WO2010005235A2 - Method for preparing optically pure lactide using an enzyme conversion reaction - Google Patents

Abstract

The present invention relates to a method for preparing optically pure lactide using an enzyme conversion reaction. In detail, the present invention relates to a method for preparing optically pure lactide by using an enzyme that uses, as a substrate, only one type of enantiometer from among optically impure lactic acid or alkyl lactate. The optically pure lactide prepared according to the present invention is useful in industry as the physical property of the copolymer polymerized by said lactide is far superior to the physical property of the copolymer polymerized by optically impure lactide.

Description

Method for preparing optical pure lactide using enzyme conversion reaction

The present invention relates to a method for producing optical pure lactide from racemic reactor using the enzyme conversion reaction. Specifically, the present invention relates to a method for preparing an optical pure lactide using an optical selective enzyme using only a specific racemic compound as a substrate.

Polylactic acid, which is used as a biodegradable polymer, is synthesized by ring-opening polymerization of lactide produced from monomer lactic acid (lactic acid). To date, much research has been conducted on lactide synthesis from lactic acid. In the US, Cargill Dow produces lactide used for high molecular weight polylactic acid polymerization by a continuous process (US Pat. No. 5,357,035). However, the lactide produced by the above method has a low yield and a high production cost, so there are many problems in using it as a substitute for a general purpose polymer. Currently, the process of synthesizing lactide directly from lactic acid and separating the produced lactide under high temperature and high pressure conditions is required. This process requires not only expensive and complicated distillation equipment but also high temperature. There is a disadvantage that the polymerization of lactide occurs in the distillation column, resulting in a decrease in yield of the resulting lactide.

In addition, the conventional chemical conversion process has a problem that can not produce an optical pure lactide. The optically pure polylactic acid obtained through the ring-opening polymerization process in the optically pure lactide is known to have excellent heat resistance and impact resistance compared to the optically non-pure polylactic acid. For example, the melting point of an optically pure polylactic acid formed from an optically pure lactide has a melting point of 170 to 180 ° C., whereas the melting point of an optically pure polylactic acid is 207 ° C (Biopolymers from Renewable Resources, DL Kaplan Ed., 397, Springer, 1999). Therefore, optically pure lactide is necessary, but no synthetic process known to date is known to synthesize such optically pure lactide.

The present invention has been made to solve the above problems, the present inventors while studying a method for producing an optical pure lactide by the enzymatic reaction, using an optical selective enzyme using only a specific racemic compound as a substrate It was found that the optical pure lactide can be prepared to complete the present invention.

Therefore, the present invention is to dissolve the reactive material consisting of a specific racemic compound in an organic solvent and to the dissolved reactive material, lipase, lipozyme, cutinase, protease and protina. It is an object of the present invention to provide a method for preparing optically pure lactide, which comprises adding and reacting at least one optical selective enzyme selected from the group consisting of proteins.

In order to solve the above problems, the present invention is the step of dissolving a reactive material consisting of a racemic compound represented by the following formula (1) in an organic solvent and a lipase (lipase, lipozyme), It provides a method for producing optically pure lactide comprising the step of adding and reacting at least one optically selective enzyme selected from the group consisting of cutinase, protease and proteinase.

Formula 1

The present invention is described in more detail below.

The term 'consisting of' in the present invention means that the main material is not included in addition to the components, for example, the phrase 'consisting of the racemic compound' in the present invention uses the racemic compound alone as a reactor and further reaction It does not include a substrate.

'Optically pure' in the present invention preferably has an optical purity of at least 80%, preferably at least 90%, more preferably at least 94% of one optical isomer among the L-type and D-type optical isomers. Say.

In the present invention, 'zeolite' is also called a molecular sieve (molecular sieve), and refers to a synthetic or natural zeolite as a porous solid that separates particles of molecular size.

