固定化木瓜蛋白酶的制备和性质研究

多孔硅球固定化木瓜蛋白酶具有热增活性 .本文在前文研究的基础上 ,用载体交联法制备了甲壳胺固定化木瓜蛋白酶和纤维素固定化木瓜蛋白酶 .考察了固定化pH值、戊二醛浓度和给酶量对固定化木瓜蛋白酶活力的影响 .研究了固定化木瓜蛋白酶的性质 ,特别是热稳定性和耐热性 ,并与溶液酶和多孔硅球固定化木瓜蛋白酶进行了比较 .所制得的甲壳胺固定化木瓜蛋白酶和纤维素固定化木瓜蛋白酶的最适反应温度均达到了 80℃ ;90℃温育 1h后固定化酶的活力保持在 95 %以上 ;70℃温育处理 5h和 6h后固定化酶的活力也仍能保持在 90 %以上 .固定化木瓜蛋白酶的热稳定性和耐热性得到了显著提高     

壳聚糖固定化总状毛霉MR137-3蛋白酶的性质

以壳聚糖为载体,戊二醛作交联剂,将总状毛霉MR137—3蛋白酶固定在壳聚糖上。研究了戊二醛浓度,给酶量,处理时间对MR137—3蛋白酶固定化的影响。同时对固定化酶与游离酶的热稳定性、最适pH、最适温度以及脲、有机溶剂、金属离子的影响等理化性质进行了探讨。     

壳聚糖固定化乳酸脱氢酶的制备及酶学性质研究

以磁性壳聚糖作为载体,戊二醛作为交联剂,对乳酸脱氢酶(LDH)进行固定化.固定化的最适条件为:戊二醛浓度6%,pH值7.5,酶的偶联时间2 h.对游离及固定化LDH酶学性质的研究表明,酶促反应的最适pH值为9.2,最适温度分别为37℃和50℃,对乳酸的表观米氏常数分别为1.6 mmol/L和0.9 mmol/L.游离酶和固定化酶在40℃放置150 min后,其活力分别为最初的56.5%和76.1%.固定化酶在4℃贮存4周后,活力仍保留50%以上.固定化酶在室温下与底物重复反应6次后,活力仍保留60%以上,说明固定化酶具有较好的热稳定性、贮存稳定性和复用性.     

Characterization of Alcohol Dehydrogenase from Permeabilized Brewer's Yeast Cells Immobilized on the Derived Attapulgite Nanofibers

Alcohol dehydrogenase (ADH) from permeabilized brewer's yeast was immobilized on derived attapulgite nanofibers via glutaraldehyde covalent binding. The effect of immobilization on ADH activity, optimum temperature and pH, thermal, pH and operational stability, reusability of immobilized ADH, and bioreduction of ethyl 3-oxobutyrate (EOB) to ethyl(S)-3-hydroxybutyrate ((S)-EHB) by the immobilized ADH were investigated. The results show the immobilized ADH retained higher activity over wider ranges of pH and temperature than those of the free. The optimum temperature and pH were 7.5 and 35 °C, respectively, and 58% of the original activity was retented after incubation at 35 °C for 32 h. More importantly, in bioreduction of EOB mediated by immobilized ADH, the conversion of substrate and enantiomeric excess (ee) of product reached 88% and 99.2%, respectively, within 2 h and retained about 42% of the initial activity after eight cycles.     

Preparation of a Cyclomaltoheptaose(β-cyclodextrin) Cross-linked Chitosan Derivative via Glyoxal or Glutaraldehyde

Chitosan has been regarded as a multifunctional polymer applicable to various purpos-es1. The potential of the material in medical applications, particularly in drug-delivery formu-lations has been demonstrated2, 3. Recently, a crosslinked chitosan with various crosslink-ing agents was prepared as adsorbents4-7. A -cyclodextrin (?CD) has been subjected to numerous studies, particular in terms of its complexing ability with a variety of components8, 9. The complexing ability of -CD has b…     

