JP7311496B2 - Modified esterase and use thereof - Google Patents

Description

The present invention relates to modified esterases. Specifically, a modified esterase with improved properties and uses thereof are provided. This application claims priority based on Japanese Patent Application No. 2018-088943 filed on May 2, 2018, the entire contents of which are incorporated by reference.

Esterases are enzymes that hydrolyze esters, and many esterases have been isolated from various organisms. Esterases derived from Acinetobacter calcoaceticus, which have unique and broad substrate specificities, are highly useful industrially. It can be used in a wide variety of applications, including decomposition of compounds (see, for example, Non-Patent Document 1).

Appl Microbial Biotechnol (2002) 60:288-292

However, since esterase is also a protein composed of amino acids, depending on the conditions of use (strong acid/strong alkali, high temperature, high concentration organic solvent, etc.), the catalytic function cannot be exhibited due to protein denaturation, so the range of application may be limited. . For example, synthetic reactions of pharmaceutical intermediates are often performed under severe conditions for enzymes (e.g., reactions at high temperatures, reactions in organic solvents, reactions in the acidic pH range), and stability is a particular problem. . Therefore, depending on the reaction conditions, sufficient enzymatic activity cannot be obtained, resulting in a decrease in reaction efficiency (yield).

Therefore, the present invention makes it possible to maximize the wide substrate specificity under various usage conditions by improving the properties of the esterase derived from Acinetobacter calcoaceticus, thereby increasing the usefulness of the esterase. The challenge is to increase the utility value. The improvement in properties brings about improvements in reaction efficiency and the like in existing uses, while enabling creation of new uses (that is, expansion of uses).

In order to solve the above problems, the present inventors attempted to modify an esterase derived from Acinetobacter calcoaceticus. After trial and error, we succeeded in identifying mutation points (amino acid residues) that are effective in improving its properties (specific activity, high-temperature stability, high-temperature reactivity, pH stability), and developed highly useful mutants ( A modified esterase) was obtained. This achievement is also important in terms of providing information and means for designing and obtaining mutants that can achieve the purpose of improving characteristics.

By the way, we often experience that the combination of two effective mutations is likely to produce an additive or synergistic effect. In addition, in view of the common general knowledge that the same type of enzyme has a high degree of similarity in structure (primary structure, three-dimensional structure) and that similar mutations are highly likely to produce similar effects, Acinetobacter having the amino acid sequence of SEQ ID NO: 1. If the useful mutations found in the esterase derived from calcoaceticus are applied to other esterases that have high structural similarity with the esterase, it is highly probable that equivalent effects will be achieved. , and those skilled in the art may recognize that such applications are useful.

The following inventions are based on the above results and considerations.
[1] In the amino acid sequence of SEQ ID NO: 1, an amino acid sequence containing one or more amino acid substitutions selected from the group consisting of K7P, F13Y, V107M, L222M, V229I and S235T, or 91% or more identical to the amino acid sequence having an amino acid sequence exhibiting a specific activity, and having improved one or more properties selected from the group consisting of specific activity, high-temperature stability, high-temperature reactivity, and pH stability, compared to the esterase consisting of the amino acid sequence of SEQ ID NO: 1. Modified esterase.
[2] The modified esterase of [1], wherein the amino acid substitution contained in the amino acid sequence of the modified esterase is S235T and the specific activity is improved.
[3] The modified esterase of [1], wherein the amino acid substitution in the amino acid sequence of the modified esterase is K7P, F13Y, L222M or V229I, and the high-temperature stability is improved.
[4] The modified esterase of [1], wherein the amino acid substitution contained in the amino acid sequence of the modified esterase is V229I or S235T, and the high-temperature reactivity is improved.
[5] The modified esterase of [1], wherein the amino acid substitutions contained in the amino acid sequence of the modified esterase are F13Y, V107M, L222M, V229I and S235T, and the pH stability is improved.
[6] An amino acid sequence of any of SEQ ID NOs: 2 to 7 or an amino acid sequence showing 91% or more identity with the amino acid sequence (however, if the identity criterion is the amino acid sequence of SEQ ID NO: 2, position 7 A position other than proline, a position other than 13th tyrosine when the identity criterion is the amino acid sequence of SEQ ID NO: 3, a position other than 107th methionine when the identity criterion is the amino acid sequence of SEQ ID NO: 4, and the identity Position other than methionine at position 222 when the reference is the amino acid sequence of SEQ ID NO: 5, position other than isoleucine at position 229 when the reference of identity is the amino acid sequence of SEQ ID NO: 6, and amino acid sequence of SEQ ID NO: 7 as the reference for identity In the case of , there is a difference in the amino acid sequence at positions other than position 235 threonine), compared to the esterase consisting of the amino acid sequence of SEQ ID NO: 1, specific activity, high temperature stability, high temperature reactivity and pH stability A modified esterase with improved one or more properties selected from the group.
[7] A gene encoding the modified esterase of any one of [1] to [6].
[8] The gene of [7], comprising any one of the nucleotide sequences of SEQ ID NOS:8-13.
[9] A recombinant DNA containing the gene of [7] or [8].
[10] A microorganism carrying the recombinant DNA of [9].
[11] The microorganism of [10], wherein the host is Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacillus aminorrhifaciens, or Brevibacillus choshinensis.
[12] An enzymatic agent containing the modified esterase of any one of [1] to [6].
[13] A method for preparing a modified esterase, comprising the following steps (I) to (III):
(I) providing a nucleic acid encoding the amino acid sequence of any of SEQ ID NOs: 2-7;
(II) expressing the nucleic acid, and
(III) Recovering the expression product.

Specific activity of mutants. The specific activity of the mutants was compared with the wild-type enzyme. High temperature stability of mutants. The residual activity rate after treatment at 80°C for 30 minutes was compared with that of the wild-type enzyme. High temperature reactivity of mutants. Relative activity (ratio of enzymatic activity at 60°C to enzymatic activity at 30°C) was compared to the wild-type enzyme. pH stability of the mutants. After 30 minutes of treatment at pH 4.0, the residual activity was compared with that of the wild-type enzyme.

For convenience of explanation, some of the terms used in connection with the present invention are defined below.
(the term)
The term "modified esterase" refers to an enzyme obtained by modifying or mutating a standard esterase (hereinafter referred to as "standard esterase"). The reference esterase is typically an esterase from Acinetobacter calcoaceticus having the amino acid sequence of SEQ ID NO:1.

The term "esterase derived from Acinetobacter calcoaceticus" refers to an esterase whose origin is Acinetobacter calcoaceticus, and the esterase produced by Acinetobacter calcoaceticus or the genetic information of the esterase is used. and esterases expressed in other microorganisms.

In the present invention, "amino acid substitution" is performed as modification or mutation. Therefore, when the modified esterase and the standard esterase are compared, some amino acid residues are different. In the present specification, the modified esterase is also referred to as modified enzyme or variant.

In this specification, according to convention, each amino acid is represented by one letter as follows.
Methionine: M, Serine: S, Alanine: A, Threonine: T, Valine: V, Tyrosine: Y, Leucine: L, Asparagine: N, Isoleucine: I, Glutamine: Q, Proline: P, Aspartic acid: D, Phenylalanine : F, glutamic acid: E, tryptophan: W, lysine: K, cysteine: C, arginine: R, glycine: G, histidine: H

In this specification, the positions of mutation points are specified by numbers assigned from the N-terminus to the C-terminus, with the N-terminal amino acid residue being the first.

In accordance with conventional practice, an amino acid residue to be subjected to amino acid substitution, that is, a "mutation point" is represented by a combination of one letter representing the type of amino acid and a number representing the position of the amino acid. In addition, "mutation" by amino acid substitution is expressed by adding one letter representing the type of amino acid after substitution to the right of the display of the mutation point. Therefore, for example, a mutation at position 7 lysine is expressed as "K7", and a mutation in which lysine at position 7 is substituted with proline is expressed as "K7P".

