Novel esterase mutants and their use as stereoselective catalysts
By identifying and expressing mutants of Arthrobacter spherical arthrobacterase, the problem of selective hydrolysis of chrysanthic acid racemates in the prior art was solved, and efficient production of (1R,3R)-chrysanthic acid was achieved and product inhibition was reduced.
Patent Information
- Application Number
- CN202280099750.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2022-07-26
- Publication Date
- 2025-05-30
AI Technical Summary
The prior art is difficult to efficiently selectively hydrolyze racemates of chrysanthetic acid ester to produce (1R,3R)-chrysanthetic acid, and the product inhibition problem of spherical arthrobacterase is difficult to solve.
Selective hydrolysis of the cherophylla racemate is achieved by identifying and expressing mutants of the arthrobacterium spherical, especially those containing mutants of specific amino acid residues in SEQ ID NO:2.
High-efficiency selective hydrolysis of chrysanthetic racemates was achieved, which significantly improved the productivity of (1R,3R)-chrysanthetic acid, reduced product inhibition, and improved the catalytic performance of the enzyme.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of enzyme mutants, in particular to Arthrobacter globiformis esterase mutants, which are suitable for asymmetric hydrolysis of cyclopropane derivatives, in particular any combination of four stereoisomers of chrysanthemic esters, to selectively obtain (1R, 3R)-chrysanthemic acid or its salts.
[0002] The present invention also describes an expression vector comprising the Arthrobacter sphaericus esterase mutant and a host microorganism transformed with the vector.
[0003] The present invention also relates to the use of an Arthrobacter sphaeroides esterase mutant for selectively obtaining (1R,3R)-chrysanthemic acid. In another aspect, the present invention relates to the use of an Arthrobacter sphaeroides esterase mutant for asymmetric hydrolysis of a cyclopropane racemate derivative having at least two chiral stereocenters, preferably for asymmetric hydrolysis of a cyclopropane racemate derivative having at least two chiral stereocenters. Background Art
[0004] Tanacetum cinerariifolium or Dalmatian chrysanthemum is a species of white flowering plant in the asteraceae family, formerly part of the genus Pyrethrum but now included in the genus Chrysanthemum.
[0005] The plant is of great economic importance as a natural source of the insecticide "pyrethrum". The flowers are ground and the active ingredients called pyrethrins contained in the seed husks are extracted and sold as an oleoresin, which can be used as a suspension in water or oil or as a powder.
[0006] Pyrethrins are a mixture of six structurally related insecticidal esters formed from two acids (chrysanthemic acid and pyrethrin) combined with different alcohols. The esters of chrysanthemic acid are known as pyrethrin I, cucurbitrin I, and jasmonate I, collectively known as pyrethrin I, while the esters of pyrethric acid are known as pyrethrin II, cucurbitrin II, and jasmonate II, collectively known as pyrethrin II. Pyrethrins attack the nervous system of all insects, inhibiting female mosquitoes from biting. Pyrethrins appear to have an insect repellent effect even at doses below those that are lethal to insects.
[0007] They are harmful to fish but are much less toxic to mammals and birds than many synthetic pesticides, are non-persistent, biodegradable, and readily break down when exposed to light. They are considered among the safest pesticides for food-related uses.
[0008] After the chemical structure of "natural pyrethroids" was elucidated, useful synthetic pyrethroids with various properties were developed, promoting the development of pyrethroid chemistry.
[0009] Chrysanthemic acid is a unique intermediate involved in a variety of natural and synthetic insecticides. In particular, pyrethroids are derivatives of chrysanthemic acid and exist in four different possible stereoisomers: (1R,3R), (1R,3S), (1S,3R), and (1S,3S).
[0010] (1R,3R)- or (+)-trans-chrysanthemic acid (one of four stereoisomers) is generally considered the most interesting and effective of the four stereoisomers:
[0011]
[0012] (1R,3R)- or (+)-trans-chrysanthemic acid
[0013] 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; Appl Environ Microbiol. 1995; 61: 3208-15"), highly stereoselective enzymatic production of (+)-trans-chrysanthemic acid would have a greater advantage over conventional chemical methods for selectively obtaining the target isomer. The esterase gene of Arthrobacter globiformis was characterized and reported to have high stereospecificity, although it was difficult to use for large-scale production of insecticides due to product inhibition.
[0014] It was therefore an object of the present invention to develop a stereoselective hydrolysis of chrysanthemic esters.
[0015] It is well known that chrysanthemic acid can be obtained by hydrolysis of a suitable chrysanthemic acid ester, in particular ethyl chrysanthemate in racemic form.
[0016] There are four isomers of chrysanthemic acid ethyl ester (or related esters):
[0017]
[0018] (1S)-trans-chrysanthemic acid ethyl ester or (1S,3S)-chrysanthemic acid ethyl ester
[0019]
[0020] (1R)-trans-chrysanthemic acid ethyl ester or (1R,3R)-chrysanthemic acid ethyl ester
[0021]
[0022] (1S)-cis-chrysanthemic acid ethyl ester or (1S,3R)-chrysanthemic acid ethyl ester
[0023]
[0024] (1R)-cis-chrysanthemic acid ethyl ester or (1R,3S)-chrysanthemic acid ethyl ester
[0025] The stereoisomer mixture is the starting material for the production of chrysanthemic acid.
[0026] Therefore, another object of the present invention is to selectively produce (1R,3R) chrysanthemic acid from ethyl chrysanthemate racemate. Summary of the Invention
[0027] The present inventors surprisingly discovered a novel esterase that can selectively hydrolyze a chrysanthemic acid ester racemate to selectively produce (1R,3R) chrysanthemic acid or a salt thereof.
[0028] Therefore, the present invention relates to an Arthrobacter sphaeroides esterase mutant, the sequence of which comprises 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.
[0029] In a second aspect, the present invention relates to an expression vector comprising a mutant nucleotide sequence of an Arthrobacter sphaeroides esterase, wherein the mutant nucleotide sequence is 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.
[0030] A third aspect of the present invention relates to a transformed host microorganism containing an expression vector comprising a mutant nucleotide sequence of an Arthrobacter sphaeroides esterase 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.
[0031] In a fourth aspect, the present invention relates to use of the Arthrobacter sphaeroides esterase mutant according to the present invention for selectively producing (1R,3R)-chrysanthemic acid.
[0032] In a preferred aspect, the present invention relates to an Arthrobacter sphaeroides esterase mutant capable of asymmetric hydrolysis of a cyclopropane racemic derivative having at least two chiral stereocenters.
