基于柠檬酸/乙二胺-碳量子点制备荧光传感器

利用柠檬酸(CA)为碳源,乙二胺(EDA)为氮源通过微波加热法合成柠檬酸/乙二胺碳点(CA/EDA-CQDs)。采用静电结合法,将CA/EDA-CQDs固定在毛细管内表面,制备了一种荧光传感器。考察了CA/EDA-CQDs与聚丙烯酸(PAA)溶液浓度、pH对固定化影响。优化的固定化条件为:CA/EDA-CQDs浓度为15 mg/L,pH为3,PAA浓度为6 mg/L,pH为3。制备的传感器对Fe⁽³⁺⁾有特异性响应。     

基于柠檬酸/乙二胺-碳量子点制备荧光传感器

利用柠檬酸(CA)为碳源,乙二胺(EDA)为氮源通过微波加热法合成柠檬酸/乙二胺碳点(CA/EDA-CQDs)。采用静电结合法,将CA/EDA-CQDs固定在毛细管内表面,制备了一种荧光传感器。考察了CA/EDA-CQDs与聚丙烯酸(PAA)溶液浓度、pH对固定化影响。优化的固定化条件为:CA/EDA-CQDs浓度为15 mg/L,pH为3,PAA浓度为6 mg/L,pH为3。制备的传感器对Fe~(3+)有特异性响应。     

基于乳化液体系脂肪酶球形酶的制备

在含有脂肪酶的油包水（W/O）乳化液体系中添加交联剂，经乳化交联后通过离心分离得到脂肪酶球形酶颗粒，研究了不同交联剂、交联时间和酶液质量浓度对球形酶活性的影响，得到制备脂肪酶球形酶的适宜条件为：以体积比1︰1的25%(v/v)戊二醛溶液与3%(v/v)聚乙烯亚胺溶液，在室温下反应5min而形成的Glu-PEI共聚物为交联剂，交联2h，酶质量浓度为50g/L时形成的球形酶酶活残留率最高达到86.3%。     

大孔树脂D380固定化硫酸软骨素裂解酶

目的以大孔树脂D380为载体,戊二醛为交联剂,进行硫酸软骨素裂解酶(ChSase)的固定化,并考察固定化酶的酶学性质。方法分别考察加酶量、吸附温度、吸附时间、吸附pH值、戊二醛交联浓度、交联时间及交联温度对ChSase固定化效果的影响,并分析该固定化酶的最适反应温度、最适反应pH值、米氏常数(Km)及其操作稳定性。结果ChSase的最佳固定化条件为:加酶量150U/g树脂,吸附温度15℃,吸附时间6h,吸附pH值7.0,戊二醛交联浓度0.01%,交联时间3h,交联温度4℃。以此条件制备的固定化酶,其酶结合效率可达79.1%。该固定化ChSase的最适反应温度为45℃;最适反应pH值为7.0;Km达1.46×10-1g/L,较游离酶高;具有较好的操作稳定性。结论以大孔树脂D380为载体固定化ChSase是可行的,所得固定化酶有较高的使用效率和稳定性,适合于工业化生产。     

氧化竹纤维固定木瓜蛋白酶

通过高碘酸钠-甲醛法对几种竹纤维氧化改性,制备了良好的竹纤维固定化酶载体,并对木瓜蛋白酶进行固定,考察了固定化酶固定条件、最适微环境以及稳定性。结果表明,在4℃、pH=7.5,给酶量为15 g/L条件下固定24 h,改性竹纤维固定化木瓜蛋白酶具有较高催化活性,活力可达805.5 U/g,其最适催化条件为50℃、pH=7.5,较游离酶有更好的耐热性和耐碱性,重复使用6次后活性仍能达到初始活力的76.9%。     

乙酰胆碱酯酶固定化方法的研究

以戊二醛为交联剂,牛血清白蛋白(BSA)为保护剂,将乙酰胆碱酯酶(AchE)交联固定到商品载体上,制备固定化酶片。对影响酶固定化的重要因素进行了考察,获得了最佳固定化条件。实验结果表明,以孔径为0．45μm的硝酸纤维素滤膜作栽体,乙酰胆碱酯酶用量10U,5％(体积分数)戊二醛2μL,1％(质量分数)BSA10μL,配成70μL的酶溶液,3℃固定8h,可获得较好的固定化效果。不同批次制备的酶片,其活力值标准偏差为3.27％～5．03％,酶片在0．1mol／L pH8．5磷酸盐缓冲溶液中3℃下可保存60d。‘     

