Electrochemical evaluation of lactate dehydrogenase immobilized in polyacrylamide gels

Lactate dehydrogenase (EC 1.1.1.27) has been immobilized in polyacrylamide gels over a platinum grid matrix. The immobilized enzyme is used to oxidize L-lactate in the presence of nicotinamide adenine dinucleotide (NAD+) and ferricyanide. The NADH produced is then chemically oxidized back to NAD+ by ferricyanide. The coupled reduction of ferricyanide ions to ferrocyanide ions results in a measurable electrochemical potential. This measurable zero-current potential is found to be Nernstian in nature and directly proportional to the logarithm values of L-lactate concentration over the range of 2 X 10(-5) to 5 X 10(-2)M. The results indicate that immobilized lactate dehydrogenase can be incorporated into a system to detect L-lactate acid in aqueous solutions.     

Immobilization of oxyreductases on inorganic supports based on alumina. Immobilization of lactate dehydrogenase on alumina by adsorption

Lactate dehydrogenase (LDH) was adsorbed on low-(γ, η) and high -(θ, α) temperature forms of alumina. θ-Al₂O₃ exhibited the greatest adsorption ability. The maximum adsorption value was 30 mg LDH/g of a carrier. The conditions for irreversible adsorption have been determined. An adsorption isotherm on θ-Al₂O₃ for pH 6.0 has been obtained; the LDH_ads surface area and the carrier surface portion accessible to the enzyme molecules have been calculated. The reaction kinetic parameter were determined by taking into account the reaction proceeding in the intradiffusional region. The specific catalytic activity (A_spec) of LDH_ads at small surface coverage of θ-Al₂O₃ is five times less than A_spec of the native enzyme and K_M^imm with respect to NADH exceeds K_M^nat by two orders or magnitude. The is evidence for a strong LDH–Al₂O₃ interaction and a considerable deformation of the enzyme globule. A_spec and K_M decrease as the amount of the enzyme attached to the carrier increases. Due to adsorption. LDH becomes thermostable and durable. The LDH_ads samples conserve 20–40% of their activity at room temperature during a year.     

Immobilization of Saccharomyces uvarum cells in porous beads of polyacrylamide gel for ethanolic fermentation

Summary A procedure which does not involve the use of an immiscible organic solvent phase is described for the entrapment of yeast cells in porous beads of polyacrylamide gel. The cells are rapidly dispersed at 4° C in an aqueous solution containing sodium alginate and acrylamide-N,Nmethylene-bis-acrylamide monomer, and the suspension is immediately dropped into a solution of calcium formate to give calcium alginate coated beads. Polyacrylamide gel forms within the bead. The calcium alginate is subsequently leached out of the composite bead with either sodium citrate or potassium phosphate buffer solution. Cells of Saccharomyces uvarum ATCC 26 602 entrapped in such polyacrylamide beads ferment cane molasses in batch mode at higher specific ethanol productivity than a free cell suspension. Their volumetric productivity in continuous fermentation is higher than that of Ca²⁺-alginate immobilized cells.NCL Communication No. 4383     

Immobilization and kinetics of lactate dehydrogenase at a rotating nylon disk

Lactate dehydrogenase (LDH) was covalently attached to an impervious nylon surface by an improved technique. The procedure allowed the kinetics of the rotating enzyme disk reactor to be successfully explored. This enzyme-disk configuration has potential applications in assays for lactic acid or pyruvic acid in fluids of biological importance (e.g., urine). In order to evaluate and understand the physics and chemistry underlying the kinetics of the heterogeneous biocatalyst, a mathematical model based on the von Karman-Levich theories of rotating electrodes, was developed. It applied well to LDH attached to a disk, under variable NADH concentrations and fixed pyruvic acid. The new theory, leads to the conclusion that the apparent Michaelis constant K(m)(app), varies linearly with f(-1/2), where f is the speed of rotation of the disk. Extrapolation of f(-1/2) to zero gives the Michaelis-Menten constant, K(m), corresponding to the diffusion-free behavior. With immobilized LDH, the diffusion-free K(m) for NADH obtained at 25 degrees C, in phosphate buffer (pH 7.5) using the extrapolation method was 84 muM. This value was in good agreement with the previously published value of 87 muM, obtained with LDH attached to the inner surface of a nylon tubing. However, when compared to the K(m) for a free enzyme system, the 84 muM was about nine times larger, indicating an inherent reduction in the activity of the bound LDH. Since, at extrapolated infinite rotation speeds, diffusion effects were assumed eliminated, the drop in the activity was thought to be due to sterric hinderances imposed on the substrate NADH as a result of having LDH bound to another polymer.     

Immobilization of hydrogenase on glass beads

Hydrogenase from $\text{Clostridium pasteurianum}$ was immobilized on glass beads by four different methods. The sensitivity of the native and bound enzyme to oxygen was examined. Hydrogenase bound to succinyl glass proved to be the most stable to oxygen. All bound enzymes were active with ferredoxin as a substrate and evolved hydrogen in a chloroplast-ferredoxin-hydrogenase system driven by light.     

