Engineering of thermomyces dupontii lipase and method of producing fatty acid ester

Abstract

Describes herein is a lipase comprising a peptide sequence with at least 80% sequence similarity to SEQ ID NO: 3 and at least one mutation of SEQ ID NO: 3 selected from Q9, N11, A14, A23, K24, N26, A30, G31, A32, T35, R37, I40, S39, M58, G59, E87, G91, N92, I93, D96, D102, S105, G106, K108, D111, S115, N122, T123, T125, R133, A152, D158, G161, N162, F169, A180, A187, L193, R209, E210, S216, T223, T226, L227, G245, N248, P250, N251, T252, A256, L259, and any combinations thereof, or the peptide sequence comprises SEQ ID NO: 3. The lipase may be mixed with a fatty acid or a triacylglycerol and an alcohol under suitable conditions to produce a fatty acid ester. The substrate specificity of a Thermomyces dupontii lipase (TDL) may be enhanced by introducing a L259 mutation into a mature peptide sequence of TDL and expressing the L259 mutant.

Description

TITLE OF THE INVENTION: Engineering of Thermomyces dupontii Lipase And Method Of Producing Fatty Acid EsterREFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to Singapore patent application number 10202400450S with a filing date of 20 February 2024 and titled “Engineering of Thermomyces dupontii Lipase And Method Of Producing Fatty Acid Ester”.FIELD OF THE INVENTION

[0002] The present invention relates to synthetic variants of Thermomyces dupontii Lipase (TDL) and its use in producing fatty acid esters.BACKGROUND OF THE INVENTION

[0003] Biodiesel, a sustainable fuel source, is typically derived from natural materials such as vegetable oils, animal fats, or recycled cooking oils, through a transesterification process. In this process, feedstocks are reacted with an alcohol, typically methanol, in the presence of a base or enzyme catalyst to produce fatty acid methyl esters (FAME). Enzymatic-catalysed biodiesel production offers several advantages over traditional chemical methods, including milder reaction conditions, which reduce the environmental impact stemming from production of the hazardous waste. This approach also enables diverse feedstocks usage, enhances energy efficiency, and reduces the need for catalysts and methanol, leading to huge cost savings and decreased waste generation.

[0004] Novozyme's Eversa Transform 2.0 (Eversa) is an affordable liquid enzyme designed for use in biodiesel production. Derived from Thermomyces lanuginosus lipase (TLL), it has demonstrated its capability to produce high quality biodiesel from soy bean, rapeseed and jatropha oil. Due to its cost-cffcctivcncss, biodiesel production with Eversa is profitable even without reusing the enzyme. A closely related lipase from Thermomyces dupontii shares a 90% sequence identity with TLL and can be expressed from Pichia pastoris to very high yield (81203 U / mL). However, its activity is lower than Eversa and it is unable to tolerate high concentration of methanol in the reaction limiting its use.SUMMARY OF THE INVENTION

[0005] In a first aspect, there is provided a lipase comprising a peptide sequence with at least 80% sequence similarity to SEQ ID NO: 3 and at least one mutation of SEQ ID NO: 3 selected from (the group consisting of) Q9, Ni l, A14, A23, K24, N26, A30, G31, A32, T35, R37, S39, 140, M58, G59, E87, G91, N92, 193, D96, D102, S105, G106, K108, Di l l, S115, N122, T123, T125, R133, A152, D158, G161, N162, F169, A180, A187, L193, R209, E210, S216, T223, T226, L227, G245, N248, P250, N251, T252, A256, L259, and any combinations thereof, or the peptide sequence comprises SEQ ID NO: 3. The position of the at least one mutation is with respect to SEQ ID NO: 3.

[0006] Preferably, the at least one mutation of SEQ ID NO: 3 is selected from (the group consisting of) Q9E, N11D, A14E, A23E, K24I, K24A, K24G, K24P, K24T, K24M, N26I, A30D, G31D, A32D, T35S, T35D, T35K, T35N, T35A, T35V, T35Y, R37K, R37G, R37D, R37E, R37S, R37F, R37W, S39D, MON, M58A, M58Q, G59D, E87K, E87R, G91A, N92D, I93F, D96G, D96K, D96A, D96I, D96Y, D102R, S105D, G106D, K108E, DI HR, S1 15E, N122C, T123D, T125H, T125R, R133S, A152S, D158K, D158R, G161N, N162D, F169Y, A180E, A187D, L193Y, L193T, R209V, E210P, S216L, T223K, T223Q, T223R, T226D, L227G, G245D, N248D, P250F, P250W, N251D, T252I, A256L, A256F, L259F, and any combinations thereof.

[0007] More preferably, the at least one mutation of SEQ ID NO: 3 is selected from (the group consisting of) Q9E, N11D, A14E, A23E, K24I, K24A, K24G, K24P, K24T, K24M, A30D, G31D, A32D, T35S, T35D, T35K, T35N, T35A, T35V, T35Y, R37K, R37G, R37D, R37E, R37S, R37W, S39D, MON, M58A, M58Q, E87K, G91A, I93F, D96G, D96K, D96A, D102R, S105D, K108E, D111R, S115E, N122C, T123D, T125R, R133S, A152S, D158K, D158R, G161N, F169Y, A180E, A187D, L193Y, L193T, R209V, S216L, T223K, T223Q, T223R, T226D, L227G, G245D, P250F, N251D, T252I, A256L, A256F, L259F, and any combinations thereof.

