铈基介孔金属有机骨架固定化酶的构建及催化性能

金属有机骨架(MOF)材料由于其孔隙率高、比表面积大以及具有发达的内联通孔道结构等优点,可以作为优良的生物分子固定化载体。通过表面活性自组装策略制备了铈基介孔MOF(Ce-MOF-F),表征结果表明,该材料有大的比表面积和呈辐射状的介孔孔道结构。以其为载体、南极假丝酵母脂肪酶B(CALB)为模型酶,通过物理吸附法制备了生物催化剂CALB@Ce-MOF-F,对该固定化酶的酶载量和催化性能进行了研究。在优化条件下,CALB的负载量为162.0mg/g载体,水解活性为899.1U/g蛋白。与游离CALB相比,CALB@Ce-MOF-F表现出对高温、酸碱和有机溶剂等有更强的耐受性;将Ce-MOF-F用于多种酶的固定化,研究其作为载体的普适性,结果表明,介孔Ce-MOF-F对洋葱伯克氏菌脂肪酶(BCL)和漆酶有良好的固定效果,可以作为良好载体,并能对酶起到较好的保护作用。     

以HPD-750大孔树脂为载体材料固定化果胶酶

HPD-750树脂是中极性大孔吸附树脂,生物相容性好,机械性能稳定,具有较大的比表面积,可用于固定化酶载体材料。本文以HPD-750大孔树脂为载体固定化果胶酶,研究各因素对固定化酶的影响,并采用正交试验对固定化条件进行优化。结果表明,当pH为4.0、固定化温度为45℃、固定化时间为4h、加酶量为0.16g/mL时,固定化酶活力可达5146U/mg。以HPD-750大孔树脂为载体材料制备的固定化酶相较于游离酶具有更好的酸碱稳定性和热稳定性。在循环使用10次后,酶活力依然保留80%以上;4℃储藏25d之后,其酶活力仍保留60%以上。与D311大孔树脂、聚丙烯酰胺和海藻酸钠微球制备的固定化酶相比,HPD-750大孔树脂固定化酶的活性、操作稳定性、机械稳定性和储存稳定性都较好。该结果说明,HPD-750大孔树脂可作为固定化酶较好的载体材料。     

Synthesis of SBA-15 from low cost silica precursor obtained from sugarcane leaf ash and its application as a support matrix for lipase in biodiesel production

Ordered mesoporous silica material was synthesized from a low-cost precursor, sugarcane leaf ash, was used as a support matrix for lipase for the production of biodiesel. The mesoporous samples were characterized using Fourier transform infra red spectroscopy. The surface topography and morphology of the mesoporous materials were studied using scanning electron microscope. The pore diameter, pore volume, Brunauer Emmett and Teller surface area of the mesoporous material were determined by N₂ gas adsorption technique. Different pore size Santa Barbara Acid-15 (SBA-15) samples were synthesized and their lipase immobilization capacity and specific enzyme activity of immobilization lipase were determined and compared. Lipase from Candida Antarctica immobilized on SBA-15 (C) had shown maximum percentage immobilization and specific enzyme activity. The immobilized lipase mesoporous matrix was used for biodiesel production from crude non-edible Calophyllum inophyllum oil. The percentage yield of fatty acid methyl ester, 97.6 % was obtained under optimized conditions: 100 mg of lipase immobilized on SBA-15, 6:1 methanol to oil molar ratio, the reaction of 2 g C. inophyllum oil with methanol.     

Electron paramagnetic resonance spin label titration: a novel method to investigate random and site-specific immobilization of enzymes onto polymeric membranes with different properties

The immobilization of biological molecules onto polymeric membranes to produce biofunctional membranes is used for selective catalysis, separation, analysis, and artificial organs. Normally, random immobilization of enzymes onto polymeric membranes leads to dramatic reduction in activity due to chemical reactions involved in enzyme immobilization, multiple-point binding, etc., and the extent of activity reduction is a function of membrane hydrophilicity (e.g. activity in cellulosic membrane?polysulfone membrane). We have used molecular biology to effect site-specific immobilization of enzymes in a manner that orients the active site away from the polymeric membrane surface, thus resulting in higher enzyme activity that approaches that in solution and in increased stability of the enzyme relative to the enzyme in solution. A prediction of this site-specific method of enzyme immobilization, which in this study with subtilisin and organophosphorus hydrolase consists of a fusion tag genetically added to these enzymes and subsequent immobilization via the anti-tag antibody and membrane-bound protein A, is that the active site conformation will more closely resemble that of the enzyme in solution than is the case for random immobilization. This hypothesis was confirmed using a new electron paramagnetic resonance (EPR) spin label active site titration method that determines the amount of spin label bound to the active site of the immobilized enzyme. This value nearly perfectly matched the enzyme activity, and the results suggested: (a) a spectroscopic method for measuring activity and thus the extent of active enzyme immobilization in membrane, which may have advantages in cases where optical methods can not be used due to light scattering interference; (b) higher spin label incorporation (and hence activity) in enzymes that had been site-specifically immobilized versus random immobilization; (c) higher spin label incorporation in enzymes immobilized onto hydrophilic bacterial cellulose membranes versus hydrophobic modified poly(ether)sulfone membranes. These results are discussed with reference to analysis and utilization of biofunctional membranes.     

