JP2015167476A - Variant hydroxynitrile lyase and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a variant hydroxynitrile lyase which exhibits excellent hydroxynitrile lyase activity and is easy to produce, and a method for efficiently producing the variant hydroxynitrile lyase.SOLUTION: The invention provides a variant hydroxynitrile lyase which has a mutation at a specific position of a specific amino acid sequence and (S)-hydroxynitrile substrate-specificity in comparison with wild type hydroxynitrile lyase, and a transformant transformed by a vector containing a variant hydroxynitrile lyase gene.

Description

  The present invention relates to a mutant hydroxynitrile lyase having excellent hydroxynitrile lyase activity and a method for efficiently producing the mutant hydroxynitrile lyase.

  Hydroxynitrile lyase is an enzyme that catalyzes the reaction of converting a carbonyl compound into cyanohydrin (α-hydroxynitrile) in the presence of a cyanide donor. Since cyanohydrin can be converted into various compounds such as α-hydroxy acid, α-hydroxy ketone, and β-amino alcohol, it is important as an intermediate in the fields of medicine and chemicals. Therefore, development of a method for producing a large amount of hydroxynitrile lyase is desired.

  Hydroxynitrile lyases are divided into two groups: (S) -selectivity and (R) -selectivity. Among them, (S) -hydroxynitrile lyase catalyzes the reaction of producing (S) -cyanohydrin from a ketone or aldehyde and a cyanide under acidic conditions. As a typical example of this reaction, there is a reaction for producing (S) -mandelonitrile from benzaldehyde and cyanogen cyanide. In addition, (S) -hydroxynitrile lyase is also used as a biocatalyst capable of producing optically active substances with high utility value as pharmaceuticals and chemical intermediates from inexpensive substrates, and is extremely useful in many fields. It is.

  In order to use hydroxynitrile lyase for industrial production of optically active cyanohydrin, it is desired to develop a method for producing a large amount of hydroxynitrile lyase having high activity per cell or protein and high stereoselectivity.

  Hydroxynitrile lyase is known to exist only in plants with cyanogenic glycosides. For example, as (R) -hydroxynitrile lyase, those derived from Rosaceae such as Almond (Prunus amygdalus) are known. In addition, as (S) -hydroxynitrile lyase, those derived from gramineous plants such as Sorghum (Sorghum bicolor), cassava (Manihot esculenta), Para rubber tree (Hevea brasiliensis), variospermum (Baliospermum), etc. Is known. However, only a trace amount of hydroxynitrile lyase could be extracted from these plants.

  In order to obtain a large amount of hydroxynitrile lyase, attempts have been made to obtain hydroxynitrile lyase by a genetic engineering method (Patent Documents 1 to 9). However, when a heterologous protein is expressed using a transformant, results such as the expression level and biochemical activity obtained by research on the same protein may not be applied as it is. That is, when expressing a heterologous protein using a transformant, it is not easy to predict in advance the behavior and expression level of the transformant, the biochemical activity of the target protein, and the like.

  In addition, depending on the type of protein to be expressed, normal protein folding may not be performed in the transformant host, and so-called inclusion bodies may be formed. Is known to be an inactive protein that has been lost. As for the hydroxynitrile lyase, for example, it has been reported that most of the hydroxynitrile lyase obtained from E. coli transformants is found in the insoluble fraction in the form of inactive inclusion bodies (Patent Document 1). In particular, (S) -hydroxynitrile lyase is known to be very difficult to express in E. coli.

  As a means for solving these problems, for example, by culturing Escherichia coli transformants expressing hydroxynitrile lyase at a low temperature, the formation of inclusion bodies is suppressed and the yield of active hydroxynitrile lyase is improved. There is a technique to try (Patent Document 8). However, this technique has problems that the culture time is long and a large amount of electric power or cooling water must be used to maintain a low temperature. Considering the industrial production of hydroxynitrile lyase, these problems cause a significant increase in production cost.

  By the way, due to recent advances in recombinant DNA technology, it has become relatively easy to create mutations by imparting mutations to amino acid sequences of proteins. Depending on the location of the mutation and the type of amino acid to be substituted, these mutants have stability, organic solvent resistance, heat resistance, acid resistance, alkali resistance, substrate specificity, It is known that performance such as substrate affinity is improved. These improvements in performance may result in a significant reduction in production costs in industrial production utilizing enzymatic reactions. Therefore, creation of useful improved enzymes having various performances improved in many enzymes.

  Mutants have also been reported for hydroxynitrile lyase. For example, it has been reported that hydroxynitrile lyase is mutated to improve affinity for aromatic aldehydes, particularly 3-phenoxybenzaldehyde (Patent Document 9, Non-Patent Document 1). The rate has not improved significantly.

  In addition, some of the M15 strain transformants expressing mutants in which the 128th tryptophan is substituted with other amino acids and mutants in which the 81st cysteine is substituted with alanine have hydroxynitrile lyase activity per cell, It is reported that the activity is higher than the hydroxynitrile lyase activity per M15 strain transformant that expresses type-hydroxynitrile lyase (Patent Document 6). However, in the same literature, the hydroxynitrile lyase activity of the M15 strain transformant expressing wild-type hydroxynitrile lyase is 1 / of that of the JM109 strain transformant expressing wild-type hydroxynitrile lyase obtained under the same culture conditions. It is about 2.

  In addition, it has been reported that the specific activity of a mutant hydroxynitrile lyase obtained by substituting serine for glycine, which is the 113th amino acid in hydroxynitrile lyase derived from Chinese cassava subspecies (Non-patent Document 2). However, the 113th amino acid of a common cassava-derived hydroxynitrile lyase is serine, which merely indicates that the amino acid is important for hydroxynitrile lyase activity.

  Recently, it has been reported that hydroxynitrile lyase derived from cassava and paraquino rubber improves hydroxynitrile lyase activity per transformant by mutation of 103rd histidine, 3 lysines in the sequence, and 2nd amino acid from the N-terminal. (Non-patent document 3, Patent document 10). The 103rd histidine is preserved in hydroxynitrile lyase derived from Euphorbiaceae plants such as cassava (Manihot esculenta), para rubber tree (Hevea brasiliensis) and bariospermum montanum. The same effect is expected. However, in reality, sufficient activity cannot be obtained by mutation of the 103rd histidine in the hydroxynitrile lyase derived from variospermum. Although the variospermum-derived hydroxynitrile lyase has been successfully expressed in E. coli using a pCold vector (Non-patent Documents 3 and 10), expression at low temperatures is essential in this expression system. In addition, due to differences in substrate specificity and amino acid sequence, it is expected to have usefulness different from cassava and paragino rubber-derived hydroxynitrile lyase in industry, and it is important to obtain a large amount of this hydroxynitrile lyase activity by E. coli. .