[0008] In an embodiment, the at least one mutation of SEQ ID NO: 3 is selected from (the group consisting of) G91 , D96, T252, and any combinations thereof. Preferably, the at least one mutation of SEQ ID NO: 3 is selected from (the group consisting of) G91A, D96G, D96K, D96A, T2521, and any combinations thereof. In an embodiment, the at least one mutation comprises the D96K mutation and the T252I mutation.

[0009] In an embodiment, the at least one mutation of SEQ ID NO: 3 is selected from (the group consisting of) T35, R37, E87, 193, K96, D102, Di l l, T125, D158, R209, T223, P250, A256, and any combinations thereof. Preferably, the at least one mutation of SEQ ID NO: 3 is selected from (the group consisting of) T35V, T35Y, R37W, E87K, I93F, K96A, D102R, D111R, T125R, D158K, D158R, R209V, T223K, T223Q, T223R, P250F, A256L, A256F, and any combinations thereof. More preferably, this is in addition to the D96K mutation and the T252I mutation in SEQ ID NO: 3.

[0010] In an embodiment, the at least one mutation of SEQ ID NO: 3 comprises a 193 mutation, preferably a 193F mutation. More preferably, the at least one mutation of SEQ ID NO: 3 comprises a L259 mutation, preferably a L259F mutation. In an embodiment, the at least one mutation of SEQ ID NO: 3 comprises the I93F mutation, the D96K mutation and the T252I mutation.

[0011] In an embodiment, the at least one mutation of SEQ ID NO: 3 is selected from (the group consisting of) Q9, Ni l , A14, A23, A30, G31 , A32, S39, 140, S105, K108, SI 15, N122, T123, R133, G161, N162, F169, A180, A187, T226, L227, G245, N251, L259, and any combinations thereof. Preferably, the at least one mutation of SEQ ID NO: 3 is selected from (the group consisting of) Q9E, N1 ID, A14E, A23E, A30D, G3 ID, A32D, S39D, MON, S 105D, K108E, S115E, N122C, T123D, R133S, G161N, N162D, F169Y, A180E, A187D, T226D, L227G, G245D, N251D, L259F, and any combinations thereof. More preferably, the at least one mutation of SEQ ID NO: 3 comprises the I93F mutation, the D96K mutation, the T252I mutation, and at least one mutation from (the group consisting of) Q9E, N1 1D, A14E, A23E, A30D, G31D, A32D, S39D, MON, S105D, K108E, S115E, N122C, T123D, R133S, G161N, N162D, F169Y, A180E, A187D, T226D, L227G, G245D, N251D, L259F, and any combinations thereof. In an embodiment, the at least one mutation of SEQ ID NO: 3 comprises the I93F mutation, the D96K mutation, the T252I mutation and the L259F mutation.

[0012]

[0011] In an embodiment, the at least one mutation of SEQ ID NO: 3 is selected from K24, T35, R37, and any combinations thereof. Preferably, the at least one mutation of SEQ ID NO: 3 is selected from (the group consisting of) K24I, K24A, K24G, K24P, K24T, K24M, T35S, T35D, T35K, T35N, T35A, R37K, R37G, R37D, R37E, R37S, and any combinations thereof. In an embodiment, the at least one mutation of SEQ ID NO: 3 comprises the I93F mutation, the D96K mutation, the T252I mutation, the L259F mutation and at leastone mutation from (the group consisting of) K24I, K24A, K24G, K24P, K24T, K24M, T35S, T35D, T35K, T35N, T35A, R37K, R37G, R37D, R37E, R37S, and any combinations thereof. In an embodiment, the at least one mutation of SEQ ID NO: 3 comprises I93F mutation, the D96K mutation, the T252I mutation, the L259F mutation, the K24A mutation, the T35K mutation, and the R37D mutation. In an embodiment, the at least one mutation of SEQ ID NO: 3 is selected from N92 and / or A152, preferably the at least one mutation of SEQ ID NO: 3 is selected from N92D and / or A152S. In an embodiment, the at least one mutation of SEQ ID NO: 3 comprises I93F mutation, the D96K mutation, the T252I mutation, the L259F mutation, the K24A mutation, the T35K mutation, the R37D mutation, and the N92D mutation and / or the A152S mutation.

[0013] In an embodiment, the at least one mutation of SEQ ID NO: 3 is selected from (the group consisting of) M58, L193, S216 and any combinations thereof, preferably the at least one mutation of SEQ ID NO: 3 is selected from (the group consisting of) M58A, M58Q, L193Y, L193T, S216L, and any combinations thereof. In an embodiment, the at least one mutation of SEQ ID NO: 3 comprises the I93F mutation, the D96K mutation, the T252I mutation, the L259F mutation, the K24A mutation, the T35K mutation, the R37D mutation, the N92D mutation, the A152S mutation, the M58Q mutation, the L193T mutation, and the S216L mutation.

