KR20130030397A - New surfactant-coated enzyme and the use thereof - Google Patents

Abstract

PURPOSE: A method for preparing a surfactant-coated enzyme is provided to obtain an enzyme with wide range of substrates. CONSTITUTION: A method for preparing a surfactant-coated enzyme comprises a step of suing an enzyme coated with a surfactant in dynamic kinetic resolution. The enzyme is selected among a purely isolated enzyme, a cell in which the enzyme is expressed, and a crushed cell product. The enzyme is lipase or protease. The surfactant-coated enzyme further contains saccharides and/or amino acid compounds.

Description

New Surfactant Coating Enzymes and Their Uses {NEW SURFACTANT-COATED ENZYME AND THE USE THEREOF}

The present invention relates to a method for preparing an enzyme having excellent activity and synthesizing an ester having optical activity from secondary alcohol through a kinetic kinetics splitting reaction using the enzyme and a metal catalyst.

Molecular synthesis methods with optical activity using enzymes as catalysts are well known [1) Hydrolases in Organic Synthesis , 2nd ed. (Eds: UT Borncheuer, RJ Kazlauskas), WILEY-VCH, Weinheim, 2006 ; 2) Asymmetric Organic Synthesis with Enzymes , (Eds: V. Gotor, I. Alfonso, E. Garcia-Urdiales), WILEY-VCH, Weinheim, 2008 ]. In particular, when a lipohydrolase is used as a catalyst, an ester having optical activity can be synthesized from a secondary alcohol. The ester synthesis using these enzymes can be divided into kinematic and dynamic kinetics. The kinematic splitting method has the disadvantage that the synthetic yield cannot theoretically exceed 50%, while the dynamic kinematic splitting method can increase the yield to 100% [H. Pellissier, Tetrahedron 2008 , 64 , 1563-1601; Tetrahedron 2011 , 67 , 3769-3802; Adv. Synth. Catal. 2011 , 353 , 659-676. Dynamic kinetic cleavage methods require racemization catalysts in addition to enzymes.

In addition, many researches over the past decade have developed various kinds of metal compounds as racemization catalysts [1) JH Lee, K. Han, M.-J. Kim, J. Park, Eur. J. Org. Chem . 2010 , 999-1015]. Representative examples are ruthenium compounds. (Korean Patent Nos. 455662, 644165, 904149)

In addition, Korean Patent Laid-Open Publication No. 10-2003-0061517 improves stereoselectivity and stability by using an enzyme coated with an ionic liquid, and by using the preparation of chiral intermediates required for the synthesis of chiral pesticides, medicines, natural compounds, etc. Disclosed are methods for use as catalysts for the process.

The enzyme used commercially in the dynamic kinematic splitting reaction is Candida antarctica lipase (trade name Novozym 435). This enzyme has good activity, selectivity, and thermal stability, but has the disadvantage of a narrow range of applicable substrates.

Therefore, there is a need for the development of enzymes with good activity and a broad range of substrates. In particular, there is a continuing need for new technologies that can dramatically increase enzyme activity.

The problem to be solved in the present invention is to provide an enzyme having a good activity and a wide range of substrates.

The problem to be solved in the present invention is to provide a method for producing a new enzyme having a good activity and a wide range of substrates.

Another problem to be solved in the present invention is to provide a variety of reaction processes, preferably dynamic kinetic optical splitting process using a new enzyme having a high activity and a wide range of substrates.

Another problem to be solved in the present invention is to provide a new surfactant used for a new enzyme having a good activity and a wide range of substrates.

