Production of fatty acid alkyl esters

Abstract

The present invention provides a process for producing fatty acid alkyl esters, the process comprising providing a substrate comprising a triglyceride, a diglyceride, a monoglyceride, a free fatty acid, or any combination thereof, and reacting the substrate with an enzyme composition comprising a sn-1,3 lipase and a sn-2 lipase to produce fatty acid alkyl esters.

Description

[Technical field]

[0001] Sequence Listing Reference This application contains a sequence listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to a process for producing fatty acid alkyl esters from a substrate using a combination of sn-1,3 lipase and sn-2 lipase that promotes the conversion of free fatty acids and / or triglycerides to fatty acid alkyl esters. [Background technology]

[0003] Biodiesel is generally classified as monoalkyl ethers of fats and oils and has recently gained attention due to its environmental benefits. Although biodiesel is currently successfully produced chemically (using, for example, NaOH and / or sodium methoxide as catalysts), there are several associated problems that limit its development, such as pretreatment of the oil due to its high content of free fatty acids, removal of chemical catalysts from the ester and glycerol phases, and removal of inorganic salts during glycerol recovery.

[0004] The drawbacks caused by chemical catalysts are largely avoided by using lipolytic enzymes as catalysts, and in recent years there has been interest in using immobilized or non-immobilized lipases in the transesterification reaction to produce biodiesel.

[0005] Fungal esterases can be used in the enzymatic production of esters and can replace catalysts such as mineral acids (e.g., sulfuric acid, hydrochloric acid and chlorosulfonic acid), amphoteric hydroxides of metals of groups I, II, III and IV, and others. The use of enzymes in ester synthesis, especially those classified as EC 3.1.1 carboxylic acid ester hydrolases according to the Enzyme Nomenclature (Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, 1992 and thereafter), has been described in the prior art.

[0006] WO 88 / 02775 discloses lipases A and B from Candida antarctica. It is stated that C. antarctica lipase B (CALB) is more effective for ester synthesis.

[0007] Cutinases are lipolytic enzymes capable of hydrolyzing the substrate cutin. Cutinases are known from various fungi (PE Kolattukudy in “Lipases”, Ed. B. Borgstrom and HLBrockman, Elsevier 1984, 471-504). The amino acid sequence of cutinases from Humicola insolens has been published (U.S. Patent No. 5,827,719).

[0008] It is well known that excess short-chain alcohols, such as methanol, significantly inactivate lipase. However, at least 3 molar equivalents of methanol based on triglyceride are required to completely convert oil to its corresponding methyl ester. Du et al. (Biotechnol. Appl. Biochem. 2003, 38:103-106) comparatively studied the effect of oil / methanol molar ratio during discontinuous batch and continuous batch operations.

[0009] To avoid inactivation of lipase, the methanol concentration has been kept low by adding methanol stepwise throughout the reaction (Shimada et al. J Mol. Catalysis Enzymatic, 2002, 17:133-142; Xu et al. 2004, Biocat. Biotransform. 22:45-48).

[0010] Boutur et al. (J. Biotechnol. 1995, 42:23-33) reported that lipase from Candida deformans can catalyze both alcoholysis of triglycerides (TG) and esterification of free fatty acids (FAA), but not under identical reaction conditions. Under the conditions described by Boutur et al., only esterification was catalyzed. Summary of the Invention [Problem to be solved by the invention]

[0011] To more economically produce fatty acid alkyl esters for biodiesel, there is a need for faster and higher yield conversion of fats and oils to their corresponding methyl esters. [Means for solving the problem]

[0012] The present invention relates to a process for producing fatty acid alkyl esters, the process comprising the steps of: a) providing a substrate comprising primarily triglycerides, diglycerides, monoglycerides, free fatty acids, or any combination thereof, and b) reacting the substrate with an enzyme composition comprising an sn-1,3 lipase and an sn-2 lipase to produce fatty acid alkyl esters.

[0013] The present invention also relates to an enzyme composition for producing fatty acid alkyl esters, comprising an sn-1,3 lipase and an sn-2 lipase.

[0014] In the present invention, a non-specific lipase that also catalyzes the reaction at the sn-2 position, which has a low specific activity, is used in combination with the non-specific lipase, which can surprisingly increase the reaction rate and conversion by partially replacing the high-speed sn-1,3 lipase.

[0015] These and further objects and advantages of the present invention will be apparent from the following description. In the following detailed description, preferred embodiments of the present invention will be described with reference to the accompanying drawings. These embodiments do not represent the full scope of the present invention. Rather, the present invention may be used in other embodiments. Therefore, to interpret the breadth of the present invention, reference should be made to the claims of this specification. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] definition Before particular embodiments of the present invention are disclosed and described, it is to be understood that the invention is not limited to the particular processes and materials disclosed herein, as such materials may vary to some extent. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be defined only by the appended claims and equivalents thereof.

[0017] In describing and claiming the present invention, the following terminology is used.

[0018] The singular forms "a," "an," and "the" include plural referents unless the context clearly requires otherwise. Thus, for example, reference to "a process" includes reference to one or more such processes.

[0019] As used herein, "substantially" when used in reference to an amount or quantity of a material, or a particular property thereof, refers to an amount sufficient for that material or property to provide the intended effect. The amount of deviation permitted may depend on the particular context, in some cases. Similarly, "substantially free" or the like refers to the absence of the identified element or agent in the composition. In particular, an element identified as "substantially free" is either completely absent from the composition or present in such a small amount that it does not adversely affect the composition.

[0020] As used herein, a reference to "about" a value or parameter includes embodiments directed to the value or parameter itself. For example, a statement referring to "about X" includes the embodiment "X." When used in conjunction with a measurement, "about" includes a range that encompasses at least the uncertainty associated with the method of measuring the particular value, and may include a range plus or minus two standard deviations around the stated value.

[0021] Similarly, reference to a gene or polypeptide "derived from" another gene or polypeptide X includes gene or polypeptide X.

[0022] It is to be understood that the embodiments described herein include "consisting of" and / or "consisting essentially of" embodiments. As used herein, unless the context requires otherwise by express words or necessary meaning, the word "comprise" or variations such as "comprises" or "comprising" are used in the inclusive sense, i.e., to specify the presence of described features, but are not used to exclude the presence or addition of further features in various embodiments.

[0023] Concentrations, amounts, and other numerical data may be presented in a range format herein. It should be understood that such range formats are used merely for convenience and brevity, and should be interpreted flexibly to include not only the numerical values ​​expressly recited as the limits of the range, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were expressly recited. For example, a weight range of about 1 percent to about 20 percent should be interpreted not only to include the explicitly recited concentration limits of 1 percent to about 20 percent, but also to include individual concentrations such as 2 percent, 3 percent, 4 percent, and subranges such as 5 percent to 15 percent, 10 percent to 20 percent, and the like.

[0024] Lipids: The term "lipids" refers to phospholipids and their derivatives, triglycerides and derivatives, sterols, stanols, cholesterol, sphingolipids, ceramides, fatty acids, fatty alcohols, glycolipids, proteolipids, lipopolysaccharides, ether lipids, polar and non-polar lipids and their derivatives.

[0025] Esterification: As used herein, the term "esterification" refers to the reaction of combining an organic acid, such as a fatty acid, with any alcohol or polyol, such as glycerol.

[0026] Hydrolysis: As used herein, the term "hydrolysis" refers to the reaction of an ester with water to produce an acid and an alcohol.

[0027] Alcohololysis: As used herein, the term "alcohololysis" refers to the reaction of an ester with a monohydric alcohol, such as ethanol, butanol, or a polyhydric alcohol, such as glycerol, to produce esters having different alkyl groups.

[0028] Acidolysis: As used herein, the term "acidolysis" refers to the reaction of an ester with an acid resulting in the exchange of an acyl group.

[0029] Intermolecular Transesterification: As used herein, the term "intramolecular transesterification" refers to the reaction of a first ester with a second ester resulting in intermixing between the acyl and alcohol moieties.

[0030] Transesterification: As used herein, the term "transesterification" refers to any of the following reactions: alcoholysis, acidolysis, and intermolecular transesterification.

[0031] Synthesis: As used herein, the term "synthesis" or "synthesis of fatty acids" refers to the covalent attachment of a fatty acid at the sn-2 position of a glyceride, preferably by a one-step reaction selected from any one of the following reactions: esterification, intermolecular transesterification, alcoholysis, acidolysis, transesterification.

[0032] The terms "alkyl" or "alkyl group" are to be interpreted in their broadest sense to describe monovalent aliphatic compounds that contain hydrocarbons.

