Note: Descriptions are shown in the official language in which they were submitted.
~93~1
~IETHOD FOR OBTAINING OF GLUCOSE ISOMERASE
The invention relates to a method for obtaining of
glucose isomerase from a streptomyces strain.
It is known that by means of the enzyme glucose
isomerase D-glucose is transmitted into D-fructose, which
finds increasingly wider application in food processing indus-
try and dietic feeding in a number of developed countries.
Glucose isomerase, in combination wit,h a complex of amylolytic
enzymes (~-amylase and glucoamylase), gives the opportunity
of obtaining of glucose-fructose syrups and of fructose
directly from starch, using enzymes.
Some methods for obtaining of glucose isomerase have
been known since 1957 when Marshall R.O. and Kooi E.R. (29)
demonstrated for the first time the possibility of direct
conversion of D-glucose into D-fructose by means of cells
-belonging to the bacterial strain Pseudomonas hydrophila N.
491 and 492. For obtaining of glucose isomerase microorganisms
of the genus Streptomyces and above all the species Str~
phaeochromogenes /34,38/, Str. venezuelae /I,24/, Str. griseus
/23!, Str. wedmorensis /3/, Str. albus /35/, Str. flavovirens,
Str. achromogenes, Str. achinatus /36/, Str. olivocinereus
/20,24,26/, Str. achromogenes, Str. olivaceus /17,18/ and
others are most widely applied. Besides the genus streptomyces
there are other active
93C)1
producers such as proactinomyces and actinomyces belonging
to other genera: Nocardia, Micromospora /21,22/, Actinoplanes
/30,31/, Thermoactinomyces, Thermopolyspora /37/ and also
some bacterial strains belonging to the genera Aerobacter
/20/ Acetobacter /28/, Lactobacillus /25/, Bacillus /33/,
Arthrobacter/28/, Flavobacterium /4/, and others.
The already known methods for obtaining of glucose
isomerase have a number of disadvantages connected mainly
with the fact that the strain producers rarely combine a high
ratio of glucose isomerase activity with a favourable tem-
perature and pH-optimum of the enzyme. The pemperature of
the isomerization process, lower than 60-65C, leads to the
danger of microbe pollution of the colony. The increase of
the temperature to 90C decreases the stability of the enzyme,
causes the coloration of the syrup and some changes in the
viscosity. As far as industrial application is concerned,
the most favourable pH is from 6.5 to 7.0, because when the
pH is over 7.0 an alkaline isomerization of glucose is carried
out with the formation of D-psicose and coloration of the
syrup, and a lower pH (below 5.5) causes an irreversible
denaturation of the enzyme.
The purpose of this invention is to establish a
method for obtaining of glucose isomerase from streptomyces
strain which has high activity with a high temperature optimum
and a favourable pH of the enzyme.
Thus, the present invention provides a method for
obtaining glucose isomerase characterized in that the enzyme
producing strain Streptomyces sp. 1339 registration No. 144 -
Bulgarian State Institute for Drugs Control, is cultivated for 36 to 72
hours in a culture medium with xylose as an inductor, the
temperature being kept from 24 to 36C and the initial pH of
cultivation being from 6.5 to 9Ø
The present invention also provides a method for
-~r~ 2 -
19301
isomerizing glucose characterized in that the isomerization
is carried out in the presence of glucose isolnerase, a-t a
temperature from 50 to 80C, a pH from 6.0 to 9.0, in the
presence of suitable concentrations of CoC12.6H2O and
MgSO4.7H2O and concentration of the substrate from 0.1 to 3M,
the glucose isomerase having been prepared by cultivating,
the enzyme producing strain Streptomyces s.p 1339 registration
No. 144 - Bulgarian State Institute for Drugs Control, for 36 to 72
hours in a culture medium with xylosc as an inductor, the
temperature being kept from 24 to 36C and the initial pH
of cultivation being from 6.5 to 9Ø
In accordance with the present invention the
culture medium used may have the following composition
xylose 1.0 - 2. n ~
maize extract 1,5 - 4.0~ (in dry weight)
MgSO4,7H2O 0.05% - 0.2
KCl 0.005% - 0.01%
CoC12,6H2O 0.008% - 0.036~
Strain of Streptomyces sp.N.1339- producer of
glucose-isomerase has been isolated from Bulgarian soil. 874
streptomyces strains have been screened for its discovery.
