Trypsin immobilization on an ethylenediamine-based monolithic minidisk for rapid on-line peptide mass fingerprinting studies

The aim of this work was to develop a trypsin-based micro-immobilized enzyme reactor prepared on a monolithic ethylenediamine BIA Separations CIM (convective interaction media) minidisk. The micro-immobilized enzyme reactor (IMER) was integrated in a liquid chromatography system hyphenated to electrospray ionization tandem mass spectrometry to carry out on-line protein digestion and identification. The performance of this IMER was compared with that obtained using a previously developed bioreactor prepared on a conventional CIM ethylenediamine disk and with that of the commercially available Poroszyme immobilized trypsin cartridge. In this work, we showed how different proteins were identified with good recoveries using a digestion time of 10 min only.     

A multimembrane enzyme reactor operating in an electric field

Summary A multimembrane enzyme reactor, operating under an electric field, is described. This reactor is based on the concept of isoelectric traps, by which a pair of membranes, of appropriate pI value, are able to trap the enzyme, the buffering ions (which have to be amphoteric), the substrate and, if needed, cofactors. Charged reaction products can thus leave the enzyme chamber and be recovered in other chambers where they are trapped by the same isoelectric mechanism (if amphoteric). The unique advantages of such reactors are absence of enzyme inhibition by a feed-back mechanism and reaction yields approaching 100%. Examples of a urease and a trypsin/casein reactor are given. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

水凝胶的制备及应用

本文回顾了“普通”和“环境敏感”水凝胶的制备方法及其作为生物材料在固定化酶、固定化细胞、免疫测试和药物控制释放等领域的应用。     

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。     

酶固定化技术用载体材料的研究进展

酶的固定化是生物技术中最为活跃的研究领域之一.作为固定化酶技术的重要组成部分,载体的结构及性能在很大程度上直接影响着所得固定化酶的催化活性及操作稳定性.综合性能优良的载体材料的设计与制备是固定化酶技术领域的一个非常重要的研究内容.通过对传统材料的改性和新型载体材料的研究开发,必将促进固定化酶在各个领域的广泛应用.本文综述了近年来国内外有关固定化酶载体材料的研究现状和发展趋势.     

Two-temperature dilute-acid prehydrolysis of hardwood xylan using a percolation process

This article outlines some of the factors influencing the choice of a suitable reactor for using immobilized biocatalysts. We have concentrated on biochemical engineering parameters of immobilized biocatalysts, which are important with respect to their application in industrial processes.     

Interaction of Rheumatoid Factor with Immobilized ss-DNA

Rheumatoid factors (RFs) are the characteristic autoantibodies of rheumatoid arthritis. Recent researches in our laboratory showed that the immobilized single-stranded DNA (ss-DNA) immunoadsorbent can selectively remove RFs from the serum of patients. In the present paper are studied the modification of argininine, tryptophan, lysine residues and carboxyl terminus of IgGRF, which was separated from patients‘ serum, with 1,2-cyclohexanedione(CHD), N-bromosuccinimide(NBS), pyridoxal 5‘-phosphate (PP) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) respectively, and their effects on the adsorption capacity of the immobilized ss-DNA immunoadsorbent for IgGRF. After the specific modification, the corresponding adsorption capacities of the adsorbents were changed from 48%, 46%, 44% and 54% to 84%, 14%, 21% and 81%, respectively. These results indicate that the electrostatic or ionic-bonding is essential for the interaction between ss-DNA and IgGRF.     

固定化酶不对称合成（S）＋—（＋）—布洛芬的研究

能够不对称水解布洛芬乙酯的酵母菌Ｔ１５８固定化在壳聚糖珠中，固定化酵母菌的活力回收达６０％，最适温度为４５℃，半衰期为７５ｄ以上。     

Modification of the immobilized metal affinity electrophoresis using sodium dodecyl sulfate polyacrylamide gel electrophoresis

Modification to the original immobilized metal affinity electrophoresis (IMAEP) technique is presented. SDS-PAGE is used instead of native PAGE for improved extraction of phosphoproteins from a mixture of proteins. Protein samples treated with 2% w/v SDS instead of native sample buffer ensure that proteins are negatively charged. These negative charges on the proteins assure that the proteins migrate electrophoretically towards the anode regardless of their pI values and hence pass through the region embedded with the metal ions. Another benefit of treating proteins with SDS is that it unfolds the phosphoproteins exposing the phosphate groups to facilitate the metal-phosphate interactions. Phosphorylated ovalbumin can only be extracted after SDS sample buffer treatment. Data show that there is no detrimental effect upon SDS treatment on the extraction of phosphoproteins from a mixture of proteins. Electrophoretic migration of phosphoproteins ceases upon encounter with metal ions like Al+3, Ti+3, Fe+3, Fe+2, and Mn+2 whereas non-phosphorylated proteins migrate freely.     

