Novel esterase that degrades trans-fatty acid-containing esters

Abstract

To provide a novel polypeptide that degrades trans-fatty acid-containing esters, and the like.SOLUTION: The novel esterase discovered by the present inventors have activity to degrade trans-fatty acid-containing esters. The esterase disclosed herein is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, 8, 12, 16, or 22, or a polypeptide encoded by the base sequence shown in SEQ ID NO: 1, 7, 11, 15, or 21, or a polypeptide having at least 70% sequence homology thereto. This esterase has the ability to degrade esters even at low and / or high temperatures, and it was also found that it has excellent thermal stability in terms of ester decomposition ability at 65°C. The esterase disclosed herein is useful for oil treatment, for example, in wastewater treatment, detergents, and fat modification / oil production technology.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to a novel esterase that decomposes trans-fatty acid-containing ester compounds and their applications (e.g., wastewater treatment, oil treatment). In another aspect, the present disclosure relates to a novel esterase that decomposes ester compounds even at low and / or high temperatures and their applications.

Background Art

[0002] Typical dirt generated in ordinary households, the food service industry, industrial facilities, etc. includes oil dirt. Oil dirt is dirt that is difficult to clean and occurs in scenes such as kitchens, sinks, kitchens, pipes, drains, ventilation fans, and washing. Since oil dirt can be a source of bad odors and pests and can also cause environmental pollution, the establishment of a revolutionary technology in oil treatment is eagerly desired from both the public health and environmental aspects.

[0003] A grease trap, which is a treatment facility for removing oil contained in kitchen wastewater in the food service industry by solid-liquid separation, is a source of bad odors and pests, and considering the labor and cost involved in the maintenance of separating, collecting, transporting, and cleaning the separated oil, the establishment of a revolutionary technology that eliminates the oil in the grease trap is eagerly desired from the industrial world, especially the food service industry.

Summary of the Invention

Means for Solving the Problems

[0004] As a result of intensive research, the present inventors have found an esterase that decomposes trans-fatty acid-containing ester compounds. This esterase has the ability to decompose ester compounds even at low and / or high temperatures, and it has also been found that it has excellent thermal stability. The present disclosure also relates to the application of the esterase of the present disclosure, such as oil treatment.

[0005] Accordingly, the present disclosure provides the following. (1) (a) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 8, 12, or 16; (b) An amino acid sequence containing one or more substitutions, additions, deletions, or combinations thereof in the amino acid sequence of (a), and having biological activity; (c) A polypeptide having at least 70% sequence identity with the amino acid sequence shown in (a) or (b) and having biological activity; (d) A polypeptide containing the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1, 7, 11, or 15; (e) A polypeptide encoded by a nucleotide sequence containing one or more substitutions, additions, deletions, or combinations thereof in the nucleotide sequence shown in (d) and having biological activity; (f) A polypeptide encoded by a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence shown in (d) or (e) and having biological activity; (g) A polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions with a polynucleotide containing the nucleotide sequence shown in any one of (d) to (f) or its complementary sequence and having biological activity; (h) A polypeptide encoded by an allelic variant of the nucleotide sequence of any one of (d) to (g) and having biological activity; or (i) A polypeptide containing a fragment of the amino acid sequence shown in (a) to (h), which is a polypeptide. (2) The polypeptide according to any of the above items, wherein the polypeptide is an esterase. (2A) The polypeptide according to any of the above items, wherein the polypeptide is a lipase. (2B) The polypeptide according to any of the above items, wherein the esterase is a lipase. (3) The polypeptide according to any of the above items, wherein the biological activity includes the ability related to the assimilation of trans-fatty acid-containing ester bodies or the ability to decompose trans-fatty acid-containing ester bodies. (3A) The biological activity is the ability related to the assimilation of the medium-chain fatty acid-containing ester form or the ability to decompose the medium-chain fatty acid-containing ester form, and the polypeptide according to any one of the above items. (4) The polypeptide is an esterase, and the substrate specificity target is a short-chain to long-chain fatty acid-containing ester form including fats and oils, and the polypeptide according to any one of the above items. (5) An esterase having the ability to decompose an ester form at 15 °C, and the polypeptide according to any one of the above items. (6) (A) (a) 75 °C, (b) 65 °C, (c) 67 °C, or (d) 70 °C having thermal stability at and / or (B) (a) 60 °C, (b) 40 °C, or (c) 65 °C having an optimum temperature at and / or (C) (a) pH 8 - 9, or (b) pH 9 having pH stability at and / or, (D) having an optimum pH at pH 9, and the polypeptide according to any one of the above items. (7) A polypeptide derived from Yarrowia lipolytica, and the polypeptide according to any one of the above items. (8) (A) A polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 1, 7, 11 or 15; (B) A polynucleotide containing a nucleotide sequence including substitution, addition, deletion of one or more nucleotides or a combination thereof in the nucleotide sequence shown in (A) and encoding a polypeptide having biological activity; (C) A polynucleotide containing a nucleotide sequence having at least 70% or more sequence identity with the nucleotide sequence shown in (A) or (B) and encoding a polypeptide having biological activity; A polynucleotide comprising a base sequence shown in any one of (D)(A) to (C) or a complementary sequence thereof, and comprising a base sequence that hybridizes under stringent conditions and encoding a polypeptide having biological activity; (E) A polynucleotide which is an allelic variant of the base sequence of any one of (A) to (D) and encodes a polypeptide having biological activity; (F) A polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 8, 12 or 16; (G) A polynucleotide comprising an amino acid sequence containing one or more substitutions, additions, deletions or combinations thereof in the amino acid sequence of (F) and encoding a polypeptide having biological activity; (H) A polynucleotide encoding a polypeptide having at least 70% or more sequence identity with the amino acid sequence shown in (F) or (G) and having biological activity; or (I) A polynucleotide comprising a fragment of the base sequence shown in (F) to (H), which is a polynucleotide. (9) The polynucleotide according to any one of the above items, wherein the polypeptide is an esterase. (9A) The polynucleotide according to any one of the above items, wherein the polypeptide is a lipase. (9B) The polynucleotide according to any one of the above items, wherein the esterase is a lipase. (10) The polynucleotide according to any one of the above items, wherein the biological activity includes the ability related to the assimilation of a trans-fatty acid-containing ester or the ability to decompose a trans-fatty acid-containing ester. (11) The polynucleotide according to any one of the above items, wherein the polypeptide is an esterase and the substrate specificity target thereof is a short-chain to long-chain fatty acid-containing ester including fats and oils. (11A) The polynucleotide according to any one of the above items, wherein the polypeptide is a lipase. (11B) The biological activity is the ability related to the assimilation of the trans-fatty acid-containing ester form or the ability to decompose the trans-fatty acid-containing ester form, and the polynucleotide according to any one of the above items. (11C) The biological activity is the ability related to the assimilation of the medium-chain fatty acid-containing ester form or the ability to decompose the medium-chain fatty acid-containing ester form, and the polynucleotide according to any one of the above items. (11D) The polynucleotide is the polynucleotide according to any one of the above items, which encodes a polypeptide having the ability to decompose the ester form at 15 °C. (11E) (A) (a) 75 °C, (b) 65 °C, (c) 67 °C, or (d) 70 °C having thermal stability at and / or (B) (a) 60 °C, (b) 40 °C, or (c) 65 °C having an optimum temperature at and / or (C) (a) pH 8-9, or (b) pH 9 having pH stability at and / or, (D) encoding a polypeptide having an optimum pH at pH 9, the polynucleotide according to any one of the above items. (11F) The polynucleotide according to any one of the above items, which is a polynucleotide derived from Yarrowia lipolytica. (12) (a) A polypeptide comprising an amino acid sequence containing one or more substitutions, additions, deletions or combinations thereof in the amino acid sequence shown in SEQ ID NO: 22; (b) A polypeptide having at least 70% sequence identity with the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 22; (c) A polypeptide encoded by a nucleotide sequence containing substitution, addition, deletion of one or more nucleotides, or a combination thereof, in the nucleotide sequence shown in SEQ ID NO: 21; (d) A polypeptide encoded by a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence shown in SEQ ID NO: 21; (e) A polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions with a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 21 or its complementary sequence; (f) A polypeptide encoded by an allelic variant of the nucleotide sequence shown in SEQ ID NO: 21; or (g) A polypeptide containing a fragment of the amino acid sequence shown in (a) to (f) which has the ability related to the assimilation of trans-fatty acid-containing ester bodies or the ability to decompose trans-fatty acid-containing ester bodies (if necessary, the above polypeptide is not a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 22). (12A) Furthermore, the polypeptide according to any one of the above items, which includes the ability related to the assimilation of medium-chain fatty acid-containing ester bodies or the ability to decompose medium-chain fatty acid-containing ester bodies. (12B) The polypeptide according to any one of the above items, wherein the target of substrate specificity includes ester bodies containing short-chain to long-chain fatty acids and triglycerides. (13) A nucleotide sequence encoding the polypeptide according to the above item, or (A) A polynucleotide containing a nucleotide sequence containing substitution, addition, deletion of one or more nucleotides, or a combination thereof, in the nucleotide sequence shown in SEQ ID NO: 21, and encoding a polypeptide having biological activity; (B) A polynucleotide containing a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence shown in SEQ ID NO: 21, and encoding a polypeptide having biological activity; (C) A polynucleotide containing a nucleotide sequence that hybridizes under stringent conditions with a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 21 or its complementary sequence, and encoding a polypeptide having biological activity; (D) A polynucleotide which is an allelic variant of the nucleotide sequence shown in SEQ ID NO: 21 and encodes a polypeptide having biological activity; (E) A polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 22; (F) A polynucleotide encoding a polypeptide having biological activity, which comprises an amino acid sequence containing substitution, addition, deletion of one or more amino acids or a combination thereof in the amino acid sequence shown in SEQ ID NO: 22; (G) A polynucleotide which has at least 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22 and encodes a polypeptide having biological activity; or (H) A polynucleotide containing a fragment of the nucleotide sequence shown in (E) to (G), wherein the biological activity includes the ability related to the assimilation of a trans-fatty acid-containing ester or the ability to decompose a trans-fatty acid-containing ester, a polynucleotide which is not a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 21 if necessary. (14) A cell or cell-free expression system containing the polynucleotide according to any one of the above items. (15) An oil-degrading agent containing the polypeptide according to any one of the above items, a polypeptide containing the amino acid sequence shown in SEQ ID NO: 22, the polynucleotide according to any one of the above items or a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 22 or a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 21, or a cell or cell-free expression system containing the cell or cell-free expression system according to any one of the above items. (16) The oil-degrading agent according to any one of the above items, wherein the oil contains a trans-fatty acid-containing ester. (16A) The oil-degrading agent according to any one of the above items, wherein the oil contains a medium-chain fatty acid-containing ester. (17) The oil-degrading agent according to any one of the above items, further containing an additional oil treatment component. (18) A kit for oil degradation, comprising a polypeptide according to any one of the above items, a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 22, a polynucleotide according to any one of the above items, a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 22 or a polynucleotide containing the nucleotide sequence set forth in SEQ ID NO: 21, a cell or cell-free expression system containing the same, a cell or cell-free expression system according to any one of the above items, or an oil-degrading agent according to any one of the above items, and a further oil treatment component. (19) An oil degradation and removal method, which includes allowing a polypeptide according to any one of the above items, a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 22, a polynucleotide according to any one of the above items, a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 22 or a polynucleotide containing the nucleotide sequence set forth in SEQ ID NO: 21, a cell or cell-free expression system containing the same, a cell or cell-free expression system according to any one of the above items, or an oil-degrading agent according to any one of the above items to act on a treatment target. (20) The method according to any one of the above items, wherein the treatment target contains a trans fatty acid or a trans fatty acid-containing ester. (21) A detergent, which includes a polypeptide according to any one of the above items, or a cell or cell-free expression system containing a polynucleotide according to any one of the above items. (22) Use of a polypeptide according to any one of the above items, a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 22, a polynucleotide according to any one of the above items, a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 22 or a polynucleotide containing the nucleotide sequence set forth in SEQ ID NO: 21, a cell or cell-free expression system containing the same, a cell or cell-free expression system according to any one of the above items, or an oil-degrading agent according to any one of the above items in fat modification and oil production technology.

[0006] In the present disclosure, it is intended that the above one or more features can be provided in further combinations in addition to the explicitly stated combinations. Further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading the following detailed description as needed.