1. Modified Esterase A first aspect of the present invention relates to a modified esterase (modified enzyme). The modified enzyme of the present invention is typically an amino acid sequence containing one or more amino acid substitutions selected from the group consisting of K7P, F13Y, V107M, L222M, V229I and S235T in the amino acid sequence of SEQ ID NO: 1. have Due to these characteristics, the specific activity, high temperature stability, high temperature reactivity, pH stability, or two or more of these are improved compared to the esterase consisting of the amino acid sequence of SEQ ID NO:1. The amino acid sequence of SEQ ID NO: 1 is the sequence of an esterase derived from Acinetobacter calcoaceticus.

In order to facilitate understanding and judgment/judgment of high-temperature stability and high-temperature reactivity of modified enzymes, the high temperature for high-temperature stability is "75-85℃" and the high temperature for high-temperature reactivity is "55-65℃". , and Moreover, pH stability means stability in an acidic pH range. In order to facilitate understanding and judgment/judgment of pH stability, the acidic pH range here is 3 to 5.

The specific activity can be evaluated, for example, based on the activity calculated by the measurement method shown in Examples below. The modified enzyme with improved specific activity has a higher specific activity than the standard esterase (that is, the esterase consisting of the amino acid sequence of SEQ ID NO: 1). The specific activity of the modified enzyme is, for example, 1.1- to 1.3-fold that of the standard esterase.

High-temperature stability can be evaluated, for example, based on residual activity after treatment at 80°C for 30 minutes. The modified enzyme with improved high-temperature stability has a higher residual activity rate than the standard esterase (that is, the esterase consisting of the amino acid sequence of SEQ ID NO: 1). The residual activity rate of the modified enzyme is, for example, 2 to 10 times the residual activity rate of the standard esterase. Preferably, it is 3 to 10 times. The active residual rate is calculated as follows.
Activity residual rate = (enzyme activity after heat treatment/enzyme activity before heat treatment) x 100 (%)

High-temperature reactivity can be evaluated, for example, based on the ratio of enzyme activity at 60°C to enzyme activity at 30°C (relative activity at 60°C based on activity at 30°C). The modified enzyme with improved high-temperature reactivity has a higher relative activity than the standard esterase (that is, the esterase consisting of the amino acid sequence of SEQ ID NO: 1). The relative activity of the modified enzyme is, for example, 1.2-2 times the relative activity of the standard esterase. Preferably, it is 1.5 times to 2 times. Relative activity is calculated as follows.
Relative activity = (enzyme activity at 60°C/enzyme activity at 30°C) x 100 (%)

pH stability can be evaluated, for example, based on the residual activity when treated at pH 4 for 30 minutes. The modified enzyme with improved pH stability has a higher residual activity rate than the standard esterase (that is, the esterase consisting of the amino acid sequence of SEQ ID NO: 1). The residual activity rate of the modified enzyme is, for example, 1.08 to 1.3 times the residual activity rate of the standard esterase. Preferably, it is 1.15 times to 3 times. The active residual rate is calculated as follows.
Activity residual rate = (enzyme activity after treatment/enzyme activity before treatment) x 100 (%)

As used herein, “including amino acid substitution” means that the mutation point (that is, the position of the amino acid residue where a specific amino acid substitution occurs) is the amino acid after substitution. Therefore, when an amino acid sequence containing an amino acid substitution (variant amino acid sequence) is compared with the amino acid sequence of SEQ ID NO: 1 not containing an amino acid substitution (reference amino acid sequence), a difference in amino acid residue is observed at the position of the amino acid substitution. It will be.

K7P represents a mutation in which the 7th amino acid (lysine) in the amino acid sequence of SEQ ID NO: 1 is substituted with proline. F13Y represents a mutation in which the 13th amino acid (phenylalanine) in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine. V107M represents a mutation in which the 107th amino acid (valine) in the amino acid sequence of SEQ ID NO: 1 is replaced with methionine. L222M represents a mutation in which the 222nd amino acid (leucine) in the amino acid sequence of SEQ ID NO: 1 is replaced with methionine. V229I represents a mutation in which the 229th amino acid (valine) in the amino acid sequence of SEQ ID NO: 1 is replaced with isoleucine. S235T represents a mutation in which the 235th amino acid (serine) in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine.

As a specific example of the modified enzyme of the present invention, an esterase consisting of the amino acid sequence of any of SEQ ID NOs: 2 to 7 (mutant 1 (K7P), mutant 2 (F13Y), mutant 3 (V107M), mutant variant 4 (L222M), variant 5 (V229I) and variant 6 (S235T) correspond). As shown in Examples below, the esterase having the amino acid sequence of SEQ ID NO: 2 (mutant 1 (K7P)) has improved high-temperature stability, and the esterase having the amino acid sequence of SEQ ID NO: 3 (mutant 2 ( F13Y)) has improved high temperature stability and pH stability, the esterase (mutant 3 (V107M)) consisting of the amino acid sequence of SEQ ID NO: 4 has improved pH stability, The esterase consisting of the amino acid sequence (mutant 4 (L222M)) has improved high temperature stability and pH stability, the esterase consisting of the amino acid sequence of SEQ ID NO: 6 (mutant 5 (V229I)) has high temperature stability, High-temperature reactivity and pH stability are improved, and the esterase (mutant 6 (S235T)) consisting of the amino acid sequence of SEQ ID NO: 7 has improved specific activity, high-temperature reactivity, and pH stability. Confirmed.

In general, when part of the amino acid sequence of a certain protein is mutated, the protein after mutation may have the same function as the protein before mutation. That is, amino acid sequence mutations do not substantially affect protein function, and protein function may be maintained before and after the mutation. On the other hand, when the amino acid sequences of two proteins are highly identical, there is a high probability that the two will exhibit similar properties. Considering these common technical knowledge, the amino acid sequence of the modified enzyme, that is, "the amino acid sequence of SEQ ID NO: 1, selected from the group consisting of K7P, F13Y, V107M, E132P, S155P, L222M, V229I and S235T An amino acid sequence containing one or more amino acid substitutions (specific examples of such amino acid sequences are the amino acid sequences of SEQ ID NOs: 2 to 7)”, but not completely identical (i.e., 100% identical), but exhibiting a high degree of identity If it has the sequence and the desired properties are improved, it can be regarded as an enzyme that is substantially the same as the modified enzyme (substantially the same esterase). The identity here is preferably 90% or more, more preferably 91% or more, still more preferably 92% or more, still more preferably 93% or more, even more preferably 95% or more, even more preferably 98% or more, and most preferably 98% or more. Preferably it is 99% or more. A slight difference in amino acid sequence will be observed when comparing the above modified enzyme with the substantially identical esterase. However, differences in amino acid sequences shall occur at positions other than the positions where the above amino acid substitutions have been made. Therefore, when the identity criterion is the amino acid sequence of SEQ ID NO: 2, positions other than proline at position 7, when the identity criterion is the amino acid sequence of SEQ ID NO: 3, positions other than position 13 tyrosine, and the identity criterion is the sequence Position other than methionine at position 107 in the case of the amino acid sequence of number 4, position other than methionine at position 222 in the case of the amino acid sequence of SEQ ID NO: 5 with the identity standard, and position other than the methionine at position 222 in the case of the amino acid sequence of SEQ ID NO: 6 with the identity standard Amino acid sequence differences occur at positions other than isoleucine at position 229, and at positions other than threonine at position 235 when the identity criterion is the amino acid sequence of SEQ ID NO:7. In other words, in the amino acid sequence showing 90% or more identity with the amino acid sequence of SEQ ID NO:2, amino acid 7 is proline. Similarly, in the amino acid sequence showing 90% or more identity with the amino acid sequence of SEQ ID NO: 3, the 13th amino acid is tyrosine, and in the amino acid sequence showing 90% or more identity with the amino acid sequence of SEQ ID NO: 4, the 107th amino acid. is methionine, amino acid 222 is methionine in the amino acid sequence showing 91% or more identity with the amino acid sequence of SEQ ID NO: 5, and 229 in the amino acid sequence showing 91% or more identity with the amino acid sequence of SEQ ID NO: 6 The amino acid at position 235 is isoleucine, and in the amino acid sequence showing 90% or more identity with the amino acid sequence of SEQ ID NO:7, the amino acid at position 235 is threonine.

