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377. This part of the report describes the state-of-the-art of enzyme engineering processes on the industrial level. Some of them could be coupled with the enzymatic production of glucose from cellulose, described in the first three volumes of this report, and/or with the subsequent microbiological conversion of glucose into nutritional or other useful products. Others can serve as examples of contemporary industrial or semi-industrial processes based on practical application of principles of enzymology and biochemical engineering.


378. Enzyme engineering is a relatively new field of science and technology dealing with the development of useful products or processes based on the catalytic action of enzymes when either isolated from their source of biological synthesis or when intact within cells that are immobilized and usually not growing. This definition (Wingard & Klyosov, 1980) omits fermentation and tissue culture systems, in which living cells are growing, but does not omit multi-enzymes in sub-cellular particles.

379. From this definition it is apparent that enzyme engineering is an applied area of research and development. However, it may not be apparent that enzyme engineering so strongly depends on the results of fundamental research in chemical enzymology, physical chemistry, microbiology, biochemistry, and polymer chemistry and that often it is not possible to differentiate clearly between enzyme engineering and these fundamental sciences. Thus, the immobilization of enzymes, the chemical modification of enzymes, studies into the mechanism of specific enzymes, and genetic manipulations to improve enzyme availability or process characteristics all are fundamental scientific topics that become part of enzyme engineering when considered in the light of a specific enzyme-based process or product. Similarly, the fundamental topics of heat and mass transfer, diffusional effects on enzyme-catalyzed reaction rates and mathematical modelling of enzyme-catalyzed reaction kinetics or overall process economics become part of enzyme engineering primarily when applied to a specific enzyme-based process or product.

380. In the final analysis an enzyme-based process or product must provide something unique if it is to find practical acceptance. Examples of suitable unique contributions include:
       (i) production of a new and useful product or the achievement of a new and useful chemical
           transformation or of an old transformation in a new environment, e.g. in vivo,
       (ii) production of a better quality product, and
       (iii) production of an existing product more economically.

381. Traditionally, enzymes have been extracted from plants and animals. Rennin, for instance, the enzyme employed to curdle milk for cheese production, is extracted from the stomach of unweaned calves. But the production of enzymes from bacteria, yeasts and fungi has rapidly become more common. As a rough estimate more than 50 enzymes are used exclusively in the food industry throughout the world: in brewing, cheese making, the production of fruit juices and wines, starch and sugar processing and as meat tenderizers (Yanchinski, 1984; Barker, 1987). Just 16 enzymes of the thousands produced in nature rank as industrial catalysts, accounting for over 90% of the total market (Biotechnology Bulletin, 1986; Linhart, 1987). Three enzymes, for instance, form the basis for an industry manufacturing liquid sweeteners from cornstarch (see below).

382. The use of enzymes as catalysts is growing remarkably. Frost & Sullivan reported recently that consumption of industrial enzymes in Western Europe will approximately double in quantity over 10 years in real terms from the 1986 sales of about $160 million (Biotechnology Bulletin, 1986). In 1983 enzyme sales worldwide were reported to be about $390 million, up nearly 25 percent from 1979 (Maugh, 1984), and in 1985 sales in the USA and Western Europe alone reached $500 million (Chaplin, 1984). A 1982 report from the Office of Technology Assessment predicted that, within 20 years, enzymes would be used in the production of $15 billion worth of chemicals and pharmaceuticals. On a dollar basis, about 80 percent of the enzymes are attributable to the food industry (Genet. Eng. Biotechnol. Mon., 1984). On a volume basis, starch conversion enzymes account for about 40 percent of sales and proteolytic enzymes used in laundry detergents account for another 30 percent. Europe dominates the world in sales, with Novo Industries in Denmark and Gist Brocades in the Netherlands controlling 60 per cent of the world market (Yanchinski, 1984). According to 1991 data, in China alone there are over 40 enzyme manufacturing factories with an annual output of over 2,000 tons (Han Ying-Shan, 1991).

383. Immobilized enzymes (a similar definition may be given in relation to immobilized cells) may be defined as enzymes whose free movement has been restricted in some manner. One of the major advantages of immobilization is to "fix" the enzyme so as to retain it in a continuous process. Use of soluble enzymes in processing has been limited in part by the cost of the enzymes due to the difficulty and expense of isolation, instability, and the fact that in a freely soluble form they usually can be used only once.

384. The use of immobilized enzymes has several advantages. An immobilized enzyme, in solid or sometimes semisolid form, is readily separable from the product solution and can be reused, thus increasing utility by a large factor. Additionally, the enzymic reaction can be terminated easily simply by removing the enzyme, allowing more precise control of the reaction. Finally, it has turned out that enzymes (or cells) are often much more stable when in fixed form than in solution, allowing an enzymatic conversion to occur over a much longer period without having to replenish the biocatalyst as compared with intact enzymes or cells. Specific examples will be given in the subsequent sections.

385. Enzymes (or cells) may be immobilized by several techniques. These include:
       (1) Physical adsorption;
       (2) Covalent attachment to an inert support;
       (3) Entrapment within a gel matrix;
       (4) Containment behind a semipermeable membrane (hollow fibers, microcapsules, etc.);
       (5) Intermolecular crosslinking of enzyme molecules.

386. Either "live" or "dead" cells can be immobilized. In dead cells most of the life processes have been destroyed more or less selectively while the enzyme of interest is allowed to survive.

387. Materials used as supports (carriers) for immobilization have included both organic and inorganic materials (Linhart, 1987; Komori et al., 1987; Bisping & Rehm, 1988). Cellulose, dextran (Sephadex), agarose (Sepharose), nylon, acrylamide-based polymers, and styrene-based polymers are typical examples of organic supports. Inorganic supports studied include porous and solid glass, diatomaceous earth, silica aluminas, nickel screening, stainless steel balls, sand, and titania. They often have better flow properties, and some are very inexpensive. Generally, the specific type of immobilization procedure and the support used are determined by the application of a given enzyme system.

388. A combination of the unique catalytic properties of enzymes when insoluble in an immobilized form has been used for the development of novel technological processes. At present these processes are related mainly to food and pharmaceuticals. The restriction of immobilized enzymes to these areas of technological applications could be explained in part by the existence of respective industrial chemical and microbiological processes with thoroughly worked-out technology. In many cases enzymes are produced industrially in relatively small quantities as yet and are still rather expensive for industrial applications; the cost for immobilization and for appropriate carriers and chemicals is often too high.

389. In the near future, immobilized enzymes will only be used industrially when the product of a process is impossible to obtain without enzymes, or if the cost of the product is so high compared with the cost of the initial materials (chemicals) that the difference can cover the expenses for the immobilization of the enzyme. That is why an interest is growing for the immobilization of enzymes by simple adsorption onto carriers and for using immobilized cells without isolation of intracellular enzymes.

390. Up to the present time, only eight processes with immobilized enzymes or microbial cells have found industrial application:

       (1) Production of high-fructose syrups by immobilized glucose isomerase.

       (ii) Optical resolution of amino acids by immobilized aminoacylase.

       (iii) Production of optically active D-amino acids by immobilized hydantoinase.

       (iv) Production of L-aspartic acid by immobilized aspartase.

       (v) Production of L-malic acid by immobilized fumarase.

       (vi) Deacylation of penicillin G and cephalosporin derivatives by immobilized penicillin

       (vii) Hydrolysis of lactose by immobilized lactase in dairy processing.

       (viii) Production of glucose-fructose syrups from saccharose by immobilized invertase.

391. Aside from these, there is the well-known process, which has already been worked out on a pilot plant scale, and is apparently not far from larger-scale applications:

       (ix) Production of glucose from starch hydrolysates by immobilized glucoamylase.


392. The production of high-fructose corn syrup (HFCS) by the isomerization of glucose to fructose, which is catalyzed by glucose isomerase of microbial origin, has been the major industrial application of immobilized enzyme technology during the past 20 years (Hagen & Pederson, 1990). The isomerization process converts about 45% of the glucose (dextrose) in a corn starch hydrolysate, which has been prepared by enzymatic hydrolysis, into fructose.

