Kinetics of Soluble and Immobilized Enzymes

The kinetics of soluble and immobilized enzymes. Involved In reactions of soluble and Insoluble substrates appears to be sufficiently well studied over the last 20 years that reactor design procedures based on fundamental kinetics rate equations may be executed with considerable confidence. The application of such emzyme kinetics forms to structured models of microbial metabolism has also progressed, as this book documents. 

Free enzyme versus immobilized enzyme can influence the yield of lOS, additionally an immobilized system would be favorable economically as the biocatalyst can be reused, enables continuous production and the end product is free of contamination. Kim et al. [275] intended to make a comparison between the reaction kinetics of free and immobilized endo-inulinases in a batch reactor however significant differences were observed in the reaction behavior and product composition due to the form of enzyme used and the initial concentration of substrate. Yun et al. [276] investigated the effect of inulin concentration on the production of lOS by free and immobilized endo-inulinase from Pseudomonas sp. Their findings corroborate those of Kim et al. [275] whereby different products are formed depending on the form of enzyme a soluble enzyme yielded inulobiose and DP3 products, whereby the immobilized form predominantly produced inulobiose. As the concentration of inulin increased the yield of lOS did not increase in the soluble system and in the immobilized the yield remained the same. Although the enzyme was derived from Pseudomonas the immobilized form required a differ-... 

Abstract An agroindustrial residue, green coconut fiber, was evaluated as support for immobilization of Candida antarctica type B (CALB) lipase by physical adsorption. The influence of several parameters, such as contact time, amount of enzyme offered to immobilization, and pH of lipase solution was analyzed to select a suitable immobilization protocol. Kinetic constants of soluble and immobilized lipases were assayed. Thermal and operational stability of the immobilized enzyme, obtained after 2 h of contact between coconut fiber and enzyme solution, containing 40 U/ml in 25 mM sodium phosphate buffer pH 7, were determined. CALB immobilization by adsorption on coconut fiber promoted an increase in thermal stability at 50 and 60 °C, as half-lives (t /2) of the immobilized enzyme were, respectively, 2- and 92-fold higher than the ones for soluble enzyme. Furthermore, operational stabilities of methyl butyrate hydrolysis and butyl butyrate synthesis were evaluated. After the third cycle of methyl butyrate hydrolysis, it retained less than 50% of the initial activity, while Novozyme 435 retained more than 70% after the tenth cycle. However, in the synthesis of butyl butyrate, CALB immobilized on coconut fiber showed a good operational stability when compared to Novozyme 435, retaining 80% of its initial activity after the sixth cycle of reaction. 

Commercial yeast invertase (Bioinvert ) was immobilized by adsorption on anion-exchange resins, collectively named Dowex (1x8 50-400,1x4 50-400, and 1x2 100-400). Optimal binding was obtained at pH 5.5 and 32°C. Among different polystyrene beads, the complex Dowex-1x4-200/invertase showed a yield coupling and an immobilization coefficient equal to 100%. The thermodynamic and kinetic parameters for sucrose hydrolysis for both soluble and insoluble enzyme were evaluated. The complex Dowex/inver-tase was stable without any desorption of enzyme from the support during the reaction, and it had thermodynamic parameters equal to the soluble form. The stability against pH presented by the soluble invertase was between 4.0 and 5.0, whereas for insoluble enzyme it was between 5.0 and 6.0. In both cases, the optimal pH values were found in the range of the stability interval. The Km and Vmax for the immobilized invertase were 38.2 mM and 0.0489 U/mL, and for the soluble enzyme were 40.3 mM and 0.0320 U/mL. 

The problem of pore diffusion is only limited to immobilized enzyme catalysts, and not enzyme catalyzed reactions in which the enzyme is used in the native or soluble form. Immobilized enzymes are supported catalysts in which the enzyme is supported or immobilized on a suitable inert support such as alumina, kiesulguhr, silica, or microencapsulated in a suitable polymer matrix. The shape of the immobilized enzyme pellet may be spherical, cylindrical, or rectangular (as in a slab). If the reaction follows Michaelis-Menten kinetics discussed previously, then a shell balance around a spherical enzyme pellet results in the following second order differential equation ... 