微胶囊固定化过氧化氢酶

以乙基纤维素为膜材，用液中干燥法将过氧化氢酶微胶囊化，研究了温度和ｐＨ值对酶活性的影响及过氧化氢和尿素对固定化酶的抑制作用，由于乙基纤维素膜的保护，明胶对ｐＨ值的缓冲作用及明胶与尿素的反应，明显地扩大了酶对温度和ｐＨ值的适应性，降低了酶活性抑制剂的影响。     

一种简化的重氮化法制备固定化酶的载体合成方法

重氮化法是固定化酶时常用的一种方法。用多孔玻璃等无机物作载体时,一般是先用g-氨丙基三乙氧基硅烷与多孔玻璃等载体反应[1,2],生成烷基胺玻璃,然后与对硝基苯酰氯反应,产物经过还原,生成带有芳胺的衍生物,最后进行重氮化。本文通过烷基胺与对氨基苯甲酸反应,直接生成芳胺的衍生物,比常用的方法缩短了一步。通过在新合成的载体上对木瓜蛋白酶进行固定化,研究了固定化条件对酶活力回收的影响,最适固定化条件如下:pH为7.0,时间为6h,酶量为240mg/g载体,并比较了固定化酶和溶液酶的有关性质,考察了固定化酶的操作稳定性。结果表明,用这种方法合成的载体固定化酶,其对热稳定性、操作稳定性及产率都比较理想。     

一体化微流控芯片的酶固定化技术

20世纪90年代,Manz等提出的微全分析系统越来越受到关注,聚二甲基硅氧烷[Poly(dimethylsiloxane),PDMS]材料具有透光性能好和绝缘等优点,并可通过浇铸法制作一体化的芯片,从而有效地解决芯片的封合问题。     

固定化酶反应器及其在蛋白质组研究中的应用

综述了近年来国内外酶固定化载体的研究进展,侧重于无机材料和有机聚合物材料上的固定化酶方法;此外,也介绍了固定化胰蛋白酶反应器与分离系统联用在蛋白质样品分析中的应用,并展望了固定化酶反应器的研究方向及其在蛋白质组中的应用前景。     

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。     

壳聚糖固定化胰蛋白酶的研究

以壳聚糖为载体，戊二醛为交联剂，采用两种方法制备了固定化胰蛋白酶。考察了固定化反应中pH值，戊二醛的浓度，以及给酶量对固定化胰蛋白酶活力的影响，并研究了这两种固定化胰蛋白酶的性质。实验结果表明，以戊二醛预交联的网状壳聚糖为载体制备的固定化胰蛋白酶具有更加优良的性能，在最佳固定化反应条件下，酶的活性加收率可达56％。此固定化胰蛋白酶的最适pH为7．0－8．5，最适温度为60℃，Km值为2．52mol/L，固定化胰蛋白酶表现出较好的热稳定性，pH贮存稳定性，以及在乙醇水溶液中的稳定性。     

Preparation and characterization of acidified chitosan immobilized in epoxidized natural rubber

Biocomposites comprising chitosan (CTS) trapped in an epoxidized natural rubber (ENR) was prepared by homogenizing CTS in ENR50 (ENR with about 50% epoxy content) latex in the presence of curing agents and acetic acid. Micrographs of CTS-t-ENR reveal no phased-out entity. Infrared spectra of CTS-t-ENR show only vibrational bands belonging to CTS and ENR, affirming that the former was not bonded but immobilized in the matrix of the latter. CTS loading up to 5 phr resulted in the increase in the tensile strength and elongation at break, modulus of the CTS-t-ENR. Thermal stability of CTS-t-ENR is higher than that of CTS but lower than that of ENR. Increase in CTS loading from 2.5 to 20 phr resulted in the decrease in toluene absorbency but increase in water uptake of CTS-t-ENR.     

PREPARATION OF CHITOSAN COATED METAL AFFINITY CHROMATOGRAPHY ADSORBENT

1NTRODUCTIONImmobilizedmetalionaffinitychromatography(IMAC)hasattractedconsiderableattentioninthepurificationofproteinssincePorathfirstlyuseditin1975Llj.IMAChassomeadvantagescomparingtootheraffinitychromatography.Forexample,IMACcanbeoperatedatanhighionstrengthandtheligandsuchasC.2 ischeapandstable.TheconventionalIMACadsorbentswereproducedbyreactingiminodiaceticacid(IDA)withpolysaccharidesuchasagaroseandthentheaffinityligandC.2 waschelatedtothe.d...b..t[2].Thepreparationwasexpensi…     

Immobilized sulfonic acid functionalized ionic liquid on magnetic cellulose as a novel catalyst for the synthesis of triazolo[4,3-a]pyrimidines

The Brønsted acidic ionic liquid 1-(propyl-3-sulfonate) vinyl imidazolium hydrogen sulfate [IL] was supported on modified magnetic cellulose. The physical structure, composition, and functional groups of the novel supported ionic liquid catalyst were characterized via XRD, FT-IR, EDS, SEM, VSM, TGA, TEM, and BET techniques. Owing to the combination of nano-support features and flexible imidazolium linkers, it acted as a “quasi-homogeneous” catalyst to catalyze the preparation of triazolo[4,3-a]pyrimidine derivatives by a one-pot three-component reaction of active methylene compounds (ethyl cyanoacetate or malononitrile), aminotriazole and aryl aldehydes. The catalyst shows good catalytic activity for the synthesis of triazolo pyrimidines after six times of recycling.     

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。     

壳聚糖微球固定化L-天门冬酰胺酶研究

以反相悬浮交联法合成了壳聚糖微球,并将其应用于固定化L-天门冬酰胺酶。探讨了不同合成条件对成球情况的影响,研究了固定化酶的性质。结果表明,壳聚糖载体可以较好地吸附L-天门冬酰胺酶。固定化酶的活力回收率较高,且稳定性得到提高。