Optimal Enzyme Loading Research Articles

Effect of operating variables on the hydrolysis rate of palm oil by lipase

Hydrolysis of palm oil by lipase enzyme in aqueous phase was studied in a well stirred bioreactor under defined mixing conditions. Agitation speed, concentration of the surfactant, palm oil, and the enzyme were varied to study their effect on the initial rate of hydrolysis. Addition of surfactant, gum arabic, increased the rate of hydrolysis and its optimum amount was found to be 25 mg l −1. The optimum enzyme loading was found to be 92.5 kLU l −1. Studies on the effect of agitation speed and substrate concentration showed that the initial rate of hydrolysis is dependent on the interfacial area between the oil phase and the aqueous phase containing the enzyme. No substrate inhibition was observed in the range of palm oil concentration 0–262 g l −1, used in this study.

Modulated insulin permeation across a glucose-sensitive polymeric composite membrane

A glucose-sensitive polymeric composite membrane was prepared based on our previously developed stimuli-responsive membrane system. Membranes were cast from a mixture of glucose oxidase (GOD), catalase, and poly(N-isopropylacrylamide-co-methacrylic acid) (poly(NIPAm/MAA)) nanoparticles dispersed in a solution of a hydrophobic polymer. High efficiency of enzyme immobilization was achieved with undetectable leakage. The bioactivity of the immobilized GOD, as measured by pH change of glucose solutions, was found to be equivalent to ∼80% of that of the free GOD. The addition of catalase markedly increased the oxidation rate of glucose. However, an optimal unit ratio of GOD to catalase and optimal enzyme loading were observed. The rate of insulin permeation through the membrane was modulated by glucose concentration due to shrinking or swelling of the embedded pH-sensitive nanoparticles. The response of insulin permeability to the change in the glucose concentration could be detected within 5–15 min. The permeability of insulin increased more than 3-fold as the glucose concentration was raised from 50 to 200 mg/dl. The average insulin permeability at 400 mg/dl of glucose was 8-fold that at 50 mg/dl in a continuous test in saline and was 6-fold in a three-cycle discontinuous test in pH 7.4 buffer.

Biosensors and flow-through system for the determination of creatinine in hemodialysate.

Biosensors for the determination of creatinine have been developed and integrated into a flow-through system. The sensors are based on a screen-printed three electrode transducer with a platinum working electrode. Applying the multi-enzyme sequence of creatininase (CA), creatinase (CI) and sarcosine oxidase (SO) hydrogen peroxide has been detected amperometrically. An optimal enzyme load was found to be 4.4 U/0.28 U/0.20 U (CA/CI/SO) and 0.28 U/0.20 U (CI/SO) per electrode for the creatinine sensor and for the creatine sensor, respectively. Among a variety of polymers Nafion has shown the highest efficiency to exclude interfering substances like ascorbic acid, acetaminophen and uric acid. First determinations of creatinine in dialysate samples obtained during hemodialysis treatments have shown a good correlation to the conventional methods, the Jaffé reaction (y=0.945x+ 2.8, R=0.9882, n=9) and the enzymatic photometric method (y=0.891x+3.5, R=0.9917, n=9).

Latex particles with thermo-flocculation and magnetic properties for immobilization of alpha-chymotrypsin.

Core-shell-type latex particles composed of styrene, N-isopropylacrylamide (NIPAAm), and N-acryloxysuccinimide (NAS) were synthesized by surfactant-free emulsion polymerization. The latex particles show thermo-flocculation behavior due to the presence of temperature-sensitive monomer NIPAAm and could be used for immobilization of alpha-chymotrypsin through covalent bonding with the reactive ester groups of NAS. Enzyme recycle could be accomplished in this immobilized enzyme system by sedimentation of the thermo-flocculated latex particles in 20 min at 30 degrees C by raising the salt (NaCl) concentration to 0.5 M. To further enhance the sedimentation rate, ultrafine magnetite particles were prepared and included during polymerization to produce magnetic temperature-sensitive latex particles (MTLP), which could be recovered 6 times faster after thermo-flocculation by applying a low magnetic field. However, a higher salt concentration was necessary to flocculate the MTLP under the same condition as a result of its increased surface hydrophilicity, which originates from different polymerization conditions and the incorporation of magnetite. The immobilized enzyme shows high activity even against macromolecular substrates (hemoglobin and casein) owing to limited diffusion resistance, with full activity retention for nonmagnetic latex but one-half reduction in activity if the magnetic property was introduced. Optimal enzyme immobilization pH and enzyme loading were determined, and properties of the immobilized enzyme were characterized. The immobilized enzyme was used in 10 repeated batch hydrolyses of casein with successive flocculation/dispersion cycles and showed less than 15% activity decrease at the end. Overall, introducing the magnetic property to the latex could effectively enhance the solid-liquid separation rate after thermo-flocculation and maintain enzyme activity after repeated use but adversely influence the activity of the immobilized enzyme.

