WO2001012834A1 - Procede de preparation et de separation simultanee de produits catalyses par des enzymes - Google Patents

Procede de preparation et de separation simultanee de produits catalyses par des enzymes Download PDF

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Publication number
WO2001012834A1
WO2001012834A1 PCT/FI2000/000702 FI0000702W WO0112834A1 WO 2001012834 A1 WO2001012834 A1 WO 2001012834A1 FI 0000702 W FI0000702 W FI 0000702W WO 0112834 A1 WO0112834 A1 WO 0112834A1
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Prior art keywords
column
process according
enzyme
xylose
arabinose
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PCT/FI2000/000702
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English (en)
Inventor
Matti Leisola
Jouni Jokela
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Hydrios Biotechnology Oy
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Publication date
Application filed by Hydrios Biotechnology Oy filed Critical Hydrios Biotechnology Oy
Priority to AU65744/00A priority Critical patent/AU6574400A/en
Publication of WO2001012834A1 publication Critical patent/WO2001012834A1/fr
Priority to FI20020288A priority patent/FI20020288A/fi

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    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • 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
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase

Definitions

  • This invention relates to enzyme technology and concerns particularly a new process for the preparation and simultaneous separation of enzyme-catalysed products from a product mixture and starting materials using a cross-linked crystalline enzyme
  • Enzymes are biocatalysts that are widely utilised in industrial processes Enzymes are used either in a disposable manner in water-soluble form (typically alfa-amylase, glucoamylase, cellulase, xylanase, and pectinase) or as immobilised catalysts bound to a solid support (glucose isomerase, lipase, dextran saccharase, and penicillin acylase) Immobilisation increases the price of an enzyme product, but makes it more durable than a soluble form of the enzyme and enables its repeated use
  • water-soluble form typically alfa-amylase, glucoamylase, cellulase, xylanase, and pectinase
  • immobilisation increases the price of an enzyme product, but makes it more durable than a soluble form of the enzyme and enables its repeated use
  • D-xylose isomerase EC 5 3 1 5
  • glucose isomerase When using xylose isomerase of bacterial origin, it is usually linked to a solid carrier in a reactor through which glucose solution flows One binds to the carrier either a pure enzyme by ionic bonds, allowing recovery of the carrier by a fresh enzyme, or a whole cell which contains an isomerase, or an enzyme by covalent cross-linking Glutaraldehyde is often used as a cross-linking and stabilising agent for the enzyme
  • the reaction catalysed by the enzyme results in an equilibrium between glucose and fructose, having an equal quantity of both sugars at about 60 °C
  • Streptomyces species (S. rubiginosus, S.albus, S. murin s) and some other bacterial species as Actinoplanes missouriensis are used for the industrial production of xylose isomerase
  • the enzyme of S. rubiginosus is produced industrially in large quantities applying a crystallisation process for purification (Visuri, U S patent No 4,699,882) and, for example, it is used as bound to an ion-exchange carrier which is prepared according to U S patent No 4,355,117
  • xylose isomerase is known to catalyse the isomerisation of D-xylose and D- glucose into D-xylulose and D-fructose, respectively It has also been shown that the Streptomyces albus isomerase is able to isomerise the reactions of D- ⁇ bose, L-arabinose, L-rhamnose and D-allose into corresponding keto sugars (Dauter, Z et al , 1989) Some sugar derivatives can be isomerised by xylose isomerase as well (de Raadt et al , 1994) Various other sugar-isomerising enzymes are known as well
  • D-xylose and L-arabinose are conventionally produced by hydrolysing carbohydrate polymers, e g hardwood, sugar beet cossette, sugar cane bagasse, etc , containing xylose and arabinose, and these sugars are chromatographically and by crystallisation purified from a mixture as generated D- ⁇ bose is produced by using microorganisms, and other pentose sugars by chemical synthesis Izumori et al , 1993, have described an enzymatic C3 -epimerisation by D-ketohexose-3-ep ⁇ merase
  • Intramolecular oxidoreductive isomerases catalysing an aldose-ketose interconversion form an enzyme group, which is responsible for the isomerisation of keto sugars into corresponding aldose products
  • isomerases belonging to the said group are e g arabmose isomerase, xylose isomerase, mannose isomerase, and ⁇ bose isomerase
  • Streptomyces albus isomerase is able to lsome ⁇ se D- ⁇ bose, L- arabinose, L-rhamnose and D-allose into corresponding keto sugars (Dauter et al , 1989)
  • a fairly new way to use enzymes is a cross-linked crystalline enzyme
  • an enzyme becomes stable, insoluble in water, and mechanically rigid It also endures organic solvents better than a soluble enzyme