Advantages of the Invention

[0007] Using the present disclosure, it has become easier to treat oils and fats containing trans fatty acids, which were difficult to remove conventionally. In addition, it has become possible to remove oils and fats and perform oil treatment even in high-temperature or low-temperature environments where it was difficult to use conventional microorganisms and enzymes. Therefore, the novel esterase (for example, lipase) of the present disclosure is useful in technical fields such as detergents, the leather industry, the food industry, purification of environmental pollution by oils and fats, treatment of food waste, composting treatment, wastewater treatment and other waste treatment and composting, pharmaceuticals such as digestive agents, and cosmetics for oily skin.

Brief Description of the Drawings

[0008]

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Mode for Carrying Out the Invention

[0009] Hereinafter, the present disclosure will be described while showing the best mode. Throughout this specification, it should be understood that singular expressions include the concepts of their plural forms unless otherwise specified. Therefore, singular articles (e.g., "a", "an", "the" in English, etc.) should be understood to include the concepts of their plural forms unless otherwise specified. Also, the terms used in this specification should be understood to be used in the meanings commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technological terms used in this specification have the same meanings as commonly understood by those skilled in the art to which the present invention pertains. In case of contradiction, this specification (including the definitions) shall prevail.

[0010] Hereinafter, the definitions of the terms particularly used in this specification and / or the basic technical content will be appropriately described. In this specification, strain names such as KH-2 strain may sometimes omit the indication of "strain", but those skilled in the art should appropriately understand that there is an indication of strain according to the context.

[0011] (Definition of terms) In this specification, "esterase" refers to a hydrolase that decomposes an ester into an acid and an alcohol by a chemical reaction with water. Since the esterase of the present disclosure has the activity of hydrolyzing esters such as fatty acid esters, it is classified as an esterase. The esterase activity possessed by the esterase can be measured by the method described in the "ability to decompose an ester form" of this specification.

[0012] In this specification, "lipase" is a kind of esterase and refers to an enzyme that reversibly catalyzes the reaction of hydrolyzing neutral fat (glycerol ester) into fatty acid and glycerol. Since the esterase of the present disclosure has the activity of an enzyme classified as EC3.1.1.3 in the enzyme number (EC number), i.e., a triglyceride lipase, it can also be said to be a "lipase", and in that case, it is also referred to as the lipase of the present disclosure.

[0013] As used herein, "oil and fat" refers to an oily substance, and the oil and fat includes an ester group-containing compound formed by dehydration condensation of a compound containing a hydroxyl group and a fatty acid. Typically, the compound containing a hydroxyl group is glycerin, but others such as polyglycerin are also included. In the present specification, in the same meaning as commonly used in this technical field, an ester group-containing compound formed by dehydration condensation of glycerin and a fatty acid is called "glyceride". When the compound containing a hydroxyl group has a plurality of hydroxyl groups, if at least one of the hydroxyl groups forms an ester by dehydration condensation with a fatty acid, it corresponds to the ester group-containing compound in the present specification.

[0014] As used herein, lipase activity refers to the activity of hydrolyzing an ester formed by dehydration condensation of glycerol and a fatty acid into glycerol and free fatty acid, and such lipase activity is also referred to as triglyceride lipase activity in the present specification. For example, whether it has lipase activity can be confirmed by the decrease of an ester (such as animal and vegetable oils such as canola oil, triolein, and trielaidin) contained in a solution added with lipase or the detection of hydrolysis products.

[0015] The ability to decompose and consume an ester and a fatty acid can be measured by analyzing the ester remaining in the medium and the free fatty acid generated by hydrolysis by thin layer chromatography. To show a specific quantification procedure, first, an equal amount of chloroform is added to the culture supernatant to extract the ester and the fatty acid. 5 μl of this extract is developed on a silica gel-coated plate using a developing solvent containing chloroform, acetone, and methanol at a volume ratio of 96:4:1, respectively. The ratio of the developing solvent and the like can be appropriately changed. The plate is treated with molybdic acid n-hydrate to develop the color of the ester.

[0016] As used herein, "fat-modifying / oil-producing ability" refers to changing the structure of fatty acids to change the properties of oils. As used herein, "fat-modifying / oil-producing ability" typically includes the activity of catalyzing the transesterification of fatty acids. The fat-modifying / oil-producing ability can be measured by analyzing the fatty acid methyl esters produced by the transesterification reaction of fatty acids and methanol by thin layer chromatography. To show a specific quantification procedure, first, the esterase, fatty acid, and methanol of the present disclosure are mixed to produce fatty acid methyl esters. By adding chloroform to the mixed solution of fatty acid methyl esters, the ester form and fatty acid are extracted. 10 μl of this extract is developed on a silica gel-coated plate using a developing solvent containing hexane, diethyl ether, and acetic acid in a volume ratio of 80:20:1, respectively. The ratio of the developing solvent, etc. can be appropriately changed. The plate is treated with molybdic acid n-hydrate to develop the color of the ester form.

[0017] As used herein, "ester form containing trans fatty acid" refers to an ester form containing trans fatty acid. "Trans fatty acid" is used in the meaning commonly used in the technical field, and means an unsaturated fatty acid having a trans-type double bond. Trans fatty acids are present in trace amounts naturally as conjugated linoleic acid and vaccenic acid, but are produced in large amounts in the oil and fat industry when producing saturated fatty acids by hydrogenation of unsaturated fatty acids, and are also included in foods such as margarine and shortening. Trans fatty acids include elaidic acid, palmitelaidic acid (palmitoelaidic acid), vaccenic acid, etc., but when mentioned in this specification, there is no particular limitation on the types of trans fatty acids. The ratio of trans fatty acids present in the ester form containing trans fatty acid is not particularly limited. Among the conventionally known Yarrowia lipases, none are known to have the activity of decomposing the ester form containing trans fatty acid.

[0018] In this specification, the "ester form containing short-chain to long-chain fatty acids" refers to an ester form of a fatty acid containing one or more of short-chain fatty acids, medium-chain fatty acids, or long-chain fatty acids. "Short-chain fatty acids", "medium-chain fatty acids", and "long-chain fatty acids" are used in the meanings commonly used in the technical field, and respectively mean fatty acids having 2 to 6 carbon atoms, 7 to 12 carbon atoms, and 13 or more carbon atoms. Short-chain fatty acids include acetic acid (2 carbon atoms), butyric acid (4 carbon atoms), caproic acid (6 carbon atoms), etc. Medium-chain fatty acids include caprylic acid (8 carbon atoms), capric acid (10 carbon atoms), lauric acid (12 carbon atoms), etc. Long-chain fatty acids include myristic acid (14 carbon atoms), palmitic acid (16 carbon atoms), stearic acid (18 carbon atoms), oleic acid (18 carbon atoms), linoleic acid (18 carbon atoms), linolenic acid (18 carbon atoms), etc.

[0019] In this specification, the "ester form containing medium-chain fatty acids" refers to an ester form containing medium-chain fatty acids. "Medium-chain fatty acids" are used in the meaning commonly used in the technical field and mean fatty acids having 7 to 12 carbon atoms. Medium-chain fatty acids are naturally contained in foods such as dairy products, palm kernel oil, and coconut oil. Medium-chain fatty acids include caprylic acid (8 carbon atoms), capric acid (10 carbon atoms), lauric acid (12 carbon atoms), etc., but when mentioned in this specification, there is no particular limitation on the types of medium-chain fatty acids. The ratio of medium-chain fatty acids present in the ester form containing medium-chain fatty acids is not particularly limited.

[0020] As used herein, the "ability related to the assimilation of trans-fatty acid-containing ester compounds" or the "ability related to the assimilation of short-chain to long-chain fatty acid-containing ester compounds" refers to the activity for bringing about the assimilation of trans-fatty acid-containing ester compounds or short-chain to long-chain fatty acid-containing ester compounds by microorganisms. As used herein, "assimilating trans-fatty acid-containing ester compounds" or "assimilating short-chain to long-chain fatty acid-containing ester compounds" is used in the meaning commonly used in the art, and means that microorganisms take in trans-fatty acid-containing ester compounds or short-chain to long-chain fatty acid-containing ester compounds as a nutrient source such as a carbon source. When "assimilating", in addition to hydrolyzing into alcohols and free fatty acids, it also includes changing into a part of other substances.

[0021] As used herein, the "ability to decompose trans-fatty acid-containing ester compounds" or the "ability to decompose short-chain to long-chain fatty acid-containing ester compounds" refers to the activity of hydrolyzing trans-fatty acid-containing ester compounds or short-chain to long-chain fatty acid-containing ester compounds into glycerol or other alcohols (such as 4-nitrophenol) and free fatty acids.

[0022] As used herein, the "ability to decompose ester compounds (at each temperature)" refers to the activity of hydrolyzing ester compounds into alcohols and free fatty acids at each temperature. As used herein, the "ability to decompose ester compounds (at each temperature)" is measured as follows. That is, the esterase is purified by the method described in this specification, and an ester compound substrate of 4-nitrophenol and a fatty acid (for example, palmitic acid, butyric acid (butyrate)) and the esterase are mixed under constant temperature at the temperature setting to be measured, and the amount of 4-nitrophenol generated by the hydrolysis reaction can be measured by measuring the absorbance at 410 nm.

[0023] As used herein, the "optimum temperature" for the ability to decompose an ester form is used in the meaning commonly used in the art, and refers to the temperature range in which the activity of decomposing the ester form is at a desired level above a certain level (for example, the temperature range in which the activity of at least 80% of the maximum level of the enzyme is maintained, although in another embodiment, it may refer to the maximum level). Specifically, the first esterase of the present disclosure has a peak activity of decomposing the ester form at about 60°C and maintains an activity of at least 80% of the peak in the range of about 55 to 65°C. The second esterase of the present disclosure has a peak activity of decomposing the ester form at about 40°C and maintains an activity of at least 80% of the peak in the range of about 35 to 50°C. The third esterase of the present disclosure has a peak activity of decomposing the ester form at about 65°C and maintains an activity of at least 80% of the peak in the range of about 53 to 68°C. The fourth esterase of the present disclosure has a peak activity of decomposing the ester form at about 65°C and maintains an activity of at least 80% of the peak in the range of about 55 to 68°C. The fifth esterase of the present disclosure has a peak activity of decomposing the ester form at about 40°C and maintains an activity of at least 80% of the peak in the range of about 33 to 47°C. In the context of referring to the optimum temperature of the esterases of the present disclosure, the optimum temperature of the first esterase of the present disclosure is intended to be about 55 to 65°C, preferably about 57 to 63°C, more preferably about 60°C. For the second esterase of the present disclosure, the optimum temperature is intended to be about 35 to 50°C, preferably about 35 to 45°C, more preferably about 40°C. For the third esterase of the present disclosure, the optimum temperature is intended to be about 53 to 68°C, preferably about 60 to 67°C, more preferably about 65°C. For the fourth esterase of the present disclosure, the optimum temperature is intended to be about 55 to 68°C, preferably about 60 to 67°C, more preferably about 65°C. For the fifth esterase of the present disclosure, the optimum temperature is intended to be about 33 to 47°C, preferably about 37 to 45°C, more preferably about 40°C.

[0024] As used herein, "thermal stability" has the same meaning as terms such as "temperature stability" and "heat resistance", and is used in the meaning commonly used in the art, meaning that the enzyme retains its activity at low and / or high temperatures. Generally, enzymes are known to be inactivated due to factors such as changes in molecular structure at a temperature of about 50°C. In contrast, in the case of the first esterase of the present disclosure, when the enzyme activity after treatment at 30°C for 30 minutes is taken as 100%, the relative enzyme activity after treatment at a temperature in the range of about 10 to about 60°C for 30 minutes is maintained at 80% or more. In the case of the second esterase of the present disclosure, when the enzyme activity after treatment at 45°C for 30 minutes is taken as 100%, the relative enzyme activity after treatment at a temperature in the range of about 15 to about 60°C for 30 minutes is maintained at 80% or more. In the case of the third esterase of the present disclosure, when the enzyme activity after treatment at 30°C for 30 minutes is taken as 100%, the relative enzyme activity after treatment at a temperature in the range of about 30 to about 65°C for 30 minutes is maintained at 80% or more. In the case of the fourth esterase of the present disclosure, when the enzyme activity after treatment at 30°C for 30 minutes is taken as 100%, the relative enzyme activity after treatment at a temperature in the range of about 30 to about 45°C for 30 minutes is maintained at 80% or more. In the case of the fifth esterase of the present disclosure, when the enzyme activity after treatment at 40°C for 30 minutes is taken as 100%, the relative enzyme activity after treatment at a temperature in the range of about 20 to about 45°C for 30 minutes is maintained at 80% or more. As used herein, expressions such as "retaining thermal stability" mean that, in the case of the first esterase, at about 30°C, in the case of the second esterase, at about 45°C, in the case of the third esterase, at 30°C, in the case of the fourth esterase, at 30°C, and in the case of the fifth esterase, at 40°C, when the enzyme activity after treatment for 30 minutes is taken as 100%, the relative enzyme activity is generally maintained at 50% or more.

