Author : Charles G .

Pheil September 25, 1968

Division : Biological Research Notebook Pages : `165901-50,

-168551-600, 175001-50,
178951-85
RDR, 1968, No . 34 Dated : March 23, 1967
through April 26, 1968

No . of Pages : 17 Previous Reports : None

CONVERSION OF D-GLUCOSE TO D-FRUCTOSE

BY ARTHROBACTER RJR 2453-2

OBJECT :

To increase the glucose isomerase activity of cells of Arthrobacter

RJR 2453-2 by cultural techniques and surface active agents .

SUMMARY :

The cells of Arthrobacter RJR 2453-2 could be synchronized by three,

eight-hour incubations prior to inoculation into the test medium . Under
these conditions, the glucose isomerizing activity was 1,175'uU per ml .
of culture broth .

A critical oxygen level was evident in the shake-flask experiments .

Various flask closures could regulate the aeration efficiency and govern
the final glucose isomerase activity .

The glucose isomerase activity could be increased by growing the

RJR 2453-2 cells using an inexpensive recrystallized bagasse xylose
fraction as a carbon source . The more expensive tryptone and yeast
extract could be replaced with inexpensive protein materials without
a significant reduction in enzymatic activity .

The glucose isomerase activity of the RJR 2453-2 cells could be

increased two to threefold by treatment with sodium dodecyl sulfate at
60° C . while a sixfold increase was observed with the same detergent at
75° C .

No significant reduction in the glucose isomerase activity of the

RJR 2453-2 cells was observed after 24 hours at 75° C . either in the
presence or absence of sodium dodecyl sulfate .

•
.
`` 11, , :.,
 2

TABLE OF CONTENTS
Page

OBJECT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

A . INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

B . EXPERI MENTAL . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

I. Analytical Method for the Determination of

Fructose and the Glucose Isomerase Activity
of Arthrobacter Cells . . . . . . . . . . . . . . . . . . . . 5
II . Growth Medium and Aeration Conditions for
Enzyme Producti on . . . . . . . . . . . . . . . . . . . . . . 5

Cultural Conditions and Nutrients for Control

of Cell Morphology and Enzymatic Activity . . . . . . . . . . 6

a . Cul tural Condi ti ons . . . . . . . . . . . . . . . . . . 6

b . Nutritional Control . . . . . . . . . . . . . . . . . . 7

IV . Effect of Aeration on Glucose Isomerase Activity . . . . . . 7

V . Incorporation of Inexpensive Substrates into the

Basal Medi um . . . . . . . . . . . . . . . . . . . . . . . . 8

a . Xylan and Xylan Containing Substrates . . . . . . . . . 8

b . Bagasse as a Xylose Source . . . . . . . . . . . . . . . 9

c . Replacement of Tryptone and Yeast Extract . . . . . . . 10

VI . Surface Active Agents and Temperature on

G1 ucose Isomerase Acti vi ty . . . . . . . . . . . . . . . . . 11

a . I ncubati on Temperature . . . . . . . . . . . . . . . . . 11

b . Assay Temperature . . . . . . . . . . . . . . . . . . . 11

c . Different Types of Surface Active Agents . . . . . . . . 12

d . Anionic Surface Active Agents . . . . . . . . . . . . . 13

e . Glucose Isomerase Activity of Culture RJR 2453-2

After 24 Hours at 750 C . . . . . . . . . . . . . . . . . 13
 3

TABLE OF CONTENTS (Cont'd)

Page

C . DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

D . CONCLUS I ONS . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

E . RECOMMENDATIONS . . . . . . . . . . . . . . . . . . . . . . . . . 15

I . Future Work . . . . . . . . . . . . . . . . . . . . . . . . . 15

II . Patentabi lity . . . . . . . . . . . . . . . . . . . . . . . . 16

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
 4

LIST OF TABLES

Table No . Title Page

I Effect of Preincubations at 30° C .

on Glucose Isomerase Activity in
XS-2 B roth . . . . . . . . . . . . . . . . . . . . 6