A "slight difference in amino acid sequence" herein is caused by deletion, substitution, addition, insertion, or a combination thereof. Typically, one to several (upper limit is, for example, 3, 5, 7, 10) amino acids constituting the amino acid sequence are deleted or substituted, or one to several (upper limit is, for example, 3, A mutation (change) in an amino acid sequence due to the addition or insertion of 5, 7, or 10) amino acids, or a combination thereof. "Minor amino acid sequence differences" are preferably caused by conservative amino acid substitutions. As used herein, "conservative amino acid substitution" refers to substitution of an amino acid residue with an amino acid residue having a side chain with similar properties. Amino acid residues can have, depending on their side chain, a basic side chain (e.g. lysine, arginine, histidine), an acidic side chain (e.g. aspartic acid, glutamic acid), an uncharged polar side chain (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine). , cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine), aromatic side chains (e.g. tyrosine, phenylalanine, Tryptophan, histidine) are classified into several families. Conservative amino acid substitutions are preferably between amino acid residues within the same family. In addition, since it is known that the amino acid residues constituting the active center of Acinetobacter calcoaceticus-derived esterase (SEQ ID NO: 1) are serine at position 97, aspartic acid at position 226 and histidine at position 256, mutation should not affect these amino acid residues when applying As an example in which a slight difference in the amino acid sequence does not substantially affect the function of the protein and the function of the protein is maintained before and after the mutation, amino acid 132 (glutamic acid) in the amino acid sequence of SEQ ID NO: 1 is proline. , an amino acid sequence in which the 192nd amino acid (glycine) in the amino acid sequence of SEQ ID NO: 1 is serine, or an amino acid sequence in which the 244th amino acid (leucine) in the amino acid sequence of SEQ ID NO: 1 is proline. As a result of comparing the specific activity and high-temperature stability of the esterases having these amino acid sequences, they were equivalent to those of SEQ ID NO:1.

By the way, the identity (%) of two amino acid sequences or two base sequences (hereinafter the term "two sequences" is used as a term including these) can be determined, for example, by the following procedure. First, the two sequences are aligned for optimal comparison (eg, gaps may be introduced in the first sequence to optimize alignment with the second sequence). When a molecule (amino acid residue or nucleotide) at a particular position in the first sequence is the same as the molecule at the corresponding position in the second sequence, the molecules at that position are said to be identical. The identity of two sequences is a function of the number of identical positions common to the two sequences (i.e., % identity = number of identical positions/total number of positions x 100), and is preferably optimized for alignment. Also take into account the number and size of gaps required for conversion.

The comparison of two sequences and determination of identity can be accomplished using a mathematical algorithm. Specific examples of mathematical algorithms that can be used for sequence comparison are described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68 and Karlin and Altschul (1993) Proc. Acad. Sci. USA 90:5873-77 includes, but is not limited to, a modified algorithm. Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) described by Altschul et al. (1990) J. Mol. Biol. 215:403-10. To obtain equivalent nucleotide sequences, for example, BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12. To obtain equivalent amino acid sequences, for example, BLAST polypeptide searches can be performed with the XBLAST program, score=50, wordlength=3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Amino Acids Research 25(17):3389-3402. When utilizing BLAST and Gapped BLAST, the default parameters of the corresponding programs (eg, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov for more information. Another example of a mathematical algorithm that can be used for sequence comparison is the algorithm described in Myers and Miller (1988) Comput Appl Biosci. 4:11-17. Such algorithms are embedded in the ALIGN program available, for example, on the GENESTREAM network server (IGH Montpellier, France) or on the ISREC server. For example, when utilizing the ALIGN program for comparing amino acid sequences, a PAM120 residue mass table can be used, gap length penalty=12, gap penalty=4.

The identity of two amino acid sequences was determined using the GAP program of the GCG software package using the Blossom 62 matrix or the PAM250 matrix, gap weight = 12, 10, 8, 6, or 4, gap length weight = 2, 3. , or can be determined as 4. The identity of two base sequences can also be determined using the GAP program of the GCG software package (available at http://www.gcg.com) with gap weight = 50 and gap length weight = 3. can.

Typically, the esterase consisting of the amino acid sequence of SEQ ID NO: 1, that is, the esterase derived from Acinetobacter calcoaceticus is mutated (one or more of the above K7P, F13Y, V107M, L222M, V229I and S235T combination) results in the modified enzyme of the present invention. With respect to the above substantially identical esterase, the esterase consisting of the amino acid sequence of SEQ ID NO: 1 is mutated (by one or a combination of two or more of the above K7P, F13Y, V107M, L222M, V229I and S235T) and is further mutated. In addition, it is equivalent to an esterase consisting of an amino acid sequence highly identical to the amino acid sequence of SEQ ID NO: 1, such as an esterase derived from the same species and genus as Acinetobacter calcoaceticus strain that produces an esterase consisting of the amino acid sequence of SEQ ID NO: 1. or by adding further mutations to those obtained by said mutations. In the "equivalent mutation" here, in the amino acid sequence highly identical to the amino acid sequence of SEQ ID NO: 1, the mutation points in the present invention (positions 7, 13, 107, 222, 229 or 235) will be substituted. An example of an esterase having an amino acid sequence highly identical to the amino acid sequence of SEQ ID NO: 1 is an esterase derived from Acinetobacter guillouiae and having the amino acid sequence of SEQ ID NO: 15 (98% amino acid sequence identity). ), an esterase derived from Acinetobacter baumannii ABNIH3 (Acinetobacter baumannii ABNIH3) and having the amino acid sequence of SEQ ID NO: 16 (97% amino acid sequence identity), derived from Acinetobacter seifertii and having SEQ ID NO: 17 Esterase having an amino acid sequence (90% amino acid sequence identity), derived from Pseudomonas aeruginosa and having an amino acid sequence of SEQ ID NO: 18 (80% amino acid sequence identity), Pseudomonas aeruginosa aeruginosa) and having the amino acid sequence of SEQ ID NO: 19 (80% amino acid sequence identity), an esterase derived from Pseudomonas aeruginosa and having the amino acid sequence of SEQ ID NO: 20 (amino acid sequence identity) sex 80%), derived from Burkholderia ubonensis, an esterase having the amino acid sequence of SEQ ID NO: 21 (81% amino acid sequence identity), derived from Paraburkholderia ferrariae , an esterase having the amino acid sequence of SEQ ID NO: 22 (81% amino acid sequence identity), an esterase derived from Burkholderia pseudomultivorans and having the amino acid sequence of SEQ ID NO: 23 (amino acid sequence identity NBRC 110496 (Acinetobacter sp. NBRC 110496), an esterase having the amino acid sequence of SEQ ID NO: 24 (81% amino acid sequence identity), derived from Acinetobacter seifertii and an esterase having the amino acid sequence of SEQ ID NO: 25 (90% amino acid sequence identity), an esterase derived from Acinetobacter sp. NIPH809 (Acinetobacter sp. NIPH809) having the amino acid sequence of SEQ ID NO: 26 (amino acid sequence 80%), derived from Pseudomonas fluorescens A506 (A506), an esterase having the amino acid sequence of SEQ ID NO: 27 (80% amino acid sequence identity), derived from Pseudomonas sp. ABAC61 (Pseudomonas sp. ABAC61) and an esterase having the amino acid sequence of SEQ ID NO: 28 (amino acid sequence identity of 79%), an esterase derived from Pseudomonas putida IFO12996 and having the amino acid sequence of SEQ ID NO: 29 (amino acid sequence identity of 76%). %), an esterase derived from Pseudomonas putida MR2068 (Pseudomonas putida MR2068) and having the amino acid sequence of SEQ ID NO: 30 (amino acid sequence identity 76%), derived from Pseudomonas aeruginosa (Pseudomonas aeruginosa) and having the amino acid sequence of SEQ ID NO: 31 Esterases with amino acid sequences (80% amino acid sequence identity) can be mentioned.

