WO1979000875A1 - Method and means for carrying out an enzymatic process - Google Patents

Method and means for carrying out an enzymatic process Download PDF

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Publication number
WO1979000875A1
WO1979000875A1 PCT/SE1979/000081 SE7900081W WO7900875A1 WO 1979000875 A1 WO1979000875 A1 WO 1979000875A1 SE 7900081 W SE7900081 W SE 7900081W WO 7900875 A1 WO7900875 A1 WO 7900875A1
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WO
WIPO (PCT)
Prior art keywords
enzyme
complex
substance
enzymes
solution
Prior art date
Application number
PCT/SE1979/000081
Other languages
English (en)
French (fr)
Inventor
K Martensson
R Axen
Original Assignee
Svenska Sockerfabriks Ab
K Martensson
R Axen
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 Svenska Sockerfabriks Ab, K Martensson, R Axen filed Critical Svenska Sockerfabriks Ab
Priority to DE19792945416 priority Critical patent/DE2945416A1/de
Publication of WO1979000875A1 publication Critical patent/WO1979000875A1/en
Priority to DK511779A priority patent/DK511779A/da

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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
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • 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
    • 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
    • C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
    • 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
    • C12P19/20—Preparation of compounds containing saccharide radicals produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
    • 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

Definitions

  • the present invention relates to a method for carrying out an enzymatic process and the invention also includes a soluble enzyme complex for use in this method.
  • Enzymes are to-day prepared and utilized industrially in several different areas. Within the foodstuff area many processes, in inter alia the starch, dairy, bakery and brewery industries, are based on enzymes, but also outside these areas the enzyme technology finds use, for example in the washing agent, tannery and pharmaceutical industries. The enzymes are also utilized to a large extent for analytical purposes in biochemical or clinical laboratories and to some extent also for medical applications. During the last fifteen years a nearly explosive development has taken place within the area of immobilized enzymes, i.e.
  • the characterizing features of an enzyme are dependent not only from the amino acids present in the active center but also from the amino acid sequence as a whole and the space configuration of the protein structure, which in turn has to do with the hydrophobic/hydrophilic balance, the charge distribution etc. This has for a result that all parts of the enzyme molecule to a larger or smaller extent effect the characteristics of the enzyme. In a corresponding manner a change in structure, for example by introducing a substance, results in a more or less pronounced change on the characteristics of the enzyme.
  • kinetic productivity characteristics which directly express the catalytic ability of the enzyme in its use as a catalyst, which in reality is a function of basic characteristics of the total enzyme system, for example substrate concentration, inhibitory concentration, enzyme quantity, temperature, pH etc., and are expressed by such traditional parameters as pH-optimum, heat stability, operational stability, Michaeli's constant (K m ), inhibitory constant (K i ), maximum reaction rate (V max ) etc., as distinguished from other characteristics of the enzyme which do not affect its catalytic function in its practical use.
  • kinetic productivity characteristics are not either meant such physical parameters in the reaction vessel as stirring rate, reactor vessel dimensions etc.
  • the present invention relates to providing a soluble enzyme complex for use in enzymetically catalyzed processes.
  • the enzyme is bound to a substance containing at least a hydrophobous part for providing the binding and at least a functional part for modifying the kinetic productivity characteristics of the enzyme.
  • hydrophobic part of the substance means a structure which has the ability of forming a practically useful stable complex together with the enzyme in question.
  • the hydrophobic part should, in order that the desired effectivity in the hydrophobic interaction with the enzyme protein shall be obtained but still a reasonably mild binding with regard to the enzyme, contained at least one structural element of a straight or branched, saturated or unsaturated alkyl or cycloalkyl chain having between 6 and 12 carbon atoms. Preferably a straight saturated alkyl chain having 8 carbon atoms is used.
  • the functional part of the substance may be any structure which has the ability of imparting to the enzyme complex such characteristics which result in modification or changing the original productivity kinetic of the free enzyme.
  • the functional part of the substance has, in this invention, an operational character and must therefore in this manner be given an operational definition in contrast with the above structural description of the hydrophobic part of the substance. Examples of different basic structures of the functional part resulting in a certain type of change of the kinetic productivity characteristics are further described under the separate sections below, which deal with displacement of pH-optimum, improvement of the stability characteristics, changes in catalytic activity etc.
  • the invention also provides for a process for the preparation of the enzyme complex described above, and in this process the enzyme in question is brought together with a chemical substance including hydrophobic and functional parts in a polar solvent to the formation of the desired enzyme complex.
