Abstract:
The production of a heat stable glucose isomerase from a microorganism belonging to the genus Ampullariella and a method for using the isomerase to convert glucose to fructose.

Description:
BACKGROUND OF THE INVENTION 
     Glucose and fructose are the monosaccharides which are liberated upon the hydrolysis of sucrose. Glucose, also known as dextrose, is an aldohexose which is produced commercially from starch, especially corn starch, by the food processing industry. Fructose, also called levulose, is a ketohexose which is not present in significant concentrations in starch-derived syrups and sugars. However, because fructose is sweeter than glucose, it is desirable to convert at least part of the glucose in the starch-derived syrup or sugar to fructose to give the product the taste characteristics of cane or beet sugar. The isomerization of glucose to fructose may be accomplished by the use of various alkaline catalysts or by means of an enzymatic conversion. Alkaline catalysis, although widely used in the past, gives rather low yields and also produces undesirable by-products which effect the quality of the syrup. As a result of these drawbacks, enzymatic conversions using a glucose isomerase have become increasingly popular. 
     A number of glucose isomerases have been isolated from different species of microorganisms. Glucose isomerase has been produced by microorganisms from the generae Streptomyces, Actinoplanes, Bacillus, Flavobacterium, Brevibacterium, Arthrobacter, Nocardia, Micromonospora, Microbispora, and Microellobospora. Disadvantages of using an enzyme conversion of glucose to fructose reside in the cost of producing the enzyme and the sensitivity of the enzyme to environmental parameters. Isomerase preparations are usually inactivated by higher temperatures, so relatively low temperatures must be used to carry out the isomerization. Thus, longer overall reaction times are necessary than would be required if the isomerization could be carried out at a higher temperature. In addition, immobilization techniques necessary to recover the isomerase during processing are limited to those employing only mild temperature conditions. Therefore, a glucose isomerase having good heat stability is especially preferred. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to a method of producing glucose isomerase from a microorganism belonging to the genus Ampullariella which comprises growing the Ampullariella in a culture medium and recovering the glucose isomerase therefrom. The glucose isomerase produced using this method has demonstrated superior thermal stability. Samples of the isomerase derived from some strains have been shown to retain essentially all of their original activity after heating at 75° C. for a period of 24 hours. 
     The present invention is also directed to a process for converting D-glucose to D-fructose which includes the step of treating an aqueous solution containing glucose with an isomerase produced by a microorganism belonging to the genus Ampullariella thereby converting a portion of the glucose to fructose. 
     Due to the difficulty of isolating members of the genus only four species of Ampullariella had been previously described in the literature. See Jour. of the Mitchell Soc., May 1963, page 53. Known species include Ampullariella lobata, Ampullariella digitata, Ampullariella campanulata, and Ampullariella regularis. All the known species have been found to produce glucose isomerase. In addition, new organisms belonging to the genus have been isolated and found to produce glucose isomerase. These isolates although belonging to the genus Ampullariella cannot be placed into any of the known species within the genus, and therefore represent previously unknown species of Ampullariella. These novel isolates are referred to herein as Ampullariella species 3876 (ATCC 31351), Ampullariella species 3877 (ATCC 31352), Ampullariella species 3965 (ATCC 31353), and Ampullariella species 3966 (ATCC 31354). Especially preferred for the production of glucose isomerase is the novel species Ampullariella species 3876, the enzyme of which has demonstrated superior heat stability. Mutant microorganisms derived from the species disclosed herein and capable of producing glucose isomerase are considered as being within the scope of the invention. Subcultures of the previously unknown isolates have been made part of the permanent collection of the American Type Culture Collection, 12301 Parklawn Dr., Rockville, Md. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The genus Ampullariella is characterized as having rod-shaped zoospores, 0.3-0.7 by 0.8 to 1.5 μm, with a single polar flagellum or from one to two lateral flagella. The agar colonies are generally yellow-brown, yellow-grey, yellow or bright lemon yellow. The most distinctive feature of the genus are the predominantly cylindrical sporangia and the zoospores which are generally arranged end to end in longitudinal rows within the sporangia. Normally, the organisms grow saprophytically on a variety of plant and animal material, especially on hair and other keratinous matter. They are found in soils. 
     Ampullariella are aerobic and grow well on various nutrient medium which contain an assimilable source of nitrogen, an assimilable source of carbon, and essential minerals. Although for the production of glucose isomerase it is preferred that the culture medium contain xylose, the isomerase will be produced in lesser amounts in the absence of xylose. The amount of xylose required in the medium to give satisfactory enzyme production for commercially recoverable quantities are generally between about 0.1 percent and 10 percent by weight. Members of the genus generally grow best at temperatures between about 15° C. and 45° C. The glucose isomerase is produced intracellularly and is generally not excreted into the surrounding culture medium. To isolate the isomerase it is, therefore, necessary to disrupt the cell walls to release the enzyme. Disruption of the cell walls may be accomplished by techniques well known in the art, such as, for example, by ultrasonic rupture. 
