(Flowsheets of the processes and other drawings are not shown on this site. Please contact the author, if needed)



608. The production of high fructose syrups from starch using amylolytic enzymes and glucose isomerase resulted in an important impact on the sugar industry (see Vol. III). Similar syrups may be obtained from the hydrolysis of sucrose by means of the enzyme invertase. The sugar mixture produced, containing glucose, fructose and sucrose, does not show any crystallization problem when compared with concentrated sucrose solution. The use of immobilized invertase allows its application in a continuous reactor process. However, as a result of the rapid expansion of the industrial production of HFCS all over the world (Volume III), interest in the industrial application of invertase has generally fallen and currently there is only one industrial and a few pilot plants for the enzymatic hydrolysis of sucrose with immobilized invertase.

1.     Background

609. The enzymatic hydrolysis of sucrose proceeds with the formation of equimolar amounts of glucose and fructose. From an economic point of view, industrial-scale enzymatic hydrolysis must be achieved with a highly concentrated sucrose solution to avoid any dilution and concentration steps (Monsan & Combes, 1984). The hydrolysis of sucrose can also be accomplished by an acidic hydrolysis process, but this method usually suffers from significant disadvantages: color and byproduct formation with subsequent high cost of refining; a low degree of hydrolysis; and the necessity for using high-purity solutions as starting material (Marconi & Morisi, 1979). Hence, the scale-up of enzyme engineering processes for sucrose inversion to an industrial scale is still considered by some experts in the field as attractive.

2.     Commercial Preparations of Immobilized Invertase

610. Snamprogetti applied its standard method of immobilization to yeast invertase, which is entrapped in spun fibers of cellulose triacetate (Marconi & Morisi, 1979). For some fiber-entrapped preparations a half-life of 5300 days is calculated after 100 days of use (Marconi & Morisi, 1979). The efficiency of the immobilized preparations decreases upon increasing the amount of invertase activity entrapped per gram of cellulose triacetate, indicating a remarkable effect of diffusional limitations.

611. Invertase immobilized on granular bone charcoal has been used in an industrial packed-bed reactor (Moroz et al., 1973). Continuous hydrolysis of sucrose on a pilot scale was performed using polystyrene-bound invertase (Mansfeld et al., 1992).

612. Immobilization of invertase (Maxinvert 200,000, Rapidase) for pilot scale continuous sucrose hydrolysis was carried out with 8.8 kg corn grits (Eurama) in a 100-liter stirred reactor. The support particle size is 0.8 mm (specific area of 1 m2/g). The glucose units of the cellulosic fraction of the support are chemically modified by sodium metaperiodate oxidation to form aldehydes, amination by condensation with ethylene diamine, reduction of the resulting imino bonds to amino bonds with sodium cyanoborohydride, and activation of the amino groups with glutaraldehyde. The immobilized enzyme, when used in a packed bed reactor for continuous sucrose inversion, showed a half-life of 365 days at 40°C with 2 M sucrose at pH 4.5, or 90 days at 45-55°C (Combes & Monson, 1982).

3.     Technological Characteristics of Processes

613. The continuous hydrolysis of highly concentrated sucrose solutions to produce invert sugar with invertase covalently coupled with corn grits (par. 612) was scaled up to a 17.6 liter pilot reactor set in a cane sugar refinery. The reactor is a stainless-steel column 15 cm in diameter and 1 m high, packed with 6 kg of immobilized invertase. The column is temperature controlled with recirculated hot water in the jacket. Industrial sucrose solutions are preheated and pH controlled within a stirred tank before and after hydrolysis. This reactor is fed with highly concentrated sucrose solutions of 65-71% (w/w) at 50-55°C. It allows a productivity equal to 9.1 kg sucrose hydrolyzed/hour, that is 517 g/l/h, with a 72% conversion rate (Monsan & Combes, 1984). An optimal working temperature was 55°C. Inversion degrees up to 93% can be obtained with a 65% (w/w) initial sucrose concentration. The performance of the pilot-scale reactor allows the processing of highly concentrated sucrose solutions (about 1 kg/l) from a sugar cane refinery (Monsan & Combes, 1984).

