WO1992013962A1 - Enzymatic process for glucosylation of glucosides - Google Patents

Enzymatic process for glucosylation of glucosides Download PDF

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Publication number
WO1992013962A1
WO1992013962A1 PCT/DK1992/000031 DK9200031W WO9213962A1 WO 1992013962 A1 WO1992013962 A1 WO 1992013962A1 DK 9200031 W DK9200031 W DK 9200031W WO 9213962 A1 WO9213962 A1 WO 9213962A1
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Prior art keywords
liquefaction
cgtase
glucoside
glucosylation
enzymatic
Prior art date
Application number
PCT/DK1992/000031
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French (fr)
Inventor
Sven Pedersen
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Novo Nordisk A/S
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Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to JP4504136A priority Critical patent/JPH06505149A/en
Publication of WO1992013962A1 publication Critical patent/WO1992013962A1/en

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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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1074—Cyclomaltodextrin glucanotransferase (2.4.1.19)
    • 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
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins

Definitions

  • This invention relates to a process for enzymatic ⁇ -1 ,4 glucosylation of a glucoside.
  • ⁇ -1 ,4 glucosylation of glucosides i.e. the process of attaching one or more glucosyl groups to the glucoside through ⁇ -1 ,4 bonds
  • coupling sugar maltooligosyl-sucrose syrup
  • ⁇ -1,4 glucosylation of the naturally occurring sweetener stevioside produces a sweetener with a more pleasant sweetness (J.Jpn.Soc.Starch Sci., vol. 34, No. 1 , p. 75-82 (1987)).
  • CGTase cyclodextrin glycosyl transferase
  • a typical prior-art process comprises liquefaction of starch slurry with ⁇ -amylase, inactivation of the ⁇ -amylase by heating, cooling, addition of the glucoside and CGTase, and enzymatic transglucosylation at 55-65°C for 1-3 days.
  • thermostable CGTase is used both for starch liquefaction and for subsequent transglucosylation, and wherein transglucosylation is conducted at elevated temperature.
  • the process is simpler than prior-art processes as the heating to inactivate ⁇ -amylase and the addition of enzyme after liquefaction can be omitted.
  • Liquefaction with CGTase instead of ⁇ - amylase avoids the formation of reducing sugars during liquefaction, and this may lead to less formation of glucose, maltose etc.
  • the higher temperature for the transglucosylation leads to higher reaction rate, so the reaction time can be reduced.
  • the invention provides a process for enzymatic ⁇ -1,4 glucosylation of a glucoside, characterized by comprising: a) liquefaction of an aqueous starch slurry by treatment with a thermostable cyclodextrin glycosyl transferase (CGTase) above 100°C, b) addition of said glucoside before or after said liquefaction, and c) enzymatic transglucosylation of the mixture with the above CGTase at 70-95°C.
  • CGTase thermostable cyclodextrin glycosyl transferase
  • the process of the invention is used for ⁇ -1 ,4 glucosylation of an ⁇ - or jS-glucoside of formula G-X to form G.-G-X (a malto-oligo-saccharide glycoside), where G is a glucopyranosyl residue, X is a sugar or non-sugar group linked to G by an ⁇ -1 or ⁇ - ⁇ glucosidic bond, n is 1 , 2 or higher, and the G's are linked by ⁇ -1 ,4 glucosidic bonds.
  • glucosides that may be glucosylated according to the invention are: Sucrose, stevioside, rubusoside, and hesperidin dihydrochalcone glucoside.
  • the process generally leads to a mixture of molecules with different values of n (degree of glucosylation), depending on the ratio between starch and glucoside and other reaction parameters.
  • n degree of glucosylation
  • an average incorporation of 1 - 2 glucosyl groups per glucoside molecule will be suitable. This can generally be achieved by using a molar ratio between the starch glucosyl groups and the glucoside of 1-3. In the case of glucosylation of sucrose this corresponds to a weight ratio between starch and sucrose of 0.5 - 1.5.
