JP7138867B2 - Decomposition of ethyl carbamate - Google Patents

Description

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

Ethyl carbamate (hereinafter sometimes abbreviated as "EC") has been used as a drug in the past, but is a compound suspected to be carcinogenic or mutagenic. There are concerns about the effects on the human body when ingested. In particular, Shaoxing wine, sherry wine, and fermented foods contain relatively large amounts of EC, and the development of means for reducing or removing EC is desired. Various attempts have been made so far, for example, a method using urethanease (see, for example, Patent Document 1 and Non-Patent Documents 1 to 3) and a method using cells of microorganisms that produce EC-degrading enzymes ( For example, see Patent Documents 2 and 3), etc. have been proposed.

JP-A-4-325079 Chinese Patent No. 102492633B Japanese Patent Application Laid-Open No. H1-24017

Kobashi K. et al., Chem. Pharm. Bull. 38(5) 1326-1328(1990) Zhao C.-J. et al., Chem. Pharm. Bull. 39(12) 3303-3306(1991) B. R. Mohapatra and M. Bapuji, Letters in Applied Microbiology 1997, 25, 393-396

The method of decomposing EC using urethane has the problem of low stability of urethane in the presence of alcohol and low thermal stability. On the other hand, the decomposition method using microbial cells is not particularly practical in terms of practicality.

Alcoholic beverages and fermented foods can be ingested on a daily and continuous basis, and their EC content is expected to become a major problem in the future. In order to cope with such a situation, an object of the present invention is to provide a highly practical method capable of efficiently decomposing ECs in foods or beverages.

In order to solve the above problems, the present inventors conducted large-scale screening of a wide range of enzymes with the aim of establishing a method for degrading ECs using enzymes. As a result, it was found that the Acinetobacter calcoaceticus-derived esterase exhibits high EC-degrading activity. Surprisingly, the esterase exhibited decomposition activity even at high alcohol concentrations. This fact indicates that the esterase derived from Acinetobacter calcoaceticus is useful as a means of degrading EC in alcoholic beverages. Esterases are classified as serine proteases, and their activity is usually inhibited by alcohol hydroxyl groups. In view of this common general knowledge, the novel use of esterase revealed by the studies of the present inventors, that is, "use of esterase for decomposition of EC in alcoholic beverages" can be said to be extremely unique.

On the other hand, when we confirmed the EC-degrading activity of an esterase-related enzyme derived from Acinetobacter calcoaceticus (with an amino acid sequence identity of 70% or more), we found that the related enzyme also exhibited degradation activity, indicating that Acinetobacter is capable of degrading EC.・It was found that it is not unique to the esterase derived from calcoaceticus.

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

The following inventions are based on the above results and considerations.
[1] Degrading ethyl carbamate in a food or beverage, characterized in that an esterase comprising an amino acid sequence that is 70% or more identical to the amino acid sequence of SEQ ID NO: 1 is allowed to act on a food or beverage containing ethyl carbamate. how to.
[2] The decomposition method according to [1], wherein the amino acid sequence of the esterase comprises any one of the amino acid sequences of SEQ ID NOS: 1-18.
[3] The decomposition method according to [1], wherein the esterase is derived from a microorganism of the genus Acinetobacter, a microorganism of the genus Pseudomonas, a microorganism of the genus Burkholderia, or a microorganism of the genus Paraburkholderia.
[4] The microorganism of the genus Acinetobacter is Acinetobacter calcoaceticus, Acinetobacter guillouiae, Acinetobacter baumannii or Acinetobacter seifertii, and the microorganism of the genus Pseudomonas is Pseudomonas aeruginosa, Pseudomonas fluorescens or Pseudomonas putida, and said Burkholderia is Burkholderia ubonensis or Burkholderia pseudomulti Burkholderia pseudomultivorans, and the paraburkholderia is Paraburkholderia ferrariae.
[5] The esterase is Acinetobacter sp. NBRC 110496, Acinetobacter sp. NIPH809, Pseudomonas fluorescens A506, Pseudomonas sp. ABAC61), Pseudomonas putida IFO12996 or Pseudomonas putida MR2068.
[6] The decomposition method according to [1], wherein the esterase has the following characteristics:
(1) optimum temperature: 20-30°C;
(2) optimal pH: pH7;
(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 decomposition method according to [6], wherein the esterase further has the following characteristics:
(6) Alcohol stability: If the alcohol concentration is 40% or less, it will not be deactivated even if it is 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 wine, stone fruit distilled liquor, whiskey, brandy, tequila, cachaça, shochu, refined sake, wine, fortified wine, plum wine, sherry, or mixed liquor. The decomposition method described in .
[10] A method for producing a food or beverage from which ethyl carbamate has been removed or reduced, comprising a step of treating with an esterase having an amino acid sequence that is 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] to [12], wherein the alcoholic beverage is Shaoxing wine, stone fruit distilled liquor, whiskey, brandy, tequila, cachaça, shochu, refined sake, wine, fortified wine, plum wine, sherry or mixed liquor; Method of manufacture as described.
[14] A food or beverage from which ethyl carbamate has been removed or reduced, obtained by the production method according to any one of [10] to [13].

