Studies of structural composition distribution heterogeneity in ethylene/1-hexene copolymers using thermal fractionation technique (SSA): Effect of catalyst structure

Investigations into the compositional heterogeneity of ethylene/1-hexene copolymers obtained with various zirconocene/MAO catalysts, either homogeneous or supported on inorganic carriers such as a complex of magnesium chloride with tetrahydrofuran or methyl alcohol, were conducted. The dependence between metallocene structure, as well as catalyst immobilization, and the compositional heterogeneity of the related products was investigated. It was found that the heterogeneity of copolymers is determined by the metallocene catalyst structure. The amount of peaks on the DSC thermograms of copolymers and their division increase with the increase of bulkiness of the ligand in the catalytic system. The immobilization of the investigated catalysts on the magnesium carrier leads to an increase of the copolymer's compositional heterogeneity. However, the modification of the MgCl₂ carrier by tetrahydrofuran or methyl alcohol seems to not have any influence on the copolymers’ CCD.     

Perfusible and non-perfusible supports with monoclonal antibodies for bioaffinity chromatography of Escherichia coli penicillin amidase within its pH stability range

Several monoclonal antibodies (mABs) have been prepared and immobilized for the biospecific isolation of penicillin amidase (PA) from Escherichia coli (EC 3.5.1.11), an enzyme without S---S bridges and a pH stability range of 4–9. During the immobilization the fluorescence emission maxima of the mABs were found to change from 336 nm to ca. 350 nm. Only one of these mABs was found to be suitable for preparative bioaffinity chromatography of PA within the pH stability range. This mAB was immobilized on different spherical supports (Eupergit C 250 L and Sepharose) and one perfusible support (Knauer Quick Disc) and used for analytical and preparative bioaffinity chromatography. Under isocratic conditions the plate height for the perfusible biospecific adsorbent was found to be an order of magnitude lower than for the other supports. The different forms of this proteolytically processed bacterial enzyme could not be separated, however, by the biospecific adsorbents. At the mAB density used in the adsorbents (10–3O μM), less than 30% of the theoretical binding capacity of the immobilized mABs could be used to adsorb the enzyme.     

Calmodulin-mediated reversible immobilization of enzymes

This work demonstrates the use of the protein calmodulin, CaM, as an affinity tag for the reversible immobilization of enzymes on surfaces. Our strategy takes advantage of the of the reversible, calcium-mediated binding of CaM to its ligand phenothiazine and of the ability to produce fusion proteins between CaM and a variety of enzymes to reversibly immobilize enzymes in an oriented fashion to different surfaces. Specifically, we employed two different enzymes, organophosphorus hydrolase (OPH) and beta-lactamase and two different solid supports, a silica surface and cellulose membrane modified by covalently attaching a phenothiazine ligand, to demonstrate the versatility of our immobilization method. Fusion proteins between CaM-OPH and CaM-beta-lactamase were prepared by using genetic engineering strategies to introduce the calmodulin tail at the N-terminus of each of the two enzymes. In the presence of Ca(2+), CaM adopts a conformation that favors interaction between hydrophobic pockets in CaM and phenothiazine, while in the presence of a Ca(2+)-chelating agent such as EGTA, the interaction between CaM and phenothiazine is disrupted, thus allowing for removal of the CaM-fusion protein from the surface under mild conditions. CaM also acts as a spacer molecule, orienting the enzyme away from the surface and toward the solution, which minimizes enzyme interactions with the immobilization surface. Since the method is based on the highly selective binding of CaM to its phenothiazine ligand, and this is covalently immobilized on the surface, the method does not suffer from ligand leaching nor from interference from other proteins present in the cell extract. An additional advantage lies in that the support can be regenerated by passing through EGTA, and then reused for the immobilization of the same or, if desired, a different enzyme. Using a fusion protein approach for immobilization purposes avoids the use of harsh conditions in the immobilization and/or regeneration steps, which could cause inactivation of the immobilized enzyme. Moreover, we have demonstrated that the CaM affinity tag allows immobilization of enzymes on a variety of surfaces without compromising their enzymatic activity substantially; for example, the immobilized OPH retained more than 80% of the activity of the free enzyme. Our results with beta-lactamase showed the feasibility of using a phenothiazine surface in several consecutive loading and regeneration cycles. This can be advantageous when expensive and/or difficult to obtain immobilization surfaces have to be employed; the immobilization surface could be reused to immobilize the same or a different enzyme using the CaM affinity tail. We also determined that the phenothiazine-modified silica particles are stable for long periods of time, i.e., up to 2 years when stored at 4 degrees C. It is envisioned that this type of reversible immobilization may find applications in the development of reversible, reusable biosensors and bioreactors endowed with the additional advantage that the biological element at the surface of the sensor or bioreactor could be replaced under mild conditions when needed to sense or process a different target molecule.     

