WO2018180187A1

WO2018180187A1 - Degradation of ethyl carbamate

Info

Publication number: WO2018180187A1
Application number: PCT/JP2018/007918
Authority: WO
Inventors: 和典吉田; 哲也 ▲高▼橋
Original assignee: 天野エンザイム株式会社; 江南大学
Priority date: 2017-03-30
Filing date: 2018-03-02
Publication date: 2018-10-04
Also published as: JP7138867B2; CN111065280A; JPWO2018180187A1

Abstract

The present invention addresses the problem of providing a practically useful method whereby ethyl carbamate in a food or beverage can be efficiently degraded. A food or beverage containing ethyl carbamate is treated with an esterase to thereby degrade ethyl carbamate.

Description

Decomposition of ethyl carbamate

The present invention relates to a method for decomposing ethyl carbamate. Specifically, the present invention relates to a method for decomposing ethyl carbamate in foods or beverages. This application claims priority based on Japanese Patent Application No. 2017-068426 filed on Mar. 30, 2017, the entire contents of which are incorporated by reference.

Ethyl carbamate (hereinafter sometimes abbreviated as “EC”) is a compound suspected to be carcinogenic or mutagenic, although it has been used as a pharmaceutical in the past. There are concerns about the effects on the human body when ingested. In particular, Shaoxing liquor, sherry liquor, or fermented foods contain a relatively large amount of EC, and development of means for reducing or removing EC is desired. Various attempts have been made so far. For example, a method using uretanase (see, for example, Patent Document 1 and Non-Patent Documents 1 to 3) and a method using a cell of a microorganism producing an EC-degrading enzyme ( For example, see Patent Documents 2 and 3).

Japanese Patent Laid-Open No. 4-325079 Chinese Patent No. 10249263B JP H1-204017

The method of degrading EC using uretanase has a problem that the stability of uretanase in the presence of alcohol is low and the thermal stability is also low. On the other hand, the decomposition method using microbial cells is not particularly satisfactory in terms of practicality.

Alcoholic beverages and fermented foods can be ingested daily and continuously, and their EC content will be a major problem in the future. In order to cope with such a situation, an object of the present invention is to provide a practical method capable of efficiently decomposing EC in food or beverage.

In order to solve the above problems, the present inventors conducted a large-scale screening for a wide range of enzymes with the aim of establishing an EC degradation method using enzymes. As a result, it has been clarified that esterase derived from Acinetobacter calcoaceticus shows high degradation activity against EC. Surprisingly, the esterase exhibited a degradation activity even under high alcohol concentration. This fact shows that esterase derived from Acinetobacter calcoaceticus is useful as a means of degrading EC in alcoholic beverages. Here, esterases are classified as serine proteases, and are usually subject to activity inhibition by alcohol hydroxyl groups. From this technical common sense, the new utilization form of esterase that has been clarified by the study of the present inventors, that is, “use of esterase for the degradation of EC in alcoholic beverages” can be said to be extremely unique.

On the other hand, when the EC-degrading activity of an esterase-related enzyme (amino acid sequence identity of 70% or more) derived from Acinetobacter calcoaceticus was confirmed, the related enzyme also showed a degrading activity, indicating that EC can be degraded.・ It became clear that it is not unique to esterases derived from Calcoaceticus.

Further investigation revealed that esterase derived from Acinetobacter calcoaceticus has high stability in alcohol and temperature stability, and is excellent in practicality. In addition, various properties of the esterase were clarified, and useful information for practical use was also provided.

The following invention is based on the above results and considerations.
[1] Decomposing ethyl carbamate in food or beverage, characterized by allowing esterase comprising 70% or more of the amino acid sequence of SEQ ID NO: 1 to act on food or beverage containing ethyl carbamate how to.
[2] The degradation method according to [1], wherein the amino acid sequence of the esterase includes any one of the amino acid sequences of SEQ ID NOS: 1 to 18.
[3] The degradation method according to [1], wherein the esterase is derived from an Acinetobacter genus microorganism, a Pseudomonas genus microorganism, a Burkholderia microorganism, or a Parabalkhorderia microorganism.
[4] The microorganism belonging to the genus Acinetobacter is Acinetobacter calcoaceticus, Acinetobacter guillouiae, Acinetobacter baumannii, or the genus Acinetobacter baumannii, Is Pseudomonas aeruginosa, Pseudomonas fluorescens or Pseudomonas putida, and the Burkholderia is Burkholderia ubonensis or Burkholderia [3] Borans (Purukholderia pseudomultivorans), and the Parabalkholderia is Paraburkholderia ferrariae [3 ] The decomposition method as described in.
[5] The esterase is Acinetobacter sp. NBRC 110496 (Acinetobacter sp. NBRC 110496), Acinetobacter sp. NIPH809 (Acinetobacter sp. NIPH809), Pseudomonas fluorescens A506, Pseudomonas sp. ABAC61), Pseudomonas putida IFO12996 (Pseudomonas putida IFO12996) or Pseudomonas putida MR2068 (Pseudomonas putida MR2068).
[6] The degradation method according to [1], wherein the esterase has the following characteristics:
(1) Optimal temperature: 20-30 ° C;
(2) Optimal pH: pH 7;
(3) Temperature stability: stable up to 70 ° C (

