Properties of a reversible soluble-insoluble cellulase and its application to repeated hydrolysis of crystalline cellulose

Cellulase was covalently immobilized on an enteric coating polymer, Eudragit L, that is reversibly soluble and insoluble depending on the pH of the medium. The hydrolysis of solid cellulose with the immobilized enzyme can take advantage of the soluble property of the immobilized enzyme itself at the most reactive pH value; on the other hand, recovery of the enzyme can take advantage of the insoluble property of the enzyme at other pH values. It was experimentally confirmed that 100% of immobilized enzyme activity in solution can be recovered by precipitation and by dissolving it again by alternative change of pH. After a period of hydrolysis, immobilized enzyme and unreacted cellulose were precipitated together to remove the product-the soluble sugar solution-by changing pH. Following this, a new buffer solution was added to the precipitate to dissolve it and resume the reaction. This was repeated several times. The hydrolysis rate of this process increased significantly compared with that of a batch process. Utilization of the reversible soluble-insoluble carrier for immobilizing enzyme is promising, not only for cellulose-cellulase systems, but also for other heterogeneous reaction systems.     

Properties of urease immobilized on the functional organic silica surface

The paper deals with kinetics of the urea hydrolysis by microbial-origin urease dissolved and immobilized on the organic silica surface. It is shown that hydrolysis kinetics for soluble urease is described by the Michaelis-Menten equation until the concentration of urea reaches 1 M. Two fractions differing in the Michaelis constant are revealed for silochrome immobilized urease. The rate of urea hydrolysis by native and immobilized urease was studied depending on the pH value in presence of the substrate in the 1 M and 5 mM concentration. The hydrolysis rate of 1 M urea in the buffer-free solution by silochrome-immobilized urease is practically independent of pH within 4.5-6.5. Application of a 2.5 mM phosphate-citrate buffer as a solvent causes an increase in the hydrolysis rate within this pH range. For a soluble urease the 1 M urea hydrolysis rate dependence on pH is ordinary at pH 5.8-6.0. If the substrate concentration is 5 mM, the pH-dependences for the rate of the urea hydrolysis by silochrome- and aerosil-immobilized urease are close and at pH above 6.0 coincide with those for a soluble enzyme. The found differences in the properties of soluble and immobilized ureases are explained by the substrate and reaction products diffusion.     

Generation of a pH gradient in an immobilized enzyme system

Several examples of two-step sequential reactions exist where, because of the poor equilibrium conversion by the first reaction, it is desirable to conduct the two reactions simultaneously. In such a scheme, the product of the first reaction is continuously removed by the second reaction, thus not allowing the first reaction to approach chemical equilibrium. Therefore, the first reaction is allowed to proceed in the desired direction at an appreciable rate. However, in many biochemical applications where enzyme catalysts are involved, the enzyme's activities are strong functions of pH. Where the pH optima of the first and second reaction differ by three to four units, the above reaction scheme would be difficult to implement. In these cases, the two reactions can be separated by a thin permeable membrane across which the desired pH gradient is maintained. In this article, it was shown, both by theory and experiment, that a thin, flat membrane of immobilized urease can accomplish this goal when one face of the membrane is exposed to the acidic bulk solution (pH(b) = 4.5) containing a small quantity of urea (0.01 M). In this particular case, the ammonia that was produced in the membrane consumed the incoming hydrogen ions and thus maintained the desired pH gradient. Experimental results indicate that with sufficient urease loading, the face of the membrane opposite to the bulk solution could be maintained at a pH that would allow many enzymes to realize their maximum activities ( approximately 7.5). It was also found that this pH gradient could be maintained even in the presence of a buffer, which greatly enhances the transport of protons into the membrane. (c) 1993 John Wiley & Sons, Inc.     