II Different Flasks and Flask Closures . . . . . . . 8

III Bagasse Xylose Fractions . . . . . . . . . . . . . 9

IV Gas Liquid Chromatography of the

Bagasse Xylose Fractions . . . . . . . . . . . . . 10

V Utilization of Inexpensive Nitrogen

and Vi tami n Sources . . . . . . . . . . . . . . . 10

VI Assay Temperature and Various

Concentrations of Sodium Heptadecyl
Sul fate . . . . . . . . . . . . . . . . . . . . . 12

VII Various Surface Active Agents . . . . . . . . . . 12

LIST OF FIGURES

Figure No . Title Page

1 Anionic Surface Active Agents and

Assay Temperatures . . . . . . . . . . . . . . . . 14
 5

A . INTRODUCTION

Enzymes which catalyze the interconversion of free aldopentose and

ketopentose have been demonstrated in several bacterial strains (12) . In
1957, Marshall and Kooi (7) reported that Pseudomonas hydrophila was able
to isomerase both xylose and glucose to the respective ketose forms . This
microorganism and certain species of Bacillus have been patented for the
process of convertin glucose to fructose using cells grown in a nutrient
containing xylose (8~ . Since this patent, there has been considerable
interest in the enzymatic conversion of D-glucose to D-fructose ; and many
microorganisms have been reported to contain this enzymatic activity (9) .

A bacterium, Arthrobacter RJR 2453, was isolated from the Idaho Potato
Processing Plant and was s own to possess high glucose isomerase activity (9) .
A stable isolate of the parent strain which exhibited higher enzymatic
activity was prepared and reclassified as Arthrobacter RJR 2453-2 . The
glucose isomerizing activity of the genus Art ro acter has not been
reported in the literature or in any patents, to date .

The genus, Arthrobacter, is characterized by its many morphological

variations during the growth cycle ; and certain cell forms have been shown
to exhibit greater glucose isomerizing activity than others . The morpho-
genesis of this organism can be controlled by variation in nutritional
conditions (2) and by s~nchronization of cell division (1) .

Surface active agents have long been known to affect enzymatic activity .
Hughes, 1950 (3), has reported acceleration of glutaminase activity with
cationic surface active agents while Wills, 1954 (11), has shown increased
activity of trypsin with the anionic detergent, sodium dodecyl sulfate .
Recently, Machida, 1966 (6), reported that the anionic surface active
agent, sodium dodecyl benzenesulfonate, increased the glucose isomerizing
activity of resting cells of Lactobacillus brevis .

B . EXPERIMENTAL

I . Analytical Method for the Determination of Fructose and the

Glucose Isomerase Activity of the Arthrobacter Cells

Fructose formed in the reaction mixtures was measured by the Seliwanoff

reaction as modified by James (4) . After centrifugation of the cells and
suitable dilutions, the samples were analyzed using a Technicon AutoAnalyzer .
The fructose content of the samples was calculated by comparison with standard
samples of fructose .

The enzymatic activity of the cells at 60° C . was determined b the

method of Lartigue (5) and the results expressed as micro-units (uU~ of
activity per ml . of culture fluid . The specific enzyme activity was expressed
as uU per mg . of wet weight cells .

II . Growth Medium and Aeration Conditions for Enzyme Production

A basal medium, which was consistently found to support the growth of

Arthrobacter RJR 2453-2 and production of glucose isomerase, served as a
 6

basic milieu to which was added various combinations of minerals, peptones,

and other test ingredients . This medium, referred to as "XS-2 broth,"
contained the following ingredients in grams per liter of distilled water :
(NH4)2HP04, 6 .0 ; KH2PO4, 0 .2 ; MgSO4•7H2O, 0 .25 ; xylose, 20 .0 ; yeast extract
(Difco), 1 .0 ; and tryptone (Difco), 5 .0 ; final pH 6 .9 . Sterilization was
accomplished by autoclaving at 121° C . for 15 minutes ; the xylose was auto-
claved separately in 50% (w/v) concentration and asceptically added to the
remainder of the medium . The inoculum consisted of a 5% (v/v) portion of
actively growing cells .

