Random transesterification lipase

Abstract

The present invention aims to provide an enzyme exhibiting random transesterification ability suitable for food applications, and to provide a lipase that exhibits high heat resistance in random transesterification of fats and oils.

Description

Technical Field

[0001] The present invention relates to random transesterification lipase and its use. This application claims priority based on Japanese Patent Application No. 2018-022487, filed on February 9, 2018, the entire contents of which are incorporated by reference. Background Art

[0002] The transesterification reaction of oils and fats is an effective method for improving the physical properties of oils and fats (melting point, crystallinity, heat resistance, etc.), and is generally divided into chemical transesterification and enzymatic transesterification (see, for example, non-patent documents 1 and 2). Chemical transesterification also has many issues such as high environmental impact and poor operational stability. In recent years, due to concerns about the health risks of trans fatty acids, the production of transesterified oils and fats as an alternative to partial hydrogenation, which is the cause of trans fatty acids, has attracted much attention, and the demand for enzymatic random transesterification has increased. It is generally believed that enzymatic random transesterification can utilize lipases from genera such as Candida, Alcaligenes, Pseudomonas, and Humicola (trade name: Novnzyme, Lipozyme TL IM) (see, for example, Patent Documents 1 to 3, Non-Patent Documents 3 and 4). It should be noted that the applicant reported a new lipase from a microorganism of the genus Geobacillus that exhibits random transesterification ability in a previous patent application (Patent Document 4).

[0003] Prior art literature

[0004] Patent Literature

[0005] Patent Document 1: International Publication No. 2006 / 059592

[0006] Patent Document 2: Japanese Patent No. 3791943

[0007] Patent Document 3: Japanese Patent Application Laid-Open No. 2008-194011

[0008] Patent Document 4: International Publication No. 2012 / 077614 Pamphlet

[0009] Non-patent literature

[0010] Non-patent document 1: Journal of the Oil Chemistry Society, 48, 1151-1159 (1999)

[0011] Non-patent document 2: Oleoscience, 6, 145-151 (2006)

[0012] Non-patent document 3: J. Am. Oil Chem. Soc., 60, 291-294 (1983)

[0013] Non-patent document 4: J. Am. Oil Chem. Soc., 75, 953-959 (1998). Summary of the Invention

[0014] In particular, there is a high demand for enzyme random transesterification in food applications. However, enzymes that can be used for food applications have not yet been put into practical use. The present invention aims to overcome this situation and provide an enzyme (random transesterification lipase) suitable for food applications.

[0015] To discover a random transesterification lipase suitable for food applications, the present inventors conducted a large-scale screening of approximately 1,500 strains of microorganisms (bacteria, yeasts, filamentous fungi, actinomycetes, etc.). As a result of this uniquely designed phased screening, they successfully obtained and identified an enzyme (a thermostable random transesterification lipase) that exhibits high random transesterification activity and excellent heat resistance in oils and fats. Furthermore, they successfully identified the amino acid sequence of this enzyme and the base sequence of the gene encoding it. The following invention is based on these findings.

[0016] [1] A lipase having the following enzymatic properties:

[0017] (1) Function: catalyze random transesterification reaction,

[0018] (2) Molecular weight: The molecular weight without N-type sugar chains is approximately 36 kDa (measured by SDS-PAGE).

[0019] (3) Temperature stability in oils and fats: Stable below 60°C (obtained by evaluation using tricaprylin and methyl stearate as substrates and the amount of methyl caprylate produced as an indicator),

[0020] (4) Reactivity in oils and fats: When reacting at 40 to 80°C, the highest reactivity is shown at 80°C (obtained by evaluation using cocoa butter as a substrate and tripalmitin production as an indicator).

[0021] [2] The lipase according to [1], further having the following enzymatic properties:

[0022] (5) pH stability: Stable within the pH range of 3 to 7 (30°C, 1 hour)

[0023] (6) Optimum pH: 6.

[0024] [3] The lipase according to [1] or [2], wherein the N-terminal amino acid sequence is VTDEPLENVPGILSHPTI (SEQ ID NO: 1).

[0025] [4] The lipase according to any one of [1] to [3], which is derived from Candida subplicata.

[0026] [5] The lipase according to [4], wherein the Candida subplicata is NBRC 0966 strain or a variant thereof.

[0027] [6] A lipase having the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence equivalent to the amino acid sequence.

[0028] [7] The lipase according to [6], wherein the equivalent amino acid sequence is an amino acid sequence that is 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2.

[0029] [8] An enzyme agent comprising the lipase according to any one of [1] to [7].

[0030] [9] A random transesterification method for fats and oils, comprising the step of allowing the lipase described in any one of [1] to [7] to act on the fats and oils.

[0031]

[10] A method for producing a lipase for random transesterification, comprising the following steps (1) and (2):

[0032] (1) a step of culturing Candida subplicata,

[0033] (2) A step of recovering lipase from the culture solution and / or bacterial cells after culture.

[0034]

[11] The production method according to

[10] , wherein the Candida subplicata is the NBRC 0966 strain.

[0035]

[12] A method for producing a randomly transesterified oil or fat, comprising the step of allowing the lipase described in any one of [1] to [7] to act on the oil or fat.

[0036]

[13] A lipase gene composed of any one of the following DNAs (a) to (c):

[0037] (a) a DNA encoding the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence equivalent thereto,

[0038] (b) a DNA consisting of the base sequence of SEQ ID NO: 4 or 5,

[0039] (c) A DNA having a base sequence equivalent to the base sequence of SEQ ID NO: 4 or 5 and encoding a protein having random transesterification activity.

[0040]

[14] A recombinant DNA comprising the lipase gene described in

[13] .

[0041]

[15] A microorganism that retains the recombinant DNA described in

[14] .

[0042]

[16] A method for producing lipase, comprising the following steps (i) and (ii):

[0043] (i) culturing the microorganism described in

[15] under conditions where the protein encoded by the gene is produced,

[0044] (ii) a step of recovering the produced protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The pH stability of a novel lipase from Candida pararugosa strain NBRC 0966.

[0046] Figure 2 This is the optimum pH for the novel lipase from Candida subplicata NBRC 0966.

[0047] Figure 3 The temperature stability of a novel lipase derived from Candida subplicata NBRC 0966 was compared in the amount of methyl octanoate produced in a reaction using tricaprylin and methyl stearate as substrates after treatment at various temperatures for 1 hour.

[0048] Figure 4 The reactivity of the novel lipase from Candida subplicata NBRC 0966 was compared based on the amount of palmitin (PPP) produced after 8 hours of reaction at various reaction temperatures.

[0049] Figure 5 This is a diagram of hydrophobic chromatography purification.

[0050] Figure 6 A shows the results of SDS-PAGE of the hydrophobic chromatography purification fraction. B shows the results of Endo-Hf treatment of the main activity peak fraction. Lane M: Molecular weight marker (GE Healthcare). Lane 1: Endo-Hf, Lane 2: Main activity peak fraction after Endo-Hf treatment, Lane 3: Main activity peak fraction without Endo-Hf treatment.

[0051] Figure 7 This is the time-dependent change in the amount of tripalmitin produced by the purified enzyme.

[0052] Figure 8 This is the time-dependent change in the amount of tripalmitin produced by the recombinant enzyme. DETAILED DESCRIPTION

[0053] 1. Terminology

[0054] In this specification, the term "isolated" is used interchangeably with "purified." The term "isolated" is used to distinguish a substance from its natural state, that is, the state in which it exists in nature. An "isolated state" is a state that is different from its natural state through the artificial process of separation. An isolated substance is clearly and decisively different from the natural substance itself.

[0055] The purity of the isolated enzyme is not particularly limited, but if it is intended for use in applications requiring high purity, the isolated enzyme preferably has a high purity.

[0056] 2. New lipase

[0057] The first aspect of the present invention relates to a novel lipase that the present inventors have successfully obtained and identified and discovered to be useful. The lipase of the present invention (hereinafter also referred to as "the present enzyme") is characterized by having the following enzymatic chemical properties.

[0058] (1) Function

[0059] This enzyme is a lipase that catalyzes random transesterification reactions. Random transesterification activity can be evaluated using a method using cocoa butter as a substrate (the "Random Transesterification Activity Assay" described below). Cocoa butter contains oleic acid, palmitic acid, stearic acid, and other fatty acids as its constituents, and its main component is a triglyceride with oleic acid bonded at the 2-position. Therefore, palmitin (PPP), in which palmitic acid is bonded to all of the 1-, 2-, and 3-positions, is produced only through random transesterification reactions (palmitic acid at position 1 or 3 needs to be exchanged with oleic acid at position 2. When using a lipase specific for positions 1 and 3 to carry out the reaction, triglycerides with palmitic acid inserted at position 2 are not produced). Therefore, the random transesterification activity can be evaluated using the amount of tripalmitin produced as an indicator. When evaluated using the random transesterification activity assay described below, if the reaction increases the production ratio of tripalmitin (C48), it can be said that the random transesterification reaction is catalyzed. The degree of increase is not particularly limited, but preferably ΔPPP(%) is 0.01 or more, more preferably ΔPPP(%) is 0.03 or more, and further preferably ΔPPP(%) is 0.05 or more.

