Use of an oxygen microsensor for the determination of intrinsic kinetic parameters of an immobilized oxygen reducing enzyme

An oxygen microsensor was used to measure internal oxygen profiles in biocatalyst particles of different diameter and activity. The particles were made of agarose gel and contained an oxygen reducing enzyme, L-lactate mono-oxygenase. The kinetics of the enzyme could be well described by the Michaelis-Menten equation. From the internal substrate concentration profile the intrinsic kinetic parameters were determined by means of fitting a simulated profile to the measurements, using Marquardt's algorithm. The intrinsic kinetic parameters found following this procedure appeared to be independent of particle radius or enzyme loading used, proving the method to be reliable. These parameters were also compared with the kinetic parameters of the free enzyme which were determined in a biological oxygen monitoring system. The intrinsic kinetic parameters showed a decrease with a factor 2.3 for V(m) value and with a factor 2.7 for the K(m) value compared to the parameters for the free enzyme. From this the conclusion can be drawn that the immobilization as such or the carrier material not only can have an effect on the maximum intrinsic conversion rate (V(m)) but also on the affinity of the enzyme (K(m)) for oxygen.     

Immobilization of enzymes in porous supports: Effects of support-enzyme solution contacting

Proteins have been immobilized in porous support particles held in a fixed-bed reactor through which protein solution is continuously circulated. Changing the recirculation flow rate alters the observed immobilization kinetics and the maximum enzyme loading which can be achieved for glucose oxidase and glucoamylase on carbodiimide-treated activated carbon and for glucoamylase immobilized on CNBr-Sepharose 4B. Direct microscopic examination of FITC-labelled protein in sectioned Sepharose particles and indirect activity-loading studies with activated carbon-enzyme conjugates all indicate that immobilized enzyme is increasingly localized near the outer surface of the support particles at larger recirculation flow rates. Restricted diffusion of enzymes may be implicated in this phenomenon. These contacting effects may be significant considerations in the scaleup of processes for protein impregnation in porous supports, since apparent activity and stability of the final preparation depend on internal protein distribution.     

氨基化二氧化硅颗粒固定木瓜蛋白酶研究

采用正硅酸乙酯与N-(β-氨乙基)氨丙基三乙氧基硅烷在油包水形成的微胶囊中同步水解的方法,一步法制备了氨基化的二氧化硅颗粒,得到的颗粒粒径在0.3～0.5μm之间,平均大小为0.37μm, 氨基含量和颗粒大小可控,氨基含量高达56mmol/g。此颗粒经戊二醛处理后,采用共价法固定木瓜蛋白酶,固定化最适pH6.5,最佳给酶量为15mg/g载体,固定化酶的最适反应温度为70℃,最适反应pH为6.5,固定化酶热稳定性,pH耐受性,贮存稳定性都明显高于游离酶,表明此颗粒可作为一种优良的酶固定化载体。     

Immobilized enzymes: Pectin esterase covalently coupled to dorous glass particles

Pectin esterase (E.C. 3.1.1.11) was covalently immobilized to porous glass particles by reaction of the native protein with pendant, benzoyl azide groups of the carrier. Enzyme loading on the carrier was 0.5 unit per ml as measured by pH stat, assay. Decreasing the size of the immobilized enzyme particles by grinding produced a 12-fold increase in activity suggesting severe internal mass transport restrictions on turnover kinetics, Gross fractionation of the citrus pectin substrate into high and low molecular weight categories and their subsequent use in kinetic characterization shows no effect of molecular weight upon the kinetic behavior of the native enzyme. In contrast the immobilized enzyme displayed a 5-fold increase in the apparent. K_m for the high molecular weight fraction relative to that of the low molecular weight fraction. A striking difference exists in the low pH profile of immobilized pectin esterase relative to the native enzyme. Carrier matrix interactions with the polyelectrolyte substrate are invoked to explain this difference. The thermal stability of the immobilized enzyme is relatively low and displays a half-life of approximately 2 weeks at 25°C.     

Kinetic behavior of immobilized Penicillin acylase

Penicillin acylase has been immobilized to carboxymethylcellulose and to the resin Amberlite XAD7. The reaction kinetics of the enzyme were affected by both intrinsic (molecular) and microenvironmental effects. The Michaelis constant for the enzyme increased after immobilization as a result of an intrinsic effect of the reagent, glutaraldehyde, used for enzyme immobilization. Microenvironmental effects were of two types: diffusional limitation of access of substrate and a reaction-generated pH depression in the support particles. This depression of internal pH was observed in all the preparations and could be reduced by addition of pH buffering salts to reactor. An adsorbed pH-indicating dyc was used to determine the surface and internal pH of particles of XAD7–penicillin acylase under various reaction conditions. The extent of diffusional rate limitation in XAD7–penicillin acylase was related to the penetration depth of protein into the porous support particles. The penetration depth of protein and thus the diffusional limitation of the reaction rate could be controlled by the conditions of preparation of the immobilized enzyme. A staining technique was used to observe the location of the protein.     