pH

7, 1 hour);
(4) pH stability: stable at pH 5-11;
(5) Molecular weight: about 85 kDa (by gel filtration).
[7] The degradation method according to [6], wherein the esterase further comprises the following characteristics:
(6) Alcohol stability: If the alcohol concentration is 40% or less, it will not be inactivated even if treated at 30 ° C. for 8 days.
[8] The decomposition method according to any one of [1] to [7], wherein the beverage is an alcoholic beverage.
[9] The alcoholic beverage is Shaoxing liquor, distilled liquor made from drupe, whiskey, brandy, tequila, cachassa, shochu, sake, wine, refined wine, plum wine, sherry liquor, or mixed liquor. [8] The disassembly method described in 1.
[10] A method for producing a food or beverage from which ethyl carbamate is removed or reduced, comprising a treatment step with an esterase comprising an amino acid sequence 70% or more identical to the amino acid sequence of SEQ ID NO: 1.
[11] The production method according to [10], wherein the esterase is the esterase defined in any one of [2] to [7].
[12] The production method according to [10] or [11], wherein the beverage is an alcoholic beverage.
[13] In the above [12], the alcoholic beverage is Shaoxing liquor, distilled liquor made from drupe, whiskey, brandy, tequila, cachassa, shochu, sake, wine, refined wine, plum wine, sherry liquor, or mixed liquor. The manufacturing method as described.
[14] A food or beverage obtained by the production method according to any one of [10] to [13] from which ethyl carbamate has been removed or reduced.

EC degradation activity of related enzymes. An esterase-related enzyme derived from Acinetobacter calcoaceticus (A. calcoaceticus) was expressed in an E. coli expression system, and the presence or absence of EC degradation ability was examined. Top: Ability to degrade EC at pH 7.0 of related enzymes No.1 to No.10. Bottom: EC degradation ability of related enzymes No. 11 to No. 16 at pH 7.0. Alcohol stability of esterase from A. calcoaceticus. A. Optimum temperature of calcoaceticus-derived esterase. A. Optimal pH of calcoaceticus-derived esterase. Temperature stability of esterase derived from A. calcoaceticus. PH stability of esterase derived from A. ticcalcoaceticus.

<Decomposition of ethyl carbamate in food or beverage>
The present invention is a method for decomposing ethyl carbamate in foods or beverages, characterized in that esterase is allowed to act on foods or beverages containing ethyl carbamates. According to the present invention, a food or beverage from which ethyl carbamate is removed or reduced can be obtained.

1. Amino acid sequence defining esterase The esterase used in the present invention is not particularly limited as long as it exhibits a degrading activity against ethyl carbamate. One preferred esterase is an esterase comprising the amino acid sequence of SEQ ID NO: 1. The esterase is an esterase derived from Acinetobacter calcoaceticus, which has been found by the present inventors, as shown in the Examples below, and has high resistance to alcohol and temperature stability. It has the feature of being excellent. On the other hand, as many as 17 esterases identified by an amino acid sequence having 70% or more identity with the amino acid sequence of SEQ ID NO: 1 have a degradation activity against ethyl carbamate, like esterase derived from Acinetobacter calcoaceticus. In view of the facts shown (see Examples described later), esterases having an amino acid sequence that is 70% or more identical to the amino acid sequence of SEQ ID NO: 1 are also suitable for the present invention. Specific examples of the corresponding esterase are derived from Acinetobacter guillouiae, derived from esterase having the amino acid sequence of SEQ ID NO: 2 (amino acid sequence identity of 98%) and Acinetobacter baumannii ABNIH3. Esterase having the amino acid sequence of SEQ ID NO: 3 (97% identity of amino acid sequence), esterase derived from Acinetobacter seifertii and having the amino acid sequence of SEQ ID NO: 4 (90% identity of amino acid sequence) , Derived from Pseudomonas aeruginosa and having the amino acid sequence of SEQ ID NO: 5 (80% identity of amino acid sequence), derived from Pseudomonas aeruginosa and having the amino acid sequence of SEQ ID NO: 6 S Terase (80% identity in amino acid sequence), esterase derived from Pseudomonas aeruginosa and having the amino acid sequence of SEQ ID NO: 7 (80% identity in amino acid sequence), Burkholderia ubonensis Esterase having the amino acid sequence of SEQ ID NO: 8 (identity of amino acid sequence 81%), derived from Paraburkholderia ferrariae and having the amino acid sequence of SEQ ID NO: 9 (of the amino acid sequence) 81% identity), an esterase derived from Burkholderia pseudomultivorans and having the amino acid sequence of SEQ ID NO: 10 (81% identity in amino acid sequence), Acinetobacter sp. NBRC 110496 (Acinetobacter sp. NBRC 110496), SEQ ID NO: Esterase having an amino acid sequence of 11 (81% identity in amino acid sequence), Esterase derived from Acinetobacter seifertii and having the amino acid sequence of SEQ ID NO: 12 (90% identity in amino acid sequence), Acinetobacter sp Derived from NIPH809 (Acinetobacter sp. NIPH809), derived from esterase having the amino acid sequence of SEQ ID NO: 13 (80% identity of amino acid sequence), Pseudomonas fluorescens A506 (A506 Pseudomonas fluorescens A506), Esterase having amino acid sequence (80% identity in amino acid sequence), Pseudomonas sp. ABAC61 (Pseudomonas sp. ABAC61), esterase having amino acid sequence of SEQ ID NO: 15 (79% identity in amino acid sequence), Pseudomonas Puchida IFO12996 (Pseudomonas putida IFO12996) Esterase derived from and having the amino acid sequence of SEQ ID NO: 16 (76% identity in amino acid sequence), Pseudomonas putida MR2068 and esterase having the amino acid sequence of SEQ ID NO: 17 (identity of amino acid sequence) 76%), an esterase derived from Pseudomonas aeruginosa and having the amino acid sequence of SEQ ID NO: 18 (80% identity in amino acid sequence). In addition, the identity of the amino acid sequence of the esterase derived from Arthrobacter ramosus (see Examples) that did not show the degradation activity against ethyl carbamate was only 21%.

