The effect of mechanical stretching of the myosin rod component (fragment LMMMM S-2) on the ATPase activity of myosin.

The binding of myosin to nylon fiber gives immobilized myosin with a considerable ATPase activity. Treatment of immobilized enzyme with papain results in the entire ATPase activity (known to be concentrated in myosin heads, (fragment HMM S-1)) being replaced from the fiber into the solution; this means that myosin is chemically bound to the fiber via its rod part (fragment LMM+HMM S-2). When nylon fiber is mechanically stretched, the ATPase activity of myosin attached to it sharply decreases; after relaxation of the fiber the enzymatic activity returns to the initial level. The detailed study of this phenomenon has shown that reversible inactivation of myosin upon fiber stretching is not the result of an altered microenvironment of the enzyme. The discovered regulatory effect is ascribed to deformation of myosin molecules induced by support stretching. Thus deformation of the myosin tail (not indispensable for ATPase since its cleaving-off does not alter the enzymatic activity) leads to decrease in the ATPase activity of the enzyme. The possible role of the above phenomenon in the mechanism of muscle contraction is discussed.     

Oligonucleotide derivatives in the hybridization analysis of nucleic acids. I. Covalent immobilization of oligonucleotide probes on nylon

The characteristics of the UV-induced immobilization of oligonucleotides on nylon membranes and the efficiency of the enzymatic labeling of immobilized probes in heterophase identifying specific DNA sequences were studied. Oligonucleotides bound to short terminal oligothymidylates (up to 10 nt) through a flexible linker based on diethylene glycol phosphodiester are proposed as probes for immobilization on nylon. The presence of this fragment allows one to enhance the immobilization efficiency and reduce the UV-dependent degradation of the sequence-specific part of the probe by decreasing the irradiation dose needed for DNA immobilization. The optimal dose of UV irradiation is evaluated to be ∼0.4 J/cm² at 254 nm, which provides a high level of the hybridization signal for immobilized probes of various nucleotide sequences. It was found that nylon amide groups play a key role in the photoinduced fixation of oligonucleotides to the polymer surface, while its primary amino groups were not as responsible for the covalent binding of DNA as previously thought. Various additives in the membrane wetting solution were demonstrated to influence both the efficiency of the UV-induced immobilization and the functional integrity of immobilized probes. Other radical generating systems alternative to UV irradiation are shown to provide the immobilization of oligonucleotides on nylon membranes.     

Direct immobilization of tyrosinase enzyme from natural mushrooms (Agaricus bisporus) on D-sorbitol cinnamic ester

Mushroom tyrosinase was immobilized from an extract onto the totally cinnamoylated derivative of D-sorbitol by direct adsorption as a result of the intense hydrophobic interactions that took place. The immobilization pH value and mass of lyophilized mushrooms were important parameters that affected the immobilization efficiency, while the immobilization time and immobilization support concentration were not important in this respect. The extracted/immobilized enzyme could best be measured above pH 3.5 and the optimum measuring temperature was 55 degrees C. The apparent Michaelis constant using 4-tert-butylcatechol as substrate was 0.38+/-0.02 mM, which was lower than for the soluble enzyme from Sigma (1.41+/-0.20 mM). Immobilization stabilized the extracted enzyme against thermal inactivation and made it less susceptible to activity loss during storage. The operational stability was higher than in the case of the tyrosinase supplied by Sigma and immobilized on the same support. The results show that the use of p-nitrophenol as enzyme-inhibiting substrate during enzyme extraction and immobilization made the use of ascorbic acid unnecessary and is a suitable method for extracting and immobilizing the tyrosinase enzyme, providing good enzymatic activity and stability.     

