Evaluation of two different Concanavalin A affinity adsorbents for the adsorption of glucose oxidase

Concanavalin A (Con A) was selected as ligand and thus immobilized onto two different supports, namely the polymeric Toyopearl and the inorganic silica, with the protection of its binding sites provided during the coupling procedure. The prepared Con A affinity adsorbents were then employed to evaluate their adsorption behaviour for the enzyme glucose oxidase (GOD). The immobilization kinetics showed that the immobilization of Con A on silica supports was much faster than that on Toyopearl supports, which could highly reduce the possibility of the denaturation of Con A. The optimal adsorption conditions for binding of GOD onto the ligand were determined in terms of the pH value and the ionic strength of the adsorption medium. The adsorption isotherms for binding GOD onto two Con A affinity adsorbents fitted well with the Langmuir equation. The maximum adsorption capacity q(m) of Toyopearl Con A and silica Con A were 7.9 mg/ml and 4.9 mg/ml, with a dissociation constant K(d) of 4.8 x 10(-7)M and 2.6 x 10(-6)M, respectively. Due to the less diffusive resistance, silica Con A showed both higher adsorption and desorption rates for GOD when compared with Toyopearl Con A. The nonspecific adsorption of GOD was less than 8% for both end-capped Toyopearl and silica supports. The dynamic adsorption of GOD for five times repeated processes showed a high stability for both prepared adsorbents. All the results indicate a good suitability of both Con A adsorbents for affinity adsorption of GOD.     

Preparation and evaluation of Ricinus communis agglutinin affinity adsorbents using polymeric supports

A practicable and efficient procedure for preparation of Ricinus communis agglutinin (RCA) affinity adsorbents has been developed. For immobilization of RCA two different polymer-based supports, Toyopearl and TSKgel (TosoHaas), were used. RCA has been successfully immobilized onto these supports with amounts of coupled ligand between 15 and 23 mg/g dry support and corresponding coupling yields of 69-93% (w/w). The prepared affinity adsorbents were characterized concerning their binding capacity for the glycoprotein asialofetuin (ASF) and accessibility of the ligand binding sites. The high accessibility of 80% showed that steric hindrance was negligible at the present ligand density. RCA-Toyopearl was successfully applied in affinity chromatography of glycoproteins indicating its high specificity. A long-term stability test proved no change in capacity for a period of at least 12 months. High-performance affinity chromatography (HPLAC) was carried out using RCA-TSKgel. Experimental results showed that the prepared adsorbents are suitable for selective separation of glycoproteins and oligosaccharides and therefore can be used for investigations of adsorption characteristics of glycoconjugates and for laboratory-scale preparations.     

Goat sperm membrane: lectin-binding sites of sperm surface and lectin affinity chromatography of the mature sperm membrane antigens.

The cell surface glycoproteins of goat epididymal maturing spermatozoa have been investigated using lectins as surface probes that interact with specific sugars with high affinity. Concanavalin A (ConA) and wheat-germ agglutinin (WGA) showed high affinity for mature cauda epididymal sperm agglutination, whereas RCA2, kidney beans lectin and peanut agglutinin caused much lower or little agglutination of the cells. The mature sperm exhibited markedly higher efficacy than the immature caput epididymal sperm for binding both ConA and WGA, as evidenced by sperm agglutination and the binding of the fluorescence isothiocyanate (FITC)-labelled lectins. FITC-ConA binds uniformly to the entire mature sperm surface whereas FITC-WGA binds to the acrosomal cap region of the head. The FITC-RCA2 mainly labelled the posterior head of mature cauda sperm. However, no WGA-specific glycoprotein receptors could be detected in sperm plasma membrane (PM) by WGA-Sepharose affinity chromatography. The data implied that the epididymal sperm maturation is associated with a marked increase in the ConA/WGA receptors and that WGA receptors may be glycolipids rather than glycoproteins. Analysis of the ConA receptors of cauda sperm PM identified by ConA-Sepharose affinity chromatography and subsequent resolution in SDS-PAGE demonstrated the presence of five glycopolypeptides of different concentrations (98, 96, 43, 27 and 17 kDa) of goat sperm membrane. The immunoblot of these ConA-specific glycopeptides with anti-sperm membrane antiserum showed that 98- and 96-kDa receptors are immunoresponsive.     