The reactive material of the present invention is composed of a racemic compound represented by the following Chemical Formula 1,

<Formula 1>

Wherein each R is independently a branched or straight chain alkyl group of Na, NH ₃ , H, or C ₁ -C ₁₂ . Examples of the alkyl group include CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , isopropyl, butyl, isobutyl, isobutyl, pentyl, hexyl, octyl ), Or nonyl.

Therefore, the racemic compound used as the reactive material of the present invention may be lactic acid, lactate or alkyl lactate. Specifically, the reactive mass of the present invention consists of the racemic compound and does not include additional reactive mass.

In the present invention, the process of preparing optically pure lactide is performed by two steps as in Scheme 1 below. Specifically, Scheme 1 below is a process for preparing D-type lactide, and the process for preparing L-type lactide is similar.

<Scheme 1>

Where R is as defined above.

The enzyme involved in the reaction refers to an optically selective enzyme, and specifically, refers to an enzyme capable of selectively recognizing L-type or D-type in a reactive group consisting of a racemic compound represented by Chemical Formula 1.

More specifically, at least one enzyme selected from the group consisting of lipase and lipozyme has activity to prepare D-type lactide by recognizing only D-type reactive material, and cutinase and protease. ) And one or more enzymes selected from the group consisting of proteinases have the activity of producing L-type lactide by recognizing only L-type reactive material.

In the present invention, the lipase used to prepare the D-type lactide may be, but is not limited to, lipase B derived from Candida antarctica and more preferably represented by the amino acid sequence of SEQ ID NO: 1. Can be. In addition, lipozymes used in the preparation of D-type lactide may be, but are not limited to, lipozyme RMIM or lipozyme TLIM, more preferably derived from SEQ ID NO: 2 and Thermomyces lanuginosus derived from Rhizomucor miehei , respectively. It may be represented by the amino acid sequence of SEQ ID NO: 3. The lipase and lipozyme are not limited thereto, and preferably, 90% to 100% of D-type lactide can be produced with respect to L-type lactide in the product. In particular, the lipase may generate 100% of D-type lactide by specifically recognizing only D-type reactive group.

In the present invention, the cutinase used in the preparation of L-type lactide may be derived from, but not limited to, Fusarium solani or Nectria haematococca , and more preferably. And amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 6, respectively. In addition, the protease used for the production of L-type lactide may be derived from Bacillus licheniformis and may be represented by the amino acid sequence of SEQ ID NO: 8 more preferably. In addition, the proteinase used in the preparation of L-type lactide may be proteinase K derived from Tritirachium album , and more preferably, may be represented by the amino acid sequence of SEQ ID NO. The cutinase, protease and proteinase are preferably, but not limited to, 94-96% of L-type lactide relative to D-type lactide in the product. Specifically, the cutinase, protease and proteinase may specifically generate L-type lactide by 94 to 96% by specifically recognizing only L-type reactive material.

D-type and L-type lactide produced in the present invention is a common name used in the art, of which D-type lactide is represented by the following Formula 2, and the IUPAC name of L-type lactide is (3S) -cis- 3,6-dimethyl-1,4-dioxane-2,5-dione ((3S) -cis-3,6-dimethyl-1,4-dioxane-2,5-dione).

Formula 2

In the present invention, the organic solvent is not limited as long as it is a hydrophobic organic solvent, preferably benzene, dichloromethane n-hexane, cyclohexane, heptane, octane ), Isooctane and toluene can be one or more organic solvents selected from the group consisting of. More preferably, the organic solvent may be at least one organic solvent selected from the group consisting of n-hexane, cyclohexane, heptane, octane, octane, isooctane, and toluene. . The reason why the hydrophobic organic solvent is used in the present invention is that when the hydrophilic organic solvent is used, the water molecules surrounding the enzyme are released to the organic solvent layer, and thus the stability of the enzyme may be impaired. In one embodiment of the present invention it was confirmed that the optically pure lactide can be produced more effectively when using a hydrophobic organic solvent than the hydrophilic organic solvent (see <Example 2>).

