EP0204805A1 - Process for producing alginates having improved physical properties, and the use of said alginates - Google Patents

Process for producing alginates having improved physical properties, and the use of said alginates

Info

Publication number
EP0204805A1
EP0204805A1 EP19860900291 EP86900291A EP0204805A1 EP 0204805 A1 EP0204805 A1 EP 0204805A1 EP 19860900291 EP19860900291 EP 19860900291 EP 86900291 A EP86900291 A EP 86900291A EP 0204805 A1 EP0204805 A1 EP 0204805A1
Authority
EP
European Patent Office
Prior art keywords
alginates
alginate
preparation
physical properties
enzyme
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP19860900291
Other languages
German (de)
French (fr)
Inventor
Bjorn Larsen
Gudmund Skjak Braek
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sintef AS
Original Assignee
Sintef AS
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
Application filed by Sintef AS filed Critical Sintef AS
Publication of EP0204805A1 publication Critical patent/EP0204805A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate

Definitions

  • the present invention relates to the preparation of alginates having improved physical properties, especially with respect to the formation of gels having inorganic or polyvalent organic ions.
  • Said modified alginates are intended to be used for immobilizing and encapsulating enzymes and/or cells for use in biotechnological processes .
  • alginate gels as an immobilizing material has several deficiencies, two of them being:
  • Calcium alginate gels are destabilized by compounds having affinity for calcium, for instance EDTA, citrate, lactate and phosphate, as well as high concentrations of
  • + + ++ cations such as Na , K and Mg ;
  • Alginates currently used have a high degree of chemical heterogenity and provide gels having pores of such great size that proteins - enzymes and other macro- molecules - can leach out, at the same time as the size distribution of the pores is difficult to control.
  • Alginate is the most important structural poly- saccharide in marine brown algae and is used for several industrial purposes wherein the properties of the polymer are utilized as a polyelectrolyte - for instance for gel formation and thickening purposes - and also for its water and ion binding capacity.
  • the purpose of the present invention is to prepare alginates having physical properties satisfying the requirements for increased gel strength and stability and better controllable pore size.
  • alginate is a polyuronide built up from two uronic acids, viz., D-mannuronic acid ( ) and the C-5-epimer L-guluronic acid (G) . They are arranged in such fashion that the polymer is further built up from three types of sequence: (G)-rich sequences called G-blocks, (M)-rich sequences called M-blocks and alter ⁇ nating structure symbolized by ( G G G) .
  • G-blocks G-blocks
  • M-rich sequences called M-blocks
  • alter ⁇ nating structure symbolized by ( G G G G) alter ⁇ nating structure symbolized by ( G G G G) .
  • the alginate's ability to form a gel by ionic binding, and the properties of said gel depends both on the relative content of the two uronic acids and on the distribution of the guluronic acid units along the chain.
  • a high content of (G) -blocks yields, for instance, an alginate with great gel-forming capacity, which, techni ⁇ cally seen, is a valuable property of the polymer.
  • the present invention is based on the following:
  • the alginate is synthesized in the alga as poly- mannuronic acid and is thereafter modified by an enzyme, mannuronan-C-5epimerase, which converts D-mannuronic acid residues into i-guluronic acid residues within the chain.
  • an enzyme mannuronan-C-5epimerase, which converts D-mannuronic acid residues into i-guluronic acid residues within the chain.
  • the invention relates to a process for producing alginates having improved physical properties such as increased gel strength, by using enzymatic modification on a polymeric level.
  • the process is characterized in that alginates derived from brown algae or bacteria are inoculated with an enzyme preparation.
  • an enzyme preparation is preferably used a C-5-epimerase preparation, more preferably an alginate lyase-free mannuronan-C-5-epimerase produced from the earth bacterium Azotobacter vinelandii.
  • the present invention also comprises the use of the thus modified alginates for immobilizing enzymes, cell organelles and cells by entrapment in gels of alginate or alginate having suitable cations, as well as by immobi ⁇ lizing biocatalysts by encapsulation in alginate poly- cation microcapsules .
  • the mannuronan-C-5-epimerase may be isolated from cultures from the earth bacterium Azotobacter vinelandii. which produces both alginate and epimerase extracellu- larily.
  • the fact that the enzyme is extracellular is a great advantage in the isolation process, and it also indicates that the enzyme may function freely in solution independent on intracellular factors, which is favourable to a technical exploitation of the invention.
  • Immobilized enzymes as catalysts has obtained still greater importance in industry and will, in the years to come, become one of the most important expansion areas for biotechnology. Immobilized enzymes are often more stable, but first and foremost, they are easier to handle than free, soluble enzymes and may be used in continuous processes.
  • the cells may serve as carriers for a single enzyme, such that isolation of the enzyme is unnecessary before immobilizing, or several enzymes may also be used in the cell in order to catalyse multistep processes (for instance synthesis of hormones, proteins, etc.).
  • the epimerization degree measured by means of high solution n.m. . -spectro- scopy, shows an increase of the guluronic acid content from 68% to 79% (see the Table) .
  • composition and GG content of the polymer before and after enzymatic modification measured by 1 high solution H-n.m.r. -spectroscopy

