TW581807B - Method for immobilizing enzyme for use as catalyst in organic reactions and hydrocolloid gel beads - Google Patents

Abstract

A method is disclosed for immobilizing enzymes for use in catalyzing organic reactions, in which the enzyme is imbibed into a dehydrated hydrocolloid polymer gel bead which has a network structure capable of swelling in aqueous media. The resulting enzyme-laden bead may then itself be dehydrated for eventual use in such organic reactions. The use of the enzyme laden beads as a catalyst for organic reactions is demonstrated in the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with propanol to N-acetyl-L-phenylalanine propyl ester in an octane solvent using subtilisin Carlsberg enzyme.

Description

581807 Revised case number 88116233 V. Description of the invention (1) The scope of patents applied for in the present invention is such as U.S. Provisional Application case number 60/101, 210, September 21, 1998 files. BACKGROUND OF THE INVENTION The present invention relates to a method for rationally fixing enzyme crops used in non-aqueous enzyme reactions. In industrial applications, the operation of using enzymes in an organic environment is often adopted, which is completely different from its use in an aqueous environment in its natural state. Since most drug synthesis is performed in organic solvents, pharmaceutical companies are (in particular) interested in using enzymes in non-aqueous environments. The application method that is particularly needed when applying enzymes in organic media is to catalyze the stereoisomeric reaction of only one isomer among several possible isomers. In organic media, the effective catalytic capacity of enzymes will be limited, so they can often only act on specific catalytic reactions, and generally only at relatively low temperatures and low pressures. It is therefore necessary to promote the ability to use enzymes to overcome these problems and to facilitate the use of various enzymes in organic reactions. The immobilization of enzymes is very important for industrial applications. When enzymes are used in a reaction device, π must be fixed in order to be separated from the product, recovered, and reused. When applied to aqueous media, enzymes have been widely used for immobilization on supports. Existing literature also describes the covalent attachment of enzymes to inorganic and organic surfaces, membranes and gels. Products that physically retain enzymes on membranes and gels have also been completed. To be successfully implemented in the industry without complicating non-aqueous enzymes in terms of activity, new methods are still needed to fix enzymes in organic media without including their activity. There is a wealth of literature on non-aqueous enzymes, especially investigating the kinetics of anhydrous enzymes under various organic solvents and reaction conditions. Many publications have been

0: \ 60 \ 60477-920904.ptc Page 5 58J807 Public CR Case No. 88116233. Years and months Amendment V. Description of the invention (2) Descriptions such as adding mechanical barriers to the medium have been adjusted and the situation of non-fixation is difficult. The existing method makes the enzyme difficult again. Except for facing the catalytic inversion only. When trying to take into account the advantages of covalent bonding in the literature is that regardless of this, the structure of the fixed structure and reaction interactions and covalent bonding of the carrier structure is significantly reduced. Out. Enzymes contain the limits of reason and construct conflict. The thermal stability of enzymes in organic solvents is attributed to the expansion of high-activity proteins, changes in substrate specificity, and the ability of these enzymes to catalyze new reactions. Water needs of enzymes in organic solvents have been checked. Enzyme restoration and reuse are based on the current state of the art enzyme restoration technology, such as the method of removing contaminated enzymes, which is ineffective, or will destroy enzyme activity during the restoration step. In addition to removing biological functional enzymes in any enzyme-catalyzed reaction mixture, when using dry enzymes in organic solvents, it is not difficult to remove them, and the enzymes have a tendency to agglomerate, which will cause the surface area that can be responded to. Reduction, thus significantly slowing the reaction rate. When enzymes are completely restored from a non-aqueous environment, the enzyme immobilization methods discussed in these issues include covalent binding, non- and physical retention. The severity of the situation where the enzyme is fixed to the carrier by a covalent bond can prevent the enzyme from leaking out of the carrier. However, the disadvantage of the pattern is that it usually changes the morphology of the active site. Non-covalent bonds such as: hydrophobic binding, polar binding electrostatic hydrogen bridge binding (adsorption) is often used to combine enzymes with non-former substances. Because this kind of binding is not as strong as covalent bonds, the yeast often does not change significantly, and therefore its enzyme reactivity is not. However, such weak binding also makes it easier for enzymes to leak from the carrier without involving any kind of chemical bond. Instead, only enzymes move through the polymer matrix. Therefore, it is completely different from enzymes. However, depending on the retention method, if the polymer conditions are too bad,

