In

Author : Donald J . Lartigue August 26, 1969

Division : Biological Research Notebook Pages : 153001-50,

161651-700, 163851-900, .
RDR, 1969, No . 29 165751-800, 167551-600,
170801-50
No . of Pages : 22 Dated : June 21, 1966 to
July 23, 1969

Previous Reports : None

ENZYMATIC ISOMERIZATION OF D-CLUCOSE

BY ARTHROBACTER CELLS

OBJECT :

To investigate the characteristics of the glucose isomerase from

RJR 2453 cells and to determine the optimum conditions for the prepara-
tion of fructose-containing corn syrups .

S UMMARY :

Cells of Arthrobacter sp . are capable of isomerizing glucose to fructose

to approximately an equimolar equilibrium . The characteristics of this
enzyme have been studied to determine the optimum conditions for assay and
for use in commercial syrup production . This work is part of a cooperative
effort and is reported only as a summary of the work done by this laboratory
and is not to be considered as indicative of the status of the entire
project .
TABLE OF CONTENTS

Page

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

S WtARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . 3

A . INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . 4

B . EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . . . . 5

I . Enzyme Assay . . . . . . . . . . . . . . . . . . . . . . . 5

II . Enzyme Characterization . . . . . . . . . . . . . . . . . 5

a . Effect of pH . . . . . . . . . . . . . . . . . . . . . 5

b . Effect of Temperature . . . . . . . . . . . . . . . . 7

c . Effect of Substrate Concentration . . . . . . . . . . 7

d . Effect of Metal Ion Concentration . . . . . . . . . . 7

e . Determination of Equilibrium Conditions . . . . . . . 10

f . Enzyme Stability . . . . . . . . . . . . . . . . . . . 10

III . Syrup Production . . . . . . . . . . . . . . . . . . . . . 10

a . Effect of pH on Syrup Production . . . . . . . . . . . 11

b . Effect of Temperature on Syrup Production . . . . . . 11

c . Effect of Cell Concentration on Syrup Production . . . 11

d . Optimum Conditions for Syrup Production . . . . . . . 11

C . CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . 15

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

APPENDIX I - Characteristics of Isomerizing Enzyme . . . . . . . . . 17

 3

LI ST OF FI GUF:ES

Figure No . Page

I Effect of pH on Isomerase Activity . . . . . . 6

II Effect of Temperature on Isomerase

Activity . . . . . . . . . . . . . . . . . . . 8

III Effect of Substrate Concentration on

Isomerase Activity . . . . . . . . . . . . . . 9

IV Effect of Temperature on Syrup Production . . 13

V Effect of Cell Concentration on Syrup

Production . . . . . . . . . . . . . . . . . . 14
 4

A . INTRODUCTION

A process for the enzymatic conversion of high DE corn syrups to

syrups containing a mixture of Oucose and fructose has been actively
investigated by the Microbiology Section since April, 1966 . One approach
initially investigated was via a phosphorylated pathway :

Pi Pi

Starch > Glucose-l-Phosphate -.1 > Glucose

Phosphorylase / Phosphatase

Phosphoglucomutase

Glucose-6-Phosphate

NOTE : Pi - inorganic Phosphohexoisomerase

phosphate

W Pi
Fructose-6-Phosphate Fructose
Phosphatase

Thus, starting from starch and using phosphorylase and a mixture of glycolytic
enzymes, a syrup containing glucose and fructose could be obtained .

This approach soon was abandoned in favor of the simpler bacterial

isomerase which converts glucose to fructose in a one-step reaction of non-
phosphorylated intermediates .

The investigation of this enzyme was a cooperative effort and the work
performed by this laboratory was in the area of enzyme characterization,
assay, and utilization .
 5

B . EXPERIMENTAL

1 . Enzvme Assay

Cells were harvested by centrifugation, suspended in water, and incubated

with glucose at 60°C . Fructose formed was measured by a modification of the
.
Seliwanoff reaction as modified by W . James . Activity was reported as
UUnits/ml whole culture, and the unit was defined as that quantity of enzyme
which would produce one gram of fructose from glucose in one minute at 60°C
using 1M glucose in 0 .1M phosphate buffer pH 7 .0 and 0 .O1M MgC12 .

