Form 2422 Rev . 5/67
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. t A FORM 2422 REV OF E M EM . Subject : Glucose Isomerase R eport (Week Ending November 2 1, 1969) To : Mr . Edward A . V assallo r C O ! Date : November 25, 1969 J F i D E n N I A L The last full scale fermentation series for enzyme production will be started this week . At the prese nt time enough enzyme iq available to convert 1,000 gallons of high DE syrup, although,•with this added series, we can use all enzyme prepared in the last six weeks . The yield of enzyme per fermentation is still low a"nd future ferme ntation series will be utilized to raise the enzyme activity back to the original yields of 700 to 800 uU per m l or high er . Preliminary experiments in shake flasks have'indi cated that the 2 % dextrose used as the substrate for the production of glucose isomerase can be r eplaced with a high DE syrup . Th e concentra tion of substrate is still 2%, h owever, this preliminary data suggest that an unfiltered syrup can be used in the fermentation . A culture of My xobacter has bee n obtained from the University of Wisconsin . Si nce this cultu re has been repor ted to conta in ly tic enzymes for Arthrobacter, its lytic action on our strain is under investigation . The Bacillus lytic enzyme system is a lso-being studied to determine t he optimum conditions for max imum glucose isomerase r elease . The isomerized syrup for animal feeding trials was rec eived from Product Development arnd .appropriate al iquots have been sent to Industrial Bio-Test Laboratories for dog feeding trials . Another batch was sent to Nut ritional Biochemicai for in corporat ion into a diet for the ra t'feedi ng trials . Th is syrup co ntained about 46% fruct ose . The progress report on the clean-up o f the enzy me isomerized syrup by th e Product Development pers onnel is attached to this report . Studies on re-use of the enzyme are continuing using a cell-free enzyme bound to an insoluble carrier in a column appr oach . Preliminary data indicate that 99% of the enzyme activity is bound to DE AE -cellulos e . When an equi valent amount of enzyme (same concentrat ion used in a one- liter batch system ) is bound to tHe carrier, 1 .3 liters of approximately 45% fructose syrup was obtaine d in 2 4 hours . Upon further incubation for another 24 hours with no additional enzyme, two liter s of ap proximately 4 1% f ructo se syrup were obtained . These fructose values were determined on a composite samp le of the syrup, hen ce, these are only average values . The conditions for optimum re-u se uf the enzyme including its heat stability over a longer period of time are under investigation .
Transcript of Form 2422 Rev . 5/67
8/14/2019 Form 2422 Rev . 5/67
http://slidepdf.com/reader/full/form-2422-rev-567 1/2
. t A. 5/67
INTER-OFFICE MEMORANDUM .
S u b j e c t : Glucose Isomerase Report(Week Ending November 21, 1969)
To: Mr . Edward A. Vassallo
rC O !
Date: November 25, 1969
J F i D E nN I A L
The last full scale fermentation series for enzyme production willbe started this week. At the present time enough enzyme iq availableto convert 1,000 gallons of high DE syrup, although,•with this addedseries, we can use all enzyme prepared in the last six weeks . The yieldof enzyme per fermentation is still low a"nd future fermentation serieswill be utilized to raise the enzyme activity back to the original yieldsof 700 to 800 uU per ml or higher .
Preliminary experiments in shake flasks have'indicated that the 2%dextrose used as the substrate for the production of glucose isomerasecan be replaced with a high DE syrup . The concentration of substrateis still 2%, however, this preliminary data suggest that an unfilteredsyrup can be used in the fermentation .
A culture of Myxobacter has been obtained from the University ofWisconsin . Since this culture has been reported to contain lytic enzymesfor Arthrobacter, its lytic action on our strain is under investigation .
The Bacillus lytic enzyme system is also-being studied to determine theoptimum conditions for maximum glucose isomerase release .
The isomerized syrup for animal feeding trials was received fromProduct Development arnd.appropriate aliquots have been sent toIndustrial Bio-Test Laboratories for dog feeding trials . Another batchwas sent to Nutritional Biochemicai for incorporation into a diet forthe rat'feeding trials . This syrup contained about 46% fructose .
The progress report on the clean-up of the enzyme isomerized syrupby the Product Development personnel is attached to this report .
Studies on re-use of the enzyme are continuing using a cell-freeenzyme bound to an insoluble carrier in a column approach . Preliminarydata indicate that 99% of the enzyme activity is bound to DEAE-cellulose .
When an equivalent amount of enzyme (same concentration used in a one-liter batch system) is bound to tHe carrier, 1 .3 liters of approximately45% fructose syrup was obtained in 24 hours . Upon further incubation foranother 24 hours with no additional enzyme, two liters of approximately41% fructose syrup were obtained . These fructose values were determinedon a composite sample of the syrup, hence, these are only average values .
The conditions for optimum re-use uf the enzyme including its heatstability over a longer period of time are under investigation .
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Acceptable analytical methods for monitoring the large scaleisomerization in Cedar Rapids were studied . Samples of glucose-fructosesyrups were taken every two hours during enzymatic isomerization and,after a short clean-up procedure, were analyzed by gas chromatography,optical rotation, and resorcinal-HC1 methods . The results indicatedthat all three methods for ketose analysis gave similar results . Aspreviously reported, the isomerase syrup contains very little (<0 .5%)unfermentable hexoketose which could cause a discrepancy between thesethree methods . Therefore, the optical rotation procedure will be usedin the Cedar Rapids experiment .
CGP :vblAttachmentCc: Mr. D . S . Peoples
Dr . Murray SenkusDr . R . E . FarrarDr . E . D . NielsonDr . R . E . BrouillardDr . E . C . RitchellMr . W . C . BlackMr . Claude SawyerDr . D . J . LartigueDr. J . L . AyresDr . Paul BenkoMr . L . E . HayesDr. Chin K. LeeMiss Margaret E. Long