O: \ 60 \ 60477-920904.ptc page 6 j case number 88116233 V. Description of the invention (3) Inferior enzymes may be damaged, or they may be blocked to reduce their reactivity. The recently completed Dailiu study uses kappa-carrageenan as a polymer matrix for both fixed cells and enzymes. / c-Antler cuisine a linear multiple St class consisting of alternating 1, 3-connections / 5-D-galacto-4-sulfate and 1-4-connections 3, 6-dehydration-D-galactose Composition (see Figure 1). o3so ch2oh ch〇 〇

OH diagram The polymer forms a gel-like network in two steps. The first step involves the binding of the polymer chain moieties into a bicyclic ring. By adding a cation (usually potassium) to associate the ring with " domain structure ", a gelatinous network is created. The gelling temperature of KK carrageenan polymer depends on the cation concentration and is relatively independent of the carrageenan concentration. The most common method for fixing biological cell suspensions to carrageenan gels is to prepare carrageenan solutions in a cation-free environment. When the solution is cooled to about 45 ° C, add the cell suspension and shape the gel into the Geometric shapes. When the gel has cooled, treat with KC1 solution. Moon #A (Biotechnol. P roc ·, 7, 5 1 6 (19 1 1)) also tried to fix the cells in a pre-formed gel by the diffusion method. The gel was treated with K C 1 solution and post-crosslinked (ρ ○ s t-cr s s 1 in ek d) to prevent cell leakage. However, cells survive damage during this step. DESCRIPTION OF THE INVENTION The present invention provides a method for immobilizing an enzyme that catalyzes an organic reaction, wherein the enzyme is absorbed into the dried hydrocolloid polymer gel and has small pores.

Q: \ 60 \ 60477-920904.ptc Page 7 581807 _Case No. 88116233 q A in April, 2009 Amendment_ V. Description of the invention (4) In the pearl. In particular, the present invention includes the following steps: (a) dehydrating the hydrocolloid gel porous beads with an average particle diameter in the range of 5 to 150 microns, and (b) allowing the dehydrated hydrocolloid gel porous beads to absorb the enzyme aqueous solution to form Absorbent gel perforated beads, and (c) Dehydrate the absorbed gel perforated beads as needed to restore the gel perforated beads, where an effective catalytic amount of enzyme has been fixed. Enzymes may be oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, decarboxylase, carboxylase, aldolase, thiolase, and synthetase. In a particularly preferred embodiment, the hydrocolloid is carrageen or kap p a carrageen. One of the preferred enzymes is subtilisin Carlsberg. In a particularly preferred embodiment, the hydrocolloid is carrageen and the enzyme is subtilisin Jia Shibo; wherein the aqueous enzyme solution contains the enzyme from 0.05 to 40% by weight. The preferred method of dehydration is freeze drying. The present invention also provides a gel perforated bead, wherein the enzyme is fixed by the above method. DETAILED DESCRIPTION OF THE INVENTION According to one aspect of the present invention, it provides a method for immobilizing an enzyme catalyzing an organic reaction system, wherein the enzyme is absorbed by dehydrated porous beads (made of a hydrocolloid polymer forming porous beads). Porous bead-forming polymers suitable for use in the present invention include various hydrocolloids that form gels upon cooling. Antlers (preferably antler) have been described in the foregoing, and other polymers that can form gel-perforated beads that have a net-like structure sufficient to absorb or retain enzymes are also suitable for use in the present invention. It is also most advantageous that the dehydrated porous beads can swell upon contact with the aqueous enzyme solution or suspension to absorb the enzymes into the dried porous beads. Other polymers suitable for use in the present invention include agar, agarose, ammonium alginate, low

