i have discovered that microorganisms which produce glucose isomerase are capable of being grown in large numbers on a medium of which the principal ingredients are beet molasses , corn steep liquor , and an inorganic nitrogen salt . the preferred microorganism is of the genus actinoplanes , but other glucose isomerizing organisms of the genera lactobacillus , pseudomonas , pasteurella , leuconostoc , streptomyces , aerobacter , arthrobacter , bacillus , nocardia , micromonospora , microbiospora , microellobospora , thermoactinomyces , thermopolyspora , thermomonospora and pseudonocardia can be grown on this medium . a seed inoculum of actinoplanes missouriensis nrrl b - 3342 is grown up by shaking at 28 ° c . for 24 hours in a 1 - liter erlenmeyer flask containing 400 ml . of inoculum media which consists of 0 . 25 % glucose , 0 . 25 % dipotassium phosphate , 0 . 8 % tryptone ( difco ), 0 . 2 % soytone ( difco ) and 1 % casein hydrolyzate ( amber ). the seed inoculum also can be grown according to the procedure set forth in u . s . pat . no . 3 , 834 , 988 . the seed inoculum is added to a growth medium which contains molasses , corn steep liquor from which the sludge has been removed , an inorganic nitrogen salt , other salts , water , and an anti - foaming agent . the medium is at a ph of about 7 . the molasses can be cane molasses , beet molasses , or mixtures of cane and beet molasses , but beet molasses is preferred . the medium can contain 2 % to 8 % beet molasses , about 0 . 6 % to 4 % corn steep liquor , and 0 . 1 % to 0 . 6 % sodium nitrate . the remainder of the medium is water . a specific preferred medium composition is composed of 3 % beet molasses , 1 % corn steep liquor , 0 . 2 % nano 3 , 0 . 025 % mgso 4 . sup .. 7 h 2 o , 0 . 1 % k 2 hpo 4 , 0 . 025 % kcl , 0 . 001 % feso 4 . sup .. 7 h 2 o , and 0 . 06 % dow corning antifoam a ( 10 % solid ). the sludge of corn steep liquor is first removed by filtration or centrifugation after mixing with molasses and adjusting the ph to 7 . 1 with naoh . the corn steep liquor can be filtered alone . other solids are added to compensate for variations in the compositions of the products which are the main constituents of the medium . the fermenter is sterilized for 60 minutes at 121 ° c . growth is started by adding 4 % inoculum to the medium in the fermentation . aeration and agitation are set at 8 liters per minute or 0 . 8 vvm and 200 - 300 rpm , respectively . temperature is set at 28 ° c . the cells are collected by centrifuging 30 ml . of the culture at 10 , 000 x g for 10 minutes . the cells are washed once with tap water , and sonified at 4 ° c . for 4 minutes in a branson sonifier j - 17a after suspending in 14 ml . of 0 . 12 m phosphate buffer ( ph 7 . 0 ). the cell - free extract is obtained by centrifugation at 27 , 000 x g for 15 minutes and used as a source of glucose isomerizing enzyme . the activity of glucose isomerase is determined as follows : to 3 . 0 ml . of assay solutions composed of 2 . 0 m glucose , 0 . 004 m mgso 4 and 0 . 0004 m coso 4 is added a crude enzyme solution in 0 . 12 m phosphate buffer of ph 7 . 0 to a final volume of 4 . 0 ml . the reaction is carried out at 75 ° 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 . 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 75 ° c . following in table i is a comparison of the production of glucose isomerase cells from this medium which contains beet molasses and corn steep liquor with a medium containing only corn steep liquor . from table i , it is apparent that bm - csl medium is superior to csl medium with respect to the production of glucose isomerase by the said organism . table i______________________________________comparison of glucose isomerase production between cellsgrowing in corn steep liquor medium ( csl ) and cells growingin beet molasses - corn steep liquor medium ( bm - csl ). ______________________________________ production of enzyme ( μ / ml . culture )* growth cells grown in cells grown inperiods ( hr .) csl medium ** bm - csl medium______________________________________24 2 . 