KR20230114870A - Family VIII Carbixylesterase For High Purity (S)-Sec-Alcohol Preparation - Google Patents

Abstract

The family VIII carboxylesterase for producing (S)-secondary alcohol of the present invention has high (S)-stereoselectivity for chiral secondary alcohol ester substrates, which can produce high-purity (S)-secondary alcohol. There are advantages. Therefore, using the family VIII carboxylesterase for producing (S) -secondary alcohol of the present invention has an effect of industrially mass-producing photochemically pure (S) -secondary alcohol.

Description

Family VIII Carbixylesterase For High Purity (S)-Sec-Alcohol Preparation}

The present invention relates to family VIII carboxylesterases for the production of high-purity (S)-secondary alcohols.

Lipase has high (R)-stereoselectivity for various chiral sec-alcohols, so that chiral sec-alcohols ((( R) - is widely used in the manufacture of secondary alcohols). The enantiomers are classified according to structural properties and optical properties, and the enantiomers according to the structural properties are classified into (R)-enantiomers and (S)-enantiomers. The (R)-enantiomer and (S)-enantiomer are obtained by prioritizing substituents attached to the chiral center according to the Cahn-Ingold-Prelog ranking rule based on atomic number and rotating the chiral center. If the priority of the remaining substituents decreases to the right (decreasing in a clockwise direction) after the substituent with the lowest priority is obscured by the chiral center, it is called an (R)-enantiomer and decreases to the left (decreasing in a counterclockwise direction). ) is called the (S)-enantiomer. Two substituents exist at the chiral center of a chiral secondary alcohol, and according to previous studies on the enantioselectivity of lipase, the substituents bind to specific binding pockets of lipase, respectively.

Candida antarctica lipase B (CAL-B) has been most widely used as an enzyme for producing chiral secondary alcohols. The CAL-B has an alcohol binding region composed of two binding pockets, the large binding region located at the entrance of the binding region and the middle binding region located deep inside the binding region. are separated by It is known that the intermediate binding site is a stereospecificity binding site, and substituents larger than ethyl-group cannot bind thereto. The binding characteristic of the intermediate binding site explains the high stereoselectivity of CAL-B for chiral secondary alcohol substrates, and is understood as the inherent (R)-stereoselectivity possessed by most lipases.

Until now, few lipases with high (S)-stereoselectivity for various chiral secondary alcohols have been reported. Therefore, if a lipase with high (S)-stereoselectivity is discovered and utilized, high purity can be obtained through dynamic resolution or dynamic resolution for chiral secondary alcohol substrates that could not be obtained through conventional lipases with (R)-stereoselectivity. It is expected to be able to prepare (S) -secondary alcohols of

Serine proteases are known as enzymes with (S)-stereoselectivity. However, serine proteases have not been widely used in the production of photochemically pure secondary alcohols because they accept only a narrow range of substrates and have a moderate or less (S)-stereoselectivity. However, serine proteases have interesting structural features compared to lipases. The catalytic triad of serine proteases is almost identical to that of lipases, but has a spatial mirror image arrangement.

According to the results of a recently reported crystal structure (PDR ID: 4IVK and 5GMX) of family VIII carboxylesterase, the catalytic residues of family VIII carboxylesterases are amino acids and the catalytic residues of serine proteases and lipases. Although different in composition, serine proteases and spatial arrangements were found to be similar to each other. That is, the three catalytic residues of family VIII carboxylesterases are spatially mirror images of the three catalytic residues of lipases. Thus, family VIII carboxylesterases are expected to show (S)-stereoselectivity for chiral secondary alcohols as well as serine proteases.

Therefore, it is expected that family VIII carboxylesterases can be used for photochemically pure (S) -secondary alcohol production through kinetic resolution or dynamic kinetic resolution. However, there has been no experimental proof of the production of chiral secondary alcohols using the family VIII carboxylesterases.

The patents and references mentioned herein are incorporated herein by reference to the same extent as if each document were individually and expressly specified by reference.

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The present invention is a new family VIII carboxylesterase capable of industrially producing high-purity (S)-secondary alcohol (sec-alcohol) with high (S)-stereoselectivity for chiral sec-alcohol ester It aims to provide

Other objects and technical features of the present invention are presented more specifically by the following detailed description, claims and drawings.

In the present invention, serine (S), the 283rd amino acid in the amino acid sequence of SEQ ID NO: 1, is aspartic acid (D), asparagine (N), valine (V), glutamic acid (E) , Glutamine (Q), Histidine (H), Leucine (L), Isoleucine (I), Methionine (M), Phenylalanine (F), Tyrosine (Y) and Provided is a variant of family VIII carboxylesterase characterized in that it is substituted with any one amino acid selected from the group of amino acids consisting of tryptophan (W).

The variant of the family VIII carboxylesterase is characterized in that it produces high-purity (S) -secondary alcohol (sec-alcohol) using a chiral sec-alcohol ester as a substrate, and the chiral Secondary alcohol esters are (±)-2-butyl acetate, (±)-2-butyl butyrate, (±)-3-methyl-2-butyl acetate, (±)-3-methyl-2-butyl butyrate, ( ±)-2-pentyl acetate, (±)-2-pentyl butyrate, (±)-3-butyn-2-yl acetate, (±)-3-butyn-2-yl butyrate, (±)-1-phenylethyl acetate, (±)-1-phenylethyl butyrate, (±)-1-(4-chlorophenyl)ethyl acetate, (±)-1-(4-chlorophenyl)ethyl butyrate, (±)-1-phenyl-2-propyl Any of the chiral secondary alcohol esters consisting of acetate, (±)-1-phenyl-2-propyl butyrate, (±)-4-phenyl-2-butyl acetate and (±)-4-phenyl-2-butyl butyrate It is characterized by being one.

The Family VIII carboxylesterase variant is characterized by showing (S)-stereoselectivity with respect to the chiral secondary alcohol ester substrate and having an Enantiomeric ratio (E) of 39 to 5500, and the chiral secondary alcohol The (S) -secondary alcohol prepared by hydrolyzing the ester substrate is characterized in that the purity is 90 to 99.9%.