O: \ 60 \ 60477-910415.ptc Page 8 581807 _Case No. 88116233 Θ Amended on April β_ V. Description of the invention (5) Methoxy pectin, gellans, furcellaran ), Curd lan, polyglucosamine, glucomannan and its different derivatives, and mixtures of two or more of the above, and hydrocolloid mixtures, such as flavonic acid / locust bean gum, Locust Bean Gum / Agar, Mountain Lentils / Agar, Mountain Lentils / Flavoric Acid, Loquat / Flavoric Acid, Carrageen / Locin Bean Gum, Loquat / Carrageen, and Coriander / Starch. Carrageen is particularly suitable as a hydrocolloid polymer due to its network structure, its ability to form very small particles of porous beads, its ability to swell in aqueous media, and its ability to interact with proteins. The use of 20 micron (or smaller) small-diameter perforated beads is believed to minimize diffusion resistance and thus facilitates the absorption and immobilization of enzymes in aqueous media, while promoting the availability of enzyme active sites. However, the average diameter of the perforated beads can range from 5 to 150 micrometers, and preferably a size of 50 to 50 micrometers can be used. Polymeric porous beads can be prepared by methods known in the art, and are particularly suitable for the method described in US Patent No. 5,6 2,682,0 (this method is incorporated herein by reference). Very fine gel with porous beads, in which a hydrocolloid sol (such as carrageen) is brought into close contact with sufficient atomizing air to immediately cool the solution below the gel temperature of the sol. The advantage of this method is that very small beads of the same gel can be made, with an average diameter in the range of about 5 to 5 micrometers, preferably in the range of 10 to about 20 micrometers. Almost all enzymes are suitable for use in the present invention. Preferred enzymes suitable for use in the present invention include oxidoreductase (including, but not limited to, dehydrogenase, oxidase, reductase, hydroxylase, monooxygenase, peroxidase, and nitrogenase), transferase (including , But not limited to) proteases, esterases, amine transferases, phosphatases, nucleases, acid diesterases, and acid filling enzymes), hydrolases, lyases

O: \ 60 \ 60477-9l0415.ptc Page 9 581807 _ Case No. 88116233 吁 丨 Mou Yue / 厶 a Amendment_ V. Description of the invention (6) (including, but not limited to, aconitase, fumarate ), Enolase, crotonase, dehydratase, and aspartase), isomerases (including, but not limited to, racemic enzymes, epimerases, and mutases), and ligases (including, But not limited to synthetases and aldehyde ligases). Other enzymes preferred for use in the present invention include, but are not limited to, decarboxylase, carboxylase, aldolase, thiolase, and synthetase. The present invention is described using the subtilisin Jia Shibo as an enzyme. Immobilization technology involves inhaling enzymes into pre-formed hydrocolloid porous beads. The carrageen mixture suitable for preparing the porous beads contains from about 5 to about 4 weight percent (preferably about 2 weight percent) of antlers, 0.05 to about 0.4 weight percent (preferably about 0.2 weight percent) potassium di 0.025 to about 0.2 weight percent (preferably about 0.1 weight percent calcium gaseous), and 0.25 to about 0.2 (eight) (preferably about 0.1% by weight) of sodium benzoate in deionized water. Knife ratio method

Uc) This method involves rapid dehydration. The resulting perforated beads are then dehydrated. There is no limit to the method of dehydration. The traditional and preferred method is freeze-drying, which can be performed in the laboratory or on a large scale, such as roughly described in enzymes = (Methods in Enzymology, »Guide to Protein Thousand Purificatlon", Vol. 182, 77-8, red and 红.Press = technology. Large-scale freeze-drying is conducive to the commercialization of the present invention, and is well known to those skilled in the art. Regardless of the scale, the cold dehydration method includes contacting gel alcohol. After dehydration, a dehydration method, xenon twist The hydration removes the water-soluble beads and water-miscible alcohols (such as ethanol or enzymes) from the acid I & @ f >