多聚半乳糖醛酸内切酶的固定化及其性质研究

棉花枯萎病菌多聚半乳糖醛酸内切酶在pH大于7时不稳定,故对它进行多种化学修饰而又不影响其活性,必须在pHd小于7的体系中进行。本文报道将PGAUase在还原剂存在下,与稀酸处理的Sepharose 4B交联,获得较高活力的固定化酶。固定化酶催化动力学表明,最适pH为4,4,最适温度为55℃,在pH1至8.0范围内稳定。和溶液酶比较,对热稳定性提高,但对碱稳定性下降。以多聚半乳糖醛酸为底物,Km为0.27mmol/L,Vmax为66.67nmol/L·min,均大于溶液酶(Km=0.07mmol/L,Vmax=28.00nmol/L·min)。在pH4.8,30℃,聚半乳糖醛酸在固相酶的柱中循环水解不同的时间降解产物经圆盘电泳和等电聚焦测定,得到不同大小的寡糖片段混合物,证明固相酶和溶液酶的作用方式相同,同时使以酶解法制备一定大小的有生物活性的寡糖分子成为可能。     

Biotechnology for the production of nutraceuticals enriched in conjugated linoleic acid: I. Uniresponse kinetics of the hydrolysis of corn oil by a Pseudomonas sp. lipase immobilized in a hollow fiber reactor

The kinetics of the hydrolysis of corn oil in the presence of a lipase from Pseudomonas sp. immobilized within the walls of a hollow fiber reactor can be modeled in terms of a three‐parameter rate expression. This rate expression consists of the product of a two‐parameter rate expression for the hydrolysis reaction itself (which is of the general Michaelis–Menten form) and a first‐order rate expression for deactivation of the enzyme. Optimum operating conditions correspond to 30°C and buffer pH values of 7.0 during both immobilization of the enzyme and the hydrolysis reaction. Under these conditions, the total fatty acid concentration in the effluent oil stream for a fluid residence time of 4 h is approximately 1.6 M. This concentration corresponds to hydrolysis of approximately 50% of the glyceride bonds present in the feedstock corn oil. The fatty acid of primary interest in the effluent stream is linoleic acid. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 568–579, 1999.     

Biocatalysis of immobilized chlorophyllase in a ternary micellar system.

The immobilization of chlorophyllase was optimized by physical adsorption on various inorganic supports, including alumina, celite, Dowex-1-chloride, glass beads and silica gel. The enzyme was also immobilized in different media, including water, Tris-HCl buffer solution and a ternary micellar system containing Tris-HCl buffer solution, hexane and surfactant. The highest immobilization efficiency (84.56%) and specific activity (0.34 mumol hydrolyzed chlorophyll mg protein-1 per min) were obtained when chlorophyllase was suspended in Tris-HCl buffer solution and adsorbed onto silica gel. The effect of different ratios of chlorophyllase to the support and the optimum incubation time for the immobilization of chlorophyllase were determined to be 1-4 and 60 min, respectively. The experimental results showed that the optimum pH and temperature for the immobilized chlorophyllase were 8.0 and 35 degrees C, respectively. The use of optimized amounts of selected membrane lipids increased the specific activity of the immobilized chlorophyllase by approximately 50%. The enzyme kinetic studies indicated that the immobilized chlorophyllase showed a higher affinity towards chlorophyll than pheophytin as substrate.     

pH gradients in immobilized amidases and their influence on rates and yields of beta-lactam hydrolysis