393. Clinton Corn Processing, a division of Standard Brands, now Nabisco Brands, was first to achieve a successful commercial production of HFCS from starch hydrolysate using immobilized glucose isomerase in 1973. The process created the largest new market developed in the ensuing decade, including $40 million/year for immobilized glucose isomerase (for further economic estimates see paragraphs 437-441).

394. The use of glucose isomerase began under a unique set of conditions: in 1974, the price of sugar in the U.S.A. increased sharply, making the cost of this conversion process competitive. By the time the cost of sugar fell, the enzyme manufacturers were able to reduce costs sufficiently to remain competitive, and presently immobilized glucose isomerase is used to produce more than 3 million tons of HFCS yearly (Maugh, 1984).

1.     Background

395. Today's typical process for production of fructose syrups uses alpha-amylase to liquefy starch followed by glucoamylase to saccharify the hydrolyzed starch to the required 94% dextrose content for the isomerization, which results in a mixture of glucose and fructose (Quax et al., 1991). The process is as follows (Hultin, 1983): a starch slurry of about 33% dry solids is liquefied with a bacterial α-amylase at a temperature ranging from 80-110°C, generally for 2-4 hrs. Since α-amylase usually requires calcium, the pH of the starch slurry is adjusted to 6-7 with calcium hydroxide. Acid hydrolysis is occasionally employed, but the glucose concentration produced after saccharification is higher if enzymic hydrolysis is utilized. The hot slurry is then cooled to about 60°C at which point the liquefied starch is saccharified by treatment with a fungal glucoamylase that produces glucose from liquefied starch.

396. This process is generally carried out at pH 4-4.5 with a "holding time" of 24-90 hr depending on conditions and the amount of enzyme used. On a dry basis this generally converts starch to 96-97% glucose. This crude syrup, high in glucose, is then decolorized with carbon and deionized with both acidic and basic resins. It is important to remove the calcium ions, which inactivate glucose isomerase.

397. The crude glucose solution may then be concentrated or blended to bring its content of solids to 40-50%. Magnesium ions are required and generally added for almost all of the glucose isomerases used presently. The pH of this crude glucose solution is made slightly alkaline (up to pH 8.5), with the exact pH depending on the enzyme and the type of reactor. The glucose content of the starting syrup for the isomerase treatment is minimally 94% on a dry basis. The conditions produce a fructose syrup containing a minimum of 42% fructose (usually 43%) and about 51% glucose with no more than 6% disaccharides or higher polymers (Hultin, 1983).

398. The syrup is then treated with activated carbon and deionized with a strong acid-cation exchange resin in the hydrogen form and a weak base-anion exchange resin in the free base form. This process should be carried out as rapidly as possible since the pH values reached with the ion exchange resins can range from 1.5 to 8, and this can induce chemical decomposition of the syrup components. The pH of the final product is generally adjusted to 4-4.5 which results in maximal stability. Finally the syrup is concentrated to 71% solids by evaporation during which process the pH is carefully controlled (Hultin, 1983).

399. The sweetness of the resultant high fructose is comparable to that of common sugar (sucrose) or the invert sugar that is produced by acid or enzymatic hydrolysis of sucrose.

400. The resultant HFCS is the functional equivalent of sucrose for many applications. It is widely used as a nutritional sweetener in the food industry, particularly in soft drinks and other beverages, as well as in desserts, baked goods, canning, and many other packaged products. For certain uses (e.g., cola beverages), HFCS with 55% or 90% fructose is preferred, made from the 42%-fructose HFCS by a process of separation analogous to liquid chromatography (Carasik & Carroll, 1983).

401. The rapid growth in demand for fructose in the last decade may be attributed to the following:
       - it can be used as an alternative to sugar since both are equally sweet,
       - it can be produced at a cost lower than sugar,
       - it is produced from starch, which is a common and widely available food material,
       - its taste is more refreshing than that of sugar,
       - consumption of fructose is said to be of lower risk for diabetics and some other metabolic

402. The amount of fructose produced by the isomerase reaction is determined by the equilibrium value of the glucose to fructose isomerization which results in roughly equal amounts of the two sugars. In order to obtain higher fructose content of the syrups, large-scale columns of strong acid-ion exchange in the form of calcium salts are often used as the stationary phase. Oligosaccharides come off the columns first, owing to a molecular exclusion effect. These are then followed by glucose and finally fructose.

403. Another alternative is to concentrate the syrup to a high enough content of solids to allow crystallization of glucose from the concentrate. The remaining liquid is then enriched in fructose. In this manner it has been possible to make syrups of up to 70% fructose (on a dry basis). The isolated glucose is recycled in an isomerization reaction. Finally, another approach is to complex fructose with borate compounds. This removes fructose from solutions and shifts the apparent equilibrium of the reaction. However, the cost of removal and recovery of the borate has so far prevented commercial adaptation of this process (Hultin, 1983).

2.     Commercial preparations of immobilized glucose isomerase

404. Clinton Corn Processing Co. developed the first industrial process HFCS production, which used the microorganisms Streptomyces sp. as the source of glucose isomerase. In contrast, Novo Laboratories Inc. selected the thermophilic microorganism Bacillus coagulans, which produces glucose isomerase without requiring the addition of an expensive inducer such as xylose in the mixture (Carasik & Carroll, 1983), and can be grown in continuous culture without contamination and with high enzyme yields. In spite of the large number of organisms that can produce glucose isomerase (Antrim et al., 1979), only a few high-producing strains are actually being utilized on a commercial basis.

405. Glucose isomerase typically is an intracellular enzyme. The enzyme is generally obtained by scaling up a submerged aerated fermentation in several stages. The three stages of development for such a production are described in U.S. Patent 3,666,628: (a) slant development, (b) culture development - two substages, (c) final fermentation stage. Glucose isomerase can be recovered in two ways: (i) recovery of the microorganisms with the enzyme entrapped in the cellular mass, and (ii) recovery of the enzyme in the soluble form after lysing the cells (Antrim et al., 1979). To prevent the loss of the enzyme from cells in the first case (as the result of the autolysis of cells during isomerization) and to allow extended use of cells in fixed-bed reactors, the enzyme is bound to the cellular matrix by heat or chemical fixation (Antrim et al., 1979).

406. Commercial immobilized glucose isomerase preparations are generally produced by various companies in a granular form and a fibrous or amorphous form. Clinton Corn Processing Company, for example, produces both a fibrous form and a granular form of glucose isomerase. Each form is designed for a particular reactor technology. The fibrous form of Clinton's enzyme preparation has a large surface area with very high enzyme potency and is designed for use in shallow-bed reactors. The granular enzyme with lower potency is designed for deep-bed reactors. Novo Enzyme Corporation produces a granular isomerase suitable for deep-bed reactors, batch isomerization, and fluidized-bed operations. The immobilized enzyme preparation from Gist Brocades (Maxazyme®) utilizes the organism A. missouriensis entrapped in cross-linked gelatin. This produces a softer particulate enzyme which can also be used in deep-bed reactors.

407. ICI produces an immobilized enzyme preparation whereby Arthrobacter cells are flocculated by polyionic reagents. This also produces a rather soft granular particle that can be used in deep-bed reactors. Sanmatsu (Japan) produces a glucose isomerase by adsorption of the enzyme on an anion-exchange resin. This yields a high-potency, particulate enzyme suitable for deep-bed reactors. Denki-Kagaku - Nagase Sangyo (Japan) has produced a polymer isomerase (Sweetase®) entrapped in hard granules for use in deep-bed reactors. The catalyst particles swell to about double their original sizes (0.4-0.8 mm) during the isomerization reaction (Okada, 1978).

408. Miles and Miles-Kali Chemie have produced a glutaraldehyde cross-linked preparation and a heat-fixed cellular preparation, respectively. Snamprogetti (Italy) produces glucose isomerase, extracted and partially purified from Streptomyces sp., and entrapped in cellulose triacetate fibers. This is carried out according to Snamprogetti's modification of the well-known technology of fiber wet spinning: an organic solution of fiber-forming polymer is emulsified with an aqueous solution of the enzyme, and the resulting emulsion is then extruded into a coagulation bath through the holes of a spinneret (Marconi & Morisi, 1979).

