链霉菌LD048硫化物氧化酶的催化机理及酶学特征

对硫化物氧化新菌株链霉菌LD048硫化物氧化酶的催化机理研究表明S2-在硫化物氧化酶的作用下首先被氧化为S2O2-3,然后生成SO2-3,最后产生末端产物SO2-4;O2对细胞生长是必须的,同时是反应过程中的电子受体.酶学特征研究说明酶的最适反应温度为大于35℃,在80℃维持4 h酶活仍能完全保留;酶的最适反应pH为8.0;金属Mn2+是酶活性的有效促进剂,在实验浓度范围内酶活提高达35.6%;酶促反应的最适底物浓度为6.0 mmol/L.     

硫化物氧化兼性自养金色链霉菌LD48培养的影响因素研究

硫化物氧化、兼性自养金色链霉菌LD48在以Na3S为唯一硫源的条件下对硫化物氧化酶产生的最适接种量为1％。在试验的pH值范围内,起始培养最适pH值为7．4。随着培养液中乙醇体积分数由1％～5％增加,菌体生长和硫化物氧化活性的积累都在下降。酵母提取物是一有效的生长刺激因子,在无机氮源(NH4)2HPO4的基础上添加0.15％的酵母提取物,能够提高菌体生物量95％。S2O3^2-与SO4^2-对菌体生长和代谢产物积累都有严重的抑制作用。培养过程分析表明,培养56h硫化物氧化酶产生达到稳定水平。     

固定化多酚氧化酶催化合成邻苯二酚

利用壳聚糖固定化蘑菇多酚氧化酶催化氧化苯酚合成邻苯二酚,考察了固定化多酚氧化酶羟基化反应条件对目的产物产率的影响.确定适宜的羟基化工艺条件为:以氯仿为溶剂、底物浓度为20 mmol·L-1,pH值为7.0,含水量为0.9%、温度为30℃、反应时间为5 h,在此条件下催化合成邻苯二酚的产率为11.02%.     

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     

多酚氧化酶的改性蛭石固定化及其催化特性研究

以马铃薯为原料,提取其中的多酚氧化酶,采用改性蛭石为载体,对其进行固定化,研究游离与固定化酶的最适催化温度、pH值,固定化酶的储存稳定性、重复使用稳定性及对苯酚的清除性能。结果表明,改性后蛭石出现了较多沟壑状孔隙,颗粒质感疏松,有利于固定化酶分子;最佳固定化条件为:改性蛭石含量0.4 g,戊二醛浓度2.0%,搅拌时间2 h,在此条件下,酶活力回收率可达到60.67%;游离与固定化酶的最适催化温度均为50℃,最适催化pH值均为7.0;固定化酶4℃下储存28 d后酶活力可保留52.1%,具有较好的储存稳定性;重复使用5次后,仍能保持61.0%的初始活性,说明固定化酶构象较稳定,固定化马铃薯多酚氧化酶对苯酚具有较好的清除性能。     

多酚氧化酶的改性蛭石固定化及其催化特性研究

以马铃薯为原料,提取其中的多酚氧化酶,采用改性蛭石为载体,对其进行固定化,研究游离与固定化酶的最适催化温度、pH值,固定化酶的储存稳定性、重复使用稳定性及对苯酚的清除性能。结果表明,改性后蛭石出现了较多沟壑状孔隙,颗粒质感疏松,有利于固定化酶分子;最佳固定化条件为:改性蛭石含量0.4 g,戊二醛浓度2.0%,搅拌时间2 h,在此条件下,酶活力回收率可达到60.67%;游离与固定化酶的最适催化温度均为50℃,最适催化pH值均为7.0;固定化酶4℃下储存28 d后酶活力可保留52.1%,具有较好的储存稳定性;重复使用5次后,仍能保持61.0%的初始活性,说明固定化酶构象较稳定,固定化马铃薯多酚氧化酶对苯酚具有较好的清除性能。     