614. Because of significant inhibition of invertase by the products of sucrose hydrolysis (glucose and fructose), reactor productivity decreases with decreasing flow rate in the column.

615. A small pilot plant for the enzymatic hydrolysis of sucrose was installed by Snamprogetti (Italy). Yeast invertase entrapped in spun fibers of cellulose triacetate, according to the standard Snamprogetti technology, "showed excellent stability and good efficiency", and its use for the production of invert sugar "compares very well with the traditional processes as far the economic and technical aspects are concerned" (Marconi & Morisi, 1979). A glass column, randomly packed with 90 gm of fibers containing invertase (50 mg protein), is continuously fed a 20% sucrose solution at room temperature; every two weeks the fibers are washed with water-glycerol (50:50, v/v). The invertase fibers were subjected to these conditions from 1968 to 1978 and during these 10 years showed only a 20% decrease in activity and hydrolyzed a total amount of 750 kg of sucrose. The possibility of operating at high sucrose concentrations with good activity made it possible to develop an efficient pilot process for completely hydrolyzing 50% (w/w) sucrose solutions at flow rates of about 6 l/hr/kg fibers, avoiding or reducing the risk of microbial contamination (Marconi & Morisi, 1979).

616. The only industrial application of immobilized invertase was a 44.6 m3 packed-bed reactor containing granular bone charcoal as a support for the enzyme (Monsan & Combes, 1984). This reactor, fed with a 65% (w/w) sucrose solution at 65°C, produced 11 g sucrose hydrolyzed/liter/hour for a 60% conversion rate. This productivity is 40 times lower than that obtained on a pilot scale (par. 613, 614).

617. The economic estimates and scale of processes for the enzymatic hydrolysis of sucrose other than those described in this section have not been disclosed in the available literature.


618. In the last 30 years the enzymatic hydrolysis of starch (mostly with soluble alpha-amylase and glucoamylase) to produce glucose syrups has become one of the most important of all industrial processes using enzymes. The main use of glucoamylase is to produce high-dextrose syrups of DE 95 from cornstarch where DE (dextrose equivalent) is a measure of reducing sugar on a dry basis, pure dextrose (glucose) being DE 100 and starch being DE 0 (Reilly, 1979). The production of such high-dextrose syrups has grown rapidly, mainly because they are used as starting material for making HFCS (see Volume III).

619. In producing high-dextrose syrups, cornstarch is liquefied and hydrolyzed to DE 10-15 with alpha-amylase. The resulting dextrin in concentration of 30-40% is mixed with A. niger glucoamylase at pH 4.0-4.5 and held in a stirred tank for 48-72 hr at 60°C. It is essential to reach as high a DE value (and therefore as high a glucose content) as possible. The question of glucose yield in the formation of high-dextrose syrup is of overriding importance, as many of the di- and oligosaccharides in the product have an unacceptable taste, and none is further hydrolyzed when high fructose syrup is being made (Reilly, 1979).

620. An efficient conversion of corn starch to glucose with immobilized glucoamylase obviously would have industrial merit (Pieters et al., 1992). Many studies have been made on this approach [see Reilly (1979)], but the process has not yet gone beyond a pilot plant scale. Up to the present time, commercial production of glucose from starch by an immobilized catalyst has been unsuccessful.

1.     Background

621. General consideration of advances in and lessons from enzyme engineering shows that chances for successful industrial application of an immobilized enzyme improves when: (i) the enzyme is expensive, (ii) it is unstable in native form, and (iii) product of better quality is obtained with the immobilized enzyme than with the native one. Glucoamylase has none of these qualities. It is relatively inexpensive. It is rather stable in the native form, but immobilization does not generally lead to further stabilization. The main factor in the stability of glucoamylase under operating conditions is stabilization by the substrate (maltodextrins) (Klyosov & Gerasimas, 1979; Klyosov et al., 1980); immobilized glucoamylase usually gives dextrose syrups of poorer quality, i.e. of lesser DE compared to the soluble enzyme.