固定化植物酯酶的制备及应用

以麦草秸秆为原料,经环氧氯丙烷和乙二胺改性,戊二醛交联,制备了植物酯酶的固定化材料,研究了固定化植物酯酶的最优条件和固定化酶的酶学性质。结果表明,加酶量为20 m L/g(改性麦杆),固定化时间为6 h,温度为35℃,p H为7.0条件下,固定化效果最好,酶活回收率可达46%。固定化酶的p H稳定性、热稳定性和贮存稳定性都明显优于游离酶。固定化酶的米氏常数为41 mmol/L,固定化酶与底物的亲合力低于游离酶。建立了酶抑制反应的标准曲线和检测敌敌畏的方法,线性范围为1.0×10-4~6.25×10-4mg/L,检测下限为0.06μg/L。     

戊二醛含量对季铵化壳聚糖耐碱性能影响的研究

以壳聚糖(CS)和环氧丙基三甲基氯化铵为原料制备季铵化壳聚糖(QCS),并以戊二醛(GA)为交联剂,制备一系列交联剂含量不同的阴离子交换膜。将其在常温下浸泡于2 mol/L Na OH溶液中24 h,考察其含水率、溶胀度、机械性能、电导率以及离子交换量的变化情况。结果表明,交联剂的加入可以提高阴离子交换膜的耐碱性能,并随着交联剂含量增加其耐碱性能随之增强。戊二醛质量分数为1.25%的膜,于2 mol/L Na OH溶液中浸泡24 h后,损失离子交换量为4.67%,在70℃条件下损失的电导率仅为7.5%。表明交联有助于提高阴离子交换膜的耐碱性。     

戊二醛含量对季铵化壳聚糖耐碱性能影响的研究

以壳聚糖(CS)和环氧丙基三甲基氯化铵为原料制备季铵化壳聚糖(QCS),并以戊二醛(GA)为交联剂,制备一系列交联剂含量不同的阴离子交换膜。将其在常温下浸泡于2 mol/L Na OH溶液中24 h,考察其含水率、溶胀度、机械性能、电导率以及离子交换量的变化情况。结果表明,交联剂的加入可以提高阴离子交换膜的耐碱性能,并随着交联剂含量增加其耐碱性能随之增强。戊二醛质量分数为1.25%的膜,于2 mol/L Na OH溶液中浸泡24 h后,损失离子交换量为4.67%,在70℃条件下损失的电导率仅为7.5%。表明交联有助于提高阴离子交换膜的耐碱性。     

海藻酸钠-明胶协同固定化S-腺苷甲硫氨酸合成酶的研究

以海藻酸钠和明胶为载体,对S-腺苷甲硫氨酸合成酶进行固定化。再用戊二醛对其进一步交联,增强固定化酶的稳定性。考察了海藻酸钠和明胶质量分数、CaCl2质量分数、酶和载体比例以及交联剂戊二醛体积分数等因素对固定化酶的影响。结果表明,最佳固定化条件为:海藻酸钠质量分数2.0%、明胶质量分数1.0%、CaCl2质量分数4.0%、固定化酶量为2.5 g/L凝胶、戊二醛体积分数0.6%。交联固定化酶热稳定性得到大幅度提高,在50℃下保温5 h仍保留72%的活力,而游离酶则完全失活。交联固定化酶在碱性溶液中的稳定性较高,在pH=8.0～9.0的缓冲液中4℃保温10 h酶活性仍保留87%以上。将交联固定化酶用于S-腺苷甲硫氨酸的合成,连续反应8批次后酶活性仍保留65%。     

Modified PMMA monosize microbeads for glucose oxidase immobilization

Glucose oxidase (GOD) was immobilized onto modified polymethylmethacrylate (PMMA) microspheres by covalent bonding. Monosize PMMA microbeads with 1.5 μm diameter were produced by dispersion polymerization of methylmethacrylate by using polyvinyl alcohol as a stabilizer. Hydroxyl groups on the microbeads were first converted to aldehyde groups by periodate oxidation. Three amino compounds, namely ammonium hydroxide, ethylene diamine and hexamethylene diamine were incorporated through the aldehyde groups. Then, GOD molecules were immobilized through the spacer-arms by using glutaraldehyde. The highest amount of immobilization and activity were obtained in which hexamethylene diamine was used as the spacer-arm with 14 atom length, and were 2.1 mg g⁻¹ polymer and 129 IU g⁻¹ polymer, respectively. The optimal conditions for GOD immobilization were obtained as follows: pH, 6.0; temperature, 30 °C; immobilization time, 60 min; and GOD initial concentration, 0.10 mg ml⁻¹. The optimal conditions for the GOD-immobilized PMMA microbeads were at pH 6.0 and at a temperature of 30 °C. The K_m and V_max values of the GOD-immobilized PMMA microbeads were, 13.79 mM and 26.31 mM min⁻¹ calculated by non-linear regression, respectively.     