The shake-flask cultures were grown in 500 ml . and 1,000 ml . Erlenmeyer

flasks containing 100 and 200 ml . of basal medium, respectively . A rolled
cotton stopper was used as a closure . The flasks were incubated at 30° C .
on a rotary shaker operating at 25012 rev ./min . to assure adequate aeration .

III . Cultural Conditions and Nutrients for Control of Cell Morphology

and Enzymatic Activity

a . Cultural Conditions

Cellular differentiation as exhibited b the genus Arthrobacter

has been described by many investigators (10~ . Organisms in the stationary
phase of growth are characteristically spherical . Upon inoculation of the
organisms into fresh medium, the cells gradually elongate into pleomorphic
rods . Cell division occurs during the rod stage and continues until the
rods fragment into smaller entities which gradually shorten into the
typical coccoid cell . It was demonstrated that the RJR 2453-2 culture
exhibited higher glucose isomerase activity in the rod form than in
the coccoid form . Thus, it was desirable to synchronize the cells in
this stage and use these cells as an inoculum . The culture synchro-
nization technique of "in-prior" treatments which consisted of various
short-time incubations was evaluated .

The cultures were not synchronized at the longer preincubation

times, 16-24 hours ; the cells exhibited many stages of development
from short rods to the undesirable coccoid forms . Approximately 60-80%
of the cells were in the rod form after the 8 and 12-hour transfers .
The degree of culture synchronization was reflected in the final
enzymatic activity (Table I) .

TABLE I

EFFECT OF PREINCUBATIONS AT 30° C .

ON GLUCOSE ISOMERASE ACTIVITY IN XS-2 BROTH

Number and Time Total Incubation Enzymatic Activity_

of Initial Transfers Time uU ml . uU/mg .

2-8 hours 65 hours 622 18 .8

3-8 " 65 " 640 19 .4
3-8 " 72 " 1,175 35 .2
2-12 " 65 " 580 18 .1
2-16 " 65 " 305 9 .5
 7

At very short preincubation times, 4-6 hours, the majority of the

cells (80-90%) were in the desired short rod form exhibiting some
branching and a binucleate morphology . However, there was not a suf-
ficient cell yield after four hours for inoculation into the test
flasks or fermentators .

b . Nutritional Control

Cellular differentiation can also be nutritionally controlled by

the incorporation of thiotone (BBL) instead of tryptone in the basal
medium or by the addition of 0 .2% succinate, 0 .2% L-asparagine, or
0 .2% citric acid to the basal medium . Under these conditions, the
cells developed into elongated, swollen, involulated forms which
failed to complete the life cycle, i .e ., no formation of coccoid
forms . However, in all cases the glucose isomerase activity of cells
was less than the controls with "XS-2" broth . Although thiotone has
been reported to be different in magnesium, iron, and calcium content,
the addition of various concentrations of these minerals did not
significantly increase glucose isomerase activity . These results
suggest that a critical nitrogen requirement is required for optimum
enzymatic activity .

IV . Effect of Aeration on Glucose Isomerase Activity

The effect of aeration and agitation was indirectly examined in shake-

flasks by different types of flask closures and baffled flasks (4-baffles-
10 mm . high and 2 mm . in diameter) . The results in Table II indicate that
a filter pad closure, which would give a higher oxygen transfer rate, yields
a higher cell activity than a cotton stopper, especially in the 1,000 ml .
flasks . With the cotton-stoppered 500 ml . flasks, no significant difference
was noted by baffling the flasks . However, excessive aeration occurred in
the baffled flasks with a filter pad closure as eVidenced by the reduction
of enzymatic activity . The use of a metal cover would reduce the aeration
rate, but the use of a baffled flask may increase the rate of oxygen transfer
into the medium .
 8

TABLE II

DIFFERENT FLASKS AND FLASK CLOSURES

Glucose Isomerase
Activit 1
Flask Size and Type Closure uU/ml . uU/m .

500 ml . - unbaffled CS2 5923 20 .9

FP 707 23 .3

300 ml . - 4-baffled CS 572 21 .6

FP 241 9 .8

1,000 ml . - unbaffled CS 377 16 .3

FP 700 24 .1

1,000 ml . - 4-baffled Metal Cover 535 21 .4

1Cells of RJR 2453-2 were incubated at 300 C . for 46 hours .