[0033] The terms "glycerol derivatives" and "glycerides" are used interchangeably herein to describe esters, ethers, and other derivatives of glycerol in which at least one of the hydrogens of any of the hydroxyl groups attached to the Cl, C2, or C3 carbons is replaced. Examples of glycerol derivatives include, among others, tristearoylglycerol (or triostearoylglycerol or glycerin tristearate, or glyceryl tristearate), 1,3-benzylideneglycerol (or 1,3-0-benzylideneglycerol), and glycerol 2-phosphate (or 2-phosphoglycerol). When the substituent is on a carbon atom rather than on the oxygen of the hydroxyl group, the compound may be considered a derivative of glycerol (e.g., 1,2,3-nonadecanetriol, which is CHCHOH-CHOH-CHOH, also considered as 1-C-hexadecylglycerol). As used herein, the term "glycerol" is intended to encompass glycerol derivatives.

[0034] Lipase: The terms "lipase", "lipase enzyme", "lipolytic enzyme", "lipid esterase", "lipolytic polypeptide" and "lipolytic protein" refer to enzymes in class EC 3.1.1 as defined by the Enzyme Nomenclature. It may have lipase activity (triacylglycerol lipase, EC 3.1.1.3), cutinase activity (EC 3.1.1.74), sterol esterase activity (EC 3.1.1.13) and / or wax-ester hydrolase activity (EC 3.1.1.50). For the purposes of the present invention, lipase activity (i.e. hydrolytic activity of lipase) may be determined in a pNP assay using substrates with various chain lengths as described in the "Materials & Methods" section.

[0035] Parent or Parent Lipase: The term "parent" or "parent lipase" refers to a lipase that is modified to produce an enzyme variant. A parent lipase can be a naturally occurring (wild-type) polypeptide, but also a variant and / or fragment thereof. In a preferred embodiment, the parent lipase can be as set forth in SEQ ID NO: 1, 2, 3 or 4.

[0036] Sequence identity: The relatedness between two amino acid sequences is described by the parameter "sequence identity."

[0037] For the purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48; 443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), version 5.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the Needle label "longest identity" (obtained using the -nobrief option) is used as the percent identity, calculated as follows: (identical residues × 100) / (length of alignment – ​​total number of gaps in alignment)

[0038] Substrate: Suitable substrates for producing fatty acid alkyl esters according to the invention are a wide variety of vegetable oils and fats, rapeseed oil and soybean oil being the most commonly used, but other crops such as mustard oil, sunflower oil, canola oil, coconut oil, hemp oil, palm oil, and even algae are promising. The substrates can be of crude quality or can be further processed (refined, bleached and deodorized). Also, animal fats can be used, such as tallow, lard, chicken oil, marine oils, and discarded vegetable and animal fats and oils commonly known as yellow and brown grease. Suitable fats and oils can be pure triglycerides or mixtures of triglycerides and free fatty acids commonly found in discarded vegetable and animal fats. The substrate can also be obtained from vegetable oil deodorizer distillates. The types of fatty acids in the substrate include those naturally occurring as glycerides in vegetable and animal fats and oils. These include oleic acid, linoleic acid, linolenic acid, palmitic acid, steric acid, and lauric acid, to name a few. The minor components in crude vegetable oils are typically phospholipids, free fatty acids and partial glycerides, i.e., mono- and diglycerides. As used herein, the phrase "fatty acid residue" refers to either free fatty acids or esterified fatty acids, e.g., triglycerides, diglycerides, monoglycerides or fatty acid alkyl esters.

[0039] Biodiesel: Fatty acid alkyl esters (FAAEs) of short chain alcohols, such as fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), are also called biodiesel, as they are used as additives to fossil diesel. Since biodiesel is produced from renewable resources, it constitutes an additive or alternative of increasing importance for diesel fuels based on fossil petroleum.

[0040] Alcohols: The alcohols used in the process of the present invention preferably have 1 to 5 carbon atoms (C 1 , C 2 , C 3 , C 4 , or C 5The lower alcohols are preferably methanol, ethanol, propanol or mixtures thereof. The alcohol content is preferably less than 4.0, 3.5, 3.0, 2.5, 2.0, 1.5 or 1.0 molar equivalents relative to the amount of fatty acids in the reaction mixture (free fatty acids and glyceride-bound fatty acids). The alcohol may be added to the reaction mixture in stages (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more stages) and / or continuously.

[0041] Fatty acid feedstock: The term "fatty acid feedstock" or "oil and / or fat" or "vegetable oil feedstock" is defined herein as a substrate comprising fatty acid derivatives. The substrate may comprise fatty acid alkyl esters, triglycerides, diglycerides, monoglycerides, free fatty acids or any combination thereof. Any oil or fat of vegetable or animal origin comprising fatty acids may be used as a substrate for producing fatty acid alkyl esters in the process of the present invention. Also, fatty acid feedstocks consisting essentially of fatty acid alkyl esters are suitable as feedstocks (biodiesel feedstocks) for the present invention. Preferably, the free fatty acid content of the fatty acid feedstock is greater than 0.25%, greater than 0.30%, greater than 0.35%, greater than 0.50%, greater than 0.75%, greater than 1.0%, greater than 5.0%, greater than 10.0%, greater than 15.0%, greater than 20.0%, greater than 25.0%, greater than 30.0%, greater than 40%, or even greater than 50.0%.

[0042] The fatty acid feedstock can be an oil selected from the group consisting of microbial oils, algal oils, canola oil, coconut oil, castor oil, coconut oil (copra oil), corn oil, cottonseed oil, flax oil, fish oil, grape seed oil, hemp oil, jatropha oil, jojoba oil, mustard oil, canola oil, palm oil, distilled corn oil, palm stearin, palm olein, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, soybean oil, sunflower oil, tall oil, and oils from halophytes, pennycress oil, camelina oil, jojoba oil, coriander seed oil, meadowfoam oil, sea mallow oil, or any combination thereof.

[0043] The fatty acid feedstock may be a fat selected from the group consisting of animal fats including tallow from porcine, bovine, and ovine, lard, chicken fat, fish oil, or any combination thereof.

[0044] The fatty acid feedstock can be crude, refined, bleached, deodorized, degummed, or any combination thereof.

[0045] Food quality oils and fats are expensive, therefore waste products and by-products from processing, as well as non-food grade oils and fats, are becoming increasingly attractive feedstocks for producing fatty acid alkyl esters. Soapstock is a fraction of oil obtained in an oil refinery by treating the oil with base to convert the free fatty acids to soap (e.g., sodium soap). Soapstock usually contains a fraction of glycerides in addition to soap. Oxidized oils are by-products from oil refineries resulting from acidifying soapstock to solubilize the soap. Oxidized oils contain primarily free fatty acids (FFA) and acylglycerols. Distillates such as palm fatty acid distillate (PFAD) are by-products from oil refining resulting from the fractionation process used to remove the free fatty acids from the oil.

[0046] The feedstock may be an intermediate, waste product or by-product of oil and fat refining selected from the group consisting of soap stock, oxidized oils, fatty acid distillates such as PFAD, soybean fatty acid distillate, rapeseed fatty acid distillate, rice bran fatty acid distillate, chicken fat fatty acid distillate, beef tallow fatty acid distillate, gums from degumming, by-products from the manufacture of omega-3 fatty acid derivatives from fish oil, fat trap grease, yellow grease, and brown grease, free fatty acids such as oleic acid, or fractions of oil obtained by physical separation, or any combination thereof.

[0047] Free fatty acids (FFA): Free fatty acids are carboxylic acids with long carbon chains. Most naturally occurring fatty acids have unbranched chains of an even number of carbon atoms between 4 and 24. Free fatty acids are usually derived from fats (triglycerides (TAG), diglycerides (DAG), monoglycerides (MAG)), phospholipids or lysophospholipids. Triglycerides are formed by combining glycerol with three fatty acid molecules. The hydroxyl (HO-) groups of glycerol and the carboxyl (-COOH) groups of the fatty acids join together to form an ester. The glycerol molecule has three hydroxyl (HO-) groups. Each fatty acid has a carboxyl group (-COOH). Diglycerides are formed by combining glycerol with two fatty acid molecules. Monoglycerides are formed by combining glycerol with one fatty acid molecule.

[0048] Biodiesel represents a promising alternative fuel for use in compression ignition (diesel) engines. Biodiesel standards require or indirectly specify that biodiesel should be fatty acid methyl esters (FAMEs). However, the term biodiesel is used broadly for fatty acid alkyl esters of short chain alcohols obtained by the following reaction: Glycerides + FFAs + Alcohols → Fatty Acid Alkyl Esters (Biodiesel) + Glycerol + Water. Short chain alcohols are alcohols with 1 to 5 carbon atoms (C 1 -C 5) The preferred short chain alcohol is ethanol or methanol.