An esterase having an amino acid sequence equivalent to the above amino acid sequence can also be used. The “equivalent amino acid sequence” here is partially different from the reference amino acid sequence (sequence of any of SEQ ID NOs: 1 to 18), but the difference is the function of the protein (here, with respect to ethyl carbamate). An amino acid sequence that does not substantially affect (degradation activity). Therefore, an enzyme having a polypeptide chain consisting of an equivalent amino acid sequence exhibits a degradation activity for ethyl carbamate. The degree of degradation activity against ethyl carbamate is preferably the same or higher than that of an enzyme having a polypeptide chain consisting of a reference amino acid sequence.

“Partial difference in amino acid sequence” typically means deletion, substitution, or addition, insertion of one to several amino acids, or a combination thereof. This means that a mutation (change) has occurred in the amino acid sequence. The difference in the amino acid sequence here is permissible as long as the decomposition activity against ethyl carbamate is retained (there may be some variation in activity). As long as this condition is satisfied, the positions where the amino acid sequences are different are not particularly limited, and differences may occur at a plurality of positions. The “plurality” herein is, for example, a number corresponding to less than about 30% of all amino acids, preferably a number corresponding to less than about 20%, more preferably a number corresponding to less than about 10%, Even more preferred is a number corresponding to less than about 5%, most preferred a number corresponding to less than about 1%. That is, the equivalent protein has a reference amino acid sequence, for example, about 60% or more, about 70% or more, about 75% or more, about 76% or more, about 79% or more, about 80% or more, about 81% or more, about 85%. More than about 90%, about 95%, about 97%, about 98% or more, or about 99% or more (the higher the percentage of identity, the better). As for the Acinebacter calcoaceticus esterase specified by the amino acid sequence of SEQ ID NO: 1, the 97th serine (Ser97), the 227th aspartic acid (Asp227) and the 256th histidine (Ser97), which are presumed to constitute the active center His256) is preferably not subject to deletion or substitution.

Preferably, an equivalent amino acid sequence can be obtained by causing a conservative amino acid substitution at an amino acid residue that is not essential for the decomposition activity against ethyl carbamate. As used herein, “conservative amino acid substitution” refers to substitution of a certain amino acid residue with an amino acid residue having a side chain having the same properties. Depending on the side chain of the amino acid residue, a basic side chain (eg lysine, arginine, histidine), an acidic side chain (eg aspartic acid, glutamic acid), an uncharged polar side chain (eg glycine, asparagine, glutamine, serine, threonine, tyrosine) Cysteine), non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine), aromatic side chains (eg tyrosine, phenylalanine, Like tryptophan and histidine). A conservative amino acid substitution is preferably a substitution between amino acid residues within the same family.

Incidentally, the identity (%) of two amino acid sequences or two nucleic acids (hereinafter, “two sequences” is used as a term including them) can be determined by the following procedure, for example. First, two sequences are aligned for optimal comparison (eg, a gap may be introduced into the first sequence to optimize alignment with the second sequence). When a molecule (amino acid residue or nucleotide) at a specific position in the first sequence is the same as the molecule at the corresponding position in the second sequence, it can be said that the molecule at that position is the same. The identity of two sequences is a function of the number of identical positions common to the two sequences (ie identity (%) = number of identical positions / total number of positions × 100), preferably the optimal alignment Take into account the number and size of gaps required for conversion.

比較 Comparison of two sequences and determination of identity can be achieved using a mathematical algorithm. Specific examples of mathematical algorithms that can be used for sequence comparison are described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 226 87: 2264-68; Karlin and Altschul (1993) Proc. Natl. There is a modified algorithm in Acad. Sci. USA 90: 5873-77, but it is not limited to this. Such an algorithm is incorporated in the NBLAST program and 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 may be carried out with the score = s100 and wordlength = 12 in the NBLAST program. In order to obtain an equivalent amino acid sequence, for example, a BLAST polypeptide search may be performed using the XBLAST program with score = 50 and wordlength = 3. In order to obtain a gap alignment for comparison, Gapped BLAST described in Altschul et al. (1997) Amino Acids Research 25 (17): 3389-3402 can be used. When utilizing BLAST and Gapped BLAST, the default parameters of the corresponding programs (eg, XBLAST and NBLAST) can be used. Please refer to http://www.ncbi.nlm.nih.gov for details. Examples of other mathematical algorithms that can be used for sequence comparison include those described in Myers and Miller (1988) Comput Appl Biosci. 4: 11-17. Such an algorithm is incorporated in the ALIGN program available on, for example, the GENESTREAM network server (IGH （Montpellier, France) or the ISREC server. When using the ALIGN program for comparison of amino acid sequences, for example, a PAM120 residue mass table can be used, with a gap length penalty = 12 and a gap penalty = 4.

