A novel esterase that degrades trans-fatty acid-containing esters

Abstract

The present disclosure provides a novel polypeptide for decomposing a trans fatty acid-containing ester. A novel esterase discovered by the inventors of the present invention exhibits activity for decomposing a trans fatty acid-containing ester. This esterase comprises a polypeptide comprising an amino acid sequence represented by SEQ ID NOS. 2, 8, 12, 16 or 22, or a polypeptide coded by a base sequence represented by SEQ ID NOS. 1, 7, 11, 15 or 21, or a polypeptide having a sequence homology of at least 70% with these sequences. This esterase was found to have the ability to decompose an ester at a low temperature and / or a high temperature, and to exhibit excellent thermal stability for ester decomposition at 65ºC. This esterase is useful in, for example, oil treatments such as waste water treatment, detergents, and techniques for modifying fats or for producing oils and fats.

Description

[Technical field]

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

[0002] Grease stains are a typical example of dirt that occurs in ordinary households, the food service industry, industrial facilities, etc. Grease stains are difficult to clean and occur in kitchens, sinks, cooking areas, pipes, drains, ventilation fans, laundry, etc. Grease stains can be a source of odors and pests, and can also cause environmental pollution, so the establishment of revolutionary technology for oil treatment is eagerly awaited from both public health and environmental perspectives.

[0003] Grease traps are treatment equipment that removes oil from kitchen wastewater in the restaurant industry by separating solids and liquids. However, they are a source of odors and pests, and the maintenance required for collecting, transporting, and cleaning the separated oil is laborious and costly. Considering this, the restaurant industry and other industrial sectors are eager to develop revolutionary technology that can eliminate oil inside grease traps. Summary of the Invention [Means for solving the problem]

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

[0005] Thus, the present disclosure provides: (1) (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 8, 12, or 16; (b) a polypeptide comprising the amino acid sequence of (a) containing one or more amino acid substitutions, additions, deletions, or combinations thereof, 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 an 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 nucleotide 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 having biological activity, which is encoded by a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences shown in (d) to (f) or a complementary sequence thereof; (h) a polypeptide encoded by an allelic variant of any one of the nucleotide sequences 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); A polypeptide comprising: (2) The polypeptide according to any one of the preceding items, wherein the polypeptide is an esterase. (2A) The polypeptide according to any one of the preceding items, wherein the polypeptide is a lipase. (2B) The polypeptide according to any one of the preceding items, wherein the esterase is a lipase. (3) The polypeptide according to any of the preceding items, wherein the biological activity includes an ability associated with the assimilation of trans fatty acid-containing ester bodies or an ability to decompose trans fatty acid-containing ester bodies. (3A) The polypeptide according to any one of the preceding items, wherein the biological activity includes an ability associated with the assimilation of medium-chain fatty acid-containing ester bodies or an ability to degrade medium-chain fatty acid-containing ester bodies. (4) The polypeptide according to any one of the preceding items, wherein the polypeptide is an esterase and has substrate specificity for short- to long-chain fatty acid-containing esters including fats and oils. (5) The polypeptide according to any one of the preceding items, which is an esterase having the ability to decompose esters at 15°C. (6) (A)(a) 75°C, (b) 65°C; (c) 67°C; or (d) 70°C and / or (B)(a) 60°C, (b) 40°C; or (c) 65℃ and / or (C)(a) pH 8–9, or (b) pH 9 and / or (D) having an optimum pH at pH 9; A polypeptide according to any of the above items. (7) The polypeptide according to any one of the preceding items, which is derived from Yarrowia lipolytica. (8) (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 nucleotide 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% 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 under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences shown in (A) to (C) or a complementary sequence thereof, and encoding a polypeptide having biological activity; (E) a polynucleotide which is an allelic variant of any one of the nucleotide sequences (A) to (D) and encodes a polypeptide having biological activity; (F) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 8, 12, or 16; (G) a polynucleotide encoding a polypeptide having biological activity, comprising an amino acid sequence of (F) containing one or more amino acid substitutions, additions, deletions, or combinations thereof; (H) a polynucleotide having at least 70% sequence identity to the amino acid sequence shown in (F) or (G) and encoding a biologically active polypeptide; or (I) A polynucleotide comprising a fragment of the nucleotide sequence shown in (F) to (H); A polynucleotide comprising: (9) The polynucleotide according to any one of the preceding items, wherein the polypeptide is an esterase. (9A) The polynucleotide according to any one of the preceding items, wherein the polypeptide is a lipase. (9B) The polynucleotide according to any one of the preceding items, wherein the esterase is a lipase. (10) The polynucleotide according to any of the preceding items, wherein the biological activity includes an ability associated with the assimilation of trans fatty acid-containing ester bodies or an ability to decompose trans fatty acid-containing ester bodies. (11) The polynucleotide according to any one of the preceding items, wherein the polypeptide is an esterase, and its substrate specificity is an ester of short- to long-chain fatty acids including fats and oils. (11A) The polynucleotide according to any one of the preceding items, wherein the polypeptide is a lipase. (11B) The polynucleotide according to any of the preceding items, wherein the biological activity includes an ability associated with the assimilation of trans fatty acid-containing ester bodies or an ability to degrade trans fatty acid-containing ester bodies. (11C) The polynucleotide according to any one of the preceding items, wherein the biological activity includes an ability associated with the assimilation of a medium-chain fatty acid-containing ester body or an ability to degrade a medium-chain fatty acid-containing ester body. (11D) The polynucleotide according to any one of the preceding items, wherein the polynucleotide encodes a polypeptide having the ability to decompose an ester at 15°C. (11E) (A) (a) 75°C, (b) 65°C; (c) 67°C; or (d) 70°C and / or (B) (a) 60°C, (b) 40°C; or (c) 65℃ and / or (C) (a) pH 8–9, or (b) pH 9 and / or (D) Encoding a polypeptide having a pH optimum at pH 9 A polynucleotide according to any of the above items. (11F) The polynucleotide according to any one of the preceding items, which is a polynucleotide derived from Yarrowia lipolytica. (12) (a) a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:22, which contains one or more amino acid substitutions, additions, deletions, or combinations thereof; (b) a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:22; (c) a polypeptide encoded by a nucleotide sequence containing one or more nucleotide 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 to 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 comprising 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 set forth in SEQ ID NO: 21; or (g) A polypeptide containing a fragment of the amino acid sequence shown in (a) to (f). a polypeptide having an ability to be involved in the assimilation of trans fatty acid-containing ester bodies or an ability to degrade trans fatty acid-containing ester bodies (optionally, the polypeptide is not a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:22). (12A) The polypeptide according to any one of the preceding items, further comprising an ability associated with the assimilation of medium-chain fatty acid-containing ester bodies or an ability to degrade medium-chain fatty acid-containing ester bodies. (12B) The polypeptide according to any one of the preceding items, whose substrate specificity includes esters and triglycerides containing short-chain to long-chain fatty acids. (13) A nucleotide sequence encoding the polypeptide according to the above item, or (A) a polynucleotide comprising a nucleotide sequence containing one or more nucleotide substitutions, additions, deletions or a combination thereof in the nucleotide sequence shown in SEQ ID NO:21, and encoding a polypeptide having biological activity; (B) a polynucleotide comprising a nucleotide sequence having at least 70% sequence identity to 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 to a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 21 or a complementary sequence thereof, and encoding a polypeptide having biological activity; (D) a polynucleotide encoding a polypeptide having biological activity, which is an allelic variant of the nucleotide sequence set forth in SEQ ID NO: 21; (E) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:22; (F) a polynucleotide encoding a polypeptide having biological activity, comprising an amino acid sequence containing one or more amino acid substitutions, additions, deletions, or combinations thereof in the amino acid sequence shown in SEQ ID NO:22; (G) a polynucleotide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22 and encoding a polypeptide having biological activity; or (H) A polynucleotide comprising a fragment of the nucleotide sequence shown in (E) to (G). The biological activity of the polynucleotide comprises an ability to associate with the assimilation of trans-fatty acid-containing esters or an ability to degrade trans-fatty acid-containing esters. (Optionally, the polynucleotide is not a polynucleotide consisting of the base sequence shown in SEQ ID NO:21). (14) A cell or a cell-free expression system comprising the polynucleotide according to any one of the preceding items. (15) A cell or cell-free expression system comprising a polypeptide described in any of the above items, a polypeptide comprising the amino acid sequence described in SEQ ID NO: 22, a polynucleotide described in any of the above items, or a polynucleotide comprising a base sequence encoding the amino acid sequence described in SEQ ID NO: 22 or the base sequence described in SEQ ID NO: 21, or an oil decomposing agent comprising a cell or cell-free expression system described in any of the above items. (16) The oil decomposing agent according to any one of the above items, wherein the oil contains a trans fatty acid-containing ester. (16A) The oil decomposing agent according to any one of the above items, wherein the oil contains a medium-chain fatty acid-containing ester. (17) An oil decomposing agent according to any of the above items, which contains a further oil treating component. (18) A kit for oil decomposition comprising a polypeptide described in any of the above items, a polypeptide comprising the amino acid sequence described in SEQ ID NO: 22, a polynucleotide described in any of the above items, or a cell or cell-free expression system comprising a base sequence encoding the amino acid sequence described in SEQ ID NO: 22 or a polynucleotide comprising the base sequence described in SEQ ID NO: 21, a cell or cell-free expression system described in any of the above items, or an oil decomposition agent described in any of the above items, and an additional oil treatment component. (19) A method for decomposing and removing oil, comprising applying to a subject to be treated a polypeptide described in any of the above items, a polypeptide comprising the amino acid sequence described in SEQ ID NO: 22, a polynucleotide described in any of the above items, or a cell or cell-free expression system comprising a base sequence encoding the amino acid sequence described in SEQ ID NO: 22 or a polynucleotide comprising the base sequence described in SEQ ID NO: 21, a cell or cell-free expression system described in any of the above items, or an oil decomposing agent described in any of the above items. (20) The method according to any one of the preceding items, wherein the object to be treated contains a trans fatty acid or a trans fatty acid-containing ester. (21) A detergent comprising a cell or a cell-free expression system comprising any one of the above-mentioned polypeptides or any one of the above-mentioned polynucleotides. (22) Use of a polypeptide described in any of the above items, a polypeptide comprising the amino acid sequence described in SEQ ID NO: 22, a polynucleotide described in any of the above items, or a cell or cell-free expression system comprising a base sequence encoding the amino acid sequence described in SEQ ID NO: 22 or a polynucleotide comprising the base sequence described in SEQ ID NO: 21, or a cell or cell-free expression system described in any of the above items, or an oil-decomposing agent described in any of the above items, in fat modification and oil production technology.

