细胞固定化方法对酶活稳定性的影响

本文以大肠杆菌为转化菌,研究了海藻酸钠包埋法细胞固定化技术,初步探讨了细胞固定化方法对产天冬氨酸酶活稳定性的影响。将固定化细胞放置于不同条件下保存,对比研究保存前后的酶活力,结果表明：海藻酸钠浓度4％,细胞浓度5％,温度40℃时保存的固定化细胞酶活稳定性最高。     

新型光交联聚氨酯固定化细胞载体的研究

本文主要研究新型光交联聚氨酯固定化载体PCPUM的交联、结构与固定化细胞的性能。结果表明:PCPUM能在室温短时光照的温和条件下交联并包埋枯草杆菌,得到固定化细胞,其产生α-淀粉酶的能力比游离细胞高30％左右,且对温度和pH的适应性比游离细胞增强;改变预聚物组成、分子量、浓度等条件,可引起载体比表面、孔容、孔径、亲疏水性等的变化,从而影响固定化细胞的产酶性能、漏菌程度和使用寿命;电镜观察发现细胞主要存在于载体孔隙内,载体表层的细胞密度较大。     

SiO2/海藻酸钠复合水凝胶作为固定化纤维素酶载体

以海藻酸钠和γ-氨丙基三乙氧基硅烷为原料,在一定条件下使二者发生交联反应生成复合水凝胶,并以此复合凝胶作为固定化纤维素酶的载体。纤维素酶的包埋率超过了85%, 酶固定前后的SEM图表明纤维素酶非常均匀地分布在载体中。探讨了pH、 温度对固定化酶和游离酶活力的影响, 结果表明固定化酶具有对pH和温度更高的稳定性, 固定化酶的最适宜pH为3.6,最佳催化温度为50℃。通过Michaelis Menten（米氏）方程计算得到的固定化酶的米式常数（K_m）值较游离酶大,表明固定化酶的可重复使用性和储藏稳定性良好,连续使用10次后依然保留>50%酶活力,储藏1个月后固定化酶依然有81%的相对酶活力。      

含纤维光交联聚氨酯固定化载体的研究

在光交联聚氨酯预聚物中加入改性短纤维，得到一种含纤维可光交联复合体。在适当条件下，通过聚氨酯末端和纤维表面的双链聚合，得到一种多相结构的亲水凝胶ＦＰＣＰＵ。由于纤维与聚氨酯之间形成化学结合，而纤维本身仍保持其结晶和取向，因此加入的纤维能产生良好的增强作用。在相同条件下，ＦＰＣＰＵ的强度是ＰＣＰＵ载体的１－２倍，用该载体包埋估草杆菌制取固定化细胞，具α－淀粉酶活力比游离细胞高出４０－５０％，而ＰＣＰ     

碳酸酐酶在硅藻土上的固定化及其性质研究

以硅藻土为载体,通过共价结合法固定化碳酸酐酶.对制备固定化晦过程中的pH值、加酶量、反应温度、反应时间几个重要的影响因素进行了研究和优化,并对固定化酶和游离酶的酶学性质进行了比较.结果表明,固定化酶的最适反应温度为30℃,比游离酶高了5℃;最适反应pH值为6.0,比游离酶低了2个pH值单位.该固定化酶的热稳定性、酸碱稳定性和操作稳定性均高于游离酶.     

固定化酵母菌填充柱对锶的吸附特性研究

锶为具有很强毒性的裂变产物.结合生物吸附和固定化技术的优点,用海藻酸钠-氯化钙包埋法制作固定酵母菌颗粒并填充成吸附柱,研究了固定化酵母菌颗拉的特性和在静态、动态吸附下的吸附率、吸附容量和柱层析参数.结果表明,酵母菌固定化颗粒具有较高的比表面积和较好的机械承受能力.静态吸附结果表明,填充柱具有较高的吸附容量,吸附平衡时平...     

