Chemically activated collagen for amyloglucosidase attachment. Use in a helicoidal reactor.

Amyloglucosidase was covalently bound to collagen sheets by a previously described method. The time of acidic methylation (first step of the collagen activation process) was important to obtain a good enzymatic surfacic activity. Homogeneity of the coupling procedure on the surface of collagen films was shown. Some properties of free enzyme were not affected after grafting; optimum pH and temperature, activation energy, and Km for maltose. Heat stability of the bound enzyme was slightly better; Km for soluble starch increased fivefold. In contrast, the maximal velocity in the presence of soluble starch remained four times that of maltose hydrolysis. Amyloglucosidase collagen membranes were used in a helicoidal reactor to produce glucose from maltose or soluble starch solutions. Tracer studies have shown that the helicoidal reactor behaved as a CSTR. The influence of maltose concentration and flow rate on conversion was studied and confirmed the absence of diffusional limitations for maltose. Recycling of concentrated solutions of maltose and soluble starch indicated strong diffusional restrictions for soluble starch. The catalytic support kept all its activity for 18 days continuous operation at 40 degrees C and 80% after 17 months storage at 4 degrees C.     

Surface-bound aspartate aminotransferase on collagen films. Compared properties with native enzyme.

Aspartate aminotransferase (L-aspartate : 2-oxoglutarate aminotransferase, EC 2.6.1.1) has been covalently bound to chemically activated collagen films. This enzyme had never previously been coupled to any other solid support. The coupling method, including acyl azide formation on the carrier, allowed coupling of many other enzymes. A systematic study of coupling conditions has been performed; influence of time of coupling and of concentration of coupling solution on the enzymatic activity retained on the film. Coupling solutions could be used for several successive couplings. To determine the yield of binding, N-[14C] ethylmaleimide-labelled enzyme was prepared fully active and bound to collagen films. After lyophilisation the film retained most of its activity when stored in buffer and the half-life of the enzymatic film was about ten months. pH Dependence and activation energy were about the same for soluble and coupled enzyme. Coupling protects against thermal denaturation and increases the stability of the enzyme; the enzymatic film could be used repeatedly. Kinetics were somewhat modified in the coupled enzyme as compared to the enzyme in solution. Glutamate appeared more available while oxaloacetate seemed to be limiting. These modifications might be due to the proteic support itself. The enzymatic films also revealed themselves as a good tool for industrial or clinical purposes as well as for studying the mechanism of enzyme action.     

Immobilization and stabilization of invertase on Cajanus cajan lectin support

Use of lectins as ligands for the immobilization and stabilization of glycoenzymes has immense application in enzyme research and industry. But their widespread use could be limited by the high cost of their production. In the present study preparation of a novel and inexpensive lectin support for use in the immobilization of glycoenzymes containing mannose or glucose residues in their carbohydrate moiety has been described. Cajanus cajan lectin (CCL) coupled covalently to cyanogen bromide activated Seralose 4B could readily bind enzymes such as invertase, glucoamylase and glucose oxidase. The immobilized and glutaraldehyde crosslinked preparations of invertase exhibited high resistance to inactivation upon exposure to enhanced temperature, pH, denaturants and proteolysis. Binding of invertase to CCL-Seralose was however found to be readily reversible in the presence of 1.0 M methyl alpha-D mannopyranoside. In a laboratory scale column reactor the CCL-Seralose bound invertase was stable for a month and retained more than 80% of its initial activity even after 60 days of storage at 4 degrees C. CCL-Seralose bound invertase exhibited marked stability towards temperature, pH changes and denaturants suggesting its potential to be used as an excellent support for the immobilization of other glycoenzymes as well.     

A mild methods of general use for covalent coupling of enzymes to chemically activated collagen films

Films of highly polymerized collagen, prepared in industrial conditions, were chosen for surface covalent binding of enzymes because of their insolubility, mechanical resistance, proteic nature, hydrophilic properties and for their abundance in chemically activable ? COOH. Untanned films, previously acid- methylated, were activated by acyl azide formation. After removal of reagents by repeated washing, the coupling of enzyme was performed by immersion of the activated film in the enzyme solutions (2 to 3 hr, 0°C). The procedure is particularly mild since the enzymes never come into contact with chemical reagents, and thus avoid all denaturing processes. All the enzymes tested were successfully bound: glutamate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, glutamate pyruvate transaminase, creatine kinase, hexokinase, trypsin, and urease. As tested with aspartate amino transferase, enzymatic activity remained constant for months (100% after 5 months) in spite of repeated use of the film at 30°C, washing and storage in buffer at 4°C between assays.     