In order to solve the above problems, the present invention provides an enzyme coated with a surfactant. Although not theoretically limited, one of the reasons for the low enzyme activity in organic solvents is that the enzymes are not dissolved, but are agglomerated in a solid state, and the enzyme activity increases when these enzymes are dispersed well in the organic solvent. The surfactant of the present invention was coated with an ionic surfactant to make it well dispersed in an organic solvent, and as a result, the enzyme activity was increased by 3000-6000 times more than that of a pure enzyme. In addition, the kinetic kinetics of secondary alcohol using an enzyme coated with a surfactant was completed, the fastest known to date [1) B. Marti-Matute, M. Edin, K. Bogar, FB Kaynak, J.-E. Backvall, J. Am. Chem. Soc . 2005 , 127 , 8817-8825; 2) JH Lee, N. Kim, M.-J. Kim, J. Park, Chem Cat Chem 2011 , 3 , 354-359. In addition, it could be applied to a wide range of substrates.

In the present invention, the surfactant is represented by the following formula (1).

In the practice of the present invention, preferred surfactants which exhibit particularly high reaction rates are represented by the general formula of the formula (2) or formula (3).

In a preferred embodiment of the invention, the surfactant is methyl 3,5-bis (2- (2- (2-ethoxyethoxy) ethoxy) ethoxy) benzoate, 3,5-bis (2- ( 2- (2-ethoxyethoxy) ethoxy) ethoxy) benzoic acid or potassium 3,5-bis (2- (2- (2-ethoxyethoxy) ethoxy) ethoxy) benzoic acid can be selected and used have.

In the practice of the present invention, the surfactant of formula (2) may be prepared by the reaction scheme 1 as follows.

The present invention also provides, in one aspect, an enzyme complex comprising an enzyme, a surfactant, a saccharide, and an amino acid compound.

In the present invention, the enzyme is not particularly limited, and may be selected from a plurality of enzymes including lipohydrolase and proteolytic enzyme. For example, a lipase that is used in the preparation of an optically active compound may be used. Preferred examples of lipases include Pseudomonas cepacia lipase (LPS), Candida antarctica lipase (CAL), Candida rugosa lipase (CRL), Aspergillus niger lipase lipase (ALN), Candida cylindracea lipase (CCL), Mucor miehei lipase, Pseudomonas fluorecens lipase (LAK), Rhizopus arrhase Rhizopus niveus lipase, Hug pancreas lipase, Candida lipolytica lipase, Mucor javanicus lipase, Penicillium locetefolia lipase roqueforti lipase, or Rhizomucor miehei lipase.

In the present invention, the enzyme may be commercially available and used, and commercial enzymes or water soluble extracts of commercial enzymes may be used.

In the present invention, the surfactant may use various surfactants such as anionic surfactants, cationic surfactants, nonionic surfactants, surfactants having an anion and a cation at the same time to disperse the enzyme in the reaction system, Preferably anionic surfactants.

In the present invention, the surfactant may have a general structure of formula (I) so that the enzyme complex is optimized for dynamic kinetic reactions.

In the present invention, the saccharides contained in the enzyme complex are used together with the amino acid compound and the surfactant to increase the activity of the enzyme complex. In the present invention, the sugars may be monosaccharides or polysaccharides, preferably polysaccharides. In the practice of the present invention, the sugars are hexosan and arabinan, such as starch, glycogen, cellulose, dextrin, inulin, chitin, and the like. Pentosan glucomannan, pectin, alginic acid, mucopolysaccharides, for example chondroitin sulfate, hyaluronic acid, such as xylan ) Can be used. In a preferred embodiment of the invention, the polysaccharide is dextrin.

In the present invention, the amino acid compound comprises an amino acid monomer, amino acid dimer, amino acid oligomer, or protein, preferably glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, cystine, methionine, aspartic acid, Asparagine, glutamic acid, diyotyrosine, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, oxyproline can be used, preferably glycine.

In the present invention, the enzyme complex may have various forms as long as the enzyme can be dispersed in a solvent together with a surfactant, an amino acid compound, and a saccharide mixture, and preferably, the enzyme is coated with a surfactant, an amino acid compound, and a saccharide. It is desirable to form.