[0014] In an embodiment, the peptide sequence comprises one of the following sequences: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76. Preferably, the peptide sequence comprises one of the following sequences: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO:58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76. More preferably, the peptide sequence comprises one of the following sequences: SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO:59, SEQ ID NO: 62 , SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76. Even more preferably, the peptide sequence comprises one of the following sequences SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76. In an embodiment, the peptide sequence consists essentially of the sequence, preferably the peptide sequence consists of the sequence. The sequence includes SEQ ID NO: 3 to 62 and SEQ ID NO: 64 to 76.

[0015] Preferably, the peptide sequence has at least 85% sequence similarity to SEQ ID NO: 3, preferably at least 90% sequence similarity to SEQ ID NO: 3, more preferably at least 95% sequence similarity to SEQ ID NO: 3.

[0016] Preferably, the peptide sequence consists essentially of the at least one mutation of SEQ ID NO: 3 described above, more preferably the peptide sequence consists of the at least one mutation of SEQ ID NO: 3 described above. The mutations of SEQ ID NO: 3 described herein provide the mutated lipase with improved properties like enzymatic activity and increase methanol tolerance. However, an additional linker moiety, additional amino acids or additional mutations could be introduced that do not affect the activity of the mutated lipase. For example, a signal peptide or a linker chain could be attached to the lipase that aids in its purification. Mutations at non-important sites that do not affect the properties of the lipase may also be introduced.

[0017] Preferably, there are at least two mutations of SEQ ID NO: 3, preferably there are at least three mutations of SEQ ID NO: 3, and more preferably there are at least four mutations of SEQ ID NO: 3. In an embodiment, there are at most 15 mutations of SEQ ID NO: 3, or at most 12 mutations of SEQ ID NO: 3.

[0018] In a second aspect, there is provided a method of producing a fatty acid ester, the method comprises mixing the lipase according to the first aspect, a fatty acid or a triacylglycerol, and an alcohol under suitable conditions to produce a fatty acid ester.

[0019] Preferably, the fatty acid or the triacylglycerol is a used product.

[0020] In an embodiment, the alcohol comprises 1 to 4 carbon atoms, preferably methanol or ethanol.

[0021] In an embodiment, the lipase is present in a dosage of 5% or less w / w.

[0022] In a third aspect, there is provided a method of enhancing substrate specificity of a Thermomyces dupontii lipase comprising introducing a L259 mutation into a mature peptide sequence of a Thermomyces dupontii lipase to produce a L259 mutant of the Thermomyces dupontii lipase; and expressing the L259 mutant of the Thermomyces dupontii lipase in a protein expression system. Preferably, the L259 mutation is a L259F mutation. In an embodiment, the mature peptide sequence comprises SEQ ID NO: 63.

[0023] The TDL variants or mutants (SEQ ID NO: 3 to 62 and 64 to 76) described have improved properties compared to the natural TDL especially in the production of biodieselfrom fatty acids or triacylglycerols. The mutants have an increased tolerance for high methanol concentrations at the beginning of the reaction, resulting in reduced production time for biodiesel. The TDL mutants described herein have comparable or higher activity to the commercially available Evcrsa lipase at the same enzyme concentration, especially the TDLI1, TDLJ1, TDLK2 and TDLL1 mutants. The L259F mutation was identified to be crucial for regulating the lipase's specificity against triacylglyccridcs (TAGs) and free fatty acids (FFAs). These findings contribute to the field of enzymatic biodiesel production and demonstrates its potential to replace chemical-based methods for sustainable biofuel generation.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure (FIG.) 1 shows the SDS-PAGE gel analysis of the TDLF2, TDLG3, TDLH7, and TDLI1 mutants expressed in Pichia pastoris.

[0025] FIG. 2 shows the methanol tolerance of the TDL wild type and TDLF2 mutant.

[0026] FIG. 3 shows the production of biodiesel from fatty matter with different enzyme dosages.

[0027] FIG. 4 shows the TLC analysis of free fatty acid after reaction of the TDL mutants with fatty matter.

[0028] FIG. 5 shows the TLC of free fatty acid after reaction of the TDL mutants with triacylglycerol in sludge palm oil.

[0029] FIG. 6 shows a comparison of the structure of TDLF2 and TDLI1.

[0030] FIG. 7 shows the effect of the rate of methanol addition to the activity of the TDL mutants.

[0031] FIG. 8 shows the thermostability of the TDL mutants.DETAILED DESCRIPTION OF THE INVENTION

[0032] In the description herein, numerous specific details are set forth in order to provide a thorough understanding of various illustrative embodiments of the invention. It will be understood, however, to one skilled in the art, that embodiments of the invention may be practiced without some or all of these specific details. Embodiments described in the context of one of the methods or devices are analogously valid for the other methods or devices.Similarly, embodiments described in the context of a method are analogously valid for a device, and vice versa.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs.

[0034] As used herein, the articles “a”, “an” and “the” as used with regard to a feature or clement include a reference to one or more of the features or elements. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. As used herein, the terms “first,” “second,” and “third,” etc. are used merely as labels, and are not intended to impose numerical requirements on their objects.