Kinetic study of catalase adsorption on disperse carbonaceous matrices

The effect of the main factors known to govern the kinetic regularities of enzyme adsorption, such as enzyme solution concentration, temperature, pH, specific surface of the adsorbent, etc., were studied. Two kinds of disperse carbonaceous materials-activated carbon NORIT and carbon black PM-100, were used as matrices for enzyme immobilization. For both immobilization matrices studied, the amount of the adsorbed enzyme was found to reach saturation at catalase (CAT) enzyme concentrations exceeding 20 mg·mL⁻¹ (∼100 μM). The pH of the solution affected the adsorption capacities of the selected immobilization matrices; larger amounts of CAT adsorbed were estimated in neutral and alkaline solutions than under acidic conditions for enzyme immobilization. UV-spectrophotometry was employed as a basic analytical approach in this study.     

Application of the FCP-activation procedure to the synthesis of a biospecific adsorbent for trypsin

The synthesis of a biospecific adsorbent for trypsin was chosen as a model to investigate the applicability of FCP activation in affinity chromatography.p-Aminobenzamidine was chosen as a ligand, directly suitable for immobilization. The nonspecific binding properties of the first series of synthesized agarose derivatives were obviated either by FCP activation of the ligand instead of the matrix, or by modifying the initial FCP-activation procedure. The adsorbents prepared in this way, however, demonstrated no selectivity between trypsin and chymotrypsin. The introduction ofe-aminocaproic acid as a spacer was ineffectual. These problems were solved by the application of glycylglycine as a spacer. The final affinity matrices had a degree of substitution of approximately 4 μ.mol of ligand per gram gel (100 μmol ligand per gram dry adsorbent). The specific activity of a current trypsin preparation was increased by 58% in a single cycle. The biospecificity of these adsorbents was demonstrated.     

Purification and some properties of tryptophan-5-monooxygenase from rabbit hindbrain

An affinity chromatography procedure for the rapid purification of tryptophan-5-monooxygenase from rabbit hindbrains was developed using e-aminocaproyl-D-tryptophan methyl ester-Sepharose-4B gels. The precise requirements for the optimal biospecific interaction between the affinity ligand and the ligate (enzyme) was established from a study of the effects of the variation in the length of the "spacer’’ on the affinity properties of the gel. The enzyme preparation isolated by this procedure was found to be essentially homogeneous and was characterized by a molecular weight of 200,000 ±20,000. SDS-polyacrylamide gel electrophoresis of the enzyme revealed it to be a dimer, the molecular weight of each subunit being approximately 90,000. The specific activity of the enzyme preparation is approxi-mately 7-10 times that of the crude homogenate, but a further fivefold enhancement in the specific activity could be obtained by limited proteolysis with trypsin. The extreme lability of the enzyme could be circumvented by its immobilization on activated Sepharose or by cross-linking with dimethyl suberimidate. The kinetic properties as well as the advantages of such stabilized enzyme preparations are presented.     