The present invention provides a polynucleotide encoding a variant of the family VIII carboxylesterase and a vector containing the polynucleotide, and any one or more of the variant, the polynucleotide, and the vector of the family VIII carboxylesterase. Provides a microorganism that produces a variant of family VIII carboxylesterase, including.

The present invention comprises the steps of culturing a microorganism producing a variant of Family VIII carboxylesterase, including any one or more of the variant of Family VIII carboxylesterase, the polynucleotide and the vector, in a medium; and recovering the variant of Family VIII carboxylesterase from the cultured medium or microorganism.

The high-purity (S)-secondary alcohol production family VIII carboxylesterase of the present invention has a high (S)-stereoselectivity for chiral secondary alcohol ester substrates, so that high-purity (S)-secondary alcohol can be prepared. There are advantages to being Therefore, using the family VIII carboxylesterase of the present invention has the effect of industrially mass-producing photochemically pure (S) -secondary alcohol.

1 shows the homology search results of the present invention.
Figure 2 shows the protein expression and purification results of eight genes selected from the homologous search results of the present invention. Panel a) shows the protein expression results of 8 genes, panel b) is a protein derived from Bradyrhizobium canariense, panel c) is a protein derived from Bradyrhizobium erythrophlei, panel d) is a protein derived from Afipia sp. Root123D2-derived protein, panel e) is Bradyrhizobium sp. Proteins from WSM1417, panel f) shows the results of purification of proteins from Proteobacteria bacterium SG_bin9.
3 shows a reaction in which chiral secondary alcohol esters are hydrolyzed and converted into (S) -secondary alcohols by Family VIII esterases of the present invention.
Figure 4 shows the structures of family VIII esterases and intermediates containing substrate 5a of the present invention. Panel a) shows the intermediate as a line, panel b) shows the fast reacting isomer and panel c) shows the slow reacting isomer.
Figure 5 shows the catalysis of the family VIII esterase of the present invention step by step.
Figure 6 shows the difference in activation energy (ΔΔG‡=-RTlnE) between enantiomers according to the volume change of the family VIII esterase variants of the present invention.
7 schematically shows a reaction in which chiral secondary alcohol esters are hydrolyzed to (S) -secondary alcohols by the Family VIII esterases and the Family VIII esterase variants of the present invention.

In the present invention, serine (S), the 283rd amino acid in the amino acid sequence of SEQ ID NO: 1, is aspartic acid (D), asparagine (N), valine (V), glutamic acid (E) , Glutamine (Q), Histidine (H), Leucine (L), Isoleucine (I), Methionine (M), Phenylalanine (F), Tyrosine (Y) and Provided is a variant of family VIII carboxylesterase characterized in that it is substituted with any one amino acid selected from the group of amino acids consisting of tryptophan (W).

The variant of the Family VIII carboxylesterase is characterized by producing (S) -secondary alcohol (sec-alcohol) using a chiral sec-alcohol ester as a substrate. The chiral secondary alcohol esters are (±)-2-butyl acetate, (±)-2-butyl butyrate, (±)-3-methyl-2-butyl acetate, (±)-3-methyl-2-butyl butyrate , (±)-2-pentyl acetate, (±)-2-pentyl butyrate, (±)-3-butyn-2-yl acetate, (±)-3-butyn-2-yl butyrate, (±)-1 -phenylethyl acetate, (±)-1-phenylethyl butyrate, (±)-1-(4-chlorophenyl)ethyl acetate, (±)-1-(4-chlorophenyl)ethyl butyrate, (±)-1-phenyl-2 Chiral secondary alcohol esters composed of -propyl acetate, (±)-1-phenyl-2-propyl butyrate, (±)-4-phenyl-2-butyl acetate, and (±)-4-phenyl-2-butyl butyrate Characterized in that it is any one of the groups.

The Family VIII carboxylesterase refers to a carboxylesterase (PBE) of Proteobacteria bacterium SG_bin9, and the PBE variant substitutes Ser381 of PBE with another amino acid (position-directed mutation) to the chiral secondary alcohol ester substrate. It is characterized in that it shows (S) -stereoselectivity and has an enantiomeric ratio (E) of 39 to 5500.

According to an embodiment of the present invention, in the case of the family VIII carboxylesterase without mutation, it has a low (S)-stereoselectivity with respect to the chiral secondary alcohol ester, so that the stereoselection ratio is only E = 1.1 to 36.1, and Since it has (R)-stereoselectivity or (S)-stereoselectivity depending on the substrate, there is a limitation in that high-purity (S)-secondary-alcohol cannot be prepared.

In contrast, the family VIII carboxylesterase variants of the present invention have high (S)-stereoselectivity with respect to the chiral secondary alcohol ester, so the enantiomeric ratio (E) is improved to 39 to 5500, so the purity is It is characterized by being able to produce high-purity (S) -secondary alcohol ranging from 90 to 99.9%.

Therefore, using the family VIII carboxylesterase variant of the present invention, photochemically pure (S) -secondary alcohol can be industrially mass-produced.

The present invention provides polynucleotides encoding variants of the family VIII carboxylesterases described above. The polynucleotide is a polymer of nucleotides in which nucleotide units are covalently linked in a long chain shape, and is a DNA or RNA strand of a certain length or more. More specifically, it refers to a polynucleotide fragment encoding the variant. .

The present invention provides a vector comprising a polynucleotide encoding a variant of the family VIII carboxylesterase described above. The vector refers to a DNA product containing a nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable control sequence so as to express the target polypeptide in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating termination of transcription and translation. After transformation into a suitable host cell, the vector can replicate or function independently of the host genome and can integrate into the genome itself. Vectors used in the present invention are not particularly limited, and any vectors known in the art may be used.

The present invention provides microorganisms that produce variants of Family VIII carboxylesterases, including any one or more of the above-described variants of Family VIII carboxylesterases, polynucleotides thereof, and vectors thereof.

The microorganism producing the variant means a microorganism endowed with the ability to produce variants of Family VIII carboxylesterase to a parent strain having no or weak ability to produce variants of Family VIII carboxylesterase. Specifically, the microorganism may be a microorganism expressing a variant of the family VIII carboxylesterase of the present invention. The type of microorganism producing the variant of the Family VIII carboxylesterase is not particularly limited as long as it can produce the Family VIII carboxylesterase or the variant of the Family VIII carboxylesterase, but recombinant microorganisms such as E. coli It may be a recombinant microorganism widely used for protein production, and in the case of yeast, specifically Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Neurospora kra Neurospora crassa) and the like.