In the practice of the present invention, the enzyme in the enzyme complex is preferably made 1-50% by weight, preferably 5-40% by weight, more preferably 10-30% by weight in order to have the optimum reaction activity . If the amount of the enzyme is too small or too high, the reaction activity may be reduced.

In the practice of the present invention, the surfactant in the enzyme complex is 1-50% by weight, preferably 5-45% by weight, more preferably 10 so that the enzyme can have the reaction activity while having the optimum dispersibility. It is good to achieve -40 wt%.

In the practice of the present invention, the saccharide in the enzyme complex in the range of 1-80% by weight, more preferably 10-75% by weight, even more preferably 30-70% by weight to have a high reaction activity It is preferable to use.

In the practice of the present invention, the amino acid compound in the enzyme complex in the range of 0.1-30% by weight, preferably 0.5-25% by weight, even more preferably 1-10% by weight so as to have a high reaction activity It is good to use.

In the present invention, the enzyme complex may be prepared by dissolving or dispersing an enzyme, a surfactant, a polysaccharide, and an amino acid compound in a medium and then freeze-drying it. The freeze drying may be performed by drying at least 24 hours after cooling with nitrogen.

In another aspect, the present invention is characterized by using an enzyme or enzyme complex coated with a surfactant in order to secure the diversity of the substrate in the dynamic kinetics optical splitting reaction and to increase the reaction rate.

In the present invention, the kinematic kinetic splitting reaction is characterized in that the substrate is reacted with an enzyme or enzyme complex and metal catalyst coated with a surfactant. In the practice of the present invention, the substrate is a racemic mixture or an optical isomer, and the dynamic kinetic cleavage reaction is, for example, a synthesis of various secondary alcohols into chiral esters.

In the practice of the present invention, the substrate is represented by the following formula (4), and the chiral ester is represented by the formula (5).

In the practice of the present invention, an enzyme is typically selected from a hydrolytic enzyme, and the one containing 20-100% of the enzyme was used. The enzyme was mixed with a surfactant and optionally a polysaccharide and / or amino acid compound at a constant weight ratio to form an aqueous solution, and then the solution was lyophilized to prepare an enzyme coated with a surfactant.

Next, the activity of the enzyme coated with the surfactant was measured in the reaction shown in Scheme 2 below. The enzyme was added to a toluene solution containing 1-phenylethyl alcohol ( 5 ) and isopropenyl acetate (IPA), and the rate of product ( 6 ) formation at 25 degrees was analyzed by HPLC. For activity comparison, the activity of the enzyme before coating as well as other commercially available enzymes was measured in the same way.

Enzymes made by mixing 20% enzyme, 25% surfactant, 50% polysaccharide and 5% glycine not only increased the activity more than 6000 times compared to the uncoated enzyme, but were also more active than Novozym 435, which is the most active among commercial enzymes. Was excellent.

The reaction of Scheme 3 was performed to evaluate the kinetic splitting reaction of secondary alcohol using an enzyme coated with a surfactant. The enzyme coated with a surfactant (ISCBCL), ruthenium metal catalysts (7-9), 1-phenylethyl alcohol (5), isopropenyl acetate was added (IPA) in toluene, was allowed to proceed the reaction at room temperature. The reaction was completed in 1-2 hours.

Next, using a enzyme coated with a surfactant, a kinetic kinetics cleavage reaction of various secondary alcohols shown in formula (6) was carried out to synthesize the corresponding chiral esters.

An enzyme having excellent activity in an organic solvent can be prepared by the method for preparing an enzyme coated with a surfactant of the present invention.

In addition, various chiral esters can be synthesized with high optical purity and good yield through dynamic kinetic cleavage reaction using the enzyme coated with a surfactant.

The chiral ester prepared by the production method of the present invention can be easily converted to chiral alcohol through a hydrolysis reaction, and can be usefully used as a material for various chiral medicines.

