Immobilization of enzymes on PTFE surfaces.

Membranes and powders prepared from PTFE (polytetrafluorethylene) were investigated for their potential use as multifunctional supports for enzymes. The obtained bioactive materials are valuable for the construction of biosensors and enzyme reactors. To allow covalent coupling of enzymes to PTFE, the surface of the material was treated with elementary sodium followed by oxidation with ozone or hydrogen peroxide.%Derivatization steps were optimized in order to achieve highest enzyme loading and short reaction times. Alliinase (EC 4.4.1.4) and L-lactic dehydrogenase (EC 1.1.1.27) were chosen as model enzymes and were either immobilized by covalent coupling or fixed indirectly by a sugar-lectin binding. For the latter method, the sugar mannan was bound to the membrane surface as an anchor for layers of the lectin concanavalin A and the alliinase. Highest alliinase loading was achieved at 0.2 microg x cm(-2). Immobilization of alliinase via the lectin concanavalin A and a bifunctional epoxide gave the best long-term stability.%L-Lactic dehydrogenase was most sufficiently immobilized by using benzoquinone as spacer. These procedures show several advantages: 1) enzymes can be immobilized under physiological conditions, 2) an enzyme-multilayer can be achieved, and 3) protein layers are renewable.     

Immobilization of enzymes with polyaziridine: II. D-amino acid oxidase

Summary A novel method of enzyme immobilization using a tri-functional aziridine to immobilize enzymes was used to immobilize D-amino acid oxidase (DAAO) with good retention of enzymatic activity (62%–89%). The stability of the immobilized DAAO in a fixed bed reactor with continuous operation using D-phenylalanine as substrate yielded a projected half-life of 69 days which is far superior to other methods of immobilization of DAAO.     

Immobilization of human intestinal mucosa enzymes on sepharose.

Digestive enzymes from human intestinal mucosa were solubilized by Triton X-100 and papain and covalently bound to eyanogen bromide-activated Sepharose 4-B gel. Triton X-100 solubilized most of the activities, and 39.1 to 63.5% were immobilized on the carrier. The other enzymes, still bound on the microvilli, were subsequently solubilized by papain but then the yield of immobilization reached only 11.0 to 17.6%. The enzyme-Sepharose gel was freeze-dried with a filler and stored without loss of activity. The rate of hydrolysis of di- and trisaccharides, dipeptides, and p-nitrophenylphosphate was measured by incubation on a small column containing less than 0.03 U of immobilized activities. The enzymatic multiplicity and catalytic properties of the intestinal mucosa enzymes were fully recovered on the carrier. This method is proposed for routine evaluation of the digestibility of dipeptides and synthetic disaccharides.     

Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors.

Two improved plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusions, have been constructed. These vectors allow fusion of any gene to the protein A moiety, giving fusion proteins which can be purified, in a one-step procedure by IgG affinity chromatography. One vector, pRIT2, is designed for temperature-inducible expression of intracellular fusion proteins in Escherichia coli and the other pRIT5, is a shuttle vector designed for secretion. The latter gives a periplasmatic fusion protein in E. coli and an extracellular protein in Gram-positive hosts such as Staphylococcus aureus. The usefulness of these vectors is exemplified by fusion of the protein A gene and the E. coli genes encoding the enzymes beta-galactosidase and alkaline phosphatase. High amounts of intact fusion protein are produced which can be immobilized on IgG-Sepharose in high yield (95-100%) without loss of enzymatic activity. Efficient secretion in both E. coli and S. aureus, was obtained for the alkaline phosphatase hybrid, in contrast to beta-galactosidase which was only expressed efficiently using the intracellular system. More than 80% of the protein A alkaline-phosphatase hybrid protein can be eluted from IgG affinity columns without loss of enzymatic activity.     

Immobilization of enzymes on magnetic particles

Trypsin (EC 3.4.4.4) was immobilized in low yield on aminoalkylsilylated magnetite (Fe₃O₄). Better results were obtained when trypsin was immobilized by crosslinking with glutaraldehyde on magnetite. The preparation contained 36 mg protein/g magnetite and the enzyme retained 46% and 11% of esterase and proteolytic activity. Immobilized trypsin was more heat stable than trypsin. Invertase (β-D -fructofuranoside fructohydrolase, EC 3.2.1.26) was cross-linked on magnetite with glutaraldehyde in low yield due to the inactivation of the enzyme. However in the presence of 1% sucrose, the total activity recovered was 79% of the initial activity and the preparation contained 4.4 mg/g of active invertase. Immobilized invertase was less active than invertase when acting on oligosaccharides of the raffinose family. The immobilized enzymes could be easily recovered, from solutions or suspensions, magnetically.     