Dual immobilization of biomolecule on the glass surface using cysteine as a bifunctional linker

A novel method for highly efficient enzyme immobilization on the glass surface, by incorporating cysteine as a linker has been demonstrated. The internal glass surface of test tube was pretreated with (3-mercaptopropyl) trimethoxysilane sol–gel and cysteine capped silver nanoparticles, to generate a cysteine layer. This, cysteine rich surface is then used to covalently immobilize alkaline phosphatase on both groups (amino and carboxyl) of cysteine through carbodiimide and glutaraldehyde treatment. The cysteine capped silver nanoparticles were synthesized with an average nanoparticle size of 61 nm as determined by particle size analyzer, while cysteine capping of nanoparticles was confirmed by Fourier transform infra-red spectroscopy. Enhanced enzymatic activity of about 73% was obtained using the dual immobilization technique, while 40% enzyme activity was recovered with carboxyl group and 51% with amino group only. The re-usability of the enzyme immobilized test tube was found to be 8 times and the enzyme retained 85% of its initial activity. With such high immobilization efficiency, cysteine provides a new approach for enhanced immobilization and its integration into different industrial processes and biosensor technology.     

Anionic exchange nylon laminated membranes for enzyme immobilization

Summary This work presents the optimization of the chemical steps involved in nylon modification with dimethyl sulphate, polyethyleneimine, glutaraldehyde and 2-diethyl aminoethylamine to obtain a weak basic anion exchange support. Activated nylon laminated membranes were utilized for aminoacylase immobilization, allowing an ionically adsorbed enzyme derivative with high activity (0.16 U/mg E·cm²) and low removed activity (<1%). Optimum immobilization conditions and kinetic parameters were also determined. This immobilized enzyme can be used in laminated enzyme membrane reactors.     

Immobilization of enzymes with polyaziridines.

A novel method of enzyme immobilization using a low molecular weight prepolymer of tri-functional aziridines which can immobilize enzymes both by covalent attachment and entrapment within a gel matrix is described. The enzymes are immobilized on a solid support and exhibit an excellent retention of enzymatic activity. The immobilization procedure is essentially a single step process which can be easily performed at room temperature or 4 degrees C in either aqueous solution or in an inert organic solvent. The polyaziridines used in the immobilization are nontoxic, available in bulk at low cost and completely miscible with water and many organic solvents, thus providing one of the most satisfactory methods of immobilization available.     

Immobilized Candida antarctica lipase B: Hydration, stripping off and application in ring opening polyester synthesis

This work reviews the stripping off, role of water molecules in activity, and flexibility of immobilized Candida antarctica lipase B (CALB). Employment of CALB in ring opening polyester synthesis emphasizing on a polylactide is discussed in detail. Execution of enzymes in place of inorganic catalysts is the most green alternative for sustainable and environment friendly synthesis of products on an industrial scale. Robust immobilization and consequently performance of enzyme is the essential objective of enzyme application in industry. Water bound to the surface of an enzyme (contact class of water molecules) is inevitable for enzyme performance; it controls enzyme dynamics via flexibility changes and has intensive influence on enzyme activity. The value of pH during immobilization of CALB plays a critical role in fixing the active conformation of an enzyme. Comprehensive selection of support and protocol can develop a robust immobilized enzyme thus enhancing its performance. Organic solvents with a log P value higher than four are more suitable for enzymatic catalysis as these solvents tend to strip away very little of the enzyme surface bound water molecules. Alternatively ionic liquid can work as a more promising reaction media. Covalent immobilization is an exclusively reliable technique to circumvent the leaching of enzymes and to enhance stability. Activated polystyrene nanoparticles can prove to be a practical and economical support for chemical immobilization of CALB. In order to reduce the E-factor for the synthesis of biodegradable polymers; enzymatic ring opening polyester synthesis (eROPS) of cyclic monomers is a more sensible route for polyester synthesis. Synergies obtained from ionic liquids and immobilized enzyme can be much effective eROPS.     

Functionalization of catalase for a photochemical immobilization on poly(ethylene terephthalate)

The enzyme catalase (EC 1.11.1.6) was covalently immobilized on textile carrier fabrics made of poly(ethylene terephthalate) (PET) by a novel combined wet chemical and photochemical process. The functionalization of catalase with allyl groups succeeds in a wet chemical treatment of the enzyme with allylglycidylether. This modified enzyme was bonded covalently to the textile material by a photochemical immobilization using a monochromatic excimer UV lamp (222 nm). Using this two-step procedure nearly 60 mg enzyme/g carrier could be fixed durably. The efficiency of the immobilization products was investigated by measuring the enzymatic decomposition of hydrogen peroxide in comparison to the free enzyme. The relative activity of the catalase after the immobilization was nearly 5% compared to the free, not fixed enzyme; however, even after 30 reuses, the modified and immobilized catalase still showed a distinct activity.     

