固定化酶法制备葡萄糖酸钙的研究

目的 用固定化葡萄糖氧化酶制备葡萄糖酸钙.方法 将葡萄糖氧化酶和过氧化氧酶共价同定到经戊二醛预处理的壳聚糖上,用所得固定化酶催化葡萄糖转化为葡萄糖酸钙.结果 固定化葡萄糖氧化酶和过氧化氧酶的同收率分别为86.9％和54.3％;反复用该固定化酶制备葡萄糖酸钙4次后,其酶活性还分别剩余26.5％和21％.结论 用固定化酶法制备葡萄糖酸钙,可提高产品纯度,降低成本.     

葡萄糖氧化酶与过氧化氢酶的共固定化研究

研究了环氧树脂在共固定化葡萄糖氧化酶(GOD)和过氧化氢酶(CAT)中的应用,利用筛选出固定化效果最好的环氧树脂ES-4,考察加酶量、p H及磷酸钠缓冲液离子浓度对共固定化效果的影响。结果表明:共固定化的最佳条件是:GOD 0.5 m L、CAT 1.0 m L、磷酸钠缓冲液p H6.5、浓度1.0 mol/L,固定24 h后固定化葡萄糖氧化酶酶活回收率为35%,过氧化氢酶酶活回收率为43%。连续操作15批次后,酶活力仍能保持初始酶活的73%。该研究为工业化利用固定化酶生产葡萄糖酸钠提供了新的思路。     

磁性Fe_3O_4协同海藻酸钠固定化葡萄糖氧化酶

采用以化学共沉淀法制备出的Fe_3O_4纳米粒子与海藻酸钠结合,制备出磁性微球,再以戊二醛作为交联剂固定化葡萄糖氧化酶,研究了该材料对葡萄糖氧化酶的固定化条件以及磁性固定化酶的酶学性质。结果表明,制备固定化葡萄糖氧化酶的最佳条件为:海藻酸钠浓度为1.5%,海藻酸钠与Fe_3O_4的质量比为5:4,CaCl_2浓度为2%,戊二醛浓度为0.5%,交联时间为1.5 h;最适反应温度提高为40℃,最适反应pH值提高为6.5;热稳定性有一定提高;重复使用5次后,相对酶活仍剩余58.91%。磁性海藻酸钠固定化葡萄糖氧化酶会提高酶的稳定性,有利于工业化生产。     

树脂固定化酶的制备及其在低pH值葡萄糖酸生产工艺中的应用

分别利用强碱性大孔树脂、氨基树脂、环氧树脂作为葡萄糖氧化酶（GOD）和过氧化氢酶（CAT）固定化载体制备固定化酶,并研究其在低pH值葡萄糖酸生产工艺中的应用效果。耐酸性试验表明固定化酶较游离酶具有更强的耐酸性能。分别利用游离酶和3种固定化酶在低pH值（pH值3.5）条件下制备葡萄糖酸,强碱性大孔树脂、氨基树脂和环氧树脂固定化酶所需反应时间分别为40 h、24 h和27 h,酶活损失率分别为46.44%、3.42%和21.84%,而游离酶在反应过程中完全失活。3种固定化酶反应液的澄清度及色度均显著优于游离酶反应液。正交试验进一步优化后氨基树脂固定化酶的制备工艺为混合酶液浓度10%、GOD:CAT=1.5:1、固定温度25 ℃。在该条件下,氨基树脂固定化酶的酶活回收率可达93.15%,转化100 g/L葡萄糖溶液所需时间为23 h,反应结束时葡萄糖酸:葡萄糖酸钠可达0.899:1（m/m）。并且氨基树脂固定化酶稳定性良好。因此,利用氨基树脂固定化酶可以建立低pH值固定化酶葡萄糖酸生产工艺,实现高品质葡萄糖酸产物的生产。     