[0006] It is contemplated that the present disclosure may provide one or more of the above features in combinations other than those specifically described. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. Effect of the Invention

[0007] By using the present disclosure, it has become easier to treat trans-fatty acid-containing oils and fats, which have been difficult to remove in the past. In addition, it has become possible to remove oils and fats and treat oils even in high-temperature or low-temperature environments, where it was difficult to use conventional microorganisms or enzymes. Therefore, the novel esterase (e.g., lipase) of the present disclosure is useful in technical fields such as detergents, leather industry, food industry, purification of environmental pollution caused by oils and fats, waste treatment and composting such as food waste treatment, composting treatment, and wastewater treatment, pharmaceuticals such as digestive agents, and cosmetics for oily skin. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows the gene numbers, predicted functions, the presence or absence of a signal peptide (SP), and predicted molecular weights of the gene products of the genes encoding the first, second, third, fourth, and fifth esterases of Yarrowia lipolytica strain KH-2. [Diagram 2] Figure 2 is a graph showing the expression levels of five types of esterases (representative sequences of esterases 1, 2, 3, 4 and 5) when the KH-2 strain was cultured in a fermenter at 15°C. The expression levels are shown as relative values ​​normalized by the expression amount of the alg9 (1,2-mannosyltransferase) gene. The horizontal axis of the graph corresponds to the cultures after 24 hours, 48 ​​hours and 72 hours of culture, from the left. The vertical axis of the graph shows the relative expression levels. [Figure 3-1]FIG. 3-1 shows the optimum temperature, thermostability, optimum pH and pH stability of recombinant proteins of representative sequences of the first and second esterases derived from the KH-2 strain. (A) to (D) in the upper row show the results of the representative sequence of the first esterase, and (E) to (H) in the lower row show the results of the representative sequence of the second esterase. (A) and (E) show the optimum temperature of the esterase, (B) and (F) show the thermostability of the esterase, (C) and (G) show the optimum pH of the esterase, and (D) and (H) show the pH stability of the esterase. The vertical axis of (A) to (H) shows the relative esterase activity to the activity at the maximum esterase activity, the horizontal axis of (A), (B), (E) and (F) shows the temperature, and the horizontal axis of (C), (D), (G) and (H) shows the pH. [Figure 3-2] FIG. 3-2 shows the optimum temperature, thermostability, optimum pH and pH stability of recombinant proteins of representative sequences of the third and fourth esterases derived from the KH-2 strain. (A) to (D) in the upper row show the results of the representative sequence of the third esterase, and (E) to (H) in the lower row show the results of the representative sequence of the fourth esterase. (A) and (E) show the optimum temperature of the esterase, (B) and (F) show the thermostability of the esterase, (C) and (G) show the optimum pH of the esterase, and (D) and (H) show the pH stability of the esterase. The vertical axis of (A) to (H) shows the relative esterase activity to the activity at the maximum esterase activity, the horizontal axis of (A), (B), (E) and (F) shows the temperature, and the horizontal axis of (C), (D), (G) and (H) shows the pH. [Figure 4]FIG. 4 shows a comparison of Novozyme lipase (Novozym® 51032) with the esterases of the present disclosure. The photographs show a greasy ventilation filter that was soaked in Novozym 51032 (N-51032), a purified esterase obtained from an E. coli strain expressing recombinant proteins of representative sequences of the first, second, third, fourth and fifth esterases of the present disclosure. The photographs show the esterase solutions and the ventilation filter after soaking in A: Novozym 51032, B: first esterase, C: second esterase, D: third esterase, E: fourth esterase, and F: fifth esterase. [Figure 5-1]Figure 5-1 shows the decomposition activity of ester bodies by the first, second and fifth esterases of the present disclosure. (A) Buffer alone (Buffer), purified esterase obtained from an E. coli strain expressing a recombinant protein having a representative sequence of the fifth esterase (fifth esterase), purified esterase obtained from an E. coli strain expressing a recombinant protein having a representative sequence of the first esterase (first esterase), or purified esterase obtained from an E. coli strain expressing a recombinant protein having a representative sequence of the second esterase (second esterase) was incubated with 0.7 g of shortening at 28°C for 24 hours. Images of the oil after incubation are shown. (B) Buffer alone (Buffer), purified esterase obtained from an E. coli strain expressing a recombinant protein having a representative sequence of a fifth esterase (Esterase No. 5), purified esterase obtained from an E. coli strain expressing a recombinant protein having a representative sequence of a first esterase (Esterase No. 1), or purified esterase obtained from an E. coli strain expressing a recombinant protein having a representative sequence of a second esterase (Esterase No. 2) was incubated with 0.5 g of lard for 24 hours at 28° C. Images of the oil after incubation are shown. (C) Buffer alone (Buffer), purified esterase obtained from an E. coli strain expressing a recombinant protein of a representative sequence of the fifth esterase (fifth esterase), purified esterase obtained from an E. coli strain expressing a recombinant protein of a representative sequence of the first esterase (first esterase), or purified esterase obtained from an E. coli strain expressing a recombinant protein of a representative sequence of the second esterase (second esterase) were incubated with triolein (Tole), trielaidin (TED), shortening, or lard, respectively, and then separated by thin-layer chromatography and detected with molybdophosphate n-hydrate. From the left, those incubated with triolein, trielaidin, shortening, and lard are shown. [Figure 5-2]FIG. 5-2 shows the decomposition activity of ester bodies by the third and fourth esterases of the present disclosure. (A) Buffer alone (Buffer), purified esterase (third esterase) obtained from an E. coli strain expressing a recombinant protein having a representative sequence of the third esterase, or purified esterase (fourth esterase) obtained from an E. coli strain expressing a recombinant protein having a representative sequence of the fourth esterase, was incubated with 0.7 g of shortening at 28° C. for 24 hours. Images of the oil and fat after incubation are shown. (B) Buffer alone (Buffer), purified esterase (third esterase) obtained from an E. coli strain expressing a recombinant protein having a representative sequence of the third esterase, or purified esterase (fourth esterase) obtained from an E. coli strain expressing a recombinant protein having a representative sequence of the fourth esterase, was incubated with 0.5 g of lard at 28° C. for 24 hours. Images of the oil and fat after incubation are shown. (C) The results are shown for buffer alone (Buffer), purified esterase (third esterase) obtained from an E. coli strain expressing a recombinant protein of a representative sequence of a third esterase, or purified esterase (fourth esterase) obtained from an E. coli strain expressing a recombinant protein of a representative sequence of a fourth esterase, incubated with triolein (Tole), trielaidin (TED), shortening, or lard, respectively, and then separated by thin-layer chromatography and detected with molybdophosphate n-hydrate. From the left, the results are shown for incubation with triolein, trielaidin, shortening, and lard. [Figure 6] FIG. 6 shows the substrate specificity of representative esterases No. 1, No. 2, No. 3, No. 4 and No. 5. The left side of the table shows the substrates used, which correspond to 4-nitrophenyl ester (pNP-ester) lipids having acetate (C2), butyrate (C4), octanoate (C8), laurate (C12) and palmitate (C16) from top to bottom. The right side of the table shows the esterase activity of esterases No. 1, No. 2, No. 3, No. 4 and No. 5, and is shown as a relative activity with the maximum activity of each substrate taken as 100%. [Figure 7] 7 shows the degradation of trans-fatty acid-containing esters by purified esterases obtained from Escherichia coli strains expressing recombinant proteins of the first, second, third, fourth and fifth representative sequences of the disclosed esterases, which were serially diluted. The data shows the results for the esterases serially diluted to 0.01 u / ml, 0.001 u / ml and 0.0001 u / ml, from the left, and each data corresponds to the results for the first, second, third, fourth and fifth representative sequences of the disclosed esterases, from the left. [Figure 8] 8 shows the degradation of trans-fatty acid-containing esters at low temperatures by purified esterases obtained from Escherichia coli strains expressing recombinant proteins of the first, second, third and fourth representative sequences of the disclosed esterases. From the left, the bands correspond to the buffer alone (Buffer), the first, second, third and fourth representative sequences of the esterases, and the arrows at the bottom correspond to the bands of the hydrolysis products (free fatty acids). [Figure 9] 9 shows the modification of triolein by purified esterases obtained from E. coli strains expressing recombinant proteins of the first, second, third and fourth representative sequences of esterases of the present disclosure. The bands shown from the left to the right are the results of thin-layer chromatography analysis of the first, second, third and fourth representative sequences of esterases, or a mixture of methanol and triolein with a buffer, and a methyl oleate sample. The signals from the top to the bottom correspond to methyl oleate, triolein, oleic acid and diacylglycerol. [Figure 10]10 shows the modification of trans fatty acids by purified esterases obtained from E. coli strains expressing recombinant proteins of the first, second, third and fourth representative sequences of esterases of the present disclosure. (A) The results of thin-layer chromatographic analysis of the first, second, third and fourth representative sequences of esterases, or buffer alone (Buffer) mixed with methanol and palmitelaidic acid and reacted, and palmitelaidic acid methyl sample are shown. The signals correspond, from top to bottom, to palmitelaidic acid methyl ester and palmitelaidic acid. (B) The results of thin-layer chromatographic analysis of the first, second, third and fourth representative sequences of esterases, or buffer alone (Buffer) mixed with methanol and vaccenic acid and reacted, and vaccenic acid methyl sample are shown. The signals correspond, from top to bottom, to vaccenic acid methyl ester and vaccenic acid. [Figure 11] 11 shows the modification of trans fatty acids by purified esterases obtained from E. coli strains expressing recombinant proteins of the representative sequences of the second and fifth esterases of the present disclosure. (A) The results of thin-layer chromatographic analysis of the representative sequences of the second and fifth esterases, or buffer alone (Buffer) mixed with methanol and palmitelaidic acid and reacted, and methyl palmitelaidic acid preparations are shown. The signals correspond, from top to bottom, to palmitelaidic acid methyl ester and palmitelaidic acid. (B) The results of thin-layer chromatographic analysis of the representative sequences of the second and fifth esterases, or buffer alone (Buffer) mixed with methanol and vaccenic acid and reacted, and methyl vaccenic acid preparations are shown. The signals correspond, from top to bottom, to vaccenic acid methyl ester and vaccenic acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present disclosure will be described below with reference to the best mode. Throughout this specification, the singular expression should be understood to include the concept of the plural form, unless otherwise specified. Therefore, the singular article (e.g., in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In case of conflict, this specification (including definitions) shall prevail.

