WO2021045234A1

WO2021045234A1 - Novel esterase for decomposing trans fatty acid-containing ester

Info

Publication number: WO2021045234A1
Application number: PCT/JP2020/033834
Authority: WO
Inventors: 克敏堀
Original assignee: 国立大学法人東海国立大学機構
Priority date: 2019-09-06
Filing date: 2020-09-07
Publication date: 2021-03-11
Also published as: JPWO2021045234A1

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

A novel esterase that decomposes trans fatty acid-containing esters

The present disclosure relates to a novel esterase that decomposes a trans fatty acid-containing ester and its application (for example, wastewater treatment, oil treatment). In another aspect, the present disclosure relates to novel esterases and their applications that degrade esters at low and / or high temperatures.

Oil stains are a typical example of stains generated in general households, the food service industry, industrial equipment, etc. Oil stains are difficult-to-clean stains that occur in kitchens, sinks, kitchens, pipes, drains, ventilation fans, laundry, and the like. Since oil stains are a source of foul odors and pests and can also cause environmental pollution, the establishment of epoch-making technology in oil treatment is eagerly desired from both public health and environmental aspects.

Grease traps, which are treatment facilities that remove oil contained in kitchen wastewater from the food service industry by solid-liquid separation, are a source of foul odors and pests, and maintenance such as recovery, transportation, and cleaning of separated oil is laborious and costly. Considering this, the establishment of an epoch-making technology for extinguishing the oil in the grease trap is eagerly desired by the industry centered on the food service industry.

As a result of diligent research, the present inventors have found an esterase that decomposes a trans fatty acid-containing ester. It was also found that this esterase has an ability to decompose an ester even at low temperature and / or high temperature, and is also excellent in thermal stability. The present disclosure also relates to applications of the esterases of the present disclosure, such as oil treatment.

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

In the present disclosure, it is intended that the above one or more features may be provided in a further combination in addition to the specified combinations. Further embodiments and advantages of the present disclosure will be appreciated by those skilled in the art upon reading and understanding the following detailed description as necessary.

Using this disclosure, the treatment of trans fatty acid-containing fats and oils, which was difficult to remove in the past, has become easier. In addition, it has become possible to remove fats and oils and treat oils even in a high temperature environment or a low temperature environment, which was difficult to use with conventional microorganisms and enzymes. Therefore, the novel esterases (eg, lipases) of the present disclosure include detergents, leather industry, food industry, purification of environmental pollution by fats and oils, food waste treatment, composting treatment, waste treatment such as wastewater treatment, composting, and digestive agents. It is useful in technical fields such as pharmaceuticals such as, and cosmetics for oily skin.

FIG. 1 shows the gene numbers, estimated functions, and signal peptides (SP) of the genes encoding the first, second, third, fourth, and fifth esterases of the Yarrowia lipolytica KH-2 strain. The presence or absence and the estimated molecular weight of the gene product are shown. FIG. 2 shows the expression of five types of esterases (typical sequences of the first, second, third, fourth and fifth esterases) when the KH-2 strain was cultured at 15 ° C. by a fermenter. It is a graph which shows a level. The expression level is indicated by a relative value normalized by the expression level of the alg9 (1,2-mannosyltransferase) gene. The horizontal axis of the graph corresponds to the culture after culturing for 24 hours, 48 hours and 72 hours in order from the left. The vertical axis of the graph shows the relative expression level. FIG. 3-1 shows the optimum temperature, thermal stability, optimum pH and pH stability of the recombinant protein of the representative sequence of the first and second esterases derived from the KH-2 strain. The upper rows (A) to (D) show the results of the representative sequence of the first esterase, and the lower rows (E) to (H) show the results of the representative sequence of the second esterase. (A) and (E) indicate the optimum temperature of esterase, (B) and (F) indicate the thermal stability of esterase, (C) and (G) indicate the optimum pH of esterase, and (D). ) And (H) indicate the pH stability of esterase. The vertical axis of (A) to (H) shows the esterase activity relative to the activity when the esterase activity is maximized, and the horizontal axes of (A), (B), (E) and (F) are temperatures. , And the horizontal axes of (C), (D), (G) and (H) indicate pH. FIG. 3-2 shows the optimum temperature, thermal stability, optimum pH and pH stability of the recombinant protein of the representative sequences of the third and fourth esterases derived from the KH-2 strain. The upper rows (A) to (D) show the results of the representative sequence of the third esterase, and the lower rows (E) to (H) show the results of the representative sequence of the fourth esterase. (A) and (E) indicate the optimum temperature of esterase, (B) and (F) indicate the thermal stability of esterase, (C) and (G) indicate the optimum pH of esterase, and (D). ) And (H) indicate the pH stability of esterase. The vertical axis of (A) to (H) shows the esterase activity relative to the activity when the esterase activity is maximized, and the horizontal axes of (A), (B), (E) and (F) are temperatures. , And the horizontal axes of (C), (D), (G) and (H) indicate pH. FIG. 4 shows a comparison between Novozymes lipase (Novozyme® 51032) and the esterases of the present disclosure. An E. coli strain expressing an oil-stained ventilation fan filter with a recombinant protein of Novozym 51032 (N-51032), a representative sequence of the first, second, third, fourth and fifth esterases of the present disclosure. It is a photograph which was soaked and washed with the purified esterase obtained from. A is Novozym 51032, B is the first esterase, C is the second esterase, D is the third esterase, E is the fourth esterase, and F is the fifth esterase. And a photograph of the ventilation fan filter is shown. FIG. 5-1 shows the decomposition activity of the ester by the first, second and fifth esterases of the present disclosure. (A) Buffer alone, purified esterase obtained from an Escherichia coli strain expressing a recombinant protein of a representative sequence of the fifth esterase (fifth esterase), representative sequence of the first esterase. Purified esterase (first esterase) obtained from an Escherichia coli strain expressing the recombinant protein of the above, or purified esterase obtained from an Escherichia coli strain expressing a recombinant protein of a representative sequence of the second esterase. (Second esterase) was incubated with 0.7 g of shortening at 28 ° C. for 24 hours. An image of fats and oils after incubation is shown. (B) Buffer alone, purified esterase obtained from an Escherichia coli strain expressing a recombinant protein of a representative sequence of the fifth esterase (fifth esterase), representative sequence of the first esterase. Purified esterase (first esterase) obtained from an Escherichia coli strain expressing the recombinant protein of the above, or purified esterase obtained from an Escherichia coli strain expressing a recombinant protein of a representative sequence of the second esterase. (Second esterase) was incubated with 0.5 g of lard at 28 ° C. for 24 hours. An image of fats and oils after incubation is shown. (C) Buffer alone, purified esterase obtained from an Escherichia coli strain expressing a recombinant protein of a representative sequence of the fifth esterase (fifth esterase), representative sequence of the first esterase. Purified esterase (first esterase) obtained from an Escherichia coli strain expressing the recombinant protein of the above, or purified esterase obtained from an Esterase strain expressing the recombinant protein of a representative sequence of the second esterase. (Second esterase) is incubated with triolein (Tole), triellaidin (TED), shortening or lard, respectively, then separated by thin layer chromatography and detected by molybtriic acid n-hydrate. From left to right, the ones incubated with triolein, trioleidin, shortening and lard are shown. FIG. 5-2 shows the decomposition activity of the ester by the third and fourth esterases of the present disclosure. (A) Buffer alone, purified esterase obtained from an Escherichia coli strain expressing a recombinant protein of a representative sequence of the third esterase (third esterase), or representative of the fourth esterase. Purified esterase (fourth esterase) obtained from an E. coli strain expressing the recombinant protein of the sequence was incubated with 0.7 g of shorting at 28 ° C. for 24 hours. An image of fats and oils after incubation is shown. (B) Buffer alone, purified esterase obtained from an Escherichia coli strain expressing a recombinant protein of a representative sequence of the third esterase (third esterase), or representative of the fourth esterase. Purified esterase (fourth esterase) obtained from an E. coli strain expressing the recombinant protein of the sequence was incubated with 0.5 g of lard for 24 hours at 28 ° C. An image of fats and oils after incubation is shown. (C) Buffer alone, purified esterase obtained from an Escherichia coli strain expressing a recombinant protein of a representative sequence of the third esterase (third esterase), or representative of the fourth esterase. Purified esterases (fourth esterases) from Escherichia coli strains expressing recombinant proteins of the sequence are incubated with triolein (Tole), triellaidin (TED), shortening or lard, respectively, followed by thin layer chromatography. The results of separation by chromatography and detection by molybtriic acid n-hydrate are shown. From left to right, the ones incubated with triolein, trioleidin, shortening and lard are shown. FIG. 6 shows the substrate specificity of representatives of the first, second, third, fourth and fifth esterases. The substrates used are shown on the left side of the table, and 4-nitrophenyl ester (pNP-ester) lipids having acetate (C2), butyrate (C4), octanoate (C8), laurate (C12) and palmitate (C16) are shown in order from the top. Corresponds to. The right side of the table shows the esterase activity of the first, second, third, fourth and fifth esterases, and the relative activity when the maximum activity among each substrate is taken as 100%. Is done. FIG. 7 is from a purified esterase obtained from an E. coli strain expressing a recombinant protein of a serially diluted representative sequence of the first, second, third, fourth and fifth esterases of the present disclosure. Decomposition of trans fatty acid-containing ester is shown. The data show the results with esterase serially diluted to 0.01u / ml, 0.001u / ml, and 0.0001u / ml from left to right, and each data is from left to right, first, second, second. Corresponds to the results with representative sequences of the third, fourth and fifth esterases. FIG. 8 shows a trans fatty acid-containing ester at low temperature by a purified esterase obtained from an Escherichia coli strain expressing a recombinant protein of a representative sequence of the first, second, third and fourth esterases of the present disclosure. Indicates decomposition of the body. From left to right, each band corresponds to a representative sequence of buffer, first, second, third and fourth esterases, with the arrow at the bottom indicating the hydrolyzate (free fatty acid). Corresponds to the band of. FIG. 9 shows the modification of triolein by a purified esterase obtained from an Escherichia coli strain expressing a recombinant protein of a representative sequence of the first, second, third and fourth esterases of the present disclosure. Each band, in order from the left, has a representative sequence of the first, second, third and fourth esterases, or a buffer that has been mixed with methanol and triolein and reacted, and olein. The results of analysis of the methyl acid acid grade by thin layer chromatography analysis are shown. Each signal corresponds to oleic acid methyl ester, triolein, oleic acid and diacylglycerol in order from the top. FIG. 10 shows the modification of trans fatty acids by a purified esterase obtained from an Escherichia coli strain expressing a recombinant protein of a representative sequence of the first, second, third and fourth esterases of the present disclosure. (A) Representative sequences of the first, second, third and fourth esterases, or buffers that have been mixed with methanol and palmitelydic acid and reacted, and palmiteraidin. The results of analysis of the methyl acid acid grade by thin layer chromatography analysis are shown. Each signal corresponds to palmitelysinic acid methyl ester and palmitelysinic acid in order from the top. (B) Representative sequences of the first, second, third and fourth esterases, or buffers that have been mixed with methanol and vaccenic acid and reacted, and methyl vaccenate evaluations. Is shown by thin layer chromatography analysis. Each signal corresponds to vaccenic acid methyl ester and vaccenic acid in order from the top. FIG. 11 shows the modification of trans fatty acids by a purified esterase obtained from an Escherichia coli strain expressing a recombinant protein of a representative sequence of the second and fifth esterases of the present disclosure. (A) Representative sequences of the second and fifth esterases, or buffers obtained by mixing and reacting with methanol and palmiterazic acid, and methyl palmiterazate grades are diluted. The result of analysis by layer chromatography analysis is shown. Each signal corresponds to palmitelysinic acid methyl ester and palmitelysinic acid in order from the top. (B) Thin-layer chromatographic analysis of representative sequences of the second and fifth esterases, or buffers obtained by mixing and reacting with methanol and buxenoic acid, and methyl buccinate grades. The result of the analysis is shown. Each signal corresponds to vaccenic acid methyl ester and vaccenic acid in order from the top.