The term "corresponding" as used herein with respect to amino acid residues means that they contribute equally to the function of the compared proteins (enzymes). For example, the amino acid sequence to be compared is arranged with respect to the amino acid sequence of the reference esterase (amino acid sequence of SEQ ID NO: 1) so as to enable optimal comparison while considering partial homology of the primary structure (amino acid sequence). (At this time gaps may be introduced to optimize the alignment if necessary), identifying the amino acid at the position corresponding to the specific amino acid in the reference amino acid sequence as the "corresponding amino acid" can be done. "Corresponding amino acids" can also be identified by comparison between conformations (three-dimensional structures) instead of or in addition to comparison between primary structures. The use of conformational information provides highly reliable comparison results. In this case, a method of performing alignment while comparing the atomic coordinates of the three-dimensional structures of a plurality of enzymes can be adopted. The three-dimensional structure information of the enzyme to be mutated can be obtained from Protein Data Bank (http://www.pdbj.org/index_j.html), for example.

An example of a method for determining protein conformation by X-ray crystal structure analysis is shown below.
(1) Crystallize the protein. Crystallization is indispensable for three-dimensional structure determination, but it is also industrially useful as a method for purifying proteins with high purity and as a method for high-density, stable storage. In this case, it is preferable to crystallize a protein bound with a substrate or an analogue thereof as a ligand.
(2) Irradiate X-rays to the produced crystal and collect diffraction data. It should be noted that there are many cases in which protein crystals are damaged by X-ray irradiation and their diffraction performance deteriorates. In that case, cryogenic measurement technology, which rapidly cools the crystal to about -173°C and collects diffraction data in that state, has recently become popular. Ultimately, high-brightness synchrotron radiation is used to collect high-resolution data for structural determination.
(3) Crystal structure analysis requires phase information in addition to diffraction data. If the crystal structure of a protein related to the target protein is unknown, it is impossible to determine the structure by the molecular replacement method, and the phase problem must be solved by the heavy atom isomorphic replacement method. The heavy atom isomorphic replacement method is a method of introducing metal atoms with large atomic numbers such as mercury and platinum into crystals and obtaining phase information by utilizing the contribution of the large X-ray scattering power of the metal atoms to the X-ray diffraction data. . The determined phase can be improved by smoothing the electron density of the solvent regions in the crystal. Since the water molecules in the solvent region fluctuate greatly, the electron density is hardly observed, so by approximating the electron density in this region to 0, the true electron density can be approached, and the phase is improved. . Also, if the asymmetric unit contains multiple molecules, averaging the electron densities of these molecules further improves the phase significantly. A model of the protein is fitted to the electron density map calculated using the phase improved in this way. This process is performed on computer graphics using programs such as QUANTA from MSI (USA). After this, structural refinement is performed using programs such as MSI's X-PLOR, and the structural analysis is completed. If the crystal structure of a protein related to the protein of interest is known, it can be determined by molecular replacement using the atomic coordinates of the known protein. Molecular replacement and structural refinement can be performed using programs such as CNS_SOLVE ver.11.

2. Nucleic Acid Encoding Modified Esterase, etc. The second aspect of the present invention provides a nucleic acid related to the modified enzyme of the present invention. That is, it can be used as a gene encoding a modified enzyme, a nucleic acid that can be used as a probe for identifying a nucleic acid encoding a modified enzyme, or a primer for amplifying or mutating a nucleic acid encoding a modified enzyme. Nucleic acids are provided that are capable of

A gene encoding a modified enzyme is typically used to prepare the modified enzyme. According to the genetic engineering preparation method using the gene encoding the modified enzyme, it is possible to obtain the modified enzyme in a more homogeneous state. In addition, the method can also be said to be a suitable method for preparing a large amount of modified enzyme. The use of genes encoding modified enzymes is not limited to the preparation of modified enzymes. For example, the nucleic acid can be used as an experimental tool for elucidating the mechanism of action of the modified enzyme, or as a tool for designing or producing further modified enzymes.

As used herein, the term "gene encoding a modified enzyme" refers to a nucleic acid from which the modified enzyme is obtained when expressed, and has a base sequence corresponding to the amino acid sequence of the modified enzyme. Nucleic acids, of course, also include nucleic acids in which a sequence that does not encode an amino acid sequence is added to such nucleic acids. Codon degeneracy is also taken into account.

Examples of sequences (nucleotide sequences) of genes encoding modified enzymes are shown in SEQ ID NOS:8-13. These sequences, as described below, encode the variants shown in the Examples below.
SEQ ID NO: 8: Mutant 1 (K7P)
SEQ ID NO: 9: Mutant 2 (F13Y)
SEQ ID NO: 10: Mutant 3 (V107M)
SEQ ID NO: 11: Mutant 4 (L222M)
SEQ ID NO: 12: Mutant 5 (V229I)
SEQ ID NO: 13: Mutant 6 (S235T)

Since the enzyme of the present invention does not have a signal sequence, it is normally not secreted outside the cells. may be introduced into the host with a gene construct to which a sequence encoding a signal peptide (signal sequence) is added. A signal sequence may be selected according to the host. Any signal sequence capable of expressing the desired mutant can be used in the present invention. Available signal sequences include the sequence encoding the signal peptide of α-factor (Protein Engineering, 1996, vol9, p.1055-1061), the sequence encoding the signal peptide of α-factor receptor, the signal peptide of SUC2 protein. sequence encoding the signal peptide of the PHO5 protein, sequence encoding the signal peptide of the BGL2 protein, sequence encoding the signal peptide of the AGA2 protein, sequence encoding the signal peptide of TorA (trimethylamine N-oxidoreductase) , sequence encoding PhoD (phosphoesterase) signal peptide derived from Bacillus subtilis, sequence encoding LipA (esterase) signal peptide derived from Bacillus subtilis, sequence encoding signal peptide of Taka-amylase derived from Aspergillus oryzae ( Japanese Unexamined Patent Application Publication No. 2009-60804), a sequence encoding a signal peptide of α-amylase derived from Bacillus amyloliquefaciens (Eur. J. Biochem. 155, 577-581 (1986)), a medium derived from Bacillus subtilis sequence encoding the signal peptide of sexual protease (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1995, p. 1610-1613 Vol. 61, No. 4), sequence encoding the signal peptide of cellulase derived from bacteria belonging to the genus Bacillus (JP-A-2007- 130012) can be exemplified.

The nucleic acid of the present invention can be obtained by referring to the sequence information disclosed in this specification or the attached sequence listing and using standard genetic engineering techniques, molecular biological techniques, biochemical techniques, chemical synthesis, etc. It can be prepared in an isolated state.