Bioelectrocatalytic Oxygen Reduction by Laccase Immobilized on Various Carbon Carriers

Laccase is an enzyme that is used for fabricating cathodes of biofuel cells. Many studies have been aimed at searching the ways for enhancing specific electrochemical characteristics of cathode with the laccase- based catalyst. The electroreduction of oxygen on the electrode with immobilized laccase proceeds under the conditions of direct electron transfer between the electrode and active enzyme center. In this work, the effect of oxygen partial pressure on the electrocatalytic activity of laccase is studied. It is shown that, at the concentrations of oxygen dissolved in the electrolyte higher than 0.28 mM, the process is controlled by the kinetics of the formation of laccase–oxygen complex, whereas at lower concentrations and a polarization higher than 0.3 V, the process is limited by the oxygen diffusion. A wide range of carbon materials are studied as the carriers for laccase immobilization: carbon black and nanotubes with various BET specific surface areas. The conditions, which provide the highest surface coverage of carbon material with enzyme in the course of spontaneous adsorptive immobilization and the highest specific characteristics when using a “floating” electrode simulating a gas-diffusion electrode, are determined: 0.2 M phosphate-acetate buffer solution; oxygen atmosphere; the carrier material (nanotubes with a BET surface area of 210 m²/g and a mesopore volume of 3.8 cm³/g); and the composition of active mass on the electrode (50 wt % of carbon material + 50 wt % of hydrophobized carbon black).     

京尼平交联磁性壳聚糖微球的制备及其脂肪酶的固定化

采用反相悬浮法与溶胶凝胶法结合制备磁性壳聚糖微球,并以此为载体,京尼平为交联剂,脂肪酶为模型酶进行固定化,研究了酶固定化的最优条件和固定化酶的性质。结果表明,在京尼平浓度为0.6 g/L、交联温度为55 ℃、交联时间8 h,固定化酶的比活力最大,为4.31 U/g。固定化酶在25~35 ℃,pH值在8.0有最大活性,其米氏常数Km为0.26 mol/L。同时,固定化酶具有良好的热稳定性及pH稳定性,可重复利用,且能进行磁分离。     

Combinatorial approach to study enzyme/surface interactions

A fast combinatorial approach to access information about the immobilization behavior and kinetics of enzymes on a variation of surfaces is presented. As a test system, Candida Antarctica Lipase B was immobilized on a self-assembled monolayer bearing a gradient of surface energy. The respective immobilization behavior was monitored by Fourier transform infrared micro-spectroscopy. In addition, the activity of the immobilized enzyme was monitored over the entire film in real time with a specially developed fluorescence activity assay embedded into a siloxane gel. It was found that the highest amount of active protein was immobilized on the hydrophilic end of the gradient surface. This effect is associated with a higher surface roughness of this area resulting in hydrophobic micro-environments in which the enzyme gets immobilized.     

Preparation of an ultrafiltration membrane from the copolymer of acrylonitrile–glycidylmethacrylate utilized for immobilization of glucose oxidase

Ultrafiltration membrane has been prepared from the copolymer of acrylonitrile–glycidylmethacrylate and the porosity of the membrane was studied. The asymmetric structure was proved by scanning electron microscopy. The basic characteristics of the membrane were measured – water permeability, water content, membrane selectivity, etc. The membrane obtained was used as a carrier for immobilization of glucose oxidase. The immobilization was carried out covalently by two methods: direct bonding of the enzyme and indirectly by a spacer (hexamethylenediamine) and cross-linking agent (glutar aldehyde). The amount of bound protein and relative activity of the immobilized glucose oxidase were determined. Temperature optimum, pH optimum and storage stability of the immobilized glucose oxidase were determined. It was proved that glucose oxidase immobilized by the direct method shows better characteristics compared with the indirect method.     