Hereinafter, the present disclosure will be described while showing the best form. Throughout the specification, it should be understood that the singular representation also includes its plural concept, unless otherwise stated. Therefore, it should be understood that singular articles (eg, "a", "an", "the", etc. in English) also include their plural concept, unless otherwise noted. It should also be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted. Thus, unless otherwise defined, all terminology and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, this specification (including definitions) takes precedence.

The definitions and / or basic technical contents of terms particularly used in the present specification will be described below as appropriate. In the present specification, a strain name such as KH-2 strain may be abbreviated as "stock" in some cases, but those skilled in the art will appropriately understand that the term "stock" is used depending on the context. ..

(Definition of terms)
As used herein, the term "esterase" refers to a hydrolase that decomposes an ester into an acid and an alcohol by a chemical reaction with water. The esterases of the present disclosure are classified as esterases because they have an activity of hydrolyzing esters such as fatty acid esters. The esterase activity of an esterase can be measured by the method described in "Ability to Decompose Esters" herein.

In the present specification, "lipase" is a kind of esterase, and refers to an enzyme that reversibly catalyzes the reaction of hydrolyzing neutral fat (glycerol ester) and decomposing it into fatty acid and glycerol. The esterase of the present disclosure can be said to be a "lipase" because it has the activity of an enzyme classified into triglycerol lipase, which is classified into EC3.1.1.3 by the enzyme number (EC number). Also referred to as disclosed lipase.

In the present specification, "fat and oil" refers to an oily substance, and the fat and oil includes an ester group-containing compound formed by dehydration condensation of a compound containing a hydroxyl group and a fatty acid. Typically, the compound containing this hydroxyl group is glycerin, but other examples include polyglycerin and the like. Similar to what is commonly used in the art, ester group-containing compounds formed by dehydration condensation of glycerin and fatty acids are referred to herein as "glycerides". When the compound containing a hydroxyl group has a plurality of hydroxyl groups, if at least one of the hydroxyl groups is dehydrated and condensed with a fatty acid to form an ester, the compound corresponds to the ester group-containing compound in the present specification. ..

In the present specification, the lipase activity refers to an activity of hydrolyzing an ester produced by dehydration condensation of glycerol and a fatty acid into glycerol and a free fatty acid, and such a lipase activity is also referred to as a triglyceride lipase activity in the present specification. Will be done. For example, whether or not it has lipase activity depends on the decrease of esters (animal and vegetable oil such as canola oil, triolein, trioleidin, etc.) contained in the solution to which lipase is added, and the detection of hydrolyzate. You can check.

The ability to decompose and consume the ester and fatty acid can be measured by analyzing the ester remaining in the medium and the free fatty acid produced by hydrolysis by thin layer chromatography. To show a specific quantification procedure, first, an ester and a fatty acid are extracted by adding an equal amount of chloroform to the culture supernatant. 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 and the like can be changed as appropriate. The plate is treated with molybdate n-hydrate to develop an ester color.

In the present specification, "fat modification / fat production capacity" refers to changing the structure of fatty acids to change the properties of oil. In the present specification, "fat modification / fat production capacity" typically includes an activity of catalyzing the transesterification of fatty acids. The fat modification / fat production capacity can be measured by analyzing the fatty acid methyl ester produced by the transesterification reaction between fatty acid and methanol by thin layer chromatography. To show a specific quantification procedure, first, the esterase, fatty acid and methanol of the present disclosure are mixed to produce a fatty acid methyl ester. The ester and fatty acid are extracted by adding chloroform to the mixed solution of fatty acid methyl ester. 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 and the like can be changed as appropriate. The plate is treated with molybdate n-hydrate to develop an ester color.

In the present specification, the "trans fatty acid-containing ester" refers to an ester containing a trans fatty acid. "Trans fatty acid" is used in the sense commonly used in the art and means an unsaturated fatty acid having a trans-type double bond. Trans fatty acids are naturally present in trace amounts 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 hydrogenation of unsaturated fatty acids, and are used in foods such as margarine and shortening. Is also included. The trans fatty acid includes elaidic acid, palmiteraidic acid (palmitoelaidic acid), vaccenic acid and the like, but the type of trans fatty acid is not particularly limited as used herein. The ratio of the trans fatty acid present in the trans fatty acid-containing ester is not particularly limited. None of the conventionally known Yarrowia lipases have the activity of degrading trans fatty acid-containing esters.

In the present specification, the "short-chain to long-chain fatty acid-containing ester" refers to an ester of a fatty acid containing one or more of a short-chain fatty acid, a medium-chain fatty acid, or a long-chain fatty acid. "Short-chain fatty acids," "medium-chain fatty acids," and "long-chain fatty acids" are used in the sense commonly used in the art and have 2-6, 7-12, and 13 or more carbon atoms, respectively. Means fatty acid. Short-chain fatty acids include acetic acid (2 carbon atoms), butyric acid (4 carbon atoms), caproic acid (6 carbon atoms) and the like. Medium-chain fatty acids include caprylic acid (8 carbon atoms), capric acid (10 carbon atoms), lauric acid (12 carbon atoms) and the like. Long-chain fatty acids include myristic acid (14 carbons), palmitic acid (16 carbons), stearic acid (18 carbons), oleic acid (18 carbons), linoleic acid (18 carbons), and linolenic acid (carbons). The number 18) and the like are included.

In the present specification, the "medium chain fatty acid-containing ester" refers to an ester containing a medium chain fatty acid. "Medium chain fatty acid" is used in the sense commonly used in the art and means a fatty acid having 7 to 12 carbon atoms. Medium-chain fatty acids are naturally found in foods such as dairy products, palm kernel oil and coconut oil. The medium-chain fatty acid includes caprylic acid (8 carbon atoms), capric acid (10 carbon atoms), lauric acid (12 carbon atoms) and the like, and when referred to herein, the type of medium chain fatty acid. There are no particular restrictions on. The ratio of the medium-chain fatty acid present in the medium-chain fatty acid-containing ester is not particularly limited.

In the present specification, "the ability related to the assimilation of a trans fatty acid-containing ester" or "the ability related to the assimilation of a short- to long-chain fatty acid-containing ester" means a trans fatty acid-containing ester or a trans fatty acid-containing ester by a microorganism. Refers to the activity for bringing about the assimilation of short- to long-chain fatty acid-containing esters. In the present specification, "assimilating a trans fatty acid-containing ester" or "assimilating a short- to long-chain fatty acid-containing ester" is used in the meaning commonly used in the art, and a microorganism is used. It means that a trans fatty acid-containing ester or a short- to long-chain fatty acid-containing ester is taken in as a nutrient source such as a carbon source. In the case of "assimilation", it includes hydrolysis to alcohols and free fatty acids, as well as conversion to some of other substances.

In the present specification, "the ability to decompose a trans-fatty acid-containing ester" or "the ability to decompose a short- to long-chain fatty acid-containing ester" means a trans-fatty acid-containing ester or a short to long-chain fatty acid-containing ester. Refers to the activity of hydrolyzing an ester into glycerol or other alcohols (such as 4-nitrophenol) and free fatty acids.

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

As used herein, the "optimal temperature" of the ability to decompose an ester is used in the sense commonly used in the art, and the activity of decomposing an ester is above a certain level desired (eg, the enzyme thereof). Refers to a temperature range in which 80% or more of the activity of the maximum level is maintained, but in another embodiment, it refers to a temperature range in which the maximum level is maintained. Specifically, the first esterase of the present disclosure has a peak activity of decomposing an ester at about 60 ° C., and retains an activity of 80% or more of the peak in the range of about 55 to 65 ° C. The second esterase of the present disclosure has a peak activity of decomposing an ester at about 40 ° C., and retains an activity of 80% or more of the peak in the range of about 35 to 50 ° C. The third esterase of the present disclosure has a peak activity of decomposing an ester at about 65 ° C., and retains an activity of 80% or more of the peak in the range of about 53 to 68 ° C. The fourth esterase of the present disclosure has a peak activity of decomposing an ester at about 65 ° C., and retains an activity of 80% or more of the peak in the range of about 55 to 68 ° C. The fifth esterase of the present disclosure has a peak activity of decomposing an ester at about 40 ° C., and retains an activity of 80% or more of the peak 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 about 55-65 ° C, preferably about 57-63 ° C, more preferably about 60 ° C. Intended. 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., more preferably about 40 ° C. The optimum temperature of the third esterase of the present disclosure is intended to be about 53-68 ° C, preferably about 60-67 ° C, more preferably about 65 ° C. The optimum temperature of the fourth esterase of the present disclosure is intended to be about 55-68 ° C, preferably about 60-67 ° C, more preferably about 65 ° C. The optimum temperature of the fifth esterase of the present disclosure is intended to be about 33-47 ° C, preferably about 37-45 ° C, more preferably about 40 ° C.

In the present specification, "thermal stability" has the same meaning as terms such as "temperature stability" and "heat resistance", and is used in the meaning commonly used in the art, and the enzyme is at a low temperature. And / or means to retain activity at high temperatures. Generally, it is known that an enzyme is inactivated at a temperature of about 50 ° C. due to factors such as a change 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 treatment at 30 ° C. for 30 minutes is 100% is at a temperature of about 10 to about 60 ° C. In the case of the second esterase of the present disclosure, which retains 80% or more after 30 minutes of treatment in the range, the relative enzyme activity when the enzyme activity after 30 minutes of treatment at 45 ° C. is 100% is about 15 to about. It retains 80% or more after 30 minutes of treatment in the temperature range of 60 ° C. In the case of the third esterase of the present disclosure, the relative enzyme activity when the enzyme activity after 30 minutes treatment at 30 ° C. is 100% is 80 after 30 minutes treatment in the temperature range of about 30 to about 65 ° C. Hold% or more. In the case of the fourth esterase of the present disclosure, the relative enzyme activity when the enzyme activity after 30 minutes treatment at 30 ° C. is 100% is 80 after 30 minutes treatment in the temperature range of about 30 to about 45 ° C. Hold% or more. In the case of the fifth esterase of the present disclosure, the relative enzyme activity when the enzyme activity after 30 minutes treatment at 40 ° C. is 100% is 80 after 30 minutes treatment in the temperature range of about 20 to about 45 ° C. Hold% or more. In the present specification, the same expression as "maintaining thermal stability" is about 30 ° C. in the case of the first esterase, about 45 ° C. in the case of the second esterase, and in the case of the third esterase. The relative enzyme activity is approximately 100% when the enzyme activity after 30 minutes of treatment at 30 ° C., 30 ° C. for the fourth esterase, and 40 ° C. for the fifth esterase is 100%. It means that it is retained at 50% or more.

As used herein, the "optimal pH" of the ability to decompose an ester is used in the sense commonly used in the art, and the activity of decomposing an ester is above a certain level (for example, the enzyme has). It refers to the pH range in which 80% or more of the activity of the maximum level is maintained, but in another embodiment, it refers to the pH at the time of the maximum level). The first, second, third and fourth esterases of the present disclosure all have a peak activity of decomposing an ester having a peak of about

pH

9, and 80% or more of the peak in the range of about pH 8.5 to pH 9.5. Retains the activity of. The fifth esterase has a peak activity of decomposing an ester at about pH 8, and retains an activity of 80% or more of the peak in the range of about pH 7.5 to pH 8.5. In the context of referring to the optimum pH of the first, second, third and fourth esterases, the optimum pH of the esterases (all of the first, second, third and fourth) of the present disclosure is pH 8. It is intended to be 5.5 to pH 9.5, preferably pH 8.8 to pH 9.3, more preferably pH 9. In the context of referring to the optimum pH of the fifth esterase, the optimum pH of the fifth esterase of the present disclosure is pH 7.5 to pH 8.5, preferably pH 7.8 to pH 8.3, more preferably pH 8. Intended to be.