In another aspect of the present invention, a nucleic acid having a partly different base sequence (hereinafter referred to as a Also referred to as a "homologous nucleic acid."Also, a nucleotide sequence that defines a homologous nucleic acid is also referred to as a "homologous nucleotide sequence." An example of a homologous nucleic acid is a nucleotide sequence containing one or more base substitutions, deletions, insertions, additions, or inversions based on the nucleotide sequence of the nucleic acid encoding the modified enzyme of the present invention. DNA encoding a protein having an enzymatic activity (ie esterase activity) characteristic of . Substitution or deletion of bases may occur at multiple sites. The term "plurality" as used herein means, for example, 2 to 40 bases, preferably 2 to 20 bases, more preferably 2 to 10 bases, although it varies depending on the positions and types of amino acid residues in the three-dimensional structure of the protein encoded by the nucleic acid. is.

Homologous nucleic acid is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, still more preferably about 90% or more, even more preferably about 90% or more, relative to the reference base sequence share 95% or greater, most preferably 99% or greater identity.

Homologous nucleic acids such as those described above can be obtained by, for example, restriction enzyme treatment, treatment with exonuclease or DNA ligase, site-directed mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York) or random mutation. It can be obtained by introducing a mutation by a mutagenesis method (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York). Homologous nucleic acids can also be obtained by other methods such as ultraviolet irradiation.

Another aspect of the present invention relates to a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of the gene encoding the modified enzyme of the present invention. Still another aspect of the present invention is that at least about 60%, 70%, 80%, 90%, 95%, Nucleic acids having 99% and 99.9% identical base sequences are provided.

Still another aspect of the present invention is a nucleic acid having a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence of the gene encoding the modified enzyme of the present invention or its homologous nucleotide sequence. Regarding. The term "stringent conditions" as used herein refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Such stringent conditions are known to those skilled in the art and can be found, for example, in Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) and Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). can be set by referring to Stringent conditions include, for example, a hybridization solution (50% formamide, 10xSSC (0.15M NaCl, 15mM sodium citrate, pH 7.0), 5x Denhardt solution, 1% SDS, 10% dextran sulfate, 10μg/ml denaturation Conditions of incubation with salmon sperm DNA, 50 mM phosphate buffer (pH 7.5) at about 42° C. to about 50° C., followed by washing with 0.1×SSC, 0.1% SDS at about 65° C. to about 70° C. can be mentioned. More preferred stringent conditions include, for example, a hybridization solution of 50% formamide, 5×SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 1×Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg/ml. denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)).

Yet another aspect of the present invention provides a nucleic acid (nucleic acid fragment) having a nucleotide sequence of a gene encoding the modified enzyme of the present invention, or a portion of the nucleotide sequence complementary thereto. Such a nucleic acid fragment can be used for detecting, identifying and/or amplifying a nucleic acid having the base sequence of the gene encoding the modified enzyme of the present invention. A nucleic acid fragment is, for example, a continuous nucleotide portion (for example, about 10 to about 100 bases long, preferably about 20 to about 100 bases long, more preferably about 30 to about 100 bases long). Nucleic acid fragments can be labeled when used as probes. For example, fluorescent substances, enzymes, and radioactive isotopes can be used for labeling.

Yet another aspect of the present invention relates to a recombinant DNA containing the gene of the present invention (the gene encoding the modified enzyme). The recombinant DNA of the present invention is provided, for example, in the form of a vector. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting a nucleic acid inserted into it into a target such as a cell.

A suitable vector is selected depending on the purpose of use (cloning, expression of protein) and in consideration of the type of host cell. Examples of E. coli host vectors include M13 phage or its variants, λ phage or variants thereof, pBR322 or variants thereof (pB325, pAT153, pUC8, etc.), pET21, etc. Yeast hosts include pYepSec1 and pMFa. , pYES2, pPIC3.5K and the like, pAc, pVL and the like for insect cell host vectors, and pCDM8, pMT2PC and the like for mammalian cell host vectors.

The vectors of the invention are preferably expression vectors. The term “expression vector” refers to a vector into which a nucleic acid inserted therein can be introduced into a target cell (host cell) and expressed in the cell. An expression vector usually contains a promoter sequence required for expression of the inserted nucleic acid, an enhancer sequence that promotes expression, and the like. Expression vectors containing selectable markers can also be used. When such an expression vector is used, the presence or absence (and degree of introduction) of the expression vector can be confirmed using a selection marker.

Insertion of the nucleic acid of the present invention into a vector, insertion of a selectable marker gene (if necessary), insertion of a promoter (if necessary), etc. can be performed using standard recombinant DNA techniques (e.g., Molecular Cloning, Third Edition, 1.84, Cold well-known methods using restriction enzymes and DNA ligase, see Spring Harbor Laboratory Press, New York).

As the host cell, from the viewpoint of ease of handling, Aspergillus oryzae (for example, Aspergillus oryzae), Bacillus spp.・Microorganisms such as Choshinensis), Escherichia coli (Escherichia coli), and budding yeast (Saccharomyces cerevisiae) can be used. is available. Escherichia coli (Escherichia coli) and budding yeast (Saccharomyces cerevisiae) are preferably used. Microorganisms of the genus Acinetobacter (for example, Acinetobacter calcoaceticus can also be used as hosts. Examples of E. coli include E. coli BL21 (DE3) when using a T7 promoter, and E. coli JM109 when not. Examples of budding yeast include budding yeast SHY2, budding yeast AH22, and budding yeast INVSc1 (Invitrogen).

Another aspect of the invention relates to microorganisms (ie, transformants) harboring recombinant DNAs of the invention. The microorganism of the present invention can be obtained by transfection or transformation using the vector of the present invention. For example, the calcium chloride method (J. Mol. Biol., Vol. 53, p. 159 (1970)), the Hanahan method (J. Mol. Biology, Vol. 166, No. 557). (1983)), the SEM method (Gene, Vol. 96, p. 23 (1990)), the method of Chung et al. (Proceedings of the National Academy of Sciences of the USA, No. 86). 2172 (1989)), calcium phosphate coprecipitation method, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. U.S.A. 81, 7161-7165 (1984)), lipofection (Felgner, et al., Proc. Natl. Acad. Sci. U.S.A. 84, 7413-7417 (1984)). In addition, the microorganism of the present invention can be used to produce the modified enzyme of the present invention.

3. Enzyme Agent Containing Modified Esterase The modified enzyme of the present invention is provided, for example, in the form of an enzyme agent. In addition to the active ingredient (the modified enzyme of the present invention), the enzymatic agent may contain excipients, buffers, suspending agents, stabilizers, preservatives, preservatives, physiological saline, and the like. As excipients, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol and the like can be used. Phosphate, citrate, acetate and the like can be used as buffers. Propylene glycol, ascorbic acid and the like can be used as stabilizers. Preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like. Ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, etc. can be used as antiseptics.

4. Use of Modified Esterase A further aspect of the present invention relates to the use of the modified enzyme and enzyme agent, and various reactions using the modified enzyme or enzyme agent of the present invention and methods for producing/manufacturing various compounds using the reaction. provided. Specifically, the modified enzyme or enzymatic agent of the present invention can be used for (1) production of organic compounds that are used in pharmaceuticals, (2) production of ester compounds that can be used in the field of fine chemicals, (3) lactone It can be used for hydrolysis of compounds, etc. As an example of (1), DL-MATI (DL-β-acetylthioisobutyrate ) by enantioselective hydrolysis (see Journal of Molecular Catalysis B: Enzymatic 38 (2006) 163-170). On the other hand, as an example of (2), production of linear ester compounds (Methyl (R)-3-bromo-2-methylpropionate, Methyl cetraxate hydrochloride, etc.) with various substituents on the side chain (Appl Microbiol Biotechnol ( 2002) 60: 288-292). As examples of (3), decomposition of aldone lactones (D-galactono-γ-lactone, L-mannono-γ-lactone, etc.) and aromatic lactones (3,4-dihydrocoumarin, homogentisic acid γ-lactone, etc.) (see Eur. J. Biochem. 267, 3-10 (2000)).