The screening has been carried out on the modified synthetic
culture medium N.l after Krasilnikov, the selection being
realized on two substrate levels with the use of xylose and
xylane. Under these conditions 18 streptomyces strains
have been discovered which can develope and produce the
enzyme
- 2a -
9 3 0 ~ L
glucose isomerase. Of these streptomyces sp. N. 1339 proves
to be the most perspective for industrial purposes. The strain
has been in the collection at the State Institute for Drugs
Control, bul. Vladimir Zaimov N. 26 since September 29, 1979
under number 144 and has the following morphological and bio-
chemical characteristics. On culture medium 1 with a mineral
source of nitrogen (after G.F. Gause and collaborators) the
colonies are usually oval in shape, with unshaped edges, flat
with convex centre like dome and slightly folded. The
sporangia are spiral with no more than 1-3 coils. On some
culture media are formed coremia. The growth is good. The
spores are elongated with sharply cut ends and uneven surface.
Their size is 0,4-0,9 micrones in length and 0,3-0,6 micrones
in width. The ends have well expressed corners. In some
cases the spores are slightly concave in the middle. They are
formed by means of fragmentation. The colonies are oval, with
unshaped edges, flat, with convex dome-shaped centre, slightly
folded, in some cases radially segmentated.
The colour of the air and the substrate mycelium is
determined according to the colour scale of A.S. Bondartsev
and the scale of Tresner - Backus.
With the different culture media the colour of the
air mycelium changes from light-grey to dark-grey (a4 - a2)
depending on the carbon and nitrogen sources. On culture
medium 1 with a mineral source of nitrogen (after G.F. Gause
and collaborators) the colour is mousy-grey (a4) to dark-grey
(a ) and on culture medium 2 with an organic source of
nltrogen (after G.F. Gause) the colour is dark-grey (a2). On
culture media with different carbon and nitrogen sources the
air mycelium is grey to dark-grey.
On culture medium 1 with a mineral source of
nitrogen (after G.F. Gause and collaborators) the substrate
~ ,, ~
~93()1
mycelium has light lemon colour to yellow-orange (d5 - d2).
On culture medium 2 with an organic source of nitrogen (after
G.F. Gause and collaborators) the substrate mycelium is
golden-yellow and after a prolonyed cultivation becomes almost
chestnut (m7-O7)
On culture media with different carbon and nitrogen
sources the substrate mycelium is from yellow to yellow-
greyish-green.
On culture medium meat-pept`on agar. Low growth. Air
mycelium-white, very scarce. Subs-trate mycelium - colourless.
On culture medium potato-glucose agar. Growth very
good. Air mycelium grey to dark-grey. Substrate mycelium
light-brown to dark-brown, on the edge of the colony-yellow.
Pigment in the centre yellow.
On culture medium Tchapek with sucrose. Medium
growth. Air mycelium light-grey. Substrate mycelium-beige.
On culture medium Tchapek with glucose. Medium
growth. Air mycelium greish. Substrate mycelium cream-
coloured.
On,culture medium with sucrose. Good growth. Air
mycelium grey. Substrate mycelium yellow.
On culture medium amylum agar. Medium growth. Air
mycelium light-grey, with pale ashy colour. Substrate
mycelium yellow, lemon-coloured.
On amylium-ammonia culture medium after Mishustin.
Good growth. Air mycelium grey to dark-grey. Substrate
mycelium yellow, light-yellow.
On synthetic medium after Krassilnikov. Growth low
to medium. Air mycelium beige grey. Substrate mycelium-
yellow. I
On CPI after N.A. Krassilnikov. Good growth. Air
mycelium milk-grey, blue-grey. Substrate mycelium yellow-
- 4 - !
93C)~
greish-green.
On CPII after N.A. Krassilnikov. Good growth. Air
mycelium grey, white exudate is separated. Substrate mycelium
cream-grey.
On CPIII after Krassilnikov. Good growth. Air
mycelium grey. Substrate mycelium yellow, lemon-coloured.
On CPIV after N.A. Krassilnikov. Medium growth.
Air mycelium light-grey, greyish. Substrate mycelium cream-
coloured.
On CPV after N.A. Krassilnikov. Low growth. Air
mycelium-white. Substrate mycelium-beige, dark-cream-coloured.
On synthetic medium after Vaxman. Medium growth.
Air mycelium-white, on separate parts-grey. Substrate
mycelium-yellow to orange.
On meat-amylum agar. Medium growth. Air mycelium-
grey. Substrate mycelium-yellow.
On peptone agar. Good growth. Air mycelium-grey to
dark-grey. Substrate mycelium - colourless with a shade of
the air mycelium.
On glucose-asparagine agar. Medium growth. Air
mycelium-light-grey. Substrate mycelium -yellow-cream-
coloured.