In addition, the method for preparing lactide of the present invention may further include a step of adding zeolite to the dissolved reactor. The zeolite (zeolite, molecular sieve) is not limited to this, but may preferably be at least one selected from the group consisting of silica gel and acrylic polymer used for moisture absorption. The acrylic polymer may be, but is not limited to, polymethyl methacrylate (PMMA).

As a result of the reaction of Scheme 1, the zeolite generates two water molecules as by-products. The zeolite may further promote the forward reaction by absorbing the generated water molecules.

The input amount of the zeolite may be added in an appropriate amount depending on the type of the reactor, it can be easily determined by those skilled in the art. Specifically, when the reactor material is alkyl lactate having a large carbon number, it is preferable to add a large amount of zeolite because the reactor material is difficult to pass through the zeolite and is well dissolved in a hydrophobic organic solvent. The zeolite is not limited thereto, but is preferably added in an amount of 1 to 100% by weight relative to the reactor quality.

On the other hand, the enzyme used in the present invention may use the enzyme itself (free-type), it is also possible to use a form immobilized on various carriers and matrices known in the art for the convenience of manipulation.

In order to react by adding the enzymes to the dissolved reactor, the reaction temperature is not limited thereto, but is preferably performed at 10 to 80 ° C. More preferably, it may be carried out at 10 ~ 60 ℃, even more preferably 20 ~ 50 ℃, most preferably at 30 ℃ (see <Example 3>). The temperature range is limited because the enzyme reaction does not occur well below the above range and a problem occurs in the stability of the enzyme above the above range. In addition, the reaction time is not limited thereto, but may be preferably 4 to 24 hours, and the time range is limited so that an enzyme reaction does not occur well below the time period and there is no additional enzyme reaction when the time range is exceeded. Because.

In addition, the amount of the enzyme is not limited thereto, but may preferably be 1/12 times to 10 times, and more preferably 1/3 to 1 times the total weight of the reactor. In one embodiment of the present invention it was confirmed that the optically pure lactide is produced by 75% when administered in 1/12 times with respect to the total weight of the reactive mass, 85% when administered in 1 / 3-fold.

In order to separate the D-type or L-type lactide produced as described above, a separation method known in the art may be used, but the present invention is not limited thereto. Can be.

Optically pure lactide prepared according to the present invention can be usefully used industrially because the physical properties of the polymer polymerized with the lactide show superior physical properties compared to the polymer polymerized with the optically non-pure lactide. have. Accordingly, the present invention is very useful in that optically pure lactide can be easily produced from a low-cost racemic lactic acid, lactate or racemic alkyl lactate with only one enzymatic reaction.

Optically pure lactide prepared according to the present invention can be usefully used industrially because the physical properties of the polymer polymerized with the lactide show superior physical properties compared to the polymer polymerized with the optically non-pure lactide. have.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples. In the following examples,% means% by weight unless otherwise specified.

<Example 1>

Enzyme that synthesizes optically pure lactide from racemic lactic acid

A 1000 ml 3-neck round bottom flask enzyme reactor with a mechanical stirrer and condenser was prepared. 500 ml of n-hexane was added to the flask, and 1.5 g of D-lactic acid and 1.5 g of L-lactic acid were added thereto to dissolve it. After adding 1.5 g of zeolite (50 μm. Sigma, USA), 1 g of different enzymes of Table 1 was added thereto, followed by stirring at 100 rpm. Among these enzymes, lipozyme RMIM and lipozyme TLIM were purchased from Novozyme, and the remaining enzymes were purchased from Sigma-Aldrich. More specifically, lipase B (from Candida Antarctica ) is SEQ ID NO: 1, lipozyme RMIM (purchased from Novozyme) is SEQ ID NO: 2, and lipozyme TLIM (purchased from Novozyme) is SEQ ID NO: 3, carboxylesterase , Bacillus stearothermophilus ) is SEQ ID NO: 4, Cutinase (from Cutinase, Fusarium solani ) is SEQ ID NO: 5, Cutinase (from Nectria haematococca ) is SEQ ID NO: 6, Proteinase K (from Tritirachium album ) Silver SEQ ID NO: 7, protease (from Protease Bacillus licheniformis ) is represented by the amino acid sequence of SEQ ID NO: 8.