The present invention is directed to culturing microorganisms producing variants of Family VIII carboxylesterases, including any one or more of the variants of the Family VIII carboxylesterases, polynucleotides thereof, and vectors containing the polynucleotides, in a medium. doing; And it provides a method for producing a variant of Family VIII carboxylesterase, comprising recovering the variant of Family VIII carboxylesterase from the cultured medium or microorganism.

The culturing means growing the microorganisms under appropriately controlled environmental conditions, and may be performed according to appropriate media and culture conditions known in the art. The culturing process can be easily adjusted and used by those skilled in the art according to the selected strain.

The medium means a material in which nutrients necessary for culturing the microorganisms are mixed as main components, and supplies nutrients and growth factors, including water essential for survival and growth. The medium controls the temperature, pH, etc. under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, phosphorous source, inorganic compound, amino acid and / or vitamin, etc. You can cultivate while doing it. Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; Amino acids such as glutamic acid, methionine, lysine, and the like may be included. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sorghum pomace and corn steep liquor can be used, specifically glucose and sterilized pretreated molasses (i.e. converted to reducing sugar). Carbohydrates such as molasses) may be used. Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, etc., organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or degradation products thereof, defatted soybean cake or degradation products thereof, etc. can be used The phosphorous source may include monopotassium phosphate, dipotassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and amino acids, vitamins, and/or appropriate precursors may be included. During cultivation of microorganisms, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner, and foaming can be inhibited by using antifoaming agents such as fatty acid polyglycol esters. there is. In addition, in order to maintain the aerobic state of the medium, oxygen or oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen or carbon dioxide gas may be injected without gas injection or to maintain an anaerobic and non-aerobic state. The temperature of the medium may be 20 °C to 50 °C, specifically 30 °C to 37 °C, and the culture period may be continued until a desired production amount of useful substances is obtained.

The variants of the Family VIII carboxylesterases of the present invention produced by the above culture may be released into the medium or remain in cells without being released. The method for producing the variant of the Family VIII carboxylesterase may include recovering the variant of the Family VIII carboxylesterase from the cultured microorganism or culture medium. The method for recovering the variant of Family VIII carboxylesterase produced in the culturing step of the present invention may use a suitable method known in the art according to the culturing method, for example, centrifugation, filtration, anion exchange chromatography Variants of family VIII carboxylesterases can be recovered from media or microorganisms using suitable methods known in the art, such as , crystallization and HPLC.

In addition, the recovery step may include a purification process, and may be performed using a suitable method known in the art, for example, filtration using a membrane.

For reference, the variant of family VIII carboxylesterase produced according to the method of the present invention can be directly used in the reaction as the enzyme itself, but it is immobilized enzyme by immobilizing it to an insoluble carrier (Matrix) by physical or chemical methods. It can be prepared and used in the form of The immobilized enzyme has the advantage of being able to be used repeatedly because it is easy to separate the reactant and the enzyme after the enzyme reaction is completed, and to increase the stability of the enzyme for reaction conditions). Techniques for fixing lipase enzymes, such as variants of Family VIII carboxylesterases of the present invention, to carriers such as acrylic resins (acrylic resins), fabric membranes, polypropylene, celite, and diatomaceous earth are widely known. The method of immobilizing the variant of VIII carboxylesterase and the selection of the carrier are not particularly limited, but the enzyme reaction rate is increased to increase the conversion rate in a short time. ) It is preferable to use it after being finely powdered to the size.

In the following, the present invention will be described in detail through examples.

Example

1. Experiment method

1) Experiment materials

Reagents used in the experiments were purchased from Sigma-Aldrich, Alfa Aesar, Acros and TCI. Pfu DNA polymerase (Polymerase) and T4 DNA ligase (Ligase) were purchased from Elpis-biotech (Daejeon, Korea). Restriction enzymes (NcoI and SalI) were purchased from New England Biolabs. Expression vector (pBADgIIIa) was purchased from Invitrogen Korea (Seoul, Korea). DNA oligomers and homologous genes were purchased from Bionics (Seoul, Korea). DNA sequencing was performed by Solgent Co. (Daejeon, Korea). CIRCLEGROW® broth was purchased from MP Biomedicals. Ni-NTA agarose resin was purchased from QIAGEN. Gas chromatography was performed using an Agilent 6890N equipped with a chiral capillary column (Cyclosil-B, 30m×0.25mm). 1H NMR analysis was performed on JNM-ECZ500R/S1 (500 MHz) using CDCl ₃ solvent. Chemical shift (Chemical shift) was expressed in ppm units using the peak of CDCl ₃ as an internal standard.

2) Protein expression vector

The protein sequence of PDB ID: 4IVK was compared with known protein sequences in the Protein Sequence Database (https://blast.ncbi.nlm.nih.gov). Eight protein sequences with a sequence identity higher than 64% were selected. Genes in which NcoI and SalI restriction sites were added to the protein sequence were synthesized by Bionics (Seoul, Korea). The synthesized gene was treated with NcoI and SalI, and then inserted into the pBADgIIIa vector to prepare a plasmid for expression. E. coli (Top 10) was transformed using the plasmid.

