Immobilized Enzyme Processes That Have Been Commercialized In The Food Industry

A. Production of High-Fructose Corn Syrup Using Immobilized Glucose Isomerase

This industry has grown from the initial commercial production in 1970 by Clinton Corn Pocessing to become the largest industrial use of an immobilized enzyme process in the world (16, 17). Most recent data indicate that the global annual production of high fructose corn syrup is 10 million metric tons dry substance(dsb) which is produced with 1500 metric tons of immobilized enzyme (J. Shetty, personal communication, 1999). Currently, the major producers of immobilized enzyme are Novo Industry A/S and Genencor. The Genencor immobilized enzyme is produced by adsorption of substantially purified enzyme on ion exchange resins, crosslinked with polyethylenei-mine and glutaraldehyde, and granulated by extrusion. This immobilized enzyme typically has a productivity of 12,000-15,000 kg dsb/kg and a half-life of 80-150 days (1920-3600 h) (J. Shetty, personal communication, 1999). The immobilized enzyme produced by Novo (Sweetzyme T) is prepared by crosslinking whole cell material from Streptomyces murinus with glutaraldehyde followed by extrusion (18). A typical bioprocess uses 1.5 m x 5 m fixed-bed bioreactors operated at 60-65°C to produce a 42% fructose iso-syrup (16, 17). More than half of this isosyrup is converted to 55% fructose using fractionation technology. Future development of a thermostable enzyme may allow direct production of a 55% fructose isosyrup by performing the isomerization at 95°C because the equilibrium is shifted in favor of fructose.

B. Production of Hydrolyzed Whey Syrups Using Immobilized p-Galactosidase

The current commercial process, Valio Hydrolysis Process, was initiated in Finland in 1980 by Valio Ltd. (19). The biocatalyst, Valio IML, consists of a mold p-galactosidase (Aspergillus oryzae) adsorbed and crosslinked on a food grade resin (19, 20; M. Harju, personal communication, 1999). The major product produced by Valio is a demineralized hydro-lyzed whey syrup containing ~ 60% solids with 72% hydrolysis of the lactose, Valio Hydroval 80 (M. Harju, personal communication, 1999). Hydrolyzed whey syrups without demineralization and with 50% demineralization are also produced. Whey from cheese-making operations is received in a liquid form (6% total solids), pasteurized, passed through fixed-bed immobilized enzyme bioreactors for lactose hydrolysis, demineralized (if desired), and concentrated to ~60% total solids. The half-life and productivity of a typical bioreactor are 20 months and 2000 kg dry matter/kg enzyme, respectively. The annual production of hydrolyzed whey syrups by this process in Finland, the United Kingdom and Norway is ~ 5000-6000 metric tons.

Bioreactors containing p-galactosidase immobilized covalently on porous silica were developed at Corning Glass Co. (21) and used commercially (6, 22) in England (Specialist Dairy Ingredient Co.), the U. S. (Nutrisearch), and France (Union Laitiere Normande). The Nutisearch plant used the hydrolyzed whey for production of baker's yeast; however, this plant is no longer in operation. The only plant still in operation is the SDI plant in the UK owned by Dairy Crest; however, they currently use Valio IML as the biocatalyst.

In addition to production of whey hydrolysates, immobilized enzyme also has been used commercially to hydrolyze lactose in milk. The yeast (Kluyveromyces lactis) enzyme, with a pH optimum near 7, was entrapped in cellulose triacetate fibers in a process developed by SNAM Progetti for production of hydrolyzed lactose milk by a plant in Milan, Italy (6, 11, 17, 23). This plant, Centrale del Latte, has a minimum capacity of 8000 L/day (23). Snow Brand Milk Products in Japan also developed an industrial process for production of hydrolyzed lactose milk using the SNAM Progetti immobilized enzyme (23). The Japanese scientists also developed a bioreactor sanitation process, involving immersion in glycerol at 10°C, that allowed them to use 60 processing cycles without an increase in microbial load.