Pore diffusion model for a two-substrate enzymatic reaction: Application to galactose oxidase immobilized on porous glass particles

An analysis of the pore diffusion model involving a two-substrate enzymatic reaction is presented. The resulting equations have been applied to the case of galactose oxidase catalyzed oxidation of galactose when the enzyme is immobilized on porous glass particles. The physical constants of the system were obtained by theoretical predictions and the enzyme concentration in the porous medium was derived from the experimental results. The calculations were performed with the assumption that the kinetic parameters of the enzyme remain unchanged upon immobilization. The theoretically calculated effectiveness factors were compared with the experimental effectiveness factors determined from the batch kinetic experiments and were found to be in agreement. The results are presented as effectiveness factor plots graphed as functions of bulk galactose and oxygen concentrations. The model was extended in order to study the effect of external mass transfer coefficients and pore enzyme concentrations on the effectiveness factors.     

多孔纳米材料固定化酶研究进展

酶是一种天然生物催化剂,有催化效率高、底物选择性强和绿色环保等优点,但酶结构不稳定且重复利用率低,制约了其产业化应用。随着技术的发展,酶的固定化可以提高酶的活性和稳定性,为生物酶的工程化应用带来了新的机遇。多孔纳米材料具有比表面积大、孔隙率高、机械和化学性能稳定等特点和优异的成本效益,是理想的固定化酶载体。本文综述了近些年来金属有机框架、共价有机框架和多孔微球等纳米材料固定化酶的研究进展和应用,重点介绍了载体固定酶的方式,并总结了每种载体的特点,最后讨论了多孔纳米材料固定化酶面临的挑战和发展趋势。     

Porous zirconia: a new support material for enzyme immobilization

Four different proteases (trypsin, chymotrypsin, papain and pepsin) were covalently attached to the surface of a new type of porous zirconia, as well as a conventional porous silica, activated with 3-isothiocyanatopropyltriethoxy silane (NCS-silane). The immobilization efficiency onto the porous zirconia material was evaluated in terms of the amount of enzyme attached to the particles and from the biological activity remaining after the immobilization step. The results were compared with the corresponding experiments with a porous silica of similar surface area/g support material. In addition, the storage stability of the modified zirconia and silica biocatalysts were evaluated. These results indicated that specific immobilized enzyme biocatalysts can be achieved with this new zirconia support material which exhibits different properties to those observed with the more conventional silica-based materials. Moreover, the results with the enzyme-zirconia biocatalysts also indicate different characteristics when compared with data for the same enzymes immobilized under similar buffer conditions to organic support materials as previously described by various other investigators. The advantages of zirconia-based immobilized enzyme biocatalysts in terms of their density and chemical robustness are also described relative to other alternative support materials currently in use.     

Galactose oxidase: applications of the covalently immobilized enzyme in a packed bed configuration.

Galactose oxidase (E.C. 1.1.3.9) was covalently immobilized to chemically modified porous silica particles by reaction of the native enzyme with pendant benzoyl azide groups on the carrier. The enzyme loading on the carrier was 100-150 units per milliliter. The immobilized enzyme was incorporated into a hardware assembly suitable for the determination of galactose or lactose concentrations in complex biological fluids. The prototype instrument as described is suitable for continuous, on-line monitoring or discrete sample analysis. Reaction conditions can be readily provided which maintain global first order kinetics within the reactor and strict linearity of the procedure over a wide range of sample concentrations. Auto-inactivation of the immobilized enzyme can be prevented by K3Fe(CN)6 and long-term reactor stability can be achieved by the periodic application of the reagent to the enzyme reactor in situ.     

Investigation of the operational stabilities and kinetics of glucoamylase immobilized on alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl₄ solution, displayed very stable behaviour, with half-lives of ～60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent K_m increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion.     