409. The main conclusion from the above table is that there are actually no covalently bonded enzymes among commercial preparations of immobilized glucose isomerase. Glucose isomerase is either adsorbed to ion exchange resins or to porous inorganic carriers (alumina, ceramics), or it is inside whole cells entrapped in a polymer matrix, usually cross-linked with glutaraldehyde or other bifunctional reagents. Even Corning Glass Works, which has developed a well-known procedure for covalent immobilization of enzymes on inorganic carriers, uses immobilization by adsorption in this particular case. The main reason for this is that immobilized glucose isomerases that have been produced by adsorption of soluble enzyme onto a solid carrier can generally be regenerated and reloaded with fresh enzyme after much of the initial activity is depleted. It is usually economically advantageous to do so, especially when the carrier used is expensive.

410. In many cases whole cells (heat-fixed or entrapped into polymer matrix) are used instead of the isolated enzyme. This can be explained both by decreased stability of isolated (extracellular) glucose isomerase (Ishikawa, 1977) and by the higher cost of isolation and immobilization of enzymes in comparison with the use of whole microbial cells.

411. These two points (par. 409 and 410) are illustrated below in the form of a comparison of the general procedures of enzyme immobilization with respect to several important commercial properties (Antrim et al., 1979).

412. The increase in HFCS penetration of the world sweetener market has been made possible in part by the evolution of immobilized glucose isomerase technology and improvements in enzyme characteristics. The dynamics can be traced using the example of Novo Laboratories Inc., which subdivided various types of immobilized glucose isomerase preparations developed by the company into the preparation of three generations of enzymes (Carasik & Carroll, 1983). These do not include the first of a series of commercial glucose isomerases marketed under the brand name Sweetzyme®, which was a spray-dried soluble product introduced in 1973. It had a high production cost and was used commercially for only a short time. When Novo's first immobilized form of the enzyme was introduced in 1974, its advantages became readily apparent and use of the spray-dried soluble form was subsequently discontinued (Carasik & Carroll, 1983).

413. The steps in the production of immobilized glucose isomerase of "three generations" (and for three types of reactors) are shown in Figure 1 and described below (Carasik & Carroll, 1983).

414. For production of the first generation enzyme (1974), in the form of a powder, the cross-linking agent selected is glutaraldehyde, which immobilized the cell protein. The initial product of cross-linking is a semigelatinous mass that has the consistency of pudding. After drying, grinding and screening, the final product has a particle size range of 100-350 microns. The product is designed for use (and re-use) in a batch reactor (Fig. 2).

415. The first-generation immobilized enzyme has several limitations. The particles gradually decrease in size as a result of the shearing effect of the agitator blades. This leads to an overall decrease in settling rate and thereby to a slowing of the process. According to Novo's data, decrease in the size of the particles of the immobilized enzyme from 0.35 to 0.25 mm and further to 0.10 mm leads to a decrease in the terminal velocities of settling particles from 9.5 to 5.0 m/hr and further to 0.8 m/hr. In addition to the degradation of the particle, there is thermal denaturation of the enzyme, decreasing its activity. Prolongation of the reaction time or addition of new enzyme to the isomerization reaction tank prior to the next cycle can compensate for this. Normal make-up is about 5% per reuse. Finally, the long holding time required for the isomerization with the first generation enzyme in turn requires operation at a relatively low pH to avoid excessive color and formation of by-products. At this pH, the addition of cobalt salts is necessary to activate the immobilized enzyme. These drawbacks of the powder form of the enzyme and the batch reactor led to the development of immobilized enzymes of the second generation, which are suitable for continuous column reactors, where the reaction can be run at a neutral to slightly alkaline pH, since the syrup contact time is reduced drastically, with no cobalt addition required.

416. The second-generation immobilized enzyme for column use was prepared by the extrusion of the wet cross-linked enzyme mass through a small orifice (< 1 mm diameter) cut into short cylindrical particles, which are then dried and screened, as shown in Figure 1. This product does not exhibit pressure-drop characteristics; upon continued operation the particles deform and the pressure drops dramatically. This is overcome by modifying the immobilized enzyme preparation through the incorporation of powdered alumina to increase the density of the particles and allow higher flow rates in the upflow mode (Carasik & Carroll, 1983). However, industrial performance is entirely satisfactory, and the deviation from laboratory data is ascribed to variations in enzyme particle size and to problems of density and flow-distribution problems.

417. For the preparation of the third-generation immobilized glucose isomerase, which should be able to resist deformation at constant syrup flow for fixed-bed operation, an important change in the immobilization process is made. The cell slurry is homogenized prior to cross-linking as shown in Figure 1. Homogenization disrupts the cell membranes and allows more of the protein surface to react with the cross-linking agent. The result is an enzyme with stronger particles that resist deformation. The improved enzyme preparation also contains additives such as magnesium oxide and dextrose, which are incorporated after crosslinking but prior to extrusion. Magnesium oxide helps to minimize the decrease in pH during isomerization, and the dextrose dissolves during operations, resulting in a more porous matrix and lowering diffusion resistance (Carasik & Carroll, 1983). The enzyme is shipped to the user in a dry granular form which, prior to use, must be hydrated in syrup. During hydration, the size of the enzyme particles expands by a factor of about two. Under operating conditions (pH 7.5-8.0, temperature 55-60°) the half-life of the enzyme is usually greater than 75 days.

3.     Technological characteristics of the processes

418. The literature contains limited information on commercially available glucose isomerase technologies. Listed below are some data on reactor configurations normally employed, and basic performance data where available. Although each system listed employs a different enzyme source (see above), a different level of enzyme purity, and a different immobilization technique, there appears to be relatively little difference in the performance of the systems for which data are available (Sweigart, 1978).

419. Major commercial manufacturers of immobilized glucose isomerase describe the application for deep-bed reactors. A deep-bed reactor is usually simpler in design than a shallow-bed model and may therefore require a lower capital expenditure. Flow may be "up" or "down" through the reactor. Column reactors operating in the "down" mode appear to be more popular. Heights up to five meters appear to be feasible. Upflow in a column is reported to be less productive, probably due to wider distribution of the "syrup residence time." Precautions must be taken with upflow to prevent loss of immobilized enzyme through the top of the reactor, and flow control is considered to be more critical than for downflow (Antrim et al., 1979). The temperature and pH recommended are almost identical (60-65°C and 7.0-8.5 respectively). Further, the initial substrate of 40-50% dry solids (92-94% dextrose, dry basis, 6-8% dry basis polysaccharides) is also much the same.

420. In continuous production of isomerized sugar by means of the immobilized enzyme process, raw material purity influences catalyst activity and therefore productivity. Thus, raw material (dextrose syrups) should be refined as much as possible. Impurities include calcium ions, peptides, oxygen, oxidation products, etc. According to the data of the Chiba Plant, Denki Kagaku Kogyo K.K. (Okada, 1978), when high purity substrates such as crystalline glucose are used as the initial substrate, the productivity of the reactor (i.e., the yield of solid high fructose syrup per unit weight of catalyst until catalyst activity decreases to a quarter of the original level) reaches 4000 kg/kg of immobilized enzyme in 100 days, and the half-life for the catalyst is 50 days. However, when purified dextrose of lower grade is used, the half-life decreases to 20 days, and productivity of the reactor to 1,500 kg fructose/kg of immobilized enzyme. In general, productivity for immobilized glucose isomerases used commercially, described by various enzyme manufacturers and used under the operating conditions recommended by them, varies from 1 to 9 tons of HFCS (dry basis) per kg of the immobilized enzyme.

