Novel esterase variants and their use as stereoselective catalysts

Abstract

The present invention relates to the field of enzyme variants, and in particular, to Arthrobacter globiformis esterase variants suitable for the asymmetric hydrolysis of cyclopropane derivatives, specifically the pyrethric acid esters of any combination of four stereoisomers, for selectively obtaining (1R,3R)-pyrethric acid or a salt thereof. The present invention further describes an expression vector containing an Arthrobacter globiformis esterase variant, and a transformed host microorganism encapsulating the vector. The present invention further relates to the use of an Arthrobacter globiformis esterase variant for selectively obtaining (1R,3R)-pyrethric acid. In a further aspect, the present invention relates to the use of an Arthrobacter globiformis esterase variant for the asymmetric hydrolysis of a racemic cyclopropane derivative having at least two chiral stereocenters, for the asymmetric hydrolysis of a racemic cyclopropane derivative having at least two chiral stereocenters.

Description

Technical Field

[0001] The present invention relates to the field of enzyme mutants, and in particular, to an Arthrobacter globiformis esterase mutant suitable for asymmetric hydrolysis of cyclopropane derivatives, specifically pyrethric acid esters of any combination of four stereoisomers, in order to selectively obtain (1R,3R)-pyrethric acid or a salt thereof.

[0002] The present invention further describes an expression vector containing an Arthrobacter globiformis esterase mutant, and a transformed host microorganism containing the vector.

[0003] The present invention further relates to the use of an Arthrobacter globiformis esterase mutant for selectively obtaining (1R,3R)-pyrethric acid. In a further aspect, the present invention relates to the use of an Arthrobacter globiformis esterase mutant for the asymmetric hydrolysis of a racemic cyclopropane derivative having at least two chiral stereocenters, preferably for the asymmetric hydrolysis of a racemic cyclopropane derivative having at least two chiral stereocenters.

Background Art

[0004] Tanacetum cinerariifolium or Dalmatian chrysanthemum is a white-flowering plant of the Asteraceae family, formerly a member of the Pyrethrum genus but now classified in the Chrysanthemum genus.

[0005] This plant is of great economic importance as it is the natural source of the "pyrethrum" insecticide. The flowers are crushed and the active ingredient called pyrethrin, contained in the seed case, is sold in the form of an oleoresin that can be applied as a suspension in water or oil, or as a powder.

[0006] Pyrethrin is a mixture of six structurally related insecticidal esters formed by the combination of two acids (chrysanthemic acid and pyrethric acid) and various alcohols. The esters of chrysanthemic acid are called pyrethrin I, cinerin I, and jasmolin I respectively, and are collectively known as pyrethrin I, while the esters of pyrethric acid are called pyrethrin II, cinerin II, and jasmolin II, and are collectively known as pyrethrin II. Pyrethrins attack the nervous systems of all insects, preventing female mosquitoes from biting. They seem to have an insect repellent effect even when present in amounts less than a lethal dose for insects.

[0007] Pyrethrins are harmful to fish but are much less toxic to mammals and birds compared to many synthetic insecticides, are not persistent, are biodegradable, and decompose easily when exposed to light. They are considered to be one of the safest insecticides for use around food.

[0008] After the chemical structure of "natural pyrethrin" was elucidated, useful synthetic pyrethroids with various characteristics were developed, leading to the advancement of pyrethroid chemistry.

[0009] Chrysanthemic acid is a special intermediate related to various natural and synthetic insecticides. In particular, pyrethroids are derivatives of chrysanthemic acid that can be found in four different possible stereoisomers, namely (1R,3R) or (1R,3S) or (1S,3R) or (1S,3S).

[0010] (1R,3R)- or (+)-trans-chrysanthemic acid (one of four stereoisomers) is generally considered to be the most interesting and effective among the four stereoisomers.

Chem.