581807 Amendment_Case No. 88116233 V. Description of the invention (7) The steps generally include immersing or suspending the dried porous beads in the enzyme aqueous solution, preferably by stirring or shaking for a period of time, sufficient to allow the porous beads to expand in the aqueous solution and entrain them. 5 至 8 小时。 Enzyme combined with pre-formed perforated beads, generally 0.5 to 8 hours. The amount and concentration of the solution used for swelling will vary depending on the polymer and enzyme applied to the porous beads. When gel-perforated beads are freeze-dried, the amount of the aqueous enzyme solution usually applied should be equal to or exceed the amount of water loss; it should be sufficient to restore the perforated beads to a completely water-containing state. Excess solution and enzyme (eg 50% excess) must be used to allow the porous beads to inhale the maximum amount of enzyme. For the carrageen perforated beads used to illustrate the present invention, it should be calculated to swell the gel perforated beads with a moisture content of about 2% by weight (before freeze-drying), plus an additional 50% solution amount. . The amount of enzyme applied ranges from 0.5 to about 0.5 grams of enzyme carrageen per gram of subtilisin Jia Shibo, preferably about 0.25 grams of enzyme per gram of antler (0.2 grams per gram of dried antler) . A suitable aqueous solution may contain from about 0.05 to about 40 weight percent enzymes, preferably from about 0.05 to about 5 weight percent, and more preferably from about 0.05 weight percent. Depending on the enzyme, the aqueous enzyme solution can also contain compatible water-soluble buffer solutions and stabilizers, especially enzymes whose activity and / or binding ability to gel-perforated beads depends on the ρ Η value. Such as the enzyme subtilisin Jia Shibo, ρ Η application of about 7.8 is more beneficial, it is best to use κ 碌 to adjust the pH to 7.8 20 milli-equivalent Lu acid Shu buffer solution. Those skilled in the art are well aware that increasing the polarity of enzymes can improve the catalytic activity of enzymes in organic solvents. Make sure that a small amount of water is in close contact with field enzymes (w i 1 d t y p e) to increase polarity. If using subtilisin Jia Shibo, it requires the equivalent of about 10 microliters of water per milligram of enzyme to exist as much as possible

O: \ 60 \ 60477-910415.ptc Page 11 581807 _Case No. 88116233_April / Mody __ 5. Description of the invention (8) The activity of the enzyme. Generally, this amount of water can be obtained from an aqueous enzyme solution that provides inhaled enzymes, and can be changed by controlling the amount of dehydration or adding additional water to the organic reaction medium. Alternatively, the increase in the polarity of the active site of the enzyme can be achieved by using enzyme genetic engineering that can directly change the polarity of the active site. In one of the specific embodiments of the present invention, the subtilisin Jia Shibo was used as the enzyme, the water content range was 10 microliters / mg, and the pH was adjusted to 7.8. After inhalation as outlined above, excess liquid is removed from the perforated beads by any suitable method, such as by centrifugation or decantation. The perforated beads can be reconstituted and used as such or further dehydrated and stored or transported for later use. The perforated beads can be freeze-dried or dehydrated by other methods described above to provide dehydrated gel perforated beads, which contain a fixed effective amount of enzyme. The following examples illustrate, but are not limited to, the method of applying the carrageen perforated beads and the enzyme subtilisin Jia Shibo according to the present invention. Example 1 The transesterification of n-acetamyl-L-phenylalanine ethyl ester in propanol using a subtilisin Jia Shibo enzyme to prepare 0.0285 g (0. 000121 mole) A mixture of ethyl-L-phenylalanine ethyl ester, 0.0164 g (0.000061 mole) of undecane, and 0.75 ml of propanol in 9.25 ml of octane. A 2 ml aliquot of this solution was placed in a 5 ml bottle. Add 0.01 g of subtilisin Jia Shibo enzyme powder and 12 microliters of water to the bottle. Shake the mixture at 40-4 2 ° C. A small number of samples are taken periodically and analyzed by gas chromatography. After 60 minutes, analysis showed the formation of 0.00486 g of n-ethylfluorenyl-L-phenylalanine propyl. Before carrying out this reaction, dissolve subtilisin Jia Shibo enzyme powder in 20 milliliters