Japanese National Patent Publication No. 11-508775 JP 2000-189159 A JP 2000-189160 A JP 2000-245486 A JP 2002-330791 A International Publication No. 01/48178 Pamphlet JP 2004-194550 A JP 2004-194551 A JP 2000-125886 A International Publication No. 2006/041226 Pamphlet

Holger Buhler et al., Chembiochem. , 4, pp. 211-216 (2003) Gonghong Yan et al., Biotechnol. Lett. , 25, pp. 1041-1047 (2003) Mohammad Dadashipour et al., Journal of Biotechnology. , 153, pp. 100-110 (2011)

  As described above, hydroxynitrile lyase can be a very important enzyme in the industry, and therefore, it is required to have a more excellent enzyme activity. However, conventional wild-type or mutant-type hydroxynitrile lyases are difficult to mass-produce. For example, when they are expressed heterogeneously, there are many cases where the activity is not obtained or decreases.

  Accordingly, an object of the present invention is to provide a mutant hydroxynitrile lyase that exhibits excellent hydroxynitrile lyase activity and is easy to produce, and a method for efficiently producing the mutant hydroxynitrile lyase.

  The inventors of the present invention have made extensive studies to solve the above problems. As a result, it was found that by adding a specific mutation to wild-type hydroxynitrile lyase derived from Variospermum, the hydroxynitrile lyase activity was improved and the heterologous expression of the active enzyme became possible, thereby completing the present invention. did.

[1] The mutant hydroxynitrile lyase according to any one of (1) to (3) below.
(1) An amino acid sequence in which the 157th asparagine in the amino acid sequence of SEQ ID NO: 2 or the 156th asparagine in the amino acid sequence of SEQ ID NO: 4 or 6 is substituted with glycine, alanine, aspartic acid or arginine ;
(2) The amino acid sequence defined in (1) above has an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the region excluding the 157th or 156th amino acid. A mutation that is a mutant-type hydroxynitrile lyase and maintains or has a higher hydroxynitrile lyase activity compared to the wild-type hydroxynitrile lyase having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 (3) having an amino acid sequence having at least 99.2% sequence identity to the amino acid sequence defined in (1) above, and SEQ ID NO: 2, SEQ ID NO: 4 or A wild-type hydroxynitrile lyase having the amino acid sequence shown in SEQ ID NO: 6; A mutant nitrile lyase having maintained or high hydroxynitrile lyase activity (however, the amino acid sequence corresponding to the 157th or 156th amino acid sequence in the amino acid sequence of the mutated hydroxynitrile lyase is not mutated). Suppose).

  [2] Further, in the above-mentioned mutant hydroxynitrile lyase (1), the 103rd histidine in the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 is substituted with cysteine. Mutant hydroxynitrile lyase.

  [3] The mutant hydroxynitrile lyase according to the above [1] or [2], which is specific to a (S) -hydroxynitrile substrate.

[4] A mutant hydroxynitrile lyase gene comprising any one of the following base sequences (4) to (6):
(4) the nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 9;
(5) The nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 9 has a deletion, substitution and / or addition of 1 to several bases, and the protein encoded by the sequence has hydroxynitrile lyase activity A base sequence maintained or higher compared to a wild-type hydroxynitrile lyase having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6;
(6) The hydroxynitrile lyase activity of the protein having at least 99.2% homology to the base sequence set forth in SEQ ID NO: 7 or 9 and encoded by the sequence is SEQ ID NO: 2, A base sequence that is maintained or higher than a wild-type hydroxynitrile lyase having the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6.

  [5] A transformant characterized by being transformed with a vector containing the mutant hydroxynitrile lyase gene according to [4].

[6] A method for producing a mutant hydroxynitrile lyase,
Culturing the transformant according to the above [5] to obtain a culture, and
And a step of purifying the mutant hydroxynitrile lyase from the culture.

  The novel mutant hydroxynitrile lyase according to the present invention has significantly higher hydroxynitrile lyase activity than that of the wild type, and the stereoselectivity of cyanohydrin in the synthesis reaction of cyanohydrin is improved. In addition, when a gene encoding the novel mutant hydroxynitrile lyase according to the present invention is used, the mutant hydroxynitrile lyase can be efficiently produced by heterologous expression or the like. Therefore, the present invention is extremely useful industrially as a technique relating to hydroxynitrile lyase that can be used for the production of cyanohydrin, which is important as an intermediate in the fields of medicine and chemicals.

It is the result of having performed alignment analysis in order to compare the amino acid sequence of the hydroxy nitrile lyase derived from cassava, a paraquino rubber, and a variospermum. The result of having compared the specific activity per transformant of the variant of the hydroxy nitrile lyase derived from variospermum is shown. The symbol in the figure indicates the amino acid in which mutation is introduced and its position in the amino acid sequence of SEQ ID NO: 6. For example, “N156D” indicates that the 156th asparagine in SEQ ID NO: 6 is mutated to aspartic acid. The figure which compared the specific activity per transformant of the mutant type | mold which introduce | transduced the saturation mutation into the 156th asparagine in addition to the 103rd histidine mutation in the hydroxy nitrile lyase derived from variospermum is shown. The vertical axis represents relative activity, and the horizontal axis represents the amino acid substituted with the 156th asparagine in one-letter code. The result of having compared the specific activity per transformant of the variant enzyme which introduce | transduced the saturation mutation into the 156th asparagine in the hydroxy nitrile lyase derived from variospermum is shown. The vertical axis shows the specific activity, and the horizontal axis shows the amino acid substituted with the 156th asparagine in one letter. The result of introducing a saturation mutation into the 157th asparagine in cassava-derived hydroxynitrile lyase is shown. The vertical axis represents the specific activity per transformant, and the horizontal axis represents the temporary number (M1 to M10) of the mutant in which the 157th asparagine is substituted. The results of analysis by SDS-PAGE of the purity of a variospermum-derived hydroxynitrile lyase wild-type, the 103rd histidine cysteine mutant, and the 156th asparagine mutant mutated to aspartic acid are shown. The result of having compared the temperature stability of the refinement | purification mutant-type hydroxy nitrile lyase by synthetic activity is shown. The horizontal axis represents temperature, and the vertical axis represents residual activity. The result of having compared the production amount of (S) -mandelonitrile and enantiomeric excess in the synthesis reaction using the purified mutant-type hydroxynitrile lyase is shown. The horizontal axis indicates the reaction time, and the vertical axis indicates the amount of production and the enantiomeric excess. Results of comparing (S) -mandelonitrile production (A) and enantiomeric excess (B) in the synthesis reaction using soluble fractions of cell-free extracts containing wild-type and mutant hydroxynitrile lyases Indicates.

  Hereinafter, first, the mutant hydroxynitrile lyase according to the present invention will be described.

<Mutant hydroxynitrile lyase>
The mutant hydroxynitrile lyase according to the present invention is any one of the above (1) to (3).

  In the above-mentioned mutant hydroxynitrile lyase (1), “amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6” are cassava (Manihot esculenta), para rubber tree (Hevea brasiliensis), and bariosperumum montanum, respectively. ) -Derived wild-type hydroxynitrile lyase. These wild-type hydroxynitrile lyases naturally have hydroxynitrile lyase activity, but an enzyme having higher activity is desired. Moreover, not only barriospermum, but at least hydroxynitrile lyase derived from Euphorbiaceae plants is difficult to express in different species. In contrast, the mutant hydroxynitrile lyase (1) can be heterologously expressed using Escherichia coli or the like.