[0011] Specifically, as also reported by Nishizawa et al. ("Stereoselective Production of (+)-trans-Chrysanthemic Acid by a Microbial Esterase: Cloning, Nucleotide Sequence, and Overexpression of the Esterase Gene of Arthrobacter globiformis in Escherichia coli", Applied and Environmental Microbiology, 1995, 61, pp. 3208-3215), the production of highly stereoselective (+)-trans-chrysanthemic acid by enzymes would have a great advantage over conventional chemical processes in selectively obtaining the target isomer. The esterase gene of Arthrobacter globiformis has been characterized and reported to be highly stereospecific but difficult to use for large-scale production of pesticides due to product inhibition.

[0012] Therefore, an object of the present invention is the development of stereoselective hydrolysis of chrysanthemic acid esters.

[0013] It is known that chrysanthemic acid can be obtained by hydrolysis of a suitable chrysanthemic acid ester, especially racemic ethyl chrysanthemate.

[0014] Ethyl chrysanthemate (or related esters) can be found as the following four isomers.

Chem.

[0015] The mixture of stereoisomers is a starting material for producing chrysanthemic acid.

[0016] Accordingly, a further object of the present invention is to selectively produce (1R,3R) chrysanthemic acid from the racemate of ethyl chrysanthemate. [Summary of the Invention]

[0017] The inventors have surprisingly found a new esterase that can selectively hydrolyze the ester of racemic chrysanthemic acid to selectively produce (1R,3R) chrysanthemic acid or a salt thereof.

[0018] Accordingly, the present invention relates to an Arthrobacter globiformis esterase mutant having a sequence containing a mutation of amino acid residue S at position 315, amino acid residue S at position 223, or amino acid residue F at position 298 of SEQ ID NO: 2.

[0019] In a second aspect, the present invention relates to an expression vector containing an Arthrobacter globiformis esterase mutant nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19.

[0020] The third aspect of the present invention relates to a transformed host microorganism containing the above-described expression vector, and the vector contains an Arthrobacter globiformis esterase mutant nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19.

[0021] In a fourth aspect, the present invention relates to the use of an Arthrobacter globiformis esterase mutant according to the present invention for the selective preparation of (1R,3R)-chrysanthemic acid.

[0022] In a preferred aspect, the present invention relates to an Arthrobacter globiformis esterase mutant capable of asymmetric hydrolysis of a racemic cyclopropane derivative having at least two chiral stereocenters.

[0023] Accordingly, in a fifth aspect, the present invention relates to the use of an Arthrobacter globiformis esterase mutant for the asymmetric hydrolysis of a racemic cyclopropane derivative having at least two chiral stereocenters.

[0024] In a preferred and advantageous aspect, the present invention relates to an Arthrobacter globiformis esterase mutant capable of asymmetric hydrolysis of (C1-C6) alkyl chrysanthemate, preferably ethyl chrysanthemate.

[0025] Accordingly, in a sixth aspect, the present invention relates to the use of an Arthrobacter globiformis esterase mutant for the asymmetric hydrolysis of (C1-C6) alkyl chrysanthemate, preferably ethyl chrysanthemate.

[0026] As will be further described in the detailed description of the present invention, the solution underlying the present invention enables the use of an innovative esterase with improved properties in obtaining the desired (1R,3R) chrysanthemic acid for the production of pyrethroid insecticides.

[0027] The problem of providing (1R,3R)-chrysanthemic acid is solved by the finding of the present invention, in particular, by identifying mutants of Arthrobacter globiformis esterase having improved enzyme kinetics, improved enzyme stability, and / or reduced product inhibition when compared to the wild-type Arthrobacter globiformis esterase of SEQ ID NO: 2, as specified in the appended claims.

[0028] The features and advantages of the present invention will become apparent from the detailed description reported below, the examples given for illustrative and non-limiting purposes, and the appended FIGS. 1 to 10.

Brief Description of the Drawings

[0029]

Figure 1

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Figure 8

Figure 9

Mode for Carrying Out the Invention

[0030] Therefore, the present invention relates to an Arthrobacter globiformis esterase variant having a sequence containing a mutation of the amino acid residue S at position 315, the amino acid residue S at position 223, or the amino acid residue F at position 298 of SEQ ID NO: 2.