EC degradation activity of related enzymes. Esterase-related enzymes derived from Acinetobacter calcoaceticus (A. calcoaceticus) were expressed in an E. coli expression system, and their ability to decompose EC was investigated. Top: EC degradation ability of analogous enzymes No. 1 to No. 10 at pH 7.0. Bottom: EC degradation ability of analogous enzymes No. 11 to No. 16 at pH 7.0. Alcohol stability of esterases from A. calcoaceticus. Optimum temperature for the esterase from A. calcoaceticus. pH optimum of esterase from A. calcoaceticus. Temperature stability of the esterase from A. calcoaceticus. pH stability of esterases from A. calcoaceticus.

<Decomposition of Ethyl Carbamate in Food or Beverage>
The present invention is a method for decomposing ethyl carbamate in food or beverage, characterized by allowing esterase to act on food or beverage containing ethyl carbamate. According to the present invention, a food or beverage from which ethyl carbamate is removed or reduced is obtained.

1. Amino Acid Sequence Defining Esterase The esterase used in the present invention is not particularly limited as long as it exhibits decomposition activity for ethyl carbamate. One preferred esterase is an esterase comprising the amino acid sequence of SEQ ID NO:1. As shown in Examples below, the esterase is derived from Acinetobacter calcoaceticus, which was found by the studies of the present inventors, and has high alcohol tolerance and temperature stability. It has the characteristics of being excellent in On the other hand, as many as 17 esterases identified by amino acid sequences showing 70% or more identity with the amino acid sequence of SEQ ID NO: 1 exhibit decomposition activity for ethyl carbamate, like the esterase derived from Acinetobacter calcoaceticus. 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 in view of the facts shown (see Examples below). Specific examples of such esterases are those derived from Acinetobacter guillouiae and having the amino acid sequence of SEQ ID NO: 2 (98% amino acid sequence identity), Acinetobacter baumannii ABNIH3. , an esterase having the amino acid sequence of SEQ ID NO: 3 (97% amino acid sequence identity), an esterase derived from Acinetobacter seifertii and having the amino acid sequence of SEQ ID NO: 4 (90% amino acid sequence identity) , an esterase derived from Pseudomonas aeruginosa and having the amino acid sequence of SEQ ID NO: 5 (80% amino acid sequence identity), derived from Pseudomonas aeruginosa and having the amino acid sequence of SEQ ID NO: 6 Esterase (80% amino acid sequence identity), an esterase derived from Pseudomonas aeruginosa and having the amino acid sequence of SEQ ID NO: 7 (80% amino acid sequence identity), Burkholderia ubonensis derived from and having the amino acid sequence of SEQ ID NO: 8 (81% amino acid sequence identity), an esterase derived from Paraburkholderia ferrariae and having the amino acid sequence of SEQ ID NO: 9 (amino acid sequence 81% identity), an esterase derived from Burkholderia pseudomultivorans and having the amino acid sequence of SEQ ID NO: 10 (81% identity of the amino acid sequence), Acinetobacter sp. NBRC 110496 (Acinetobacter sp. NBRC 110496) and having the amino acid sequence of SEQ ID NO: 11 (81% amino acid sequence identity), an esterase derived from Acinetobacter seifertii and having the amino acid sequence of SEQ ID NO: 12 (amino acid sequence identity 90%), Acinetobacter sp. NIPH809 (Acinetobact er sp. NIPH809) and having the amino acid sequence of SEQ ID NO: 13 (80% amino acid sequence identity), Pseudomonas fluorescens A506 (A506) derived from Pseudomonas fluorescens A506, having the amino acid sequence of SEQ ID NO: 14 (80% amino acid sequence identity), Pseudomonas sp. ABAC61 and having the amino acid sequence of SEQ ID NO: 15 (79% amino acid sequence identity), Pseudomonas putida IFO12996 ( Pseudomonas putida IFO12996) and an esterase having the amino acid sequence of SEQ ID NO: 16 (76% amino acid sequence identity), an esterase derived from Pseudomonas putida MR2068 (Pseudomonas putida MR2068) and having the amino acid sequence of SEQ ID NO: 17 ( 76% amino acid sequence identity), an esterase derived from Pseudomonas aeruginosa and having the amino acid sequence of SEQ ID NO: 18 (80% amino acid sequence identity). The amino acid sequence identity of the Arthrobacter ramosus-derived esterase (see Examples) that did not exhibit degrading activity on ethyl carbamate was only 21%.

