Abstract:
There is provided a process for producing glucose-isomerase in high yield by culturing under aerobic conditions, the strain Streptomyces griseoflavus (NCIB 11542).

Description:
PURPOSE OF THE INVENTION 
     This invention describes a process for the preparation of glucose-isomerase from a strain of the genus Streptomyces, as well as a culture medium in which the strain is capable of producing the enzyme in continuous culture. 
     TECHNICAL-ECONOMICAL JUSTIFICATION 
     The demand for fructose has increased in the latest years, since because of its sweetening power, low calories and profitability, it can compete with the sugar commonly used, sucrose. 
     It is known that the enzyme glucose-isomerase can be used for the conversion (epimerization) of glucose (dextrose) into fructose (levulose). Different micro-organisms, capable of producing the enzyme glucose-isomerase have been described, including bacteria of the genders Bacillus, Escherichia, Aerobacter, Lactobacillus, Arthrobacter, and Pseudomonas. Other micro-organisms capable of achieving said transformation are some species of the Order Actinomycetales, such as Actinoplanes missouriensis, Streptomyces phaeochromogenes, Streptomyces albus, Streptomyces flavovirans. 
     This patent discloses the production of the enzyme glucose-isomerase by a strain of the species Streptomyces griseoflavus. 
     This species, isolated from soil samples, presents as its greatest advantage a high specific speed of growth, which implies a lower risk of contamination during its culture in the fermenter. At the same time, it permits its culture at high speeds of dilution, which leads towards a production of enzyme comparable to that of other micro-organisms of a higher specific activity. 
     The present invention also comprises a description of the species, a method for the production of the enzyme glucose-isomerase in culture media which contain, among others, soy flour, tryptone and xylans. The latter modification was introduced with the purpose of lowering the cost of the culture medium, due to the high price of xylose. 
     DESCRIPTION OF THE INVENTION 
     A strain of the species Streptomyces griseoflavus, isolated from a soil sample by the present inventors, has been studied. 
     The taxonomic characteristics of the strain Streptomyces griseoflavus A-40 (National Collection of Industrial Bacteria, Aberdeen Scotland, NCIB-11542) are as follows: 
     1. Morphologic properties: 
     a. Abundant aerial grey mycelium. 
     b. Morphology of the aerial mycelium: Chain of spores, with the shape of a spiral. Spores with a thorny surface. 
     c. Melanoyd pigment: Negative. 
     2. Culture properties: 
     Aspect after ten days at 25° C. 
     
                       TABLE I______________________________________                                 Pig-                                 ment                                 Dif-         Aerial       Mycelium   fus-Medium        Mycelium     Substrate  ible______________________________________Malt-yeast agar         Dark grey    BrownSalts-starch agar         Somewhat grey                      Pale yellow                                 --Glucose-asparagine agar         No aerial hyphes                      Pale yellowGlyceral-asparagine         No aerial hyphes                      Pale yellow                                 --agarOatmeal agar  grey         Yellowish  --                      orangeWater sucrose nitrate         No aerial    Pale yellow                                 --         hyphesAgar tyrosine Somewhat     Pale yellow                                 --         pale greyNutrient agar Pale grey    Pale yellow                                 --Agar peptone iron         No aerial    Pale yellow                                 --         hyphesAgar melanoyd pigment         No aerial    Antenned         hyphesTemperature of growth:          It grows between 25° C. and 37° C.pH of culture: It grows between 4.5 and 9Demand of oxygen:          Aerobic______________________________________ 
    
     3. Physiological and Biochemical Properties: 
     a. Hydrolyzes gelatine, casein and starch 
     b. Action in litmus milk: weak 
     c. The use of the following carbonated compounds, as the only source of carbon in Pridham and Gottlieb&#39;s medium, is positive in the cases of glucose, xylose, arabinose, ramnose, fructose, galactose, mannitol, inositol and sucrose, while neither raffinose nor salicin are used. 
     Said strain of Streptomyces griseoflavus A-40 has been made grow in aerobic conditions in a culture medium containing a source of carbon, a source of nitrogen and inorganic salts. As the source of carbon, several sugars have been added, the best results being obtained by the addition of xylose to the medium as the substance to induce the production of glucose-isomerase. In some cases, the xylose has been totally or partially substituted by xylans. Other sources of carbon that can be used are: lactic serum, milk, molasses, sucrose, lactose, maltose, glucose, fructose and glycerol. 
     The sources of Nitrogen used in the culture medium were: Triptone, soy flour, yeast extract, ammonium salt, meat extract, liquid of macerated corn, and their mixtures and combinations. 
     All the culture media present the following composition as far as mineral salts are concerned: 
     