  • a common cross-linking agent is glutaraldehyde Visuri (U S Patent No 5,437,933) prepared the first industrial cross-linked crystalline enzyme product from xylose isomerase
  • Cross-linked xylose isomerase is insoluble in water, wherefore it can be used in an enzyme reactor without a separate carrier
  • Such a reactor, filled merely with enzyme has a significantly greater volumetric efficiency than a traditional enzyme reactor which mainly includes carrier material Altus Biologies Inc has manufactured several crystalline enzyme products and registered the trademark CLECTM (cross-linked enzyme-crystal)
  • CLECTM cross-linked enzyme-crystal
  • the invention relates to a new way to use cross-linked crystalline enzymes for performing enzyme-catalysed reactions and simultaneous separation of products This enables increase in the amount of products, a change in product composition, and/or decrease in product inhibition caused by the products.
  • the invention concerns a process for the preparation of enzyme-catalysed products, particularly monosaccharides, as well as natural and synthetic derivatives thereof, such as isomers and/or C2-epimers, and for at least partial separation of these by cross-linked crystalline enzymes.
  • a chromatographic column is packed with cross-linked crystalline enzyme, whereafter a suitable substrate, as an oligo- or monosaccharide, is applied to the column, and the formed and at least partially separated products are collected.
  • the invention concerns a process for the preparation and simultaneous separation of enzyme-catalysed products from a product mixture and substrates, the process being characterized in that cross-linked crystalline enzyme is packed into a chromatographic column, whereafter a suitable substrate is applied to the column, and the formed and at least partially separated products are collected from the effluent The substrate solution can also be recycled in the column until the reaction reaches an equilibrium, or until the concentration of a desired sugar is sufficient
  • the invention concerns particularly a process for the preparation and separation of hexoses, pentoses and tetroses as well as natural and synthetic derivatives thereof, such as isomers and/or C2-epimers, the process being characterized in that cross-linked crystalline xylose isomerase is packed into a liquid chromatographic column, whereafter hexose, pentose, or tetrose is applied into the column and at least partially separated products are collected from the effluent.
  • Crosslinked crystalline enzyme is preferably xylose isomerase, endoglucanase III, xylanase, proteinase k, or ⁇ -glucosidase.
  • peptides in a peptide mixture can also be separated from each other.
  • a peptide mixture e.g. Lys-Tyr-Lys tripeptide
  • xylose can be prepared from the mixtures of xylooligomers and cellobiose from the mixtures of cellooligomers.
  • Figure 1 Isomerisation of L-arabinose into L-ribulose and L-ribose, and its partial separation in a column containing cross-linked xylose isomerase.
  • xylose isomerase is able to catalyse some previously unknown isomerisation reactions of tetrose, pentose and hexose sugars, and that the said enzyme also catalyses a previously unknown C2-ep ⁇ me ⁇ sat ⁇ on reaction
  • cross-linked crystalline enzyme can be used for the simultaneous catalysis and separation of products and substrates, whereby the reaction proceeds beyond the state of balance and product inhibition is decreased
  • Xylose isomerase is also an example of simultaneous catalysis, separation, and a change in the relative amounts of products following the separation
  • Table 1 The observed isomerisation and epimerisation reactions catalysed by xylose isomerase (Table 1) and the separation event can be combined Then in the reaction more product is formed than in a normal reaction which has reached its equilibrium
  • Table 2 The phenomenon is based on two facts Firstly, the sugars in Table 1 have different binding efficiencies to the cross-linked crystalline enzyme In consequence of this, elution of the sugars from the immobilised enzyme reactor takes place in different rates (Table 2) Secondly, each reaction has a different specific reaction rate Therefore separated products do not form immediately an equilibrium
  • a column is packed with cross-linked glucose isomerase prepared according to the U S patent No 5,437,993 (Visuri, 1998)
  • a column is placed into an oven which is at 50°C and L-arabinose solution is applied to the column
  • L- ⁇ bulose binds more strongly to the enzyme crystals, wherefore it passes through the column slower than L-arabmose Since the rate of the reaction is faster when L-arabinose turns into L- ⁇ bulose than vice versa, besides the reaction separation and a shift in the balance of the reaction take place L- ⁇ bose is also formed in the reaction Its binding to the enzyme is the strongest of all, and the reaction rate back into L-arabinose and L- ⁇ bulose is substantially slower than reactions between them This leads to the separation of L- ⁇ bose from the other two sugars, and to its slow elution as the last one