Esterases having amino acid sequences equivalent to the above amino acid sequences can also be used. The term "equivalent amino acid sequence" as used herein refers to a partial difference from the reference amino acid sequence (any of SEQ ID NOS: 1 to 18), but the difference is the function of the protein (here, for ethyl carbamate). It refers to an amino acid sequence that does not substantially affect the degradation activity). Therefore, an enzyme having a polypeptide chain consisting of equivalent amino acid sequences exhibits degrading activity for ethyl carbamate. The degree of decomposition activity for ethyl carbamate is preferably the same as 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 or substitution of 1 to several amino acids, or addition or insertion of 1 to several amino acids, or a combination thereof It means that mutation (change) has occurred in the amino acid sequence due to A difference in the amino acid sequence here is permissible as long as the decomposition activity for ethyl carbamate is maintained (the activity may vary slightly). As long as this condition is satisfied, the positions where the amino acid sequences differ are not particularly limited, and differences may occur at multiple positions. Here, "plurality" 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%, and most preferred is a number corresponding to less than about 1%. That is, the equivalent protein is, 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% of the reference amino acid sequence. greater than or equal to about 90%, greater than or equal to about 95%, greater than or equal to about 97%, greater than or equal to about 98%, or greater than or equal to about 99% (higher percentages of identity are preferred). In addition, for the Acinebacter calcoaceticus esterase specified by the amino acid sequence of SEQ ID NO: 1, serine at position 97 (Ser97), aspartic acid at position 227 (Asp227) and histidine at position 256 (Asp227), which are presumed to constitute the active center, His256) is preferably not subject to deletion or substitution.

Preferably, equivalent amino acid sequences are obtained by making conservative amino acid substitutions at amino acid residues that are not essential for the degradative activity towards ethyl carbamate. As used herein, "conservative amino acid substitution" refers to substitution of an amino acid residue with an amino acid residue having a side chain with similar properties. Amino acid residues can have, depending on their side chain, a basic side chain (e.g. lysine, arginine, histidine), an acidic side chain (e.g. aspartic acid, glutamic acid), an uncharged polar side chain (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine). , cysteine), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine), aromatic side chains (e.g. tyrosine, phenylalanine, Tryptophan, histidine) are classified into several families. Conservative amino acid substitutions are preferably between amino acid residues within the same family.

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

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

The identity of two amino acid sequences was determined using the GAP program of the GCG software package using the Blossom 62 matrix or the PAM250 matrix, gap weight = 12, 10, 8, 6, or 4, gap length weight = 2, 3. , or can be determined as 4. The degree of homology between two nucleic acid sequences can also 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. can.