胰蛋白酶定向固定化亲和配基的理性设计

在胰蛋白酶三维(3D)结构的基础上, 首先利用分子对接从ZINC 数据库中筛选获得了与胰蛋白酶具有较高亲和性的小分子配基2-硝基苯基-β-D-葡糖苷, 并分析了该配基与蛋白质之间的相互作用力主要为范德华和氢键相互作用. 并利用分子动力学模拟进一步验证了2-硝基苯基-β-D-葡糖苷与胰蛋白酶之间具有较强的亲和作用. 分子动力学(MD)模拟结果表明, 配基-目标蛋白质之间形成稳定的复合物且它们之间的距离基本没有变化. 此外, 一个水分子通过氢键在配基和目标蛋白质的结合腔之间架桥. 最后制备了偶联有该配基的亲和载体, 进行了胰蛋白酶的定向固定化, 并考察了该固定化酶的活性. 研究结果表明, 利用修饰2-硝基苯基-β-D-葡糖苷配基的亲和载体固定化胰蛋白酶的酶活达到340.8 U·g^-1, 比活达到300.3 U·mg^-1, 分别是未修饰亲和配基载体的10倍和5倍, 具有明显的优势. 上述结论证明了结合分子对接和分子动力学模拟理性设计定向固定化亲和配基的方法是可行的, 具有一定的理论和实用价值.     

Immobilization and catalytic properties of candida lipolytic lipase on surface of organic intercalated and modified MgAl-LDHs

In this study, MgAl-LDHs (layered double hydroxides) intercalated with sodium dodecyl sulfate and outside surface modified with (3-aminopropyl)triethoxysilane (KH550) were prepared. The existence of organic part in LDHs improved immobilization efficiency and activity recovery of candida lipolytic lipase loaded. Also the positive charge in framework of LDHs was found to be beneficial to the enzyme immobilization. An immobilization efficiency of 56.4% and an activity recovery over 69.2% of the enzyme were obtained after it was loaded on the intercalated and modified LDHs, and catalytic activity of the immobilization can be kept at least five times. Moreover, the immobilized enzyme was found to have higher temperature resistance, wider pH value and better thermostability in reactive activity.     

Dynamic thermodynamic and dynamic kinetic resolution of 2-lithiopyrrolidines

Dynamic resolution has been studied as a method for the asymmetric synthesis of 2-substituted pyrrolidines. Highly enantioselective electrophilic substitutions of racemic 2-lithiopyrrolidines in the presence of a chiral ligand have been achieved. The organolithium compounds were prepared by tin-lithium exchange from the corresponding tributylstannanes and n-butyllithium or by deprotonation of N-(tert-butyloxycarbonyl)pyrrolidine with sec-butyllithium. A range of N-substituents and chiral ligands were investigated for the dynamic resolution. Electrophilic quench of the resolved diastereomeric 2-lithiopyrrolidine-chiral ligand complexes provided the enantiomerically enriched 2-substituted pyrrolidines. With N-alkyl derivatives, the resolution occurs conveniently at (or just below) room temperature and either enantiomer of the product can be formed by appropriate choice of the chiral ligand. The asymmetric induction occurs as a result of a thermodynamic preference for one of the diastereomeric complexes. The minor complex was found to have a faster rate of reaction with the electrophile. The use of N-allylic derivatives provides a means to prepare the N-unsubstituted pyrrolidine products. Best results were obtained with the N-2,3-dimethylbut-2-enyl derivative, and this N-substituent could be cleaved using 1-chloroethyl chloroformate. With N-Boc-2-lithiopyrrolidine, the enantioselectivity arises by a kinetic resolution and high levels of asymmetric induction in the presence of excess n-butyllithium can be obtained. Dynamic kinetic resolution of the N-Boc derivative is limited in the scope of electrophile that can be used.     

壳聚糖亲和磁性毫微粒的制备及其对蛋白质的吸附性能研究

以壳聚糖为包裹材料包埋自制的磁流体 ,制备了具有核 壳结构的磁性毫微粒 ,并偶联色素配基CibacronBlue 3GA(偶联量 1 4 .5μmol/mL)得到了一种新型亲和磁性毫微粒 .结果表明 ,所得亲和磁性微球具有较窄的粒径分布、形状规整 .以牛血清白蛋白 (BSA)和溶菌酶 (Lys)为目标蛋白 ,考察了该亲和磁性毫微粒的吸附性能 ,发现其对BSA和Lys的吸附量分别为 4和 2 8mg/g,吸附行为满足Langmuir吸附等温式 ,且对时间依赖性小而对溶液离子强度敏感 .     