Covalent attachment of microbial lipase onto microporous styrene-divinylbenzene copolymer by means of polyglutaraldehyde

A novel method for immobilization of Thermomyces lanuginosus lipase onto polyglutaraldehyde-activated poly(styrene-divinylbenzene) (STY-DVB), which is a hydrophobic microporous support has been successfully developed. The copolymer was prepared by the polymerization of the continuous phase of a high internal phase emulsion (polyHIPE). The concentrated emulsion consists of a mixture of styrene and divinylbenzene containing a suitable surfactant and an initiator as the continuous phase and water as the dispersed phase. Lipase from T. lanuginosus was immobilized covalently with 85% yield on the internal surface of the hydrophobic microporous poly(styrene-divinylbenzene) copolymer and used as a biocatalyst for the transesterification reaction. The immobilized enzyme has been fully active 30 days in storage and retained the activity during the 15 repeated batch reactions. The properties of free and immobilized lipase were studied. The effects of protein concentration, pH, temperature, and time on the immobilization, activity, and stability of the immobilized lipase were also studied. The newly synthesized microporous poly(styrene-divinylbenzene) copolymer constitutes excellent support for lipase. It given rise to high immobilization yield, retains enzymatic activity for 30 days, stable in structure and allows for the immobilization of large amount of protein (11.4mg/g support). Since immobilization is simple yet effective, the newly immobilized lipase could be used in several application including oil hydrolysis, production of modified oils, biodiesel synthesis, and removal of fatty acids from oils.     

Determination of active diaphorase immobilized at electrode surfaces

The activity of diaphorase (from Bacillus stearothermophilus) immobilized on glassy carbon (GC) electrodes was determined by analyzing the catalytic currents for oxidation of the immobilized enzyme using digital simulation techniques, which gives the concentration of the active enzyme at the electrode surface. Results show that the immobilization by the cross-linking reaction with glutaraldehyde deactivates the enzyme and only about 10% of the total enzyme remains active at the electrode surface.     

A General Strategy for Site‐Directed Enzyme Immobilization by Using NiO Nanoparticle Decorated Mesoporous Silica

Mesoporous materials have recently gained much attention owing to their large surface area, narrow pore size distribution, and superior pore structure. These materials have been demonstrated as excellent solid supports for immobilization of a variety of proteins and enzymes for their potential applications as biocatalysts in the chemical and pharmaceutical industries. However, the lack of efficient and reproducible methods for immobilization has limited the activity and recyclability of these biocatalysts. Furthermore, the biocatalysts are usually not robust owing to their rapid denaturation in bulk solvents. To solve these problems, we designed a novel hybrid material system, mesoporous silica immobilized with NiO nanoparticles (SBA‐NiO), wherein enzyme immobilization is directed to specific sites on the pore surface of the material. This yielded the biocatalytic species with higher activity than free enzyme in solution. These biocatalytic species are recyclable with minimal loss of activity after several cycles, demonstrating an advantage over free enzymes.     

Immobilization of Penicillin G Acylase on Calcined Layered Double Hydroxides

A hydrotalcite-like Mg2 /Al3 layered double hydroxide (LDH) material was prepared by means of amodified coprecipitation method involving a rapid mixing step followed by a separate aging process. LDH calcined at 500℃ , denoted as CLDH, was characterized by XRD, IR and BET surface area measurements.CLDH has a poor crystalline MgO-like structure with a high surface area and porosity. CLDH was used as asupport for the immobilization of penicillin G acylase(PGA). The effect of varying the immobilization conditions, such as pH, contact time and the ratio of enzyme to support, on the activity of the immobilized enzymein the hydrolysis of penicillin G has been studied. It was found that the activity of the immobilized enzyme decreased slightly with decreasing pH and reached a maximum after a contact time of 24 h. The activity of theimmobilized enzyme increased with increasing the ratio of enzyme to support. It was found that the adsorption of PGA inhibited the expected reaction of CLDH with an aqueous medium to regenerate a LDH phase. Itsoriginal activity(36%) after 15 cycles of reuse of the immobilized enzyme was retained, but no further loss in the activity was observed.     