421. A multiplicity of reactor designs has been described for use with immobilized glucose isomerase (six different design types, according to one particular classification, i.e. batch, packed-bed, continuous-flow stirred-tank, continuous-flow stirred-tank/ultrafiltration membrane, and others, including recycle reactors and tubular reactors with enzymatically active walls) (Antrim et al., 1979). However, most glucose isomerase reactors now in commercial operation are of the packed-bed type. Novo Industri has described batch reuse of Type A, a glutaraldehyde cross-linked homogenate of B. coagulans. Although they subsequently found performance advantages with a continuous system, in 1976 they reported that Type A had been used commercially in large-scale batch reuse since 1974 [cit. in Antrim et al. (1979)]. Gist-Brocades has also described conditions for reuse of their Maxazyme GI-Immob in a batch reactor.

422. Numerous investigators have compared performance and economics of batch versus continuous reactor systems. Usually in a packed-bed reactor the concentration of active glucose isomerase is high compared to a batch reactor and contact time between substrate and enzyme is relatively short, usually 2-4 hr as compared with 20-60 hr for a batch reactor (Antrim et al., 1979). The short contact time helps to minimize formation of colored materials and nonfructose isomerization compounds. Additionally, enzyme usage (comparative amounts of enzyme activity), is found to be considerably higher (1.4-4.0 times) for batch versus continuous packed-bed reactors, primarily due to loss of active enzyme through multiple batch recovery operations (Antrim et al., 1979).

423. U.S. Patents 3,847,740 and 3,847,741 disclose a process for regulating production by means of temperature control as well as for increasing the productivity of the enzyme. One example demonstrates that by increasing the temperature in the reactor by 2°C increments from 60° to 70°C over a period of 14 days, the enzyme productivity increases 42% over a 14-day isothermal run at 60°C.

424. Novo Laboratories Inc. used the 'first-generation' immobilized glucose isomerase (see par. 413) in a batch reactor (Fig. 2). Typically the enzyme is added to a tank containing purified high-dextrose syrup (> 93%, dry basis) adjusted to certain conditions (pH 7.0, temperature 60-65°C, Co+2 3.5x10-4M, MgSO4x7H2O 0.1-1.0 g/liter). The typical reaction time is 20-24 hr, after which the enzyme is recovered by allowing the particles to settle and the supernatant liquor drawn off. The entire process is then restarted. The limitations of this mode of process operation are described in paragraph 415.

425. Glucose isomerase of the second generation, i.e. crosslinked with glutaraldehyde and extruded (see par. 416), has led to non-satisfactory pressure-drop characteristics of the column reactor in the downflow operation mode. Ultimately, the entire enzyme bed will compress to a point where flow is reduced drastically and the operation is stopped. Therefore, Novo Labs at that stage of development (1975) attempted to use an expanded- or fluidized-bed reactor, in which the dextrose syrup flows into the bottom of the column through a flow distributor and the immobilized enzyme is suspended in the flowing syrup (Carasik & Carroll, 1983). In the fluidized-bed operation, the substrate solution passes upward through the enzyme bed. In this mode the most critical factor is uniform flow distribution. In commercial reactors this is achieved with a shallow bed of heavy inert material (alumina) on a screen.

426. With third-generation immobilized glucose isomerases, which have stronger particles that resist deformation (par. 417), after the hydration the enzyme slurry is transferred to the column reactor, which runs in an upflow (fluid-bed) mode for about 12-24 hr. This permits the enzyme to come into equilibrium with syrup at column operating conditions without compacting. After this "fluid" period, the bed is allowed to settle briefly, then fluid is run in the downflow mode. This process is currently in wide industrial use in many parts of the world (Carasik & Carroll, 1983). The enzyme bed height at optimum operating conditions is 5 m maximum, diameter 1.5 m maximum (height:diameter ratio is 3:1 minimum), temperature 55-60°C at inlet, pH 7.5-8.0 at 25°C, dextrose concentration at inlet 94% minimum (dry basis), feed solids 45%, Mg+2 content equals 20x Ca+2. Half-lives are usually more than 75 days.

427. To achieve high enzyme productivity (in this particular case above 4,000 kg of dry solids/kg of enzyme, at 45% conversion of dextrose to fructose, see also paragraph 420) on a continuing basis, Novo recommends (Carasik & Carroll, 1983) that careful attention be paid to details of plant design and operation. Thus, in reactor design, flow distribution is important for operation over a wide range of flow rates. The enzyme is often used until the flow rate (activity) falls to 10% of the initial flow. Feed purity is of great importance to avoid cumulative enzyme poisoning (cf. paragraph 420). Filtration, decolorization (active carbon treatment), deionization, and evaporation must be designed and operated carefully. It is important to provide a purified feed of constant composition with consistent control of parameters.

428. Data on the isomerization process are also available from Kyowa Hakko Kogyo Co., Ltd (Yokote et al., 1975). Glucose isomerase used in their process was immobilized on a phenol formaldehyde resin Duolite A7. The flow chart of the process is shown in Figure 3. A 40% (w/v) glucose solution at 60°C is fed continuously through the immobilized enzyme columns in series at 60°C, pH 8.2. Before feeding, the substrate solution is passed through pretreatment columns that contain Duolite A7 (buffered) to eliminate some impurities and air bubbles. The effluent solution from the immobilized enzyme column is passed successively through a cation exchange (Diaion SK-1A, H-type) and an anion exchange (Diaion WA20, OH-type) column in order to eliminate salts. Microbial contamination was not observed throughout the whole operation period due most likely to the high glucose concentration (40%) and the high operating temperature (60°C). The half-life of the enzyme catalyst under their operating conditions is 34-40 days, depending on the flow rates. According to the company's data, 1 liter of the immobilized enzyme (an adsorption type) isomerizes 288 kg of glucose within 30 days. On the other hand, 1 liter of a covalently immobilized enzyme (with triazinyl chloride as a coupling agent) isomerizes 576 kg of glucose in the same period.

429. In Clinton Corn Processing's approach (flow sheet shown in Figure 4), the immobilized enzyme constitutes a thin bed of 2.5-12.5 cm depth (Davis, 1974). Several of these beds are staged to form a multibed processing unit resembling a pressure leaf filter (see also paragraph 419). A major advantage of having more than one bed is to minimize the effect of channeling that easily occurs in shallow beds. For bed dimensions, Clinton Corn recommends a depth-to-width ratio of about 0.02 to 0.05 (Antrim et al., 1979; Davis, 1974). Single beds can be removed for regeneration during processing without interruption of the process flow. Carbon treatment and ion exchange are used to purify the 42%-fructose effluent, and it is concentrated to syrup by evaporation. Enzyme half-life, according to Clinton Corn, is "several hundred hours" (Davis, 1974).

430. In the mid 1970s Sanmatsu Kogyo was the world's first manufacturer of sugar obtained by isomerization with soluble glucose isomerase. Mitsubishi Chemical Industries Ltd and Seikagaku Kogyo jointly developed an ion exchange resin that selectively adsorbs glucose isomerase "without affecting its activity" (Ishikawa, 1977). These three companies signed a license contract for this process that led to a commercial test plant that was put into operation in 1975. On the basis of satisfactory test results, Sanmatsu Kogyo decided to switch its whole production process to this system in 1976. Later on that year Sanmatsu factories in Chiba and Fukuoka were remodelled to utilize Mitsubishi's technology. Since then these factories have operated commercially. The production process is summarized as follows (Ishikawa, 1977). The microbial product of glucose isomerase is filtered for culture liquor separation, repulped in water and treated with additives for several hours to extract the enzyme. After separation of the residual cells, the enzyme liquor is allowed to contact the ion exchange resin, which adsorbs almost all the glucose isomerase within several hours. Since the carrier selectively adsorbs glucose isomerase, refining the enzyme liquor is unnecessary. The isomerization itself takes place in a simple column packed with the immobilized enzyme, glucose concentration of 45-50%, reaction temperature 55-70°C, pH 6.5-8.5, space velocity (initial) 1.5-4 hr-1. The glucose conversion level is 45%. At these operating conditions the half-life of the immobilized enzyme is 50 days if crystalline glucose is used as raw material, but as low as 30 days if the raw material is a saccharified glucose solution subjected to conventional purification. The isomerized glucose syrup is almost colorless and has few byproducts. Simple treatment of the syrup with ion exchange resin completely desalts and decolorizes the product. After being used the immobilized enzyme is washed away from its carrier in the reactor through a regeneration step employing "some chemical solutions". The regenerated carrier is reused for isomerization after adsorbing new, active enzyme.