CO2转化酶的固定化研究

CO2是导致温室效应的主要气体,固定和转化CO2的研究对于温室效应的减缓和环境保护方面具有重要意义。近年来CO2转化的研究取得了迅猛发展,其中生物法固定CO2由于其反应条件温和且绿色无污染的优点而备受关注。本文对转化CO2有关的乳酸脱氢酶（LDH）、苹果酸脱氢酶（MDH）和草酰乙酸脱羧酶（OAADC）进行了初步的固定化分析。首先以碳纳米管、壳聚糖和海藻酸钠为原料,制备了包埋上述CO2转化酶的微胶囊固定化体系,然后分别比较了游离酶和固定化酶的操作稳定性和储存稳定性。研究结果表明,固定化的CO2转化酶的操作稳定性和储存稳定得到明显的提高。本研究对CO2的转化和应用方面具有重要参考价值。     

Immobilization of enzymes on globulin]

Immobilization of lipase and amylosubtilisin on water-insoluble proteins, i. e. globulins, was studied. It was found that immobilization results in the enzyme stabilization. The immobilized enzyme can be transformed from the soluble state into the insoluble one and vice versa by changing the ionic strength of the solution. The advantages of immobilization on globulin in the reactions with water-insoluble substrates are demonstrated.     

Immobilization of enzymes and microbial cells using carrageenan as matrix.

Conditions for the gelation k-carrageenan, which is a new polymer for immobilization of enzymes and microbial cells, were investigated in detail. k-Carrageenan was easily induced to gel by contact with metal ions, amines, amino acid derivatives, and water-miscible organic solvents. By using this property of k-carrageenan, the immobilization of enzymes and microbial cells was investigated. Several kinds of enzymes and microbial cells were easily immobilized with high enzyme activities. Immobilized preparations were easily tailor-made to various shape such as cube, bead, and membrane. The obtained immobilized preparations were stable, and columns packed with them were used for continuous enzyme reaction for a long period. Their operational stabilities were enhanced by hardening with glutaraldehyde and hexamethylenediamine.     

Immobilization of enzymes on chitin and chitosan

During the last few years, d-glucose isomerase, glucoamylase, β-d-galactosidase (lactase), β-d-glucosidase, d-glucose oxidase, AMP deaminase, urease, pronase, subtilisin, trypsin, papain, alkaline phosphatase, acid phosphatase, pepsin, chymotrypsin and lysozyme have been immobilized on chitin and on some of its derivatives, mainly with glutaraldehyde. The preparation and performances of the immobilized enzymes are described.     

Immobilization of enzymes by radiopolymerization of acrylamide

A new and simple method for immobilization of enzymes by the aerobic radio-polymerization of acrylamide was developed. Irradiation treatment of acrylamide in the frozen state produces a spongy immobilized enzyme membrane without the addition of carriers. Aerobic polymerization yields of acrylamide in the frozen state were increased by the addition of starch and also by lyophilization. Glucose oxidase (activity recovery was 12.3–33.7%), invertase (69.2%), D -amono acid oxidase (25.0–70.5%), aminoacylase (39.2–43.7%), mold α-amylase (18.0%), malt β-amylase (4.1%), glucoamylase (6.5%), alkaline protease (5.3%), and neutral protease (10.5%) were immobilized by this method. Invertase entrapped by this method had a wider optium pH range and was active at higher temperatures.     

Immobilization of enzymes on heterofunctional epoxy supports

Immobilization of enzymes and proteins on activated supports permits the simplification of the reactor design and may be used to improve some enzyme properties. In this sense, supports containing epoxy groups seem to be useful to generate very intense multipoint covalent attachment with different nucleophiles placed on the surface of enzyme molecules (e.g., amino, thiol, hydroxyl groups). However, the intermolecular reaction between epoxy groups and soluble enzymes is extremely slow. To solve this problem, we have designed "tailor-made" heterofunctional epoxy supports. Using these, immobilization of enzymes is performed via a two-step process: (i) an initial physical or chemical intermolecular interaction of the enzyme surface with the new functional groups introduced on the support surface and (ii) a subsequent intense intramolecular multipoint covalent reaction between the nucleophiles of the already immobilized enzyme and the epoxy groups of the supports. The first immobilization may involve different enzyme regions, which will be further rigidified by multipoint covalent attachment. The design of some heterofunctional epoxy supports and the performance of the immobilization protocols are described here. The whole protocol to have an immobilized and stabilized enzyme could take from 3 days to 1 week.     

Immobilization of enzymes in microtiter plate scale

Different immobilization methods were adapted to the 96-well microtiter plate scale using esterases as model enzymes. The methods tested were based on adsorption, coprecipitation, aggregation and covalent bonding. The protein covered microcrystals proved to be the best method in terms of yield and expressed activity for the test reaction, which was the alcoholysis of p-nitrophenyl acetate with 1-propanol under anhydrous conditions.     