漆酶在纳米多孔金上的固定化及其酶学性质研究

利用纳米材料为载体对酶等生物大分子进行固定化近年来引起人们的浓厚兴趣. 以Au/Ag合金为原料, 通过控制浓硝酸的腐蚀时间再辅以退火处理得到了不同孔径的纳米多孔金(NPG), 利用扫描电镜(SEM)和N2气体吸附仪对孔性质进行了表征. 以NPG为载体, 用α-硫辛酸和N-乙基-N’-(3-二甲基氨基丙基)碳酰二亚胺/N-羟基琥珀酰亚胺(EDC/NHS)对金表面进行活化, 通过化学共价偶联的方法对产自Trametes versicolor的漆酶进行了固定化. 比较了孔径大小对酶固定化量及比活力的影响. 发现小孔径更有利于对该漆酶的固定化. 与游离酶相比, 固定化酶的最适pH没有改变, 但最适温度却从原来的40 ℃升到了60 ℃. 固定化后, 漆酶的pH和热稳定性都明显提高了. 重复使用8次仍能保持初始活力的65%, 且在4 ℃下保存1个月几乎观察不到酶活力的下降. 此外, 失活的固定化酶经浓硝酸处理后, NPG载体可重复利用. 本结果初步显示出了NPG在生物技术领域中的应用潜力.     

Preparation,characterization, and luminescence of (SBA-15) immobilized pepsin

SBA-15 mesoporous silica was synthesized by hydrothermal method and its surface was methylated by treatment with methyltrimethoxysilane. Pepsin was immobilized on the obtained materials giving host-guest composite materials (SBA-15)-pepsin and (methylated SBA-15)-pepsin. The optimum conditions for preparation of these materials were established. Methylated SBA-15 (M-SBA-15) has improved immobilization efficiency of enzyme compared to initial SBA-15 silica. It was shown that with the gradual increase of NaCl solution ionic strength the immobilized amount of enzyme was reduced. Powder X-ray diffraction and Fourier transform infrared spectroscopy showed that the host frameworks in the prepared host-guest composite materials are intact and the ordered structure was retained. Scanning electron microscopic studies revealed fibrous morphologic characteristics of the SBA-15 and the immobilized pepsin composite materials. The average particle diameter of (SBA-15)-pepsin composite was 338 ± 10 and 343 ± 10 nm for (M-SBA-15)-pepsin. The low temperature N₂ adsorption-desorption study at 77 K showed that the pore sizes and specific surface areas of the host-guest composite materials were smaller than those before the introduction of the enzyme, suggesting that the immobilized enzyme occupied a definite position in the host material pore channels. The UV-vis solid diffuse reflectance and luminescence studies showed that the enzyme was successfully immobilized on to the host material and that after the immobilization of enzyme on SBA-15 the conformation of pepsin macromolecule has not been changed.     

氨基酸分子改性的SiO2杂化材料用于胰蛋白酶固定化

为改善二氧化硅载体材料本身的生物相容性和疏水性,维持包埋生物分子的活性,本文对水解前驱体3-氨基丙基三甲氧基硅烷进行氨基酸分子改性。具体过程包括N-Fmoc-L-缬氨酸和氯化亚砜反应生成N-Fmoc-L-缬氨酰氯,再和3-氨基丙基三甲氧基硅烷反应生成N-（3-三甲氧基硅基）丙基-N′-Fmoc-L-缬氨酰胺后。然后去除Fmoc,得到N-（3-三甲氧基硅基）丙基-L-缬氨酰胺作为氨基酸修饰的硅源前驱体。通过IR、MS、1H-NMR等分析测试手段对合成得到的各个化合物的结构进行了表征。利用正硅酸甲酯（TMOS）和N-（3-三甲氧基硅基）丙基-L-缬氨酰胺为复合硅源,经过溶胶-凝胶过程来包埋了胰蛋白酶,研究得到最适的固定化条件为,N-（3-三甲氧基硅基）丙基-L-缬氨酰胺的含量为15mol%。在该条件下,固定化胰蛋白酶活力的绝对值是199U,游离酶的酶活力的绝对值是103U, 四甲氧基硅烷直接包埋的固定化酶活力的活性是38 U。在该条件下,杂化硅源得到的固定化酶的活性是以四甲氧基硅烷水解前驱体的固定化酶活性的5倍,杂化硅源固定化胰蛋白酶的最相比游离酶,酶的最高活力提高的几乎2倍。这些结果表明氨基酸分子对水解前驱体修饰以后,水解产生的固定化载体具有良好的生物相容性。通过改性载体制备的固定化酶,对甲醇变性剂的稳定性,对酸碱的抵抗性及热稳定性也有明显地提高。     

