Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
The invention relates to a method of obtaining oE 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 industry and dietic feeding
in a number of developed countries. ~lucose isomerase, in
combination with 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 the possibility of direct conversion
of D-glucose into D-fructose through the cells of the bacterial
strain Pseudomonos hydrophila N~491 and N.492 was demonstrated
for the first tlme. For obtaining of glucose isomerase micro-
organisms of ~he genus strePtomycesl are most widely applied.
It was found out that the presence of different mineral salts
in the cultivation medium is a necessary condition for the
.
biosynthesis of the enzyme glucose isomerase. In the culture
media, described in literature and in patents magnesium salts
are present in the iorm of MgSO4.7H2O and cobalt in the form
of COC12.6H2O (18). The concentrations in which these salts
are introduced into the cultivation medium depend on the kind
of the producing microorganism and vary most frequently from
0.02 to 0.5~ for MgSO4.7H2O and from 0.005 to 0.024% for
COC12.6H2O (14). With some streptomyces species cobalt is
absolutely necessary for the formation of glucose isomerase,
through the activation is carried out due to the magnesium
ions. (13, 16, 17).
It is necessary that some strains and mutants-producers
of glucose isomerase should be found out, which are able to
produce a sufficient quantity of enzyme in the absence of cobalt
ions in the cultivation~medium. The mutants of CPC International
~7~6~
Inc ~2) are known, as well as some species Artrobacter, studied
by R~Jo Reynolds Tobacco (9), which don't require cobalt for
the biosynthesis of glucose isomerase.
The addition of cobalt and magnésium ions to the glucose
solutions during the isomeri~ation also influences strongly
the enzyme activity. It was proved, that these metals are
cofactors of the enzyme and that glucose isomerase can be
referred to aa a metal enzymes (3). It was found that one
molecule of glucose isomerase belonging to Str.sp.YT N.5
contains ~,1 cobalt atoms and 33 magnesium atoms. It is
supposed (3) that the combination of Mg+~ and Co + with
glucose isomerase is necessary for its transformation into an
active ~orm. It is also considered that during this process
a change in the conformation o the enzime takes place. Some
authers assume that the introduction of cobalt ions, especially
together with magnesium ions considerably increases the
thermostability of glucose isomerase (3,10).
The presence of higher quantities of Co in fructose
syrups is undesirable (5), although traces of Co are
necessary for the nutrition of man (12). The contents of Co
ions in the fr~ctose syrup is about lmM~l), but it is proved
that this concentration causes certain toxic e~fects on rats.
For the separation of the cobalt ions from the glucose isomerase
syrups when izomerization process is over they must necessarily
be cultivated with ion-exchange resins till thei~ maximum
separation. It is necessary that industrial automatic ion
-exchange systems should be constructed for this additional
process. Mi-Car International company uses some completely
automated ion-exchange systems consisting of two cation-anion
parts / strongl~ acidic cation-exchange resin Duolite C25-D
and ~eakly basic anion resin Duolite S-56/. This ion-exchange
system needs periodical regeneration with acids or bases and
-- 2 --
~7~
great additional quantities of clean water .
The contents of cobalt ions in the glucose-fructose
" syrups , which have been processed iIl the ion-exchange system,
is controlled with the help of an automatic abso'rption spectro-
~, v photometer , by means of tests , constantly taken from the
stream . Thus the operation of the cation-exchange system is
regulated .~In this processing Co ~ ions must be removed
effectively so that they shouldn't exceed 5 ppb with 25~S.(8).
This process complicates the production and influences
the effecti~ity and the prime-cost of the product .
It would be advantageous to have a method for obtaining
of glucose isomerase from strain-producer with industrial
importance , wherein cobalt'ions are not necessary for the
biosynthesis of the enzyme and for the izomerization of D-
glucose into D-fructose ~.~
The present invention provides a method for obtaining
' glucose isomerase~characterized in that the enzyme producing
strain Streptomyces sp.N.765 registration N. 143 - State
,,Institute for Drugs Control , is cultivated for 36 to 96 hours ,
~20 e.g. 36 to 72 hours , in a culture medium with xylose as
an inductor , the temperature being kept from 24 to 36C and
the initial p~ of cultivation being from 6.5 to 9.0 .