[0060] (2) Molecular weight

[0061] When the enzyme is in its native form (when produced by Candida subplicata), it contains sugar chains (i.e., glycoproteins). The molecular weight of the enzyme after removal of N-type sugar chains, i.e., when it does not contain N-type sugar chains, is approximately 36 kDa (molecular weight measured by SDS-PAGE).

[0062] (3) Temperature stability in oils and fats

[0063] Temperature stability in fats and oils can be evaluated using a method (described in detail below) using tricaprylin and methyl stearate as substrates and the amount of methyl caprylate produced as an indicator. The enzyme of the present invention maintains 90% or more of its activity when treated in tricaprylin for one hour at temperatures below 60°C. This enzyme, exhibiting excellent temperature stability, is suitable for food applications requiring processing at relatively high temperatures (e.g., processing of fats and oils with high melting points).

[0064] (4) Reactivity in oils and fats

[0065] Reactivity in oils and fats can be evaluated using a method (described below) using cocoa butter as a substrate and tripalmitin production as an indicator. This enzyme exhibits peak reactivity at 80°C when reacting in oils and fats at temperatures between 40°C and 80°C. This high reactivity at high temperatures makes it suitable for food applications requiring relatively high temperature processing (e.g., processing of oils and fats with high melting points).

[0066] The enzyme can be further characterized using the following enzymatic chemical properties (5) and (6).

[0067] (5) pH stability

[0068] The present enzyme exhibits stable activity at pH 3 to 7. For example, if the pH of the enzyme solution used for treatment is within the range of 3 to 7, it exhibits 80% or more of its maximum activity after treatment at 30°C for 1 hour. The pH stability is determined based on the results of measurements in glycine-HCl buffer for the pH range of 2 to 3, in citric acid buffer for the pH range of 3 to 6, in phosphate buffer for the pH range of 6 to 8, in Tris-HCl buffer for the pH range of 8 to 9, and in sodium carbonate buffer for the pH range of 9 to 11.

[0069] (6) Optimum pH

[0070] The optimal pH of the present enzyme is 6. The optimal pH can be determined based on the results of measurement in a Britan-Robinson buffer (pH 2 to 9), for example.

[0071] The present enzyme can be further characterized by its source, namely "Candida pararugosa". "Derived from Candida pararugosa" refers to a lipase produced by a microorganism classified as Candida pararugosa (which can be a wild strain or a variant), or a lipase obtained by genetic engineering methods using the lipase gene of Candida pararugosa (which can be a wild strain or a variant). Therefore, a recombinant produced by a host microorganism into which a lipase gene obtained from Candida pararugosa (or a gene that has changed the gene) or a gene with an equivalent base sequence is introduced also belongs to the lipase from Candida pararugosa. For the sake of convenience, the Candida pararugosa that will become the source of this enzyme is referred to as the source bacteria of this enzyme. In addition, the microorganism used to produce this enzyme (Candida pararugosa, host microorganism) is referred to as the production bacteria.

[0072] A specific example of Candida subplicata is NBRC 0966. NBRC 0966 is a strain deposited at the Institute of Technology and Evaluation (2-5-8, Kazusa Kamata, Kisarazu-shi, Chiba Prefecture) (listed as NBRC 0966 in the NBRC Culture Catalogue) and can be obtained through prescribed procedures.

[0073] Amino acid sequence analysis of the purified enzyme (lipase) from NBRC strain 0966 revealed the N-terminal amino acid sequence to be VTDEPLENVPGILSHPTI (SEQ ID NO: 1). Based on this fact, the enzyme can be further characterized using this N-terminal amino acid sequence. The enzyme is characterized by containing the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence equivalent thereto, at its N-terminus.

[0074] The inventors of the present invention conducted further research and determined the amino acid sequence of the lipase produced by the NBRC 0966 strain (sequence number 2). Therefore, one embodiment of the present invention has the characteristic of being composed of a protein having the amino acid sequence shown in sequence number 2. Here, generally speaking, when a part of the amino acid sequence of a certain protein is changed, sometimes the changed protein has the same function as the protein before the change. That is, sometimes the change in the amino acid sequence does not have a substantial effect on the function of the protein, and the function of the protein is maintained before and after the change. Therefore, the present invention provides, as another embodiment, a protein composed of an amino acid sequence equivalent to the amino acid sequence shown in sequence number 2 and having random ester exchange activity (hereinafter also referred to as "equivalent protein").

[0075] The "equivalent amino acid sequence" of this specification refers to an amino acid sequence that is partially different from the sequence used as a benchmark (the amino acid sequence of sequence number 1 or the amino acid sequence of sequence number 2), but the difference does not substantially affect the function of the protein (here, the random ester exchange ability). Therefore, when the amino acid sequence of sequence number 1 is a benchmark, the enzyme having an equivalent amino acid sequence as the N-terminal amino acid sequence catalyzes a random ester exchange reaction. The degree of activity is not particularly limited as long as it can only function as a random ester exchange lipase. However, it is preferably the same as or higher than the enzyme having the sequence used as a benchmark at the N-terminus. Similarly, when the amino acid sequence of sequence number 2 becomes the benchmark, the enzyme having an equivalent amino acid sequence catalyzes a random ester exchange reaction. The degree of activity is not particularly limited as long as it can function as a random ester exchange lipase. However, it is preferably the same as or higher than the enzyme having the amino acid sequence (sequence number 2) used as a benchmark.

[0076] When the amino acid sequence of SEQ ID NO: 1 is used as a reference, "part of the amino acid sequence is different" typically refers to the deletion or substitution of 1 to several (for example, the upper limit is 2, 3, or 4) amino acids constituting the amino acid sequence, or the addition or insertion of 1 to several (for example, the upper limit is 2, 3, or 4) amino acids, or a combination thereof, resulting in a variation (change) in the amino acid sequence. The difference in the amino acid sequence here is allowed as long as the random transesterification ability is maintained (the activity may also vary slightly). As long as this condition is met, the position where the amino acid sequence differs is not particularly limited, and differences can occur at multiple positions. That is, when the amino acid sequence of SEQ ID NO: 1 is used as a reference, the equivalent amino acid sequence has, for example, a homology of about 75% or more, preferably about 80% or more, more preferably about 85% or more, even more preferably about 90% or more, and most preferably about 95% or more. It should be noted that the difference in the amino acid sequence can occur at multiple positions.

[0077] When the amino acid sequence of SEQ ID NO: 2 is used as a reference, "partial difference in the amino acid sequence" typically refers to a variation (change) in the amino acid sequence due to deletion or substitution of one to several (for example, the upper limit is 2, 3, 4, 5, 6, 7, 8, or 9) amino acids constituting the amino acid sequence, or addition or insertion of one to several (for example, the upper limit is 2, 3, 4, 5, 6, 7, 8, or 9) amino acids, or a combination thereof. Differences in the amino acid sequence are permitted as long as the ability to perform random transesterification is maintained (even if the activity may vary slightly). As long as this condition is met, the position at which the amino acid sequence differs is not particularly limited, and differences may occur at multiple positions. "Multiple" here refers to, for example, a number corresponding to less than about 30% of all amino acids, a number corresponding to less than about 20%, a number corresponding to less than about 15%, or a number corresponding to less than about 10%, preferably a number corresponding to less than about 5%, more preferably a number corresponding to less than about 3%, even more preferably a number corresponding to less than about 2%, and most preferably a number corresponding to less than about 1%. That is, equivalent proteins share, for example, about 70% or more, about 80% or more, about 85% or more, or about 90% or more, preferably about 95% or more, more preferably about 97% or more, even more preferably about 98% or more, and most preferably about 99% or more (the higher the percentage of homology, the more preferred) with the reference amino acid sequence. It should be noted that amino acid sequence differences may occur at multiple positions.

[0078] Equivalent amino acid sequences are preferably obtained by making conservative amino acid substitutions at amino acid residues that are not essential for random transesterification. "Conservative amino acid substitutions" herein refer to the replacement of an amino acid residue with an amino acid residue having a side chain with similar properties. Amino acid residues are classified into several families based on their side chains, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (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), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions are preferably substitutions between amino acid residues within the same family.