离子交换树脂共固定葡萄糖氧化酶-过氧化氢酶

从5种大孔阴离子交换树脂中,筛选出固定化效果较好的大孔强碱性苯乙烯系阴离子交换树脂D 201为载体,以戊二醛为交联剂,通过先吸附后交联的方法共固定化葡萄糖氧化酶(GOD)和过氧化氢酶(CAT),研究了固定化酶的制备条件和酶学性质。结果表明,共固定化的最佳条件是:GOD∶CAT=1∶1(酶活力之比),吸附p H值为7.5,吸附温度30℃,吸附时间为8 h;交联剂戊二醛质量分数为1%,交联温度4℃,交联时间8 h。在此条件下固定化,以GOD计,最高酶活回收率为30.8%。与游离酶相比,共固定化GOD-CAT树脂的热稳定性、p H稳定性均增强,间歇操作10批次后酶活力仍然保持在初始活力的90%以上。     

磁性生物活性玻璃共固定葡萄糖氧化酶-过氧化氢酶的研究

生物活性玻璃具有良好的生物相容性和生物活性,利用其进行酶的固定化是一种新的实验理念。本文对生物活性玻璃进行改性,加入一定量乙二醇利用溶胶-凝胶的方法制备了表面具一定磁性的磁性生物活性玻璃微球,并利用该微球对葡萄糖氧化酶和过氧化氢酶进行固定。实验表明,同时固定两种酶比单独固定效果更为显著。之后将GOD-CAT以一定的比例分别在传统水相和有机相二恶烷中进行共固定化,比较水相共固定化酶和有机相共固定化酶的酶比活力和酶学性质,找到了最佳的固定介质。实验表明,戊二醛浓度为0.4%,加酶活力比GOD:CAT为1:2,二恶烷含水量1.5%时,GOD的表观酶活回收率达到90.25%;而传统水相中,GOD的表观酶活回收率最大仅为72.18%。连续使用10次后,有机相固定化酶活为初始值的67.30%,而传统水相中酶活仅为初始值的44.33%。     

葡萄糖氧化酶的固定化及其性能的研究

本文对葡萄糖氧化酶的固定化进行了研究,考查了载体、缓冲溶液pH值,以及酶担载量对固定化酶性能的影响,确定了固定化过程的工艺条件,并对固定化葡萄糖氧化酶的自由态葡萄氧化酶的性能进行了比较。研究结果表明,以TiCl_4处理的5A分子筛作载体所制备的固定化葡萄糖氧化酶具有良好的化学与物理性能,在主要指标上优于文献报道的结果。     

壳聚糖微球固定化葡萄糖氧化酶的研究

以壳聚糖微球为载体,戊二醛为交联剂,固定葡萄糖氧化酶,对葡萄糖氧化酶的固定化条件及固定化酶的各种性质进行了研究,确定了酶固定的最佳条件为0.1g壳聚糖微球与5ml5%戊二醛交联,固定6mg葡萄糖氧化酶,在此条件下酶活力回收可达60%。固定化酶的最适温度为50℃,最适pH为6.0,通过Lineweaver-Burk作图,确定动力学参数Km值为18.3mmol/L,表观米氏常数较游离酶有所降低,固定化酶的热稳定性较游离酶明显提高,该固定化酶具有良好的操作及保存稳定性。     

碳纤维共固定葡萄糖氧化酶-过氧化氢酶的研究

研究了经不同方法改性的碳纤维(CF)在共固定化葡萄糖氧化酶(GOD)和过氧化氢酶(CAT)中的应用。筛选出固定化效果较好的HNO3-H2SO4法改性CF,研究双酶配比、戊二醛含量、交联时间对共固定化效果的影响。结果表明,共固定化的最佳条件是:GOD∶CAT活力比1∶2、戊二醛体积分数0.2%、交联时间15 min,此条件下固定化GOD-CAT的酶活回收率为30.6%。固定化酶的热稳定性、p H稳定性、贮藏稳定性及重复操作性能均有显著提高。连续使用10次后,固定化表观酶活为初始酶活的72.4%。该研究为固定化酶在葡萄糖酸盐工业生产中的应用提供了新思路。     

