WO1986000336A1 - Concentre stable de glucose-isomerase et un procede de preparation de celui-ci - Google Patents

Concentre stable de glucose-isomerase et un procede de preparation de celui-ci Download PDF

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Publication number
WO1986000336A1
WO1986000336A1 PCT/FI1985/000057 FI8500057W WO8600336A1 WO 1986000336 A1 WO1986000336 A1 WO 1986000336A1 FI 8500057 W FI8500057 W FI 8500057W WO 8600336 A1 WO8600336 A1 WO 8600336A1
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WO
WIPO (PCT)
Prior art keywords
glucose isomerase
glucose
solution
concentrate
isomerase
Prior art date
Application number
PCT/FI1985/000057
Other languages
English (en)
Inventor
Kalevi Juhani Visuri
Original Assignee
Suomen Sokeri Oy
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from FI842549A external-priority patent/FI842549A/fi
Application filed by Suomen Sokeri Oy filed Critical Suomen Sokeri Oy
Priority to HU853121A priority Critical patent/HU194937B/hu
Publication of WO1986000336A1 publication Critical patent/WO1986000336A1/fr

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Classifications

    • 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)
    • 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
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Definitions

  • the present invention relates to a concentrate of a glucose isomerase enzyme which is chemically and microbiologically stable without addition of microbi- cides and which is very useful as such for the immobilization of the enzyme on the carrier material in a reactor column.
  • the invention also relates to a process for the preparation of such a concentrate.
  • the use of glucose isomerase for the isome- rization of glucose to fructose is a well known indus ⁇ trial process, in which the enzyme is always used in the immobilized form.
  • the immobilized enzyme preparation is prepared as a separate process, in which either the enzyme is immobilizied on the carrier by means of the adsorption technique or the entire microbial cell mass with the enzymes is immobilized to make a matrix.
  • the reactor column is made up of ready immobilized enzyme. When the enzyme has been used up, the column is emptied and again made up of fresh immobilized enzyme preparation.
  • a precipitate or crystal mass obtained from glucose isomerase purification processes based on precipitation or crystallization contains at least 60 per cent by weight of water, which cannot be removed without destruction of the enzyme structure.
  • the crystal mass as such is not microbiologically stable, it is difficult to handle and to dose.
  • An alternative is dissolution of the crystal mass to a solution, the concentration of which should preferably be as high as possible, in view of storage, stability and transport. The most natural way is to dissolve the enzyme crystals in water or in a dilute salt solution. In practice, however, it has been noticed that, owing to the rela- tively low solubility, it is not possible to prepare a sufficiently concentrated solution of the isomerase by dissolving crystals in water.
  • a diluted water solution of the isomerase is highly instable and loses its activity as a result of microbiological and chemical deterioration in a few days.
  • fractional sulfate precipitation ammonium and/or magnesium sulfate
  • the sulfate concentrations used in these purification processes for isomerases have usually been relatively high.
  • the unneces ⁇ sary proteins are first precipitated from the iso- merase solution at the saturation degree of 40 % of ammonium sulfate (about 240 g (NH ⁇ J-SO. in a litre of solution) , and then the isomerase itself is precipitated at a higher saturation degree (up to 60 %) .
  • ammonium sulfate concentrations are used in fractional precipitation, an abundance of other proteins always precipitate along with the iso ⁇ merase, the more other proteins the higher the con ⁇ centration of ammonium sulfate used, unless the isomerase has already been prepurified in some way by some other methods.
  • the amorphous precipitate is, as a rule, a mixed deposit, which, besides the isomerase, also contains other proteins if the raw-material is a microbial-cell liquid which has not been prepurified. Such an amorphous precipitate is difficult to separate from the mother liquor, and precipitation does not give the desired purification effect with respect to the isomerase.
  • the isomerase has first been purified by means of some methods (extraction with an organic solvent, DEAE-Sephadex-column chromatograph , ammonium sulfate precipitation, dialysis) , whereupon crystallization of purified isomerase from pure water solution has been carried out with ammonium sulfate, phosphate buffer, or acetone.
  • the present invention relates to a stable glucose isomerase concentrate, which is characterized in that it contains glucose isomerase dissolved in a concentrated carbohydrate water solution.
  • the concentrate according to the invention is both chemically and microbiologi- cally stable and particularly useful in processes in which the immobilization of glucose isomerases on the carrier material is carried out by the regeneration technique in a reactor column.
  • Suitable carbohydrates are all non-toxic carbohydrates readily soluble in water, such as sugars and polyols. Particularly suitable are sugars that are used as foods as such as well as sugar alcohols which are allowed as food additives. Since the end product of isomerization is a glucose-fructose syrup, the mixture of these sugars, i.e. invert sugar, is excellently suitable for the purpose.
  • the total dry solids content in the concentrate must be such that it is in itself microbiologically stable, i.e.