Esterases for use in the invention may be part of a larger protein (eg, a fusion protein). Additional sequences in the fusion protein include, for example, sequences that aid in purification such as multiple histidine residues, additional sequences that ensure stability during recombinant production, and the like.

An esterase having the above amino acid sequence can be easily prepared by genetic engineering techniques. For example, it can be prepared by transforming a suitable host cell (eg, E. coli) with a DNA encoding the esterase of interest and recovering the protein expressed in the transformant. The recovered protein is appropriately purified depending on the purpose. Various modifications are possible if the target enzyme is obtained as a recombinant protein in this way. For example, by inserting a DNA encoding an enzyme of interest and other suitable DNA into the same vector and using the vector to produce a recombinant protein, the recombinant protein linked to any peptide or protein can It is possible to obtain the desired enzyme. In addition, addition of sugar chains and/or lipids, or modification that causes N-terminal or C-terminal processing may be performed. Such modifications enable extraction of recombinant proteins, simplification of purification, addition of biological functions, and the like.

2. Origin of Esterase The origin of the esterase is not particularly limited. For example, Acinetobacter microorganisms such as Acinetobacter calcoaceticus, Acinetobacter guillouiae, Acinetobacter baumannii, Acinetobacter seifertii, Pseudomonas aeruginosa Pseudomonas microorganisms such as Pseudomonas fluorescens and Pseudomonas putida, Burkholderia ubonensis and Burkholderia pseudomultivorans Esterases derived from microorganisms such as Delia microorganisms or Paraburkholderia microorganisms such as Paraburkholderia ferrariae can be used. As specific examples of the Acinetobacter microorganisms, Acinetobacter baumannii ABNIH3, Acinetobacter sp. NBRC 110496 and Acinetobacter sp. Examples include Pseudomonas fluorescens A506, Pseudomonas sp. ABAC61, Pseudomonas putida IFO12996 and Pseudomonas putida MR2068.

Here, the term "esterase derived from microorganisms" refers to esterases produced by microorganisms of the above genera (wild strains or mutant strains), or esterases produced by microorganisms of the above genera (wild strains). It means that it is an esterase obtained by a genetic engineering method using the esterase gene of . 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 above is introduced is also an esterase derived from a microorganism classified above.

Esterase-producing strains may be wild strains (isolated strains from nature, which have not undergone mutation or modification treatment such as genetic manipulation) or mutant strains. A transformant obtained by introducing an esterase gene isolated from the original producing strain into an appropriate host microorganism (Escherichia coli, Bacillus, Aspergillus oryzae, budding yeast (Saccharomyces cerevisiae), etc.) may be used as producing bacteria.

3. Properties of Esterase Derived from Acinetobacter calcoaceticus The esterase derived from Acinetobacter calcoaceticus, identified by the amino acid sequence of SEQ ID NO: 1, is an esterase particularly suitable for the degradation method of the present invention. The present inventors also succeeded in identifying various properties of this esterase as follows.

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

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

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

(4) pH stability of 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 of the 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). In addition, it forms a homotrimer.

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

4. Foods or Beverages on which Esterase Acts Subjects on which esterase acts (hereinafter sometimes referred to as "treatment subjects"), that is, foods or beverages, are not particularly limited. Examples of foods and beverages that can be treated include various fermented foods (eg yogurt, cheese, pickles, soy sauce, miso), bread, Shaoxing wine, stone fruits (cherries, peaches, plums, apricots, etc.). Liquor, whiskey, brandy, tequila, cachaça, shochu, sake, wine, fortified wine (sherry, port wine, Madeira wine, Marsala wine, etc.), plum wine, sherry, mixed liquor (liqueur, vermouth, medicinal liquor, etc.) be. In a preferred embodiment, alcoholic beverages such as Shaoxing wine, sherry wine, and refined sake are treated.

Foods or beverages in the process of being manufactured (that is, intermediate products) may be processed as well as finished foods or beverages.