[0035] Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the invention. Conversely, where different elements or groups of elements are disclosed, combinations thereof are also disclosed. Where any element of an invention is disclosed as having a plurality of alternatives, examples of that invention in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of an invention can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0036] Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the invention. Where a value being discussed has inherent limits, for example where a component can be present at a concentration of from 0 to 100%, or where the pH of an aqueous solution can range from 1 to 14, those inherent limits are specifically disclosed. Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the invention, as are ranges based thereon.

[0037] Although each of these terms has a distinct meaning, the terms “comprising”, “consisting of’ and “consisting essentially of’ may be interchanged for one another throughout the instant application. The term “having” has the same meaning as “comprising” and may be replaced with either the term “consisting of’ or “consisting essentially of’. The terms“consisting essentially of’ means that specific further components can be present, namely those not materially affecting the essential characteristics of the lipase or lipase variant in the context herein. For example, addition mutations may be made that do not affect the properties of the lipase or additional linker or amino acids may be added for purification or identification of the lipase.

[0038] The terms “polypeptide” and “protein”, used interchangeably herein, refer to a polymer of amino acids without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude chemical or post-expression modifications of the polypeptides of the invention, although chemical or post-expression modifications of these polypeptides may be included or excluded as specific embodiments. Therefore, for example, modifications to polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Further, polypeptides with these modifications may be specified as individual species to be included or excluded from the present invention. The natural or other chemical modifications, such as those listed in examples above can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Modifications include acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross -linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, aminoacids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems, etc.), polypeptides with substituted linkages, as well as other modifications known in the ait, both naturally occurring and non-naturally occurring.

[0039] The terms “sequence similarity", “percentage of sequence identity" and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Identity is evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, CLUSTAL W, FASTDB [Pearson and Lipman, (1988), Proc. Natl. Acad. Sei. USA 85(8):2444-2448; Altschul et al., (1990), J. Mol. Biol. 215(3):403-410; Thompson et al. (1994), Nucleic Acids Res. 22(2):4673-4680; Higgins et al., (1996), Meth. Enzymol. 266:383-402; Altschul et al., (1993), Nature Genetics 3:266-272; Brutlag et al. (1990) Comp. App. Biosci. 6:237-24], the disclosures of which are incorporated by reference in their entireties.

[0040] Biodiesel is a sustainable and environmentally friendly alternative to conventional diesel fuel, typically derived from renewable sources through a transesterification process. Enzymatic biodiesel production offers significant advantages over chemical methods due to its milder reaction conditions and a broader range of compatible feedstocks.

[0041] Described herein is the engineering of a Thermomyces dupontii lipase (TDL) to modify and enhance the enzyme's performance in converting fatty matter, a waste feed enriched in fatty acids, to fatty acid methyl esters (FAME) that may be used as biodiesel. A number of mutants were synthesised with several iterations of mutations to determine the mutations that provide desirable properties in the TDL variants or mutants.

[0042] Expression of TDLF2 for biodiesel production

[0043] The TDL lipase (Uniprot: F6LQK7, SEQ ID NO: 2) shares an 89.6% sequence identity with TLL lipase (Uniprot: 059952, SEQ ID NO: 1). TDLF2 (SEQ ID NO: 3) is a variant of TDL with 13 point mutations (E56K / S58M / R74K / L95F / G99E / D101N / S158D / L21 IF / T231R / K232R / N233R / D245G / D254S) with respect to the mature peptide of TDL (SEQ ID NO: 63). TDLF2 can be expressed to high yield and purity in Pichia pastoris, with a yield of around 230 mg / L when produced from a 30 mL culture (FIG. 1). TDLF2 was generated by random mutagenesis and rationale design and showed improved tolerance of methanol. FIG. 2 shows a comparison of the TDL wild type (TDLWT) and TDLF2 at different methanol concentrations. For the TDLWT, it may be observed from FIG. 2 that there is no enzyme activity when the methanol concentration is 50% or higher, whereas TDLF2 still possess enzyme activity even up to 70% methanol concentration as evident by the observance of the methyl ester peak on the TLC in FIG. 2. To assess its ability to produce biodiesel from waste fatty matter, titration was conducted to determine the remaining amount of fatty acids in the feed after 8 hours of incubation. When 0.3% w / w enzyme was added, TDLF2 reduced free fatty acids (FFA) from 40% w / w to 18% w / w (FIG. 3 and FIG. 4).

[0044] For comparison, 0.3% w / w Eversa can reduce FFA to 3.8% w / w as shown in FIG. 3 and FIG. 4. Interestingly, when sludge palm oil was used as a feed, a significant amount of conversion from triacylglycerol (TAG) to FAME was observed, even with the addition of 0.15% w / w of TDLF2 (FIG. 5) This suggests that TDLF2 exhibits a preference for sludge palm oil as a feed for biodiesel production, indicating the need to further engineer the enzyme to enhance its specificity against acid-rich feedstocks.