[0025] As used herein, the "optimal pH" for the ability to decompose an ester form is used in the meaning commonly used in the art, and refers to the pH when the activity of decomposing the ester form is at a certain level or higher (for example, the pH range in which the activity of 80% or more of the maximum level of the enzyme is maintained, but in another embodiment, it may refer to the maximum level). The first, second, third, and fourth esterases of the present disclosure all have a peak activity of decomposing the ester form at about pH 9 and maintain an activity of 80% or more of the peak in the range of about pH 8.5 to pH 9.5. The fifth esterase has a peak activity of decomposing the ester form at about pH 8 and maintains an activity of 80% or more of the peak in the range of about pH 7.5 to pH 8.5. In the context of referring to the optimal pH of the first, second, third, and fourth esterases, the optimal pH of the esterases (any of the first, second, third, and fourth) of the present disclosure is intended to be pH 8.5 to pH 9.5, preferably pH 8.8 to pH 9.3, more preferably pH 9. In the context of referring to the optimal pH of the fifth esterase, the optimal pH of the fifth esterase of the present disclosure is intended to be pH 7.5 to pH 8.5, preferably pH 7.8 to pH 8.3, more preferably pH 8.

[0026] In this specification, the "pH stability" of the ability to decompose the ester form has the same meaning as terms such as "pH tolerance", "acid resistance", and "alkali resistance", and is used in the meaning commonly used in this technical field, referring to the pH range in which the activity of decomposing the ester form is above a certain level. In the case of the first esterase of the present disclosure, when the enzyme activity after incubation treatment in a buffer solution at pH 9 for 30 minutes is taken as 100%, the relative enzyme activity in the treatment at about pH 7.5 to 9.5 is maintained at 80% or more. In the case of the second esterase of the present disclosure, when the enzyme activity after incubation treatment in a buffer solution at pH 8 for 30 minutes is taken as 100%, the relative enzyme activity in the treatment at about pH 7.5 to 9.5 is maintained at 80% or more. In the case of the third esterase of the present disclosure, when the enzyme activity after incubation treatment in a buffer solution at pH 9 for 30 minutes is taken as 100%, the relative enzyme activity in the treatment at about pH 8.5 to 9.2 is maintained at 80% or more. In the case of the fourth esterase of the present disclosure, when the enzyme activity after incubation treatment in a buffer solution at pH 9 for 30 minutes is taken as 100%, the relative enzyme activity in the treatment at about pH 7.8 to 9.2 is maintained at 80% or more. In the case of the fifth esterase of the present disclosure, when the enzyme activity after incubation treatment in a buffer solution at pH 8 for 15 hours is taken as 100%, the relative enzyme activity in the treatment at about pH 4 to 8.5 is maintained at 80% or more. In this specification, expressions such as "maintaining pH stability" mean that, in the case of the first esterase, when the enzyme activity after incubation at pH 9 for a certain period of time or more is taken as 100%, the relative enzyme activity after incubation at the test pH for the same period of time is generally maintained at 50% or more; in the case of the second esterase, it is at pH 8; in the case of the third esterase, it is at pH 9; in the case of the fourth esterase, it is at pH 9; and in the case of the fifth esterase, it is at pH 8.

[0027] As used herein, "Yarrowia lipolytica" refers to the species Yarrowia lipolytica in the biological taxonomy. Yarrowia lipolytica is a type of alkane-assimilating yeast and has been identified as being characterized by a high ability to assimilate hydrophobic hydrocarbon chains such as n-alkanes and fats. The Yarrowia lipolytica strain KH-2 (a microbial strain identified by the accession number NITE BP-02732) in this specification has genes encoding at least five types of esterases (i.e., the representative sequences of the "first esterase", the "second esterase", the "third esterase", the "fourth esterase", and the "fifth esterase" of the present disclosure). It has been found in the present disclosure that these representative esterases of the present disclosure have characteristics not found in conventionally known esterases. These remarkable characteristics are detailed elsewhere in this specification. The esterases of the present disclosure include, but are not limited to, those produced by the KH-2 strain belonging to Yarrowia lipolytica.

[0028] As used herein, the "cell-free expression system" is used in the meaning commonly used in the art and refers to a system that uses the transcription and translation mechanism of biomolecules extracted from cells to produce a recombinant protein of interest in vitro. The cell-free expression system for producing the esterases of the present disclosure is not particularly limited, but a cell-free expression system derived from prokaryotes such as Escherichia coli can be used.

[0029] As used herein, the "oil treatment component" means a component that aids in the assimilation and decomposition of ester forms. Specifically, it includes components that promote the dispersion of ester forms, such as surfactants, components that decompose ester forms into fatty acids and alcohols, components that decompose fatty acids, components that decompose alcohols, and components that adsorb oil and remove it from the object to be treated.

[0030] As used herein, the "oil-degrading agent" refers to a formulation capable of decomposing ester compounds, which contains as an active ingredient the Yarowia lipolytica KH-2 strain of the present disclosure or the first, second, third, fourth, or fifth esterase of the present disclosure produced by this microbial strain. In the present disclosure, the oil-degrading agent may be used in combination with an oil treatment component. In this case, the timing of the combined use of the oil-degrading agent and the oil treatment component may be simultaneous or either one may be used first. Furthermore, the oil-degrading agent may further contain components that enhance the activity of the microbial strain or esterase derived from the microbial strain used (e.g., carbon source, nitrogen source), surfactants, drying protectants, components for maintaining the microorganism for a long period, preservatives, excipients, strengthening agents, antioxidants, and the like.

[0031] The oil-degrading agent provided by the present disclosure is provided in a liquid, solid, or dried state. Examples of the liquid form include a microbial culture solution (which may be concentrated or diluted as necessary), a product obtained by adsorbing an enzyme component derived from the culture solution onto a support, and a product obtained by separating and purifying the enzyme from the culture solution and dissolving it in a buffer or solvent. Examples of the solid form include a product obtained by suspending or dissolving it in a solvent containing a protective agent such as glycerol, freezing it, a product immobilized on a carrier (a product adsorbed onto the carrier by covalent bonding, electrostatic interaction, hydrophobic interaction, etc., a product obtained by molecular cross-linking the carrier, etc.), a product dehydrated by centrifugation, press compression, etc., and further a dried product. In a preferred embodiment, the oil-degrading agent is provided in a liquid form, powder, or granule form and may be provided as a detergent or as a component of a detergent, together with other components.

[0032] As used herein, "derivatives", "analogs", or "variants" (such as esterases) preferably, but not by way of limitation, include molecules containing regions substantially homologous to the protein of interest (e.g., an esterase), and such molecules, in various embodiments, are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical over an amino acid sequence of the same size, or when compared to an aligned sequence aligned by a computer homology program known in the art, or the nucleic acid encoding such a molecule is hybridizable to the sequence encoding the component protein under highly stringent conditions, moderately stringent conditions, or non-stringent conditions. This means that each is a product obtained by modifying a protein by amino acid substitution, deletion, and addition, and the derivative is a protein that shows that the biological function of the original protein does not necessarily have to be the same degree, preferably a protein having biological activity equal to or higher than that degree. For example, it is also possible to examine the biological function of such a protein by an appropriate and available in vitro assay described herein or known in the art. As used herein, "functionally active" or "having functional activity" refers to having a structural function, regulatory function, or biochemical function of a protein, such as biological activity, in accordance with the aspects related to the polypeptides of the present disclosure, i.e., fragments or derivatives.

[0033] In the present disclosure, a fragment of an esterase is a polypeptide containing any region of the esterase, and does not necessarily have all of the biological functions of the natural esterase as long as it functions for the purposes of the present disclosure (e.g., decomposition of trans fatty acid-containing esters or decomposition of medium-chain fatty esters).

[0034] As used herein, "protein", "polypeptide", "oligopeptide" and "peptide" are used interchangeably herein and refer to polymers of amino acids of any length. Such polymers may be linear, branched or cyclic. The amino acids may be natural, non-natural or modified amino acids. The term may also encompass assemblies of multiple polypeptide chains into complexes. The term also encompasses amino acid polymers that have been modified naturally or artificially. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (e.g., conjugation with a labeling component). This definition also encompasses, for example, polypeptides containing one or more analogs of amino acids (e.g., including non-natural amino acids, etc.), peptide-like compounds (e.g., peptoids) and other modifications known in the art. As used herein, "amino acid" is a general term for organic compounds having an amino group and a carboxyl group. When a protein or enzyme according to an embodiment of the present disclosure contains a "specific amino acid sequence", any amino acid in the amino acid sequence may be chemically modified. Also, any amino acid in the amino acid sequence may form a salt or a solvate. Also, any amino acid in the amino acid sequence may be of the L-form or the D-form. Even in such cases, it can be said that the protein according to the embodiment of the present disclosure contains the above "specific amino acid sequence". Chemical modifications that amino acids in a protein undergo in vivo include, for example, N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), or side chain modifications (e.g., phosphorylation, sugar chain addition, etc.). The amino acids may be natural or non-natural as long as the objectives of the present disclosure are met.

[0035] As used herein, "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably and refer to polymers of nucleotides of any length. This term also includes "oligonucleotide derivatives" or "polynucleotide derivatives". "Base sequence" or "nucleic acid sequence" means the order of consecutive nucleobases in a "polynucleotide", "oligonucleotide" or "nucleic acid". "Oligonucleotide derivative" or "polynucleotide derivative" refers to an oligonucleotide or polynucleotide that contains derivatives of nucleotides or in which the bonds between nucleotides are different from normal, and are used interchangeably. Such oligonucleotides specifically include, for example, 2'-O-methyl-ribonucleotides, oligonucleotide derivatives in which the phosphodiester bonds in the oligonucleotide are converted to phosphorothioate bonds, oligonucleotide derivatives in which the phosphodiester bonds in the oligonucleotide are converted to N3'-P5' phosphoramidate bonds, oligonucleotide derivatives in which the ribose and phosphodiester bonds in the oligonucleotide are converted to peptide nucleic acid bonds, oligonucleotide derivatives in which uracil in the oligonucleotide is replaced by C-5 propynyluracil, oligonucleotide derivatives in which uracil in the oligonucleotide is replaced by C-5 thiazoleuracil, oligonucleotide derivatives in which cytosine in the oligonucleotide is replaced by C-5 propynylcytosine, oligonucleotide derivatives in which cytosine in the oligonucleotide is replaced by phenoxazine-modified cytosine, oligonucleotide derivatives in which ribose in DNA is replaced by 2'-O-propylribose, and oligonucleotide derivatives in which ribose in the oligonucleotide is replaced by 2'-methoxyethoxyribose. Unless otherwise indicated, a particular base sequence is also intended to include conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly recited sequence.Specifically, degenerate codon substitutions can be achieved by creating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). As used herein, "nucleic acid" is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. As used herein, "nucleotide" may be natural or non-natural.

[0036] As used herein, "gene" refers to a factor that defines a genetic trait, and "gene" may refer to "polynucleotide", "oligonucleotide", and "nucleic acid".

[0037] As used herein, the "homology" of a gene refers to the degree of identity between two or more gene sequences with respect to each other. Generally, having "homology" means having a high degree of identity or similarity. Therefore, the higher the homology between two genes, the higher the identity or similarity of their sequences. Whether two types of genes have homology can be examined by direct comparison of the sequences or, in the case of nucleic acids, by the hybridization method under stringent conditions. When directly comparing two gene sequences, if the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical, those genes have homology. Therefore, as used herein, a "homolog" or "homologous gene product" means a protein in another species, preferably a microorganism, more preferably yeast, that exhibits the same biological function as the protein components of the complex further described herein. Such homologs may also be referred to as "orthologous gene products". It is understood that such homologs, homologous gene products, orthologous gene products, etc. can also be used as long as they meet the objectives of the present disclosure. As used herein, the "similarity" of a gene or base sequence refers to the degree of similarity between two or more gene sequences with respect to each other, meaning that the degree of similarity of other sequences in addition to identity is high. "Similarity" is a numerical value that takes into account similar bases in addition to identity, where similar bases refer to cases where there is partial agreement in degenerate bases (e.g., R = A + G, M = A + C, W = A + T, S = C + G, Y = C + T, K = G + T, H = A + T + C, B = G + T + C, D = G + A + T, V = A + C + G, N = A + C + G + T).

