固定化葡萄糖氧化酶活性的X射线微区分析

利用X射线微区分析方法,对固定化活性葡萄糖氧化酶进行了定位分析;葡萄糖作为底物,FeSO4和KI作为捕捉剂,底物经固定化葡萄糖氧化酶催化产生H2O2,后者和捕捉剂反应生成沉淀,可以确定固定化葡萄糖氧化酶的催化活性部位。结果表明：颗粒越小,酶活越高,活性葡萄糖氧化酶在凝胶内分布均匀,且绝大多数葡萄糖氧化酶固定在凝胶的内部。作者还研究了固定化活性葡萄糖氧化酶定位的最佳条件。     

固定化乳酸脱氢酶活性的X射线微区分析

利用X射线微区分析，对吸附法得到的固定化乳酸脱氢酶的活性进行了分析；以乳酸为底物，铁氰化钾及铜离子作为捕捉剂，底物经乳酸脱氢酶及氧化型辅酶NAD^ 的作用下催化产生丙酮酸，反应生成的还原性辅酶NADH使铁氰化物还原为亚铁氰化物，后者和铜离子反应产生具有高电子密度的亚铁氰化铜沉淀，可以确定固定化乳酸脱氢酶的催化活性部位；结果表明，以合成出的大孔吸附树脂为固定化酶载体，其酶活性较高，活性乳酸脱氢酶分布较均匀，得到了固定化乳酸脱氢酶的活性定位的最佳条件，并对不同结构载体固定化酶活性进行了研究。     

多孔壳聚糖膜固定葡萄糖氧化酶活性的X射线微区分析

甲壳素(chitin)是无脊椎动物,特别是节肢动物,如虾、蟹及昆虫等的外骨骼重要组成部分,其学名为β(1,4)-2-乙酰氨基-2-脱氧-D-葡萄糖。壳聚糖是甲壳素脱乙酰基的衍生物,其结构上具有许多—NH2、—OH等反应基团,对蛋白质具有极高的亲和性,且具有生物相容性好、无毒、可生物降解等     

同步辐射微束X射线荧光分析及其在生物医学中的应用

介绍了同步辐射微束Ｘ射线荧光分析的特点、主要的仪器设备和方法。介绍了在头发和病变组织中微量元素成分的扫描分析，细胞元素谱及其在外界物理、化学条件下的变化分析等。展示了同步辐射微束Ｘ射线荧光分析在生物医学研究中的广阔前景。     

固定化L-天门冬酰胺酶活性的X射线微区分析

1　引　　言固定化L 天门冬酰胺酶的研究长期以来倍受人们的关注。共价法是一种常用的方法 ,该方法通过载体与酶之间形成共价键而将酶固定于载体上 ,这样可防止酶的脱落 ,故而起到了催化作用。对固定化L 天门冬酰胺酶进行活性定位的研究 ,将有助于L 天门冬酰胺酶的固定化 ,其活性定位的基本原理如下 :底物 酶 产物 +捕捉剂 (金属离子 )活性部位沉淀利用聚焦电子束对固定化酶表面进行激发 ,有活性的部位发射出沉淀金属的特征X 射线 ,利用X 射线能谱分析即可定位酶的活性部位。本文利用MgCl2 作为捕捉剂 ,以L 天门冬酰胺为底物 ,经固定化L…     

有机相中固定化脂肪酶催化合成植物甾醇酯

酶法合成植物甾醇酯具有反应条件温和、产物纯度和产量高等优点,但非水相酶催化的活性和稳定性普遍较低.本文以大孔树脂固定化脂肪酶为催化剂,并在催化过程中添加乳糖的类似物,构建了有机相高效合成植物甾醇酯的工艺过程.以酯化率为考察指标,对脂肪酶和反应溶剂进行筛选,对酯化条件进行优化,同时考察了糖的种类及添加量对酶催化性能的影响.结果表明,大孔树脂NKA吸附固定化的褶皱假丝酵母(Candida rugosa)脂肪酶(NKA-CRL)为最适宜的催化剂,以正己烷为反应介质,在酸醇摩尔比为2和添加酶蛋白质量7.5%的海藻糖的条件下,40°C反应10 h,酯化率达到96.6%.连续6次催化后,植物甾醇的酯化率仍维持在85.0%以上.     

微乳液凝胶固定化脂肪酶催化α-单硬脂酸甘油酯水解反应活性

微乳液凝胶固定化脂肪酶催化α-单硬脂酸甘油酯水解反应活性;微乳液凝胶;固定化脂肪酶;催化活性;水解反应;α-单硬脂酸甘油酯     

黑曲霉脂肪酶的耦合固定化及特性

研究了吸附-絮凝耦合的方法固定脂肪酶的工艺条件.结果表明:在33℃下,用0.03mol/L的磷酸二氢钾?氢氧化钠缓冲液控制体系pH为7.0,酶与树脂(质量比1∶8)作用吸附1h后,用0.2mL絮凝剂聚丙烯酰胺(w(PAM)=0.5%)处理,得到活力较高的固定化脂肪酶.固定化酶最适pH9.0,最适温度为45℃,活力为405U/g,酶活回收率可以达到40%.固定化脂肪酶制备简便,可重复使用,稳定性较高.     