[0079] However, the homology (%) of two amino acid sequences or two nucleic acids (hereinafter, "two sequences" is used as a term including them) can be determined, for example, by the following steps. First, the two sequences are arranged in a manner that enables optimal comparison (for example, a gap can be introduced into the first sequence to optimize its alignment with the second sequence). When the molecule (amino acid residue or nucleotide) at a specific position of the first sequence is the same as the molecule at the corresponding position of the second sequence, it can be said that the molecules at that position are the same. The homology of two sequences is a function of the number of identical positions shared by the two sequences (i.e., homology (%) = number of identical positions / total number of positions × 100), preferably taking into account the number and size of the gaps required for optimization of the alignment.

[0080] The comparison of two sequences and the determination of homology can be achieved using a mathematical algorithm. As specific examples of mathematical algorithms that can be used for sequence comparison, there are algorithms described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-68 and modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-77, but are not limited thereto. Such algorithms have been incorporated into the NBLAST program and the XBLAST program (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10. In order to obtain a nucleotide sequence equivalent to the nucleic acid molecule of the present invention, for example, a BLAST nucleotide search can be performed in the NBLAST program with score = 100 and wordlength = 12. To obtain an amino acid sequence equivalent to the present enzyme, for example, a BLAST peptide search can be performed using the XBLAST program with score = 50 and wordlength = 3. To obtain gapped alignments for comparison, Gapped BLAST as described in Altschul et al. (1997) Amino Acids Research 25(17): 3389-3402 can be used. When using BLAST and Gapped BLAST, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. For details, please refer to http: / / www.ncbi.nlm.nih.gov. As an example of another mathematical algorithm that can be used for sequence comparison, there is the algorithm described in Myers and Miller (1988) Comput Appl Biosci. 4: 11-17. Such an algorithm is, for example, incorporated into the ALIGN program available on the GENESTREAM web server (IGH Montpellier, France) or the ISREC server. When utilizing the ALIGN program for amino acid sequence comparisons, for example, the PAM120 residue mass table, gap length penalty=12, and gap penalty=4 can be used.

[0081] The homology between two amino acid sequences can be determined using the GAP program of the GCG software package using a Blossom 62 matrix or a PAM250 matrix with a gap weight of 12, 10, 8, 6, or 4 and a gap length weight of 2, 3, or 4. Alternatively, the degree of identity between two nucleic acid sequences can be determined using the GAP program of the GCG software package (available at http: / / www.gcg.com) with a gap weight of 50 and a gap length weight of 3.

[0082] The enzyme may be part of a larger protein (e.g., a fusion protein). Examples of sequences added to the fusion protein include sequences useful for purification such as multiple amino acid residues and additional sequences to ensure stability during recombinant production.

[0083] This enzyme with above-mentioned amino acid sequence can be easily prepared by genetic engineering method.For example, can be by transforming suitable host cell (for example Escherichia coli) with the DNA of coding this enzyme, reclaim the protein expressed in transformant and prepare.The protein that is reclaimed can be suitably purified according to purpose.If so obtain this enzyme as recombinant protein, then can carry out various modifications.For example, if the DNA of coding this enzyme and other suitable DNA are inserted into identical vector and use this vector to carry out the production of recombinant protein, then can obtain this enzyme that is constituted by the recombinant protein that is connected with arbitrary peptide or protein.In addition, can implement the such modification of processing that adds or produces N-terminal or C-terminal of sugar chain and / or lipid.Through as above modification, can simplify the extraction, purification, or additional biological function etc. of recombinant protein.

[0084] 3. Enzymes

[0085] This enzyme can be provided, for example, in the form of an enzyme agent. In addition to the active ingredient (this enzyme), the enzyme agent can also contain excipients, buffers, suspending agents, stabilizers, preservatives, preservatives, physiological saline, etc. The degree of purification of the enzyme as the active ingredient is not particularly limited. It can be a crude enzyme or a purified enzyme. As excipients, lactose, sorbitol, D-mannitol, maltodextrin, white sugar, etc. can be used. As buffers, phosphates, citrates, acetates, etc. can be used. As stabilizers, propylene glycol, ascorbic acid, etc. can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methyl parahydroxybenzoate, etc. can be used. As preservatives, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. can be used.

[0086] 4. Nucleic acid encoding the enzyme, etc.

[0087] The second aspect of the present invention provides nucleic acids related to the present enzyme, that is, nucleic acids that can be used as probes for identifying genes encoding the present enzyme, nucleic acids encoding the present enzyme, and nucleic acids that can be used as primers for amplifying or mutating nucleic acids encoding the present enzyme.

[0088] The gene of encoding this enzyme is typically utilized in the preparation of this enzyme. According to the genetic engineering preparation method of the gene using this enzyme of encoding, this enzyme of more homogeneous state can be obtained. In addition, this method can also be said to be a suitable method when preparing a large amount of these enzymes. It should be noted that the purposes of the gene of encoding this enzyme are not limited to the preparation of this enzyme. For example, this nucleic acid can also be utilized as a tool for experiment with the mechanism of action of illustrating this enzyme etc. or as a tool for designing or making the variant of this enzyme.

[0089] In this specification, "gene encoding this enzyme" refers to a nucleic acid that can obtain this enzyme when expressed, and naturally includes nucleic acids having a base sequence corresponding to the amino acid sequence of this enzyme, and also includes nucleic acids formed by adding a sequence that does not encode an amino acid sequence in such a calculation. In addition, the degeneracy of codons is also considered. About nucleic acids having a base sequence that does not contain a start codon, this enzyme can be obtained by expressing on the basis of adding a start codon or a signal peptide containing a start codon.

[0090] Examples of sequences of genes encoding the present enzyme are shown in SEQ ID NO: 4 (cDNA sequence encoding the sequence of the mature form (amino acid sequence of SEQ ID NO: 2) and excluding the signal sequence) and SEQ ID NO: 5 (cDNA sequence encoding the sequence containing the signal peptide (amino acid sequence of SEQ ID NO: 3) and including the signal sequence).

[0091] The nucleic acid of the present invention can be prepared in an isolated state by using standard genetic engineering methods, molecular biological methods, biochemical methods, etc., with reference to the sequence information disclosed in this specification or the attached sequence listing.

[0092] In another embodiment of the present invention, a nucleic acid is provided which, when compared with the base sequence of the gene encoding the enzyme, has the same function as the protein encoded by it, but has a different base sequence in part (hereinafter also referred to as "equivalent nucleic acid". In addition, the base sequence of the specified equivalent nucleic acid is also referred to as "equivalent base sequence"). As an example of an equivalent nucleic acid, there can be cited a DNA which is composed of a base sequence based on the base sequence of the nucleic acid encoding the enzyme and containing one or more base substitutions, deletions, insertions, additions, and inversions and which encodes a protein having a characteristic enzymatic activity (i.e., random transesterification activity) for the enzyme. Substitutions, deletions, etc. of bases can occur at multiple sites. The "multiple" here also varies according to the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the nucleic acid, for example, 2 to 40 bases, preferably 2 to 20 bases, and more preferably 2 to 10 bases.

[0093] The equivalent nucleic acid has, for example, a homology of 60% or more, preferably 70% or more, more preferably 80% or more, further preferably 85% or more, further preferably about 90% or more, further preferably 95% or more, and most preferably 99% or more with respect to the base sequence serving as a reference (the sequence of sequence number 4 or the sequence of sequence number 5).

[0094] The equivalent nucleic acid described above can be obtained, for example, by treatment with restriction endonucleases, treatment with exonucleases, DNA ligases, etc., by introduction of mutations based on the site-directed mutagenesis method (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York), or the random mutagenesis method (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York). In addition, equivalent nucleic acids can also be obtained by other methods such as ultraviolet irradiation.

[0095] Another embodiment of the present invention relates to a nucleic acid having a base sequence complementary to the base sequence of a gene encoding the present enzyme. Still another embodiment of the present invention provides a nucleic acid having a base sequence that is at least about 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% identical to the base sequence of a gene encoding the present enzyme of the present invention or a base sequence complementary thereto.

[0096] Another embodiment of the present invention relates to a nucleic acid having a base sequence that hybridizes the base sequence of the gene encoding this enzyme or a base sequence complementary to its equivalent base sequence under stringent conditions." stringent conditions" herein refer to conditions that form so-called specific hybridization and do not form non-specific hybridization. Such stringent conditions are well known to those skilled in the art, and for example, can be set with reference to Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York), Current protocols in molecular biology (edited by Frederick M.Ausubel et al., 1987). Examples of stringent conditions include incubation at about 42°C to 50°C using a hybridization solution (50% formamide, 10×SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 5× Denhardt's solution, 1% SDS, 10% dextran sulfate, 10 μg / ml of modified salmon sperm DNA, and 50 mM phosphate buffer (pH 7.5)), followed by washing at about 65°C to 70°C using 0.1×SSC and 0.1% SDS. More preferred stringent conditions include conditions using a hybridization solution of 50% formamide, 5×SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 1× Denhardt's solution, 1% SDS, 10% dextran sulfate, 10 μg / ml of modified salmon sperm DNA, and 50 mM phosphate buffer (pH 7.5).

