Abstract:
This disclosure relates to an improved medium for growing organisms (preferably Actinoplanes missouriensis) which produce glucose isomerase. Use of the medium results in increased yields of enzyme in shorter fermentation times. The improved medium contains molasses and particulate soy material, preferably soy flour.

Description:
BACKGROUND OF THE INVENTION 
     In U.S. Pat. Nos. 3,813,320 and 3,834,988 (of which the applicant herein is a co-inventor), there are shown various media and various microorganisms which produce glucose isomerase. U.S. Pat. No. 3,813,320 involves the use of Aerobacter levanicum in a two-stage fermentation procedure using unpurified hardwood sulfite liquor as part of the medium in the second stage where the glucose isomerizing enzyme is produced. U.S. Pat. No. 3,834,988 shows the production of glucose isomerizing enzymes from an organism of the Actinoplanes genus in a medium whose principal constituent is corn steep liquor which has had the sludge removed. In the patent and scientific literature there are disclosures of other microorganisms which produce glucose isomerizing enzymes. Some of these enzymes convert D-glucose to D-fructose through one or more chemical intermediates (e.g. D-glucose-6-phosphate) but these enzymes do not appear to be practical for industrial use at the present time. 
     More promising are enzymes known as glucose isomerase that convert D-glucose to D-fructose directly. A number of these enzymes have been prepared from microorganisms of the genera Lactobacillus, Pseudomonas, Pasteurella, Leuconostoc, Streptomyces and Aerobacter (see review by Yamaka in Biochem. Biophys. Acta 154, 670-680 [1968]). In order that a significant quantity of glucose isomerase be formed by any of the foregoing microorganisms, xylose or xylan must be present in the growth medium to induce the enzyme. Pure xylose is relatively expensive, and when xylan is used in the growth medium, the microorganism must also produce enzymes capable of hydrolyzing the xylan. 
     In order to overcome the expense of growing the microorganism in a xylose or xylan containing medium, efforts have been expended to obtain a bacterium that will produce the enzymes constitutively. Lee, Hayes and Long (U.S. Pat. No. 3,645,848) have disclosed that certain strains of microorganisms belonging to the genus Arthrobacter are capable of producing enzymes that directly convert glucose or xylose to the corresponding ketose when grown in a medium in which xylose or xylan is absent. However, relatively small amounts of isomerase are produced and the growth medium requires relatively expensive nitrogen sources, such as yeast extract and meat protein. 
     REFERENCE TO PRIOR APPLICATIONS 
     In co-pending application Ser. No. 548,537 of Shieh entitled IMPROVED MEDIUM CONTAINING MOLASSES AND CORN STEEP LIQUOR FOR PRODUCING GLUCOSE ISOMERASE filed of even date herewith are disclosed media containing beet molasses, corn steep liquor and an inorganic nitrogen salt. 
     SUMMARY OF THE INVENTION 
     One of the principal objects of the present invention is to provide a method of growing microorganisms possessing enzymes for converting aldoses to ketoses using a medium which is relatively inexpensive and which results in good yields of enzyme. 
     This invention comprises a method of producing glucose isomerizing enzyme in a medium comprising molasses and soy flour. This medium has the advantage of not requiring filtration or purification to remove sludge as do media containing corn steep liquor. 
     Use of the present media also results in higher yields of enzyme compared to conventional media. 
    
    
     DETAILED DESCRIPTION 
     The following examples illustrate the present invention. 
     EXAMPLE NO. 1 
     Production of glucose isomerase from Actinoplanes missouriensis NRRL B-3342 and the effect of beet molasses and soy flour concentrations in the culture medium. 
     A. preparation of inoculum 
     Inoculum was prepared by inoculating a culture of Actinoplanes missouriensis NRRL B-3342 into a 250 ml. Erlenmeyer flask containing 100 ml. of a sterile medium composed of the ingredients described in Table I. 
     