[0038] Amino acids may be referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be referred to by generally recognized one-letter codes. In this specification, comparisons of amino acid sequences and nucleotide sequences for similarity, identity, and homology are calculated using default parameters with the BLAST sequence analysis tool. The identity search can be performed, for example, using NCBI's BLAST 2.7.1 (released on October 19, 2017). The "identity" value in this specification usually refers to the value obtained when aligned under default conditions using the above BLAST. However, if a higher value is obtained by changing the parameters, the highest value shall be taken as the identity value. When identity is evaluated in multiple regions, the highest value among them shall be taken as the identity value. "Similarity" is a numerical value that takes into account similar amino acids in addition to identity.

[0039] In one embodiment of the present disclosure, "several" may be, for example, 10, 8, 6, 5, 4, 3, or 2, and may be less than any of those values. It is known that a polypeptide that has undergone deletion, addition, insertion, or substitution with another amino acid of one or several amino acid residues maintains its biological activity (Mark et al., Proc Natl Acad Sci USA. 1984 Sep;81(18): 5662-5666., Zoller et al., Nucleic Acids Res. 1982 Oct 25;10(20):6487-6500., Wang et al., Science. 1984 Jun 29;224(4656):1431-1433.). A protein with deletions or the like can be produced, for example, by site-directed mutagenesis, random mutagenesis, or biopanning using a protein phage library. As the site-directed mutagenesis method, for example, the KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.) can be used. It is possible to select a protein with the same activity as the wild type from the mutant protein into which deletions or the like have been introduced by performing various characterizations such as FACS analysis and ELISA.

[0040] In one embodiment of the present disclosure, the numerical value such as "70% or more" for identity or the like may be, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% or more, or may be within the range of any two values of these starting values. The above-mentioned "identity" is calculated according to a known method as described above, which is the ratio of the number of identical amino acids in an amino acid sequence between two or more. Specifically, before calculating the ratio, the amino acid sequences of the amino acid sequence groups to be compared are aligned, and gaps are introduced into a part of the amino acid sequence if necessary to maximize the ratio of identical amino acids. Methods for alignment, methods for calculating ratios, comparison methods, and computer programs related thereto are well-known in the art (for example, BLAST described above). In this specification, "identity" and "similarity" can be represented by values measured by NCBI's BLAST unless otherwise specified. When comparing amino acid sequences by BLAST, the algorithm Blastp can be used with default settings. The measurement results are quantified as Positives or Identities. In this case, when using "similarity" instead of "identity", it is a numerical value that also takes into account those that meet the definition of "similar" "amino acids" or "bases" described in this specification.

[0041] As used herein, the term "polynucleotide that hybridizes under stringent conditions" refers to well-known conditions commonly used in the art. Such polynucleotides can be obtained by using, as a probe, a polynucleotide selected from the polynucleotides of the present disclosure, and employing methods such as colony hybridization, plaque hybridization, or Southern blot hybridization. Specifically, it means a polynucleotide that can be identified by performing hybridization at 65°C in the presence of 0.7 - 1.0 M NaCl using a filter immobilized with DNA derived from colonies or plaques, and then washing the filter under 65°C conditions using an SSC (saline-sodium citrate) solution at a concentration of 0.1 - 2 times (the composition of a 1× SSC solution is 150 mM sodium chloride and 15 mM sodium citrate). "Stringent conditions" can employ, for example, the following conditions: (1) using low ionic strength and high temperature for washing (e.g., at 50°C, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate), (2) using a denaturing agent such as formamide during hybridization (e.g., at 42°C, 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5, and 750 mM sodium chloride, 75 mM sodium citrate), or (3) incubating overnight at 37°C in a solution containing 20% formamide, 5× SSC, 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 mg / ml of denatured and sheared salmon sperm DNA, and then washing the filter with 1× SSC at about 37 - 50°C. Note that the formamide concentration may be 50% or higher. The washing time may be 5, 15, 30, 60, or 120 minutes, or longer.Multiple factors such as temperature and salt concentration can affect the stringency of the hybridization reaction. For details, refer to Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). Examples of "highly stringent conditions" are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65 - 68 °C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide, 42 °C. Hybridization can be carried out according to the methods described in experimental manuals such as Molecular Cloning 2nd ed., Current Protocols in Molecular Biology, Supplement 1 - 38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). Here, sequences that hybridize under stringent conditions preferably exclude sequences containing only A sequences or only T sequences. Moderately stringent conditions can be easily determined by those skilled in the art based on, for example, the length of the DNA, as shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Vol. 1, 7.42 - 7.45 Cold Spring Harbor Laboratory Press, 2001, and for nitrocellulose filters, include the use of a pre-washing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of approximately 40 - 50 °C in approximately 50% formamide, 2×SSC - 6×SSC (or other similar hybridization solutions such as Stark's solution in approximately 50% formamide at approximately 42 °C), and washing conditions of approximately 60 °C, 0.5×SSC, 0.1% SDS.Accordingly, the polypeptides used in the present disclosure include polypeptides encoded by nucleic acid molecules that hybridize to nucleic acid molecules encoding the polypeptides specifically described in the present disclosure under highly or moderately stringent conditions.

[0042] The esterases of the present disclosure can preferably be "purified" or "isolated". As used herein, a "purified" substance or biological agent (e.g., nucleic acid or protein, etc.) refers to a substance or biological agent from which at least a part of the factors naturally associated with the substance or biological agent has been removed. Accordingly, usually, the purity of the biological agent in the purified biological agent is higher (i.e., concentrated) than the state in which the biological agent usually exists. The term "purified" as used herein preferably means that there is at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of the same type of biological agent. The substances or biological agents used in the present disclosure are preferably "purified" substances. As used herein, an "isolated" substance or biological agent (e.g., nucleic acid or protein, etc.) refers to a substance or biological agent from which the factors naturally associated with the substance or biological agent have been substantially removed. Since the term "isolated" as used herein varies depending on the purpose, it does not necessarily have to be expressed in terms of purity, but if necessary, preferably means that there is at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of the same type of biological agent. The substances used in the present disclosure are preferably "isolated" substances or biological agents.

[0043] As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 with respect to a full-length polypeptide or polynucleotide (length n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length can be, for polypeptides, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids, and lengths represented by integers not specifically listed here (such as 11, etc.) can also be appropriate as the lower limit. Also, for polynucleotides, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides, and lengths represented by integers not specifically listed here (such as 11, etc.) can also be appropriate as the lower limit. It is understood that in this specification, such fragments are within the scope of the present disclosure as long as, for example, when the full-length one functions as an esterase-degrading molecule, the fragment itself also has the function of an esterase-degrading molecule.

[0044] As used herein, the term "biological function" refers to a specific function that a gene, nucleic acid molecule, or polypeptide related thereto may have in vivo or in vitro. Examples of such functions include, but are not limited to, the degradation of ester forms (e.g., the degradation of trans-fatty acid-containing ester forms). In the present disclosure, for example, in addition to the degradation of trans-fatty acid-containing ester forms, the degradation of medium-chain fatty acid-containing ester forms, the degradation of cis-fatty acid-containing ester forms, the degradation of saturated fatty acid-containing ester forms without double bonds, etc. can be mentioned, but are not limited thereto. In this specification, the biological function can be exerted by the corresponding "biological activity". As used herein, the term "biological activity" refers to the activity that a factor (e.g., polynucleotide, protein, etc.) may have, and includes activities that exert various functions (e.g., the degradation activity of trans-fatty acid-containing ester forms). The "biological activity" may be an activity exerted in vivo or an activity exerted in vitro by secretion or the like. For example, when a factor is an enzyme, its biological activity includes its enzyme activity. Such biological activity can be measured by techniques well known in the art. Therefore, "activity" refers to any measurable indicator that indicates or reveals binding (either direct or indirect); affects a response (i.e., has a measurable effect in response to some exposure or stimulus), such as the affinity of a compound that directly binds to the polypeptide or polynucleotide of the present disclosure, or for example, the amount of a protein upstream or downstream or other similar measures of function after some stimulus or event may also be included.

[0045] As used herein, the "expression" of a gene, polynucleotide, polypeptide, etc. refers to the process by which such a gene, etc. undergoes a certain action in vivo and changes into another form. Preferably, it refers to the process by which a gene, polynucleotide, etc. is transcribed and translated into a polypeptide form, but the production of mRNA by transcription is also one aspect of expression. Therefore, as used herein, the "expression product" includes such polypeptides or proteins, or mRNA. More preferably, such a polypeptide form may have undergone post-translational processing. For example, the expression level of esterase can be determined by any method. Specifically, the expression level of esterase can be known by evaluating the amount of esterase mRNA, the amount of esterase protein, and the biological activity of the esterase protein. The amount of esterase mRNA or protein can be determined by methods as detailed elsewhere in this specification or other methods known in the art.

[0046] As used herein, "functional equivalent" refers to any entity that has the same intended function but a different structure with respect to the original entity of interest. Thus, a functional equivalent of the "esterase" of the present disclosure is not the esterase of the present disclosure itself, but a variant or modified form thereof (e.g., an amino acid sequence variant, etc.) that has the biological activity of the esterase, and that can be changed into a variant or modified form that has the biological activity of the esterase at the time of action (e.g., a nucleic acid encoding the variant or modified form, and a vector, cell, etc. containing the nucleic acid) is understood to be included. In the present disclosure, it is understood that a functional equivalent of an esterase can be used in the same manner as the esterase even if not specifically mentioned. A functional equivalent can be found by searching a database or the like. As used herein, "search" refers to finding another nucleic acid base sequence having a specific function and / or property using a certain nucleic acid base sequence electronically, biologically, or by other means. Examples of electronic searches include, but are not limited to, BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85:2444-2448 (1988)), Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147:195-197 (1981)), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)). Examples of biological searches include, but are not limited to, stringent hybridization, macroarray or microarray (microarray assay) in which genomic DNA is attached to a nylon membrane or the like or a glass plate, PCR, and in situ hybridization. As used herein, it is intended that the genes used in the present disclosure should include corresponding genes identified by such electronic and biological searches.

[0047] As functional equivalents of the present disclosure, in the amino acid sequence, one or more amino acids can be inserted, substituted, or deleted, or added to one or both ends thereof. In the present specification, "insertion, substitution, or deletion of one or more amino acids in the amino acid sequence, or addition to one or both ends thereof" means that modification has been made by a well-known technical method such as site-directed mutagenesis or by natural mutation, and substitution of a plurality of amino acids to such an extent that can occur naturally. The modified amino acid sequence can be, for example, one in which 1 to 30 amino acids, preferably 1 to 20 amino acids, more preferably 1 to 9 amino acids, still more preferably 1 to 5 amino acids, and particularly preferably 1 to 2 amino acids have been inserted, substituted, or deleted, or added to one or both ends thereof. The modified amino acid sequence preferably has an amino acid sequence in which one or more (preferably 1 or more or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) conservative substitutions are made in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22. Here, "conservative substitution" means substituting one or more amino acid residues with another chemically similar amino acid residue so as not to substantially modify the function of the protein. For example, when substituting a certain hydrophobic residue with another hydrophobic residue, or substituting a certain polar residue with another polar residue having the same charge. Functionally similar amino acids for which such substitutions can be made are known in the art for each amino acid. Specific examples include, as nonpolar (hydrophobic) amino acids, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc. As polar (neutral) amino acids, glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. As positively charged (basic) amino acids, arginine, histidine, lysine, etc. Also, as negatively charged (acidic) amino acids, aspartic acid, glutamic acid, etc.

[0048] As used herein, the term "kit" refers to a unit that is usually divided into two or more compartments and in which the parts to be provided (for example, enzymes, lipolytic agents, buffers, instructions, etc.) are provided. When the purpose is to provide a composition that should not be provided in a mixed state for reasons such as stability and is preferably mixed and used immediately before use, this kit form is preferred. Such a kit preferably includes an instruction manual or description that describes how to use the provided parts (for example, enzymes or lipolytic agents), or how to handle the reagents or waste liquids after use. When the kit is used as a reagent kit in this specification, the kit usually includes an instruction manual that describes how to use enzymes, lipolytic agents, etc.