Comparison of two experimental methods for the determination of Michaelis-Menten kinetics of an immobilized enzyme

For the application of immobilized enzymes, the influence of immobilization on the activity of the enzyme should be Known. This influence can be obtained by determining the intrinsic kinetic parameters of the immobilized enzyme, and by comparing them with the kinetic parameters of the suspended enzyme. This article deals with the determination of the intrinsic kinetic parameters of an agarose-gel bead immobilized oxygen-consuming enzyme: L-lactate 2-monooxygenase. The reaction rate of the enzyme can be described by Michaelis-Menten kinetics. Batch conversion experiments using a biological oxygen monitor, as well as steady-state profile measurements within the biocatalyst particles using an oxygen microsensor, were performed. Two different mathematical methods were used for the batch conversion experiments, both assuming a pseudosteady-state situation with respect to the shape of the profile inside the bead. One of the methods used an approximate relation for the effectiveness factor for Michaelis-Menten kinetics which interpolates between the analytical solutions for zero- and first-order kinetics. The other mathematical method was based on a numerical solution and combined a mass balance over the reactor with a mass balance over the bead. The main difference in the application of the two methods is the computer calculation time; the completely numerical calculation procedure was about 20 times slower than the other calculation procedure.The intrinsic kinetic parameters resulting from both experimental methods were compared to check the reliability of the methods. There was no significant difference in the intrinsic kinetic parameters obtained from the two experimental methods. By comparison of the kinetic parameters for the suspended enzyme with the intrinsic kinetic parameters for the immobilized enzyme, it appeared that immobilization caused a decrease in the value of V(m) by a factor of 2, but there was no significant difference in the values obtained for K(m).     

Immobilization of lipase from Candida rugosa on a polymer support

Lipase from Candida rugosa was immobilized by adsorption onto a macroporous copolymer support. Under optimum conditions the maximum amount of protein bound was 15.4 mg/g and the immobilization efficiency was 62%. The kinetics of lipase binding to the selected polymer carrier was assessed by using a general model of topochemical reactions. The effect of temperature on adsorption was thoroughly investigated, as was the adsorption mechanism itself. Analysis of the proposed kinetic model and the specific kinetic parameters measured suggest that surface kinetics control the adsorption process. According to the activation energy (E _a) and the rate constant, k, the enzyme has rather a high affinity for the support's active sites. The immobilized enzyme was used to catalyse the hydrolysis of palm oil in a lecithin/isooctane reaction system, in which the enzyme's activity was 70% that of the free enzyme. Kinetic parameters such as maximum velocity (V _max) and the Michaelis constant (K _m) were determined for the free and the immobilized lipase. Following repeated use, the immobilized lipase retained 56% of its initial activity after the fifth hydrolysis cycle. Received: 3 April 1998 / Received revision: 28 July 1998 / Accepted: 29 July 1998     

Silver nanoparticle (AgNPs) doped gum acacia-gelatin-silica nanohybrid: an effective support for diastase immobilization

An effective carrier matrix for diastase alpha amylase immobilization has been fabricated by gum acacia-gelatin dual templated polymerization of tetramethoxysilane. Silver nanoparticle (AgNp) doping to this hybrid could significantly enhance the shelf life of the impregnated enzyme while retaining its full bio-catalytic activity. The doped nanohybrid has been characterized as a thermally stable porous material which also showed multipeak photoluminescence under UV excitation. The immobilized diastase alpha amylase has been used to optimize the conditions for soluble starch hydrolysis in comparison to the free enzyme. The optimum pH for both immobilized and free enzyme hydrolysis was found to be same (pH=5), indicating that the immobilization made no major change in enzyme conformation. The immobilized enzyme showed good performance in wide temperature range (from 303 to 323 K), 323 K being the optimum value. The kinetic parameters for the immobilized, (K(m)=10.30 mg/mL, V(max)=4.36 μmol mL(-1)min(-1)) and free enzyme (K(m)=8.85 mg/mL, V(max)=2.81 μmol mL(-1)min(-1)) indicated that the immobilization improved the overall stability and catalytic property of the enzyme. The immobilized enzyme remained usable for repeated cycles and did not lose its activity even after 30 days storage at 40°C, while identically synthesized and stored silver undoped hybrid lost its ~31% activity in 48 h. Present study revealed the hybrids to be potentially useful for biomedical and optical applications.     