Hereinafter, preferred embodiments of the present invention will be described in detail, but the present invention is not limited only to the following examples.

Synthesis of Surfactant

Synthesis of bromo-2- (2- (2-ethoxyethoxy) ethoxy) ethane

Into a 50 mL round bottom flask, triethylene glycol monoethyl ether (13.5 mmol, 1.0 eq) and triphenylphosphine (1.3 eq) were put in a vacuum, filled with argon gas, THF (20 mL), and carbon tetrabromide (1.3). eq) was added and stirred at room temperature for 12 hours. The organic solvent was removed and concentrated to give a liquid product by column chromatography using ethyl acetate and hexane (1: 4 volume ratio). (2.3 g, 71.1% yield).

¹ H NMR (300 MHz, CDCl ₃ , ppm): δ 3.82 (t, J = 6.36 Hz, 2H), 3.68-3.45 (m, 12H), 1.21 (t, J = 7.00 Hz, 3H). ¹³ C NMR (75 MHz, CDCl ₃ , ppm): 71.2, 70.7, 70.6, 70.5, 69.8, 66.6, 30.3, 15.1

Methyl 3,5-bis (2- (2- (2-ethoxyethoxy) ethoxy) ethoxy) benzoate  2 Synthesis of

Into a 250 mL round bottom flask, 3,5-dihydroxy methyl benzoate (1.7 mmol, 1.0 eq), potassium carbonate (1.5 eq), 18-crown-6-ether (0.1 eq) were added, and vacuum was filled with argon gas. Acetone (50 mL) was added and 1-bromo-2- (2- (2-ethoxyethoxy) ethoxy) ethane (2.3 eq) was added to reflux overnight. The organic solvent was removed and concentrated to give a liquid product by column chromatography using ethyl acetate and hexane (1: 4 volume ratio). (638 mg, 77% yield).

¹ H NMR (300 MHz, CDCl ₃ , ppm): δ 7.19 (d, J = 2.34 Hz, 2H), 6.69 (t, J = 2.34 Hz, 1H), 4.14-4.12 (m, 4H), 3.78-3.75 ( m, 4H), 3.89-3.86 (m, 3H), 3.72-3.51 (m, 20H), 1.20 (t, J = 7.02 Hz, 6H). ¹³ C NMR (75 MHz, CDCl ₃ , ppm): 166.9, 159.9, 132.1, 108.2, 107.1, 71.1, 70.9, 70.8, 70.0, 69.8, 67.9, 66.8, 52.4, 15.3

3,5-bis (2- (2- (2-ethoxyethoxy) ethoxy) ethoxy) benzoic acid  3 Synthesis of

To a 50 mL round bottom flask, add methyl 3,5-bis (2- (2- (2-ethoxyethoxy) ethoxy) ethoxy) benzoate (500 mg) and THF (1 mL) and ethanol (7 mL) To this was added potassium hydroxide (2.0 eq) and stirred at room temperature overnight. 1Normal concentration Hydrogen chloride solution (2.5eq) was added and acidified, the solvent was removed, extracted with methylene chloride and concentrated to obtain a pure product without separate separation process. (474 mg, 99% yield).

¹ H NMR (300 MHz, CDCl ₃ , ppm): δ 7.97 (bs, 1H), 7.23 (d, J = 2.31 Hz, 2H), 6.72 (t, J = 2.28 Hz, 1H), 4.14 (m 4H), 3.87 (m, 4H), 3.74-3.53 (m, 20H), 1.23 (t, J = 7.08 Hz, 6H). ¹³ C NMR (75 MHz, CDCl ₃ , ppm): 170.2, 159.7, 131.5, 108.4, 107.4, 70.8, 70.7, 70.6, 69.6, 69.4, 67.7, 66.6, 15.1

Potassium 3,5-bis (2- (2- (2-ethoxyethoxy) ethoxy) ethoxy) benzoic acid  4 Synthesis of

To a 25 mL round bottom flask was added 3,5-bis (2- (2- (2-ethoxyethoxy) ethoxy) ethoxy) benzoic acid (0.21 mmol), THF (0.5 mL) and potassium hydroxide (0.1M, 2.3 mL) was added and the mixture was stirred at room temperature for 1 hour, after which the solvent was removed and vacuumed to obtain a product.