[0010] The definitions of terms particularly used in this specification and / or basic technical contents are appropriately explained below. In this specification, the name of a strain such as KH-2 strain may be omitted from the term "strain", but those skilled in the art will appropriately understand that the term "strain" is used depending on the context.

[0011] (Definition of terms) As used herein, "esterase" refers to a hydrolase that decomposes esters into acids and alcohols through a chemical reaction with water. The esterase of the present disclosure is classified as an esterase because it has the activity of hydrolyzing esters such as fatty acid esters. The esterase activity of the esterase can be measured by the method described in the "Ability to decompose esters" section of the present specification.

[0012] As used herein, the term "lipase" refers to a type of esterase, an enzyme that reversibly catalyzes the reaction of hydrolyzing neutral fats (glycerol esters) and decomposing them into fatty acids and glycerol. The esterase of the present disclosure has the activity of an enzyme classified as triglycerol lipase, which is classified as EC3.1.1.3 in terms of enzyme number (EC number), and therefore can also be called a "lipase," in which case it is also called the lipase of the present disclosure.

[0013] In this specification, "oils and fats" refers to oily substances, and oils and fats include ester group-containing compounds formed by dehydration condensation of a hydroxyl group-containing compound and a fatty acid. A typical example of this hydroxyl group-containing compound is glycerin, but other examples include polyglycerin. In this specification, an ester group-containing compound formed by dehydration condensation of glycerin and a fatty acid is called a "glyceride" in the same sense as in the ordinary use in this technical field. When this hydroxyl group-containing compound has multiple hydroxyl groups, if at least one of the hydroxyl groups is dehydration condensed with a fatty acid to form an ester, it corresponds to the ester group-containing compound in this specification.

[0014] In this specification, lipase activity refers to the activity of hydrolyzing esters produced by dehydration condensation of glycerol and fatty acids into glycerol and free fatty acids, and such lipase activity is also referred to as triglyceride lipase activity in this specification.For example, whether or not lipase activity is present can be confirmed by the reduction of esters (animal and vegetable oils such as canola oil, triolein, trielaidin, etc.) contained in a solution to which lipase is added, or the detection of hydrolysis products.

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

[0016] In this specification, "fat modification and oil production ability" refers to changing the structure of fatty acids to change the properties of oil. In this specification, "fat modification and oil production ability" typically includes the activity of catalyzing fatty acid transesterification. Fat modification and oil production ability can be measured by analyzing fatty acid methyl esters produced by transesterification of fatty acids and methanol by thin layer chromatography. A specific quantitative procedure is as follows: First, the esterase of the present disclosure, fatty acids, and methanol are mixed to produce fatty acid methyl esters. Esters and fatty acids are extracted by adding chloroform to the mixture of fatty acid methyl esters. 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 can be changed as appropriate. The plate is treated with molybdic acid n-hydrate to color the esters.

[0017] In this specification, the term "trans fatty acid-containing ester" refers to an ester containing trans fatty acid. "Trans fatty acid" is used in the sense commonly used in this technical field, and refers to an unsaturated fatty acid having a trans-type double bond. Trans fatty acids exist in small amounts in nature as conjugated linoleic acid and vaccenic acid, but in the oil and fat industry, they are produced in large quantities when producing saturated fatty acids by hydrogenating unsaturated fatty acids, and are also contained in foods such as margarine and shortening. Trans fatty acids include elaidic acid, palmitellidic acid (palmitoelaidic acid), vaccenic acid, etc., but there is no particular limit to the type of trans fatty acid referred to in this specification. The ratio of trans fatty acid present in the trans fatty acid-containing ester is not particularly limited. Among the conventionally known Yarrowia lipases, there are no known ones that have the activity of decomposing trans fatty acid-containing esters.

[0018] In this specification, "short-long chain fatty acid-containing ester" refers to an ester 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 acid", "medium chain fatty acid" and "long chain fatty acid" are used in the sense usually used in this 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 the present specification, the term "medium-chain fatty acid-containing ester" refers to an ester containing medium-chain fatty acids. "Medium-chain fatty acid" is used in the sense commonly used in this technical field, and means a fatty acid 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 (carbon number 8), capric acid (carbon number 10), lauric acid (carbon number 12), etc., but when referred to in the present specification, there is no particular limitation on the type of medium-chain fatty acid. There is no particular limitation on the ratio of medium-chain fatty acids present in the medium-chain fatty acid-containing ester.

[0020] In this specification, "ability to assimilate trans fatty acid-containing ester bodies" or "ability to assimilate short- to long-chain fatty acid-containing ester bodies" refers to activity for assimilating trans fatty acid-containing ester bodies or short- to long-chain fatty acid-containing ester bodies by microorganisms. In this specification, "assimilate trans fatty acid-containing ester bodies" or "assimilate short- to long-chain fatty acid-containing ester bodies" are used in the sense commonly used in this technical field, and mean that microorganisms take in trans fatty acid-containing ester bodies or short- to long-chain fatty acid-containing ester bodies as a nutrient source such as a carbon source. When "assimilates", it includes not only hydrolysis into alcohols and free fatty acids, but also conversion into part of other substances.

[0021] In this specification, the term "ability to decompose trans fatty acid-containing esters" or "ability to decompose short-chain to long-chain fatty acid-containing esters" refers to the activity of hydrolyzing trans fatty acid-containing esters or short-chain to long-chain fatty acid-containing esters into glycerol or other alcohols (such as 4-nitrophenol) and free fatty acids.

[0022] In this specification, the "ability to decompose esters" (at each temperature) refers to the activity of hydrolyzing esters into alcohols and free fatty acids at each temperature. In this specification, the "ability to decompose esters" (at each temperature) is measured as follows. That is, an esterase is purified by the method described in this specification, and an ester substrate of 4-nitrophenol and a fatty acid (e.g., palmitic acid, butyric acid (butyrate)) is mixed with the esterase at a constant temperature set 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] In this specification, the "optimum temperature" of the ability to decompose esters is used in the sense commonly used in the technical field, and refers to a temperature range in which the activity of decomposing esters is at a certain level or higher than the desired level (for example, the temperature range in which 80% or more of the maximum level of the enzyme's activity is maintained, but in another embodiment, it may refer to the maximum level). Specifically, the first esterase of the present disclosure has a peak of activity for decomposing esters at about 60°C, and maintains 80% or more of the peak activity in the range of about 55 to 65°C. The second esterase of the present disclosure has a peak of activity for decomposing esters at about 40°C, and maintains 80% or more of the peak activity in the range of about 35 to 50°C. The third esterase of the present disclosure has a peak of activity for decomposing esters at about 65°C, and maintains 80% or more of the peak activity in the range of about 53 to 68°C. The fourth esterase of the present disclosure has a peak of activity for decomposing esters at about 65°C, and maintains 80% or more of the peak activity in the range of about 55 to 68°C. The fifth esterase of the present disclosure has a peak activity of decomposing ester bodies at about 40°C, and retains 80% or more of the peak activity in the range of about 33 to 47°C. In the context of referring to the optimum temperature of the esterase 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, and more preferably about 60°C. The optimum temperature of the second esterase of the present disclosure is intended to be about 35 to 50°C, preferably about 35 to 45°C, and more preferably about 40°C. The optimum temperature of the third esterase of the present disclosure is intended to be about 53 to 68°C, preferably about 60 to 67°C, and more preferably about 65°C. The optimum temperature of the fourth esterase of the present disclosure is intended to be about 55 to 68°C, preferably about 60 to 67°C, and more preferably about 65°C. The optimum temperature for the fifth esterase of the present disclosure is intended to be about 33 to 47°C, preferably about 37 to 45°C, and more preferably about 40°C.

[0024] In this specification, the term "thermostable" has the same meaning as terms such as "thermostable" and "thermostable", and is used in the sense commonly used in the technical field, and means that an enzyme retains activity at low and / or high temperatures. In general, it is known that enzymes are inactivated at a temperature of about 50°C due to factors such as changes in molecular structure. In contrast, in the case of the first esterase of the present disclosure, the relative enzyme activity when the enzyme activity after 30 minutes of treatment at 30°C is taken as 100%, is retained at 80% or more after 30 minutes of treatment at a temperature range of about 10 to about 60°C, and in the case of the second esterase of the present disclosure, the relative enzyme activity when the enzyme activity after 30 minutes of treatment at 45°C is taken as 100%, is retained at 80% or more after 30 minutes of treatment at a temperature range of about 15 to about 60°C. In the case of the third esterase of the present disclosure, the relative enzyme activity when the enzyme activity after 30 minutes of treatment at 30°C is taken as 100%, is retained at 80% or more after 30 minutes of treatment at a temperature range of about 30 to about 65°C. In the case of the fourth esterase of the present disclosure, the relative enzyme activity, when the enzyme activity after 30 minutes of treatment at 30° C. is taken as 100%, is maintained at 80% or more after 30 minutes of treatment at a temperature range of about 30 to about 45° C. In the case of the fifth esterase of the present disclosure, the relative enzyme activity, when the enzyme activity after 30 minutes of treatment at 40° C. is taken as 100%, is maintained at 80% or more after 30 minutes of treatment at a temperature range of about 20 to about 45° C. In this specification, expressions such as "maintaining thermostability" mean that the relative enzyme activity, when the enzyme activity after 30 minutes of treatment at about 30° C. for the first esterase, about 45° C. for the second esterase, 30° C. for the third esterase, 30° C. for the fourth esterase, and 40° C. for the fifth esterase, is maintained at about 50% or more.