     As recognized by one skilled in the art, the conversion of D-glucose to D-fructose using the glucose isomerase produced above is generally carried out in an aqueous medium. Aqueous solution useful in the isomerization of glucose, as for example corn syrup, may contain from about 5 percent to 80 percent glucose by weight, with about 30 percent to 60 percent being preferred. The final fructose concentration of the isomerized solution will depend on a number of factors well understood in the art, such as for example the amount of glucose isomerase used, the period of treatment, the temperature employed, pH of the solution, etc. In addition, as the equilibrium between the aldo and keto hexoses is approached the rate of isomerization will decrease; therefore, the process is generally terminated when the percent weight of fructose is between about 40 to 50 percent of the total combined dry weight of glucose and fructose. 
     Pursuant to the present invention, the syrup may be treated with glucose isomerase using either a batch or continuous process. In either case, it is generally preferred that the enzyme be immobilized by a water-insoluble support, usually a polymer, either by physical absorption covalent bonding, or by entrapment. In the first two methods of immobilization, a glucose isomerase/water-insoluble polymer conjugate is formed which makes retention and recycling of the enzyme possible. Conjugates formed by the covalent bonding of the isomerase to the water-insoluble polymer include conjugates resulting from the reaction between the isomerase and a water-insoluble, functionalized polymer; the incorporation of the isomerase into a growing polymer chain; or the intermolecular crosslinking of the enzyme with a multifunctional, low molecular weight reagent. Synthetic supports which have been used for covalently bonding to enzymes and which would be suitable for use with the present invention include polymers based upon acrylamide, maleic anhydride, methacrylic acid, and styrene. Well known natural supports include agarose, cellulose, dextran and starch. Commonly used adsorbents which have been used to immobilize enzymes include alumina, ion-exchange resins, calcium carbonate, carbon, cellulose, clays, collagen, collodion, conditioned metal or glass surfaces, diatomaceous earth, hydroxylapatite and the like. Various methods of forming such glucose isomerase/water-insoluble polymer conjugates are discussed in texts such as Immobilized Enzymes,  by O. Zaborky (CRC Press, Ohio) 1973. See also U.S. Pat. Nos. 3,519,538; 3,788,945; 3,933,587; and 3,933,589. 
     Another suitable method for the immobilization of the glucose isomerase is by use of a hollow fiber reactor. This type of system may be preferable to immobilization on an active support where the possibility of active chemical residues should be avoided, as for example in the production of foodstuffs. See Jour. of Food Science 42, 258 (1977). 
     It is understood that the amount of glucose isomerase employed in the isomerization of the syrup can vary considerably. Although greater or lesser amounts of glucose isomerase may be used in carrying out the method of this invention, in practice productivity ranges of between about 50 pounds and 5000 pounds of fructose/glucose mixture as dry solids per pound of immobilized glucose isomerase are preferred. As used herein, one glucose isomerase unit is defined as the amount of glucose isomerase that will convert one micromole of glucose to fructose per minute in a solution containing two moles of glucose/liter, 0.02 moles of magnesium sulfate/liter and 0.001 mole of cobalt chloride/liter at a pH of 6.85 (0.2 M sodium maleate) at 60° C. Assay for D-glucose isomerase is done at 70° C. with 5 percent D-glucose solution containing 2 mM cobalt chloride hexahydrate (CoCl 2 .6H 2  O) and 50 mM magnesium sulfate heptahydrate (MgSO 4 .7H 2  O). After appropriate dilution (0-50 μg fructose/ml) the fructose concentration produced by the isomerase is determined by the cysteine-carbazole reaction at 60° C. for 10 minutes. See J. Biol. Chem. 192, 583 (1951) and Science 125, 648 (1957). A control stopped with 7 percent perchlorate is run to determine background levels of glucose in the sample. 
     The conditions under which the isomerization may be carried out may show wide variation, although operable temperature and pH ranges generally vary from about 45° C. to about 85° C. and from about pH 6 to about 8.5, respectively; more preferably the process is carried out at a temperature of from about 50° C. to about 75° C. and at a pH of from about 6.5 to about 7.5. 
     Thermal stabilizing agents may be added to the syrup to inhibit the inactivation of the isomerase at the elevated temperature used to improve the efficiency of the process. Multivalent cations which are commonly used as thermal stabilizers and/or cofactors include cobalt, magnesium and manganese ions. Optimal quantities of these ions may be readily determined by comparing the optimal temperature for isomerization to different concentrations of the thermal stabilizer and/or cofactor followed by an assay to determine the percent retention of isomerase activity after a given time period. 
    