O: \ 60 \ 60477-9l0415.ptc Page 12 581807 _ Case No. 88116233 9 丨 March 4th __ V. Description of the invention (9) The amount of potassium phosphate buffer solution (concentration: 5 mg / ml). After adjusting pΗ to 7.8 with potassium hydroxide, freeze-dry the solution with liquid nitrogen, and then place the sample under -4.5 ° C, 2.67 Pa (Pa) under vacuum for 30 hours. Example 2 Preparation of Carrageena Gel Porous Beads with Bacillus subtilisin Enzyme Jasper's Enzyme Embedded The method described in US Patent No. 5, 6 6 2, 8 4 0 Gel has perforated beads. A solution containing 2.5 weight percent carrageen, 0.2 weight percent potassium vaporized, 0.1 weight percent gasified calcium, and 0.1 weight percent sodium benzoate stored in deionized water was prepared. The resulting solution was heated and maintained at a temperature of about 9 2-9 3 ° C, while stirring to prevent gelation. The solution was pumped through a heating tube to a high-pressure nozzle at a flow rate of 24 ml / min. Before reaching the nozzle, 25 milligrams of potassium phosphate buffer solution per milliliter of 25 mg subtilisin Jia Shibo enzyme cold solution (whose p Η has been adjusted to 7.8 with potassium hydroxide) at 6 ml / Infused into carrageen solution at a minute rate. In this way, a solution containing 2% by weight of carrageen and 5 mg of subtilisin Jia Shibo per ml can be produced. At the same time, a stream of air was passed through the nozzle at a pressure of 5 1 · 7 psi (75 psi), and when the carrageenan / enzyme solution left the nozzle, it was pressurized and the aqueous solution was atomized. The pressure difference immediately dispersed the carrageenan / enzyme solution into droplets, immediately cooled and solidified into perforated beads with an average diameter of about 20 microns. The resulting subtilisin-containing protease Jia Shibo enzyme carrageenan perforated beads were freeze-dried and stored under anhydrous conditions for later use. After calculation, it was determined that 25 mg of the dried porous beads contained 5 mg of enzyme. Example 3

O: \ 60 \ 60477-9l0415.ptc Page 13 581807 Amendment No. 88116233 V. Description of the invention (10) Use of the subtilisin enzyme Jiashibo Enzyme in propanol embedded in carrageenan in propanol- Preparation of transesterification of ethyl L-phenylalanine containing 0.0280 g (0.00119 mol) of ethyl n-ethylfluorenyl-L-phenylalanine, 0.0145 g (0.000061 mol) of 19 carbons A mixture of alkanes and 0.75 ml of propanol in 9.25 ml of octane. A 2 ml aliquot of this solution was placed in a 5 ml bottle. 250 microliters of water was added to 5 mg of lyophilized perforated beads made in Example 2 to rehydrate the perforated beads to their state before lyophilization. Add these rehydrated perforated beads to the reaction flask and shake at 40 ° C. Periodically, a small sample of the reaction mixture is taken and analyzed by gas chromatography. After 60 minutes, the analysis showed that 0.01 g of n-ethylfluorenyl-L-benzyl alanine had formed. Carrageen beads with subtilisin Jia Shibo enzyme showed absorption of 25% of n-ethylamyl-L-phenylalanine ethyl ester, and the original mass of the original mixture available for transesterification Reduced to 0.004 g. Example 4 Preparation of a subtilisin enzyme solution A 20 milli-equivalent potassium phosphate buffer solution was prepared and the pH was adjusted to 7.8 with potassium hydroxide. Add 0.27 67 g of subtilisin Jia Shibo enzyme powder to 55.5 ml of the buffer solution. The resulting solution contained 0.49 weight percent enzymes. Stir the enzyme solution in the freezer for 30 minutes before use. For long-term storage, freeze-dry the solution as described in Example 1 and reconstitute it as needed when used. Example 5 Preparation of Carrageen Porous Beads with Absorbed Subtilase Protease Jia Shibo Enzyme