The pH gradients developing within immobilized biocatalysts during hydrolysis of penicillin G and glutaryl-7-aminocephalosporanic acid have been estimated both theoretically and experimentally. For the latter a fluorimetric method for the direct measurement of the average pH value within the carrier during reaction has been developed using the pH-dependent fluorescence intensity of an enzyme-bound fluorophore determined with a fiber bundle. The theoretical calculations were based on a model for the hydrolysis with immobilized enzymes using a kinetic expression with five pH-dependent, measurable kinetic and equilibrium constants. The transport reaction differential equation which considers the laminar boundary layer has been solved numerically for the key component. The calculated values agreed well with the experimental data. Under the typical reaction conditions of penicillin G hydrolysis the average pH value in the carrier was 1 and 2.5 pH units below the bulk pH (=8) with and without buffer, respectively. The corresponding changes for the hydrolysis of glutaryl-7-aminocephalosporanic acid at bulk pH 8 in the presence of buffer was 0.5. This demonstrates the existence of considerable pH gradients in carriers during hydrolytic reactions, even in buffered systems with negligible mass transfer resistance. The low pH value causes suboptimal reaction rates, reduced equilibrium conversion, and reduced enzyme stability. These pH gradients can be minimised by using buffers with pK values approximately equal to the bulk pH used for the hydrolysis. The prediction quality of the model has been tested applying it to fixed bed reactor design. The reduction in rate and yield due to concentration and pH gradients can be overcome with simple measures such as high initial pH value and pH adjustments in segmented or recycling fixed bed reactors. Thus, enzymatic conversions with high yield and high operational effectiveness are achieved.     

Chemical stability of prostacyclin (PGI2) in aqueous solutions.

The rate constant for the hydrolysis of prostacyclin (PGI2) to 6-keto-PGF1alpha was measured by monitoring the UV spectral change, over a pH range 6 to 10 at 25 degrees C and the total ionic strength of 0.5 M. The first-order rate constant (kdegreesobs) extrapolated to zero buffer concentration follows an expression, kdegreesobs = kH+ (H+), where kH+ is a second-order rate constant for the specific acid catalyzed hydrolysis. The value of kH+ obtained (3.71 x 10(4) sec-1 M-1) Is estimated approximately 700-fold greater than a kH+ value expected from the hydrolysis of other vinyl ethers. Such an unusually high reactivity of PGI2 even for a vinyl ether is attributed to a possible ring strain release that would occur upon the rate controlling protonation of C5. A Br?nsted slope (alpha) of 0.71 was obtained for the acid (including H3O+) catalytic constants, from which a pH independent first-order rate constant for the spontaneous hydrolysis (catalyzed by H2O as a general acid) was estimated to be 1.3 x 10(-6) sec-1. An apparent activation energy (Ea) of 11.85 Kcal/mole was obtained for the hydrolysis at pH 7.48, from which a half-life of PGI2 at 4 degrees C was estimated to be approximately 14.5 min. when the total phosphate concentration is 0.165 M (cf. 3.5 min. at 25 degrees C).     

Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP) nerve agents was developed. The basic element of this enzyme electrode was a pH electrode modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking OPH with bovine serum albumin (BSA) and glutaradehyde. OPH catalyses the hydrolysis of organophosphorus pesticides to release protons, the concentration of which is proportional to the amount of hydrolysed substrate. The sensor signal and response time was optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and units of OPH immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 500 IU of OPH and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, ethyl parathion, methyl parathion and diazinon. The biosensor was completely stable for at least one month when stored in pH 8.5, 1 mM HEPES + 100 mM NaCl buffer at 4 degrees C.     

Lipase applications in oil hydrolysis with a case study on castor oil: a review

Lipase (triacylglycerol acylhydrolase) is a unique enzyme which can catalyze various types of reactions such as hydrolysis, esterification, alcoholysis etc. In particular, hydrolysis of vegetable oil with lipase as a catalyst is widely studied. Free lipase, lipase immobilized on suitable support, lipase encapsulated in a reverse micelle and lipase immobilized on a suitable membrane to be used in membrane reactor are the most common ways of employing lipase in oil hydrolysis. Castor oil is a unique vegetable oil as it contains high amounts (90%) of a hydroxy monounsaturated fatty acid named ricinoleic acid. This industrially important acid can be obtained by hydrolysis of castor oil. Different conventional hydrolysis processes have certain disadvantages which can be avoided by a lipase-catalyzed process. The degree of hydrolysis varies widely for different lipases depending on the operating range of process variables such as temperature, pH and enzyme loading. Immobilization of lipase on a suitable support can enhance hydrolysis by suppressing thermal inactivation and estolide formation. The presence of metal ions also affects lipase-catalyzed hydrolysis of castor oil. Even a particular ion has different effects on the activity of different lipases. Hydrophobic organic solvents perform better than hydrophilic solvents during the reaction. Sonication considerably increases hydrolysis in case of lipolase. The effects of additives on the same lipase vary with their types. Nonionic surfactants enhance hydrolysis whereas cationic and anionic surfactants decrease it. A single variable optimization method is used to obtain optimum conditions. In order to eliminate its disadvantages, a statistical optimization method is used in recent studies. Statistical optimization shows that interactions between any two of the following pH, enzyme concentration and buffer concentration become significant in presence of a nonionic surfactant named Span 80.     