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

The esterase used in the present invention may be a part of a larger protein (for example, a fusion protein). Examples of sequences added in the fusion protein include sequences useful for purification, such as multiple histidine residues, and additional sequences that ensure stability during recombinant production.

The esterase having the amino acid sequence can be easily prepared by a genetic engineering technique. For example, it can be prepared by transforming a suitable host cell (for example, E. coli) with DNA encoding the target esterase and recovering the protein expressed in the transformant. The recovered protein is appropriately purified according to the purpose. Thus, various modifications are possible if the desired enzyme is obtained as a recombinant protein. For example, if a DNA encoding the target enzyme and another appropriate DNA are inserted into the same vector and a recombinant protein is produced using the vector, the recombinant protein to which any peptide or protein is linked can be obtained. The target enzyme can be obtained. In addition, modification may be performed so that addition of sugar chain and / or lipid, or processing of N-terminal or C-terminal may occur. By the modification as described above, extraction of recombinant protein, simplification of purification, addition of biological function, and the like are possible.

2. Origin of esterase The origin of esterase is not particularly limited. For example, Acinetobacter calcoaceticus, Acinetobacter guillouiae, Acinetobacter baumannii, Acinetobacter seudotii, Acinetobacter seifertii , Pseudomonas fluorescens, Pseudomonas putida, and other Pseudomonas microorganisms, Burkholderia ubonensis, Burkholderia pseudomultivorans, Burkholderia pseudomultivorans, etc. Microorganisms derived from microorganisms, such as microorganisms belonging to the genus Delia or Paraburkholderia ferrariae such as Paraburkholderia ferrariae A sterase can be used. As specific examples of microorganisms belonging to the genus Acinetobacter, Acinetobacter baumannii ABNIH3 (Acinetobacter baumannii ABNIH3), Acinetobacter sp. NBRC 110496 (Acinetobacter sp. NBRC 110496) and Acinetobacter sp. Pseudomonas fluorescens A506, Pseudomonas sp. ABAC61 (Pseudomonas sp. ABAC61), Pseudomonas putida IFO12996 (Pseudomonas putida IFO12996) and Pseudomonas putida MR2068 (Pseudomonas putida MR2068).

The “esterase derived from a microorganism” as used herein means an esterase produced by a microorganism of each genus (which may be a wild strain or a mutant), or a microorganism of each genus (a wild strain). It may be an esterase obtained by genetic engineering techniques using an esterase gene (which may or may not be a mutant). Therefore, an esterase produced by a host microorganism into which an esterase gene (or a gene obtained by modifying the gene) obtained from a microorganism classified as described above is also an esterase derived from the microorganism classified as described above.

The esterase-producing bacterium may be a wild strain (an isolated strain that has not been subjected to mutation / modification treatment such as gene manipulation) or a mutant strain. In addition, a transformant obtained by introducing an esterase gene isolated from the original production strain into an appropriate host microorganism (such as Escherichia coli, Bacillus bacteria, Neisseria gonorrhoeae, or budding yeast (Saccharomyces cerevisiae)). May be used as production bacteria.

3. Various properties of esterase derived from Acinetobacter calcoaceticus The esterase derived from Acinetobacter calcoaceticus specified by the amino acid sequence of SEQ ID NO: 1 is a particularly suitable esterase in the degradation method of the present invention. The present inventors also succeeded in specifying various properties of the esterase as follows.

(1) Optimal temperature for esterase activity The optimal temperature for this esterase is 20-30 ° C. The optimum temperature can be evaluated based on the measurement result under the condition of pH 7 (for example, using 50 mM phosphate buffer).

(2) Optimal pH for esterase activity
The optimum pH of this esterase is 7. The optimum pH is determined based on, for example, the results of measurement in a 100 mM McIlvaine buffer in the pH range of 3.0 to 8.0 and in a 100 mM Atkins buffer in the pH range of pH 8.0 to 11.0.

(3) Temperature stability regarding esterase activity As for the temperature stability of this esterase, 80% or more of the activity remains even after treatment for 1 hour at 70 ° C. or less in a 50 mM phosphate buffer (pH 7).

(4) pH stability regarding esterase activity This esterase is stable at pH 5-11. That is, if the pH of the enzyme solution to be treated is within this range, 90% or more activity remains after treatment at 37 ° C. for 1 hour.

(5) Molecular weight The molecular weight of this esterase is about 85 kDa (by gel filtration). A homotrimer is formed.

(6) Alcohol stability This esterase exhibits high stability in alcohol. If the alcohol concentration is 40% or less, it will not be inactivated even when treated at 30 ° C for 8 days (residual activity is 90% or more).

4). Foods or beverages on which esterases are allowed to act The targets on which esterases are allowed to act (hereinafter sometimes referred to as “treatment targets”), that is, foods or beverages are not particularly limited. Examples of foods and beverages that can be processed include various fermented foods (eg yogurt, cheese, pickles, soy sauce, miso), bread, Shaoxing liquor, and nuclear fruits (cherry, peach, plum, apricot, etc.) Liquor, whiskey, brandy, tequila, cachassa, shochu, sake, wine, refined wine (sherry, port wine, madeira wine, marsala wine, etc.), plum wine, sherry, mixed liquor (liqueur, vermouth, medicinal liquor, etc.) is there. In a preferred embodiment, alcoholic beverages such as Shaoxing liquor, sherry liquor, and sake are treated.