[0049] The present invention relates to a process for producing fatty acid alkyl esters, the process comprising: a) providing a substrate comprising a triglyceride, a diglyceride, a monoglyceride, a free fatty acid, or any combination thereof, and b) reacting the substrate with an enzyme composition comprising an sn-1,3 lipase and an sn-2 lipase to produce fatty acid alkyl esters.

[0050] In one aspect, the enzyme composition comprises an sn-1,3 lipase, wherein the lipase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO:1; and an sn-2 lipase, wherein the lipase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO:3, wherein the lipase is in liquid form.

[0051] In another aspect, the present invention further comprises reduction of the free fatty acids in the fatty acid alkyl esters produced in step b) by saponification and / or esterification in the presence of an enzyme or an acid, followed by separation and optional recovery of glycerol.

[0052] In one embodiment of the invention, alcohol and optionally water are added during step b) of the process.

[0053] In one aspect of the invention, the alcohol is a C1-C5 alcohol, preferably methanol, ethanol, propanol, or a mixture thereof.

[0054] An excess of alcohol may drive the equilibrium reaction towards complete conversion. For the purposes of this invention, the amount of alcohol is expressed in equivalents (eq.), which is the molar ratio of methanol to fatty acids (FA) present in the substrate (MetOH:FA), whether it is glycerol-bound FA or free FA.

[0055] In certain embodiments, the present invention relates to a process for producing fatty acid ethyl esters, wherein the molar ratio of methanol to fatty acid (MetOH:FA) in the substrate is about 1.0-4.0 molar equivalents, such as 1.3-3.5 molar equivalents, such as 1.5-2.5 molar equivalents.

[0056] Proteins are generally destabilized in the presence of short-chain alcohols, such as methanol and ethanol, and the inactivation of lipolytic enzymes occurs rapidly as soon as they come into contact with the insoluble alcohol present as droplets in the oil. Therefore, it is often recommended to keep the amount of alcohol below its solubility limit in oil. This can be obtained by adding alcohol continuously and / or stepwise.

[0057] In one aspect of the invention, the substrate is derived from one or more of algal oil, canola oil, coconut oil, castor oil, coconut oil, copra oil, corn oil, distilled corn oil, cottonseed oil, flax oil, fish oil, grape seed oil, hemp oil, jatropha oil, jojoba oil, mustard oil, canola oil, palm oil, palm stearin, palm olein, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, soybean oil, sunflower oil, tall oil, oil from halophytes, and / or animal fats including tallow, lard, chicken fat, fish oil from pigs, cattle, and sheep, palm oil free fatty acid distillate, soybean oil free fatty acid distillate, soapstock fatty acid material, yellow grease, used cooking oil, palm oil mill effluent, and brown grease, or any combination thereof.

[0058] The triglyceride-containing substrate is mixed with an alcohol, preferably methanol or ethanol, and heated to 30-60°C, preferably 40°C, on a reciprocating water shaking bath (200 rpm). Preferably, water is added, the solution is mixed and further heated to the desired temperature. The enzyme composition is added, the solution is vigorously mixed and left to react at the desired temperature, preferably 40°C and 200 rpm, on a reciprocating water shaking bath. The phases of the reaction mixture can be mixed by using a high shear mixer, for example of the type used in enzymatic degumming of vegetable oils (manufactured by Silverson or IKA Labortechnik) (Clausen, K. (2001), European Journal of Lipid Science and Technology, vol. 103, 333-340).

[0059] In certain embodiments, the present invention relates to a process for producing fatty acid ethyl esters, wherein methanol is added continuously and / or in a stepwise manner.

[0060] The alcohol can be added stepwise and / or continuously to the reaction over time. Water can be added separately or into the aqueous enzyme composition solution. The final concentration of water in the reaction mixture can be 0-50% (w / w), preferably 0-10% (w / w), more preferably 0.5-5% (w / w). The substrate comprises 0.00001-100% (w / w) triglycerides. Additionally, the substrate can comprise 0-100% (w / w) free fatty acids. Also, mono- and diglycerides and phospholipids can be present (the higher the glyceride content of the feedstock, the more efficient the process).

[0061] Depending on the total amount of methanol used in the conversion reaction, the number of steps of the staged addition may vary, and therefore may comprise at least 1 step, at least 2 steps, at least 3 steps, at least 4 steps, at least 5 steps, at least 6 steps, at least 7 steps, at least 8 steps, at least 9 steps, or at least 10 steps.

[0062] Lipolytic enzymes are generally rather thermotolerant in oils, and commercial processes for enzymatic intermolecular transesterification are generally carried out at 70°C. However, short-chain alcohols have a negative effect on the stability and thus the activity of lipolytic enzymes, and this destabilizing effect increases with increasing temperature. The destabilizing effect of alcohols on lipolytic enzymes appears to decrease as the molecular weight of the alcohol increases. A relationship between the solubility of alcohols in oils and the destabilizing effect of oils has been noted by several groups.

[0063] Several cases have been described where the higher alcohol dosage showed a positive effect: inactivation is not an issue when the enzyme is very robust or when using large alcohols that do not have inactivating properties. In that case, the high alcohol concentration is favorable for driving the equilibrium reaction towards complete conversion.

[0064] Complete conversion of triglyceride substrate results in the formation of glycerol as a by-product. Glycerol has been shown to inactivate especially immobilized lipolytic enzymes, possibly by physically blocking the access of the substrate to the enzyme. It has been suggested that high concentrations of alcohol may help to keep glycerol in solution, thus preventing it from inactivating lipolytic enzymes. It has been shown that glycerol adsorbed on spent silica particles can be removed by ethanol, followed by drying ("Near-quantitative production of fatty acid alkyl esters by lipase-catalyzed alcoholysis of fats and oils with adsorption of glycerol by silica gel" Stevenson et al. (1994) Enzyme Microb. Technol., vol. 16, p. 478-484).

[0065] The presence of water has been repeatedly pointed out as being important for maintaining the activity of lipolytic enzymes, and most of the currently known methods provide for the addition of water to the reaction. Surprisingly, it has been found that the method of the present invention can be carried out without the addition of water.

[0066] The process of the invention can be applied to any oils and fats consisting mainly of triglycerides (triacylglycerols), for example vegetable oils and animal fats, which typically contain more than 90% by weight (for example more than 95% by weight) of triglycerides. The acyl groups in the triglycerides can typically be linear fatty acyl groups having from 4 to 24 carbon atoms, in particular from 12 to 22 carbon atoms. They can be saturated or unsaturated, containing one or more double bonds. The triglycerides can in particular be triglycerides of unsubstituted acyl groups, i.e. acyl groups of the general formula R-CO, where R is a hydrocarbyl group. The process can be carried out at moderate temperatures and can advantageously be applied even to thermostable triglycerides, for example triglycerides with polyunsaturated acyl groups. The process results in high yields of glycerol and fatty acids, which can be separated and purified by conventional methods.

[0067] In one aspect of the invention, the process is carried out in batch or continuous mode.

[0068] The process can be carried out by contacting the substrate with the enzyme composition in its original (soluble) form in a stirred tank.

[0069] Stirred tanks can be used in batch or continuous mode. The effluent from the stirred tank (whether batch or continuous) can be separated into an oil phase containing triglycerides and fatty acids and an aqueous phase containing glycerol and lipase. The water and lipase of the aqueous phase can be recycled, optionally after separation of the glycerol, to more efficiently utilize the lipase.

[0070] Typical conditions for stirred tank operation are temperatures of 30 to 60° C., particularly 40 to 55° C., and reaction times of 6 to 72 hours, particularly 12 to 48 hours.

[0071] In one aspect of the invention, at least 80%, at least 85%, at least 90%, or such as at least 95% of the fatty acyl groups or free fatty acids in the substrate are converted to fatty acid alkyl esters.

[0072] In one embodiment of the invention, the total time of step b) is from 5 to 72 hours, such as from 10 to 70 hours, such as from 12 to 68 hours, such as from 18 to 60 hours, such as from 24 to 48 hours in a batch process.

[0073] In one embodiment of the invention, the total time of step b) is from 5 to 80 hours, such as from 10 to 75 hours, such as from 12 to 72 hours, such as from 18 to 60 hours, such as from 24 to 48 hours in a continuous process.

[0074] In one aspect of the invention, optionally the amount of water added in step b) ranges from 0.01% to 10% of the total substrate.