聚丙烯酸甲酯功能基团的微区分析

研究了一种测定聚丙烯酸甲酯上酰肼基团分布的新方法。将功能化的聚丙丙烯酸甲酯类树脂上的酰肼基团与Cu^2 络合,利用X射线能谱微区分析对树脂上Cu^2 的存在部分及分布进行分析,从而确定树脂上酰肼基团的存在部位及分布情况。结果表明：在交联度及其他成分相同时,树脂表面及断面的酰肼化程度有很大差异。     

聚丙烯酸甲酯类大孔树脂对猪胰脂肪酶的固定化研究

本文合成了一系列大孔丙烯酸甲酯－二乙烯苯交联共聚物及它们的功能基化产物。考察了交联度，致孔剂量及不同胺化试剂对载体固定化猪胰脂肪酶的影响，选择出一最佳载体并比较了自由和固定化猪胰脂肪酶的酶学性质。     

Activity and Enantioselectivity of Lipase Immobilized in CTAB Microemulsion-based Gels

Introduction The formation of gelatin-containing microemulsionbased gels(MBGs)was first described in1986[1,2]and the physical/structural characterization was carried out by a number of groups[3,4]with a variety of techniques including tracer diffusion,electrical conductivity,NMR,X-ray and small angle neutron scattering.     

Amperometric Sensor and Immobilized Enzyme Electrode for the Determination of Enzymatic Activities

Abstract

A sensitive method for the rapid determination of activities of soluble or immobilized enzymes, based on the electrochemical detection of hydrogen peroxide is described. Kinetic studies (Vmax and K_M determinations) can be performed for all H₂O₂ generating enzymes (i.e. most of the oxidases) using an amperometric probe with a platinum anode at a fixed potential.

When associated with an immobilized glucose oxidase membrane, this sensor constitutes a glucose electrode and the activity of any hydrolase which releases glucose can be measured. There is no need for other auxiliary enzymes and no preincubation step is required. The possibility to carry out continuous analysis constitutes the main advantage of the described method.     

Immobilized enzyme reactor chromatography: optimization of protein retention and enzyme activity in monolithic silica stationary phases

Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol-gel processing route. While such columns proved to be useful for applications such as screening of protein-ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low μL/min range. Using the enzyme γ-glutamyl transpeptidase (γ-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns with different silica morphologies as a function of flowrate and backpressure using the enzyme reactor assay mode. It was found that the apparent activity of the enzyme was highest in mesoporous columns that retained high levels of enzyme. In such columns, enzyme activity increased by ∼2-fold with increases in both flowrate (from 250 to 1000 nL/min) and backpressure generated (from 500 to 2100 psi) during the chromatographic activity assay owing to increases in k_cat and decreases in K_M, switching from diffusion controlled to reaction controlled conditions at ca. 2000 psi. These results suggest that columns with minimal macropore volumes (<5%) are advantageous for the entrapment of soluble proteins for bioaffinity and bioreactor chromatography.     

有机相中蚕丝固定化脂肪酶催化酯化反应性能研究

研究了有机相中蚕丝固定化脂肪酶催化酶化反应的催化活性。考究了有机溶剂,底物、反应温度,ＰＨ值和体系含水量等因素对固定化脂肪酶催化活性的影响。结果表明,以异辛烷为有机溶剂,在反应温度为５０℃ＰＨ值为７．４时,酶催化活性最好。     

A Trypsin Immobilized Sol-Gel for Protein Indentification in MALDI-MS Applications

The proteolytic enzyme trypsin was chemically immobilized to an amine-functionalized sol-gel using adipoyl chloride under nonaqueous conditions and a nitrogen atmosphere. In the synthesis of the sol-gel, tetraethyl orthosilicate (TEOS), and 3-(2-aminoethylamino) propyldimethoxymethylsilane (AEAPMS) (50:50, v/v) were used, which provided convenient physical and chemical conditions to maintain catalytic activity of immobilized trypsin molecules for the digestion of proteins in proteomics applications. Bovine serum albumin was used as a model protein to perform enzymatic digestion using the trypsin immobilized sol-gel. The resulting peptides were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry to evaluate the digestion performance and specificity of the sol-gel material. The trypsin immobilized sol-gel showed superior enzymatic activity in protein digestion and it was determined that the sol-gel material could be repeatedly used at least 25 times without significant activity loss in long-term use. Additionally, autocatalysis was prevented by immobilization of trypsin. The peptide digest having the highest purity was obtained for protein identification studies.     

聚丙烯偕胺肟-聚乙烯醇大孔球状载体的合成及固定化嗜热菌蛋白酶的研究

以线型聚醋酸乙烯酯为致孔剂,由悬浮聚合制得了丙烯腈-醋酸乙烯酯-二乙烯基苯大孔树脂(MR-AV树脂),通过MR-AV树脂的化学改性,合成了聚丙烯偕胺肟-聚乙烯醇大孔球状载体(MR-AVa载体),用FT-IR、SEM、压汞仪和元素分析法研究了树脂及载体的结构与组成,结果表明MR-AVa载体含氮量较高,具有一定的亲水性,且平均孔径大于8,000(?),嗜热菌蛋白酶用戊二醛固定于所得载体之上。以酪蛋白为底物,考察了固定化嗜热菌蛋白酶的催化水解活性及其影响因素,与可溶酶比较,固定化嗜热菌蛋白酶的热稳定性和抗抑制能力显著提高,当载体孔径较大时,所得固定化酶显示出较好的催化活性。