Glucose isomerase from fungi of the basidiomycetes class

Glucose isomerase is produced by cultivating fungi of the Basidiomycetes class and is used for isomerizing glucose to fructose.

BACKGROUND OF THE INVENTION 
This invention relates to enzymatic processes for converting glucose 
(dextrose) to fructose (levulose). 
Most food grade glucose is provided as an enzymatic hydrolysate of corn 
starch, i.e., the corn syrup of commerce. Glucose is generally rated as 
being 60 to 80% as sweet as sucrose and therefore sells at a 
correspondingly lower price. It has long been known to isomerize glucose 
to fructose which is even sweeter than sucrose by employing an enzyme 
having glucose isomerase activity, preferably one which has been 
immobilized by adsorption onto insoluble supports, such as crosslinking 
the enzyme with the support matrix and entrapment in a polymer matrix 
support such as diethylaminoethylcellulose or porous glass. The 
isomerization of glucose provides an equilibrium mixture typically 
containing 42-50% fructose and is referred to as high fructose corn syrup 
(HFCS). 
It is known that glucose isomerase can be isolated from a substantial 
number of microorganisms including species of Streptomyces, Bacillus, 
Arthrobacter, Nocardia, Lactobacillus, Ampullariella, and various other 
genera of microorganisms, and the enzyme has been employed in the 
commercial production of fructose from glucose by known isomerization 
techniques to provide mixtures of glucose and fructose. In the commercial 
process most commonly in present use, cornstarch is liquefied, 
enzymatically or chemically, and then treated with glucoamylase to produce 
glucose which is thereafter isomerized using glucose isomerase to mixtures 
containing both fructose and glucose. Higher concentrations of fructose 
are particularly desirable and may be obtained by the use of more active 
enzymes and/or the use of high isomerization temperatures. 
Detailed descriptions of the enzymatic conversion of glucose to fructose 
employing glucose isomerase can be found in Hamilton, et al. "Glucose 
Isomerase: A Case Study of Enzyme-Catalyzed Process Technology", 
Immobilized Enzymes in Food and Microbial Processes, Olson et al., Plenum 
Press, New York, (1974), pp. 94-106, 112, 115-137; Chen, et al., "Glucose 
Isomerase (a Review)", Process Biochem., (1980), pp. 30-35; Chen, et al., 
"Glucose Isomerase (a Review)", Process Biochem., (1980), pp. 36-41; 
Nordahl, et al., "Fructose Manufacture from Glucose by Immobilized Glucose 
Isomerase", Chem. Abstracts, vol. 82, (1975), Abs. No. 110316h; and, 
Takasaki, "Fructose Production by Glucose Isomerase", Chem. Abstracts, 
vol. 81, (1974), Abs. No. 76474a. In addition, there are numerous patents 
relating to glucose isomerization of which U.S. Pat. Nos. 3,616,221; 
3,623,953 (Reissue 28,885); 3,964,313; 3,708,397; 3,715,276; 3,788,945; 
3,909,354; 3,960,663; and, 4,308,349 are representative. 
Because of the economics involved in producing glucose isomerase, it is of 
the utmost importance to use the isomerase under conditions whereby 
maximum yields of fructose are produced using minimum quantities of 
glucose isomerase. Moreover, the conditions for isomerization should be 
such that minimal quantities of objectionable by-products are produced. 
SUMMARY OF THE INVENTION 
It has now been surprisingly discovered that fungi of the class 
Basidiomycetes produce significant quantities of glucose isomerase. In 
particular, species of Flammulina, Phellinus, Irpex, Mucronella, Stereum, 
Perenniporia, Ramaricium, Sebacina, Lentinus, Coriolus and Pannelus 
accumulate isomerase activity which is produced in the mycelia of these 
organisms. The glucose isomerase can be separated from the mycelia by the 
usual extraction techniques e.g. using sonic treatment or chemical lysing 
or alternatively the mycelia can be used directly. 