431. According to Zhang (1982), Chinese biotechnologists used glucose isomerase from Streptomyces roseoruber, adsorbed on a strong basic anion exchange resin 290 (made by Nankai University, China), for making HFCS. Pilot plant scale experiments have been carried out with 1.25-2.2 tons of HFCS produced per kg of dry immobilized enzyme. A general overview of biotechnology programs in China is given in Mang (1991) and Han Ying-Shan (1991).

432. In the Snamprogetti (Italy) process glucose isomerase entrapped in fibers is used for the isomerization of industrial glucose solutions. Under operating conditions (65°C, 50% w/w glucose syrup) the half-life of the immobilized enzyme preparations is about 70 days. The product is colorless, and activated carbon treatment is not required. The best process performances were achieved with tubular and radial reactors (Marconi & Morisi, 1979). In the tubular reactor the packing is obtained with fibers placed in an ordered manner parallel to the long axis of the column, a packing degree in the range of 0.2-0.25 kg of dry fibers per liter of reactor volume results in a stable catalyst bed practically incompressible and with negligible channelling and relatively low pressure drop. On the other hand, the radial reactor fits very well with the filamentous structure of the fibers. It is prepared by rolling the fibers around a perforated pipe in an ordered manner, as on a bobbin; the reactor mixture flows from the holes of the central pipe, passes through the fiber layer where the enzymatic reaction takes place, and exits. Packing degrees up to 0.35 kg of dry fibers per liter of reactor volume are reached, making it possible to increase the efficiency of the fiber-entrapped glucose isomerase and to decrease the residence time. According to Snamprogetti, the radial reactors have the great advantage that they can be easily prepared with standard equipment of the textile industry, such as winding machines. The total productivity during two half-lives of the immobilized enzyme in the reactors is around 5-6 tons of HFCS (dry mass) per kilogram of fibers (Marconi & Morisi, 1979).

433. Figure 5 shows a flow sheet for the Hungarian industrial process with a daily processing capacity of 400 tons of native corn, or 120,000 tons/year (Hollo et al., 1984). The plant based on the wet milling of maize was built in 1983 in Szabadegyhaza, Hungary, and has been operating since then. The first step of the process is the continuous enzymatic liquefaction of the starch slurry, based on the technology of Miles/DDS-Kroer and using thermostable alpha-amylase Optitherm® from Miles Kali-Chemie. The hydrolysate leaves the dextrinizing tank with a DE value of 15. Liquefaction is followed by the enzymatic saccharification at 60°C and pH 4.5 in batch mixing tanks of 200 m3. For this step, the Aspergillus niger glucoamylase of Miles Kali Chemie (Optidex®) is used. After a saccharification time of 60 hr, the DE value of the resulting glucose solution is 96-98. The glucose solution is purified by separating the germ oil by rotary drum filtration in the presence of active carbon and by full ionization with ion exchange resins. Following the evaporation step, pH and temperature correction, and the addition of magnesium ions the solution is pumped for continuous isomerization into the columns, packed with immobilized glucose isomerase Takasweet®, Miles-USA. A recent general overview of biotechnology in Hungary is given in Dibner & Burrill (1988) and Wagner & Groo (1992).

434. In the Hungarian process two columns are fed by down-flow and operated in parallel; new columns are put into operation at the necessary time, when the activity of the immobilized glucose isomerase decreases below the control level. The reaction process proceeds at a temperature of 60-62°C, pH 7.8, and yields 2.5 tons of the syrup (dry mass) per 1 kg of the immobilized enzyme; the enzyme half-life is 40 days. The bed volume of enzyme catalyst in the column is 20 m3, the overall consumption of the immobilized enzyme equals 35 tons/year (Hollo et al., 1983). The isomerization is followed by ion exchange, filtration with active carbon and finally concentration in a 4-stage falling film evaporator to 71% DS with a standard composition of 42% fructose, 52% glucose and 6% malto-oligosaccharides.

435. Novo's plant-scale design recommendations (Hemmingsen, 1979), based on the criteria for their Sweetzyme® such as initial enzyme activity 200 IU/gm, half-life 825 hr, running time 2 half-lives, and wet bulk density 0.3 gm/cm3, are as follows:

As Novo indicates, similar data have been employed as a basis for the design and engineering of a number of fructose syrup plants in Europe, the Far East, the United States, Canada, and Latin America (Hemmingsen, 1979).

436. In 1977-82 Cetus Corporation, the U.S. R&D company headquartered in Berkeley, California, developed a patented two-step process for making 100% pure fructose from glucose syrups (U.S. Patents 4,247,641; 4,284,723), which is "ready for scale-up" (McGraw-Hill's Biotechnology Newswatch, 1982a). The project had funding of some $8 million from the Standard Oil Company of California (Socal), which, having retained its 17% corporate investment in Cetus, had left the latter with full title to all patents and know-how generated. Cetus' proprietary new technology converts the glucose to 100% fructose in two steps - one enzymatic (the oxidation of glucose to D-gluconolactone by means of immobilized glucose oxidase derived from Polyporus obtusus), the other chemical (the reduction of the gluconolactone to fructose by hydrogen with palladium as a catalyst). The hydrogen peroxide that appears as a by-product of the enzymatic glucose oxidation is utilized to transform ethylene or propylene into the corresponding oxide by the consecutive action of the two other enzymes, haloperoxidase and halohydrin epoxidase (Katchalski-Katzir & Freeman, 1982; Borglum & Marshall, 1984). A Cetus representative termed the process developed for the transformation of glucose as "the third-generation technology for fructose production" (McGraw Hill's Biotechnology Newswatch, 1982a). Cetus expected to develop the process to a full-scale facility of 250,000 to 500,000 tons annual capacity (Borglum & Marshall, 1984). Apparently the method was never scaled up since the enzyme has not become available in large quantities (Jensen & Rugh, 1987), and Cetus seems to have abandoned the concept.

4.     Economic estimates

437. The increase in high fructose syrup penetration of the world sweetener market was made possible in part by the evolution of immobilized glucose isomerase technology and improvement in enzyme characteristics and process conditions. Each improvement enhanced cost effectiveness. Shown below, for example, are the dynamics of decreasing the relative enzyme cost for producing high fructose corn syrup by Novo (Carasik & Carroll, 1983).

438. Economic estimates are available for the Kyowa Hakko process of isomerization of glucose by means of glucose isomerase immobilized on phenol-formaldehyde resin (see paragraph 428), which can be compared with data obtained from the use of native (soluble) glucose isomerase (Yokote et al., 1975). The cost estimate is based on a process in which 50 tons of glucose are converted monthly to an isomerized sugar mixture containing 45% fructose. In order to isomerize 1,000 kg of glucose, 21 kg of glucose isomerase containing fungal mycelia are required in the batch process with native enzyme. In the case of the immobilized enzyme systems 9.8 liters of immobilized enzyme can be obtained from the same amount of mycelia. Being adsorbed on Duolite A7, 9.8 liters of the immobilized enzyme can isomerize 2,822 kg of glucose within 30 days when operated at 60°C with 40% glucose solution as substrate. It is also presumed that an immobilized enzyme system is operated with two columns in series and that older columns are replaced with fresh columns every 30 days. MgSO4 and CaCl2 requirements in the immobilized glucose isomerase systems are 10% and 5% respectively of the necessary amounts of MgSO4 and CaCl2 with the native enzyme systems, thereby decreasing the volume of ion exchange resins required for deionization of the isomerized sugar mixture. Coloration of the isomerized sugar mixture in the immobilized enzyme system is much smaller, i.e. about 1/100, compared with that of the native enzyme system, thus also decreasing the cost for decolorization. Labor costs are also reduced in the immobilized enzyme system compared with the batch system. Taking into account all of these factors, the relative projected costs of these two systems are calculated (Figure 6). The cost of the Duolite A7 system is 61.5% of the cost of the batch system.