In the present specification, "pH stability" of the ability to decompose an ester has the same meaning as terms such as "pH resistance", "acid resistance" and "alkali resistance", and is commonly used in the art. It is used in the sense that it is used, and refers to the pH range in which the activity of decomposing an ester is above a certain level. In the case of the first esterase of the present disclosure, the relative enzyme activity after 30 minutes incubation treatment in a buffer solution of pH 9 is about pH 7.5 to 9.5 when the enzyme activity is 100%. In the case of the second esterase of the present disclosure, which retains 80% or more, the relative enzyme activity when the enzyme activity after 30 minutes incubation treatment in a buffer solution of pH 8 is 100% is about pH 7.5. Retains 80% or more in the process of ~ 9.5. In the case of the third esterase of the present disclosure, the relative enzyme activity when the enzyme activity after incubation treatment in a buffer solution of pH 9 for 30 minutes is 100% is about pH 8.5 to 9.2. Hold 80% or more. In the case of the fourth esterase of the present disclosure, the relative enzyme activity when the enzyme activity after incubation treatment in a buffer solution of pH 9 for 30 minutes is 100% is about pH 7.8 to 9.2. Hold 80% or more. In the case of the fifth esterase of the present disclosure, the relative enzyme activity when the enzyme activity after incubation treatment in a buffer solution of pH 8 for 15 hours is 100% is 80% in the treatment of about pH 4 to 8.5. Hold the above. In the present specification, the same expressions as "maintaining pH stability" include pH 9 in the case of the first esterase, pH 8 in the case of the second esterase, and pH 9 in the case of the third esterase. In the case of the fourth esterase, the relative enzyme activity at pH 9 and in the case of the fifth esterase at pH 8 after incubating for a certain period of time or longer is 100%, and the relative enzyme activity is the same time at the test pH. It means that it is retained at about 50% or more when incubated.

In the present specification, "Yarrowia lipolytica" means a species of the genus Yarrowia in terms of biotaxonomy. Yarrowia lipolytica is a type of alkane assimilating yeast, and has been identified as having a high ability to assimilate hydrophobic hydrocarbon chains such as n-alkane and fat. The Yarrowia lipolytica KH-2 strain (microorganism strain specified by accession number NITE BP-02732) in the present specification is a representative sequence of at least five types of esterases (that is, the "first esterase" of the present disclosure, "No. 1". It has a gene encoding a representative sequence of "2 esterase", a representative sequence of "third esterase", a representative sequence of "fourth esterase", and a representative sequence of "fifth esterase"). It has been found in the present disclosure that these representative esterases of the present disclosure have characteristics not found in conventionally known esterases. These salient features are detailed elsewhere herein. The esterases of the present disclosure include, but are not limited to, those produced by the KH-2 strain belonging to Yarrowia lipolytica.

As used herein, the term "cell-free expression system" is used in the sense commonly used in the art, and uses a transcriptional translation mechanism of a biomolecule extracted from a cell to in vitro the desired recombination. Refers to a system that produces proteins. The cell-free expression system for producing the esterase of the present disclosure is not particularly limited, but a cell-free expression system derived from a prokaryote such as Escherichia coli can be used.

In the present specification, the "oil treatment component" means a component that assists the assimilation and decomposition of the ester. Specifically, in addition to components that promote the dispersion of esters such as surfants, components that decompose esters into fatty acids and alcohols, those that decompose fatty acids, those that decompose alcohols, and oils. Includes those that are adsorbed and removed from the object to be treated.

As used herein, the term "oil-degrading agent" refers to 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. It refers to a preparation that can decompose the ester form as an ingredient. In the present disclosure, the oil decomposing agent may be used in combination with an oil treatment component. In this case, the timing of the combined use of the oil decomposing agent and the oil treatment component may be the simultaneous use or one of them may be used first. Further, the oil decomposing agent includes a microbial strain to be used or a component that enhances the activity of an esterase derived from the microbial strain (for example, a carbon source or a nitrogen source), a surfactant, a desiccant protective agent, a component for maintaining the microorganism for a long period of time, Preservatives, excipients, fortifiers, antioxidants and the like may be further contained.

The oil degrading agent provided in the present disclosure is provided in a liquid or solid or dry state. The liquid form includes a culture solution of microorganisms (which may be concentrated or diluted if necessary), a substance in which an enzyme component derived from the culture solution is adsorbed on a support, and a buffer solution obtained by separating and purifying the enzyme from the culture solution. Examples thereof include those dissolved in a solvent. For solids, those suspended or dissolved in a solvent containing a protective agent such as glycerol and frozen, and those immobilized on a carrier (covalent bond to the carrier, adsorbed by electrostatic interaction / hydrophobic interaction, etc.) Examples thereof include those which have been subjected to molecular cross-linking on a carrier, those which have been dehydrated by centrifugation, press compression, etc., and those which have been dried and dried. In a preferred embodiment, the oil degrading agent is provided in liquid form, powder, granule form and may be provided as a detergent or contained with other ingredients as a component of the detergent.

The "derivatives", "similars" or "variants" used herein (such as esterases) are preferably, but not intended to be limiting, substantially to the protein of interest (eg, esterases). Such molecules include, in various embodiments, sequences that are aligned over amino acid sequences of the same size or by computer homology programs known in the art. Nucleic acids that are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical when compared, or encode such molecules (highly) ) It is possible to hybridize to a sequence encoding a component protein under stringent, moderately stringent, or non-stringent conditions. This is the product of modifying a protein by amino acid substitution, deletion and addition, respectively, and the derivative of which still exhibits the biological function of the original protein, though not necessarily to the same extent. Preferably, it means a protein having the same or higher biological activity. For example, it is also possible to investigate the biological function of such proteins by appropriate and available in vitro assays described herein or known in the art. As used herein, "functionally active" or "having functional activity" is used herein to include biological activity, etc., according to the embodiments in which the polypeptides of the present disclosure, ie fragments or derivatives, are associated. Refers to having a structural function, a regulatory function, or a biochemical function of a protein.

In the present disclosure, an esterase fragment is a polypeptide containing an arbitrary region of esterase, as long as it functions as the object of the present disclosure (for example, decomposition of a trans fatty acid-containing ester or decomposition of a medium chain fatty acid ester). It does not necessarily have all of the biological functions of natural esterases.

In the present specification, "protein", "polypeptide", "oligopeptide" and "peptide" are used interchangeably in the present specification and refer to a polymer of amino acids of any length. The polymer may be linear, branched or cyclic. The amino acid may be natural or non-natural, or may be a modified amino acid. 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 (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids), peptide-like compounds (eg, peptoids) and other modifications known in the art. Will be done. In the present specification, "amino acid" is a general term for organic compounds having an amino group and a carboxyl group. When the protein or enzyme according to the embodiment of the present disclosure contains a "specific amino acid sequence", any amino acid in the amino acid sequence may be chemically modified. Moreover, any amino acid in the amino acid sequence may form a salt or a solvate. Further, any amino acid in the amino acid sequence may be L-type or D-type. Even in such cases, it can be said that the protein according to the embodiment of the present disclosure contains the above-mentioned "specific amino acid sequence". Chemical modifications that amino acids contained in proteins undergo in vivo include, for example, N-terminal modification (eg, acetylation, myristoylation, etc.), C-terminal modification (eg, amidation, glycosylphosphatidylinositol addition, etc.), or side chain. Modifications (eg, phosphorylation, glycosylation, etc.) are known. Amino acids may be natural or non-natural as long as they meet the purposes of the present disclosure.

In the present specification, "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably in the present specification and refer to a polymer of nucleotides of arbitrary length. The term also includes "oligonucleotide derivatives" or "polynucleotide derivatives". "Nucleobase sequence" or "nucleic acid sequence" means the sequence of successive nucleic acid bases in "polynucleotide", "oligonucleotide" or "nucleic acid". The "oligonucleotide derivative" or "polynucleotide derivative" refers to an oligonucleotide or polynucleotide containing a derivative of a nucleotide or having an unusual bond between nucleotides, and is used interchangeably. Specifically, such an oligonucleotide includes, for example, 2'-O-methyl-ribonucleotide, an oligonucleotide derivative in which a phosphate diester bond in an oligonucleotide is converted into a phosphorothioate bond, and a phosphate diester bond in an oligonucleotide. Is an oligonucleotide derivative converted to N3'-P5'phosphoroamidate bond, an oligonucleotide derivative in which ribose and phosphate diester bond in the oligonucleotide are converted into peptide nucleic acid bond, and uracil in the oligonucleotide is C- 5 Oligonucleotide derivatives substituted with propynyl uracil, oligonucleotide derivatives in which uracil in the oligonucleotide is replaced with C-5 thiazole uracil, oligonucleotide derivatives in which cytosine in the oligonucleotide is replaced with C-5 propynyl cytosine, oligonucleotides Oligonucleotide derivatives in which cytosine in nucleotides is replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which ribose in DNA is replaced with 2'-O-propylribose, and ribose in oligonucleotides are 2 Examples thereof include oligonucleotide derivatives substituted with'-methoxyethoxyribose. Unless otherwise indicated, a particular base sequence is also a conservatively modified variant (eg, a degenerate codon substitution) and a complementary sequence, as well as the explicitly indicated sequence. Is intended to be included. Specifically, the degenerate codon substituent creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosin residue. It can be achieved by (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 genes, cDNAs, mRNAs, oligonucleotides, and polynucleotides. As used herein, the "nucleotide" may be natural or non-natural.

In the present specification, the "gene" refers to a factor that defines a genetic trait, and the "gene" may refer to a "polynucleotide", an "oligonucleotide", and a "nucleic acid".

In the present specification, "homology" of a gene means the degree of identity of two or more gene sequences to each other, and generally, having "homology" means a high degree of identity or similarity. Say. Therefore, the higher the homology of two genes, the higher the identity or similarity of their sequences. Whether or not the two genes are homologous can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization under stringent conditions. When comparing two gene sequences directly, the DNA 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% if they are identical, the genes are homologous. Thus, as used herein, a "homologous" or "homologous gene product" is another species, preferably a microorganism, more preferably, which exerts the same biological functions as the protein components of the complex further described herein. Means protein in yeast. Such homologues are also sometimes referred to as "ortholog gene products." It is understood that such homologues, homologous gene products, ortholog gene products and the like can also be used as long as they meet the purposes of the present disclosure. As used herein, the term "similarity" of a gene or base sequence refers to the degree of similarity between two or more gene sequences to each other, and the high degree of similarity of other sequences of identity. "Similarity" is a numerical value that takes into account similar bases in addition to identity. Here, similar bases are mixed bases (for example, 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).

Amino acids may be referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can also be referred to by the generally accepted one-letter code. In the present specification, comparison of amino acid sequence and base sequence similarity, identity and homology is calculated using default parameters using BLAST, which is a tool for sequence analysis. The identity search can be performed using, for example, NCBI's BLAST 2.7.1 (issued October 19, 2017). The value of "identity" in the present specification usually refers to the value when the above BLAST is used and aligned under the default conditions. However, if a higher value is obtained by changing the parameter, the highest value is set as the identity value. When identity is evaluated in multiple regions, the highest value among them is set as the identity value. "Similarity" is a numerical value that takes into account similar amino acids in addition to identity.

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

In one embodiment of the present disclosure, "70% or more", which is a numerical value such as identity, is, 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, and may be within the range of any two of the numerical values that are the starting points thereof. The above "identity" is calculated by calculating the ratio of the number of amino acids homologous in an amino acid sequence between two or a plurality of amino acids according to a known method as described above. Specifically, before calculating the proportion, align the amino acid sequences of the amino acid sequences to be compared and introduce a gap in a part of the amino acid sequence if necessary to maximize the proportion of the same amino acid. To do. Methods for alignment, method of calculating proportions, methods of comparison, and related computer programs are well known in the art (eg, BLAST, described above). As used herein, "identity" and "similarity" can be expressed as values measured by NCBI's BLAST unless otherwise specified. Blastp can be used by default for algorithms when comparing amino acid sequences with BLAST. The measurement results are quantified as Positives or Identities. In this case, the term "similarity" instead of "identity" is a numerical value that also considers those that fall under the definition of "similar" "amino acid" or "base" described in the present specification.