The present invention also provides a method for
isomerizing glucose characterized in that the isomarization is
carried out in the~presence o glucose isomerase , at a tempe-
rature from 50 to 90C , a pH from 6.0 to 9.0 , in the presence
of MgSO4.7~I2O in a concentration of from 1.10 4 to 1.10 M
and a substrate concentration from 0.1 to 3M , the glucose
isomerase having been prepared by cultivating the enzyme
producing strain Streptomyces sp.N. 765 registration N. 143 -
, State Institute for Drugs Control , for 36 to 72 hours in a
" ~"~ 3
,/1, ,~ . _
culture medium with xylose as an inductor , the temperature
being kept from 24 to 36~C and the initial pH oE cultivation
being from 6.5 to 9.0 .
In accordance with the present invention the culture
medium which is used may have the~following composition :
xylose 1.0 - 2.0 %
maize extract 1.5 - ~.0 ~ (in dry weight)
Na-acetate .025 0 1.0 %
Strain Streptomyces sp.N.765-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 a modified synthetic culture medium N. 1 after
Krassilnikov , the selection being realized on two substrate
levels with the use of xylose and xylane . Under these condi-
tions 18 streptomyces strains have been discovered which can
develop and produce the enzyme glucose isomerase . Among them is
the strain Streptomyces sp. N. 765 which is capable of producing
the enzyme glucose isomerase in the absence of Co + in the culti-
vation medium . The strain is put on deposit in the State
- Institute for Drugs Control - bul.Vladimir Zaimov N. 26 on
September 29th 1979 under N. 143 and has the following morpho-
logical and biochemical characteristics :
On culture medium 1 with a mineral source of nitrogen
(after G.G. Gause abd collaborators) the colonies are usually
oval in shape with unshaped edges , convex in the centre
swollen _ _
,/ , .
- 3a -
l:l3710~Lq~
with a crater like cavity with strongly expressed radial folds.
The growth is good. The sporangia in young cultures are elon-
gated, monopodially situated with 3-5 coils and in older cul-
tures they are condensed in sorgnum.
, , .
The spores are elongated with rounded ends: when they are
magnified more than 16 000 times some hair-like formations can
be observed on their surace. On a weaker magnification the
spores look smooth. Their size varies between 0,9 - 1,2
micrones in length and 0,4 - 0,6 micrones in width.
The colour of the air mycelium and substrate mycelium is
determined according to the colour scale of A.C. Bondartsev
and the scale of Tresner and Backus. With the different
culture media the colour of the air-mycelium changes from white
(dl) light-grey to violet (a5-d3) depending on the carbon and
nitrogen sources. On culture medium 1 with a mineral source
of nitrogen (after G.F.Gause and collaboratores) the colour is
light-grey to mousy-grey ~(a5-a3) and on a culture medium with
an organic source of nitrogen (after G.F.Gause and col.) the
colour is dark-grey to grey-~iolet (a2-a3).
~ 20 On culture media with different carbon and nitrogen sources
; the air mycelium is grei~sh-green.
On culture medium 1 with a mineral source of nitrogen
~after G.F.Gause and collaboratores) the substrate mycelium is
dove coloured to ultramarine (l~-v1~,
On culture medium 2 with an organic source of nitrogen
~after G.F.Gause and collaboratores) it is ultramarine (vl),
and after prolonged cultivation becomes black (al).
On culture media with different carbon and nitrogen sources
the substrate mycelium is from dark-red to black.
On culture medium meat-pepton agar. Low growth. Air mycelium
- pink and brick-red along the edge. Substrate mycelium brick-
red.
-- 4 --
On culture medium potato glucose agar. Growth very good.
Air mycelium - srey-blue. Substrate mycelium - blue to dark-
blue. On culture medium Tchapek with sucrose. Low growth.
Air mycelium-pink. Substrate mycelium - light-brick.
On culture medium Tchapek with glucose. Medium growth.
Air mycelium - light-blue. Substrate mycelium colourless with
a shade of the air mycelium.
On culture medium wi~h sucrose. Good growth. Air mycelium
- sky-blue. Substrate mycelium ~ dark-blue to black.