D-氨基酸氧化酶的分离纯化及固定化研究

在工业生产的原料当中,D-氨基酸氧化酶属于一种具有重要的工业价值的原料,可以良好构建出大肠杆菌中经过乳糖诱导发酵的表达式,从而对其中的菌体进行提取粗酶液,在进行固化提纯的过程当中,需要保证其中的活性物质的活性没有明显的损失,提升其底物部分的转化率,因此就需要对氨基酸氧化酶的分离提纯技术进行研究。首先对D-氨基酸氧化酶进行了概述,其次对其提纯固化的方式进行了研究。     

硫化物的生物催化降解研究进展

综述了对硫化物生物催化降解的自养微生物、兼性营养菌与异养菌的分离与性能研究进展,固定化功能性细胞或酶对硫化物的生物催化降解以及硫化物氧化酶的催化机理及酶学特征,并展望了硫化物生物催化降解方法的应用前景.     

一种新型固定化黄嘌呤氧化酶的制备与性能评价

为了使黄嘌呤氧化酶（XOD）可回收且能够长时间多次使用,本研究以色谱级硅球（SiO2）为载体,采用共价键和的方式用碳纳米管（CNTs）对硅球进行包裹,并将XOD在碳纳米管包裹的硅胶球上进行固定,制备了一种新型的以碳纳米管为介质的固定化黄嘌呤氧化酶（XOD@CNTs@SiO2）。采用TEM和FTIR对固定化酶进行表面形貌和反应基团变化的表征;以产物尿酸为指标进行相对酶活性的评价;采用Bradford法对载体的担载量进行评价。结果表明,硅球表面经CNTs包裹后,XOD的担载量和相对酶活性分别提高2倍和2.6倍。XOD@CNTs@SiO2在60℃储存时,储存时间至400 min后,活性仍能保持至最初的80%。4℃避光保存时,第11d时相对活性仍为60%~70%。室温反应条件下,连续使用至9次时,XOD@CNTs@SiO2-300的相对酶活性仍能保留66.1%。     

Influence of chitosan derivatization on its physicochemical characteristics and its use as enzyme support

Chitosan was derivatized by two methodologies for analyzing their effect on chitosan physicochemical characteristics and its applicability as carrier for Bacillus circulans β‐galactosidase immobilization. Glutaraldehyde (GA) and epichlorohydrin (EPI) were used for crosslinking and activation of chitosan, producing the corresponding supports (C‐GA and C‐EPI‐EPI) after a one‐step and a two‐step process, respectively. The spherical shape and mean diameter of chitosan particles was not significantly affected by polymer derivatization, while Fourier transform infrared analysis showed that in both cases, chitosan polymer was chemically modified. TGA analysis indicated that C‐EPI‐EPI was the most thermally stable. The high degree of activation of C‐EPI‐EPI (586 μmol of aldehydes/g) resulted in the highest loss of activity during immobilization; hence a support with 100 μmol of aldehydes/g was produced (C‐EPI‐EPI₁₀₀). The highest expressed activity (89.3 IU/g) was obtained with the enzyme immobilized in C‐GA, while the biocatalyst with highest thermal stability at 60°C was obtained with C‐EPI‐EPI₁₀₀ (half‐life was 84‐fold higher than the one of the soluble enzyme). The best compromise between biocatalyst expressed activity and thermal stability corresponded to β‐galactosidase immobilized in C‐EPI‐EPI₁₀₀. According to this study, chitosan derivatized with EPI is a thermally stable carrier appropriate for producing highly stable immobilized B. circulans β‐galactosidase. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40171.     

