微胶囊固定化过氧化氢酶的制取及对H2O的分解作用

以乙基纤维素为膜材，用溶液干燥法制取了微胶囊固定化过氧化氢酶，探讨了制取过程中明胶的加入对微胶囊产率及固定化过化氢酶活性的影响，同时论述了存放时间、温度以及环境ＰＨ值对微胶囊固定化过氧化氢酶稳定性的影响，深入研究了微胶囊固定化过氧化氢酶对Ｈ２Ｏ的分解作用，获得了十分有意义的结果。     

乙基纤维素微胶囊对过氧化氢酶固定化的研究

以乙基纤维素作膜材,用液中干燥法使过氧化氢酶微胶囊化。研究了微胶囊化操作条件对酶活性的影响。通过测定微胶囊化酶的释放曲线,证明微胶囊膜对过氧化氢酶具有较好的固定性能力。固定化酶用于催化底物过氧化氢分解,测定米氏常数为０．５５ｍｏｌ／Ｌ。     

乙基纤维素微胶囊对过氧化氢酶固定化的研究

以乙基纤维素作模材,用液中干燥法使过氧化氢酶微胶囊化,研究了微胶囊化操作条件对酶活性影响。通过测定微胶囊化酶的释放曲线,证明微胶囊膜对过氧化氢酶具有较好的固定性能力,固定化酶用于催化底物过氧化氢分解,测定米氏常数为０．５５ｍｏｌ／Ｌ。     

SA/CS-CaCl2/PMCG微胶囊的生物相容性研究

介绍了一种新型多组份生物微胶囊体系－SA／CS－CaCl2/PMCG微胶囊。考察了PMCG和SA／CS－CaCl2/PMCG微胶囊体系对大肠杆菌和酿酒酵母生长的影响,并用SA／CS－CaCl2/PMCG微胶囊进行了固定化培养大肠杆菌和酿酒酵母的研究。结果表明,与其它合成聚阳离子类似,PMCG组分对细胞生长有明显的抑制作用,但是在制胶囊过程中以及在用SA／CS－CaCl2/PMCG微胶囊对大肠杆菌和酿酒酵母培养过程中,都显示了良好的生物相容性,因此作为整个体系来说,该微胶囊可用于微生物细胞的固定化培养。     

过氧化氢酶对固定化葡萄糖氧化酶稳定性的影响

本文通过重氮盐共价键合法把葡萄糖氧化酶接到交联琼脂糖上制备固定化酶。为了提高固定化酶的使用稳定性,把过氧化氢酶和葡萄糖氧化酶同时接到载体上,并研究了这两种酶在不同比例时对固定化葡萄糖氧化酶活力和稳定性的影响,随着加入过氧化氢酶量的增加,固定化葡萄糖氧化酶稳定性显著增加。所制得的固定化葡萄精氧化酶-过氧化氢酶在25℃下连续使用36h,活力几乎不变,失活半衰期可达1155h。     

海藻酸钠明胶协同固定化的研究

目的:研究不同因素对固定化微胶囊的影响以及不同物质向微胶囊扩散的规律。方法:采用海藻酸钠与明胶协同固定化制备微胶囊,考察了海藻酸钠、明胶浓度等因子对微胶囊直径与机械强度的影响,以及牛血清蛋白与葡萄糖向微胶囊扩散的状况,并利用非稳态传递模型计算了这两种物质在微胶囊中的有效扩散系数。结果:随着海藻酸钠质量浓度的升高,微胶囊的直径与机械强度逐渐增大。制备的最优条件是CaCl2浓度为10%,滴定速度为180滴/min,最优浸泡时间为30min。在此条件下,葡萄糖与牛血清蛋白的有效扩散系数分别为4.63×10-5cm2/min、1.01×10-7cm2/min。结论:海藻酸钠明胶协同固定化制备的微胶囊作为一种生物载体,非常适合细胞或酶的固定化。     

SA/CS-CaCl2/PMCG微胶囊的生物相容性研究

介绍了一种新型多组份生物微胶囊体系——SA/CS-CaCl₂/PMCG微胶囊。考察了PMCG和SA/CS-CaCl²/PMCG微胶囊体系对大肠杆菌和酿酒酵母生长的影响,并用SA/CS－CaCl²/PMCG生物微胶囊进行了固定化培养大肠杆菌和酿酒酵母的研究。结果表明,与其它合成聚阳离子类似,PMCG组分对细胞生长有明显的抑制作用,但是在制胶囊过程中以及在用SA/CS-CaCl²/PMCG微胶囊对大肠杆菌和酿酒酵母培养过程中,都显示了良好的生物相容性,因此作为整个体系来说,该微胶囊可用于微生物细胞的固定化培养。       