In addition to the aforementioned microorganisms, the present invention 
contemplates the use of mutants and variants thereof as well as 
genetically transformed microorganisms derived therefrom by introduction 
of the respective glucose isomerase genes into other microorganisms 
including mesophilic and preferably thermophilic microorganisms. Of 
particular importance are those genetically transformed microorganisms 
produced by introduction of mutated glucose isomerase genes into 
preferably thermophilic microorganisms. The mutated glucose isomerase 
genes selected for such use are those which provide glucose isomerase 
which is stable at elevated temperatures, especially above 90.degree. C. 
and preferably up to about 110.degree. C. Such genes can be prepared by 
the usual techniques used for mutation of microorganisms such as 
irradiation or chemical means. Thus, isolated glucose isomerase genes 
which produce glucose isomerase of moderate thermal stability, on in vitro 
mutagenesis will undergo mutation, and selection of the appropriate 
mutated genes is accomplished by reintroduction of the mutated gene into 
either the parent or other organism, preferably a thermophilic organism 
followed by replication of the organism and testing of the thermal 
stability of the resulting glucose isomerase. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The glucose which is isomerized to fructose in accordance with the present 
invention can be derived from any of the known sources for this sugar. For 
reasons of economy, the glucose will usually be derived from the 
hydrolysis of starch or cellulose employing acid and/or enzyme, preferably 
the latter, in accordance with known procedures. Glucose obtained in this 
way will typically contain minor quantities of polysaccharides, sugar 
oligomers, etc., depending upon the carbohydrate source employed and the 
hydrolysis method utilized. Cereal grains such as corn, milo, wheat, rye, 
and the like, and amylaceous roots and tubers such as potatoes, yams, 
carrots, cassava (manioc), and the like, are excellent sources of starch 
for conversion to the glucose starting material of this invention. In the 
United States, corn starch is especially preferred due to its 
comparatively low cost and ready availability. Since the production of 
food grade glucose favors the use of enzymatic starch hydrolysis 
procedures, such procedures are preferred herein. Enzyme hydrolysis 
methods are described in U.S. Pat. Nos. 4,017,363, 3,912,590; 3,922,196, 
3,922,197-201 and 4,284,722, the disclosures of which are incorporated by 
reference herein. Glucose can be isomerized to fructose in accordance with 
the present invention employing any of the known procedures, including 
contacting glucose solutions with whole cells, or passing the solutions 
through a bed containing bound, or immobilized, glucose isomerase. 
Materials and procedures used for the immobilization of enzymes are well 
known and are described in a number of publications including Wang, et 
al., Fermentation & Enzyme Technology, John Wiley & Sons, Inc., New York 
(1979), pp. 318--318 and Kirk-Othmer, Encyclopedia of Chemical Technology, 
3rd Ed., John Wiley & Sons, Inc., New York, (1980) Vol. 9, pp. 148-172, 
the disclosures of which are incorporated by reference herein. 
Particularly preferred species of the aforesaid glucose isomerase producing 
Basidiomycetes for use in the present invention include: 
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Fungus ATCC Number 
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Stereum striatum 20633 
Irpex mollis 20634 
Lentinus edodes 20635 
Perenniporia compacta 
20636 
Ramaricium albofdanescens 
20637 
Sebacina calcea 20638 
Coriolus versicolor 
20639 
Panellus stipticus 
20640 
Mucronella aggregata 
20641 
Flammulina velutipes 
20642 
Phellinus torulosus 
20632 
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Cultures of strains of these preferred species of fungi have been deposited 
with the American Type Culture Collection where these organisms were 
accorded the indicated accession numbers, i.e. ATCC number. 
The determination of other glucose-isomerase-producing fungi of the 
Basidiomycetes class can be carried out using a simple test procedure. 
Cultures of the test organism are incubated for 7 days at 25.degree. C. 
with vigorous shaking in a growth medium containing cornsteep liquor, 
magnesium sulfate, potassium phosphate, xylose and agar in shake flasks. 
These cultures are then checked for glucose isomerase activity using 
fructose determination methods, such as the acid carbazol-cysteine test, 
or xylulose determination methods, using gas chromatography or high 
pressure liquid chromatography (HPLC). 
Using these test procedures, or obvious modifications thereof, various 
species of fungi can be tested to determine the presence of the desired 
glucose isomerase activity. 
The selected fungi can be grown in accordance with known methods of 
propagation. One such method employs xylose as carbohydrate source as well 
as other ingredients usually present in such media such as cornsteep 
liquor, inorganic salts and the like. 