In addition to foods and beverages as finished products, foods or beverages in the process of production (ie, intermediate products) may be treated.

5). Action conditions In order to make an esterase act on a food or a beverage, for example, the esterase is added to a food or beverage to be treated and reacted for a predetermined time. Alternatively, the food or beverage to be treated is brought into contact with the esterase immobilized on the carrier to cause an enzymatic reaction. The amount of enzyme to be used (enzyme concentration), temperature conditions, reaction time, etc. may be determined through preliminary experiments.

<Method for producing food or beverage>
As is apparent from the above description, by incorporating the decomposition method of the present invention into the production process, a food or beverage from which ethyl carbamate is removed or reduced can be obtained. Then, the 2nd aspect of this invention provides the manufacturing method of the foodstuff or drink using the decomposition | disassembly method of this invention. In the production method of the present invention, a treatment step with esterase (hereinafter sometimes referred to as “enzyme treatment step”) is performed during the production process. As long as the effects specific to the present invention, that is, the decomposition of ethyl carbamate by the action of esterase, is exerted, the position of the enzyme treatment process in the entire production process (that is, the order of other production processes) is particularly It is not limited. However, except in exceptional cases, the enzyme treatment process is carried out after these processes, considering that ethyl carbamate is produced in the fermentation process or the aging / storage process, which is the final or final stage of the production process. It is preferable to do. Therefore, in a preferred embodiment, the enzyme treatment step is performed after the fermentation step, or the enzyme treatment step is performed after the aging / storage step.

Instead of providing a dedicated enzyme treatment step, esterase may be added and allowed to act during a specific step. In this case, an enzyme reaction occurs in parallel with the progress of a specific process. Examples of the “specific process” here include a fermentation process, an aging process, and a storage process.

Except for the steps specific to the present invention (enzyme treatment step), a normal production method may be used. The term “normal manufacturing method” is used to distinguish from the manufacturing method to which the present invention is applied, and is not intended to be limited to a specific manufacturing method. Therefore, the “normal manufacturing method” to which the present invention is applicable is not particularly limited.

1. Screening for ethyl carbamate (EC) degrading enzymes In order to find enzymes capable of degrading ethyl carbamate (EC), including 130 enzymes (23 lipases or esterases, as well as various hydrolases such as protease and amylase) ) And a large-scale screening for microorganisms. The screening method is shown below. First, prepare an EC reaction solution (100 mM phosphate buffer pH 7.0: 2 mL, EC: 10 mM, enzyme: 2 mg) for each enzyme to be screened (the culture solution was used instead of the enzyme for microorganisms), and the reaction (30 ° C., stirring with a stirrer, 48 hours). After completion of the reaction, 1N HCl 0.1 mL and chloroform 0.6 mL (Wako) were added to 0.4 mL of the reaction solution, and then mixed well. After the mixture was centrifuged (15,000 rpm × 5 minutes, 4 ° C.), the chloroform layer was collected in a vial and used as a GC analysis sample. EC was detected by GC analysis using the sample thus prepared. The EC decomposition rate was calculated from the peak area (RT = 8.9 minutes) of ethyl carbamate (EC).
(GC analysis conditions)
Gas chromatography (GC7700, Agilent) was used for analysis under the following conditions.
Column: DB-WAX (60m x 0.25mm x 0.25um) (Agilent J & W)
Injector: 250 ℃
Detector: FID, 250 ℃
Oven: 100 ° C (0 min) → Temperature rise at 10 ° C / min → Hold at 250 ° C for 5 min Flow rate: 2.0 mL / min Injection volume: 5 μL

As a result of GC analysis, it was discovered that esterase derived from A. calcoaceticus can degrade EC. On the other hand, lipase and Arthrobacter bramosus-derived esterase were not able to degrade EC, and the degradation activity against EC was found to be a characteristic property of A. calcoaceticus-derived esterase.

2. Degradation of EC by esterase derived from A. calcoaceticus (Shaoxing Sake)
The ability of A. calcoaceticus-derived esterase found by screening to examine EC degradation in Shaoxing liquor was examined. EC reaction solution (Shaoxing liquor (pH 5.0): 60 mL, EC: 1.3 ppm, E-2 enzyme preparation: 3 g (final concentration 800 U / mL)) was prepared and reacted in a 300 mL Erlenmeyer flask (30 ° C, 100 rpm, 9th) After the reaction, the EC reaction solution was centrifuged (7,000 g × 10 minutes, 4 ° C.), subjected to membrane filtration (0.45 μm or 0.2 μm), and then analyzed for EC concentration by gas chromatography-mass spectrometry.

As a result of the analysis, it was confirmed that esterase derived from A. calcoaceticus can degrade EC in Shaoxing liquor (pH 5.0) (Table 1).

3. Examination of EC degradation ability of related enzymes It was confirmed that the esterase derived from A. calcoaceticus can degrade EC in Shaoxing liquor. The following examination was conducted to determine whether the A. calcoaceticus-derived esterase-related enzyme has EC-degrading ability.