Immobilization of proteins on organic polymer beads

A new method is described for the immobilization of biologically active proteins onto several types of organic polymer beads. First, the soluble protein is modified by reaction with an excess of a hydrophobic imidoester, for example methyl 4-phenyl-butyrimidate, at ca. pH 9 and 0 degrees . Excess imidoester and side products resulting from imidoester hydrolysis are separated from the hydrophobic protein derivative by exclusion chromatography or dialysis. A suspension of polymer beads (e.g. Amberlite XAD-7) is then added to a solution of the modified protein at room temperature or below and stirred gently for 1-2 h. The polymer beads are allowed to settle, separated from the solution by decantation or filtration, washed, and resuspended in an appropriate buffer. Quantitative adsorption of protein to the polymer beads is observed under such conditions. The synthesis of seven hydrophobic imidoesters and their use for the immobilization of trypsin onto several types of porous polymer beads is described. The immobilizations of trypsin, yeast alcohol dehydrogenase, and E. coli asparaginase by this procedure with high recoveries of catalytic activity, suggests that it will be applicable to a large number of biologically active proteins.     

Immobilization of proteins on aldehyde-activated polyacrylamide supports

A method has been developed for the immobilization of proteins on derivatized polyacrylamide gels. Aminoethyl Bio-Gel P-150 was converted to its stable N-2,3-dihydroxypropyl derivative by borohydride reduction of the Schiff base formed with glyceraldehyde. Periodate oxidation of the modified gel provided a reactive aldehyde, which was subsequently coupled to protein by reductive amination with sodium cyanoborohydride. Coupling efficiencies were found to be >90% for concanavalin A and bovine serum albumin, and the gels contained as much as 5 and 20 mg of protein/ml of gel, respectively. Immobilized concanavalin A retained 89% of its binding capacity and was demonstrated to be chemically stable with variations in pH, and changes in concentrations of Triton X-100 and sodium dodecyl sulfate (at concentrations <0.1%). Bovine β-hexosaminidase and β-glucuronidase, higher molecular weight proteins, were also bound with retention of activity, but with less efficiency. This procedure provides an efficient method for the covalent immobilization of proteins.     

Immobilization of tyrosinase on chitosan-clay composite beads

Tyrosinase was immobilized on glutaraldehyde crosslinked chitosan-clay composite beads and used for phenol removal. Immobilization yield, loading efficiency and activity of tyrosinase immobilized beads were found as 67%, 25% and 1400 U/g beads respectively. Optimum pH of the free and immobilized enzyme was found as pH 7.0. Optimum temperature of the free and immobilized enzyme was determined as 25-30 °C and 25 °C respectively. The kinetic parameters of free and immobilized tyrosinase were calculated using l-catechol as a substrate and K(m) value for free and immobilized tyrosinase were found as 0.93 mM and 1.7 mM respectively. After seven times of repeated tests, each over 150 min, the efficiency of phenol removal using same immobilized tyrosinase beads were decreased to 43%.     

Immobilized enzymes: the catalytic properties of lactate dehydrogenase convalently attached to glass beads

Chick LDH (H₄ and M₄) has been covalently attached to aryl and alkyl amine glass using sodium nitrite and glutaraldehyde respectively. These immobilized enzymes remain active for months at 0°C and exhibit Km values similar to those of the soluble enzyme; however, they have pH-rate profiles that are independent of pH and show decreased substrate inhibition. Disaggregation followed by reassociation indicate the enzymes are bound by all four subunits and the resulting activity restored to the native, aryl amine and glutaraldehyde bound enzyme are 33, 25 and 90% respectively. At a pH of 3.2 and 25°, the soluble and aryl amine glass LDH's are rapidly denatured while the glutaraldehyde bound enzyme shows no loss of activity for at least 35 days.     

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.     

Immobilization of proteins on partially hydrolyzed agarose beads

Treatment of agarose beads with mild acid (0.2 M HCl, 55 degrees C, several hours) hydrolyzes some of the glycosidic bonds between D-galactosyl residues and 3,6-anhydro-L-galactosyl residues, and thus produces aldehydo-groups useful for immobilization of amino compounds by reductive amination with NaCNBH3. More than 20 mg (0.3 mumol) of bovine serum albumin could be coupled per gram of partially hydrolyzed agarose beads. Arthrobacter neuraminidase immobilized by this method was useful for desialylation of sialyl glycoconjugates, and was found not to leach from the gel and to be much more thermostable than the free enzyme.     

Immobilization of tyrosinase within polyacrylamide gels

The enzyme tyrosinase (E.G. 1.14.18.1) has been immobilized in a polyacrylamide gel and intermittently assayed for enzyme activity over a period of 19 days using phenol as the substrate. The results of these studies indicate that the immobilized enzyme could be incorporated into a system to detect phenol and related compounds that are found in industrial effluents and as surface water contaminants.     

Immobilization of lipase onto micron-size magnetic beads

A novel and economical magnetic poly(methacrylate-divinylbenzene) microsphere (less than 8 microm in diameter) was synthesized by the modified suspension polymerization of methacrylate and cross-linker divinylbenzene in the presence of magnetic fluid. Then, surface aminolysis was employed to obtain a high content of surface amino groups (0.40-0.55 mmolg(-1) supports). The morphology and properties of these magnetic supports were characterized with scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy and a vibrating sample magnetometer. These magnetic supports exhibited superparamagnetism with a high specific saturation magnetization (sigma(s)) of 14.6 emicrog(-1). Candida cylindracea lipase was covalently immobilized on the amino-functionalized magnetic supports with the activity recovery up to 72.4% and enzyme loading of 34.0 mgg(-1) support, remarkably higher than the previous studies. The factors involved in the activity recovery and enzymatic properties of the immobilized lipase prepared were studied in comparison with free lipase, for which olive oil was chosen as the substrate. The results show that the immobilized lipase has good stability and reusability after recovery by magnetic separation within 20s.