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C O N T I ; E ~ , , f i f I A L

A u t h o r : Chin K . Lee April 14, 1969Division : Biological Research Notebook Pages : 195720, 25, 28-3,6

203651, 55-63, 68-72, 76-97, 99 ;RDR, 1969, No . 11 204855-57) 011 '?t / - r)5'r

D a t e d : November 19, 1968 toNo . o f P a g e s : 23 March 25, 1969

Previous Reports : None

DEVELOPMENT OF MUTANT OF ARTHROBACTER PRODUCING GLUCOSE ISOMERASEWHICH IS CONSTITUTIVE AND INSENSITIVE TO CATABOLITE REPRESSION

OBJECT :

To develop a mutant of Arthrobacter which produces high levels ofglucose isomerase in the presence of D-glucose without requiring D-xyloseas an inducer .

SUMMARY:

RJR strains of Arthrobacter, 2453-2 and 2453-Y, produces glucose isomeraseonly when D-xylose is provided in the medium . Addition of glucose or othercarbohydrates severely represses the enzyme synthesis . These problems were

resolved by the development of a double mutant which is both constitutive andinsensitive to catabolite repression . The mutant produces 700 to 800 u units/ml .of glucose isomerase in a newly formulated media containing inexpensive sourcesof vitamin and organic nitrogen and 2% D-glucose as the principal source ofcarbon and energy .

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TABLE OF CONTENTS

Page

OBJECT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , 1

S U M M A R Y . . . . . . . . ..

. . . . . . . . . . . . . . . . . . . . . .1

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

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

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

I . Materials and Method . . . . . . . . . . . . . . . . . . . . . 5

a . Culture Media . . . . . . . . . . . . . . . . . . . . . 5

b . Organisms . . . . . . . . . . . . . . . . . . . . . . . . 6

c . Determination of Enzyme Activity . . . . . . . . . . . . . 6

d . Measurement of Growth . . . . . . . . . . . . . . . . ..

7

II . Induction and Repression of Glucose Isomerase in RJR 2453-2 . . 7

III . Development of the Constitutive Mutant from RJR 2453-2 . . . . 8

a . Selection Method of the Constitutive Mutant . . . . . . . 8

b . Properties of the Constitutive Mutant . . . . . . . . . . 9

IV . Development of the Constitutive Mutant from RJR 2453-Y . . . . 10

a . Selection and Properties of the Mutants. . . . . . . . . 10

b . Controlled Addition of Glucose to the Mutant Culture . . . 1 2

V . The Effect of Phosphate Concentration on the IsomeraseS y n t h e s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4

VI . Development of a Constitutive and Catabolite Repression-Insensitive :futant (Double Mutant) . . . . . . . . . . . . . . 1 4

a . Screening of the Mutant . . . . . . . . . . . . . . . . . 1 4

b . Glucose Isomerase Production by F-Mutant . . . . . . . . . 1 5

VII . New Sources of Vitamins and Organic Nitrogen . . . . . . . . . 1 7

VIII . Isomerase Production by F-Mutant in the 30-Gallon Fermentor . . 1 7

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TABLE OF CONTENTS (Cont'd)

P a g e

C . DISCUSSION ..

.. . . . . . . . . . . . .

. . . . . . . . . . . . 20

D .

CONCLUSIONS. . . . . . . . . . . . . . . . . .

. . . . . . . . . 20

E . R E C O I ~ . " 4 E N D A T I O N S . . . .. . . . . . . . . . . . . . . .

. . . . . 21

I . Future Work . . . . . . . . . . . . . . . . . . . . . . . . . 21

II . Patentability . . . . . . . . . . . . . . . . . . . . . . . .21

F . B I B L I O G R A P H Y . . . . . . .. . . . . . . . . . . . . . . . . .