On cultural medium amylium agar. Good growth. Air mycelium
- grey. Substrate mycelium - blueO
On amylum ammonia culture, after Mishustin, Very good
growth. Air mycelium - grey. Substrate mycelium wine-red to
dark wine-red~
On synthetic medium after N.AOKrassilnikov. Medium
growth. Air mycelium light-asky-coloured. Substrate mycelium -
pink-violet. -
On CPI after N.A.Krassilnlkov. Good growth~ Air mycelium -
grey-blue. Substrate mycelium - red-brown.
On CPII after N.A. Krassilnlkov. Growth low to medium.
Air mycelium - blue. Substrate mycelium dark cream-coloured.
On CPIII after N.A.Krassilnikov. Growth good. Air mycelium
~- grey to ultramarine. Substrate mycelium wine-red.
On CPIV after N.A.Krassilnikov. Low growth. Air mycelium
light-grey. Substrate mycelium colourless, with a shade of
the air mycelium colour.
On CPV after N.A. Krassilnikov. Good growth. Air mycelium~-
grey. Substrate mycelium - dark-violet.
On synthetic culture medium after Vaxman. Medium growth.
Air mycelium grey to mousy-grey. Substrate mycelium dark cream
-coloured to red-brown.
On meat-amylum agar. Low growth. Air mycelium ~ white.
-- 5 --
9.C9
Substrate cream-coloured.
On peptone agar. Good growth. Air mycelium - grey~
Substrate colourless with a grey shade of the air mycelium
colour.
On glucose-asparagine agar. Very good growth. Air
mycelium-grey to ultramarine. Substrate mycelium hlue-violet
to dark-blue.
On glycerine-asparagine agar. Good growth. Air mycelium-
ultramarine. Substrate mycelium dark-violet to black.
-On tyrosine culture medium. Good growth. Air mycelium-
blue-grey, ultramarine. Substrate mycelium - red-brown.
On tyrosine-caseine-nitrate agar. Low growth. Air mycelium
-white. Substrate mycelium - cream~coloured.
On glucose-tyrosine agar. Medium growth. Air mycelium -
cream grey. Substrate mycelium - violet.
On saccharese-nitrate agara Good growth. Air mycelium -
grey to ultramarine. Substrate mycelium ~ blue to dark blue.
On glycerol-calcium malate agarO Good growth. Air
mycelium-blue to ultramarine.
On peptone-beef agar. Medium good growth~ Air mycelium-
grey. Substrate mycelium - colourless with a grey shade of the
air mycelium.
On oats agar. Medium growth. Air mycelium - grey.
Substrate mycelium - wine-red.
On tomato agar. Good growth. Air mycelium - grey.
Substrate mycelium - dark-beige, terracotta colour.
On lead-acetate agar. Low growth. Air m,ycelium brown.
Substrate mycelium colourless with a shade of the air mycelium
colour~
On iron peptone agar. ~ood growth. Air mycelium grey.
Substrate mycelium - colourless with a grey shade of the air
mycelium.
, ,,
~7~Q~
O~ yeast-malt agar~ Medium growth. Air mycelium - grey,
mousy grey. Substrate mycelium dark cream-coloured.
Strain tolerance towards NaCl. ~t shows low tolerance
towards the concentration of sodium chloride in the medium.
The maximum concentration is 4%. Under this concentration the
strain growth is low. The air mycelium is light-blue. The
substrate mycelium is dark-blue. A concentration higher than
2~ NaCl has a negative influence on the degree of sporulation.
It coagulates fattless milk. It doesn't condense gelatine.
It grows well on sucrose medium, but doesn't invert sucrose.
It grows very well on an amylum agar and hydrolyzes starch well.
It doesn't decompose cellulose and reduces nitrates to
nitrites.
It liberates hydrogen sulphide. It grows on potatos.
Hemolysis - negative. Tyrosinase - positive, it forms mela-
noids.
It has been found out that on basic culture medium of
Pridham and Gottlieb the growth is good in the presence of
the following carbon sources: glucose, fructose, lactose,
levulose, xylose, manose, cellulose, galactose, mannite, inosite,
arabinose, dextrine, ribose and glycerol.