Preparation and characterization of PEGyated Concanavalin A for affinity chromatography with improved stability

In order to improve its stability, immobilized Concanavalin A (Con A) on Toyopearl adsorbents was conjugated with monomethoxy poly(ethylene glycol) succinimidyl propionate (mPEG-SPA) with different molecular weight. A colorimetric method using ninhydrin is proposed to determine the degree of PEGylation; this method has proved to be easy applicable and reproducible. The PEGylation reaction was studied in detail to elucidate how parameters such as molar ratio of mPEG-SPA to Con A and molecular weight of mPEG-SPA affect the degree of PEGylation. The adsorption isotherms of glucose oxidase (GOD) onto native and PEGylated Con A adsorbents showed that the modification did not alter substantially the specificity of the carbohydrate binding ability of Con A. However, the binding capacity for GOD was slightly reduced probably due to the steric hindrance caused by mPEG chains. Adsorption kinetic studies revealed a lower adsorption rate after PEGylation which was attributed to the steric effect. The dynamic adsorption capacity for modified Con A depended very much on the degree of PEGylation and the molecular weight of mPEG derivatives. The adsorption capacity could be highly preserved for Toyopearl Con A modified by mPEG2k (90% of the original adsorption capacity) even with a degree of PEGylation up to 20% (the ratio of primary amino groups of PEGylated immobilized Con A to that of native immobilized Con A). Studies show that the binding capacity of PEGylated Con A was highly preserved under mild process conditions. PEGylated Con A also exhibited obviously higher stability against more stressful conditions such as the exposure to organic solvents and high temperatures. Conjugation of Con A with mPEG2k provided better adsorption performance thus has greater potential for application in affinity separation processes compared with mPEG5k. The fact that PEGylation stabilizes the properties of Con A may greatly expand the range of applications of unstable proteins to bioprocessing (e.g. biocatalysis and downstream separation) as well as other protein applications (e.g. medication, industrial use, etc.).     

Superparamagnetic adsorbents for high-gradient magnetic fishing of lectins out of legume extracts

This work presents the development, testing, and application in high-gradient magnetic fishing of superparamagnetic supports for adsorption of lectins. Various approaches were examined to produce affinity, mixed mode, and hydrophobic charge induction type adsorbents. In clean monocomponent systems affinity supports created by direct attachment of glucose or maltose to amine-terminated iron oxide particles could bind concanavalin A at levels of up to approximately 280 mg g(-1) support with high affinity ( approximately 1 microM dissociation constants). However, the best performance was delivered by adsorbents featuring coupled tentacular dextran chains displaying a maximum binding capacity of 238 mg g(-1) and a dissociation constant of 0.13 microM. Adsorbents derivatized with mixed mode or hydrophobic charge induction ligands likewise demonstrated very high capacities for both concanavalin A and Lens culinaris agglutinin (> or = 250 mg g(-1)) with dissociation constants in the micromolar range, though neither of these systems showed any selectivity for lectins in leguminous extracts. When the affinity supports were applied to carbohydrate containing legume extracts only the dextran-linked adsorbents supplied sufficient competition to dissolved sugars to selectively bind concanavalin A in an extract of jack beans. The dextran-linked supports were employed in a high-gradient magnetic fishing experiment, in which concanavalin A was purified to near homogeneity from a crude, unclarified extract of jack beans.     

Isolation of a cytotoxic glycoprotein from the Scyphozoa Cyanea lamarckii by lectin-affinity chromatography and characterization of molecule interactions by surface plasmon resonance

A biospecific lectin-affinity-based isolation process for a novel glycoprotein (ClGp1) from the venom of the pelagic jellyfish Cyanea lamarckii, is described and the isolated glycoprotein is chemically and biologically characterized according to size, molecular interaction and toxicity. The molecular mass of the isolated protein is 25.7 kDa as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF). The carbohydrate content was calculated after enzymatic deglycosylation as 6.85 kDa. The glycoprotein is cytotoxic and could be isolated from cnidocysts of mesenteric and fishing tentacles. The binding behaviour of the glycoprotein to the lectins Concanavalin A (ConA) and Wheat Germ Agglutinin (WGA) was analyzed by surface plasmon resonance (SPR) and affinity constants in the range of K(D)=3.0 x 10(-7) M for ConA and 2.1 x 10(-6) M (pH 5.0) and 2.6 x 10(-6) M (pH 7.4) for WGA were obtained.     