[0033] Therefore, in a fifth aspect, the present invention relates to the use of an Arthrobacter sphaericus esterase mutant for the asymmetric hydrolysis of a cyclopropane racemic derivative having at least two chiral stereocenters.
[0034] In a preferred and advantageous aspect, the present invention relates to an Arthrobacter sphaeroides esterase mutant capable of asymmetrically hydrolyzing (C1-C6)alkyl chrysanthemate, preferably ethyl chrysanthemate.
[0035] Therefore, in a sixth aspect, the present invention relates to the use of an Arthrobacter sphaeroides esterase mutant for asymmetric hydrolysis of chrysanthemic acid (C1-C6) alkyl esters (preferably chrysanthemic acid ethyl ester).
[0036] As will be further described in the detailed description of the present invention, the basic technical solution of the present invention is to provide innovative esterases with improved properties to obtain (1R,3R) chrysanthemic acid required for the production of pyrethroid insecticides.
[0037] The present invention solves the problem of providing (1R,3R)-chrysanthemic acid, in particular by identifying mutants of the Arthrobacter sphaeroides esterase having improved enzyme kinetics, improved enzyme stability and / or reduced product inhibition compared to the wild-type Arthrobacter sphaeroides esterase of SEQ ID NO: 2 as identified in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] The characteristics and advantages of the present invention will be apparent from the detailed description reported below, the examples given for illustrative and non-limiting purposes, and the accompanying drawings. Figure 1-1 0 is obvious, among which:
[0039] Figure 1 : Comparison of specific activities of crude extracts from cells expressing different esterase variants.
[0040] The activity is expressed as a percentage of the specific activity of the wild-type enzyme and is determined using 10% (v / v) racemate of ethyl chrysanthemate as substrate. The formation of (1R,3R)chrysanthemic acid is determined by HPLC analysis.
[0041] Figure 2Schematic diagram of the gene library generation kit's functions: 1. Megaprimer mutant synthesis: Amplify the gene encoding the esterase S315M using a DNA polymerase that is receptive to insertional mutagenesis to obtain purified gene variants. 2. The vector library containing the mutant gene generated in step 1 is used as a megaprimer to amplify target vectors using a high-fidelity DNA polymerase to obtain a library of vectors containing mutant versions of the S315M esterase gene.
[0042] Figure 3 : Representative plots of the specific activity of each enzyme affected by product inhibition as a function of product concentration.
[0043] Figure 4 : Schematic representation of the selection scheme for enzyme variants obtained by random mutagenesis.
[0044] Figure 5 : Percent increase in residual activity of 61 selected variants compared to the starting enzyme S315M.
[0045] Figure 6 : Percent increase in specific activity and therefore inhibition of 61 selected variants in the presence of (1R,3R)chrysanthemic acid relative to the starting enzyme S315M.
[0046] Figure 7 : Percent increase in residual activity of the 25 selected variants compared to the starting enzyme S315M.
[0047] Figure 8 : Chromatograms obtained by HPLC analysis involving a 20-hour reaction with the enzyme S315M and the mutant variants V274L-S315M-S331C (triple mutant), S315F, S223M.
[0048] Figure 9 : Calibration line of (1R,3R)-chrysanthemic acid: Amount of (1R,3R)-chrysanthemic acid (g / L) at constant pH and % bioconversion yield of the enzyme variant according to the invention and of the S315M enzyme used as starting point for random mutagenesis. DETAILED DESCRIPTION
[0049] Therefore, the present invention relates to an Arthrobacter sphaeroides esterase mutant, the sequence of which comprises 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.
[0050] Without being bound by any theory, the inventors believe that the Arthrobacter sphaeroides esterase mutants claimed herein allow for higher yields in the selective conversion of cyclopropane derivatives having at least two chiral centers (preferably racemates of (C1-C6) alkyl chrysanthemates, more preferably racemates of ethyl chrysanthemates) to (1R,3R)-chrysanthemic acid or its salts by affecting the following three aspects of catalysis:
[0051]
[0052] i) Improved enzyme stability at various temperatures and pH values: The enzyme exhibits good operational stability under alkaline conditions (pH 9.0 to 11.00). Optimal conversion yields are achieved within this range, preferably within the pH range of 10.0-10.5, with significant decreases below 8.5-9.0.
[0053] ii) improving kinetic parameters; and
[0054] iii) reduced product inhibition when compared to the wild-type Arthrobacter sphaeroides esterase having the amino acid sequence set forth in SEQ ID NO: 2. The described mutants exhibit improved catalytic performance, achieving conversions as high as 88-95% compared to the WT enzyme having the amino acid sequence reported in SEQ ID NO: 2 (conversions did not exceed 40-49% using a pH-stat during the reaction). These mutations increase the affinity for the substrate without altering the enzyme's high stereospecificity and reduce product inhibition, which was still evident in the WT enzyme.
[0055] For the purposes of this invention, the terms:
[0056] - "(1R,3R)-chrysanthemic acid" as used herein is 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 has a CAS number of 4638-92-0;
[0057] - "(1R,3R)-chrysanthemic acid or its salt" refers to a salt selected from alkali metals and alkaline earth metals such as potassium, sodium, calcium, magnesium, etc.;
[0058] - "Cyclopropane derivatives having at least two chiral centers" refers to cyclopropane compounds having at least two asymmetric carbon atoms, thus having at least two stereoisomers, preferably four stereoisomers;
[0059] - "Chrysanthemic acid (C1-C6) alkyl ester" refers to a linear or branched alkyl chain (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc.) ester of chrysanthemic acid having 1 to 6 carbon atoms;
[0060] As used herein, "chrysanthemic acid ethyl ester" is synonymous with "ethyl 2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropane-1-carboxylate" and has a CAS number of 97-41-6. According to the present invention, chrysanthemic acid ethyl ester can be provided in any ratio of trans / cis form, for example, in a ratio of 60 / 40 to 100 / 0, preferably 65 / 35, 70 / 30, 80 / 20, 90 / 10, 92 / 8, or 98 / 2.
[0061] In a preferred aspect, in the Arthrobacter sphaeroides 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 a non-polar amino acid residue selected from the group consisting of M, F, L, W, A, and I. The inventors surprisingly found that by substituting the amino acid residue at position 315, 223, or 298 of SEQ ID NO: 2 with a non-polar amino acid, the properties of the esterase are improved while maintaining the ability to asymmetric hydrolyze racemic chrysanthemic acid esters.