[0097] Another aspect of the present invention provides a nucleic acid (nucleic acid fragment) having a base sequence of a gene encoding this enzyme or a part of a base sequence complementary thereto. Such nucleic acid fragment can be used to detect, identify and / or amplify nucleic acids having a base sequence of a gene encoding this enzyme, etc. Nucleic acid fragments are, for example, designed to at least comprise a part that hybridizes with 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) in the base sequence of the gene encoding this enzyme. When used as a probe, nucleic acid fragments can be labeled. Labeling can, for example, use fluorescent substances, enzymes, radioactive isotopes.

[0098] Another aspect of the present invention relates to a recombinant DNA containing a gene of the present invention (a gene encoding the enzyme). The recombinant DNA of the present invention is provided, for example, in the form of a vector. The term "vector" as used herein refers to a nucleic acid molecule capable of delivering a nucleic acid inserted into the vector to a target cell or the like.

[0099] An appropriate vector can be selected based on the intended use (cloning, protein expression) and the type of host cell. Examples of vectors using Escherichia coli as a host include M13 phage or its variants, lambda phage or its variants, pBR322 or its variants (pB325, pAT153, pUC8, etc.); examples of vectors using yeast as a host include pYepSec1, pMFa, and pYES2; examples of vectors using insect cells as a host include pAc and pVL; and examples of vectors using mammalian cells as a host include pCDM8 and pMT2PC.

[0100] The vector of the present invention is preferably an expression vector. An "expression vector" refers to a vector that can introduce a nucleic acid inserted into the vector into a target cell (host cell) and express it in the cell. An expression vector generally contains a promoter sequence required for expression of the inserted nucleic acid, an enhancer sequence that promotes expression, and the like. An expression vector containing a selection marker can also be used. When using such an expression vector, the presence (and extent) of the expression vector can be confirmed by using a selection marker.

[0101] 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 carried out using standard recombinant DNA techniques (for example, see Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York, for well-known methods using restriction endonucleases and DNA ligases).

[0102] As host cells, bacteria (Escherichia coli, Bacillus subtilis, etc.), yeast (Saccharomyces cerevisiae, etc.), Pichia pastoris, etc.), filamentous fungi (Aspergillus oryzae, etc.) are preferably used from the viewpoint of ease of manipulation. oryzae) etc.), actinomycetes (Streptomyces (Streptomyces) etc.), as long as it is a host cell in which the recombinant DNA can be replicated and the gene of this enzyme can be expressed, just can be used. As an example of Escherichia coli, when utilizing T7 promoter, Escherichia coli BL21 (DE3) can be mentioned, and when not utilizing T7 promoter, Escherichia coli JM109, DH5α can be mentioned. In addition, as an example of yeast, budding yeast SHY2, budding yeast AH22 or budding yeast INVSc1 (In vitrogen), yeast Pichia pastris GS115 (Thermo Fisher Scientific) can be mentioned.

[0103] Another aspect of the present invention relates to a microorganism (i.e., a transformant) having the recombinant DNA of the present invention. The microorganism of the present invention can be obtained by transfection or transformation using the vector of the present invention. For example, the method can be carried out by calcium chloride method (Jarnal of molecura biology (J.Mol.Biol.), Vol. 53, p. 159 (1970)), Hanahan method (Jarnal of molecura biology, Vol. 166, p. 557 (1983)), SEM method (Gene, Vol. 96, p. 23 (1990)), Chung et al. method (Procidings of the nashonal academy of science of the USA, Vol. 86, p. 2172 (1989)), calcium phosphate coprecipitation method, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), lipofection (Felgner, P Lett. It can be carried out by, for example, saccharin ...

[0104] 5. Method for producing the enzyme

[0105] A further aspect of the present invention provides a method for producing the present enzyme. In the first embodiment of the production method of the present invention, a step of culturing a microorganism that produces the present enzyme (step (1)) and a step of recovering lipase from the culture solution and / or bacterial cells after culturing (step (2)) are performed. The microorganism that produces the present enzyme is Candida subtilis, preferably Candida subtilis NBRC 0966 strain or a variant thereof. The variant can be obtained by irradiation with ultraviolet rays, X-rays, gamma rays, etc., treatment with nitrous acid, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, etc. There is no limitation on the variant as long as it produces the present enzyme. Examples of the variant include strains that increase the productivity of the present enzyme, strains that reduce the productivity of inclusions, strains that are easier to culture, strains that are easier to recover from the culture solution, etc.

[0106] The culture conditions and culture methods are not particularly limited as long as they can produce this enzyme. That is, the method and culture conditions for cultivating the microorganisms suitable for use can be appropriately set as long as the enzyme can be produced. As the culture method, either liquid culture or solid culture can be used, preferably liquid culture. Taking liquid culture as an example, its culture conditions are described.

[0107] As a culture medium, there is no particular limitation as long as it is a culture medium in which the microorganisms used can grow. For example, a carbon source such as glucose, sucrose, gentiobiose, soluble starch, glycerol, dextrin, molasses, or an organic acid can be added, and a nitrogen source such as ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or peptone, yeast extract, corn steep liquor, casein hydrolyzate, wheat bran, or meat extract can be added, and a culture medium such as an inorganic salt such as potassium salt, magnesium salt, sodium salt, phosphate, manganese salt, iron salt, and zinc salt can be added. In order to promote the growth of the transformant used, vitamins, amino acids, etc. can be added to the culture medium. The pH of the culture medium is, for example, adjusted to about 3 to 8, preferably about 4 to 7, and the culture temperature is generally about 20 to 40°C, preferably about 25 to 35°C, and cultured under aerobic conditions for 1 to 20 days, preferably about 3 to 10 days. As a culture method, for example, a shaking culture method or an aerobic submerged culture method using a fermenter can be used.

[0108] After culturing under the above conditions, the target enzyme is recovered from the culture solution or the bacterial cell (step (2)). When recovered from the culture solution, for example, after removing insoluble matter by filtering the culture supernatant, centrifuging, etc., various chromatography methods such as concentration of ultrafiltration membrane, salting out such as ammonium sulfate precipitation, dialysis, ion exchange resin, etc. are appropriately combined to separate and purify, thereby obtaining this enzyme. On the other hand, when recovered from the bacterial cell, for example, after the bacterial cell is crushed by pressure treatment, ultrasonic treatment, etc., separation and purification are carried out in the same manner as above, thereby obtaining this enzyme. It should be noted that the above-mentioned series of steps (crushing, separation, purification of bacterial cell) can also be carried out after the bacterial cell is recovered from the culture solution in advance by filtering, centrifuging, etc.

[0109] The degree of enzyme purification is not particularly limited, and can be purified to a specific activity of 0.5 to 20 (U / mg), preferably 5 to 20 (U / mg). The final form can be liquid or solid (including powder).

[0110] In another embodiment of the present invention, the above-mentioned transformant is used to manufacture the present enzyme. In the manufacturing method of this embodiment, first, the above-mentioned transformant is cultured under conditions that produce a protein encoded by the gene introduced therein (step (i)). Regarding various vector-host systems, the culture conditions of the transformant are well known, and those skilled in the art can easily set appropriate culture conditions. Then, after the culture step, the produced protein (i.e., the present enzyme) is recovered (step (ii)). For recovery and subsequent purification, it is sufficient to proceed in the same manner as in the above-mentioned embodiment.

[0111] The purified enzyme obtained as described above can also be provided as a powder by, for example, freeze drying, vacuum drying, or spray drying. In this case, the purified enzyme can be dissolved in acetate buffer, phosphate buffer, triethanolamine buffer, tris-hydrochloric acid buffer, or GOOD buffer in advance. Preferably, acetate buffer, phosphate buffer, or triethanolamine buffer can be used. It should be noted that, as GOOD buffer, PIPES, MES, or MOPS can be mentioned.

[0112] Typically, as described above, gene expression and recovery of the expression product (the enzyme) are performed using an appropriate host-vector system, but a cell-free synthesis system can also be used. Here, a "cell-free synthesis system (cell-free transcription system, cell-free transcription / translation system)" refers to a system that does not use living cells, but instead uses ribosomes, transcription / translation factors, etc. from living cells (or obtained through genetic engineering methods) to synthesize the mRNA and protein encoded by the mRNA in vitro from nucleic acids (DNA, mRNA) serving as templates. In a cell-free synthesis system, a cell extract is generally used, which is obtained by purifying a cell lysate as needed. The cell extract generally contains various factors such as ribosomes and initiation factors necessary for protein synthesis, and various enzymes such as tRNA. When synthesizing proteins, various amino acids, energy sources such as ATP and GTP, and other substances necessary for protein synthesis such as creatine phosphate are added to the cell extract. Of course, when synthesizing proteins, ribosomes, various factors, and / or various enzymes prepared separately can be supplemented as needed.

