CA1171010A - Method for obtaining of glucose-isomerase - Google Patents
Method for obtaining of glucose-isomeraseInfo
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- CA1171010A CA1171010A CA000365821A CA365821A CA1171010A CA 1171010 A CA1171010 A CA 1171010A CA 000365821 A CA000365821 A CA 000365821A CA 365821 A CA365821 A CA 365821A CA 1171010 A CA1171010 A CA 1171010A
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- mycelium
- glucose
- culture medium
- glucose isomerase
- xylose
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
- C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
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Abstract
ABSTRACT OF THE DISCLOSURE
The present invention relates to a method for obtaining of glucose isomerase, wherein the enzyme-producing strain Streptomyces sp.No 765 registration N.143 - State Institute for Drugs Control, is cultivated for 36 - 72 hours in a culture medium with xylose as an inductor, the temperature being kept from 24 to 36°C, the initial pH of cultivation is from 6,5 to 9,0; the temperature of izomerization is from 50 to 90°C, pH
is from 6,0 to 9,0 in the presence of MgSO4.7H2O in concentration from:1.10 4 to 1.10-2M and substrate concentration from 0,1 to 3M.
The present invention relates to a method for obtaining of glucose isomerase, wherein the enzyme-producing strain Streptomyces sp.No 765 registration N.143 - State Institute for Drugs Control, is cultivated for 36 - 72 hours in a culture medium with xylose as an inductor, the temperature being kept from 24 to 36°C, the initial pH of cultivation is from 6,5 to 9,0; the temperature of izomerization is from 50 to 90°C, pH
is from 6,0 to 9,0 in the presence of MgSO4.7H2O in concentration from:1.10 4 to 1.10-2M and substrate concentration from 0,1 to 3M.
Description
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 ~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.
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.
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
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 ~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.
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.
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 ~ ,.
!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 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
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
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 ~ ,.
!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 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
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.
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.
Claims (4)
1. 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 72 hours in a culture medium with xylose as an inductor , the temperature being kept from 24 to 36°C
and the initial pH of cultivation being from 6.5 to 9.0 .
and the initial pH of cultivation being from 6.5 to 9.0 .
2. Method for isomerizing glucose characterized in that the isomerization is carried out in the presence of glucose isomerase , at a temperature from 50 to 90°C , a pH
from 6.0 to 3.0 , in the presence of MgSO4.7H2O in a concentration of from 1.10-4 to 1.10-2M 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 culture medium with xylose as an inductor , the temperature being kept from 24 to 36°C and the initial pH
of cultivation being from 6.5 to 9.0 .
from 6.0 to 3.0 , in the presence of MgSO4.7H2O in a concentration of from 1.10-4 to 1.10-2M 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 culture medium with xylose as an inductor , the temperature being kept from 24 to 36°C and the initial pH
of cultivation being from 6.5 to 9.0 .
3. Method according to claim 1 wherein the culture medium which is used has the following composition :
xylose 1.0 - 2.0 %
maize extract 1.5 - 4.0 % (in dry weight) Na-acetate 0.25 - 1.0 % .
xylose 1.0 - 2.0 %
maize extract 1.5 - 4.0 % (in dry weight) Na-acetate 0.25 - 1.0 % .
4. 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 in a culture medium with xylose as an inductor , the temperature being kept from 24 to 36°C
and the initial pH of cultivation being from 6.5 to 9.0 .
and the initial pH of cultivation being from 6.5 to 9.0 .