[0045] Construction of TDL mutants

[0046] Golden-Gate cloning was used to create the various TDL mutants and has been described (see Chow JY and Nguyen GKT, Int. I. Mol. Sci. 2022, 23(17), 9515). To create each specific mutant, the following templates were used for amplification by the respective primers shown in Table 1. Primers for the other mutants may be prepared in the same manner. The PCR products were then gel-purified and assembled in pFAi2-Aoxlp.0047] Table 1 : Examples of Primers used to create TDL mutantsThe nucleotides in bold represent the site of the mutations. The underlined nucleotides represent the 4 base pairs overhang used for GoldenGate cloning to generate the mutants.

[0048] Expression of TDL in Pichia pastoris

[0049] Various mutants of TDL were expressed in P. pastoris under the expression of the AOX1 promoter. Cells harbouring the construct were inoculated into 30 mL BMGY medium (1% w / v yeast extract, 2% w / v peptone, 100 mM potassium phosphate, pH 6.0, 1.34% w / v yeast nitrogen base (Formedium), 1% v / v glycerol, 0.00004% w / v biotin (Sigma)) and grown at 30 °C overnight for the cells to reach stationary phase The cells were then diluted to an OD of 6 in BMMY medium (1% w / v yeast extract, 2% w / v peptone, 100 mM potassium phosphate, pH 6.0, 1.34% w / v yeast nitrogen base, 1% v / v methanol, 0.00004% w / v biotin) and incubated at 30 °C with shaking for 3 days. 1% v / v methanol was added to the culture twice, on day 1 and 2 of the induction. After 3 days of induction, the supernatant was harvested, washed with 50 mM Tris, pH 7 and concentrated to 30 mg / mL with a lOkDa cutoff centrifugal fdter units (Pall Corporation). The signal peptide will be cleaved prior to secretion out of the cell and the sequences of the TDL mutants described are the mature peptides. Protein concentration was measured by comparison with BSA standards using the Bradford assay (Bio-rad).

[0050] Titration assay against fatty matter

[0051] To measure the biodiesel conversion efficiency of TDL against fatty matter, 1.5 pL (0.3% w / w) enzyme and 13.5 μL water was added to 0.5 g fatty matter and mixed in a 2 mL tube at 40 °C with shaking (2000 rpm). Methanol was then slowly added at various time intervals to reduce the exposure of the enzymes to high methanol concentration. At the beginning of the reaction, 40 μL (6 %w / w) methanol was added. At the Ih, 2h, 3h and 4h timepoint, 35 μL, 25 μl, 25 μl, 25 μL methanol was added to the reaction, respectively, to achieve a final concentration of 24 % w / w methanol. After 8 hours of incubation, the reaction was stopped by adding 2.2 mL isopropanol and 0.1 % w / v phenolphthalein. To determine the amount of fatty acids remaining in the feed, the reaction mix was titrated with 50 mM NaOH. The % FFA (w / w) remaining in the feed is calculated by equation (1).

[0052] % FFA — (1)where VNaOH is the volume of NaOH added in mL, MWKOH is the molecular weight of KOH (56.1 g / mol), CNaon is the concentration of NaOH in M (0.05 M) and Wpeed is the weight of the feed in g (0.5 g).

[0053] TLC analysis of biodiesel reaction

[0054] To monitor the progress of the reaction at various time points, 10 μL of the reactionmix was removed and stopped with 90 μL isopropanol 1 μl of the sample is spotted onto a TLC plate and the products were separated by running with a solvent system that contains hexane, diethyl ether and formic acid in a 80:20: 1 ratio. After 7 mins, the TLC plate was air dried and the products were stained with iodine for detection.

[0055] Construction and expression of TDL mutants including TDLG3 (SEQ ID NO: 9), TDLH7 (SEQ ID NO: 34) and TDLI1 (SEQ ID NO: 62) mutants for biodiesel production

[0056] The mutations listed for TDLF2 is with respect to the TDL mature peptide (SEQ ID NO: 63). The TDL mutants described herein is described with respect to the TDLF2 sequence (SEQ ID NO: 3) unless otherwise stated, for example TDLG3-XXXX and TDLH7-XXXX means that the mutation is with respect to TDLG3 (SEQ ID NO: 9) and TDLH7 (SEQ ID NO: 34) respectively. The mutations are listed with the first alphabet indicating the single letter abbreviation of the original amino acid residue in the sequence in question, the number indicating amino acid position, and the second alphabet indicating the single letter abbreviation of the new amino acid residue. The enzyme sequences are provided in Table 9 and the accompanying sequence listing. A summary of the key mutant lipases which serve as the basis for future iterations is provided in Table 10.

[0057] The activity of the TDL mutants is shown in Table 2, Table 3 and Table 4 based on the free fatty acid titration assay. Two single mutants of TDLF2 (D96K, SEQ ID NO: 7 and T252I, SEQ ID NO: 8) were identified, which produced a higher yield of biodiesel from fatty matter compared to TDLF2. These mutations were combined to create TDLG3 (SEQ ID NO: 9). TDLG3 exhibits increased expression levels (270 mg / L) when expressed in Pichia pastoris and can reduce FFA in fatty matter to 10% w / w after 8 hours of incubation (FIG. 3 and FIG. 4). The G91 A and D96G single mutations also have comparable activity (90% or greater activity) to TDLF2 as shown in Table 2.