                       TABLE II______________________________________(NH.sub.4).sub.2 SO.sub.4                0.3%    p/vMgSO.sub.4.7 H.sub.2 O                0.025%  p/vNa.sub.2 HPO.sub.4   0.2%    p/vKH.sub.2 PO.sub.4    0.02%   p/vSolution of traces   1%      v/vAntifoam             0.1%    v/v______________________________________ 
    
     The composition of the solution of traces is the following: 
     
                       TABLE III______________________________________MgSO.sub.4.7 H.sub.2 O              0.6%       p/vCaCl.sub.2.2 H.sub.2 O              1.5 × 10.sup.-3 %                         p/vFeSO.sub.4.7 H.sub.2 O              2.8 × 10.sup.-3 %                         p/vZnSO.sub.4.7 H.sub.2 O              1.4 × 10.sup.-2 %                         p/vCuSO.sub.4.5 H.sub.2 O              2.5 × 10.sup.-3 %                         p/vNaMoO.sub.4.2 H.sub.2 O              2.4 × 10.sup.-2 %                         p/vCoCl.sub.2.2 H.sub.2 O              2.4 × 10.sup.-3 %                         p/vMnSO.sub.4.H.sub.2 O              8.4 × 10.sup.-3 %                         p/v______________________________________ 
    
     The conditions of inoculum and culture for all the examples are the following: 
     A. Preparation of the inoculum: 
     The inoculum was prepared from a culture in agar xylose of Streptomyces griseoflavus A-40, by inoculating an erlenmeyer of 250 ml. containing 100 ml. of sterile medium. The composition of the medium for cuture was modified according to the type of medium used for the continuous culture. The medium was sterilized after adjusting the pH at 7.2. The erlenmeyer was inoculated with 5% (volume/volume) of inoculum, and incubated for 17-24 hours at 34° C. in an orbital incubator at 150-200 r.p.m. After 3-4 successive passes in the same medium, a cellular suspension representing 10% of the total volume of the culture medium of the fermenter was used as inoculum of same. 
     B. Continuous culture. 
     For the continuous production of glucose-isomerase of Streptomyces griseoflavus A-40, 2- and 15-liter fermenters with aeration, agitation, pH and temperature under control have been used. The inoculation was done as described in the above paragraph. The cuture media used are described in the examples. 
     The conditions of culture were: 
     Temperature: The temperature of culture was 30°-37° C. 
     pH: The pH was maintained at 7-7.5 with HCl 1 N. 
     Aeration-agitation: Both the aeration and agitation used were variable due to the fact that the transfer of Oxygen is different in the fermenters of 2 liters and 15 liters. In the case of the 2-liters fermenter, the agitation rate was 750 r.p.m. and the aeration 1 volume/volume/minute; whereas in the 15-liters fermenter the agitation rate was 400 r.p.m. and the aeration 1 v/v/min. 
     Dilution speed: 12-17 hours after inoculation of fermenters with the strain Streptomyces griseoflavus, it was put in continuous. The speed of dilution ranged between 0.05 and 0.4 h -1 , varying according to investigation needs. 
     Determination of activity Glucose-Isomerase. 
     1. Collection of cells. 
     The cells are separated from the culture medium by filtering through a cellulose acetate filter with pores 0.8μ diameter. They are twice washed with imidazole buffer (see Table IV); the volume of imidazole buffer used was double the volume of culture broth filtered. 
     
                       TABLE IV______________________________________Imidazole             5.10.sup.-2 MCoCl.sub.2.6 H.sub.2 O                 10.sup.-3 MMgSO.sub.4.7 H.sub.2 O                 10.sup.-2 M______________________________________ 
    
     The washed cells are collected and resuspended in imidazole buffer, the volume thereof used being double the volume of filtered culture broth. 
     The resuspension of cells is homogenized in a homogenizer at room temperature, for 30 seconds. 
     2. Isomerization reaction. 
     In a test tube containing 1 ml. of the cellular suspension described above, 3 ml. of isomerization medium are added. (see Table V). 
     The reaction takes place at 60° C. during one hour. After this time, the reaction of isomerization is stopped by adding 1 ml. of HCl 1 N and quickly cooling to 4° C. The glucose freed in the isomerization medium is determined in an automatic glucose analyzer. 
     The unit of activity is defined as glucose micromoles obtained per minute for a definite amount of enzyme, under the conditions described above. 
     
                       TABLE V______________________________________Fructose              45%MgSO.sub.4.7 H.sub.2 O                 5.10.sup.-3 MCoCl.sub.2.6 H.sub.2 O                 5.10.sup.-4 MImidazole             5.10.sup.-2 M______________________________________ 
    
     pH=7.8 at room temperature 
     The following examples illustrate typical aspects of the invention. It is understood, however, that they are presented with the purpose of illustrating and must not be considered as restrictive. 
    