  • the reaction mixture may also contain unreacted glucoside G-X, glucose G and maltooligosaccharides G ⁇ .
  • this reaction mixture can be used as such after purification; if desired, the components of the mixture can be separated by known methods.
  • thermostable CGTase used in the invention is one that has retains at least 80% of initial activity after 50 minutes incubation at 95°C in 5% Lintner starch at optimum pH (e.g. pH 5.0) with 50 ppm Ca ++ .
  • a preferred CGTase is one derived from a strain of Thermoanaerobacter or Thermoanaerobium (these two genera believed to be indistinguishable), described in WO 89/03421.
  • Specific examples are the CGTases derived from strains ATCC 53,627 or one of the strains NCIB 40,053 through NCIB 40,059. The strains were deposited by the inventors under the terms of the Budapest Treaty, as specified in WO 89/03421.
  • CGTase is derived from a strain of Clostridium, e.g. from C. thermoamylolyticum ATCC 39,252 or C. thermohydrosulfuricum ATCC 53,016. Both strains are freely available. US 4,578,352 and US 4,613,570 describe aciduric alpha-amylase enzymes from these organisms, but we have discovered CGTase produced by these two strains. Further details about characterization and production of this CGTase is given in our pending applications US 07/455,188 and PCT/DK 90/ 00338.
  • the CGTase enzyme may be produced by anaerobic cultivation of a CGTase-producing strain of Thermoanaerobacter, Thermoanaerobium or Clostridium (such as those listed above or a mutant or variant thereof) under anaerobic conditions, or by aerobic cultivation of a transformant containing the appropriate genetic information from such strain and, thereafter recovering the CGTase enzyme from the culture medium.
  • Process conditions may be produced by anaerobic cultivation of a CGTase-producing strain of Thermoanaerobacter, Thermoanaerobium or Clostridium (such as those listed above or a mutant or variant thereof) under anaerobic conditions, or by aerobic cultivation of a transformant containing the appropriate genetic information from such strain and, thereafter recovering the CGTase enzyme from the culture medium.
  • Suitable conditions for the liquefaction are 1-60 minutes reaction time at 100-1 5°C. Heating of the starch slurry is preferably done in a jet-cooker.
  • the reaction time of the transglucosylation may be 6-48 hours, preferably below 24 hours, e.g. 12-24 hours.
  • the temperature is preferably 80-90°C. Lower temperature may be used with a heat-labile glucoside.
  • the pH is preferably kept in the range 4-7 throughout the process. pH adjustment after liquefaction is generally not needed. pH in the range 4.0-5.5 is optimum for the CGTase and can be used if the glucoside is sufficiently acid-stable, but with an acid-unstable substrate like sucrose it is preferred to use pH in the range 5.5-7.0.
  • the enzyme dosage is preferably 5-20 Phadebas Units (unit defined in WO 89/03421). Due to the good thermostability of the CGTase, re-dosage of enzyme after liquefaction can generally be avoided. If needed, 20-100 ppm of a calcium salt may be added to the starch slurry for enzyme stabilization.
  • the concentration of the starch slurry is preferably 15-30 % dry substance. In the case of glucosylation of sucrose, the concentration will generally be 30-40% dry substance during transglucosylation.
  • the glucoside may be added before or after the liquefaction. With a sufficiently heat-stable substrate like sucrose it is convenient to add the glucoside to the starch slurry before liquefaction. If the glucoside is unstable at high temperature, it can be added after the liquefaction, conveniently in dry form.

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Abstract

The invention provides a process for enzymatic α-1,4 glucosylation of a glucoside, characterized by comprising: a) liquefaction of an aqueous starch slurry by treatment with a thermostable cyclodextrin glycosyl transferase (CGTase) above 100 °C, b) addition of said glucoside before or after said liquefaction, and c) enzymatic transglucosylation of the mixture with the above CGTase at 70-95 °C. This process is simple, avoids the formation of reducing sugars during liquefaction, and the reaction time is short.