    
     The present invention will be further illustrated with the following examples; however, these examples should not be interpreted as a limitation upon the scope of the present invention. 
     EXAMPLE 1 
     As noted above, a new species of Ampullariella, named Ampullariella species 3876 ATCC 31351 has been discovered. This species along with strains from the known species within the genus are capable of producing glucose isomerase. Ampullariella species 3876 is characterized morphologically by sporangia with irregular contours having a shape varying from oval to bottle shaped which vary in size from about 9 to 11 microns wide to about 14 to 18 microns long. Aerial mycelia are always absent, however, in some media the sporangial mass takes up the appearance of an aerial mycelium. 
     The cultural properties of Ampullariella species 3876 ATCC 31351 on different media suggested by Shirling and Gottlieb in Int. J. Syst. Bacterial. 16, pages 313-340 (1966) and Waksman in the Actinomycetes, Vol. 2 (Williams and Wilkins Co., Baltimore 1966) pages 328-334 are shown in Table 1. The cultural characteristics were determined after 6 to 14 days of incubation at 30° C. 
     
                                           TABLE 1__________________________________________________________________________The Number Accompanying Some of the Culture MediaRefers to Those Given By Shirling and GottliebCulture Media         Cultural Characteristics__________________________________________________________________________Medium n°2 (yeast extract - malt agar)                 Abundant growth, crusty surface                 orange to 10 E6*Medium n°3 (oatmeal agar)                 Moderate growth, crusty surface                 orange lightMedium n°4 (inorganic salts starch agar)                 Abundant growth, crusty surface                 orangeMedium n°5 (glycerol asparagine agar)                 Abundant growth, smooth surface                 light orange 10 J 7* -Medium n°6 (peptone                 yeast extract iron agar) Moderate growth, smooth                 surface                 brown, deep brown pigmentMedium n°7 (tyrosine agar)                 Abundant growth, crusty surface                 orange, light brown pigmentOatmeal agar (according to Waksman)                 Abundant growth, wrinkled surface                 orange 10 I 8* yellowish pigmentHickey and Tresner&#39;s agar                 Moderate growth, wrinkled surface                 brown, light brown pigmentCzapek glucose agar   Abundant growth, crusty surface                 orange 11 E 6*Glucose asparagine agar                 Abundant growth, crusty surface                 orange 9 K 9*Nutrient agar         Moderate growth, smooth surface                 brown, light brown pigmentPotato agar           Abundant growth, wrinkled surface                 brown, brown pigmentBennett&#39;s agar        Abundant growth, wrinkled surface                 amber-brownCalcium malate agar   Very scant growth, smooth surface                 hyalineSkim milk agar        Abundant growth, wrinkled surface                 brown 15 C 12*Czapek agar           Scant growth, smooth surface                 light orangeEgg agar              Moderate growth, smooth surface                 light orangePeptone glucose agar  Moderate growth, crusty surface                 orange to reddish 4 H 10* brown                 pigment -Agar Very scant growth, hyalineLoeffler serum        Very scant growthPotato                Abundant growth, wrinkled surface                 orange to brown, brown pigmentGelatin               Scant growth, cream to light                 orangeCellulose agar        Very scant growth, thin, hyaline__________________________________________________________________________ *Index numbers refer to A Dictionary of Color, edited by A. Maerz and M. Rea Paul (McGrawHill, 1950). 
    