Immobilization of trypsin onto 1,4-diisothiocyanatobenzene-activated porous glass for microreactor-based peptide mapping by capillary electrophoresis: Effect of calcium ions on the immobilization procedure

The immobilization conditions and kinetic behaviour of trypsin, covalently immobilized via the 1,4-diisothiocyanatobenzene (DITC) linker onto aminopropylated controlled pore glass (CPG) particles, have been evaluated to establish a rapid and efficient protocol for fabrication of an immobilized enzyme microreactor (IMER) for protein hydrolysis and subsequent peptide mapping. Addition of calcium ions to either the immobilization reaction solution or hydrolysis assay was studied for a synthetic substrate. Activity was slightly higher when immobilization was carried out in the presence of Ca²⁺ whereas more enzyme could be immobilized in its absence. A protocol requiring less than 3 h was devised to obtain maximal enzymatic activity with the lowest ratio of soluble trypsin to DITC-CPG particles. The resulting immobilized enzyme was found to retain an acceptable percentage (ca. 35%) of its activity after immobilization. The particles were dry-packed into a capillary to make a microscale IMER. Repeatability, reusability and digestion efficiency of the μIMER were investigated for the substrate β-casein using capillary electrophoretic-based peptide mapping. In initial tests, a single device showed reproducible peptide maps for 21 digestions lasting 2 h each, carried out over a period of 2 months. Complete digestion of β-casein could be achieved in a few minutes (86 s residence time in the μIMER followed by a wash step).     

聚脲多孔材料的简单制备及在酶固定和手性拆分中的应用

以甲苯二异氰酸酯(TDI)为单体,在水与丙酮混合溶剂中通过沉淀聚合一步法制备了富含胺基的聚脲多孔材料(PPU),通过扫描电镜和压汞法对其表面形貌和孔结构进行了表征.PPU经戊二醛(GA)活化后用于荧光假单胞菌脂肪酶(PFL)的固定,考察了GA活化过程中GA浓度对酶固定量及固定酶活性的影响.结果表明,PPU是一种粒子尺寸分布在30~50μm范围的形状不规则的多孔粒子,孔径在2 nm~100μm之间呈连续分布.在pH=8.0的缓冲溶液中用0.17 mol/L的GA对PPU进行改性,将改性后的PPU用于PFL的固定,当酶溶液浓度为2.56 mg/m L时,得到酶的最大固定量为95.2 mg/g,固定酶的活性为375 U/mg,相对活性为76%.将此固定酶作为催化剂,用于1-苯乙醇外消旋化合物的手性拆分,并与游离酶催化的结果相比较.结果表明,固定酶的反应活性和立体选择性都明显优于游离酶.通过沉淀聚合制备的聚脲多孔材料在酶固定及手性分子拆分方面具有应用前景.     

Galactosyl-biomimetic dye-ligands for the purification of Dactylium dendroides galactose oxidase

Two anthraquinone galactosyl-biomimetic dye-ligands comprising, as terminal biomimetic moiety, galactose analogues (1-amino-1-deoxy-beta-D-galactose and D(+)-galactosamine) were designed for the enzyme galactose oxidase (GAO), using molecular modelling, synthesized and characterized. The biomimetic ligands were immobilized on agarose beads and the affinity adsorbents, together with a non-biomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their ability to purify GAO from Dactylium dendroides. Both biomimetic adsorbents showed higher purifying ability for GAO compared to the non-biomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent comprising, as terminal biomimetic moiety, 1-amino-1-deoxy-beta-D-galactose (BM1) exhibited the highest purifying ability for GAO. This affinity adsorbent did not bind galactose dehydrogenase, glucose dehydrogenase, alcohol dehydrogenase, or glucose oxidase. The dissociation constant (K(D)) of the immobilized BM1 ligand with GAO was found to be equal to 45.8 microM, whereas the binding capacity was equal to 709 U per ml adsorbent. Therefore, the BMI adsorbent was integrated in a facile two-step purification procedure for GAO. The purified enzyme showed a specific activity equal to 2038 U/mg, the highest reported so far, approximately 74% overall recovery and a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis.     