Description

ENZYMATIC PROCESS FOR GI_O_X)SYLATI0N OF GLUCOSIDES
TECHNICAL FIELD
This invention relates to a process for enzymatic α-1 ,4 glucosylation of a glucoside.
BACKGROUND ART
α-1 ,4 glucosylation of glucosides, i.e. the process of attaching one or more glucosyl groups to the glucoside through α-1 ,4 bonds, can be used to prepare various useful products. Thus, α-1,4 glucosylation of sucrose produces maltooligosyl-sucrose syrup (called coupling sugar), which has been proposed as a low-cariogenic sweetener (GB 1,471,613). Further, it has been reported that α-1,4 glucosylation of the naturally occurring sweetener stevioside produces a sweetener with a more pleasant sweetness (J.Jpn.Soc.Starch Sci., vol. 34, No. 1 , p. 75-82 (1987)).
It is known that enzymatic α-1 ,4 glucosylation can be achieved by transglucosylation of the glucoside with starch or starch hydrolysate in the presence of a cyclodextrin glycosyl transferase (E.C. 2.4.1.9, hereinafter called CGTase) derived from various Bacillus species (GB 1,471,613; J.Jpn.Soc.Starch Sci., loc.cit; Handbook of Amylases and Related Enzymes, p. 226-228, Pergamon Press, 1988). A typical prior-art process comprises liquefaction of starch slurry with α-amylase, inactivation of the α-amylase by heating, cooling, addition of the glucoside and CGTase, and enzymatic transglucosylation at 55-65°C for 1-3 days.
It is the object of this invention to provide an improved process.
STATEMENT OF THE INVENTION
We have developed a process wherein thermostable CGTase is used both for starch liquefaction and for subsequent transglucosylation, and wherein transglucosylation is conducted at elevated temperature. The process is simpler than prior-art processes as the heating to inactivate α-amylase and the addition of enzyme after liquefaction can be omitted. Liquefaction with CGTase instead of α- amylase avoids the formation of reducing sugars during liquefaction, and this may lead to less formation of glucose, maltose etc. Further, the higher temperature for the transglucosylation leads to higher reaction rate, so the reaction time can be reduced.
Accordingly, the invention provides a process for enzymatic α-1,4 glucosylation of a glucoside, characterized by comprising: a) liquefaction of an aqueous starch slurry by treatment with a thermostable cyclodextrin glycosyl transferase (CGTase) above 100°C, b) addition of said glucoside before or after said liquefaction, and c) enzymatic transglucosylation of the mixture with the above CGTase at 70-95°C.
DETAILED DESCRIPTION OF THE INVENTION
Glucoside
The process of the invention is used for α-1 ,4 glucosylation of an α- or jS-glucoside of formula G-X to form G.-G-X (a malto-oligo-saccharide glycoside), where G is a glucopyranosyl residue, X is a sugar or non-sugar group linked to G by an α-1 or β-\ glucosidic bond, n is 1 , 2 or higher, and the G's are linked by α-1 ,4 glucosidic bonds.
(Examples of glucosides that may be glucosylated according to the invention are: Sucrose, stevioside, rubusoside, and hesperidin dihydrochalcone glucoside. The process generally leads to a mixture of molecules with different values of n (degree of glucosylation), depending on the ratio between starch and glucoside and other reaction parameters. For many purposes, an average incorporation of 1 - 2 glucosyl groups per glucoside molecule will be suitable. This can generally be achieved by using a molar ratio between the starch glucosyl groups and the glucoside of 1-3. In the case of glucosylation of sucrose this corresponds to a weight ratio between starch and sucrose of 0.5 - 1.5.
The reaction mixture may also contain unreacted glucoside G-X, glucose G and maltooligosaccharides Gπ. In many cases, this reaction mixture can be used as such after purification; if desired, the components of the mixture can be separated by known methods.