  In the present invention, “hydroxynitrile lyase activity” refers to an activity that catalyzes a reaction for producing cyanohydrin from a cyanide compound and a ketone compound or an aldehyde compound (hereinafter referred to as “synthesis activity”), and an activity that catalyzes the reverse reaction ( Hereinafter referred to as “degradation activity”). In the present invention, for example, the synthetic activity can be calculated by measuring the amount of mandelonitrile produced from benzaldehyde. The production amount of mandelonitrile can be quantified by, for example, HPLC. The decomposition activity can be calculated by measuring the amount of benzaldehyde produced from mandelonitrile as a substrate. The amount of benzaldehyde produced can be quantified, for example, by measuring the increase in absorbance at a wavelength of 280 nm when hydroxynitrile lyase and racemic mandelonitrile are added to a sodium citrate buffer.

  The mutant hydroxynitrile lyase of the present invention is preferably a (S) -hydroxynitrile substrate-specific one. In this case, the activity is preferably determined by quantifying (S) -mandelonitrile using a chiral column or the like.

  The mutant-type hydroxynitrile lyase according to the present invention includes, for example, an improvement in specific activity per protein, an improvement in the ability to form an active conformation, and a transformant, particularly a transformant, compared to a wild-type hydroxynitrile lyase. An increase in the expression level in the soluble fraction of the crushed bacterial cells of Therefore, the present invention includes a hydroxynitrile lyase with improved activity per transformant, a hydroxynitrile lyase with improved specific activity per protein, and an active conformation forming ability by introducing a mutation into wild-type hydroxynitrile lyase. Hydroxynitrile lyase with improved expression or hydroxynitrile lyase with improved expression level per transformant.

  In the present invention, “activity per transformant” means the activity of hydroxynitrile lyase per amount of protein in the enzyme solution obtained from the transformant. Hydroxynitrile lyase activity means hydroxynitrile lyase activity and specific activity per unit solution amount such as enzyme solution such as protein amount in enzyme solution.

  As used herein, “specific activity” means hydroxynitrile lyase activity per unit protein amount.

  In the mutant hydroxynitrile lyase (1) according to the present invention, the 157th asparagine in the amino acid sequence of SEQ ID NO: 2 or the 156th asparagine in the amino acid sequence of SEQ ID NO: 4 or 6 is glycine, It has an amino acid sequence substituted with alanine, aspartic acid or arginine. The protein obtained by substituting the 157th or 156th asparagine with the specific amino acid is a wild-type protein having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, as shown in the Examples. Hydroxynitrile lyase activity is higher than that of type hydroxynitrile lyase. Each amino acid of the hydroxynitrile lyase (1) is preferably L-form.

  In the present invention, an enzyme having “(specific) amino acid sequence” means that the amino acid sequence of the enzyme only needs to contain the specified amino acid sequence, and the function of the enzyme is maintained. To do. Examples of sequences other than the amino acid sequence specified in the enzyme include a histidine tag, a linker sequence for immobilization, and a crosslinked structure such as a disulfide bond.

  As the mutant hydroxynitrile lyase (1), one in which the 103rd histidine in the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 is substituted with cysteine is preferable. A mutant hydroxynitrile lyase having such a mutation has a high production efficiency such that the hydroxynitrile lyase activity is further improved and it can be produced by heterologous expression while maintaining the active conformation.

  In the mutant hydroxynitrile lyase (2) of the present invention, “the region excluding the 157th or 156th amino acid” refers to the 1st to 156th positions and the 1st to 156th positions in the amino acid sequence of SEQ ID NO: 2, respectively. This refers to the 158th and subsequent regions, and the first to 155th and 157th and subsequent regions in the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6.

  In addition, the range of “1 to several” in the “amino acid sequence in which one or several amino acids are deleted, substituted and / or added” is a wild type in which a hydroxynitrile lyase having a deletion or the like has a hydroxynitrile lyase. It is not particularly limited as long as it has the same or better hydroxynitrile lyase activity as hydroxynitrile lyase. The range of “1 to several” can be, for example, 1 or more and 30 or less, preferably 1 or more, 20 or less, more preferably 1 or more, 10 or less, and still more preferably 1 The number may be 1 or more, 7 or less, more preferably 1 or more, 5 or less, and particularly preferably 1 or more, 3 or less, 1 or more, 2 or less, and about 1.

  In the mutant hydroxynitrile lyase (3) of the present invention, the “sequence identity” in the “amino acid sequence having at least 99.2% sequence identity to the amino acid sequence defined in (1) above” is: The mutant hydroxynitrile lyase having the same amino acid sequence identity is an enzyme having hydroxynitrile lyase activity equivalent to or higher than that of the wild-type hydroxynitrile lyase having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. As long as there is, it is not specifically limited. The sequence identity of the amino acid sequence is not particularly limited as long as it is 99.2% or more, preferably 99.5% or more or 99.7% or more, more preferably 99.8% or more, and still more preferably. It is 99.9% or more. In the present invention, the term “sequence identity” refers to the degree of amino acid identity with respect to each other of two or more amino acid sequences. Therefore, the higher the identity of two amino acid sequences, the higher the identity or similarity of those sequences. Whether or not two kinds of amino acid sequences have identity can be analyzed by direct comparison of the sequences, and specifically, can be analyzed using commercially available sequence analysis software or the like.

  Further, in the present invention, “mutation” in “however, the amino acid corresponding to the 157th or 156th amino acid sequence in the amino acid sequence of the mutant hydroxynitrile lyase shall not be mutated” Means amino acid deletion or substitution. That is, in the amino acid sequence of the mutant hydroxynitrile lyase (3), it corresponds to the 157th or 156th amino acid in the amino acid sequence of the mutant hydroxynitrile lyase (1) serving as a criterion for determining sequence identity. This means that the amino acid is the same as the 157th or 156th amino acid in the amino acid sequence of the mutant hydroxynitrile lyase (1).

  In the amino acid sequence of the mutant hydroxynitrile lyase (2) and (3), the amino acid at the 157th or 156th position in the amino acid sequence of the mutant hydroxynitrile lyase (1) serving as a criterion for determining the sequence identity Amino acids corresponding to can be examined by the above-described analysis of sequence identity or homology. Specifically, using commercially available sequence analysis software or the like, the analysis target is the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 or the amino acid sequence of the mutant hydroxynitrile lyase (1). It is possible to search for an amino acid corresponding to the 157th or 156th amino acid. Such alignment analysis techniques are widely known to those skilled in the art.

  In the amino acid sequence of the mutant hydroxynitrile lyase (2) and (3), “hydroxynitrile lyase as compared with the wild-type hydroxynitrile lyase having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6” “The activity is maintained or high” means that the hydroxynitrile lyase activity of the protein of interest is compared with the wild-type hydroxynitrile lyase having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 above. Is relatively expensive. Specifically, the two or more enzymes to be compared are subjected to the above synthesis reaction or decomposition reaction under the same conditions, the amounts of the products are compared, and the amino acid sequences of the above-described mutant hydroxynitrile lyases (2) and (3) are included. It is sufficient if the enzyme activity is higher than that of the wild-type enzyme.