. . 2 3

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LIST OF TABLE S

Table No . Tt l e Pag e

I Effect of Var ious Car bon Source o

the Synthesis of Glucose Isomerase i nStrain 2453-2 . . . . . . . . . . . . . . . . 7

II Synthesi s of Gucose Isomer ase by W l dTypes (2453-2) and Mutant with and withou tInducer . . . . . . . . . . . . . . . . . . . 9

III Effect of Var ious Car bohydrat es o nth e.Constitutive Synthesis of Glucose Isomeras e

by C-3 Mutant . . . . . . . . . . . . . . . . 1 0

IVSynthesi s of Gucose I somerase i nth ePresence and Absence of Xylose by Wild Typ eand Mu t ant s . . . . . . . . . . . . . . . . . 1 1

VSynthesi s of Gucose Isomer ase i n2%Gucos eby Wild Type and Mutants . . . . . . . . . . 1 2

VI Schedule of Glucose Addition to EYC-2-b . . . 1 3

VI Respo nse o f EYC2- btoControlled Gucos

Addition . . . . . . . . . . . . . . . . . . 13

VII Effect of Phosphat e Concentrat iononth eGlucose Isomerase Synthesis in EYC-2-b . . . 1 4

IXSchedule of Gucose AdditiontoF-Mutant . . 1 6

XSynthesi s of Gucose I somer ase by F -Mutant . . 1 6

XI Compar ison of Vt amns an dOrgan ic Ntroge nSources . . . . . . . . . . . . . . . . . . . 17

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A . INTRODL'CT10N

RJR strains of Arthrobacter (2,3) are capable of producing intracellularglucose isomerase in complex media supplemented with xylose . The enzymeformation by these strains, however, is severely repressed by the presence ofmore readily metabolizable carbohydrates such as glucose . This absoluterequirement for xylose as the inducer, and the inability of glucose to replace

xylose, place this species of organisms at an economically unfavorableposition for the commercial production of glucose isomerase . The key to thesolution of the problem seems to lie in either finding a new organism ordeveloping a mutant in which the enzyme synthesis is not dependent on xylose(constitutive) and is not repressed by the glucose (catabolite repression-insensitive) . Only the new organism or the mutant cell of such propertieswould be expected to produce high levels of glucose isomerase at economicallyfeasible costs

.

In this report, the procedure leading to obtaining such a mutant and theresult of fermentation by the mutant thus obtained are described .

B . EXPERIMENTAL

I . Materials and Method

a . Culture Media

The compositions of various media are as follows :

Medium I :

g/ 1

( N H 4 ) 2 H P 0 4 6 .0

KH2P04 0 . 2

MgSO47H20 0 . 25

T r y p t o n e ( D i f c o ) 5 .0

Ye a s t e x t r a c t ( D i f c o ) 1 .0

Medium II :

Same as Medium I, without tryptone and yeast extract .

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M e d i u m I I I

g/l

( N H 4 ) 2 H P 0 46 . 0

K H 2 P 0 42 .0

M g S O 4 7 H 2 00 . 25

Trvptone (Difco) 5 . 0

Yeast extract (Difco) 1 .0

Medium IV :

Same as Medium III, without tryptone and yeast extract .

Medium V :

g/l

( N H 4 ) 2 H P 0 46 .0

K H 2 P 0 42 .0

MgSO47H20 0 . 2 5

O . M . HAP* 8 . 4g .

B Y F - 1 0 0 * * 1 .0

* Hydrolyzed meat protein (Amber Laboratories)** Brewers yeast fraction (Amber Laboratories)

Carbohydrates and magnesium salts were separately autoclaved as a50% and 2 .5% solution, respectively ; and the proper amount was added tothe medium for the desired final concentration .

b . _ O r _ & a n i s m s

RJR Strain 2453-2 and 2453-Y were used as the parent culture for thedevelopment of mutants . In most experiments on enzyme synthesis, cultureswere inoculated in 100 ml . of medium in 500-m1 . Erlenmeyer flasks and in-cubated at 30C . on the shaker rotating at 250 RPM with a 2-inch stroke .

c ._ Determination of Enzy,me Activity_

2 .5 to 5 . 0 m l . of samples were taken from the flasks periodicallyand cells were centrifuged at 12,000 x g . for 10 minutes at roomtemperature . The cells were then resuspended in water and assayed for

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isomerase activities by the method previously described (1) . A u unitof the enzyme activity is defined as that amount of enzyme producing1 u gram of D-fructose per minute at the standard condition . In thisreport, enzyme activities were expressed as p units/ml .

d . Measurement of Growth

The growth was measured with a Klett-Summerson photo-colorimeterwith the red filter using centrifuged and resuspended cells .

