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TABLE OF CONTENTS

Page

OBJECT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4A . INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 5B . EXPERI MENTAL . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

I . Analytica l Method for the Determination of

Fructose and the Glucose Isomerase Activity

of Arthrobacter Cells . . . . . . . . . . . . . . . . . . . . 5

I I . Growth Medium and Aeration Conditions for

Enzyme Producti on . . . . . . . . . . . . . . . . . . . . . .

Cultural C onditions and Nutrients for Control

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

5

6

a . Cul tural C ondi ti ons . . . . . . . . . . . . . . . . . . 6b . Nutritional Control . . . . . . . . . . . . . . . . . . 7

I V . Effect of A eration on Glucose Isomerase Activity . . . . . . 7V . Incorporation of Inexpensive Substrates into the

Basal Medi um . . . . . . . . . . . . . . . . . . . . . . . . 8a . Xylan and Xylan Containing Substrates . . . . . . . . . 8b . Bagasse as a X ylose Source . . . . . . . . . . . . . . . 9c . Replacement of Tryptone and Yeast Extract . . . . . . . 10

V I . Surface Active Agents and Temperature on

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

a . I ncubati on T emperature . . . . . . . . . . . . . . . . . 11

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

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

d . Anionic Surface Active A gents . . . . . . . . . . . . . 13

e . Glucose Isomerase Activity of Culture RJR 2453-2

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

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TABLE OF CONTENTS (Cont'd)

Page

C . DISCUSSIO N . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

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

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

I . Future Work . . . . . . . . . . . . . . . . . . . . . . . . . 1 5

I I . Patentabi li ty . . . . . . . . . . . . . . . . . . . . . . . . 1 6

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 7

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LIST OF TABLES

Table No. Title PageI Effect of Preincubations at 30 C.

on Glucose Isomerase Activity i n

XS-2 B roth . . . . . . . . . . . . . . . . . . . . 6II Different Flasks and Flask Closures . . . . . . . 8III Bagasse Xylose Fractions . . . . . . . . . . . . . 9IV Gas Liquid Chromatography of the

Bagasse Xylose Fractions . . . . . . . . . . . . . 1 0

V Utilization of Inexpensive Nitrogenand Vi tami n Sources . . . . . . . . . . . . . . . 10

VI Assay Temperature and VariousConcentrations of Sodium Heptadecyl

S u l f a t e . . . . . . . . . . . . . . . . . . . . . 1 2

VII Various Surface Active Agents . . . . . . . . . . 1 2

LIST OF FIGURES

Figure No. Title Page1 Anionic Surface Active Agents and

Assay Temperatures . . . . . . . . . . . . . . . . 1 4

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A . INTRODUCTION

Enzymes which catalyze the interconversion of free aldopentose and

ketopentose have been demonstrated in several bacterial strains (12) . In1957, 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 fructo se 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 d etermined 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 .

I I . Growth Medium and Aeration Conditions for Enzyme Produc tion

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

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

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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 ; MgSO47H2O, 0 . 2 5 ; 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 .

I I I . 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 elong ate into pleomorphic

rods . Cell division occurs during the rod stage and continu es 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 1 2-hour transfers .

The degree of culture synchronization was reflected in the final

enzymatic activity (Table I) .

TABLE I

EFF ECT OF PREINCUBATIONS AT 30 C .

ON GLUCOSE ISOMERASE ACTIVITY IN XS-2 BROTH

Number and Time Total Incubation Enzymatic Activity_of Initial Transfers Time u U m l . uU/mg .2-8 hours 65 hours 622 1 8 . 83-8 " 65 " 640 1 9 . 43-8 " 72 " 1,175 3 5 . 22-12 " 65 " 580 1 8 . 12-16 " 65 " 305 9 . 5

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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 .

I V . 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 .

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TABLE II

DIFFERENT FLASKS AND FLASK CLOSURES

Glucose Isomerase

Activit 1

Flask Size and Type Closure uU/ml . uU/m .

5 0 0 m l . - unbaffled CS2 5923 2 0 . 9

FP 707 2 3 . 3

3 0 0 m l . - 4-baffled C S 5 7 2 2 1 . 6

F P 2 4 1 9 . 8

1 , 0 0 0 m l . - unbaffled C S 3 7 7 1 6 . 3

F P 700 2 4 . 1

1 , 0 0 0 m l . - 4-baffled Metal Cover 535 2 1 . 4

1Cells of RJR 245 3-2 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 h igh enzymatic activity .