葡萄糖酸钠的双酶催化制备法

利用生物酶的特异性,采用葡萄糖氧化酶和过氧化氢酶进行葡萄糖酸钠的双酶催化法制备研究。首先分析了葡萄糖氧化酶和过氧化氢酶的理化性质,测试不同温度和不同pH时两支酶的相对活力,得到葡萄糖氧化酶的最适温度范围为20～50℃,最适pH范围为4.0～6.5,过氧化氢酶的最适温度范围为20～50℃,最适pH范围为4.0～8.0。结合酶的理化性质,从催化反应机理出发,以20h时的糖转化率和体系中H2O2量为指标,分析了通风和搅拌、温度、pH、葡萄糖氧化酶的用量、葡萄糖氧化酶和过氧化氢酶的用量比等不同因素的影响,获得了最佳的双酶催化制备工艺:罐压0.05MPa、风量1.5m3/h、转速400r/min、40℃、pH5.5、300g/L的葡萄糖液、30U/g糖量的GOD、GOD:CAT=1∶85。在此条件下,葡萄糖在20h时的糖转化率为99.96%。     

葡萄糖酸钠酶法生产工艺研究

以市售的葡萄糖氧化酶及过氧化氢酶为基础,探索出最佳的酶法生产葡萄糖酸钠的工艺条件为温度32 ℃、转速190 r/min、通风量1 600 m3/h、葡萄糖氧化酶添加量4 mL/g,过氧化氢酶添加量在3 mL/g。并从反应进程、最终残糖、过滤时间、脱色效果、结晶产品质量等方面对酶法及黑曲霉发酵法生产葡萄糖酸钠工艺进行分析比较,确定了酶法生产葡萄糖酸钠优于微生物发酵法生产葡萄糖酸钠。     

Glucose Oxidation by Modified Mould Mycelium

A new strain of Aspergillus niger containing a high intracellular glucose oxidase and catalase activity could be isolated. The glucose oxidase of this strain showed some favourable properties with regard to enzymatic glucose oxidation in industrial scale. The mycelium was permeabilized by treatment with iso-propanol. Then, additional catalase was bound to the mycelium so that a coimmobilizate, resulted. The enzymatic characteristics of this coimmobilizate, as compared with former immobilized glucose oxidase/catalase preparations, showed higher temperature stability and lower K_m-value for glucose. Some basic trials were done for glucose oxidation in a stirred tank reactor where oxygen supply was carried out by controlled addition of hydrogen peroxide instead of conventional aeration. The influence of temperature on the stability of both, glucose oxidase and catalase, was investigated under these application conditions.     

Neue Erkenntnisse bei der Glucoseoxidation mit immobilisierter Glucoseoxidase

New Findings in the Oxidation of Glucose by Means of Immobilized Glucose Oxidase Based on proceeding publications the present paper shows recent aspects, which seem to be important for an economical and technical use of the immobilized glucose oxidase/catalase system. The results of trials in continuous processing were negative, since the conversion rates were unsufficient and technical difficulties arose in keeping back the enzyme particles in the reaction vessel. Batchwise processing with repeated enzyme application showed the major importance of optimal oxygen supply and keeping low temperatures of about 2°C. Different types of stirred tank reactors were tested and classified concerning their suitability. The chances for large scale application of the immobilized glucose oxidase/catalase system are judged to be good.     