  When the 103rd histidine in the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 is substituted with cysteine in the mutant hydroxynitrile lyase (1), the mutation is expressed by the mutant hydroxynitrile. It is also maintained in lyases (2) and (3), that is, the 103rd cysteine is not mutated any more.

<Mutant hydroxynitrile lyase gene>
The mutant hydroxynitrile lyase gene according to the present invention has any one of the base sequences (4) to (6) and encodes any one of the mutant hydroxynitrile lyases (1) to (3). Is. The definition of the word defining the mutant hydroxynitrile lyase gene according to the present invention shall apply the definition of the word defining the mutant hydroxynitrile lyase (1) to (3) according to the present invention. .

  The above-mentioned mutant hydroxynitrile lyase of the present invention is improved by introducing a mutation into wild-type hydroxynitrile lyase, and the origin of the wild-type hydroxynitrile lyase that is the basis thereof is not particularly limited, For example, those derived from plants are preferred. As used herein, “wild-type hydroxynitrile lyase” refers to a hydroxynitrile lyase that can be isolated from a natural organism such as a plant. In the amino acid sequence constituting the enzyme, an intentional or unintentional deletion of an amino acid, It means a hydroxynitrile lyase that does not have other amino acid insertions or substitutions and retains its natural properties. Wild type hydroxynitrile lyase includes (S) -hydroxynitrile lyase and (R) -hydroxynitrile lyase, and (S) -hydroxynitrile lyase is preferred in the present invention. Examples of the derived plant include Euphorbiaceae plants such as cassava (Manihot esculenta), para rubber tree (Hevea brasiliensis), and bariosperumum montanum. For example, the amino acid sequence of cassava-derived wild-type hydroxynitrile lyase is disclosed in GenBank / EMBL accession number Z29091 and is represented by SEQ ID NO: 2. In addition, the amino acid sequence of wild-type hydroxynitrile lyase derived from Para rubber tree is disclosed in GenBank / EMBL accession number U40402 and is represented by SEQ ID NO: 4. The amino acid sequence of wild-type hydroxynitrile lyase derived from Variospermum is disclosed in GenBank / EMBL accession number BAI50630 and is represented by SEQ ID NO: 6.

  The base sequence of the mutant hydroxynitrile lyase gene according to the present invention can be designed to encode the amino acid sequence of the mutant hydroxynitrile lyase (1) to (3). Specifically, for example, it is a gene that expresses a mutation of the above-mentioned mutant hydroxynitrile lyase (1) to (3) and hybridizes with a nucleic acid encoding wild-type hydroxynitrile lyase under stringent conditions. What is necessary is just to design a base sequence and to screen for an enzyme whose expression activity is superior to that of wild-type hydroxynitrile lyase. Examples of the nucleic acid encoding wild-type hydroxynitrile lyase include nucleic acids having the base sequences of SEQ ID NOs: 1, 3, and 5.

  The mutant hydroxynitrile lyase gene of the present invention includes, for example, a mutant hydroxynitrile lyase gene DNA having a base sequence of SEQ ID NO: 7 or SEQ ID NO: 9 or a complementary sequence thereof, or a fragment thereof as a probe, colony hybridization, Southern It can be obtained from a cDNA library and a genomic library by a known hybridization method such as blotting. As the library, a library prepared by a known method can be used, or a commercially available cDNA library and genomic library can be used.

  In the present invention, “stringent conditions” are conditions at the time of washing after hybridization, wherein the salt concentration is 300 mM or more and 2000 mM or less, the temperature is 40 ° C. or more and 75 ° C. or less, preferably the salt concentration is 600 mM or more, It means a condition of 900 mM or less and a temperature of 65 ° C. For example, conditions such as 50 ° C. can be given for 2 × SSC. A person skilled in the art will consider the mutant hydroxynitrile lyase of the present invention in consideration of other conditions such as the concentration of the probe, the length of the probe, and the reaction time in addition to the conditions such as the salt concentration and temperature of the buffer. Conditions for obtaining a gene encoding can be set.

  For the detailed procedure of the hybridization method, “Molecular Cloning, A Laboratory Manual 2nd ed.” Cold Spring Harbor Laboratory Press (1989) and the like can be referred to. Examples of the hybridizing nucleic acid include a nucleic acid containing a base sequence having at least 99.2% identity to the base sequence of SEQ ID NO: 7 or 9 or a partial fragment thereof. The homology is preferably 99.5% or more or 99.7% or more, more preferably 99.8% or more, and further preferably 99.9% or more.

  In the present invention, the method for preparing the mutant hydroxynitrile lyase gene is not particularly limited, and can usually be performed by a known method. For example, using a commercially available kit based on the wild-type hydroxynitrile lyase gene, a method for generating site-specific substitution, or selectively cleaving the gene DNA, and then removing and adding the selected oligonucleotide The method of connecting etc. is mentioned.

  These site-directed mutagenesis methods are described in “Molecular Cloning, A Laboratory Manual 2nd ed.” Cold Spring Harbor Press (1989), “Current Protocols in Molecular Biology” John Wiley 97, S & M. Natl. Acad. Sci. USA, 82, pp. 488-92 (1985), Kramer and Fritz Method. Enzymol. , 154, pp. 350-67 (1987), Kunkel, Method. Enzymol. , 85, pp. 2763-6 (1988). In recent years, kits for mutagenesis using site-directed mutagenesis based on the Kunkel method or the Gapped duplex method, such as QuikChangeTM Site-Directed Mutagenesis Kit (Stratagene), GeneTailorTM Site-Directed Mutagen Strain And TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc .: manufactured by Takara Bio Inc.).

  In addition, when the target mutation introduction site is present in the vicinity of a restriction enzyme site that can be easily digested and linked in the target gene sequence, PCR is performed using a primer (synthetic oligo DNA) into which the target mutation has been introduced, A gene DNA fragment into which the target mutation has been introduced can be easily obtained. Furthermore, it can also be obtained as a synthetic gene by extending by a PCR method (assembling PCR) combining synthetic oligo DNA.

  In addition, random methods such as a method of contacting and acting a drug that is a mutation source such as hydroxylamine or nitrous acid, a method of inducing a mutation by ultraviolet irradiation, a method of randomly introducing a mutation using PCR (polymerase chain reaction), etc. A mutation-type hydroxynitrile lyase gene can also be obtained from a wild-type hydroxynitrile lyase gene by a mutation introduction method.