The ef fects of increasing cell density on aeration rates, as measured

by dissolved oxygen activity, w ere determined using an oxygen probe . Thedata was expressed as dissolved oxygen activity (DOA) realizing how ever

that w e are actually measuring oxygen partial pressure and/or oxygen

activity rath er than the actual concentration of dissolved oxygen . TheDOA is high initially, 90- 100, with an aeration rate of 2 .1 L/min . ; but

as the cells reach the logarithmetric phase of growth, th e DOA was reduced

to 70 ; and during the stationary phase of growth, the D OA 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 D OA to 80 . Th e agitation rate was constant

during these experiments .

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

increased aeration rates may be requi red to increase the ox ygen transfer

rate ; and th e rate of solution of oxygen may not equal or exceed the rate

at which oxygen is required by the aerobic Arthrob acter cells .

V . Incorporation of Inexpensive Substrates into the Basal Medium

a ._ ~ylan and ~ylan Containing Substrates

The h igh cost of pure xylose prohibits its use in a commercial

fermentation . Therefore, inexpensive substrates were evaluated for

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

b ._ Bauasse as a Xylose Source_Whole and ground bagasse stalks could support the growth ofArthrobacter; however, the glucose isomerase activity was less than100 uU/ml .The effect of crude xylose from bagasse and two recrystallizedbagasse xylose (Pfanstiehl No . 1, No. 2, and No. 3) samples wasevaluated for their ability to support the production of glucose

isomerase in cells of RJR 2453-2 . The crude xylose (P-1) yieldedreduced enzymatic activity while the recrystallized fractionsincreased the enzymatic activity (Table III) .

TABLE IIIB A G A S S E X Y L O S E F R A C T I O N S

Glucose IsomeraseActivitylXylose Source (2%) uU ml . uU m .Pure Xylose 44 7 16 . 3Bagasse Xylose (P-1) 1 46 4 . 7Bagasse Xylose (P-2) 614 22 . 4Bagasse Xylose (P-3) 76 1 27 . 81Cells were assayed after 69 hours at 300 C .

Combinations of commercial and bagasse xylose (P-1) yielded cellswith lower enzyme activity than the equivalent concentration of regularxylose. The data in Table IV suggest that the inhibitory substance inP-1 is glucose while the increased enzymatic activity of the cells grownin P-2 and P-3 is due to the absence of glucose .

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1 0

TABLE IV

GAS LI UID CHROMATOGRAPHY

OF THE BAGASSE XYLOSE FRACTIONS

Relative %

Bagasse Xylose Ribose and

Fractions Xylose Glucose Mannose Ar abinose

P - 1 6 9 . 4 1 % - - 1 %

P- 2 9 7 . 7 N D 1 Trace Trace

P- 3 97 . 3 ND ND Trace

1ND signifies none det ecte d .

c ._ Replac ement of Tryptone and Yeast Extra ct

Enzyme hydrolyzed case in (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 t he yeast extra ct ($8 .40/lb .) could be replac ed

by BFY yeast (Amber Labs - $0 .34/lb . ) . Higher gluc ose isomerase ac tivity

was noted with the case in than with the animal protein hydrolysate while

no differenc e was observed with the BFY yeast when casein was used, but

significantl y lower acti vity was observed with the BFY yeast and animal

protein .

TABLE V

UTILIZA TION OF INEXPENSIVE NITROGEN AND VITAMIN SOURCES

Glucose Isomerase Activi tyl

Media u U / m l . uU m .

Basal - XS-2 broth 4 4 7 1 6 . 3

Casein + yeast extract 4 9 5 1 9 . 0

Animal protei n + yeast extrac t 2 5 1 1 1 . 1

Tryptone + BFY yeast 524 1 9 . 7

Casein + BFY yeast 5 0 9 1 6 . 8

Animal protein + BFY yeast 2 9 9 9 . 1

1RJR 2453- 2 cel ls were assayed after 70 hours at 30 C .

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V I . 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 . Th e add itio n of

this detergent increased the enzymatic activity at all temperatures ;

however, the two effects, assay temperature and detergent concentration,

were not additive .