3) protein expression

The transformed E. coli (Top 10) was inoculated into CIRCLEGROW® medium (10 ml) containing 10 μl of 100 mg/ml ampicillin, and then cultured overnight at 37° C. and 200 rpm. 5 ml of the overnight culture medium was diluted in 500 ml of CIRCLEGROW® medium containing 500 μl of 100 mg/ml Ampicillin, and cultured at 37° C. and 200 rpm until the OD ₆₀₀ reached 0.5. Protein expression was induced by adding L-(+)-arabinose (500 μl of 2% w/v) to the culture medium, and further incubated for 20 hours at 20°C and 200 rpm, followed by 450°C at 4,500 rpm and 4°C. E. coli cells were pelleted by centrifugation for 1 minute, and an E. coli cell suspension was prepared using 20 ml of a lysozyme solution (1 mg/ml lysozyme, 5 mM BES buffer, pH 7.2). The E. coli cell suspension was incubated on ice for 45 minutes, then frozen and thawed, passed through a 20-gauge syringe needle to disrupt E. coli cells, and then centrifuged at 10,000 rpm and 4° C. for 30 minutes to obtain a solution The fraction and the insoluble fraction were separated. The insoluble fraction was dissolved in 20 ml of urea buffer (100 mM NaH ₂ PO4, 10 mM Tris-Cl, 8 M urea, pH 8.0), and the soluble fraction was Ni-NTA agarose resin (50% emulsion) in 20% ethanol) to prepare a solution fraction-Ni-NTA mixture, and mixed for 1 hour at 4 ℃. The solution fraction-Ni-NTA mixture was loaded and passed through a Poly-Prep chromatography column (Bio-Rad) to purify the protein. The buffer solution was flowed through the Poly-Prep chromatography column to wash the resin, and then 5 ml of dissolution buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole, pH 8.0) was flowed three times. Then, 5 ml of washing buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 20 mM imidazole, pH 8.0) was flowed three times, and then the elution buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 250 mM imidazole, pH 8.0) 5 ml was further flowed to elute the protein. The protein eluate was exchanged with BES buffer (5 mM, pH 7.2) using a centrifugal filter (Viva spin 20, GE healthcare), and SDS-PAGE analysis was performed on each fraction. Final protein concentration was quantified using the Bradford assay.

4) Preparation of Substrate

Secondary alcohol esters used as substrates in the present invention were purchased from Sigma-Aldrich or prepared through synthesis. The synthesis of secondary alcohol esters was prepared according to the following method. 20 mmol acetic anhydride or butyric anhydride was mixed with 30 mmol triethylamine and methylene chloride with 20 mg 4-dimetylaminopyridine (DMAP) and 10 mmol alcohol. chloride) solution was added to 50 ml to prepare a reaction mixture. The reaction mixture was stirred at 25° C. for 16 hours in a nitrogen gas atmosphere. Thereafter, the organic layer was prepared by washing twice with 50 ml of 1% hydrochloric acid solution and twice more with 50 ml of saturated NaHCO ₃ solution. The organic layer was dried with anhydrous MgSO ₄ (10 g) and the solvent was removed using a rotary evaporator. The compound was analyzed by NMR (Jeol, JNM-ECZ500R/S1). Table 1 shows the secondary alcohol ester substrates of the present invention.

name Formal name of substrate compound How to prepare Chemical shift of 1H NMR (500 MHz, CDCl ₃ ) Chemical shift of 13C NMR (125 MHz, CDCl ₃ ) (±)-1a (±)-2-butyl acetate synthesis δ 4.86-4.80 (m, 1H), 2.03 (s, 3H), 1.62-1.49 (m, 2H), 1.20 (d, 3H), 0.89 (t, 3H) δ 170.94, 72.30, 28.85, 21.45, 19.53, 9.75. (±)-1b (±)-2-butyl butyrate synthesis δ 4.87-4.81 (m, 1H), 2.25 (t, 2H), 1.67-1.62 (m, 2H), 1.58-1.49 (m, 2H), 1.19 (d, 3H), 0.94 (t, 3H), 0.89 (t, 3H) δ 173.54, 71.97, 36.71, 28.90, 19.59, 18.66, 13.73, 9.78. (±)-2a (±)-3-methyl-2-butyl acetate synthesis δ 4.74-4.69 (m, 1H), 2.03 (s, 3H), 1.80-1.73 (m, 1H), 1.15 (d, 3H), 0.90 (d, 6H) δ 170.93, 75.29, 32.69, 21.41, 18.13, 18.07, 16.75. (±)-2b (±)-3-methyl-2-butyl butyrate synthesis δ 4.76-4.71 (m, 1H), 2.26 (t, 2H), 1.74-1.78 (m, 1H), 1.66-1.63 (m, 2H), 1.15 (d, 3H), 0.94 (t, 3H), 0.89 (d, 6H); δ 173.48, 74.96, 36.75, 32.774, 18.68, 18.15, 18.10, 16.84, 13.75. (±)-3a (±)-2-pentyl acetate purchase - - (±)-3b (±)-2-pentyl butyrate purchase - - (±)-4a (±)-3-butyn-2-yl acetate synthesis δ 5.45-5.40 (m, 1H), 2.45 (d, 1H), 2.08 (s, 3H), 1.50 (d, 3H) δ 169.96, 82.20, 72.88, 60.11, 21.28, 21.10. (±)-4b (±)-3-butyn-2-yl butyrate synthesis δ 5.47-5.42 (m, 1H), 2.44 (d, 1H), 2.30 (t, 2H), 1.67-1.62 (m, 2H), 1.49 (d, 3H), 0.95 (t, 3H) δ172.58, 82.35, 72.76, 59.81, 36.20, 21.31, 18.45, 13.65. (±)-5a (±)-1-phenylethyl acetate purchase - - (±)-5b (±)-1-phenylethyl butyrate purchase - - (±)-6a (±)-1-(4-chlorophenyl)ethyl acetate synthesis δ 7.32-7.26 (m, 4H), 5.86-5.82 (m, 1H), 2.06 (s, 3H), 1.51 (d, 3H) δ170.33, 140.29, 133.70, 128.76, 127.61, 71.68, 22.23, 21.38. (±)-6b (±)-1-(4-chlorophenyl)ethyl butyrate synthesis δ 7.31-7.26 (dd, 4H), 5.86-5.82 (q, 1H), 2.30-2.27 (m, 2H), 1.67-1.60 (q, 2H), 1.50-1.49 (d, 3H), 0.93-0.90 ( t, 3H) δ 172.93, 140.47, 133.62, 128.73, 127.56, 71.39, 36.51, 22.30, 18.52, 13.71. (±)-7a (±)-1-phenyl-2-propyl acetate synthesis δ 7.30-7.18 (m, 5H), 5.14-5.08 (m, 1H), 2.95-2.73 (m, 2H), 2.00 (s, 3H), 1.22 (d, 3H) δ 170.67, 137.70, 129.50, 128.41, 126.55, 71.56, 2.32, 21.41, 19.53. (±)-7b (±)-1-phenyl-2-propyl butyrate synthesis δ 7.29-7.18 (m, 5H), 5.16-5.10 (m, 1H), 2.93-2.73 (m, 2H), 2.22 (t, 2H), 1.63-1.55 (m, 2H), 1.22 (d, 3H) , 0.88 (t, 3H) δ 173.26, 137.77, 129.50, 128.38, 126.50, 71.24, 42.39, 36.61, 19.63, 18.53, 13.66. (±)-8a (±)-4-phenyl-2-butyl acetate synthesis δ 7.30-7.16 (m, 5H), 4.96-4.92 (m, 1H), 2.71-2.58 (m, 2H), 2.03 (s, 3H), 1.94-1.81 (m, 2H), 1.25 (d, 3H) δ 170.89, 141.63, 128.51, 128.39, 126.00, 70.65, 37.67, 31.92, 21.44, 20.12. (±)-8b (±)-4-phenyl-2-butyl butyrate synthesis δ 7.28-7.15 (m, 5H), 4.96-4.92 (m, 1H), 2.41-2.44 (t, 2H), 2.24-2.27 (t, 2H), 1.95-1.88 (m, 2H), 1.72-1.65 ( m, 2H), 1.24-1.23 (d, 3H), 1.00-0.97 (t, 3H) δ 173.45, 141.69, 128.50, 128.40, 125.98, 70.29, 37.19, 36.67, 31.93, 20.17, 18.65, 13.78.