622. Glucoamylase is 25% cheaper than invertase, and 30 times cheaper than lactase (Daniels, 1984). In addition, soluble glucoamylase is currently used to process batches of concentrated dextrin solutions at temperatures sufficiently high, and pH sufficiently low, to discourage microbial contamination. Therefore, any immobilized form of the enzyme would have to have truly superior qualities compared to the native enzyme to be adopted for industrial use (Reilly, 1979). Unfortunately, there are none, or at least they have not been found up to now. For example the thermal stability of the enzyme at pasteurization temperatures is poor. According to Klyosov and coworkers (1980), immobilized glucoamylase that has a half-life of about 3 to 4 weeks at 65°C could be considered a technologically feasible preparation with respect to thermostability. However, in spite of a great number of papers published on the immobilization of this enzyme, the half-life of the most stable insoluble preparation at 65°C does not exceed 5 to 7 days, and it seems that this value may be the limit of thermostability of glucoamylase (Klyosov & Gerasimas, 1979).

623. Because of the poor thermal stability of commercially available glucoamylase, it is necessary to operate immobilized reactors at about 40°C and to maintain the total system in a sterile condition. Corn sweetener producers, however, show concern about being able to maintain such conditions in large-scale production (Sweigart, 1978).

624. Lower maximum yields of glucose, which reduce the quality of high-dextrose syrups with immobilized but not with native glucoamylase, were found to be due to slow diffusion of the large substrate molecules into porous carriers, and by slow outward diffusion of the glucose product. Higher concentrations of glucose were therefore found in the pores than in the bulk solution; these higher concentrations led to a greater incidence of reversion reactions, giving, e.g., isomaltose and other disaccharides (Reilly, 1976, 1979). As a result, immobilized glucoamylase struggles to produce a 90-92 DE product when a 97 DE product is needed for commercial viability and actually is provided by native glucoamylase under industrial conditions (Klyosov et al., 1980). Thus, it is the lower operating temperature and potential lower yield that are the chief objections to the use of immobilized glucoamylase.

2.     Preparations of Immobilized Glucoamylase

625. Despite the hurdles indicated above, the immobilization of glucoamylase has been studied extensively (Schwardt, 1990). The enzyme is immobilized on a multitude of different carriers by entrapment, adsorption, ion exchange, and covalent bonding (Reilly, 1979; Schafhauser & Storey, 1992, 1993). The greatest number of attempts to immobilize glucoamylase have been by covalent attachment to organic and inorganic carriers, including low-cost magnetic support (Pieters & Bardeletti, 1992). Among these attempts, the most common method is to employ nitrous acid or glutaraldehyde to link the enzyme to amine-activated porous glass or silica.

626. The immobilized glucoamylase for use in the pilot plant at Iowa State University (see par. 629) is prepared in place by applying 1.8 kg of A. niger glucoamylase (Novo) to 14.3 kg of Corning controlled pore alkylamine silica, previously treated with glutaraldehyde. Approximately 45% of the original activity is retained upon immobilization. The silica-bound enzyme is extremely stable at 40°C, the operating temperature of the pilot plant column, and in 30% dextrin solutions, the extrapolated half-life is several years. The measured half-lives ranged from 7.5 hr at 70°C to 519 hr at 55°C (Reilly, 1976).

627. Snamprogetti (Italy) used glucoamylase (Diazyme®, Miles Laboratories) entrapped in cellulose triacetate fibers (Marconi & Morisi, 1979). Fibers contained from 90 to 360 mg of Diazyme® per gram of polymer, and the expressed activity of the immobilized enzyme ranges from 10 to 60% according to the type and the concentration of the substrate. The storage stability of the entrapped enzyme is very good, showing unaltered activity after 6 months at room temperature. With the glucoamylase fibers it is possible to operate the hydrolysis of maltose batchwise for 360 days with little loss of activity (Marconi & Morisi, 1979).

628. Recently the British Charcoals and Macdonalds (Greenock), a Scottish biotechnology company that is part of the Tate & Lyle sugar group, developed an immobilized glucoamylase. The method of the immobilization and the carrier are undisclosed. It was claimed that the immobilized enzyme completed the process in under 10 min (Genet. Engin. Biotechnol. Mon., 1984).

