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''FORM 2422 REV .

5/67

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Subject : Glucose Isomerase Date : March 11, 1970


(Period Covering February 23 to
march 6, 1970)

To : ASr . Edward A . Vassallo

CONFIDENTIAL
The glucose isomerase activity in the last six 30-gallon
fermentations has ranged from 860 to 1,060 uU per ml . This is a sub-
stantial increase in activity and should reduce the enzyme production
costs . The fermentation series this week should give us enough enzyme
for the pilot run in Cedar Rapids .

The two columns containing an acetone fraction and the cell-


free preparations are still active after 26 days, and the percent
conversion,is, still, about 45% using 2M buffered glucose as a substrate .
Another column using rehydrated total sugar as a substrate has run six
days and the conversion is about 45% . This particu`lar total sugar
sample contains some precipitate, similar to filter aid . This type
material and any carbon particles must be removed from the syrup prior
to isomerization .

The isomerized syrup from the above columns using glucose as


a substrate were analyzed by Product Development to determine the
approximate thru put capabilities of a mixed bed ion exchange system .
The mixed bed system contained 6 parts IRC-120 and 10 parts IRA-93
resins . The average thru put per cycle was 7 .95 liters at 30 .4% solids .
Th3.s gave an average thru put of approximately 910 gallons per cubic
f oot of resin . The high thru put value was about 935 gallons per
cu . f t . and the low value was about 860 gallons per cu . ft .

Samples of isomerized total sugar will be sent to Product


Development f or mixed bed resin studies . This substrate more closely
u
approximates the syrup to be used in the pilot experiment .

Batch experiments have demonstrated that the magnesium con-


tent can be reduced from 0 .01M to 0 .002M . This lower level will be
tested on a column run to exhaustion for verification of these results .
This lowered magnesium content should further extend the life of the
mixed bed resins . The magnesium content is quite critical since any
concentration above 0 .02M will remove the enzyme from the column
, . . . ... .. . .... . . . . ..... . . . . . . . . . . ... .. . . .. .
support .

Further optimalization of the flow rate, amount of enzyme


bound to the cellulose, and other process variables, has changed the
column dimensions for the pilot run . The new column dimensions are
4 inches in diameter and a bed depth of 32 inches . Two columns will
Mr . Vassallo
Page 2
March 11, 1970

be set up in series so that maximum flow rate data can be obtained . The
optimum temperature is 60-62°C and the column temperature must be closely
regulated . At 70°C, an8% increase in conversion is obtained, but there
is a 33% reduction in enzymatic activity .

The concentration of cells during lysis has been increased to


15% . At this concentration, 100% of the total activity was released in
cell-free extract (CFX) after sonification . Before sonification, only
28% was released and only 21 vU of enzyme could be bound to the support .
Therefore, the sonification-procedure must be used to release the enzyme
and reduce the viscosity . The industrial sonifier should be in operation
by March 23, 1970 (delivery promised by March 19 or 20) . F1ow rate data
have been collected for the small sonifier which will assist in scaling
up to the large unit . As soon as the industrial unit is in operation,
a more realistic target date can be set for the pilot run in Cedar
Rapids . ' - --*

CGP :msc

cc : Mr . D . S . Peoples
Dr . Murray Senkus
Dr . S . .O . Jones
Dr . C . E . Teague, Jr .
~D£ E . D . Nielson
Dr . R . E . Brouillard
Dr . E . C . Ritchell
Mr . W . C . Black
Mr . Claude Sawyer
Dr . D . J . Lartigue
Dr . J . L . Ayres
Dr . Paul Benko
Mr . L . E . Hayes
Dr . Chin K . Lee
Miss Margaret E . Long
i
Dr . C . W . Nystrom . I

Dr . W . S . Simmons