  • the glucose isomerase concentrate according to the invention preferably contains 5 to 15 wt. % of glucose isomerase, 30 to 60 wt. % of a carbohydrate soluble in water, such as glucose, maltose, fructose, saccharose, sorbitol, xylitol, or of a mixture thereof, e.g. invert sugar, glucose syrup or isomerized glucose syrup, no more than 15 wt. % of an appropriate salt, such as ammonium and/of magnesiu 'sulfate, and/or a buffer (pH 6.0 to 8.0), e.g. sodiumpo ' tassium- phosphate buffer, carbonate buffer, or a buffer made of an organic salt (e.g. salt of an aminoacid) or a mixture of buffers, and balance water.
  • a buffer pH 6.0 to 8.0
  • GlU glucose isomerase units
  • composition of the concentrate may, however, be varied as required, while taking care that the total concentration is sufficiently high and the solution, yet, in liquid form and easy to handle.
  • the glucose isomerase concentrates according to the invention are pure, stable, and easy to handle and dose.
  • the enzyme activity can be adjusted. In view of the use, a suitable range of activity is 2000 to 5000 GlU/g preparation.
  • the sugars, sugar alcohols and salts to be used are preferably of food grade (e.g., Food Chemicals Codes standard) .
  • the enzyme shall be purified adequate- ly by crystallization or by any other method.
  • the glucose isomerase can be readily adsorbed from the concentrate directly onto the carrier material placed in the reactor column.
  • the concentrate is diluted with water and the dilute solution is fed into the column, whereby the enzyme is adsorbed onto the carrier.
  • the technique resembles the regeneration of an ion-exchange column. Alter the enzyme has been in use and thus inactivated, the protein is washed off the column by means of an alkali, whereinafter fresh enzyme can be fed into the column so as to regenerate it.
  • a suitable carrier material is a material with anion-exchange capacity, e.g. glass beads, ion- exchange resin or a silica-based carrier. Particularly suitable are diethylaminoethyl (DEAE) derivatives. which are known to adsorb proteins, such as DEAE-cellu- lose and DEAE-dextran. There is a large number of carrier materials described in the literature.
  • DEAE diethylaminoethyl
  • the invention also relates to a process for the preparation of the stable glucose-isomerase concentrate described above.
  • the process is characterized by a) adding a suitable salt to a ultrafiltered glucose isomerase solution obtained from fermentation, so as to crystallize the glucose isomerase, b) cooling the solution so as to promote crystallization of the glucose isomerase, and separating the crystal mass formed and then if desired, effecting one or several recrystallizations, and c) adding a carbohydrate or a concentrated water solution thereof to the obtained crystal mass, which dissolves, whereby a stable glucoseisomerase concentrate is obtained.
  • the isomerase enzyme can be purified highly efficiently by crystal ⁇ lizing it from a salt solution.
  • Suitable salts are all such non-toxic salts, which do not inactivate the enzyme.
  • ammonium and/or magnesium sulfate is used.
  • Precipitation of glucose isomerase by means of a salt is in itself a conventional procedure, being described, e.g., in Agr. Bio. Chem. 29 (1965) pp. 1129-1134 (Isumure and Sato) .
  • an amorphous precipitate or a crystalline precipitate can be obtained from the isomerase by using the same chemical, ammonium sulfate or magnesium sulfate. It is generally known hat numerous enzymes behave in the same way.
  • a characteristic and surprising feature of the process according to the invention is that the iso ⁇ merase precipitates as a crystalline substance and before all other substances that may precipitate.
  • precisely selected conditions and such a low ammonium sulfate concentration are used that no other substances precipitate from the solution.
  • the process differs essentially from what has been described in the literature in this, field. Earlier isomerase has not been crystallized directly and alone as the only precipitating component from a cell liquid or a cell liquid concentrate of a production microbe. The process also gives a very high yield, which differs essentially from what has been earlier stated in the literature.
  • enzyme preparations can be increased, e.g., by means of glycerol, polyalcohols and sugars.
  • Such enzyme preparations are usually prepared by adding the said substances to a concentrated enzyme solution.
  • the preparation of the glucose isomerase con- centrate is preferably carried out as follows: a) A cell liquid is prepared from the organism Streptomyces rubiginosus by means of lysis (US Patent 4,410,627), and from the cell liquid, by ultrafiltration, a concentrate containing isomerase is prepared for raw-material for the crystallization process,* a preferred isomerase concentration of the concentrate is 200 to 800 GlU/g. b) The pH of the isomerase solution is adjusted to the range of 5.7 to 8.0, preferably pH 7.0. c) The solution is cooled to 16 C or below. d) Ammonium and/or magnesium sulfate is added to the solution, preferably 50 to 170 g per litre of solution.
  • the sulfate quantity to be added depends on the original isomerase concentration and on the final temperature of crystallization.
  • the most preferable quantity of sulfate to be added is such a quantity with which only the isomerase is crystallized but the other proteins do not yet start precipitating.
  • the addition of the sulfates is preferably effected gradually so that the addition of the whole quantity takes 2 to 4 hours, even though a acceptable result may also be obtained by adding the whole quantity all at once, but in such a case the size of the isomerase crystals remains unfavourably small