5) Measurement of hydrolysis activity for p-nitrophenyl acetate and p-nitrophenyl butyrate

20 μl of 200 mM p-nitrophenyl acetate or p-nitrophenyl butyrate solution (solvent: acetonitrile) and 870 μl of acetonitrile were mixed with 5 mM BES buffer solution (pH 7.2) 11,110 To prepare an assay solution, 5 μl of a 0.1 mg/ml enzyme solution was added to 100 μl of the assay solution, and the absorbance was measured at 404 nm for 5 seconds for 10 minutes.

6) Measurement of stereoselection ratio (E)

Reaction containing 100 μl of 1M ester substrate solution (solvent: acetonitrile), 600 μl of acetonitrile and 0.1 mg/ml enzyme solution (solvent: 5 mM BES, pH 7.2) in 9.2 ml of BES buffer (100 mM, pH7.2) A mixture was prepared. The reaction mixture was stirred at 500 rpm at 25 °C. 100 μl of the stirred reaction mixture was taken at about 40% conversion and extracted with 1 ml of methyl t-butyl ether. After drying the organic layer with anhydrous Na ₂ SO ₄ , it was analyzed by gas chromatography (GC) equipped with a chiral column. GC analysis conditions for quantification of the substrate and reactant are shown in Table 2 below. The retention time of the enantiomer was confirmed by comparison with the retention time of the enantiomer sample.

7) Securing homology model of carboxyl esterase (PBE) of Proteobacteria bacterium SG_bin9

Amino acid sequences were deposited in the automatic mode of SWISS-MODEL (http://swissmodel.expasy.org/) to secure a homology model of carboxylesterase (PBE) of Proteobacteria bacterium SG_bin9.

8) Molecular Modeling

Molecular modeling was performed using the SYBYL-X 2.11 package. Coordinates for water molecules were adopted from the crystal structure of family VIII carboxylesterases (PDB ID: 4IVK). Some water molecules (H2O601, H2O626 and H2O927) were removed to avoid steric collisions in the model. The tetrahedral intermediate was manually generated based on the inhibitor contained in the crystal structure. Atomic types and partial charges of enzymes and substrates are specified by default settings in the AMBER99 Force field. The generic AMBER99 force field was used to define the missing atom type and parameters of the tetrahedral intermediate. A non-junction cut-off distance of 8 Å and a distance-dependent dielectric function were used for all calculations. Energy minimization was performed by the Powell method until the rms derivative was less than 0.005 kcal·mol ^-1 ·Å ^-1 . The structure of the model was expressed through PYMOL (http://www.pymol.org).

9) PBE site-directed mutations

Mutant genes were prepared by performing overlap extension PCR with the primers shown in Table 3 below. The mutant gene prepared through PCR was treated with restriction enzymes NcoI and SalI, and then inserted into the pBADgIIIa vector treated with the same restriction enzymes to prepare a plasmid, and the plasmid was transformed into E. coli (Top 10).

2. Experiment results and discussion

In the present invention, a new family VIII esterase was discovered and presented. To this end, a homologous search was performed using the protein sequence of family VIII esterase (PDB ID: 4IVK) whose three-dimensional structure was already known in the protein database. Figure 1 shows the homology search results, and Table 4 shows that the amino acid sequence identity among the homology search results is 60% or more.

When the amino acid sequence identity is 50% or more, it is known to have an almost identical three-dimensional structure. After optimizing the gene codons of the 8 selected sequences, they were inserted into the pBADgIIIa expression vector to transform E. coli (Top 10). Protein expression using E. coli (Top 10) was performed by adding L-arabinose to the cultured E. coli (Top 10). It was confirmed that only five genes out of eight genes were successfully expressed, and among them, Proteobacteria bacterium carboxylesterase (Proteobacteria bacterium SG_bin9, PBE) was confirmed to have the highest yield (see Fig. 2 and Table 5) .

The hydrolytic activity of p-nitrophenyl acetate (pNPAc) and p-nitrophenyl butyrate (pNPBu) was measured for the carboxylesterases of the five genes successfully expressed. did The hydrolytic activity was determined by measuring the absorbance change at 404 nm for 15 minutes at 25° C. to calculate the reaction rate. As a result of the experiment, it was confirmed that all five enzymes showed higher activity against pNPBu than pNPAc, and in the case of PBE, it was confirmed that it showed high activity against both pNPAc and pNPBu (see Table 5). As shown in the above results, it was confirmed that proteobacteria bacterium carboxylesterase (Proteobacteria bacterium SG_bin9, PBE) was expressed in the highest yield and also showed the highest activity. Therefore, in the present invention, dynamic resolution was analyzed for chiral secondary-alcohols using only the PBE.

The stereoselectivity of PBE was determined through the enantiomeric ratio (E) of hydrolysis of a series of secondary alcohol esters including but-3-yn-2-yl ester. The but-3-yn-2-yl ester is a useful building block for the synthesis of bioactive compounds, and is known as a substrate for which it is difficult to separate enantiomers using enzyme-based dynamic resolution. Figure 3 shows a reaction in which secondary alcohol esters are hydrolyzed by PBE to (S) -secondary alcohols. Table 6 shows the results of analyzing the enantiomeric selectivity of the PBE.

