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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
  • 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
• • 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
• • 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
  • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
  • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions

Definitions

• This invention relates to enzyme compositions and their use as catalysts.
• Desired enantiomers of chiral chemicals are in most cases synthesised most conveniently by the use of biocatalysts such as enzymes.
• Enzyme-catalysed reactions have not been universally adopted by the pharmaceutical or chemical industries, since in a large number of cases the reactions have proved difficult to scale up. This is particularly true where the reaction involves a catalytic process in which the reaction must take place in an organic solvent. Processes in organic solvents have many advantages, for the chemistry, downstream processing, and often for the catalysis itself. However, enzyme catalysis in solvents can cause considerable reduction in the activity of the enzyme and reduce the speed of the process to an unusable or uneconomic level.
• Reactions catalysed by enzymes in solvents also tend to be physically unstable, especially where water has to be added as a substrate for a hydrolytic reaction. Such reactions can lead to the flocculation of the enzyme, precipitation, and irreversible cessation of the catalytic process.
• Enzyme reactions in aqueous solution can be potentiated by the addition of various chemicals. These may include surfactants, salts, phospholipids and lipid micelles; see, for example, Hebb era/, Biochem. Pharmac. (1975) 24:1007-12, Mann ef a/, Biochem. Pharmac. (1975) 24: 1013-17, and Mann, J. Neurochem. (1978) 31:747-9.
• inhibitory substances may be removed from a reaction medium by the presence of various materials.
• An obvious example is the removal of acid products of the reaction using simple buffers.
• the use of salts or other materials in this manner would appear not to be possible with an unrefined catalyst in organic solvents. However, this is one situation where there is a particular need for activation of the catalyst and the removal of any inhibitory processes.
• Enzyme-catalysed reaction are often substrate-dependent, and may be inhibited by the product. Thus, although high conversion may be desirable, it cannot be achieved because the substrate and/or product concentration must be kept low.
• the use of diluents to reduce or distribute the activity of an enzyme is well- established; for example, enzymes may be spray-dried on bran or another, similar material. Similarly, dehydrated enzymes may be prepared for use in organic synthesis in solvents by freeze-drying; this process can be costly.
• the present invention is based on the realisation that, e.g. in the case where an enzymatic reaction requires the presence of water and is conducted in organic solvent, and water bound to the enzyme should be sufficient to allow the catalytic reaction to proceed, the enzyme has to be used in excess merely to accommodate the water required for the hydrolytic reaction.
• This invention addresses such a problem by the addition of a material capable of forming a matrix and thereby retaining additional water in the proximity of the enzyme. More generally, the material can form a matrix within which the reaction can occur, or retain a substrate (which includes the possibility of more than one substrate) for reaction.
• the invention describes a mean of formulating an enzyme that solves many of the problems that occur with enzyme-catalysed reactions in organic solvents.
• the formulation therefore has the effect of making enzymatic catalysis in solvents robust, while preserving or enhancing the enzyme's activity, increasing stability, and facilitating the manipulation of the enzyme's immediate environment.
• the matrix-forming material provides a reservoir of water to act as a substrate in the reaction.
• the invention also has the advantages of economy and, because it is protected, longer utility for the enzyme. Description of the Invention
• the term "enzyme” is used to include both conventional enzymes, e.g. based on proteins and amino acids, and mimetics thereof; the “enzyme” should thus have the capability of catalysing the conversion of a primary substrate to a product (a secondary substrate, e.g. water, will usually also be involved).
• the process may be hydrolytic, in which case the enzyme may be, for example, a lipase or protease. It may also be non-hydrolytic, e.g. using glucose dehydrogenase as the enzyme.
• Other enzymes that may be used are oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.
• This invention utilises materials or "diluents" capable of maintaining a natural or an artificial environment around the enzyme catalyst. It is exemplified by the use, especially, of carbohydrate-containing polysaccharidessuchasdextrins, cyclodextrins and poly-galacturonans. Other materials such as proteins, surfactants, lipids and phospholipids may be used, but in each case the effect is to produce an environment around the catalyst that permits the catalyst to be robust within the reaction and to be manipulated so that maximal activity may be obtained.
• the efficiency of the reaction and the enzyme's activity in organic solvents can be increased by the use of specific materials. These materials may be considered diluents in that the activity of the enzyme is lowered within the formulation. Nevertheless, in organic solvents, the enzyme may demonstrate increased overall activity together with a considerable increase in efficiency and economic effectiveness.
• diluents are typified by polysaccharides of sufficient length to produce a framework or matrix wherein the enzyme may function. This matrix effectively protects the enzyme from the dehydrating and denaturing effects of the solvent and provides an environment which can be manipulated, to maximise the enzyme's effect.