Removal of fluoride from aqueous solution by TiO2 and TiO2–SiO2 nanocomposite

Adsorption plays an important role in the removal of pollutants such as fluoride from aqueous solutions. With the rapid development of environmental technology, TiO₂ particle has become promising material to adsorb fluoride ion because of its low cost, non-toxic, good chemical stability, and good sorption ability. This work used sol-gel and hydrothermal synthesis methods to prepare TiO₂ particles and load them onto SiO₂ particles. The physicochemical properties such as heat stability, particle size, and surface area of the resulting TiO₂ adsorbents were characterized with various analytical methods. In addition, their adsorption abilities to fluoride were determined under various conditions including different initial fluoride concentration, pH and coexisting ions. The maximum adsorption capacity of the TiO₂ adsorbents can reach up to 94.3 mg/g. The adsorption isotherms of fluoride onto the TiO₂ adsorbents can be closely described by the Langmuir model, suggesting the monolayer adsorption process.     

Silica-encapsulated magnetic nanoparticles: enzyme immobilization and cytotoxic study

Silica-encapsulated magnetic nanoparticles (MNPs) were prepared via microemulsion method. The products were characterized by high resolution transmission electron microscopy (HRTEM) and energy-dispersive X-ray spectrum (EDS). MNPs with no observed cytotoxic activity against human lung carcinoma cell and brine shrimp lethality were used as suitable support for glucose oxidase (GOD) immobilization. Binding of GOD onto the support was confirmed by the FTIR spectra. The amount of immobilized GODs was 95 mg/g. Storage stability study showed that the immobilized GOD retained 98% of its initial activity after 45 days and 90% of the activity was also remained after 12 repeated uses. Considerable enhancements in thermal stabilities were observed for the immobilized GOD at elevated temperatures up to 80°C and the activity of immobilized enzyme was less sensitive to pH changes in solution.     

Adsorption of the uremic toxin p-cresol onto hemodialysis membranes and microporous adsorbent zeolite silicalite

Para-cresol CH3C6H4OH is a protein-bound solute which is not eliminated efficiently by hemodialysis systems. In this study, we present adsorption of p-cresol as a complementary process to hemodialysis. The kinetics and isotherms of adsorption onto cellulose-based membranes (cellulose diacetate and triacetate), synthetic membranes (polyamide, polysulfone, polyacrylonitrile and polymethylmethacrylate) and microporous zeolite silicalite (MFI), have been evaluated in static conditions. The results indicate that p-cresol has a low affinity to all membranes but polysulfone and polyamide and that the times to reach equilibrium conditions are slow. In contrast, equilibration time on silicalite is fast (2 min to eliminate 90%) while adsorption levels are high (maximum adsorption about 106 mg g(-1)). Adsorption onto microporous adsorbents could be a novel way to eliminate uremic toxins from blood.     

Lectin-reactive components of the microsporidian Glugea plecoglossi and their relation to spore phagocytosis by head kidney macrophages of ayu Plecoglossus altivelis.

We investigated the reactivity of lectins to spores of Glugea plecoglossi from ayu Plecoglossus altivelis. Smear preparations of purified spores were treated with 8 kinds of lectins. Lectin blots were used to detect glycoproteins of spore lysates. In addition, lectin-treated spores were applied to head kidney macrophages of ayu, and the percentage of phagocytosis (PP) was calculated and compared with the control. Two lectins (ConA, WGA) reacted with the surface of the spores, and a major band (55 kDa) and some minor bands were visualized on blots after treatment with these. PP was decreased after ConA treatment. From these results, we suggest that G. plecoglossi spores can be phagocytized by ayu head kidney macrophages via ConA-reactive glycoprotein-mediated recognition.     