固定化黄孢原毛平革菌木素过氧化物酶的研究

用大孔吸附树脂进行黄孢原毛平革菌来源的木素过氧化物酶固定化试验,筛选出固定化效果较好的XAD7HP大孔树脂,研究了其固定化条件。结果表明,当树脂1.0g,酶液pH4.5,加酶量87.2U,吸附温度25℃,吸附4h,戊二醛质量分数0.2%,戊二醛处理时间120min,可获得最佳的固定化效果,固定化酶活力可达到16U/g(对载体)。     

<Emphasis Type="Italic">Penicillium</Emphasis> sp. ZD-Z1 chitosanase immobilized on DEAE cellulose by cross-linking reaction

Chitosanase obtained fromPenicillium sp.ZD-Z1 was immobilized on DEAE cellulose with glutaraldehyde by cross-linking reaction. The optimal conditions of immobilization were as follows: 0.1 g DEAE cellulose was treated with 5 ml 5% glutaraldehyde solution; then 2.3 mg chitosanase was immobilized on the carrier. The optimal temperature and pH was 60 °C and 4.0, and the K _m value was 18.87 g/L. Under optimal conditions, the activity of immobilized enzyme is 1.5 U/g, and the recovery of enzyme activity is 81.3%. After immobilization, the optimal temperature and K _m value increased (from 50 °C to 60 °C, from 2.49 g/L to 18.87 g/L), whereas the optimal pH was reduced (from 5.0 to 4.0). The enzyme activity loss was less than 20% after 10 times batch reaction; the immobilized enzyme showed good operation stability.     

醇脱氢酶在聚乙烯膜表面的固定化研究

以聚丙烯酸（PAA）改性的聚乙烯（PE）膜为载体，研究了醇脱氢酶（ADH）的两种固定化路线，并以甲醛为底物考察了固定化酶的催化性能。路线1用聚乙烯亚胺（PEI）进一步改性，使用戊二醛（GA）固定化ADH。最优固定化pH为6.0，温度为5～15℃，酶浓度为1.0 mg/ml，GA浓度为0.01%(质量)；固定化酶的最适反应pH为6.5，温度为15～30℃，反应速率最高为9.6 μmol/(L·min)；重复利用10次后可保持47.3%的活性。路线2以PAA-PE为载体，用1-(3-二甲氨基丙基)-2-乙基碳二亚胺盐酸盐（EDC）和N-羟基琥珀酰亚胺（NHS）为活化剂，固定化ADH。EDC和NHS最优摩尔比为1∶0.5，固定化时间为24 h；固定化酶的最适反应pH为6.5，温度为20～37℃，反应速率为15.58 μmol/(L·min)；重复利用10次后可保持53.8%的活性。     

Immobilization of alcohol dehydrogenase on the surface of polyethylene membrane

Using polyacrylic acid (PAA) modified polyethylene (PE) membrane as a carrier, two immobilization routes of alcohol dehydrogenase (ADH) were studied, and the catalytic performance of immobilized enzyme was investigated using formaldehyde as a substrate. In the first route, PAA-PE membrane was further modified by polyethyleneimine (PEI) and then ADH was covalently linked by glutaraldehyde (GA) to the surface of PEI/PAA-PE. The results show that the optimal immobilization pH was 6.0, immobilization temperature was 5—15℃, ADH and GA concentrations were 1.0mg/ml and 0.01%(mass). For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 15—30℃, and the highest reaction rate was 9.6 μmol/(L·min), the remaining activity was 47.3% after 10 use cycles. In the second route, ADH was immobilized on PAA-PE membrane with 1-(3-dimethylaminopropyl)-2-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as activators. The results show that the optimal molar ratio of EDC and NHS was 1∶0.5, and the immobilization time was 24 h. For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 20—37℃, and the highest reaction rate was 15.58 μmol/(L·min), 53.8% activity was remained after 10 cycles.     