Application of ammonium bicarbonate buffer as a smart microenvironmental pH regulator of immobilized cephalosporin C acylase catalysis in different reactors

In a stirred tank reactor, during catalysis with immobilized cephalosporin C acylase (CCA), the microenvironmental pH dropped to 7.2 in a nonbuffered system (with the pH maintained at 8.5 by adding alkali) due to the existence of diffusional resistance. Moreover, the immobilized CCA only catalyzed five batch reactions, suggesting that the sharp pH gradient impaired the enzyme stability. To buffer the protons produced in the hydrolysis of cephalosporin C by CCA, phosphate and bicarbonate buffers were introduced. When CCA was catalyzed with 0.1 M ammonium bicarbonate buffer, no obvious gradient between the bulk solution and intraparticle pH was detected, and the catalysis of 15 batch reactions was achieved. Accordingly, with 0.2 M ammonium bicarbonate buffer in a packed bed reactor, the immobilized CCA exhibited continuous catalysis with high conversion rates (≥95%) for 21 days. Reactions with ammonium bicarbonate buffer showed significant increases in the stability and catalytic efficiency of the immobilized CCA in different reactors compared to those in nonbuffered systems.     

Biotechnology for the production of nutraceuticals enriched in conjugated linoleic acid: II. Multiresponse kinetics of the hydrolysis of corn oil by a Pseudomonas sp. lipase immobilized in a hollow-fiber reactor

The hydrolysis of corn oil in the presence of a lipase from Pseudomonas sp. immobilized within the walls of a hollow-fiber reactor was studied at 30 degrees C. To assess the selectivity of this immobilized enzyme, the effluent concentrations of five different free fatty acids were measured using high-performance liquid chromatography (HPLC). Several rate expressions associated with a generic ping-pong bi-bi mechanism were used to fit the experimental data for this lipase-catalyzed reaction. A multiresponse nonlinear regression method was employed to determine the kinetic parameters associated with these rate expressions. Quasi-optimum operating conditions corresponded to 30 degrees C and a buffer pH value of 7.0. Under these conditions, the concentration of free linoleic acid (C18:2) (the fatty acid of primary interest) in the effluent oil stream for a fluid residence time of 6 h was approximately 0.5 M.     

Mechanisms of reaction of benzo(a)pyrene-7,8-diol-9,10-epoxide with DNA in aqueous solutions