[0113] The development of a reconstructed transcription / translation system for various molecules (factors) necessary for protein synthesis has also been reported (Shimizu, Y. et al.: Nature Biotech., 19, 751-755, 2001). In this synthetic system, genes for 31 factors, including three initiation factors, three elongation factors, four factors involved in termination, 20 amino acid acyl-tRNA synthetases that bind each amino acid to tRNA, and methionyl-tRNA formylation, that constitute the bacterial protein synthesis system, were amplified from the Escherichia coli genome and used to reconstruct the protein synthesis system in vitro. This reconstructed synthetic system can also be utilized in the present invention.

[0114] The term "cell-free transcription / translation system" can be used interchangeably with cell-free protein synthesis system, in vitro translation system, or in vitro transcription / translation system. In an in vitro translation system, RNA is used as a template to synthesize protein. As template RNA, total RNA, mRNA, in vitro transcription products, etc. can be used. In other in vitro transcription / translation systems, DNA is used as a template. The template DNA should contain a ribosome binding region and preferably contains an appropriate terminator sequence. It should be noted that in an in vitro transcription / translation system, conditions are set to allow the transcription reaction and translation reaction to proceed continuously by adding the factors necessary for each reaction.

[0115] 6. Application of this enzyme (random transesterification of oils and fats)

[0116] A further aspect of the present invention relates to a use of the present enzyme, providing a method for random transesterification of fats and oils using the present enzyme. In the random transesterification method of the present invention, the present enzyme is used as a catalyst. In other words, the present invention involves a step of allowing the present enzyme to act on the fat, thereby performing random transesterification of the fat, i.e., reorganizing (rearranging) the constituent fatty acids of triacylated glycerols (TG or TAG) in the fat.

[0117] Examples of fats and oils that can be treated by the random transesterification method of the present invention include vegetable oils and oils such as soybean oil, rapeseed oil, rice oil, corn oil, sunflower seed oil, cottonseed oil, peanut oil, safflower oil, palm oil, palm olein, palm fractionated oil, palm kernel oil, coconut oil, and cocoa butter, animal fats and oils such as fish oil, lard, tallow, and milk fat, and synthetic fats and oils such as their fractionated oils, hydrogenated oils, trilaurin, triolein, and tripalmitin. In addition to transesterification of fats and oils, the random transesterification method of the present invention can also be used for transesterification between fats and oils and fatty acids or fatty acid esters. Examples of fatty acids are stearic acid, palmitic acid, lauric acid, arachidic acid, behenic acid, oleic acid, and linoleic acid, and examples of fatty acid esters are ethyl stearate, ethyl palmitate, ethyl oleate, and ethyl linoleate.

[0118] In the random transesterification method of the present invention, the enzyme is added to fats and oils (an enzyme agent containing the enzyme may also be added), and the reaction is carried out at, for example, 30 to 100° C., preferably 35 to 80° C., for a predetermined time (e.g., 5 to 48 hours). To promote the reaction, stirring may be performed during the reaction.

[0119] As the enzyme used in the reaction, an immobilized enzyme can be used. In the reaction using the immobilized enzyme, a batch stirred tank reactor, a flow stirred tank reactor, a packed bed reactor, a fluidized bed reactor, etc. can be used.

[0120] The random transesterification method of the present invention is useful for modifying and improving the physical properties of fats and oils or processed fat products (e.g., shortening and margarine). For example, the random transesterification method of the present invention can be applied to improve spreadability, enhance emulsion stability, optimize solid fat content (SFC), enhance solidification properties, selectively enrich specific fatty acids, and produce low-trans acid fats and oils or processed fat products containing low-trans acid fats and oils. Improved physical properties of fats and oils obtained using the random transesterification method of the present invention, or processed fats and oils containing the same, compared to pre-treatment, have been confirmed, indicating high industrial utility.

[0121] As can be seen from the above description, according to the random transesterification method of the present invention, random transesterified oils and fats can be produced. That is, the present invention also provides a method for producing random transesterified oils and fats. Typically, in the method for producing random transesterified oils and fats of the present invention, the following steps are carried out: a step of preparing an acceptor substrate (oils and fats (triglycerides), glycerol fatty acid esters (diglycerides, monoglycerides), glycerol) and a donor substrate (fatty acids, ester compounds (fatty acid esters, etc.) or oils and fats (which may be the same as the acceptor substrate)) and a step of allowing the enzyme of the present invention to act (i.e., an enzyme reaction in the presence of the acceptor substrate and the donor substrate). The oils and fats, fatty acids, and fatty acid esters used in the acceptor substrate and the donor substrate may be the above-mentioned oils and fats, fatty acids, and fatty acid esters. The conditions for the enzyme reaction may be the above-mentioned conditions (for example, reacting for a specified time (such as 5 hours to 48 hours) under the conditions of 30 to 100 °C, preferably 35 to 80 °C).

[0122] Examples

[0123] <Method for measuring lipase activity>

[0124] Unless otherwise specified, the measurement of lipase activity (hydrolytic activity) is carried out using a lipase kit S (manufactured by DSPharma) in accordance with the manual attached to the kit. Among them, the buffer solution used is the attached buffer solution adjusted to pH 7, and the reaction stop solution used is acetone. The lipase activity is calculated from the following formula.

[0125] U / mL = (A412 sample - A412 blank) × 1 / 0.05 × n

[0126] (where A412 sample is the absorbance at 412 nm of the sample, A412 blank is the absorbance at 412 nm of the blank, 0.05 is the sample addition amount (mL), and n is the dilution factor)

[0127] <Measurement of pH stability>

[0128] The following buffers were used, namely 1M glycine-hydrochloric acid buffer (pH 2, 3), 1M citrate buffer (pH 3, 4, 5, 6), 1M phosphate buffer (pH 6, 7, 8), 1M Tris-hydrochloric acid buffer (pH 8, 9), and 1M sodium carbonate buffer (pH 9, 10, 11). The enzyme solution was mixed with an equal amount of each buffer and treated at 30°C for 1 hour. The treated sample was used to measure the lipase activity by the following method. First, 0.05mL of the lipase kit S colorimetric stock solution and 0.05mL of the lipase kit S substrate solution were added to 0.45mL of 0.5M PIPES solution (pH 7) and heated at 30°C for 5 minutes. Next, 0.025mL of the enzyme solution was added, and after reacting for 15 minutes, 1mL of ethanol was added to stop the reaction. The supernatant was recovered by centrifugation and the absorbance was measured (412nm). The lipase activity was calculated using the above formula.

[0129] <Determination of Optimum pH>

[0130] Use 20mM Bryant-Robinson buffer (pH 2, 3, 4, 5, 6, 7, 8, 9) as a buffer. Add 0.05mL of lipase kit S colorimetric stock solution and 0.05mL of lipase kit S substrate solution to 0.45mL of each buffer and heat at 30°C for 5 minutes. Add 0.025mL of enzyme solution, react for 15 minutes, and then add 1mL of lipase kit S reaction stop solution to stop the reaction. After adding 1mL of 1M Tris-HCl (pH 9), immediately measure the absorbance (412nm). Calculate the lipase activity using the above calculation formula.

[0131] <Random transesterification activity assay>

[0132] The enzyme solution was freeze-dried, and 1 mL of cocoa butter (Daito Cocoa Co., Ltd.) and 2 μL of ultrapure water were added per 1 U (lipase activity) of enzyme, and the mixture was reacted while stirring at 60°C. The reaction solution was sampled at each measurement time, and 10 μL was dissolved in 1 mL of hexane to prepare a sample for gas chromatography. The composition ratio of tripalmitin (C48) (PPP) in the triglyceride molecular species was calculated by gas chromatography analysis (column: DB-1HT (J&W, 5m×0.25mm, df0.1μm), temperature conditions: 120°C, held for 4 minutes, then heated to 150°C at a rate of 20°C / min, further heated to 315°C at a rate of 30°C / min and held for 3 minutes, then heated to 325°C at a rate of 1.5°C / min, further heated to 370°C at a rate of 30°C / min and held for 2 minutes, detector: FID, carrier gas: helium). The random transesterification ability of each enzyme was evaluated using the increase in the production ratio of C48, i.e., "(composition ratio of PPP in the triglyceride fraction of the reaction oil) - (composition ratio of PPP in the triglyceride fraction of the substrate oil)" (ΔPPP (%)) as an indicator.