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

Ce procédé consiste à inoculer des alginates dérivés d'algues brunes ou de bactéries, avec une préparation d'enzyme telle qu'une préparation de mannuronane-C-5-épimérase à partir d'Azotobacter vinelandii. Les alginates modifiés sont utilisés pour immobiliser des enzymes, des organites cellulaires ou des cellules ainsi que pour la microencapsulation de biocatalyseurs.This process consists in inoculating alginates derived from brown algae or bacteria, with an enzyme preparation such as a preparation of mannuronan-C-5-epimerase from Azotobacter vinelandii. The modified alginates are used to immobilize enzymes, cellular organelles or cells as well as for the microencapsulation of biocatalysts.

Description

Process for producing alginates having improved physical properties, and the use of said alginates.
The present invention relates to the preparation of alginates having improved physical properties, especially with respect to the formation of gels having inorganic or polyvalent organic ions. Said modified alginates are intended to be used for immobilizing and encapsulating enzymes and/or cells for use in biotechnological processes .
Using alginate gels as an immobilizing material has several deficiencies, two of them being:
1 ) Calcium alginate gels are destabilized by compounds having affinity for calcium, for instance EDTA, citrate, lactate and phosphate, as well as high concentrations of
+ + ++ cations such as Na , K and Mg ;
2) Alginates currently used have a high degree of chemical heterogenity and provide gels having pores of such great size that proteins - enzymes and other macro- molecules - can leach out, at the same time as the size distribution of the pores is difficult to control.
Alginate is the most important structural poly- saccharide in marine brown algae and is used for several industrial purposes wherein the properties of the polymer are utilized as a polyelectrolyte - for instance for gel formation and thickening purposes - and also for its water and ion binding capacity.
The purpose of the present invention, thus, is to prepare alginates having physical properties satisfying the requirements for increased gel strength and stability and better controllable pore size.
Chemically seen, alginate is a polyuronide built up from two uronic acids, viz., D-mannuronic acid ( ) and the C-5-epimer L-guluronic acid (G) . They are arranged in such fashion that the polymer is further built up from three types of sequence: (G)-rich sequences called G-blocks, (M)-rich sequences called M-blocks and alter¬ nating structure symbolized by ( G G G) . The alginate's ability to form a gel by ionic binding, and the properties of said gel depends both on the relative content of the two uronic acids and on the distribution of the guluronic acid units along the chain.
A high content of (G) -blocks yields, for instance, an alginate with great gel-forming capacity, which, techni¬ cally seen, is a valuable property of the polymer.
The present invention is based on the following:
The alginate is synthesized in the alga as poly- mannuronic acid and is thereafter modified by an enzyme, mannuronan-C-5epimerase, which converts D-mannuronic acid residues into i-guluronic acid residues within the chain. When said enzyme affects the alginate, both the relative content and the uronic acid sequence will be changed and, consequently, its physical properties.
Thus, the invention relates to a process for producing alginates having improved physical properties such as increased gel strength, by using enzymatic modification on a polymeric level. The process is characterized in that alginates derived from brown algae or bacteria are inoculated with an enzyme preparation.
As such an enzyme preparation is preferably used a C-5-epimerase preparation, more preferably an alginate lyase-free mannuronan-C-5-epimerase produced from the earth bacterium Azotobacter vinelandii.