  • concentration range should be selected in such a way that a sufficiently great amount of substance is bound to the enzyme so that its kinetic productivity characteristics will be changed to the desired degree but at the same time micell formation of the substance will be avoided. Micell formation is often no hindrance to the formation of the complex but results in an unnecessary waste of substance which cannot be utilized for the intended purpose.
  • the concentration of substance should therefore lie above the limit for cooperative binding if such can take place but below CMC.
  • Initiation of complex formation may in certain cases take place by smooth heating of the reaction mixture, alternatively by adding a minor quantity of denaturing substance, for example urea.
  • the enzyme complex formed may either be used directly in the solution obtained or may if desired be isolated and recovered.
  • Enzymes may depending on the type of reaction to be catalyzed be systematically divided-up into the following six main groups: Oxidoreductasae, hydrolasae, isomerasae, transferasae, lyasae and ligasae. Since the hydrophobic complex formation described in connection with this invention is general and thus is applicable to in principle all proteins it is also generally useful to modify the kinetic productivity characteristics of all enzymes. The types of enzymes utilized in practical use may, however, almost exclusively be systematically found in the groups oxidoreductasae, hydrolasae or isomerasae, in view of which the invention is considered to have the highest value for enzymes belonging to any of said groups.
  • the principle described above may be used also to displace the isoelectric point of an enzyme and to change kinetic parameters, such as Michaelis constant, inhibitor constant etc. in view of a corresponding change of affinity towards electrostatically charged substrates, inhibitors etc.
  • stabilizing agents of for example the above mentioned types may be introduced in the enzyme structure by means of hydrophobic interaction.
  • the functional part of the substance used is thereby for example any of the above-mentioned structures, whereas the hydrophobic part in accordance with this invention consists of a structure in accordance with what has been previously described which is chemically bound to the functional part in question.
  • substances that may be used to improve the stability characteristics of enzymes in accordance with this invention are octyl dextrane, hexyl saccharose, octyl polyethylene glycol, decyl glutation, hexylthiol, octyl-EDTA, phenyl cystein etc. Change in the catalytic activity of an enzyme.
  • the catalytic activity of an enzyme is, of course, the most central factor among the characteristics possessed by the enzyme, and the practical value of an increase of the catalytic ability is easily understood. In special cases a change of the affinity of an enzyme towards different substrates may also be desirable.
  • General methods of providing a directed change in the form of for example an increase in activity cannot be formulated, but suitable measures must be judged from case to case and adapted to each single enzyme in dependence on the substrate in question, possible inhibitors etc.
  • different measures of the type described under the applications mentioned above may in different ways change the activity and the specificity of an enzyme in view of change in charge distribution, solubility, surface structure etc.
  • an enzyme may, for example, be protected against macromolecular inhibitor to advantage for a low molecular substrate by the fact that the functional part as stated above consists of a polymer which sterically blocks the surface of the enzyme from the attack of the inhibitor.
  • the introduction of a compound, the functional part of which is electrostatically charged, may in a corresponding manner attract the substrate of opposite charge, so that the activity against this substrate will be selectively increased.
  • the functional part introduced is strongly hydrophilic the water-solubility of the enzyme may be increased and thereby also the affinity towards particularly hydrophilic substrates.
  • xylose isomerase in 0.02 M tris-HCl-buffer (pH 8.5) (10 ml; 20 mg/ml) is added hexyl dextrane (T500) (1.0 g), the solution being than shaken a room temperature for three hours.
  • T500 hexyl dextrane
  • the enzyme-dextrane complex formed is then adsorbed onto DEAE-cellulose (What- man DE-52) (1.0 g) by adding the cellulose to the enzyme solution obtained according to the above under careful mixing which is maintained for 2 hours at room temperature.
  • the immobilized enzyme dextrane complex hereby obtained is then packed in a column ( ⁇ 9 mm) and then a substrate solution of glucose in 0.02 M tris-HCl-buffer (pH 8.5) (40 % weight/weight), which is 0.4 mM with regard to M g SO 4 . 7H 2 0, is continuously pumped through the column at 60°C.
  • the flow through the column is continuously controlled in such a manner that the conversion degree with regard to a fructose formed is kept constant at 44 %.
  • the immobilized enzyme dextrane complex hereby shows a half time of 30 days with a total productivity of 18.7 g of fructose per mg of enzyme calculated at the ratio between quantity of fructose formed and quantity of utilized enzyme until the enzyme shows 25 % of its initial activity.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
PCT/SE1979/000081 1978-04-03 1979-04-02 Method and means for carrying out an enzymatic process WO1979000875A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
DE19792945416 DE2945416A1 (de) 1978-04-03 1979-04-02 Method and means for carrying out an enzymatic process
DK511779A DK511779A (da) 1978-04-03 1979-11-30 Es fremgangsmaade og middel til udfoerelse af en enzymatisk proc