The physical and chemical reaction pathways of the metabolite model compound benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) in aqueous (double-stranded) DNA solutions was investigated as a function of temperature (0-30 degrees C), pH (7.0-9.5), sodium chloride concentration (0-1.5M) and DNA concentration in order to clarify the relationships between the multiple reaction mechanisms of this diol epoxide in the presence of nucleic acids. The reaction pathways are (1) noncovalent intercalative complex formation with DNA, characterized by the equilibrium constant K, and Xb the fraction of molecules physically bound; (2) accelerated hydrolysis of BPDE bound to DNA; (3) covalent binding to DNA; and (4) hydrolysis of free BPDE(kh). The DNA-induced hydrolysis of BPDE to tetraols and the covalent binding to DNA are parallel pseudo-first-order reactions. Following the rapid (millisecond time scale) noncovalent complex formation between BPDE and DNA, a much slower (approximately minutes) H+-dependent (either specific or general acid catalysis) formation of a DNA-bound triol carbonium ion (rate constant k3) occurs. At pH 7.0 the activation energy of k3 is 8.7 +/- 0.9 kcal/mol, which is lower than the activation energy of hydrolysis of free BPDE in buffer solution (14.2 +/- 0.7 kcal/mol), and which thus partially accounts for the acceleration of hydrolysis of BPDE upon complexation with DNA. The formation of the triol carbonium ion is followed by a rapid reaction with either water to form tetraols (rate constant kT), or covalent binding to DNA (kc). The fraction of BPDE molecules which undergo covalent binding is fcov approximately equal to kc/(kc + kT) = 0.10 and is independent of the overall BPDE reaction rate constant k = kh(1 - Xb) + k3Xb if Xb----1.0, or is independent of Xb as long as k3Xb much greater than kh(1 - Xb). Thus, at Xb = 0.9, fcov is independent of pH (7.0-9.5) even though k exhibits a 70-fold variation in this pH range and k----kh above pH 9 (k3 = kh). Similarly, fcov is independent of temperature (0-30 degrees C), while k varies by a factor of approx. 3. In the range of 0-1.5 M NaCl, fcov decreases from 0.10 to 0.04. These variations are attributed to a combination of salt-induced variations in the factors k3, Xb and the ratio kc/kT.     

A fluorescent biosensor based on acetylcholinesterase and 5-oxazolone derivative immobilized in polyvinylchloride (PVC) matrix

A new 2-phenyl-4-[4-(1,4,7,10-tetraoxa-13-azacyclopentadecyl)benzylidene]-5-oxazolone (CPO) derivative was utilized to develop an optical acetylcholinesterase (AChE) biosensor in which the azlactone derivative was embedded in plasticized polyvinylchloride (PVC) matrix. The sensor system was calibrated for the detection of acetylcholine (ACh) and donepezil which is a competitive cholinesterase (ChE) inhibitor. Two different biosensing systems were developed by using AChE enzyme in solution and immobilized together with the fluorescent derivative (CPO) doped in transparent PVC membrane. The enzymatic hydrolysis of ACh was monitored by following changes in the pH induced fluorescence intensity. When AChE enzyme was immobilized in PVC matrix together with CPO, the sensitivity of the measuring system has increased approximately three times for ACh, in comparison to the sensing system where AChE enzyme was in solution phase.

The photophysical parameters of CPO were also examined in solvents of tetrahydrofuran (THF), acetonitrile (ACN) and dichloromethane (DCM) and in solid matrix of PVC. The azlactone derivative exhibits excellent photostability in PVC matrix.     

Strategies for the depyrogenation of contaminated immunoglobulin G solutions by histidine-immobilized hollow fiber membrane

The depyrogenation of different IgG solutions using the histidine-linked hollow fiber membrane developed in our laboratory is presented here. Three strategies for endotoxin (ET) removal were investigated according to the immobilized histidine's ability to bind different immunoglobulins: (1) ET removal from 1 mg/ml non histidine-binding mouse monoclonal IgG₁ (MabCD4) solution was achieved in the presence of acetate buffer (pH 5.0) without any protein loss. (2) For contaminated human IgG, combined adsorption of ET and IgG in the presence of MOPS or Tris buffer was tested, followed by differential elution using increasing salt concentrations. This attempt was not successful since ET were quantitatively found in the IgG elution fraction. (3) Alternatively, it was proposed to adsorb selectively ET in the presence of acetate buffer (pH 5.0) under non binding conditions for human IgG. Human IgG could then be purified if necessary with the same membrane in the presence of MOPS buffer (pH 6.5). With a 1 m² histidine-PEVA module under these operating conditions, it is estimated that the depyrogenation of 3 1 of 1 mg/ml IgG (human or murine) solution containing 80 EU/ml of ET should be possible.     