[0062] For purposes of the present invention, amino acid residues are represented by their "single-letter code," where "M" corresponds to Met or methionine, "F" corresponds to Phe or phenylalanine, "S" corresponds to Ser or serine, etc., as understood by those of ordinary skill in the art.
[0063] In a more preferred aspect, in the Arthrobacter sphaeroides esterase mutant of the present invention, the 315th amino acid residue S, the 223rd amino acid residue S and the 298th amino acid residue F of SEQ ID NO: 2 are substituted by a non-polar amino acid residue selected from the group consisting of M, F, L or W.
[0064] In a more preferred aspect, the Arthrobacter sphaericus esterase mutant of the present invention has one of the following mutations in the amino acid sequence of SEQ ID NO: 2: S315M, S315F, S223M, S223L, S223F or F298W.
[0065] In a more preferred aspect, the Arthrobacter sphaericus esterase mutant of the invention has one additional mutation (double mutation) or two additional mutations (triple mutation), preferably selected from the group consisting of:
[0066] - a double mutant having mutations S315M and S223M in the amino acid sequence of SEQ ID NO: 2;
[0067] - a double mutant having mutations S315F and S223M in the amino acid sequence of SEQ ID NO: 2; and
[0068] - a triple mutant having mutations S315M, V274L and S331C in the amino acid sequence of SEQ ID NO: 2.
[0069] Preferred Arthrobacter sphaericus esterase mutants have the following amino acid sequence: 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.
[0070] More preferably, the Arthrobacter sphaeroides esterase mutant according to the present invention has the amino acid sequence of SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:12.
[0071] More preferably, the Arthrobacter globosa esterase mutant according to the present invention has the amino acid sequence of SEQ ID NO: 12 and the corresponding nucleotide sequence, for example, SEQ ID NO: 11. The mutant is an S315M mutant of Arthrobacter globosa esterase.
[0072] The present invention also provides nucleotide sequences of Arthrobacter sphaeroides esterase mutants, which have nucleotide sequences as described 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.
[0073] In a second aspect, the present invention relates to an expression vector comprising a mutant nucleotide sequence of an Arthrobacter sphaeroides esterase, wherein the mutant nucleotide sequence is 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.
[0074] A third aspect of the present invention relates to a transformed host microorganism containing an expression vector comprising a mutant nucleotide sequence of an Arthrobacter sphaeroides esterase 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.
[0075] In a fourth aspect, the present invention relates to use of the Arthrobacter sphaeroides esterase mutant according to the present invention for selectively preparing (1R,3R)-chrysanthemic acid or a salt thereof.
[0076] In a preferred aspect, the present invention relates to an Arthrobacter sphaeroides esterase mutant capable of asymmetric hydrolysis of a cyclopropane racemic derivative having at least two chiral stereocenters.
[0077] Therefore, in a fifth aspect, the present invention relates to the use of an Arthrobacter sphaericus esterase mutant for the asymmetric hydrolysis of a cyclopropane racemic derivative having at least two chiral stereocenters.
[0078] In a preferred and advantageous aspect, the present invention relates to an Arthrobacter sphaeroides esterase mutant capable of asymmetrically hydrolyzing (C1-C6)alkyl chrysanthemate, preferably ethyl chrysanthemate.
[0079] Therefore, in a sixth aspect, the present invention relates to the use of an Arthrobacter sphaeroides esterase mutant for asymmetric hydrolysis of chrysanthemic acid (C1-C6) alkyl esters (preferably chrysanthemic acid ethyl ester).
[0080] For the purposes of this disclosure, each Arthrobacter sphaeroides esterase sequence (wild type and mutant) has the following corresponding SEQ ID NO, wherein for each mutant sequence, the mutated nucleotide triplet or amino acid is indicated in bold and underlined:
[0081] SEQ ID NO: 1 corresponds to the following nucleotide sequence:
[0082] Carboxylate ester hydrolase Arthrobacter sphaericus esterase Q44050 (wild type)
[0083] ATGGACGCGCAGACCATTGCGCCGGGTTTTGAGAGCGTGGCGGAACTGTTCGGCCGTTT