[0025] In the present specification, the "optimum pH" of the ability to degrade esters is used in the sense commonly used in the technical field, and refers to the pH at which the activity of degrading esters is at a certain level or higher (for example, the pH range in which 80% or more of the maximum level of the enzyme's activity 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 for degrading esters at about pH 9, and maintain 80% or more of the peak activity in the range of about pH 8.5 to pH 9.5. The fifth esterase has a peak activity for degrading esters at about pH 8, and maintain 80% or more of the peak activity 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 (all 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, and more preferably pH 9. In the context of referring to the pH optimum of the fifth esterase, it is intended that the pH optimum of the fifth esterase of the present disclosure is between pH 7.5 and pH 8.5, preferably between pH 7.8 and pH 8.3, and more preferably pH 8.

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

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

[0028] As used herein, the term "cell-free expression system" is used in the sense commonly used in the art, and refers to a system for producing a recombinant protein of interest in vitro using a transcription and translation mechanism of a biomolecule extracted from a cell. The cell-free expression system for producing the esterase of the present disclosure is not particularly limited, but may be a cell-free expression system derived from a prokaryote such as Escherichia coli.

[0029] In this specification, the term "oil treatment component" refers to a component that assists the assimilation and decomposition of esters. Specifically, it includes components that promote the dispersion of esters, such as surfactants, components that decompose esters 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 of treatment.

[0030] In this specification, the term "oil decomposer" refers to a preparation capable of decomposing ester bodies, which contains as an active ingredient the Yarrowia 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 this disclosure, the oil decomposer may be used in combination with an oil treatment component. In this case, the timing of the combined use of the oil decomposer and the oil treatment component may be simultaneous or one of them may be used first. Furthermore, the oil decomposer may further contain a component (e.g., carbon source, nitrogen source) that enhances the activity of the microbial strain or the esterase derived from the microbial strain used, a surfactant, a drying protection agent, a component for maintaining the microorganism for a long period of time, a preservative, an excipient, a strengthening agent, an antioxidant, etc.

[0031] The oil decomposing agent provided in the present disclosure is provided in a liquid, solid or dried form. Examples of liquid forms include a culture solution of a microorganism (which may be concentrated or diluted as necessary), an enzyme component derived from the culture solution adsorbed on a support, and an enzyme separated and purified from the culture solution and dissolved in a buffer solution or solvent. Examples of solid forms include a suspension or solution in a solvent containing a protective agent such as glycerol and frozen, an immobilized on a carrier (adsorbed on a carrier by covalent bonding, electrostatic interaction, hydrophobic interaction, etc., molecular cross-linked on a carrier, etc.), a dehydrated product by centrifugation, press compression, etc., and a dried product. In a preferred embodiment, the oil decomposing agent is provided in a liquid form, a powder, or a granular 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 include, but are not limited to, molecules that contain a region substantially homologous to the protein of interest (e.g., esterases), which in various embodiments are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical over the same size of the amino acid sequence or when compared to sequences aligned by computer homology programs known in the art, or the nucleic acid encoding such a molecule is hybridizable to the sequence encoding the component protein under (highly) stringent, moderately stringent or non-stringent conditions. This refers to proteins that are products of protein modification by amino acid substitution, deletion and addition, respectively, and whose derivatives still exhibit the biological function of the original protein, although not necessarily to the same degree, and preferably have the same or greater biological activity. For example, the biological function of such proteins can be examined by suitable and available in vitro assays described herein or known in the art. As used herein, "functionally active" or "having functional activity" refers herein to a polypeptide of the present disclosure, i.e., a fragment or derivative, having a structural, regulatory, or biochemical function of a protein, such as biological activity, according to the relevant aspect.

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

[0034] As used herein, "protein," "polypeptide," "oligopeptide," and "peptide" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified. The term may also include those assembled into a complex of multiple polypeptide chains. The term also includes naturally or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeling moiety). The definition also includes, for example, polypeptides containing one or more analogs of amino acids (e.g., including non-natural amino acids), peptide-like compounds (e.g., peptoids), and other modifications known in the art. As used herein, "amino acid" is a general term for an organic compound having an amino group and a carboxyl group. When a protein or enzyme according to an embodiment of the present disclosure includes a "specific amino acid sequence," any amino acid in the amino acid sequence may be chemically modified. Any of the amino acids in the amino acid sequence may form a salt or a solvate. Any of the amino acids in the amino acid sequence may be L-type or D-type. In such cases, the protein according to the embodiment of the present disclosure may contain the above-mentioned "specific amino acid sequence". Examples of chemical modifications that amino acids contained in proteins undergo in vivo include N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), and side chain modifications (e.g., phosphorylation, sugar chain addition, etc.). The amino acids may be natural or unnatural as long as they satisfy the purpose of the present disclosure.

[0035] As used herein, "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of nucleotides of any length. This term also includes "oligonucleotide derivative" or "polynucleotide derivative". "Base sequence" or "nucleic acid sequence" refers to the order of consecutive nucleic acid bases in a "polynucleotide", "oligonucleotide" or "nucleic acid". "Oligonucleotide derivative" or "polynucleotide derivative" refers to an oligonucleotide or polynucleotide that contains a derivative of nucleotides or has a bond between nucleotides that is different from the usual one, and are used interchangeably. Specific examples of such oligonucleotides include 2'-O-methyl-ribonucleotides, oligonucleotide derivatives in which the phosphodiester bond in an oligonucleotide has been converted to a phosphorothioate bond, oligonucleotide derivatives in which the phosphodiester bond in an oligonucleotide has been converted to an N3'-P5' phosphoramidate bond, oligonucleotide derivatives in which the ribose and phosphodiester bond in an oligonucleotide have been converted to a peptide nucleic acid bond, oligonucleotide derivatives in which the uracil in an oligonucleotide has been replaced with C-5 propynyl uracil, oligonucleotide derivatives in which the uracil in an oligonucleotide has been replaced with C-5 thiazole uracil, oligonucleotide derivatives in which the cytosine in an oligonucleotide has been replaced with C-5 propynyl cytosine, oligonucleotide derivatives in which the cytosine in an oligonucleotide has been replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in DNA has been replaced with 2'-O-propyl ribose, and oligonucleotide derivatives in which the ribose in an oligonucleotide has been replaced with 2'-methoxyethoxy ribose. Unless otherwise indicated, a particular base sequence is also intended to encompass conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (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, the term "gene" refers to a factor that determines a genetic trait, and may refer to a "polynucleotide," an "oligonucleotide," and a "nucleic acid."

[0037] As used herein, the term "homology" of a gene refers to the degree of identity between two or more gene sequences, and generally, "homology" refers to a high degree of identity or similarity. Thus, the higher the homology between two genes, the higher the identity or similarity between their sequences. Whether two genes have homology can be determined by direct comparison of the sequences, or, in the case of nucleic acids, by hybridization methods under stringent conditions. When two gene sequences are compared directly, the genes have homology if the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical. Thus, as used herein, a "homolog" or "homologous gene product" refers to a protein in another species, preferably a microorganism, more preferably a yeast, that exerts the same biological function as a protein component of a complex as further described herein. Such a homolog may also be referred to as an "orthologous gene product." 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. In this specification, the "similarity" of a gene or base sequence refers to the degree of similarity between two or more gene sequences, and refers to a high degree of similarity between other sequences of identity. "Similarity" is a numerical value that takes into account not only identity but also similar bases, where similar bases refer to cases where a portion of mixed 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) match.

[0038] Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one-letter codes. In this specification, comparisons of amino acid and nucleotide sequence similarity, identity, and homology are calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST2.7.1 (published 2017.10.19). The value of "identity" in this specification usually refers to the value when aligned under default conditions using the above BLAST. However, if a higher value is obtained by changing the parameters, the highest value is taken as the identity value. If identity is evaluated in multiple regions, the highest value among them is taken as the identity value. "Similarity" is a 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, or any of these values ​​or less. It is known that a polypeptide that has one or several amino acid residues deleted, added, inserted, or substituted with other amino acids 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. For site-directed mutagenesis, for example, KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.) can be used. Proteins with the same activity as the wild type can be selected from mutant proteins into which deletions or other modifications have been introduced by performing various characterizations such as FACS analysis and ELISA.

[0040] In one embodiment of the present disclosure, the numerical value of identity, etc., "70% or more" 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 of the starting numerical values. The above-mentioned "identity" is calculated by calculating the ratio of the number of homologous amino acids in two or more amino acid sequences according to a known method as described above. Specifically, before calculating the ratio, the amino acid sequences of the amino acid sequences 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 the ratio, methods for comparison, and computer programs related thereto are conventionally well known in the art (for example, BLAST, etc., as described above). In this specification, "identity" and "similarity" can be expressed as values ​​measured by BLAST of NCBI, unless otherwise specified. The algorithm for comparing amino acid sequences with BLAST can be Blastp with default settings. The measurement results are quantified as Positives or Identities. In this case, when "similarity" is used instead of "identity," it is a numerical value that takes into account those that fall under 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 colony hybridization, plaque hybridization, Southern blot hybridization, or the like, using a polynucleotide selected from the polynucleotides disclosed herein as a probe. Specifically, the term refers to a polynucleotide that can be identified by hybridizing a filter on which DNA derived from colonies or plaques is immobilized in the presence of 0.7 to 1.0 M NaCl at 65°C, and then washing the filter at 65°C using a 0.1 to 2-fold concentration of SSC (saline-sodium citrate) solution (the composition of a 1-fold concentration SSC solution is 150 mM sodium chloride and 15 mM sodium citrate). For example, the following conditions can be adopted as the "stringent conditions." (1) using low ionic strength and high temperature for washing (e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C), (2) using a denaturing agent such as formamide during hybridization (e.g., 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer pH 6.5, with 750 mM sodium chloride, 75 mM sodium citrate at 42°C), or (3) incubating overnight at 37°C in a solution containing 20% ​​formamide, 5x SSC, 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured sheared salmon sperm DNA, followed by washing the filters with 1x SSC at approximately 37-50°C. The formamide concentration may be 50% or more, and the washing time may be 5, 15, 30, 60, or 120 minutes or more.Factors that affect the stringency of the hybridization reaction include temperature, salt concentration, and more. For details, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). Examples of "highly stringent conditions" are 0.0015M sodium chloride, 0.0015M sodium citrate, 65-68°C, or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide, 42°C. Hybridization can be performed according to the method described in experimental books 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 that contain only A or T sequences. Moderately stringent conditions can be readily determined by one of skill in the art based on, for example, the length of the DNA, and are set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, No. 3, Vol. 1, 7.42-7.45 Cold Spring Harbor Laboratory Press, 2001, and include the use of, for nitrocellulose filters, a pre-wash solution of 5xSSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50% formamide, 2xSSC-6xSSC (or other similar hybridization solutions, such as Stark's solution in about 50% formamide at about 42°C) at about 40-50°C, and wash conditions of 0.5xSSC, 0.1% SDS at about 60°C.Thus, a polypeptide as used in this disclosure also includes a polypeptide encoded by a nucleic acid molecule that hybridizes under high or moderate stringency conditions to a nucleic acid molecule encoding a polypeptide specifically described in this disclosure.