CO2转化酶的固定化研究

CO2是导致温室效应的主要气体,固定和转化CO2的研究对于温室效应的减缓和环境保护方面具有重要意义。近年来CO2转化的研究取得了迅猛发展,其中生物法固定CO2由于其反应条件温和且绿色无污染的优点而备受关注。本文对转化CO2有关的乳酸脱氢酶（LDH）、苹果酸脱氢酶（MDH）和草酰乙酸脱羧酶（OAADC）进行了初步的固定化分析。首先以碳纳米管、壳聚糖和海藻酸钠为原料,制备了包埋上述CO2转化酶的微胶囊固定化体系,然后分别比较了游离酶和固定化酶的操作稳定性和储存稳定性。研究结果表明,固定化的CO2转化酶的操作稳定性和储存稳定得到明显的提高。本研究对CO2的转化和应用方面具有重要参考价值。     

以Fe(Ⅲ)改性胶原纤维为载体固定过氧化氢酶

将胶原纤维用三价铁改性后作为载体,通过戊二醛的交联作用将过氧化氢酶固定在该载体上.制备的固定化过氧化氢酶蛋白固载量为16.7 mg/g,酶活收率为35％.研究了固定化酶与自由酶的最适pH、最适温度、热稳定性、贮存稳定性及操作稳定性.结果表明:过氧化氢酶经此法固定化后,最适pH及最适温度与自由酶相同,分别为pH 7.0和25℃;但固定化酶的热稳定性显著提高,在75℃保存5 h后,仍能保留30％的活力,而自由酶则完全失活;固定化酶在室温下保存12 d后,酶活力仍保持在88％以上,而自由酶在此条件下则完全失活;此外,固定化过氧化氢酶还表现出了良好的操作稳定性,在室温下连续反应26次后,相对活力为57％.该研究表明胶原纤维可作为固定化过氧化     

M.natoriense TNJL143-2菌的D-氨基酰化酶的固定化研究

D-氨基酰化酶可用于D-氨基酸的生产,本研究利用来源于Microbacterium natoriense TNJL143-2的D-氨基酰化酶,分别通过琼脂糖包埋、介孔二氧化硅MCM-41和SBA-15吸附,制备了三种固定化酶,并对三种固定化酶的固定化条件、酶学性质、活性保持时间、重复使用次数、米氏常数等参数进行了研究。结果表明,MCM-41载体固定化酶的蛋白固定率为91.6%,SBA-15载体固定化酶的蛋白固定率为88.0%,琼脂糖包埋法蛋白固定率为79.5%。MCM-41、SBA-15以及琼脂糖三种载体固定化酶最适反应pH均为7.0,最适反应温度范围均为37℃。在固定化酶的活性保持时间以及重复利用活性方面,SBA-15固定化酶同样优于其他两种固定化酶。以D型苯丙氨酸(D-Phe)为底物时,琼脂糖包埋固定化酶的Km为28.8 mol/L,SBA-15固定化酶的Km为25.9 mol/L,MCM-41固定化酶的Km为25.0 mol/L。同时本文还探索了三种固定化酶的pH使用范围及酸碱稳定性、温度使用范围及热稳定性,结果显示,SBA-15作为固定化载体均表现出较广的适用范围及较高的稳定性。在不同条件的反应体系中,SBA-15固定化酶的蛋白损失率始终小于其他两种固定化酶。     

不同壁材微胶囊饲料对黄姑鱼稚鱼生长和消化酶活力的影响

研究采用湿法制粒流化床包衣工艺, 分别以明胶、乙基纤维素、玉米醇溶蛋白为壁材制备微胶囊饲料, 比较其对黄姑鱼稚鱼生长和消化酶活力的影响. 粒径(178-590) m的3种微胶囊饲料质量均大于50%. 扫描电镜观察微胶囊饲料的表面均有一层较为致密的包衣薄膜. 壁材明胶、乙基纤维素、玉米醇溶蛋白微胶囊饲料的包含率分别为95.4%、95.6%和95.8%; 脂类包埋率分别为72.6%、76.5%和64.3%; 氮保留率分别为53.5%、62.3%和54.6%. 将3种微胶囊饲料分别饲喂15日龄黄姑鱼稚鱼30d. 明胶组和玉米醇溶蛋白组稚鱼的体重、全长均显著高于乙基纤维素组(P0.05), 但成活率差异不显著(P0.05); 明胶组稚鱼的体重、全长和成活率均高于玉米醇溶蛋白组, 但差异均不显著(P0.05). 明胶组稚鱼的胰蛋白酶活力显著高于乙基纤维素组和玉米醇溶蛋白组(P0.05), 但淀粉酶和碱性磷酸酶活力的差异均不显著(P0.05). 与乙基纤维素、玉米醇溶蛋白相比, 明胶更适合作为黄姑鱼稚鱼微胶囊饲料壁材.       