Site-directed and random immobilization of subtilisin on functionalized membranes: Activity determination in aqueous and organic media

Kinetic comparisons have been made between a randomly immobilized and a site-specifically immobilized subtilisin BPN' on microfiltration membranes of varying hydrophilicities in both aqueous and organic media. Site-directed mutagenesis was employed to introduce a single cysteine into the amino acid sequence of subtilisin at a location away from the active site. Immobilization of this mutant enzyme was then carried out using the single cysteine residue to orient the active site of the enzyme away from the membrane surface. Kinetic comparison of the immobilized mutant enzyme with the randomly immobilized wild-type enzyme in aqueous media showed an activity enhancement on both hydrophilic silica-containing and hydrophobic poly(ether)sulfone membranes. Higher loading efficiencies were observed for the site-directed enzyme on immobilization. Optimal enzyme loading values were calculated for the randomly immobilized enzyme. An enhancement of activity was also observed for the site-directed immobilized systems using nearly anhydrous hexane as the solvent.

Site‐directed and random immobilization of subtilisin on functionalized membranes: Activity determination in aqueous and organic media

Kinetic comparisons have been made between a randomly immobilized and a site-specifically immobilized subtilisin BPN′ on microfiltration membranes of varying hydrophilicities in both aqueous and organic media. Site-directed mutagenesis was employed to introduce a single cysteine into the amino acid sequence of subtilisin at a location away from the active site. Immobilization of this mutant enzyme was then carried out using the single cysteine residue to orient the active site of the enzyme away from the membrane surface. Kinetic comparison of the immobilized mutant enzyme with the randomly immobilized wild-type enzyme in aqueous media showed an activity enhancement on both hydrophilic silica-containing and hydrophobic poly(ether)sulfone membranes. Higher loading efficiencies were observed for the site-directed enzyme on immobilization. Optimal enzyme loading values were calculated for the randomly immobilized enzyme. An enhancement of activity was also observed for the site-directed immobilized systems using nearly anhydrous hexane as the solvent. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 608–616, 1998.

Stereoselective bioconversions in continuously operated fixed bed reactors: Modeling and process optimization

The nonaqueous lipase-catalyzed kinetic resolution of racemic 2-methyl-1-pentanol in a continuously operated fixed bed reactor was theoretically and experimentally studied. A 2-dimensional mathematical model was developed to predict the performance of the heterogeneous enantioselective biotransformation and to optimize the productivity and effective stereoselectivity of the process by taking pore diffusion, solid(SINGLEBOND)liquid mass transfer, convection, and axial dispersion into account. Experimental investigations were conducted with lipase from Pseudomonas sp. immobilized by ionic binding in the pores of the anion exchange resin Duolite A 568. As determined in prior initial rate experiments, the specific activities of the immobilized lipase were maximal at a water activity of approximately a(w) = 0.67 and revealed a significant dependence on the amount of enzyme bound to the carrier material, with enantiomeric ratios slightly increasing with increasing water activities. Continuous resolution processes were carried out at a wide range of enzyme loadings. By controlled immobilization according to the theoretically evaluated optimal enzyme loading, the continuous racemic resolution could be optimized to obtain (R)-2-methyl-1-pentanol at high productivity and enantiopurity (ee > 95%). The steady-state characteristics of the system could be generally predicted by the model, despite the necessity to reevaluate the kinetic properties of the immobilized lipase to account for the complex non-aqueous microkinetics in a heterogeneous environment. Further model extension introducing competitive inhibition of free water in solutions as well as water diffusion and adsorption to the biocatalyst were useful in providing a more accurate description of the experimental results. (c) 1996 John Wiley & Sons, Inc.