(1) Construction of E. coli expression system of esterase derived from A. calcoaceticus and related strain (1-1) Total synthesis of esterase gene (E.coli optimization)
In constructing the E.coli expression system, esterase genes derived from A. calcoaseticus and related strains were optimized after codon optimization for E.coli expression. The amino acid sequence and gene sequence (after codon optimization) of esterase derived from A. calcoaseticus and related strains are shown below.
<Esterase from A. calcoaseticus>
Amino acid sequence: SEQ ID NO: 1
Gene sequence: SEQ ID NO: 19
<Related enzyme No.1>
Origin: Acinetobacter guillouiae
Amino acid sequence: SEQ ID NO: 2
Gene sequence: SEQ ID NO: 20
<Related enzyme No.2>
Origin: Acinetobacter baumannii ABNIH3
Amino acid sequence: SEQ ID NO: 3
Gene sequence: SEQ ID NO: 21
<Related enzyme No.3>
Origin: Acinetobacter seifertii
Amino acid sequence: SEQ ID NO: 4
Gene sequence: SEQ ID NO: 22
<Related enzyme No. 4>
Origin: Pseudomonas aeruginosa
Amino acid sequence: SEQ ID NO: 5
Gene sequence: SEQ ID NO: 23
<Related enzyme No. 5>
Origin: Pseudomonas aeruginosa
Amino acid sequence: SEQ ID NO: 6
Gene sequence: SEQ ID NO: 24
<Related enzyme No. 6>
Origin: Pseudomonas aeruginosa
Amino acid sequence: SEQ ID NO: 7
Gene sequence: SEQ ID NO: 25
<Related enzyme No. 7>
Origin: Burkholderia ubonensis
Amino acid sequence: SEQ ID NO: 8
Gene sequence: SEQ ID NO: 26
<Related enzyme No. 8>
Origin: Paraburkholderia ferrariae
Amino acid sequence: SEQ ID NO: 9
Gene sequence: SEQ ID NO: 27
<Related enzyme No. 9>
Origin: Burkholderia pseudomultivorans
Amino acid sequence: SEQ ID NO: 10
Gene sequence: SEQ ID NO: 28
<Related enzyme No. 10>
Origin: Acinetobacter sp. NBRC 110496 (Acinetobacter sp. NBRC 110496)
Amino acid sequence: SEQ ID NO: 11
Gene sequence: SEQ ID NO: 29
<Related enzyme No. 11>
Origin: Acinetobacter seifertii
Amino acid sequence: SEQ ID NO: 12
Gene sequence: SEQ ID NO: 30
<Related enzyme No. 12>
Origin: Acinetobacter sp. NIPH809
Amino acid sequence: SEQ ID NO: 13
Gene sequence: SEQ ID NO: 31
<Related enzyme No. 13>
Origin: Pseudomonas fluorescens A506
Amino acid sequence: SEQ ID NO: 14
Gene sequence: SEQ ID NO: 32
<Related enzyme No. 14>
Origin: Pseudomonas sp. ABAC61
Amino acid sequence: SEQ ID NO: 15
Gene sequence: SEQ ID NO: 33
<Related enzyme No. 15>
Origin: Pseudomonas putida IFO12996 (Pseudomonas putida IFO12996)
Amino acid sequence: SEQ ID NO: 16
Gene sequence: SEQ ID NO: 34
<Related enzyme No. 16>
Origin: Pseudomonas putida MR2068
Amino acid sequence: SEQ ID NO: 17
Gene sequence: SEQ ID NO: 35
<Related enzyme No. 17>
Origin: Pseudomonas aeruginosa
Amino acid sequence: SEQ ID NO: 18
Gene sequence: SEQ ID NO: 36

(1-2) Obtaining an E. coli expression plasmid and constructing an E. coli expression system PCR (PrimeSTAR GXL) using a primer that adds a linker sequence (Eco RI, Hind III) using a fully synthesized esterase gene as a template DNA Polymerase (Takara)). PCR conditions were as follows.
<PCR conditions>
Composition of the reaction mixture: 10 μl of 5 × PrimeSTAR GXL Buffer, 4 μl of dNTP Mixture (2.5 mM each), 10 pmol of forward primer, 10 pmol of reverse primer, 10 ng of template, PrimeSTAR GXL DNA Polymerase (Takara) 1μl (adjusted to 50μl with sterilized distilled water)
Reaction conditions: 30 cycles of 98 ° C for 10 seconds, 60 ° C for 30 seconds, and 68 ° C for 1.5 minutes

The amplified product was purified (NucleoSpin Geland PCR Clean-up (MACHEREY-NAGEL)) to obtain each gene fragment. Each gene fragment and pUC18 (Takara) were treated with restriction enzymes (Eco RI (Takara), Hind III (Takara)), and then ligated (DNA Ligation Kit <Mighty Mix> (Takara)) to obtain E.coli DH5α Each E. coli recombinant was obtained by transformation into (Takara). Each E. coli recombinant is inoculated into LB Broth Base (invitrogen) + Amp: 100μg / mL: 5mL and shaken (37 ℃, 16h, 140rpm), then NucleoSpin Plasmid EasyPure (MACHEREY-NAGEL) To obtain an E. coli expression plasmid. Each obtained E. coli expression plasmid was transformed into E. coli BL21 (DE3) (Nippongene) to obtain each E. coli expression strain. In addition, each E.coli expression using Sequence 用い Primer (M13 M4 Primer: 5'-GTTTT CCCAGTCACGAC-3 '(SEQ ID NO: 37), M13 RV Primer: 5'- CAGGAAACAGCTATGAC-3' (SEQ ID NO: 38)) The sequence of the plasmid was confirmed.