As used herein, the term "polynucleotide that hybridizes under stringent conditions" refers to well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using a polynucleotide selected from the polynucleotides of the present disclosure as a probe and using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. Specifically, using a filter on which DNA derived from colonies or plaques is immobilized, hybridization is performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl, and then the concentration is 0.1 to 2 times higher. It means a polynucleotide that can be identified by washing the filter under 65 ° C. conditions using an SSC (saline-sodium citrate) solution (the composition of the 1-fold SSC solution is 150 mM sodium chloride and 15 mM sodium citrate). .. For the "stringent condition", for example, the following conditions can be adopted. (1) use low ion intensity and high temperature for washing (eg, at 50 ° C., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulphate), (2) during hybridization Use a denaturant such as formamide (eg, at 42 ° C., 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% ficol / 0.1% polyvinylpyrrolidone / 50 mM sodium chloride buffer, pH 6.5, And 750 mM sodium chloride, 75 mM sodium citrate), or (3) 20% formamide, 5 x SSC, 50 mM sodium phosphate (pH 7.6), 5 x denhard solution, 10% dextran sulfate, and 20 mg / ml denaturation. Incubate overnight at 37 ° C. in a solution containing sheared salmon sperm DNA, then wash the filter with 1 × SSC at about 37-50 ° C. The formamide concentration may be 50% or more. The wash time may be 5, 15, 30, 60, or 120 minutes, or longer. Multiple factors such as temperature and salt concentration can be considered as factors that affect the stringency of the hybridization reaction. For details, refer to Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). .. Examples of "highly stringent conditions" are 0.0015M sodium chloride, 0.0015M sodium citrate, 65-68 ° C, or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide, 42. ℃. Hybridization, 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), etc. It can be performed according to. Here, sequences containing only the A sequence or only the T sequence are preferably excluded from the sequences that hybridize under stringent conditions. Moderate stringent conditions can be easily determined by those skilled in the art, eg, based on DNA length, Sambrook et al., Molecular Cloning: A Laboratory Manual, No. 3, Vol. 1, 7.42-7.45 Cold Spring Harbor Laboratory Press, 2001, and for nitrocellulose filters, 5 x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) prewash solution, Other similar hybridizations such as Stark's solution in about 50% formamide at about 40-50 ° C., 2xSSC-6xSSC (or about 50% formamide at about 42 ° C.). Includes the use of solution) hybridization conditions and washing conditions of about 60 ° C., 0.5 × SSC, 0.1% SDS. Accordingly, the polypeptides used in the present disclosure are encoded by nucleic acid molecules that hybridize under highly or moderately stringent conditions to the nucleic acid molecules encoding the polypeptides specifically described herein. Polypeptides are also included.

The esterases of the present disclosure can preferably be "purified" or "isolated". As used herein, a "purified" substance or biological factor (eg, nucleic acid or protein) is one in which at least some of the factors naturally associated with the substance or biological factor have been removed. .. Therefore, the purity of the biological factor in the purified biological factor is usually higher (ie, enriched) than in the state in which the biological factor is normally present. The term "purified" as used herein is 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. It means that the same type of biological factor is present. The substance or biological factor used in the present disclosure is preferably a "purified" substance. As used herein, an "isolated" substance or biological factor (eg, nucleic acid or protein) is substantially depleted of a factor naturally associated with that substance or biological factor. Say something. The term "isolated" as used herein varies depending on its purpose and therefore does not necessarily have to be expressed in purity, but if necessary, preferably at least 75% by weight, more preferably. Means that there is 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. The substance used in the present disclosure is preferably an "isolated" substance or biological factor.

As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 with respect to a full-length polypeptide or polynucleotide (length n). The length of the fragment can be appropriately changed according to its purpose. For example, in the case of a polypeptide, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Amino acids such as 15, 20, 25, 30, 40, 50 and above are mentioned, and lengths represented by integers not specifically listed here (eg, 11) are also suitable as lower limits. obtain. In the case of polynucleotides, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides are mentioned, and are specifically listed here. A length represented by a non-integer (eg, 11) can also be a suitable lower bound. In the present specification, such a fragment is within the scope of the present disclosure, for example, when a full-length one functions as an ester-degrading molecule, as long as the fragment itself also functions as an ester-degrading molecule. Is understood.

As used herein, when referring to a gene or a nucleic acid molecule or polypeptide related thereto, the term "biological function" refers to a specific function that the gene, nucleic acid molecule or polypeptide may have in vivo or in vitro. That is, for example, decomposition of an ester (for example, decomposition of a trans fatty acid-containing ester) and the like can be mentioned, but the present invention is not limited thereto. In the present disclosure, for example, in addition to decomposition of trans fatty acid-containing ester, decomposition of medium-chain fatty acid-containing ester, decomposition of cis fatty acid-containing ester, decomposition of saturated fatty acid-containing ester containing no double bond, and the like are mentioned. Can, but is not limited to them. As used herein, a biological function can be exerted by a corresponding "biological activity". As used herein, the term "biological activity" refers to the activity that a certain factor (for example, polynucleotide, protein, etc.) can have, and has various functions (for example, decomposition activity of a trans fatty acid-containing ester). Activities that exert the above are included. The "biological activity" may be an activity exerted in vivo or an activity exerted in vitro by secretion or the like. For example, if a factor is an enzyme, its biological activity comprises that enzymatic activity. Such biological activity can be measured by techniques well known in the art. Thus, "activity" indicates or reveals binding (either direct or indirect); affects the response (ie, has a measurable effect in response to some exposure or stimulus). Various measurable indicators, such as the affinity of a compound that binds directly to a polypeptide or polynucleotide of the present disclosure, or, for example, the amount of upstream or downstream protein or other after some stimulation or event. Scales of similar function may also be included.

In the present specification, "expression" of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo and becomes another form. Preferably, a gene, polynucleotide, or the like is transcribed and translated into the form of a polypeptide, but transcribing to produce mRNA is also an aspect of expression. Thus, as used herein, an "expression product" includes such a polypeptide or protein, or mRNA. More preferably, the form of such a polypeptide can be post-translationally processed. For example, the expression level of esterase can be determined by any method. Specifically, the expression level of esterase can be known by evaluating the amount of esterase mRNA, the amount of esterase protein, and the biological activity of esterase protein. The amount of esterase mRNA or protein can be determined by methods as detailed elsewhere herein or by other methods known in the art.

In the present specification, the "functional equivalent" means an arbitrary entity having the same target function but a different structure with respect to the target entity. Therefore, the functional equivalent of the "esterase" of the present disclosure is not the esterase itself of the present disclosure, but a variant or a variant thereof (for example, an amino acid sequence variant), and the biological equivalent of the esterase. Those having an action and those capable of being transformed into a variant or a variant having a biological action of the esterase at the time of action (for example, a nucleic acid encoding the variant or the variant, and a nucleic acid thereof). It is understood that vectors containing nucleic acids, including cells, etc.) are included. It is understood that in the present disclosure, functional equivalents of esterases can be used in the same manner as esterases, without special mention. Functional equivalents can be found by searching databases and the like. As used herein, the term "search" refers to finding another nucleobase sequence having a specific function and / or property by utilizing one nucleobase sequence electronically, biologically, or by some other method. Say. Electronic searches include 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)), but not limited to them. Biological searches include stringent hybridization, macroarrays in which genomic DNA is attached to a nylon membrane or the like, or microarrays (microarray assays) in which genomic DNA is attached to a glass plate, PCR and in situ hybridization, and the like. Not limited to. As used herein, it is intended that the genes used in the present disclosure should also include corresponding genes identified by such electronic and biological searches.

As the functional equivalent of the present disclosure, one or more amino acids inserted, substituted or deleted, or added to one or both ends of the amino acid sequence can be used. In the present specification, "insertion, substitution or deletion of one or more amino acids in an amino acid sequence, or addition to one or both ends" is a well-known technical technique such as a site-specific mutagenesis method. It means that the modification has been made by a method or by a natural mutation, such as by substituting a plurality of amino acids that can occur naturally. The modified amino acid sequence includes, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 9, still more preferably 1 to 5, and particularly preferably 1 to 2 amino acids inserted, substituted, or missing. It can be lost, or added to one or both ends. The modified amino acid sequence preferably has one or more (preferably one or more) amino acid sequences in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22. Amino acids with conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) It may be an array. As used herein, "conservative substitution" means substituting 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, there is a case where one hydrophobic residue is replaced with another hydrophobic residue, a case where one polar residue is replaced with another polar residue having the same charge, and the like. Functionally similar amino acids capable of making such substitutions are known in the art for each amino acid. Specific examples include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

In the present specification, the "kit" usually refers to a unit in which parts to be provided (for example, an enzyme, a fat-decomposing agent, a buffer solution, an instruction manual, etc.) are provided by dividing into two or more sections. The form of this kit is preferred when the purpose is to provide a composition that should not be mixed and provided for stability and the like, but is preferably mixed and used immediately before use. Such a kit preferably describes how to use the provided portion (eg, enzyme or oil degrading agent) or how to treat the reagent or waste liquid after use. Or it is advantageous to have instructions. When the kit is used as a reagent kit in the present specification, the kit usually includes an instruction sheet or the like describing how to use an enzyme, a fat-decomposing agent, or the like.

In the present specification, the "instruction" describes the method for using the present disclosure to the user. This instruction sheet contains words indicating how to use the polypeptides, polynucleotides, cells, etc. of the present disclosure. If necessary, this instruction may be provided by the regulatory agency of the country in which this disclosure is implemented (eg, Ministry of Health, Labor and Welfare or Ministry of Agriculture, Forestry and Fisheries in Japan, Food and Drug Administration (FDA), Department of Agriculture (USDA) in the United States. ) Etc.), and it is clearly stated that it has been approved by the regulatory agency. Instructions may be provided in paper media, but are not limited to, and may also be provided in the form of, for example, electronic media (eg, homepages provided on the Internet, email).

(Preferable embodiment)
The preferred embodiments of the present disclosure will be described below. It is understood that the embodiments provided below are provided for a better understanding of the present disclosure and 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 consideration of the description in the present specification. It is also understood that the following embodiments of the present disclosure may be used alone or in combination.

(enzyme)
In one aspect, the present disclosure provides a novel polypeptide and a nucleic acid encoding it. This polypeptide typically has esterase activity. This polypeptide has lipase activity in one embodiment. The polypeptides of the present disclosure have prominent properties that differ from conventionally known enzymes.

In certain embodiments, the present disclosure typically describes a representative sequence of a first esterase, a representative sequence of a second esterase, a third esterase, typically obtained from a KH-2 strain of the genus Yarrowia lipolytica. Provided are a representative sequence, a representative sequence of a fourth esterase, and a representative sequence of a fifth esterase and derivatives thereof.

In one aspect, the polypeptides of the present disclosure are the first to fourth esterases, i.e.
(A) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 2, 8, 12 or 16;
(B) A polypeptide having biological activity, which comprises an amino acid sequence containing one or more amino acid substitutions, additions, deletions or combinations thereof in the amino acid sequence of (a);
(C) A polypeptide having at least 70% or more sequence identity with the amino acid sequence shown in (a) or (b) and having biological activity;
(D) A polypeptide containing an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1, 7, 11 or 15;
(E) In the base sequence shown in (d), a polypeptide having biological activity encoded by a base sequence containing one or more nucleotides of substitution, addition, deletion or a combination thereof;
(F) A polypeptide having biological activity encoded by a base sequence having at least 70% or more sequence identity with the base sequence shown in (d) or (e);
(G) Biological activity encoded by a nucleotide sequence that hybridizes under stringent conditions with a polynucleotide containing the nucleotide sequence shown in any one of (d) to (f) or a complementary sequence thereof. Polypeptide with
(H) A polypeptide having biological activity encoded by an allelic variant of any one of the base sequences of (d) to (g); or the amino acid sequence shown in (i) (a) to (h). Polypeptide, including fragments of
Is a polypeptide.