On glycerine-asparagine agar. Good growth. Air
mycelium-light-grey, becoming grey in the process of ageing.
Substrate mycelium-dark yellow.
On tyrosine agar. Medium growth. Air mycelium-grey,
in some colonies to greyish-yellow. Substrate mycelium-dark-
yellow to orange.
On tyrosine-caseine-nitrate agar. Medium growth.
Air mycelium-ashy-grey. Substrate mycelium-yellow.
On glucose-tyrosine agar. Good growth. Air
mycelium-cream-grey to grey. Substrate mycelium-yellow-brown.
~93~;)1
On saccharose-nitrate agar. Medium growth. Air
mycelium-grey. Substrate mycelium-cream-yellow.
On glycerol-calcium-malate agar. Good growth. Air
mycelium-light-grey to grey. Substrate mycelium-yellow-
orange.
On peptine-beef agar. Good growth. Air mycelium-
grey. Substrate mycelium-colourless to beige.
On oats agar. Growth- very good. Air mycelium-
grey. Substrate mycelium- light yellow.
On tomato agar. Good growth. Air mycelium -dark-
grey. Substrate mycelium -terracotta-colour.
On lead-acetate agar. Low growth. Air mycelium
greish. Substrate mycelium -yellow-brown.
On iron-peptone agar. Medium growth. Air mycelium-
mousy-grey. Substrate mycelium -colourless with a grey shade
from the air mycelium.
On yeast -malt agar. Growth good. Air mycelium
-grey. Substrate mycelium -orange.
Strain tolerance towards NaCl. It shows low
tolerance towards the concentration of sodium chloride in the
medium. The maximum concentration is 4~. Under this concen-
tration the strain is low. Air mycelium is white. The
substrate mycelium is yellow. A concentration higher than 1%
has a negative influence on the degree of sporulation.
It peptonizes fatless milk. In the beginning of
the peptonozation the reaction is acidic, and after a while
turns to alkaline. It waters down gelatine. It grows very
well on a sucrose mcdium, but doesn't invert sucrose. It
~ grows very well on a amylum agar and hydrolyzes starch well.
It doesn't decompose cellulose and doesn't reduce
nitrates to nitrites. It liberates hydrogen sulphide. It
grows on potatoes. Hemolysis - positive. Tyrosinase -
-- 6
~93~1
negative.
It has been found out that on basic culture medium
of Pridham and Gottlieb the growth is good in the presence of
the following carbone sources: gIucose, fructose, levulose,
xylose, maltose, cellobiose, galactose, mannitol, arabinose,
dextrose, ribose and glycerol.
The strain absorbs salicin on a small scale.
It doesn't absorb carbon sources such as lactose,
sorbite, inosite, sucrose and raffinose.
Some differences in the pigmentation of the air and
substrate mycelium are found out depending on the source of
carbon.
It has been found out that on modified basic culture
medium of Pridham and Gottlieb the growth of the strain is
good in the presence of the following sources of nitrogen:NH4CL
(NH4)2So4~ (NH4)2HPo4~ NH4H2PO4~ NH4NO3, Na2Hpo4 and carbamide.
The growth is moderate on a culture medium with
NaNO3. The strain doesn't grow on a culture medium with NaNO2.
A very good growth of the strain has been observed
on culture media with the following amino - acids: asparaginic
acid, asparagine, proline, cystine, tyrosine. The growth is
moderate on a culture medium with valine, hydroxyproline,
phenylalanine, leucine and allanine. The strain doesn't grow
on a culture medium with glutamic acid.
Depending on the source of nitrogen some differences
in the pigmentation of the air and the substrate mycelium are
founded out.
According to some characteristics streptomyces strain
resembles Streptomyces griseoflavus, belonging to the series
Gray after Bergey's (1974) - Actinomyces griseoflavus of the
Flavus group after N.A. Krassilnikov - 1970. The latter is
distinguished by a number of morphological - cultural and
9301
physiological - biochemical properties described in the
species characterization by Bergey's (1974) and N.A.
Krassilnikov (1970). Streptomyces griseoflavus, for example,
has elongated rod-like and oval spores with smoth surface; it
shows tolerance towards NaCl from 7 to 10~. It hydrolyses
starch weakly. It absorbs sucrose and sorbite and doesn't
absorb galactose. Therefor streptomyces strain N 1339 is not
identical with the similar Streptomyces griseoflavus
(Actinomyces griseoflavus).
. .
- 7a -
.~
3~1
That is why it is reffered to as Streptomyces sp.
N 1339, belonging to the Gray series after Bergey's ~1974)
and the Flavus group after N.A. Krassilnikov (1970).