[0049] As used herein, an "instruction manual" describes instructions for users on how to use the present disclosure. This instruction manual describes words that instruct on how to use the polypeptides, polynucleotides, cells, etc. of the present disclosure. If necessary, this instruction manual is prepared in accordance with the format specified by the regulatory authorities of the country where the present disclosure is implemented (for example, the Ministry of Health, Labour and Welfare or the Ministry of Agriculture, Forestry and Fisheries in Japan, the Food and Drug Administration (FDA), the United States Department of Agriculture (USDA), etc. in the United States), and it is specified that approval has been obtained from the regulatory authorities. The instruction manual can be provided in a paper medium, but is not limited thereto, and can also be provided in forms such as electronic media (for example, a homepage provided on the Internet, an e-mail).

[0050] (Preferred Embodiment) Preferred embodiments of the present disclosure are described below. It is understood that the embodiments provided below are for better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present disclosure by referring to the descriptions in this specification. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0051] (Enzyme) In one aspect, the present disclosure provides novel polypeptides and nucleic acids encoding the same. This polypeptide typically has esterase activity. In one embodiment, this polypeptide has lipase activity. The polypeptides of the present disclosure have remarkable properties different from those of conventionally known enzymes.

[0052] In certain embodiments, the present disclosure provides representative sequences of a first esterase, a second esterase, a third esterase, a fourth esterase, and a fifth esterase typically obtained from the KH-2 strain of the genus Yarrowia lipolytica, as well as derivatives thereof.

[0053] In one aspect, the polypeptides of the present disclosure are the first to fourth esterases, namely, (a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 8, 12, or 16; (b) a polypeptide comprising an amino acid sequence containing one or more substitutions, additions, deletions, or combinations thereof in the amino acid sequence of (a) and having biological activity; (c) a polypeptide having at least 70% sequence identity with the amino acid sequence shown in (a) or (b) and having biological activity; (d) a polypeptide comprising the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1, 7, 11, or 15; (e) a polypeptide encoded by a nucleotide sequence containing one or more substitutions, additions, deletions, or combinations thereof in the nucleotide sequence of (d) and having biological activity; (f) a polypeptide encoded by a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence shown in (d) or (e) and having biological activity; A polypeptide that comprises a polynucleotide containing the nucleotide sequence shown in any one of (g) or the complementary sequence thereof, and is encoded by a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence and has biological activity; (h) A polypeptide encoded by an allelic variant of the nucleotide sequence of any one of (d) to (g) and having biological activity; or (i) A polypeptide comprising a fragment of the amino acid sequence shown in (a) to (h), which is a polypeptide.

[0054] In an alternative aspect, the polypeptide of the present disclosure is a fifth esterase, namely, (a) A polypeptide comprising an amino acid sequence containing one or more substitutions, additions, deletions, or combinations thereof in the amino acid sequence shown in SEQ ID NO: 22; (b) A polypeptide having at least 70% sequence identity with the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 22; (c) A polypeptide encoded by a nucleotide sequence containing one or more substitutions, additions, deletions, or combinations thereof in the nucleotide sequence shown in SEQ ID NO: 21; (d) A polypeptide encoded by a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence shown in SEQ ID NO: 21; (e) A polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 21 or the complementary sequence thereof; (f) A polypeptide encoded by an allelic variant of the nucleotide sequence shown in SEQ ID NO: 21; or (g) A polypeptide comprising a fragment of the amino acid sequence shown in (a) to (f) is. In one embodiment, the polypeptide of the present disclosure is not a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 22. In one embodiment, the polypeptide of the present disclosure is a polypeptide having biological activity.

[0055] In another aspect, the present disclosure provides a nucleotide sequence encoding a novel polypeptide. The polynucleotide of the present disclosure is a polynucleotide encoding the first to fourth esterases, that is, (A) a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1, 7, 11, or 15; (B) a polynucleotide comprising a nucleotide sequence containing one or more substitutions, additions, deletions, or combinations thereof in the nucleotide sequence shown in (A), and encoding a polypeptide having biological activity; (C) a polynucleotide comprising a nucleotide sequence having at least 70% or more sequence identity with the nucleotide sequence shown in (A) or (B), and encoding a polypeptide having biological activity; (D) a polynucleotide comprising a nucleotide sequence that hybridizes with the polynucleotide comprising the nucleotide sequence shown in any one of (A) to (C) or its complementary sequence under stringent conditions, and encoding a polypeptide having biological activity; (E) an allelic variant of the nucleotide sequence of any one of (A) to (D), and encoding a polypeptide having biological activity; (F) a polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 8, 12, or 16; (G) a polynucleotide comprising an amino acid sequence containing one or more substitutions, additions, deletions, or combinations thereof in the amino acid sequence of (F), and encoding a polypeptide having biological activity; (H) a polynucleotide having at least 70% or more sequence identity with the amino acid sequence shown in (F) or (G), and encoding a polypeptide having biological activity; or (I) a polynucleotide comprising a fragment of the nucleotide sequence shown in (F) to (H), and can be a polynucleotide.

[0056] In another aspect, the present disclosure provides a nucleotide sequence encoding a novel polypeptide. The polynucleotide of the present disclosure is a polynucleotide encoding the fifth esterase, that is, (A) A polynucleotide comprising a nucleotide sequence containing substitutions, additions, deletions of one or more nucleotides, or combinations thereof, in the nucleotide sequence shown in SEQ ID NO: 21; (B) A polynucleotide comprising a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence shown in SEQ ID NO: 21 and encoding a polypeptide having biological activity; (C) A polynucleotide comprising a nucleotide sequence that hybridizes under stringent conditions with a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 21 or its complementary sequence and encoding a polypeptide having biological activity; (D) An allelic variant of the nucleotide sequence shown in SEQ ID NO: 21, encoding a polypeptide having biological activity; (E) A polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 22; (F) A polynucleotide comprising an amino acid sequence containing substitutions, additions, deletions of one or more amino acids, or combinations thereof, in the amino acid sequence shown in SEQ ID NO: 22 and encoding a polypeptide having biological activity; (G) A polynucleotide having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 22 and encoding a polypeptide having biological activity; or (H) A polynucleotide containing a fragment of the nucleotide sequences shown in (E) to (G). In one embodiment, the polynucleotide of the present disclosure is not a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 21. The biological activity includes the ability related to the assimilation of trans-fatty acid-containing ester bodies or the ability to decompose trans-fatty acid-containing ester bodies.

[0057] In one embodiment, the biological activity of the polypeptide of the present disclosure can be at least one of any properties of the first, second, third, fourth, or fifth esterase of the present disclosure. For example, it may include the ability related to the assimilation of trans-fatty acid-containing ester bodies, or the ability to decompose trans-fatty acid-containing ester bodies or both. Without wishing to be bound by theory, the conventionally known Yarrowia-derived esterase has not been observed to have activity against trans-fatty acid-containing ester bodies. At least in that sense, the esterase of the present disclosure has remarkable characteristics.

[0058] Therefore, in one embodiment, the polypeptide of the present disclosure or the polypeptide encoded by the polynucleotide may have the ability related to the assimilation of trans-fatty acid-containing ester bodies, or the ability to decompose trans-fatty acid-containing ester bodies or both.

[0059] In other embodiments, the biological activity of the polypeptide of the present disclosure may also include, for example, the ability related to the assimilation of short-chain to long-chain fatty acid-containing ester bodies, or the ability to decompose short-chain to long-chain fatty acid-containing ester bodies or both.

[0060] Therefore, in one embodiment, the polypeptide of the present disclosure or the polypeptide encoded by the polynucleotide may have the ability related to the assimilation of medium-chain fatty acid-containing ester bodies, or the ability to decompose medium-chain fatty acid-containing ester bodies or both.

[0061] In one preferred embodiment, the polypeptide of the present disclosure or the polypeptide encoded by the polynucleotide has the ability to decompose ester bodies at 15°C or 10°C. The temperature range at which the activity of the conventionally known Yarrowia-derived esterase is observed is mostly relatively high, and it was unexpected that significant activity was observed at low temperatures.

[0062] In another preferred embodiment, for the polypeptide which is the first esterase of the present disclosure or the polypeptide encoded by a polynucleotide, 55 to 65°C, preferably 57 to 63°C, or 60°C is the optimum temperature for the ability to decompose the ester form. For the polypeptide which is the second esterase of the present disclosure or the polypeptide encoded by a polynucleotide, 35 to 50°C, preferably 35 to 45°C, more preferably 40°C is the optimum temperature for the ability to decompose the ester form. For the polypeptide which is the third esterase of the present disclosure or the polypeptide encoded by a polynucleotide, 53 to 68°C, preferably 60 to 67°C, most preferably 65°C is the optimum temperature for the ability to decompose the ester form. For the polypeptide which is the fourth esterase of the present disclosure or the polypeptide encoded by a polynucleotide, 55 to 68°C, preferably 60 to 67°C, most preferably 65°C is the optimum temperature for the ability to decompose the ester form. For the polypeptide which is the fifth esterase of the present disclosure or the polypeptide encoded by a polynucleotide, typically, preferably 33 to 47°C, more preferably 37 to 45°C, most preferably 40°C is the optimum temperature for the ability to decompose the ester form.

[0063] In one embodiment, the polypeptide encoded by the polypeptide or polynucleotide that is the first esterase of the present disclosure can be stable at 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, or 75°C or higher, and can be stable up to 50°C or lower, 55°C or lower, 60°C or lower, 65°C or lower, 70°C or lower, 75°C or lower, or 80°C or lower. The polypeptide encoded by the polypeptide or polynucleotide that is the first esterase of the present disclosure can preferably be stable at 50°C or higher and can be stable up to 70°C or lower. The polypeptide encoded by the polypeptide or polynucleotide that is the second esterase of the present disclosure can be stable at 45°C or higher, 50°C or higher, 55°C or higher, or 60°C or higher, and can be stable up to 50°C or lower, 55°C or lower, 60°C or lower, 65°C or lower, or 70°C or lower. The polypeptide encoded by the polypeptide or polynucleotide that is the second esterase derived from the KH-2 strain of the present disclosure can preferably be stable at 45°C or higher and can be stable up to 60°C or lower. The polypeptide encoded by the polypeptide or polynucleotide that is the third esterase of the present disclosure can be stable at 30°C or higher, 40°C or higher, 45°C or higher, 50°C or higher, or 60°C or higher, and can be stable up to 60°C or lower, 65°C or lower, 70°C or lower, or 75°C or lower. The polypeptide encoded by the polypeptide or polynucleotide that is the third esterase derived from the KH-2 strain of the present disclosure can preferably be stable at 45°C or higher and can be stable up to 65°C or lower. The polypeptide encoded by the polypeptide or polynucleotide that is the fourth esterase of the present disclosure can be stable at 45°C or higher, 50°C or higher, 55°C or higher, or 60°C or higher, and can be stable up to 50°C or lower, 55°C or lower, 60°C or lower, 65°C or lower, or 70°C or lower. The polypeptide encoded by the polypeptide or polynucleotide that is the fourth esterase derived from the KH-2 strain of the present disclosure can preferably be stable at 45°C or higher and can be stable up to 50°C or lower. The polypeptide encoded by the polypeptide or polynucleotide that is the fifth esterase derived from the KH-2 strain of the present disclosure can preferably be stable at 40°C or higher and can be stable up to 20°C or lower.However, by "stable" is meant that the activity after treatment at each temperature for 30 minutes is maintained at 80% or more compared to the activity when treated at about 30°C.

[0064] In one specific embodiment, the polypeptide encoded by the polypeptide or polynucleotide of the present disclosure may be, but is not limited to, derived from Yarrowia lipolytica. In a preferred embodiment, it may be, but is not limited to, an enzyme derived from the Yarrowia lipolytica KH-2 strain.

[0065] In a further aspect, the present disclosure provides an oil-degrading agent comprising a cell or cell-free expression system containing any of the above polypeptides or polynucleotides.

[0066] The cells of the present disclosure contain the polypeptide of the first, second, third, fourth or fifth esterase of the present disclosure, or a polynucleotide encoding the polypeptide of the first, second, third, fourth or fifth esterase of the present disclosure is expressibly incorporated therein. The cell-free expression system is provided with a polynucleotide encoding the polypeptide of the first, second, third, fourth or fifth esterase of the present disclosure, and the polypeptide is expressed by an appropriate mechanism to exhibit an oil-degrading effect.