C. Production of L-Amino Acids Using Immobilized Aminoacylase

This process for enzymatic resolution of D, L-amino acids was developed by Chibata and co-workers at Tanabe-Seiyaku Co. in Japan, and its commercialization in 1969 represented the first industrial use of an immobilized enzyme (6, 24-26). Chemically synthe sized acyl-D,L-amino acids are asymmetrically hydro-lyzed to produce the L-amino acid and acyl-D-amino acid that are easily separated by differences in solubility. The acyl-D-amino acid is then racemized and passed back through the bioreactor for resolution. Bioreactors are prepared by immobilization of the enzyme from Aspergillus oryzae by adsorption on DEAE-Sephadex. A typical half-life of a bioreactor is 65 days at 50°C. Operating as a fixed-bed bioreactor, the productivity ranges between 13 and 46 kg amino acid/L/half-life, depending on the amino acid being produced. According to Uhlig (17), the annual production in 1988 was 1000 metic tons of Phe, Met, Tyr, and Val. The overall cost of amino acid production using the immobilized enzyme was 60% of that for use of the soluble form (24).

D. Production of Specific Amino Acids

L-Aspartic acid has been produced from ammonium fumarate commercially in Japan since 1973 using immobilized L-aspartate ammonia lyase (aspartase) (EC 4.3.1.1.) prepared by entrapment of Escherichia coli cells in k-carrageenan (27). In 1988, ~ 1000 metric tons of L-aspartate was produced (17). The production of L-alanine from ammonium fumarate using two bioreactors with different immobilized cell preparations was successfully commercialized in 1983 in Japan by Tanabe Seiyaku Co. (28). The first bioreactor contained E. coli cells that were pH-treated to inactivate alanine racemase and fumarase activities and entrapped in carageenan, and the second bioreactor contained pH-treated and glutaraldehyde-crosslinked Pseudomonas dacunhae also entrapped in carrageenan. Thus, the first bioreactor provided the aspartase activity while the second provided L-aspartate p-decarbox-ylase activity. This bioprocess represents the first industrial use of sequential bioreactors.

Because of its demand as a percursor in the production of aspartame (a-L-aspartyl-L-phenylalanine methyl ester), various methods for enzymatic production of L-phenylalanine have been developed (29). Purification Engineering (Rhone Poulenc) (11,29) developed an immobilized enzyme bioprocess using phenylpyruvate as the starting material. A mutant of

E. coli having exceptionally high activities of transami-nases with the required specificities was immobilized by entrapment in a polyazetidine layer on Amberlite IRA-938 porous beads. Bioreactors typically exhibited a half-life>8 months. A 600 metric ton per year plant was constructed (29).

Industrial processes for production of aspartame using immobilized enzymes also have been developed (10, 11, 30, 31). The process developed in Japan by Toya Soda Co. (30) utilized immobilized Thermoase (thermolysin) in a biphasic system to stereospecifically couple N-protected aspartate with D, L-phenylalanine methyl ester to give «-aspartame after hydrogenolysis to remove the protecting group. The enzymatic process eliminates the need to protect and deprotect the p-car-boxyl group of aspartate to prevent formation of the bitter isomer p-aspartame and allows use of the less expensive phenylalanine methyl ester racemate. The technology has been licensed to Holland Sweetener Co., a joint venture between Tosoh Corp. and DSM. More recently, another industrial process has been reported that uses enzymes in immobilized cells to make phenylalanine from cinnamic acid and ammonia followed by direct coupling to aspartate (31).

E. Production of 5 -Ribonucleotides

A market exists for 5 '-ribonucleotides as flavor enhancers in foods and as precursors for pharmaceuticals. A process was developed in Germany by Keller and co-workers at Hoechst Aktiengesellschaft for hydrolysis of RNA using 5 '-phosphodiesterase immobilized on oxirane acrylic beads (Eupergit C from Rohm Pharma) (32). Elimination of the necessity of fractionation of RNA and DNA was accomplished because DNA could not penetrate the matrix and thus was not hydrolyzed. In a pilot plant operation using crude nucleic acid feed stocks at 60°C, the immobilized enzyme was used continuously for 500 days with no detectable loss of activity and produced 10 metric tons per year (32). Earlier work in Japan also resulted in a process for production of 5 '-ribonucleo-tides, including IMP, using a mixed bed of 5'-phospho-diesterase and 5 '-adenylate deaminase immobilized on porous silica (33).