Protective Effects of Soluble Collagen during Ultraviolet-A Crosslinking on Enzyme-Mediated Corneal Ectatic Models

Collagen crosslinking is a relatively new treatment for structural disorders of corneal ectasia, such as keratoconus. However, there is a lack of animal models of keratoconus, which has been an obstacle for carefully analyzing the mechanisms of crosslinking and evaluating new therapies. In this study, we treated rabbit eyes with collagenase and chondroitinase enzymes to generate ex vivo corneal ectatic models that simulate the structural disorder of keratoconus. The models were then used to evaluate the protective effect of soluble collagen in the UVA crosslinking system. After enzyme treatment, the eyes were exposed to riboflavin/UVA crosslinking with and without soluble type I collagen. Corneal morphology, collagen ultrastructure, and thermal stability were evaluated before and after crosslinking. Enzyme treatments resulted in corneal curvature changes, collagen ultrastructural damage, decreased swelling resistance and thermal stability, which are similar to what is observed in keratoconus eyes. UVA crosslinking restored swelling resistance and thermal stability, but ultrastructural damage were found in the crosslinked ectatic corneas. Adding soluble collagen during crosslinking provided ultrastructural protection and further enhanced the swelling resistance. Therefore, UVA crosslinking on the ectatic model mimicked typical clinical treatment for keratoconus, suggesting that this model replicates aspects of human keratoconus and could be used for investigating experimental therapies and treatments prior to translation.     

Aspartate aminotransferase immobilized on collagen films. Activity of dissociated subunits.

Aspartate aminotransferase has been covalently bound to insoluble films of collagen. The immobilized subunits of holoenzyme obtained after alkaline dissociation were inactive but their activity was recovered by incubation with either active enzyme, reduced holoenzyme, or apoenzyme. It was also possible to reduce the bound subunits by sodium borohydride and to reassociate them with native subunits of holoenzyme.     

Biochemical and photometric studies of modification of collagen structure induced by UV irradiation

The influence of UV irradiation (270–380 nm) on the biochemical, fluorescence and colorimetric properties of collagen was studied. The long-term UV irradiation (120 h) was accompanied by the increase of the structural stability of collagen to specific and nonspecific proteolytic enzymes, by formation of new additional fluorophore containing compounds, by the increased amount of carbonyl groups in the collagen, and by significant changes in the distribution pattern of products of alkaline hydrolysis during gel chromatography. The coordinates of color of the collagen films have been also changed. These changes of collagen suggest that UV irradiation induces photomodification and photooxidation processes in collagen.     

Thermal stabilization of amylolytic enzymes by covalent coupling to soluble polysaccharides

The immobilization of pullulanase and beta-amylase on soluble polysaccharides (dextrans and amylose) has been carried out. The method used for coupling the enzymes to the carbohydrate support involves limited periodate oxidation of the polysaccharide followed by reductive alkylation with sodium cyanoborohydride or borohydride. The influence of the degree of functionalization of the carbohydrate, the incubation time, the nature of the reducing agent and, for the dextrans studied, their molecular weight, on the properties of the conjugate were studied. We have observed an apparent correlation between the molecular weight of the glycoprotein conjugates formed and their thermal stability, resistance to urea denaturation and their kinetic parameters. By selecting the proper experimental conditions leading to conjugates with maximum thermal stabilities, it has also been shown that beta-amylase conjugates can hydrolyze starch at a temperature 20 degrees C higher than the corresponding value for the native enzyme. This result demonstrates that conjugation may result in modified enzymes leaving a high operational stability at elevated temperatures. We suggest that the immobilization method presented in this article represents an approach to the stabilization of enzymes employed at an industrial level, which may be of general application.     

Effect of irradiation on immobilized enzymes compared with that on enzymes in solution

Summary Glucose oxidase and catalase were immobilized by attaching them to nylon fibers that had been treated with triethyloxonium-tetrafluoroborate, diaminohexane and glutardialdehyde according to Morris, Campbell and Hornby (1975). This method assures that the enzymes are bound to a side chain of the polyamide structure. Enzyme activity (as measured by the O₂-uptake and by microcalorimetry) was found to be unchanged after 2 years. The apparent K_m-constants of the immobilized enzymes with glucose were the same as those for enzymes in solution. GOD and catalase immobilized in poly(acrylamide) gel had the same K_m-value.Despite the high stability during storage, the radiation induced inactivation of enzymes immobilized on gel or chromosorb, an inorganic carrier, was of the same order of magnitude as that of the dissolved enzymes. The enzymes bound to nylon fibers showed a higher radiation sensitivity. This might have been caused by an additional attack on the binding site of the carrier.Dedicated to Professor Dr. Dr. U. Hagen on the occasion of his 60^th birthdayDAAD-Fellow from AustriaDAAD-Fellow from South-Korea     