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。     

Influence of Silica-Derived Nano-Supporters on Cellobiase After Immobilization

Core shell magnetite nanoparticle (CSMN) was successfully synthesized with diameter around 125 nm according to the determination with scanning electronic microscopy. SBA-15 with diameter around 31 nm was synthesized in our previous work as another supporter for immobilized degradation enzymes. The aim of this study was to investigate the influence of silica-derived nano-supporters on cellobiase after immobilization. With covalent method, glutaraldehyde was introduced to immobilize cellobiase. The immobilized enzyme efficiency, specific activity, and its characterization, including optimum pH, pH stability, optimum temperature for enzyme reaction, and enzyme thermal stability were investigated. Results show that the method of enzyme immobilization on both nano-supporters could improve cellobiase stability under low pH and high temperature conditions compared with the free enzyme. In the aspect of immobilization efficiency, SBA had higher amount of bounded protein than that of CSMN, but had lower specific enzyme activity than CSMN, assumably due to the change in silica surface properties caused by process of supporter synthesis.     

Amperometric Biosensor Based on Immobilization of Oxalate Oxidase in a Mucin/Chitosan Matrix

An amperometric electrode for oxalate determination immobilizing the oxalate oxidase in a mucin/chitosan (muc/chit) gel with glutaraldehyde as crosslinking agent is presented. The effect of muc/chit weight ratio and volume percent (vol.%) of glutaraldehyde was studied. A very low dynamic response was observed in the case of 100% chitosan with 5 vol.% crosslinking agent. The addition of mucin to chitosan for enzyme immobilization resulted in a biosensor with much better performance, concerning to dynamic response, sensitivity, and stability, with 75% of the initial response after two months. The ratio muc/chit 70/30 was considered optimum for the immobilization. A slight crosslinking and the incorporation of mucin largely influences the swelling and diffusion of the analyte; a direct effect of these properties on the calibration slope was found; the hydrophilic environment for the biomolecule also favor the enzymatic activity through a higher enzyme‐substrate interaction.     

壳聚糖固定化血管紧张素转化酶及其性质

以壳聚糖微球为载体, 戊二醛为交联剂固定化血管紧张素转化酶, 研究了酶固定化的最优条件和固定化酶的性质. 结果表明, 在戊二醛质量分数为2.5%、给酶量为8 mg/mL时, 固定化酶的比活性最大, 为0.085 U/g. 固定化酶在40~50 ℃, pH在7~9之间有最大活性, 其米氏常数Km为2.39 mmol/L. 同时, 固定化酶具有良好的稳定性, 可重复利用.     

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定化研究

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。     

磁性壳聚糖微球的制备及对Cr(Ⅵ)的吸附性能研究

用壳聚糖包埋磁流体,用戊二醛交联制成磁性壳聚糖微球,并用红外光谱表征其结构。用制备的磁性壳聚糖微球吸附Cr(Ⅵ)离子,考察了其对Cr(Ⅵ)离子的吸附性能;探讨了吸附时间、溶液pH值、吸附剂用量、温度、Cr(Ⅵ)起始浓度以及其他离子存在对Cr(Ⅵ)离子去除率的影响。实验结果表明,磁性壳聚糖微球吸附Cr(Ⅵ)离子的最佳条件为:吸附平衡时间40 min,最佳吸附pH值6左右,磁性壳聚糖微球用量10 mg,温度升高有利于提高磁性壳聚糖微球的吸附效率,Cr(Ⅵ)离子起始质量浓度为12μg/mL,无机盐的存在引起磁性壳聚糖微球的吸附性能降低。并且考察了吸附剂的再生性能,实验结果表明磁性壳聚糖微球具有良好的重复使用性。     

固定化乙酰胆碱酯酶的制备及其特性研究

本研究以期研制出能重复使用的固定化乙酰胆碱酯酶(AChE),为天然产物复杂体系中AchE抑制剂筛选新方法的发展奠定基础.以氨基化硅胶(APS-Si)微球为载体,戊二醛为交联剂对乙酰胆碱酯酶进行交联固定化,并研究了酶的最佳固定化条件和固定化酶的性质.结果表明,0.05 g氨基化硅胶微球载体,用戊二醛溶液活化6 h后,在给酶量5 U,28℃固定16 h条件下,得到固定化酶的活性最大.固定化酶在常温(20～40℃),以及较宽pH范围内(pH 6～10)均具有较高的活性,并且具有良好的保存稳定性和可重复利用率,为基于固定化靶酶亲和-色谱质谱联用分析快速筛选乙酰胆碱酯酶抑制剂新方法的发展奠定了基础.     