¹ H NMR (300 MHz, CDCl ₃ , ppm): δ 7.12 (d, J = 2.31 Hz, 2H), 6.52 (t, J = 2.24 Hz, 1H), 4.07-4.04 (m, 4H), 3.78-3.75 ( m, 4H), 3.68-3.45 (m, 20H), 1.16 (t, J = 7.01 Hz, 6H). ¹³ C NMR (75 MHz, CDCl ₃ , ppm): 171.6, 159.3, 138.5, 108.3, 104.9, 70.8, 70.7, 70.6, 69.9, 69.8, 67.6, 66.9, 15.3

(Example 2)

[Preparation and activity measurement of the lipid hydrolases coated with a surfactant;

Enzyme, surfactant, and other additives (polysaccharide, glycine, etc.) are mixed with water and dioxane 1: 1 mixture in an appropriate weight ratio, quenched with liquid nitrogen, and lyophilized for 1 day to prepare an enzyme coated with an anionic surfactant. Get At this time, the enzyme uses a commercial enzyme or a water-soluble extract of a commercial enzyme. In case of using the water-soluble extract of commercial enzyme Lipase PS, the extract and the surfactant were mixed in an appropriate ratio and lyophilized.

The activity of the lyophilized enzyme was measured as follows.

Toluene solution containing phenylethyl alcohol ( 5 , 0.5M) and isopropenyl acetate (1.5M) was added to the enzyme coated with a surfactant, the reaction proceeded at 25 ° C, and the yield of product ( 6 ) was obtained by HPLC. Analysis over time.

The activities of commercial enzymes Lipase PS, Lipase PS-C, and Nobozym 435 were also examined. The results are shown in Table 1 below.

order Kind of enzyme content Total activity (mMmg ^-1 h ^-1 ) Enzyme only activity
(mMmg ^-1 h ^-1 ) Enzyme-only Relative Activity Comparison Etc One Commercial Enzyme (Lipase PS) Enzyme 1% 0.24 24 63 2 Commercial Enzyme (Lipase PS-C) Enzyme 10% 11 110 290 3 Commercial Enzymes (Novozym 435) Enzyme 20% 120 600 1580 4 Commercial enzymes 80% of enzymes,
Glycine 20% 1.2 1.5  4 5 Enzyme Coated with Surfactant Enzyme 18%, Surfactant 23% 250 1390 3600 6 Enzyme Coated with Surfactant Enzyme 20%, Surfactant 25%, Polysaccharide 50%, Glycine 5% 500 5500 6600 7 Pure enzyme 100% Enzyme 0.38 0.38  One

The enzyme coated with the surfactant of step 5 was prepared with a water-soluble extract of Lipase PS, the commercially available enzyme of step 1, and the enzyme coated with the surfactant of step 6 was prepared using the commercial enzyme of step 4 as it is. .

The enzymes coated with the surfactants of Steps 5 and 6 were much more active than commercial enzymes, especially the Novozym 435 of Step 3, which is the most active among commercial enzymes. And, when compared with the pure enzyme of step 7 activity was increased more than 3000-6000 times.