[0075] Enzyme Composition The present invention uses two lipolytic enzymes or lipases (triacylglycerol lipases), i.e. enzymes that catalyze the hydrolysis of ester bonds of triglycerides (triacylglycerols). They are classified as EC 3.1.1.3 according to enzyme nomenclature. The two lipases are characterized by position specificity, i.e. specificity for the acyl group at the 3-position of the triglyceride. Microbial position specific (or 1,3-specific) lipases hydrolyze acyl groups at the 1- and 3-positions and show little activity at the 2-position, whereas position non-specific lipases hydrolyze all acyl groups at the 3-positions at a similar rate. The position specificity of lipases can be determined as described in WO 8802775, WO 8901032 or WO 9414940, Example 8. The present invention uses a position non-specific lipase and a position specific lipase. Each lipase can be used in its original (solubilized) form or in immobilized form.

[0076] In the context of the present invention, lipolytic enzymes are classified in EC 3.1.1 and include true lipases, esterases, phospholipases and lysophospholipases. More specifically, lipolytic enzymes can be lipases classified in EC 3.1.1.3, EC 3.1.1.23 and / or EC 3.1.1.26, esterases classified in EC 3.1.1.1, EC 3.1.1.2, EC 3.1.1.6, EC 3.1.1.7 and / or EC 3.1.1.8, phospholipases classified in EC 3.1.1.4 and / or EC 3.1.1.32, lysophospholipases classified in EC 3.1.1.5 and cutinases classified in EC 3.1.1.74. The two or more lipolytic enzymes can also be a mixture of two or more lipases. The two or more lipolytic enzymes can include lipases and phospholipases. The two or more lipolytic enzymes include a lipase in EC 3.1.1.3. The two or more lipolytic enzymes include a lipase with activity on triglycerides, diglycerides, and monoglycerides. The two or more lipolytic enzymes include a sn-1,3 lipase and a sn-2 lipase.

[0077] In one aspect, the present invention relates to an enzyme composition for producing fatty acid alkyl esters, comprising an sn-1,3 lipase and an sn-2 lipase.

[0078] The lipolytic enzyme is preferably of microbial origin, in particular of bacterial, fungal or yeast origin. In a particular embodiment the lipolytic enzyme used is selected from the group consisting of strains of Absidia, in particular strains of Absidia blakesleena and Absidia corymbifera, strains of Achromobacter, in particular strains of Achromobacter iophagus, strains of Aeromonas, strains of Alternaria, in particular strains of Alternaria brassiciola, strains of Aspergillus, in particular strains of Aspergillus niger and Aspergillus flavus, strains of Achromobacter, in particular strains of Achromobacter iophagus, strains of Aeromonas, strains of Alternaria, in particular strains of Alternaria brassiciola, strains of Aspergillus, in particular strains of Aspergillus niger and Aspergillus flavus, strains of Achromobacter, in particular strains of Achromobacter iophagus, strains of Aeromonas, strains of Achromobacter, in particular strains of Achromobacter iophagus strains, Aureobasidium, in particular Aureobasidium pullulans strains, Bacillus, in particular Bacillus pumilus, Bacillus stearothermophilus and Bacillus subtilis strains, Beauveria strains, Brochothtix, in particular Brochothtix thermosphacta strains, Candida, in particular Candida cylindracea (Candida rugosa), Candida paralipolytica (Candida paralipolytica, Candida tsukubaensis, Candida auriculariae, Candida humicola, Candida foliarumfoliarum, strains of Candida cylindracea (Cadida rugosa) and Candida antarctica, Chromobacter, in particular strains of Chromobacter viscosum, Coprinus, in particular strains of Coprinus cinerius, Fusarium, in particular Fusarium oxysporum, Fusarium solani, Fusarium solani pisi and Fusarium roseum culmorum strains, Geotricum, in particular Geotricum penicillatum strains, Hansenula, in particular Hansenula anomala strains, Humicola, in particular Humicola brevispora, Humicola lanuginosa, Humicola brevis var. thermoidea and Humicola insolens strains, Hyphozyma strains, Lactobacillus, in particular Lactobacillus curvatus strains, strains of Penicillium, in particular Penicillium cyclopium, Penicillium crustosum and Penicillium expansum;Strains of Pseudomonas, especially Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas cepacia (synonym Burkolderia cepacia), Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas maltophilia, Pseudomonas mendocina, Pseudomonas mephitica lipolytica, Pseudomonas alcaligenes, Pseudomonas alcaligenes, Pseudomonas plantari, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas stutzeri and Pseudomonas wisconsinensis strains, Rhizoctonia, in particular strains of Rhizoctonia solani, Rhizomucor, in particular strains of Rhizomucor miehei, Rhizopus, in particular strains of Rhizopus japonicus, Rhizopus microsporus, strains of Rhodosporidium, especially strains of Rhodosporidium toruloides, Rhodotorula, especially strains of Rhodotorula glutinis,glutinis strains, Sporobolomyces, in particular Sporobolomyces shibatanus strains, Thermomyces, in particular Thermomyces lanuginosus strains (formally Humicola lanuginosa), Thiarosporella, in particular Thiarosporella phaseolina strains, Trichoderma, in particular Trichoderma harzianum and Trichoderma reesei strains, and / or Verticillium strains.

[0079] In one aspect of the invention, the sn-1,3 lipase and the sn-2 lipase are of microbial origin, in particular of fungal or bacterial origin.

[0080] In one embodiment of the present invention, the sn-1,3 lipase and the sn-2 lipase are selected from the group consisting of Aspergillus lipase, Aspergillus niger lipase, Thermomyces lanuginosa lipase, Candida Antarctica lipase A, Candida Antarctica lipase B, Candida cylindracea lipase, Candida deformans lipase, Candida lipolytica lipase, Candida parapsilosis lipase, Mucor miehei, Candida rugosa, and the like. rugosa lipase, Corynebacterium acnes lipase, Humicola lanuginosa, Cryptococcus species S-2 lipase, Fusarium culmorum lipase, Fusarium heterosporum lipase, Fusarium oxysporum lipase, Mucorjavanicus lipase, Rhizomucor miehei lipase, Rhizomucor deremar delemar lipase, Burkholderia cepacia lipase, Pseudomonas species ATCC 21808, Pseudomonas camembertii lipase, Pseudomonas fluorescens lipase, Rhizopus lipase, Rhizopus alitus lipasearrhizus lipase, Staphylococcus aureus lipase, Geotrichium candidum lipase, Hyphozyma species lipase, Klebsiella oxytoca lipase.

[0081] sn-1,3 lipase: In a preferred embodiment, the sn-1,3 lipase of the present invention comprises a lipase derived from a strain of Thermomyces, in particular a strain of Thermomyces lanuginosus (TLL) (synonym Humicola lanuginosa), or a variant thereof. In a specific embodiment, the sn-1,3 lipase is as set forth in SEQ ID NO: 1 or a variant thereof.

[0082] In one embodiment, the sn-1,3 lipase is i) a lipase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO:1; ii) A variant of a parent lipase having lipase activity that has at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the lipase set forth in SEQ ID NO:1; iii) a fragment of a lipase of (i) or (ii) having lipase activity, The variant comprises T231R+N233R and substitutions at positions corresponding to at least one or more (e.g., multiple) of D96E, D111A, D254S, G163K, P256T, G91T, and G38A of SEQ ID NO:1.

[0083] In a particular embodiment, the sn-1,3 lipase used in the present invention is a variant of a parent lipase, the variant having lipase activity with at least 60%, in particular at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity with SEQ ID NO:1, D96E+T231R+N233R, N33Q+D96E+T231R+N233R, N33Q+D111A+T231R+N233R, N33Q+T231R+N233R+P256T, N33Q+G38A+G91T+G163K+T231R+N233R+D254S, N33Q+G38A+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T, D27R+N33Q+G38A+D96E+D111A+G163K+T231R+N233R+D254S+P256T, D27R+N33Q+G38A+G91T+D96E+D111A+G163K+T231R+N233R+P256T, D27R+N33Q+G38A+G91T+D96E+D111A+G163K+T231R+N233R+D254S, D27R+G38A+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T, D96E+T231R+N233R+D254S, T231R+N233R+D254S+P256T, G163K+T231R+N233R+D254S, <h2 style=";text-align:left;direction:ltr">D27R+N33Q+G38A+G91T+D96E+G163K+T231R+N233R+D254S+P256T、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D27R+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D96E+G163K+T231R+N233R+D254S、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D27R+G163K+T231R+N233R+D254S、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D27R+G38A+G91T+D96E+D111A+G163K+T231R+N233R+D254S、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D27R+G38A+G91T+D96E+G163K+T231R+N233R+D254S+P256T、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D27R+G38A+D96E+D111A+G163K+T231R+N233R+D254S+P256T、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D27R+D96E+G163K+T231R+N233R+D254S、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D27R+D96E+D111A+G163K+T231R+N233R+D254S+P256T、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D27R+G38A+D96E+G163K+T231R+N233R+D254S+P256T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D111A+G163K+T231R+N233R+D254S+P256T、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D111A+T231R+N233R<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D111A+T231R+N233R+D254S+P256T、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D27R+D96E+D111A+G163K+T231R+N233R<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D27R+D96E+D111A+T231R+N233R<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D27R+N33Q+G38A+D96E+D111A+T231R+N233R+D254S+P256T、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D27R+G38A+D96E+D111A+G163K+E210Q+T231R+N233R+D254S+P256T、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D27R+T231R+N233R+D254S+P256T、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D96E+D111A+G163K+T231R+N233R<h2 style=";text-align:left;direction:ltr"> D96E+D111A+G163K+T231R+N233R+D254S+P256T, D96E+D111A+G163K+T231R+N233R+P256T, D96E+D111A+T231R+N233R, D96E+D111A+T231R+N233R+D254S, D96E+D111A+T231R+N233R+D254S+P256T D96E+D111A+T231R+N233R+P256T, D96E+G163K+T231R+N233R+D254S+P256T, D96E+T231R+N233R+D254S+P256T, D96E+T231R+N233R+P256T, G38A+D96E+D111A+T231R+N233R, G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T, G91T+D96E+D111A+T231R+N233R, G91T+D96E+T231R+N233R, G91T+T231R+N233R+D254S+P256T, N33Q+D96E+D111A+G163K+T231R+N233R+D254S+P256T, T231R+N233R+D254S+P256T, T231R+N233R+P256T It contains a substitution selected from the group consisting of:

[0084] In another embodiment, the sn-1,3 lipase is a variant of a parent lipase, said variant comprising: (a) comprising a modification at at least one position corresponding to positions E1, V2, N33, F51, E56, L69, K98, V176, H198, E210, Y220, L227, and K237 of SEQ ID NO:1, and optionally further comprising a modification at at least one position corresponding to positions D27, G38, D96, D111, G163, T231, N233, D254, and P256 of SEQ ID NO:1; (b) having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:1; (c) having lipase activity;

[0085] In one embodiment, the sn-1,3 lipase is a variant of a parent lipase, wherein the parent lipase is a) a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO:1; b) a fragment of the polypeptide of SEQ ID NO:1 is selected from the group consisting of:

[0086] In one embodiment, the sn-1,3 lipase is a variant having lipase activity and at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:1.

[0087] In one embodiment, the sn-1,3 lipase variant comprises modifications at at least one of the following positions corresponding to E1, V2, D27, N33, G38, F51, E56, L69, D96, K98, D111, G163, V176, H198, E210, Y220, L227, T231, N233, K237, D254, and P256, where the numbering is according to SEQ ID NO:1. More preferably, the lipase variant comprises at least one of the following modifications corresponding to E1C, V2Y, D27R, N33K, N33Q, G38A, F51V, E56K, L69R, D96E, D96L, K98I, K98Q, D111A, G163K, V176L, H198S, E210K, Y220F, L227G, T231R, N233R, N233C, K237C, D254S, and P256T, numbering according to SEQ ID NO:1.

[0088] In one embodiment, the lipase variant further comprises one of the substitutions selected from the group consisting of S54T, S83T, G91A, A150G, I255A, and E239C.

[0089] In a preferred embodiment, the sn-1,3 lipase variant comprises a substitution corresponding to E1C+N233C in SEQ ID NO:1, and optionally one or more additional substitutions.

[0090] In specific embodiments, the variant has lipase activity and has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% but less than 100% sequence identity to SEQ ID NO:1 and comprises or consists of substitutions corresponding to one of the following sets of substitutions using SEQ ID NO:1 for numbering:

[0091] [Table 1]

[0092] [Table 2]

[0093] [Table 3]

[0094] sn-2 lipase: The sn-2 lipase may be derived from a microorganism, for example a fungus or a bacterium, in particular from the following genera and species described in the indicated publications: Candida, C. rugosa (also called C. cylindracea), C. antarctica lipase A or B (WO 8802775), Pseudomonas, P. cepacia (WO 8901032), Streptomyces (WO 9414940). It may also be a variant obtained by substitution, deletion or insertion of one or more amino acids in one of the indicated lipases, as described, for example, in WO 9401541.

[0095] In a preferred embodiment, the sn-2 lipase has at least 60%, such as at least 70%, for example at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, for example 100% identity to the mature portion of the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 herein, and is preferably derived from a strain of the genus Moesziomyces, for example a strain of Moesziomyces antarcticus (Candida antarctica).

[0096] In one embodiment, the sn-2 lipase comprises or consists of the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, or an allelic variant thereof, or a fragment thereof having sn-2 lipase activity. In another embodiment, the sn-2 lipase comprises or consists of the mature polypeptide of SEQ ID NO:3 or SEQ ID NO:4, or a variant of the mature polypeptide of SEQ ID NO:3 or SEQ ID NO:4 comprising substitutions, deletions, and / or insertions at one or more positions.

[0097] In a more preferred embodiment, the sn-2 lipase used according to the invention is derived from a strain of Bacillus pumilus, a strain of Bacillus stearothermophilus, a strain of Candida cylindracea, Candida antarctica, in particular Candida antarctica lipase A (obtained as described in WO 88 / 02775).

[0098] Commercially available lipase preparations suitable for use in the process of the present invention include LIPOZYME® TL 100L, CALLERA™ TRANS and Eversa® Transform, Eversa® Transform 2.0, Novocor ADL (all available from Novozymes A / S), or mixtures thereof.

[0099] Lipase activity: In the context of the present invention, lipolytic activity can be determined as Lipase Units (LU) using tributyrate as substrate. The method is based on the enzymatic hydrolysis of tributyrin, during which the alkali consumption to keep the pH constant is recorded as a function of time. [ka]

[0100] One lipase unit (LU) may be defined as the amount of enzyme that liberates 1 micromole of titratable butyric acid per minute under standard conditions (i.e., 30° C., pH 7.0, with 0.1% (w / v) gum arabic as emulsifier and 0.16 M tributyrin as substrate).

[0101] Alternatively, lipolytic activity can be determined as long-chain lipase units (LCLU) using the substrate pNP-palmitic acid (C:16) when incubated at pH 8.0, 30° C., the lipase hydrolyzes the ester bond and releases pNP, which is yellow in color and can be detected at 405 nm. [ka]

[0102] Dosage of lipolytic enzymes Enzyme dosage is expressed for the purposes of the present invention as weight / weight percentage (w / w%) of lipolytic enzyme added to the reaction mixture relative to the substrate. For example, 10% enzyme in 100 g of oil would result in 10 g of enzyme added. In general, increased amounts of lipolytic enzyme will reduce conversion times, but from an economical point of view it is desirable to operate at reduced enzyme dosages.

[0103] In certain embodiments, the invention relates to a process for producing fatty acid ethyl esters, wherein the enzyme composition is administered at about 0.01-1.0 g enzyme protein (EP) / kg substrate.

[0104] Enzyme sources and formulations: The two or more lipolytic enzymes used in the process of the invention may be derived from or available from any of the sources mentioned herein. In this context, the term "derived" means that the enzyme can be isolated from the organism in which it originally occurs, i.e. the identity of the amino acid sequence of the enzyme is the same as the original enzyme. The term "derived" also means that the enzyme can be recombinantly produced in a host organism, where the recombinantly produced enzyme has the same identity as the original enzyme or has a modified amino acid sequence, e.g. one or more amino acids are deleted, inserted and / or substituted, i.e. the recombinantly produced enzyme is a mutation and / or fragment of the original amino acid sequence. Within the meaning of the original enzyme, naturally occurring variants are included. Furthermore, the term "derived" includes enzymes that are synthetically produced, e.g. by peptide synthesis. The term "derived" also encompasses enzymes that have been modified, e.g. by glycosylation, phosphorylation, etc., whether in vivo or in vitro. In this context, the term "available" means that the enzyme has the same amino acid sequence as the original enzyme. The term encompasses enzymes isolated from the organism in which they originate, or recombinantly expressed in the same or another organism, or synthetically produced, for example, by peptide synthesis. With respect to recombinantly produced enzymes, the terms "available" and "derived from" refer to the identity of the enzyme, not the identity of the host organism from which it is recombinantly produced.

[0105] Thus, the two or more lipolytic enzymes may be obtained from a microorganism by using any suitable technique. For example, an enzyme preparation may be obtained by fermenting a suitable microorganism and then isolating the enzyme preparation from the resulting fermentation broth or the microorganism by methods known in the art. The enzymes may also be obtained by using recombinant DNA techniques. Such methods usually involve culturing a host cell transformed with a recombinant DNA vector containing a DNA sequence encoding the enzyme of interest, the DNA sequence being operably linked to appropriate expression signals so as to express the enzyme in the culture medium under conditions allowing expression of the enzyme, and recovering the enzyme from the culture. The DNA sequence may also be integrated into the genome of the host cell. The DNA sequence may be of genomic, cDNA or synthetic origin, or any combination thereof, and may be isolated or synthesized according to methods known in the art.