A poly(N-isopropylacrylamide-co-N-acryloxysuccinimide-co-2-hydroxyethyl methacrylate) composite hydrogel membrane for urease immobilization to enhance urea hydrolysis rate by temperature swing*

A composite membrane made of cross-linked poly(N-isopropylacrylamide-co-N-acryloxysuccinimide-co-2-hydroxyethyl methacrylate) (p(NIPAAm-NAS-HEMA)) hydrogel on polyester nonwoven support has been synthesized. The composite membrane shows temperature-responsive properties similar to conventional PNIPAAm hydrogels beads, which reversibly swells below and de-swells above the lower critical solution temperature of PNIPAAm (around 32 to 33 degrees C). Diffusion of urea through the membrane was temperature-dependent with the effective diffusion coefficient at 20 degrees C being 18 times that at 60 degrees C. Urease was immobilized directly to the membrane by forming covalent bonds between its amino groups and the succinimide ester groups of the membrane. Membrane prepared with NIPAAm to NAS molar ratio of 9, and then reacted in pH 7 buffer with 6 mg of urease gave the best immobilized enzyme, where 0.102 mg protein and 5.71 U activity per cm(2) membrane, and 55% relative specific activity could be obtained. There was negligible internal mass transfer resistance for this preparation judging from the calculated effectiveness factor. Urease shows enhanced thermal stability after immobilization with the first-order inactivation rate constant at 70 degrees C decreased to 1/8 of that of free urease. Membrane-immobilized urease could be utilized in a two-compartment membrane reactor with temperature swing to substantially enhance urea hydrolysis rate. The best operating condition of the membrane reactor was with temperature cycling between 60 to 20 degrees C and with temperature change every 10 min, where concentration of product ammonia after 3 h reaction increased 3.8-folds when compared with isothermal operation at 60 degrees C.     

Engineering sensitive glutathione transferase for the detection of xenobiotics

Cytosolic glutathione transferases (GSTs) are a major reserve of high-capacity ligand binding proteins which recognise a large variety of hydrophobic compounds. In the present study, the binding of non-substrate xenobiotic compounds (herbicides and insecticides) to maize GST I was investigated by employing kinetic inhibition studies, site-directed mutagenesis and molecular modelling studies. The results showed that the xenobiotics bind at the substrate binding site. Based on in silico docking analysis, two residues were selected for assessing their contribution to xenobiotic binding. The mutant Gln53Ala of GST I Exhibits 9.2-fold higher inhibition potency for the insecticide malathion, compared to the wild-type enzyme. A potentiometric assay was developed for the determination of malathion using the Gln53Ala mutant enzyme. The assay explores the ability of the xenobiotic to promote inhibition of the GST-catalysing 1-chloro-2,4-dinitrobenzene (CDNB)/glutathione (GSH) conjugation reaction. The sensing scheme is based on the pH change occurring in a low buffer system by the GST reaction, which is measured potentiometrically using a pH electrode. Calibration curve was obtained for malathion, with useful concentration range 0-20muM. The method's reproducibility was in the order of +/-3-5% and malathion recoveries were 96.7+/-2.8%. Immobilized Gln53Ala mutant GST was used to assemble a biosensor for malathion. The enzyme was immobilized by crosslinking with glutaraldehyde and trapped behind a semipermeable membrane in front of the pH electrode. The results demonstrated that the immobilized enzyme behaved similar to free enzyme.     