2CS = rolled cotton stopper ; FP = 1 milk filter pad .

3A11 values are the average of three replications .

These results suggest that a critical oxygen level is required for the
production of cells with high enzymatic activity .

The effects of increasing cell density on aeration rates, as measured

by dissolved oxygen activity, were determined using an oxygen probe . The
data was expressed as dissolved oxygen activity (DOA) realizing however
that we are actually measuring oxygen partial pressure and/or oxygen
activity rather than the actual concentration of dissolved oxygen . The
DOA is high initially, 90-100, with an aeration rate of 2 .1 L/min . ; but
as the cells reach the logarithmetric phase of growth, the DOA was reduced
to 70 ; and during the stationary phase of growth, the DOA was reduced to 40 .
When the aeration rate was increased to 4 L/min ., no increase in the DOA was
noted . The combination of 0 .1 L/min . of "pure" oxygen and 1 .2 L/min . of air
were required to increase the DOA to 80 . The agitation rate was constant
during these experiments .

Since the DOA is lowered as the concentration of cells increases,

increased aeration rates may be required to increase the oxygen transfer
rate ; and the rate of solution of oxygen may not equal or exceed the rate
at which oxygen is required by the aerobic Arthrobacter cells .

V . Incorporation of Inexpensive Substrates into the Basal Medium

a ._ ~ylan and ~ylan Containing Substrates

The high cost of pure xylose prohibits its use in a commercial

fermentation . Therefore, inexpensive substrates were evaluated for
 9

their ability to support the growth and enzymatic activity of cells

of RJR 2453-2 . Raw and hydrolyzed ground corn hulls, peanut shells,
and seaweed were evaluated since all these materials contain a xylan
fraction . These substrates could support the growth of these cells ;
however, the glucose isomerase activity was less than 100 uU/ml . A
pure xylan (Nutritional Biochemicals) could also act as a carbon
source, but the enzymatic activity was still less than 100 uU/ml .
Chemical analysis of fermentation broths showed the presence of low
levels of glucose, 2 .2-8 .7 mg . % . The addition of 1 mg . % glucose to
the basal broth ('XS-2" broth) will significantly reduce the glucose
isomerase activity ; therefore, these higher levels of glucose are
probably responsible for the reduction in enzymatic activity .

b ._ Bauasse as a Xylose Source _

Whole and ground bagasse stalks could support the growth of

Arthrobacter ; however, the glucose isomerase activity was less than
100 uU/ml .

The effect of crude xylose from bagasse and two recrystallized

bagasse xylose (Pfanstiehl No . 1, No . 2, and No . 3) samples was
evaluated for their ability to support the production of glucose
isomerase in cells of RJR 2453-2 . The crude xylose (P-1) yielded
reduced enzymatic activity while the recrystallized fractions
increased the enzymatic activity (Table III) .

TABLE III

BAGASSE XYLOSE FRACTION S

Glucose Isomeras e
Activity l
Xylose Source (2%) uU ml . uU m .

Pure Xylose 447 16 . 3

Bagasse Xylose (P-1) 146 4 .7

Bagasse Xylose (P-2) 614 22 . 4

Bagasse Xylose (P-3) 761 27 . 8

1Cells were assayed after 69 hours at 300 C .

Combinations of commercial and bagasse xylose (P-1) yielded cells

with lower enzyme activity than the equivalent concentration of regular
xylose . The data in Table IV suggest that the inhibitory substance in
P-1 is glucose while the increased enzymatic activity of the cells grown
in P-2 and P-3 is due to the absence of glucose .
 10

TABLE IV

GAS LI UID CHROMATOGRAPHY

OF THE BAGASSE XYLOSE FRACTIONS

Relative %
Bagasse Xylose Ribose and
Fractions Xylose Glucose Mannose Arabinose

P-1 69 .4 1% -- 1%

P-2 97 .7 ND1 Trace Trace

P-3 97 .3 ND ND Trace

1ND signifies none detected .