439. Purdue University presented another set of economic estimates (Emery et al., 1976) concerning an investigation of the optimum cycle time and relative costs of operation for soluble and immobilized glucose isomerase in which the latter is covalently bonded "to an expensive but good carrier", i.e., agarose activated by cyanogen bromide. The goals of the investigation were to bind the enzyme to the carrier, measure activity and stability of the preparation, and design a plant to process 450 tons/year of dry HFCS. Optimum cycle time is calculated to give results at minimum annual cost of the reaction. One-year carrier life is assumed. A batch process (soluble enzyme) is also designed for comparison purposes. For the batch process, the enzyme is the major cost, as shown below, while for the immobilized-enzyme process, the expensive carrier and reagents are the major costs. Even though these latter costs are high, the immobilized enzyme process is less costly because the expensive enzyme is used in the continuous process many times more frequently than in the batch. It is noteworthy that the actual isomerization step, including cost of the enzyme, usually accounts for less than a quarter of the total production cost, with pretreatment of feedstock and product clean up accounting for the remainder.

Glucose isomerase was bound to agarose by cyanogen bromide.
One-year carrier life was assumed.

440. According to Jensen & Rugh (1987), the cost of conversion of glucose to HFS is roughly 10-20 cents per 100 lb of HFS dry substance.

441. Economic estimates produced in Hungary in the early 1980's indicate that although the sugar yield of beets per kg was much higher than that of maize, the total monetary value of maize products should be at least 25% higher. The raw material cost of producing sugar from maize was 3.53 Hung. forint (1986 U.S. $0.28) per kg, whereas beet sugar cost 5.40 Hung. forint (1986 U.S.$0.44) per kg. As a result, the new maize processing plant was built in Szabadegyhaza, Hungary (see paragraphs 433 and 447) with a daily processing capacity of 400 tons of native corn (120,000 tons/year). The investment costs for the plant were reported as about 2.1 billion Hung. forint (1986 U.S. $169 mil.), and payback time based on the standards of the national economy was calculated as 7.3 years (Hollo et al., 1984).

5.     Scale of the processes

442. For the industrial production of high fructose syrup, dry solid capacities ranging from 30-100 tons (Okada, 1978) to 400 tons (Hemmingsen, 1979) a day are considered optimum. Owing to the rapid development of the new technology using immobilized glucose isomerase, high fructose syrup from corn starch has become a rapidly expanding business and will undoubtedly represent the most successful use of an immobilized enzyme in food chemistry. Currently, the United States accounts for most of the world's HFCS production utilizing immobilized glucose isomerase, with Japan as the second-largest producer. The growing utilization of HFCS in these two countries is shown below.

443. By the end of the 1970's, high fructose syrup filled 10% of the demand for sugar consumption in Japan, which was estimated between 2.4 and 3 million tons a year (Okada, 1978). In the U.S., usage of HFCS in 1978 reached 6 kg (dry basis) per person (12% of sugar consumption) (Antrim et al., 1979) and a steady increase in per capita usage is projected to reach 30-40% by the year 2000. Significant commercial production facilities are also in operation in Canada, Argentina, Austria, South Korea, and several European countries (see below). In 1982 HFCS accounted for 4% of the world's production of caloric sweeteners (Food Technology, 1986b). Penetration of world market of industrial sugars by HFCS had reached as much as 32% in 1981, and 36% in 1982 (Carasik & Carroll, 1983).

444. The glucose isomerase market is dominated by three manufacturers: Novo Industri (Denmark), Gist Brocades (The Netherlands), and Miles Laboratories (U.S.A.). A rough estimate of the 1983 market was 1,625 tons of glucose isomerase which in turn has created a $40 million/year market for the immobilized enzyme.

445. In 1982 approximately 2.15 million tons of 42% HFCS and 1.45 million tons of 55% HFCS was produced (Poulsen, 1985). Average productivity for commercially used immobilized glucose isomerase was approximately 2,000 kg of HFCS per kg of the enzyme. HFCS manufacturers produced an estimated 1.2 million tons of the product in 1977 and 3.7 million tons in 1980. Production increased to about 6.7 million tons in 1984. It is also noteworthy that the U.S. fructose market in 1982 amounted to about $11 billion a year and was marked by high price stability (McGraw Hill's Biotechnology Newswatch, 1982b).

446. Today, several companies are producing fructose corn syrup in the United States with an estimated production volume of over 3 million metric tons. The producers and brand names of their products are listed below. A comparison of the economics of fructose syrup production versus beet sugar production leads one to the conclusion that there is an advantage to processing corn starch into fructose syrup in the U.S.A.

447. In 1983 in Szabadegyhaza (Hungary) a new maize processing plant was put in operation with a processing capacity of 400 tons of native corn daily (120,000 tons/year). The yearly production in 1988 was: 50,000 tons of HFCS (42% fructose), 30,000 tons of protein feed, and 20 million liters of ethanol. The plant's annual consumption is 95 m3 of thermostable alpha-amylase, 90 m3 of glucoamylase, and 35 tons of immobilized glucose isomerase (Hollo et al., 1983, 1984).

448. As one more example in this area, in 1985 Cargill Incorporated began full operations at its new $100 million wet-corn mill in Eddyville, Iowa. The plant, capable of producing up to half a million tons of HFCS, employs 100 people and processes more than 20 million bushels of corn (Food Technol., 1985a).

449. Fructose syrup is produced in Japan (paragraph 442) and Europe although the present markets are somewhat limited in comparison with the United States. In Europe, where the product is known as isoglucose, and particularly in the European Economic Community, development of fructose syrup production has been slowed due to the strong political influence of the sugar industry and a subsidy on exported beet sugar (Antrim et al., 1979). It is difficult to process European corn by wet milling, and therefore a major part of the corn needed for fructose syrup production would have to be imported without the luxury of a subsidy (Antrim et al., 1979). Even so, 1976 production of fructose syrup in Europe was estimated to be about 100,000 metric tons (United Kingdom, 35,000 tons; Spain, 25,000 tons; West Germany, 21,000 tons; Belgium, 14,000 tons; the Netherlands, 10,000 tons), with a projected 1980 production of 0.75-1.0 million tons. Since then additional fructose syrup plants were constructed in France (Societe des Products du Maise and Roquette Freres), Ireland, Italy (Liquichemica under license from Miles Laboratories and Cargill), the Netherlands, United Kingdom (in Tilbury, under a joint venture between Schotten/Honig of the Netherlands and Tate & Lyle of the U.K.), Yugoslavia (under a joint venture of AIPK Poljoprivreda, Miles Laboratories, and MI-Car International, a Miles affiliate), Canada (two plants, one under joint venture of John Labatt Ltd., Toronto and Redpath Industries Ltd., a unit of Tate & Lyle, Ltd; and the other by Canada Starch Co., a unit of CPC International). Total world production of high-fructose syrup by 1984 has been estimated to be about 6.7 million metric tons (Jensen & Rugh, 1987).


450. Utilization of L-amino acids for medicine, food, and animal feed has developed rapidly in recent years. In 1982, the world production of amino acids was estimated as 500,000 tons, with a market value of about US $1.3 billion (Kieslich, 1985). Of this amount 300,000 tons were used in the food industry as monosodium glutamate, 100,000 tons of DL-methionine were used as animal feed additives, and 40,000 tons of L-lysine were used as food and fodder constituent.

451. These data are generally consistent with those for 1979, according to which the world production of amino acids amounted to 424,340 tons: 270,000 tons of L-glutamic acid, 100,000 tons of DL-methionine, and 29,000 tons of L-lysine (Hitosi, 1983). The biggest producer of methionine is Degussa AG, the Frankfurt (West Germany) chemical and precious metals company, which produced 75,000 tons/year of that essential amino acid, which is used in pharmaceuticals, medical infusion solutions, and special diet foodstuffs. Japan, primarily Ajinomoto and Kyowa Hakko, produces about two-thirds of the world volume of amino acids (Kieslich, 1985). Another significant producer of amino acids is China with a total output of 75,000 tons annually (Han Ying-Shan, 1991).