[0042] The esterase of the present disclosure may preferably be "purified" or "isolated". As used herein, a "purified" substance or biological factor (e.g., nucleic acid or protein, etc.) refers to a substance or biological factor from which at least a portion of naturally associated factors have been removed. Thus, the purity of the biological factor in a purified biological factor is usually higher (i.e., concentrated) than the state in which the biological factor is normally present. As used herein, the term "purified" means that the same type of biological factor is present in an amount of preferably 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. The substance or biological factor used in the present disclosure is preferably a "purified" substance. As used herein, an "isolated" substance or biological factor (e.g., nucleic acid or protein, etc.) refers to a substance or biological factor from which naturally associated factors have been substantially removed. The term "isolated" as used herein does not necessarily denote purity, as this can vary depending on the purpose, but, if necessary, preferably means that 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 factor is present. The materials used in this disclosure are preferably "isolated" materials or biological factors.

[0043] As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 relative to the full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed depending on the purpose. For example, the lower limit of the length of a polypeptide can be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more amino acids, and a length represented by an integer not specifically listed here (e.g., 11, etc.) can also be a suitable lower limit. In addition, in the case of a polynucleotide, the lower limit can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, or more nucleotides, and a length represented by an integer not specifically listed here (e.g., 11, etc.) can also be a suitable lower limit. In the present specification, such a fragment is understood to be within the scope of the present disclosure, for example, if the full-length fragment functions as an ester decomposition molecule, as long as the fragment itself also functions as an ester decomposition molecule.

[0044] As used herein, the term "biological function" refers to a specific function that a gene, a nucleic acid molecule, or a polypeptide related thereto may have in vivo or in vitro, and includes, but is not limited to, the degradation of esters (e.g., the degradation of trans-fatty acid-containing esters). In the present disclosure, examples of the biological function include, but are not limited to, the degradation of medium-chain fatty acid-containing esters, the degradation of cis-fatty acid-containing esters, and the degradation of saturated fatty acid-containing esters not containing double bonds. In the present specification, a biological function may be exerted by a corresponding "biological activity". As used herein, "biological activity" refers to an activity that a certain factor (e.g., polynucleotide, protein, etc.) may have, and includes an activity that exerts various functions (e.g., the decomposition activity of trans-fatty acid-containing esters). 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 certain factor is an enzyme, its biological activity includes its enzymatic activity. Such biological activity can be measured by a technique well known in the art. Thus, "activity" refers to various measurable indicators that indicate or reveal binding (either directly or indirectly); affect a response (i.e., have a measurable effect in response to some exposure or stimulus); and can also include, for example, the affinity of a compound to bind directly to a polypeptide or polynucleotide of the present disclosure, or, for example, a measure of the amount of an upstream or downstream protein or other similar function after some stimulus or event.

[0045] As used herein, the term "expression" of a gene, polynucleotide, polypeptide, etc. refers to the gene, etc. undergoing a certain action in vivo to become a different form. Preferably, the term refers to the gene, polynucleotide, etc. being transcribed and translated to become a polypeptide, but transcription to produce an mRNA is also an embodiment of expression. Thus, as used herein, the term "expression product" includes such a polypeptide or protein, or mRNA. More preferably, such a polypeptide form may be one that has undergone post-translational processing. For example, the expression level of an esterase can be determined by any method. Specifically, the expression level of an 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 detailed elsewhere in this specification or other methods known in the art.

[0046] As used herein, the term "functional equivalent" refers to any entity that has the same intended function but a different structure compared to the original entity of interest. Thus, it is understood that the functional equivalent of the "esterase" of the present disclosure is not the esterase itself of the present disclosure, but includes its mutant or variant (e.g., amino acid sequence variant, etc.) that has the biological action of the esterase, as well as those that can be changed into a mutant or variant that has the biological action of the esterase at the time of acting (e.g., including a nucleic acid that codes for the mutant or variant, and a vector, cell, etc. that contains the nucleic acid). In the present disclosure, it is understood that the functional equivalent of an esterase can be used in the same way as an esterase, even if not specifically mentioned. The functional equivalent can be found by searching a database, etc. As used herein, "search" refers to using a certain nucleic acid base sequence to find other nucleic acid base sequences that have a specific function and / or property, electronically, biologically, or by other methods. 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, macroarrays in which genomic DNA is attached to a nylon membrane or the like, or microarrays in which genomic DNA is attached to a glass plate (microarray assay), PCR, and in situ hybridization. As used herein, it is intended that the gene used in the present disclosure should also include corresponding genes identified by such electronic and biological searches.

[0047] As the functional equivalent of the present disclosure, an amino acid sequence in which one or more amino acids have been inserted, substituted or deleted, or which has been added to one or both termini thereof can be used. In this specification, "an amino acid sequence in which one or more amino acids have been inserted, substituted or deleted, or which has been added to one or both termini thereof" means that the amino acid sequence has been modified by a well-known technical method such as site-directed mutagenesis, or by natural mutation, by substitution of a number of amino acids to the extent that would occur naturally. The modified amino acid sequence can be, for example, one in which 1 to 30, preferably 1 to 20, more preferably 1 to 9, even more preferably 1 to 5, and particularly preferably 1 to 2 amino acids have been inserted, substituted or deleted, or which has been added to one or both termini thereof. The modified amino acid sequence may preferably be an amino acid sequence having one or more (preferably one 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 in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19 or 20) in SEQ ID NO: 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Here, "conservative substitution" means replacing one or more amino acid residues with another chemically similar amino acid residue so as not to substantially alter the function of the protein. For example, a hydrophobic residue may be replaced with another hydrophobic residue, a polar residue may be replaced with another polar residue having the same charge, etc. Functionally similar amino acids that can be replaced in this way are known in the art for each amino acid. Specific examples of non-polar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include arginine, histidine, and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0048] As used herein, the term "kit" refers to a unit in which the parts to be provided (e.g., enzymes, lipolytic agents, buffer solutions, instructions, etc.) are provided, usually divided into two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for stability reasons, but is preferably mixed and used immediately before use. Such a kit is advantageously provided with instructions or instructions that preferably describe how to use the parts to be provided (e.g., enzymes and lipolytic agents) or how to dispose of the reagents or waste liquid after use. When the kit is used as a reagent kit in this specification, the kit usually includes instructions that describe how to use the enzymes, lipolytic agents, etc.

[0049] In this specification, "instructions" refers to a description of a method for using the present disclosure to a user. The instructions contain a statement instructing how to use the polypeptide, polynucleotide, cell, etc. of the present disclosure. If necessary, the instructions are prepared in accordance with a format specified by the supervisory agency of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare or the Ministry of Agriculture, Forestry and Fisheries in Japan, the Food and Drug Administration (FDA), the Department of Agriculture (USDA), etc. in the United States), and it is clearly stated that the instructions have been approved by the supervisory agency. The instructions may be provided in paper form, but are not limited thereto, and may also be provided in the form of, for example, electronic media (e.g., a homepage provided on the Internet, e-mail).

[0050] (Preferred embodiment) The preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description 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 a novel polypeptide and a nucleic acid encoding the same. The polypeptide typically has esterase activity. In one embodiment, the polypeptide has lipase activity. The polypeptide of the present disclosure has significant properties different from conventionally known enzymes.