As a result of the analysis, it was confirmed that the PBE showed low or medium stereoselectivity (stereoselectivity ratio E = 1.1 to 10.1) for most secondary alcohol esters except for 1-phenylethyl alcohol ester (E = 9.9 or 36.1).

Interestingly, the enantiomeric preference of PBE was confirmed to vary depending on the substituent. For example, PBE is a secondary alcohol ester with a branched alkyl chain (substrate 4a), a secondary alcohol ester with an acetylene substituent (substrate 4b) and a secondary alcohol ester with a phenyl substituent (substrate 5a, substrate 5b). and (S)-stereoselectivity for substrate 6a), and (R)-stereoselectivity for substrate 1a, substrate 3a, substrate 3b, substrate 7a and substrate 8a having a methylene group in the substituent was confirmed to show Additionally, the acyl group has been found to influence the enantiomeric preference of PBE, for example it tends to have (S)-stereoselectivity for reactants with longer acyl butyl groups. The PBE is found to exhibit (S)-stereoselectivity towards certain substrates. However, since the stereoselectivity of PBE is not high enough, it is judged that industrial application of high-purity (S) -secondary alcohol production using dynamic resolution will be difficult.

In order to improve the stereoselectivity of PBE, molecular modeling was performed for the reaction of PBE with (S)-1-phenylethyl acetate or (R)-1-phenylethyl acetate as a substrate (entry 9 in Table 6). First, the homologous structure of the PBE model was prepared using the SWISS-Model website. In addition, more accurate energy minimization was performed by adding water molecules to the homologous structure using the coordinates of water molecules included in the known protein crystal structure (PDB code: 4IVK). Since the 4IVK crystal structure contains the inhibitor cephalothin, the tetrahedral intermediate of (S)- and (R)-1-phenylethyl acetate based on the cephalothin included in the 4IVK structure was generated and energy minimization and analysis were performed (see FIG. 4 and Table 7)

It is believed that the catalytic reaction by family VIII carboxylesterase occurs in acylation and deacylation steps similar to conventional lipase catalysis (FIG. 5). According to the known crystal structure (PBD ID: 4IVK), the catalyst serine attacks the carbonyl carbon to form an acyl-enzyme intermediate, and in this process, the carbonyl oxygen of the open β-lactam ring is Ser100 (catalytic serine) and It is confirmed that hydrogen bonds to the oxyanion hole composed of Ala384. Ser100 (catalyst serine) and Ala384 of the known crystal structure (PBD ID: 4IVK) correspond to Ser98 and Ala383 of the PBE. However, the reaction mechanism of family VIII carboxylesterases is not fully understood because the catalytic triresidues are not identical to those of conventional lipases or serine proteases.

The molecular modeling shows hydrogen bonding between the catalytic triresidues (Ser98-Lys101-Tyr216) and between the oxyanion and the oxyanion hole composed of Ser98 and Ala383. The above results explain the role of amino acids constituting the catalytic triresidue (see panel a of FIG. 4). In particular, the catalyst Lys accepts a Ser proton and increases the hydroxyl group nucleophilicity of the latter. Lys in the above molecular model is the equivalent of the catalyst His among three standard Ser-His-Asp functional groups of lipase and serine hydrolase. However, the Lys cannot transfer a proton from Ser to a leaving group corresponding to the alcohol of the ester substrate. This is because the distance between the Nε atom of Lys and the O atom of alcohol is 4.4 Å, which is outside the normal hydrogen bond distance (see Table 7). However, in order for the reaction to proceed, the proton of the catalyst Ser must be transferred to the leaving group (oxygen atom of the alcohol moiety) in the same manner as His. According to the molecular modeling results, the catalytic Tyr is judged to act as a proton bridge. The hydrogen of Tyr also bonds with the catalyst Lys and the oxygen of the alcohol moiety. Thus, Tyr accepts a proton from the catalytic Lys and donates a proton to the alcohol moiety. The mechanism described above is supported by the results of site-directed mutagenesis experiments on family VIII carboxylesterases. According to the experimental results, it was confirmed that when a mutation occurs in the three catalytic residues including the catalytic Tyr, the activity of family VIII carboxylesterase is rapidly reduced.

According to the molecular modeling results, the methyl substituents of the two enantiomers bind to different pockets, whereas the larger substituent (phenyl group) is located in the same entrance pocket. It is also confirmed that the methyl substituent of the fast-reacting enantiomer binds to the pocket composed of Leu349, Trp380, Ser381 and Thr409, whereas the methyl substituent of the slow-reacting enantiomer binds to the pocket composed of Ser381, Ala383 and Arg408 (Fig. see panels b) and c) of 4). The size of the binding pocket of the slow-reacting enantiomer's methyl group is believed to be smaller than that of the fast-reacting enantiomer, indicating that the slow-reacting enantiomer is less likely to bind than the fast-reacting enantiomer. . The difference between the two pocket sizes is believed to explain the (S)-stereoselectivity of PBE for substrate 5a.

In the present invention, it was determined that the enantioselectivity of PBE would be improved if the binding of the methyl substituent of the slowly reacting enantiomer was further hindered. Among the residues in the binding pocket for the slow-reacting enantiomer, the side chains of Ser381 and Arg408 were confirmed to face the active site. These results indicate that the enantiomeric selectivity of PBE will change when two residues are substituted.

In the present invention, Ser381 was selected from two residues of PBE and its variants were generated through site-directed mutagenesis. The volume of Ser is 94.1 Å ³ , which is smaller than that of Arg (192.8 Å ³ ). Therefore, it was determined that the size of the binding pocket could be changed more effectively when the Ser was substituted with an amino acid having a larger bulk. First, the Ser381 was substituted with Leu (volume = 164.6Å ³ ) and Ile (volume = 164.9Å ³ ) (see Table 8). The reason for selecting Leu and Ile is that they have a volume larger than Ser and have a volume value intermediate among amino acids having a volume larger than Ser. In addition, Leu and Ile have amino acid side chains that do not have polar functional groups and do not carry electric charges. It was judged that it would not affect other characteristics other than the size of the binding pocket. The mutant gene was prepared through overlapping extension polymerase chain reaction, inserted into pBADgIIIa, transformed into E. coli (Top10), and expressed as a protein. The expressed PBE mutant protein was purified using Ni-NTA resin and then hydrolyzed with substrate 5a. And the reaction solution was analyzed by gas chromatography to analyze the enantioselectivity of the PBE variant.