In an alternative aspect, the polypeptides of the present disclosure are a fifth esterase, ie.
(A) In the amino acid sequence shown in SEQ ID NO: 22, a polypeptide containing an amino acid sequence containing one or more amino acid substitutions, additions, deletions or combinations thereof;
(B) A polypeptide having at least 70% or more sequence identity with the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 22;
(C) In the base sequence shown in SEQ ID NO: 21, a polypeptide encoded by a base sequence containing one or more nucleotides of substitution, addition, deletion or a combination thereof;
(D) A polypeptide encoded by a base sequence having at least 70% or more sequence identity with the base sequence shown in SEQ ID NO: 21;
(E) A polypeptide encoded by a nucleotide sequence containing the nucleotide sequence shown in SEQ ID NO: 21 or a complementary sequence thereof and a nucleotide sequence that hybridizes under stringent conditions;
(F) A polypeptide encoded by an allelic variant of the nucleotide sequence shown in SEQ ID NO: 21; or a polypeptide containing a fragment of the amino acid sequence shown in (g) (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.

In another aspect, the present disclosure provides a base sequence encoding a novel polypeptide. The polynucleotides of the present disclosure are polynucleotides encoding first to fourth esterases, ie
(A) A polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 1, 7, 11 or 15;
(B) In the base sequence shown in (A), a polynucleotide comprising a base sequence containing one or more nucleotides of substitution, addition, deletion or a combination thereof and encoding a polypeptide having biological activity;
(C) A polynucleotide comprising a base sequence having at least 70% or more sequence identity with the base sequence shown in (A) or (B) and encoding a polypeptide having biological activity;
(D) A polynucleotide containing the base sequence shown in any one of (A) to (C) or a complementary sequence thereof, and a base sequence that hybridizes under stringent conditions, and has biological activity. A polynucleotide encoding a polypeptide having
(E) A polynucleotide that is an allelic variant of the base sequence of any one of (A) to (D) and encodes a polypeptide having biological activity;
(F) A polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 8, 12 or 16;
(G) In the amino acid sequence of (F), a polynucleotide encoding a polypeptide having biological activity, which comprises an amino acid sequence containing one or more amino acid substitutions, additions, deletions or combinations thereof;
(H) A polynucleotide encoding a polypeptide having at least 70% or more sequence identity with the amino acid sequence shown in (F) or (G) and having biological activity; or (I) (F) to A polynucleotide containing a fragment of the nucleotide sequence shown in (H),
Can be a polynucleotide.

In another aspect, the present disclosure provides a base sequence encoding a novel polypeptide. The polynucleotides of the present disclosure are those encoding a fifth esterase, ie
(A) In the base sequence shown in SEQ ID NO: 21, a polynucleotide containing a base sequence containing one or more nucleotides of substitution, addition, deletion or a combination thereof;
(B) A polynucleotide comprising a base sequence having at least 70% or more sequence identity with the base sequence shown in SEQ ID NO: 21 and encoding a polypeptide having biological activity;
(C) A polynucleotide encoding a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 21 or a complementary sequence thereof and a nucleotide sequence that hybridizes under stringent conditions and has biological activity. ;
(D) A polynucleotide that is an allelic variant of the nucleotide sequence shown in SEQ ID NO: 21 and encodes a polypeptide having biological activity;
(E) A polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 22;
(F) A polynucleotide encoding a bioactive polypeptide containing 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 encoding a polypeptide having at least 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22 and having biological activity; or (H) (E) to (G). A polynucleotide, which contains a fragment of the nucleotide sequence shown.
Is. In one embodiment, the polynucleotides of the present disclosure are not polynucleotides consisting of the nucleotide sequence set forth in SEQ ID NO: 21. The biological activity includes the ability associated with the assimilation of trans fatty acid-containing esters, or the ability to degrade trans fatty acid-containing esters.

In one embodiment, the biological activity of the polypeptide of the present disclosure is at least one of any properties possessed by the first, second, third or fourth, or fifth esterase of the present disclosure. It may include, for example, the ability associated with the assimilation of trans fatty acid-containing esters, and / or the ability to decompose trans fatty acid-containing esters. Although not bound by theory, conventionally known Yarrowia-derived esterases have not been observed to be active against trans fatty acid-containing esters, and at least in that sense, the esterases of the present disclosure have distinctive features. ..

Thus, in one embodiment, the polypeptide of the present disclosure or the polynucleotide encoded by the polynucleotide has the ability to relate to the assimilation of the trans fatty acid-containing ester, and / or to degrade the trans fatty acid-containing ester. Can have.

In other embodiments, the biological activity of the polypeptides of the present disclosure includes, for example, the ability associated with the assimilation of short- to long-chain fatty acid-containing esters, or short- to long-chain fatty acid-containing esters. It may also include the ability to break down the body or both.

Thus, in one embodiment, the polypeptides or polynucleotides encoded by the disclosures are capable of relating to the assimilation of medium-chain fatty acid-containing esters, or degrading medium-chain fatty acid-containing esters, or the ability thereof. You can have both.

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

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

In one embodiment, the polypeptide or polynucleotide encoded by the first esterase of the present disclosure is 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. It can be stable even at ° C. or higher, and can be stable up to 50 ° C. or lower, 55 ° C. or lower, 60 ° C. or lower, 65 ° C. or lower, 70 ° C. or lower, 75 ° C. or lower, or 80 ° C. or lower. The first esterase of the present disclosure, a polypeptide or a polypeptide encoded by a polynucleotide, can preferably be stable above 50 ° C. and below 70 ° C. The second esterase of the present disclosure, a polypeptide or a polypeptide encoded by a polynucleotide, can be stable at 45 ° C. or higher, 50 ° C. or higher, 55 ° C. or higher, or 60 ° C. or higher, and can be stable at 50 ° C. or higher, 55 ° C. or higher. Below, it can be stable up to 60 ° C. or lower, 65 ° C. or lower, or 70 ° C. or lower. The polypeptide or polynucleotide-encoded polypeptide, which is the second esterase derived from the KH-2 strain of the present disclosure, can preferably be stable above 45 ° C. and below 60 ° C. The third esterase of the present disclosure, a polypeptide or a polypeptide encoded by a polynucleotide, can be stable at 30 ° C. or higher, 40 ° C. or higher, 45 ° C. or higher, 50 ° C. or higher, or 60 ° C. or higher, and can be 60 ° C. or higher. Below, it can be stable up to 65 ° C. or lower, 70 ° C. or lower, or 75 ° C. or lower. The polypeptide or polynucleotide-encoded polypeptide, which is the third esterase derived from the KH-2 strain of the present disclosure, can preferably be stable above 45 ° C. and below 65 ° C. The fourth esterase of the present disclosure, a polypeptide or a polypeptide encoded by a polynucleotide, can be stable at 45 ° C. or higher, 50 ° C. or higher, 55 ° C. or higher, or 60 ° C. or higher, and can be stable at 50 ° C. or higher, 55 ° C. or higher. Below, it can be stable up to 60 ° C. or lower, 65 ° C. or lower, or 70 ° C. or lower. The polypeptide or polynucleotide-encoded polypeptide, which is the fourth esterase derived from the KH-2 strain of the present disclosure, can preferably be stable above 45 ° C. and below 50 ° C. The polypeptide or polynucleotide-encoded polypeptide, which is the fifth esterase from the KH-2 strain of the present disclosure, can preferably be stable above 40 ° C and stable below 20 ° C. .. However, "stable" means that the activity after treatment at each temperature for 30 minutes is maintained at about 80% or more as compared with the case of treatment at about 30 ° C.

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

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

The cells of the present disclosure contain a polypeptide of the first, second, third, fourth or fifth esterase of the present disclosure, or the first, second, third, fourth or fifth of the present disclosure. A polynucleotide encoding an esterase polypeptide is expressively incorporated. In the cell-free expression system, a polynucleotide encoding a polypeptide of the first, second, third, fourth or fifth esterase of the present disclosure is provided so as to be expressible, and the polypeptide is expressed by an appropriate mechanism. Demonstrates an oil decomposition effect.

In one embodiment, the oil decomposing agent comprises an additional oil treatment component. Examples of the oil treatment component include, but are not limited to, other enzymes (eg, esterase, etc.) or microorganisms.

In another aspect, the present disclosure comprises a polypeptide of the present disclosure, or a cell-free or cell-free expression system of the present disclosure, or an oil-degrading agent, and a kit for oil-decomposing agent or oil-fat treatment, comprising an additional oil-treating component. I will provide a. It is understood that the oil degrading agents and oil treatment components included in the kits of the present disclosure may be of any kind as described elsewhere herein in any combination.

In another aspect, the present disclosure provides a method for removing oil decomposition, which comprises allowing the polypeptide of the present disclosure, or the cell- or cell-free expression system of the present disclosure, or the oil-degrading agent of the present disclosure to act on a treatment subject. To do. In the oil decomposition removal method of the present disclosure, the treatment target preferably includes, but is not limited to, a trans fatty acid-containing ester. It is shown herein that even a subject that does not contain a trans fatty acid-containing ester can perform oil decomposition very efficiently when the esterase of the present disclosure is used.

In another aspect, the disclosure provides a detergent comprising the polypeptides of the present disclosure, the cell or cell-free expression system of the present disclosure, or the oil degrading agents of the present disclosure.

In yet another aspect, an application in a fat modification / fat production technique (transesterification, etc.) using the polypeptide of the present disclosure, the cell-free or cell-free expression system of the present disclosure, or the oil-decomposing agent of the present disclosure is provided. Examples of fat modification / fat production techniques (transesterification, etc.) include reactions that produce esters such as methyl esters and ethyl esters, substitution reactions of fat-containing fatty acids, and production of diacylglycerols and monoacylglycerols.

(Enzyme production)
In one embodiment, the esterases of the present disclosure are produced by a secretory production system that uses microorganisms.

Specifically, the esterases of the present disclosure are:
(A) Nucleic acid molecules encoding the full-length sequences of the first, second, third, fourth or fifth esterases of the present disclosure (eg, SEQ ID NOs: 5, 9, 13, 17, 19 or modified sequences thereof). Steps of gene transfer into a pEZZ18 plasmid (GE Healthcare) and expression in Escherichia coli HB101 strain as protein A fusion recombinant protein, and (b) IgG Sepharose 6 Fast Flow or Butyl Sepharose 6 It can be produced by a method that includes a step of purifying with Fast Flow. Illustratively, the nucleotide sequences encoding the first, fourth and fifth esterases of the present disclosure are inserted into the XbaI and KpnI sites within the pEZZ18 vector, and the nucleotide sequences encoding the second esterase are PstI and The nucleotide sequence inserted into the KpnI site and encoding the third esterase is inserted into the EcoRI and XbaI sites. In addition to the above-mentioned secretory production system using Escherichia coli, the esterase recombinant protein of the present disclosure uses a CORYNEX® system (Ajinomoto) using Corynebacterium glutamicum, a Pichia pastoris expression system (ThermoFisher), and insect cells. It can be produced by an expression system such as a baculovirus expression system (ThermoFisher) or a protein secretion expression system using Aspergillus oryzae (Ozeki). In the method for producing esterase using this microorganism, detailed conditions such as a method for expressing esterase, a host for expressing esterase, and a method for purifying esterase secreted from the host are appropriately adjusted by those skilled in the art. Further, the esterase of the present disclosure can be expressed by using various expression systems such as an intracellular expression system and a cell-free expression system in addition to the secretory production system of microorganisms.

Alternatively, the esterases of the present disclosure are:
(A) Mutations are introduced into the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21 or other basic nucleotide sequences, and SEQ ID NOs: 1, 3, 3, A step of obtaining a modified sequence of the base sequence shown in 5, 7, 9, 11, 13, 15, 17, 19 or 21 or another basic base sequence.
(B) A suitable host as a recombinant protein (if necessary, fused with a tag sequence (for example, protein A tag) that facilitates purification) by introducing the above-mentioned modified sequence into a plasmid as appropriate. For example, the step of expressing in Escherichia coli HB101 strain) and (c) the protein produced in the host are purified by an appropriate separation method (for example, chromatography such as IgG Sepharose 6 Fast Flow or Butyl Sepharose 6 Fast Flow). It can be produced by a method involving steps. Illustratively, the variants 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 are the above-mentioned restriction enzyme sites in the pEZZ18 vector. Is inserted into. The step of introducing a mutation into the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21 is a commonly used partial-specific mutagenesis method, mutagenesis method. Alternatively, it is appropriately carried out by using a molecular evolution method using error prone PCR or a commercially available kit.