Immobilization and direct electrochemistry of glucose oxidase on a tetragonal pyramid-shaped porous ZnO nanostructure for a glucose biosensor

A tetragonal pyramid-shaped porous ZnO (TPSP-ZnO) nanostructure is used for the immobilization, direct electrochemistry and biosensing of proteins. The prepared ZnO has a large surface area and good biocompatibility. Using glucose oxidase (GOD) as a model, this shaped ZnO is tested for immobilization of proteins and the construction of electrochemical biosensors with good electrochemical performances. The interaction between GOD and TPSP-ZnO is examined by using AFM, N(2) adsorption isotherms and electrochemical methods. The immobilized GOD at a TPSP-ZnO-modified glassy carbon electrode shows a good direct electrochemical behavior, which depends on the properties of the TPSP-ZnO. Based on a decrease of the electrocatalytic response of the reduced form of GOD to dissolved oxygen, the proposed biosensor exhibits a linear response to glucose concentrations ranging from 0.05 to 8.2mM with a detection limit of 0.01mM at an applied potential of -0.50V which has better biosensing properties than those from other morphological ZnO nanoparticles. The biosensor shows good stability, reproducibility, low interferences and can diagnose diabetes very fast and sensitively. Such the TPSP-ZnO nanostructure provides a good matrix for protein immobilization and biosensor preparation.     

Effect of organic Cr(III) complexes on chromium speciation

Abstract

Chromium speciation in the presence of organic chromium(III) complexes was investigated using solid-phase extraction. The adsorptions of Cr(VI) and Cr(III) on alumina and pumice powder were studied. Maximum sorption of Cr(VI) was obtained by alumina (90.22%), while Cr(III) was highly adsorbed onto pumice powder (86.65%). This result shows that pumice may be a new and promising adsorbent for Cr(III). The experimental equilibrium data for Cr(VI) adsorption onto alumina and Cr(III) sorption onto pumice were analysed using Langmuir and Freundlich isotherms. The separation and adsorption of Cr(VI), Cr(III) and five organic chromium(III) complexes onto pumice and alumina at different pH values were evaluated. Ethylenediaminetetraacetate (EDTA), oxalate, citrate, glycine, alanine and 8-hydroxyqinoline were used as ligands. Sorption of alanine and ethylenediaminetetraacetate complexes was higher onto alumina than pumice at pH>3. The enhancement of adsorption of chromium(III) complexes onto pumice was achieved by surface modification of pumice using a surfactant, namely hexadecyltrimethylammoniumbromür (HDTMA). The presence of surfactant enhanced the adsorption of Cr(III) citrate, oxalate, glycine and 8-hydroxyquinoline complexes onto pumice. However, the adsorption of EDTA and alanine complexes decreased, with ratio of 13.40% and 4.00% respectively. Here we demonstrate that chromium speciation methods depending on adsorption onto various adsorbents including alumina may lead erroneous results. Analytical measurements were performed by flame AAS, data were obtained by standard addition method.     

Bilirubin removal from human plasma by Cibacron Blue F3GA using immobilized microporous affinity membranous capillary method

A novel affinity sorbent system for direct bilirubin removal from human plasma was developed. These new adsorbents comprise Cibacron Blue F3GA as the specific ligand, and microporous membranous poly(tetrafluoroethylene) capillary (modified by coating with a hydrophilic layer of poly(vinyl alcohol) after activation) as the carrier matrix. The affinity adsorbents carrying 126.5 micromol Cibacron Blue F3GA/g polymer was then used to remove bilirubin in a flow-injection system. Non-specific adsorption on the poly(vinyl alcohol) coated capillary remains low, and higher affinity adsorption capacity, of up to 76.2 mg/g polymer was obtained after dye immobilization. The bilirubin adsorption capacity of the affinity capillary decreased with increase in the recirculation rate of plasma. The adsorption capacity increased with increase the temperature while decreased with increase the ionic strength. The maximum adsorption was only observed in neutral solution (pH 6-7). The adsorption isotherm fitted the Langmuir model well. These new adsorbents have higher velocity of mass transfer, better adsorption capacity, less fouling, longer service life and good reusability. The results of blood tests suggested the dye affinity capillary has good blood compatibility.     