Immobilization of the α-amylase of Bacillus amyloliquifaciens TSWK1-1 for the improved biocatalytic properties and solvent tolerance

The α-amylase of Bacillus amyloliquifaciens TSWK1-1 (GenBank Number, GQ121033) was immobilized by various methods, including ionic binding with DEAE cellulose, covalent coupling with gelatin and entrapment in polyacrylamide and agar. The immobilization of the purified enzyme was most effective with the DEAE cellulose followed by gelatin, agar and polyacrylamide. The K _m increased, while V _max decreased upon immobilization on various supports. The temperature and pH profiles broadened, while thermostability and pH stability enhanced after immobilization. The immobilized enzyme exhibited greater activity in various non-ionic surfactants, such as Tween-20, Tween-80 and Triton X-100 and ionic surfactant, SDS. Similarly, the enhanced stability of the immobilized α-amylase in various organic solvents was among the attractive features of the study. The reusability of the immobilized enzyme in terms of operational stability was assessed. The DEAE cellulose immobilized α-amylase retained its initial activity even after 20 consequent cycles. The DEAE cellulose immobilized enzyme hydrolyzed starch with 27 % of efficiency. In summary, the immobilization of B. amyloliquifaciens TSWK1-1 α-amylase with DEAE cellulose appeared most suitable for the improved biocatalytic properties and stability.     

Improving of Catalase Stability Properties by Encapsulation in Alginate/Fe3O4 Magnetic Composite Beads for Enzymatic Removal of H2O2

The aim of this study was enhancing of stability properties of catalase enzyme by encapsulation in alginate/nanomagnetic beads. Amounts of carrier (10–100 mg) and enzyme concentrations (0.25–1.5 mg/mL) were analyzed to optimize immobilization conditions. Also, the optimum temperature (25–50°C), optimum pH (3.0–8.0), kinetic parameters, thermal stability (20–70°C), pH stability (4.0–9.0) operational stability (0–390 min), and reusability were investigated for characterization of the immobilized catalase system. The optimum pH levels of both free and immobilized catalase were 7.0. At the thermal stability studies, the magnetic catalase beads protected 90% activity, while free catalase maintained only 10% activity at 70°C. The thermal profile of magnetic catalase beads was spread over a large area. Similarly, this system indicated the improving of the pH stability. The reusability, which is especially important for industrial applications, was also determined. Thus, the activity analysis was done 50 times in succession. Catalase encapsulated magnetic alginate beads protected 83% activity after 50 cycles.     

Improved Properties of Bacillus coagulans β‐Galactosidase through Immobilization

Thermostable β‐galactosidase from Bacillus coagulans RCS3 was purified by successive column chromatography using DEAE‐cellulose and Sephadex G‐50. Immobilization of the purified enzyme was studied with DEAE‐cellulose and calcium alginate. The efficiency of β‐galactosidase retention was 87 % with DEAE‐cellulose (17 mg protein/mL of matrix) and 80 % with calcium alginate (2.2 mg protein/g bead). Comparative studies of immobilization displayed a shift in the optimum temperature from 65 °C to 70 °C provoked by DEAE‐cellulose, although no effect was observed with calcium alginate. The heat inactivation curve revealed an improvement in the stability (t_1/2 of 14.5 h for the immobilized enzyme as compared to 2 h for the free enzyme at 65 °C) in a calcium alginate system. This immobilized enzyme has a wide pH stability range (6.5–11). β‐Galactosidase immobilized by DEAE‐cellulose and calcium alginate allowed a 57 and 70 % lactose hydrolysis, respectively, to be achieved within 48 h after repeated use for twenty times.     

Expression of human catalase in acatalasemic murine SV-B2 cells confers protection from oxidative damage

Reactive oxygen species have been implicated in aerobic organisms as causative agents in damage to DNA, proteins, and lipids. Catalase is a major enzyme in the defense against such oxidant damage. To determine whether increased catalase expression confers greater resistance to oxidant stress, a eukaryotic expression vector harboring a human catalase cDNA clone was constructed. Acatalasemic murine fibroblasts were then co-transfected with the catalase expression vector and pSV2-neo, and successfully transfected cells were identified by their ability to grow in the presence of geneticin. Clones that contained integrated copies of the catalase expression vector were identified by Polymerase Chain Reaction (PCR) analysis. Stably transfected geneticin-resistant cell lines that overexpressed catalase in potentially positive cell lines were confirmed by catalase enzyme assays. To examine the physiological relevance of catalase overexpression, cells were exposed to oxidant stresses (hydrogen peroxide and hyperoxia), and survival rates were determined. Results demonstrated a significant resistance to oxidative stress in cells overexpressing catalase when compared to controls. These transfected cell lines will provide important models for further evaluation of the role of catalase in protecting cells against the toxic effects of oxygen-derived free radicals and their derivatives.     