[0031] Although not bound by any theory, the inventors believe that the Arthrobacter globiformis esterase variant described in the claims enables higher yields to be obtained in the following reaction: the selective conversion of cyclopropane derivatives having at least two chiral centers, preferably racemic chrysanthemic acid (C1-C6) alkyl esters, more preferably the ethyl ester of racemic chrysanthemic acid, to (1R,3R)-chrysanthemic acid or a salt thereof: [Chemical formula] This is thought to be due to its influence on the following three aspects of catalysis. i) Improved enzyme stability at different temperatures and pH values: The enzyme has good operational stability under basic conditions in the pH range of 9.0 - 11.00. The maximum conversion yield is also obtained in this range, preferably in the range of 10.0 - 10.5, and significantly decreases below 8.5 - 9.0. ii) Improved kinetic parameters, and iii) Reduction of product inhibition when compared to the wild-type Arthrobacter globiformis esterase having the amino acid sequence set forth in SEQ ID NO: 2. The described variants have undergone changes such that their catalytic performance is improved. During the reaction, in a pH-stat, a maximum conversion yield of 88 - 95% was achieved compared to the conversion yield (a value not exceeding 40 - 49%) achievable with the wild-type enzyme having the amino acid sequence reported in SEQ ID NO: 2. These mutations increased the affinity for the substrate without changing the high stereospecificity of the enzyme and reduced the product inhibition that was apparent in the wild-type enzyme.

[0032] For the purposes of the present invention, the term - "(1R,3R)-chrysanthemic acid" as used herein is intended to be synonymous with "(1R)-trans-chrysanthemic acid", "(+)-trans-chrysanthemic acid", and "(1R,3R)-2,2-dimethyl-3-(2-methyl-1-propenyl)cyclopropane-1-carboxylic acid" and to have the CAS number 4638-92-0 - "(1R,3R)-chrysanthemic acid or its salt" means a salt of a metal selected from alkali metals and alkaline earth metals such as potassium, sodium, calcium, magnesium, and others, - "a cyclopropane derivative having at least two chiral centers" means a cyclopropane compound having at least two asymmetric carbon atoms such that it has at least two stereoisomers, preferably four stereoisomers, - "(C1-C6) alkyl ester of chrysanthemic acid" means a linear or branched ester of chrysanthemic acid having 1 to 6 carbon atoms with an alkyl group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-pentyl, hexyl, and others, - "ethyl ester of chrysanthemic acid", as used herein, is synonymous with "ethyl 2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropane-1-carboxylate" and is intended to have the CAS number 97-41-6. According to the present invention, the ethyl ester of chrysanthemic acid can be provided in any ratio of trans / cis isomers, such as 60 / 40 to 100 / 0, preferably 65 / 35, 70 / 30, 80 / 20, 90 / 10, 92 / 8, 98 / 2.

[0033] In a preferred embodiment, in the Arthrobacter globiformis esterase mutant according to the present invention, the amino acid residue S at position 315, the amino acid residue S at position 223, and the amino acid residue F at position 298 of SEQ ID NO: 2 are substituted with non-polar amino acid residues selected from the group consisting of M, F, L, W, A, I, P, or V. Surprisingly, by substituting the amino acid residues present at positions 315, 223, or 298 of SEQ ID NO: 2 with non-polar amino acids, the present inventors have found that the characteristics of the esterase enzyme are improved and the ability to asymmetrically hydrolyze the ester of racemic chrysanthemic acid is maintained.

[0034] For the purposes of the present invention, amino acid residues are indicated by the "one-letter code", where "M" corresponds to Met, i.e., methionine, "F" corresponds to Phe, i.e., phenylalanine, "S" corresponds to Ser, i.e., serine, etc., as will be understood by those skilled in the art.

[0035] In a more preferred embodiment, in the Arthrobacter globiformis esterase variant of the present invention, the amino acid residue S at position 315, the amino acid residue S at position 223, and the amino acid residue F at position 298 of SEQ ID NO: 2 are replaced with non-polar amino acid residues selected from the group consisting of M, F, L, or W.