  • f) The solution is cooled preferably during several hours, preferably close to the freezing point of the mixture concerned, the freezing point being at the lowest tested sulfate concentrations about -2 C and at the highest ones -6°C; the cooling may be started either simultaneously with the beginning of the sulfate addition or only upon completion of the sulfate addition; by means of gradual cooling, a cooling-crystallization effect is obtained that increases the size of the isomerase crystals advantageously, besides the fact that cooling has a solubility lowering effect, which again increases the yield.
  • the isomerase crystals are separated from the solution by allowing them to settle to the bottom of the vessel, by filtering them or, on a large scale most preferably by centrifuging them by means of a continuous separator. h) The separated crystal mass is dissolved by adding to it a dr carbohydrate or its concentrated water solution, whereby the isomerase crystals are dissolved and a stable glucose-isomerase concentrate is produced.
  • the crystallization may be repeated, in which case the crystal mass must be dissolved after the separation (step g) into an abundant quantity of water at a relatively high temperature (20 to 30°C) .
  • a suitable quantity of water is such that the isomerase activity of the solution is 500 to 2000 Gl ⁇ /ml, in other words, the weight of the quantity of water used is typically 4 to 10 times the weight of the crystal mass.
  • a batch of about 40 cubic metres of Streptomyces rubiginosus microbe was fermented in a way known per se (reference US Pat. 4,410,627).
  • the cell mass was lysed in a known way (same reference) .
  • the cell residues and the other solid matter were removed by filtration by means of a conventional siliceous- earth drum filter, whereby 32 tons of isomerase-con- taining filtrate was obtained.
  • This filtrate was filtered by means of a PCI (Patterson-Candy Inc.) ultrafilter, whereby 3000 kg of isomerase-containing concentrate was obtained, the activity of which was 960,000,000 GIU.
  • the permeate that had passed through the ultrafiltration membrane was removed.
  • To the crystal mass 45 kg of glucose and 45 kg of fructose as well as 20 kg of invert sugar having a" dry solids content of 70 per cent by weight were added.
  • 200 kg of an enzyme preparation was obtained, the composition of which was as follows: 29.2 wt. % water 52.0 wt. % sugars 14.5 wt. % glucose isomerase 4.3 wt. % salts (magnesium-ammonium sulfate)
  • the glucose-isomerase activity of the enzyme concentrate was 4500 GlU/g.
  • An ultrafiltered fermentate was prepared in the way described in Example 1. To 4000 kg of ultra- filtered fermentate, 244 kg of crystalline ammonium sulfate was added, followed by an ammonium sulfate solution in which 600 kg of salt had been dissolved into 900 kg of water. The solution was cooled to 13°C and kept at this temperature for 20 hours. The crystal mass formed was separated by means of a
  • the glucose-isomerase activity of the concentrate was 3000 GIU per gram.
  • An ultrafiltered fermentate was prepared in the way described in Example 1.
  • 4000 litres of ultrafiltered fermentate having an activity of 2,400,000,000 GIU was used.
  • the pH of the solution was adjusted by means of a 5 % NaOH solution to pH 7.0, and the temperature of the solution was adjusted to 12 C.
  • 500 kg of ammonium sulfate dissolved in 750 litres of water was added to the solution during two hours with an even rate of feed. Then the solution was cooled to -2 C, and the solution was stirred for 24 hours.
  • the glucose isomerase crystals were separated by means of a Westfalia NA-7 separator.
  • the yield was 390 kg of crystal mass having a dry solids content of 23.6 per cent by weight and an activity of 2,300,000,000 GIU.
  • 30 kg of sodium chloride and 180 kg of glucose were added to the crystal mass, and the pH of the enzyme concentrate obtained was adjusted, by means of a 5 % NaOH solution, to 7.0 (the glucose was partly isomerized) .
  • 600 kg cf stable enzyme concentrate was obtained, the composition of which was as follows:
  • An ultrafiltered fermentate was prepared in the way described in Example 1.
  • 4000 litres of ultrafiltered fermentate having an activity of 2,400,000,000 GIU was used.
  • the pH of the solution was adjusted by means of a 5 % NaOH solution to pH 7.0.
  • the temperature of the solution was adjusted to 12°C.
  • 500 kg of ammonium sulfate dissolved in 750 litres of water was added to the solution during two hours with an even rate of feed. The solution was then cooled to -2°C, and stirred for 24 hours.
  • the glucose isomerase crystals were separated by decanting.
  • the yield was 230 kg of a crystal mass having a dry solids content of 40.0 per cent by weight and an activity of 2,300,0C ⁇ 000 GIU.
  • To the crystal mass 115 kg of glucose and 115 kg of fructose as well as 50 kg of invert sugar having a dry solids content of 70 % were added. In this way, 510 kg of an enzyme preparation was obtained, the composition of which was as follows:
  • Example 5 An ultrafiltered isomerase concentrate was prepared in the way described in Example 1. 50 g of ammonium sulfate was added to 0.95 litre of an isomerase concentrate having an activity of 600 GlU/ml and a temperature of 25 C. No precipitate was formed in the solution at this stage. The solution was cooled during 16 hours to 0 C, and it was kept at that tem ⁇ perature under gentle stirring constantly.
  • the isomerase started crystallizing in two days, and the crystallization continued so that after five days, 97.5 per cent by weight of the isomerase was in crystalline form and 2.5 per cent by weight still in dissolved form in the mother liquor.
  • the crystals were separated from the solution by means of a laboratory centrifuge. Thereby 56 grams of wet crystal mass was recovered.