磁性壳聚糖微球的合成及其在固定化血管紧张素转化酶的应用

以化学共沉淀法合成Fe3O4纳米粒子为磁核,采用乳化交联法制备磁性壳聚糖微球,并对其形貌、结构和磁饱和强度等性质进行了表征。以磁性壳聚糖微球作为载体,固定化猪肺粗提物中的血管紧张素转化酶,并对固定化条件进行研究。结果表明,固定化血管紧张素转化酶的最佳条件为：pH值为8.3,最佳温度为50 ℃,最佳时间为1.5 h,最佳酶溶液蛋白浓度为6 mg/mL,此时固定化酶活力最高为0.048 U/g微球。与游离酶相比,固定化酶的pH值稳定性和热稳定性均得到提高。固定化酶重复使用10次,仍然保持40%以上相对活力,说明磁性壳聚糖微球是固定化血管紧张素转化酶的良好载体。     

Immobilization of a nonspecific chitosan hydrolytic enzyme for application in preparation of water‐soluble low‐molecular‐weight chitosan

A nonspecific chitosan hydrolytic enzyme, cellulase, was immobilized onto magnetic chitosan microspheres, which was prepared in a well spherical shape by the suspension crosslinking technique. The morphology characterization of the microspheres was carried out with scanning electron microscope and the homogeneity of the magnetic materials (Fe₃O₄) in the microspheres was determined from optical micrograph. Factors affecting the immobilization, and the properties and stabilities of the immobilized enzyme were studied. The optimum concentration of the crosslinker and cellulase solution for the immobilization was 4% (v/v) and 6 mg/mL, respectively. The immobilized enzyme had a broader pH range of high activity and the loss of the activity of immobilized cellulase was lower than that of the free cellulase at high temperatures. This immobilized cellulase has higher apparent Michaelis–Menten constant K_m (1.28 mg/mL) than that of free cellulase (0.78 mg/mL), and the maximum apparent initial catalytic rate V_max of immobilized cellulase (0.39 mg mL^?1 h^?1) was lower than free enzyme (0.48 mg mL^?1 h^?1). Storage stability was enhanced after immobilization. The residual activity of the immobilized enzyme was 78% of original after 10 batch hydrolytic cycles, and the morphology of carrier was not changed. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 1334–1339, 2006     

Immobilization of hybridoma cells in chitosan alginate beads

A new method for immobilizing hybridoma cells using chitosan-stabilized calcium alginate beads was developed. The ionotropic gelation of chitosan and calcium with alginate resulted in the formation of highly cross-linked, porous beads that were mechanically and chemically stable in phosphate buffered medium. Hybridomas entrapped in these beads were cultured semi-continuously using periodic medium exchange. Viable population densities in the order of 5 × I0⁷ cells/mL were attained within the beads and up to two-fold increases in volumetric monoclonal antibody (MAb) productivity over batch suspension cultures were observed. Oxygen mass transfer limitations within the chitosan-alginate beads were evaluated by considering biokinetics and diffusive transport. Model equations were developed and used to evaluate the effect of bead diameter on the contained cells. The predictions were consistent with experimental observations of maximum viable population densities attained in beads of various size.     

Immobilization of chitosan on nylon 6,6 and pet granules through hydrolysis pretreatment

Chitosan has been increasingly studied as an adsorbent for removing heavy metal ions and organic compounds from aqueous solutions. Most of the studies used chitosan in the form of flakes, powder, or hydrogel beads. This research investigates the immobilization of chitosan on other granular materials to overcome the poor mechanical property of chitosan and offers the potential for chitosan to be used as a regenerable adsorbent. Nylon 6,6 and poly(ethylene terephthalate) (PET) granules were partially hydrolyzed under an acidic or alkaline condition to allow chitosan to be coated or immobilized on the granules' surfaces. The surface morphologies of nylon 6,6 or PET granules before and after hydrolysis and those with immobilized chitosan layer were examined by scanning electron microscopy (SEM), and their surface properties were characterized through ζ‐potential analysis and X‐ray photoelectron spectroscopy. The immobilization of chitosan on nylon 6,6 or PET granules was identified to be through the formation of the salt structure (–NH…^?OOC–) between the surfaces of hydrolyzed nylon 6,6 or PET granules and the chitosan layer. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 3973–3979, 2003     

Effect of substrate (ZnO) morphology on enzyme immobilization and its catalytic activity