II . Induction and Repression of Glucose Isomerase in RJR 2453-2

Strain 2453-2 was inoculated in Medium I containing various carbonsources, and the enzyme activities were assayed at various time intervals .T h e r e s u l t s a r e s u m m a r i z e d i n T a b l e I .

TABLE I

EFFECT OF VARIOUS CARBON SOURCES ON THE S1T THESISOF GLUCOSE ISOMERASE IN STRAIN 2453-2

Enzyme Activities/ml .

Carbon-Source(s)* 17 Hours 41 Hours 65 Hours

N o n e 0 0 0

X y l o s e3 3 1 1 9 1 2 8

Glucose 0 0 0

X y l o s e a n d G l u c o s e 0 526

F r u c t o s e 160 0

Xylose and Fructose 19 26 53

G l y c e r o l0 0 0

X y l o s e a n d G l y c e r o l 1412 14

* C-sources were used at 2% each .

.

In the absence of added carbohydrate, the culture grew at the expenseof tryptone and yeast extract . However, no enzyme activities were detected .The medium with xylose produced the highest level of the enzyme .

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i

All of the other carbohydrates tested failed to produce enzyme activity ; a n d ,when used with xylose, inhibitory effects were observed . These resultssuggest that, (1) glucose isomerase is an inducible enzyme, (2) xylose isthe inducer, (3) other carbohydrates function as catabolic repressors .

II I . Development of the Constitutive Mutant from RJR 2453-2

a .- Selection Method of the Constitutive Mutant- ----------------To eliminate the need for the highly costly inducer by the organism,

a constitutive mutant was required . Success in obtaining a mutant,however, depends largely on the availability of a fast and accuratescreening method . Such a method was developed based on the facts that,(1) glucose isomerase catalyzes the reversible reaction of D-fructoseto D-glucose, and (2) D-glucose can be specifically assayed by theglucose oxidase system . Therefore, by coupling the two enzyme systems,the activity of glucose isomerase was made readily visible by thedevelopment of a red-brown color on the indicator plate . The detailsof the procedures are as follows :

A culture of 2453-2 was grown at 30C . for 10 hours in 100 ml . ofMedium I . 5 . 0 m l . of this culture was then transferred to 100 ml . offresh medium and incubated for 3 hours . At this point, 10 mg . ofN-methyl-N'-nitro-N-nitrosoguanidine was introduced to the flask andincubation continued for 30 minutes . A 1% transfer was then made tothe new medium and this was grown for 24 hours . These cells wereproperly diluted in 0 .5% tryptone broth and spread on agar plates ofMedium I (without xylose) at a density of about 200 to 300 coloniesper plate . After 48 hours of incubation of 30C . the entire platewas replica-plated to the indicator plate of the following composition :

gramsF r u c t o s e 1 4 . 0

A g a r 2 .0

M g C 1 2 0 .1

F e r m c o z y m e 9 5 2 D M 2 2 5 ( m l . )

o - D i a n i s i d i n e 0. 0 0 7 5

W a t e rT o 1 0 0 m l .

1 Fructose was first treated with glucose oxidase in order to removetraces of glucose present which would yield a false-positive test .

1 1

2 Fermcozyme 952 DM was diluted as described in the Fermco Laboratoriesbulletin .

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The plates .were incubated at 50C . for 12 hours . Those colonieswhich produced a brown color around them were suspected to be consti-tutive mutants and were isolated for further studies

.

b ._ Prooerties of Constitutive Mutants_

The mutants (C-Mutant) obtained by the above procedures weregrown in Medium I and examined for constitutive production of isomerase .The results are shown in Table II .