[0036] In an even more preferred embodiment, the Arthrobacter globiformis esterase variant of the present invention has one of the mutations S315M, S315F, S223M, S223L, S223F, or F298W in the amino acid sequence of SEQ ID NO: 2.

[0037] In an even more preferred embodiment, the Arthrobacter globiformis esterase variant of the present invention has one additional mutation (double mutant) or two additional mutations (triple mutant), preferably - a double mutant having the mutations S315M and S223M in the amino acid sequence of SEQ ID NO: 2, - a double mutant having the mutations S315F and S223M in the amino acid sequence of SEQ ID NO: 2, and - a triple mutant having the mutations S315M, V274L, and S331C in the amino acid sequence of SEQ ID NO: 2 selected from the group consisting of.

[0038] A preferred Arthrobacter globiformis esterase mutant has an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20.

[0039] More preferably, the Arthrobacter globiformis esterase mutant according to the present invention has an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12.

[0040] Even more preferably, the Arthrobacter globiformis esterase mutant according to the present invention has an amino acid sequence of SEQ ID NO: 12 and a nucleotide sequence such as the corresponding SEQ ID NO: 11. This mutant is the S315M mutant of Arthrobacter globiformis esterase.

[0041] The present invention also provides a nucleotide sequence of an Arthrobacter globiformis esterase mutant, which mutant has a nucleotide sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19.

[0042] In a second aspect, the present invention relates to an expression vector comprising an Arthrobacter globiformis esterase mutant nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19.

[0043] The third aspect of the present invention relates to a transformed host microorganism containing the above expression vector, and the vector contains an Arthrobacter globiformis esterase mutant nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19.

[0044] In a fourth aspect, the present invention relates to the use of an Arthrobacter globiformis esterase mutant according to the present invention for the selective preparation of (1R,3R)-chrysanthemic acid or a salt thereof.

[0045] In a preferred aspect, the present invention relates to an Arthrobacter globiformis esterase mutant capable of asymmetric hydrolysis of a racemic cyclopropane derivative having at least two chiral stereocenters.

[0046] Accordingly, in a fifth aspect, the present invention relates to the use of an Arthrobacter globiformis esterase mutant for the asymmetric hydrolysis of a racemic cyclopropane derivative having at least two chiral stereocenters.

[0047] In a preferred and advantageous aspect, the present invention relates to an Arthrobacter globiformis esterase mutant capable of asymmetric hydrolysis of a (C1-C6) alkyl ester of chrysanthemic acid, preferably ethyl ester of chrysanthemic acid.

[0048] Accordingly, in a sixth aspect, the present invention relates to the use of an Arthrobacter globiformis esterase mutant for the asymmetric hydrolysis of a (C1-C6) alkyl ester of chrysanthemic acid, preferably ethyl ester of chrysanthemic acid.

[0049] For the purposes of the present disclosure, each Arthrobacter globiformis esterase sequence (wild-type and mutant) has the corresponding SEQ ID NOs as follows, and for each mutant sequence, the triplet of mutant nucleotides or amino acids is shown in bold and underlined. SEQ ID NO: 1 corresponds to the following nucleotide sequence. Carboxylic-ester hydrolase Arthrobacter globiformis esterase Q44050 (wild-type)

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[0050] The various embodiments and aspects of the invention described above and claimed in the following claims of the patent find experimental support in the following examples.

Example

[0051] Here, reference is made to the following examples which illustrate some embodiments of the invention together with the above description.

[0052] Example 1. Preparation of a gene library of variants encoding esterase enzymes For the selection of enzyme variants with reduced product inhibition by high-throughput screening (HTS), 1045 variants of the esterase enzyme were generated by random mutagenesis.

[0053] Specifically, the most promising variant, the S315M variant (also referred to as variant 19), was obtained.