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Abstract

Concentré stable de glucose-isomérase où la glucose-isomérase est dissoute dans une solution aqueuse concentrée d'hydrates de carbone. La présente invention a également trait à un procédé de préparation d'un tel concentré de glucose-isomérase où a) un sel approprié est ajouté à la solution ultra-filtrée de glucose-isomérase obtenue à partir de la fermentation, afin de cristalliser la glucose-isomérase b) la solution est refroidie de manière à promouvoir la cristallisation de la glucose-isomérase et la masse cristalline formée est séparée, après quoi, si on le désire, une ou plusieurs recristallisations sont effectuées, et c) on ajoute un hydrate de carbone ou une solution aqueuse concentrée de celui-ci à la masse cristalline obtenue, ladite masse étant dissoute, permettant d'obtenir un concentré stable de glucose-isomérase. Le concentré de glucose-isomérase conforme à la présente invention est utilisé pour immobiliser la glucose-isomérase sur un matériau porteur dans une colonne de réacteur.
PCT/FI1985/000057 1984-06-25 1985-06-19 Concentre stable de glucose-isomerase et un procede de preparation de celui-ci WO1986000336A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
HU853121A HU194937B (en) 1984-06-25 1985-06-19 Stable concentrate of glycose isomerase and process for producing them

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
FI842549A FI842549A (fi) 1984-06-25 1984-06-25 Stabilt glukosisomeraskoncentrat och foerfarande foer dess framstaellning.
FI842549 1984-06-25
FI852270 1985-06-06
FI852270A FI78117C (fi) 1984-06-25 1985-06-06 Foerfarande foer framstaellning av ett stabilt glukosisomeraskoncentrat.

Publications (1)

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WO1986000336A1 true WO1986000336A1 (fr) 1986-01-16

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FI (1) FI78117C (fr)
HU (1) HU194937B (fr)
SU (1) SU1575946A3 (fr)
WO (1) WO1986000336A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0383569A2 (fr) * 1989-02-16 1990-08-22 Pafra Limited Stockage de matériaux
US5565318A (en) * 1994-09-02 1996-10-15 Pharmacia Biotech, Inc. Room temperature stable reagent semi-spheres
US5593824A (en) * 1994-09-02 1997-01-14 Pharmacia Biotech, Inc. Biological reagent spheres
US5801022A (en) * 1990-08-03 1998-09-01 Vertex Pharmaceuticals, Incorporated Method of producing a product with crosslinked crystals of thermolysin
US6426210B1 (en) 1991-06-26 2002-07-30 Inhale Therapeutic Systems, Inc. Storage of materials
US6589560B2 (en) 1995-04-14 2003-07-08 Nektar Therapeutics Stable glassy state powder formulations
USRE38385E1 (en) * 1989-02-16 2004-01-13 Nektar Therapeutics Storage of materials