5. Method for Preparing Modified Esterase A further aspect of the present invention relates to a method for preparing a modified enzyme. In one aspect of the preparation method of the present invention, the modified enzyme successfully obtained by the present inventors is prepared by genetic engineering techniques. In this embodiment, a nucleic acid encoding an amino acid sequence of any of SEQ ID NOS: 2-7 is provided (step (I)). Here, the "nucleic acid encoding a specific amino acid sequence" is a nucleic acid that, when expressed, yields a polypeptide having the amino acid sequence. That is, extra sequences (which may or may not encode amino acid sequences) may be added to such nucleic acids. Codon degeneracy is also taken into account. "Nucleic acid encoding any one of the amino acid sequences of SEQ ID NOS: 2 to 7" refers to the sequence information disclosed in this specification or the attached sequence listing, standard genetic engineering techniques, molecular biological techniques, It can be prepared in an isolated state by using biochemical techniques and the like. Here, the amino acid sequences of SEQ ID NOs: 2 to 7 are all mutated amino acid sequences of Acinetobacter calcoaceticus-derived esterases. Therefore, a nucleic acid (gene) encoding any of the amino acid sequences of SEQ ID NOs: 2 to 7 can also be obtained by adding necessary mutations to the gene encoding the Acinetobacter calcoaceticus-derived esterase. A number of methods for site-specific nucleotide sequence replacement are known in the art (see, for example, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York), and an appropriate method is selected from among them. can be used A site-directed amino acid saturation mutagenesis method can be employed as the site-directed mutagenesis method. The site-specific amino acid saturation mutagenesis method is a "semi-rational, semi-random" technique that presumes the positions involved in the desired function based on the three-dimensional structure of the protein and introduces amino acid saturation mutations (J.Mol. Biol. 331, 585-592 (2003)). For example, a site-specific amino acid saturation mutation can be introduced using a kit such as Quick change (Stratagene) or Overlap extention PCR (Nucleic Acid Res. 16, 7351-7367 (1988)). Taq polymerase etc. can be used for the DNA polymerase used for PCR. However, it is preferable to use highly accurate DNA polymerases such as KOD-PLUS- (Toyobo) and Pfu turbo (Stratagene).

Following step (I), the prepared nucleic acid is expressed (step (II)). For example, first, an expression vector into which the above nucleic acid is inserted is prepared and used to transform host cells.

Next, the transformant is cultured under conditions in which the expression product, the modified enzyme, is produced. Cultivation of transformants may be carried out according to conventional methods. The carbon source used in the medium may be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, molasses, and pyruvic acid. The nitrogen source may be any available nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkaline extract, and the like. In addition, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.

On the other hand, the culture temperature can be set within the range of 30°C to 40°C (preferably around 33°C to 37°C). The culture time can be set in consideration of the growth characteristics of the transformant to be cultured, the production characteristics of the modified enzyme, and the like. The pH of the medium is adjusted within the range in which transformants grow and enzymes are produced. The pH of the medium is preferably about 6.0 to 9.0 (preferably around pH 7.0).

Subsequently, the expression product (modified enzyme) is collected (step (III)). The culture solution containing the cultured cells can be used as it is, or after concentration or removal of impurities, it can be used as an enzyme solution. If the expression product is a secretory protein, it can be recovered from the culture medium, otherwise it can be recovered from the cells. When collecting from the culture medium, for example, the culture supernatant is filtered and centrifuged to remove insoluble matter, and then concentrated under reduced pressure, membrane concentration, salting out using ammonium sulfate or sodium sulfate, methanol, ethanol, or acetone. Various chromatography methods such as fractional sedimentation, dialysis, heat treatment, isoelectric point treatment, gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography (for example, Sephadex gel (GE Healthcare Bioscience) Separation using a combination of gel filtration, DEAE Sepharose CL-6B (GE Healthcare Bioscience), Octyl Sepharose CL-6B (GE Healthcare Bioscience), CM Sepharose CL-6B (GE Healthcare Bioscience), etc. A purified product of the modified enzyme can be obtained by purification. On the other hand, when collecting from the cells, the cells are collected by filtering or centrifuging the culture solution, and then the cells are subjected to mechanical methods such as pressure treatment, ultrasonic treatment, physical crushing treatment, or lysozyme. After disruption by an enzymatic method such as the above, the purified modified enzyme can be obtained by separating and purifying in the same manner as described above.

The purified enzyme obtained as described above can be powdered by, for example, freeze-drying, vacuum-drying, or spray-drying. At that time, the purified enzyme may be dissolved in advance in a phosphate buffer, triethanolamine buffer, Tris-HCl buffer, or GOOD buffer. Phosphate buffer and triethanolamine buffer are preferably used. The GOOD buffer includes PIPES, MES or MOPS.

As described above, an appropriate host-vector system is usually used to express a gene and recover an expression product (modified enzyme), but a cell-free synthesis system may also be used. Here, the "cell-free synthesis system (cell-free transcription system, cell-free transcription/translation system)" does not use living cells, but ribosomes derived from living cells (or obtained by genetic engineering techniques), It refers to the in vitro synthesis of mRNA or protein encoded by a template nucleic acid (DNA or mRNA) using transcription/translation factors. A cell-free synthesis system generally uses a cell extract obtained by purifying a cell lysate as necessary. Cell extracts generally contain various factors such as ribosomes and initiation factors necessary for protein synthesis, and various enzymes such as tRNA. When synthesizing proteins, other substances necessary for protein synthesis such as various amino acids, energy sources such as ATP and GTP, and creatine phosphate are added to the cell extract. Of course, ribosomes, various factors, and/or various enzymes prepared separately may be supplemented as necessary during protein synthesis.

Development of a transcription/translation system in which each molecule (factor) required for protein synthesis is reconstituted has also been reported (Shimizu, Y. et al.: Nature Biotech., 19, 751-755, 2001). In this synthesis system, 3 types of initiation factors, 3 types of elongation factors, 4 types of factors involved in termination, 20 types of aminoacyl-tRNA synthetases that bind each amino acid to tRNA, which constitute the protein synthesis system of bacteria, and Genes of 31 factors consisting of methionyl-tRNA formyltransferase have been amplified from the E. coli genome and used to reconstruct the protein synthesis system in vitro. The present invention may utilize such a reconstituted synthetic system.

The term "cell-free transcription/translation system" is used interchangeably with cell-free protein synthesis system, in vitro translation system or in vitro transcription/translation system. In vitro translation systems use RNA as a template to synthesize proteins. Total RNA, mRNA, in vitro transcripts and the like are used as template RNA. The other in vitro transcription/translation system uses DNA as a template. The template DNA should contain a ribosome binding region and preferably contains a suitable terminator sequence. In the in vitro transcription/translation system, conditions are set such that factors necessary for each reaction are added so that the transcription reaction and the translation reaction proceed continuously.

<Search for mutation points effective for improving characteristics>
Aiming to improve the properties of Acinetobacter calcoaceticus-derived esterase (SEQ ID NO: 1), the mutation point ( Amino acid residues to be substituted) were selected, and 15 types of mutants (modified esterases) were designed. Each designed mutant was produced by the following method, and its properties were evaluated. The amino acid sequence of SEQ ID NO: 1 is the mature esterase (without signal peptide) derived from Acinetobacter calcoaceticus.