TABLE II

SYNTHESIS OF GLUCOSE ISOMERASE BY WILD TYPE (2453-2) ANDMUTANT WITH AND WITHOUT INDUCER

Culture Inducer Enzyme Activities/ml .

1 6 H o u r s4 0 H o u r s 6 4 H o u r s

2 4 5 3 - 2X y l o s e 24 2 4 7 3 0 1

2 4 5 3 - 2 N o n e 1 00 0

C - 1 X y l o s e8 5 1 3 0 154

C- 1 N o n e 1 1 3 1 5 8 1 1 1

C- 3 X y l o s e35 11 0 12 1

C- 3 N o n e54 18 0 10 8

C- 4 X y l o s e - - - 1 1 0 1 4 3

C - 4 N o n e 23 10 7 79

It is clear from the table that mutants produce the enzyme whetheror not xylose is added to the medium . These constitutive mutants, however,did not grow well without an adequate supply of carbon and energy source .

Various carbohydrates were tested for this purpose at 2% concentration .The results of this experiment are shown in Table III .

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TABLE III

EFFECT OF VARIOUS CARBOHYDRATES ON THE CONSTITUTIVESYNTHESIS OF GLUCOSE ISOMERASE BY C-3 MUTANT

Carbohydrates Enzyme Activities/ml . Klett Reading S .A .2

N o n e 1 8 12 5 0 0 .7 3

G l y c e r o l70 1 1 6 0 0 . 06

P o t a s s i u m g l u c o n a t e2 67 1 7 0 0 0 . 1 5 7

F r u c t o s e 1 6 31 8 0 0 0

. 0 9 0

G l u c o s e1 0 4

1 3 5 0 0. 0 7 7

R i b o s e1 3 8

1 8 0 0 0 . 0 77

1 Enzyme activities and Klett readings are checked at 43 hours.

2 S .A . = Specific activity = units of isomerase/Klett units .

The mutant grew well in all the carbohydrates as shown by Klettreadings and produced enzymes at significant levels . However, thespecific activities of the cultures grown in the presence of added

carbohydrates is almost one-tenth of the control . This fact meansthat isomerase synthesis in the mutant is still under the influenceof catabolite repression . The higher level of enzyme activities inthe C-3 mutant compared to those in 2453-2 (Table I) seems to suggestthe mutant is partially insensitive to catabolite repression

.

By this time, L . Hayes of the Biological Division isolated anew strain of Arthrobacter called 2453-Y . This strain was reportedto produce a higher level of isomerase than that by 2453-2 . There-fore, all the subsequent studies were conducted with this new strain

.

IV . Development of a Constitutive Mutant from RJR 2453-Y

a ._ Selection and Properties of the Mutants

Cultures of 2453-Y grown in Medium I were treated with eitherethyl methane sulfonate or nitrosoguanidine and screened for a consti-tutive mutant by the method described for 2453-2 . A number of positivecolonies appeared on the indicator plate . The mutants obtained fromethyl methane sulfonate treatment were designated EYC- and thoseobtained from nitrosoguanidine were designated YC- . The enzymesynthesis by the mutants in Medium I with and without 2% xylose wereshown in Table IV .