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE7803732A SE416314B (sv) 1978-04-03 1978-04-03 Enzymatiskt forfarande, varvid enzymet foreligger i ett losligt komplex med hydrofob bindning och en del som modifierar enzymetsproduktivitetskinetiska egenskaper samt det losliga komplexet
SE7803732 1978-04-03

Publications (1)

Publication Number Publication Date
WO1979000875A1 true WO1979000875A1 (en) 1979-11-01

Family

ID=20334481

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE1979/000081 WO1979000875A1 (en) 1978-04-03 1979-04-02 Method and means for carrying out an enzymatic process

Country Status (6)

Country Link
JP (1) JPS55500188A (enrdf_load_stackoverflow)
DK (1) DK511779A (enrdf_load_stackoverflow)
FR (1) FR2421909A1 (enrdf_load_stackoverflow)
GB (1) GB2043076B (enrdf_load_stackoverflow)
SE (1) SE416314B (enrdf_load_stackoverflow)
WO (1) WO1979000875A1 (enrdf_load_stackoverflow)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020159314A1 (ko) * 2019-01-31 2020-08-06 고려대학교 산학협력단 효소-담체 복합체

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4006059A (en) * 1974-07-29 1977-02-01 Purdue Research Foundation Hydrophobic noncovalent binding of proteins to support materials

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA986441A (en) * 1971-10-22 1976-03-30 Aslam Khwaja Enzyme treatment
JPS4924235A (enrdf_load_stackoverflow) * 1972-07-01 1974-03-04
JPS4962686A (enrdf_load_stackoverflow) * 1972-10-23 1974-06-18
GB1463513A (en) * 1974-08-13 1977-02-02 Beecham Group Ltd Enzymes
JPS5282782A (en) * 1975-12-26 1977-07-11 Mitsubishi Chem Ind Ltd Glucose isomerase-containing liquid having high storage stability
JPS5294482A (en) * 1976-01-28 1977-08-09 Mitsubishi Chem Ind Ltd Glucoseisomerase concentrate of good storage stability
JPS51104087A (ja) * 1976-02-02 1976-09-14 Toyo Boseki Kosososeibutsunoseizohoho

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4006059A (en) * 1974-07-29 1977-02-01 Purdue Research Foundation Hydrophobic noncovalent binding of proteins to support materials

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Methods in Enzymology Volume XLIV, Immobilized Enzymes, Ed K Mosbach, N Y, published 1976, see pages 357 - 8, 397 - 403, 406 - 412, 436, 438, 444 - 50 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020159314A1 (ko) * 2019-01-31 2020-08-06 고려대학교 산학협력단 효소-담체 복합체
US11441143B2 (en) 2019-01-31 2022-09-13 Korea Univercity Research and Business Foundation Enzyme-carrier complex

Also Published As

Publication number Publication date
GB2043076B (en) 1982-08-18
GB2043076A (en) 1980-10-01
FR2421909B1 (enrdf_load_stackoverflow) 1983-12-09
JPS55500188A (enrdf_load_stackoverflow) 1980-04-03
FR2421909A1 (fr) 1979-11-02
SE416314B (sv) 1980-12-15
DK511779A (da) 1979-11-30
SE7803732L (sv) 1979-10-04

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