[0067] In one embodiment, the above oil-degrading agent further contains an additional oil treatment component. Examples of the oil treatment component include, but are not limited to, other enzymes (such as esterases, etc.) or microorganisms.

[0068] In another aspect, the present disclosure provides an oil-degrading agent or a kit for oil or grease treatment, comprising the polypeptide of the present disclosure, or the cell or cell-free expression system of the present disclosure, or the oil-degrading agent, and an additional oil treatment component. It is understood that the oil-degrading agent and the oil treatment component included in the kit of the present disclosure can be used in any combination of any types described elsewhere in this specification.

[0069] In another aspect, the present disclosure provides an oil degradation and removal method, which includes allowing the polypeptide of the present disclosure, or the cell or cell-free expression system of the present disclosure, or the oil-degrading agent of the present disclosure to act on a treatment target. In the oil degradation and removal method of the present disclosure, the treatment target preferably includes, but is not limited to, a trans-fatty acid-containing ester form. It is shown herein that even when the target does not contain a trans-fatty acid-containing ester form, oil degradation can be performed very efficiently when the esterase of the present disclosure is used.

[0070] In another aspect, the present disclosure provides a detergent containing the polypeptide of the present disclosure, the cell or cell-free expression system of the present disclosure, or the oil-degrading agent of the present disclosure.

[0071] In yet another aspect, the present disclosure provides an application in fat modification and oil production technology (such as transesterification) using the polypeptide of the present disclosure, the cell or cell-free expression system of the present disclosure, or the oil-degrading agent of the present disclosure. Examples of fat modification and oil production technology (such as transesterification) include reactions that produce ester forms, such as methyl esters and ethyl esters, substitution reactions of fatty acids contained in oils and fats, and production of diacylglycerol and monoacylglycerol.

[0072] (Production of Enzyme) In one embodiment, the esterase of the present disclosure is produced by a secretory production system using microorganisms.

[0073] Specifically, the esterase of the present disclosure is (a) introducing a nucleic acid molecule encoding the full-length sequence of the first, second, third, fourth, or fifth esterase of the present disclosure (for example, SEQ ID NO: 5, 9, 13, 17, 19 or a modified sequence thereof) into the pEZZ18 plasmid (GE Healthcare) and expressing it in Escherichia coli HB101 strain as a Protein A fusion recombinant protein, and (b) purifying the protein secreted from Escherichia coli HB101 strain by IgG Sepharose 6 Fast Flow or Butyl Sepharose 6 Fast Flow It can be produced by a method including. Exemplarily, the nucleotide sequences encoding the first, fourth, and fifth esterases of the present disclosure are inserted into the XbaI and KpnI sites in the pEZZ18 vector, the nucleotide sequence encoding the second esterase is inserted into the PstI and KpnI sites, and the nucleotide sequence encoding the third esterase is inserted into the EcoRI and XbaI sites. In addition to the secretion production system using the above-mentioned Escherichia coli, the esterase recombinant protein of the present disclosure can be produced by expression systems such as the CORYNEX (registered trademark) system (Ajinomoto) using Corynebacterium glutamicum, the Pichia pastoris expression system (ThermoFisher), the baculovirus expression system (ThermoFisher) using insect cells, and the protein secretion expression system (Ozeki) using Aspergillus oryzae. In the method for producing an esterase using this microorganism, detailed conditions such as a method for expressing the esterase, a host for expressing the esterase, and a method for purifying the esterase secreted from the host are appropriately adjusted by those skilled in the art. Furthermore, the esterase of the present disclosure can also be expressed using various expression systems such as an intracellular expression system and a cell-free expression system in addition to the secretion production system of microorganisms.

[0074] Alternatively, the esterase of the present disclosure is (a) a step of introducing a mutation into the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21 or another basic nucleotide sequence to obtain a modified sequence of the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21 or another basic nucleotide sequence, (b) a step of appropriately introducing the modified sequence into a plasmid and expressing it in a suitable host (e.g., Escherichia coli HB101 strain) as a recombinant protein (optionally fused with a tag sequence (e.g., protein A tag) that facilitates purification), and (c) Purifying the protein produced in the host by an appropriate separation method (e.g., chromatography such as IgG Sepharose 6 Fast Flow or Butyl Sepharose 6 Fast Flow, etc.) It can be produced by a method comprising. Exemplarily, variants of the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21 of the present disclosure are inserted into the aforementioned restriction enzyme sites within the pEZZ18 vector. The step of introducing mutations into the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21 is appropriately carried out using commonly used site-directed mutagenesis methods, mutagenesis methods, or molecular evolution methods using error-prone PCR, etc., or commercially available kits.

[0075] Alternatively, the esterase of the present disclosure can be purified, for example, from the Yarrowia lipolytica KH-2 strain. Specifically, the esterase of the present disclosure (a) Culturing the KH-2 strain in a medium containing 1% (v / v) canola oil at 28 °C for 24 hours (b) Sterilizing the culture supernatant of (a) with a 0.45 μm pore filter and applying it to Butyl Sepharose 6 Fast Flow (GE Healthcare) pre-equilibrated with 20 mM Tris-HCl (pH 7.0) buffer containing approximately 10 times the column volume of 0.5 M NaCl and 2 mM CaCl2 (hereinafter referred to as Tris buffer), and allowing it to stand for about 1 hour to adsorb hydrophobic proteins to the column (c) Washing the column of (b) with approximately 10 times the column volume of Tris buffer, and then washing it 3 times with Tris buffer without NaCl in an amount approximately equal to the column volume to elute proteins weakly bound to the column carrier, and (d) Washing the column of (c) 3 times with Tris buffer containing 1% Triton (registered trademark) X-100 in an amount approximately equal to the column volume to elute proteins strongly bound to the column It can be purified by a method including this. In the enzyme purification method, detailed conditions such as the culture conditions of the KH-2 strain and the conditions of column chromatography are appropriately adjusted by those skilled in the art.

[0076] (Use of Enzyme) In one aspect, the esterase of the present disclosure is useful for treating fats and oils and can be used for treating wastewater and waste liquid containing fats and oils. In certain embodiments, the esterase of the present disclosure is used in wastewater treatment. When the esterase of the present disclosure is used for wastewater treatment, it can be used in applications such as industrial wastewater, kitchen wastewater, and domestic wastewater.

[0077] As another aspect, the esterase of the present disclosure is used as a detergent. When the esterase of the present disclosure is used as a detergent, it can be used in applications such as laundry detergents, kitchen detergents, cleaning detergents, and industrial detergents. Detergents containing the esterase of the present disclosure are particularly useful for drain cleaners such as pipe cleaners.

[0078] In other aspects, the esterase of the present disclosure is used in fat modification and oil production technologies. When the esterase of the present disclosure is used in fat modification and oil production technologies, it can be used in applications such as for food, industry, and fuel.

[0079] In other aspects, the esterase of the present disclosure can be used as a measure against environmental pollution. When the esterase of the present disclosure is used in measures against environmental pollution, it can be used for removing pollutants in soil pollution, groundwater pollution, and marine pollution caused by oil.

[0080] In other aspects, the esterase of the present disclosure is used in waste treatment and composting. When the esterase of the present disclosure is used in waste treatment and composting, it can be used in applications such as the treatment of perishable waste including the disappearing type, composting, the conversion of agricultural and food waste into feed, and the volume reduction of oily sludge from pressurized flotation separation devices and grease traps.

[0081] In other aspects, the esterase of the present disclosure is used as a pharmaceutical. When the esterase of the present disclosure is used as a pharmaceutical, it can be used in applications such as digestive agents and fat decomposition accelerators.

[0082] In other aspects, the esterase of the present disclosure is used as a cosmetic. When the esterase of the present disclosure is used as a cosmetic, it can be used in applications such as cosmetics for improving, preventing or treating oily skin.

[0083] When the esterase of the present disclosure is used in various applications, it may be used as a component containing the isolated enzyme, or as a component containing the microorganism itself. Those skilled in the art can appropriately use the esterase of the present disclosure in an appropriate form.

[0084] (General technology) The molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well-known and commonly used in the art. For example, Savli, H., Karadenizli, A., Kolayli, F., Gundes, S., Ozbek, U., Vahaboglu, H. 2003. Expression stability of six housekeeping genes: A proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J.Med.Microbiol. 52:403-408., Marie-Ange Teste, Manon Duquenne, Jean M Francois and Jean-Luc Parrou 2009. Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae. BMC Molecular Biology 10:99, Seiji Ishii, Hiroshi Okumura, Chiyo Matsubara, Fumi Ninomiya, Hiroshi Yoshioka, 2004, "Simple method for measuring oil in water using thermosensitive polymers", Vol 46, No.12, "Water Supply and Sewerage", etc. These are incorporated herein by reference in their entirety (which may be all) to the extent relevant.

[0085] (Note) In this specification, "or" is used when at least one or more of the items listed in the text can be adopted. The same applies to "or else". When it is specified in this specification "within the range of two values", the range includes the two values themselves.

[0086] References such as scientific literature, patents, patent applications, etc. cited in this specification are incorporated herein by reference in their entirety to the same extent as each is specifically described.

[0087] As described above, the present disclosure has been described by showing preferred embodiments for ease of understanding. Hereinafter, the present disclosure will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only and not for the purpose of limiting the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described in this specification, but is limited only by the claims.

Example

[0088] Examples are described below. The handling of organisms used in the following examples complied with the standards defined in the Nagoya University, regulatory authorities, and the Cartagena Act, if necessary. Specifically, the products described in the examples were used for the reagents, but equivalents from other manufacturers (Sigma-Aldrich, Fujifilm Wako Pure Chemical, Nacalai Tesque, R&D Systems, USCN Life Science INC, Thermo Fisher Scientific, Kanto Chemical, Funakoshi, Tokyo Chemical Industry, Merck, etc.) can also be substituted. Also, unless otherwise specified, the addition concentration of various oils, fats, fatty acids, etc. to the medium refers to the final concentration of the substance in the medium, and for percentage notation, oils and fats or fatty acids with a liquid property are represented by volume / volume (v / v%), and those with a solid property are represented by weight / volume (w / v%).

[0089] (Example 1: Summary of the gene and amino acid sequence encoding esterase derived from KH-2 strain) In this example, a summary of the gene and amino acid sequence encoding the esterase of the present disclosure is shown.

[0090] (Experimental method) The whole genome sequence of the KH-2 strain was performed. The genes encoding the first esterase (gene number G4021), the second esterase (gene number G2944), the third esterase (gene number G2601), the fourth esterase (gene number G5971), and the fifth esterase (gene number G3702), their estimated functions, the presence or absence of signal peptides (SP), and the estimated molecular weights of the gene products are shown (Figure 1).

[0091] (Results) The first, second, third, fourth, and fifth esterases found in Example 1 were estimated to have cholesterol esterase activity, triglyceride lipase activity, patatin and phospholipase activity, patatin and phospholipase activity, and triglyceride lipase activity, respectively. The first, second, third, fourth, and fifth esterases were all estimated to have a signal peptide (SP) sequence, and their respective molecular weights were estimated to be 63.5 kDa, 61 kDa, 62.9 kDa, 69.6 kDa, and 36.7 kDa.

[0092] (Example 2: Expression analysis of esterase genes in the KH-2 strain) In this example, the expression of the five types of esterase genes identified in Example 1 was analyzed.