Example 3

[ Kinematic Kinetics of 1-phenylethyl Alcohol Using Enzyme Coated with Surfactant]

The kinematic reactions shown in [Formula 7] were performed. Enzyme coated with surfactant (10 mg), ruthenium metal catalyst (4.0 mol%) , phenylethyl alcohol (1.0 mmol) and isopropenyl acetate (1.5 equiv) were added to toluene (2.0 mL) and the reaction proceeded at room temperature. I was. The reaction results are shown in the table below. When ruthenium metal catalyst 7 was used, the reaction was completed in 1 hour, and when ruthenium metal catalysts 8 and 9 were used, the reaction took 2 hours to complete. The fastest known kinematic response took three hours to complete, because [1] B. Martin-Matute, M. Edin, K. Bogar, FB Kaynak, J.-E. Backvall, J. Am. Chem. Soc . 2005 , 127 , 8817-8825; 2] JH Lee, N. Kim, M.-J. Kim, J. Park, ChemCat Chem 2011 , 3 , 354-359], it can be seen that the reaction of the present invention is the fastest.

order Ru catalyst Time (h) Yield (%) ^b Optical purity (ee%) ^c One 7 One 95 99 2 8 1 (2) 75 (95) 99 3 9 1 (2) 85 (97) 99

(Example 5)

[Dynamic Kinetic Splitting Reaction of Gamma-Chlorohydrin Using Enzyme Coated Enzyme]

In a pear-shaped Schlenk flask, gamma chlorohydrin (0.3 mmol), ruthenium metal catalyst 7 (12.5 mg, 4 mol%), potassium carbonate (41.5 mg), and an enzyme coated with a surfactant (3 mg) After vacuum drying and filling with argon gas, 500 μl of purified toluene was added thereto, followed by stirring at room temperature for 30 minutes. After stirring for 30 minutes, 49.6 μl of acyljugaine isopropenyl acetate was added thereto, and the mixture was stirred at room temperature for 24 h. The reaction vessel was cooled to room temperature, methylene chloride was added to the reaction solution, and then diluted. The reaction mixture was filtered through celite, concentrated, and concentrated to separate chiral gamma chlorohydrin acetate through silica gel column chromatography.

The optical purity of the product was measured using high resolution liquid chromatography equipped with a chiral column. The separation yield and optical splitting result of the obtained product are shown in Table 3 below.

order temperament product Separation yield (%) Optical purity (%) One

96 98 2

98 95 3

94 98 4

90 97 5

98 96 6

85 > 99

Example 6

[ Kinematic Kinetic Splitting Reaction of Homoallyl Alcohol Using Enzyme Coated with Surfactant]

The method of Example 4 was the same as that of Example 3, except that 8 was used as the metal catalyst, and the separation yield and optical splitting result of the obtained product are shown in the following table.

Table 4 shows the separation yield and optical purity of the product synthesized by dynamic kinetic partitioning.

order temperament product Separation yield (%) Optical purity (ee%) One

94 > 99 2

97 82 3

96 82 4

97 81 5

97 96 6

96 > 99 7

93 > 99 8

96 99 9

91 > 99

(Example 7)

[Dynamic Kinetic Splitting Reaction of Alpha-Aryl-Propazyl Alcohol Using Surfactant-coated Enzymes]

Into a pear-shaped Schlenk flask, 15 mg of ruthenium metal catalyst 8 , 50 mg of sodium carbonate, 2 mg of coated lipase was dried in vacuo, filled with argon gas, 400 μl of purified toluene, Stir for minutes. 5 μl of 1.0 M potassium tert -butoxide contained in THF solution was added to the reaction vessel and stirred until it changed from orange to dark brown. Then, 0.1 mmol of propazyl alcohol substrate was added and stirred for 5 minutes. 17 μl of isopropenyl acetate was added thereto, and the mixture was stirred at 60 ° C. for 48 hours. The reaction vessel was cooled to room temperature, methylene chloride was added to the reaction solution, and then diluted. The reaction mixture was filtered through celite, concentrated, and concentrated to separate chiral propargyl acetate through silica gel column chromatography.

The optical purity of the product was measured using high resolution liquid chromatography equipped with a chiral column.

The separation yield and optical splitting results of the obtained product are shown in Table 5 below.