Kinetic models to describe lipase-catalyzed reaction mechanisms have been proposed, and most have been extensions of the model developed by Michaelis and Menten (1913). However, normal Michaelis-Menten kinetics do not apply to lipase-induced changes, because the substrates (lipids) are not water-soluble and the enzyme operates at an interface (Brockman, 1984). However, rate expressions for the hydrolysis of emulsified lipids catalyzed by immobilized lipases resemble the rate expressions modeled with Michaelis-Menten mechanisms (Benzonana and Desnuelle, 1965). The kinetics and mechanisms of reactions catalyzed by immobilized lipases have been reviewed by Malcata et al. (1990 1992). 

In Figure 10.1 the time course of thermodynamically and kinetically controlled processes catalysed by biocatalysts are compared. The product yield at the maximum or end point is influenced by pH, temperature, ionic strength, and the solubility of the product. In the kinetically controlled process (but not in the thermodynamically controlled process) the maximum yield also depends on the properties of the enzyme (see next sections). In both processes the enzyme properties determine the time required to reach the desired end point. The conditions under which maximum product yields are obtained do not generally coincide with the conditions where the enzyme has its optimal kinetic properties or stability. The primary objective is to obtain maximum yields. For this aim it is not sufficient to know the kinetic properties of the enzyme as functions of various parameters. It is also necessary to know how the thermodynamically or the kinetically controlled maximum is influenced by pH, temperature and ionic strength, and how this may be influenced by the immobilization of the biocatalysts on different supports. 

The performance of cellulase and amylase immobilized on siliceous supports was investigated. Enzyme uptake onto the support depended on the enzyme source and immobilization conditions. For amylase, the uptake ranged between 20 and 60%, and for cellulase, 7-10%. Immobilized amylase performance was assessed by batch kinetics in 100-300 g/L of com flour at 65°C. Depending on the substrate and enzyme loading, between 40 and 60% starch conversion was obtained. Immobilized amylase was more stable than soluble amylase. Enzyme samples were preincubated in a water bath at various temperatures, then tested for activity. At 105°C, soluble amylase lost -55% of its activity, compared with -30% loss for immobilized amylase. The performance of immobilized cellulase was evaluated from batch kinetics in 10 g/L of substrate (shredded wastepaper) at 55°C. Significant hydrolysis of the wastepaper was also observed, indicating that immobilization does not preclude access to and hydrolysis of insoluble cellulose. 

Kinetics studies were conducted at 65 1°C in a jacketed batch reactor. Five hundred milliliters or 1 L of buffer was added to the reactor and heated to the assay temperature. The buffer pH was chosen according to the optima specified by the enzyme manufacturers. Corn flour (100-300 g/L) was then added to the reactor, along with a specified quantity of either soluble or immobilized amylase to initiate hydrolysis. Samples were collected at regular intervals over 30-60 min, and centrifuged to separate solids. The supernatant was analyzed for sugar content by measuring the %Brix with an optical refractometer. 

Immobilized cellulase and amylase are able to hydrolyze cellulose and starch. However, the immobilized enzymes possess only about 1-6% of the activity of the soluble forms. In addition, immobilization clearly enhanced the thermal stability of amylase. Immobilized amylase retained more than half of its activity, even after incubation at 125°C. By comparison, soluble amylase was almost completely inactivated under these conditions. Furthermore, kinetics modeling indicates that the susceptibility to product inhibition is dependent on the amylase source. Finally, immobilization can reduce the susceptibility to product inhibition fQ was less for each of the immobilized forms, compared with their soluble counterparts. 

The mechanism and theory of bioelectrocatalysis is still under development. Electron transfer and variation of potential in the electrodeenzyme-electrolyte system has therefore to be investigated. Whether the enzyme is soluble and the electron transfer process occurs through a mediator, or whether there is direct enzyme immobilization on the electrode surface, the homogeneous process in the enzyme active centre has to be described by the laws of enzyme catalysis, and the heterogeneous processes on the electrode surface by the laws of electrochemical kinetics. Besides this there are other aspects outside electrochemistry or... 