c ._ Replacement of Tryptone and Yeast Extract

Enzyme hydrolyzed casein (EHC Amber Labs - $0 .79/lb .) or an animal

protein hydrolysate (Amber Labs - $0 .22/lb .) could replace the commercial
tryptone ($4 .40/lb .) while the yeast extract ($8 .40/lb .) could be replaced
by BFY yeast (Amber Labs - $0 .34/lb .) . Higher glucose isomerase activity
was noted with the casein than with the animal protein hydrolysate while
no difference was observed with the BFY yeast when casein was used, but
significantly lower activity was observed with the BFY yeast and animal
protein .

TABLE V

UTILIZATION OF INEXPENSIVE NITROGEN AND VITAMIN SOURCES

Glucose Isomerase Activityl

Media uU/ml . uU m .

Basal - XS-2 broth 447 16 .3

Casein + yeast extract 495 19 .0

Animal protein + yeast extract 251 11 .1

Tryptone + BFY yeast 524 19 .7

Casein + BFY yeast 509 16 .8

Animal protein + BFY yeast 299 9 .1

1RJR 2453-2 cells were assayed after 70 hours at 30° C .

 11

VI . Surface Active Agents and Temperature on Glucose Isomerase

Activity

The effects of anionic, cationic, and nonionic surface active agents

on glucose isomerase activity were studied using resting cells of RJR 2453-2 .
The centrifuged cells from fermentations Nos . 17 and 22 were resuspended in
distilled water and standardized to a cell concentration of 70 mg . (wet weight)
per ml . Since this cell concentration is not obtained during the actual
fermentations, it is superfluous to report the enzymatic activity as uU/ml . ;
therefore, all data is expressed as uU of glucose isomerase activity per mg .
of cells (wet weight) . The various detergent concentrations are expressed
as pg . of detergent per mg . of cells (wet weight) . Certain anionic surface
active agents, e .g ., sodium dodecyl sulfate, have been approved for food use ;
hence, these agents would be most desirable .

Since the definition of pU of enzyme activity refers to an assay temperature

of 60° C . (5), the enzyme activity of cells at higher temperatures (70-80° C .)
are expressed as relative pU/mg . of cells . All enzymatic assays were corrected
for thermal isomerization .

a ._ Incubation Temperature_

The resting cell suspensions at a pH of 6 .8f0 .02 were refrigerated

at various temperatures in the presence of sodium heptadecyl sulfate
(HDS) . The average enzymatic activity at 1 and 8° C . was 9-11 pU/mg .
while at 25 and 30° C . it was 4 .5-5 .0 pU/mg . The HDS (4 ug ./mg .) had
no effect on the enzymatic activity at 25 and 30° C . However, the
activity was increased from 8 to 14 pU/mg . and 11 to 18 pU/mg . at 1 and
8° C ., respectively . Agitation during the incubations did not increase
the activity over those cells held in a stationary flask . No significant
increase in enzymatic activity was observed at higher concentrations of
the HDS (5-16 pg ./mg .) .

b ._ Assay Temperature

The influence of the assay temperature and sodium heptadecyl sulfate

concentration on the glucose isomerase activity in the RJR 2453-2 cells
is shown in Table VI . The relative enzymatic activity at 70 to 80° C .
was significantly greater than those values at 60° C . The addition of
this detergent increased the enzymatic activity at all temperatures ;
however, the two effects, assay temperature and detergent concentration,
were not additive .
 12

TABLE VI

ASSAY TEMPERATURE AND VARIOUS CONCENTRATIONS

OF SODIUM HEPTADECYL SULFATE

Detergent Glucose Isomerase Activity (pU/mg .)

Concentration Assay Temperature °C .
(ug ./mg .) 60 65 70 75 80

0 9 .4 14 .0 21 .0 26 .6 29 .5

2 .3 11 .1 16 .5 26 33 33 .2

4 .0 19 .4 -- 39 36 32

20 .0 16 .8 18 .5 33 .8 -- --

c ._ Different Types of Surface Active Aqents_

The effect of various types of surface active agents on resting

cells of RJR 2453-2 was evaluated at two assay temperatures (Table VII) .
Generally, the anionic agents, especially sodium dodecyl sulfate, yielded
a greater increase in the enzymatic activity .