[0084] TCTGAGCGAGGATCGTGAATACAGCGCGCAACTGGCGGCGTATCACCGTGGTGTGAAG
[0085] GTTCTGGACATCAGCGGTGGCCCGCACCGTCGTCCGGATAGCGTGACCGGCGTTTTTAG
[0086] CTGCAGCAAAGGTGTGAGCGGCCTGGTTATTGCGCTGCTGGTTCAGGACGGTTTCCTGG
[0087] ACCTGGATGCGGAAGTGGTTAAGTACTGGCCGGAGTTTGGTGCGGAAGGCAAAGCGAC
[0088] CATTACCGTGGCGCAGCTGCTGAGCCACCAAGCGGGTCTGCTGGGCGTTGAAGGTGGC
[0089] CTGACCCTGGCGGAGTACAACAACAGCGAACTGGCTGCGGCGAAGCTGGCGCAAATGC
[0090] GTCCGCTGTGGAAACCGGGTACCGCGTTCGGCTATCACGCGCTGACCATCGGTGTGTTT
[0091] ATGGAGGAACTGTGCCGTCGTATTACCGGCAGCACCCTGCAGGAGATCTACGAACAACG
[0092] TATTCGTAGCGTTACCGGTGCGCACTTCTTTCTGGGTCTGCCGGAGAGCGAGGAACCGC
[0093] GTTATGCGACCCTGCGTTGGGCGGCGGACCCGAGCCAGCCGTGGATTGATCCGGCGAGC
[0094] CACTTTGGTCTGAGCGCGAACAGCGCGGTGGGTGACATTCTGGATCTGCCGAACCTGCG
[0095] TGAGGTTCGTGCGGCGGGTCTGAGCAGCGCGGCGGGCGTGGCGAGCGCGGAGGGTATG
[0096] GCGCGTGTTTATGCTGCGGCGCTGACCGGTCTGGCGGCGAACGGTGACCGTGCGGCGG
[0097] TGGCGCCGCTGCTGAGCGAGGAAACCATCCAGACCGTGACCGCGGAGCAAGTTTTCGG
[0098] CATTGATCGTGTGTTTGGTGAAACCAGCTGCTTCGGCACCGTTTTTATGAAGAGCCACGC
[0099] GCGTAGCCCGTACGGTAGCTATCGTGCGTTCGGTCATGATGGTGCGAGCGCGAGCCTGG
[0100] GTTTCGCGGATCCGGTGTACGAGCTGGCGTTTGGCTATGTGCCGCAGCAAGCGGAGCCG
[0101] GGTGGCGCGGGTTGCCGTAACCTGGAACTGAGCGCGGCGGTGCGTAAAGCGGTTACCG
[0102] AACTGGCGCAATGA
[0103] SEQ ID NO:2 corresponds to the following amino acid sequence:
[0104] Carboxylate ester hydrolase Arthrobacter sphaericus esterase Q44050 (wild type)
[0105] MDAQTIAPGFESVAELFGRFLSEDREYSAQLAAYHRGVKVLDISGGPHRRPDSVTGVFSCS
[0106] KGVSGLVIALLVQDGFLDLDAEVVKYWPEFGAEGKATITVAQLLSHQAGLLGVEGGLTLAE
[0107] YNNSELAAAKLAQMRPLWKPGTAFGYHALTIGVFMEELCRRITGSTLQEIYEQRIRSVTGA
[0108] HFFLGLPESEEPRYATLRWAADPSQPWIDPASHFGLSANSAVGDILDLPNLREVRAAGLSSA
[0109] AGVASAEGMARVYAAALTGLAANGDRAAVAPLLSEETIQTVTAEQVFGIDRVFGETSCFGT
[0110] VFMKSHARSPYGSYRAFGHDGASASLGFADPVYELAFGYVPQQAEPGGAGCRNLELSAAV
[0111] RKAVTELAQ
[0112] SEQ ID NO:3 corresponds to the following nucleotide sequence:
[0113] Arthrobacter sphaeroides esterase mutant S223M (variant 9)
[0114] ATGGACGCGCAGACCATTGCGCCGGGTTTTGAGAGCGTGGCGGAACTGTTCGGCCGTTT
[0115] TCTGAGCGAGGATCGTGAATACAGCGCGCAACTGGCGGCGTATCACCGTGGTGTGAAG
[0116] GTTCTGGACATCAGCGGTGGCCCGCACCGTCGTCCGGATAGCGTGACCGGCGTTTTTAG
[0117] CTGCAGCAAAGGTGTGAGCGGCCTGGTTATTGCGCTGCTGGTTCAGGACGGTTTCCTGG
[0118] ACCTGGATGCGGAAGTGGTTAAGTACTGGCCGGAGTTTGGTGCGGAAGGCAAAGCGAC
[0119] CATTACCGTGGCGCAGCTGCTGAGCCACCAAGCGGGTCTGCTGGGCGTTGAAGGTGGC
[0120] CTGACCCTGGCGGAGTACAACAACAGCGAACTGGCTGCGGCGAAGCTGGCGCAAATGC
[0121] GTCCGCTGTGGAAACCGGGTACCGCGTTCGGCTATCACGCGCTGACCATCGGTGTGTTT
[0122] ATGGAGGAACTGTGCCGTCGTATTACCGGCAGCACCCTGCAGGAGATCTACGAACAACG
[0123] TATTCGTAGCGTTACCGGTGCGCACTTCTTTCTGGGTCTGCCGGAGAGCGAGGAACCGC
[0124] GTTATGCGACCCTGCGTTGGGCGGCGGACCCGAGCCAGCCGTGGATTGATCCGGCGAGC
[0125] CACTTTGGTCTGAGCGCGAAC ATG GCGGTGGGTGACATTCTGGATCTGCCGAACCTGCG
[0126] TGAGGTTCGTGCGGCGGGTCTGAGCAGCGCGGCGGGCGTGGCGAGCGCGGAGGGTATG
[0127] GCGCGTGTTTATGCTGCGGCGCTGACCGGTCTGGCGGCGAACGGTGACCGTGCGGCGG
[0128] TGGCGCCGCTGCTGAGCGAGGAAACCATCCAGACCGTGACCGCGGAGCAAGTTTTCGG
[0129] CATTGATCGTGTGTTTGGTGAAACCAGCTGCTTCGGCACCGTTTTTATGAAGAGCCACGC
[0130] GCGTAGCCCGTACGGTAGCTATCGTGCGTTCGGTCATGATGGTGCGAGCGCGAGCCTGG
[0131] GTTTCGCGGATCCGGTGTACGAGCTGGCGTTTGGCTATGTGCCGCAGCAAGCGGAGCCG
[0132] GGTGGCGCGGGTTGCCGTAACCTGGAACTGAGCGCGGCGGTGCGTAAAGCGGTTACCG
[0133] AACTGGCGCAATGA
[0134] SEQ ID NO:4 corresponds to the following amino acid sequence:
[0135] Arthrobacter sphaeroides esterase mutant S223M (variant 9)
[0136] MDAQTIAPGFESVAELFGRFLSEDREYSAQLAAYHRGVKVLDISGGPHRRPDSVTGVFSCS
[0137] KGVSGLVIALLVQDGFLDLDAEVVKYWPEFGAEGKATITVAQLLSHQAGLLGVEGGLTLAE
[0138] YNNSELAAAKLAQMRPLWKPGTAFGYHALTIGVFMEELCRRITGSTLQEIYEQRIRSVTGA
[0139] HFFLGLPESEEPRYATLRWAADPSQPWIDPASHFGLSAN M AVGDILDLPNLREVRAAGLSSA
[0140] AGVASAEGMARVYAAALTGLAANGDRAAVAPLLSEETIQTVTAEQVFGIDRVFGETSCFGT
[0141] VFMKSHARSPYGSYRAFGHDGASASLGFADPVYELAFGYVPQQAEPGGAGCRNLELSAAV
[0142] RKAVTELAQ
[0143] SEQ ID NO:5 corresponds to the following nucleotide sequence:
[0144] Arthrobacter sphaeroides esterase mutant S223L (variant 15)
[0145] ATGGACGCGCAGACCATTGCGCCGGGTTTTGAGAGCGTGGCGGAACTGTTCGGCCGTTT
[0146] TCTGAGCGAGGATCGTGAATACAGCGCGCAACTGGCGGCGTATCACCGTGGTGTGAAG
[0147] GTTCTGGACATCAGCGGTGGCCCGCACCGTCGTCCGGATAGCGTGACCGGCGTTTTTAG
[0148] CTGCAGCAAAGGTGTGAGCGGCCTGGTTATTGCGCTGCTGGTTCAGGACGGTTTCCTGG
[0149] ACCTGGATGCGGAAGTGGTTAAGTACTGGCCGGAGTTTGGTGCGGAAGGCAAAGCGAC
[0150] CATTACCGTGGCGCAGCTGCTGAGCCACCAAGCGGGTCTGCTGGGCGTTGAAGGTGGC
[0151] CTGACCCTGGCGGAGTACAACAACAGCGAACTGGCTGCGGCGAAGCTGGCGCAAATGC