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

[0053] In one aspect, the polypeptide of the present disclosure is a first to fourth esterase, i.e., (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 8, 12, or 16; (b) a polypeptide comprising the amino acid sequence of (a) containing one or more amino acid substitutions, additions, deletions, or combinations thereof, 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 an 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 nucleotide 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 having biological activity, which is encoded by a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences shown in (d) to (f) or a complementary sequence thereof; (h) a polypeptide encoded by an allelic variant of any one of the nucleotide sequences 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); It 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 as set forth in SEQ ID NO:22, which contains one or more amino acid substitutions, additions, deletions, or combinations thereof; (b) a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:22; (c) a polypeptide encoded by a nucleotide sequence containing one or more nucleotide 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 to 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 comprising 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 set forth in SEQ ID NO: 21; or (g) A polypeptide containing a fragment of the amino acid sequence shown in (a) to (f). In one embodiment, the polypeptide of the present disclosure is not a polypeptide consisting of the amino acid sequence set forth 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 base sequence encoding a novel polypeptide. The polynucleotide of the present disclosure is a polynucleotide encoding the first to fourth esterases, i.e. (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 nucleotide 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% 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 under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences shown in (A) to (C) or a complementary sequence thereof, and encoding a polypeptide having biological activity; (E) a polynucleotide which is an allelic variant of any one of the nucleotide sequences (A) to (D) and encodes a polypeptide having biological activity; (F) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 8, 12, or 16; (G) a polynucleotide encoding a polypeptide having biological activity, comprising an amino acid sequence of (F) containing one or more amino acid substitutions, additions, deletions, or combinations thereof; (H) a polynucleotide having at least 70% sequence identity to the amino acid sequence shown in (F) or (G) and encoding a biologically active polypeptide; or (I) A polynucleotide comprising a fragment of the nucleotide sequence shown in (F) to (H); The polynucleotide may be a polynucleotide having the formula:

[0056] In another aspect, the present disclosure provides a nucleic acid sequence encoding a novel polypeptide. The polynucleotide of the present disclosure is a polynucleotide encoding a fifth esterase, i.e. (A) a polynucleotide containing a nucleotide sequence as shown in SEQ ID NO:21, which contains one or more nucleotide substitutions, additions, deletions, or combinations thereof; (B) a polynucleotide comprising a nucleotide sequence having at least 70% sequence identity to 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 to a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 21 or a complementary sequence thereof, and encoding a polypeptide having biological activity; (D) a polynucleotide encoding a polypeptide having biological activity, which is an allelic variant of the nucleotide sequence set forth in SEQ ID NO: 21; (E) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:22; (F) a polynucleotide encoding a polypeptide having biological activity, comprising an amino acid sequence containing one or more amino acid substitutions, additions, deletions, or combinations thereof in the amino acid sequence shown in SEQ ID NO:22; (G) a polynucleotide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22 and encoding a polypeptide having biological activity; or (H) A polynucleotide comprising a fragment of the nucleotide sequence shown in (E) to (G). In one embodiment, the polynucleotide of the present disclosure is not a polynucleotide consisting of the base sequence shown in SEQ ID NO: 21. The biological activity includes an ability to associate with the assimilation of trans-fatty acid-containing ester bodies or an ability to degrade trans-fatty acid-containing ester bodies.

[0057] In one embodiment, the biological activity of the polypeptide of the present disclosure may be at least one of any of the properties of the first, second, third, fourth, or fifth esterase of the present disclosure, and may include, for example, the ability to assimilate trans-fatty acid-containing esters, or the ability to degrade trans-fatty acid-containing esters, or both. Without wishing to be bound by theory, no activity against trans-fatty acid-containing esters has been observed in conventionally known Yarrowia-derived esterases, and at least in this sense, the esterase of the present disclosure has a distinctive feature.

[0058] Thus, in one embodiment, a polypeptide encoded by a polypeptide or polynucleotide of the present disclosure may have the ability to participate in the assimilation of trans fatty acid-containing esters, or the ability to degrade trans fatty acid-containing esters, or both.

[0059] In other embodiments, the biological activity of the polypeptides of the present disclosure may include, for example, the ability to associate with the assimilation of short- to long-chain fatty acid-containing esters, or the ability to degrade short- to long-chain fatty acid-containing esters, or both.

[0060] Thus, in one embodiment, a polypeptide encoded by a polypeptide or polynucleotide of the present disclosure may have the ability to participate in the assimilation of medium-chain fatty acid-containing esters, or the ability to degrade medium-chain fatty acid-containing esters, or both.

[0061] In a preferred embodiment, the polypeptide encoded by the polypeptide or polynucleotide of the present disclosure has the ability to degrade esters at 15° C. or 10° C. The activity of conventionally known Yarrowia-derived esterases is observed mostly at relatively high temperatures, and it was unexpected that significant activity was observed at low temperatures.

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

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

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

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

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

[0067] In one embodiment, the oil decomposer further comprises an oil-treating component, which may include, but is not limited to, other enzymes (e.g., esterases, etc.) or microorganisms.

[0068] In another aspect, the present disclosure provides a kit for oil decomposition or oil and fat treatment, comprising the polypeptide of the present disclosure, or the cell or cell-free expression system of the present disclosure, or the oil decomposition agent and additional oil treatment components. It is understood that the oil decomposition agent and oil treatment components included in the kit of the present disclosure can be any type described elsewhere herein in any combination.

[0069] In another aspect, the present disclosure provides an oil decomposition and removal method, which comprises the polypeptide of the present disclosure, or the cell or cell-free expression system of the present disclosure, or the oil decomposition agent of the present disclosure acting on the subject of treatment.In the oil decomposition and removal method of the present disclosure, the subject of treatment preferably contains trans-fatty acid-containing esters, but is not limited thereto.This specification shows that even if the subject does not contain trans-fatty acid-containing esters, when the esterase of the present disclosure is used, oil decomposition can be performed very efficiently.

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

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

[0072] (Enzyme production) In one embodiment, the esterases of the present disclosure are produced by a secretory production system using a microbial organism.

[0073] Specifically, the esterases of the present disclosure include: (a) introducing a nucleic acid molecule encoding the full-length sequence of the first, second, third, fourth or fifth esterase of the present disclosure (e.g., SEQ ID NO: 5, 9, 13, 17, 19, or a modified sequence thereof) into a pEZZ18 plasmid (GE Healthcare) and expressing the same in Escherichia coli HB101 strain as a Protein A fusion recombinant protein; and (b) purifying the protein secreted from E. coli HB101 strain using IgG Sepharose 6 Fast Flow or Butyl Sepharose 6 Fast Flow The esterase can be produced by a method including the steps of: exemplarily, the base 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 base sequence encoding the second esterase is inserted into the PstI and KpnI sites; and the base sequence encoding the third esterase is inserted into the EcoRI and XbaI sites. In addition to the secretion production system using Escherichia coli described above, 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 this method of producing esterase using a microorganism, detailed conditions such as the method of expressing the esterase, the host for expressing the esterase, and the method of purifying the esterase secreted from the host can be 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 secretory production system of a microbial organism.

[0074] Alternatively, the esterase of the present disclosure may comprise: (a) 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) introducing the modified sequence into a plasmid as appropriate and expressing it as a recombinant protein (fused, if necessary, with a tag sequence (e.g., protein A tag) that facilitates purification) in a suitable host (e.g., E. coli HB101 strain); and (c) purifying the protein produced in the host by a suitable separation technique (e.g., chromatography such as IgG Sepharose 6 Fast Flow or Butyl Sepharose 6 Fast Flow) The method includes the steps of: 1. Exemplarily, a variant of the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21 of the present disclosure is inserted into the above-mentioned restriction enzyme site in the pEZZ18 vector. The 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 is appropriately carried out using a conventional site-directed mutagenesis method, a mutagenesis method, or a molecular evolution method using error-prone PCR, or a commercially available kit.

[0075] Alternatively, the esterase of the present disclosure can be purified from, by way of example only, Yarrowia lipolytica KH-2 strain. (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, applying it to Butyl Sepharose 6 Fast Flow (GE Healthcare) that has been pre-equilibrated with 20 mM Tris-HCl (pH 7.0) buffer (hereinafter referred to as Tris buffer) containing 0.5 M NaCl and 2 mM CaCl2 in an amount of about 10 times the column volume, and leaving it to stand for about 1 hour to adsorb hydrophobic proteins to the column; (c) washing the column of (b) with about 10 column volumes of Tris buffer, and then washing three times with about 1 column volume of NaCl-free Tris buffer to elute proteins weakly bound to the column carrier; and (d) washing the column of (c) three times with a Tris buffer solution containing 1% Triton® X-100 in an amount equivalent to approximately one column volume to elute proteins tightly bound to the column; In the purification method of the enzyme, detailed conditions such as culture conditions for the KH-2 strain and column chromatography conditions can be appropriately adjusted by those skilled in the art.

[0076] (Use of enzymes) In one aspect, the esterase of the present disclosure is useful for treating fats and oils, and can be used for treating wastewater or waste liquid containing fats and oils. In an embodiment, the esterase of the present disclosure is used in wastewater treatment. When used for wastewater treatment, the esterase of the present disclosure can be used in applications such as industrial wastewater, kitchen wastewater, and residential wastewater.

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

[0078] In another aspect, the esterase of the present disclosure is used in fat modification and oil production technology. When the esterase of the present disclosure is used in fat modification and oil production technology, it can be used in food, industrial, fuel, and other applications.

[0079] In another aspect, the esterases of the present disclosure can be used as an environmental pollution countermeasure. When used in environmental pollution countermeasures, the esterases of the present disclosure can be used to remove contaminants in oil-contaminated soil, groundwater, marine pollution, and the like.

[0080] In another aspect, 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 food waste treatment including the disappearing type, composting, conversion of agricultural and food waste into feed, and reduction of the volume of oily sludge from a pressurized flotation separator or a grease trap.

[0081] In another aspect, the esterase of the present disclosure is used as a pharmaceutical. When used as a pharmaceutical, the esterase of the present disclosure can be used in applications such as digestive aids and lipolysis promoters.

[0082] In other aspects, the esterases of the present disclosure are used as cosmetics. When used as cosmetics, the esterases of the present disclosure 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 for various applications, it may be used as a component containing an isolated enzyme or as a component containing the microorganism itself, and those skilled in the art can use the esterase of the present disclosure in an appropriate form as appropriate.

[0084] (General technology) The molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in 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, Ishii Seiji, Okumura Hiroshi, Matsubara Chiyo, Ninomiya Fumi, Yoshioka Hiroshi, 2004, "Simple method for measuring oil content in water using thermosensitive polymers," Vol. 46, No. 12, "Water and Wastewater," and the relevant parts (which may be in their entirety) are incorporated herein by reference.

[0085] (Note) In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when it is specified that "within a range of two values," the range includes the two values ​​themselves.

[0086] All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth.

[0087] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided 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 scope of the claims. EXAMPLES

[0088] Examples are described below. The handling of the organisms used in the following examples complied with the standards stipulated by Nagoya University, the regulatory authorities, and the Cartagena Protocol, if necessary. The reagents used were specifically the products described in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Fujifilm Wako Pure Chemical Industries, Nacalai Tesque, R&D Systems, USCN Life Science INC, Thermo Fisher Scientific, Kanto Chemical, Funakoshi, Tokyo Kasei, Merck, etc.) can also be used. In addition, unless otherwise stated, the concentration of various oils and fatty acids added to the medium refers to the final concentration of the substance in the medium, and the percentage notation indicates volume / volume (v / v%) for oils and fatty acids that are liquid, and weight / volume (w / v%) for oils and fatty acids that are solid.