5 9 . 636 -- 23 . 248 8 . 7 39 . 060 16 . 7 50 . 572 20 . 9 51 . 0______________________________________ * ph of cultures was not controlled ** csl medium contained 4 % corn steep liquor supplemented with 0 . 1 mm co ++ and 0 . 05 mm cu + table ii which follows shows that removal of sludges from corn steep liquor is essential to the production of glucose isomerase from actinoplanes missouriensis . it also shows that a molasses medium will support growth of the organism to produce glucose isomerase , but that the combination of molasses and corn steep liquor greatly increases the production of isomerase . table ii______________________________________effect of corn steep liquor sludge on the production ofglucose isomerase from actinoplanes missouriensis ( shakergrown cells ) ______________________________________ production of enzymemedia ( μ / ml .) culture______________________________________molasses medium ( nocorn steep liquor ) 17 . 9molasses and corn steepliquor * ( sludges havenot been removed ) 0 ( no growth ) molasses and corn steepliquor * ( sludges have beenremoved ) 32 . 0______________________________________ * molasses : 3 . 2 %; corn steep liquor : 1 . 0 %. grown for 4 days . as mentioned , the optimal concentration of beet molasses for production of glucose isomerase is between about 2 % to about 8 %. the preferred concentration is about 3 %. the production of enzyme decreases sharply in the presence of more than 10 % molasses . table iii which follows demonstrates these data . the media contained 1 . 5 % corn steep liquor except for run 5 . the amounts of beet molasses varied as indicated . the rest of the media were as set forth hereinbefore under ` medium composition `. table iii__________________________________________________________________________effect of beet molasses concentrations on the production of glucoseisomerasegrowth periodsenzymeproduction % beet molasses in each fermentation run . sup . 1 and run - 1 run - 2 run - 3 run - 4 run - 5 run - 6 run - 7 run - 8 run - 9 run - 10hrs . growth 0 1 . 0 2 . 0 3 . 0 3 . 0 4 . 0 5 . 0 6 . 0 8 . 0 10 . 0__________________________________________________________________________24 activity . sup . 2 nd . sup . 4 nd nd 11 . 0 7 . 1 15 . 4 12 . 2 7 . 8 16 . 9 nd growth . sup . 3 nd nd nd 1 . 3 0 . 6 1 . 6 1 . 5 1 . 0 1 . 2 nd36 activity nd nd 9 . 8 33 . 3 15 . 1 -- 24 . 4 23 . 0 21 . 1 6 . 2 growth nd nd 1 . 1 3 . 1 1 . 4 -- 2 . 6 2 . 1 2 . 3 0 . 848 activity nd nd 26 . 7 47 . 3 24 . 1 35 . 7 35 . 9 27 . 4 32 . 2 9 . 2 growth nd nd 2 . 9 3 . 5 1 . 8 3 . 0 3 . 2 2 . 5 3 . 1 1 . 360 activity nd nd 48 . 4 59 . 2 28 . 6 57 . 3 50 . 3 41 . 3 44 . 1 12 . 3 growth nd nd 3 . 6 3 . 7 2 . 3 3 . 8 4 . 2 3 . 6 3 . 6 1 . 672 activity 15 . 1 19 . 6 56 . 7 63 . 8 25 . 8 66 . 5 68 . 2 45 . 8 50 . 9 14 . 0 growth 0 . 8 1 . 2 3 . 3 3 . 5 2 . 3 4 . 3 4 . 9 4 . 0 3 . 9 1 . 784 activity -- -- 62 . 0 -- 32 . 4 78 . 8 63 . 0 60 . 2 61 . 1 10 . 8 growth -- -- 3 . 0 -- 2 . 5 4 . 4 5 . 10 4 . 20 4 . 2 1 . 796 activity 16 . 3 33 . 4 69 . 2 -- 31 . 4 74 . 1 59 . 0 62 . 2 62 . 9 13 . 9 growth 0 . 7 1 . 8 3 . 2 -- 2 . 7 4 . 3 4 . 9 5 . 3 4 . 4 1 . 6__________________________________________________________________________ . sup . 1 all fermentation runs contained 1 . 5 % corn steep liquor except run - in which csl was omitted . . sup . 2 μ / ml culture . sup . 3 mg extractable protein / ml culture . sup . 