[0152] GTCCGCTGTGGAAACCGGGTACCGCGTTCGGCTATCACGCGCTGACCATCGGTGTGTTT
[0153] ATGGAGGAACTGTGCCGTCGTATTACCGGCAGCACCCTGCAGGAGATCTACGAACAACG
[0154] TATTCGTAGCGTTACCGGTGCGCACTTCTTTCTGGGTCTGCCGGAGAGCGAGGAACCGC
[0155] GTTATGCGACCCTGCGTTGGGCGGCGGACCCGAGCCAGCCGTGGATTGATCCGGCGAGC
[0156] CACTTTGGTCTGAGCGCGAAC TTG GCGGTGGGTGACATTCTGGATCTGCCGAACCTGCG
[0157] TGAGGTTCGTGCGGCGGGTCTGAGCAGCGCGGCGGGCGTGGCGAGCGCGGAGGGTATG
[0158] GCGCGTGTTTATGCTGCGGCGCTGACCGGTCTGGCGGCGAACGGTGACCGTGCGGCGG
[0159] TGGCGCCGCTGCTGAGCGAGGAAACCATCCAGACCGTGACCGCGGAGCAAGTTTTCGG
[0160] CATTGATCGTGTGTTTGGTGAAACCAGCTGCTTCGGCACCGTTTTTATGAAGAGCCACGC
[0161] GCGTAGCCCGTACGGTAGCTATCGTGCGTTCGGTCATGATGGTGCGAGCGCGAGCCTGG
[0162] GTTTCGCGGATCCGGTGTACGAGCTGGCGTTTGGCTATGTGCCGCAGCAAGCGGAGCCG
[0163] GGTGGCGCGGGTTGCCGTAACCTGGAACTGAGCGCGGCGGTGCGTAAAGCGGTTACCG
[0164] AACTGGCGCAATGA
[0165] SEQ ID NO:6 corresponds to the following amino acid sequence: Arthrobacter sphaeroides esterase mutant S223L (variant 15)
[0166]
[0167] SEQ ID NO:7 corresponds to the following nucleotide sequence: Arthrobacter sphaeroides esterase mutant S223F (variant 16)
[0168]
[0169] SEQ ID NO:8 corresponds to the following amino acid sequence: Arthrobacter sphaeroides esterase mutant S223F (variant 16)
[0170]
[0171] SEQ ID NO:9 corresponds to the following nucleotide sequence: Arthrobacter sphaeroides esterase mutant S315F (variant 18)
[0172]
[0173] TCTGAGCGAGGATCGTGAATACAGCGCGCAACTGGCGGCGTATCACCGTGGTGTGAAG
[0174] GTTCTGGACATCAGCGGTGGCCCGCACCGTCGTCCGGATAGCGTGACCGGCGTTTTTAG
[0175] CTGCAGCAAAGGTGTGAGCGGCCTGGTTATTGCGCTGCTGGTTCAGGACGGTTTCCTGG
[0176] ACCTGGATGCGGAAGTGGTTAAGTACTGGCCGGAGTTTGGTGCGGAAGGCAAAGCGAC
[0177] CATTACCGTGGCGCAGCTGCTGAGCCACCAAGCGGGTCTGCTGGGCGTTGAAGGTGGC
[0178] CTGACCCTGGCGGAGTACAACAACAGCGAACTGGCTGCGGCGAAGCTGGCGCAAATGC
[0179] GTCCGCTGTGGAAACCGGGTACCGCGTTCGGCTATCACGCGCTGACCATCGGTGTGTTT
[0180] ATGGAGGAACTGTGCCGTCGTATTACCGGCAGCACCCTGCAGGAGATCTACGAACAACG
[0181] TATTCGTAGCGTTACCGGTGCGCACTTCTTTCTGGGTCTGCCGGAGAGCGAGGAACCGC
[0182] GTTATGCGACCCTGCGTTGGGCGGCGGACCCGAGCCAGCCGTGGATTGATCCGGCGAGC
[0183] CACTTTGGTCTGAGCGCGAACAGCGCGGTGGGTGACATTCTGGATCTGCCGAACCTGCG
[0184] TGAGGTTCGTGCGGCGGGTCTGAGCAGCGCGGCGGGCGTGGCGAGCGCGGAGGGTATG
[0185] GCGCGTGTTTATGCTGCGGCGCTGACCGGTCTGGCGGCGAACGGTGACCGTGCGGCGG
[0186] TGGCGCCGCTGCTGAGCGAGGAAACCATCCAGACCGTGACCGCGGAGCAAGTTTTCGG
[0187] CATTGATCGTGTGTTTGGTGAAACCAGCTGCTTCGGCACCGTTTTTATGAAGAGCCACGC
[0188] GCGT TTT CCGTACGGTAGCTATCGTGCGTTCGGTCATGATGGTGCGAGCGCGAGCCTGG
[0189] GTTTCGCGGATCCGGTGTACGAGCTGGCGTTTGGCTATGTGCCGCAGCAAGCGGAGCCG
[0190] GGTGGCGCGGGTTGCCGTAACCTGGAACTGAGCGCGGCGGTGCGTAAAGCGGTTACCG
[0191] AACTGGCGCAATGA
[0192] SEQ ID NO:10 corresponds to the following amino acid sequence:
[0193] Arthrobacter sphaeroides esterase mutant S315F (variant 18)
[0194] MDAQTIAPGFESVAELFGRFLSEDREYSAQLAAYHRGVKVLDISGGPHRRPDSVTGVFSCS
[0195] KGVSGLVIALLVQDGFLDLDAEVVKYWPEFGAEGKATITVAQLLSHQAGLLGVEGGLTLAE
[0196] YNNSELAAAKLAQMRPLWKPGTAFGYHALTIGVFMEELCRRITGSTLQEIYEQRIRSVTGA
[0197] HFFLGLPESEEPRYATLRWAADPSQPWIDPASHFGLSANSAVGDILDLPNLREVRAAGLSSA
[0198] AGVASAEGMARVYAAALTGLAANGDRAAVAPLLSEETIQTVTAEQVFGIDRVFGETSCFGT
[0199] VFMKSHAR F PYGSYRAFGHDGASASLGFADPVYELAFGYVPQQAEPGGAGCRNLELSAAV
[0200] RKAVTELAQ
[0201] SEQ ID NO:11 corresponds to the following nucleotide sequence:
[0202] Arthrobacter sphaeroides esterase mutant S315M (variant 19)
[0203] ATGGACGCGCAGACCATTGCGCCGGGTTTTGAGAGCGTGGCGGAACTGTTCGGCCGTTT
[0204] TCTGAGCGAGGATCGTGAATACAGCGCGCAACTGGCGGCGTATCACCGTGGTGTGAAG
[0205] GTTCTGGACATCAGCGGTGGCCCGCACCGTCGTCCGGATAGCGTGACCGGCGTTTTTAG
[0206] CTGCAGCAAAGGTGTGAGCGGCCTGGTTATTGCGCTGCTGGTTCAGGACGGTTTCCTGG
[0207] ACCTGGATGCGGAAGTGGTTAAGTACTGGCCGGAGTTTGGTGCGGAAGGCAAAGCGAC
[0208] CATTACCGTGGCGCAGCTGCTGAGCCACCAAGCGGGTCTGCTGGGCGTTGAAGGTGGC
[0209] CTGACCCTGGCGGAGTACAACAACAGCGAACTGGCTGCGGCGAAGCTGGCGCAAATGC
[0210] GTCCGCTGTGGAAACCGGGTACCGCGTTCGGCTATCACGCGCTGACCATCGGTGTGTTT
[0211] ATGGAGGAACTGTGCCGTCGTATTACCGGCAGCACCCTGCAGGAGATCTACGAACAACG
[0212] TATTCGTAGCGTTACCGGTGCGCACTTCTTTCTGGGTCTGCCGGAGAGCGAGGAACCGC
[0213] GTTATGCGACCCTGCGTTGGGCGGCGGACCCGAGCCAGCCGTGGATTGATCCGGCGAGC
[0214] CACTTTGGTCTGAGCGCGAACAGCGCGGTGGGTGACATTCTGGATCTGCCGAACCTGCG
[0215] TGAGGTTCGTGCGGCGGGTCTGAGCAGCGCGGCGGGCGTGGCGAGCGCGGAGGGTATG
[0216] GCGCGTGTTTATGCTGCGGCGCTGACCGGTCTGGCGGCGAACGGTGACCGTGCGGCGG
[0217]
[0218] SEQ ID NO: 12 corresponds to the following amino acid sequence: Arthrobacter sphaeroides esterase mutant S315M (variant 19)