  In order to express the mutant hydroxynitrile lyase gene of the present invention obtained by the above method in a host, an expression cassette is constructed by inserting a transcription promoter upstream of the gene and a terminator downstream, and this cassette is used as an expression vector. Can be inserted. Alternatively, when the transcription promoter and terminator already exist in the expression vector into which the improved hydroxynitrile lyase gene is introduced, the mutant gene is used between the promoter and terminator in the vector without constructing the expression cassette. Can be inserted. In order to insert the improved hydroxynitrile lyase gene into a vector, a method using a restriction enzyme, a method using topoisomerase, or the like is used. Further, if necessary at the time of insertion, an appropriate linker may be added. In the present invention, such an integration operation can also be performed in conjunction with an operation for preparing a mutant hydroxynitrile lyase gene. That is, PCR is performed using a primer having a base sequence substituted with a base sequence encoding another amino acid, using a recombinant vector in which a wild-type hydroxynitrile lyase gene has been cloned as a template, and the resulting amplification product is incorporated into the vector. be able to.

  The type of promoter is not particularly limited as long as it allows appropriate expression in the host. Examples of the promoter include trp promoter of tryptophan operon derived from E. coli, lac promoter of lactose operon, PL promoter derived from lambda phage, and Examples include PR promoter, Bacillus subtilis-derived gluconate synthase promoter (gnt), alkaline protease promoter (apr), neutral protease promoter (npr), α-amylase promoter (amy), and the like. In addition, modified and designed sequences such as tac promoter and trc promoter can be used.

  The terminator is not necessarily required, and the type of the terminator is not particularly limited, and examples thereof include ρ-factor-independent ones such as lipoprotein terminator, trp operon terminator, rrnB terminator and the like.

  Ribosome binding sequences such as an SD sequence and a Kozak sequence are known as base sequences important for translation into amino acids, and these sequences can be inserted upstream of the mutant gene. An SD sequence may be added by a PCR method or the like when a prokaryote is used as a host, and a Kozak sequence may be added when a eukaryotic cell is used as a host. Examples of the SD sequence include sequences derived from Escherichia coli or Bacillus subtilis, but are not particularly limited as long as the sequence functions in a desired host such as Escherichia coli or Bacillus subtilis. For example, a consensus sequence in which a sequence complementary to the 3 'terminal region of 16S ribosomal RNA is continuous for 4 or more bases may be prepared by DNA synthesis and used.

  In general, a vector contains a factor (selection marker) for selecting a target transformant. Examples of selectable markers include drug resistance genes, auxotrophic complementary genes, and assimilative genes, and can be selected according to the purpose and host. For example, drug resistance genes used as selection markers in E. coli include ampicillin resistance gene, kanamycin gene, dihydrofolate reductase gene, neomycin resistance gene and the like.

  The vector used in the present invention is not particularly limited as long as it retains the above mutant gene, and a vector suitable for each host can be used. Examples of the vector include plasmid DNA, bacteriophage DNA, retrotransposon DNA, artificial chromosome DNA and the like. For example, when Escherichia coli is used as a host, pTrc99A having a region capable of autonomous replication in Escherichia coli (Centralalbueau floor Schemes (CBS), the Netherlands; http://www.cbs.knaw.nl/), pUC19 (Takara Bio, Japan), pKK233-2 (Centralalureau voor Schulmelculures (CBS), Netherlands; http://www.cbs.knaw.nl/), pET-12 (Novagen, Germany), pET-26b (Noge) (Germany) can be used. Moreover, what modified these vectors as needed can also be used. An expression vector with high expression efficiency such as an expression vector pTrc99A or pKK233-2 having a trc promoter or a lac operator can also be used.

  Recombinant vectors containing the improved hydroxynitrile lyase gene are within the scope of the present invention.

<Transformant>
A transformant can be produced by transforming or transducing the recombinant vector of the present invention into a host. Such transformants are also included in the scope of the present invention.

  The host used in the present invention is not particularly limited as long as the target mutant hydroxynitrile lyase can be expressed after the introduction of the above recombinant vector. Examples of the host include bacteria such as Escherichia coli and Bacillus subtilis; fungi such as yeast (Pichia and Saccharomyces) and fungi (Aspergillus); animal cells; insect cells; plant cells and the like.

  In the case of using bacteria as a host, Escherichia coli can be used as a particularly preferable host in the present invention. Examples of Escherichia coli include Escherichia coli K12 strain and B strain, or JM109 strain, XL1-Blue strain, and C600 strain, which are derived from these wild strains. In particular, when the lac promoter of the lactose operon as described above and its derived promoter are used as expression promoters, expression becomes inducible when a host having a lacI repressor gene is used (induced by IPTG or the like), and the lacI repressor gene is present. If a non-host is used, the expression becomes a constitutive type, so that a host as required can be used. These strains are easily available from, for example, the American Type Culture Collection (ATCC). Examples of Bacillus subtilis include Bacillus subtilis. The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method.

  When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and the like are used. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.

  When animal cells are used as hosts, monkey cells COS-7, Vero, CHO cells, mouse L cells, rat GH3, human FL cells and the like are used. Examples of methods for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.

  When insect cells are used as hosts, Sf9 cells, Sf21 cells and the like are used. As a method for introducing a recombinant vector into insect cells, for example, calcium phosphate method, lipofection method, electroporation method and the like are used.

  When a plant cell is used as a host, tobacco BY-2 cells and the like can be mentioned, but are not limited thereto. As a method for introducing a recombinant vector into a plant cell, for example, an Agrobacterium method, a particle gun method, a PEG method, an electroporation method, or the like is used.

<Method for producing mutant hydroxynitrile lyase>
The mutant hydroxynitrile lyase of the present invention can be produced by culturing the transformant and purifying it from the obtained culture.

  In the present invention, “culture” means any of culture supernatant, cultured cells, cultured cells, or disrupted cells or cells. A culture obtained by culturing the transformant of the present invention is included in the scope of the present invention.

  Culturing of the transformant of the present invention is carried out according to a usual method used for host culture. The target mutant hydroxynitrile lyase accumulates in the culture.

  The medium for culturing the transformant of the present invention contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the host, and can be a natural medium as long as the transformant can be cultured efficiently. Any of synthetic media may be used. Examples of the carbon source include carbohydrates such as glucose, galactose, fructose, sucrose, raffinose and starch; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol. Examples of the nitrogen source include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, or other nitrogen-containing compounds. In addition, peptone, yeast extract, meat extract, corn steep liquor, various amino acids, and the like may be used. Examples of inorganic substances include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, copper sulfate, and calcium carbonate. Moreover, you may add an antifoamer in order to prevent foaming during culture | cultivation as needed. Moreover, you may add a vitamin etc. suitably as needed. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.

  During the culture, the vector and the target gene may be cultured under selective pressure in order to prevent the loss of the vector and the target gene. That is, a drug corresponding to the case where the selectable marker is a drug resistance gene may be added to the medium, and a nutrient factor corresponding to the case where the selectable marker is an auxotrophic complementary gene may be removed from the medium. When the selectable marker is an assimilation gene, a corresponding assimilation factor can be added as a sole factor as necessary. For example, when culturing E. coli transformed with a vector containing an ampicillin resistance gene, ampicillin may be added to the medium as needed during the culture.