[0058] To further enhance the activity of TDLG3, a number of point mutations of TDLG3 were synthesised (TDLG3-XXXX in Table 3) and their free fatty acid activity assessed and shown in Table 3. The activity of the TDLG3 mutants is shown normalised to the TDLG3 parent. Several of these mutations have comparable activity (90% or greater activity) to TDLG3 with DI 11R and I93F having the greatest improvement in activity.

[0059] Based on this iteration of mutations, I93F was added to create TDLH7 (SEQ ID NO:34). TDLH7 shows decreased expression level (180 mg / L) in Pichia pastoris, likely due to the increased hydrophobicity of the enzyme's surface when isoleucine is mutated to phenylalanine as shown in FIG. 6 (TDLI1 is shown with the I93F mutation as TDLH7). However, TDLH7 demonstrates a significant improvement in its esterification activity, further reducing the percentage FFA remaining in fatty matter to 7% w / w (FIG. 3 and FIG. 4).

[0060] To further improve the TDL mutants activity and create a mutant that matches Eversa's activity, another iteration of mutations (TDLH7-XXXX in Table 4) was performed and shown in Table 4. Two of the mutations (N122C and L259F) showed significant improvement in the activity. Most of the other mutations had lower activity compared to TDLH7, however these mutations still had comparable (90-101%) or higher activity compared to TDLF2.

[0061] Based on this iteration, L259F was added to TDLH7 to create TDLI1 (SEQ ID NO: 62). The TDLI1 mutant has a reduced expression level, approximating 130mg / L, likely due to the addition of another hydrophobic residue to the enzyme's surface. The TDLI1 activity is the highest against fatty matter, resulting in a decrease in FFA to 3.9% w / w, close to the 3.8% w / w observed for Eversa (FIG. 3 and FIG. 4).

[0062] Site-saturation mutagenesis of TDLI1

[0063] Since TDLI1 (SEQ ID NO: 62) exhibits lower expression levels compared to its predecessor, combinatorial saturation mutagenesis was used to enhance both its expression yield and enzyme activity. Three key residues - K24, T35, and R37 - were targeted to identify the optimal combination of mutations that may synergistically improve the enzyme's performance. After screening the variants using a phenol-red assay and further validating results via titration against fatty matter, nine mutants that outperformed TDLI1 were identified as shown in Table 5. The most promising mutant, TDLI1 (SEQ ID NO: 66), containing the mutations K24A, T35K, and R37D, demonstrated a 24% increase in activity relative to TDLI1 (SEQ ID NO: 62).

[0064] Random mutagenesis of TDLK2

[0065] Random mutagenesis via error-prone PCR was further employed to identify additional potential hot spots crucial for enhancing both the activity and yield of the enzyme. After mutagenizing TDLJ 1 and performing a single round of screening, two mutations - N92D and A152S - were identified as shown in Table 6 that significantly contributed to improvedactivity against fatty matter. This mutant, designated TDLK2 (SEQ ID NO: 75), exhibited a 19% increase in activity.

[0066] In addition, a few other beneficial mutations at positions M58, L193, and S216 were identified. To identify the optimal combination of mutations that would further enhance TDLK2's activity, these three positions were targeted for combinatorial saturation mutagenesis. The resulting library was screened using the phenol-red assay to identify the best performing mutants. Two mutants, M58A / L193Y / S216L (SEQ ID NO: 76) and M58Q / L193T / S216L (TDLL2, SEQ ID NO: 77), exhibited superior performance, with activity increases of 10% and 19%, respectively, compared to TDLK2 and are shown in Table 7. TDLL2 (SEQ ID NO: 77) exhibited activity increase of about 210% (calculated based on the indicated mutant as the control) compared to the TDLF2 (SEQ ID NO: 3) enzyme.

[0067] A total of 51 amino acids residues in the TDLF2 (SEQ ID NO: 3) were mutated and described herein which is 18.9% of the amino acid residues in TDLF2. Whilst not all the individual point mutations may improve the activity of the TDL mutant compared to TDLF2, it is possible that the mutations have other improvements or uses, or a combination of the mutations may lead to a mutant with improved properties (e.g. activity, expression levels, substrate specificity, alcohol or methanol tolerance, thermostability). Thus, mutants with at least 80% or 81% sequence similarity to TDLF2 may be synthesised and used, preferably 85% or 90% or higher sequence similarity.