In this study, zinc oxide (ZnO) nanocrystals with different morphologies were synthesized and used as substrates for enzyme immobilization. The effects of morphology of ZnO nanocrystals on enzyme immobilization and their catalytic activities were investigated. The ZnO nanocrystals were prepared through a hydrothermal procedure using tetramethylammonium hydroxide as a mineralizing agent. The control on the morphology of ZnO nanocrystals was achieved by varying the ratio of CH₃OH to H₂O, which were used as solvents in the hydrothermal reaction system. The surface of as-prepared ZnO nanoparticles was functionalized with amino groups using 3-aminopropyltriethoxysilane and tetraethyl orthosilicate, and the amino groups on the surface were identified and calculated by FT-IR and the Kaiser assay. Horseradish peroxidase was immobilized on as-modified ZnO nanostructures with glutaraldehyde as a crosslinker. The results showed that three-dimensional nanomultipod is more appropriate for the immobilization of enzyme used further in catalytic reaction.     

Immobilization of horseradish peroxidase on chitosan for use in nonaqueous media

Chitosan, a natural polysaccharide, was used for the covalent immobilization of horseradish peroxidase, an enzyme of high synthetic utility, with the carbodiimide method. Of the enzyme, 62% was immobilized on chitosan when 1‐ethyl‐3‐(3‐dimethylaminopropyl carbodiimide) was used as the peptide coupling agent. The influence of different parameters, such as the enzyme concentration, carbodiimide concentration, and incubation period, on the activity retention of the immobilized enzyme was investigated. Kinetic studies using horseradish peroxidase immobilized on chitosan revealed the effects of several parameters, such as the substrate hydrophilicity and hydrophobicity, the solubility of substrates in the medium, the solvent hydrophobicity, and the support aquaphilicity, on the catalytic activity of the immobilized enzyme in nonaqueous media. General rules for the optimization of solvents for nonaqueous enzymology based on the partitioning of the solvent were not applicable for the immobilized horseradish peroxidase. The catalytic efficiency was greatest when o‐phenylene diamine was used as the substrate and least when guaiacol was used. The aquaphilicity of the support played an important role in the kinetics of the immobilized horseradish peroxidase in water‐miscible solvents. The results were promising for the future development of chitosan‐immobilized enzymes for use in organic media. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 1456–1464, 2003     

碳纳米管负载壳聚糖络合铂配合物的制备及其催化硅氢加成性能

将壳聚糖(CS)负载到碳纳米管(CNT)上得到CNT-CS,再与Pt配位,制得CNT负载CS络合铂配合物(CNT-CS-Pt).将后者用作烯丙基缩水甘油醚和三乙氧基硅烷的硅氢加成反应的催化剂,并考察了该配合物的催化性能.结果表明,Pt与CS中的N原子配位形成Pt-N配位键;该催化剂在烯丙基缩水甘油醚和三乙氧基硅烷的反应中表现出极好的区域选择性及良好的催化活性,β-加成产物的选择性为100%,130 ℃下反应180 min,产物的收率达到70.4%.该催化剂可通过简单的方法回收但重复使用性能有一定下降,重复使用4次后产物的收率下降到24.0%.并对催化机理进行了初步的探讨.     

Immobilization of palladium nanoparticles on latex supports and their potential for catalytic applications

Palladium nanoparticles were reduced in the presence of several latex dispersions possessing different hydrophobicities. Various reduction methods were investigated, specifically the slower methods of refluxing the alcoholic solution and the more rapid reduction by potassium tetrahydridoborate. In several cases the latexes showed the ability to adsorb and immobilize the palladium nanoparticles on their surface. Transmission electron microscopy was employed to show the immobilization of the metal nanoparticles on the latex surfaces, and their nanosize dimensions. The latex-metal dispersions showed catalytic activity for the hydrogenation of cyclohexene as a model reaction. A selection of water-soluble protective polymers was included to explore whether the metal nanoparticles were still immobilized. In the case of the more hydrophobic latexes, the accumulation and immobilization of the metal nanoparticles was preserved both before and after their use as hydrogenation catalysts.