  When cultivating a transformant transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when a transformant transformed with an expression vector having a promoter inducible with isopropyl-β-D-thiogalactoside (IPTG) is cultured, IPTG or the like can be added to the medium. In addition, when culturing a transformant transformed with an expression vector using a trp promoter inducible with indoleacetic acid (IAA), LAA or the like can be added to the medium.

  The culture conditions for the transformant are not particularly limited as long as the productivity of the desired improved hydroxynitrile lyase and the growth of the host are not hindered. Usually, the culture temperature is 10 ° C. or higher and 45 ° C. In the following, it is preferably carried out at 10 ° C. or more and 40 ° C. or less, more preferably 15 ° C. or more and 40 ° C. or less, and even more preferably 20 ° C. or more and 37 ° C. or less. Also good. The culture time can be about 5 hours or more and 120 hours or less, preferably 5 hours or more and 100 hours or less, more preferably 10 hours or more and 100 hours or less, and even more preferably about 15 hours or more and 80 hours or less. Do. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like, and adjusted to 6 or more and 9 or less for E. coli. Examples of the culture method include solid culture, stationary culture, shaking culture, and aeration and agitation culture.

  In particular, when culturing E. coli transformants, it is preferable to culture under aerobic conditions by shaking culture or aeration and agitation culture (jar fermenter). In particular, in order to culture an E. coli transformant, it may be cultured by an ordinary solid culture method, but it is preferable to employ a liquid culture method as much as possible. Examples of the medium used for the culture include one or more nitrogen sources such as yeast extract, tryptone, polypeptone, corn steep liquor, soybean or wheat bran leachate, sodium chloride, monopotassium phosphate, dipotassium phosphate. In addition, one or more inorganic salts such as magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate are added, and if necessary, saccharide raw materials, vitamins and the like are appropriately added. The initial pH of the medium is suitably adjusted to 7 or more and 9 or less. Further, the culture is performed at 5 ° C. or higher and 40 ° C. or lower, preferably 10 ° C. or higher and 37 ° C. or lower for 5 hours or longer and 100 hours or shorter. It is preferably carried out by aeration and agitation deep culture, shaking culture, static culture, fed-batch culture or the like.

  The mutant hydroxynitrile lyase according to the present invention is purified from the culture, but the degree of purification is not particularly limited. For example, the above culture may be homogenized, and a solution from which insolubles have been removed by filtration or centrifugation may be used as it is, or further purified by column chromatography to obtain a crude enzyme solution or enzyme solution. .

<Enzyme reaction>
Hydroxynitrile lyase is an enzyme that catalyzes the reaction of converting aldehydes and ketones to cyanohydrin in the presence of a cyanide donor, and catalyzes the decomposition reaction of cyanohydrin, which is the reverse reaction.

  In the present invention, examples of the method for measuring the hydroxynitrile lyase activity include a method in which benzaldehyde is used as a substrate in the presence of potassium cyanide, and the amount of mandelonitrile produced is measured by HPLC. As a simpler method, there may be mentioned a method in which racemic mandelonitrile is used as a substrate and the decomposition is detected by measuring an absorption wavelength of 280 nm with an absorptiometer. As the solvent, it is preferable to use a sodium citrate buffer in order to make it more acidic than neutral due to the stability of cyanohydrin. The synthesis reaction is preferably carried out at a pH of about 4.0 or more and 4.2 or less, and the decomposition reaction is carried out at a pH of about 5 or more and 5.5 or less.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.

  Hereinafter, the hydroxynitrile lyase is abbreviated as “HNL”, the cassava-derived wild-type hydroxynitrile lyase is abbreviated as “MeHNL”, the paraquino rubber-derived one is abbreviated as “HbHNL”, the one derived from variospermum is abbreviated as “BmHNL”, The genes encoding hydroxynitrile lyase may be abbreviated as “MeHNL gene”, “HbHNL gene”, and “BmHNL gene”, respectively.

Example 1
(1) Acquisition of HNL gene Amplification of each HNL gene using pColdI vector or pET15b vector introduced with cassava-derived hydroxynitrile lyase MeHNL gene or bariospermum-derived hydroxynitrile lyase BmHNL gene as templates The MeHNL gene and BmHNL gene were amplified by PCR using the following primers. After purifying the PCR product by agarose gel electrophoresis, it was subjected to restriction enzyme treatment with BamHI and SphI and ligated to the simultaneously treated pUC19 vector. Hereinafter, the pUC19 vector into which the MeHNL gene has been introduced is referred to as “pUC19-MeHNL”, and the pUC19 vector into which the BmHNL gene has been introduced is referred to as “pUC19-BmHNL”.

5'-atatgcatgctaaggagatagaaaatggtaactgcacattttgtt-3 '(SEQ ID NO: 11)
5'-atatggatcctcaagcatatgcatcagccac-3 '(SEQ ID NO: 12)
5'-atatgcatgctaaggagatagaaaatggtgagcgctcattttat-3 '(SEQ ID NO: 13)
5'-atatggatcctcatgcgacagccagcaagt-3 '(SEQ ID NO: 14)

(2) Construction of H103 mutant Using the pUC19-BmHNL constructed in (1) above as a template, a saturation mutation was introduced into the 103rd histidine using the following primers and QuikChange Lightening Site-Directed Mutagenesis Kit (manufactured by Agilent). . PCR reaction solution is 10 pmol forward primer 0.15 μL, 10 pmol reverse primer 0.15 μL, 100 ng template DNA solution 0.2 μL, 10 × PCR buffer 1 μL, dNTP mix 0.2 μL, Quick solution reagents 0.3 μL, QuickChange Lightning Enzyme 0.2 μL and sterilized water 7.8 μL were mixed to prepare. PCR conditions were 94 ° C for 30 seconds, (i) 95 ° C for 2 minutes, (ii) 60 ° C for 10 seconds, (iii) 68 ° C for 3 minutes, (i) to (iii) repeated 16 times. It was. Thereafter, the PCR reaction solution was treated with the restriction enzyme DpnI to remove the template DNA. Escherichia coli JM109 strain was transformed by the heat shock method using the obtained PCR reaction solution. Subsequently, the plasmid was extracted from E. coli by the alkaline SDS method.