The present invention also comprises the use of the thus modified alginates for immobilizing enzymes, cell organelles and cells by entrapment in gels of alginate or alginate having suitable cations, as well as by immobi¬ lizing biocatalysts by encapsulation in alginate poly- cation microcapsules .
The mannuronan-C-5-epimerase may be isolated from cultures from the earth bacterium Azotobacter vinelandii. which produces both alginate and epimerase extracellu- larily. The fact that the enzyme is extracellular is a great advantage in the isolation process, and it also indicates that the enzyme may function freely in solution independent on intracellular factors, which is favourable to a technical exploitation of the invention.
The use of immobilized enzymes as catalysts has obtained still greater importance in industry and will, in the years to come, become one of the most important expansion areas for biotechnology. Immobilized enzymes are often more stable, but first and foremost, they are easier to handle than free, soluble enzymes and may be used in continuous processes.
In addition to immobilizing simple enzymes there has also been developed techniques for immobilizing whole cells. The cells may serve as carriers for a single enzyme, such that isolation of the enzyme is unnecessary before immobilizing, or several enzymes may also be used in the cell in order to catalyse multistep processes (for instance synthesis of hormones, proteins, etc.).
We have tried out the epi erizing of a plurality of high polymer alga and bacterium alginates having varying block structures and formulation, and the conclusions are that all of the alginates can be epimerized to a substantial degree. The epimerization degree varies from 60 to 90 percent depending on the original block structure of the alginates and for some alginates this yields more than a doubling of the gel strength measured in 2 percent homogenous Ca-alginate gels .
Examples
Example 1
Sodium alginate derived from Laminaria diσitata, in an amount of 0.07% by weight, was dissolved in cationic buffer, 0.05 collidine pH 7.0 and Ca2+ 6.8mM. This was incubated with a lyase-free C-5-epimerase preparation from A. vinelandii at 30°C for 8 hours. The epimerization degree, measured by means of high solution n.m.r. -spectro- scopy, shows an increase of the guluronic acid content from 41% to 69% (see the Table) . Example 2
Sodium alginate from Macrocvstis pyrifera, in an amount of 0.07% by weight, was dissolved in cationic buffer, 0.05M collidine pH 7.0 and Ca2+ 6.8mM. This was incubated with a lyase-free C-5-epimerase preparation from A. vinelandii at 30°C for 8 hours. The epimerization degree, measured by means of high solution n.m.r.-spectro- scopy, shows an increase of the guluronic acid content from 37% to 62% (see the Table).
Example 3
Sodium alginate from Laminaria hvperborea, in an amount of 0.07% by weight was dissolved in a cationic
2+ buffer, 0.05M collidine pH 7.0 and Ca 6.8mM. This was incubated with a lyase-free C-5-epimerase preparation from
A. vinelandii at 30°C for 8 hours. The epimerization degree, measured by means of high solution n.m. . -spectro- scopy, shows an increase of the guluronic acid content from 68% to 79% (see the Table) .
Example 4
Sodium alginate from Laminaria diαitata containing 40° guluronic acid is treated with C-5-epimerase from A. vinelandii at pH 7.0 and Ca2+ 0.68mM for 6 hours at 30°C. The modified alginate contains 63% guluronic acid. Gel strength measurements on homogenous 2% calcium gels show a gel strength of 3.8 N/cm 2 and 9.6 N/cm2 in native and enzyme modified alginate, respectively (see Figure 1) . TABLE
Composition and GG content of the polymer before and after enzymatic modification measured by 1 high solution H-n.m.r. -spectroscopy
Before After
Source epimerization epimerization
M GG M GG
Laminaria dicritata 0. .41 0.59 0. .25 0.69 0.31 0.54
Laminaria hvoerborea 0. .68 0.32 0. .57 0.79 0.21 0.67
Macrocvstis pyrifera 0. .37 0.63 0. .14 0.62 0.38 0.32
Elachistae so. 0. .68 0.32 0. .64 0.89 0.11 0.85
Dichtiosvphon foenicula. 0 0.. .6677 0.33 0. .61 0.81 0.19 0.75
Ascophyllum nodosum 0. .36 0.64 0. .16 0.63 0.37 0.39
Azotobacter vinelandii 0. .45 0.55 0. ,41 0.69 0.33 0.54 deacetylated