     Table 2 compares the ability of Ampullariella species 3876 ATCC 31351 and the known species to utilize carbon sources according to the method of Pridham et al. (Intern. J. Syst. Bact. 56, 107, 1948). Table 3 compares physiological characteristics of the various species of Ampullariella. 
     From the morphological characteristics (namely the predominantly cylindrical sporangia, the rod-shaped spores, and the arrangement of the spores end to end in longitudinal rows within the sporangium) strain ATCC 31351 is recognized as a member of the genus Ampullariella. However, the cultural characteristics, the pattern of pigmentation on different agars, the physiological characteristics (namely failure to form H 2  S, to reduce nitrate and ability to peptonize litmus milk) and the pattern of carbon compound utilization (namely the ability to grow on the complex carbohydrate raffinose) separate this strain from any of the previously known species. 
     
                       TABLE 2______________________________________MINIMAL MEDI-UM CARBONCOMPOUND    Species A. di-  A.    A. cam-                                    A. reg-UTILIZATION 3876*   gitata* lobata*                             panulata*                                    ularis*______________________________________Inositol    -       No      No    -      -               growth  growthFructose    +       No      No    -      +               growth  growthRhamnose    +       No      No    +      +               growth  growthMannitol    +       No      No    +      -               growth  growthXylose      +       No      No    +      +               growth  growthRaffinose   +       No      No    -      -               growth  growthArabinose   +       No      No    +      +               growth  growthCellulose   -       No      No    -      -               growth  growthSucrose     +       No      No    -      +               growth  growthGlucose     +       No      No    +      +               growth  growthMannose     +       No      No    +      +               growth  growthLactose     +       No      No    -      +               growth  growthSalicin     +       No      No    -      +               growth  growth______________________________________ * + represents growth - represents no growth 
    
     
                                           TABLE 3__________________________________________________________________________                 Species 3876                          A. digitata                                A. lobata                                     A. campanulata                                             A. regularis__________________________________________________________________________CULTURAL CHARACTERISTICSVegetative mycelium   orange to brown                          brown orange                                     deep orange                                             orange                 (some media)        (brown in                                     Czapek agar)Pigment               brown on brown absent                                     absent  yellow                 various agar                          (N.6-7)            (oatmeal)                          agar               agarPHYSIOLOGICAL CHARACTERISTICS*Hydrolysis of starch  +        +     +    +       +H.sub.2 S formation   -        +     +    +       +Melanin production    +        +     -    -       -Tyrosine reaction     -        -     -    -       -Casein hydrolysis     -        -     +    -       -Solubilization of calciummalate                -        -     -    -       -Nitrate reduction     -        +     +    +       +Liquefaction of gelatine                 ±     ±  -    -       -coagulation           -        +     -    -       -Litmus milkpeptinization         +        -     -    -       -Cellulose decomposition                 -        -     -    -       -__________________________________________________________________________ *+ strongly positive - strongly negative ± weakly positive 
    
     In addition to Ampullariella species 3876, three other new isolates which each represent new species within the genus have been found to produce glucose isomerase. These isolates are designated as Ampullariella species 3877 (ATCC 31352), Ampullariella species 3965 (ATCC 31353), and Ampullariella species 3966 (ATCC 31354). 
     The utilization of carbon sources for each of these above species is shown in Table 4. The physiological characteristics of the new species are shown in Table 5. 
     