5'-ccagagaaagtttctgccttagttttcnnkaatgcattgatgcctgacattgatcac-3 '(SEQ ID NO: 15)
5'-gtgatcaatgtcaggcatcaatgcattmnngaaaactaaggcagaaactttctctgg-3 '(SEQ ID NO: 16)

(3) Construction of Vario Enzyme Gene Library of Variospermum-Derived Hydroxynitrile Lyase An expression plasmid constructed as described above, into which a gene encoding BmHNL H103C in which the histidine at position 103 of BmHNL has been replaced with cysteine was introduced (hereinafter referred to as “the plasmid”). Error-prone PCR was performed for mutagenesis using “pUC19-BmHNL H103C” as a template. The PCR reaction solution was 10 μmol forward primer 1.0 μL, 10 pmol reverse primer 1.0 μL, 500 μg pUC19-BmHNL H103C 1 μL or 0.2 μL, 10 mM dATP 1.0 μL, 10 mM dGTP 1.0 μL, 10 mM dTTP 1.0 μL, Sterile water was added to a mixture of 1.0 μL of 10 mM dCTP, 5 μL of 10 × PCR buffer (−MgSO ₄ ), 1.0 μL of 25 mM MgSO ₄ aqueous solution, 4 μL or 6 μL of 5 mM MnCl ₂ aqueous solution, and 1.0 μL of Dream Taq Polymerase (manufactured by Fermentas). In addition, the total amount was adjusted to 50 μL. The PCR reaction conditions were as follows: (i) 30 seconds at 94 ° C, (i) 94 ° C for 20 seconds, (ii) 55 ° C for 30 seconds, (iii) 1 minute at 72 ° C for 30 minutes. Repeated times. After the PCR reaction, the PCR product was purified by agarose gel electrophoresis. The purified PCR product was treated with the restriction enzymes BamHI and SphI and ligated to the pUC19 vector treated simultaneously.

M13-reverse primer: 5'-aacagctatgaccatg-3 '(SEQ ID NO: 17)
M13-forward primer: 5'-gtaaaacgacggccagt-3 '(SEQ ID NO: 18)

  Among the obtained mutants, those with increased activity were screened by the following experiment.

(4) Sample preparation Samples were prepared using 96-well plates. 160 μL each of LB medium (containing 80 μg / mL ampicillin and 0.1 mM IPTG) was dispensed into 0.8 mL 96-well sterilized plates (manufactured by ABgene), and mutant colonies were inoculated from the master plate. The colonies were cultured with shaking using BioShaker (manufactured by TAITEC, “M-BR-024”) at 37 ° C. and 1,200 rpm for 12 hours. After culturing, the culture broth was collected by centrifugation at 5,000 rpm for 10 minutes. After removing the supernatant and inverting on a newspaper to remove the medium as much as possible, the obtained cells were suspended in 100 μL of 0.85% NaCl aqueous solution using BioShaker, and then centrifuged (manufactured by Hitachi, Ltd.). , “Himac CR20”, “Rotor R6S”), and collected by centrifugation at 4,500 rpm for 10 minutes at 4 ° C. The supernatant was removed and the water was removed by inverting it on newspaper. Separately, a Lysozyme solution containing Lysozyme derived from egg white (Seikagaku Corporation) 10 mg / mL, KPB (pH 7.0) 100 mM and EDTA 10 mM was prepared. Lysozyme treatment was performed by adding 5 μL of the solution to the cells and incubating at 37 ° C. for 1 hour to protoplastize E. coli. The obtained Escherichia coli was freeze-thawed at −40 ° C. and 37 ° C., and 100 μL of hypotonic solution (10 mM KPB (pH 7.0), 5 mM MgCl ₂ ) was added for lysis. This solution is centrifuged at 4,500 rpm, 4 ° C. for 10 minutes using a centrifuge (manufactured by Hitachi, “himac CR20”, “Rotor R6S”) to precipitate the genome of Escherichia coli and cell walls, etc. A supernatant was obtained as an enzyme solution and used in the following experiments.

(5) Activity measurement About the obtained crude enzyme liquid, the decomposition activity of the mandelonitrile which is a substrate of a hydroxy nitrile lyase was measured by the production amount of benzaldehyde. The activity was measured by adding sodium citrate buffer to a 96-well UV plate (manufactured by Greiner bio-one) at 25 ° C. Next, the crude enzyme solution was added and suspended by pipetting. Next, after adding a racemate of mandelonitrile, pipetting and shaking, an increase in absorbance at a wavelength of 280 nm using a microplate reader (manufactured by Tecan Japan, “GENios”) at 25 ° C. for 5 minutes. It was measured. For the analysis, data processing software (“LS-PLATE manager 2001 (Win)” manufactured by Wako Pure Chemical Industries, Ltd.) was used. The results are shown in FIG.

  As shown in FIG. 2, several mutants showing increased activity were obtained. Furthermore, the specific activity per transformant was compared between BmHNL H103C in which histidine No. 103 used as a template was mutated to cysteine and each mutant, and the expression level was compared by SDS-PAGE electrophoresis. Among them, the highest specific activity improvement was observed in the 156th asparagine mutant. Further, since a mutation was confirmed at this position in many active mutants, it was considered that the mutation at the 156th asparagine is important for improving the specific activity per transformant.

Example 2
(1) Construction of N156 Mutant and Sample Preparation Saturation mutation was introduced into the 156th asparagine using pUC19-BmHNL H103C and pUC19-BmHNL as templates in the same manner as described above. The obtained mutant was inoculated into 5 mL of LB medium (containing 80 μg / mL ampicillin and 0.1 mM IPTG) and cultured at 37 ° C. for 12 hours. The cells were collected from 1.5 mL of the culture solution by centrifugation, the obtained cells were suspended in 300 μL of 20 mM KPB, and the cells were disrupted by ultrasonic disruption. The supernatant after centrifugation was used as a soluble fraction, and the precipitate was used as an insoluble fraction.

5'-gtgacaggactttagcggagnnkatttttagcaattcgcc-3 '(SEQ ID NO: 19)
5'-ggcgaattgctaaaaatmnnctccgctaaagtcctgtcac-3 '(SEQ ID NO: 20)

(2) Comparison of activity between BmHNL H103C N156 mutant and BmHNL N156 mutant The hydroxynitrile lyase activity was compared by a synthesis reaction using the soluble fraction obtained in (1) above. The reaction solution was adjusted to a total volume of 500 μL by adding MilliQ water to a mixture of 150 μL of 1M sodium citrate buffer (pH 4.0), 50 μL of crude enzyme solution, 50 μL of 1M KCN and 20 μL of benzaldehyde. The reaction was performed at room temperature for 1, 3 and 5 minutes. 100 μL was taken from the reaction solution, 900 μL of hexane: 2-propanol = 85: 15 mixed solution was added to stop the reaction, the organic layer of the supernatant was analyzed by HPLC, and the amount of mandelonitrile produced was measured. The results are shown in FIGS.

  As shown in FIG. 3 and FIG. 4, the specific activity was improved even in the 156th asparagine mutation alone. In addition, the addition of the 156th asparagine mutation to the H103C mutation further improved the activity. In particular, amino acids with small side chains such as glycine and alanine were considered preferable.

Example 3
(1) Construction of MeHNL N157 Mutant and Sample Preparation In the same manner as described above, a saturation mutation was introduced into the 157th asparagine using pUC19-MeHNL as a template and the primers shown below. The obtained mutant was inoculated into 5 mL of LB medium (containing 80 μg / mL ampicillin and 0.1 mM IPTG) and cultured at 37 ° C. for 12 hours. The cells were collected from the culture solution 1.5 mL by centrifugation. The obtained bacterial cells were suspended in 300 μL of 20 mM KPB, and the bacterial cells were crushed by ultrasonic crushing. The supernatant after centrifugation was used as a soluble fraction, and the precipitate was used as an insoluble fraction.