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TABLE IV

SYNTHESIS OF GLUCOSE ISOMERASE IN THE PRESENCE ANDABSENCE OF XYLOSE BY WILD TYPE AND MUTANTS


22 Hours 46 Hours

Isolate None Xylose None X l y o s e

E Y C - 2 - a 1 5 5 2 0 1 1 2 6 3 8 5

E Y C - 2 - b 1 6 0 2 0 6 1 4 23 7 5

E Y C - 2 - c 1 2 9 1 9 31 1 4 3 0 1

E Y C - 2 - d 1 5 9 2 1 51 1 8

20 4

Y C - 6 1 8 0 1 7 1 1 0 5 3 5 1

Y C - 9 - a 1 48 1 6 9 1 2 4 22 6

Y C - 9 - b 1 4 1 201 1 0 5 2 7 3

Y C - 9 - c 1 3 8 1 5 1 1 0 1 1 9 6

Y C - 9 - d 1 2 6 1 9 1 1 0 1 2 5 1

Y C - 9 - e 1 3 8 1 8 41 0 9 2 3 1

2 4 5 3 - Y 01 22

0 31 4

The above results show that the mutants are indeed constitutivefor isomerase . However, without adequate supply of carbohydrate,the organisms did not grow well and consequently the enzyme synthesisceased after 22 hours . Therefore, synthesis of the isomerase in 2%glucose medium was studied with the mutant and wild type . The results areshown in Table V .

.

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TABLE V

SYNTHESIS OF GLUCOSE ISOMERASE IN 2% GLUCOSEBY WILD TYPE AND MUTANTS

.


Culture C-Source 18 Hours 42 Hours 70 Hours

2 4 5 3 - Y G l u c o s e 0 00

2453-Y Xylose 110 2 98 450

E Y C - 2 - aG l u c o s e 0 43

58

E Y C - 2 - b G l u c o s e 037 49

E Y C - 2 - c G l u c o s e0 1 3 1 4

EYC-2-d Glucose 0 31 35

Y C - 6G l u c o s e 0

2 6 2 9

Y C - 9 - a G l u c o s e0 89

1 81

YC-9-b Glucose 0 96 193

Y C - 9 - cG l u c o s e 0 6 8 1 7 3

Y C - 9 - d G l u c o s e 095

1 7 0

Y C - 9 - eG l u c o s e 0 93

1 64

The above results clearly indicate that mutants derived from2453-Y are, though constitutive, still repressed by glucose ; a n denzyme synthesis starts slowly as derepression takes place followingconsumption of the glucose by the cells .

b. Controlled Addition of Glucose to the Mutant Culture_

The results presented in Table V pointed to the possibilitythat enzyme formation in the mutant cells might be increased bycarefully controlling the level of glucose in the media . Therefore,a 50% solution of glucose was added to the Medium I at various timesof growth to the total amount of 2% . The time table of glucoseaddition to the mutant culture (EYC-2-b) and the results of enzymeassays are shown in Table VI and VII, respectively .

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TABLE VI

SCHEDULE OF GLUCOSE ADDITION TO EYC-2-b1

Flask No.

0 Hours 20 Hours 44 Hours 68 Hours

1 0 1 . 02 . 0 1 . 0

21 .0 1 . 0 1 . 0

1 .0

32 .0 0 2 .0

0

4 2 .00 1 . 0 1 . 0

54 .0 0

0 0

6 2 4 .00 0

0

J

1 Values are ml . of 50% glucose solution added to 100 ml . ofMedium I .

2 50% xylose solution is used for Flask No . 6 .

TABLE VII

RESPONSE OF EYC-2-b TO CONTROLLED GLUCOSE ADDITION


F l a s k N o . 20 Hours 44 Hours 6 8 H o u r s 9 2 H o u r s

11 3 8 1 84 2 5 9 3 1 1

2 2 330 59

55

3 022 2 4 1 3 9 0

40

21 7 1 6 4

50

2 2 0 0

61 2 6 1 8 4

2 82 280 1-'1

The results shown in Table VII demonstrate that isomerase ~synthesis by the mutant is greatly influenced by the timing of athe glucose addition . In a well-controlled system, EYC-2-b can G,be a very productive culture

.

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V . The Effect of Phosphate Concentration on the Isomerase Svnthesis

It has been noticed that the levels of the enzyme activities in bothwild type and mutant cultures were rather inconsistent . It was alsoobserved that the adjusting of the pH of the Medium I to 6 .9 using phos-phoric acid often resulted in increased levels of enzyme activities

.

Medium I contains 0 .02% of monobasic phosphate and this value appeared tobe too low . Therefore, the effect of phosphate on the isomerase formationin the mutant culture (EYC-2-b) was tested by using various amounts ofmonobasic potassium phosphate to the Medium I supplemented with 2% xylose

.