环氧基功能化块体SiO_2大孔材料对漆酶的固定化

采用后嫁接法在溶剂热条件下对新型块体SiO2大孔材料进行环氧基化改性,以环氧基功能化SiO2大孔材料作为载体,通过共价结合法固定化诺维信(Novozymes)工业级漆酶,对固定化条件进行了优化,研究了固定化酶与游离酶的酶学性质。结果表明,在固定化时间为4 h、pH值为4.5、初始酶液浓度为25 mg/mL时,固定化效果最好,固定化漆酶活力达到101.7 U/g;固定化漆酶的最适pH值为4.0,最适温度为50℃,其pH值稳定性和热稳定性都显著优于游离漆酶。固定化漆酶具有可重复操作的性质,与底物反应反复操作10批次后剩余活性为43.4%。     

多孔二醋酸超细纤维膜的固定化酶及染料降解性能

通过静电纺丝技术,调控溶剂种类和溶液浓度,制备了圆柱状无孔纤维、条带状多孔纤维和圆柱状多孔纤维。通过扫描电镜观察纤维形貌,通过物理吸附仪探究纤维比表面积。并将3种纤维膜进行固定化酶,探究其最适反应温度、最适反应pH、存储稳定性和重复使用稳定性。最终进行了染料降解性能测试。结果表明:多孔有利于提高纤维的比表面积。固定化酶和游离酶的最适反应温度为50℃,最适反应pH=3.5,固定化酶有利于提高酶的存储稳定性和重复使用稳定性。较游离酶和其他形貌纤维,圆柱状多孔纤维固定化酶后具有最好的染料降解性能。     

聚乙烯醇凝胶固定化脲酶及载体生物相容性的评价

本文以聚乙烯醇（ＰＶＡ）凝胶为载体，用包埋法对脲酶进行了固定化，并与聚丙烯酰胺固定化脲酶进行了对比研究。结果表明，ＰＶＡ凝胶固定化脲酶具有较高的活力收率（７９％）。其急性毒性及溶血试验合格，表明该材料具有良好的生物相容性。     

氧化硅介孔泡沫材料固定木瓜蛋白酶的研究

以氧化硅介孔泡沫材料(Siliceous Mesocellular Foam, MCF)为载体对木瓜蛋白酶进行了固定, 并对固定化酶的性能和影响因素以及酶的稳定性作了系统的研究. 结果表明, 木瓜蛋白酶在MCF上能获得较大的负载量（334mg/g MCF）. 固定化木瓜蛋白酶的最适反应温度较游离酶提高了10℃, 最适pH向碱性方向偏移0.5个单位. 固定化木瓜蛋白酶的米氏常数（Km）为6.99×10^-3mol/L, 在八批次操作后酶活保留65.1%. 在4℃条件下放置60d后, 固定化酶的剩余活性仍保持75%以上, 而游离酶的活性只有初始活性的53.6%. 与游离酶相比, 固定化木瓜蛋白酶的pH稳定性、热稳定性、操作稳定性和储藏稳定性都有明显改善, 有利于酶的重复使用和储藏. 介孔泡沫材料是一种良好的木瓜蛋白酶固定载体.     

HRP immobilized microporous Poly(styrene-divinylbenzene-polyglutaraldehyde) monolith for forced flow injected phenol biosensing