Immobilization of enzymes on porous silica supports

Four silica supports differing in pore dimensions were activated by treatment with SiCl₄ and then with ethylenediamine to obtain alkylamine groups on the silica surface. Three enzymes, peroxidase from cabbage, glucoamylase from Aspergillus niger C and urease from soybean were immobilized on these supports using glutaraldehyde as coupling agent. It was found that the protein content, the retained enzymatic activity and the storage stability of the silica supported enzymes were considerably affected by support pore size and enzyme molecular weight, the factors which are supposed to alter protein distribution inside the support pores. The highest activity was found for peroxidase and glucoamylase attached to the silica with the widest pores, but their loss in activity during storage was considerable. The urease retained less activity after immobilization, but its storage stability was excellent.     

Immobilization of enzymes masks their active site

We studied the effect of immobilizing cellulase to carboxycellulose sodium by radiation polymerization on the masking of the active site of the enzyme. Masking of the enzyme during the preparation of immobilized enzyme was assayed at tow temperature. The activity of immobilized enzyme was retained during repeated batch reactions, indicating that the enzyme was firmly trapped in the polymer matrix. Various compounds (designated monomers) were used to dissolve the carboxymethylcellulose; enzyme activity was affected by the nature of the monomer, by the monomer concentration, and by the solubility of the substrate in monomer.     

Immobilization of microbial cells and enzymes with hydrophobic photo-crosslinkable resin prepolymers

Summary The accumulation of cadmium from aqueous systems by various green microalgae was investigated with focus, on Chlorella regularis as it is known to concentrate large amounts of heavy metals. The amount of cadmium absorbed by Chlorella cells was rapid during the first 30 min following addition of cadmium and then continued to be absorbed more slowly. The uptake of cadmium by Chlorella was not markedly affected by temperature or metabolic inhibitors. Most of the cadmium absorbed by Chlorella cells was easily released by EDTA. The amount of cadmium absorbed differed markedly with the pH value of the solution and was inhibited by the presence of other divalent cations. Heat-killed Chlorella cells took up cadmium to a greater degree than living ones. From these results, it was considered that the uptake of cadmium into Chlorella cells was not directly mediated by metabolic processes, rather it appeared completely dependent upon physico-chemical adsorption on the cell surface.The ability to accumulate cadmium was species specific and found to be (in decreasing order); Chlamydomonas reinhardtii>Chlorella regularis> Scenedesmus bijuga>Scenedesmus obliquus>Chlamydomonas angulosa> Scenedesmus chlorelloides.Studies on the Accumulation of Heavy Metal Elements in Biological Systems Part XIV     

Immobilization of the hydantoin cleaving enzymes from Arthrobacter aurescens DSM 3747.

The immobilization procedure of the two industrially important hydantoin cleaving enzymes--hydantoinase and L-N-carbamoylase from Arthrobacter aurescens DSM 3747--was optimized. Using different methods (carbodiimide, epoxy activated carriers) it was possible to immobilize the crude hydantoinase from A. aurescens DSM 3747 to supports containing primary amino groups with a yield of up to 60%. Immobilization on more hydrophobic supports such as Eupergit C and C 250 L resulted in lower yields of activity, whereas the total protein coupled remained constant. All attempts to immobilize the crude L-N-carbamoylase resulted in only low activity yields. Therefore, the enzyme was highly purified and used in immobilization experiments. The pure enzyme could easily be obtained in large amounts by cultivation of a recombinant Escherichia coli strain following a three step purification protocol consisting of cell disruption, chromatography on Streamline diethylaminoethyl and Mono Q. The immobilization of the L-N-carbamoylase was optimized with respect to the coupling yield by varying the coupling method as well as the concentrations of protein, carrier and carbodiimide. Using 60 mM of water-soluble carbodiimide, nearly 100% of the enzyme activity and protein could be immobilized to EAH Sepharose 4B.     

Immobilization of perfluoroalkylated enzymes in a biologically active state onto Perflex support.

Perflex has been introduced by E. I. du Pont de Nemours and Co., Inc., as a new fluorocarbon-based technology for protein immobilization. Due to the hydrophobic character of the support, however, significant loss of enzymatic activity may occur upon immobilization of certain enzymes, which appears to be due to a large conformational change of the protein ("inversion"). Pretreatment of the Perflex support with a neutral fluorosurfactant lessened the surface hydrophobicity, thus decreasing the hydrophobic interaction between the support and the protein. Modification of enzymes with a high number of fluorocarbon residues, which forms a hydrophobic "envelope" around the protein, also appears to prevent enzyme inactivation upon immobilization on Perflex support. Moreover, preactivation of the support with either perfluorooctylpropylisocyanate or reactive poly(fluoroalkyl) sugar reagents greatly improves the enzyme particle activity by increasing the amount of immobilized enzyme. Fluorosurfactant treatment of the support activated with perfluorooctylpropylisocyanate improves the retention of activity for sensitive enzymes such as alpha-chymotrypsin and increases the wetability and ease of handling of the Perflex particles.