                       TABLE I______________________________________Ingredients       Amount (DSB)______________________________________Tryptone (Difco)  1.7%Soytone (Difco)   0.3%Glucose           0.25%K.sub.2 HPO.sub.4 0.25%______________________________________ 
    
     The medium ws sterilized after the pH of the medium was adjusted to 7.1 with hydrochloric acid. The flask was inoculated with 5% inoculum (volume by volume) and incubated for 40 to 48 hours at 33° C. on a reciprocating shaker. 
     B. production of the Enzyme 
     Production of glucose isomerase from Actinoplanes missouriensis NRRL B-3342 was carried out in 250 ml. Erlenmeyer flasks each containing 100 ml. of sterile medium. The media in a first set of six flasks were composed of 2% beet molasses, 0.15% dipotassium hydrogen phosphate, 0.05% magnesium sulfate heptahydrate. To a first flask soy flour was not added. To the other five, soy flour was added in the following quantities respectively: 0.5, 1.0, 1.5, 2.0 and 3.0 g. per flask. 
     The media in a second, third, fourth and fifth set of six flasks contained 3.0, 4.0, 5.0, and 6.0% beet molasses respectively, and the same amounts of ingredients as described in the first set of six flasks. Media containing no beet molasses, but 0.8, 3, and 7% soy flour also were prepared. 
     In preparing the foregoing described media, cane molasses or mixtures of cane and beet molasses can be used in place of beet molasses, but beet molasses is preferred. 
     The pH of the media was adjusted to 7.1 with hydrochloric acid prior to sterilization. 
     Each flask was inoculated with 8 ml. inoculum and incubated for 68 to 72 hours at 32° C. on a reciprocating shaker. 
     C. determination of glucose isomerase activity 
     1. Harvest of cells 
     Cells are harvested from a culture by centrifugation at 10,000 times gravity for 15 minutes after the culture has been pasteurized at a temperature between 60° C. to 75° C. for 20 minutes at a pH between 7 and 9. The cells are washed once with tap water and dried at room temperature. 
     2. Extraction of cell-free enzyme 
     To 0.2 g. of dry cells is added 14 ml. of sodium phosphate buffer pH 7.0 (0.0375 M). The cell suspension is sonified at 4° C. for 4 minutes in Branson Sonifier J-17A. The cell-free extract is obtained by centrifugation at 27,000 times gravity for 15 minutes and used as a source of glucose isomerase. 
     3. Assay of isomerase activity 
     To each assay tube add 3.0 ml. of 1.33 M glucose solution buffered with 0.03 M sodium phosphate (pH 7.0), 0.2 ml. of salt solution composed of 0.06 M MgSO 4  . 7 H 2  O and 0.006 M CoSO 4  . 7 H 2  O, and 0.5 ml. of 0.0375 M sodium phosphate buffer (pH 7.0). At zero time add 0.3 ml. of enzyme preparation to give a final volume of 4.0 ml. The reaction is carried out at 70° C. Aliquots are taken at 10, 15, 20 and 25 minutes and diluted in 0.02 M HCl. 
     The fructose content of the samples is assayed in an automatic analyzer by adapting the skatole-HCl method described by Pogell [J. Biol. Chem. 211:143 (1954)]. The color development is carried out at 52° C. as opposed to 37° C. Activity of glucose isomerizing enzyme is calculated from the slope and expressed in units (u). A unit of activity is defined as that quantity of enzyme which will produce 1 micromole of fructose from the glucose in 1 minute at 70° C. 
     D. presentation of the results 
     Table II shows the effect of concentration of beet molasses and soy flour on the production of glucose isomerase from Actinoplanes missouriensis. The presence of soy flour in the growth medium in addition to molasses stimulates the growth of the organism as well as the enzyme production. The optimal concentrations of molasses and soy flour for the production of enzyme are 2 to 4% and 1 to 2% respectively. However, concentrations of molasses from 1% to 6% and concentrations of soy flour from 0.4% to 7% can be used. The organism can utilize soy flour as a sole source of carbon and nitrogen for growth and production of significant amount of glucose isomerase, but a combination of molasses and soy flour gives increased yields and is preferred. 
     The terms particulate soy material, soy flour, soy meal, etc., are used interchangeably throughout this application. Soy flour is generally considered to have a smaller average particle size than soy meal, but both are useful in this invention. Soy flour is preferred. 
     In addition to soy flour and molasses, it is preferred to have a nitrate source in the medium. The amount of nitrate is equivalent to that contained in NaNO 3  in a concentration of 0.1% to about 0.6% NaNO 3 . 
     