After growth for a sufficient period of time, e.g. to about 120 hours, the 
mycelia are harvested usually by filtration followed by washing with water 
buffered to a pH in the range of 6 to 7. The enzyme is then extracted by 
known physical or chemical procedures, such as using sonication, cell 
homogenization, lytic enzymes, surfactants, etc. The extract may be passed 
through a Sephadex column (G-25) for purification. The enzyme extract can 
now be used in the isomerization reaction. Alternatively, as previously 
mentioned, the mycelia can be used as the source of the enzyme in the 
isomerization mixture. 
In order to describe more clearly the nature of the present invention, a 
specific example will hereinafter be described. It should be understood, 
however, that this is done solely by way of example and is intended 
neither to delineate the scope of the invention nor limit the ambit of the 
appended claims.

EXAMPLE 
Preparation of glucose isomerase 
Mucronella aggregata ATCC 20641 was grown in accordance with the following 
procedure: 
A. Culture Maintenance: After incubating the cultures on malt agar slants 
for 7 days at 30.degree. C., the isolates were inoculated into shaker 
flasks or maintained under refrigeration (about 10.degree. C.). 
B. Shake Flask Propagation: Inoculation medium was made up as follows: 
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Ingredient % By Weight 
______________________________________ 
Cornsteep liquor 2.0 (d.b.) 
Xylose 5.0 
KH.sub.2 PO.sub.4 0.1 
MgSO.sub.4.7H.sub.2 O 
0.15 
Agar 0.4 
adjust pH to 6.5 
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80 Ml aliquots of the above medium were placed in 500 ml Erlenmyer flasks 
together with 20 ml of a 25% glucose solution (sterilized) for the inoclum 
fermentation. Production flasks ere similarly charged except no agar was 
added. 
First Stage (test tube) Propagation 
In a sterile hood, approximately one half of the mycelia from a slant is 
transferred with a metal loop to a test tube with 10 ml of the inoculation 
medium and about six 3-mm glass beads (sterile). The tubes are vortexed 
for 30-60 seconds or until the mycelia are dispersed. The tubes are then 
placed on a G-50 shaker at 200 rpm, 30.degree. C., for 7 days. 
Second stage (inoculum) Propagation 
After 7 days, 5 ml are transferred to a 500 ml Erlenmeyer shake flask, and 
1 ml is transferred into brain heart infusion to check sterility. These 
inoculation flasks are placed on a G-50 shaker at 200 rpm, 30.degree. C., 
for 7 days. 
Third stage (production) Propagation 
After 7 days, 5 ml are transferred from the inoculation flask to several 
fermentation flasks. The fermentation flasks are placed on the G-50 shaker 
at 200 rpm, 30.degree. C., for 9 days. 
C. Harvesting Cell Biomass 
After the 9-day incubation period, the pH of each shake flask was measured; 
the cell biomass was filtered and washed twice with pH 7.0 phosphate 
buffer. After the second filtration, the harvested cell biomass from each 
culture was weighed and frozen for bioconversion experiments. 
Cell-Free Extract Preparation 
Mycelia (4 g. wet weight) in phosphate buffer (pH 6.5) are blended in a 
Waring blender at low speed for 15 seconds. The buffered homogenate is 
then transferred to a 50 ml. glass Duran Sample Flask containing 50 g. 
(about 80% by volume) glass beads of a diameter of 0.45 to 0.5 mm. The 
chamber is then vigorously agitated with a Braun Mechanical Cell for 1 
minute while cold carbon dioxide is allowed to flow past the chamber to 
minimize heating. 
Alternatively, the low speed blended mycelia in buffer is placed in a 
plastic centrifuge tube in an ice bath and then sonicated with a Heat 
Systems Ultrasonics Cell Disrupter, Model 350, set at 50% duty cycle, 
output control at 6, continuous mode, in 5 cycles of 15 seconds on and 15 
seconds off. 
Isomerization of glucose to fructose 
The isomerization mixture containing 10% by weight glucose 
(maleate-buffered to pH 6.7), MgCl.sub.2 (10 mM), Co.sup.+2 (1 mM) and 
enzyme solution (50 mg of protein) was incubated at 60.degree. C. for 3 
hours. 
Assay of the mixture, actually aliquots thereof, showed the presence of 
fructose in addition to glucose. The assays employed were gas 
chromatography and the cysteine carbazole method (N. E. Lloyd, Cereal 
Chem., 49, #5, pp. 544-553, 1972).