Fundamentals of Biochemical Engineering Part 1 - Bailey J., Ollis D. 1989

Application of enzyme-catalyzed reactions
Technological processes involving immobilized enzymes
Industrial processes

Immobilized enzyme-based catalysts are already utilized to varying degrees in A number of large-scale industrial processes. Among The most significant Applications of Immobilized Enzymes are The production of high-fructose corn syrup from corn starch and the manufacture of L-Amino acids through the resolution of racemic mixtures (consisting of L- and D-isomers). Additionally, immobilized penicillin acylase is employed in the production of Semisynthetic Penicillins. From an economic perspective, the first of these applications is by far the most important.

Sugar (sucrose) cannot be replaced by D-glucose because glucose is less sweet. Furthermore, the crystallization of concentrated glucose solutions can complicate their subsequent Processing and storage. These issues can be largely mitigated by partially isomerizing glucose into fructose using the enzyme glucose isomerase:

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At 50 °C, the Equilibrium Constant for this reaction is close to unity, and Temperature variations have virtually no effect on it, as the heat of the isomerization reaction is only about 1 kcal/mol. Consequently, the reaction product is a mixture of glucose and fructose in an approximately 1:1 ratio. Such a mixture is significantly sweeter than pure glucose and can successfully replace sugar in A wide variety of applications, including the production of soft drinks, the preparation of various food products, and the baking industry. Even sweeter mixtures can be obtained by chromatographic Separation (Chap. 11) of the isomerization products, leading to a fructose-enriched mixture.

FIG. 4.10. Flow diagram of the high-fructose corn syrup production process using glucose isomerase. [Reprinted from: Harden J. D. On-Line Control Optimizes Processing; Food Eng., 44, 59 (1972).]

Glucose isomerase is an intracellular enzyme produced by a number of microorganisms, primarily certain strains of Arthrobacter and Streptomyces. The need to disrupt Cells using sufficiently mild Methods that do not cause irreversible Enzyme inactivation leads to a significantly higher cost for glucose isomerase compared to, for example, extracellular Hydrolases. Moreover, glucose isomerase is highly sensitive to a variety of inhibitors. Both of these factors suggest the advisability of immobilizing glucose isomerase and conducting the process under strictly controlled conditions. Various methods of glucose isomerase immobilization have been studied, including a method based on The Use of whole enzyme-containing cells immobilized in turn within Collagen or by means of some other flocculating and binding agent. This technique serves as an example of yet another approach to the immobilization of intracellular enzymes, which consists in immobilizing the cells producing this enzyme (typically treated to reduce Cell membrane resistance to mass transfer) or lysates of such cells. We will return to Structure/149.html">The problem of cell immobilization in Chapter 9.

Fig. 4.10 shows a process flow diagram for the production of high-fructose corn syrup based on the USE OF IMMOBILIZED glucose isomerase. Here, The Need for numerous separation and intermediate processing operations between the saccharification and isomerization stages is dictated by The properties of the enzyme systems. For instance, to increase the thermal stability of $\alpha$-amylase used for starch Hydrolysis (this process is typically carried out at a temperature of about 105°C), Calcium Ions are added. The latter, however, inhibit glucose isomerase and are therefore removed using ion-exchange resins before the dextrose solution enters the isomerization Reactor.

Example 4.2. Process parameters for glucose isomerization in a reactor in the presence of immobilized glucose isomerase*. Selecting the catalyst type, reactor design, and operational conditions for the catalytic process required a thorough study of numerous interrelated parameters. Table 4S2.1 lists some of the parameters and criteria previously considered in the design of reactors for the production of high-fructose corn syrup using immobilized glucose isomerase. Table 4S2.2 outlines the Main Properties of the catalyst selected As a result of analyzing these parameters during the design development by the H. J. Heinz Company. The chosen catalyst particle size satisfies two mutually exclusive requirements: on the one hand, the catalyst particles are small enough to prevent diffusion rates from limiting the overall process rate, and on the other hand, they are large enough to minimize the pressure drop across a packed-bed Column reactor containing the immobilized enzyme.

The optimal reaction conditions found are summarized in Table 4S2.3. Note the necessity of a high glucose concentration in the feed mixture entering the reactor; the presence of more than 10% Oligosaccharides in the latter significantly reduces enzyme activity. The wide range of contact time between the catalyst and the substrate is due to the gradual inactivation of the catalyst. Over time, enzyme activity declines, and at a constant reactant flow rate, the substrate conversion degree gradually decreases. Therefore, to ensure the required product quality as the enzyme inactivates, its contact time with the substrate is increased by lowering the flow rate. The corresponding mathematical expressions that allow for a quantitative Assessment of the relationship between activity, contact time, and conversion degree for various types of enzyme catalysts will be discussed in Chapter 9.

* Venkatasubramanian K., Harrow H. S., Design and Operation of a Commercial Immobilized Glucose Isomerase Reactor System, Ann. N.Y. Acad. Sci., 326, 141 (1979).

Table 4S2.1. Parameters studied during the design of an immobilized glucose isomerase reactor^a

^a Reproduced with permission from: Venkatasubramanian K., Harrow H. S., Design and Operation of a Commercial Immobilized Glucose Isomerase Reactor System, Ann. N. Y. Acad. Sci., 326, 141 (1979).