At this time, the reaction temperature was maintained at 50 ℃ and the reaction time was fixed at 12 hours, the sample was taken after 12 hours, the consumption rate of lactic acid and the production rate of lactide was measured by GC-Mass (Agilent 6890, Agilent, USA). The GC column used here is J & W Scientific Cyclosil-B (length: 30 m, inner diameter: 0.32 mm, film: 0.25 μm). Specific GC-Mass analysis conditions are as follows: 50 ~ 100 ℃, 5 ℃ / min temperature rise, 100 ~ 200 ℃, 10 ℃ / min temperature rise, inlet temperature 230 ℃. The carrier gas was helium, and the flow rate was 1 ml / min.

Table 1 Enzyme for synthesizing optically pure lactide from optical non-pure lactic acid Enzyme Name L-lactic acid consumption rate (%) D-lactic acid consumption rate (%) L-lactide production rate (%) D-lactide production rate (%) Lipase B 0 9 0 85 Repozyme RMIM 2 5 0.5 43 Repozyme TLIM 3 5 One 10 Carboxylesterase 0 0 0 0 Cutinase (Fusarium solani) 15 2 8 0.5 Cutinase (Nectria haematococca) 25 One 9 0.5 Proteinase 20 2 11 0.5 Protease 17 2 8 0.5

Table 1 shows the consumption rate of the substrate and the production rate of lactide when the enzyme reaction was performed under the conditions of <Example 1> using various enzymes.

As shown in Table 1, the carboxyesterase does not use any type of lactic acid as a substrate and cannot produce lactide, but lipase or lipozyme uses D-lactic acid much better than L-lactic acid, and as a result D -Overwhelmingly generated a lot of lactide was confirmed.

In the case of cutinase, proteinase K, or protease, it was confirmed that L-lactide can be generated much more than D-lactide by using more L-lactic acid as a substrate than D-lactic acid.

<Example 2>

Effect of Organic Solvents on D-Lactide Synthesis

In this example, the experiment was performed under the same conditions as in <Example 1>. However, when synthesizing D-lactide using lipase B derived from Candida antarctica showing the best D-lactide synthesis ability in <Example 1>, the consumption rate of the substrate and the type of organic solvent used were changed. The production rate was measured.

As shown in Table 2, it was observed that D-lactide was produced in high yield in a hydrophobic solvent such as n-hexane. In contrast, hydrophilic organic solvents showed relatively low production rates.

It appears that the water molecules surrounding the enzyme in the hydrophilic organic solvent are released to the organic solvent layer, and as a result, the enzyme stability is inhibited. In addition, the solubility of the substrate and the resulting D-lactide also appears to affect the degree of reaction.

TABLE 2 Effect of Organic Solvents on D-Lactide Synthesis Organic solvent L-lactic acid consumption rate (%) D-lactic acid consumption rate (%) L-lactide production rate (%) D-lactide production rate (%) n-hexane 0 99 0 85 Acetonitrile 0 0 0 0 toluene 0 99 0 88 Dichloromethane 0 82 0 52 Tetrahydrofuran 0 53 0 0 t-butanol 0 0 0 0 Dimethylformamide 0 0 0 0 Acetone 0 0 0 0 benzene 0 83 0 55 Cyclohexane 0 99 0 88 Heptane 0 99 0 90 octane 0 99 0 95 Isooctane 0 99 0 82

<Example 3>

Effect of reaction temperature on D-lactide synthesis

In this example, the experiment was performed under the same conditions as in <Example 1>. However, when synthesizing D-lactide using Candida antarctica-derived lipase B showing the best D-lactide synthesis ability in <Example 1>, the consumption rate and the production rate of the substrate were measured while changing the reaction temperature.