TABLE VII

VARIOUS SURFACE ACTIVE AGENTS

Relative Glucose Isomerase Activity

(pU/mg•)l
Temperature
60° C . 750 C .
Surface Active Agent Type Concentration ug ./mq .
0 4 0

Sodium heptadecyl sulfate Anionic 15 .3 15 .2 45 .1 41 .5

Sodium dodecyl sulfate Anionic 18 25 .0 37 .4 63 .3

Sodium dioctyl sulfosuccinate Anionic 11 .9 11 .6 34 .9 34 .9

Cetyl trimethyl ammonium Cationic 12 .9 12 .6 36 .0 28 .0

bromide

n-Dodecylamine acetate Ampholytic 11 .6 12 .4 38 .0 37 .4

Polyoxyethylene sorbitan Nonionic 11 .4 11 .3

monostearate

Sorbitan monostearate Nonionic 11 .1 10 .4

1 The nontreated controls of cells of RJR 2453-2 exhibited enzymatic activities
of 10 .6 and 28 .9 pU/mg . at 60 and 750 C ., respectively . ''
 13

d .- Anionic Surface Active Agents

The effects of the two most active surface active a ents, sodium
dodecyl sulfate (SDS) and sodium heptadecyl sulfate (HDS~, were evalu-
ated at a wide range of detergent concentration . The results in
Figure 1 indicate that the SDS exhibits a greater stimulation of
enzymatic activity than did the HDS . It is evident that SDS has a
marked optimum concentration for increasing the enzymatic activity .
It was observed in these experiments that the surface active agent
temperature effects were also influenced by the age and morphology of
the cells . Older cells in the rod form yielded greater increases in
enzymatic activity than young cells in the coccoid or involuted forms .

e . Glucose Isomerase Activity of Culture RJR 2453-2 After 24

Hours at 75° C .
-------------------------------
The effect of sodium dodecyl sulfate (SDS) at a concentration of
10 pg ./mg . on the enzymatic activity (60° C .) of RJR 2453-2 cells in a
2M glucose syrup was determined at 75° C . The glucose syrup was buffered
at pH 7 .0 with phosphate buffer . The initial enzymatic activity of the
cells was 3 .6 and 5 .1 pU/mg . with and without SDS, respectively . After
24 hours at 75° C ., the enzymatic activity was 6 .0 and 5 .8 uU/mg . with
and without SDS, respectively . The percent conversion of glucose to
fructose .was 14 .3% with no cells, 20 .7% with cells alone, and 25 .4%
with cell and SDS . The conditions for conversion were not optimum
since no attempt was made to control the pH . These results indicate
that a 24-hour treatment of the cells at 750 C . does not significantly
affect the enzymatic activity either in the presence or absence of SDS .

C . DISCUSSION

The production of glucose isomerase by Arthrobacter RJR 2453-2 can be

regulated by culture and nutritional conditions . The technique of culture
activation utilizing short-time incubations increased the enzymatic activity
of final cells . The pleomorphic characteristics of this organism make it
imperative that cells in the same morphological state be used as an inoculum
for enzyme production . The increased enzymatic activity using inexpensive
bagasse xylose as a replacement for pure xylose ($17 .50/lb .) and the inex-
pensive nitrogen and vitamin sources would reduce the medium costs . This
fermentation could then be comparable in cost to other glucose isomerase
fermentations .

Since this is an aerobic fermentation, the rate of oxygen transfer into

the medium is very important in controlling the production of glucose isomerase .
Indirect measurements have indicated that a critical oxygen level is necessary
for high enzymatic activity . These results also explain the difficulties in
scale-up from shake flasks to the 30-gallon fermentators .

The surface active agent, sodium dodecyl sulfate, increased the glucose
isomerase activity of cells of RJR 2453-2 by 2 .5 to 6-fold dependent on the
assay temperature . Since this anionic detergent is approved for food use,
 •
14

FIGURE 1

701 ANIONIC SURFACE ACTIVE AGENTS

AND ASSAY TEMPERATURES

rn
E

r
CJ

. Sodium Heptadecyl Sulfate - 60° C .