[0054] In particular, as described herein, the S315M esterase variant is characterized by higher activity towards the substrate of chrysanthemic acid ester and is used as the starting point (template) for the random mutagenesis cycle.

[0055] The gene library was prepared by random mutagenesis using the commercially available kit Genemorph II Random Mutagenesis Kit (Agilent, catalog number 200550) with the S315M esterase gene as the template (Figure 2).

[0056] As described in Figure 2, the method is divided into two stages. 1. The gene encoding the S315M esterase enzyme is used as the template for a PCR reaction carried out using error-prone Mutazyme II DNA polymerase, as well as DNA and amplification cycles in an amount that determines a low mutation frequency, i.e., 0 to 4.5 mutations / Kbase. The gene variants thus obtained are loaded onto a gel, the corresponding bands are excised and then purified. 2. The gene variant of the S315M enzyme prepared in Step 1 was used as a megaprimer for amplifying a target vector (generally a vector having a T7 promoter) using a high-fidelity DNA polymerase to avoid introducing mutations into the vector backbone. Subsequently, the mutant vector library was purified and then used for transformation of Escherichia coli (E. coli) cells.

[0057] Example 2. Gene Library Expression in Overproducing Escherichia coli (E. coli) Cells First, the entire "esterase variant" vector library was amplified in Escherichia coli (E. coli) DH5α. After amplification, plasmid DNA extraction and transformation of overexpressing Escherichia coli (E. coli) cells suitable for gene expression were performed.

[0058] The cells transformed in this way were spread on a selective medium and incubated at 37°C overnight, and 10,832 colonies were obtained.

[0059] Of these, 1045 were randomly selected and then advanced to gene expression and subsequently to HTS.

[0060] Each colony was inoculated into 1 mL of ZYM5052 medium containing lactose in a deep well. This can induce the expression of the gene encoding the variant of the esterase enzyme inserted into the vector.

[0061] After incubation at 37°C for 24 hours, the cells were collected by centrifugation and the pellet was processed for subsequent analysis.

[0062] Example 3. High-Throughput Screening (HTS) for Selection of Enzyme Variants with Reduced Product Inhibition In an enzyme reaction, the enzyme can be inhibited by the substrate or the product.

[0063] Substrate inhibition: In this case, there is a decrease in the initial specific activity as a function of the substrate concentration.

[0064] Product inhibition: In this case, the specific activity decreases to zero only after the product has accumulated to a certain concentration, and Figure 3 represents this phenomenon. As a direct result, incomplete bioconversion from substrate to product can occur.

[0065] Specifically, as reported by Nishizawa et al. ("Stereoselective Production of (+)-trans-Chrysanthemic Acid by a Microbial Esterase: Cloning, Nucleotide Sequence, and Overexpression of the Esterase Gene of Arthrobacter globiformis in Escherichia coli", Applied and Environmental Microbiology, 1995, 61, pp. 3208-3215), since the esterase enzyme targeted in this study is susceptible to product inhibition, in mutant screening, the chromogenic substrate p-nitrophenyl butyrate was given, and the specific activity was evaluated in both the absence and presence of the product (1R,3R)-chrysanthemic acid. The concentration of the tested (1R,3R)-chrysanthemic acid was equal to 100 mM, and the value obtained at the end of the bioconversion performed by the S315M enzyme was selected.

[0066] Next, the inhibition was evaluated by converting to the percentage of residual activity, or the ratio of the percentage of specific activity with acid to the percentage of specific activity without acid. Therefore, the higher the percentage of residual activity, the lower the degree of inhibition.

[0067] Experimental protocol: The previously collected cell pellet was resuspended in 200 μL of Cellytic™ (enzyme cocktail in detergent) and lysed by incubating at room temperature for 2 hours with stirring. The resulting lysate was clarified by centrifugation, and the supernatant, i.e., the total crude extract, was used for screening after dilution.

