号响应模式,包括单酶-荧光材料响应、多酶级联响应和酶活性靶向小分子荧光探针响应,还分别总结了不同方法的目标物响应机制、检测效果及应用现状,最后对酶抑制农药残留快速检测的发展方向进行了展望。

超声波-微波辅助酶法提取花生红衣白藜芦醇的工艺研究

以花生红衣为原料,以白藜芦醇得率为研究指标,采用超声波-微波辅助酶法提取技术,在单因素试验的基础上,采用响应面法对花生红衣白藜芦醇的提取工艺进行优化,得到最佳工艺条件为:酶添加量2.00%,超声波功率260 W,协同时间20 min,微波功率200 W,此工艺条件下花生红衣白藜芦醇得率为0.998 mg/100 g。研究结果为花生红衣的废物利用和工业化生产提供了理论依据及数据支撑。

不同耕作方式对东北旱地土壤养分和生物学性质的影响

为提高黑土区土壤质量和作物产量,并促进区域农业可持续发展,进行了4年田间定位试验,设置了深松、免耕以及传统耕作3种耕作方式,研究了不同耕作方式对土壤速效养分、土壤微生物数量以及土壤酶活性的影响。结果表明,与传统耕作相比,深松能够显著提高上层土壤(0~20 cm)碱解氮、有效磷以及速效钾含量(分别提高23.6%、49.6%和63.3%),同时,显著提高了下层土壤(20~40 cm)有效磷含量(86.2%);深松处理上层土壤pH显著提高,下层土壤各处理间pH变化较小;深松和免耕能够显著增加上层土壤细菌和真菌的数量,深松显著增加放线菌数量;下层土壤各处理间微生物数量差别较小,表现为深松>免耕>传统耕作;深松处理上层土壤蔗糖酶和过氧化氢酶活性最高,免耕增加了土壤脲酶和磷酸酶活性;除过氧化氢酶外,其他3种酶活性均为上层土壤高于下层。深松和免耕处理能增加土壤速效养分含量、微生物数量及酶活性,有助于改善土壤化学和生物学性状,进而提高土壤质量,其中,深松方式效果最为显著,对于培肥土壤和维护土壤生物的生存环境等方面具有重要作用。

乳中嗜冷菌检测和分析方法的研究进展

乳是适宜微生物生长的优良培养基,易被来自环境的微生物污染。在生乳低温贮存过程中,嗜冷菌是最主要的微生物,并且嗜冷菌在低温环境中会产生耐热的蛋白酶和脂肪酶,热处理过后仍保持活性,在液态乳贮藏过程中分解蛋白质和脂肪,影响乳的品质。目前我国行业标准规定的乳中嗜冷菌计数方法需要将试样在6.5℃条件下培养10 d,耗时过长,在实际生产中应用价值较低。因此,开发针对嗜冷菌及其耐热酶的检测技术对保障乳及乳制品品质十分必要。本文综述了国内外针对乳中嗜冷菌鉴定与分析开发的多种方法,涉及的技术包括聚合酶链式反应、测序技术、荧光原位杂交、环介导等温扩增等,为开发可用于检测乳中嗜冷菌的技术提供参考。

均相酶免疫法检测血清维生素D含量的性能验证

目的建立均相酶免疫法测定末梢血25-羟维生素D(25(OH)D)的方法。方法采用苏州博源医疗生产的均相酶法总25-羟维生素D检测试剂,验证其正确度、精密度、线性范围、可报告范围、灵敏度、方法比对及干扰试验等分析性能指标。结果四个不同浓度(8.00 ng/mL,17.50 ng/mL,35.00 ng/mL,70.00 ng/mL)25(OH)D校准品的偏倚分别为0.44%、-1.91%、1.51%和-0.23%;低值、高值血清样品的批内精密度和总精密度分别为2.22%、1.69%和2.42%、2.52%,小于厂商声明的1/4 TEa(6.25%)和1/3 TEa(8.33%);线性范围为8.0~70 ng/mL,可报告范围上限为210 ng/mL;分析灵敏度符合厂商声明标准(2.9 ng/mL)。方法学比对收集110例临床血清样品,分别使用均相酶免疫法(试验方法,Y)与罗氏发光(参比方法,X)的25(OH)D试剂进行检测,Deming回归方程为Y=0.9988X-0.8575,R2=0.9281。结论均相酶免疫法检测总25-羟维生素D试剂各项分析验证均符合要求,性能良好,满足临床应用要求。

基于响应面法优化双酶同步酶解的全豌豆乳的稳定性及营养特性研究

豌豆作为一种大宗豆类,具有营养价值高及低致敏性等特点,但全豌豆乳体系易絮凝失稳,限制其在食品中的应用。本文以脱皮豌豆为原料,采用高压均质耦合生物酶解(中温α-淀粉酶和纤维素酶)处理,通过离心沉淀率等指标,以及Box-Behnken设计优化酶解工艺条件,揭示最优工艺下全豌豆乳的稳定性和营养特性变化规律。结果表明,相较于传统过滤、单一酶解、双酶分步酶解等方法,中温α-淀粉酶和纤维素酶双酶同步酶解制备的全豌豆乳稳定性更优,最佳的酶解工艺参数为:中温α-淀粉酶:纤维素酶为4.5:5.5(酶活力比,U/g淀粉),添加总量为12 U/g,酶解时间为65 min,该条件下制得的全豌豆乳的离心沉淀率最低(27.70%),在不经过滤和不添加稳定剂的情况下,贮藏60 d内无明显的沉淀分层。与传统单体营养素复配工艺得到的豌豆乳相比,该优化工艺下制得的全豌豆乳稳定性显著提高,淀粉的消化程度降低了12.74%,蛋白质的消化程度提高了16.41%。研究结果为浓浆类的植物乳开发提供一定的理论指导。

酶底物光谱法与酶底物法检测水中大肠菌群的比较分析

采用新型酶底物光谱法和酶底物法对不同类型水样中总大肠菌群、大肠埃希氏菌及粪大肠菌群指标进行同步检测。两种方法各有优势,采用SPSS软件对检测结果处理后进行配对t检验,结果显示:两组总大肠菌群的数据P=0.057,相关系数r=0.904;两组大肠埃希氏菌的数据P=0.593,相关系数r=0.972;两组粪大肠菌群的数据P=0.136,相关系数r=0.986,表明两种检测方法具有相关性,且没有统计学意义上的显著性差异。采用酶底物光谱法检测有证质控样品,总大肠菌群、大肠埃希氏菌、粪大肠菌群的检测结果均在质控样真值范围内,证明该方法检测准确度符合要求。酶底物光谱法与酶底物法相比,反应原理类似,操作更加简便,具有仪器自动化和智能化快速检测、判读、存储、联网传输数据等优点,可满足实验室常规检测需求,在应急突发事件现场检测方面有较强的运用前景和优势。