[0093] (Experimental method) The KH-2 strain was cultured overnight in LB medium, and the culture was washed twice with TBS buffer (137 mM NaCl, 2.68 mM KCl, 25 mM Tris, pH 7.4) to remove the LB medium. The washed KH-2 strain was inoculated into 3 L of BS medium with 1% (v / v) canola oil added so that the final concentration OD 660 = 0.05, and fermentation culture was carried out at 15 °C. Total RNA was extracted from the cultures at 24 hours, 48 hours, and 72 hours after culture using the High Pure RNA Isolation Kit. Using 200 ng of total RNA as a template, PrimeScript TMGenomic DNA was removed and cDNA was synthesized using the RT reagent Kit with gDNA Eraser Perfect Real Time (Takara Bio Inc.). The cDNA was diluted by adding an equal volume of pure water and used. Quantitative real-time RT-PCR was performed using Applied Biosystems® StepOnePlus TM (Applied Biosystems) with synthetic primers specific to each esterase-encoding gene TM in a 20 μl solution containing PowerUp SYBR® Green Master Mix (Thermo Fisher Scientific) (10 μl), each primer (final concentration 0.5 μM), and cDNA (1 μl). The PCR reaction was carried out with a program of 1 cycle of denaturation at 95 °C for 20 seconds, followed by 40 cycles of 95 °C for 1 second and 60 °C for 20 seconds. The expression level was normalized to the expression level of 1,2-mannosyltransferase (alg9). The data were analyzed by the comparative Ct method (ΔΔCt method) after confirming that the melting curve had a single peak. The expression levels of the genes encoding the first, second, third, fourth, and fifth esterases were calculated as relative expression levels when the expression level of each esterase gene expressed when cultured in LB medium without oil was set to 1 as a control

[0094] (Results) By providing oil as a carbon source to the KH-2 strain, induction of the expression of genes encoding four types of esterases (those with the representative sequences of the first esterase, the second esterase, the third esterase, and the fourth esterase) was observed at the 24-hour time point. Induction of the expression of these esterases was also observed 72 hours after culturing. In contrast, the gene encoding the fifth esterase showed an expression level equivalent to that in culture in LB medium, suggesting that this gene is constitutively expressed regardless of the carbon source

[0095] (Example 3: Production of Recombinant Escherichia coli Expressing the First, Second, Third, Fourth, and Fifth Esterases)

[0096] (Experimental Procedure) In this example, to express the recombinant proteins of the representative sequences of the first, second, third, fourth, and fifth esterases in Escherichia coli cells, the pEZZ18 Protein A Gene Fusion Vector system (GE Healthcare) was used. The nucleotide sequences of the genes encoding the representative sequences of the first, second, third, fourth, and fifth esterases were amplified by PrimestarMax, respectively, and the amplified transgenes were inserted into the XbaI and KpnI sites (for the first, fourth, and fifth esterases), PstI and KpnI sites (for the second esterase), and EcoRI and XbaI sites (for the third esterase) in the pEZZ18 vector. The Escherichia coli strain containing the transgene was cultured in 200 ml of LB medium containing ampicillin at 37 °C for 48 hours and then at 42 °C for 3 hours. The cells were removed by centrifugation, and the recombinant protein was adsorbed onto a pre-equilibrated Butyl Sepharose 6 Fast Flow column. Then, the recombinant protein was eluted with an elution buffer of 20 mM Tris-HCl (pH 7.0) containing 2 mM CaCl2 and 0.5% Triton® X-100.

[0097] (Example 4: Measurement of Optimal Temperature, Thermal Stability, Optimal pH, and pH Stability) In this example, the optimal temperature, thermal stability, optimal pH, and pH stability of the representative sequences of the first, second, third, and fourth esterases produced by recombinant Escherichia coli and roughly purified are shown.

[0098] Escherichia coli expressing the representative sequences of the first, second, third, and fourth recombinant esterases was cultured in 1500 ml of LB medium for 48 hours using a fermenter. An equal volume of acetone was added to the culture supernatant and left standing at 4°C for 24 hours. Subsequently, centrifugation was performed to obtain a pellet, and the crude purified esterase obtained by suspending the pellet in 1 ml of buffer (20 mM Tris-HCl (pH 7.4), 2 mM CaCl2 with 0.5% Triton® X-100) was used as the analysis target. (A and E) The optimal temperature was examined by mixing the crude purified esterase with buffer adjusted to each temperature between 0°C and 100°C and measuring the esterase activity. (B and F) The thermal stability was examined by mixing the crude purified esterase with buffer adjusted to each temperature between 0°C and 100°C, incubating for 30 minutes, and then measuring the esterase activity. (C and G) The optimal pH was examined by mixing the crude purified esterase with acetate buffer (pH 3.0 - 5.0), sodium phosphate buffer (pH 5.0 - 7.0), Tris-HCl buffer (pH 7.0 - 9.0), or CAPS buffer (pH 9.0 - 11.0) and measuring the esterase activity. (D and H) The pH stability was examined by mixing the crude purified esterase with acetate buffer (pH 3.0 - 5.0), sodium phosphate buffer (pH 5.0 - 7.0), Tris-HCl buffer (pH 7.0 - 9.0), or CAPS buffer (pH 9.0 - 11.0), incubating for 30 minutes, and then measuring the esterase activity.

[0099] (Results) The optimal temperature of the representative sequence of the first esterase was about 60°C and it showed thermal stability in the temperature range of about 10°C to about 60°C. The optimal pH of the representative sequence of the first esterase was pH 9.0 and it showed pH stability in the pH range of about pH 7.5 to about pH 9.5. The optimal temperature of the representative sequence of the second esterase was about 40°C and it showed thermal stability in the temperature range of about 10°C to about 50°C. The optimal pH of the representative sequence of the second esterase was pH 9.0 and it showed pH stability in the pH range of about pH 7.5 to about pH 9.2. The optimal temperature of the representative sequence of the third esterase was about 65°C and it showed thermal stability in the temperature range of about 30°C to about 65°C. The optimal pH of the representative sequence of the third esterase was pH 9.0 and it showed pH stability in the pH range of about pH 8 to about pH 9.5. The optimal temperature of the representative sequence of the fourth esterase was about 65°C and it showed thermal stability in the temperature range of about 30°C to about 70°C. The optimal pH of the representative sequence of the fourth esterase was pH 9.0 and it showed pH stability in the pH range of about pH 7 to about pH 9.5.

[0100] (Example 5: Cleaning effect of the first, second, third, fourth and fifth esterases and N-51032 enzyme on the ventilation fan filter oil stain) In this example, the cleaning effect of the oil stain on the ventilation fan filter was measured for the representative sequences of the first, second, third, fourth and fifth esterases of the present disclosure derived from strain KH-2 and Novozym 51032 lipase (Novozymes).

[0101] (Experimental method) The representative sequences of the first, second, third, fourth, and fifth esterases of the present disclosure derived from the KH-2 strain were obtained according to the method described in Example 3. Novozym 51032 lipase (N-51032) was purchased from Novozymes. Each esterase or lipase was dissolved in 20 mM Tris-HCl, 2 mM CaCl2 buffer (pH 7.4) containing 0.25% Triton® X-100. The concentration of Novozym 51032 lipase was adjusted to 15 U / ml, the concentration of the representative sequences of the first and second esterases was adjusted to 0.5 U / ml, and the concentration of the representative sequences of the third, fourth, and fifth esterases was adjusted to 0.1 U / ml. At that time, palmitate (C16) was used as the substrate for the representative sequences of the first and second esterases, and butyrate (C4) 4-nitrophenyl ester (pNP-ester) was used as the substrate for the representative sequences of the third, fourth, and fifth esterases. Based on the enzyme activity quantified by the absorbance at 410 nm of the amount of 4-nitrophenol produced by the hydrolysis of the ester by the esterase or lipase, the concentration of the enzyme solution was adjusted. The ventilation fan filter with oil stain deposition was cut into 2 cm squares and placed on a plate, and the solution of each esterase or lipase was added to each plate and immersed for 30 minutes.

[0102] (Results) Figure 4 shows the ventilation fan filter after immersion in the esterase or lipase solution and the esterase or lipase solution after immersion. All of the ventilation fan filters immersed in the representative sequences of the first, second, third, and fourth esterases of the present disclosure had significantly decomposed oil stains compared to Novozym 51032 lipase, and it was also found that the decomposition of oil stains was improved to some extent in the fifth esterase.

[0103] (Example 6: Degradation of oils and fats by the recombinant proteins of the first, second, third, fourth, and fifth esterases of the present disclosure) In this example, the degradation of lard, shortening, triolein, and trielaidin by the recombinant proteins of the first, second, third, fourth, and fifth esterases of the present disclosure is shown.

[0104] (Experimental method) (A: Degradation of shortening by the first, second, third, fourth, and fifth esterases) Each purified recombinant esterase produced by the steps of Example 3 was prepared to a final concentration of 0.2 u / ml. 2 ml of the solution of each esterase of the present disclosure was placed in a plate, and 0.7 g of shortening was added thereto. While appropriately stirring to mix with the solution, it was incubated at 28 °C for 24 hours. A control was treated with only the elution buffer described above.

[0105] (B: Degradation of lard by the first, second, third, fourth, and fifth esterases) Each purified recombinant esterase produced by the steps of Example 3 was prepared to a final concentration of 0.2 u / ml. 2 ml of the solution of each esterase of the present disclosure was placed in a plate, and 0.5 g of lard was added thereto. While appropriately stirring to mix with the solution, it was incubated at 28 °C for 24 hours. A control was treated with only the elution buffer described above.

[0106] (C: Degradation of lard, shortening, triolein, and trielaidin by the first, second, third, fourth, and fifth esterases) Each purified recombinant esterase produced by the steps of Example 3 was mixed with lard, shortening, triolein, and trielaidin, respectively, and treated at 37 °C for 48 hours. The treatment was carried out by putting 0.1 ml of each purified recombinant esterase and 1 ml of elution buffer into a tube and shaking at 130 rpm. The treated sample was applied to a silica gel plate and developed with a chloroform:acetone:methanol (96:4:2) solution. After development, detection was performed by coloring with molybdotungstic acid n-hydrate (2.4 g / 60 ml EtOH).

[0107] (Result) The results of the 1st, 2nd, and 5th recombinant esterases are shown in Fig. 5-1, and the results of the 3rd and 4th recombinant esterases are shown in Fig. 5-2, respectively. From the results of (A) to (C), it was shown that all of the recombinant proteins of the representative sequences of the 1st, 2nd, 3rd, and 4th esterases of the present disclosure have the activity to decompose all of the fats and oils of lard, shortening, triolein, and trielaidin. Therefore, it became clear that the recombinant proteins of the representative sequences of the 1st, 2nd, 3rd, and 4th esterases of the present disclosure have the activity to hydrolyze both triolein, which is a cis-form triglyceride, and trielaidin, which is a trans-form triglyceride. Furthermore, it was shown that the decomposition activity against shortening is remarkably high. Furthermore, although inferior to the decomposition activity of these esterases, it was shown that the recombinant protein of the representative sequence of the 5th esterase also has the decomposition activity of these fats and oils.

[0108] (Example 7: Comparison of Substrate Specificities of the 1st, 2nd, 3rd, 4th, and 5th Esterases) In this example, the decomposition activities of each substrate by the 1st, 2nd, 3rd, 4th, and 5th esterases of the present disclosure were compared.

[0109] (Experimental Method) Those of the representative sequences of the first, second, third, fourth, and fifth esterases of the present disclosure were obtained according to the method described in Example 3. The measurement of esterase activity was performed by quantifying the amount of 4-nitrophenol generated by the hydrolysis of the ester by the esterase using 4-nitrophenyl esters (pNP-ester) of five types of fatty acids (acetate (C2), butyrate (C4), octanoate (C8), laurate (C12), and palmitate (C16)) as substrates at the absorbance at 410 nm. As the substrate solution, a solution obtained by mixing 0.05 mol of each substrate with 12 ml of a 3% (v / v) Triton® X-100 aqueous solution and melted at 70 °C was used. 60 μl each of each substrate solution, 150 mM GTA buffer (pH 7.0), and each sample were mixed, and the absorbance at 410 nm immediately after mixing and the absorbance at 410 nm 30 minutes after mixing were measured. Then, the esterase activity was calculated by subtracting the absorbance at 410 nm immediately after mixing from the absorbance at 410 nm 30 minutes after mixing and further subtracting the value measured under the same conditions by mixing the buffer alone with the substrate. For the activity of the fifth esterase, the esterase activity was calculated by measuring the absorbance at 410 nm for 1 minute after mixing the substrate solution with the sample solution containing the fifth esterase. The esterase activity was defined as the amount of enzyme that liberates 1 μM of 4-nitrophenol as 1 unit, and the number of units per 1 ml of the sample was calculated, and the relative activity was calculated with the one showing the maximum activity in each substrate as 100%.

[0110] (Results) The results of Example 7 are shown in FIG. 6. The first esterase had the highest esterase activity against lipids having an octanoate (C8) carbon chain and also showed high esterase activity against the laurate (C12) carbon chain. The second esterase had the highest esterase activity against lipids having a butyrate (C4) carbon chain and also showed high esterase activity against octanoate (C8). The third esterase had the highest esterase activity against lipids having a butyrate (C4) carbon chain. The fourth esterase had the highest esterase activity against lipids having a butyrate (C4) carbon chain and also showed high esterase activity against acetate (C2) and octanoate (C8). The fifth esterase had the highest esterase activity against lipids having a butyrate (C4) carbon chain and also showed high esterase activity against acetate (C2).

