a. Field of the Invention
This invention relates to a relatively pure and stable glucose isomerase enzyme concentrate in a water soluble form having a high specific activity and improved storage stability and the process for preparing the stabilized enzyme concentrate.
B. Description of the Prior Art
Transformation of dextrose to levulose, known as isomerization provides a product which in practice is a replacement for commercial invert sugar. Here one starts with dextrose, a monosaccharide sugar about 70% as sweet as sucrose or invert sugar, and converts up to one-half of it to a sugar about 140% as sweet as sucrose or invert sugar. It is this extra sweetness quality which interests the corn wet-milling industry in levulose-bearing corn syrup (commonly known today as high fructose corn syrups [HFCS]) and their manufacture.
Research on the enzymatic isomerization process began in 1955 and from this work came U.S. Pat. No. 2,950,228 to Marshall and the article by Marshall and Kooi in Science, 125, 648 (1957). From this point in time, the literature and patents literally exploded with descriptions of the work done by researchers spurred into activity by the initial work described by Marshall and Kooi.
One of the more significant papers was that of Drs. Sato and Tsumura of the Japanese Food Research Institute in their preliminary print of the Annual Meeting of the Agricultural Chemical Society of Japan held at Sapporo on July 20, 1964. In this 1964 preliminary print and its progeny appearing in Japanese Patent No. 17,640, published Oct. 7, 1966, Drs. Sato and Tsumura disclosed that several strains of microorganisms of the genus Streptomyces, when cultured on xylose, produce the enzyme, glucose isomerase, and reported also that it was effective in isomerizing glucose to fructose. In a subsequent development, another group of scientists working with Dr. Takasaki of the Japanese Fermentation Research Institute, which is an agency of the Ministry of International Trade and Industry (MITI), found that certain strains of microorganisms of the Streptomyces genus, which assimilate xylan (a polymer of xylose), can be cultivated on xylan with the production of glucose isomerase. The Takasaki et al. process and its progeny have been described in Japanese Patent No. 27,525 and in U.S. Pat. No. 3,616,221.
Manufacturers of high fructose corn syrup have found that the enzymatic isomerization of glucose should preferably be conducted on a continuous basis in order to economically produce a commercially acceptable high fructose corn syrup product. Generally, in such a process, a glucose syrup is contacted with a massive amount of a glucose isomerase enzyme preparation which has been located onto an inert substance, i.e., an immobilized glucose isomerase enzyme preparation. An essential aspect in such a process involves utilizing an immobilized glucose isomerase enzyme preparation which possesses a high concentration of isomerase activity per unit volume to thereby render the enzyme suitable in a continuous enzymatic isomerization process.
Glucose isomerase is generally produced intracellularly, i.e., the major portion of the glucose isomerase enzyme is located within and/or on the cell walls of the microorganisms from which it is produced. Some continuous isomerization processes contemplate the use of the whole cells containing the glucose isomerase. Such processes generally require special treatment of the cells to prevent extraction of the glucose isomerase from the cells so that the enzyme, in effect, is immobilized within the cells themselves, and/or special equipment to accommodate hydraulic problems encountered in driving the glucose-containing solution through the bed of cells containing the glucose isomerase enzyme. Examples of such processes which employ the whole cells containing glucose isomerase are described in U.S. Pat. Nos. 3,694,314; 3,753,858; 3,779,869; 3,817,832; 3,821,086; German OS 2,317,680; and German OS 2,345,186. These processes which employ the whole cells containing glucose isomerase suffer a number of disadvantages. These disadvantages include hydraulic problems, the formation of impurities due to the presence of non-isomerase enzymes, nucleic acids and other materials within the cell walls and longer contact times of the glucose solution with the cellular enzyme preparation (the latter of which under alkaline conditions will cause the formation of undesirable alkaline-catalyzed reaction products of fructose such as psicose and hydroxymethylfurfural [HMF]). The foregoing problems result in higher manufacturing costs due to the refining necessary to remove the impurities produced and/or the production of an inferior product.
In an effort to solve the problems encountered in using whole cells containing intracellular glucose isomerase, the prior art workers have proposed processes where the glucose isomerase is removed from the cells to place it in soluble form and thereafter immobilized on a water insoluble inert carrier. The immobilized enzyme is then suitable for use in continuously converting glucose to a high fructose corn syrup. Examples of such processes are described in U.S. Pat. Nos. 3,708,397; 3,788,945; 3,850,751; 3,868,304; Belgium Patent No. 819,859; U.S. Application No. 505,823 now U.S. Pat. No. 3,960,663, granted June 1, 1976 (Belgium Pat. No. 810,480).