[0219]
[0220] SEQ ID NO: 13 corresponds to the following nucleotide sequence: Arthrobacter sphaeroides esterase mutant F298W (variant 20)
[0221]
[0222] SEQ ID NO: 14 corresponds to the following amino acid sequence: Arthrobacter sphaeroides esterase mutant F298W (variant 20)
[0223]
[0224]
[0225] SEQ ID NO: 15 corresponds to the following nucleotide sequence: Arthrobacter sphaeroides esterase double mutant S315M+S223M
[0226]
[0227] SEQ ID NO: 16 corresponds to the following amino acid sequence: Arthrobacter sphaeroides esterase double mutant S315M+S223M
[0228]
[0229] SEQ ID NO: 17 corresponds to the following nucleotide sequence: Arthrobacter sphaeroides esterase double mutant S315F+S223M
[0230]
[0231]
[0232] SEQ ID NO: 18 corresponds to the following amino acid sequence:
[0233] Arthrobacter sphaeroides esterase double mutant S315F+S223M
[0234]
[0235] SEQ ID NO: 19 corresponds to the following nucleotide sequence:
[0236] Arthrobacter sphaeroides esterase triple mutant S315M+V274L+S331C
[0237]
[0238] SEQ ID NO:20 corresponds to the following amino acid sequence:
[0239] Arthrobacter sphaeroides esterase triple mutant S315M+V274L+S331C
[0240]
[0241] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
[0242] Example
[0243] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention.
[0244] Example 1. Generation of a gene library of mutants encoding esterases
[0245] 1045 esterase variants were generated by random mutagenesis in order to select enzyme variants with reduced product inhibition by high throughput screening (HTS).
[0246] In particular, the most promising mutant, the S315M mutant (also known as variant 19), was obtained.
[0247] In particular, the S315M esterase mutant, characterized by a higher activity towards chrysanthemate substrates, was used as the starting point (template) for random mutagenesis cycles, as described herein.
[0248] Random mutagenesis was performed using the commercial kit Genemorph II random mutagenesis kit (Agilent cat. N 200550) to generate a gene library. The S315M esterase gene was used as a template ( Figure 2 ).
[0249] like Figure 2 As shown, the method is divided into two stages:
[0250] 1. The gene encoding S315M esterase was used as a template for a PCR reaction using the error-prone Mutazyme II DNA polymerase. The DNA amount and amplification cycle number were used to determine the low mutation frequency (i.e., 0-4.5 mutations / kilobase (kbase)). The gene variants thus obtained were loaded onto a gel, the corresponding bands were excised, and then purified.
[0251] 2. The S315M enzyme variant generated in stage 1 is used as a megaprimer to amplify the target vector (typically a vector with a T7 promoter) using a high-fidelity DNA polymerase to avoid introducing mutations into the vector backbone. The mutant vector library is then purified and subsequently used to transform E. coli cells.
[0252] Example 2. Expression of gene libraries in high-yielding E. coli cells
[0253] The "esterase mutant" vector library was first fully amplified in E. coli DH5α. After amplification, plasmid DNA was extracted and transformed into overexpressing E. coli cells suitable for gene expression.
[0254] The thus transformed cells were plated on a selective medium and cultured at 37°C overnight to obtain 10,832 colonies.
[0255] Of these, 1045 colonies were randomly selected and then subjected to gene expression followed by HTS.
[0256] Each colony was inoculated into a deep well of 1 mL of ZYM5052 medium containing lactose, which can induce the expression of the gene encoding the esterase variant inserted into the vector.
[0257] After culturing at 37°C for 24 hours, cells were harvested by centrifugation and the pellets were processed for subsequent analysis.
[0258] Example 3. High Throughput Screening (HTS) to Select Enzyme Variants with Reduced Product Inhibition
[0259] In an enzymatic reaction, the enzyme may be inhibited by either the substrate or the product.
[0260] Substrate inhibition: In this case, the initial specific activity decreases with increasing substrate concentration.
[0261] Product inhibition: In this case, the specific activity will drop to zero only when a certain concentration of product accumulates. Figure 3 is a representative of this phenomenon. As a direct consequence, incomplete biotransformation of substrates into products may occur.