[0068] The screening was set up in a 96-well multiplate and carried out in 100 mM sodium phosphate buffer with the chromogenic substrate p-nitrophenyl butyrate at a final concentration of 4 mM, in the presence and absence of (1R,3R) pyrethric acid at a final concentration of 100 mM, at 25 °C for 10 minutes. The absorbance was monitored over time by taking readings at 405 nm every 16 seconds.

[0069] Therefore, the enzyme activity (U / mL) is defined as follows.

Equation

[0070] To calculate the specific activity (U / mg) of the enzyme variant in the presence or absence of acid, the total protein in the crude extract was quantified by the Bradford method using BSA as the reference protein for the calibration curve.

[0071] Twenty-five enzyme variants could be obtained and selected by the HTS procedure.

[0072] Example 4. Quantification of (1R,3R) pyrethric acid by HPLC in the 25 selected enzyme variants Ethyl pyrethrate was added as a substrate to the 25 enzyme variants selected by HTS and the production of (1R,3R) pyrethric acid was evaluated. Specifically, the acid product was quantified by reverse-phase HPLC analysis with gradient elution of a solution composed of 50% methanol, 50% acetonitrile, and 0.1% TFA.

[0073] By quantification of (1R,3R) pyrethric acid, i)

Equation

[0074] This value is calculated by determining the ratio of the acid produced (g / L) to the maximum amount of acid that can be obtained (g / L).

[0075] Figure 4 summarizes the selection scheme implemented and discussed below.

[0076] After providing the chromogenic substrate p-nitro-phenyl butyrate and performing HTS on 1045 samples, 61 variants of the starting enzyme S315M were found to exhibit improved characteristics in terms of: · An increase in the percentage of residual activity or a decrease in inhibition from the product · An increase in specific activity (U / mg) in the presence of (1R,3R)-chrysanthemic acid Specifically, with respect to the percentage of residual activity, all 61 selected enzymes have an increase in residual activity of at least 25% compared to the starting enzyme.

[0077] Figures 5 and 6 show the increase rates of residual activity and specific activity (in the presence of (1R,3R)-chrysanthemic acid) of each variant relative to the starting enzyme S315M.

[0078] Of these 61 enzyme variants, the inventors selected 25 and re-tested them by "scaling up" the procedure.

[0079] Specifically, the growth of 25 E. coli clones expressing these 25 enzyme variants was carried out in 250 mL flasks containing 25 mL of auto-induction medium instead of 1 mL deep wells.

[0080]

[0081] ​Therefore, by providing the chromogenic substrate p-nitrophenyl butyrate, the specific activities in the presence and absence of acid were re-evaluated, and the increase rate of the residual activity relative to the starting enzyme S315M was calculated (Figure 7).

[0082] All 25 enzyme variants (resulting from flask-scale bacterial growth) showed an increase rate of residual activity compared to the S315M enzyme, with some variation in the absolute values, which supports what was observed in HTS.

[0083] These 25 variants were also tested by providing the ethyl ester substrate of chrysanthemic acid.

[0084] Bioconversion of three selected enzyme variants (V274L-S315M-S331C-S315F-S223M) at pH-stat From the previous 25 mutants, the selection of three mutants to be compared with the S315M reference was performed by using a chromogenic substrate, but in the presence of chrysanthemic acid in the reaction mixture, by performing small-scale bioconversions. And mutants with greater activity in the presence of potential inhibitors (product inhibition), and thus showing less influence by acid, were selected. Then, of course, the performance was evaluated using the substrate of interest. Specifically, the reaction at pH-stat is carried out in a final volume of 20 mL in the presence of 10% (v / v) ethyl ester of racemic chrysanthemic acid as the substrate. The reaction is carried out at 45 °C for 20 hours, during which the pH value is maintained at 9.5 by the addition of 1 M NaOH. At the end of the reaction, the amount of (1R,3R)-chrysanthemic acid is evaluated by HPLC analysis.

[0085] Results and conclusions Figure 8 shows the HPLC chromatogram of the previous bioconversion obtained after a 20-hour reaction (T20) using 10% ethyl chrysanthemate (v / v). Specifically, the peak at about 2 minutes refers to (1R,3R) chrysanthemic acid, and the peak at about 4.7 minutes relates to all ethyl chrysanthemate that was not converted.