A colorimetric detection system based on horseradish peroxidase (HRP) immobilized microporous Poly(styrene-divinylbenzene-polyglutaraldehyde) monolith was developed for phenol biosensing. The incorporation of the aldehyde groups to the backbone of the microporous (90%) monolith was achieved by the polymerization of the continuous phase of a high internal phase emulsion. HRP was chemically immobilized onto the inner walls of the micropores of the monolith via the aldehyde groups. HRP immobilized microporous monolith (HRP-MM) was used as a biocatalytic cell under forced flow conditions in a flow injection system. A carrier stream containing 4-aminoantipyrine (4-AAP) as color reagent was flowed through the HRP-MM at various flow rates ranged between 0.1-15 mL min^-1. The analytical parameters of response time, detection limit and linear range varied at the range of 1-60 s, 1-250 μM, and 0.25-2 mM, respectively. The results showed that the most accurate phenol biosensing was obtained at the flow rate of 1 mL min^-1. The microarchitected structure of the monolith improved the mass transferring conditions for the enzymatic reaction due to the forced flow through the micropores. The detection system presented a satisfactory precision evaluated by relative standard deviation of 1.1% (n = 13) and recovery of 100% at the flow rate of 1 mL min^-1. The system showed 60% of its initial signal from 12th day up to the two months.     

Biosorption characteristics of Bacillus sp. ATS-2 immobilized in silica gel for removal of Pb(II)

The bacterial strain Bacillus sp. ATS-2 isolated from Pb(II) polluted soil was immobilized with a silica matrix and Pb(II) biosorption properties of immobilized biosorbent were examined. Optimum biosorption conditions were investigated in the fixed bed column with the variation in the parameters of pH, bed length, flow rate and influent concentration. The Pb(II) biosorption equilibrium was attained within 60 min and the maximum biosorption yield for silica gel immobilized Bacillus sp. ATS-2 was determined as 91.73% at pH 4.0. The higher biosorption yields were observed at flow rates of 60 and 180 ml h(-1). The optimum bed length for the column was found as 10 cm. Data obtained from batch studies were evaluated by Freundlich, Langmuir and Dubinin-Radushkevich (D-R) isotherm models. The maximum monolayer capacity of Bacillus sp. ATS-2 for Pb(II) was 2.36 x 10(-5) mol g(-1). The involvement of the functional groups on the surface of immobilized cells in biosorption process was also evaluated by FTIR spectral analysis.     

Synthesis of Pt-immobilized on silica and polystyrene-encapsulated silica and their applications as electrocatalysts in the proton exchange membrane fuel cell

Nano sized Pt particles were successfully immobilized onto SiO₂ and polystyrene-encapsulated silica core shell (SiO₂@PS). To make the immobilization of Pt onto both silica and polystyrene-encapsulated silica core shell, SiO₂ was first functionalized with -NH₂ using 3-amino propyl trimethoxysilane (APTMS) while for core shell, the negatively charged surface of polystyrene (PS) was changed with positive charge by cationic surfactant such as cetyltrimethylammonium chloride (CTACl) to make the formation of SiO₂ shell on preformed PS sphere. Transmission electron micrograph (TEM) images shows that Pt nanoparticles immobilized onto SiO₂ and SiO₂@PS were to be 3-4 nm without agglomeraiton. The energy dispersive spectroscope (EDS) shows that Pt contents on both SiO₂ and SiO₂@PS were to be 21.45% and 20.28%, respectively. In case of Pt-SiO₂@PS, it is believed that Pt should have been immobilized onto PS surface and pore within SiO₂ shell as well as SiO₂ surface. The MEA fabricated with Pt-SiO₂@PS shows better cell performance than of Pt-SiO₂.     