Characterization of hydrophobic sol-gel materials containing entrapped lipases

The entrapment of lipases in hydrophobic sol-gel materials of RSi(OCH₃)₃ or mixtures of RSi(OCH₃)₃ and Si(OCH₃)₄ results in heterogeneous biocatalysts having dramatically enhanced enzyme activities as measured by the esterification of lauric acid by n-octanol in isooctane. These materials have been characterized by solid state NMR studies, revealing the degree of cross-linking. It is shown that this parameter generally does not correlate with relative enzyme activity. Likewise, the specific surface area or the pore size does not seem to be the decisive factor in determining the relative enzyme activities of the lipase-containing hybrid gels and the corresponding SiO₂-gels obtained from Si(OCH₃)₄. Scanning electron microscopic studies (SEM) show that the hybrid gels all have a similar morphology. On the basis of these studies a model is proposed according to which most of the lipase enzyme is entrapped near the surface of the gel particles, where it is readily accessible by substrate molecules.     

Synthesis with Glutaraldehyde and Characterization of Uniformly Coated Alkaline Phosphatase-Nanoparticle Bioconjugates

Alkaline phosphatase from raw milk was immobilized on cysteine-functionalized silver nanoparticles with high efficiency. The nanoparticles were characterized by ultraviolet–visible spectroscopy, X-ray diffraction, and transmission electron microscopy. The surface functionalization was confirmed by infrared spectroscopy. The spherical nanoparticles were from 40 to 60?nm in size and used for the covalent immobilization of alkaline phosphatase on the surface with glutaraldehyde treatment. As compared to soluble enzyme, an enhanced enzymatic activity of 79.87% was obtained with a percentage immobilization of 75.41%. The immobilization process did not significantly affect the structure and size of the nanoparticles, while providing a uniform coating of the enzyme on the nanoparticle as characterized by electron microscopy. The bioconjugates were reusable for up to eight times with 85% retention of the initial enzymatic activity. The synthesis of these enzyme–nanoparticle bioconjugates with high activity and stability suggests their use in biological applications.     

A new method to prepare macroporous copolymer of glycidyl methacrilate and ethylene dimethacrilate as enzyme carrier

Macroporous copolymer of glycidyl methacrylate and ethylene dimethacrylate containing surface epoxy groups was firstly synthesized with dodecyl alcohol and cyclohexanol as liquid pore-forming agents and nanosize calcium carbonate as solid porogenic agent. The scanning electron microscopy was used to characterize their surface structure. Under the optimum conditions, β-galactosidase from Aspergillus oryzae was immobilized on the support obtained above, and the basic property and the kinetic data of the reaction on immobilized enzyme were determined. These data were compared with those obtained for the enzyme immobilized on the support prepared only with the liquid solution as pore-forming agent. Satisfactory results were obtained in enzyme activity, immobilization yield, pH stability, thermal stability, operational stability, and Michaelis constants K _M. The results indicated that the copolymer of glycidyl methacrilate and ethylene dimethacrylate newly prepared was more suitable to immobilize enzyme than the carrier synthesized with traditional method.     

Single-step purification and immobilization of penicillin acylase using hydrophobic ligands

Five differenthydrophobic ligands immobilized on 4% (4XL) and 6% (6XL) crosslinked agarose were used to study the single-step purification of penicillin acylase from cell lysate. The 4XL gels showed relatively higher specific activity and recovery than the 6XL gels. In single-step purification, highly active enzyme (42 U/mg) was obtained using moderately hydrophobic ligand (octyl). The crude enzyme immobilized on octyl gel by adsorption showed significant operational stability over a period of 30 d at room temperature. Reactor studies demonstrated the feasibility of hydrophobic ligands as a medium for immobilization.     

A Trypsin Immobilized Sol-Gel for Protein Indentification in MALDI-MS Applications

The proteolytic enzyme trypsin was chemically immobilized to an amine-functionalized sol-gel using adipoyl chloride under nonaqueous conditions and a nitrogen atmosphere. In the synthesis of the sol-gel, tetraethyl orthosilicate (TEOS), and 3-(2-aminoethylamino) propyldimethoxymethylsilane (AEAPMS) (50:50, v/v) were used, which provided convenient physical and chemical conditions to maintain catalytic activity of immobilized trypsin molecules for the digestion of proteins in proteomics applications. Bovine serum albumin was used as a model protein to perform enzymatic digestion using the trypsin immobilized sol-gel. The resulting peptides were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry to evaluate the digestion performance and specificity of the sol-gel material. The trypsin immobilized sol-gel showed superior enzymatic activity in protein digestion and it was determined that the sol-gel material could be repeatedly used at least 25 times without significant activity loss in long-term use. Additionally, autocatalysis was prevented by immobilization of trypsin. The peptide digest having the highest purity was obtained for protein identification studies.