[0133] <Evaluation of Thermal Stability in Oils and Fats>

[0134] (1) Evaluation of temperature stability

[0135] To 1 U of freeze-dried enzyme powder, 0.5 mL of tricaprylin (Wako Pure Chemical Industries, Ltd.) was added and thoroughly suspended. Heat treatment was performed at each temperature for 1 hour. After pre-incubation at 60°C for 10 minutes, 0.5 mL of methyl stearate (Wako Pure Chemical Industries, Ltd.) preheated at 60°C was added and thoroughly suspended. The enzyme reaction was then carried out at 60°C for 2 hours. 30 μL of the reaction solution was dissolved in 1 mL of hexane to prepare a gas chromatography sample.

[0136] The area value of the amount of methyl octanoate produced as a result of the enzyme reaction (ester exchange) was determined by gas chromatography analysis (column: DB-1HT (J&W, 5m×0.25mm, df 0.1μm), temperature conditions: 50°C, held for 1 minute, then heated to 370°C at a rate of 40°C / min, detector: FID, carrier gas: helium), and the residual activity was compared with that of the untreated sample.

[0137] (2) Evaluation of reactivity in oils and fats

[0138] The enzyme powder (freeze-dried product) equivalent to 5 U was suspended in cocoa butter, and 2 μL of purified water was added per 1 mL of cocoa butter to start the reaction. The tripalmitin production at 8 hours after the start of the reaction was compared between the reaction temperatures.

[0139] 1. Screening

[0140] To identify bacteria exhibiting random transesterification activity, approximately 1,500 strains, including bacteria, yeast, filamentous fungi, and actinomycetes, were screened. In the first screening, bacteria were grown on plates containing 0.5% soybean oil until halos formed. The plates were then incubated at 55°C, and strains with expanded halos were selected as thermostable lipase-producing bacteria.

[0141] In the second screening, the culture broth obtained by liquid culture of the strains selected in the first screening was powdered by vacuum drying at 60°C, 1 mL of cocoa butter and 2 μl of water were added, and the mixture was reacted for 8 days. Strains that could be confirmed by gas chromatography (GC) for the production of tripalmitin (Table 1), an indicator of random transesterification, were used in the following screening.

[0142] [Table 1]

[0143] NO. strain ΔPPP (%) 1 Candida nitrativorans 0.73 2 Candida sp 0.03 3 Pichia cf silvicola 0.09 4 Penicillium Resticulosum 0.09 5 Rhizopus cf.oryzae 0.09 6 Thermomyces cf.lanuginosus 0.41 7 Pichia xylosa 0.08 8 Pichia anomala 0.04 9 Candida pararugosa 0.35 10 Rhodotorula glutinis 0.05 11 Geobacillus sp. 0.07 12 Rhizopus oryzae 1.41 13 Rhizomucor miehei 0.21

[0144] In the third screening, the enzyme activity was maintained at a constant level (6 U) and the tripalmitin production was evaluated (reaction for 12 days) in the same manner as in the second screening. As a result, Candida pararugosa NBRC0966 was found to have a high random transesterification activity.

[0145] 2. Obtaining crude enzyme

[0146] 100 mL of the pre-culture medium was placed in a 500 mL Sakaguchi flask, and one platinum loop of steam-sterilized Candida pararugosa NBRC 0966 strain was inoculated and cultured at 27°C for 4 days to obtain a pre-culture solution.

[0147] <Pre-culture medium>

[0148] Yeast extract. (manufactured by Becton Dickinson Co., Ltd.) 0.3%

[0149] Malto extract. (manufactured by Becton Dickinson Co., Ltd.) 0.3%

[0150] Bacto Peptone (manufactured by Becton Dickinson Co., Ltd.) 0.5%

[0151] Hydrous crystalline glucose 1.0%

[0152] Soybean salad oil 0.5%

[0153] pH 6.2

[0154] The main culture medium was placed in a 30 L fermenter and steam sterilized, and then the pre-culture solution was added. The culture was cultured for 4 days at 27°C, with a stirring speed of 250 rpm and an aeration rate of 0.5 vvm to obtain a culture solution.

[0155] <Main culture medium>

[0156] Yeast extract. (manufactured by Becton Dickinson Co., Ltd.) 0.9%

[0157] Malto extract. (manufactured by Becton Dickinson Co., Ltd.) 0.9%

[0158] Peptone (manufactured by Becton Dickinson Co., Ltd.) 1.5%

[0159] Hydrous crystalline glucose (San-ei Sucrochemical) 3.0%

[0160] Soybean salad oil (Nisshin Oillio) 1.5%

[0161] pH 6.2

[0162] The resulting culture solution is centrifuged to recover the culture supernatant. The culture supernatant is filtered through diatomaceous earth to obtain a clarified solution. The clarified solution is concentrated and desalted by ultrafiltration, and the resulting desalted solution (crude enzyme solution) is freeze-dried to produce a crude enzyme powder. A portion of the crude enzyme solution is used for enzyme purification.

[0163] 3. Evaluation of enzyme chemical properties

[0164] (1) pH stability and optimal pH

[0165] The crude enzyme powder was dissolved in water to prepare an enzyme solution. The pH stability and optimum pH of the enzyme solution were determined by the above-mentioned determination method. The determination results are shown in Figure 1 (pH stability) and Figure 2 (Optimum pH) High activity is maintained at pH 3-7 (more than 80% remains after treatment at 30°C for 1 hour), and the optimal pH is 6.

[0166] (2) Temperature stability and reactivity in oils and fats

[0167] The crude enzyme powder was used to evaluate the temperature stability and reactivity in oils and fats by the above-mentioned evaluation method. The results are shown in Figure 3 (temperature stability) and Figure 4(Reactivity): Stable up to 60°C (in tricaprylin, after treatment for 1 hour, it maintains over 90% activity at temperatures below 60°C), demonstrating excellent temperature stability. Furthermore, within the 40-80°C range for oils and fats, it exhibits peak reactivity at 80°C.

[0168] 4. Enzyme Purification and Molecular Weight Determination

[0169] The crude enzyme solution was subjected to hydrophobic chromatography (Butyl-HP (manufactured by GE Health Care)). The column was equilibrated with 20 mM phosphate buffer, pH 7.0, and eluted with a linear gradient of ammonium sulfate (0.5 → 0 M) over 30 CV. The lipase activity (hydrolytic activity) was used as an indicator for fractionation. The purification diagram is shown in FIG. Figure 5 Since multiple fractions containing lipase activity could be identified, these fractions were subjected to SDS-PAGE. Meanwhile, the fraction with the activity peak (main activity peak fraction) was treated with Endo-Hf (manufactured by New England Biolabs) (to remove N-type sugar chains), and the molecular weights before and after treatment were compared by SDS-PAGE.

[0170] The results of SDS-PAGE of the purified fractions are shown in Figure 6 A. In addition, the results of Endo-Hf treatment of the active main peak fraction are shown in Figure 6 B. The molecular weight of the target enzyme (lipase) after Endo-Hf treatment (after removal of N-linked sugar chains) is approximately 36 kDa. Amino acid sequence analysis of the purified enzyme revealed the N-terminal amino acid sequence to be VTDEPLENVPGILSHPTI (SEQ ID NO: 1). Database searches based on this N-terminal sequence revealed no identical proteins.

[0171] 5. Confirmation of Random Transesterification Activity of Purified Enzyme

[0172] The random transesterification activity was measured using the purified fractions (fractions 48 to 52 were recovered and concentrated and desalted). The formation of tripalmitin (PPP) was also confirmed in the purified fractions ( Figure 7 ), indicating that the target enzyme was successfully purified.

[0173] 6. Gene cloning

[0174] The Candida rugosa NBRC 0966 strain was cultured in YM medium (Yeast extract. (Bacto) 0.9%, Malto extract. (Bacto) 0.9%, Peptone (Bacto) 1.5%, hydrated crystalline glucose 3.0%, soybean salad oil 1.5%) (pH 6.2) supplemented with 0.5% soybean salad oil for one day, and the cells were recovered by centrifugation. The recovered cells were thoroughly washed with TE buffer (pH 8.0). The obtained cells were ground using a mortar and pestle under liquid nitrogen. Total RNA was extracted from the ground cells using RNAisoPlus (manufactured by Takara Bio) according to the attached instructions. Next, Oligotex TM -dT30 <super>mRNA was prepared from the extracted RNA using mRNA Purification Kit (manufactured by Takara Bio) according to the attached instructions.