[0262] 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; Appl Environ Microbiol. 1995; 61: 3208-15"), the esterase target in this study was subject to product inhibition, so in the mutant screening, specific activity was assessed in the absence and presence of the product (1R, 3R)-chrysanthemic acid, and the chromogenic substrate p-nitrophenylbutyrate was provided. The concentration of (1R, 3R)-chrysanthemic acid tested was equal to 100 mM, a value selected as the value obtained at the end of the bioconversion using the S315M enzyme.
[0263] The inhibition was then assessed based on the percentage of residual activity, or the ratio of the specific activity with acid to the specific activity without acid. Thus, the higher the percentage of residual activity, the lower the degree of inhibition.
[0264] Experimental plan:
[0265] by resuspending in 200 μL Cellytic Tm (enzyme mixture in detergent) and incubated with stirring at room temperature for 2 hours to dissolve the previously collected cell pellet. The obtained lysate was clarified by centrifugation and the supernatant, i.e. the diluted total crude extract, was used for screening.
[0266] Screening was performed in a 96-well plate format at 25°C for 10 minutes in 100 mM sodium phosphate buffer in the absence or presence of (1R,3R)chrysanthemic acid at a final concentration of 100 mM. The substrate, p-nitrophenylbutyrate, was provided at a final concentration of 4 mM in the sodium phosphate buffer. Changes in absorbance over time were monitored by taking readings at 405 nm every 16 seconds.
[0267] Therefore, enzyme activity (U / mL) is defined as:
[0268]
[0269] where ε is the molar extinction coefficient of p-nitrophenol.
[0270] To calculate the specific activity (U / mg) of the enzyme variants in the presence or absence of acid, total protein in the crude extracts was quantified by the Bradford method using BSA as the reference protein for the calibration curve.
[0271] Twenty-five enzyme variants were obtained and screened through the HTS program.
[0272] Example 4. Quantification of (1R,3R)chrysanthemic acid in 25 selected enzyme variants by HPLC
[0273] The production of (1R,3R)-chrysanthemic acid was evaluated against 25 enzyme variants screened in HTS, with ethyl chrysanthemate added as a substrate. Specifically, the acid product was quantified by reverse-phase HPLC analysis using a gradient elution solution consisting of 50% methanol, 50% acetonitrile, and 0.1% TFA.
[0274] The quantification of (1R,3R)chrysanthemic acid can be used for:
[0275] i) Calculate the specific enzyme activity (U / mg) of chrysanthemic acid ethyl ester, which is defined as
[0276]
[0277] ii) The experiment was carried out at a constant pH (pH stat) and the bioconversion yield of chrysanthemic acid ethyl ester substrate in (1R,3R) chrysanthemic acid was calculated.
[0278] This value is calculated by taking the ratio between the acid produced (g / L) and the maximum amount of acid that can be obtained (g / L).
[0279] Figure 4 The options implemented and discussed below are summarized.
[0280] After HTS of 1045 samples (provided with the chromogenic substrate p-nitrophenylbutyrate), 61 variants of the starting enzyme S315M were found to exhibit improved properties in terms of:
[0281] Increased residual activity %, or decreased product inhibition;
[0282] The specific activity (U / mg) was increased in the presence of (1R,3R)-chrysanthemic acid.
[0283] In particular, regarding the % residual activity, all 61 selected enzymes increased the residual activity by at least 25% compared to the starting enzyme.
[0284] Figure 5 and Figure 6 The residual activity and the % increase in specific activity (in the presence of (1R,3R)-chrysanthemic acid) of each mutant relative to the starting enzyme S315M are shown.
[0285] Of these 61 enzyme variants, we selected 25 that were retested using a “scale-up” procedure.
[0286] Specifically, 25 E. coli colonies expressing 25 enzyme variants were grown in 250 mL flasks with 25 mL autoinduction medium instead of 1 mL deep wells.
[0287] Therefore, the specific activities in the presence and absence of acid were re-evaluated by providing the chromogenic substrate p-nitrophenylbutyrate and the % increase in residual activity relative to the starting enzyme S315M was calculated ( Figure 7 ).
[0288] All 25 enzyme variants (obtained from flask-scale bacterial growth) showed an increased % residual activity compared to the S315M enzyme, confirming what was observed in the HTS, although the absolute values varied.
[0289] The 25 variants were also tested by providing ethyl chrysanthemate substrate.
[0290] Biotransformation of three selected enzyme variants (V274L-S315M-S331C, S315F, S223M) under pH stat
[0291] Three comparative mutants with an S315M reference were selected from the previous 25 mutants by performing a small-scale biotransformation using a chromogenic substrate but in the presence of chrysanthemic acid in the reaction mixture. Mutants with greater activity in the presence of potential inhibitors (product inhibition) were then selected, demonstrating that the effect of the acid was smaller. Subsequently, the performance was naturally estimated using the target substrate. Specifically, the pH stat reaction was carried out in a final volume of 20 mL using chrysanthemic acid ethyl ester racemate as a substrate at a concentration of 10% (v / v). The reaction was carried out at 45°C for 20 hours, during which the pH was maintained at 9.5 by adding 1 M NaOH. After the reaction, the amount of (1R,3R)-chrysanthemic acid was evaluated by HPLC analysis.
[0292] Results and Conclusions
[0293] Figure 8 The HPLC chromatogram of a previous bioconversion obtained after 20 hours (T20) using 10% chrysanthemic acid ethyl ester (v / v) is shown. Specifically, the peak at approximately 2 minutes corresponds to (1R,3R) chrysanthemic acid, while the peak at approximately 4.7 minutes is associated with all unconverted chrysanthemic acid ethyl ester.
[0294] To quantify the (1R,3R) chrysanthemic acid produced at the end of the reaction, a calibration curve was prepared using the standards of the above acids ( Figure 9 ), which relates peak areas to known acid concentrations (expressed in g / L) (Table 1).
[0295] To determine the bioconversion yield, we first calculated the theoretically achievable maximum yield. Analysis of the starting isomeric mixture of chrysanthemic acid ethyl ester revealed a concentration of 404.6 g / L of the enantiomer (1R,3R)-chrysanthemic acid ethyl ester. This mixture was used at a 10% (v / v) concentration, resulting in a reaction solution containing 40.46 g / L of (1R,3R)-chrysanthemic acid ethyl ester.