The results are shown in Table VIII .

TABLE VIII

EFFECT OF PHOSPHATE CONCENTRATION ON THE GLUCOSEISOMERASE SYNTHESIS IN EYC-2-b


% KH P0 18 Hours 42 Hours 60 Hours 90 Hours4

0 . 0 2 1 2 2 2 0 0 28 430 5

0 . 1 1 3 5 2 5 4 6 4 370 3

0 . 21 4 3

4 4 8 7 6 8 7 4 4

0 .3 15 54 4 6

72 47 3 0

0 .4 1 3 5 3 7 6 7 2 479 0

Increasing the phosphate concentration to ten-fold resulted in morethan a two-fold increase of enzyme activity . Further increase of phosphateconcentration did not produce any significant change in the enzyme level .Based on this result, a new medium containing 0 .2% of KH2PO4 was formulated(Medium III) . It was subsequently tested and confirmed that all the mutantsand wild types produce higher enzyme activities in Medium III .

VI

. Development of a Constitutive and Catabolite Repression-InsensitiveMutant (Double Mutant)

a ._ Screenin .& of the Mutant

It was reasoned that since EYC- and YC- mutants produce the isomeraseconstitutively but are repressed by catabolites, a catabolite repression-insensitive mutant derived from constitutive parent culture would sub-stantially increase the yield of activity in glucose-supplemented media .

Such a mutant was screened from the mutagenized culture of YC-9-c .

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The YC-9-c culture was first grown in Medium II with 2% glucose,then treated with nitrosoguanidine as described before and grown inthe same media for 24 hours . The properly diluted culture was platedon the selection media of the following composition :

B / 1 0 0 m l .

( N H 4 ) 2 H P 0 40 . 3 6 3

K H 2 P 0 40 . 01 2

A g a r1 . 5

Fructose 1 . 5

M g C 1 2 0 .0 3

F e r m c o z y m e 9 5 2 D M 2 0 m l .

0-Dianisidine 0 . 0 0 6 2

This selection media was designed based on the previous observa-tions that, (1) isomerase synthesis is extremely low in mineral saltmedium (Medium II), and (2) catabolite repression is more pronouncedin the culture growing in this medium . In the-selection medium,fructose serves as a source of catabolite repression and also as asubstrate for glucose isomerase . Since there is no xylose present inthis media, only the constitutive mutants which overcome the repression

_by fructose can manifest the enzyme activities by oxidizing theo-dianisidine in the coupled reaction . After four days of incubationat 30C . a few colonies turned brown and these were isolated anddesignated as F-mutants .

b .- Glucose Isomerase Production-bY F-Mutant-- --------------F-mutants were tested for their constitutive production of the

enzyme and their sensitivity to the presence of glucose . For thelatter purpose, 50% solution of glucose was added at various stagesof growth to the total final concentration of 2% . The time scheduleof glucose addition and the results of enzyme assay are summarized inTables IX and X .

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TABLE IX

SCHEDULE OF GLUCOSE ADDITION TO F-MUTANT *

Flask No . 0 Hours 15 Hours 39 Hours 63 Hours

1 0 1 2 1

2 11

1 1

3 2 0 2 0

4 2 01 1

5 4 0 00

6 4 ( X y l o s e ) 0 00

* Values are ml . of 50% glucose solution added to 100 ml . of culture,except in Flask No . 6, where xylose was used .

TABLE X

SYNTHESIS OF GLUCOSE ISOMERASE BY F-MUTANT


Flask No . 15 Hours 39 Hours 63 Hours 87 Hours

1 1 2 4 2 1 5 4 0 2 5 8 7

2 1 3 0 3 2 15 1 7 6 4 5

3 1 1 2 3 4 0 5 1 6 5 9 9

4 1 3 6 3 3 5 4 6 7 6 3 3

5 1 1 5 3 1 6 5 2 6 6 0 8

6 1 2 7 4 2 5 5 4 8 6 3 9

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Table X clearly demonstrates that the F-mutant retained itsconstitutivity and acquired a new property of being insensitiveto catabolite repression . Both glucose- and xylose-grown culturesproduced the same final level of enzyme activity, and littledifference was noticed in the pattern of enzyme synthesis among theflasks . In a separate experiment, the F-mutant was grown in Medium I

with 2% glucose or xylose . About 330 u units/ml . of enzyme wasproduced in both glucose and xylose media confirming that the F-mutant also produces higher levels of enzyme in Medium III .