[0086] To quantify the (1R,3R) chrysanthemic acid produced at the end of the reaction, a calibration curve (Figure 9) correlating the peak area with the known acid concentration (Table 1) expressed in g / L was run using the above acid as a standard.

[0087] To determine the bioconversion yield, the maximum yield that could theoretically be obtained was first calculated. From the analysis of the starting mixture of isomers of ethyl chrysanthemate, the ethyl ester of the enantiomer (1R,3R) chrysanthemic acid was present at a concentration of 404.6 g / L. Since 10% (v / v) of this mixture was used, the reaction solution contained 40.46 g / L of ethyl (1R,3R)-chrysanthemate.

[0088] Taking into account the molecular weight of the (1R,3R)-chrysanthemic acid obtained, the maximum acid concentration that could be obtained starting from 40.46 g / L of the ester is 34.67 g / L. Table 1 shows the data for the (1R,3R)-chrysanthemic acid obtained (g / L) and the relative percentage yield of bioconversion (the percentage represents the maximum yield that could theoretically be obtained). In addition, as a control, the values of the previously obtained wild-type enzyme are reported.

Table 1

[0089] As reported above, the introduction of the point mutation S315M allows for the obtaining of a catalyst that is superior to the wild-type enzyme in terms of both specific activity (Figure 1 - Mutant 19) and reduced inhibition by the product, and the yield of bioconversion of ethyl (1R,3R)-chrysanthemate actually increased from 49.5% to 94.1% (Table 1).

[0090] Furthermore, as shown in Table 1, the performance of the S223M variant is similar to that of the S315M enzyme, and actually, after 20 hours of incubation, a high proportion of the substrate was converted at a yield of approximately 89% compared to the maximum yield that could theoretically be obtained.

[0091] In conclusion, among the tested variants, the S315M, S223M, and S315F variants were proven to be the optimal variants that selectively catalyze the bioconversion of (1R,3R)-ethyl ester of chrysanthemic acid to (1R,3R)-chrysanthemic acid from a mixture of four possible stereoisomers.

[0092] Example 5. Enzymatic hydrolysis of ethyl ester of racemic chrysanthemic acid 92 / 8 Ethyl chrysanthemate 92 / 8 consisting of four stereoisomers was provided, and (1R,3R)-chrysanthemic acid was produced by an enzymatic hydrolysis reaction with the S315M variant.

[0093] The enzymatic hydrolysis reaction was carried out in batch mode. In addition to the 92 / 8 trans / cis ester mixture, glycine, the enzyme, and an aqueous sodium hydroxide solution were loaded into the reactor. The function of the aqueous sodium hydroxide solution was to maintain an alkaline environment throughout the reaction. At the end of the reaction, the enzyme denatured by decomposition in an acidic environment with hydrochloric acid and by trichloroacetic acid was removed by filtration. Toluene was added to the remaining solution containing the unconverted ester and the desired (1R,3R) chrysanthemic acid to dissolve the ester, and an aqueous sodium hydroxide solution (10% w / w) was added to dissolve the chrysanthemic acid as the sodium salt in water. From the separation of these two phases, a toluene solution containing the unconverted ester was obtained and subjected to the next work-up step. The product (1R,3R) chrysanthemic acid was recovered from the acidified aqueous phase by extraction with toluene and subsequent evaporation of the solvent.

[0094] The reaction was carried out using 16.7 kg of glycine, 444 kg of the enzyme dissolved in water, and 413 kg of a 10% aqueous sodium hydroxide solution with respect to 400 kg of racemic ethyl chrysanthemate 92 / 8 in 3556 kg of water. At the end of the reaction, the raw materials were acidified with 116 kg of 37% hydrochloric acid and treated with 11.6 kg of trichloroacetic acid to denature the enzyme so that it could be filtered. Approximately 17.4 kg of the denatured enzyme was obtained from the filtration, which was discarded, and a solution of toluene and water was introduced to wash the filter, and the ester was extracted using approximately 868 kg of this solution.