The strain absorbssalicin on a smalL scale.
It doesn't grow on culture medium with sorbite, sucrose
and raffinose. Some differences in the pigmentation of the
air mycelium and the substrate mycelium are observed, depending
on the source of carbon.
The growth of the strain 06 good on modified basic culture
medium of Prudham and Gottlieb with the following sources of
nitrogen: NH4cl~ (N~4)2SO4 ; (NH4)2HPo4; NH4H2p 4
The growth is moderate on a culture medium with NH4NO3 and
Ma2HPO4. The strain does not grow at all on culture medium with
NaN03 and NaN02.
-
A very good growth of the strain is observed on culture
media with the following aminoacids:
glutamic acid, asparayi~lc acid, alanine, valine, asparagine~
The growth is moderate on a eulture medium with leueide, cystine,
proline, xydroxyproline, phenylalanine and tyrosine. Depending
on the source oE nitrogen, some differences in the pigmentation
of the air mycelium and the substrate mycelium are observed
According to some eharacteristies streptomyces strain
N.765 resembles Streptomyces eoelicolor, belonging to the series
gray after Bergey's - 1974 (Actinomyees coelieolor of the
Coelieolor growp after N.A.Krassilnikov - 1970). The latter is
distinguishecl by some morphologieal - eultural and physiological-
bioehemical properties, deseribed in the spebies'characterization
by Bergeys (]97~) and N.A.Krassilnikov (1970). Streptomyces
coelicolor forexample, has from 1 to 3 eoils of the spirals, it
condenses gelatine slowly and peptonizes fatless milk. Its
tyrosinase is negative. Therefor streptomices strain N. 765 is not
identical with the similar Streptomyces coelicolour (Actinomyces
eoelieolor) and that is why it is referred to as Streptomyces
sp, N. 76~ belonging to the Gray series after Bergey's (1974)
and the Coelieolor group after N.A.Krassilnikov (1970).
The strain-producer may be cultivated in Erlenmeyer flasks
of 500 ml containmg 50 ml of fermentation eulture medium using xylose as
"~.. .
inductor , for 36 to 96 hours at a temPerature of 24 to 36C , with an
~nltial pH of eultivation ranging from 6.5 to 9.0,using a shaker
at 180-320 revolutions per minute .
Isomeriæation of glueose to fruetose by means of glucose
; isomerase of the strain Streptomyces sp. N.765 can be carried
out through a direet treatment with fresh mycelium (separatecl
through eentrifugation at 12,000 revolutions per minute and
washed three times with 0 05 M phosphate buffer at pH 7-0)
or with dried mycelium (air-dried or aceton-dried cells) with
- 8 -
~ ,.
!l7~ LQ
enzyme solution ~obtained after supersound desintegration or
autolysis of cell material and separation of supernatant
through centrifugation at 15,000 rev./mln. with cultural r
centrifugation, containing extracellular isomerase or cells
made immobile on a hard carrier.
The fructose, formed in the reacting mixture determined
according to the cystein-carbasole method (4) and the activity
of the strain is expressed in mg. of fructose per ml.cultural
liquid or in International glucose isomerase units (GIU). One
10GIU is equal to the quantity of enzyme which under 7:0C and
pH 7.0 lM glucose solution in 0.05M phosphate buffer, and
2.10 2M MgSO4.7H2O transformes in one minute lMmol glucose
into lMmolO fructose.
The advantages of the method according to the invention,
are the following :
The strain Streptomyces sp. No765 produces the enzyme
glucose-isomerase in the absence of cohalt ions in the ferment-
; ation medium, The obtained e~zyme transforms D-glucose into D-
-fructose in the absence of cobalt ions in the isomerization mix-
20 ture, which considerably facilitates the technological process
and it is not necessary to use ion-exchange systems for the
separation of cobalt from the fructose~containing syrups. F
As compared with the strains, microorganisms and mutant- E
.,.~ ,.
producers of glucose isomerase known in patent literature, which c
do not require cobalt ions in the cultivation medium and in
izomerization mixtures (2a, b, c; 9a, b), Streptomyces sp. N.