4 not determined due to poor growth table iii shows that in the absence of beet molasses , the production of enzyme in the 72 hour old culture is only 24 % of that obtained from the medium containing 3 % molasses but the specific activity of enzyme from both media are almost the same ( see run - 1 and run - 4 in table iii ). in the absence of corn steep liquor , the production of enzyme from the medium containing 3 % molasses is only 41 % of that containing 1 . 5 % corn steep liquor in addition to 3 % molasses , but the specific activity of the former enzyme preparation is only 62 % of the latter ( see run - 5 in table ii ). this result indicates that the corn steep liquor promotes the production of enzyme , whereas molasses stimulates the growth of the organism . combinations of both molasses and corn steep liquor therefore increase the yields of the enzyme . the amount of corn steep liquor that can be used is from about 0 . 6 % to about 4 . 0 %. above 4 % the growth may be poor depending on the quality of corn steep liquor . it preferably is about 1 . 5 % as shown in table iv which follows : table iv______________________________________effect of corn steep liquor ( csl ) concentration on theproduction of glucose isomerase in the beet molasses medium *( fermentor grown cells 60 hours old ) concentration of csl production of enzyme ( μ / ml . culture ) ______________________________________0 28 . 60 . 6 29 . 01 . 0 44 . 01 . 5 59 . 5______________________________________ * the composition of this medium has been described hereinbefore under ` medium composition `. cane molasses , beet molasses and mixtures of cane and beet molasses can be used to produce glucose isomerase . this is illustrated as follows : actinoplanes missouriensis is cultured in 100 ml . of medium of 3 % cane molasses , 1 . 5 % desludged corn steep liquor mixture at a ph of 7 . 1 supplemented with minerals as hereinbefore described . the culture is grown in 250 ml . erlenmeyer flasks on a rotary shaker at 28 ° c . for 4 days . the enzyme yields are then determined . the results are set forth in table v . table v______________________________________molasses % enzyme yields ( μ / ml . culture ) ______________________________________cane 3 % 29 . 7beet 3 % 39 . 2cane andbeet 1 . 5 % each 34 . 6______________________________________ from the foregoing table v , it is apparent that cane molasses can also be used to produce glucose isomerase . inorganic nitrogen salts , as well as urea , also are effective in increasing enzyme production by a factor of 48 % to 128 %. in preparing the following table vi , actinoplanes missouriensis is grown in 100 ml . of filtrate of 3 % beet molasses , 1 . 5 % corn steep liquor mixture ( ph 7 . 1 ), supplemented with minerals as previously described . the filtrate is placed in 250 ml . erlenmeyer flasks and sufficient amounts of inorganic nitrogen salts such as nano 3 , nh 4 no 3 , ( nh 4 ) 2 so 4 , nh 4 h 2 po 4 , nano 2 , and urea are added to the medium to give a final concentration of 5 . 9 mm of nitrogen atom . after sterilization , the medium is inoculated , the flasks are placed on a rotary shaker , and the organism is grown in the flasks for 4 days at 28 ° c . if nano 3 is the nitrogen salt , from about 0 . 1 % to about 0 . 6 % is used . other nitrate salts can be used which give equivalent amounts of nitrate . the other nitrogen salts are used in amounts effective to give nitrogen equivalent to that in from 0 . 1 % to 0 . 6 % nano 3 . table vi______________________________________effectof inorganic nitrogen salts and urea on the productionof glucose isomerase of actinoplanes missouriensisinorganic nitrogen salts enzyme production μ / ml . culture______________________________________none 19 . 4nano . sub . 3 32 . 5nano . sub . 2 28 . 6 ( nh . sub . 4 ). sub . 2 so . sub . 4 44 . 0nh . sub . 4 no . sub . 3 37 . 8nh . sub . 4 h . sub . 2 po . sub . 4 34 . 7urea 33 . 3______________________________________