[0095] This 5808 kg of crude reaction product was a two-phase system consisting of a toluene solution of the ester and an aqueous phase of the acid. 3055 kg of acidic water was discharged from the bottom of the reactor for treatment, and 61.8 kg of a 50% aqueous sodium hydroxide solution was added to basify the environment and dissolve all the acid as sodium salts in water.

[0096] After phase separation, 1091 kg of toluene solution and 1724 kg of aqueous phase containing chlorinated chrysanthemic acid were obtained. When 77 kg of 37% (w / w) hydrochloric acid was added to this aqueous phase, the desired (1R,3R) chrysanthemic acid was obtained, and 253 kg of toluene was added to form an organic phase. After separation of 1666 kg of water to be discarded, the toluene in the organic phase was evaporated, and finally 135 kg of (1R,3R) chrysanthemic acid was separated in a purity exceeding 95% as the only (1R,3R) form.

[0097] From the above description and the above examples, the advantages described in accordance with the present invention and achieved by the resulting products are clear.

Claims

**Claim 1** An Arthrobacter globiformis esterase variant having a sequence containing a mutation of the amino acid residue S at position 315, the amino acid residue S at position 223, or the amino acid residue F at position 298 of SEQ ID NO:

2. **Claim 2** The Arthrobacter globiformis esterase variant according to claim 1, which is capable of asymmetric hydrolysis of a racemic ester of chrysanthemic acid. **Claim 3** The Arthrobacter globiformis esterase variant according to any one of claims 1 or 2, wherein the amino acid residue S at position 315, the amino acid residue S at position 223, and the amino acid residue F at position 298 of SEQ ID NO: 2 are substituted with non-polar amino acid residues selected from the group consisting of M, F, L, W, A, I, P, or V. **Claim 4** The Arthrobacter globiformis esterase variant according to claim 3, wherein the non-polar amino acid residue is selected from the group consisting of M, F, L, or W. **Claim 5** The Arthrobacter globiformis esterase variant according to any one of claims 1 to 4, having one of the mutations S315M, S315F, S223M, S223L, S223F, or F298W in the amino acid sequence of SEQ ID NO:

2. **Claim 6** - A double mutant having mutations S315M and S223M in the amino acid sequence of SEQ ID NO: 2, - A double mutant having mutations S315F and S223M in the amino acid sequence of SEQ ID NO: 2, and - A triple mutant having mutations S315M, V274L, and S331C in the amino acid sequence of SEQ ID NO: 2 The Arthrobacter globiformis esterase variant according to claim 5, having one or two additional mutations selected from the group consisting of. **Claim 7** An Arthrobacter globiformis esterase variant according to any one of claims 1 to 6, having an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO:

20.

8. An Arthrobacter globiformis esterase variant according to any one of claims 1 to 7, having an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO:

12.

9. An Arthrobacter globiformis esterase variant according to any one of claims 1 to 8, having an amino acid sequence of SEQ ID NO:

12.

10. An Arthrobacter globiformis esterase variant according to any one of claims 1 to 9, having a nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO:

19.

11. An expression vector comprising an Arthrobacter globiformis esterase variant nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO:

19.

12. A transformed host microorganism comprising the expression vector according to claim 11, which comprises the Arthrobacter globiformis esterase variant nucleotide sequence.

13. Use of an Arthrobacter globiformis esterase variant according to any one of claims 1 to 10 for the selective preparation of (1R,3R)-chrysanthemic acid or a salt thereof.

14. Use of an Arthrobacter globiformis esterase variant according to any one of claims 1 to 10 for the asymmetric hydrolysis of a racemic cyclopropane derivative having at least two chiral stereocenters.

15. For the asymmetric hydrolysis of (C １ ~C ６ ) alkyl esters, preferably ethyl esters of chrysanthemic acid, use of the Arthrobacter globiformis esterase mutant according to any one of claims 1 to 10.

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