5. Action Conditions In order to allow esterase to act on food or beverages, for example, esterase is added to the food or beverage to be treated and allowed to react for a predetermined period of 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 clear from the above description, by incorporating the decomposition method of the present invention into the production process, it is possible to obtain foods or beverages from which ethyl carbamate has been removed or reduced. Therefore, a second aspect of the present invention provides a method for producing food or beverage using the decomposition method of the present invention. In the production method of the present invention, a treatment step with an esterase (hereinafter sometimes referred to as an "enzyme treatment step") is carried out during the production process. As long as the effect peculiar to the present invention, i.e., the decomposition of ethyl carbamate by the action of esterase, is exhibited, the position of the enzymatic treatment step in the overall production process (i.e., the order with other production steps) is particularly Not limited. However, except in exceptional cases, considering that ethyl carbamate is generated in the fermentation process or aging/storage process, which are the latter or final stages of the manufacturing process, the enzymatic treatment process should be carried out after these processes. preferably. 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 enzymatic treatment step, esterase may be added and allowed to act during a specific step. In this case, an enzymatic reaction will occur in parallel with the progress of the specific process. A fermentation process, an aging process, and a storage process can be illustrated as a "specific process" here.

Other than the step (enzyme treatment step) that is unique to the present invention, a normal production method may be followed. The term "usual 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 "usual manufacturing method" to which the present invention can be applied is not particularly limited.

1. Screening for Ethyl Carbamate (EC) Degrading Enzymes In order to discover enzymes that can degrade ethyl carbamate (EC), 130 types of enzymes (including 23 types of lipases or esterases, as well as various hydrolases such as proteases and amylases) ) and microorganisms. The screening method is shown below. First, an EC reaction solution (100 mM phosphate buffer pH 7.0: 2 mL, EC: 10 mM, enzyme: 2 mg) was prepared for each enzyme to be screened (for microorganisms, culture solution was used instead of enzyme), and the reaction was performed. (30°C, stirrer stirring, 48 hours). After completion of the reaction, 0.1 mL of 1N HCl and 0.6 mL of chloroform (Wako) were added to 0.4 mL of the reaction solution, followed by thorough mixing. After centrifuging the mixture (15,000 rpm×5 minutes, 4° C.), the chloroform layer was collected in a vial and used as a GC analysis sample. Using the sample thus prepared, EC was detected by GC analysis. The EC decomposition rate was calculated from the peak area of ethyl carbamate (EC) (RT = 8.9 minutes).
(GC analysis conditions)
Analysis was performed using Gas chromatography (GC7700, Agilent) under the following conditions.
Column: DB-WAX (60m×0.25mm×0.25um) (Agilent J&W)
Injector: 250℃
Detector: FID, 250℃
Oven: 100°C (0 min) → heat 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 found that an esterase derived from Acinetobacter calcoaceticus (A. calcoaceticus) was capable of degrading EC. On the other hand, neither lipase nor Arthrobacter ramosus-derived esterase was able to decompose EC, indicating that the EC-degrading activity was a characteristic characteristic of A. calcoaceticus-derived esterase.

2. Degradation of EC by esterase from A. calcoaceticus (Shaoxing wine)
The ability of the A. calcoaceticus-derived esterase found by screening to decompose EC in Shaoxing wine was investigated. EC reaction solution (Shaoxing wine (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 min, 4° C.), membrane filtered (0.45 μm or 0.2 μm), and the EC concentration was analyzed by gas chromatography-mass spectrometry.

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

3. Examination of EC degradation ability of related enzymes 2 . confirmed that A. calcoaceticus-derived esterase can degrade EC in Shaoxing wine. The following examination was conducted to determine whether the analogous enzyme of the esterase derived from A. calcoaceticus has the ability to decompose EC.