[0106] The two or more lipolytic enzymes may be applied as any suitable formulation, for example as a lyophilized powder or a liquid / aqueous solution.

[0107] Furthermore, the present invention relates to a batch process and / or a continuous, stepwise process for producing fatty acid alkyl esters using the first and second lipolytic enzymes described above, in which the alcohol is added continuously and / or stepwise and the enzyme is recycled or used only once. If the enzyme is in the aqueous phase, this phase can be separated from the fat phase by a decanter, settler or centrifuge. In a continuous process, the two phases, the oil phase and the aqueous phase, can be treated in countercurrent, respectively.

[0108] Cloning of DNA sequences encoding lipolytic enzymes. The DNA sequence encoding the parent lipolytic enzyme can be isolated from any cell or microorganism that produces the lipolytic enzyme of interest using various methods well known in the art. First, a genomic DNA and / or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the lipolytic enzyme to be studied. Then, if the amino acid sequence of the lipolytic enzyme is known, a labeled oligonucleotide probe can be synthesized and used to identify the clones encoding the lipolytic enzyme from the genomic library prepared from the organism of interest. Alternatively, a labeled oligonucleotide probe containing a sequence that is homologous to other known lipolytic enzyme genes can be used as a probe using low stringency hybridization and washing conditions to identify the clones encoding the lipolytic enzyme.

[0109] Yet another method for identifying clones encoding lipolytic enzymes involves inserting genomic DNA fragments into an expression vector, e.g. a plasmid, transforming cutinase-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria on agar containing a substrate for the lipolytic enzyme (i.e. triglycerides), thereby allowing identification of clones which express the lipolytic enzyme.

[0110] Alternatively, the DNA sequence encoding the enzyme can be prepared synthetically by established standard methods, such as the phosphoramidite method described by SLBeaucage and M HCaruthers, (1981), Tetrahedron Letters 22, p. 1859-1869, or the method described by Matthes et al., (1984), EMBO J. 3, p. 801-805. In the phosphoramidite method, oligonucleotides are synthesized, for example, by an automatic DNA synthesizer, purified, annealed, ligated and cloned in a suitable vector.

[0111] Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by joining fragments of synthetic, genomic or cDNA origin (where appropriate, fragments corresponding to various parts of the total DNA sequence) according to standard techniques. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for example as described in U.S. Pat. No. 4,683,202 or RK Saiki et al., (1988), Science 239, 1988, pp. 487-491.

[0112] Expression vector The recombinant expression vector carrying the DNA sequence encoding the lipolytic enzyme of the present invention can be any vector that can be conveniently subjected to recombinant DNA procedures, and the choice of vector often depends on the host cell into which it is introduced.When the vector is introduced into the host cell, it can be integrated into the host cell genome and replicated together with the chromosome into which it is integrated.An example of a suitable expression vector includes pMT838.

[0113] The expression vector of the present invention may also contain a suitable transcription terminator and, in eukaryotes, a polyadenylation sequence operably connected to the DNA sequence encoding the lipolytic enzyme of the present invention. The termination and polyadenylation sequences may suitably be derived from the same source as the promoter.

[0114] The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.

[0115] Vectors may also contain a selectable marker, for example a gene whose product complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene conferring antibiotic resistance, for example, ampicillin, kanamycin, chloramphenicol or tetracycline. Additionally, vectors may contain Aspergillus selection markers, such as amdS, argB, niaD and sC, i.e., markers conferring hygromycin resistance, or selection may be achieved by cotransformation, for example as described in WO 91 / 17243.

[0116] The procedures used to ligate the DNA constructs of the present invention, each encoding a cutinase variant, promoter, terminator and other elements, and insert them into a suitable vector containing the information necessary for replication, are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989).

[0117] promoter In the vector, the DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.

[0118] Examples of promoters suitable for directing the transcription of a DNA sequence encoding the lipolytic enzyme of the invention, particularly in bacterial hosts, are the promoter of the lac operon of E. coli, the promoter of the Streptomyces coelicolor agarose gene dagA, the promoter of the Bacillus licheniformis α-amylase gene (amyL), the promoter of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoter of the Bacillus amyloliquefaciens α-amylase (amyQ), the promoter of the Bacillus subtilis xylA and xylB genes, etc. For transcription in fungal hosts, examples of useful promoters include the promoters from A. oryzae TAKA amylase, S. cerevisiae (Alber et al. (1982), J. Mol. Appl. Genet 1, p. 419-434), Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger acid-stable α-amylase, A. niger glucoamylase, Rhizomucor miehei miehei lipase, A. oryzae alkaline protease, A. oryzae triosephosphate isomerase, or A. nidulans acetamidase.

[0119] host cell The cell of the present invention, comprising either the DNA construct or expression vector of the present invention as defined above, is advantageously used as a host cell in the recombinant production of the lipolytic enzyme of the present invention. The cell can be transformed with the DNA construct of the present invention encoding the lipolytic enzyme, advantageously by integrating the DNA construct (in one or more copies) into the host chromosome. Integration is generally considered to be advantageous, since the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA construct in the host chromosome can be carried out according to conventional methods, for example by homologous or heterologous recombination. Alternatively, the cell can be transformed with an expression vector as described above for the different types of host cells.

[0120] The cell of the invention may be a cell of a higher organism, such as a mammalian or insect cell, in particular a microbial cell, such as a bacterial or fungal (including yeast) cell.

[0121] Examples of suitable bacteria include gram-positive bacteria, such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophillus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, Bacillus thuringiensis or Streptomyces lividans or Streptomyces murinus, or a Gram-negative bacterium, for example E. coli. The transformation of bacteria can be effected, for example, by protoplast transformation or by using competent cells in a manner known per se.

[0122] The yeast organism may suitably be selected from Saccharomyces or Schizosaccharomyces, for example Saccharomyces cerevisiae.

[0123] Host cells may also be filamentous fungi, such as strains belonging to the species Aspergillus, in particular Aspergillus oryzae or Aspergillus niger, or strains of Fusarium, such as Fusarium oxysporum, Fusarium graminearum (synonymous with the full name Gibberella zeae, formerly Sphaeria zeae, Gibberella roseum and Gibberella roseum f. species cerealis), or Fusarium sulphureum. It may be a strain of Fusarium sulphureum (synonymous with the full names Gibberella puricaris, Fusarium trichothecioides, Fusarium bactridioides, Fusarium sambucinum, Fusarium roseum, and Fusarium roseum var. graminearum), Fusarium cerealis (synonymous with Fusarium crookwellense), or Fusarium venenatum.

[0124] In a particular embodiment of the invention, the host cell is a protease-deficient or protease-minus strain, which can be, for example, the protease-deficient strain Aspergillus oryzae JaL125, in which the alkaline protease gene with the name "alp" has been deleted. This strain is described in WO 97 / 35956 (Novo Nordisk).

[0125] Filamentous fungal cells can be transformed by a process involving the formation of protoplasts and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. The use of Aspergillus as a host microorganism is described in EP 238023 (Novo Nordisk A / S), the contents of which are incorporated herein by reference.

[0126] Production of lipolytic enzymes by culturing transformants The present invention relates inter alia to a method for producing a lipolytic enzyme of the invention, comprising culturing a host cell under conditions conducive to the production of the lipolytic enzyme and recovering the lipolytic enzyme from the cells and / or the culture medium.

[0127] The medium used to culture the cells may be any conventional medium suitable for growing the host cells in question and obtaining expression of the lipolytic enzymes of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).

[0128] Lipolytic enzymes secreted from the host cells may be conveniently recovered from the culture medium by well-known procedures including separation of the cells from the medium by centrifugation or filtration and precipitation of protein components from the medium with a salt, e.g., ammonium sulfate, followed by the use of chromatographic procedures, e.g., ion exchange chromatography, affinity chromatography, and the like.

[0129] Enzymatic biodiesel process design The process setup is very important as technical issues such as homogeneity of the reaction / product mixture, alcohol solubility, enzyme stability, enzyme recovery etc. must be considered. There are several process designs to consider: batch stirred tank reactors and continuous stirred tank reactors. These are briefly outlined in the following paragraphs.

[0130] Batch processes are typical processes used in laboratories because of their ease of setup. The process can be operated in bulk, i.e., with all ingredients added from the beginning, or stepwise and / or continuous addition of alcohol is recommended. Batch processes are useful for collecting data about the process, such as enzyme productivity. The negative aspects of this large-scale process setup are the large tank volumes required, the long reaction times, and the fact that the process is not continuous. Another very important fact to consider is the gradual decrease in enzyme activity as the number of reuses increases. When enzyme activity decreases, the reaction time must be increased accordingly to maintain a constant conversion, or additional enzyme can be added to compensate for the decrease in activity.