共固定化生产高含量低聚果糖的研究

生产低聚果糖过程中，副产物葡萄糖抑制酶反应，降低了产物中低聚果糖的含量。本文采用两种固定化方法，通过去除或转化葡萄糖，从而制备出高含量的低聚果糖。首先以２％戊二醛和０．１％丹宁于２０℃时处理固定化黑曲霉菌体与葡萄糖氧化酶，将菌体与酶共包埋得到固定化颗粒，所得到的共包埋产物于５０℃ｐＨ５．０条件下与５０％蔗糖溶液摇瓶反应２４ｈ，得到７１％的低聚果糖；又采用固定化黑曲霉增殖细胞与固定化葡萄糖异构酶协同作用方法，将５０％蔗糖溶液通入柱式反应器，连续生产得到高含量低聚果糖，产物中低聚果糖和果糖含量分别为６３％和１６％。     

定向固定化葡萄糖氧化酶及其酶学性质的研究

戊二醛将伴刀豆球蛋白(ConA)和载体壳聚糖膜交联,然后利用ConA 与葡萄糖氧化酶糖链的特异性结合作用,实现酶的定向固定化。定向固定化的最适条件为戊二醛浓度0.1%、ConA 浓度0.02mg/ml、葡萄糖氧化酶浓度0.08mg/ml。定向固定化葡萄糖氧化酶的最适pH4.0、最适温度57℃,米氏常数Km 为15.84mmol/L,与游离酶及非定向固定化葡萄糖氧化酶比较,定向固定化葡萄糖氧化酶的最适pH 值向酸性范围发生了偏移并有更宽的pH 值适用范围,最适温度提高,与底物的亲和力较大。     

葡萄糖氧化酶的生产及应用

葡萄糖氧化酶能够利用氧气将葡萄糖氧化成葡萄糖酸而有效去除氧,因而被广泛用作抗氧化剂、葡萄糖酸、尿糖试纸和生物传感器的生产。综述了葡萄糖氧化酶作为生物去氧剂的作用机理、生产工艺条件以及在食品、医药等行业的广泛应用。     

Conversion of Glucose into Gluconic Acid by Means of Immobilized Glucose Oxidase

The properties of a glucose oxidase from Aspergillus niger var., immobilized by different methods were studied in a batch process. Dependencies between formation of gluconic acid and concentration of enzyme and glucose, supply with oxygen, pH-value and temperature were determined and the optimal parameters of reaction fixed. In this connexion consideration of the oxygen demand of the system was of special importance. Different “oxygen carriers” (pure oxygen, air, hydrogen peroxide) were used and their influences investigated not only on glucose oxidase but also on catalase, which is a conclusive fact in the reaction process. This led to very informative experiences on stability, re-usability and productivity of the carrier bound enzyme system. The results obtained should be applied to develop a continuous process for gluconic acid production and an enzyme preparation adapted to this process.     

Utilization of Glucose Oxidase for Extending the Shelf-Life of Fish

The preservative benefits of the enzymic glucose oxidase/catalase system were measured in refrigerated, fresh, whole winter flounder (Pseudopleuronectes americanus Waldbaum) and winter flounder fillets. The enzyme system was applied as a dip, as an ice and immobilized in algin blankets. Advantages were an extended period of sensory acceptance and delayed onset of putrefactive odors, 21 days vs 15 days for controls. The pattern of endogenous hypoxanthine accumulation and decline was unaffected, but parameters thought to measure proteolytic activity were modified: creatinine turnover was slowed and ammonia generation was retarded.     

Determination of Glucose in Food by the Ionic Liquid and Carbon Nanotubes Modified Dual-Enzymatic Sensors

A sensitive electrochemical glucose biosensor based on chitosan (CS)/glucose oxidase (GOx)/catalase (CAT)/CS?+?carboxylic multi-walled carbon nanotubes (MWCNTs-COOH)?+?ionic liquid (IL) film modified glassy carbon electrode has been developed and its characteristics were investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Hydrogen peroxide (H₂O₂) generated during the enzymatic reaction of GOx with glucose could be decomposed by catalase, resulting in the higher sensitivity. The linear range and detection limit were found to be 0.5–100 and 0.2 μmol L^?1, respectively. The proposed biosensor was successfully applied for the determination of glucose in drink and food samples with the spiked recoveries in the range of 95.5 to 101.5 %.