Thermostable CGTase
The thermostable CGTase used in the invention is one that has retains at least 80% of initial activity after 50 minutes incubation at 95°C in 5% Lintner starch at optimum pH (e.g. pH 5.0) with 50 ppm Ca++.
A preferred CGTase is one derived from a strain of Thermoanaerobacter or Thermoanaerobium (these two genera believed to be indistinguishable), described in WO 89/03421. Specific examples are the CGTases derived from strains ATCC 53,627 or one of the strains NCIB 40,053 through NCIB 40,059. The strains were deposited by the inventors under the terms of the Budapest Treaty, as specified in WO 89/03421.
Another preferred CGTase is derived from a strain of Clostridium, e.g. from C. thermoamylolyticum ATCC 39,252 or C. thermohydrosulfuricum ATCC 53,016. Both strains are freely available. US 4,578,352 and US 4,613,570 describe aciduric alpha-amylase enzymes from these organisms, but we have discovered CGTase produced by these two strains. Further details about characterization and production of this CGTase is given in our pending applications US 07/455,188 and PCT/DK 90/ 00338.
The CGTase enzyme may be produced by anaerobic cultivation of a CGTase-producing strain of Thermoanaerobacter, Thermoanaerobium or Clostridium (such as those listed above or a mutant or variant thereof) under anaerobic conditions, or by aerobic cultivation of a transformant containing the appropriate genetic information from such strain and, thereafter recovering the CGTase enzyme from the culture medium. Process conditions
Suitable conditions for the liquefaction are 1-60 minutes reaction time at 100-1 5°C. Heating of the starch slurry is preferably done in a jet-cooker.
The reaction time of the transglucosylation may be 6-48 hours, preferably below 24 hours, e.g. 12-24 hours. The temperature is preferably 80-90°C. Lower temperature may be used with a heat-labile glucoside.
With CGTase from Thermoanaerobacter, Thermoanaerobium or Clostridium, the pH is preferably kept in the range 4-7 throughout the process. pH adjustment after liquefaction is generally not needed. pH in the range 4.0-5.5 is optimum for the CGTase and can be used if the glucoside is sufficiently acid-stable, but with an acid-unstable substrate like sucrose it is preferred to use pH in the range 5.5-7.0.
The enzyme dosage is preferably 5-20 Phadebas Units (unit defined in WO 89/03421). Due to the good thermostability of the CGTase, re-dosage of enzyme after liquefaction can generally be avoided. If needed, 20-100 ppm of a calcium salt may be added to the starch slurry for enzyme stabilization.
The concentration of the starch slurry is preferably 15-30 % dry substance. In the case of glucosylation of sucrose, the concentration will generally be 30-40% dry substance during transglucosylation. The glucoside may be added before or after the liquefaction. With a sufficiently heat-stable substrate like sucrose it is convenient to add the glucoside to the starch slurry before liquefaction. If the glucoside is unstable at high temperature, it can be added after the liquefaction, conveniently in dry form.
EXAMPLE 1
10.6 g amylopectin was mixed with 24 g deionized water and 5.3 g sucrose. CGTase from Thermoanaerobacter sp. ATCC 53,627 was added (14.3 Phadebas unit/g starch). pH was adjusted to 5.0. The mixture was liquefied at 105°C for 14 minutes followed by incubation at 90°C for 24 hours. An HPLC-analysis of the product gave the following approximate sugar composition of the product. Fructose ~ 2%
Glucose - 2%
Sucrose - 18%
Maltose ~ 2%
Glucosyl sucrose ~ 6.5%
Maltotriose ~ 2%
Maltosyl sucrose - 6%
Higher maltooligosaccharides ~ 40%
Higher "coupling sugars" - 22%