Characterization of Soy Polysaccharide and Its In Vitro and In Vivo Evaluation for Application in Colon Drug Delivery

The objective of the present investigation was to establish potential of commercially available soy polysaccharide (Emcosoy®) for colon drug delivery. The soy polysaccharide–ethyl cellulose films were fabricated and characterized. The effect of the pectinase enzyme on the tensile strength and surface morphology of the film was evaluated. The permeation of chlorpheniramine maleate (CPM), a model hydrophilic drug from pectinase enzyme treated and untreated films was measured in pH 7.4 buffer. The soy polysaccharide–ethyl cellulose films were also incubated with Lactobacillus sp. culture for a specific duration, and effect on the CPM permeation was evaluated. The CPM capsules were coated with the soy polysaccharide–ethyl cellulose mixture, and Eudragit S100 was applied as a secondary coat. The coated CPM capsules were radiolabelled, and their in vivo transit was evaluated in human volunteers on oral administration. The pectinase enzyme had a significant influence on the tensile strength and surface morphology of the soy polysaccharide–ethyl cellulose films. The permeability of pectinase enzyme-treated and Lactobacillus sp.-treated films was significantly higher than that of untreated films. The CPM capsules were coated with the soy polysaccharide–ethyl cellulose mixture and Eudragit S100 and were successfully radiolabelled by a simple method. Gamma scintigraphic studies in human volunteers showed that the radiolabelled capsules maintained integrity for at least 9 h after oral administration. Thus, the soy polysaccharide has a potential in colon drug delivery.     

Ultraviolet protection of nucleopolyhedrovirus through microencapsulation with different polymers

A microencapsulated formulation of Helicoverpa armigera nuclear polyhedrosis virus (HaNPV) was produced by emulsion technique to improve its stability under ultraviolet (UV) radiation. The polymers used include sodium alginate, gelatin and starch at concentration levels of 3% and 5% w/v. Except for the starch microencapsulated formulation (3% w/v), the difference in mortality of treated insects between microencapsulated and non-microencapsulated suspensions before irradiation was not significant according to variance analysis (Duncan test, P < 0.05, df = 6). This indicates that microencapsulation of HaNPV does not affect viral activity. Among the three polymers, gelatin performed the best and provided the most stable formulation. The Original Activity Remaining (OAR) percentage for the gelatin formulations did not change from its initial value after 24 h of irradiation. There was no significant difference between the OAR percentage values of 3% and 5% gelatin formulations after 72 h of UVA exposure (90 and 94, respectively; Duncan test, P < 0.05, df = 6). The OAR percentage for the gelatin microencapsulated formulation was 90 after 30 minutes of exposure to UVC radiation. However, for the non-microencapsulated virus suspension, the OAR percentage value declined sharply to 16 after 30 minutes of exposure to UVC radiation. Concerning the in vitro release behaviour of gelatin microparticles (MPs), virus release initiated quickly, but continued at a slower rate until it reached 100% after 1 h of exposure to the release media. The experimental data for the gelatin MPs showed good correlations with the Korsmeyer–Peppas semi-empirical model, indicating that the transport mechanism is primarily consistent with Fickian diffusion.     

Immobilized glutamate oxaloacetate transaminase. Steady state kinetic analysis and stability studies.

1. Glutamate oxaloacetate transaminase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) was immobilized on amino ethyl cellulose using the bifunctional reagent diethyl adipimidate. 2. The steady state kinetic analysis was performed for the particulate and the free enzyme, and the Michaelis constants measured for the amino ethyl cellulose derivative were not greatly different from those measured for the free glutamate oxaloacetate transaminase, while the latter were in good agreement with values in the literature. 3. The amino ethyl cellulose-glutamate oxaloacetate transaminase was slightly more stable than the free enzyme at 65 degrees C, but was stabilised less by polyethylene glycol than the free enzyme.     

Complex Formation Between Chymotrypsin and Ethyl Cellulose as a Means to Solubilize the Enzyme in Active Form in Toluene

Chymotryspin and ethyl cellulose were mixed in an aqueous phosphate buffer solution and freeze dried. Due to complex formation between the substances it was possible to dissolve or at least finely disperse these preparations in toluene. The chymotrypsin-ethyl cellulose complexes were characterized by light scattering measurements. Complexes were also formed by mixing enzyme powder in toluene containing ethyl cellulose and buffer but this was a slow process. Experiments with radioactively labelled bovine serum albumin showed that this protein was also solubilized in toluene in the presence of ethyl cellulose and buffer salts. Chymotrypsin complexes were used to catalyze the esterification of N-acetyl-L-phenylalanine with ethanol in toluene. The presence of buffer salts greatly increased the initial reaction rate of the esterification reaction. The complexes were consideraly more active and stable than enzyme powder in toluene.