VI I . New Sources of Vitamins and Organic Nitrogen

Yeast extract and tryptone provided by Difco Company are expensiveand unavailable in large quantities . Therefore, the alternate sources weretested ; these were autolyzed brewers yeast fraction(BYF-100) and hydrolyzedmeat protein (HAP) produced by Amber Laboratories, Juneau, Wisconsin . Theeffect of these on glucose isomerase synthesis was tested with the F-mutant

.

The results of the test are shown in Table XI .

TABLE XI

COMPARISON OF VITAMINS AND ORGANIC NITROGEN SOURCES *

E n z y m e A c t i v i t i e s /m l .

Vitamins & Nitrogen Source 15 Hours 39 Hours 63 Hours 87 Hours

Y e a s t E x t r a c t a n d T r y p t o n e 1 8 43 9 7 5 4 2 6 0 0

Y e a s t E x t r a c t a n d H A P 2 1 34 5 8 5 1 5 6 0 0

B Y F - 1 0 0 a n d T r y p t o n e 1 7 1 37 44 0 2 5 1 9

B Y F - 1 0 0 a n d H A P 1 9 9 58 5 6 3 6 4 8 5

* Medium IV served as the basal medium ; 2% glucose was added to all media.

It appears that BYF-100 and HAP can perform as well as yeast extract and

tryptone . In fact, slightly higher enzyme activities were observed in mostexperiments . This medium was named Medium V .

VIII . Isomerase Production b~the F-Mutant in the 30-Gallon Fermentor

Isomerase production by the F-mutant was tested in a pilot plant scaleby growing the cells in the 30 gallons of Medium V, supplemented with 2%glucose .

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The inoculum was prepared by first growing the F-mutant in 100 ml.

of the medium for 24 hours and transferring it to 6 liters of themedium in the New Brunswick fermentor . This was incubated 24 hours

and 5 .5 liters of the culture was used to inoculate the 30-gallonfermentor . Samples were taken at various times and the enzyme activitiescell yield, and pH of the medium were recorded . The results are shown inFigure I . The maximum activity of 720 U units/ml . was reached in 60 hours .

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FIGURE I

SYNTHESIS OF GLUCOSE ISOMERASE BY THE F-MUTANT IN A 30-GALLON FERMENTOR

6 0 0

2 00

Hours (Incubation)

r 7 .0

F 6. 0 P H

r 5 . 0

3 0 0 0

- 2 0 0 0

h-1000

The medium contained HAP, BYF-100, and glucose in addition to salts.

O-Klett Unit

O-Enzyme Activity

A-pH

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C . DISCUSSION

In the earlier stage of this work, it became clear that there were thefollowing three major problems to be solved :

1 . How to avoid the requirement for xylose by the organism.

2 . Can glucose or another readily available inexpensivecarbohydrate be used to produce the enzyme?

3 . What are inexpensive and effective sources of vitamins andorganic nitrogen?

The success of the whole project depended on the finding of the solutionsto these problems . All the previous efforts to by-pass the xylose requirementby using xylan or xylan-rich material seemed to confirm that xylose alone isthe only effective inducer for the enzyme and all other carbohydrates testedare repressors of the enzyme . Therefore, the solutions to the problems weresought at genetic levels . The genetic regulation of glucose isomerase inArthrobacter was viewed analogous to the well-studied B-galactosidase systemin Escherichia coli . Both enzymes are inducible and catabolite repressible .