[0175] Using SuperScript TM cDNA was synthesized using the III First-Strand Synthesis System (manufactured by Thermo Fisher Scientific) according to the attached instructions. The resulting cDNA was used as a template, and PCR was performed using primers with homology upstream of the structural gene (RELFUP: 5'-CACATCTCAATAGCATCA-3': SEQ ID NO: 6) and downstream of the structural gene (RELEDOWN: 5'-AGCTGGGTATGTTCAGAAGTTAA-3': SEQ ID NO: 7) using PrimeSTAR (registered trademark) Max DNA Polymerase (manufactured by Takara Bio) according to the attached instructions. The approximately 1000 bp amplification product, confirmed to be amplified, was cloned using the Mighty TA-cloning Kit (manufactured by Takara Bio), and the sequence was confirmed (SEQ ID NO: 5).

[0176] 7. Recombinant Expression

[0177] Utilize Pichia expression system, confirm that the cDNA obtained is the gene of target enzyme.Utilize PCR to increase from the initiator codon of cDNA sequence (sequence number 5) to the terminator codon, clone into the snaBI site of pPick3.5k (ThermoFisher Science system) according to ordinary method, prepare expression vector pPick3.5k-REL.Utilize restriction endonuclease SalI to cut off pPick3.5k-REL, reclaim linear DNA by ethanol precipitation.Use the DNA that reclaims, according to standard method, Pichia pastris GS115 (Thermo Fisher Science system) is transformed by electroporation, obtain transformant. The transformants were cultured in BMGY medium (2% bacto-peptone, 1% yeast extract, 1% glycerol, 0.67% yeast nitrogen base (YNB (containing ammonium sulfate, no amino acids)), 100 mM potassium phosphate buffer (pH 6.0), 0.00004% D-biotin) at 30°C for 2 days. The resulting cells were suspended in BMMY medium (2% bacto-peptone, 1% yeast extract, 0.5% methanol, 0.67% yeast nitrogen base (YNB (containing ammonium sulfate, no amino acids)), 100 mM potassium phosphate buffer (pH 6.0), 0.00004% D-biotin) and cultured at 30°C for 3 days while adding appropriate amounts of methanol. The supernatant was recovered by centrifugation to obtain a crude enzyme solution. The lipase activity of the resulting crude enzyme solution was confirmed according to the above method.

[0178] 8. Confirmation of Random Transesterification Activity

[0179] The crude enzyme solution obtained was used to confirm the random transesterification activity according to the above method. The results are shown in Figure 8 The formation of tripalmitin (PPP) was confirmed ( Figure 8 ), it was confirmed that the sequence of SEQ ID NO: 5 was the gene (cDNA) for the target enzyme (lipase exhibiting random transesterification activity). The amino acid sequence of the target enzyme encoded by the sequence of SEQ ID NO: 5 is shown in SEQ ID NO: 3. The 63 bases at the 5' end of the sequence of SEQ ID NO: 5 constitute a signal sequence, and the sequence excluding the signal sequence (SEQ ID NO: 4) encodes the amino acid sequence of the mature enzyme (excluding the signal peptide) (SEQ ID NO: 2).

[0180] Industrial applicability

[0181] The random transesterification lipase of the present invention has high heat resistance and is suitable for use in food applications. The random transesterification lipase of the present invention is used, for example, to modify edible oils and fats such as margarine and shortening.

[0182] The present invention is not limited in any way by the description of the embodiments and examples of the invention described above. Various modifications that can be easily conceived by those skilled in the art are also included in the present invention without departing from the scope of the claims. The contents of papers, published patent publications, patent publications, etc. explicitly mentioned in this specification are incorporated by reference in their entirety. Sequence Listing <110> Amano Enzyme Products Co., Ltd. <120> Random transesterification lipase <130> AE17005P <150> JP P2018-022487 <151> 2018-02-09 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> PRT <213> Candida subplicata <400> 1 Val Thr Asp Glu Pro Leu Glu Asn Val Pro Gly Ile Leu Ser His Pro 1 5 10 15 Thr Ile <210> 2 <211> 324 <212> PRT <213> Candida subplicata <400> 2 Val Thr Asp Glu Pro Leu Glu Asn Val Pro Gly Ile Leu Ser His Pro 1 5 10 15 Thr Ile His Gly Phe Pro Ser Gly Gly Tyr Gly Pro Ser Gln Asp Thr 20 25 30 Phe Ile Ala Ala Ser Leu Tyr Ala Lys Gly His Ile Gly Tyr Ala Pro 35 40 45 Pro Gly Ser Gly Asn Ala Ser Cys Val Val Gly Glu Gly Gln Tyr Pro 50 55 60 Val Val Leu Val Pro Gly Thr Val Glu Asp Ala Tyr Ser Asn Trp Ala 65 70 75 80 Tyr Tyr Ser Gln Lys Leu Thr Asp Gln Gly Leu Cys Val Tyr Thr Phe 85 90 95 Asn His Asn Pro Met Ser Phe Phe Gly Gln Ser Glu Val Leu Gly Ile 100 105 110 Ser Leu Glu Ala Trp Pro Phe Ala Gly Asp Ile Lys Asp Ser Ala Ala 115 120 125 Ala Leu Ser Gln Val Val Asp Tyr Val Leu Gln Ile Thr Gly Ala Ser 130 135 140 Lys Val Asp Leu Val Gly His Ser Gln Gly Gly Gly Ala Leu Pro Ser 145 150 155 160 Trp Tyr Ile Lys Lys Leu Gly Gly Ala Pro Lys Val Asn Lys Met Val 165 170 175 Ala Leu Ala Gly Asp Tyr Lys Gly Thr Asn Pro Val Gly Leu Gly Ala 180 185 190 Leu Leu Thr Glu Val Gly Leu Gly Thr Val Ala Asp Thr Ile Leu Asn 195 200 205 Glu Ser Ile Asn Ala Glu Gly Leu Thr Gln Gln Leu Thr Gly Ser Asp 210 215 220 Phe Met Lys Glu Leu Asn Asp Gly Asp Gly Pro Gly Val Ala Gly Val 225 230 235 240 Arg Tyr Thr Asn Ile Ala Thr Met Tyr Asp Glu Ile Leu Ile Pro Phe 245 250 255 Thr Asn Thr Trp Phe Thr Gln Asp Gly Val Asp Val Asn Asn Ile Lys 260 265 270 Ile Gln Asp Tyr Cys Ala Leu Asp Phe Thr Asp His Ile Gly Phe Ala 275 280 285 Tyr Asp Pro Val Ala Tyr Gln Ile Val Glu Asn Val Leu Leu Asp Lys 290 295 300 Asn Asp Lys Ile Lys Cys Thr Tyr Val Pro Pro Val Phe Gln Lys Arg 305 310 315 320 Glu Phe Glu Ala <210> 3 <211> 345 <212> PRT <213> Candida parapsilosis <400> 3 Met Ile Ser Ser Val Phe Leu Ser Ala Leu Ser Leu Ala Ala Ser Phe 1 5 10 15 Pro Leu Asn Lys Arg Val Thr Asp Glu Pro Leu Glu Asn Val Pro Gly 20 25 30 Ile Leu Ser His Pro Thr Ile His Gly Phe Pro Ser Gly Gly Tyr Gly 35 40 45 Pro Ser Gln Asp Thr Phe Ile Ala Ala Ser Leu Tyr Ala Lys Gly His 50 55 60 Ile Gly Tyr Ala Pro Pro Gly Ser Gly Asn Ala Ser Cys Val Val Gly 65 70 75 80 Glu Gly Gln Tyr Pro Val Val Leu Val Pro Gly Thr Val Glu Asp Ala 85 90 95 Tyr Ser Asn Trp Ala Tyr Tyr Ser Gln Lys Leu Thr Asp Gln Gly Leu 100 105 110 Cys Val Tyr Thr Phe Asn His Asn Pro Met Ser Phe Phe Gly Gln Ser 115 120 125 Glu Val Leu Gly Ile Ser Leu Glu Ala Trp Pro Phe Ala Gly Asp Ile 130 135 140 Lys Asp Ser Ala Ala Ala Leu Ser Gln Val Val Asp Tyr Val Leu Gln 145 150 155 160 Ile Thr Gly Ala Ser Lys Val Asp Leu Val Gly His Ser Gln Gly Gly 165 170 175 Gly Ala Leu Pro Ser Trp Tyr Ile Lys Lys Leu Gly Gly Ala Pro Lys 180 185 190 Val Asn Lys Met Val Ala Leu Ala Gly Asp Tyr Lys Gly Thr Asn Pro 195 200 205 Val Gly Leu Gly Ala Leu Leu Thr Glu Val Gly Leu Gly Thr Val Ala 210 215 220 Asp Thr Ile Leu Asn Glu Ser Ile Asn Ala Glu Gly Leu Thr Gln Gln 225 230 235 240 Leu Thr Gly Ser Asp Phe Met Lys Glu Leu Asn Asp Gly Asp Gly Pro 245 250 255 Gly Val Ala Gly Val Arg Tyr Thr Asn Ile Ala Thr Met Tyr Asp Glu 260 265 270 Ile Leu Ile Pro Phe Thr Asn Thr Trp Phe Thr Gln Asp Gly Val Asp 275 280 285 Val Asn Asn Ile Lys Ile Gln Asp Tyr Cys Ala Leu Asp Phe Thr Asp 290 295 300 His Ile Gly Phe Ala Tyr Asp Pro Val Ala Tyr Gln Ile Val Glu Asn 305 310 315 320 Val Leu Leu Asp Lys Asn Asp Lys Ile Lys Cys Thr Tyr Val Pro Pro 325 330 335 Val Phe Gln Lys Arg Glu Phe Glu Ala 340 345 <210> 4 <211> 975 <212> DNA <213> I'm sorry <400> 4 gttacggacg aacccttgga aaatgtccct ggaatactgt ctcaccccac tatccatggc 60 ttccctagtg gaggctatg tccctctcaa gatactttca tagctgcttc gttgtatgcc 120 aagggccata ttggatatgc tcctcctggc tctggtaacg cttcctgcgt tgttggggag 180 gggcagtatc ctgtcgttct cgtacctggt actgtggagg acgcctactc aaactgggcg 240 tactactccc agaaattaac tgaccagggt ctctgtgttt acactttcaa ccacaaccca 300 atgagtttct ttgggcagag cgaggttctc ggtatctctt tggaggcatg gccattgct 360 ggagatatta aagatagtgc agctgctctt tctcaagtag tggactatgt tttgcagatt 420 accggtgcga gcaaggttga tctcgttgga cactctcagg gtggtggagc attgccaagt 480 tggtacatca agaaacttgg aggcgcccct aaggttaaca agatggttgc tcttgctggt 540 gactacaagg gcaccaaccc ggttggtctt ggggctctct tgaccgaggt aggacttggt 600 acggtggcag acacaatttt gaatgagtct attaatgctg agggtttgac ccaacaactc 660 actgggtcag acttcatgaa ggaactcaat gacggtgacg gacctggagt tgcaggggtt 720 cgctacacta acatcgctac aatgtatgat gagattctca tcccgtttac aaatacttgg 780 ttcactcaag atggagtgga tgtcaacaac atcaaaatcc aggattactg tgcactcgat 840 tttactgatc acatcggctt tgcctatgat cccgttgcat atcagatcgt tgagaacgtt 900 ttgctggaca agaacgataa gatcaagtgc acttatgtac ctcctgtgtt ccagaagcgc 960 gaatttgaag cctaa 975 <210> 5 <211> 1038 <212> DNA <213> Candida pararugosa <400> 5 atgatctcga gcgtcttcct ctctgcactc tctcttgctg cctcctttcc gctgaacaag 60 cgtgttacgg acgaaccctt ggaaaatgtc cctggaatac tgtctcaccc cactatccat 120 ggcttcccta gtggaggcta tggtccctct caagatactt tcatagctgc ttcgttgtat 180 gccaagggcc atattggata tgctcctcct ggctctggta acgcttcctg cgttgttggg 240 gaggggcagt atcctgtcgt tctcgtacct ggtactgtgg aggacgccta ctcaaactgg 300 gcgtactact cccagaaatt aactgaccag ggtctctgtg tttacacttt caaccacaac 360 ccaatgagtt tctttgggca gagcgaggtt ctcggtatct ctttggaggc atggccattt 420 gctggagata ttaaagatag tgcagctgct ctttctcaag tagtggacta tgttttgcag 480 attaccggtg cgagcaaggt tgatctcgtt ggacactctc agggtggtgg agcattgcca 540 agttggtaca tcaagaaact tggaggcgcc cctaaggtta acaagatggt tgctcttgct 600 ggtgactaca agggcaccaa cccggttggt cttggggctc tcttgaccga ggtaggactt 660 ggtacggtgg cagacacaat tttgaatgag tctattaatg ctgagggttt gacccaacaa 720 ctcactgggt cagacttcat gaaggaactc aatgacggtg acggacctgg agttgcaggg 780 gttcgctaca ctaacatcgc tacaatgtat gatgagattc tcatcccgtt tacaaatact 840 tggttcactc aagatggagt ggatgtcaac aacatcaaaa tccaggatta ctgtgcactc 900 gattttactg atcacatcgg ctttgcctat gatcccgttg catatcagat cgttgagaac 960 gttttgctgg acaagaacga taagatcaag tgcacttatg tacctcctgt gttccagaag 1020 cgcgaatttg aagcctaa 1038 <210> 6 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Forward primer <400> 6 cacatctcaa tagcatca 18 <210> 7 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Reverse primer <400> 7 agctgggtat gttcagaagt taa 23< / super>