Size-exclusion effect of a substrate upon kinetics of trypsin immobilized on porous bead cellulose. 1. Influence of distribution coefficient of a substrate

A model of heterogeneous biocatalysis, in which kinetics and partition effects are connected via the size-exclusion principle, was worked up experimentally and theoretically. The present paper shows that the maximum relative activity of trypsin (EC 3.4.21.4) immobilized on porous bead (spherical) cellulose is directly proportional to the available distribution coefficient of the substrate. Providing that the excess of substrate is not sufficient (e.g.S/K_m ≈ 1) to safeguard saturated enzyme kinetics, the originally linear relationship of R_a versus K_av turns to an exponential one, without any dependence upon the manner of enzyme immobilization. It is suggested that the above may be a result of partition resistance and that the main factors determining the shape of the R_a versus K_av relation in conditions of substrate shortage are the size and geometry of the matrix. The physical characteristics of the porous carrier as well as the manner of covalent immobilization of the enzyme are all reflected in the constants applied in the derived equations.     

Enhanced recombinant protein production and differential expression of molecular chaperones in sf-caspase-1-repressed stable cells after baculovirus infection

Background

Industrial-scale biocatalytic synthesis of fine chemicals occurs preferentially as continuous processes employing immobilized enzymes on insoluble porous carriers. Diffusional effects in these systems often create substrate and product concentration gradients between bulk liquid and the carrier. Moreover, some widely-used biotransformation processes induce changes in proton concentration. Unlike the bulk pH, which is usually controlled at a suitable value, the intraparticle pH of immobilized enzymes may deviate significantly from its activity and stability optima. The magnitude of the resulting pH gradient depends on the ratio of characteristic times for enzymatic reaction and on mass transfer (the latter is strongly influenced by geometrical features of the porous carrier). Design and selection of optimally performing enzyme immobilizates would therefore benefit largely from experimental studies of the intraparticle pH environment. Here, a simple and non-invasive method based on dual-lifetime referencing (DLR) for pH determination in immobilized enzymes is introduced. The technique is applicable to other systems in which particles are kept in suspension by agitation.

Results

The DLR method employs fluorescein as pH-sensitive luminophore and Ru(II) tris(4,7-diphenyl-1,10-phenantroline), abbreviated Ru(dpp), as the reference luminophore. Luminescence intensities of the two luminophores are converted into an overall phase shift suitable for pH determination in the range 5.0-8.0. Sepabeads EC-EP were labeled by physically incorporating lipophilic variants of the two luminophores into their polymeric matrix. These beads were employed as carriers for immobilization of cephalosporin C amidase (a model enzyme of industrial relevance). The luminophores did not interfere with the enzyme immobilization characteristics. Analytical intraparticle pH determination was optimized for sensitivity, reproducibility and signal stability under conditions of continuous measurement. During hydrolysis of cephalosporin C by the immobilizate in a stirred reactor with bulk pH maintained at 8.0, the intraparticle pH dropped initially by about 1 pH unit and gradually returned to the bulk pH, reflecting the depletion of substrate from solution. These results support measurement of intraparticle pH as a potential analytical processing tool for proton-forming/consuming biotransformations catalyzed by carrier-bound immobilized enzymes.

Conclusions

Fluorescein and Ru(dpp) constitute a useful pair of luminophores in by DLR-based intraparticle pH monitoring. The pH range accessible by the chosen DLR system overlaps favorably with the pH ranges at which enzymes are optimally active and stable. DLR removes the restriction of working with static immobilized enzyme particles, enabling suspensions of particles to be characterized also. The pH gradient developed between particle and bulk liquid during reaction steady state is an important carrier selection parameter for enzyme immobilization and optimization of biocatalytic conversion processes. Determination of this parameter was rendered possible by the presented DLR method.     

Evaluation of intrinsic immobilized kinetics in hollow fiber reactor systems.

Immobilized cell and enzyme hollow fiber reactors have been developed for a variety of biochemical and biomedical applications. Reported mathematical models for predicting substrate conversion in these reactors have been limited in accuracy because of the use of free-solution kinetic parameters. This paper describes a method for determining the intrinsic kinetics of enzymes immobilized in hollow fiber reactor systems using a mathematical model for diffusion and reaction in porous media and an optimization procedure to fit intrinsic kinetic parameters to experimental data. Two enzymes, a thermophilic beta-galactosidase that exhibits product inhibition and L-lysine alpha-oxidase, were used in the analysis. The intrinsic kinetic parameters show that immobilization enhanced the activity of the beta-galactosidase while decreasing the activity of L-lysine alpha-oxidase. Both immobilized enzymes had higher Km values than did the soluble enzyme, indicating less affinity for the substrate. These results are used to illustrate the significant improvement in the ability to predict substrate conversion in hollow fiber reactors.     