The strain - producer is cultivated in Erlenmeyer
flasks of 500 ml. containing 50 ml. fermentation culture
medium from 36 to 96 hours under a temperature of 24 to 36C,
initial pH of cultivation from 6.5 to 9.0 on a shaker with
180-320 revolutions per minute.
Isomerization of glucose to fructose by means of
; 10 glucose isomerase of the strain Streptomyces sp. N 1339
can be carried out through a direct treatment with fresh
mycelium (separated through centrifugation at 12000 revolutions
per minute and washed three times with 0,05M phosphate
buffer with pH 7,0) or with dried mycelium (air- dried
or aceton- dried cells): with enzyme solution (obtained after
a supersound desintegration or autolysis of cell material
and separation of supernatant through centrifugation at
15 000 rev/min. with cultural centrifugate, containing
extracellular isomerase or cells made immobile on a hard carrier.
The fructose, formed in the reacting mixture is
determined according to the cystein-carbazole method (19)
and the activity of the strain is expressed in mg. of fructose
per ml. cultural liquid or in International glucose- isomerae
units (GIU). One GIU is equal to the quantity of enzyme
which under 70C and pH 7,0 lM glucose solution in 0,05M
phosphate buffer, 1.10 M CoC12. 6H2O and 1.10 MgSO4. 7H2O
transformed in one minute 1~ mol glucose into 1~ mol fructose.
'l'he a~vantages of tlle metl~od according to the invention
are the following:-
For obtaining of glucose-isomerase the strain
streptomyces sp. N. 1339 was used, which possesses high
biosynthctic abilities of -the enzyme (12000 - 20000 GIO~ which
-- 8 --
~93~1
exceeds from 4 to 20 times the activity of s-train~producers,
known in patent literature (5-6). The enzyme which is obtained
is marked by a high temperature optimum (70) under low
optimum concentrations of Co++ (1.10 4M) and Mg+l (1.10 2M)
in the isomerization mixture. It has favourable pH-optimum
(7.0) and considerable thermostability between 40 and 65C
The invention is illustrated by the following
examples:
Example 1. The strain producer is ~aintained in test-tubes
with slant agar on culture media with the following composition:
1. Xylose culture medium
xylose 20g
agar 20g
KN03 l.Og
K2HP04 0.5g
MgS04.7H20 0.5 g
NaCL 0.5g
CaC03 l.Og
FeS04 O.OOlg
water up to 1 litre.
2. Potato -glucose agar:
Potato extract from 300g. boiled potatoes
glucose lOg
agar 20g
water up to 2 ]itres.
It is recommended that for maintaince of the strain both culture
media should be alternated.
To a well germinated material from 10-15 days culture
on culture medium 1 or 2 (1 is recommended~ 6ml inoculation
culture medium is added, having the following composition:
~9301
xylose 1,0%
tryptone 2,0%
g 47 2 0,1%
K2HPO4 0,25~
A wash-out of the cell mass is carried out and the
test-tube is put on a shaker for 24 hours under 30C and at
240 revolutions per minute. From thus adapted culture
inoculation medium with the following composition is sown.
xylose 1,0~
maize extract 3,0~ (in dry weight)
MgSO4.7H2O 0,1%
K2HPO4 0,25%
agar 0,1~
After 60 hours of cultivation under the above
mentioned conditions 5% of the inoculum is introduced into a
fermentation culture medium with the following composition:
xylose 1%
maize extract 3,0% (in dry weight)
MgS04 . 7H20 0, lg6
KCl 0,0075%
CoC12.6H2O 0,024%
The cultivation is carried out in Erlenmeyer flasks
of 500 ml. containing 50 ml fermentation culture medium. It
is carried out for 60 hours under 30C on a shaker at 240
revolutions per minute. The initial pH of cultivation is 8,5.
After 60 hours of cultivation of Streptomyces
sp.N.1339 it is obtained 140-220 g humid biomass per 11 of
cultural liquid.
~ Example 2. Isomerization of glucose to fructose by
means of glucose - isomerase of the strain Streptomyces sp.
N1339 is carried out under a temperature of 70C, pH 7,0 in
the presence of COC126H20
-- 10 --
~r
93~
in concentration 1.10 4M, MgSO4.7H2O in concentration 1.10 2M
and substrate concentration lM.
The activity of the strain Streptomyces sp. N 1339
is 130-215mg fructose per lml cultural liquid or 12 000-
20 000 GIU per 1 litre cultural liquid.
9301
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38. Tsumura M., Sato T. Ibid., 1129