[0070] Table 4: Activity of TDLH7 mutants

[0071] Table 5: Activity of TDLT1 mutants

[0072] Table 6: Activity of TDLJ 1 mutants

[0073] Table 7 : Activity of TDLK2 mutants

[0074] Substrate specificity of TDL mutants

[0075] When the TDL mutants were tested with sludge palm oil, it was observed that TDLF2 (SEQ ID NO: 3), TDLG3 (SEQ ID NO: 9), TDLH7 (SEQ ID NO: 34), and Eversa all exhibited high activity against the feed, even at a 0.15% w / w enzyme dosage (FIG. 5). However, the TDLI1 (SEQ ID NO: 62) mutant was unable to fully hydrolyse the TAG-rich feed, with a significant amount of TAG remaining in the feed after 8 hours of incubation at 0.6% w / w enzyme dosage. This suggests that the L259F mutation plays a crucial role in controlling the substrate specificity of the lipase. A closer examination of the enzyme's structure revealed that the L259F mutation is located at the entrance of the substrate pocket, and the substitution with a bulkier hydrophobic residue prevents larger TAG molecules from entering the active site (FIG. 6). Interestingly, the L259F mutation along with other mutations were previously identified in TDL as essential for the efficient synthesis of 2-carboxyethyl-3-cyano- 5-methylhexanoic acid ethyl ester, a crucial precursor for the synthesis of the drug Pregablin.

[0076] Methanol tolerance of TDL mutants

[0077] To assess how the rate of methanol addition impacts the activity of TDL mutants and their ability to produce biodiesel, the speed at which methanol is added to the reaction was varied to examine its effects. Methanol was added in 6%, 12% or 20% w / w at the start of reaction, with the concentration gradually increasing to 24% w / w for all samples after 4 hours.

[0078] For the methanol tolerance assay, various amounts of methanol were added to the reaction at 1 to 4 hours timepoints, according to Table 8 below. The final concentration of methanol added to all samples are fixed at 24 % w / w.Table 8: Methanol Tolerance Assay

[0079] A significant decrease in activity was observed for TDLF2, TDLG3 and TDLH7 as the initial methanol concentration increased (FIG. 7). However, the amount of FFA remaining in the feed remained consistent (3.5 — 4.5% w / w) for TDLI1 and Eversa, even with a high amount of methanol (20% w / w) at the beginning of the reaction. This indicates that both TDLI1 and Eversa can effectively utilise methanol or have increased tolerance to high methanol concentrations. This simplifies the conversion process and reduces the conversion time of the starting material to the product.

[0080] Thermostability of TDL mutants

[0081] The thermostability of each enzyme was determined by incubating them at the respective temperature for 15 mins before running the reaction with fatty matter. When the temperature was higher than 40°C, the activity of all TDL mutants and Eversa significantly decreased as shown in FIG. 8, suggesting that the mutations did not improve the thermostability of the enzyme.Table 9: Lipase Sequences (bold letters indicate the mutations with respect to TDL (SEQ ID NO: 2) in TDLF2 and TDLF2 (SEQ ID NO:3) in the additional mutated lipases)Table 10: Sequence of TDLF2 and mutations of selected lipases

Claims

CLAIMS

1. A lipase comprising a peptide sequence with at least 80% sequence similarity to SEQ ID NO: 3 and at least one mutation of SEQ ID NO: 3 selected from Q9, Nil, A14, A23, K24, N26, A30, G31, A32, T35, R37, S39, 140, M58, G59, E87, G91, N92, 193, D96, D102, S105, G106, K108, Dil l, S115, N122, T123, T125, R133, A152, D158, G161, N162, F169, A180, A187, L193, R209, E210, S216, T223, T226, L227, G245, N248, P250, N251, T252, A256, L259, and any combinations thereof, or the peptide sequence comprises SEQ ID NO: 3.

2. The lipase according to claim 1, wherein the at least one mutation of SEQ ID NO: 3 is selected from Q9E, N11D, A14E, A23E, K24I, K24A, K24G, K24P, K24T, K24M, N26I, A30D, G31D, A32D, T35S, T35D, T35K, T35N, T35A, T35V, T35Y, R37K, R37G, R37D, R37E, R37S, R37F, R37W, S39D, MON, M58A, M58Q, G59D, E87K, E87R, G91A, N92D, I93F, D96G, D96K, D96A, D96I, D96Y, D102R, S105D, G106D, K108E, DI HR, S115E, N122C, T123D, T125H, T125R, R133S, A152S, D158K, D158R, G161N, N162D, F169Y, A180E, A187D, L193Y, L193T, R209V, E210P, S216L, T223K, T223Q, T223R, T226D, L227G, G245D, N248D, P250F, P250W, N251D, T252I, A256L, A256F, L259F, and any combinations thereof.

3. The lipase according to claim 2, wherein the at least one mutation of SEQ ID NO: 3 is selected from Q9E, N1 ID, A14E, A23E, K24I, K24A, K24G, K24P, K24T, K24M, A30D, G31D, A32D, T35S, T35D, T35K, T35N, T35A, T35V, T35Y, R37K, R37G, R37D, R37E, R37S, R37W, S39D, MON, M58A, M58Q, E87K, G91A, 193F, D96G, D96K, D96A, D102R, S105D, K108E, D111R, S115E, N122C, T123D, T125R, R133S, A152S, D158K, D158R, G161N, F169Y, A180E, A187D, L193Y, L193T, R209V, S216L, T223K, T223Q, T223R, T226D, L227G, G245D, P250F, N251D, T252I, A256L, A256F, L259F, and any combinations thereof.

4. The lipase according to claim 2, wherein the at least one mutation of SEQ ID NO: 3 is selected from G91, D96, T252, and any combinations thereof.