Claims

Claims
1. A process for producing alginates having improved physical properties, for instance increased gel strength, comprising inoculating alginates derived from brown algae or bacteria with an enzyme preparation.
2. The process of claim 1, wherein said enzyme prepa¬ ration is a C-5-epimerase preparation.
3. The process of claim 1 or 2, wherein said mannuronan-C-5epimerase preparation is derived from Azotobacter vinelandii.
4. The use of alginates modified by the process according to any one of the preceding claims, for immobilizing enzymes, cell organelles and cells by gel entrapment in alginate gels or microcapsules of alginate and poly- cations.
EP19860900291 1984-12-17 1985-12-16 Process for producing alginates having improved physical properties, and the use of said alginates Withdrawn EP0204805A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NO845059A NO845059L (en) 1984-12-17 1984-12-17 PROCEDURE FOR PREPARING ALGINATES WITH CHANGED PHYSICAL PROPERTIES AND USING THESE ALGINATES.
NO845059 1984-12-17

Publications (1)

Publication Number Publication Date
EP0204805A1 true EP0204805A1 (en) 1986-12-17

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ID=19887997

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Application Number Title Priority Date Filing Date
EP19860900291 Withdrawn EP0204805A1 (en) 1984-12-17 1985-12-16 Process for producing alginates having improved physical properties, and the use of said alginates

Country Status (3)

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EP (1) EP0204805A1 (en)
NO (1) NO845059L (en)
WO (1) WO1986003781A1 (en)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NO160374C (en) * 1986-10-17 1989-04-12 Protan As PROCEDURE FOR MODIFICATION OF ALGINATES (POLYURONIDES) TO GET CHANGED PHYSICAL PROPERTIES.
GB9221163D0 (en) * 1992-10-08 1992-11-25 Nobipol And Protan Biopolymer Dna compounds
FR2849056B1 (en) * 2002-12-19 2007-07-13 Centre Nat Rech Scient MANNURONANE C5-EPIMERASES OF BROWN ALGAE, METHODS OF OBTAINING AND USES
NO320691B1 (en) * 2004-06-14 2006-01-16 Ntnu Technology Transfer As New contrast release system.
BRPI0517800A (en) * 2004-11-12 2008-10-21 Fmc Biopolymer As process for preparing a modified alginate polymer, modified alginate polymer, and alginate gel or fiber
US8178130B2 (en) 2006-07-04 2012-05-15 Elisabeth Kommisrud Preservation and controlled delivery / release of spermatozoa
US9446168B2 (en) 2010-06-07 2016-09-20 Beta-O2 Technologies Ltd. Multiple-layer immune barrier for donor cells
WO2010032242A1 (en) 2008-09-17 2010-03-25 Beta O2 Technologies Ltd. Optimization of alginate encapsulation of islets for transplantation
WO2012167223A1 (en) 2011-06-02 2012-12-06 Massachusetts Institute Of Technology Modified alginates for cell encapsulation and cell therapy
GB201120368D0 (en) 2011-11-24 2012-01-04 Sperm Vital As Methods for the preparation of hydrogels
WO2018104160A1 (en) 2016-12-05 2018-06-14 Spermvital As Sustained release composition
IL278899B2 (en) 2018-06-04 2024-03-01 Spermvital As Functionalized kit for preparing hydrogels

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU742434A1 (en) * 1978-10-11 1980-06-25 Ленинградский ордена Трудового Красного Знамени институт текстильной и легкой промышленности им.С.М.Кирова Method of preparing immobilized peroxydase
JPS5974984A (en) * 1982-10-21 1984-04-27 Sumitomo Chem Co Ltd Preparation of immobilized enzyme or microorganism

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8603781A1 *

Also Published As

Publication number Publication date
WO1986003781A1 (en) 1986-07-03
NO845059L (en) 1986-06-18

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