3.     Technological Characteristics of the Processes

629. The best known pilot scale process has a nominal capacity of 450 kg/day of glucose and operated at Iowa State University in the 1970's (Reilly, 1976). A flow sheet of that facility is shown in Fig. 1. Starch is slurried in a 1000-liter mix tank with deionized water and its pH adjusted with Ca(OH)2. The slurry is pumped to an open receiving vessel, from which it is fed to a Hydroheater jet, where it is contacted with steam. After remaining for 3 to 4 min in a coil under pressure and removing the steam by flash condensation, the cooked starch is collected in a 1000-liter holding tank where it is further cooked in the presence of alpha-amylase for prolonged periods. Following this treatment, the pH of the dextrin is adjusted with concentrated HCl and the fat, gluten, fiber and uncooked or retrograded starch are allowed to separate. The dextrin is then heated to 120°C in a coil with a residence time of 4 min and filtered. The filtered dextrin is pumped to either end of a 1.8 m column of 15 cm diameter, filled to 1.5 m depth with 16 kg of the silica-bound glucoamylase (see par. 626). The residence time, at 450 kg/day glucose production and 30% (w/w) solids content, is 9.0 min, whereas in the reaction with soluble glucoamylase it is 72 hr under similar conditions. The column is mounted on a wheeled base and connected to feed and drain lines with quick-connect fittings, so it can be removed from the system and stored in a cold room between runs (Lee et al., 1978).

630. The pilot plant runs confirmed that feeds of higher DE led to lower final DE's and glucose concentrations. In all cases higher levels of glucose and DE are obtained when free glucoamylase rather than the immobilized form is employed. Generally, the yields attained are 1-1.5 DE units or 1-1.5% glucose yields, lower than with native glucoamylase under the same conditions. No enzyme deactivation is noted during an 80-day run. When microbial contamination becomes heavy, it is controlled by washing the column with a saturated aqueous solution of chloroform (Reilly, 1976).

631. One of the principal designers of the pilot plant, Dr. Peter J. Reilly, suggests that the two main operational objections to the use of immobilized glucoamylase for glucose production - the decreased yield and possibility of microbial contamination - can be overcome, and, as an alternative, very small or pellicular particles of the enzyme carrier and biocidal solutions could be used (Reilly, 1979).

632. A pilot plant, which included a 50 liter stirred tank reactor and a column reactor, has been used in the USSR for the hydrolysis of 25 to 30% maize starch solution preliquefied by acid up to a DE of 35 to 40 (Nakhapetyan & Menyailova, 1980). The reactors contain glucoamylase immobilized on alkylaminated porous silica. Over "long time" continuous operation, glucose syrups containing 88 to 93% glucose are obtained. Besides, the hydrolysis of 35 to 40% potato starch preliquefied by acid to DE 40 is carried out in a 50-liter immobilized enzyme reactor under batch conditions. As a result, samples of glucose pellets containing 78% dry matter and 60% reducing compounds, are obtained by vacuum concentration of the prepared glucose syrups, and these pellets are used in confectionery applications (Nakhapetyan & Menyailova, 1980).

633. It was reported (Chem. Econ. Engin. Rev., 1986) that the Saccharide Operation Development Association, consisting of eight Mitsui group companies, including Mitsui Co. and Mitsui Sugar, will construct a bioreactor test plant for continuous production of glucose from starch with immobilized saccharifying enzymes to produce 2 ton/hr. Also, its Saccharide Fermentation Laboratory (Kawasaki City) is currently working on producing saccharifying enzymes with high thermal stability.

4.     Economic Estimations

634. In 1975, Corning Glass Works conducted preliminary studies on the economics of a pilot plant capable of producing 4.5 thousand tons of glucose per year. It was reported that under the conditions of (i) using seven columns in sequence, (ii) an operational temperature of 50°C, (iii) a 100-day half-life, and (iv) immobilized-enzyme cost of $11/kg, the cost of 1 kg of glucose would be between 2.2 and 3.37¢ (Kilara & Shahani, 1979).

635. In the table below, the cost of saccharification by immobilized glucoamylase is shown for a plant capable of producing 45 thousand tons of glucose per year (Weetall et al., 1975). The data are based on experimental observation of performance. The yield of dextrose is estimated as 16.8 kg solids per kg of immobilized glucoamylase per day at 40°C. Immobilized enzyme life is estimated as 13.8 days at 60°C, 51.2 days at 50°C, and 402 days (projected) at 40°C. Labor costs are estimated to be about $4.00/m3 for a plant of this size.