Encapsulation of glucose oxidase microparticles within a nanoscale layer-by-layer film: immobilization and biosensor applications

We report on an immobilization strategy utilizing layer-by-layer encapsulated microparticles of enzymes within a nanoscale polyelectrolyte film. Encapsulation of glucose oxidase (GOD) microparticles was achieved by the sequential adsorption of oppositely charged polyelectrolytes onto the GOD biocrystal surface. The polyelectrolyte system polyallylamine/polystyrene sulfonate was used under high salt conditions to preserve the solid state of the highly water soluble GOD biocrystals during the encapsulation process. The resulting polymer multilayer capsule of about 15 nm wall thickness is permeable for small molecules (glucose), but non-permeable for macromolecules thus preventing the enzyme from leakage and at the same time shielding it from the outer environment e.g., from protease or microbial activity. Decrease of the buffer salt concentration leads to the dissolution of the enzyme under formation of μ-bioreactors. The spherical μ-bioreactors are bearing an extremely high loading of biocompound per volume. Encapsulated GOD was subsequently used to construct a biosensor by nanoengineered immobilisation of μ-bioreactor capsules onto an electrode surface. The presented approach demonstrates a general method to encapsulate highly soluble solid biomaterials and an immobilization strategy with the potential to create highly active thin and stable films of biomaterial.     

Optimization of evanescent-field fluorescence-assisted lectin microarray for high-sensitivity detection of monovalent oligosaccharides and glycoproteins

Lectin microarray is an emerging technique, which will accelerate glycan profiling and discovery of glycan-related biomarkers. One of the most important stages in realizing the potential of the technique is to achieve sufficiently high sensitivity to detect even the low concentrations of some target glycoproteins which occur in sera or tissues. Previously, we developed a lectin microarray based on an evanescent-field fluorescence-assisted detection principle that allows rapid profiling of glycoproteins. Here, we report optimization of procedures for lectin spotting and immobilization to improve the sensitivity and reproducibility of the lectin microarray. The improved microarray allows high-sensitivity detection of even monovalent oligosaccharides that generally have a low affinity with lectins (K(d)>10(-6) M). The LOD observed for RCA120, a representative plant lectin, with asialofetuin, and an asialo-biantennary N-glycan probe were determined to be 100 pg/mL and 100 pM, respectively. With the improved lectin microarray system, closely related structural isomers, i.e., Le(a) and Le(x), were clearly differentiated by the difference in signal patterns on relevant multiple lectins, even though specific lectins to detect these glycan structures were not available. The result proved a previously proposed concept of lectin-based glycan profiling.     

Screening for and purification of novel self-aggregatable lectins reveal a new functional lectin group in the bark of leguminous trees

A solubility-insolubility transition assay was used to screen the bark and stems of seven leguminous trees and plants for self-aggregatable lectins. Novel lectins were found in two trees, Robinia pseudoacacia and Wisteria floribunda, but not in the leguminous plants. The Robinia lectin was isolated from coexisting lectin by combined affinity chromatographies on various sugar adsorbents. The purified lectins proved to be differently glycosylated glycoproteins. One lectin exhibited the remarkable characteristics of self-aggregatable lectins: localization in the bark of legume trees, self-aggregation dissociated by N-acetylglucosamine/mannose, and coexistence with N-acetylgalactosamine/galactose-specific lectins, which are potential endogenous receptors. Self-aggregatable lectins are a functional lectin group that can link enhanced photosynthesis to dissociation of glycoproteins.     

Combining various strategies to increase the coverage of the plant cell wall glycoproteome

Glycoproteomics recently became a very active field, mostly in mammals. The first part of this paper consists of a mini-review on the strategies used in glycoproteomics, namely methods for enrichment in glycoproteins and mass spectrometry (MS) techniques currently used. In a second part, these strategies are applied to the cell wall glycoproteome of etiolated hypocotyls of Arabidopsis thaliana, showing their complementarity. Several sub-glycoproteomes were obtained by: (i) affinity chromatography on concanavaline A (ConA) and analysis of glycoproteins by MALDI-TOF MS; (ii) multidimensional lectin chromatography (using AIL, PNA, ConA and WGA lectins) and subsequent identification of glycoproteins by MALDI-TOF MS and LC-MS/MS; (iii) boronic acid chromatography followed by identification of glycoproteins by MALDI-TOF MS. Altogether, 127 glycoproteins were identified. Most glycoproteins were found to be putative N-glycoproteins and N-glycopeptides were predicted from MS data using the ProTerNyc bioinformatics software.     