452. L-lysine, L-tryptophan and L- or DL-methionine are the most common amino acid animal feed supplements, and their world-wide demand is increasing both in the health food industry and in bioresearch. A world wide market survey of amino acid production shows that the synthetic DL-methionine and its hydroxy analog used as feed supplements are both produced commercially from petrochemicals, not via fermentation. L-lysine and L-tryptophan, on the other hand, are usually produced by fermentation (Eldib et al., 1985). The Japanese companies Ajinomoto and Bio-Kyowa were the only producers of L-lysine in the early 1970's. Since then, the South Korean firm Miwon, the French company Eurolysine and the Mexican company Fermex have joined the ranks. Currently, no American company produces L-lysine. In fact in the 1960's and early 1970's Du Pont, Monsanto, and Stauffer Chemical Co. were the only large American companies producing any amino acids. Stauffer closed its monosodium glutamate plant in 1983 because of severe competition from foreign suppliers (Eldib et al., 1985).

453. The Japanese companies Ajinomoto, Kyowa Hakko, Tanabe Seiyaku, Mitsui Toatsu, and Showa Denko produce L- or DL-tryptophan on an industrial scale. In Europe tryptophan is produced by Degussa only in semi-commercial quantities. There are no U.S. companies that produce tryptophan (Eldib et al., 1985). In 1983 two Japanese firms announced plans to produce L-lysine at plants in the U.S.A.: Bio-Kyowa, in Cape Giradeau, Missouri (projected yearly production capacity of 15,000 tons) and Ajinomoto, in Eddyville, Iowa, (projected yearly production capacity of 6,000 tons). The current U.S. demand for L-lysine is 24,000 tons/year. American producers sell lysine at the current market price of $1.40/pound ($3.11/kg), and L-tryptophan at almost $9/pound ($20/kg) (Process Biochem., 1985).

454. The above data relate primarily to microbiological processes for producing amino acids. During the last two decades, however, a new approach to the production of optically active amino acids has been developed using immobilized aminoacylase, or L-amino acid acylase. The first industrial application of immobilized enzymes occurred in 1969 when the Tanabe Seiyaku Company, Ltd. initiated its process for the production of L-methionine.

1.     Background

455. Chemical synthesis of amino acids generally produces an optically inactive racemic mixture, i.e. both the L- and D-isomers. To obtain natural L-amino acid from the chemically synthesized DL-form, optical resolution is required. Among the many optical resolution methods, the enzymatic method with microbial aminoacylase is one of the most advantageous procedures, yielding optically pure L-amino acids. The enzyme is specific for the L-form and thus a chemically synthesized acyl-DL-amino acid is hydrolyzed asymmetrically by aminoacylase to give L-amino acid and unhydrolyzed acyl-D-amino acid. Both products are separated easily by their differing charges and solubilities. The acyl-D-amino acids are then racemized by heat treatment into a racemic mixture of acyl-D- and acyl-L-amino acids, and reused for the resolution procedure. Since the substrate specificity of mold aminoacylase is broad and attacks acyl-L-amino acids with various side-chains, the enzymatic resolution of racemates can be applied to various amino acids (Chibata, 1980).

456. From 1954 to 1969, this enzymatic resolution method was employed by Tanabe Seiyaku Co. Ltd. using soluble Aspergillus oryzae aminoacylase for the industrial production of several L-amino acids. The enzyme reaction is carried out batchwise. This procedure, however, had some disadvantages inherent to a batch process using soluble enzymes in that in order to isolate L-amino acids from the reaction mixture, it is necessary to remove enzyme protein by pH and/or heat treatments (Chibata, 1978a). This results in uneconomical use of enzyme, lowered yield of L-amino acids, and increased labor. Therefore, as a result of extensive studies of the continuous optical resolution of DL-amino acids using immobilized aminoacylase, the industrial production of L-amino acids was switched to the immobilized enzyme process in 1969 to produce L-methionine.

2.     Commercial preparations of immobilized aminoacylase

457. As in the case of immobilized glucose isomerase (see paragraphs 404-417), covalently bonded enzymes are not prevalent among preparations of immobilized aminoacylase intended for industrial application. The best known preparations include aminoacylase immobilized by ionic binding to DEAE-Sephadex (developed by Tanabe Seiyaku Co.), in which the enzyme is trapped by means of fiber wet spinning (developed by Snamprogetti, see also paragraph 408) into fibers of cellulose triacetate as microdroplets of its aqueous solution. Tanabe indicates that the preparation of their immobilized enzyme is easy, the activity is "stable and high", and the regeneration of deteriorated immobilized enzyme preparations is possible. This last point is particularly important since DEAE-Sephadex is a very expensive carrier.

458. The immobilized enzyme is prepared as follows. 1,000 liters of DEAE Sephadex A-25 and 1,100-1,700 liters of aqueous solution of aminoacylase are mixed and stirred at 35°C, pH 7.0, for 10 hr, before filtration and washing with water. The yield of activity compared with that of the initial enzyme preparation is 50-60%. The half-life of the DEAE-Sephadex aminoacylase is equal to 65 days at 50°C, as compared with 48 days at 37°C for aminoacylase entrapped in polyacrylamide gel (Chibata, 1978a). According to Snamprogetti's data (Marconi & Morisi, 1979), the entrapped preparations of aminoacylase from hog kidney and microorganisms show "very good" stability under operating conditions in the course of resolving racemic mixtures of N-acetylmethionine in that the loss of enzyme activity is only 25-30% after 50 days of operation.

459. Rohm GmbH (Darmstadt, FRG) used macroporous beads made of plexiglas-like material to immobilize amino acid acylase. This carrier has a porosity of 3-4 mg/g and the enzyme is bound covalently by oxirane groups. Because of their rigid structure, the 0.1-0.3 mm diameter beads are pressure stable and show "good flow properties". The brand name of the immobilized aminoacylase is Plexazym AC, and 1 g of it is as effective as 0.4 g of the original preparation under operating conditions (Plainer & Sprossler, 1982).

460. Recently Chinese researchers reported the immobilization of aminoacylase from A. oryzae by adsorption on synthetic modified polyacrylamide. "High immobilized enzyme activity and high operational stability" is mentioned regarding the immobilized preparation (loss of activity at 30 min at 70°C is 90% for soluble enzyme, 37% for the immobilized enzyme) (Wang et al., 1992).

3.     Technological characteristics of the processes

461. The flow diagram for the continuous production of L-amino acids by Tanabe Seiyaku Co. is shown in Figure 7. Substrate, i.e., N-acetyl-DL-amino acid solution, is charged continuously into an enzyme column at a constant flow rate through a filter and heat exchanger by means of a chemical pump. The substrate is converted to L-amino acid and N-acetyl-D-amino acid while passing through the column. Enzyme column effluent is concentrated, and the L-amino acid is crystallized. Acetyl D-amino acid is racemized by heating in a racemization tank and reused for optical resolution. The system is controlled automatically and operated continuously. The reaction takes place at pH 7.0 (in the presence of 5x10-4 M cobalt salts), 50°C with a flow velocity (in a downflow operation) of 900-20,000 l/hr and a column volume of 1 m3.

462. The aminoacylase column maintained approximately 70% of its initial activity after 30 days of operation, and the half-life of the enzyme in the column is estimated to be approximately 65 days. The ratio of height to diameter does not influence the efficiency of the process. A deteriorated column is completely reactivated simply by addition of the amount of aminoacylase needed to replace the deteriorated activity. The stability of the water insoluble carrier DEAE-Sephadex is very high, and according to Tanabe Seiyaku (Chibata, 1978a) has been used for more than 8 years without significant loss of binding capacity or physical decomposition.

463. As an example, continuous production of L-methionine in a 1000-liter enzyme column is as follows (Chibata & Tosa, 1976). A solution of 0.2 M acetyl-DL-methionine (pH 7.0, 5x10-4M Co+2) is passed through the aminoacylase column at a flow rate of 2000 l/hr at 50°C. Two thousand liters of the effluent are evaporated, and the separated crude L-methionine is collected by centrifugation and recrystallized from water. The yield is 27 kg (91% of the theoretical maximum yield). The residual acetyl-D-methionine is heated at 60°C with acetic anhydride to achieve racemization. The reaction mixture is adjusted to pH 1.8 and the separated acetyl-DL-methionine is collected and reused as substrate. The yield is 36 kg (94% of the theoretical maximum yield).