Heterogeneity in the binding of chymotrypsin and related proteins to affinity chromatographic media

The binding of a series of related proteins, namely, α-chymotrypsin, chymotrypsinogen A, DIP-chymotrypsin, and TPCK-chymotrypsin,² to affinity gels consisting of 4-phenylbutylamine or ε-aminocaproyl-d-tryptophanmethylester covalently attached to Sepharose 4B was investigated. Considerable heterogeneity in the binding was observed, both at the level of the affinity adsorbent and with respect to the proteins themselves. Both the aromatic moiety of the ligand and the positively charged isourea group introduced during the coupling of the ligand to the gel play a part in the stabilization of the binding of α-chymotrypsin. At least a part of the binding involves the substrate binding site of the enzyme. The existence of secondary binding sites on the proteins, capable of interaction with gel-bound ligand, is suggested by the observation that at fairly high levels of substitution of gel by ligand the zymogen and covalently modified enzyme species are also bound quite strongly. Since the level of gel substitution is difficult to control, the results emphasize the necessity of a thorough investigation of the binding properties of a given affinity medium prior to use in the separation of active chymotrypsin from inactive derivatives.     

Enzymatic ring-opening polymerization of 2,2-dimethyltrimethylene carbonate catalyzed by PPL immobilized on silica nanoparticles

Silica nanoparticles were first used as the carrier for the porcine pancreas lipase (PPL) immobilization. The result of transmission electron microscopy (TEM) showed that the immobilized lipase was still in nanosize after enzyme immobilization. The ring-opening polymerization of 2,2-dimethyltrimethylene carbonate (DTC) catalyzed by this immobilized PPL (IMPPL) was explored. ¹H NMR spectra suggested no evidence of decarboxylation during propagation. Influences of IMPPL concentration and reaction temperature on the molecular weight and yield of poly(DTC) were studied. The recovery and reuse of IMPPL for the ring-opening polymerization of DTC was also investigated. The recycling IMPPL showed even higher catalytic activity and a higher molecular weight of polycarbonate could be achieved.     

Supermacroporous cryogel matrix for integrated protein isolation. Immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line

A new type of supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing the specific capture of urokinase from conditioned media of human fibrosarcoma cell line HT1080. The affinity adsorbent was designed with the objective of using it as a capture column in an integrated perfusion/protein separation bioreactor setup. A comparative study between the utility of this novel cryogel based matrix and the conventional Sepharose based affinity matrix for the continuous capture of urokinase in an integrated bioreactor system was performed. Cu(II)-ion was coupled to epoxy activated polyacrylamide cryogel and Sepharose using iminodiacetic acid (IDA) as the chelating ligand. About 27-fold purification of urokinase from the conditioned culture media was achieved with Cu(II)-IDA-polyacrylamide cryogel column giving specific activity of about 814 Plough units (PU)/mg protein and enzyme yields of about 80%. High yields (95%) were obtained with Cu(II)-IDA-Sepharose column by virtue of its high binding capacity. However, the adsorbent showed lower selectivity as compared to cryogel matrix giving specific activity of 161 PU/mg protein and purification factor of 5.3. The high porosity, selectivity and reasonably good binding capacity of Cu(II)-IDA-polyacrylamide cryogel column make it a promising option for use as a protein capture column in integrated perfusion/separation processes. The urokinase peak pool from Cu(II)-IDA-polyacrylamide cryogel column could be further resolved into separate fractions for high and low molecular weight forms of urokinase by gel filtration chromatography on Sephacryl S-200. The selectivity of the cryogel based IMAC matrix for urokinase was found to be higher as compared to that of Cu(II)-IDA-Sepharose column.     