As shown in Table 3 below, the reaction temperature showed the highest D-lactide production rate at 30 ° C. In particular, the consumption of D-lactic acid at this temperature was similar to that of 40 ° C and 50 ° C, but the production rate was relatively high.

On the other hand, at too low a temperature, the consumption rate of D-lactic acid itself is relatively low, and thus the production rate of D-lactide is low. At too high a temperature, the consumption rate of the substrate also decreases because the stability of the enzyme seems to decrease at a high temperature, and the decrease in the production rate of D-lactide appears to be due to a decrease in ring formation reaction efficiency.

TABLE 3 Effect of reaction temperature on D-lactide synthesis Reaction temperature (℃) L-lactic acid consumption rate (%) D-lactic acid consumption rate (%) L-lactide production rate (%) D-lactide production rate (%) 10 0 83 0 75 20 0 90 0 85 30 0 99 0 92 40 0 99 0 88 50 0 99 0 85 60 0 85 0 70 70 0 70 0 55

<Example 4>

Effect of Enzyme Amount on D-Lactide Synthesis

In this example, the experiment was performed under the same conditions as in <Example 1>. However, when synthesizing D-lactide using Candida antarctica-derived lipase B showing the best D-lactide synthesis ability in <Example 1>, the consumption rate and the production rate of the substrate were measured while changing the amount of enzyme.

According to Table 4, it can be seen that the consumption rate of D-lactic acid and the production rate of D-lactide gradually increase as the dosage of enzyme increases. In particular, when more than 1.0 g of enzyme is consumed, D-lactic acid is completely consumed, but D-lactide gradually increases.

This may be due to the two-step reaction in order to form lactide from lactic acid as shown in Scheme 1. That is, even though the D-lactic acid was completely consumed, it is judged that some of the D-lactide still exists as an intermediate compound.

Table 4 Effect of Enzyme Amount on D-Lactide Synthesis Enzyme amount (g) L-lactic acid consumption rate (%) D-lactic acid consumption rate (%) L-lactide production rate (%) D-lactide production rate (%) 0.25 0 83 0 75 0.5 0 90 0 78 1.0 0 99 0 85 1.5 0 99 0 88 2.0 0 99 0 93

Example 5

Effect of Zeolite on D-Lactide Synthesis

In this example, the experiment was performed under the same conditions as in <Example 1>. However, when synthesizing D-lactide using lipase B derived from Candida antarctica showing the best D-lactide synthesis ability in <Example 1>, the substrate was changed while changing the zeolite (50 μm. Sigma, USA) dosage. The consumption rate of and the production rate of the reactor was measured.

In Table 5, it can be seen that the consumption rate of D-lactic acid and the production rate of D-lactide gradually increased as the input amount of zeolite increased. In particular, when 1.0 g or more of zeolite is consumed, D-lactic acid is completely consumed, but D-lactide gradually increases. This is believed to be due to a two-step reaction to form lactide from lactic acid as described above.

The reason why the production rate of D-lactide decreases when the amount of zeolite decreases is due to an increase in the reverse reaction due to the presence of water molecules. Specifically, two water molecules are produced as by-products by the reaction of Scheme 1. If the generated water molecules are not removed, a reverse reaction by water molecules, that is, a decrease in yield of the product lactide due to the hydrolysis reaction will occur. Therefore, it is necessary for the efficient production of D-lactide that zeolite is added in sufficient amount.

However, some of the produced D-lactide was not soluble in the organic solvent as a reaction medium, so it was observed to be separated from the reaction system. In this case, since it is not greatly affected by the reverse reaction, it can be explained that a large amount of D-lactide is produced even when experimented with a small amount of zeolite.

However, this effect is also halved in the case of alkyl lactate having a long carbon chain in which the reaction mixture is not lactic acid. This is because alkyl lactates with long carbon chains dissolve well in hydrophobic solvents such as hexanes and are difficult to pass through zeolites, which will cause adverse reactions. Therefore, it is preferable to add an appropriate amount of zeolite depending on the reactor quality, which will be easily implemented by those skilled in the art.