O: \ 60 \ 60477-9l0415.ptc Page 14 581807 Case No. 88116233 9 Year, month and month ¥ Amendment V. Description of the invention (11) In the same way as in Example 2, a step containing 2% by weight of antlers (but no enzyme) was prepared. Antlers with perforated beads. These perforated beads were freeze-dried in the manner described in Example 1. 0.75 15 g of perforated beads were added to 55. 5 ml of the enzyme solution prepared in Example 4, and the mixture was stirred in a freezer for 2 hours. At the end of this period, the mixture was transferred to a centrifuge tube and centrifuged at 10 ° C for 15 minutes at 4,000 rpm. During centrifugation, carrageenan formed sticky pellets, allowing 30.7 ml of supernatant to be poured out. Carrageen balls are transferred to a freeze-dried bottle for freeze-drying. Analyze the supernatant with a 280 nm ultraviolet spectrophotometer (such as the method of Pantolino (Pant ο 1 ia η 〇) and others. (Biochemistry, 28, 7205 (1989)). The enzyme concentration was found to be 0.46% by weight. From the amount of the supernatant, it is known that the solution absorbed by the dried carrageen beads is sufficient to restore the perforated beads to 3 wt% carrageenan gel containing 0.1 7 3 grams of enzyme per gram of carrageen. These perforated beads were freeze-dried as described above and stored for later use. Example 6 The ester of n-acetamido-phenylalanine ethyl ester in propanol using subtilase protease Jiashibo enzyme in inhaled carrageen beads. 25mL of octane prepared by radical transfer containing 0.0282 g (0,000120 mol) of n-ethylfluorenyl-L-phenylalanine, 0.0170 g (0.000063 mol) of nonadecane, and stored in 9.25 ml of octane 0.75 ml of a mixture of propanol. 2 ml aliquots of the solution were placed in a 5 ml bottle. 193 microliters of water was added to 5.8 mg of freeze-dried perforated beads prepared in Example 5. Porous beads are rehydrated to their pre-lyophilized state. These are rehydrated The perforated beads were added to the reaction flask and shaken at 40 ° C. A small sample of the reaction mixture was periodically taken for gas chromatography analysis. After 60 minutes, analysis showed that 0.00268 grams of

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Claims (1)

581807, l:, (q: /, v., 4; r:.,;... ^ &Quot; " 1 乂 Γ Prime No. 88116233 _ ^^ Amended on February 2, 2006._ Scope of Patent Application 1 A method for immobilizing an enzyme used as a catalyst in an organic reaction, comprising: (a) a hydrocolloid having an average particle diameter ranging from 5 to 150 micrometers and having a network structure capable of expanding in an aqueous medium; The gel perforated beads are dehydrated; (b) the dehydrated hydrocolloid gel perforated beads absorb the enzyme aqueous solution to form gel perforated beads that have absorbed the enzyme; (c) if necessary, the gel perforated beads that have absorbed the enzyme are Dehydration; and (d) recovering gel perforated beads in which an effective catalytic amount of enzyme has been immobilized. 2. The method according to item 1 of the scope of patent application, wherein the dehydration is used in step (a), or at the same time ( a) and step (c). 3. The method according to the scope of the patent application, wherein the hydrogel system is antlers. 4. The method, such as the scope of the patent application, the first method, wherein the hydrogel system is kappa. Antler dishes 5. If patented The method of the first item, wherein the enzyme is selected from the group consisting of oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, decarboxylase, carboxylase, aldolase, sulfur Hydrolytic enzymes, synthetic enzymes. 6. The method according to item 5 of the patent application, wherein the enzyme is Carlsberg, a subtilisin protease. 7. The method, item 1 in the patent application, wherein hydrolyzed gum carrageen And the enzyme is subtilisin Jia Shibo, in which the aqueous enzyme solution contains fermentation Q: \ 60 \ 60477-930210.ptc Page 18 581807 Case No. 88116233 said Amendment VI. Patent Application Range The prime range is 0.05 to 40 weight percent. 8. A kind of hydrocolloid gel perforated beads with an average particle diameter ranging from 5 to 150 microns and a network structure capable of expanding in an aqueous medium, wherein per gram of gel perforated beads have absorbed 0.5 to 5 5 grams of enzyme, and wherein the enzyme is selected from the group: oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, decarboxylase, carboxylase, aldolase, thiolase Synthetic enzymes. 9. The porous colloidal gel with porous beads as described in item 8 of the patent application scope, wherein the enzyme is subtilisin Jia Shibo. 10. The hydrocolloid gel with porous beads as described in item 8 of the scope of patent application, among which the hydrocolloid system carrageen. 1 1. The method according to item 1 of the scope of patent application, wherein the method for dehydrating the hydrocolloid gel porous beads or the absorbed gel porous beads is dehydration; cold drying; Q: \ 60 \ 60477-930210.ptc Page 19