Citrate determination with aconitase immobilized on solid support

Pig heart aconitase has been immobilized on Enzacryl AA solid support and its kinetic behaviours were studied by using a stirred bath reactor with continuous recycling. In this reactor, the best flow rate has been determined to eliminate diffusional problems. Kinetics constants, thermic stability and pH variations have been compared between the soluble and immobilized aconitase for determination of enzyme-Enzacryl AA effectivity. Stability of the soluble and immobilized aconitase was also studied after repeated use and long-time storage. While the soluble form loses its activity after 24 hr storage, the immobilized form preserves its full activity after repeated usage and long-lasting storage. Finally, an easily measurable parameter has been found to quantitate citrate. The maximum increase of absorbance, is proportional to citrate concentration in a range between 0.2 and 3.2 mM. In conclusion, these results show that the immobilized aconitase system can be used for the determination of citrate with reproductility and great sensitivity. In addition to the simplicity of the assay, great economy in enzyme consumption has been demonstrated, in contrast to the traditional methods of quantitative citrate analysis.     

Kinetics of collagen-bound sorbitol dehydrogenase in the rotating membrane reactor: Opposite variations of affinity constants under diffusional limitations

Sorbitol dehydrogenase was bound to the surface of acyl-azide-activated collagen membranes and its kinetics was investigated as a model of two-substrate or cofactor-requiring enzyme reactions. The study was performed with the “rotating membrane reactor” especially designed to obtain a precise variation of the external mass-transfer coefficient, and thus the direct visualization of diffusional effects on the bound enzyme behavior. Diffusional limitations for NADH were found to decrease the apparent affinity for NADH, but to increase the apparent affinity for fructose. Such opposite effects of diffusional limitations on apparent affinities are generally applicable to reactions involving two substrates or a substrate and a cofactor of widely different affinities.     

Homogeneous Biocatalysis in Organic Solvents and Water-Organic Mixtures

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.     

Homogeneous biocatalysis in organic solvents and water-organic mixtures

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.     

Effect of buried potential barrier in label-less electrochemical immunodetection of glutathione and glutathione-capped gold nanoparticles

The influence of potential barriers, introduced to the immunoglobulin-based sensory films, on voltammetric signals of a redox ion probe has been investigated. Films with positive and negative barriers have been examined by depositing charged self-assembled thiol monolayers as the basal layers of a sensory film. The studies performed with monoclonal anti-glutathione antibody-based sensors using ferricyanide ion probe have shown stronger sensor response to the layer components, as well as to the glutathione-capped gold nanoparticles acting as the antigen, for films with positive potential barrier buried deep in the film than for negative barrier films. The larger changes in differential resistance, peak separation and peak heights observed for films with positive barrier have been attributed to different depth and width of the charge distributions in these films. A buried positive barrier with narrow charge distribution width provides the best conditions for film stability and prevents fouling (less ion-exchanges with the medium). This conclusion has been confirmed by calculations of the electric field distribution and potential profiles in immunosensing films performed by numerical integration of Poisson equation for Gaussian distributions of fixed charges of covalently bound components. The proposed fixed-charge model can aid in rapid evaluation of sensory films in sensor development work. The implications of potential barriers in sensory film design are discussed.     

以Fe(III) 改性胶原纤维为载体固定过氧化氢酶

将胶原纤维用三价铁改性后作为载体,通过戊二醛的交联作用将过氧化氢酶固定在该载体上。制备的固定化过氧化氢酶蛋白固载量为16.7 mg/g,酶活收率为35％。研究了固定化酶与自由酶的最适pH、最适温度、热稳定性、贮存稳定性及操作稳定性。结果表明：过氧化氢酶经此法固定化后,最适pH及最适温度与自由酶相同,分别为pH 7.0和25 ℃;但固定化酶的热稳定性显著提高,在75 ℃保存5 h后,仍能保留30％的活力,而自由酶则完全失活;固定化酶在室温下保存12 d后,酶活力仍保持在88％以上,而自由酶在此条件下则完全失     

Influence of collagen and chondroitin sulfate (CS) coatings on poly-(lactide-co-glycolide) (PLGA) on MG 63 osteoblast-like cells