The dimensions of a column-type reactor are determined by the hydromechanical Properties of the catalyst bed. Under downflow conditions of the reaction mixture, the immobilized enzyme catalyst bed is subject to compression under pressure, resulting in increased resistance to flow. Fig. 4P2.1 illustrates the dependence of the pressure drop across the column on the catalyst bed height at the maximum flow rate permissible for a fresh catalyst. If the pressure drop across the column exceeds 0.2 kg/cm², the column resistance increases sharply. It follows that the maximum catalyst bed height should be approximately 4.6 m. Considering the known catalyst-substrate contact time and the required productivity of the entire plant, it is straightforward to determine the dimensions of the necessary column reactors and their number. Owing to the aforementioned catalyst inactivation processes, it is advisable to install multiple columns so that they undergo inactivation (and replacement) sequentially; this allows for the maximum utilization of the catalyst while maintaining a constant high productivity of the plant as a whole.

Table 4P2.2. Physical and catalytic properties of Immobilase, a catalyst with glucose isomerase activity (an ICI product)^a

^a Reproduced from: Venkatasubramanian K., Harrow H. S., Design and Operation of a Commercial Immobilized Glucose Isomerase Reactor System, Ann. N. Y. Acad. Sci., 326, 141 (1979).

^b One unit of activity is defined as The amount of catalyst capable of converting 10^-9 moles of substrate per 1 min at 60 °C and pH 8.0.

Table 4P2.3. Reaction conditions for the isomerization of glucose syrup in a packed-bed column reactor with an immobilized enzyme^a

^a Reproduced with permission from: Venkatasubramanian K., Harrow H. S., Design and Operation of a Commercial Immobilized Glucose Isomerase Reactor System, Ann. N. Y. Acad. Sci., 326, 141 (1979).

The demand for L-amino acids for food and medical applications is steadily increasing. Consequently, considerable attention is being focused on developing both microbiological and chemical Methods for the production of L-amino acids. A major drawback of chemical methods is the racemic nature of synthetic amino acids. In general, D-isomers have no nutritional value; therefore, it is desirable to produce exclusively physiologically active L-amino acids.

FIG. 4P2.1. Dependence of the pressure drop across the column on the catalyst bed height; downflow column packed with immobilized glucose isomerase granules. [Reproduced with permission from: Venkatasubramanian K., Harrow H. S., Design and Operation of a Commercial Immobilized Glucose Isomerase Reactor System; Ann. N. Y. Acad. Sci., 326, 1141 (1979).]

The stated goal was achieved using a process developed by Tanabe Seiyaku Co., Ltd. (Osaka, Japan), which marked the first industrial-scale application of immobilized enzymes and was documented in the literature. The process is based on the optical isomer separation method utilizing the following enzyme-catalyzed aminoacylase reaction:

FIG. 4.11. Packed-bed reactors with immobilized enzyme used by Tanabe Seiyaku Co., Ltd. for the production of L-amino acids. (Photograph courtesy of Dr. I. Shibata, Tanabe Seiyaku Co., Ltd.)

This reaction is carried out in a packed-bed reactor containing immobilized aminoacylase (Fig. 4.11). Afterward, the main reaction product, the L-amino acid, is separated from the unhydrolyzed D-acylamino acid based on their differing solubilities. The D-acylamino acid is then racemized into the DL-acylamino acid, which is recycled back into the aminoacylase column. The overall process flow diagram is shown in Fig. 4.12.

To determine the optimal immobilized enzyme form for this process, Shibata, Tosa, and their coworkers at Tanabe Seiyaku conducted extensive research, partially published in their paper [16]. Table 4.12 summarizes their findings on the properties of various immobilized enzyme preparations. These data once again demonstrate that when selecting a catalyst for industrial applications, many factors beyond initial activity must be taken into account. In this case, the researchers ultimately chose aminoacylase immobilized via ionic bonding on DEAE-Sephadex due to its high activity, ease of preparation, regenerability, and operational stability. In 1978, representatives of Tanabe Seiyaku reported that this catalyst had been in continuous use for over five years without any mechanical degradation or loss of binding activity.

FIG. 4.12. Flowsheet of the immobilized aminoacylase process implemented by Tanabe Seiyaku. [Reprinted with permission from: Chibata I. et al., Preparation and Industrial Application of Immobilized Aminoacylases, in Fermentation Technology Today, Terui G. (ed.), p. 387, Society of Fermentation Technology, Japan, Osaka, Japan, 1972.]

In 1983, the first industrial process utilizing enzymes immobilized by semipermeable membranes was reported. This involved The conversion of N-acetyl-DL-Methionine to L-methionine using an acylase enzyme. The potential applications of immobilized enzymes in numerous other chemical and technological processes have also been investigated (Table 4.13). Some of these applications have already been commercialized, while others remain in the research and development stages.

Table 4.12. Properties of Various Forms of aminoacylase (substrate: acetyl-DL-methionine)^a

^a Reprinted from: Mori T., Sato T., Tosa T., Chibata I., Studies on Immobilized Enzymes X: Preparation and Properties of Aminoacylase Entrapped into Acrylamide Gel-Lattice, Enzymologia, 43, 213 (1972).