5'-gcttcgtacttctgagggaannkttatttaccaaatgcactg-3 '(SEQ ID NO: 21)
5'-cagtgcatttggtaaataamnnttccctcagaagtacgaagc-3 '(SEQ ID NO: 22)

(2) Comparison of activity in MeHNL N157 mutant Using the soluble fraction obtained in (1) above, hydroxynitrile lyase activity was compared by a synthesis reaction. The reaction solution was prepared by adding MilliQ water to a mixture of 150 μL of 1M sodium citrate buffer (pH 4.0), 50 μL of crude enzyme solution, 50 μL of 1M KCN and 20 μL of 1.25M benzaldehyde to make a total volume of 500 μL. The reaction was performed at room temperature for 1, 3 and 5 minutes. 100 μL was taken from the reaction solution, 900 μL of hexane: 2-propanol solution = 85: 15 mixed solution was added to stop the reaction, and the amount of mandelonitrile produced in the organic layer of the supernatant was measured by HPLC. The results are shown in FIG.

  As shown in the results shown in FIG. 5, the specific activity was improved in several mutants as in the variospermum. From these facts, in other plant-derived hydroxynitrile lyases, an improvement in activity can be expected by mutation of asparagine corresponding to the 156th asparagine in the amino acid sequence of BmHNL of variospermum (SEQ ID NO: 6).

Example 4
(1) Acquisition of purified enzymes of BmHNL H103C mutant and BmHNL H103C N156D mutant pUC19-BmHNL H103C and pUC19-BmHNL H103C N156D strain E. coli strain JM109 holding LB medium (80 μg / mL ampicillin and 0.1 mM IPTG) Contained) was inoculated into 500 mL and cultured at 37 ° C. for 12 hours. Thereafter, the cells were collected and the obtained cells were suspended in 20 mM KPB (pH 7.0) and crushed by ultrasonic crushing to obtain a cell-free extract. The enzyme was purified by ammonium sulfate fractionation and various chromatography. The degree of purification in each type of chromatography is shown in FIG.

(2) Comparison of activity using purified enzyme The specific activities (U / mg) of the BmHNL H103C mutant and the BmHNL H103C N156D mutant after gel filtration purification were compared. The results are shown in Table 1.

  As shown in Table 1, the specific activity was improved about 4 to 5 times by N156D mutation introduction.

(3) Temperature stability of purified enzyme Using purified enzyme, the temperature stability of BmHNL H103C and BmHNL H103C N156D was compared. Incubated from 10 ° C. to 90 ° C. every 10 ° C. for 60 minutes and cooled in ice for 10 minutes. The residual activity of the enzyme was compared by synthetic activity. The results are shown in FIG.

  As shown in FIG. 7, both mutants maintained an activity of about 70% at 60 ° C. Wild-type BmHNL is also known to be a heat-stable enzyme, and from these results, the heat stability was not affected by mutations in H103C and N156D.

(4) Synthesis of (S) -mandelonitrile using purified enzyme and comparison of enantiomeric excess rate Using purified enzyme, synthesis reaction was performed and the amount of (S) -mandelonitrile produced in the reaction and enantiomer The excess rate was compared. For the reaction, the same reaction system as the conventional synthesis reaction was used, and 200 μg of each purified enzyme was used. Every 10 minutes, the product in the reaction solution and its enantiomeric excess were quantified by HPLC and compared. The results are shown in FIG.

  As shown in FIG. 8, the H103C mutant had a product amount of 25 mM and an enantiomeric excess of 35%, but the N156D mutation was introduced to improve the product amount to 35 mM and the enantiomeric excess of 50%.

(5) Synthesis of (S) -mandelonitrile in various soluble fractions of BmHNL variants and comparison of enantiomeric excess ratios Using the soluble fraction obtained by the expression method shown above, (S)- The amount of mandelonitrile produced and the enantiomeric excess were compared. The mutant enzymes used were BmHNL, BmHNL H103C, BmHNL N156D, BmHNL H103C N156D, and BmHNL H103C N156G, all of which were expressed under the same conditions. For the reaction, the same reaction system as in the previous synthesis reaction was used, and 14 mg of the crude enzyme solution of the soluble fraction was used. FIG. 9 shows the result.

  As shown in the results shown in FIG. 9, it is considered that wild-type BmHNL and BmHNL N156D are low in production amount and enantiomeric excess and are probably not expressed soluble. BmHNL H103C was produced in a yield of 30 mM and an enantiomeric excess of 35%. BmHNL H103C N156D and BmHNL H103C N156G have a production amount of 40 mM and an enantiomeric excess of 70 to 80%. In 30 minutes, 80% of benzaldehyde in the reaction system is converted to mandelonitrile, and the enantiomeric excess is also 70 to 70%. As high as 80%, (S) -mandelonitrile was obtained with high optical purity.

Claims (6)

The mutant hydroxynitrile lyase according to any one of (1) to (3) below.
(1) An amino acid sequence in which the 157th asparagine in the amino acid sequence of SEQ ID NO: 2 or the 156th asparagine in the amino acid sequence of SEQ ID NO: 4 or 6 is substituted with glycine, alanine, aspartic acid or arginine ;
(2) The amino acid sequence defined in (1) above has an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the region excluding the 157th or 156th amino acid. A mutation that is a mutant-type hydroxynitrile lyase and maintains or has a higher hydroxynitrile lyase activity compared to the wild-type hydroxynitrile lyase having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 (3) having an amino acid sequence having at least 99.2% sequence identity to the amino acid sequence defined in (1) above, and SEQ ID NO: 2, SEQ ID NO: 4 or A wild-type hydroxynitrile lyase having the amino acid sequence shown in SEQ ID NO: 6; A mutant nitrile lyase having maintained or high hydroxynitrile lyase activity (however, the amino acid sequence corresponding to the 157th or 156th amino acid sequence in the amino acid sequence of the mutated hydroxynitrile lyase is not mutated). Suppose).   Furthermore, in the mutant hydroxynitrile lyase (1), the 103rd histidine in the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 is substituted with cysteine. Nitrile lyase.   The mutant hydroxynitrile lyase according to claim 1 or 2, which is specific to (S) -hydroxynitrile substrate. A mutant hydroxynitrile lyase gene having any one of the following base sequences (4) to (6):
(4) the nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 9;
(5) The nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 9 has a deletion, substitution and / or addition of 1 to several bases, and the protein encoded by the sequence has hydroxynitrile lyase activity A base sequence maintained or higher compared to a wild-type hydroxynitrile lyase having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6;
(6) The hydroxynitrile lyase activity of the protein having at least 99.2% homology to the base sequence set forth in SEQ ID NO: 7 or 9 and encoded by the sequence is SEQ ID NO: 2, A base sequence that is maintained or higher than a wild-type hydroxynitrile lyase having the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6.   A transformant which has been transformed with a vector containing the mutant hydroxynitrile lyase gene according to claim 4. A method for producing a mutant hydroxynitrile lyase comprising:
Culturing the transformant according to claim 5 to obtain a culture, and
And a step of purifying the mutant hydroxynitrile lyase from the culture.