Alternatively, the esterases of the present disclosure can be purified, for example, from the Yarrowia lipolytica KH-2 strain. Specifically, the esterases of the present disclosure are:
(A) A step of culturing the KH-2 strain in a medium containing 1% (v / v) canola oil at 28 ° C. for 24 hours.
(B) The culture supernatant of (a) is sterilized with a 0.45 μm pore filter and buffered with 20 mM Tris-HCl (pH 7.0) containing _{0.5 M NaCl and 2 mM CaCl 2 in an amount about 10 times the column volume.} A step of applying to Butyl Sepharose 6 Fast Flow (GE Healthcare) pre-equilibrium with a solution (hereinafter referred to as Tris buffer) and allowing it to stand for about 1 hour to adsorb hydrophobic proteins on a column.
(C) The column of (b) is washed with a Tris buffer solution having an amount of about 10 times the column volume, and then washed three times with a Tris buffer solution containing no NaCl in an amount of about 1 times the column volume, and is weak against the column carrier. The step of eluting the bound protein and the columns of (d) and (c) were washed 3 times with Tris buffer containing 1% Triton® X-100, which is about 1 times the volume of the column, and the columns. It can be purified by a method including a step of eluting a protein strongly bound to. In the method for purifying the enzyme, detailed conditions such as culture conditions for the KH-2 strain and column chromatography conditions are appropriately adjusted by those skilled in the art.

(Use of enzyme)
In one aspect, the esterases of the present disclosure are useful in the treatment of fats and oils, and can be used in the treatment of wastewater containing fats and oils, waste liquids and the like. In certain embodiments, the esterases of the present disclosure are used in wastewater treatment. When used for wastewater treatment, the esterases of the present disclosure can be used in applications such as factory wastewater, kitchen wastewater, and residential wastewater.

In another aspect, the esterase of the present disclosure is used as a detergent. When used as a detergent, the esterases of the present disclosure can be used in applications such as laundry detergents, kitchen detergents, cleaning detergents, and industrial detergents. The detergents containing esterases of the present disclosure are particularly useful for drain cleaners such as pipe cleaners.

In other aspects, the esterases of the present disclosure are used in fat modification and fat production techniques. When used in fat modification / fat production technology, the esterases of the present disclosure can be used in applications such as food, industrial, and fuel.

In other aspects, the esterases of the present disclosure can be used as an environmental pollution control measure. When used in environmental pollution control, the esterases of the present disclosure can be used to remove pollutants in soil pollution, groundwater pollution, marine pollution, etc. by oil.

In other aspects, the esterases of the present disclosure are used in waste treatment and composting. When used in waste treatment and composting, the esterases of the present disclosure include food waste treatment including annihilation type, composting, feed conversion of agricultural products and food waste, oily sludge from pressurized levitation separators and grease traps. It can be used in applications such as volume reduction.

In other aspects, the esterases of the present disclosure are used as pharmaceuticals. When used as a pharmaceutical product, the esterases of the present disclosure can be used in applications such as digestive agents and lipolysis promoters.

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.

When the esterase of the present disclosure is used for various purposes, 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 will appropriately use the present invention in an appropriate form. The disclosed esterases can be used.

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

(Note)
As used herein, "or" is used when "at least one" of the matters listed in the text can be adopted. The same applies to "or". When the specification "within the range of two values" is specified in the present specification, the range also includes the two values themselves.

References such as scientific literature, patents, and patent applications cited herein are incorporated herein by reference in their entirety to the same extent as they are specifically described.

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

Examples are described below. The handling of organisms used in the following examples complied with the standards stipulated by Nagoya University, regulatory agencies and the Cartagena Law, if necessary. Specifically, the reagents described in the examples were used, but other manufacturers (Sigma-Aldrich, Fujifilm Wako Pure Chemical Industries, Inc., Nacalai Tesque, R & D Systems, USCN Life Science INC, Thermo Fisher Scientific, Funakoshi , Tokyo Kasei, Merck, etc.) can be substituted. Unless otherwise specified, the concentration of various fats and fatty acids added to the medium refers to the final concentration of the substance in the medium, and the percentage notation indicates the volume / volume of the liquid fats and fatty acids. In terms of volume (v / v%), solid properties represent weight / volume (w / v%).

(Example 1: Summary of gene and amino acid sequence encoding esterase derived from KH-2 strain)
This example provides a summary of the genes and amino acid sequences encoding the esterases of the present disclosure.

(Experimental method)
The whole genome sequence of the KH-2 strain was performed. The gene encoding the first esterase (gene number G4021), the gene encoding the second esterase (gene number G2944), the gene encoding the third esterase (gene number G2601), the gene encoding the fourth esterase The estimated function of (gene number G5791) and the gene encoding the fifth esterase (gene number G3702), the presence or absence of the signal peptide (SP), and the estimated molecular weight of the gene product are shown (FIG. 1).

(result)
The first, second, third, fourth and fifth esterases found in Example 1 are cholesterol esterase activity, triglycerol lipase activity, patatin and phospholipase activity, patatin and phospholipase activity, and triglycerol lipase, respectively. It was presumed to have activity. The first, second, third, fourth and fifth esterases are all presumed to have a signal peptide (SP) sequence, and their molecular weights are 63.5 kDa, 61 kDa, 62.9 kDa, 69. It was estimated to be 6 kDa and 36.7 kDa.

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

(Experimental method)
The KH-2 strain was cultured overnight in LB medium, and the culture was washed twice with TBS buffer (137 mM NaCl, 2.68 mM KCl, 25 mM Tris, pH 7.4) to remove the LB medium. The washed KH-2 strain was inoculated into 3 L of BS medium by adding canola oil having a volume ratio of 1% to a final concentration of OD ₆₆₀ = 0.05, and subjected to fermenter culture at 15 ° C. Total RNA was extracted from the cultures 24 hours, 48 hours and 72 hours after culture using the Hypure RNA Isolation Kit.
Using 200 ng of total RNA as a template, genomic DNA was removed and cDNA was synthesized using ^{PrimeScript TM RT reagent Kit with gDNA Laser Perfect Real Time (Takara Bio Inc.).} The cDNA was used by adding twice the amount of pure water and diluting it. Quantitative real-time RT-PCR was performed by Applied Biosystems® ^{StepOnePlus TM} (Applied Biosystems) using synthetic primers specific for each esterase-encoding gene. The PCR reaction was ^{performed in a 20 μl solution containing PowerUp TM} 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 by a program in which one cycle of denaturation at 95 ° C. for 20 seconds was followed by a cycle of 95 ° C. for 1 second and 60 ° C. for 20 seconds repeated 40 times. The expression level was normalized by the expression level of 1,2-manosyltransphase (arg9). The data were analyzed by the comparative Ct method (ΔΔCt method) after confirming that the melting curve had a single peak. Genes encoding the first, second, third, fourth and fifth esterases when the expression level of each esterase gene expressed when cultured in LB medium containing no fat was set to 1 as a control. The expression level was calculated as a relative expression level.

(result)
By feeding the KH-2 strain with fat and oil as a carbon source, four types of esterases (a representative sequence of the first esterase, a representative sequence of the second esterase, and a third esterase) at 24 hours. Induction of expression of the gene encoding the representative sequence of the above and the representative sequence of the fourth esterase) was observed. Expression induction of these esterases was observed even 72 hours after culturing. On the other hand, the expression level of the gene encoding the fifth esterase was equivalent to that obtained by culturing in LB medium, suggesting that this gene is constitutively expressed regardless of the carbon source.

(Example 3: Production of first, second, third, fourth and fifth esterases by recombinant Escherichia coli)

(Experimental method)
In this example, the pEZZ18 Protein A Gene Fusion Vector System (GE) is used to express recombinant proteins of representative sequences of the first, second, third, fourth and fifth esterases in E. coli cells. Healthcare) was used. The nucleotide sequences of the genes encoding the representative sequences of the first, second, third, fourth and fifth esterases are amplified by PrimestarMax, respectively, and the amplified transgenes are XbaI and KpnI in the pEZZ18 vector. It was inserted into sites (first, fourth and fifth esterases), PstI and KpnI sites (second esterases), EcoRI and XbaI sites (third esterases). The E. coli strain containing the transgene was cultured in 200 ml of LB medium containing ampicillin at 37 ° C. for 48 hours and then at 42 ° C. for 3 hours. The cells were removed by centrifugation and the recombinant protein was adsorbed on 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 CaCl 2 and 0.5% Triton® X-100.}

(Example 4: Measurement of optimum temperature, thermal stability, optimum pH and pH stability)
In this example, the optimum temperature, thermal stability, optimum pH and pH stability of the representative sequences of the first, second, third and fourth esterases produced by recombinant Escherichia coli and crudely purified. Is shown.

Escherichia coli expressing representative sequences of the first, second, third and fourth recombinant esterases was 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 the mixture was allowed to stand at 4 ° C. for 24 hours. Centrifugation is then performed to obtain pellets, which are suspended in 1 ml buffer (20 mM Tris-HCl (pH 7.4), 0.5% Triton® X-100 in _{2 mM CaCl 2).} The crude esterase obtained in the above was used as an analysis target. The optimum temperature was examined by mixing (A and E) crude esterase with a buffer solution adjusted to each temperature between 0 ° C. and 100 ° C. and measuring the esterase activity. The crude esterase (B and F) was mixed with a buffer adjusted to each temperature between 0 ° C. and 100 ° C. and incubated for 30 minutes, and then the esterase activity was measured to examine the thermal stability. (C and G) crudely purified esterase was used in acetate buffer (pH 3.0 to 5.0), sodium phosphate buffer (pH 5.0 to 7.0), and Tris-HCl buffer (pH 7.0 to 9.). Optimal pH was determined by mixing with 0) or CAPS buffer (pH 9.0 to 11.0) and measuring esterase activity. (D and H) crudely purified esterase was used in acetate buffer (pH 3.0 to 5.0), sodium phosphate buffer (pH 5.0 to 7.0), and Tris-HCl buffer (pH 7.0 to 9.). After mixing with 0) or CAPS buffer (pH 9.0 to 11.0) and incubating for 30 minutes, pH stability was examined by measuring esterase activity.

(result)
The optimum temperature of the representative sequence of the first esterase was about 60 ° C., and showed thermal stability in the temperature range between about 10 ° C. and about 60 ° C. The optimum pH of the representative sequence of the first esterase was pH 9.0, and 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 showed thermal stability in the temperature range between about 10 ° C. and about 50 ° C. The optimum pH of the representative sequence of the second esterase was pH 9.0, and 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 showed thermal stability in the temperature range between about 30 ° C. and about 65 ° C. The optimum pH of the representative sequence of the third esterase was pH 9.0, and showed pH stability in the pH range of about pH 8 to about pH 9.5. The optimum temperature of the representative sequence of the fourth esterase was about 65 ° C., and showed thermal stability in the temperature range between about 30 ° C. and about 70 ° C. The optimum pH of the representative sequence of the fourth esterase was pH 9.0, and showed pH stability in the pH range of about pH 7 to about pH 9.5.

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

(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. Novozyme 51032 lipase (N-51032) was purchased from Novozymes. Each esterase or lipase was dissolved in 20 mM Tris-HCl, 2 mM CaCl ₂ buffer (pH 7.4) containing 0.25% Triton® X-100 to a concentration of 15 U / ml for Novozym 51032 lipase. A concentration of 0.5 U / ml for the representative sequences of the first esterase and the second esterase, and 0.1 U / ml for the representative sequences of the third, fourth and fifth esterases. Adjusted to concentration. At that time, palmitate (C16) for the representative sequences of the first and second esterases, and butyrate (C4) 4-nitrophenyl for the representative sequences of the third, fourth and fifth esterases. The concentration of the enzyme solution was adjusted based on the enzyme activity in which the amount of 4-nitrophenol produced by the hydrolysis of the ester by esterase or lipase was quantified by the absorbance at 410 nm using the ester (pNP-ester) as a substrate. A ventilation fan filter on which oil stains were deposited was cut into 2 cm squares and placed in a plate, a solution of each esterase or lipase was added to each plate, and the mixture was immersed for 30 minutes.