[0089] Example 1: Summary of genes and amino acid sequences encoding esterases from KH-2 strain This example provides a summary of the genes and amino acid sequences encoding the esterases of the disclosure.

[0090] (Experimental Method) We performed whole genome sequencing of strain KH-2. The predicted functions of the first esterase-encoding gene (gene number G4021), the second esterase-encoding gene (gene number G2944), the third esterase-encoding gene (gene number G2601), the fourth esterase-encoding gene (gene number G5971) and the fifth esterase-encoding gene (gene number G3702), the presence or absence of signal peptides (SPs) and the predicted molecular weights of the gene products are shown (Figure 1).

[0091] (result) The first, second, third, fourth and fifth esterases found in Example 1 were predicted to have cholesterol esterase activity, triglycerol lipase activity, patatin and phospholipase activity, patatin and phospholipase activity, and triglycerol lipase activity, respectively. The first, second, third, fourth and fifth esterases were predicted to have signal peptide (SP) sequences, and their molecular weights were predicted to be 63.5 kDa, 61 kDa, 62.9 kDa, 69.6 kDa and 36.7 kDa, respectively.

[0092] (Example 2: Expression analysis of esterase genes in 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 added to 3 L of BS medium supplemented with 1% canola oil by volume, and the final concentration of OD 660 = 0.05, and fermenter culture was carried out at 15° C. Total RNA was extracted from the cultures 24 hours, 48 ​​hours, and 72 hours after culture using a High Pure RNA Isolation Kit. 200 ng of total RNA was used as a template and PrimeScript TMGenomic DNA was removed and cDNA was synthesized using the RT reagent Kit with gDNA Eraser Perfect Real Time (Takara Bio). The cDNA was diluted with 2 volumes of pure water before use. Synthetic primers specific to the genes encoding each esterase were used to synthesize cDNA using Applied Biosystems (registered trademark) StepOnePlus TM Quantitative real-time RT-PCR was performed using the Applied Biosystems PCR kit. TM The PCR was carried out in a 20 μl solution containing 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 consisting of one 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 with the expression level of 1,2-mannosyltransferase (alg9). The data was 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 fat or oil was set to 1 as a control.

[0094] (result) By feeding KH-2 strain with fats and oils as a carbon source, 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 induced at 24 hours. The expression of these esterases was also induced 72 hours after cultivation. In contrast, the expression level of the gene encoding the fifth esterase was equivalent to that in the culture in LB medium, suggesting that this gene is constitutively expressed regardless of the carbon source.

[0095] (Example 3: Production of the first, second, third, fourth and fifth esterases by recombinant E. coli)

[0096] (Experimental Method) In this example, the pEZZ18 Protein A Gene Fusion Vector system (GE Healthcare) was used to express recombinant proteins of the representative sequences of the first, second, third, fourth and fifth esterases in Escherichia coli cells. The base 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 (first, fourth and fifth esterases), PstI and KpnI sites (second esterase), and EcoRI and XbaI sites (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 cultured at 42°C for 3 hours. The cells were removed by centrifugation, and the recombinant proteins were adsorbed onto an equilibrated Butyl Sepharose6 Fast Flow column. The recombinant protein was then eluted with 20 mM Tris-HCl (pH 7.0) elution buffer containing 2 mM CaCl2 and 0.5% Triton® X-100.

[0097] (Example 4: Measurement of optimum temperature, thermal stability, optimum pH and pH stability) In this example, the optimum temperature, thermostability, optimum pH and pH stability of representative sequences of the first, second, third and fourth esterases produced in recombinant E. coli and roughly purified are shown.

[0098] E. coli expressing the representative sequences of the first, second, third and fourth recombinant esterases were cultured in 1500 ml of LB medium using a fermenter for 48 hours. An equal amount of acetone was added to the culture supernatant and allowed to stand at 4°C for 24 hours. After that, the crude esterase obtained by centrifugation and suspending the pellet in 1 ml of buffer (20 mM Tris-HCl (pH 7.4), 2 mM CaCl2, 0.5% Triton (registered trademark) X-100) was analyzed. (A and E) The crude esterase was mixed with buffers adjusted to each temperature between 0°C and 100°C, and the esterase activity was measured to examine the optimum temperature. (B and F) The crude esterase was mixed with buffers adjusted to each temperature between 0°C and 100°C, incubated for 30 minutes, and then the esterase activity was measured to examine the thermal stability. (C and G) The optimum pH of the crude esterase was examined by mixing it 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 of the crude esterase was examined by mixing it 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 incubating for 30 minutes, and then measuring the esterase activity.

[0099] (result) The optimum temperature of the representative sequence of the first esterase was about 60°C, and it showed thermostability in the temperature range of about 10°C to about 60°C. The optimum 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 optimum temperature of the representative sequence of the second esterase was about 40°C, and it showed thermostability in the temperature range of about 10°C to about 50°C. The optimum 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 optimum temperature of the representative sequence of the third esterase was about 65°C, and it showed thermostability in the temperature range of about 30°C to about 65°C. The optimum 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 representative sequence of the fourth esterase had an optimum temperature of about 65° C., and exhibited thermostability in a temperature range of about 30° C. to about 70° C. The representative sequence of the fourth esterase had an optimum pH of pH 9.0, and exhibited pH stability in a pH range of about pH 7 to about pH 9.5.

[0100] (Example 5: Effect of esterases 1, 2, 3, 4 and 5 and N-51032 enzyme on cleaning oil stains on ventilation fan filters) In this example, the cleaning effects of representative sequences of the first, second, third, fourth and fifth esterases of the present disclosure derived from the KH-2 strain and Novozym 51032 lipase (Novozymes) on oil stains on ventilation fan filters were measured.

[0101] (Experimental Method) 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 (registered trademark) X-100, and adjusted to a concentration of 15 U / ml for Novozym 51032 lipase, 0.5 U / ml for the representative sequences of the first and second esterases, and 0.1 U / ml for the representative sequences of the third, fourth and fifth esterases. In this experiment, the concentration of the enzyme solution was adjusted based on the enzyme activity quantified by absorbance at 410 nm for the amount of 4-nitrophenol produced by hydrolysis of the esterase or lipase, using 4-nitrophenyl ester (pNP-ester) of palmitate (C16) as a substrate for the representative sequences of esterases 1 and 2, and butyrate (C4) as a substrate for the representative sequences of esterases 3, 4, and 5. An oil-soiled ventilation fan filter was cut into 2 cm squares and placed on a plate, and each plate was filled with a solution of esterase or lipase, and soaked for 30 minutes.

[0102] (result) The ventilation fan filters after soaking in the esterase or lipase solution and the esterase or lipase solution after soaking are shown in Figure 4. It was found that the ventilation fan filters soaked with the representative sequences of the first, second, third and fourth esterases of the present disclosure all decomposed oily soils significantly better than Novozym 51032 lipase, and the fifth esterase also improved the decomposition of oily soils to some extent.

[0103] (Example 6: Decomposition of fats and oils by 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 first, second, third, fourth and fifth esterase recombinant proteins of the present disclosure is shown.

[0104] (Experimental Method) (A: Decomposition of shortening by esterases 1, 2, 3, 4 and 5) Each purified recombinant esterase produced by the process of Example 3 was prepared to a final concentration of 0.2 u / ml. 2 ml of each solution of the disclosed esterase was placed in a plate, to which 0.7 g of shortening was added. The plate was incubated at 28° C. for 24 hours while stirring appropriately to mix with the solution. The control was treated with only the above-mentioned elution buffer.

[0105] (B: Decomposition of lard by esterases I, II, III, IV and V) Each purified recombinant esterase produced by the process of Example 3 was prepared to a final concentration of 0.2 u / ml. 2 ml of each esterase solution of the present disclosure was placed in a plate, to which 0.5 g of lard was added. The plate was incubated at 28°C for 24 hours while stirring appropriately to mix with the solution. The control was treated with only the above-mentioned elution buffer.

[0106] (C: Decomposition of lard, shortening, triolein and trielaidin by esterases 1, 2, 3, 4 and 5) Each purified recombinant esterase produced by the process 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 placing 0.1 ml of each purified recombinant esterase and 1 ml of elution buffer in 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 carried out by color development with molybdophosphate n-hydrate (2.4 g / 60 ml EtOH).

[0107] (result) The results of the first, second and fifth recombinant esterases are shown in FIG. 5-1, and the results of the third and fourth recombinant esterases are shown in FIG. 5-2. From the results (A) to (C), it was shown that the recombinant proteins of the representative sequences of the first, second, third and fourth esterases of the present disclosure all have the activity of decomposing all fats and oils, including lard, shortening, triolein and trielaidin. Therefore, it was revealed that the recombinant proteins of the representative sequences of the first, second, third and fourth esterases of the present disclosure have the activity of hydrolyzing both triolein, which is a cis triglyceride, and trielaidin, which is a trans triglyceride. Furthermore, it was shown that the decomposition activity against shortening was remarkably high. Furthermore, it was shown that the recombinant protein of the representative sequence of the fifth esterase also has the decomposition activity against these fats and oils, although it is inferior to the decomposition activity of these esterases.

[0108] Example 7: Comparison of substrate specificity of the first, second, third, fourth and fifth esterases In this example, the decomposition activities of the first, second, third, fourth and fifth esterases of the present disclosure for each substrate were compared.

[0109] (Experimental Method) 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 esterase activity was measured by quantifying the amount of 4-nitrophenol generated by hydrolysis of esters by esterase using 4-nitrophenyl esters (pNP-esters) of five types of fatty acids (acetate (C2), butyrate (C4), octanoate (C8), laurate (C12) and palmitate (C16)) as substrates, based on the absorbance at 410 nm. As a substrate solution, 0.05 mol of each substrate was mixed with 12 ml of 3% (v / v) Triton (registered trademark) X-100 aqueous solution, and melted at 70°C. Each substrate solution was mixed with 150 mM GTA buffer (pH 7.0) and each sample in an amount of 60 μl, and the absorbance at 410 nm immediately after mixing and 30 minutes after mixing were measured. 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. The activity of the fifth esterase 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 calculated by calculating the number of units per 1 ml of sample, with the amount of enzyme that liberates 1 μM of 4-nitrophenol defined as 1 unit, and the relative activity was calculated by taking the maximum activity in each substrate as 100%.