Electrospun polyacrylonitrile nanofibrous membranes for lipase immobilization

Polyacrylonitrile (PAN) nanofibers could be fabricated by electrospinning with fiber diameter in the range of 150–300 nm, providing huge surface area for enzyme immobilization and catalytic reactions. Lipase from Candida rugosa was covalently immobilized onto PAN nanofibers by amidination reaction. Aggregates of enzyme molecules were found on nanofiber surface from field emission scanning electron microscopy and covalent bond formation between enzyme molecule and the nanofiber was confirmed from FTIR measurements. After 5 min activation and 60 min reaction with enzyme-containing solution, the protein loading efficiency was quantitative and the activity retention of the immobilized lipase was 81% that of free enzyme. The mechanical strength of the NFM improved after lipase immobilization where tensile stress at break and Young's modulus were almost doubled. The immobilized lipase retained >95% of its initial activity when stored in buffer at 30 °C for 20 days, whereas free lipase lost 80% of its initial activity. The immobilized lipase still retained 70% of its specific activity after 10 repeated batches of reaction. This lipase immobilization method shows the best performance among various immobilized lipase systems using the same source of lipase and substrate when considering protein loading, activity retention, and kinetic parameters.     

Activation of polyvinyl chloride sheet surface for covalent immobilization of oxalate oxidase and its evaluation as inert support in urinary oxalate determination

Polyvinyl chloride (PVC) sheets are a promising material for enzyme immobilization owing to the PVC’s properties such as being chemically inert, corrosion free, weather resistant, tough, lightweight, and maintenance free and having a high strength-to-weight ratio. In this study, this attractive material surface was chemically modified and exploited for covalent immobilization of oxalate oxidase using glutaraldehyde as a coupling agent. The enzyme was immobilized on activated PVC surface with a conjugation yield of 360 μg/cm². The scanning electron micrographs showed the microstructures on the PVC sheet surface revealing the successful immobilization of oxalate oxidase. A colorimetric method was adopted in evaluating enzymatic activity of immobilized and native oxalate oxidase. The immobilized enzyme retained 65% of specific activity of free enzyme. Slight changes were observed in the optimal pH, incubation temperature, and time for maximum activity of immobilized oxalate oxidase. PVC support showed no interference when immobilized oxalate oxidase was used for estimation of oxalic acid concentration in urine samples and showed a correlation of 0.998 with the values estimated with a commercially available Sigma kit. The overall results strengthen our view that PVC sheet can be used as a solid support for immobilization of enzymes and in the field of clinical diagnostics, environmental monitoring and remediation.     

Laccase immobilization on enzymatically functionalized polyamide 6,6 fibres

Polyamide matrices, such as membranes, gels and non-wovens, have been applied as supports for enzyme immobilization, although in literature the enzyme immobilization on woven nylon matrices is rarely reported. In this work, a protocol for a Trametes hirsuta laccase immobilization using woven polyamide 6,6 (nylon) was developed. A 2⁴ full factorial design was used to study the influence of pH, spacer (1,6-hexanediamine), enzyme and crosslinker concentration on the efficiency of immobilization. The factors enzyme dosage and spacer seem to have played a critical role in the immobilization of laccase onto nylon support. Under optimized working conditions (29 U mL⁻¹ of laccase, 10% of glutaraldehyde, pH = 5.5, with the presence of the spacer), the half-life time attained was about 78 h (18% higher than that of free enzyme), the protein retention was 30% and the immobilization yield was 2%. The immobilized laccase has potential for application in the continuous decolourization of textile effluents, where it can be applied into a membrane reactor.     