While these processes which utilize cell-free immobilized glucose isomerase solve some of the problems encountered with the use of the whole cells, the processes require the step of solubilizing the glucose isomerase (i.e., removing the glucose isomerase from the whole cells). Processes for solubilizing the glucose isomerase are well-known. However, it is desirable to obtain cell-free and soluble glucose isomerase in a highly purified form which would possess a high level of isomerase activity per unit volume. The use of a highly purified and concentrated enzyme increases the efficiency with which the enzyme can be located on the insoluble carrier.
Processes for extracting and purifying glucose isomerase from cells of microorganisms in a concentrated and purified form have heretofore involved costly and complicated procedures, only suitable in small scale laboratory operations.
One such laboratory procedure for extracting and purifying solubilized glucose isomerase has been described by Danno et al. in Agr. Biol. Chem., Vol. 31, pp. 284-292 (1967). Danno et al. described a process for purifying glucose isomerase derived from Bacillus coagulans strain HN-68 by subjecting ethylene diamine tetraacetic acid (EDTA) treated cells to lysozyme to prepare a cell-free extract followed by treatment of the cell-free extract with manganese sulfate to produce a precipitate of unwanted materials. The supernatant was repeatedly treated with ammonium sulfate to precipitate a proteinaceous glucose isomerase fraction which was further purified by dialysis and chromatography on DEAE-Sephadex A-50 (a registered trademark). A 60-fold increase in concentration was reported.
Another laboratory procedure for purifying glucose isomerase has been described by Takasaki et al., (Agri. Biol. Chem., Vol. 33, pp. 1527-1534 [1969] and "Fermentation Advances", Edited by D. Perlman, pp. 561-589 Academic Press, Inc., New York, New York [1969]). Takasaki et al. disclosed that intracellular glucose isomerase derived from Streptomyces albus could be released from the cells by treatment with the cationic surface active agent, cetyl pyridinum chloride and then fractionated by sequential treatment of the cell-free extract with acetone, dialysis treatment, followed by DEAE-cellulose and DEAE-Sephadex column chromotography and further dialysis. The purified cell-free extract was further purified by dialysis in a solution containing 0.005 M magnesium sulfate and 0.0002 M CoCl.sub.2. Acetone was gradually added to the resulting solution to a concentration of 40, 45 and finally 50% to crystallize the glucose isomerase enzyme. This process is too complicated and time-consuming to be applicable on a large scale operation.
U.S. Pat. No. 3,708,397 to Sipos described a process of extracting glucose isomerase from EDTA treated cells of the microorganism Streptomyces phaeochromogenes grown on wheat bran followed by treatment with a solution containing lysozyme, toluene, magnesium chloride, buffer and water. The lysed slurry was treated with magnesium chloride and ultimately, the enzyme was precipitated by the gradual addition of cold acetone. This enzyme precipitate was dissolved in water and immobilized on DEAE-cellulose for use as a bio-catalyst to convert glucose to a high fructose corn syrup.
In U.S. Pat. No. 3,788,945 to Thompson et al., there has been described a process wherein intracellular glucose isomerase derived from Streptomyces sp ATCC 21,175 grown on xylose and xylan hydrolysate was treated with a cationic detergent (Arquad 18-50), followed by treatment with DEAE-cellulose to remove non-isomerase material. The filtrate containing glucose isomerase was then immobilized on DEAE-cellulose or a synthetic anion exchange resin to prepare the biocatalyst to continuously convert glucose to a high fructose corn syrup.
In U.S. Pat. Nos. 3,847,740 and 3,847,741 to Heady et al., there has been described a process for treating cells containing intracellular glucose isomerase with a small amount of the surfactant, Tween 80, (a registered trademark) in a buffered glycine solution. After thorough mixing, the cellular debris was removed and a filtrate containing 17.5 U/ml of isomerase activity was obtained.
U.S. Pat. No. 3,847,740 has provided a process for immobilizing purified glucose isomerase preparations on a basic magnesium carbonate carrier and U.S. Pat. Nos. 3,850,751 and 3,868,304 have provided processes for immobilizing glucose isomerase preparations on controlled porous carriers of alumina. The use of these immobilized enzyme preparations provide superior processes for producing high fructose corn syrups, but require a highly purified and concentrated glucose isomerase for their efficient and economical commercialization.
In spite of the processes previously described, there is still a need for an economically feasible process, adaptable for large scale operation, for producing a substantially soluble and purified glucose isomerase preparation have a high level of activity per unit volume and a high level of stability suitable for binding to water insoluble carriers for use in continuous processes in the manufacture of high fructose corn syrups, but also useful per se, and in other immobilization processes.