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

[0111] (Example 8: Comparison of decomposition activity of trans fatty acid-containing esters by first, second, third, fourth and fifth esterases by serial dilution) In this example, the decomposition activity of trans fatty acid-containing esters by the first, second, third, fourth and fifth esterases of the present disclosure was compared by serial dilution.

[0112] (Experimental Method) 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 decomposition activity of trans-fatty acid-containing esters was measured by using trielaidin as an ester, serially diluting 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 them by thin layer chromatography according to the following experimental conditions. Ester type: 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: Place 100 μl of enzyme solution in an Eppendorf tube. 10x stock of each ester was added at 1 / 10 volume (0.02u / ml: NP-butyrate as substrate). Processing temperature: 37℃ Processing time: 48 hours Oil extraction: Extracted with half the amount of chloroform, and 10 μl was applied to thin layer chromatography. · Development plate: Silica gel coated plate Developing solvent: chloroform:acetone:methanol=96:4:2 Detection: Color development with molybdophosphate n-hydrate (2.4 g / 60 ml EtOH)

[0113] (result) The representative sequence of the fifth esterase of the present disclosure showed activity when treated for 48 hours at a concentration of 0.01u / ml, but almost completely lost activity when treated for 48 hours at a concentration of 0.001u / ml. The representative sequences of the first, second, third and fourth esterases showed activity even when diluted to 0.001u / 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 of the representative sequences of the first, second, third and fourth esterases.

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

[0115] (Experimental Method) 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 decomposition activity of trans-fatty acid-containing esters was measured by thin-layer chromatography at a treatment temperature of 15° C., using trielaidin as an ester, and following the experimental conditions below. Ester type: 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: Place 100 μl of enzyme solution in an Eppendorf tube. 10x stock of each ester was added at 1 / 10 volume (0.02u / ml: NP-butyrate as substrate). Processing temperature: 15℃ Processing time: 72 hours Oil extraction: Extracted with half the amount of chloroform, and 10 μl was applied to thin layer chromatography. · Development plate: Silica gel coated plate Developing solvent: chloroform:acetone:methanol=96:4:2 Detection: Color development with molybdophosphate n-hydrate (2.4 g / 60 ml EtOH)

[0116] (result) All of the esterases of the first, second, third and fourth representative sequences of the present disclosure showed activity in decomposing trans fatty acid-containing esters at 15°C.

[0117] Example 10: Modification of triolein In this example, triolein was modified (methyl esterified) using the first, second, third, and fourth esterases of the present disclosure.

[0118] (Experimental Method) 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 the addition of the esterase solution was also performed at the same time as a control. Then, 100 μl of each solution was dispensed into a screw-capped tube and left to stand in an incubator at 37° C. for 96 hours. Then, 100 μl of water and 50 μl of chloroform were added, and the mixture was thoroughly mixed with a vortex mixer, followed by centrifugation 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 the developing solvent.

[0119] (result) The results of Example 10 are shown in Figure 9. All of the esterases of the representative sequences of the first, second, third and fourth esterases catalyzed the transesterification of triolein, which is a cis-triglyceride, to produce methyl oleate.

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

[0121] (Experimental Method) 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 (A) palmitelaidic acid or (B) vaccenic acid monomer in methanol to a concentration of 0.5%. 30 μl of each trans fatty acid stock solution and 70 μl of enzyme solution were dispensed into screw-capped tubes and left to stand in an incubator at 37°C for 72 hours. A control group was treated simultaneously with a buffer instead of the esterase solution. Then, 100 μl of water and 50 μl of chloroform were added, and the mixture was thoroughly mixed with a vortex mixer and 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] (result) The results of Example 11 are shown in Figure 10. All of the esterases of the representative sequences of the first, second, third and fourth esterases catalyzed the transesterification of trans-fatty acids palmitelaidic acid and vaccenic acid to produce palmitelaidic acid methyl ester and vaccenic acid methyl ester, respectively.

[0123] Example 12: Comparison of the second and fifth esterases in the modification of trans fatty acids In this example, the second and fifth esterases of the present disclosure were used to modify (methyl esterify) trans fatty acids.

[0124] (Experimental Method) 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 (A) palmitelaidic acid or (B) vaccenic acid monomer in methanol to a concentration of 0.5%. 30 μl of each trans fatty acid stock solution and 70 μl of enzyme solution were dispensed into screw-capped tubes and left to stand in an incubator at 37°C for 72 hours. A control group was treated simultaneously with a buffer instead of the esterase solution. Then, 100 μl of water and 50 μl of chloroform were added, and the mixture was thoroughly mixed with a vortex mixer and 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.

[0125] (result) The results of Example 12 are shown in Figure 11. The representative sequence of the fifth esterase was not observed to have catalytic activity for transesterification of palmitelaidic acid, unlike the representative sequence of the second esterase of the present disclosure, which showed catalytic activity for methyl esterification of palmitelaidic acid. When vaccenic acid was used as a trans fatty acid, the representative sequence of the fifth esterase had lower catalytic activity for transesterification than the representative sequence of the second esterase of the present disclosure, but still had catalytic activity for transesterification.

[0126] (Example 13: Preparation of modified esterase) In this example, variants of the esterase of the present disclosure are made. Methods and Materials A construct is prepared that contains one or more bases inserted, substituted or deleted, or added to one or both ends of the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21. The construct is designed using the same method as in the above-mentioned Examples, or using experimental techniques commonly performed in this technical field known to those skilled in the art, or using commercially available kits. Alternatively, an oligonucleotide encoding a polypeptide in which amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 are substituted is artificially synthesized. The oil or ester decomposition activity of these various modified forms is analyzed using the same method as in the above-mentioned Examples. (Construction 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 mutated by the above-mentioned mutation-introducing technique are prepared. (Thermal stability, optimum temperature, optimum pH and pH stability) According to Example 4, the thermal stability, optimum temperature, optimum pH and pH stability of the mutants thus prepared are measured. (Effective in cleaning dirt from ventilation fan filters and decomposing esters) Tests are carried out according to Examples 5 and 6, respectively, to measure the decomposition activity. (substrate specificity) A test is carried out according to Example 7 to measure substrate specificity. (result) As a result of the above tests, it is found that it is possible to obtain a mutant strain having the activity discovered in the present disclosure (e.g., the ability related to the assimilation of trans fatty acid-containing oils or the ability to decompose trans fatty acid-containing oils) possessed by a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22.

[0127] (Note) As described above, the present invention has been illustrated using preferred embodiments of the present invention, but it is understood that the scope of the present invention should be interpreted only by the claims. It is understood that the patents, patent applications and other documents cited in this specification should be incorporated by reference to this specification in the same manner as if the contents themselves were specifically described in this specification. This application claims priority to Japanese Patent Application No. 2019-163343 filed on September 6, 2019 at the Japan Patent Office, and it is understood that the entire contents of the application should be incorporated by reference to this specification. [Industrial Applicability]

[0128] INDUSTRIAL APPLICABILITY The present disclosure is useful for decomposing trans fatty acid-containing esters and for treating wastewater containing trans fatty acids, which is a problem in food factories and the like. [Accession number]

[0129] NITE BP-02732 [Sequence List Free Text]

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

Claims

1. An oil decomposing agent for decomposing oil in a treatment target containing a trans fatty acid-containing ester body, comprising: The polypeptide is (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 8, 12, 16 or 22; (b) a polypeptide comprising an amino acid sequence of (a) containing 1 to 30 amino acid substitutions, additions, deletions or combinations thereof, and having esterase activity; (c) a polypeptide having at least 90% sequence identity with the amino acid sequence shown in (a) and having esterase activity; (d) a polypeptide comprising an amino acid sequence encoded by the base sequence shown in SEQ ID NO: 1, 7, 11, 15 or 21; (e) a polypeptide having esterase activity, encoded by a base sequence containing a substitution, addition, deletion or a combination thereof of 1 to 90 nucleotides in the base sequence shown in (d); or (f) a polypeptide encoded by a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence shown in (d), and having esterase activity; It is an oil decomposer.

2. An oil decomposition agent for decomposing oil in a subject to be treated that contains a trans fatty acid-containing ester body, the agent comprising: a cell or a cell-free expression system containing a polynucleotide; The polynucleotide comprises: (A) a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1, 7, 11, 15 or 21; (B) a polynucleotide comprising a base sequence containing a substitution, addition, deletion or a combination thereof of 1 to 90 nucleotides in the base sequence shown in (A), and encoding a polypeptide having esterase activity; (C) a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence shown in (A) and encoding a polypeptide having esterase activity; (D) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 8, 12, 16 or 22; (E) a polynucleotide encoding a polypeptide having an esterase activity, comprising an amino acid sequence of (D) containing a substitution, addition, deletion, or combination thereof of 1 to 30 amino acids; or (F) a polynucleotide having at least 90% sequence identity with the amino acid sequence shown in (D) and encoding a polypeptide having esterase activity; It is an oil decomposer.

3. The polypeptide has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, 8, 12 or 16 and has esterase activity; or The oil decomposition agent according to claim 1 or 2, wherein the polynucleotide is a polynucleotide that contains a base sequence having at least 90% sequence identity with the base sequence shown in SEQ ID NO: 1, 7, 11 or 15 and encodes a polypeptide having esterase activity.

4. The oil decomposing agent according to any one of claims 1 to 3, further comprising an oil treating component.

5. A kit for decomposing oil comprising the oil decomposing agent according to any one of claims 1 to 3 and a further oil treatment component.

6. An oil decomposition method (excluding methods within the human body), comprising applying the oil decomposition agent according to any one of claims 1 to 3 to an object to be treated.

7. The oil decomposing agent according to any one of claims 1 to 3, which is a detergent.

8. Use of the oil decomposing agent according to any one of claims 1 to 3 in fat modification and oil production technology (excluding use inside the human body).

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