    
     EXAMPLE I 
     This example describes in detail the results obtained in a continuous process of the strain Streptomyces griseoflavus A-40. This micro-organism was cultivated in a medium the mineral salts of which were described in the tables II and III above. The source of Carbon used was 0.7% of xylose, and as source of Nitrogen, 0.5% of soy flour and 0.01% of meat extract. 
     The concentration of soy flour was determined in prior studies, in a non-continuous process, at concentrations ranging between 0.25% and 1.5%. The best results are obtained at concentrations between 0.4 and 0.6%. 
     The soy is subjected to a pre-treatment at pH=8.6 for 20 minutes at 121° C., and further filtering. The rest of the components of the medium are added to the filtrate, with the exception of the xylose. The pH is adjusted to 7.2 and is sterilized at 121° C. for 20 minutes. 
     Once the culture medium is at room temperature, xylose previously sterilized by filtering is added. 
     The culture conditions are: 
     pH=7.2 
     Temperature=34° C. 
     Agitation=750 r.p.m. 
     The putting in continuous was done 17 hours after inoculation. 
     The results obtained are shown in TABLE VI. 
     
                       TABLE VI______________________________________Cultivation      Speed of          Units/gram oftime (hours)      dilution   pH     complete cells______________________________________17         --         7.3    27823         0.1        7.11   40641         0.1        7.16   31848         0.1        7.14   26365         0.2        7.15   25368         0.2        7.18   28770         0.2        7.18   283143        0.2        7.17   276161        0.2        7.14   208167        0.2        7.10   222185        0.2        7.16   215191         0.25      7.15   270209        0.3        7.18   269215        0.3        7.14   253______________________________________ 
    
     The specific speed of growth, μ max , determined by the &#34;wash-out&#34; technique, was 0.732 h -1  calculated according to the formula: 
     
         ln X.sub.t =ln X.sub.o +(μ.sub.max -D)t 
    
     where: 
     X o  =initial cellular concentration (gm/1) 
     X t  =final cellular concentration (gm/1) 
     D=Speed of dilution 
     t=Time in which the count goes from X o  to X t   
     EXAMPLE 2 
     In this example the strain of Streptomyces griseoflavus A-40 was cultivated in a medium the sources of Carbon of which were xylose and sucrose, and the sources of Nitrogen, tryptone and yeast extract, though these can be used as sources of Carbon. The mineral salts of the medium were the same as in example 1. 
     The culture conditions were: 
     pH: 7.2 to 7.5 
     Temperature: 34° C. 
     Agitation: 40 r.p.m. 
     Pressure: 0.05 bar. 
     Oxygen dissolved, no less than 20% 
     Table VII shows the effect of the concentration of sucrose, xylose, tryptone, yeast extract, in the production of Glucose-Isomerase by the strain Streptomyces griseoflavus A-40. The presence of tryptone in the culture medium besides the yeast extract, stimulates the growth of the micro-organism, as well as the production of enzyme. The sucrose not only inhibits the growth, but the production of enzyme as well. 
     
                       TABLE VII______________________________________                          Cell.  μ/gComposition of culture medium  Con-   of (%)                           cen-   com-                   Yeast Speed      tra- pleteSucrose  Xylose  Tryptone Ext.  Dilut.                               pH   tion cells______________________________________--      1      1        --    0.05  7    3.07 410  1    1       1        --    0.05  7    3.38 3340.5    0.5     0.5      0.25  0.18  7    5.78 2100.5    0.5     1        0.25  0.18  7    6.01 251--     1       1        0.5   0.18  7    7.40 347--     1       1.3      0.85  0.18    7.5                                    9.90 334______________________________________ 
    
     The results show an average of the values obtained along 48-72 hours of cultivation. 
     EXAMPLE 3 
     The culture medium presents as its source of Nitrogen 1.5% of tryptone and 1% of yeast extract. As a source of Carbon, different concentrations of xylose and xylans. The mineral salts of this culture medium are the ones described above, (Tables II and III). 
     The culture conditions were: 
     pH=7.2 
     Temperature=34° C. 
     Agitation=750 r.p.m. 
     17 hours after inoculating the fermenter, the culture was put in continuous. 
     
                       TABLE VIII______________________________________Composition of medium        Dilution          μ/100 cultureXylose   Xylans  Speed      pH   medium______________________________________0.5      0.5     0.1        7.2  1070.2      0.8     0.1        7.2  1860.2      0.8     0.2        7.2  1100.1      0.9     0.1        7.2  177--       1       0.1        7.2  128______________________________________