II . Enzyme Characterization

a ._ Effect of pH

Cells were incubated with 1M glucose in the presence of 0 .1M buffers

and 0 .O1M MgC12 at 60°C for intervals up to 1 hour . Seven buffers were
employed and the range from pH 3 .5 to 10 .0 was studied . The results,
shown in Figure I, are corrected for alkaline isomerization and illustrate
that the enzyme is inactive at pH's below 5 .5, gives the same activity
in arsenate buffer as in phosphate buffer, and is inhibited by buffers
containing nitrogen . No clearly defined optimum pH was observed with
the uninhibited enzyme, but it is obvious that the optimum lies above pH 8 .
 6

FIGURE I

EFFECT OF pH ON ISOMERASE ACTIVITY

300 I I

Activity 200
(ug Fructose
per minute)

100

I
I

L----~---~+--b-~--~ ~.~. W .~i...a1.~

3 .0 4 .0 5 .0 6 .0 7 .0 8 .0 9 .0 10 .0

pH
Buffers : Conditions :

a -e n-^etate pH 3 .5 - 6 .0 Substrate - LM Glucose

d-- A°'alate pH 5 .5 - 7 .0 Buffers - 0 .1M

0 -Ori:osp'.:ate pH 6 .0 - 8 .0 `'gC12 - 0 .01M

r_,. --O'.rsenzte •HC1 pH 6 .0 - 8 .0 Temperature - 60°C

C--sir:icazole •HC1 pH 6 .0 - 8 .0

O---O ; lycine •' :aOH pH 7 .5 - 10 .0

4__ ATris •HC1 pH 7 .0 - 9 .0

 7

b .- Effect of Temperature

Cells were incubated under standard conditions at temperatures

between 40 and 70°C . The amounts of fructose formed at the different
temperatures are shown in Figure II . A maximum temperature was not
observed under these conditions, for thermal isomerization becomes
.
important at temperatures in excess of 70°C . Little activity was
observed at temperatures below 50°C .

c . Effect of Substrate Concentration

-------------------

The amount of glucose present in the standard assay was varied

from 0 .01M to 1 .0M . Incubation with whole cells gave the results
shown in Figure III . If we assign a maximum velocity value (Vmax)
as the plateau region of the curve, a Michaelis constant (Km) for the
enzyme can be estimated since Km is defined as that concentration of
substrate which yields half the maximum velocity . A Km of 0 .15 M is
thus obtained which is in good agreement with reported values .

This experiment was run to insure that a 1M substrate concentration

as used in the standard assay was adequate . Further work on deter-
mining Km's using whole cells and cell-free preparations and using both
xylose and glucose as substrates were run . 'This work was reported in
a special memorandum which is reproduced in Appendix I* .

d . Effect of Metal Ion Concentration

-------------------

A concentration of 0 .01M_ MgCl2 was used in all of this work .

This concentration was in excess of the enzyme's requirement and
adequate to insure maximum activity . Minimum concentrations required
may be readily determined once a standard cell preparation is obtained .

*Since the work in Appendix I was done on the mutant strain of

Arthrobacter (RJR 2453-F), it is not included in the body of this
report which deals with the parent culture .
 CouqrGroua 12 Ds2CL ;psq ru ZS :cG

;LsmoetsGnts (oC)

50

10

66t 50 wr,unGsa)
(113 EtncGoas

ycGrnr,CX

QO

80

EE.bEC .L OE ,LEiIbE6Y ..r.CKE Oi! I20iIEdi2fi YC .LIi1ILd

~I(:~~E II
 FIGURE III

EFFECT OF SUBSTRATE CONCENTRATION ON ISOMERASE ACTIVITY

Rate of Reaction or v (See Text f6r Discussion)

(uK Fructose/min .)

i
i
30~ • ~ -, ...
Vmax . -- - ~-- - ~- -
1 0

/
I
I 0
/
/
/

/
20`-- / %O

1
/

9
U .7 .8 ' .9
.1 .2 .3 .4 .S .6

LZ/- tj oc,pp ; ~ Molar Glucose Concentration [sl 19 1 . #

 10

e ._ Deternination of_Eguilibrium Conditions

Whole cells were incubated with mixtures of analytical grade

glucose and fructose at a total concentration of 360 g hexose per
liter for 48 hours at 60°C . The amount of enzyme employed was
0 .044 units/100 g substrate and the pH was maintained at pH 7 .4 f 0 .2
by 0 .1M phosphate buffer . Fructose was determined by optical rotation
and the results are given in Table I . Thus, an equilibrium concentra-
tion of about 50% fructose is obtained as expected by the similarity
of the free energy of the two carbohydrates .

TABLE I

EQUILIBRIUM CONCENTRATIONS IN ISOMERIZED SYRUPS

Initial Concentration (%)

- - - Concentration of
Syrup No . Glucose Fructose Fructose after 48 Hours (%)

28 65 35 47 .8
29 60 40 48 .5
30 55 45 49 .0
27 50 50 48 .8
31 45 55 49 .5
32 40 60 49 .6
33 35 65 50 .0

f ._ Enzyme Stability

The harvested cells could be stored at refrigerator temperature

(4°C) for about a week or at freezer temperature for several months .
During storage, a slight increase in activity was observed .