F. Production of Isomaltulose

Isomaltulose (6-O-a-D-glucopyranosyl-D-fructofura-nose) is a natural component of honey with a sweetness one-third that of sucrose. However, it appears to be noncariogenic and it encourages the growth of Bifidobacter. Several processes for its commercial production using immobilized enzymes have been developed (34-36). Although only one enzyme, isomaltulose synthase, is required for conversion of sucrose to iso-maltulose, normally whole cells were immobilized rather than incur the cost of purification. In the pro cess developed by Cheetham (34) in England, Erwinia rhapontici cells were entrapped in calcium alginate gels and formed into pellets by extrusion. The enzyme expressed by these cells was specific for sucrose and followed an unusual intramolecular mechanism in which both the glucose and fructose moieties remain enzyme bound (34). Operating as a fixed-bed bioreac-tor, the immobilized enzyme exhibited a half-life of 1 year and had a productivity of 1500 times its own weight in crystalline isomaltulose.

In another process developed at the South Germany Sugar Co. Protaminobacter rubrum cells were flocculated and crosslinked with glutaraldehyde (34, 35). Palatinit AG, a subsidiary of South Germany Sugar Co. and Bayer AG, used this process for commercial production of isomaltulose.

Using a similar procedure, except that the cells entrapped in calcium alginate gels were crosslinked with polyethyleneimine and glutaraldehyde, Mitsui Sugar Co. in Japan commercialized a process using Serratia plymuthica or P. rubrum cells (34, 36). The productivity of a fixed-bed bioreactor containing the immobilized P. rubrum enzyme was 260 kg/L of bioca-talyst/half-life. The company initiated production with a 600 metric ton per year plant in Okayama, Japan.

G. Production of Invert Sugar (Hydrolysis of Sucrose)

Invertase immobilized by adsorption on ion exchangers (31) or covalently bound to a macroporous Plexiglas (17), Plexazyme IN (Rohm GmbH), has been commercialized in Europe for hydrolysis of sucrose obtained from sugar beets or cane sugar. The productivity of the Plexazyme IN bioreactor is 6000 kg dry weight/kg biocatalyst when producing a syrup with 90% conversion (17). Enzymatic hydrolysis of sucrose is less expensive than the HCl chemical process because of side reactions in the latter that lower the yield and decrease product quality. The annual production of invert sugar using immobilized invertase was 1000 metric tons in 1988 (17).

H. Production of Modified Triacyglycerols (Fats)

Chemically catalyzed interesterification results in random interchange of fatty acids among the three positions of the glycerol backbone. However, enzyme catalysis using a 1,3-specific lipase confines acyl migration to the 1- and 3-positions yielding a mixture of triacylglycerols not obtainable by any other means. Several commercial processes have been reported for immobilization of lipase and its use for fat modification (37-40). Workers at Unilever (37, 39) immobilized lipases from Aspergillus, Mucor and Rhizopus spp. by adsorption from acetone, methanol, or ethanol solutions on inorganic matrices such as diatomaceous earth, hydroxylapatite, or alumina and used these preparations in fixed-bed bioreactors to interesterify shea oil or the mid fraction of palm oil. The substrates were dissolved in petroleum ether or hexane and passed through the bioreactor under carefully controlled water concentrations to maximize inter- or transester-ification. Transesterification of the midfraction of palm oil with myristic acid produced a cocoa butter substitute from these less expensive starting materials (37). Scientists at Fuji Oil Co. Ltd, in Japan also have developed an immobilized lipase process for production of a cocoa butter-like fat using the palm midfraction as a starting material (41). Similarly, the triacylglycerols in shea oil or shea oleine have been modified by interes-terification using these adsorbed lipases in two-phase systems (11, 38).

Tsukishima Kikai Co. (17, 42) developed a unique countercurrent two-phase multistage bioreactor for triglyceride hydrolysis using an immobilized thermostable lipase from a Pseudomonas sp. for production of glycerin and fatty acids. Workers at AKZO Chemicals (17, 43) also produced glycerin and fatty acids using a lipase from Candida sp. immobilized by adsorption on high-density polyethylene. The productivity of the CSTR bioreactor was 1100 kg of fatty acid/kg biocatalyst resulting in a cost that was competitive with the cost of steam splitting, and the product was of superior quality (43).

Eigtved (40), at Novo Industri A/S, developed an immobilized lipase bioreactor that could be used in a solvent-free system. Lipase from Mucor miehei was adsorbed from aqueous solution onto macroporous phenolformaldehyde resin and dried to a water content of 2-20%. The support has a relatively large particle size, allowing it to be employed with melted triglycerides in a fixed-bed bioreactor without excessive pressure drop. Operating to interesterify liquid fat (olive oil) at 60°C, the bioreactor exhibited a half-life of 3100 h and a productivity of 6.5 metric tons of modified triglyceride/kg of biocatalyst.

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