Properties of invertase immobilized on the poly(ethylene-co-vinyl alcohol) hollow fiber membrane

Invertase was ionically immobilized on the poly(ethylene-co-vinyl alcohol) hollow fiber inside surface, which was aminoacetalized with 2-dimethylaminoacetaldehyde dimethyl acetal. Immobilization and enzyme reaction were carried out by letting the respective solutions pass or circulate through the inside of the hollow fiber, and the activity of invertase was determined by the amount of glucose produced enzymatically from sucrose. Immobilization conditions were examined with respect to the enzyme concentration and to the time, and consequently the preferable conditions at room temperature were found to be 5 mug/mL of enzyme concentration and 4 h of immobilization time. Under those conditions the immobilization yield and the ratio of the activity of the immobilized invertase to that of the native one were 89 and 80%, respectively. For both repeating and continuous usages, the activity fell to ca. 60% of the initial activity in the early stage and after that almost kept that value. The apparent Michaelis constant K(m) (') for the immobilized invertase decreased with increasing the flow rate of the substrate solution, to be close to the value for the native one. Furthermore, the possibility of the separation of the enzymatically formed glucose from the reaction mixture through the hollow fiber membrane was preliminarily examined.     

Covalent immobilization of acid phosphatase on amorphous AlPO4 support

Covalent attachment of acid phosphatase enzyme, AP, on the surface of amorphous AlPO₄, used as inorganic support, was studied. Immobilization of the enzyme was carried out by the ε-amino group of lysine residues through an aromatic Schiff's-base (linker A), as well as through an `azo' linkage to a p-OH-benzene group of tyrosine residues of the proteins (linker B). Activation of the supports in both cases was developed through the reaction of appropriate molecules with support surface –OH groups. The enzymatic activities in the 1-naphthyl phosphate hydrolysis of native, the different immobilized AP systems, and the filtrates, were obtained by a spectrophotometric method. According to the results, immobilization through linker A gave E_imm=99% while the residual activity, E_res, at different temperatures was in the range 0.2–0.8%. On the other hand, in the immobilization by linker B, through a diazonium salt, E_imm was in the range 35–46%, but residual and specific activity values, E_res and E_spe, were between 19% and 46%. Thus, instead of linker A was more effective in the enzyme immobilization, the highest enzymatic activity after immobilization was obtained with linker B because with linker A a strong deactivation was developed.     

Gluco-oligosaccharide synthesis by free and immobilized beta-glucosidase

Gluco-oligosaccharides were synthesized through the enzymatic condensation of D-glucose at high concentration using a commercial almond beta-glucosidase. The synthesis reactions were carried out with both free and immobilized enzyme, with or without sorbitol, an efficient depressor of water activity (a(w)) in the presence of different glucose concentrations. The yield and the composition of the gluco-oligosaccharides produced changed with the reaction mixture and the form of the enzyme used (free or immobilized). The use of 5 M glucose solution permitted only disaccharides to be obtained, whereas with a glucose concentration of 7.5 M glucose, di-, tri-, and tetrasaccharides were produced. A 7.5 M glucose solution used with 4.4 M sorbitol gave three times more disaccharides than the same solution without sorbitol. Moreover, the immobilized enzyme was much more active in synthesis. The synthesis yield (oligomers mg/mL . mg of enzyme) after immobilization was 573% compared to that of the free enzyme, when a 7.5 M glucose solution was tested. The effects of substrate concentration, sorbitol addition and enzyme immobilization were investigated. (c) 1993 John Wiley & Sons, Inc.     

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.     

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.     

Novel sol-gel immobilization of horseradish peroxidase employing a detergentless micro-emulsion system

A novel sol-gel immobilization method employing a detergentless micro-emulsion system that consisted ofn-hexane/iso-propanol/water was developed and used to immobilize a horseradish peroxidase (HRP). Micro-sized gel powder containing enzymes was generated in the ternary solution without drying and grinding steps or the addition of detergent, therefore, the method described in this study is a simple and straightforward process for the manufacture of gel powder. The gel powder made in this study was able to retain 84% of its initial enzyme activity, which is higher than gel powders produced through other immobilization methods. Furthermore, the HRP immobilized using this method, was able to maintain its activity at or above 95% of its initial activity for 48h, whereas the enzyme activities of free HRP and HRP that was immobilized using the other sol-gel method decreased dramatically. In addition, even when in the presence of excess hydrogen peroxide, the enzyme immobilized using the novel sol-gel method described here was more stable than enzymes immobilized using the other method.     