Table 5 shows the separation yield and optical purity of the product synthesized by dynamic kinetic partitioning.

order temperament product Separation yield (%) Optical purity (ee%) One

95 > 99 2

91 96 3

90 96 4

91 96 5

94 > 99 6

92 > 99 7

90 98 8

91 98 9

89 97

The invention is not limited to the specific embodiments described above, and various modifications can be made by those skilled in the art without departing from the gist of the present invention. Therefore, the scope of the present invention should not be construed as being limited to the above embodiments, but should be determined by not only the following claims but also equivalents thereof.

Claims (20)

In a kinematic kinetic splitting reaction for preparing a chiral ester compound using a secondary alcohol, a method characterized by using an enzyme coated with a surfactant. The method of claim 1, wherein the enzyme is one selected from purely isolated enzymes, cells expressing enzymes, or lysates of cells expressing enzymes. The method according to claim 1 or 2, wherein the enzyme is lipohydrolase or proteolytic enzyme. The method of claim 1 wherein the kinematic kinetic splitting reaction comprises a ruthenium-based catalyst. The method of claim 3, wherein the lipohydrolase is Burkholderia cepacia lipase, Pseudomonas cepacia lipase (LPS), Pseudomonas fluorecens lipase, Candida anthica Lipases A and B ( Candida Antarctica lipase A and B), Candida rugosa lipase, Aspergillusniger lipase, Candida cylindracea lipase, Mucormiehei lipase ), Hog pancreas lipase, Candida lipolytica lipase, Mucorjavanicus lipase, Penicillium roqueforti lipase, and Rizomucomihei the referents ah at least one selected from the group consisting of such as (Rhizomucormiehei lipase) Characterized in that a. The method of claim 1, wherein the surfactant is represented by the following formula (1).

The method of claim 1, wherein the surfactant is represented by the following formula (2) or (3).

The method according to claim 1, wherein the secondary alcohol is represented by the following formula (4) and the chiral ester compound is represented by the following formula (5).

The method of claim 1, wherein the enzyme coated with the surfactant further comprises a saccharide and / or an amino acid compound. The method of claim 9, wherein the sugars are starch, glycogen, cellulose, dextrin, inulin, chitin, arabinan, xylan, glucomannan ( glucomannan), pectin (pectin), arginic acid (alginic acid), and mucopolysaccharide (mucopolysaccharide) characterized in that it is selected from the group consisting of. The method of claim 9, wherein the amino acid compound is selected from one or more of amino acids, amino acid oligomers, and proteins. Enzyme coated with a surfactant represented by the formula (1).

The enzyme according to claim 12, wherein the surfactant is represented by the following Chemical Formula 2.

The method of claim 12, wherein the surfactant
Methyl 3,5-bis (2- (2- (2-ethoxyethoxy) ethoxy) ethoxy) benzoate,
3,5-bis (2- (2- (2-ethoxyethoxy) ethoxy) ethoxy) benzoic acid,
An enzyme, characterized in that at least one selected from potassium 3,5-bis (2- (2- (2-ethoxyethoxy) ethoxy) ethoxy) benzoic acid. Enzyme complexes comprising enzymes, surfactants, sugars, and amino acid compounds. The enzyme complex according to claim 15, wherein the enzyme is a lipohydrolase. The enzyme complex according to claim 15, wherein the surfactant is at least one selected from anionic surfactants, cationic surfactants, and nonionic surfactants. The enzyme complex according to claim 15, wherein the enzyme complex comprises 1-50% by weight of enzyme, 1-50% by weight of surfactant, 1-80% by weight of saccharide, and 1-20% by weight of amino acid compound. The enzyme complex according to claim 15, wherein the enzyme complex is lyophilized after dissolving or dispersing an enzyme, a surfactant, a sugar, and an amino acid compound in a medium. The enzyme complex according to claim 15, wherein the enzyme complex is coated with a surfactant, a saccharide, and an amino acid compound.