  • L-arabinose applied to a reactor is converted into both L- ⁇ bulose and L- ⁇ bose while passing through the immobilised enzyme
  • L-arabinose comes out from the reactor first, next the mixture of L-arabinose and L- ⁇ bulose in which the proportion of L-arabinose decreases, and finally growing concentrations of L- ⁇ bose
  • the relative proportion of L- ⁇ bose can be over 60 % in last fractions
  • a reactor can also be used in another way
  • a solution of L- ⁇ bulose is applied to it, a fast conversion into L-arabinose takes place
  • the affinity of L-arabinose to the enzyme is weaker than that of L- ⁇ bulose, thus it passes through the reactor substantially faster
  • the balance of the reaction shifts and L- ⁇ bulose and L-arabinose are separated L- ⁇ bose is also formed slowly in the reaction and again it binds most strongly of all to the immobilised enzyme
  • the other two sugars leave it behind and it is eluted again as the last one from the reactor Its reaction backwards is very slow, and so in the last eluted fractions its concen- tration rises distinctly higher than in an equilibrium reaction
  • Another example of the same phenomenon is p/o-nitrophenyl-cellobiose applied to a reactor containing endoglucanase III enzyme As the enzyme's substrate it is bound to the active centre The products p-nitrophenol and cellobiose are eluting in different rates, which results in decreasing product inhibition and in separation of the products The same thing occurs when a solution containing cellooligomers is used as substrate The reaction proceeds to its end and pure cellobiose is eluted from the reactor
  • cellobiose formed in the reactor can be hydrolysed completely and efficiently into glucose It is also possible to combine endoglucanase III and ⁇ -glucosidase crystals, in which case the reaction from cellooligomers into glucose proceeds quickly to its end
  • a solution of L-ribulose is recycled in a thermo-controlled enzyme column, which contains cross-linked xylose isomerase crystals
  • a mixture of three sugars is formed by xylose isomerase from L-ribulose, and when the reaction has reached equilibrium the reaction mixture contains 50% of L-arabinose, 25% of L-ribose and 25% of L-ribulose
  • the proportion of L-arabinose raises to a sufficient level (about 30%) the recycled solution can be directed to the separation of L-arabinose and the sugar is purified by crystallisation.
  • Crystalline protease can be used by the same principle for the hydrolysis of peptides and for the separation of the products formed
  • the temperature of a chromatographic column is preferably 30-70 °C, most preferably 40- 60 °C
  • the temperature used depends primarily on the cross-linked enzyme A person skilled in the art is able to choose the most suitable temperature and pH, as the case may be
  • Example 3 7 3 mg of L- ⁇ bulose in a water solution were applied to a reactor described in Example 1 The temperature of the reactor was 55°C and the flow rate was 0 5 ml/min The elution was carried out as described in Example 1 L- ⁇ bulose reacted quickly into L-arabinose, which was eluted from the reactor so that in the beginning the effluent contained mostly L-arabi- nose (Fig 2) Next, L- ⁇ bulose and finally the slowly forming L- ⁇ bose were eluted Example 3
  • Xylanase II produced by the fungus Trichoderma reesei was crystallised according to the publication Torronen et al. , 1993 Enzyme crystals were cross-linked according to Visuri 's U S patent No 5,437,993 using glutaraldehyde The material was packed into a chromatographic column according to Example 1 The temperature of the reactor was 45°C 5 mg of a xylooligomer mixture were applied to the reactor, the degree of of the mixture being about five. The cross-linked crystalline enzyme hydrolysed the solution, and the simultaneous separation based on the molecular weight resulted in the loss of product inhibition and in complete hydrolysis.
  • Bacillus subtilis protease k was crystallised by a known process, cross-linked with glutaraldehyde and packed into a column according to Example 1 , the temperature of which was 35°C and pH 7.5. A substrate solution containing peptide bonds was applied to the column. A reaction mixture was formed through hydrolysis of peptide bonds, the mixture being separated in the enzyme column. Lys-Tyr-Lys tripeptide was utilised as this kind of substrate and through its hydrolysation lysine, tyrosine as well as the dipeptides Lys-Tyr and Tyr-Lys were formed. The aromatic amino acid tyrosine was separated from lysine and the dipeptides in the enzyme column. Depending on the conditions used and the flow rate, tri- and dipeptides as well as tyrosine and lysine is obtained from the reactor.
  • Glucose isomerase (EC 5 3 1 5) as a reagent in carbohydrate synthesis success and failures with the isomerisation of non-natural derivatives of D-glucose into the corresponding 2-ketoses, Catalysis Today 12 549-561
  • Torronen, A Rouvinen, J , Ahlgren, M , Harkki, A and Visuri, K , 1993, Crystallization and preliminary characterization of two major xylanases from Trichoderma reesei, J. Mol. Biol. 233 313-316