(2) Acquiring each esterase culture solution (cultured cells) and cell extract Using each constructed esterase gene recombinant E.coli expression strain (host: E.coli BL21 (DE3)) An attempt was made to obtain (cultured cells). The culture solution (cultured cells) of each esterase gene recombinant E. coli expression strain was obtained in two stages of culture. First, each esterase gene recombinant E. coli expression strain was inoculated into 5 mL of L Broth (inbitrogen) (Amp: 100 μg / mL) and cultured for 16 hours (140 rpm, 37 ° C) in a shaking incubator. Teriffic Broth (Invitrogen) (Amp: 100 μg / mL) 0.5 mL was inoculated into 50 mL. Thereafter, the cells were cultured for 48 hours under the conditions of 200 rpm and 33 ° C., and the expression of the enzyme was induced by adding 0.1 mM IPTG to the culture solution 24 hours after the start of the culture. After centrifuging 50 mL of the culture solution (7,500 g × 10 minutes, 4 ° C.), the cultured cells were recovered by removing the centrifugal supernatant.

Suspend each obtained bacterial cell in 20 mM phosphate buffer (pH 7.0) 20 mL, add 10 g beads (0.1 mm) (Yasui Kikai), and add beads shocker (2,500 rpm, on: 60 seconds, off: 破砕 30 seconds, 15 cycles, 4 ℃) (Yasui Kikai). The physical disruption solution was centrifuged (7,500 g × 10 minutes, 4 ° C.), and the supernatant was collected to obtain a cell extract.

(3) Degradation of EC by related enzymes (buffer pH 7.0)
Using the obtained bacterial cell extract, EC decomposition ability in a buffer solution (pH 7.0) was confirmed. EC reaction solution (buffer solution (100 mM phosphate buffer pH 7.0): 1.3 mL, EC: 10 mM, each bacterial cell extract: 1.3 mL) was prepared and reacted (30 ° C., 100 rpm, 9 days) . During the reaction period, the reaction solution was appropriately sampled (0.4 mL) to prepare an analysis sample. The analytical sample was analyzed by GC. As a result of the analysis, it was confirmed that all the related enzymes have EC-decomposing ability although the activity is strong and weak (FIG. 1).

4). Alcohol stability of A. calcoaceticus-derived esterase The alcohol stability of A. calcoaceticus-derived esterase that showed excellent EC degradation ability was examined. First, a treatment solution was prepared by mixing 9 mL of each concentration of alcohol solution (ethanol-reagent grade (Wako)) (0 to 70%) and 1 g of A. calcoaceticus esterase. Each treatment solution was allowed to stand at 30 ° C. and sampled (1 mL) after 16 hours, 4 days, and 8 days. The enzyme activity of each sample was measured by the following measurement method.
(Activity measurement method using 3,4-dihydrocoumarin as substrate)
50 mL phosphate buffer solution (pH 7.0) 2.1 mL, 5 mM 3,4-dihydrocoumarin (Sigma-aldrich (Code: D104809)) solution (containing 40% EtOH) 0.3 mL, enzyme solution 0.6 mL are mixed and reacted. It was. In this measurement method, the change in absorbance of the product (3- (2-hydroxyphenyl) propionic acid) produced by the reaction is measured by kinetics (Abs. 270 nm, 30 ° C., 5 minutes). The amount of enzyme that produces the product (3- (2-hydroxyphenyl) propionic acid) is defined as 1 unit (U). In this measurement, ΔOD for obtaining an accurate measurement value is in the range of ΔOD = 0.01 to 0.05 / 2 min (Abs. 250 nm). If necessary, the enzyme solution can be used with 50 mM phosphate buffer pH 7.0. Dilute.

The residual activity was calculated from the measured value (activity value), and the residual activity of each treatment solution was compared. As a result, it was confirmed that A. calcoaceticus-derived esterase was not inactivated even when treated at 30 ° C. for 8 days when the alcohol concentration was 40% or less (FIG. 2).

5). Various properties of A. calcoaceticus-derived esterase Using the above "activity measurement method using 3,4-dihydrocoumarin as a substrate" (sample pretreatment conditions are described separately), enzymatic properties of A. calcoaceticus-derived esterase Identified.

(1) Optimum temperature The activity was measured by setting the sample block of the absorptiometer to a predetermined temperature, and the optimum temperature was determined. A 50 mM phosphate buffer (pH 7.0) was used to dilute the enzyme to match the measurement range. As a result of the measurement, the optimum temperature was 20-30 ° C. (FIG. 3).

(2) Optimum pH
Activity was measured with a measurement system in which “50 mM phosphate buffer pH 7.0 2.1 mL” in the reaction solution was replaced with a buffer adjusted to a predetermined pH, and the optimum pH was determined. Two types of buffer solutions (McIlvaine, Atkins) adjusted to each pH for measurement at a predetermined pH (McIlvaine buffer solution: pH3.0, pH4.0, pH5.0, pH6.0, pH7.0, pH 8.0, Atkins buffer: pH 8.0, pH 9.0, pH 10.0, pH 11.0) were prepared. A 50 mM phosphate buffer (pH 7.0) was used to dilute the enzyme to match the measurement range. As a result of the measurement, the optimum pH was pH 7 (FIG. 4).