Microfluidic flow cell for sequential digestion of immobilized proteoliposomes

We have developed a microfluidic flow cell where stepwise enzymatic digestion is performed on immobilized proteoliposomes and the resulting cleaved peptides are analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The flow cell channels consist of two parallel gold surfaces mounted face to face with a thin spacer and feature an inlet and an outlet port. Proteoliposomes (50-150 nm in diameter) obtained from red blood cells (RBC), or Chinese hamster ovary (CHO) cells, were immobilized on the inside of the flow cell channel, thus forming a stationary phase of proteoliposomes. The rate of proteoliposome immobilization was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D) which showed that 95% of the proteoliposomes bind within 5 min. The flow cell was found to bind a maximum of 1 μg proteoliposomes/cm(2), and a minimum proteoliposome concentration required for saturation of the flow cell was determined to be 500 μg/mL. Atomic force microscopy (AFM) studies showed an even distribution of immobilized proteoliposomes on the surface. The liquid encapsulated between the surfaces has a large surface-to-volume ratio, providing rapid material transfer rates between the liquid phase and the stationary phase. We characterized the hydrodynamic properties of the flow cell, and the force acting on the proteoliposomes during flow cell operation was estimated to be in the range of 0.1-1 pN, too small to cause any proteoliposome deformation or rupture. A sequential proteolytic protocol, repeatedly exposing proteoliposomes to a digestive enzyme, trypsin, was developed and compared with a single-digest protocol. The sequential protocol was found to detect ~65% more unique membrane-associated protein (p < 0.001, n = 6) based on peptide analysis with LC-MS/MS, compared to a single-digest protocol. Thus, the flow cell described herein is a suitable tool for shotgun proteomics on proteoliposomes, enabling more detailed characterization of complex protein samples.     

Determination of trace lead in water samples by graphite furnace atomic absorption spectrometry after preconcentration with nanometer titanium dioxide immobilized on silica gel

Nanometer titanium dioxide immobilized on silica gel (immobilized nanometer TiO2) was prepared by sol-gel method and characterized using X-ray diffraction (XRD) and scanning electron microscope (SEM). The adsorptive capability of immobilized nanometer TiO2 for lead was assessed in this work using column method. It was found that lead can be quantitatively retained by immobilized nanometer TiO2 in the pH range 4-7, then eluted completely with 1.0molL(-1) HCl. The adsorption capacity of immobilized nanometer TiO2 for Pb was found to be 3.16mgg(-1). A new method has been developed for the determination of trace lead based on preconcentration with a microcolumn packed with immobilized nanometer TiO2 prior to its determination by graphite furnace atomic absorption spectrometry (GFAAS). The detection limit of this method for Pb was 9.5ngL(-1) with an enrichment factor of 50, and the relative standard deviations (R.S.D.s) was 3.2% at the 10ngmL(-1) Pb level. The method was validated using a certified reference material, and was applied for the determination of trace lead in water samples.     

Phenol degradation by Aureobasidium pullulans FE13 isolated from industrial effluents

The degradation of phenol (2-30 mM) by free cells and by alginate-immobilized cells of Aureobasidium pullulans FE13 isolated from stainless steel effluents was studied in batch cultures with saline solution not supplemented with nutrients or yeast extract. The rate at which the immobilized cells degrade phenol was similar to the rate at which the suspended cells could degrade phenol, for a concentration of up to 16 mM of phenol. The maximum phenol volumetric degradation rate for 16 mM phenol was found to be 18.35 mg l(-1)h(-1) in the assays with free cells and 20.45 mg l(-1)h(-1) in the assays with alginate-immobilized cells, 18 mM phenol and cellular concentration of 0.176 g/l. At concentrations higher than this, an inhibitory effect was observed, resulting in the lowering of the phenol degradation rates. The immobilization was detrimental to the catechol 1,2-dioxygenase activity. However, the immobilized cells remained viable for a longer period, increasing the efficiency of phenol degradation. The yeast showed catechol 1,2-dioxygenase activity only after growth in the phenol, which was induced at phenol concentrations as low as 0.05 mM and up to 25 mM at 45 h of incubation at 30 degrees C. Phenol concentrations higher than 6mM were inhibitory to the enzyme. Addition of glucose, lactate, succinate, and benzoate reduced the rate at which phenol is consumed by cells. Our results suggest that inoculants based on immobilized cells of A. pullulans FE13 has potential application in the biodegradation of phenol and possibly in the degradation of other related aromatic compounds.     