Table 5 Effect of Zeolite Amount on D-Lactide Synthesis Zeolite amount (g) L-lactic acid consumption rate (%) D-lactic acid consumption rate (%) L-lactide production rate (%) D-lactide production rate (%) 0.25 0 82 0 75 0.5 0 85 0 78 1.0 0 99 0 84 1.5 0 99 0 85

<Example 6>

Effect of Reactor Mass on D-Lactide Synthesis

In this example, the experiment was performed under the same conditions as in <Example 1>. However, when using lipase B derived from Candida antarctica showing the best D-lactide synthesis ability in <Example 1>, and using an optical non-pure alkyl lactate as a substrate rather than an optical non-pure lactic acid as a substrate, The consumption rate of the substrate and the production rate of the reactor material were measured. As can be seen in Table 6 below, when methyl lactate or ethyl lactate was used as the reactor rather than lactic acid, the substrate consumption was high, and the production rate of D-lactide was also slightly increased. This is because ethyl lactate is more soluble in the reaction solvent n-hexane as the length of the hydrophobic alkyl group is longer than that of lactic acid or methyl lactate. Therefore, it is thought that alkyl lactate having a longer hydrocarbon chain in addition to such ethyl lactate can produce D-lactide more efficiently.

Table 6 Effect of Reactor Mass on D-Lactide Synthesis temperament L-consumption rate (%) D-consumption rate (%) L-lactide production rate (%) D-lactide production rate (%) Legacy 0 99 0 85 Methyl lactate 0 99 0 90 Ethyl lactate 0 99 0 92

Optically pure lactide prepared according to the present invention can be usefully used industrially because the physical properties of the polymer polymerized with the lactide show superior physical properties compared to the polymer polymerized with the optically non-pure lactide. have.

Claims (14)

Dissolving a reactive material composed of a racemic compound represented by Formula 1 in an organic solvent, and Reacting the dissolved reactive material by adding at least one optical selective enzyme selected from the group consisting of lipase, lipozyme, cutinase, protease and proteinase Method for producing optically pure lactide comprising the step <Formula 1>

Wherein each R is independently a branched or straight chain alkyl group of Na, NH ₃ , H, or C ₁ -C ₁₂ . The optically pure lactide of claim 1, wherein the dissolved reactive mass is added with one or more enzymes selected from the group consisting of lipase and lipozyme to prepare D-type lactide. Manufacturing method. The method of claim 2, wherein the D-type lactide is contained in an amount of 90 to 100% by weight based on the total weight of the produced lactide. The preparation of optically pure lactide according to claim 1, wherein L-lactide is prepared by reacting the dissolved reactor with at least one enzyme selected from the group consisting of cutinase, protease and proteinase. Way. The method of claim 4, wherein the L-type lactide is produced in an amount of 94 to 96% by weight based on the total weight of the produced lactide. The method of claim 1, wherein the organic solvent is benzene, dichloromethane n-hexane, cyclohexane, heptane, octane, isooctane and Toluene (toluene) is at least one selected from the group consisting of a method for producing optically pure lactide. The method of claim 1, further comprising the step of introducing a zeolite (zeolite) to the dissolved reactor material. 8. The method of claim 7, wherein the zeolite is at least one selected from the group consisting of silica gel and acrylic polymer. The method of claim 1, wherein the lipase is lipase B derived from Candida antarctica . The method of claim 1, wherein the lipozyme is lipozyme RMIM or lipozyme TLIM. The method of claim 1, wherein the cutinase is derived from Fusarium solani or Nectria haematococca . The method of claim 1, wherein the protease is derived from Bacillus licheniformis . The method of claim 1, wherein the proteinase is proteinase K derived from Tritirachium album . The method of claim 1, wherein the reaction is carried out at 10 ~ 80 ℃.