[0111] (Example 8: Comparison of the degradation activities of trans-fatty acid-containing ester compounds by the first, second, third, fourth, and fifth esterases by serial dilution) In this example, by performing serial dilution, the degradation activities of trans-fatty acid-containing ester compounds by the first, second, third, fourth, and fifth esterases of the present disclosure were compared.

[0112] (Experimental method) Those of the representative sequences of the first, second, third, fourth, and fifth esterases of the present disclosure were obtained according to the method described in Example 3. The measurement of the degradation activity of the trans-fatty acid-containing ester compound was performed by using trielaidin as the ester compound and serially diluting those of the representative sequences of the first, second, third, fourth, and fifth esterases to 0.01 u / ml, 0.001 u / ml, and 0.0001 u / ml, and analyzing by thin-layer chromatography according to the following experimental conditions. · Type of ester compound: Trielaidin · Reaction solution: 20 mM Tris-HCl (pH 7.0), 2 mM CaCl2, 0.5% Triton® X100 · Final concentration of ester compound: 0.2% ·Treatment method: Put 100 μl of enzyme solution into an Eppendorf tube, Add 1 / 10 volume of 10x each ester stock. (0.02 u / ml: NP-butyrate as substrate) ·Treatment temperature: 37 °C ·Treatment time: 48 hours ·Oil extraction: Extracted with half volume of chloroform and 10 μl was applied for thin layer chromatography. ·Developing plate: Silica gel coated plate ·Developing solvent: chloroform:acetone:methanol = 96:4:2 ·Detection: Color development with molybdotungstic acid n-hydrate (2.4 g / 60 ml EtOH)

[0113] (Results) For the representative sequence of the fifth esterase of the present disclosure, activity was observed when treated at a concentration of 0.01 u / ml for 48 hours, but when treated at a concentration of 0.001 u / ml for 48 hours, the activity almost completely disappeared. For the representative sequences of the first, second, third, and fourth esterases, activity was observed even when diluted to 0.001 u / ml. From this, it was estimated that the representative sequence of the fifth esterase has about 1 / 10 of the trans-fatty acid decomposition activity compared to the representative sequences of the first, second, third, and fourth esterases.

[0114] (Example 9: Decomposition of trans-fatty acid-containing esters at low temperature) In this example, the decomposition activities of the trans-fatty acid-containing esters by the first, second, third, and fourth esterases of the present disclosure at 15 °C were compared.

[0115] (Experimental method) The representative sequences of the first, second, third, and fourth esterases of the present disclosure were obtained according to the method described in Example 3. The measurement of the decomposition activity of the trans-fatty acid-containing esters was analyzed by thin layer chromatography according to the following experimental conditions using trielaidin as the ester at a treatment temperature of 15 °C. · Type of ester: trielaidin · Reaction solution: 20 mM Tris-HCl (pH 7.0), 2 mM CaCl2, 0.5% Triton® X100 · Final concentration of ester: 0.2% · Treatment method: Put 100 μl of enzyme solution into an Eppendorf tube, Add 1 / 10 volume of 10x stock of each ester. (0.02 u / ml: NP-butyrate as substrate) · Treatment temperature: 15°C · Treatment time: 72 hours · Oil extraction: Extracted with half volume of chloroform and applied 10 μl for thin layer chromatography. · Development plate: Silica gel coated plate · Developing solvent: chloroform:acetone:methanol = 96:4:2 · Detection: Color development with molybdotriphosphoric acid n-hydrate (2.4 g / 60 ml EtOH)

[0116] (Results) Any of the esterases of the representative sequences of the first, second, third, and fourth esterases of the present disclosure showed activity to decompose the trans-fatty acid-containing ester at 15°C.

[0117] (Example 10: Modification of triolein) In this example, modification (methyl esterification) of triolein was performed with the first, second, third, and fourth esterases of the present disclosure.

[0118] (Experimental procedure) The representative sequences of the first, second, third, and fourth esterases of the present disclosure were obtained according to the method described in Example 3. Each esterase solution was adjusted to 0.1 u / ml, and triolein and methanol were added thereto. A treatment without adding the esterase solution was also performed simultaneously as a control group. Thereafter, 100 μl of each solution was dispensed into tubes with screw caps and allowed to stand in an incubator at 37° C. for 96 hours. Then, 100 μl of water and 50 μl of chloroform were added, and after thoroughly stirring with a vortex mixer, centrifugation was performed at 12,000 g for 5 minutes. 10 μl of the lower chloroform layer was loaded, and thin layer chromatography analysis was performed using hexane-diethyl ether-acetic acid (80:20:1) as a developing solvent.

[0119] (Results) The results of Example 10 are shown in FIG. 9. Any of the esterases having the representative sequences of the first, second, third, and fourth esterases of the present disclosure catalyzed the transesterification reaction of triolein, which is a cis triglyceride, to produce methyl oleate.

[0120] (Example 11: Modification of trans fatty acids) In this example, modification (methyl esterification) of trans fatty acids was performed using the first, second, third, and fourth esterases of the present disclosure.

[0121] (Experimental procedure) The representative sequences of the first, second, third, and fourth esterases of the present disclosure were obtained according to the method described in Example 3. Each esterase solution was adjusted to 0.1 u / ml, and a stock solution was prepared by dissolving monomers of (A) palmitelaidic acid or (B) vaccenic acid in methanol to a concentration of 0.5%. 30 μl of each trans-fatty acid stock solution and 70 μl of the enzyme solution were dispensed into a tube with a screw cap and left standing in an incubator at 37°C for 72 hours. A treatment using Buffer instead of the esterase solution was carried out simultaneously as a control. Thereafter, 100 μl of water and 50 μl of chloroform were added, stirred well with a vortex mixer, and then centrifuged at 12,000 g for 5 minutes. 5 μl of the lower chloroform layer was loaded, and thin layer chromatography analysis was performed using hexane-diethyl ether-acetic acid (80:20:1) as the developing solvent.

[0122] (Results) The results of Example 11 are shown in FIG. 10. Any of the esterases with the representative sequences of the first, second, third, and fourth esterases catalyzed the transesterification reaction of the trans-fatty acids palmitelaidic acid and vaccenic acid, producing methyl palmitelaidate and methyl vaccenate, respectively.

[0123] (Example 12: Comparison with the second and fifth esterases in the modification of trans-fatty acids) In this example, the modification (methyl esterification) of trans-fatty acids was carried out using the second and fifth esterases of the present disclosure.

[0124] (Experimental procedure) Those of the representative sequences of the second and fifth esterases of the present disclosure were obtained according to the method described in Example 3. Each esterase solution was adjusted to 0.1 u / ml, and a stock solution was prepared by dissolving monomers of (A) palmitelaidic acid or (B) vaccenic acid in methanol to a concentration of 0.5%. 30 μl of each trans-fatty acid stock solution and 70 μl of the enzyme solution were dispensed into tubes with screw caps and left standing in an incubator at 37 °C for 72 hours. A treatment using Buffer instead of the esterase solution was carried out simultaneously as a control. Then, 100 μl of water and 50 μl of chloroform were added, and after thorough stirring with a vortex mixer, centrifugation was performed at 12,000 g for 5 minutes. 5 μl of the lower chloroform layer was loaded, and thin-layer chromatography analysis was performed using hexane-diethyl ether-acetic acid (80:20:1) as the developing solvent.

[0125] (Results) The results of Example 12 are shown in Fig. 11. Those of the representative sequence of the fifth esterase, unlike those of the representative sequence of the second esterase of the present disclosure that showed methyl esterification catalytic activity for palmitelaidic acid, did not show catalytic activity for the transesterification reaction of palmitelaidic acid. When vaccenic acid was used as the trans-fatty acid, those of the representative sequence of the fifth esterase showed catalytic activity for the transesterification reaction, although the catalytic activity was lower compared to those of the representative sequence of the second esterase of the present disclosure.

[0126] (Example 13: Preparation of modified esterases) In this example, modified esterases of the present disclosure are prepared. (Methods and materials) In the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21, constructs are prepared that include the insertion, substitution or deletion of one or more nucleotides, or those added to one or both ends thereof. The constructs are designed using the same method as in the above-described examples, or experimental techniques or commercially available kits commonly used in the art known to those skilled in the art. Alternatively, oligonucleotides encoding polypeptides in which the amino acids of the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22 are substituted are artificially synthesized. The decomposition activities of the various modified forms of fats, oils or esters thereof are analyzed by the same method as in the above-described examples. (Preparation of Expression Vector) The expression vector is prepared according to the method described in Example 3 above. That is, expression vectors containing mutants having various nucleotide sequences with mutations are prepared by the method of introducing the above mutations. (Thermal Stability, Optimal Temperature, Optimal pH and pH Stability) According to Example 4, the thermal stability, optimal temperature, optimal pH and pH stability of the prepared mutants are measured. (Ventilation Fan Filter Dirt Cleaning Effect and Decomposition of Ester Forms) Tests are performed according to Examples 5 and 6, respectively, and their decomposition activities are measured. (Substrate Specificity) Tests are performed according to Example 7, and substrate specificity is measured. (Results) As a result of the above tests, it can be seen that mutant strains having the activities found in the present disclosure (for example, the ability related to the assimilation of trans-fatty acid-containing fats and oils, or the ability to decompose trans-fatty acid-containing fats and oils, etc.) possessed by the polypeptides containing the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22 can be obtained.

[0127] (Note) As described above, the present invention has been exemplified using the preferred embodiments of the present invention. However, it is understood that the scope of the present invention should be construed only by the claims. It is understood that patents, patent applications, and other documents cited herein should be incorporated by reference into this specification as if the contents thereof were specifically set forth herein. This application claims priority to Japanese Patent Application No. 2019-163343 filed with the Japan Patent Office on September 6, 2019, and all of its contents should be incorporated by reference into this specification.

Industrial Applicability

[0128] The present disclosure is useful in decomposing trans-fatty acid-containing ester compounds and treating trans-fatty acid-containing wastewater, which is a problem in food factories and the like.

Deposit Number

[0129] NITE BP-02732

Sequence Listing Free-Text

[0130] SEQ ID NO: 1 Mature sequence of the representative nucleotide sequence of the first esterase of the present disclosure SEQ ID NO: 2 Mature sequence of the representative amino acid sequence of the first esterase of the present disclosure SEQ ID NO: 3 Nucleotide sequence containing nucleotides encoding the pre-sequence in the representative nucleotide sequence of the first esterase of the present disclosure SEQ ID NO: 4 Amino acid sequence containing the pre-sequence in the representative amino acid sequence of the first esterase of the present disclosure SEQ ID NO: 5 Full-length sequence of the representative nucleotide sequence of the first esterase of the present disclosure SEQ ID NO: 6 Full-length sequence of the representative amino acid sequence of the first esterase of the present disclosure SEQ ID NO: 7 Mature sequence of the representative nucleotide sequence of the second esterase of the present disclosure SEQ ID NO: 8 Mature sequence of the representative amino acid sequence of the second esterase of the present disclosure Full-length sequence of the representative base sequence of the second esterase of the present disclosure, SEQ ID NO: 9 Full-length sequence of the representative amino acid sequence of the second esterase of the present disclosure, SEQ ID NO: 10 Mature sequence of the representative base sequence of the third esterase of the present disclosure, SEQ ID NO: 11 Mature sequence of the representative amino acid sequence of the third esterase of the present disclosure, SEQ ID NO: 12 Full-length sequence of the representative base sequence of the third esterase of the present disclosure, SEQ ID NO: 13 Full-length sequence of the representative amino acid sequence of the third esterase of the present disclosure, SEQ ID NO: 14 Mature sequence of the representative base sequence of the fourth esterase of the present disclosure, SEQ ID NO: 15 Mature sequence of the representative amino acid sequence of the fourth esterase of the present disclosure, SEQ ID NO: 16 Full-length sequence of the representative base sequence of the fourth esterase of the present disclosure, SEQ ID NO: 17 Full-length sequence of the representative amino acid sequence of the fourth esterase of the present disclosure, SEQ ID NO: 18 Full-length sequence of the representative base sequence of the fifth esterase of the present disclosure, SEQ ID NO: 19 Full-length sequence of the representative amino acid sequence of the fifth esterase of the present disclosure, SEQ ID NO: 20 Mature sequence of the representative base sequence of the fifth esterase of the present disclosure, SEQ ID NO: 21 Mature sequence of the representative amino acid sequence of the fifth esterase of the present disclosure, SEQ ID NO: 22

Claims

【Claim 1】 The invention described in part of the specification of this application.

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