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

(1-2) Acquisition of E. coli Expression Plasmid and Construction of E. coli Expression System Using the fully synthesized esterase gene as a template, PCR (PrimeSTAR GXL DNA Polymerase (Takara)) was performed. PCR conditions were as follows.
<PCR conditions>
Composition of reaction solution: 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) 1 µl (adjusted to 50 µl with sterile distilled water)
Reaction conditions: 30 cycles of 98°C for 10 seconds, 60°C for 30 seconds, 68°C for 1.5 minutes

The amplified product was purified (NucleoSpin Gel and 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 E. coli DH5α. (Takara) to obtain each E. coli recombinant. Each E.coli recombinant was inoculated into LB Broth Base (invitrogen) + Amp: 100μg/mL: 5mL, cultured with shaking (37°C, 16h, 140rpm), and NucleoSpin Plasmid EasyPure (MACHEREY-NAGEL) was added. 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. Each E. coli expression was performed using Sequence Primer (M13 M4 Primer: 5'-GTTTT CCCAGTCACGAC-3' (SEQ ID NO: 37), M13 RV Primer: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 38)). We confirmed the sequence of the plasmid used.

(2) Acquisition of each esterase culture medium (cultured cells) and bacterial cell extract (cultured cells) was attempted. The culture solution (cultured cells) of each esterase gene-recombinant E.coli-expressing strain was obtained by two-step culture. First, each esterase gene recombinant E.coli expression strain was inoculated into 5 mL of L Broth (Invitrogen) (Amp: 100 μg/mL) and cultured for 16 hours in a shaking incubator (140 rpm, 37°C). , Teriffic Broth (Invitrogen) (Amp: 100 μg/mL) 50 mL was inoculated with 0.5 mL. Then, the cells were cultured at 200 rpm and 33° C. for 48 hours, and 0.1 mM IPTG was added to the culture medium 24 hours after the start of the culture to induce expression of the enzyme. After centrifuging 50 mL of the culture solution (7,500 g×10 minutes, 4° C.), the cultured cells were recovered by removing the centrifugal supernatant.

After suspending each of the obtained cultured cells in 20 mL of 20 mM phosphate buffer (pH 7.0), add 10 g of beads (0.1 mm) (Yasui Instruments) and shake with a bead shocker (2,500 rpm, on: 60 seconds, off: 30 seconds, 15 cycles, 4°C) (Yasui Kiki Co., Ltd.). After the physical homogenate was centrifuged (7,500 g×10 minutes, 4° C.), the centrifugal supernatant was collected and used as a cell extract.

(3) Degradation of EC by related enzymes (buffer pH 7.0)
Using the obtained cell extract, the ability to decompose EC in a buffer solution (pH 7.0) was confirmed. An EC reaction solution (buffer (100 mM phosphate buffer pH 7.0): 1.3 mL, EC: 10 mM, each 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. An analytical sample was analyzed by GC. As a result of the analysis, it was confirmed that all of the related enzymes have the ability to decompose EC, although their activities differed (Fig. 1).

4. Alcohol Stability of Esterase Derived from A. calcoaceticus The alcohol stability of the esterase derived from A. calcoaceticus, which showed excellent EC degradation ability, was examined. First, treatment solutions were prepared by mixing 9 mL of alcohol solutions (ethanol-reagent special grade (Wako)) (0-70%) of various concentrations and 1 g of esterase derived from A. calcoaceticus. Each treatment solution was allowed to stand at 30° C., and samples (1 mL) were taken after 16 hours, 4 days, and 8 days. The enzymatic activity of each sample was measured by the following measuring method.
(Activity measurement method using 3,4-dihydrocoumarin as substrate)
2.1 mL of 50 mM phosphate buffer (pH 7.0), 0.3 mL of 5 mM 3,4-dihydrocoumarin (Sigma-aldrich (Code: D104809)) solution (containing 40% EtOH), and 0.6 mL of enzyme solution were mixed and allowed to react. rice field. In this measurement method, kinetics measurement (Abs. 270 nm, 30°C, 5 minutes) of the product (3-(2-hydroxyphenyl)propionic acid) produced by the reaction yields 1 nmol per minute. One unit (U) is defined as the amount of enzyme that produces the product (3-(2-hydroxyphenyl)propionic acid). The ΔOD at which an accurate measurement value can be obtained in this measurement is in the range of ΔOD = 0.01 to 0.05/2 min (Abs. 250 nm). 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 after treatment at 30°C for 8 days if the alcohol concentration was 40% or less (Fig. 2).