Amperometric bi-enzyme based biosensor for the detection of lactose--characterization and application.

Based on the glucose oxidase-beta-galactosidase sequence an enzyme probe for the specific determination of lactose has been developed. beta-Galactosidases from different sources have been compared, the sensor containing beta-galactosidase from Curvularia inaequalis has been characterized in respect of optimal pH, enzyme loading, apparent activity and functional stability. The response of the bi-enzyme probe depends linearly on lactose concentration between 0.02 and 3.00 mmol dm-3. The application to different milk and foodstuff samples resulted in good correlations toward enzymatic photometric (y = (0.956x-1.67) mmol dm-3) and infrared detection (y = (1.0772x-0.3909)%). Using a measuring frequency of 100 h-1 the serial imprecision is about 2% for diluted milk, urine, or foodstuff samples.

Theoretical and experimental studies of glucose sensitive membranes

A mathematical model has been developed to describe the steady-state behavior of two types of glucose sensitive membranes. Both membranes are synthetic hydrogels containing immobilized glucose oxidase enzyme (GluOx). The formation of gluconic acid from glucose and oxygen, catalyzed by GluOx, is the key to the functioning of either type of membrane since it causes a pH decrease within the membrane. In amine group-containing membranes, the pH decrease enhances the swelling (and permeability) of the gels, allowing control of insulin delivery in response to glucose concentrations. In membranes containing immobilized pH indicator dyes the pH decrease causes a color change, thus providing the basis for a glucose sensor. The model's predictions of the response of amine-containing membranes to glucose show that the pH decrease is often limited by O 2 depletion and that the occurrence of O 2 depletion is strongly influenced by enzyme loading and membrane thickness. At a given thickness, an optimal enzyme loading exists which results in the maximum response to glucose over a given range of glucose concentrations. The influence of buffer and amine concentrations, amine pK, solute diffusivities, and flowrate past the membrane also have been examined. For polyacrylamide membranes containing phenol red (but no amines), substrate turnover rates, oxygen depletion, and the pH decrease within the membrane have been calculated using the model and found to agree qualitatively with experimentally determined values of these parameters. Quantitative agreement is lacking, however. In particular, the model predicts that as GluOx loading is increased (all other parameters, including glucose concentration, held constant) the pH decrease should asymptotically approach a maximum value. However, it is found experimentally that as GluOx loading is increased, the measured pH decrease increases progressively over the full range of GluOx concentrations that we have studied. Possible reasons for the quantitative disagreements between simulated and observed results will be discussed. Implications of the model with respect to optimizing membrane designs will be presented. One finding of particular interest is that a glucose sensor using immobilized GluOx will achieve a maximum response at sub-physiological concentrations of glucose, and not respond to higher glucose concentrations, unless the enzyme loading is made sufficiently low.

ENZYMATIC HYDROLYSIS OF CELLULOSE: DETERMINATION OF KINETIC PARAMETERS

Abstract The effects of cellulose substrate concentration, cellulase enzyme concentration, and product concentration on the kinetic parameters involved in the enzymatic hydrolysis of cellulose have been studied. The Michaelis constant showed a decreasing trend with a decreasing crystallinity of cellulose substrate while the maximum reaction rate showed an increasing trend. These kinetic parameters were found to be significantly larger when the enzyme concentration was increased. The adsorption kinetic parameters showed an increasing trend as the crystallinity is decreased. It was found that the optimal enzyme loading should be determined by the source, concentration, and crystallinity of cellulose substrate and the initial specific rate of cellulose hydrolysis which is, in large part, influenced by the degree of crystallinity of cellulose substrate. The inhibition constant for cellulase-by cellobiose and that for cellobiase were also determined. These kinetic parameters determined experimentally can be applied to kinetic modeling and simulation of cellulose hydrolysis.