1. Obtaining Mutants (Modified Esterases) (1) Introduction of Mutations and Transformation In order to introduce random mutations at each mutation introduction point, a plasmid (pET21 (Esterase)) into which the esterase gene was inserted was used as a template, and random primers were used. PCR was performed using PrimeSTAR GXL DNA Polymerase (Takara Bio Inc.). After PCR, the template plasmid was treated with Dpn I (Takara Bio Inc.) (37°C, 3 hours), and the ligation reaction was performed using T4 Kinase (Toyobo Co., Ltd.) and Ligation High (Toyobo Co., Ltd.) (16°C). , overnight), and then transformed into E. coli BL21 (DE3) to obtain random mutant strains in which each mutation introduction point was randomly mutagenized.

<PCR conditions>
Composition of reaction solution: 5x PrimeSTAR GXL Buffer 5 μl, dNTP Mixture (2.5 mM each) 2 μl, forward primer 0.5 μl, reverse primer 0.5 μl, template 0.25 μl, PrimeSTAR GXL DNA Polymerase (Takara Bio Inc.) Company) 1 μl (Adjusted to 25 μl with sterilized distilled water)
Reaction conditions: 15 cycles of 98°C for 10 seconds, 60°C for 15 seconds, 68°C for 2 minutes

(2) Cultivation of transformants and acquisition of enzyme solution (cell extract) bacteria) was attempted. A culture medium (cultured cells) of each esterase gene-recombinant E.coli-expressing strain was obtained by a two-stage culture. First, inoculate 5 mL of L Broth (Invitrogen) (Amp: 100 μg/mL) with each esterase gene-recombinant E. coli expression strain, and culture (150 rpm, 37°C) for 16 hours in a shaking incubator. , Teriffic Broth (Invitrogen) (Amp: 100 μg/mL) 50 mL was inoculated with 0.5 mL. Then, the cells were cultured at 200 rpm and 33° C. for 48 hours, and 0.1 mM IPTG was added to the culture medium 24 hours after the start of the culture to induce expression of the enzyme. After centrifuging 50 mL of the culture solution (7,000 g×10 minutes, 4° C.), the cultured cells were recovered by removing the centrifugal supernatant.

After suspending each of the obtained cultured cells in 25 mL of 100 mM phosphate buffer (pH 7.0), add 10 g of beads (0.1 mm) (Yasui Instruments) and shake with a bead shocker (2,500 rpm, on: 60 seconds, off: 30 seconds, 10 cycles, 4°C) (Yasui Kiki Co., Ltd.). After the physical homogenate was centrifuged (7,000 g×10 minutes, 4° C.), the centrifugal supernatant was collected and used as a cell extract.

(3) Acquisition of Purified Enzyme The cell extract was filtered and diluted to 20 mL. Next, ammonium sulfate precipitation (collection of 40-80% fraction) was carried out by the following procedure. First, ammonium sulfate was added to 40% saturation. After centrifugation and collection of the supernatant, ammonium sulfate was added to 80% saturation. Centrifugation was performed again, and the centrifugal precipitate was recovered. The recovered precipitate was suspended in 10 mL of 10 mM phosphate buffer (pH 7.0).

After ammonium sulfate precipitation, the enzyme solution was desalted and concentrated by ultrafiltration to lower the conductivity to about 2 mS/cm. Subsequently, it was purified by the following two-step chromatography (DEAE-Sepharose column chromatography, phenyl-Sepharose column chromatography). The fraction collected by DEAE-Sepharose column chromatography was subjected to phenyl-Sepharose column chromatography after NaCl was added to adjust the conductivity to 2M.

(i) DEAE-Sepharose column chromatography Column used: Deae-sepharoseFF 1 mL (GE Healthcare)
Buffer: 10 mM phosphate buffer (pH 7.0)
Elution: Elute the target enzyme with a linear NaCl gradient (0 → 0.3 M) Charge volume: 5 mL (total amount of desalted and concentrated sample)
Flow rate: 1mL/min

(ii) Phenyl-Sepharose column chromatography Column used: Phenyl-HP 1 mL (GE Healthcare)
Buffer: 10 mM phosphate buffer (pH 7.0)
Elution: Elute target enzyme with NaCl linear gradient (2M→0M) Charge volume: 10 mL (total amount of DEAE-purified fraction)
Flow rate: 1mL/min

Eluted fractions from phenyl-Sepharose column chromatography were collected and used as purified enzyme samples.

2. Mutant Characterization <Activity Measurement Method>
50 mM phosphate buffer (pH 7.0) 2.1 mL, 5 mM 3,4-dihydrocoumarin (Tokyo Kasei (TCI)) solution (50 mM phosphate buffer (pH 7.0) (containing 40% EtOH)) 0.3 mL, enzyme 0.6 mL of the solution was mixed and allowed to react. In this measurement method, kinetics measurement (Abs. 270 nm, 30°C, 5 minutes) of the product (3-(2-hydroxyphenyl)propionic acid) produced by the reaction yields 1 nmol per minute. One unit (U) is defined as the amount of enzyme that produces the product (3-(2-hydroxyphenyl)propionic acid). The ΔOD at which an accurate measurement value can be obtained in this measurement is in the range of ΔOD = 0.01 to 0.05/2 min (Abs. 270 nm). Dilute.

(1) Evaluation of specific activity Specific activity was evaluated using purified enzyme samples. The enzyme activity of the purified enzyme of each mutant was measured by the activity measurement method described above, and the specific activity was calculated. The protein concentration was calculated by measuring the absorbance at a wavelength of 280 nm and setting the protein concentration to 1 mg/ml when the absorbance at A280 nm was 1.0. The specific activity of each mutant is shown in FIG. Mutant 6 (S235T) was found to have improved specific activity.

(2) Evaluation of high-temperature stability High-temperature stability was evaluated using purified enzyme samples. First, the purified enzyme of each mutant was heat-treated (80°C, 30 minutes). The sample after heat treatment and the sample before heat treatment (untreated) were diluted to an appropriate concentration with a diluent (50 mM phosphate buffer (pH 7.0)), and the enzyme activity was measured by the activity measurement method described above. From the measured value (enzyme activity), the residual activity was calculated as follows to evaluate the high-temperature stability.
Activity residual rate = (enzyme activity after heat treatment/enzyme activity before heat treatment) x 100 (%)

The evaluation results are shown in FIG. Four kinds of mutants (mutant 1 (K7P), mutant 2 (F13Y), mutant 4 (L222M), and mutant 5 (V229I)) showed improved high-temperature stability. In particular, mutant 1 (K7P), mutant 4 (L222M), and mutant 5 (V229I) have high high-temperature stability.

(3) Evaluation of high-temperature reactivity High-temperature reactivity was evaluated using purified enzyme samples. First, after diluting the purified enzyme of each mutant with a diluent (50 mM phosphate buffer (pH 7.0)) to an appropriate concentration, the enzyme activity was measured at each temperature (30°C, 60°C). High-temperature reactivity was evaluated by relative activity (ratio of enzyme activity at high temperature (60°C) to enzyme activity at 30°C) calculated by the following formula.
Relative activity = (enzyme activity at high temperature (60°C)/enzyme activity at 30°C) x 100 (%)

The evaluation results are shown in FIG. Two types of mutants (mutant 5 (V229I) and mutant 6 (S235T)) were found to have improved high-temperature reactivity. In particular, mutant 5 (V229I) has high high-temperature reactivity.

(4) Evaluation of pH stability pH stability was evaluated using purified enzyme samples. First, the purified enzyme of each mutant was treated with low pH (pH 4.0, 30 minutes). After diluting the treated sample and the untreated (untreated) sample with a diluent (50 mM phosphate buffer (pH 7.0)) to an appropriate concentration, the enzyme activity was measured by the activity measurement method described above. From the measured value (enzyme activity), the residual activity was calculated as follows to evaluate the high-temperature stability.
Activity residual rate = (enzyme activity after low pH treatment/enzyme activity before treatment) x 100 (%)

The evaluation results are shown in FIG. Seven mutants (mutant 2 (F13Y), mutant 3 (V107M), mutant 4 (L222M), mutant 5 (V229I), and mutant 6 (S235T)) showed improved pH stability. rice field. In particular, mutant 2 (F13Y), mutant 3 (V107M), mutant 4 (L222M), and mutant 5 (V229I) have high pH stability.