The first objective was to obtain a constitutive mutant which would notrequire xylose . Such a mutant became a reality after a fast and sensitivescreening method was developed . The usefulness of the screening method wouldnot only be limited to finding a mutant of Arthrobacter but could also beapplied in searching for the constitutive- or the hyper-producer of glucoseisomerase of a wide range of microorganisms .

Development of a new media which contains a ten-fold increase in phosphateplayed an important role in increasing the enzyme synthesis . A few other mediawere also tested, but none proved better than Medium III . BYF-100 and HAP hadbeen used for wild type (2453-Y) culture and found to be half as effective asyeast extract and tryptone in producing the glucose isomerase . Since growth ofthe wild type on BYF-100 and HAP was not hampered but only the enzyme synthesiswas repressed, the presence of carbohydrates as impurity in the preparationwere suspected for the difference of enzyme synthesis between the wild typeand mutant .

Now that all the three main problems are resolved by the development ofthe mutant, new medium formula, and new source of vitamins and nitrogen, theeconomic outlook for the industrial production of glucose isomerase shouldbe greatly improved .

D . CONCLUSIONS

1 . RJR strains of Arthrobacter, 2453-2 and 2453-Y, produce glucoseisomerase which is inducible with xylose and repressible with glucose .

2 . A fast and unambiguous screening method for glucose isomerase wasdeveloped .

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3 . The wild types (2453-2 and 2453-Y) were treated with mutagens andconstitutive mutants were obtained .

4 . The double mutants, which are both constitutive and insensitive tocatabolite repression, were obtained from the mutagenized cultures of theconstitutive mutant .

5 . A new medium containing a high phosphate content and an inexpensivesource of vitamins and organic nitrogen was developed .

6 . The double mutant (F-mutant) produced an average of 750 u units/ml.

o f g l u c o s e i s o m e r a s e i n t h i s n e w m e d i a w i t h 2 % g l u c o s e a s a c a r b o n s o u r c e.

7 . The results in the flasks were reproduced in a large scale fermentor(30-gallon size) .

E . RECOMMENDATIONS

I . Future Work

F-mutants appear to have all the desirable genetic qualifications forthe commercial production of glucose isomerase . The maximum level of enzymeproduction, however, may be significantly increased by studying the optimumconditions for the fermentation, such as media, pH control, aeration, etc .Work along this line should be emphasized in the future

.

Cost of the enzyme production can be further reduced if less expensivesources of vitamins and organic nitrogen are found . The use of hydrolyzedcells of F-mutant after use in syrup conversion might be able to solve thisproblem in addition to that of waste disposal .

Now that quick screening methods are available, a search into thethermophillic organism, which may have heat-stable glucose isomerase, couldbe a profitable venture .

II . Patentability

A p a t e n t s h o u l d b e a p p l i e d f o r t h e F - m u t a n t i m m e d i a t e l y .

(See next page for distribution)

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D i s t r i b u t i o n :

Dr . Murray Senkus Dr . Herbert J . BluhmDr . R . E . Farrar Mr . Manford R . HaxtonMr .Dr .

E .

K .H .

J .

HarwoodMysels

Dr.

Dr . Eldon D . NielsonChin K . LeeLibrary (2)Dr . C . G . Pheil

/ YeXQ4 1& lJ7a . z y c . .~c~~5 ~a /-3/- 7,a

S u b m i t t e d : A p r i l 1 1 , 1 9 6 9

C o m p l e t e d : A p r i l 1 4 , 1 9 6 9

From manuscript : v b l

~~-~ - ~ . . ~Approved : ' ~ ~j . ~ . E ~ ~ - . ~ - ~ . .

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BIBLIOGRAPHY

1 . James, W . B ., Automatic Analysis of Fructose . RDM, 1968, No . 2(January 5) .

2

. Hayes, L. E

., Biosynthesis of Glucose Isomerase by a Strain ofArthrobacter . RDM, 1969, No . 2 (January 23) .

.

3 . Long, M . E ., Enzymatic Conversion of D-Glucose to D-Fructose :Isolation and Identification of an Active New Bacterial Species

.

RDR, 1968, No . 33 (September 13) .

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