Acrylamide grafted poly(ethylene terephthalate) fibers activated by glutaraldehyde as support for urease

Urease was covalently immobilized on acrylamide-grafted poly (ethylene terephthalate) fibers after glutaraldehyde activation. Ureasecontaining fibers showed a very high operational stability and reusability, with about 85% of the initial activity after 90 d. The thermostability of the bound urease was positively influenced, and a slight change in optimum temperature was observed after immobilization, when compared with the free enzyme. The pH optimum of both types of urease was found to be the same, but immobilized urease showed an increased stability in a broader range of pH. The kinetic studies exhibited a slightly higherK _m value for the bound enzyme, with a value of 4.50 mmol dm^-3, when compared with the free enzyme (2.82 mmol dm^-3), which demonstrated that the immobilization procedure did not cause an unfavorable conformation for the substrate-product formation and a hindered diffusion. The graft yield was also found effective on maximum activity of immobilized urease. Twenty-five percent of the acrylamide-grafted fibers exhibited the highest enzymatic activity together with the highest water uptake. Higher graft yields were not suitable for the immobilization of the enzyme molecules as a result of crosslinks formed between the poly(acrylamide) chains and glutaraldehyde.     

Dehydrogenase activity measurement in yeast fermentation

The dehydrogenase activity was used as a measure of active biomass in preference to other biochemical parameters because of the simple, but accurate nature of the dehydrogenase test. After a consierable amount of experimental work on the dehydrogenase activity measurement technique and the consideration of utilization of the technique as a measure of the active biomass in yeast fermentation systems, an analytical procedure was developed. The developed procedure was first tested on two yeast cultures (Kluyveromyces fragilis andCandida pseudotropicalis) and then used for the determination of active yeast cells in single-cell protein reactors operating on cheese whey. The dehydrogenase enzymes appeared to be sensitive to the pH, the optimum being 7±0.2. Because the optimum pH for the propagation of these yeasts is about 4.5±0.2 and a pH variation of 3 U impairs enzyme activity, the pH of the medium must be adjusted. The incubation time is also a critical factor in determining the accuracy of the TTC test, especially at low-biomass concentrations. An incubation period of 80 min was found to be reasonable.     

Preparation and characterization of lipase immobilized on reversibly soluble-insoluble N-(2-carboxylbenzoyl) chitosan

N-(2-carboxylbenzoyl) chitosan (CBC), a reversibly soluble-insoluble polymer with pH change, was prepared by modifying chitosan backbone with phthalic anhydride and employed as carrier for lipase immobilization. The obtained CBC exhibited reversible solubility in aqueous solution; it was soluble at pH above 3.8 and precipitated at pH below 3.4. The porcine pancreatic lipase was covalently immobilized on CBC with glutaraldehyde as the crosslinking agent. Under the optimal immobilization condition, the retention activity of the immobilized lipase was found to be 69.8 %. The maximum activity of lipase immobilized on CBC was observed at 40 °C, pH 8.0, while the free lipase presented maximum activity at 37 °C, pH 7.5. The lipase immobilized on CBC exhibited improved thermal and storage stabilities and retained 58.7 % of its initial activity after 9 times of repeated use.     