0 Sodium Heptadecyl Sulfate - 75° C :
o Sodium Dodecyl Sulfate - 60° C .
a Sodium Dodecyl Sulfate - 75° C .

.A,00
a
Concentration pg ./mg .
0
 15

there shouldn't be any problem in clearing it for enzymatic use especially

in view of the very low concentration required to enhance enzymatic activity .
The action of the surface active agents is probably on alteration of the
permeability of cell to glucose, either by changing the cell surface or
the activation of a glucose permease which would allow a better diffusion
of the substrate into the cells . These suppositions are supported, in part,
by the greater increase in enzymatic activity in rod shaped cell . Coccoid
cells are generally known to have more compact cell walls, and these cells
exhibited lower enzymatic activity with or without detergent treatment .

D . CONCLUSIONS

1 . The glucose isomerase activity of cells of RJR 2453-2 can be increased

by culture activation techniques .

2 . Variations in aeration rates will affect the glucose isomerase

activity of RJR 2453-2 cells .

3 . A recrystallized form of bagasse xylose could replace expensive

xylose with no loss of enzymatic activity . Inexpensive nitrogen and vitamin
materials could also replace the commercial tryptone and yeast extract .

4 . The anionic surface active agent significantly increased the glucose

isomerase activity of culture RJR 2453-2 . Sodium dodecyl sulfate exhibited
greater increases in activity than the other anionic detergents . Other types
of surface active agents did not exhibit any significant increase in enzymatic
activity .

5 . The enzymatic activity was increased two to threefold after correction

for thermal isomerization when the assay temperature was increased from 60 to
75° C .

6 . No significant reduction of enzymatic activity in cells of culture

RJR 2453-2, either in the presence or absence of sodium dodecyl sulfate, was
observed after a 24-hour treatment in 2 M glucose at 75° C .

E . RECOMMENDATIONS

I . Patentability

1 . The genus, Arthrobacter, is not mentioned in any glucose isomerase

patents ; therefore, this organism and process should be patented .

2 . The process of utilizing anionic surface active agents for increasing

the glucose isomerase activity of whole cells of Arthrobacter RJR 2453-2 is
patentable .

es . Phei

(See next page for Di stri buti on) Approved

: 0,(~ i)` LAe'.'
 16

Distribution :

Dr . Murray Senkus Mr . Manford R . Haxton

Dr . R . E . Farrar Dr . Herbert J . Bluhm
Mr . E . H .HarwodD
. Eldon D . Nielson
Dr . Charles G . Pheil
Library (2)
Dr . William C . Squires

Submitted : September 23, 1968

Completed : September 26, 1968

From manuscript :pws
 17

BIBLIOGRAPHY

1 . Campbell, Allen, S nchronization of Cell Division . BACTERIOL . REV .,

21, 263-272 (1957 .

2 . Ensign, J . C . and Wolfe, R . S ., Nutritional Control of Mor ho enesis

in Arthrobacter crystallopoietes . J . BACTERIOL ., 87, 924-932 (19647 .

3 . Hughes, D . E ., The Effect of Surface Active Agents on Bacterial

Glutamic Decarbox lose and Glutaminose . BIOCHEM . J ., 46, 231-236
1950 .

4 . James, W . B ., Automated Analysis of Fructose . RDM, 1968, No . 2

(January 5) .

5 . Lartigue, D . J ., Definition of Glucose Isomerase Unit . R . J .

Reynolds Research Notebook, 1967, 161677 February .

6 . Machida, Y ., Isomerization of Glucose b Microor anisms . CHEM . ABSTR .,

65, 4295d (August 1, 1966 .

7 . Marshall, R . 0 . and Kooi, E . R ., Enz atic Conversion of D-Glucose

to D-Fructose . SCIENCE, 125, 648-649 (1957) .

8 . Marshall, R . 0 ., Enzymatic Process . U . S . Patent 2,950,228

(August 23, 1960) .

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