Recycle use of immobilized glucoamylase by tangential flow filtration unit

Various properties of glucoamylase immobilized onto corn stover supporting material and separation of immobilized enzyme by tangential flow filtration unit were studied. Optimum pH and temperature of immobilized enzyme were 3.5 and 60 degrees C, respectively. Enzyme stability was studied in a packed-bed column. The starch conversion rate was attained at 81% for 15 days; after that, the hydrolysis rate gradually decreased. Size of supporting material proved to be an important factor, with higher activity and good loading yield resulting from smaller supporting material. Glucoamylase immobilized onto supporting material less than 44 mum was used for hydrolysis of 10% soluble starch at pH 3.5 and 40 degrees C for 3 h. Then immobilized glucoamylase was separated from the product by means of a tangential flow filtration unit using a 0.2-mum pore size Nylon 66 membrane filter. This operation was continued until 180 ml filtrate was obtained from a 260-mL starting volume. Then, the next batch was started by adding 180 mL starch substrate into the reactor. The batchwise experiments were repeated 20 times. The average filtration rate of each batch was determined and found to sharply decline during the first four batches. Thereafter, it gradually decreased from batch to batch. The cause of decreasing filtration rate appeared to be due to retrogradation of starch. The percentage of starch hydrolysis within 20 batches was in the range 89-96%. The filtration rate becomes higher if the hydrolyzation time is extended to 14 h. Resistance to filtration was also investigated. Almost all of the total resistance is related to insoluble materials, with the significant part of this from the resistance due to insoluble materials deposited on a surface of membrane and boundary layer resistance. Using a microscopic method, no microorganisms were found in the filtrate.     

Immobilization of cane invertase on bentonite

Sugar-cane invertase (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26) immobilized on bentonite clay in 0.05 m acetate buffer, pH 4.5, has been shown to be capable of hydrolysing sucrose. The bentonite-invertase (BI) complex gave 55.5% retention of enzyme activity on the surface. A further 17 and 22% increase in retention of enzyme activity was obtained using the covalent linking agents, cyanuric chloride and thionyl chloride, giving bentonite-cyanuric chloride-invertase (BCCI) and bentonite-thionyl chloride-invertase (BTCI) complexes. Concentrations of acetate buffer >0.2 M disrupt the bentonite-invertase complexes. The immobilized invertase complexes showed high temperature optima (60–65°C) and high thermal stability compared to the free enzyme. The pH profiles of the free and immobilized enzyme were the same. The rate of hydrolysis of sucrose was increased using immobilized enzymes, which required a higher substrate concentration than the free enzyme. The insoluble enzyme conjugate-carrier complexes when used for sucrose hydrolysis in a batch process showed 53.1 (BI), 57.4 (BCCI) and 59.6% (BTCI) conversions, respectively, in 12 h, compared to 42.3% conversion in 24 h with the free enzyme. The immobilized invertase complexes can be used for sucrose inversion for about five cycles. The application of this immobilization procedure may help in the removal of invertase from cane juice to reduce sugar losses in industry.     

Urea hydrolysis by immobilized urease in a fixed-bed reactor: analysis and kinetic parameter estimation

Urea hydrolysis by urease immobilized onto ion exchange resins in a fixed-bed reactor has been studied. A modified Michaelis-Menten rate expression is used to describe the pH-dependent, substrate- and product-inhibited kinetics. Ionic equilibria of product and buffer species are included to account for pH changes generated by reaction. An isothermal, heterogeneous plug-flow reactor model has been developed. An effectiveness factor is used to describe the reaction-diffusion process within the particle phase. The procedure for covalent immobilization of urease onto macroporous cation exchangers is described. Urea conversion data are used to estimate kinetic parameters by a simplex optimization method. The best-fitted parameters are then used to predict the outlet conversions and pH values for systems with various inlet pH values, inlet urea and ammonia concentrations, buffers, particle sizes, and spacetimes. Very good agreement is obtained between experimental data and model predictions. This immobilized urease system exhibits quite different kinetic behavior from soluble urease because the pH near the enzyme active sites is different from that of the pore fluid. This effect results in a shift of the optimal pH value of the V(max) (pH) curve from 6.6 (soluble urease) to ca. 7.6 in dialysate solution, and ca. pH 8.0 in 20mM phosphate buffer. The reactor model is especially useful for estimating intrinsic kinetic parameters of immobilized enzymes and for designing urea removal columns.