[0131] A continuous stirred tank reactor is a vessel that is continuously fed and removed. This design requires multiple tanks in series to ensure the same degree of conversion in the same reaction time, which means that the total tank volume is likely to increase compared to a similar batch system due to the increased total residence time in a continuous system. The advantage of such a continuous system is that the plant capacity is typically increased because the time required for emptying / filling is eliminated. Another advantage of a continuous design is that separation steps can be introduced between the tanks, such as continuously removing the glycerol formed. Furthermore, continuous systems reach a steady state where most process parameters remain fairly constant, assuming the input parameters are constant. This allows the continuous system to be tuned in a way that allows optimal operation of the enzymes by providing better steady state operating conditions than evolutionary batch systems where the operating conditions, especially the component concentrations, naturally change throughout the reaction time.

[0132] In certain embodiments, the present invention relates to a process for producing fatty acid ethyl esters, the process being selected from the group consisting of process designs containing batch, continuous stirred tank reactors, and others.

[0133] Feedstock for enzymatic production of biodiesel Fatty acid ethyl esters can be prepared from several types of vegetable oils. In the world vegetable oil production, palm oil holds the top position in terms of profits and has the highest yields compared to the yields of other vegetable oils, and therefore, economically speaking, it is intuitive to consider palm oil as an advantageous feedstock for biodiesel production. However, since edible oils are not in surplus supply, some argue in favor of using non-edible oils such as jatropha oil. Examples of plants that can serve as feedstocks for vegetable oils to be used as substrates in the production of fatty acid ethyl esters include, for example, babassu, borage, canola, coconut, corn, cotton, hemp, jatropha, karanji, mustard, palm, peanut, rapeseed, rice, soybean, and sunflower.

[0134] Microalgae are also considered as a feedstock for biodiesel production due to the higher photosynthetic efficiency of microalgae compared to plants, potentially resulting in higher productivity per unit area.

[0135] Alternatively, fatty acid ethyl esters can be prepared from non-plant feedstocks such as animal fats, including lard, tallow, butterfat and chicken or marine oils.

[0136] It is estimated that 60-90% of the cost of biodiesel is attributable to the cost of feedstock oil, and the utilization of cheap waste oils will have a significant impact on the cost reduction of biodiesel. In addition, it is considered as an important step in the reduction and recycling of waste oils. Fresh vegetable oils and their wastes differ in their water and free fatty acid contents. Unlike the conventional chemical route for the synthesis of diesel fuel, the biocatalytic route allows the transesterification of a wide variety of oil feedstocks in the presence of acidic impurities such as free fatty acids. Thus, fatty acid distillates (from deodorization / fatty acid stripping), oxidized oils (from soapstock splitting in chemical petroleum refineries), waste oils and used oils can serve as feedstocks in the production of biodiesel.

[0137] Thus, the feedstock can be of crude quality or further processed (refined, bleached and deodorized). Suitable oils and fats can be pure triglycerides or mixtures of triglycerides, diglycerides, monoglycerides, and free fatty acids commonly found in discarded vegetable oils and animal fats. The feedstock can also be obtained from vegetable oil deodorizer distillates. The types of fatty acids in the feedstock include those naturally occurring as glycerides in plant and animal fats and oils. These include oleic acid, linoleic acid, linolenic acid, palmitic acid, steric acid, and lauric acid, to name a few. The minor components in crude vegetable oils are typically phospholipids, free fatty acids, and partial glycerides, i.e., monoglycerides and diglycerides.

[0138] In certain embodiments, the present invention relates to a process for producing fatty acid ethyl esters, wherein the substrate is selected from the group containing babassu oil, borage oil, canola oil, coconut oil, corn oil, cotton oil, hemp oil, jatropha oil, karanji oil, mustard oil, palm oil, peanut oil, rapeseed oil, rice oil, soybean oil, and sunflower oil, oil from microalgae, animal fat, tallow, lard, butterfat, chicken oil, marine oil, tuna oil, fatty acid distillates, oxidized oil, waste oil, used oil, partial glycerides, and any combination thereof.

[0139] Reuse of lipolytic enzyme compositions in the production of fatty acid ethyl esters In certain embodiments, the present invention relates to the reuse of the enzyme composition in the production of fatty acid ethyl esters obtained by reacting methanol with a substrate comprising triglycerides, diglycerides, monoglycerides, free fatty acids or any combination thereof, in which the molar ratio of methanol to fatty acids (MetOH:FA) in the substrate is about 1.0-4.0 equivalents, the enzyme dosage is less than 1% w / w (enzyme protein to oil) with respect to the substrate, and the enzyme after use in the conversion reaction is separated from the resulting reaction mixture and directly reused in the next conversion reaction without modification. By modification is meant any treatment or activity, such as activation, washing, drying, etc., apart from separating the lipolytic enzyme from the reaction mixture.

[0140] In certain embodiments, the present invention relates to reuse of the enzyme composition in the production of fatty acid ethyl esters, wherein the sn-1,3 lipase and the sn-2 lipase are selected from the group consisting of Thermomyces lanuginosa lipase, Candida Antarctica B lipase, Candida deformans lipase, Candida lipolytica lipase, Candida parapsilosis lipase, Candida rugosa lipase, Cryptococcus species S-2 lipase, Rhizomucor miehei lipase, Rhizomucor deremarc. delemar lipase, Burkholderia (Burkholderia) lipase, Pseudomonas (Pseudomonas) cepacia lipase, Pseudomonas camembertii lipase, Pseudomonas fluorescens lipase, Geotrichium candidum lipase, Hyphozyma species lipase, Klebsiella oxytoca lipase, and variants thereof.

[0141] In certain embodiments, the invention relates to the reuse of the enzyme composition in the production of fatty acid ethyl esters, the enzyme composition being administered in the range of 0.01-1.0 g enzyme protein (EP) / kg substrate.

[0142] In certain embodiments, the present invention relates to the reuse of the enzyme composition in the production of fatty acid ethyl esters, where methanol is added continuously or stepwise.

[0143] In certain embodiments, the present invention relates to the reuse of the enzyme composition in the production of fatty acid ethyl esters, the process being selected from the group consisting of process designs containing batch, continuous stirred tank reactor, and others.

[0144] In certain embodiments, the present invention relates to at least reuse of the enzyme composition in the production of fatty acid ethyl esters, wherein the substrate is selected from the group containing babassu oil, borage oil, canola oil, coconut oil, corn oil, cotton oil, hemp oil, jatropha oil, karanji oil, mustard oil, palm oil, peanut oil, rapeseed oil, rice oil, soybean oil, and sunflower oil, oil from microalgae, animal fat, tallow, lard, butterfat, chicken oil, marine oil, tuna oil, fatty acid distillates, oxidized oil, waste oil, used oil, partial glycerides, and any combination thereof.

[0145] In certain embodiments of the invention, reuse of the enzyme composition is facilitated by recycling all or part of the heavy phase resulting from the reaction. In particular, in the case of liquid enzyme composition formulations, the enzyme composition is found almost entirely in the heavy phase and can be reused by separation of the light and heavy phases by gravity settling principles, for example by centrifugation or decantation.

[0146] The invention is described in the following numbered paragraphs.

[0147] 1. A process for producing fatty acid alkyl esters, comprising: a) providing a substrate comprising a triglyceride, a diglyceride, a monoglyceride, a free fatty acid, or any combination thereof; b) reacting the substrate with an enzyme composition containing sn-1,3 lipase and sn-2 lipase to produce a fatty acid alkyl ester; The process includes:

[0148] 2. The process of paragraph 1, further comprising reduction of the free fatty acids in the fatty acid alkyl esters produced in step b) by saponification and / or esterification in the presence of an enzyme or an acid, followed by separation and optional recovery of glycerol.

[0149] 3. The process according to paragraph 1 or 2, wherein an alcohol and optionally water are added during step b).

[0150] 4. The process of any one of paragraphs 1 to 3, wherein the alcohol is a C1-C5 alcohol, preferably methanol, ethanol, propanol, or a mixture thereof.

[0151] 5. The process of any one of paragraphs 1 to 4, wherein the molar ratio of methanol to fatty acid (MetOH:FA) in the substrate is about 1.0 to 4.0 molar equivalents, such as 1.3 to 3.5 molar equivalents, such as 1.5 to 2.5 equivalents.

[0152] 6. The process of any one of paragraphs 1 to 5, wherein the process is conducted in a batch or continuous mode.