765 excels them with its glucose isomerase activity, favourable
pH optimum (7.0), higher temperature optimum (80) of the enzyme
and its considerable thermostability between 40 and 70C.
l. Xylose culture medium: i
Xylose 20 g.
agar
- - : , _ 9 _
o
KNO3 1.0 g,
K2HPO4 0 5 g~ ~
MgSO4.7H2O 0.5 g~ 'I
NaCl 0.5 gO
Caco3 1.,0 g.
FeSO4 0.001 g.
water up to 1. 1.
2. ~otato-glucose agar :
Potato extract from 300 g. boiled potatoes
glucose 10 g.
agar 20 g.
water up to 1. 1.
It is recommended that for the maintenance of the strain
both culture media should be alternated.
To a well germinated material from a 10-15 days culture on
culture medium 1 or 2 (1 is recommended) , 6 ml of inoculation
culture medium wasadded, having the following composition:
xylose 1,%
beef extract 2 %
MgSO~.7H2O 0.1
K2HPO4
A wash-out of the cell mass was carried out and the test-
tube was put on a shaker for 24 hours 30C and at 240 revolu-
tions per minute . From thus adapted culture inocula-tion medium
with the following composition is shown :
Xylose 1 ~
maize extract 3 % (in dry weight)
Na-acetate 0.5 %
The cultivation was carried out in Erlenmeyer flasks of
500 ml fermentation culture medium. It was carried out for
60 hours at 30C using a shaker operating at 240 revolution~ per minute .
The initial pH of cultivation was 8.5 .
~'~'J~ - lO -
....
... ,.. _. _. _. _~. , ~......... ,._ .. _... .. , . . ,.. .. ,__ .__
After 60 hours of cultivation of Streptomyces sp.N.765
160 to 240 g. of humid biomass we.re obtained per liter of
cultural liquid .
Izomerization of glucose to fructose by means of glucose
isomerase of the strain Streptomyces sp.N.765 was carried out
at a temperature of 70C and a pH of 7 in the presence of
MgSO4.7H2O in a concentration of 2.10 2M and of a substrate
in a concentration of lM .
The activity of the strain Streptomyces sp. N.765 was
10 of 75-130 mg of fructose per 1 ml of cultural liquid , or
7 000-12 000 GIU per liter of cultural liquid .
~,
, . . .
~7~
REFERENCES
1. Cotter WnP~ ~ Lloyd N.E., Hinman C.W. 1971. Patent USA
3623953
2. a/ CPC international Inc. 1975. British Patent 1411763
b/ CPC International Inc. 1975. British Patent 1411764
c/ CPC International Inc. 1975. British Patent 1411765
3. Danno G. Ibid~ 1971,35 ,7,997
4. Dishe Z., Borenfreud E. 1951. J. Biol. Chem., 192,583
Jacbbziner H., Raybin H.W. 1961. Arch.Pediat 78,200
6. Levy H., Levinson V~, Shade A. 1950 Arch. Biochem., 27,34
7. Marschall R.O., Rooi E.R. 1957. Science, 125, N.3249,648
8. Mi-Car International Inc. Isomerized Syrup process. 1975
MFS Information.
9. a/ R.J. Reynolds Tobacco Co. 1973 British Patent 1328970
b/ R.J. Reynolds Tobacco Co. 1974-British Patent 1362365
10. Sergio S., Kare L. 1975. Appl. Microbiol., 29,6,745
11. Somers E. 197~.J. Food Sci., 39,215
12. Schraeder H.A.~ Nason A.P., Tipton I.H. 1967. J, Chronic.
Dis., 20,869.
13. Stradberg G.W., Smiley K,L. 1971. Appl. Microb., 21,588
14. Takasaki Y. Ibid. 1966, 30jl2,1247
]5. Takasaki Y. Kosegu Y., Kanbayashi A. 1969. Agr, Biol.Chem
33, 11,1527
-16. Takasaki Y,,Kosogu Y. 1969. Academic Press Inc. N.Y.p.561.
17. Tsumura N., Hagi N., Sato T. 1967. Ayr. Biol. Chem., 31,902
18. Yamanaka K. 1961. Agr. Biol. Chem., 25,4,272.
- 12 -