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Abstract

La présente invention concerne la technologie enzymatique et notamment un nouveau procédé de préparation et de séparation simultanée de produits catalysés par des enzymes, à partir d'un mélange de produits et de matières premières, par utilisation d'une enzyme cristalline réticulée.
PCT/FI2000/000702 1999-08-18 2000-08-18 Procede de preparation et de separation simultanee de produits catalyses par des enzymes WO2001012834A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU65744/00A AU6574400A (en) 1999-08-18 2000-08-18 Process for the preparation and simultaneous separation of enzyme-catalysed products
FI20020288A FI20020288A (fi) 1999-08-18 2002-02-13 Menetelmä etsyymikatalysoitujen tuotteiden valmistamiseksi ja samanaikaiseksi erottamiseksi

Applications Claiming Priority (4)

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FI19991747 1999-08-18
FI991747 1999-08-18
FI19991883 1999-09-03
FI991883 1999-09-03

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002017392A (ja) * 2000-07-07 2002-01-22 Hayashibara Biochem Lab Inc Ｄ−キシロース・イソメラーゼを用いるアルドヘキソースの製造方法
WO2002088155A1 (fr) * 2001-04-27 2002-11-07 Danisco Sweeteners Oy Procede pour produire du xylitol
WO2003099410A1 (fr) * 2002-05-29 2003-12-04 Teknillinen Korkeakoulu Procede de separation chromatographique de nucleosides
US6894199B2 (en) 2001-04-27 2005-05-17 Danisco Sweeteners Oy Process for the production of xylitol
US6991923B2 (en) 2001-07-16 2006-01-31 Arla Foods Amba Process for manufacturing of tagatose
EP2161034A1 (fr) * 2008-09-04 2010-03-10 Sciotec Diagnostic Technologies GmbH Traitement de malabsorptions de fructose
WO2010025483A1 (fr) 2008-09-04 2010-03-11 Sciotec Diagnostic Technologies Gmbh Traitement de la malabsorption du fructose
US10759727B2 (en) 2016-02-19 2020-09-01 Intercontinental Great Brands Llc Processes to create multiple value streams from biomass sources

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5238826A (en) * 1989-06-16 1993-08-24 Roquette Freres Process for manufacturing xylose
WO1997044445A1 (fr) * 1996-05-24 1997-11-27 Altus Biologics Inc. Formulations de proteines cristallines reticulees et leur utilisation en tant que catalyseurs dans des solvants organiques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5238826A (en) * 1989-06-16 1993-08-24 Roquette Freres Process for manufacturing xylose
WO1997044445A1 (fr) * 1996-05-24 1997-11-27 Altus Biologics Inc. Formulations de proteines cristallines reticulees et leur utilisation en tant que catalyseurs dans des solvants organiques

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002017392A (ja) * 2000-07-07 2002-01-22 Hayashibara Biochem Lab Inc Ｄ−キシロース・イソメラーゼを用いるアルドヘキソースの製造方法
WO2002088155A1 (fr) * 2001-04-27 2002-11-07 Danisco Sweeteners Oy Procede pour produire du xylitol
WO2002088154A1 (fr) * 2001-04-27 2002-11-07 Danisco Sweeteners Oy Procede de production de xylose
US6894199B2 (en) 2001-04-27 2005-05-17 Danisco Sweeteners Oy Process for the production of xylitol
US6991923B2 (en) 2001-07-16 2006-01-31 Arla Foods Amba Process for manufacturing of tagatose
WO2003099410A1 (fr) * 2002-05-29 2003-12-04 Teknillinen Korkeakoulu Procede de separation chromatographique de nucleosides
EP2161034A1 (fr) * 2008-09-04 2010-03-10 Sciotec Diagnostic Technologies GmbH Traitement de malabsorptions de fructose
WO2010025483A1 (fr) 2008-09-04 2010-03-11 Sciotec Diagnostic Technologies Gmbh Traitement de la malabsorption du fructose
EP2764872A1 (fr) * 2008-09-04 2014-08-13 Sciotec Diagnostic Technologies GmbH Traitement de malabsorptions de fructose
US9144602B2 (en) 2008-09-04 2015-09-29 Sciotec Diagnostic Technologies Gmbh Treatment of fructose malabsorption
EP3269383A1 (fr) * 2008-09-04 2018-01-17 Stada Arzneimittel Ag Traitement de malabsorption du fructose
US10759727B2 (en) 2016-02-19 2020-09-01 Intercontinental Great Brands Llc Processes to create multiple value streams from biomass sources
US11840500B2 (en) 2016-02-19 2023-12-12 Intercontinental Great Brands Llc Processes to create multiple value streams from biomass sources

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