Construction of a choline biosensor through enzyme immobilization on a poly(2‐hydroxyethyl methacrylate)‐grafted Teflon film

An amperometric choline biosensor was constructed by immobilizing choline oxidase (ChO) on poly(2‐hydroxyethyl methacrylate) (PHEMA)‐grafted Teflon (polytetrafluoroethylene, PTFE) film. Grafting was achieved by γ irradiation. PHEMA‐grafted Teflon films were activated with epichlorohydrin or glutaraldehyde to achieve covalent immobilization of enzyme onto the film. To decrease the diffusional barrier caused by the enzyme‐immobilized film, the film was stretched directly on the electrode. The PHEMA‐grafted Teflon film, therefore, had to have appropriate mechanical properties. Glucose oxidase (GOD) was used in the determination of optimum immobilization conditions, then these were applied to ChO. With GOD, the effect of activation type and film position in electrode on enzyme activity was studied and the highest catalytic activity was obtained when the enzyme was immobilized using glutaraldehyde and the film was stretched over the electrode surface. Further studies revealed that the films activated with glutaraldehyde, immobilized in 2 mg/mL ChO concentration, and stretched directly on the electrode were suitable (specific activity, 0.427 ± 0.068 U mg^?1) for use in the choline biosensor. The linear working range of this biosensor was found to be 52–348 μM, with a 40 ± 5 μM minimum detection limit. The response of the sensor, however, decreased linearly upon repeated use. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007     

聚乙烯亚胺/多巴胺改性氧化硅固定碳酸酐酶

利用聚乙烯亚胺（PEI）/多巴胺（DA）共沉积法改性氧化硅，并以此为载体固定化碳酸酐酶（CA）。考察了PEI/DA质量比、沉积时间对沉积率的影响，用傅里叶红外光谱（FTIR）和扫描电子显微镜（SEM）对改性前后的微球进行了表征；研究了沉积率、载体用量、酶浓度及戊二醛（GA）浓度对固定化酶活回收率的影响；考察了固定化酶的储存稳定性和重复利用性。结果表明，随PEI/DA质量比增加，沉积率先增加后降低，质量比为1∶1时最大；随沉积时间增加，沉积率线性增长，10 h后PEI/DA体系沉积率为单独DA沉积改性的2.66倍，但沉积时间对N元素含量和酶活回收率影响不大；酶固定化时载体用量存在饱和值，CA和GA浓度的最优值分别为0.8 mg/ml和0.1%(质量)，此时酶活回收率可达78.8%。在25℃下储存30 d后，固定化酶的保留活性为77.2%，而游离酶只有12%；重复使用10次后，固定化酶仍能保持88.3%的相对活性。     

利用酰胺活化聚乙烯醇作为载体固定漆酶的研究

交联聚乙烯醇经羧基化、酰胺化制成一种活性固定化酶的载体,并在温和的条件下对漆酶进行了固定。比较不同的固定化时间,pH值,温度和离子强度对固定化效果的影响,发现在12 h,40℃,pH值为3.2所制得的固定化酶的活力最高;在0.05~1.0 mol/L的范围内,随着作为固定化反应介质的缓冲溶液浓度的增加,所制得的固定化酶的活力有所下降。还发现固定化酶较游离酶的酶催化反应最适pH值有所升高。     

Bisacryloylpiperazine as vinylating agent for enzyme immobilization by graft-copolymerization onto polysaccharides

A method of enzyme immobilization by graft-copolymerization onto polysaccharides is reported. Bisacryloylpiperazine has been used as a vinylating reagent and the reaction product with several enzymes (HRP, GOD, Am, ChT, Cel) was copolymerized onto different matrices (cellulose, Sepharose, Sephadex, starch). Immobilization parameters which influence the copolymer activity have been studied for the insolubilization of horseradish peroxidase onto cellulose. These parameters are pH, time, and temperature of bisacryloylpiperazine enzyme activation reaction. Under the best immobilization conditions copolymer activity linearly depends on enzyme concentration. Enzyme coupling efficiency depends on the type of enzyme and it ranges from 7 to 20%. The most important characteristics of these immobilized enzyme systems were tested and compared with those of similar systems obtained by glycidylmethacrylate enzyme activation (stability in continuous washing, kinetic characteristics, and storage, thermal, and operational stability). Immobilized enzyme graft copolymers have kinetic behaviour very close to that of the free enzymes. Diffusion is not seriously limited because immobilization reaction does not alter the enzymatic activity. By means of bisacryloylpiperazine it was possible to immobilize chymotrypsin with better results than those previously obtained, particularly coupling efficiency and long term continuous working.     

烟草多酚氧化酶固定化新工艺

引　言多酚氧化酶是一类广泛存在于自然界 (包括从细菌到哺乳动物 )的含铜蛋白 ,它们的共同特征是在分子氧存在下具有催化氧化酚到醌的能力[1] .酚类物质及其衍生物的毒性和危害性以及它们在工业废水中含量日益增加的事实 ,已引起人们的重视[2 ] .人们开始寻求用过氧化物酶或多