                       TABLE II______________________________________Effect of Beet Molasses and Soy Flour Concentrations on theProduction of Glucose Isomerase by Actinoplanes Missouriensis______________________________________Compositionsof media     Solids               Enzyme    %       harvested  Sp. act.                               yields% beet   soy     (g/100 ml. (u/g.   (u/ml.molasses flour   cultures)  dry solid)                               culture)______________________________________2        0       0.21       2900     6.12        0.5     0.59       3926    23.22        1.0     0.83       4662    38.92        1.5     1.15       4550    52.52        2.0     1.23       4043    49.62        3.0     1.39       3744    52.03        0       0.30       3705    11.13        0.5     0.66       3777    24.83        1.0     0.89       4563    40.63        1.5     1.20       4342    52.03        2.0     1.29       3146    40.53        3.0     1.42       2691    38.24        0       0.21       3600    7.64        0.5     0.55       3510    19.34        1.0     1.05       4043    42.44        1.5     1.25       3887    48.54        2.0     1.14       2704    30.84        3.0     1.14       3538    40.25        0       0.32       3874    12.45        0.5     0.65       4388    28.45        1.0     0.83       4088    33.85        1.5     1.02       4232    43.05        2.0     1.10       3874    42.55        3.0     1.28       3504    45.06        0       0.34       3662    12.46        0.5     0.60       3835    23.06        1.0     0.80       3822    30.56        1.5     1.06       3738    39.66        2.0     1.02       3256    33.26        3.0     1.34       3120    41.60        0.8     0.21       6572    13.80        3.0     0.93       4320    40.00        7.0     1.46       2600    38.0______________________________________ 
    
     EXAMPLE NO. 2 
     Production of glucose isomerase from Actinoplanes missouriensis growing in a fermenter, and comparison of glucose isomerase production between cells growing in beet molasses-soy flour medium (BM-SF) and cells growing in beet-molasses corn steep liquor medium (BM-CSL). 
     A. preparation of seed culture 
     The seed culture was prepared by inoculating a culture of Actinoplanes missouriensis into 1 liter Erlenmeyer flask containing 400 ml. of sterile seed media composed of the following ingredients described below in Table III. 
     
                       TABLE III______________________________________Ingredients       Amount (DSB)______________________________________Beet molasses     1.0%Soy flour         0.4%Na.sub.2 HPO.sub.4             0.05%K.sub.2 HPO.sub.4 0.05%MgSO.sub.4 . 7 H.sub.2 O             0.05%pH                7.1______________________________________ 
    
     The organism was grown for 48 hours at 33° C. on a reciprocating shaker. 
     B. production of glucose isomerase in a fermenter 
     Glucose isomerase was produced by Actinoplanes missouriensis growing in a 14 liter fermenter containing 8 liters beet molasses-soy flour medium (BM-SF medium) or beet molasses-corn steep liquor medium (BM-CSL medium). The composition of BM-SF medium and BM-CSL medium were described in Table IV and Table V, respectively. The fermenter was made by the New Brunswick Co., New Brunswick, New Jersey. 
     