As described above, we succeeded in obtaining eight types of mutants with improved characteristics. The amino acid sequence of each mutant is shown below.
Mutant 1 (K7P): SEQ ID NO:2
Mutant 2 (F13Y): SEQ ID NO:3
Variant 3 (V107M): SEQ ID NO:4
Mutant 4 (L222M): SEQ ID NO:5
Mutant 5 (V229I): SEQ ID NO:6
Mutant 6 (S235T): SEQ ID NO:7

The properties (specific activity, high temperature reactivity, high temperature stability, pH stability) of the modified esterase of the present invention are improved. Therefore, its use or application range is wide, and an improvement in reaction efficiency can be expected.

The present invention is by no means limited to the description of the above embodiments and examples of the invention. Various modifications are also included in the present invention within the scope of those skilled in the art without departing from the description of the claims. The contents of articles, published patent publications, patent publications, etc., identified herein are incorporated by reference in their entirety.

Claims (9)

Modified esterase of any one of (1) to (4) below:
(1) In the amino acid sequence of SEQ ID NO: 1 , it has an amino acid sequence containing an amino acid substitution of S235T or an amino acid sequence showing 91% or more identity with the amino acid sequence (provided that S235T is conserved) , and SEQ ID NO: 1 A modified esterase having an improved specific activity compared to an esterase consisting of an amino acid sequence of
(2) an amino acid sequence containing a K7P amino acid substitution in the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence showing 91% or more identity with the amino acid sequence (provided that K7P is conserved), the amino acid sequence of SEQ ID NO: 1 In the amino acid sequence containing the amino acid substitution of F13Y or an amino acid sequence showing 91% or more identity with the amino acid sequence (however, F13Y is conserved), the amino acid sequence of SEQ ID NO: 1, the amino acid containing the amino acid substitution of L222M A sequence or an amino acid sequence showing 91% or more identity with said amino acid sequence (however, L222M is conserved), or an amino acid sequence containing an amino acid substitution of V229I in the amino acid sequence of SEQ ID NO: 1, or 95% with said amino acid sequence A modified esterase having an amino acid sequence exhibiting the above identity (however, V229I is conserved) and having improved high-temperature stability compared to the esterase consisting of the amino acid sequence of SEQ ID NO: 1;
(3) an amino acid sequence containing an amino acid substitution of V229I in the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence showing 95% or more identity with the amino acid sequence (provided that V229I is conserved), or the amino acid of SEQ ID NO: 1 In the sequence, it has an amino acid sequence containing an amino acid substitution of S235T or an amino acid sequence showing 91% or more identity with the amino acid sequence (however, S235T is conserved), and compared to an esterase consisting of the amino acid sequence of SEQ ID NO: 1 modified esterase with improved high-temperature reactivity, and
(4) an amino acid sequence containing an F13Y amino acid substitution in the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence showing 91% or more identity with the amino acid sequence (provided that F13Y is conserved), the amino acid sequence of SEQ ID NO: 1 In the amino acid sequence containing the amino acid substitution of V107M or an amino acid sequence showing 91% or more identity with the amino acid sequence (however, V107M is conserved), the amino acid sequence of SEQ ID NO: 1, the amino acid containing the amino acid substitution of L222M A sequence or an amino acid sequence showing 91% or more identity with said amino acid sequence (however, L222M is conserved), an amino acid sequence containing a V229I amino acid substitution in the amino acid sequence of SEQ ID NO: 1, or 95% or more with said amino acid sequence (V229I is conserved), or an amino acid sequence containing an amino acid substitution of S235T in the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence showing 91% or more identity with the amino acid sequence ( However, S235T is conserved), and has improved pH stability compared to the esterase consisting of the amino acid sequence of SEQ ID NO:1. Modified esterase of any one of (1) to (4) below:
(1) the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence showing 91% or more identity with the amino acid sequence ( however , if the identity criterion is the amino acid sequence of SEQ ID NO: 7, a position other than position 235 threonine, A difference in the amino acid sequence occurs in ), and a modified esterase with improved specific activity compared to the esterase consisting of the amino acid sequence of SEQ ID NO: 1 ,
(2) the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence exhibiting 91% or more identity with the amino acid sequence (however, if the identity criterion is the amino acid sequence of SEQ ID NO: 2, an amino acid at a position other than position 7 proline) sequence difference occurs), the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence showing 91% or more identity with the amino acid sequence (however, if the identity criterion is the amino acid sequence of SEQ ID NO: 3, tyrosine at position 13 The amino acid sequence of SEQ ID NO: 5 or an amino acid sequence showing 91% or more identity with the amino acid sequence (provided that the identity criterion is the amino acid sequence of SEQ ID NO: 5 In the case of , there is a difference in the amino acid sequence at positions other than position 222 methionine), or the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence showing 95% or more identity with the amino acid sequence (however, the criteria for identity is the amino acid sequence of SEQ ID NO: 6, there is a difference in the amino acid sequence at positions other than isoleucine at position 229), and the modification that improves the high temperature stability compared to the esterase consisting of the amino acid sequence of SEQ ID NO: 1 type esterase,
(3) the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence showing 95% or more identity with the amino acid sequence (however, if the identity criterion is the amino acid sequence of SEQ ID NO: 6, amino acids at positions other than isoleucine at position 229) sequence difference occurs), or the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence showing 91% or more identity with the amino acid sequence (however, if the identity criterion is the amino acid sequence of SEQ ID NO: 7, position 235 a difference in amino acid sequence occurs at positions other than threonine), and a modified esterase having improved high-temperature reactivity compared to the esterase consisting of the amino acid sequence of SEQ ID NO: 1;
(4) the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence showing 91% or more identity with said amino acid sequence (however, if the identity criterion is the amino acid sequence of SEQ ID NO: 3, amino acids at positions other than 13th tyrosine) sequence difference occurs), the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence showing 91% or more identity with the amino acid sequence (however, if the standard of identity is the amino acid sequence of SEQ ID NO: 4, methionine at position 107 The amino acid sequence of SEQ ID NO: 5 or an amino acid sequence showing 91% or more identity with the amino acid sequence (provided that the identity criterion is the amino acid sequence of SEQ ID NO: 5 In the case of , there is a difference in the amino acid sequence at positions other than position 222 methionine), the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence showing 95% or more identity with the amino acid sequence (however, the identity criterion is In the case of the amino acid sequence of SEQ ID NO: 6, there is a difference in the amino acid sequence at positions other than isoleucine 229), or the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence showing 91% or more identity with the amino acid sequence ( However, when the standard of identity is the amino acid sequence of SEQ ID NO: 7, there is a difference in the amino acid sequence at positions other than position 235 threonine), and compared with the esterase consisting of the amino acid sequence of SEQ ID NO: 1, pH A modified esterase with improved stability . A gene encoding the modified esterase according to claim 1 or 2 . 4. The gene according to claim 3 , comprising any one of the base sequences of SEQ ID NOS:8-13. A recombinant DNA containing the gene according to claim 3 or 4 . A microorganism carrying the recombinant DNA according to claim 5 . 7. The microorganism according to claim 6 , wherein the host is Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacillus aminorrhifaciens, Brevibacillus choshinensis. An enzyme agent containing the modified esterase according to claim 1 or 2 . A method for preparing a modified esterase, comprising the following steps (I)-(III):
(I) providing a nucleic acid encoding the amino acid sequence of any of SEQ ID NOs: 2-7;
(II) expressing the nucleic acid, and
(III) Recovering the expression product.

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