Biosorption of heavy metal ions on immobilized white-rot fungus Trametes versicolor

Trametes versicolor mycelia were immobilized in carboxymethylcellulose, CMC, beads via entrapment, and the bead containing immobilized fungus spores were incubated at 30 degrees C for 3 days to attain uniform growth on the bead surface. After incubation, the live and heat inactivated immobilized fungus on the CMC beads were used for the biosorption of Cu(2+), Pb(2+) and Zn(2+) ions.Plain CMC beads were used as a control system. The biosorption of Cu(2+), Pb(2+) and Zn(2+) ions by the CMC and both live and inactivated immobilized preparations increased as the initial concentration of Cu(2+), Pb(2+) and Zn(2+) ions in the medium increased. The maximum biosorption capacities for both immobilized live and heat inactivated Trametes versicolor were 1.51 and 1.84mmol Cu(2+), 0.85 and 1.11mmol Pb(2+) and 1.33 and 1.67mmol Zn(2+) per g of dry biosorbents, respectively. Biosorption equilibrium was established in about 1.0h and the equilibrium was well described by Langmuir and Freundlich isotherms. A temperature change in the range of 15-45 degrees C did not affect the biosorption capacity. The affect of pH was also investigated and the maximum adsorption of Cu(2+), Pb(2+) and Zn(2+) ions on the CMC and both live and inactivated immobilized fungal biomass was observed between pH 4.0 and 6.0. The CMC beads with the immobilized fungus can be regenerated using 10mM HCl, with up to 97% recovery of the metal ions; the biosorbents reused up to five biosorption-desorption cycles without any major loss in the biosorption capacity.     

A new nano-TiO2 immobilized biodegradable polymer with self-cleaning properties

This study concentrated on the direct immobilization of anatase nano titanium dioxide particles (TiO(2), 10nm particle size) into or onto a biodegradable polymer, polycaprolactone, by solvent-cast processes. The self-cleaning, namely photocatalytic properties of the produced materials were tested by photocatalytic removal of methylene blue as model compound and antimicrobial properties were investigated using Candida albicans as model microorganism. Produced TiO(2) immobilized polymer successfully removed methylene blue (MB, 1 × 10(-5)M) from aqueous solution without additional pH arrangement employing a UV-A light (365 nm) source. Almost 83.2% of dye was removed or decomposed by 5 wt% TiO(2) immobilized into PCL (0.08 g) and removal percentage reached to 94.2% with 5 wt% TiO(2) immobilized onto PCL after a 150 min exposure period. Although removal percentage decrease with increased ionic strength and usage of a visible light source, produced materials were still effective. TiO(2) immobilized onto PCL (5 wt%) was quite effective killing almost 54% of C. albicans (2 × 10(6)CFU/mL) after only 60 min exposure with a near visible light source. Control experiments employing PCL alone in the presence and absence of light were ineffective under the same condition.     

Picomolar peroxide detection using a chemically activated redox mediator and square wave voltammetry

A method for low-level, low-potential electrochemical detection of hydrogen peroxide using a chemically activated redox mediator is presented. This method is unique in that it utilizes a mediator, Amplex Red, which is only redox-active when chemically oxidized by H2O2 in the presence of the enzyme horseradish peroxidase (HRP). When employed in concert with microelectrode square wave voltammetry to optimize sensing at ultralow concentrations (<1 microM), this method exhibits marked improvements in analytical sensitivity and detection limits (limit of detection as low as 8 pM) over existing protocols. Sensing schemes incorporating both freely diffusing and immobilized HRP are evaluated, and the resulting analytical sensitivities are 1.22 +/- 0.04 and (2.1 +/- 0.6) x 10(-1) microA/(microM mm2), respectively, for peroxide concentrations in the high picomolar to low micromolar range. A second linear region exists for lower peroxide concentrations. Furthermore, quantitative enzyme kinetics analysis using Michaelis-Menten parameters is possible through interpretation of data collected in this scheme. Km values for soluble and immobilized HRP were 84 +/- 13 and 504 +/- 19 microM, respectively. This method is amenable to any biological detection scheme that generates hydrogen peroxide as a reactive product.