636. Weetall and coworkers (1975) concluded that since the estimated cost of purified glucoamylase is less than $4.00 per kg of the immobilized enzyme, most of the latter is attributable to carrier and immobilization cost. Besides, it was indicated that at the lower temperatures (40°C and below) immobilized glucoamylase (IG) costs as high as $40/kg could be competitive with the soluble batch process ($17.8/m3, with $4.4/m3 enzyme cost, $4.0/m3 labor cost and $9.4/m3 capital cost, if installed in a new plant) while at 60°C an unrealistically low IG cost of $6/kg would be necessary. It was noted that increased plant size or capacity above 45,000 ton/yr would only slightly decrease costs, primarily through labor cost savings. Estimated column capacities and plant costs for 45,000 ton/yr plants under various operating conditions are shown below.

637. Shown below is another comparison of batch (soluble glucoamylase) and continuous (immobilized glucoamylase) processes in terms of the reaction cost (Emery et al., 1976). As one can see, for the batch process the enzyme is the major cost. For the immobilized-enzyme process, the expensive carrier and reagent are the major cost. Because glucoamylase itself is very inexpensive, the batch process is much less costly than the continuous immobilized-enzyme process. It can be concluded from these data that inexpensive enzymes like glucoamylase require inexpensive carriers (Emery et al., 1976).

638. A comparison of the glucoamylase process with that for glucose isomerase (see Volume III) shows that immobilized glucose isomerase made possible the production of an important new product, i.e. HFCS, while the glucoamylase process is aimed at displacing a well-established, highly optimized soluble enzyme system for high volume production of glucose. In the latter situation, system performance and economics should be dramatically superior in order to attract serious commercial attention. At present, the potential economic benefits of immobilized glucoamylase appear marginal, except for possible capacity expansion or new product application involving low temperature processing (i.e., light beer) (Sweigart, 1978).

5.     Scale of the Processes

639. According to Poulsen (1985), presumably less than 5000 tons of glucose syrup is produced per year, with the estimated immobilized enzyme amount less than 1 ton/year. The largest producer of immobilized enzyme is Tate & Lyle, U.K. (Poulsen, 1985).


640. Some of the future directions of enzyme engineering that appear to have high potentials for industrial developments are listed below. A few comments on each of these directions is given in the following paragraphs.


     Application of multi-enzyme systems
     Enzymatic degradation of polymeric substrates (esp. cellulosic materials)
     Bioelectrocatalysis and energy transfer
     Enzymatic amplification of weak signals
     Enzyme-catalyzed synthesis of fine chemicals
     Enzymes in organic solvents, non-miscible with water; enzymes in reverse micelles
     Thermostable enzymes; stabilization of enzymes
     Recombinant DNA and cell fusion as tools for designing enzymes and enzyme systems with predetermined technological properties

641. In biological systems, one of the unique consequences of enzyme catalysis is the high degree of specificity and high rate at which sequences of chemical reactions are carried out, with each reaction catalyzed by a different enzyme. Efforts to adapt such multi-enzyme systems for in vitro use require considerably more research; but the possibilities merit considerable effort. Work needs to be continued on the stabilization of biologically produced multi-enzyme systems as found in chloroplasts and liver microsomes, for in vitro use. At present, the non-biological preparation of multi-enzyme systems is limited to random distribution of different enzymes on the surface of solid supports or in solution (see, for example, Komori et al., 1987; Bisping & Rehm, 1988). An experimental approach for the non-biological preparation of an ordered sequence of different enzymes immobilized on the surface of a solid support would constitute a major breakthrough with great potential for the in vitro application of multi-enzyme systems. One of the most advanced groups in this direction is that headed by Klaus Mosbach from Lund, Sweden, who has been working with co-immobilization of several enzymes, arranged in an oriented fashion by their crosslinking, leading to preparations with the active sites of the enzymes juxtaposed to one another (Mansson et al., 1983). Another approach of this group is to fuse two enzymes by ligating their structural genes using recombinant DNA techniques. It was demonstrated experimentally that the translated gene product was able to catalyze the sequential reaction normally carried out by the separate native enzymes (Bulow et al., 1985; Bulow & Mosbach, 1991).