[0296] Considering the molecular weight of the obtained (1R,3R)-chrysanthemic acid, the maximum acid concentration achievable starting from 40.46 g / L of ester is equal to 34.67 g / L. Table 1 shows the obtained (1R,3R)-chrysanthemic acid data (g / L) and the relative yield (%) of the bioconversion (relative to the theoretical maximum yield). In addition, as a control, the values previously obtained for the WT enzyme are reported.
[0297] Table 1:
[0298]
[0299] As reported above, the introduction of the point mutation S315M resulted in a better catalyst than the wild-type enzyme, both in terms of specific activity ( Figure 1 - mutant 19), also in terms of lower product inhibition: in fact the bioconversion yield of (1R,3R)-ethyl chrysanthemate has increased from 49.5% to 94.1% (Table 1).
[0300] Furthermore, as shown in Table 1, the S223M variant performed similarly to the S315M enzyme; in fact, it converted a high percentage of the substrate, with a yield of approximately 89%, after 20 hours of incubation, compared to the theoretically achievable maximum yield.
[0301] In summary, among the tested mutants, the S315M, S223M, and S315F variants have been shown to be the best variants for catalyzing the selective bioconversion of (1R,3R)-chrysanthemic acid ethyl ester from a mixture of four possible stereoisomers to (1R,3R)-chrysanthemic acid.
[0302] Example 5. Enzymatic hydrolysis of racemic chrysanthemic acid ethyl ester 92 / 8
[0303] Chrysanthemic acid ethyl ester 92 / 8, consisting of four stereoisomers, was provided for enzymatic hydrolysis of the S315M mutant to produce (1R,3R)-chrysanthemic acid.
[0304] The enzymatic hydrolysis reaction was carried out in batches; glycine, enzyme, and aqueous sodium hydroxide were charged to the reactor, in addition to a 92 / 8 trans / cis ester mixture, which maintained an alkaline environment throughout the reaction. After the reaction, the enzyme, which was degraded by hydrochloric acid and denatured by trichloroacetic acid in the acidic environment, was removed by filtration. The remaining solution, containing the unconverted ester and the desired (1R,3R)chrysanthemic acid, was added to toluene to dissolve the ester, and aqueous sodium hydroxide (10% w / w) was added to dissolve the chrysanthemic acid in the water as its sodium salt. Separation of the two phases yielded a toluene solution containing the unconverted ester, which was fed to the subsequent workup stage. After acidification, the product (1R,3R)chrysanthemic acid was recovered from the aqueous phase by extraction with toluene followed by evaporation of the solvent.
[0305] The reaction was conducted in a mixture of 400 kg of racemic chrysanthemic acid ethyl ester 92 / 8, 16.7 kg of glycine, 444 kg of enzyme dissolved in water, and 3556 kg of 10% aqueous sodium hydroxide solution (containing 413 kg of sodium hydroxide). After the reaction, the starting material was acidified with 116 kg of 37% hydrochloric acid, treated with 11.6 kg of trichloroacetic acid to denature the enzyme, and filtered. Approximately 17.4 kg of denatured enzyme was obtained from the filtration and sent for disposal. A toluene and water solution was introduced to wash the filter, and approximately 868 kg of this solution was used to extract the ester.
[0306] The 5808 kg crude reaction product was a two-phase system consisting of a toluene solution of the ester and an acidic aqueous phase. 3055 kg of acidic water was discharged from the bottom of the reactor for treatment, and 61.8 kg of 50% aqueous sodium hydroxide solution was added to alkalize the environment and convert any acid in the water into sodium salts.
[0307] Phase separation yielded 1091 kg of toluene solution and 1724 kg of an aqueous phase containing chlorinated chrysanthemic acid. 77 kg of 37% (w / w) hydrochloric acid was added to this aqueous phase to yield the desired (1R,3R) chrysanthemic acid and 253 kg of toluene, which then passed into the organic phase. After separating 1666 kg of water for disposal, the toluene in the organic phase was evaporated, ultimately yielding 135 kg of (1R,3R) chrysanthemic acid, the sole (1R,3R) form, with a purity greater than 95%.
[0308] From the above description and the above examples, the advantages obtained by the products described and obtained according to the invention are evident.
Claims
1. A Arthrobacter globiformis esterase mutant, wherein the sequence of the mutant comprises a mutation of the 315th amino acid residue S, the 223rd amino acid residue S or the 298th amino acid residue F of SEQ ID NO:
2.
2. The Arthrobacter globiformis esterase mutant according to claim 1, wherein the mutant is capable of asymmetrically hydrolyzing racemic chrysanthemic acid ester.
3. The Arthrobacter globiformis esterase mutant according to any one of claims 1 or 2, wherein the 315th amino acid residue S, the 223rd amino acid residue S and the 298th amino acid residue F of SEQ ID NO:2 are replaced by non-polar amino acid residues selected from M, F, L, W, A, I, P or V.
4. The Arthrobacter globiformis esterase mutant according to claim 3, wherein the non-polar amino acid residue is selected from the group consisting of M, F, L or W.
5. The Arthrobacter globiformis esterase mutant according to any one of claims 1 to 4, which has one of the following mutations in the amino acid sequence of SEQ ID NO:2: S315M, S315F, S223M, S223L, S223F or F298W.
6. The Arthrobacter globiformis esterase mutant according to claim 5, which has one or two additional mutations selected from the group consisting of: 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.
7. The Arthrobacter globiformis esterase mutant according to any one of claims 1 to 6, wherein the mutant has the 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. The Arthrobacter globiformis esterase mutant according to any one of claims 1 to 7, wherein the mutant has the amino acid sequence of SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:
12.
9. The Arthrobacter globiformis esterase mutant according to any one of claims 1 to 8, wherein the mutant has the amino acid sequence of SEQ ID NO:
12.
10. The Arthrobacter globiformis esterase mutant according to any one of claims 1 to 9, wherein the mutant has the 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 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.
12. A transformed host microorganism comprising the expression vector according to claim 11, said expression vector comprising an Arthrobacter globiformis esterase mutant nucleotide sequence.
13. Use of the Arthrobacter globiformis esterase mutant 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 the Arthrobacter globiformis esterase mutant according to any one of claims 1 to 10 for the asymmetric hydrolysis of a cyclopropane racemic derivative having at least two chiral stereocenters.
15. Use of the Arthrobacter globiformis esterase mutant according to any one of claims 1 to 10 for asymmetric hydrolysis of chrysanthemic acid (C 1 -C 6 ) alkyl esters, preferably for the asymmetric hydrolysis of ethyl chrysanthemate.