Enzymatic activity of glucose oxidase covalently wired via viologen to electrically conductive polypyrrole films

The surface functionalization of an electrically conductive polypyrrole film (PPY) with a viologen, (N-(2-carboxyl-ethyl)-N'-(4-vinyl-benzyl)-4,4'-bipyridinium dichloride, or CVV) for the covalent immobilization of glucose oxidase (GOD) has been carried out. The viologen was first synthesized and graft polymerized on PPY film. It then served as an anchor via its carboxyl groups for the covalent immobilization of GOD. The surface composition of the as-functionalized substrates was characterized by X-ray photoelectron spectroscopy (XPS). The effects of the CVV monomer concentration on the CVV-graft polymer concentration and the amount of GOD immobilized on the surface were investigated. The activity of the immobilized GOD was compared with that of free GOD and the kinetic effects were also obtained. The cyclic voltammetric (CV) response of the GOD-functionalized PPY substrates was studied in a phosphate buffer solution under an argon atmosphere. The CV results support the mechanism in which CVV acts as a mediator to transfer electron between the electrode and enzyme, and hence regenerating the enzyme in the enzymatic reaction with glucose. High sensitivity and linear response of the enzyme electrode was observed with glucose concentration ranging from 0 to 20 mM.     

Enzymatic synthesis of glucuronides using lipophilic hollow fiber membranes

A lipophilic hollow fiber membrane preparation was used for the enzymatic glucuronidation of lipophilic aromatic compounds. A crude solubilized microsomal enzyme preparation was circulated on the external side of the lipophilic membrane while the phenol containing buffer solution was circulated through the internal side of the hollow fiber membrane. Phenols, which accumulate in and penetrate the lipophilic membrane, were converted by UDP-glucuronyltransferase to the corresponding glucuronides. During this process the lipophilic compounds are converted to hydrophilic substances, which are not able to rediffuse through the lipophilic membrane into the donor side of the hollow fiber module. The produced glucuronide is separated by means of a coupled dialysis with a module of hydrophilic surface (cellulose acetate), while the enzyme protein is retained.On the stripping side of the dialysing module the glucuronide can be separated by solid phase extraction (Lichroprep RP-18) while a continuous substitution of cofactor into this compartment is possible. UDPGA follows its own concentration gradient and migrates into the enzymatic mixture, where it is utilized. This new technique using hollow fiber modules offers completely new possibilities for long-term high-capacity, highly specific glucuronidation of phenolic compounds. Fields of application are not only the economical production of special glucuronides, but also the specific elimination of phenols from waste fluids or from serum and blood of patients.For the production of glucuronides by this technique the use of highly purified enzymes is not essential. Cheap crude enzyme preparations are quite adequate for an optimal reaction. Using a crude enzyme preparation with a specific batch activity of 13 nMol/min per mg of protein, the activity in the reactor system was observed to be 4.6 nMol/min of 2-naphtol glucuronide formed per mg of protein. This corresponds to 3.6 nMol/h of product formed per mg of protein per cm² of hollow fiber surface.Using a membrane surface of 0.5 m² the production of 18 mMol of glucuronide per h and mg protein can be achieved.     

Determination of concentration and activity of immobilized enzymes

Methods that directly measure the concentration of surface-immobilized biomolecules are scarce. More commonly, the concentration of the soluble molecule is measured before and after immobilization, and the bound concentration is assessed by elimination, assuming that all bound molecules are active. An assay was developed for measuring the active site concentration, activity, and thereby the catalytic turnover rate (k_cat) of an immobilized dihydrofolate reductase as a model system. The new method yielded a similar first-order rate constant, k_cat, to that of the same enzyme in solution. The findings indicate that the activity of the immobilized enzyme, when separated from the surface by the DNA spacers, has not been altered. In addition, a new immobilization method that leads to solution-like activity of the enzyme on the surface is described. The approaches developed here for immobilization and for determining the concentration of an immobilized enzyme are general and can be extended to other enzymes, receptors, and antibodies.