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2773002A (en) * 1955-03-31 1956-12-04 Nat Dairy Res Lab Inc Purification of lactase enzyme and spray-drying with sucrose
GB1076750A (en) * 1964-09-16 1967-07-19 Takeda Chemical Industries Ltd Enzyme preparations in liquid form
US3413198A (en) * 1966-06-30 1968-11-26 Calbiochem Reagents and method for assaying biological samples
DE2038103A1 (de) * 1970-07-31 1972-02-10 Henkel & Cie Gmbh Waessrige Reinigungsmittelkonzentrate mit einem Gehalt an stabilisierten Enzymen
DE2712007A1 (de) * 1976-03-19 1977-09-29 Cpc International Inc Stabilisiertes glucoseisomerase- enzymkonzentrat und verfahren zu seiner herstellung
US4152211A (en) * 1977-08-23 1979-05-01 Novo Industri A/S Iron containing cell mass glucose isomerase preparation
US4237231A (en) * 1979-11-13 1980-12-02 Uop Inc. Method of purifying glucose isomerase and a composition for storing same
GB2090599A (en) * 1981-01-05 1982-07-14 Novo Industri As Stabilized plasmin compositions and method for preparation thereof

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2773002A (en) * 1955-03-31 1956-12-04 Nat Dairy Res Lab Inc Purification of lactase enzyme and spray-drying with sucrose
GB1076750A (en) * 1964-09-16 1967-07-19 Takeda Chemical Industries Ltd Enzyme preparations in liquid form
US3413198A (en) * 1966-06-30 1968-11-26 Calbiochem Reagents and method for assaying biological samples
DE2038103A1 (de) * 1970-07-31 1972-02-10 Henkel & Cie Gmbh Waessrige Reinigungsmittelkonzentrate mit einem Gehalt an stabilisierten Enzymen
DE2712007A1 (de) * 1976-03-19 1977-09-29 Cpc International Inc Stabilisiertes glucoseisomerase- enzymkonzentrat und verfahren zu seiner herstellung
US4152211A (en) * 1977-08-23 1979-05-01 Novo Industri A/S Iron containing cell mass glucose isomerase preparation
US4237231A (en) * 1979-11-13 1980-12-02 Uop Inc. Method of purifying glucose isomerase and a composition for storing same
GB2090599A (en) * 1981-01-05 1982-07-14 Novo Industri As Stabilized plasmin compositions and method for preparation thereof

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE38385E1 (en) * 1989-02-16 2004-01-13 Nektar Therapeutics Storage of materials
EP0383569A3 (en) * 1989-02-16 1990-11-22 Pafra Limited Storage of materials
US5098893A (en) * 1989-02-16 1992-03-24 Pafra Limited Storage of materials
EP0383569A2 (fr) * 1989-02-16 1990-08-22 Pafra Limited Stockage de matériaux
USRE39497E1 (en) * 1989-02-16 2007-02-27 Nektar Therapeutics Storage of materials
USRE37872E1 (en) * 1989-02-16 2002-10-08 Inhale Therapeutics Systems, Inc. Storage of materials
US5801022A (en) * 1990-08-03 1998-09-01 Vertex Pharmaceuticals, Incorporated Method of producing a product with crosslinked crystals of thermolysin
US6825031B2 (en) 1991-06-26 2004-11-30 Nektar Therapeutics Storage of materials
US6426210B1 (en) 1991-06-26 2002-07-30 Inhale Therapeutic Systems, Inc. Storage of materials
US5565318A (en) * 1994-09-02 1996-10-15 Pharmacia Biotech, Inc. Room temperature stable reagent semi-spheres
US5763157A (en) * 1994-09-02 1998-06-09 Pharmacia Biotech Inc. Biological reagent spheres
US5593824A (en) * 1994-09-02 1997-01-14 Pharmacia Biotech, Inc. Biological reagent spheres
US6589560B2 (en) 1995-04-14 2003-07-08 Nektar Therapeutics Stable glassy state powder formulations

Also Published As

Publication number Publication date
FI852270L (fi) 1985-12-26
FI78117C (fi) 1989-06-12
SU1575946A3 (ru) 1990-06-30
FI852270A0 (fi) 1985-06-06
HU194937B (en) 1988-03-28
FI78117B (fi) 1989-02-28
HUT40463A (en) 1986-12-28

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