642. The enzymatic degradation of insoluble polymeric substrates, such as cellulose, lignin, chitin, continue to present both fundamental and practical challenges for the controlled use of such a process on a large industrial scale. The behavior of enzymes in the degradation of these substrates is complex, especially since multi-enzyme systems are usually (if not always) involved. Adsorption of these substrates and other surface phenomena as well as the non-regular supramolecular structure of polymers influence the kinetics of their degradation. The practical demonstration of economical processes must receive major inputs from innovative research. Since cellulose represents a replenishable substrate for energy, food and other necessities of life, there is a large impetus to explore the possibilities for development of in vitro processes for the degradation of it (and other polymeric substrates) via enzymatic catalysis (see Volume I of this series).

643. Bioelectrocatalysis, biosensors and energy transfer are other directions where our present knowledge of biological energy transfer mechanisms suggests that there may be some unique in vitro systems capable of development. Studies so far have shown that some enzymes can be incorporated into functioning electrodes and can be used thereby to catalyze electrochemical and energy transfer reactions (Achtnich et al., 1992; Aizawa, 1990; Alcock et al., 1992; Arquint et al., 1992; Atanasov et al., 1992; Bonting, 1992; Brooks et al., 1992; Bubriak et al., 1992; Cattaneo et al., 1992a,b; Dempsey et al., 1992; Garguilo et al., 1993; Geckeler & Muller, 1993; see other references at the end of this volume). Various immobilization schemes and electron transfer mediators need to be explored, and the theoretical quantitative description of the rates of reaction and rates of energy transfer need to be developed for different electrochemical reactor configurations. The experimental demonstration of enzyme-catalyzed fuel cells, the enzyme-catalyzed biophotolysis of water, the microbial enzymatic conversion of carbohydrates by hydrogen, and the electrosynthesis of energy-rich products are specific examples where a great deal of fundamental as well as practical research is needed. This direction has been studied fruitfully by several groups (Berezin & Varfolomeev, 1980; Van Brunt, 1987; Ghindilis et al., 1992a,b; Kauffmann & Guilbault, 1992; Phadke, 1992; Shiono et al., 1992; Sorochinskii & Kurganov, 1992) including those who study hydrogen electrooxidation and oxygen electroreduction on electrodes with immobilized hydrogenase and laccase respectively; enzymes immobilized in semi-conductive matrices as a new type of heterogeneous bioanalytical sensors; and new ways for bioelectrosynthesis of organic compounds including complex biological substances (see references at the end of this volume).

644. The in vitro enzymatic amplification of weak signals is a concept that has its in vivo counterpart in the cascade theory of blood clotting, wherein a very small concentration of clot-promoting tissue factor causes the eventual precipitation of a large quantity of fibrin particles. Both light and ultrasonic signals are suggested as inputs for enzymatic amplifiers; the former approach has been developed to the point of semi-industrial testing of light-sensitive enzyme-containing materials in the USSR (Berezin et al., 1980; Kazanskaya et al., 1983; Berezin & Kazanskaya, 1983; see also Elcin & Akbulut, 1992).

645. The enzyme-catalyzed synthesis of fine chemicals is an area wherein alternative processes often exist and the selection of commercial processes is highly dependent on processing economics. The synthesis of complex compounds having physiologic or pharmacologic activity, including antibiotics, prostaglandins and neuropeptides, may be an appropriate avenue for enzyme-based processes; however, each product must be considered separately and in competition with alternative routes of fermentation, non-enzymatic chemical synthesis, or extraction from biological tissue or fluids. The development of economically practical processes for the synthesis or regeneration of high energy phosphate compounds, and the demonstration of ways to couple the hydrolysis of these compounds to the synthesis of organic compounds could lead to a wider scope of in vitro synthesis reactions where enzymatic catalysis might have an advantage (Laane, 1987; Chenault & Whitesides, 1987; Chenault et al., 1988; Blanco et al., 1992; Chang et al., 1992).