As a result of the analysis, in the case of PBE (wild type PBE) without mutation, the stereoselection ratio (E) was confirmed to be E = 36.1, but in the case of PBE S381L and PBE S381I, which are variants of PBE, the stereoselection ratio (E) was E = 515 and 515 respectively. 212, a significant increase was confirmed. In addition, in the case of PBE S381A in which Ser381 is substituted with Ala (side chain volume = 90.1Å ³ ) having the same side chain characteristics as the Leu and Ile but having the smallest volume, the stereoselection ratio (E) is E = 18.4, which is higher than that of wild type PBE. found to be lowered. This result means that the increase in the size of the binding pocket and the stereoselectivity for substrate 5a are inversely proportional to each other (see Table 8).

PBE variants using other bulky amino acids than Ser were additionally prepared, and their stereoselectivity was analyzed (see entries 2-8 and 11-16 in Table 8). Among the PBE variants in Table 8, the stereoselectivity (stereoselection ratio, E) of all PBE variants except for PBE S381P, PBE S381K, and PBE S381R and PBE S381A, in which the reaction was too slow to calculate the stereoselection ratio, was found to increase. Confirmed. It is believed that the reason why the reaction among the variants was too slow to calculate the stereoselection ratio was due to a structural change due to Pro substitution or a rapid change in catalytic mechanism due to substitution of a positively charged amino acid.

It is believed that the improvement in stereoselectivity of the PBE variant is achieved by hindering the intraenzyme binding of the (S)-enantiomer. PBE S381F is confirmed to show the highest enantioselectivity (E = 882), and PBE S381Y and PBE S381W are also confirmed to show high stereoselectivity due to their large volume. Figure 6 shows the activation energy difference (ΔΔG‡=-RTlnE) between enantiomers according to the volume change of the PBE variant of the present invention. According to FIG. 6, it was confirmed that the change in the activation energy difference according to the volume of the substituted amino acid showed the aspect of a saturation curve. This result means that enantioselectivity also increases when the volume of substituted amino acids increases.

In order to further expand the substrate range, the enantioselectivity of PBE S381F, PBE S381Y, and PBE S381W was analyzed. As a result of the analysis, it was confirmed that all three variants showed stereoselectivity related to the hydrolysis of the secondary alcohol ester series (see Table 9).

In particular, the PBE S381F, PBE S381Y, and PBE S381W were confirmed to exhibit high (S)-stereoselectivity (E=162 to 5,450) with respect to substrate 1a, substrate 3a, substrate 7a, and substrate 8a. This is in contrast to wild type PBE showing low (R)-stereoselectivity (E = 1.1 to 4.8).

The PBE S381F was found to exhibit high stereoselectivity for substrates other than substrate 2a (E = 83) and substrate 7a (E = 167), and the PBE S381W showed high stereoselectivity to substrate 2a (E = 345) and substrate 7a (E = 167). 459) is confirmed to show higher stereoselectivity. Substrates 1a and 4a are known as difficult-to-resolve substrates for enantiomeric separation by dynamic resolution. For example, in the case of Candida antarctica lipase B (CAL-B), which is one of the lipases widely used for kinetic resolution of secondary alcohol, for substrate 1a and substrate 4a (R ) -selectivity was confirmed to show low stereoselectivity. In contrast, PBE S381F was confirmed to exhibit high stereoselectivity (E = 162 for substrate 1a and E = 2,001 for substrate 4a) with (S) -selectivity for substrate 1a and substrate 4a, which are difficult to separate.

Therefore, the PBE variant of the present invention is considered to be applicable to preparing high-purity (S) -secondary alcohol from various secondary-alcohol ester substrates.

3. Conclusion

Conventionally, only lipases having (R)-stereoselectivity for secondary alcohol substrates have been widely used, and hydrolases with high (S)-stereoselectivity and broad substrate specificity have been rarely reported, and their dynamic resolution Also, nothing is known about its use.

In the present invention, a novel (S)-stereoselective family VIII carboxylesterase (carboxylesterase from Proteobacteria bacterium SG_bin9, PBE) was reported, and a new family VIII carboxylesterase with high (S)-stereoselectivity was analyzed through variant analysis based on molecular modeling. esterases have been reported.

In the present invention, the substrate binding pocket was confirmed through molecular modeling, and it was experimentally confirmed that the enantioselectivity of PBE changes when amino acids in the binding pocket are substituted. Specifically, it was experimentally confirmed that stereoselectivity for various chiral secondary alcohol esters is improved when S381 among the binding pocket residues is substituted with a bulky amino acid.

The above result was supported by the results showing a saturation curve as a result of graphically analyzing the difference in activation energy of the enantiomers and the volume of the introduced amino acid, and the binding pocket for the methyl substituent of the slow enantiomer must have a limited space, which is This means that the enhanced stereoselectivity with the reduction should reach a maximum value. Therefore, it is believed that the enhancement of enantioselectivity of the PBE variants of the present invention is due to the effect of the size of the substituted amino acid. In conclusion, it is believed that using the PBE variant of the present invention, it is possible to prepare high-purity (S) -secondary alcohol from various chiral secondary-alcohol ester substrates.

The specific embodiments described in this specification are meant to represent preferred embodiments or examples of the present invention, and the scope of the present invention is not limited thereby. It will be apparent to those skilled in the art that variations and other uses of the present invention do not depart from the scope of the invention described in the claims.