                       TABLE 4______________________________________MINIMAL MEDIUM CARBON                Ampullariella speciesCOMPOUND UTILIZATION 3877*   3695*   3966*______________________________________Inositol             -       -       -Fructose             +       +       +Rhamnose             +       +       +Mannitol             +       +       +Xylose               +       +       +Raffinose            -       -       +Arabinose            +       +       +Cellulose            -       -       -Sucrose              +       +       +Glucose              +       +       +Mannose              +       +       +Lactose              -       +       +Salicin              +       +       +______________________________________ *+ Represents growth - Represents no growth 
    
     
                       TABLE 5______________________________________PHYSIOLOGICAL        Ampullariella speciesCHARACTERISTICS*     3877    3965    3966______________________________________Hydrolysis of starch +       +       +H.sub.2 S formation  +       +       +Melanin production   +       +       +Tyrosine reaction    -       -       -Casein hydrolysis    -       -       -Solubilization of calcium malate                -       -       -Nitrate reduction    -       -       -Liquefaction of gelatine                ±    ±    ±coagulation          -       -       -Litmus milkpeptonization        +       -       -Cellulose decomposition                -       -       -______________________________________ *+ represents strongly positive - represents strongly negative ± represents weakly positive 
    
     EXAMPLE 2 
     A nutrient broth generally suitable for the maintenance of Ampullariella cultures is as follows: 
     
         ______________________________________3 grams            beef extract5 grams            peptone1 liter            distilled water______________________________________ 
    
     For the production of glucose isomerase, an Emerson xylose growth media was found to be satisfactory. The ingredients are as follows: 
     
         ______________________________________4.0 grams           beef extract4.0 grams           peptone2.5 grams           sodium chloride1.0 gram            yeast extract10.0 grams          xylose1.0 liter           distilled water______________________________________ 
    
     EXAMPLE 3 
     Two ml of a nutrient broth culture of Ampullariella species 3966 was used as inoculum in a 500 ml baffled flask containing 100 ml of Emerson xylose growth medium. The culture was incubated three days at 30° C. on a shaker. The cells were harvested by centrifugation at 16,300×g for 3 minutes and washed with 100 ml of 0.013 M phosphate buffer, pH 7.0. The cells were centrifuged a second time at 27,000×g for 30 minutes. The button of wet cells was resuspended in 0.05 M Tris-HCl buffer, pH 7.4 at a concentration of 10 ml buffer per gram of wet cells. The cells were ruptured at a temperature of 4° C. using a Branson Sonifier® Cell Disruptor producing two, three-minute pulses with a three-minute rest between pulses. Cellular debris was removed by centrifugation at 27,000×g for 30 minutes. A sample of the crude extract was assayed for glucose isomerase activity by treating a 5% glucose solution with the extract and testing for the presence of fructose using the method described hereinbefore. The extract was found to contain 0.69 glucose isomerase units per milliliter of extract. 
     A second sample of the extract was heated for 24 hours at 75° C. Assay of the heated sample showed 0.54 glucose isomerase units per milliliter indicating the extract retained about 78 percent of the original glucose isomerase activity of the unheated control. 
     EXAMPLE 4 
     Ampullariella species 3876 (130 wet grams) grown in a 14 liter New Brunswick fermenter with Emerson xylose medium was harvested by centrifugation. The cells were washed with distilled deionized water and centrifuged at 4° C., 27,000×g for 30 minutes. The pellet of wet cells was resuspended in 0.025 m Tris-HCl buffer, pH 7.4 containing 0.5 mM cobalt chloride at a concentration of 5 ml buffer per gram of wet cells. The cells were ruptured at 4° C. using a Branson Sonifier® Cell Disruptor at 50 watts to produce five three-minute pulses with a three-minute rest between pulses. Cellular debris was removed by centrifugation at 27,000×g for 30 minutes. A sample of the crude extract was assayed for glucose isomerase activity and protein concentration. The sample was found to contain 1.59 glucose isomerase units per milliliter of extract, and 5.6 mg protein per milliliter of extract (0.283 glucose isomerase units per milligram protein). 
     Crude glucose isomerase was concentrated two-fold by rotary evaporation at 48° C. and purified two-fold (to 0.580 glucose isomerase units per milligram protein) by heating at 70° C. for one hour at 150 rpm; centrifugation at 78,480×g, 4° C. for 30 minutes removed precipitated protein from the isomerase-containing supernatant. Exhaustive dialysis of the isomerase against 0.05 M Tris-HCl, pH 7.4 was done to remove cobalt from the enzyme. 
     Assay of the final enzyme product using the method described in Example 3 above, showed 2.67 glucose isomerase units per milliliter. Samples of the enzyme which were heated at 75° C. for 6 and 24 hours retained 112 percent and 104 percent, respectively, of their original activity as compared to a glucose isomerase unheated control.