                       TABLE IV______________________________________Composition of BM-SF MediumIngredients       Amount (DSB)______________________________________Beet molasses     3.0%Soy flour         1.5%Starch            0.2%NaNO.sub.3        0.2%K.sub.2 HPO.sub.4 0.15%MgSO.sub.4 . 7 H.sub.2 O             0.05%KCl               0.025 %FeSO.sub.4 . 7 H.sub.2 O             0.001%DC Antifoam A     0.05%pH                7.1______________________________________ 
    
     
                       TABLE V______________________________________Composition of BM-CSD MediumIngredients       Amount (DSB)______________________________________Beet molasses     3.0%Corn steep liquor 1.5%Starch            0.2%NaNO.sub.3        0.2%K.sub.2 HPO.sub.4 0.15%MgSO.sub.4 . 7 H.sub.2 O             0.05%KCl               0.025%FeSO.sub.4 . 7 H.sub.2 O             0.001%DC Antifoam A     0.05%pH                7.1______________________________________ 
    
     After the corn steep liquor was mixed with the beet molasses and the pH was adjusted to 7.1, the sludge of the corn steep liquor was removed by filtration. 
     The fermenter was sterilized for 60 minutes at 121° C. Fermentation was started by adding 10% seed culture into the medium in the fermenter. Aeration and agitation were set at 4 liter per minute or 0.4 vvm and 300 rpm respectively. Temperature was set at 33° C. The enzyme activity was determined according to the methods described in Example No. 1. 
     C. presentation of results 
     Table VI shows the results concerning the yields of cells and enzyme from BM-SF medium and BM-CSL medium. The yield of enzyme is greater using BM-SF medium as compared to a BM-CSL medium. The medium of this invention is preferred over a corn steep liquor medium for Actinoplanes missouriensis because the medium does not have to be filtered to remove sludge as does a corn steep liquor medium. 
     
                                           TABLE VI__________________________________________________________________________Comparative Study of Enzyme Yield in Fermenter Using Beet Molasses-CornSteep Liquor Medium andBeet Molasses-Soy Flour Medium as a Fermentation Medium.__________________________________________________________________________   Beet Molasses-   Corn Steep Liquor Medium                          Beet Molasses-Soy Flour Medium   Solid             Enzyme                          Solid          Enzyme   Harvested         Yields                          Harvested                                   Sp. act.                                         YieldsFermentation   (g. dry wt./            Sp. act. (u/ml.                          (g. dry wt./                                   (u./g. dry                                         (u/ml.Periods (hr.)   100 ml. cult.)            (u/g. dry solid)                     culture)                          100 ml. cult.)                                   solid)                                         culture)__________________________________________________________________________24      0.63     3062     19.6 0.91     2665  24.648      0.94     3718     35.0 1.23     4400  54.060      0.90     4550     41.0 1.28     5252  67.072      0.87     4797     42.0 1.25     5398  67.5__________________________________________________________________________ 
    
     Example No. 3 
     Effect of growth temperatures on the production of glucose from Actinoplanes missouriensis. 
     Actinoplanes missouriensis was grown in a 14 liter fermenter containing 8 l. beet molasses-soy flour medium at 28° C., 33° C. or 36° C. The composition of growth medium and fermentation conditions were similar to that described in Example No. 2. Table VII shows that the preferred temperature for production of the enzyme is at around 33° C., but at 28° and 36° C. the organism is also able to produce a good quantity of enzyme. 
     
                                           TABLE VII__________________________________________________________________________Effect of Fermentation Temperature on the Yield of Glucose Isomerase    Fermentation Temperature (° C.)__________________________________________________________________________    28             33             36    Growth Periods (hr.)                   Periods (hr.)  Growth Periods (hr.)     24        36           48              60                 72                    24                       36                          48                             60                                72                                   24                                      36                                         48                                            60                                               72__________________________________________________________________________SolidHarvestedg. dry wt./100 ml. cult.   0.76      1.11         1.14            1.25               1.32                  0.91                     1.19                        1.23                           1.28                              1.25                                 0.91                                    1.26                                       1.07                                          1.10                                             1.08Sp. act.u/g. drysolids  1272      2778         3159            3452               3523                  2664                     3504                        4400                           5252                              5398                                 2600                                    3250                                       4452                                          4641                                             5252Enzyme Yieldsu/ml. cult.   14.2      30.8         36.0            43.0               46.5                  24.6                     41.6                        54.0                           67.0                              67.5                                 23.9                                    41.0                                       47.0                                          51.0                                             56.7__________________________________________________________________________