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FORM 2422 REV . SJ67

INTER-OFFICE MEMORANDUM

Subject : Glucose Isomerase Report Date : February 25 ., 1'970

(Period Covering February 9 - 20, 1970)

To : Mr . Edward A . Vassallo

r
CONFIDENTIAL
The glucose isomerase activity in the 30-gallon fermentators is
in the 700-800 pU per ml range . Attempts to consistently increase the
activity further have been usually unsuccessful . About three more
fermentations are required to produce the enzyme for the pilot experi-
ment in Cedar Rapids . The equipment breakdown delayed the production
of enzyme for this test .

Since the enzyme preparation used in the 24-day experiment was

obtained from an acetone fractionation, it was necessary to repeat
these experiments using enzyme recovered from the culture broth with
no acetone treatment . After 13 days, both enzyme preparations, with
and without acetone fractionation,exhibit approximately the same rate
of conversion using glucose as the substrate . Approximately five gallons
of syrup was collected from these columns and this syrup will be sent
to Product Development for an evaluation of the cleanup of the syrup
using a mixed bed resin .

Further studies of mechanical methods to release the enzyme from

the cell after the lysozyme treatment have been unsuccessful . An
Omni Mixer and the Cowles Dissolver which are shear-type mixers did
not adequately rupture the cells . The rupture of the cells after the
lysozyme treatment is one of the critical steps in this process : Although
sonification can be used to prepare the enzyme for laboratory studies,
we do not have a procedure or equipment as yet to prepare the enzyme
for a pilot scale study . This problem is not only one of cell rupture
but also removal or resuspension of other contaminating substances such
as nucleic acids which interfere with enzyme binding to the carrier .
Other procedures, including large volume sonifiers are under investigation
to prepare the enzyme for these studies . Until this problem is solved,
it will be very difficult to prepare enough enzyme for the pilot run
in Cedar Rapids by March 1, 1970 . Therefore, the target date for the
pilot run has been rescheduled for March 16, 1970 .

There have been improvements in the amount of DEAE-cellulose that

is required to bind the enzymes,in the projected flow rates, and the
total cubic feet of media required . Therefore, t;he process varia3~les
have not been completely finalized .

Cc : Mr . D . S . Peoples Dr .
Dr . Murray Senkus Mr .
Dr . S . .0 . Jones Mr .
Dr . C . E . Teague, Jr . Dr .
Dr . E . D . Nielson Dr .
Dr . R . E . Brouillard -'EF-r .
`Charles G . Pheil
E . C . Ritchell Mr . L . E . Hayes
W . C . Black Dr . Chin K . Lee
Claude Sawyer Miss Margaret E . Long
D . J . Lartigue
J . L . Ayres
Paul Benko