III . Syrup Production

Over 100 laboratory-scale conversions were run during the course of

this study . Initially, syrups were run at 60°C and pH 7 .65, conditions
where thermal and alkaline conversions were reduced to a minimum ; thus,
the enzymatic conversion only was observed . Under these conditions 0 .035
units per 100 g glucose would result in about a 40% conversion in 24 hours .
The prnduct had a light amber color and a pleasant taste . No appreciable
loss of carbohydrate concentration was detected .
 11

a .- Effect of PH on Svrup Production-

Glucose syrups (2M) were incubated with 0 .035 units of enzyme

per 100 g of glucose at 60°C and at pH 7 .65, 8 .0, 8 .5, 9 .0, and 9 .5
to determine the effect of pH . Enzyme-free controls were run to
determine the extent of alkaline conversion observed under these
conditions . The results indicated that a pH of 8 .0 to 8 .5 is most
satisfactory since color development is reduced and adequate enzyme
activity is retained . At pH 9 .5, a 41 .2% conversion of glucose was
effected in 24 hours when cells were present and a 36 .2% conversion
in the absence of cells . Both syrups were extremely dark and a 14%
loss in total carbohydrates was observed .

b .- Effect of Temperature on SYrupProduction

Glucose solutions (2M) were incubated with 0 .035 units per 100 g
of substrate at pH 7 .65 and at 60, 65, 70, 75, and 80°C . Enzyme-free
controls were run and the glucose conversion was followed by optical
rotation . The results are shown in Figure IV . Therefore, a tempera-
ture of 70°C is considered optimum .

c .- Effect of Cell Concentration on ~ruP Production

- -- --- -------- ---

Glucose solutions (2M) were incubated at 60°C and pH 7 .65 using

different concentrations of enzyme . The results are shown in
Figure V . The objective was to produce a 40% conversion in 24 hours .
Therefore, an enzyme concentration of 0 .035 units per 100 g substrate
was chosen .

d .- .Qptimum Conditions for Sy_rupProduction

The above studies in syrup conversions were run to determine the

optimum conditions of conversion . The results obtained were as follows :

1 . pH = 8 .0 to 8 .5
2 . temperature = 70 f 5°C

3 . substrate concentration = 1 to 2M glucose

4 . time = 8 to 10 hours

Under these conditions the enzyme concentration required remained un-

changed from that previously employed, and the product obtained was
not significantly different from that previously obtained .

High DE syrups prepared by various techniques were all satisfactory

but did give a slightly lower degree of conversion than did pure glucose
at 2'•i concentration . A typical syrup conversion was conducted as follows :

A glucose syrup prepared by the dual enzyme conversion which had

a DE of 97+, a solids concentration of 28 to 40% on a dry weight basis,
and a pH of 4 .0 was first adjusted to pH 8 .25 using 10% KOH .

The ' ;gd+ cc: .iccntration was brought to 0 .0L'•1 by the :dc' t,on of the
roquired ~711ount of .~.,OIC12 dissolved in - :inimum ;;~.ter . The t-:,,erature of
 J-1

the syrup was maintained at 70°C . An aqueous slurry of whole cells

containing the required units calculated on the basis of glucose
concentration as determined by DE and solids content was slowly added,
and the pH was maintained at 8 .25 by the automatic addition of 10% KOH .
Samples were taken periodically and the fructose content was determined
by the optical rotation procedure . After 8 to 10 hours of incubation,
the fructose concentration was about 40%, the syrup was an amber color,
and a total loss of carbohydrate of about 6% was experienced .
 13

FIGL'RE I V

EFFECT OF TEMPERATURE ON SYRUP PRODUCTIO N

..
50 1 .I I
_Q,
Total
i '• GJ Conversion
X Conversion r
\

40

30

20

Conversion in
Cell-Free
10 Control s

~ 60 65 70 75 80

Trmnerature (-°C )

See Text for Discussion

~_J
 14

FIGURE V

EFFECT OF CELL CONCENTRATION ON SYRUP PRODUCTION

(See Text for Discussion)

50 I 1 1 1 1

% Conversion

40

30

2C

10

5 10 15 20 25 T 0

Hours

A = Syrup 48-1
0 .014 units/100 g

B = Syrup 48-2
0 .028 units/100 g

C = Syrup 48-3
0 .042 units/100 g
 15

C . CONCLUSIONS

The majority of the work described in this report was done prior to
October, 1967 . Despite considerable advancements in other aspects of this
project, these observations are still indicative of the enzyme involved .

Donald 3! Lartigue

Distribution :

Mr . Edward A . %'assallo Mr . Manford R . Haxton

Dr . Murray Senkus Dr . Herbert J . Bluhm
Dr . R . E . Farrar P 'ro/i , S-1c-7o
7Gr . !'IerSf^ c ( /~4TL
Mr . E . H . Harwood
Dr . Eldon D . Nielson ? ~ . 4 e A- r . M . Lo•j-~ ~1-G-7Y
Dr . C . G . Pheil
Dr . D . J . Lartigue
Library (2)

Submitted : August 25, 1969

Completed : August 26, 1969

From manuscript/vbl

r
Approved :
 16

REFERENCES

The literature on isomerases has been studied in detail and will

not be reviewed in this report .