5. The lipase according to claim 4, wherein the at least one mutation of SEQ ID NO: 3 is selected from G91A, D96G, D96K, D96A, T252I, and any combinations thereof, preferably the at least one mutation comprises the D96K mutation and the T252I mutation.

6. The lipase according to any one of claims 1 to 5, wherein the at least one mutation of SEQ ID NO: 3 is selected from T35, R37, E87, 193, K96, D102, Di l l, T125, D158, R209, T223, P250, A256, and any combinations thereof.[Claim 7 J The lipase according to claim 6, wherein the at least one mutation of SEQ ID NO: 3 is selected from T35V, T35Y, R37W, E87K, I93F, K96A, D102R, DI HR, T125R, D158K, D158R, R209V, T223K, T223Q, T223R, P250F, A256L, A256F, and any combinations thereof.

8. The lipase according to claim 4 or claim 5, wherein the at least one mutation of SEQ ID NO: 3 comprises a 193 mutation, preferably a I93F mutation.

9. The lipase according to any one of claims 1 to 8, wherein the at least one mutation of SEQ ID NO: 3 is selected from Q9, Ni l, A14, A23, A30, G31, A32, S39, 140, S105, K108, SI 15, N122, T123, R133, G161, N162, F169, A180, A187, T226, L227, G245, N251, L259, and any combinations thereof.

10. The lipase according to claim 9, wherein the at least one mutation of SEQ ID NO: 3 is selected from Q9E, N11D, A14E, A23E, A30D, G31D, A32D, S39D, MON, S105D, K108E, S115E, N122C, T123D, R133S, G161N, N162D, F169Y, A180E, A187D, T226D, L227G, G245D, N251D, L259F, and any combinations thereof.

11. The lipase according to claim 8, wherein the at least one mutation of SEQ ID NO: 3 comprises a L259 mutation, preferably a L259F mutation.

12. The lipase according to any one of claims 1 to 11, wherein the at least one mutation of SEQ ID NO: 3 is selected from K24, T35, R37, and any combinations thereof.

13. The lipase according to claim 12, wherein the at least one mutation of SEQ ID NO: 3 is selected from K24I, K24A, K24G, K24P, K24T, K24M, T35S, T35D, T35K, T35N, T35A, R37K, R37G, R37D, R37E, R37S, and any combinations thereof.

14. The lipase according to any one of claims 1 to 13, wherein the at least one mutation of SEQ ID NO: 3 is selected from N92 and / or A152, preferably the at least one mutation of SEQ ID NO: 3 is selected from N92D and / or A152S.

15. The lipase according to any one of claims 1 to 14, wherein the at least one mutation of SEQ ID NO: 3 is selected from M58, L193, S216, and any combinations thereof, preferably the at least one mutation of SEQ ID NO: 3 is selected from M58A, M58Q, L193Y, L193T, S216L, and any combinations thereof.

16. The lipase according to any one of claims 1 to 15, wherein the peptide sequence has at least 85% sequence similarity to SEQ ID NO: 3, preferably at least 90% sequence similarity to SEQ ID NO: 3, more preferably at least 95% sequence similarity to SEQ ID NO: 3.

17. The lipase according to any one of claims 1 to 15, wherein the peptide sequence consists essentially of the at least one mutation of SEQ ID NO: 3

18. The lipase according to any one of claims 1 to 15, wherein the peptide sequence consists of the at least one mutation of SEQ ID NO: 3.

19. The lipase according to any one of claims 1 to 18, wherein there are at least two mutations of SEQ ID NO: 3, preferably there are at least three mutations of SEQ ID NO: 3, and more preferably there are at least four mutations of SEQ ID NO: 3.

20. The lipase according to claim 1, wherein the peptide sequence comprises one of the following sequences: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76.

21. The lipase according to claim 16, wherein the peptide sequence comprises one of the following sequences: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76.

22. The lipase according to claim 16, wherein the peptide sequence comprises one of the following sequences: SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 28, SEQ IDNO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76, preferably the peptide sequence comprises one of the following sequences SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 74, and SEQ ID NO: 76.

23. The lipase according to any one of claims 20 to 22, wherein the peptide sequence consists essentially of the sequence, preferably the peptide sequence consists of the sequence.

24. A method of producing a fatty acid ester, the method comprises mixing the lipase according to any one of claims 1 to 23, a fatty acid or a triacylglycerol, and an alcohol under suitable conditions to produce a fatty acid ester.

25. The method according to claim 24, wherein the fatty acid or the triacylglycerol is a used product.

26. The method according to claim 24 or claim 25, wherein the alcohol comprises 1 to 4 carbon atoms, preferably methanol or ethanol.

27. The method according to any one of claims 24 to 26, wherein the lipase is present in a dosage of 5% or less w / w.

28. A method of enhancing substrate specificity of a Thermomyces dupontii lipase comprising introducing a L259 mutation into a mature peptide sequence of the Thermomyces dupontii lipase to produce a L259 mutant of the Thermomyces dupontii lipase; and expressing the L259 mutant of the Thermomyces dupontii lipase in a protein expression system.

29. The method according to claim 28, wherein the L259 mutation is a L259F mutation.

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