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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 7 0 75 80

0 9 . 4 1 4 . 0 2 1 . 0 2 6 . 6 2 9 . 5

2 . 3 1 1 . 1 1 6 . 5 26 33 33 . 2

4 . 0 1 9 . 4 - - 3 9 3 6 3 2

20 . 0 1 6 . 8 1 8 . 5 3 3 . 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 tempe ratures (Table VII) .

Generally, the anionic agents, especially sodium dodecyl sulfate, yielded

a greater incre ase in the enzymatic activity .

TABLE VII

VARIOUS SURFACE ACTIVE AG ENTS

Relative Glucose Isomerase Activity

(pU/mg)l

Temperature

60 C . 750 C .

Surface Active Agent Type C o n c e n t r a t i o n ug ./mq .

0 4 0

Sodium heptadecy l sulfate Anionic 1 5 . 3 1 5 . 2 4 5 . 1 4 1 . 5

Sodium dodecyl sulfat e Anionic 1 8 2 5 . 0 3 7 . 4 6 3 . 3

So di um di oc ty l s ul fo su cc in at e An io ni c 1 1 . 9 1 1 . 6 3 4 . 9 3 4 . 9

Cetyl trimethyl ammonium

bromide

Cationic 1 2 . 9 1 2 . 6 3 6 . 0 28 . 0

n-Dodecylamine ace tate Ampholytic 1 1 . 6 1 2 . 4 3 8 . 0 3 7 . 4

Polyoxyethylene sorbitan

monostearate

Nonionic 1 1 . 4 1 1 . 3

Sorbitan monostearate Nonionic 1 1 . 1 1 0 . 4

1

The nontreate d controls of cells of RJR 2453-2 exhibited enzymatic activitie s

of 10 .6 and 28 .9 pU/mg . at 60 and 750 C ., respectively . ''

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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 increas es in

enzymatic activity than young cells in the coccoid or involuted forms .

e . Glucose Iso meras e Acti vity 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 a ctivity (60 C .) o f RJR 2453-2 cells in a

2M glucose syrup was determine d at 75 C . T h e g l u c o s e s y r u p w a s b u f f e r e d

at pH 7 .0 with phosphate buffer . T h e i n i t i a l e n z y m a t i c a c t i v i t y o f t h e

cells was 3 .6 and 5 .1 pU/mg . with and without SDS, respectively . After

24 hou rs a t 75 C ., the enzymatic activity was 6 .0 and 5 .8 uU/ mg . with

and without SDS, respectively . T h e p e r c e n t c o n v e r s i o n o f g l u c o s e t o

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 t he cells at 750 C . does not significantly

affect the enzymatic activit y either in the presence or absence of SDS .

C . D ISCUSSION

The producti on of gluc ose isome rase by Art hrobacter RJR 2453-2 can be

regulated by culture and nutritional conditions . The tec hnique of cu lture

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 . T h e i n c r e a s e d e n z y m a t i c a c t i v i t y u s i n g i n e x p e n s i v e

bagasse xylose as a re placement for pure xylose ($17 .50/lb .) and the inex-

pensive nitrogen and vitamin sources would reduce the medium costs . T h i s

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 indicat ed that a critical oxygen level is necessar y

for high enzymatic activity . These results also explain the difficulties in

scale-up from shake flasks to the 30-gallon fermentator s .

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,

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AND ASSAY TEMPERATURES

FIGURE 1

701 ANIONIC SURFACE ACTIVE AGENTS

rnE

rCJ

1 4

. Sodium Heptadecyl Sulfate - 60 C .

0 Sodium Heptadecy l Sulfate - 75 C :

o Sodium Dodecyl Sulfate - 60 C .

a Sodium Dodecyl Sulfate - 75 C .

Concentration pg ./mg .

. A ,00

,

a

0

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1 5

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 ac tivity 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 expensi ve

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 corr ection

for thermal isomerization when the assay temperature was increased from 60 to

75 C .

6 . No significant reduction of enzyma tic 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 incre asing

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

patentable .

e s . P h e i

(See next page for Di stri buti on) Approved : 0,(~ i)` LAe'. '

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Distribution :

Dr. Murray Senkus Mr. Manford R . HaxtonDr . R . E. Farrar Dr Herbert J . BluhmMr . E . H

. Eldon D . Nielson

Dr . Charles G . Pheil

Library (2)

Dr . William C . Squires

Submitted : Septemb er 23, 1968

Completed : September 26, 1968

From manuscript : p w s

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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

1 9 5 0 .

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 .

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