• the matrix-forming material will typically be polymeric, e.g. having a degree of polymerisation (DP) of between 10 and 400. Proteins have the necessary characteristics. The water content of proteins may, however, be quite limited, and they are generally less preferred.
• Short-chain polysaccharides such as sucrose, trehalose and lactose may be used, but better results are obtained, in most cases, by using polysaccharides having a DP of greater than 10 carbohydrate units.
• a suitable example is dextrin or dextran of sufficient polymerisation. For the purposes of illustration, a simple dextrin containing little water-soluble material will be used.
• the formulation may be prepared in a number of ways, but adequate results may be obtained by mixing the dextrin with enzyme until a homogeneous blend is obtained.
• the diluted enzyme is placed in the organic medium or solvent, including substrate, and then hydrated by careful addition of water.
• a non-hydrolytic process only a small amount of water (if any) may need to be added.
• the enzyme protein forms a matrix or latticework with the dextrin. Small amounts of water hold this enzyme-dextrin matrix together during the reaction. This matrix is not only a physical support for the enzyme but, in the case of hydrolytic processes (e.g.
• the carbohydrate itself becomes a co- substrate in that it is the matrix that supplies the water as a substrate for the reaction.
• the carbohydrate itself becomes a co- substrate in that it is the matrix that supplies the water as a substrate for the reaction.
• the enzyme protein and the dextrin.
• hydrolytic reaction it can be assumed that there is a continuum and a dynamic relationship between the two pools of water. Water can pass from one to the other as the hydrolytic reaction proceeds. Such hydrolytic reactions remove water from the enzyme's active centre only to be replenished from another pool. The provision of water is however not the sole benefit or function of the matrix.
• the matrix may effectively support the enzyme in the organic phase in which the primary substrate (i.e. a substrate other than water) is dissolved.
• the substrate moves through the matrix and is presented to the enzyme in an environment where sufficient water, but often no more, is present for the catalytic reaction.
• the maintenance of the matrix is therefore crucial to the successful completion of any reaction. It is this physical relationship between the enzyme and, say, dextrin, that provides the physical robustness to the catalytic process and provides a "framework" in which other materials that can potentiate the catalytic reaction may be added.
• Reactions that are not hydrolytic may also be supported in this way. In some cases, there may be sufficient water, even in freeze-dried enzymes, to support the matrix. In others, small amounts of water may need to be added.
• the degree of polymerisation of the polysaccharide or dextrin should exceed 10. It has been found that, in the most effective dextrins, more than 60% of the dextrin has a DP lying between 10 and 350, with little soluble material. In the construction of a successful matrix, the amount of water can, in theory, be determined empirically. In practice, however, since enzymes may themselves vary in their ability to absorb water, the amount of dextrin and water needed to achieve a stable matrix, in any position, is probably best achieved by experimentation. This can readily be done by the skilled person.
• non-hydratable matrix formers may have to be used. In this case, only those very small amounts of water remaining within the enzyme will add to preserve enzyme functionality. It is unlikely that significant amounts of water will then need to be added, and could even be detrimental.
• the enzyme exists primarily in a form protected by the matrix from undesirable effects of the solvent.
• additional materials in addition to or instead of water, which may enhance the catalytic reactions, is now possible.
• a component that potentiates the enzyme-mediated reaction the component being relatively compatible with the enzyme and/or the material, and relatively incompatible with the reaction medium.
• salts in particular water-soluble salts, e.g. salts containing sodium, potassium, calcium, ammonium or phosphate ions
• surfactant is another example of a suitable additive.
• suitable additional materials that can be added, to ensure further activity, include those that remove inhibitory substances or unwanted products.
• a matrix- forming polysaccharide, protein, or similar material are: to formulate the enzyme and thereby enhance catalytic efficiency, protect the enzyme, and introduce both robustness and flexibility of manipulation into the process; while providing support, to act as a co-substrate by providing water for hydrolytic reactions; and to increase the efficiency of non-hydrolytic reactions by stabilising the enzyme, increase the maximal substrate concentration, and physically support the enzyme, which will, in turn, allow further manipulation of the enzyme's environment to maximise efficiency.
• this Example shows that the desired (S)-acetate is obtained in a yield of 0% in water, 8.2% in toluene without dextrin, and 32.6% in toluene with dextrin.
• a racemic mixture of a pharmaceutical precursor is resolved, when the same lipase as in Example 1 is used to hydrolyse one of a pair of enantiomers containing a halogen at a chiral centre, thus releasing a halogenic acid.
• the original reaction was subject to considerable problems on scaling up, both chemically, physically and in terms of efficiency.
• a dilution of the enzyme with dextrin produced excellent results in terms of recovery and ee while producing a stable and reproducible process, capable of scale-up.

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• 1999
  • 1999-04-12 GB GBGB9908328.9A patent/GB9908328D0/en not_active Ceased
• 2000
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