(result)
The ventilation fan filter after immersion in the esterase or lipase solution and the esterase or lipase solution after immersion are shown in FIG. All of the ventilation fan filters immersed in the representative sequences of the first, second, third and fourth esterases of the present disclosure have significantly more oil stains than Novozym 51032 lipase, and the fifth esterase. It was also found that the decomposition of oil stains was improved to some extent.

(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 trioleidin by recombinant proteins of the first, second, third, fourth and fifth esterases of the present disclosure is shown.

(Experimental method)
(A: Decomposition of shorting by first, second, third, fourth and fifth esterases)
Each purified recombinant esterase produced by the step of Example 3 was prepared so as to have a final concentration of 0.2 u / ml. 2 ml of each esterase solution of the present disclosure was placed in a plate and 0.7 g of shortening was added thereto. Incubation was carried out at 28 ° C. for 24 hours with appropriate stirring and blending with the solution. The one treated with only the above-mentioned elution buffer was used as a control.

(B: Decomposition of lard by first, second, third, fourth and fifth esterases)
Each purified recombinant esterase produced by the step of Example 3 was prepared so as to have a final concentration of 0.2 u / ml. 2 ml of each esterase solution of the present disclosure was placed in a plate and 0.5 g of lard was added thereto. Incubation was carried out at 28 ° C. for 24 hours with appropriate stirring and blending with the solution. The one treated with only the above-mentioned elution buffer was used as a control.

(C: Degradation of lard, shortening, triolein and trioleidin by first, second, third, fourth and fifth esterases)
Each purified recombinant esterase produced by the step of Example 3 was mixed with lard, shortening, triolein and trioleidin, 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 the mixture 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 developing a color with molybtriic acid n-hydrate (2.4 g / 60 ml EtOH).

(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, respectively. From the results of (A) to (C), the recombinant proteins of the representative sequences of the first, second, third and fourth esterases of the present disclosure are all of lard, shortening, triolein and trioleidin. It has been shown to have the activity of decomposing all fats and oils. Therefore, the recombinant proteins of the representative sequences of the first, second, third and fourth esterases of the present disclosure are both triolein, which is a cis triglyceride, and trioleidin, which is a trans triglyceride. It was revealed that it has an activity of hydrolyzing. Furthermore, it was shown that the decomposition activity for shortening was remarkably high. Furthermore, it was shown that the recombinant protein of the representative sequence of the fifth esterase also has the degrading activity of these fats and oils, although it is inferior to the degrading activity of these esterases.

(Example 7: Comparison of substrate specificity of first, second, third, fourth and fifth esterases)
In this example, the degrading activity of each substrate by the first, second, third, fourth and fifth esterases of the present disclosure was compared.

(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 using 4-nitrophenyl ester (pNP-ester) of five fatty acids (acetate (C2), butyrate (C4), octanoate (C8), laurate (C12) and palmitate (C16)) as substrates. , The amount of 4-nitrophenol produced by the hydrolysis of the ester by esterase was measured by quantifying the absorbance at 410 nm. As the substrate solution, 0.05 mol of each substrate was mixed with 12 ml of a 3% (v / v) Triton® X-100 aqueous solution and melted at 70 ° C. was used. Each substrate solution was mixed with 150 mM GTA buffer (pH 7.0) and 60 μl of each sample, and the absorbance at 410 nm immediately after mixing and the absorbance at 410 nm 30 minutes after mixing were measured. Then, the esterase activity was calculated by subtracting the absorbance at 410 nm immediately after mixing from the absorbance at 410 nm 30 minutes after mixing, mixing the buffer solution alone with the substrate, and further subtracting the value measured under the same conditions. Regarding the activity of the fifth esterase, the esterase activity was calculated by measuring the absorbance at 410 nm for 1 minute after mixing the substrate solution and the sample solution containing the fifth esterase. For esterase activity, the amount of enzyme that liberates 1 μM 4-nitrophenol is defined as 1 unit, the number of units per 1 ml of sample is calculated, and the relative activity is 100% with the one showing the maximum activity in each substrate as 100%. Was calculated.

(result)
The results of Example 7 are shown in FIG. 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 chains. The second esterase had the highest esterase activity on lipids having a butyrate (C4) carbon chain, and also showed high esterase activity on octanoate (C8). The third esterase had the highest esterase activity on lipids having a butyrate (C4) carbon chain. The fourth esterase had the highest esterase activity on lipids having a butyrate (C4) carbon chain, and also showed high esterase activity on 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).

(Example 8: Comparison of decomposition activity of trans fatty acid-containing ester by first, second, third, fourth and fifth esterases by serial dilution)
In this example, the degrading 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.

(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. For the measurement of the degrading activity of the trans fatty acid-containing ester, 0.01u / u / of the representative sequences of the first, second, third, fourth and fifth esterases using triellaidin as the ester. It was serially diluted to ml, 0.001u / ml, and 0.0001u / ml and analyzed by thin layer chromatography according to the following experimental conditions.
-Type of ester: Triellaidin-Reaction solution: 20 mM Tris-HCl (pH 7.0), 2 mM CaCl ₂ , 0.5% Triton® X100
・ Ester final concentration: 0.2%
-Treatment method: Put 100 μl of enzyme solution in an Eppendorf tube and put it in.
1/10 amount of each 10x ester stock was added. (0.02u / ml: NP-butyrate as substrate)
・ Processing temperature: 37 ℃
-Treatment time: 48 hours-Oil extraction: Extraction was performed with half the amount of chloroform, and 10 μl was applied to perform thin layer chromatography.
-Development plate: Silica gel coat plate-Development solvent: chloroform: acetone: methanol = 96: 4: 2
-Detection: Color development by molybtriic acid n-hydrate (2.4 g / 60 ml EtOH)

(result)
The representative sequence of the fifth esterase of the present disclosure was found to be active when treated at a concentration of 0.01 u / ml for 48 hours, but when treated at a concentration of 0.001 u / ml for 48 hours. , Almost completely lost activity. The representative sequences of the first, second, third and fourth esterases were found to be active even when diluted to 0.001 u / ml. From this, the representative sequence of the fifth esterase has about 1/10 of the trans fatty acid degrading activity of the representative sequences of the first, second, third and fourth esterases. Was presumed.

(Example 9: Decomposition of trans fatty acid-containing ester at low temperature)
In this example, the degrading activity of the trans fatty acid-containing ester by the first, second, third and fourth esterases of the present disclosure at 15 ° C. was compared.

(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 the trans fatty acid-containing ester was measured by thin layer chromatography at a treatment temperature of 15 ° C. using trielaidic acid as the ester according to the following experimental conditions.
-Type of ester: Triellaidin-Reaction solution: 20 mM Tris-HCl (pH 7.0), 2 mM CaCl ₂ , 0.5% Triton® X100
・ Ester final concentration: 0.2%
-Treatment method: Put 100 μl of enzyme solution in an Eppendorf tube and put it in.
1/10 amount of each 10x ester stock was added. (0.02u / ml: NP-butyrate as substrate)
・ Processing temperature: 15 ℃
-Treatment time: 72 hours-Oil extraction: Extraction was performed with half the amount of chloroform, and 10 μl was applied to perform thin layer chromatography.
-Development plate: Silica gel coat plate-Development solvent: chloroform: acetone: methanol = 96: 4: 2
-Detection: Color development by molybtriic acid n-hydrate (2.4 g / 60 ml EtOH)

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

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

(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, to which triolein and methanol were added. Treatment without addition of esterase solution was also performed at the same time to prepare a control group. Then, 100 μl of each solution was dispensed into a tube with a screw cap and allowed to stand in an incubator at 37 ° C. for 96 hours. Then 100 μl of water and 50 μl of chloroform were added, stirred well with a vortex mixer, and then centrifuged at 12,000 g for 5 minutes. A thin layer chromatographic analysis was performed by loading 10 μl of the lower chloroform layer and using hexane-diethyl ether-acetic acid (80: 20: 1) as the developing solvent.

(result)
The results of Example 10 are shown in FIG. Any of the representative sequences of the first, second, third and fourth esterases catalyzed the transesterification reaction of triolein, which is a cis triglyceride, to produce oleic acid methyl ester.

(Example 11: Modification of trans fatty acid)
In this example, the trans fatty acid was modified (methyl esterified) by the first, second, third and fourth esterases of the present disclosure.

(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 in which the monomer of (A) palmiterazic acid or (B) vaccenic acid was dissolved in methanol so as to be 0.5%. 30 μl of each trans fatty acid stock solution and 70 μl of the enzyme solution were dispensed into a tube with a screw cap and allowed to stand in an incubator at 37 ° C. for 72 hours. Treatment using Buffer instead of esterase solution was performed at the same time to prepare a control group. Then, 100 μl of water and 50 μl of chloroform were added, and the mixture was stirred well with a vortex mixer and then centrifuged at 12,000 g for 5 minutes. A thin layer chromatography analysis was performed by loading 5 μl of the lower chloroform layer and using hexane-diethyl ether-acetic acid (80: 20: 1) as the developing solvent.

(result)
The results of Example 11 are shown in FIG. Any esterase of the representative sequences of the first, second, third and fourth esterases catalyzes the transesterification reactions of the trans fatty acids palmiteraidic acid and baxenoic acid, respectively. And produced a methyl ester of baxenoic acid.

(Example 12: Comparison with the second and fifth esterases in the modification of trans fatty acids)
In this example, the trans fatty acid was modified (methyl esterified) using the second and fifth esterases of the present disclosure.

(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 in which the monomer of (A) palmiterazic acid or (B) vaccenic acid was dissolved in methanol so as to be 0.5%. 30 μl of each trans fatty acid stock solution and 70 μl of the enzyme solution were dispensed into a tube with a screw cap and allowed to stand in an incubator at 37 ° C. for 72 hours. Treatment using Buffer instead of esterase solution was performed at the same time to prepare a control group. Then, 100 μl of water and 50 μl of chloroform were added, and the mixture was stirred well with a vortex mixer and then centrifuged at 12,000 g for 5 minutes. A thin layer chromatography analysis was performed by loading 5 μl of the lower chloroform layer and using hexane-diethyl ether-acetic acid (80: 20: 1) as the developing solvent.

(result)
The results of Example 12 are shown in FIG. The representative sequence of the fifth esterase is different from that of the representative sequence of the second esterase of the present disclosure showing the methyl esterification catalytic activity of palmiterazic acid, and is different from that of the transesterification reaction of palmiterazic acid. No catalytic activity was observed. When vaccenic acid is used as the trans fatty acid, the representative sequence of the fifth esterase has a lower catalytic activity for the transesterification reaction than that of the representative sequence of the second esterase of the present disclosure. , The catalytic activity of the transesterification reaction was confirmed.

(Example 13: Preparation of modified esterase)
In this example, a variant of the esterase of the present disclosure is prepared.
(Method and material)
In the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21, one or more bases are inserted, substituted or deleted, or one or both ends thereof. Make a construct containing the added one. The construction is designed using the same method as in the above embodiment, or by using experimental methods or commercially available kits commonly used in the art known to those skilled in the art. Alternatively, an oligonucleotide encoding a polypeptide in which the amino acid of the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22 is substituted is artificially synthesized. The decomposing activity of the fats and oils or esters of these various variants is analyzed by the same method as in the above-mentioned examples.
(Preparation of expression vector)
The expression vector is prepared according to the method described in Example 3 above.
That is, an expression vector containing mutants having various base sequences containing mutations is prepared by the above-mentioned method of inserting mutations.
(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 prepared mutants are measured.
(Ventilation fan filter dirt cleaning effect and decomposition of ester)
Tests are carried out according to Examples 5 and 6, respectively, and their degrading activity is measured.
(Substrate specificity)
The test is carried out according to Example 7 to measure the substrate specificity.
(result)
As a result of the above tests, the activity found in the present disclosure (eg, eg) of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22. It can be seen that a mutant strain having an ability related to assimilation of a trans fatty acid-containing fat or oil, or an ability to decompose a trans fatty acid-containing fat or oil) can be obtained.