646. In biological systems enzymes catalyze both synthesis and degradation of lipid materials. The mechanistic role of enzymes in a lipid environment is not well understood, since many enzymes are assumed to function primarily in an aqueous environment (see, however, below). With lipids, many enzymes may work at the aqueous-lipid interface rather than within the lipid environment (Carrea, 1984; Kirchner et al., 1985; Fletcher et al., 1985; Semenov et al., 1987; Valivety et al., 1992a,b,c). This topic requires in-depth fundamental research in order to learn how to adapt lipid enzymatic systems for use in vitro. Significant applications in food processing, waste conversion and chemical synthesis can be envisioned (Wingard & Klyosov, 1980; Kilara, 1985).

647. In water, the equilibrium of many important enzymatic processes is shifted to a great extent towards starting materials. This shift relates, first of all, to the processes in which the starting materials are ionized and, therefore, strongly hydrated, and secondly to those in which water is formed as a resultant product (peptide synthesis, etc.). The unfavorable thermodynamic conditions can be improved by performing the enzymatic reaction in a biphasic "water/water-immiscible organic solvent" system (Adlercreutz & Matiasson, 1987; Adlercreutz et al., 1990; Doddema et al., 1987; Grunwald et al., 1986; Halling, 1987a,b, 1990, 1991; Inada et al., 1986; Khmelnitsky et al., 1991, 1992a; Russell & Klibanov, 1988, 1989; Semenov et al., 1987). Here the chemical equilibrium can be shifted by lowering the water content in the reaction medium or by choosing an organic solvent that can extract the product efficiently. A micellar medium represents, in fact, a variation of the biphasic water/organic solvent system. Investigations into the problems have attracted a number of research groups, particularly those working in the USA, England, France, Netherlands, Japan and USSR (Burke et al., 1992; Dekker et al., 1989; Fukui & Tanaka, 1985; Hilhorst et al., 1984; Kabakov et al., 1992; Kabanov et al., 1988; Khmelnitsky et al., 1990, 1992b; Klibanov, 1986, 1989; Martinek, 1989; Martinek & Semenov, 1981; Martinek et al., 1986, 1987, 1989; Mozhaev et al., 1991; Nicot et al., 1985; Semenov et al., 1987; Zaks & Klibanov, 1985).

648. Scale-up in the area of enzyme engineering is often hindered because many enzymes even in an optimally immobilized form are not stable enough and are rapidly denatured. Hence, the problem of enzyme stabilization or reactivation (from their denatured state) is of great importance for the future of enzyme engineering. This is also determined by the fact that in many cases the end product of an enzyme engineering process is subjected to fast microbial degradation (e.g. glucose), and sometimes, the least expensive way to decrease the chances of microbial contamination of an enzyme reactor is to run the reaction process at the pasteurization temperature (around 65°C) for a long time if the process is continuous. Very few industrial (and other) enzymes can survive at this temperature for longer than a week (and usually only for several minutes). Less vigorous conditions are not enough, as a rule, for good protection from bacterial contamination in a continuous enzymatic process.

649. On the other hand, there is at present very little nonempirical data on the stability of immobilized enzymes. It will be necessary to establish the molecular basis of stability and inactivation of individual enzymes in both soluble and immobilized forms and then, probably, of multienzyme systems. At the same time (or as a consequence), it is necessary to know how to modulate the thermostability of individual enzymes on a molecular level and how to reactivate them from their denatured state (at least for specific cases). A major area for future development will be a better understanding of the denaturation process especially as it is affected by immobilization and under the conditions to which immobilized enzymes ar subjected in reactors. Two groups are working fruitfully in this direction, one in the USA and one in the USSR (Ahern & Klibanov, 1985; Ahern et al., 1987; Belova et al., 1991; Klibanov, 1979; Levitskii et al., 1990, 1992; Melik-Nubarov et al., 1990; Mozhaev & Martinek, 1984; Mozhaev et al., 1986, 1988; Tomazic & Klibanov, 1988; Khmelnitsky et al., 1991; Volkin & Klibanov, 1992).

650. Considering problems that hamper industrial application of enzyme engineering developments, one rather unexpected problem should be mentioned: sometimes the most suitable enzyme, on the basis of microbial screening, turns out to be quite unsatisfactory upon scaling up the process. In practice the search for a suitable enzyme preparation for a subsequent industrial application by means of screening of the corresponding strains is directed as a rule to enzyme activity determination, that is to the amount of the enzyme in the system under study (a culture fluid, a cell lysate and so on). On the other hand, in enzyme engineering processes other qualities of the enzyme are often more important, like thermal stability, sensitivity to product inhibition, catalytic constant, the ability to adsorb to a solid support, etc. In other words, a highly active culture liquid does not necessarily give a technologically useful enzyme preparation (Klyosov, 1988).