<110> SUNGSHIN R&DB FOUNDATION <120> Family VIII Carbixylesterases For High Purity (S)-Sec-Alcohol Preparation <130> MP21-0209 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 426 <212> PRT <213> unknown <220> <223> Proteobacteria bacterium SG_bin9 <400> 1 Met Lys Thr Ser Ala Lys Phe Leu Ser Phe Ala Val Ser Phe Val Leu 1 5 10 15 Leu Ile Ile Ala Ser Thr Ser Phe Ala Glu Gly Pro Val Thr Ala Thr 20 25 30 Lys Pro Lys Glu Ala Gly Phe Thr Ser Glu Gly Leu Ala Arg Ile Asp 35 40 45 Ala Tyr Leu Lys Asn Glu Ile Gln Ala Lys Thr Met Pro Gly Ala Val 50 55 60 Met Met Ile Lys Arg Asn Gly Glu Thr Ala Tyr Phe Ser Ser Phe Gly 65 70 75 80 Leu Arg Asp Pro Asp Thr Lys Glu Pro Met Thr Ala Glu Thr Ile Phe 85 90 95 Arg Ile Tyr Ser Met Ser Lys Pro Ile Thr Thr Val Ala Ala Met Met 100 105 110 Leu Val Glu Glu Gly Lys Leu Gln Leu Asp Glu Pro Val Ser Lys Tyr 115 120 125 Ile Pro Ser Phe Ala Asn Val Lys Val Gly Val Glu Thr Lys Gly Glu 130 135 140 Asn Gly Met Ala Leu Glu Thr Gly Pro Val Lys Arg Ala Ile Thr Ile 145 150 155 160 Gln Asp Leu Met Arg His Thr Ser Gly Ile Thr Tyr Gly Phe Val Gly 165 170 175 Asp Gly Leu Val Lys Lys Ala Tyr Ile Ala Ser Asn Leu Phe Asp Gly 180 185 190 Asp Phe Asp Asn Ala Glu Phe Ala Glu Arg Ile Ala Lys Leu Pro Leu 195 200 205 Val Tyr Gln Pro Gly Thr Thr Trp Asp Tyr Gly His Ser Thr Asp Ile 210 215 220 Leu Gly Arg Val Val Glu Val Val Ser Gly Lys Ser Leu Tyr Gln Phe 225 230 235 240 Glu Lys Glu Arg Leu Leu Asp Pro Leu Gly Met Lys Asp Thr Gly Phe 245 250 255 Tyr Val Thr Asp Pro Ala Lys Lys Ser Leu Val Ala Glu Ala Met Pro 260 265 270 Asn Asp Arg Lys Ile Gly Gly Ser Glu Met Phe Asp Pro Arg Val Gln 275 280 285 Lys Lys Trp Glu Pro Gly Gly Gln Gly Met Val Ser Thr Ile Gly Asp 290 295 300 Tyr Ala Arg Phe Thr Gln Met Val Leu Asn Gly Gly Thr Leu Asp Gly 305 310 315 320 Lys Arg Tyr Leu Ser Pro Lys Thr Ile Ala Tyr Met Gly Ser Asn His 325 330 335 Ile Pro Gln Ala Ser Gly Ile Val Pro Gly Ala Tyr Tyr Leu Pro Gly 340 345 350 Pro Gly Val Gly Phe Gly Leu Gly Phe Ala Val Arg Thr Glu Ala Gly 355 360 365 Val Thr Pro Val Glu Gly Ser Val Gly Asp Leu Ser Trp Gly Gly Ala 370 375 380 Gly Gly Thr Val Phe Trp Ile Asp Pro Lys Glu Asn Leu Thr Val Val 385 390 395 400 Phe Met Ala Pro Met Val Ser Pro Arg Ala Arg Val Trp Arg Thr Leu 405 410 415 Arg Asn Ile Val Tyr Gly Ala Phe Asp Arg 420 425

Claims (9)

Serine (S), the 283rd amino acid in the amino acid sequence of SEQ ID NO: 1, is aspartic acid (D), asparagine (N), valine (V), glutamic acid (E), glutamine ( Glutamine (Q), Histidine (H), Leucine (L), Isoleucine (I), Methionine (M), Phenylalanine (F), Tyrosine (Y) and Tryptophan , W) is a variant of family VIII carboxylesterase, characterized in that substituted with any one amino acid selected from the group consisting of amino acids.
The variant of claim 1, wherein the family VIII carboxylesterase variant uses a chiral secondary alcohol ester (sec-alcohol ester) as a substrate to produce (S) -secondary alcohol (sec-alcohol). A variant of a family VIII carboxylesterase that does.
The method of claim 2, wherein the chiral secondary alcohol ester is (±)-2-butyl acetate, (±)-2-butyl butyrate, (±)-3-methyl-2-butyl acetate, (±)-3-butyl acetate, methyl-2-butyl butyrate, (±)-2-pentyl acetate, (±)-2-pentyl butyrate, (±)-3-butyn-2-yl acetate, (±)-3-butyn-2-yl butyrate , (±)-1-phenylethyl acetate, (±)-1-phenylethyl butyrate, (±)-1-(4-chlorophenyl)ethyl acetate, (±)-1-(4-chlorophenyl)ethyl butyrate, (±) -1-phenyl-2-propyl acetate, (±)-1-phenyl-2-propyl butyrate, (±)-4-phenyl-2-butyl acetate and (±)-4-phenyl-2-butyl butyrate A variant of the family VIII carboxylesterase, characterized in that it is any one of the chiral secondary alcohol ester group.
The method of claim 2, wherein the variant of the family VIII carboxylesterase shows (S)-stereoselectivity for the chiral secondary alcohol ester substrate and an enantiomeric ratio (E) of 39 to 5500. A variant of the family VIII carboxylesterase.
The method of claim 2, wherein the (S) -secondary alcohol prepared by hydrolyzing the chiral secondary alcohol ester substrate with the variant of the family VIII carboxylesterase has a purity of 90 to 99.9%. Variants of family VIII carboxylesterases.
A polynucleotide encoding a variant of the family VIII carboxylesterase of any one of claims 1 to 5.
A vector comprising the polynucleotide of claim 6.
A microorganism producing a variant of Family VIII carboxylesterase, including any one or more of the variant of any one of claims 1 to 5, the polynucleotide of claim 6, and the vector of claim 7. .
A microorganism producing a variant of Family VIII carboxylesterase, including any one or more of the variant of any one of claims 1 to 5, the polynucleotide of claim 6, and the vector of claim 7. culturing in a medium; and
A method for producing a variant of Family VIII carboxylesterase comprising the step of recovering the variant of Family VIII carboxylesterase from the cultured medium or microorganism.