(Note)
As described above, the present invention has been illustrated using the preferred embodiments of the present invention, but it is understood that the scope of the present invention should be interpreted only by the scope of claims. The patents, patent applications and other documents cited herein should be incorporated herein by reference in their content as they are specifically described herein. Is understood. This application is accompanied by a priority claim for Japanese Patent Application No. 2019-163343 submitted to the Japan Patent Office on September 6, 2019, the contents of which are all incorporated by reference in this specification. Is understood.

The present disclosure has usefulness in the treatment of trans fatty acid-containing wastewater, which is a problem in food factories and the like, in decomposing trans fatty acid-containing esters.

NITE BP-02732

SEQ ID NO: 1 Mature sequence number of the representative base sequence of the first esterase of the present disclosure SEQ ID NO: 2 Mature sequence number of the representative amino acid sequence of the first esterase of the present disclosure SEQ ID NO: 3 Representative base of the first esterase of the present disclosure Nucleotide sequence containing a nucleotide encoding a pre-sequence in the sequence SEQ ID NO: 4 Amino acid sequence number containing a pre-sequence in the representative amino acid sequence of the first esterase of the present disclosure 5 The representative base sequence of the first esterase of the present disclosure Full-length SEQ ID NO: 6 Full-length sequence of the representative amino acid sequence of the first esterase of the present disclosure SEQ ID NO: 7 Mature sequence of the representative base sequence of the second esterase of the present disclosure SEQ ID NO: 8 Representative of the second esterase of the present disclosure Mature Nucleotide Sequence SEQ ID NO: 9 Full-length Nucleotide Sequence of the Representative Nucleotide Sequence of the Second Esterase of the Present Disclosure SEQ ID NO: 10 Full-length Nucleotide Sequence of the Representative Nucleotide Sequence of the Second Esterase of the Disclosure 11 Mature Nucleotide Sequence No. 12 of the Representative Nucleotide Sequence of Esterase of No. 12 Mature Nucleotide Sequence No. 12 of the Representative Nucleotide Sequence of the Third Esterase of the present Disclosure. Full-length sequence of the representative amino acid sequence of the third esterase of the disclosure SEQ ID NO: 15 Mature sequence of the representative nucleotide sequence of the fourth esterase of the present disclosure SEQ ID NO: 16 Mature sequence of the representative amino acid sequence of the fourth esterase of the present disclosure SEQ ID NO: 17 Full-length sequence number of the representative base sequence of the fourth esterase of the present disclosure SEQ ID NO: 18 Full-length sequence number of the representative amino acid sequence of the fourth esterase of the present disclosure SEQ ID NO: 19 Representative base of the fifth esterase of the present disclosure Full-length sequence of the sequence SEQ ID NO: 20 Full-length sequence of the representative amino acid sequence of the fifth esterase of the present disclosure SEQ ID NO: 21 Mature sequence of the representative base sequence of the fifth esterase of the present disclosure SEQ ID NO: 22 The fifth esterase of the present disclosure Mature sequence of typical amino acid sequence of

Claims

(A) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 2, 8, 12 or 16;
(B) A polypeptide having biological activity, which comprises an amino acid sequence containing one or more amino acid substitutions, additions, deletions or combinations thereof in the amino acid sequence of (a);
(C) A polypeptide having at least 70% or more sequence identity with the amino acid sequence shown in (a) or (b) and having biological activity;
(D) A polypeptide containing an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1, 7, 11 or 15;
(E) In the base sequence shown in (d), a polypeptide having biological activity encoded by a base sequence containing one or more nucleotides of substitution, addition, deletion or a combination thereof;
(F) A polypeptide having biological activity encoded by a base sequence having at least 70% or more sequence identity with the base sequence shown in (d) or (e);
(G) Biological activity encoded by a nucleotide sequence that hybridizes under stringent conditions with a polynucleotide containing the nucleotide sequence shown in any one of (d) to (f) or a complementary sequence thereof. Polypeptide with
(H) A polypeptide having biological activity encoded by an allelic variant of any one of the base sequences of (d) to (g); or the amino acid sequence shown in (i) (a) to (h). Polypeptide, including fragments of
Is a polypeptide.
The polypeptide according to claim 1, wherein the polypeptide is an esterase.
The polypeptide according to claim 1 or 2, wherein the biological activity includes an ability associated with assimilation of a trans fatty acid-containing ester or an ability to decompose a trans fatty acid-containing ester.
The polypeptide according to any one of claims 1 to 3, wherein the polypeptide is an esterase, and the target of its substrate specificity is a short- to long-chain fatty acid-containing ester containing fats and oils.
The polypeptide according to any one of claims 1 to 4, which is an esterase having an ability to decompose an ester at 15 ° C.
(A)
(A) 75 ° C,
(B) 65 ° C,
(C) 67 ° C or (d) 70 ° C
Has thermal stability in and / or (B)
(A) 60 ° C,
(B) 40 ° C or (c) 65 ° C
Has the optimum temperature in and / or (C)
(A) pH 8-9, or (b) pH 9
Have pH stability in and / or
(D)
Has optimal pH at pH 9,
The polypeptide according to any one of claims 1 to 5.
The polypeptide according to any one of claims 1 to 6, which is a polypeptide derived from Yarrowia lipolytica.
(A) A polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 1, 7, 11 or 15;
(B) In the base sequence shown in (A), a polynucleotide comprising a base sequence containing one or more nucleotides of substitution, addition, deletion or a combination thereof and encoding a polypeptide having biological activity;
(C) A polynucleotide comprising a base sequence having at least 70% or more sequence identity with the base sequence shown in (A) or (B) and encoding a polypeptide having biological activity;
(D) A polynucleotide containing the base sequence shown in any one of (A) to (C) or a complementary sequence thereof, and a base sequence that hybridizes under stringent conditions, and has biological activity. A polynucleotide encoding a polypeptide having
(E) A polynucleotide that is an allelic variant of the base sequence of any one of (A) to (D) and encodes a polypeptide having biological activity;
(F) A polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 8, 12 or 16;
(G) In the amino acid sequence of (F), a polynucleotide encoding a polypeptide having biological activity, which comprises an amino acid sequence containing one or more amino acid substitutions, additions, deletions or combinations thereof;
(H) A polynucleotide encoding a polypeptide having at least 70% or more sequence identity with the amino acid sequence shown in (F) or (G) and having biological activity; or (I) (F) to A polynucleotide containing a fragment of the nucleotide sequence shown in (H),
Is a polynucleotide.
The polynucleotide according to claim 8, wherein the polypeptide is an esterase.
The polynucleotide according to claim 8 or 9, wherein the biological activity includes an ability associated with assimilation of a trans fatty acid-containing ester or an ability to decompose a trans fatty acid-containing ester.
The polynucleotide according to any one of claims 8 to 10, wherein the polynucleotide is an esterase, and the target of the substrate specificity is a short- to long-chain fatty acid-containing ester containing fats and oils.
(A) In the amino acid sequence shown in SEQ ID NO: 22, a polypeptide containing an amino acid sequence containing one or more amino acid substitutions, additions, deletions or combinations thereof;
(B) A polypeptide having at least 70% or more sequence identity with the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 22;
(C) In the base sequence shown in SEQ ID NO: 21, a polypeptide encoded by a base sequence containing one or more nucleotides of substitution, addition, deletion or a combination thereof;
(D) A polypeptide encoded by a base sequence having at least 70% or more sequence identity with the base sequence shown in SEQ ID NO: 21;
(E) A polypeptide encoded by a nucleotide sequence containing the nucleotide sequence shown in SEQ ID NO: 21 or a complementary sequence thereof and a nucleotide sequence that hybridizes under stringent conditions;
(F) A polypeptide encoded by an allelic variant of the nucleotide sequence shown in SEQ ID NO: 21; or a polypeptide containing a fragment of the amino acid sequence shown in (g) (a) to (f) and containing a trans fatty acid. A polypeptide having the ability to assimilate an ester or decompose a trans-amino acid-containing ester.
The base sequence encoding the polypeptide according to claim 12, or
(A) In the base sequence shown in SEQ ID NO: 21, a polynucleotide comprising a base sequence containing one or more nucleotides of substitution, addition, deletion or a combination thereof and encoding a polypeptide having biological activity;
(B) A polynucleotide comprising a base sequence having at least 70% or more sequence identity with the base sequence shown in SEQ ID NO: 21 and encoding a polypeptide having biological activity;
(C) A polynucleotide encoding a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 21 or a complementary sequence thereof and a nucleotide sequence that hybridizes under stringent conditions and has biological activity. ;
(D) A polynucleotide that is an allelic variant of the nucleotide sequence shown in SEQ ID NO: 21 and encodes a polypeptide having biological activity;
(E) A polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 22;
(F) A polynucleotide encoding a bioactive polypeptide containing 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 encoding a polypeptide having at least 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22 and having biological activity; or (H) (E) to (G). A polynucleotide, which contains a fragment of the nucleotide sequence shown.
A polynucleotide, wherein the biological activity comprises the ability to assimilate a trans fatty acid-containing ester, or to degrade a trans fatty acid-containing ester.
Is a polynucleotide.
A cell- or cell-free expression system comprising the polynucleotide according to any one of claims 8-11 or 13.
The polynucleotide according to any one of claims 1 to 7 or 12, the polypeptide containing the amino acid sequence according to SEQ ID NO: 22, the polynucleotide or sequence according to any one of claims 8 to 11 or 13. Includes a cell or cell-free expression system comprising a nucleotide sequence encoding the amino acid sequence of No. 22 or a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 21, or the cell or cell-free expression system of claim 14. Oil decomposing agent.
The oil decomposing agent according to claim 15, wherein the oil contains a trans fatty acid-containing ester.
The oil decomposing agent according to claim 15 or 16, further comprising an oil treatment component.
The polynucleotide according to any one of claims 1 to 7 or 12, the polypeptide containing the amino acid sequence according to SEQ ID NO: 22, the polynucleotide or sequence according to any one of claims 8 to 11 or 13. A cell or cell-free expression system comprising a nucleotide sequence encoding the amino acid sequence set forth in No. 22 or a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 21, a cell or cell-free expression system according to claim 14, or a claim. A kit for oil decomposition, comprising the oil decomposing agent according to any one of Items 15 to 17 and an additional oil treatment component.
The polynucleotide according to any one of claims 1 to 7 or 12, the polypeptide containing the amino acid sequence according to SEQ ID NO: 22, the polynucleotide or sequence according to any one of claims 8 to 11 or 13. A cell or cell-free expression system comprising a nucleotide sequence encoding the amino acid sequence set forth in No. 22 or a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 21, a cell or cell-free expression system according to claim 14, or a claim. A method for removing oil decomposition, which comprises allowing the oil decomposing agent according to any one of Items 15 to 17 to act on a treatment target.
The method according to claim 19, wherein the treatment target contains a trans fatty acid or a trans fatty acid-containing ester.
The polypeptide according to any one of claims 1 to 7 or 12, the polypeptide containing the amino acid sequence of SEQ ID NO: 22, or the polynucleotide according to any one of claims 8 to 11 or 13. A detergent comprising a cell or cell-free expression system comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 22 or a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 21.
The polypeptide according to any one of claims 1 to 7 or 12, the polypeptide containing the amino acid sequence according to SEQ ID NO: 22, or any one of claims 8 to 11 or 13 in the fat modification / fat production technique. A cell or cell-free expression system containing the polynucleotide according to the item or the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 22 or the polynucleotide containing the nucleotide sequence set forth in SEQ ID NO: 21, the cell according to claim 14. Alternatively, the cell-free expression system, or the use of the oil degrading agent according to any one of claims 15 to 17.