5. Various properties of esterase derived from A. calcoaceticus Using the above-mentioned "Activity measurement method using 3,4-dihydrocoumarin as a substrate" (pretreatment conditions of samples, etc. are described individually), the enzymatic properties of esterase derived from A. calcoaceticus were evaluated. identified.

(1) Optimum temperature The sample block of the absorbance meter was set at a predetermined temperature, the activity was measured, 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) Optimal pH
Activity was measured using a measurement system in which "2.1 mL of 50 mM phosphate buffer pH 7.0" in the reaction solution was replaced with a buffer adjusted to a predetermined pH, and the optimum pH was determined. Two types of buffers (McIlvaine, Atkins) were adjusted to each pH (McIlvaine buffer: pH3.0, pH4.0, pH5.0, pH6.0, pH7.0, pH6.0, pH7.0, pH 8.0.Atkins buffer solutions: 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 measurement, the optimum pH was pH 7 (Fig. 4).

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

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

(5) Molecular Weight Molecular weight was measured under non-denaturing conditions 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: Superdex 200HR 10/30 manufactured by Amersham pharmacia (currently GE Healthcare)
Buffer used: 50mmol/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.

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

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

SEQ ID NO: 37: Description of Artificial Sequence: Primer SEQ ID NO: 38: Description of Artificial Sequence: Primer

Claims (13)

Applying an esterase consisting of an amino acid sequence that is 85% or more identical to any of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 17 and exhibiting activity to decompose ethyl carbamate on a food or beverage containing ethyl carbamate. A method for decomposing ethyl carbamate in food or beverage, characterized by: 2. The degradation method according to claim 1, wherein the amino acid sequence of said esterase comprises any one of the amino acid sequences of SEQ ID NOS: 1-18. 2. The decomposition method according to claim 1, wherein the esterase is derived from a microorganism belonging to the genus Acinetobacter, a microorganism belonging to the genus Pseudomonas, a microorganism belonging to the genus Burkholderia, or a microorganism belonging to the genus Paraburkholderia. The Acinetobacter microorganism is Acinetobacter calcoaceticus, Acinetobacter guillouiae, Acinetobacter baumannii or Acinetobacter seifertii, and the Pseudomonas microorganism is Pseudomonas Pseudomonas aeruginosa, Pseudomonas fluorescens or Pseudomonas putida, and the Burkholderia is Burkholderia ubonensis or Burkholderia pseudomultibolans ( Burkholderia pseudomultivorans) and the Paraburkholderia is Paraburkholderia ferrariae. NBRC 110496, Acinetobacter sp. NIPH809, Pseudomonas fluorescens A506, Pseudomonas sp. ABAC61, Pseudomonas sp. 2. Degradation method according to claim 1, derived from Pseudomonas putida IFO12996 or Pseudomonas putida MR2068. 2. Degradation method according to claim 1, wherein said esterase has the following characteristics:
(1) optimum temperature: 20-30°C;
(2) optimal pH: pH7;
(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 decomposition method of claim 6, wherein said esterase further comprises the following characteristics:
(6) Alcohol stability: If the alcohol concentration is 40% or less, it will not be deactivated even if it is 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 wine, stone fruit spirit, whiskey, brandy, tequila, cachaça, soju, sake, wine, fortified wine, plum wine, sherry or mixed wine. Method. Consisting of an amino acid sequence that is 85% or more identical to any of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 17, ethyl carbamate is removed or reduced, including a treatment step with an esterase that exhibits decomposition activity for ethyl carbamate Food or beverage manufacturing method. The production method according to claim 10, wherein the esterase is an esterase defined in any one of claims 2-7. 12. The production method according to claim 10 or 11, wherein said beverage is an alcoholic beverage. 13. The preparation of claim 12, wherein the alcoholic beverage is Shaoxing wine, stone fruit spirit, whiskey, brandy, tequila, cachaça, soju, sake, wine, fortified wine, plum wine, sherry or mixed wine. Method.

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