Modelling of solid-liquid adsorption: effects of adsorbent heterogeneity

Effects of adsorbent heterogeneity on the adsorption of cobalt phthalocyanine dye on activated carbon have been studied. Adsorption experiments were carried out by varying the temperature and adsorbent mass in batch adsorbers and, in addition, the adsorbent particle size and fluid flow rate in a continuous stirred tank reactor (CSTR)-type adsorber in order to investigate the equilibrium and the kinetics of adsorption.The Brunauer-Emmett-Teller (BET), Langmuir with uniform distribution (LUD) and Langmuir-Freundlich equations are able to represent the equilibrium data with similar accuracy within the range of measurements. Reasonably large values of the heterogeneity parameter (2.69–2.86) show that the carbon surface is energetically heterogeneous.A mathematical model that describes the adsorption dynamics, including film-, pore- and concentration-dependent surface diffusion on an energetically and structurally heterogeneous adsorbent, is presented here and fitted to the experimental concentration vs. time curves obtained in the continuously stirred tank adsorber.Structural heterogeneity of the carbon, if not accounted for in the kinetic model, can be responsible for the very strong concentration dependence of the surface diffusion coefficient and for the variation in the parameter D_o with particle size and adsorber porosity as shown in this work.     

Glucose oxidase/hexokinase electrode for the determination of ATP

A hydrogen peroxide based enzyme electrode for the determination of ATP has been developed by the immobilization of glucose oxidase and hexokinase. Competition between the enzymes for the substrate glucose allowed the measurement of ATP. Different immobilization procedures and different types of hexokinase have been tested. Using a BSA-glutaraldehyde procedure and hexokinase from an overproducing strain of bakers' yeast, ATP was measured in the 0.05–0.5 mmol l⁻¹ range with a detection limit of 0.01 mmol l⁻¹. ATP concentrations comparable to those reported in the literature and a good recovery were obtained when the enzyme electrode was used with human erythrocyte hemolysate.     

Detection of phenolic compounds in flow systems based on tyrosinase-modified reticulated vitreous carbon electrodes

The fabrication and performance of a reticulated vitreous carbon (RVC)-based tyrosinase flow-through electrode, in which the enzyme was covalently immobilized, is reported. The bioelectrode was tested as an amperometric detector for phenolic compounds. Variables affecting the construction of the enzyme flow-through electrode such as the RVC chemical pretreatment procedure, the enzyme immobilization method in the RVC matrix, the enzyme loading and the pH value of the buffer solution used, were optimized by flow-injection with amperometric detection. A good immobilization of the enzyme in the RVC matrix, in spite of the hydrodynamic conditions, was found. The same tyrosinase-RVC electrode could be used with no significant loss of the amperometric response for around 20 days, and reproducible responses could be achieved with different electrodes constructed in the same manner. Moreover, the operational stability of the bioelectrode was tested under continuous monitorization conditions. Calibration plots by flow injection with amperometric detection at -0.20 V were obtained for phenol, 2,4-dimethylphenol; 3-chlorophenol; 4-chlorophenol; 4-chloro-3-methylphenol and 2-aminophenol, with detection limits ranging from 2 mug l(-1) (4-chloro-3-methylphenol) to 2 mg l(-1).     

Anisotropic membranes with carboxypeptidase G1

Anisotropic polysulfone membranes were prepared with carboxypeptidase G₁ embedded in the polymer structure. The enzymatically active flat and hollow-fiber membranes were obtained by precipitating the polymer from solution in an organic mixture in which an aqueous solution of the enzyme had been dispersed. The process has been found to be particularly suitable for the immobilization of enzymes in anisotropic hollow fibers that exhibited no detectable enzyme leakage upon perfusion. The pH profiles measured with the enzyme in free solution and in the embedded form were similar. Kinetic parameters of multitubular enzyme reactors were investigated by measuring the rate of hydrolysis of glutamate from folic acid or methotrexate at different flow rates and substrate concentrations. The relatively slow mass transfer in such reactors was found to affect strongly the observed kinetics. The results of in vitro experiments with 5000 fiber reactors suggest that hollow fiber cartridges prepared with such membranes have clinical potential for the extracorporeal removal of methotrexate from blood.