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| BG7945720A BG32192A1 (en) | 1979-11-29 | 1979-11-29 | Method for obtaining of glucoseisomerase |
| BG45720 | 1979-11-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1171010A true CA1171010A (en) | 1984-07-17 |
Family
ID=3906780
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000365821A Expired CA1171010A (en) | 1979-11-29 | 1980-11-28 | Method for obtaining of glucose-isomerase |
Country Status (15)
| Country | Link |
|---|---|
| JP (1) | JPS56131383A (en) |
| AT (1) | AT378204B (en) |
| BE (1) | BE886407A (en) |
| BG (1) | BG32192A1 (en) |
| CA (1) | CA1171010A (en) |
| CH (1) | CH648592A5 (en) |
| DD (2) | DD161153A3 (en) |
| DE (1) | DE3044357A1 (en) |
| DK (1) | DK151270C (en) |
| ES (1) | ES497272A0 (en) |
| FR (1) | FR2473549B1 (en) |
| GB (1) | GB2063884B (en) |
| IT (1) | IT1145317B (en) |
| NL (1) | NL8006509A (en) |
| YU (1) | YU297780A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3224024C2 (en) * | 1982-06-28 | 1986-09-11 | Uop Inc., Des Plaines, Ill. | Method for immobilizing glucose isomerase and immobilized glucose isomerase system |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1103394A (en) * | 1965-05-11 | 1968-02-14 | Agency Ind Science Techn | A method of manufacturing syrup containing fructose from glucose by the use of an enzymatic process |
| IL38715A (en) * | 1971-04-22 | 1974-11-29 | Miles Lab | Production of glucose isomerase |
| BE788843A (en) * | 1971-09-17 | 1973-03-15 | Cpc International Inc | PRODUCTION OF ENZYMATIC PREPARATIONS OF XYLOSE ISOMERASE (DEXTROSE) |
| US3957587A (en) * | 1973-11-21 | 1976-05-18 | Cpc International Inc. | Production of xylose (dextrose) isomerase enzyme preparations |
-
1979
- 1979-11-29 BG BG7945720A patent/BG32192A1/en unknown
-
1980
- 1980-11-24 AT AT0573680A patent/AT378204B/en not_active IP Right Cessation
- 1980-11-24 YU YU02977/80A patent/YU297780A/en unknown
- 1980-11-25 DE DE19803044357 patent/DE3044357A1/en not_active Withdrawn
- 1980-11-26 DK DK503480A patent/DK151270C/en not_active IP Right Cessation
- 1980-11-26 CH CH8782/80A patent/CH648592A5/en not_active IP Right Cessation
- 1980-11-28 FR FR8025320A patent/FR2473549B1/en not_active Expired
- 1980-11-28 NL NL8006509A patent/NL8006509A/en not_active Application Discontinuation
- 1980-11-28 DD DD80225621A patent/DD161153A3/en unknown
- 1980-11-28 BE BE0/202978A patent/BE886407A/en not_active IP Right Cessation
- 1980-11-28 DD DD80225622A patent/DD161124A3/en unknown
- 1980-11-28 IT IT50273/80A patent/IT1145317B/en active
- 1980-11-28 ES ES497272A patent/ES497272A0/en active Granted
- 1980-11-28 CA CA000365821A patent/CA1171010A/en not_active Expired
- 1980-11-29 JP JP16903680A patent/JPS56131383A/en active Pending
- 1980-12-01 GB GB8038498A patent/GB2063884B/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| BE886407A (en) | 1981-03-16 |
| NL8006509A (en) | 1981-07-01 |
| DE3044357A1 (en) | 1981-07-02 |
| BG32192A1 (en) | 1982-06-15 |
| CH648592A5 (en) | 1985-03-29 |
| DD161153A3 (en) | 1985-02-27 |
| ES8406088A1 (en) | 1984-07-01 |
| ES497272A0 (en) | 1984-07-01 |
| FR2473549B1 (en) | 1985-07-05 |
| DK151270B (en) | 1987-11-16 |
| DD161124A3 (en) | 1985-01-09 |
| GB2063884A (en) | 1981-06-10 |
| ATA573680A (en) | 1984-11-15 |
| JPS56131383A (en) | 1981-10-14 |
| DK151270C (en) | 1988-05-16 |
| FR2473549A1 (en) | 1981-07-17 |
| AT378204B (en) | 1985-07-10 |
| YU297780A (en) | 1983-02-28 |
| DK503480A (en) | 1981-05-30 |
| IT1145317B (en) | 1986-11-05 |
| IT8050273A0 (en) | 1980-11-28 |
| GB2063884B (en) | 1983-04-20 |
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