464. Since 1969 Tanabe Seiyaku has operated several series of enzyme reactors industrially in the company's plants to produce L-methionine, L-valine, L-phenylalanine and other L-amino acids.

  Yield of amino acids (kg)

465. A basically similar approach has been applied by Snamprogetti (Italy) for batchwise resolution of a racemic mixture of N-acetyl-DL-tryptophan in a small pilot plant (Marconi & Morisi, 1979; Bartoli et al., 1978). Amino acid acylase entrapped in cellulose triacetate fibers was used to produce L-tryptophan and N-acetyl-D-tryptophan. In the pilot process the feed tank contains 9.85 kg acetyl-DL-tryptophan, 23.8 g CoCl2x6H2O, 1.6 kg NaOH (for pH 7.0) and 200 liters water at 45°C. This solution is recycled for 5.5 hr through the enzyme reactor at flow velocity of 350 l/hr (after which the hydrolysis is practically complete). The reactor is 77 by 19 cm with 4 kg of dry fiber, containing 0.28 kg protein/kg dry fiber. The hydrolyzed material is evaporated to 15 liters under vacuum and separated, based on solubility differences, to yield 3.9 kg of L-tryptophan (95% yield) and 15 liters of acetyl D-tryptophan solution. The latter is mixed with 3 l of acetic anhydride and held for 5 hr at 45°C to yield 4.7 kg of racemized and precipitated product (yield 96%). The racemized acetyl-D-tryptophan is recycled to the feed tank. The small pilot plant, having a capacity of about 1 kg of L-tryptophan/hr, has operated for several months. The activity of the aminoacylase fibers decreased by 20% during this period, and their productivity was about 400 kg of L-tryptophan per kilogram of fiber. The same approach was also used for the resolution of N-acetyl derivatives of DL-valine and DL-methionine (Marconi & Morisi, 1979).

466. In 1983 Degussa AG (West Germany) reported a plan to develop a new technology for the production of optically and chemically pure L-amino acids, i.e., arginine, isoleucine, threonine, proline, and tryptophan, by 1985-1988 (Harper, 1983). For the optical resolution of racemates the company uses a membrane reactor fed with the soluble catalyst aminoacylase instead of immobilized enzymes. A Degussa-produced N-acetyl racemic amino acid solution is cycled through the membrane reactor where acylase, produced by Amano Pharmaceutical Co., Ltd. in Nagoya, Japan, produces deacylated L-amino acids. The latter, of lower molecular weight as compared with N-acetyl-D- and N-acetyl-L-amino acids, pass through the reactor's membrane. Because of its high molecular weight, the enzyme is also retained in the vessel (McGraw Hill's Biotechnology Newswatch, 1982c). Separated L-amino acids are collected by ion exchange or crystallization. After racemization, the remaining solution is recycled with more of the original N-acetyl-DL-amino acid substrate. According to a Degussa representative, the continuous-production process with the membrane reactor is more efficient than other methods, including those with carrier-fixed enzymes (McGraw Hill's Biotechnology Newswatch, 1982c).

467. Another process developed by Degussa became the first industrial application of a two-enzyme system with regeneration of a cofactor and the production of some L-amino acids from cheap keto acids. The regeneration of cofactors like NAD or ATP, which dissociate from their apoenzymes, is a serious problem in enzyme engineering, since dissociating coenzymes must retain a degree of mobility in order to have access to the active centers of the two apoenzymes. The alternative, i.e. continuous replacement of the cofactor, would be very expensive. In the Degussa process NAD+ is coupled to polyethylene glycol and retained in the membrane reactor with keto acid and formate dehydrogenase (Hartmeier, 1985).

468. In a Rohm GmbH pilot process N-acetyl-DL-amino acids are converted to L-amino acids in a packed bed reactor at 37°C by Plexazym AC (see paragraph 459). N-acetyl-DL-methionine, 0.7 M at pH 8, is hydrolyzed 80% at a space velocity of 6 hr-1. The rate of hydrolysis decreases to about 30% when the space velocity is increased to 25 hr-1. A yield of 500 kg L-methionine/kg Plexazym AC is reached in a 90 day reactor run (Plainer & Sprossler, 1982).

469. Recently, Tanabe Seiyaku announced a new approach for the continuous production of L-alanine from L-aspartic acid developed by the company (Chibata, 1982). That approach used immobilized Pseudomonas dacunhae cells with high L-aspartate beta-decarboxylase activity. The decarboxylase enzyme shows high enantiomer selectivity reacting only with L-aspartic acid. Thus, L-alanine and D-aspartic acid can be produced simultaneously from D,L-aspartic acid. D-Aspartic acid is used as an important intermediate for semisynthetic penicillin. However, this continuous decarboxylase system generates problems associated with evolution of CO2 gas during the reaction. It was difficult both to maintain the plug flow of the substrate solution under normal pressure and to keep a constant pH of the reaction mixture in the reactor because of CO2 effervescence. The company therefore designed a closed column reactor that performs the enzyme reaction at an elevated pressure of 10 kg/cm2. Since liberated CO2 gas is mixed into the reaction mixture, the complete plug-flow of the substrate solution is maintained and the pH of the reaction mixture is not changed appreciably. Moreover, the efficiency of immobilized cells to produce L-alanine in the closed column system at high pressure increases by 1.5 times (from 250 to 360 mmole/l/hr) as compared with the efficiency at the normal pressure conditions; the stability of the immobilized cells is not affected by pressure elevation (Chibata, 1982). Tanabe Seiyaku established a continuous production process for L-alanine in 1982 and succeeded in making it a functioning commercial enterprise.

4.     Economic estimations

470. A comparison of the cost for production of L-amino acids by soluble and immobilized amino acid acylase in shown in Figure 8. With the immobilized enzyme, the purification procedure for product become simpler and the yield is higher than in the case of the soluble enzyme. Therefore, less substrate is required for the production of a unit amount of L-amino acid. As the immobilized aminoacylase is stable, the cost of enzyme is reduced markedly compared with that of the soluble enzyme. In the case of immobilized enzyme, the process is controlled automatically, and the labor cost is also reduced substantially. As a result the overall production cost of the immobilized enzyme process is about 60% of that of a conventional batch process using soluble enzyme (Chibata, 1978a). The cost of the enzyme in this process is only approximately 1-2 per cent of the overall operating budget, as estimated in 1984 (Chaplin, 1984).

5.     Scale of the processes

471. By 1971 the reported capacity of the Tanabe Seiyaku process (using immobilized aminoacylase) was greater than 700 kg of L-amino acids per day (Suckling, 1977). Immobilized aminoacylase columns usually produce up to 750 kg of product per day (Chaplin, 1984). Based on 1984 data, this enzyme technology can be used to produce over 50,000 tons of L-amino acids annually (Chaplin, 1984). However, it was reported in 1984 that presumably less than 250 tons of L-amino acids is produced by this technology per year, and the estimated immobilized enzyme amount is less than 1 ton/year (Poulsen, 1985). Amano (Japan) also uses immobilized aminoacylase in industry (Poulsen, 1985).

472. In 1981 Degussa installed an experimental 5 ton/year plant in Konstanz, West Germany, producing L-alanine, -methionine, -valine, -phenylalanine, and -tryptophan. In 1982 the production volume of the plant increased to 60 tons/year as a result of a new method of separating amino acids from protein hydrolysates by means of ion exchangers. The company planned to install a new 6,000 tons/year plant for L-lysine production by fermentation method at Valencia de Don Juan, Spain in 1984 (Harper, 1983; McGraw Hill's Biotechnology Newswatch, 1982c), and produced 10-15 tons per month of L-methionine, L-valine and L-phenylalanine by means of the two-enzyme system with cofactor regeneration (Hartmeier, 1985) (see also par. 467).


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