Several studies have demonstrated the improved stability of peroxidases when they were subjected to immobilization. Akhtar and Husain observed that bitter gourd peroxidase (BGP) was able to remove higher percentage of phenols over a wider range of pH when immobilized on a bioaffinity support [37]. Sasaki et al. highlighted an improvement of thermal stability of MnP immobilized on FSM-16 mesoporous material [59]. Furthermore, some other studies demonstrated a protective effect of peroxidase immobilization against inactivation by H202 [7, 20]. The different behavior of immobilized peroxidases with respect to soluble ones points out the necessity of an optimization of the process conditions when immobilized enzyme is used. Nevertheless, the possible improvement in stability should balance the usual decrease in kinetic rates, due to substrate transfer limitations to reach the enzyme inside the support. 

Water affects the reaction rate through its effect on reaction kinetics and protein hydration, which is required for optimal enzyme conformation and activity. Enzymes need a small amount of water to maintain their activity however, increasing the water content can decrease the reaction rate as a result of hydrophilic hin-drance/barrier to the hydrophobic substrate, or because of denaturation of the enzyme (189). These opposite effects result in an optimum water content for each enzyme. In SCFs, both the water content of the enzyme support and water solubilized in the supercritical phase determine the enzyme activity. Water content of the enzyme support is, in turn, determined by the distribution/partition of water between the enzyme and solvent, which can be estimated from water adsorption isotherms (141, 152). The solubility of water in the supercritical phase, operating conditions, and composition of the system (i.e., ethanol content) can affect the water distribution and, hence, determine the total amount of water that needs to be introduced into the system to attain the optimum water content of the support. The optimum water content of the enzyme is not affected by the reaction media, as demonstrated by Marty et al. (152), for esterification reaction using immobilized lipase in n-hexane and SCC02- Enzyme activity in different solvents should, thus, be compared at similar water content of the enzyme support. 

Shiraishi et al. [49,50] immobilized glucoamylase of Rhizopus delemar in monolith structures and used them for saccharification of soluble starch. The process was studied at first in a batch reactor at SOX and 4.5 bar. The simplified kinetic model was developed. A continuous process was realized in a monolith reactor consisting of 10 pieces stacked on top of each other, where the blocks were rotated by ir/4 on their axes. The reaction rate at a glucose concentration of 460 g dm" was approximately two times higher than in a conventional industrial process. Conversion of 47% was reached at a space time of 12 hr. The half-life of enzyme was 79 days. 

In many cases it is not possible or desirable to register the reaction catalyzed by an enzyme. It may be that the product is difficult to detect or that the sensitivity in the analysis one needs to apply is not high enough. Then the use of one or more additional enzymes is quite common. The strategy has been worked out for soluble enzymes, and, in the flow systems, the enzymes are either immobilized separately, or coimmobilized. The latter approach has certain advantages in the sense that a better kinetic performance can be observed in a coimmobilized enzyme sequence as compared to when the enzymes are immobilized separately [52]. However, since most assays are based on the use of an excess of immobilized enzymes, no dramatic differences are observed. 

The many circumstances leading to the Henri equation for enzyme conversion of soluble substrates are first noted, followed by some kinetic forms for particulate and polymer hydrolysis. Effects common to immobilized enzyme systems are summarized. Illustrative applications discussed Include metabolic kinetics, lipid hydrolysis, enzymatic cell lysis, starch liquefaction, microenvironment influences, colloidal forces, and enzyme deactivation, all topics of interest to the larger themes of kinetics and thermodynamics of microbial systems. 

The influence of colloidal forces on reactions involving Immobilized enzymes acting on Insoluble substrates has received less attention, yet it appears to offer some clear examples of fundamental phenomena important in enzyme kinetics. Datta examined lysis of Micrococcus Ivsodelktlcus by soluble and (polyacrylamide) immobilized lysozyme. He noted that the decrease in soluble enzyme activity with decreasing ionic strength (Table 1) paralleled the measured decrease in cell lysis measured in flow through a packed bed reactor of Immobilized enzyme. 

Soluble copolymers of albumin and L-glutamate dehydrogenase have been prepared by glutaraldehyde cross-linking. The kinetic and electron microscopic properties of the soluble derivatives were compared with data available concerning the enzyme immobilized within proteic films. 

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