Adsorptive immobilization of a Pseudomonas strain on solid carriers for augmented decolourization in a chemostat bioreactor

Pseudomonas GM3, a highly efficient strain in cleavage of azo bonds of synthetic dyes under anoxic conditions, was immobilized via adsorption on two types of carriers, porous glass beads and solid PVA particles. The cells were cultivated in a nutrient medium, adsorbed on sterile carriers, stabilized as biofilms in repeated batch cultures, and introduced into a chemostat activated sludge reactor for augmented decolourization. The microbial cells were quickly adsorbed and fixed on the PVA surface, compared to a slow and linear immobilization on the glass surface. The porous structure of glass beads provided shelter for the embedded cells, giving a high biomass loading or thick biofilm (13.3 mg VS ml-1 carrier) in comparison with PVA particles (4.8 mg VS ml-1 carrier), but the mass transfer of substrate in the biofilm became a significant limiting factorin the thicker biofilms (effectiveness factor eta = 0.31). The microbial decolourization rate per volume of carriers was 0.15 and 0.17 mg dye ml-1 of glass beads and PVA particles, respectively. In augmented decomposition of a recalcitrant azo dye (60 mg l-1), the immobilized Pseudomonas cells in porous glass beads gave a stable decolourization efficiency (80-81%), but cells fixed on solid PVA particles showed an initial high colour removal of 90% which then declined to a stable removal efficiency of 81%. In both cases, the colour removal efficiency of the chemostat bioreactor was increased from < 10% by an activated sludge to approximately 80% by the augmented system.     

Distribution of immobilized enzymes on porous membranes

In this article, the results from a theoretical and experimental investigation of enzyme immobilization in porous membranes are reported. A theoretical model of the immobilization process, which accounts for restricted diffusion of enzyme in the pores of the membrane, has been developed. The model predicts the effect of immobilization kinetics and time of immobilization on the enzyme distribution in the pores of the membrane. The immobilization of glucose oxidase and glucose oxidase-biotin conjugate on porous alumina membranes was experimentally investigated. Enzyme uptake data was correlated to the theory to determine the rate constant of imobilization and the distribution of the enzyme in the pore. Immobilization studies were carried out for enzyme adsorption and for enzyme attachment by covalent coupling. The distribution of enzyme was experimentally studied by assembling five membranes in the diffusion cell. Following immobilization, the membranes were separated and each was assayed for activity. The amount of active enzyme present in each membrane yielded a discrete distribution that compared well with that predicted by theory. (c) 1992 John Wiley & Sons, Inc.     

Reversible covalent enzyme immobilization methods for reuse of carriers

Reversible immobilization techniques which allow for multiple use of the carrier are relevant for applications, such as enzymatic microreactors, biosensors with specific setups and for expensive carriers such as superparamagnetic particles. The activity of immobilized enzymes reduces with time, so that the introduction of fresh immobilized enzyme becomes necessary. Thus, methods for reversible immobilization and multiple carrier reuse can help to reduce purchase costs and facilitate reactor construction. In this work, we present a method that makes use of the reduction and oxidation of cystamine, a cleavable linker with disulfide bond and amine functionality. For a proof of principle, α-chymotrypsin was immobilized on polyethylene glycol with terminal epoxy groups using cystamine as a crosslinker. The enzyme was highly active and could be used in repeated cycles. After the enzymatic reaction was demonstrated, α-chymotrypsin was cleaved off the particle by reducing agents. The resulting thiols on the particle surface were oxidized to disulfides by means of cysteamine, the reduction product of cystamine. This way, an almost complete oxidation of surface thiols with cysteamine was possible, restoring amine functionalization for further reactions. Reduction and oxidation were repeated several times without a decrease in the extent of amine coupling. Finally, immobilization of α-chymotrypsin could be repeated with results comparable to first run.     

Preparation and properties of immobilized amyloglucosidase on nonporous PS/PNaSS microspheres

Nonporous polystyrene/poly(sodium styrene sulfonate) (PS/PNaSS) microspheres were used for immobilization of amyloglucosidase and the properties of immobilized enzyme was studied and compared with those of free enzyme. Sulfonated groups on the PS/PNaSS microspheres present a very simple, mild, and time-saving process for enzyme immobilization. Nonporous microspheres provide their surface for immobilization of enzyme and prevent the diffusion limitation problem in the pore. Despite the high concentration of bound enzyme the influence of immobilization on kinematic parameters, K(m) and V(max), is relatively low compare to other porous supports. Simple and time-saving immobilization procedure as well as the effects of pH and temperature on immobilized enzyme also showed that the PS/PNaSS microspheres could be good support.