Claims

1. A process for enzymatic α-1 ,4 glucosylation of a glucoside, characterized by comprising: a) liquefaction of an aqueous starch slurry by treatment with a thermostable cyclodextrin glycosyl transferase (CGTase) above
100°C, b) addition of said glucoside before or after said liquefaction, and c) enzymatic transglucosylation of the mixture with the above CGTase at 70-95°C.
2. The process of Claim 1, wherein the liquefaction is conducted at 100-
115°C, pH 4-7 and reaction time 1-60 minutes.
3. The process of Claim 1 or 2, wherein said enzymatic transglucosylation is conducted at pH 4-7 and reaction time 6-48 hours, preferably 12-24 hours.
4. A process according to any of Claims 1 - 3, wherein said enzymatic transglucosylation is done substantially without intermediate pH adjustment after the liquefaction
5. A process according to any preceding claim, wherein an enzyme dosage of 5-20 Phadebas units per g dry starch is used in the liquefaction, and said enzymatic transglucosylation is done without re-dosing the liquefied starch with CGTase.
6. The process of any preceding claim, wherein the CGTase is derived from a strain of Thermoanaerobacter, Thermoanaerobium or Clostridium, preferably from Thermoanaerobacter sp. ATCC 53,627 or any of NCIB 40,053-40,059 or from C. thermoamylolyticum ATCC 39,252 or C. thermohydrosulfuricum ATCC 53,016.
7. A process according to any preceding claim for preparing maltooligosyl- sucrose by glucosylation of sucrose.
PCT/DK1992/000031 1991-01-31 1992-01-30 Enzymatic process for glucosylation of glucosides WO1992013962A1 (en)

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JP4504136A JPH06505149A (en) 1991-01-31 1992-01-30 Enzyme treatment for glucosylation of glucosides

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EP91610005.0 1991-01-31
EP91610005 1991-01-31

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0690170B1 (en) * 1994-06-29 2000-09-06 Coöperatieve Verkoop- en Productievereniging van Aardappelmeel en Derivaten 'AVEBE' B.A. A process for surface sizing or coating paper
WO2002010427A1 (en) * 2000-07-28 2002-02-07 Novozymes A/S Method for producing maltose syrup by using a hexosyltransferase
EP1743693A1 (en) 2005-07-12 2007-01-17 Coöperatie Avebe U.A. Emulsifier

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3819484A (en) * 1970-12-16 1974-06-25 Hayashibara Ken Process for preparing sucrose-starch sweetner free from reducing sugar
WO1989003421A1 (en) * 1987-10-15 1989-04-20 Novo Industri A/S Thermostable cyclodextrin glycosyl transferase, its production and use
WO1991009962A1 (en) * 1989-12-22 1991-07-11 Novo Nordisk A/S A method for enzymatically converting starch into cyclodextrins

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3819484A (en) * 1970-12-16 1974-06-25 Hayashibara Ken Process for preparing sucrose-starch sweetner free from reducing sugar
WO1989003421A1 (en) * 1987-10-15 1989-04-20 Novo Industri A/S Thermostable cyclodextrin glycosyl transferase, its production and use
WO1991009962A1 (en) * 1989-12-22 1991-07-11 Novo Nordisk A/S A method for enzymatically converting starch into cyclodextrins

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FOOD; DISINFECTANTS; DETERGENTS, Vol. 82, No. 34, August 1975, HAYASHIBARA SEIBUTS: "(2-Alpha)-maltosyl:glucose prepn.", *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0690170B1 (en) * 1994-06-29 2000-09-06 Coöperatieve Verkoop- en Productievereniging van Aardappelmeel en Derivaten 'AVEBE' B.A. A process for surface sizing or coating paper
WO2002010427A1 (en) * 2000-07-28 2002-02-07 Novozymes A/S Method for producing maltose syrup by using a hexosyltransferase
EP1743693A1 (en) 2005-07-12 2007-01-17 Coöperatie Avebe U.A. Emulsifier
US8178323B2 (en) 2005-07-12 2012-05-15 Coöperatie Avebe U.A. Emulsifier prepared using a glycosyl transferase

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