(3) Temperature stability Temperature stability was evaluated by measuring residual activity after treating the enzyme at each temperature. First, an enzyme diluted solution was prepared using 50 mM phosphate buffer pH 7.0, then treated at a predetermined temperature for 1 hour, and cooled with ice water for 5 minutes. After this treatment, the activity was measured by diluting with 50 mM phosphate buffer pH 7.0 to be within the measurement range. As a result, it was stable up to 70 ° C. (residual activity of 80% or more) (FIG. 5).

(4) pH stability The pH stability was evaluated by measuring the residual activity after treating the enzyme at each pH. First, an enzyme dilution solution was prepared using a buffer solution adjusted to each pH, and then treated at 37 ° C. for 1 hour (pH treatment). Two types of buffer solution (McIlvaine, Atkins) adjusted to each pH as the buffer solution used for enzyme pH treatment (McIlvaine buffer solution: pH3.0, pH4.0, pH5.0, pH6.0, pH7. 0, pH 8.0 Atkins buffer: pH 8.0, pH 9.0, pH 10.0, pH 11.0) was used. After the pH treatment, the pH treatment was stopped by adding an equal volume of 1M phosphate buffer pH 7.0 to the enzyme dilution. The activity was measured after diluting with 50 mM phosphate buffer pH 7.0 to be within the measurement range. As a result of the measurement, it was stable at pH 5 to 11 (residual activity of 90% or more) (FIG. 6).

(5) Molecular weight The molecular weight under non-denaturing conditions was measured by gel filtration chromatography under the following conditions. As a result of the measurement, the molecular weight was estimated to be about 85 kDa.
(Experimental conditions)
Column used: Supermers 200HR 10/30 manufactured by Amersham pharmacia (currently GE Healthcare)
Buffer used: 50 mmol / L NaCl phosphate buffer (pH 7.0) + 0.15 mol / L NaCl
Molecular weight marker: High Molecular Weight Gel Filtration Calibration Kit (manufactured by Amersham) (Aldolase 158 k, Catalase 232 k, Ferritin 440 k, Thyroglobulin 669 k) was used.

According to the present invention, ethyl carbamate (EC) in food or beverage can be decomposed. In particular, the present invention is useful for removing or reducing EC in alcoholic beverages and fermented foods.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

SEQ ID NO: 37: Description of artificial sequence: primer SEQ ID NO: 38: Description of artificial sequence: primer

Claims

A method for decomposing ethyl carbamate in a food or beverage, comprising causing an esterase comprising an amino acid sequence 70% or more identical to the amino acid sequence of SEQ ID NO: 1 to act on a food or beverage containing ethyl carbamate.
The degradation method according to claim 1, wherein the amino acid sequence of the esterase comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 18.
The degradation method according to claim 1, wherein the esterase is derived from an Acinetobacter genus microorganism, a Pseudomonas genus microorganism, a Burkholderia microorganism, or a Parabalkhorderia microorganism.
The microorganism belonging to the genus Acinetobacter is Acinetobacter calcoaceticus, Acinetobacter guillouiae, Acinetobacter baumannii, or the microorganism belonging to the genus Acinetobacter baumannii, Acinetobacter ii Erginosa (Pseudomonas aeruginosa), Pseudomonas fluorescens or Pseudomonas putida, the Burkholderia or Burkholderia ubonensis (Burkholderia ubonensis) Burkholderia pseudomultivorans), and the Parabark holderia is Paraburkholderia Ferrariae, Decomposition method according to Motomeko 3.
The esterase is Acinetobacter sp. NBRC 110496 (Acinetobacter sp. NBRC 110496), Acinetobacter sp. The decomposition method of Claim 1 which originates in Pseudomonas putida IFO12996 (Pseudomonas putida IFO12996) or Pseudomonas putida MR2068 (Pseudomonas putida MR2068).
The degradation method of claim 1, wherein the esterase comprises the following characteristics:
(1) Optimal temperature: 20-30 ° C;
(2) Optimal pH: pH 7;
(3) Temperature stability: stable up to 70 ° C (pH 7, 1 hour);
(4) pH stability: stable at pH 5-11;
(5) Molecular weight: about 85 kDa (by gel filtration).
The degradation method of claim 6, wherein the esterase further comprises the following characteristics:
(6) Alcohol stability: If the alcohol concentration is 40% or less, it will not be inactivated even if treated at 30 ° C. for 8 days.
The decomposition method according to any one of claims 1 to 7, wherein the beverage is an alcoholic beverage.
9. The decomposition according to claim 8, wherein the alcoholic beverage is Shaoxing liquor, distilled liquor made from drupe, whiskey, brandy, tequila, cachassa, shochu, sake, wine, refined wine, plum wine, sherry liquor or mixed liquor. Method.
A method for producing a food or beverage from which ethyl carbamate has been removed or reduced, comprising a treatment step with an esterase comprising an amino acid sequence 70% or more identical to the amino acid sequence of SEQ ID NO: 1.
The production method according to claim 10, wherein the esterase is the esterase defined in any one of claims 2 to 7.
The manufacturing method according to claim 10 or 11, wherein the beverage is an alcoholic beverage.
The production according to claim 12, wherein the alcoholic beverage is Shaoxing liquor, distilled liquor made from drupe, whiskey, brandy, tequila, cachassa, shochu, sake, wine, refined wine, plum wine, sherry liquor or mixed liquor. Method.
A food or beverage obtained by the production method according to any one of claims 10 to 13, wherein ethyl carbamate is removed or reduced.