Immobilization of glucose oxidase and lactate dehydrogenase onto magnetic nanoparticles for bioprocess monitoring system

Glucose oxidase (GOD) and lactate dehydrogenase (LDH) were immobilized onto magnetic nanoparticles, viz. Fe₃O₄, via carbodiimide and glutaraldehyde. The immobilization efficiency was largely dependent upon the immobilization time and concentration of glutaraldehyde. The magnetic nanoparticles had a mean diameter of 9.3 nm and were superparamagnetic. The immobilization of GOD and LDH on the nanoparticles slightly decreased their saturation magnetization. However, the FT-IR spectra showed that GOD and LDH were immobilized onto the nanoparticles by different binding mechanisms, the reason for which was not well explained. The optimum pH values of the immobilized GOD and LDH were changed to 8 and 10, respectively. The free and immobilized enzyme kinetic parameters (K_m and V_max) were determined by Michaelis-Menten enzyme kinetics. The Km values for free and immobilized GOD were 0.168 and 0.324 mM, respectively, while those for free and immobilized LDH were 0.19 and 0.163 mM for NAD, and 2.976 and 4.785 mM for lactate, respectively. High operational stability was observed, with more than 80% of the initial enzyme activity being retained for the immobilized GOD up to 12 h and for the immobilized LDH up to 24 h. The immobilized GOD was applied to a sequential injection analysis system for the application of bioprocess monitoring.     

Preparation of immobilized enzyme with high activity using affinity tag based on proteins A and G

Affinity tag AG consisting of immunoglobulin G (lgG)-binding domains of protein A from Staphylococcus aureus (EDABC) and those of protein G from Streptococcus strain G148 (C2C3) were used to facilitate immobilization of beta-galactosidase (betagal) from Escherichia coli. Poly(methylmethacrylate/N-isopropylacrylamide/methacrylic acid) [P(MMA/NIPAM/MAA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] latex particles, which show thermosensitivity, were used as support materals to prepare affinity adsorbents. Human gamma-globulin (HgammaGb), whose major fraction is lgG, was used as an affinity ligand and was covalently immobilized onto the both latex particles by the carbodiimide method under various conditions. A fusion protein, AGbetagal, was immobilized at pH 7.3 by the specific binding of affinity tag to these affinity adsorbents. The amount of adsorbed AGbetagal per unit amount of immobilized HgammaGb, namely, efficiency of ligand utilization, was strongly affected by the type of latex particles and pH value for HgammaGb immobilization. The efficiency of ligand utilization was maximum in the affinity adsorbents prepared at pH 6.0 to 7.0, and that in the HgammaGb-P(MMA/NIPAM/MAA) latex particles was high. This result could be explained by the conformation and orientation of immobilized HgammaGb molecules. Immobilized AGbetagal retained approximately 75% of its activity in solution and the binding is stable enough to allow repeated use. These results clearly demonstrate that combination of the affinity tag AG and the affinity adsorbents, based on the thermosensitive latex particles, offers a simple and widely applicable method for preparation of immobilized enzyme with high activity. (c) 1995 John Wiley & Sons, Inc.     

葡萄糖氧化酶的有机相共价固定化

将葡萄糖氧化酶(GOD)在最适pH条件下冻干后,以戊二醛活化的壳聚糖为载体,分别在传统水相和1,4-二氧六环、乙醚、乙醇三种不同的有机相中进行共价固定化。通过比较水相固定化酶和有机相固定化酶的酶比活力、酶学性质及酶动力学参数,考察酶在有机相中的刚性特质对酶在共价固定化过程中保持酶活力的影响。结果表明,戊二醛浓度为0.1%、加酶量为80 mg/1 g载体、含水1.6%的1,4-二氧六环有机相固定化GOD与水相共价固定化GOD相比,酶比活力提高2.9倍,有效酶活回收率提高3倍;在连续使用7次后,1,4-二氧六环有机相固定化GOD的酶活力仍为相应水相固定化酶的3倍。在酶动力学参数方面,不论是表观米氏常数,最大反应速度还是转换数,1,4-二氧六环有机相固定化的GOD(Kmapp=5.63 mmol/L,Vmax=1.70μmol/(min.mgGOD),Kcat=0.304 s-1)都优于水相共价固定化GOD(Kmapp=7.33 mmol/L,Vmax=1.02μmol/(min.mg GOD),Kcat=0.221 s-1)。因此,相比于传统水相,GOD在合适的有机相中进行共价固定化可以获得具有更高酶活力和更优催化性质的固定化酶。该发现可能为酶蛋白在共价固定化时因构象改变而丢失生物活性的问题提供解决途径。