Claims

1. A method for random transesterification of fats and oils for food use, comprising the step of allowing a lipase having the following enzymatic chemical properties to act on the fats and oils, wherein the lipase comprises the amino acid sequence shown in SEQ ID NO:

2. (1) Function: catalyze random transesterification reaction, (2) Molecular weight: The molecular weight without N-type sugar chains is approximately 36 kDa, which is the molecular weight measured by SDS-PAGE. (3) Temperature stability in oils and fats: Stable below 60°C, which is obtained by evaluation using tricaprylin and methyl stearate as substrates and the amount of methyl caprylate produced as an indicator. (4) Reactivity in oils and fats: When reacting at 40-80°C, the highest reactivity is shown at 80°C. This was obtained by evaluation using cocoa butter as a substrate and tripalmitin production as an indicator.

2. The random transesterification method for fats and oils for food use according to claim 1, wherein The lipase further has the following enzymatic chemical properties, (5) pH stability: Stable in the range of pH 3 to 7 at 30°C for 1 hour. (6) Optimum pH:

6.

3. The random transesterification method for fats and oils for food use according to claim 1 or 2, wherein: The N-terminal amino acid sequence is VTDEPLENVPGILSHPTI, i.e., sequence number 1.

4. The random transesterification method for fats and oils for food use according to any one of claims 1 to 3, wherein The lipase is from Candida subplicata.

5. The random transesterification method for fats and oils for food use according to claim 4, wherein The Candida subplicata is the NBRC 0966 strain.

6. A method for producing a lipase for random transesterification, comprising the following steps (I) and (II): (1) a step of culturing Candida subplicata, (II) a step of recovering lipase from the culture solution and / or bacterial cells after culture, The lipase has the following enzymatic chemical properties. The lipase is composed of the amino acid sequence shown in SEQ ID NO:

2. (1) Function: catalyze random transesterification reaction, (2) Molecular weight: The molecular weight without N-type sugar chains is approximately 36 kDa, which is the molecular weight measured by SDS-PAGE. (3) Temperature stability in oils and fats: Stable below 60°C, which is obtained by evaluation using tricaprylin and methyl stearate as substrates and the amount of methyl caprylate produced as an indicator. (4) Reactivity in oils and fats: When reacting at 40-80°C, the highest reactivity is shown at 80°C. This was obtained by evaluation using cocoa butter as a substrate and tripalmitin production as an indicator.

7. The manufacturing method according to claim 6, wherein: The Candida subplicata was the NBRC 0966 strain.

8. A method for producing a randomly transesterified oil, comprising the step of allowing a lipase having the following enzymatic properties to act on the oil, wherein the lipase comprises the amino acid sequence shown in SEQ ID NO:

2. (1) Function: catalyze random transesterification reaction, (2) Molecular weight: The molecular weight without N-type sugar chains is approximately 36 kDa, which is the molecular weight measured by SDS-PAGE. (3) Temperature stability in oils and fats: Stable below 60°C, which is obtained by evaluation using tricaprylin and methyl stearate as substrates and the amount of methyl caprylate produced as an indicator. (4) Reactivity in oils and fats: When reacting at 40-80°C, the highest reactivity is shown at 80°C. This was obtained by evaluation using cocoa butter as a substrate and tripalmitin production as an indicator.

9. A lipase gene consisting of any one of the DNAs selected from the following (a) and (b): (a) a DNA encoding the amino acid sequence of SEQ ID NO: 2, (b) A DNA consisting of the base sequence of SEQ ID NO: 4 or 5.

10. A recombinant DNA comprising the lipase gene according to claim 9.

11. A microorganism harboring a recombinant DNA containing the lipase gene according to claim 9.

12. A method for producing lipase, comprising the following steps (i) and (ii): (i) culturing the microorganism according to claim 11 under conditions where the protein encoded by the lipase gene is produced, (ii) a step of recovering the produced protein.

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