Preparation and application of triangular silver nanoplates/chitosan composite in surface plasmon resonance biosensing

This paper reports the utilization of triangular silver nanoplates (TSNPs) to enhance the sensitivity of surface plasmon resonance (SPR) biosensor. TSNPs modified with 3-mercaptopropinic acid (MPA) were simply mixed with chitosan and glutaraldehyde to form TSNPs/chitosan composite. The composite was deposited on Au film as immobilization substrate for SPR biosensor. The novel structures of TSNPs are preserved against etching by MPA and chitosan polymer. Moreover, chitosan cross-linked by glutaraldehyde enables antibody to be immobilized on fabricated substrate directly via Schiff alkali reaction. In the optimized conditions, the resulting biosensor based on TSNPs/chitosan composite shows a satisfactory response to bovine IgG in the concentration range of 0.075–40.00 μg mL⁻¹. While the biosensor based on chitosan without TSNPs shows a response in the concentration range of 0.6–40 μg mL⁻¹ and the biosensor based on Au film shows a response in the concentration range of 2.5–40 μg mL⁻¹. The experiment results show that the sensitivity of SPR biosensor based on TSNPs/chitosan composite was significantly enhanced and the immobilization procedure of antibody was simplified.     

Properties of fructosyltransferase from <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> immobilized on an acrylic carrier

Optimization of immobilization conditions was carried out for covalent binding of Aureobasidium pullulans fructosyltransferase to a copolymer of butyl acrylate and ethylene glycol dimethacrylate using a glutaraldehyde method. It was found that the highest activity of the preparation could be obtained for the immobilization pH 6.0 and initial protein amount 8.5 g per dm³ of the carrier. Effects of the reaction pH, temperature, and initial sucrose concentration on the activity and stability of the preparation were analyzed. Further investigations involved storage stability and operational stability in a mechanically stirred-tank reactor.     

β-Galactosidase from <Emphasis Type="Italic">Penicillium canescens</Emphasis>. Properties and immobilization

β-galactosidase from Penicillium canescens was immobilized on chitosan, sepharose-4B, foamable polyurethane and some other carriers. The highest yield of immobilization (up to 98%) was obtained by using chitosan as a carrier. The optimum pH and temperature were not significantly altered by immobilization. High stability of immobilized β-galactosidase during storage was demonstrated. Efficient lactose saccharification (over 90%) in whey was achieved by using immobilized β-galactosidase.     

Preparation of Crosslinked Chitosan/Silk Fibroin Blend Films for Drug Delivery System

Crosslinked chitosan/silk fibroin blend films were prepared by a solution casting technique using glutaraldehyde as crosslinking agent. Drug release characteristics of the blend films with various blend compositions were investigated. Theophylline, diclofenac sodium, amoxicillin trihydrate, and salicylic acid were used as model drugs. The release studies were performed at 37 °C in buffer solutions at pH 2.0, 5.5, and 7.2. It was found that the blend films with 80% chitosan content showed the maximum amount of model drug release at pH 2.0 for all the drugs studied here. This result corresponded to the swelling ability of the blend films. From a swelling study, the maximum degrees of swelling of the drug‐loaded blend films were obtained at this pH and blend composition. The amount of drugs released from the films with 80% chitosan content, from the highest to the lowest values, occurred in the following sequence: salicylic acid > theophylline > diclofenac sodium > amoxicillin.

Comparison of the amounts of drug released from chitosan and the blend film with 80% chitosan content at pH 2.0: (filled) chitosan film, and (blank) blend film with 80% chitosan content (SAL = salicylic acid, THEO = theophylline, DFS = diclofenac sodium, AMX = amoxicillin).     

Mono-Enzyme Biosensor for the Detection of Organophosphorous Compounds

Organophosphorous compounds are important neuroactive molecules whose presents significant analytical challenges. New monoenzyme biosensor introduced here, is used for the measurement of the level of two organophosphorous compounds, paraoxon (POX) and ethyl parathion (EPA), in phosphate buffer. Prussian blue electrodepositionized on a graphite electrode served as a template for immobilization of monoenzyme layer. A monoenzyme layer containing choline oxidase was immobilized along with nafion and bovine serum albumin, by cross-linking with glutaraldehyde. The results revealed that the response of the proposed biosensor was linear between 5 μM and 80 μM in the presence of POX and EPA. Detection limits were 0.4 μM in both cases. Also, for the optimization of the biosensor performance, temperature and pH dependency, concentration of choline chloride as substrate of choline oxidase and incubation time were determined. The results showed that the optimum values for temperature, pH, substrate concentration and incubation time were room temperature, pH 7, 4 mM and 30 min, respectively.