651. There are at least two ways to resolve the general situation: either to look for new natural producers of the desired enzymes by means of "molecular screening", that is to estimate quantitatively specific properties of enzymes in the course of screening that are particularly important for the new technology, or to modulate molecular characteristics of the "next generation" enzymes by means of specific changes in the genetic apparatus of strains with the help of genetic or protein engineering and related methods. Both routes could lead to the discovery or design of enzymes with predetermined technological properties (see Volume II, Cellulases of the fourth generation).

652. Concerning specific changes in genetic apparatus of strains and the improvement of strains in general, it should be mentioned that enzyme engineering should gain in the near future from the recent advances of biotechnology and molecular biology: recombinant DNA techniques, gene amplification and protoplast fusion. The first two approaches deal with transfer of a gene (or genes) via a plasmid (or other genetic vehicle on a molecular level) to a host that becomes a producer of the desired biomolecule. The second approach deals specifically with the transfer into a bacterium for amplification of the portion of the genome that codes for a useful enzyme. Protoplast fusion is particularly useful for the improvement of strains, by combining a high-producing but slow-growing mutant with a poorly producing but vigorously growing strain to form a healthy overproducing recombinant strain (Bryan, 1987; Bialy, 1987; Bulow et al., 1985).


653. The analysis of the current state of enzyme engineering at the industrial level may allow a number of rather general conclusions and recommendations with emphasis on lessons to be learned. Apparently commercial success of some enzyme technologies and failure of others is connected more or less directly with the following questions:

     (i) How new is the end product that is going to be produced by means of immobilized enzymes and what is the demand for it on the market (world-wide or regional)?
     (ii) What is the cost of the enzyme that is going to be used in an immobilized form as the basis of the new technology?
     (iii) What is the cost of the carrier and the immobilization methods?
     (iv) Is the heat stability of the immobilized enzyme high enough to run a continuous process at the pasteurization temperature (e.g., around 65°C) to minimize microbial growth?
     (v) What is the efficiency of the new technology (i.e. degree of conversion to the end product) compared to conventional processes based on soluble enzymes or on use of chemicals?
     (vi) What is the quality (i.e. purity, etc.) of the end product obtained by means of the new technology compared with conventional processes?

654. The importance of the above questions is particularly glaring when considering characteristics of the glucose isomerase systems that contributed to its commercial success (see Volume IV). This success is the result of the following:

     (a) Immobilized glucose isomerase made possible the production of an important new product, HFCS; that is, technology of HFCS production has developed as a result of the commercial introduction of immobilized enzyme technology. Thus, there was no competition between this process and any conventional mature technology (chemical, enzymatic, or microbiological) since there were none.
     (b) Glucose isomerase is a rather expensive enzyme, and its multiple uses in an immobilized form are especially beneficial even if immobilization costs are taken into consideration. This contributes a great deal to the commercial success of immobilized glucose isomerase technology as compared with the application of the soluble enzyme.
     (c) High heat stability of glucose isomerase has allowed the continuous isomerization process to run at the pasteurization temperature (60-70°C), which is strongly preferred for continuous food processing to minimize microbial contamination. This problem is particularly serious in many processes of enzyme engineering.
     (d) Conversion efficiency is high when immobilized glucose isomerase is used and is not less than that with the soluble enzyme. This problem happened to be a critical one in the immobilized glucoamylase-catalyzed process, where the enzyme struggles to produce a 90-92 DE glucose syrup (see par. 624) when a 97 DE product is needed for commercial viability, and usually the latter figure can be achieved using soluble glucoamylase.

655. All these conclusions reinforce the importance of economics to the commercial success of enzyme engineering processes. Thus, the economic evaluation of any immobilized enzyme system on a laboratory or pilot scale level is a prerequisite step in the process development. This is the basis for the future success of a new enzyme technology at the industrial level.


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