Poly-(lactide-co-glycolide) (PLGA) is an FDA-approved biodegradable polymer which has been widely used as a scaffold for tissue engineering applications. Collagen has been used as a coating material for bone contact materials, but relatively little interest has focused on biomimetic coating of PLGA with extracellular matrix components such as collagen and the glycosaminoglycan chondroitin sulfate (CS). In this study, PLGA films were coated with collagen type I or collagen I with CS (collagen I/CS) to investigate the effect of CS on the behaviour of the osteoblastic cell line MG 63. Collagen I/CS coatings promoted a significant increase in cell number after 3 days (in comparison to PLGA) and after 7 days (in comparison to PLGA and collagen-coated PLGA). No influence of collagen I or collagen I/CS coatings on the spreading area after 1 day of culture was observed. However, the cells on collagen I/CS formed numerous filopodia and displayed well developed vinculin-containing focal adhesion plaques. Moreover, these cells contained a significantly higher concentration of osteocalcin, measured per mg of protein, than the cells on the pure collagen coating. Thus, it can be concluded that collagen I/CS coatings promote MG 63 cell proliferation, improve cell adhesion and enhance osteogenic cell differentiation.     

胶原蛋白水解物在化妆品中的功能特性

研究了胶原蛋白经酶水解后所得产物的功能特性:吸水性、保水性、溶解性都得到提高;吸油性、起泡性和泡沫稳定性均明显下降。可根据不同分子量范围的胶原水解物的功能特性满足不同化妆品的不同需求。     

Pulse technique for the electrochemical deposition of polymer films on electrode surfaces

Often, electrochemically-induced deposition of conducting polymer films on electrode surfaces fails using potentiostatic, galvanostatic or multisweep deposition procedures if bulky substituents at the monomer, nucleophilic attack at intermediate radical cations, hindered diffusional mass transport of the monomer to the electrode surface or the copolymerization of monomers with different oxidation potentials prevent fast chain propagation. A pulse profile for the electrochemical deposition of conducting polymer films has been developed based on the rationalization of the limiting steps and the concentration profiles in front of the electrode surface. The pulse deposition method could be advantageously applied for the localized deposition of conducting polymers using scanning electrochemical microscopy, for the copolymerization of pyrrole/[Os(2,2′-bipyridine)₂(3-{pyrrole-1-ylmethyl}pyridine)Cl]⁺ and for the entrapment of enzymes within the growing ramified network of the polymer.     

The use of forced protein evolution to investigate and improve stability of family 10 xylanases. The production of Ca2+-independent stable xylanases

Metal ions such as calcium often play a key role in protein thermostability. The inclusion of metal ions in industrial processes is, however, problematic. Thus, the evolution of enzymes that display enhanced stability, which is not reliant on divalent metals, is an important biotechnological goal. Here we have used forced protein evolution to interrogate whether the stabilizing effect of calcium in an industrially relevant enzyme can be replaced with amino acid substitutions. Our study has focused on the GH10 xylanase CjXyn10A from Cellvibrio japonicus, which contains an extended calcium binding loop that confers proteinase resistance and thermostability. Three rounds of error-prone PCR and selection identified a treble mutant, D262N/A80T/R347C, which in the absence of calcium is more thermostable than wild type CjXyn10A bound to the divalent metal. D262N influences the properties of the calcium binding site, A80T fills a cavity in the enzyme, increasing the number of hydrogen bonds and van der Waals interactions, and the R347C mutation introduces a disulfide bond that decreases the free energy of the unfolded enzyme. A derivative of CjXyn10A (CfCjXyn10A) in which the calcium binding loop has been replaced with a much shorter loop from Cellulomonas fimi CfXyn10A was also subjected to forced protein evolution to select for thermostablizing mutations. Two amino acid substitutions within the introduced loop and the A80T mutation increased the thermostability of the enzyme. This study demonstrates how forced protein evolution can be used to introduce enhanced stability into industrially relevant enzymes while removing calcium as a major stability determinant.     

Magnetic supports for immobilized enzymes and bioaffinity adsorbents

Magnetic separation of immobilized enzymes or bioaffinity adsorbents allows their selective recovery from liquors containing other suspended solids, and gives easier handling of large numbers of samples in analysis. Non-porous magnetic supports seem to be more resistant to fouling, diffusional limitation and attrition than conventional porous supports. A variety of magnetic powders and linkage methods have been used in the preparation of supports, though it is not clear how many of these have the required properties for potential applications (including linkage stability, particle size and magnetic properties). Non-porous magnetic supports are attractive for use in liquors containing fouling materials or suspended solids, either for bioaffinity adsorbents or for immobilized enzymes acting on small molecular substrates. Magnetic supports also offer considerable advantages in analysis based on bioaffinity interactions, such as immunoassay.