[0153] 7. The process of any one of paragraphs 1-6, wherein the substrate is derived from one or more of algal oil, canola oil, coconut oil, castor oil, coconut oil, copra oil, corn oil, distilled corn oil, cottonseed oil, flax oil, fish oil, grape seed oil, hemp oil, jatropha oil, jojoba oil, mustard oil, canola oil, palm oil, palm stearin, palm olein, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, soybean oil, sunflower oil, tall oil, oil from halophytes, and / or animal fats including tallow, lard, chicken fat, fish oil from pigs, cattle, and sheep, palm oil free fatty acid distillate, soybean oil free fatty acid distillate, soapstock fatty acid material, yellow grease, used cooking oil, palm oil mill effluent, and brown grease, or any combination thereof.

[0154] 8. The process according to any one of paragraphs 1 to 7, wherein the total time of step b) is from 5 to 72 hours, such as from 10 to 70 hours, for example from 12 to 68 hours, such as from 18 to 60 hours, for example from 24 to 48 hours in a batch process.

[0155] 9. The process according to any one of paragraphs 1 to 8, wherein the total time of step b) is from 5 to 80 hours, such as from 10 to 75 hours, for example from 12 to 72 hours, such as from 18 to 60 hours, for example from 24 to 48 hours, in a continuous process.

[0156] 10. Optionally, the process of any one of paragraphs 1 to 9, wherein the amount of water added in step b) is in the range of 0.01% to 10% of the total substrate.

[0157] 11. The process of any one of paragraphs 1 to 10, wherein at least 80%, at least 85%, at least 90%, or for example at least 95% of the fatty acyl groups or free fatty acids in the substrate are converted to fatty acid alkyl esters.

[0158] 12. An enzyme composition for producing fatty acid alkyl esters, comprising an sn-1,3 lipase and an sn-2 lipase.

[0159] 13. A composition according to paragraph 12, in which the sn-1,3 lipase and the sn-2 lipase are of microbial origin, in particular of fungal or bacterial origin.

[0160] 14. sn-1,3 lipase and sn-2 lipase are selected from the group consisting of Aspergillus lipase, Aspergillus niger lipase, Thermomyces lanuginosa lipase, Candida Antarctica lipase A, Candida Antarctica lipase B, Candida cylindracea lipase, Candida deformans lipase, Candida lipolytica lipase, Candida parapsilosis lipase, Mucor miehei lipase, Candida rugosa lipase, and the like. rugosa lipase, Corynebacterium acnes lipase, Humicola lanuginosa, Cryptococcus species S-2 lipase, Fusarium culmorum lipase, Fusarium heterosporum lipase, Fusarium oxysporum lipase, Mucorjavanicus lipase, Rhizomucor miehei lipase, Rhizomucor deremar delemar lipase, Burkholderia cepacia lipase, Pseudomonas species ATCC 21808, Pseudomonas camembertii lipase, Pseudomonas fluorescens lipase, Rhizopus lipase, Rhizopus arrhizus lipase, Staphylococcus aureus lipase14. The composition according to paragraph 12 or 13, wherein the lipase is selected from the group consisting of: Citrobacter aureus lipase, Geotrichium candidum lipase, Hyphozyma species lipase, Klebsiella oxytoca lipase.

[0161] 15. sn-1,3 lipase i) a lipase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO:1; ii) A variant of a parent lipase having lipase activity that has at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the lipase set forth in SEQ ID NO:1; 15. The composition according to any one of paragraphs 12 to 14, which is a fragment of the lipase of (i) or (ii) having lipase activity.

[0162] 16. The composition of any one of paragraphs 12 to 15, wherein the sn-1,3 lipase is a variant comprising T231R+N233R and optionally substitutions at positions corresponding to at least one or more (e.g., a plurality) of D96E, D111A, D254S, G163K, P256T, G91T and G38A in SEQ ID NO:1.

[0163] 17. The sn-1,3 lipase is a variant of a parent lipase, wherein the variant has lipase activity with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to SEQ ID NO:1, and is selected from the group consisting of T231R+N233R and a.D96E+T231R+N233R, b.N33Q+D96E+T231R+N233R, c.N33Q+D111A+T231R+N233R, d.N33Q+T231R+N233R+P256T, e.N33Q+G38A+G91T+G163K+T231R+N233R+D254S, f.N33Q+G38A+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T, g.D27R+N33Q+G38A+D96E+D111A+G163K+T231R+N233R+D254S+P256T, h.D27R+N33Q+G38A+G91T+D96E+D111A+G163K+T231R+N233R+P256T, i.D27R+N33Q+G38A+G91T+D96E+D111A+G163K+T231R+N233R+D254S, j.D27R+G38A+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T, k.D96E+T231R+N233R+D254S, l.T231R+N233R+D254S+P256T, m.G163K+T231R+N233R+D254S, n.D27R+N33Q+G38A+G91T+D96E+G163K+T231R+N233R+D254S+P256T, o.D27R+G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T, p.D96E+G163K+T231R+N233R+D254S、 q.D27R+G163K+T231R+N233R+D254S、 r.D27R+G38A+G91T+D96E+D111A+G163K+T231R+N233R+D254S、 s.D27R+G38A+G91T+D96E+G163K+T231R+N233R+D254S+P256T、 t.D27R+G38A+D96E+D111A+G163K+T231R+N233R+D254S+P256T、 u.D27R+D96E+G163K+T231R+N233R+D254S、 v.D27R+D96E+D111A+G163K+T231R+N233R+D254S+P256T、 w.D27R+G38A+D96E+G163K+T231R+N233R+D254S+P256T x.D111A+G163K+T231R+N233R+D254S+P256T、 y.D111A+T231R+N233R、 z.D111A+T231R+N233R+D254S+P256T、 aa.D27R+D96E+D111A+G163K+T231R+N233R、 bb.D27R+D96E+D111A+T231R+N233R、 cc.D27R+N33Q+G38A+D96E+D111A+T231R+N233R+D254S+P256T、 dd.D27R+G38A+D96E+D111A+G163K+E210Q+T231R+N233R+D254S+P256T、 ee.D27R+T231R+N233R+D254S+P256T、 ff.D96E+D111A+G163K+T231R+N233R、 gg.D96E+D111A+G163K+T231R+N233R+D254S+P256T、 hh.D96E+D111A+G163K+T231R+N233R+P256T, ii.D96E+D111A+T231R+N233R, jj.D96E+D111A+T231R+N233R+D254S, kk.D96E+D111A+T231R+N233R+D254S+P256T ll.D96E+D111A+T231R+N233R+P256T, mm.D96E+G163K+T231R+N233R+D254S+P256T, nn.D96E+T231R+N233R+D254S+P256T, oo.D96E+T231R+N233R+P256T, pp.G38A+D96E+D111A+T231R+N233R, qq.G91T+D96E+D111A+G163K+T231R+N233R+D254S+P256T, rr.G91T+D96E+D111A+T231R+N233R, ss.G91T+D96E+T231R+N233R, tt.G91T+T231R+N233R+D254S+P256T, uu.N33Q+D96E+D111A+G163K+T231R+N233R+D254S+P256T, vv.T231R+N233R+D254S+P256T, ww.T231R+N233R+P256T 17. The composition of any one of paragraphs 12 to 16, comprising a substitution at a position corresponding to at least one or more (e.g., a plurality) of D96E, D111A, D254S, G163K, P256T, G91T and G38A of SEQ ID NO:1 selected from the group consisting of:

[0164] 18. The sn-1,3 lipase is a variant of a parent lipase, said variant comprising: (a) comprising a modification at at least one position corresponding to positions E1, V2, N33, F51, E56, L69, K98, V176, H198, E210, Y220, L227, and K237 of SEQ ID NO:1, and optionally further comprising a modification at at least one position corresponding to positions D27, G38, D96, D111, G163, T231, N233, D254, and P256 of SEQ ID NO:1; (b) has at least 60% but less than 100% sequence identity to SEQ ID NO:1; (c) having lipase activity 18. The composition according to any one of paragraphs 12 to 17.

[0165] 19. The composition according to any one of paragraphs 12 to 18, wherein the sn-1,3 lipase variant comprises a modification in at least one of the following positions: E1, V2, D27, N33, G38, F51, E56, L69, D96, K98, D111, G163, V176, H198, E210, Y220, L227, T231, N233, K237, D254, and P256, the numbering being according to SEQ ID NO:1.

[0166] 20. The composition according to paragraph 18 or 19, wherein the sn-1,3 lipase variant comprises at least one of the following modifications: E1C, V2Y, D27R, N33K, N33Q, G38A, F51V, E56K, L69R, D96E, D96L, K98I, K98Q, D111A, G163K, V176L, H198S, E210K, Y220F, L227G, T231R, N233R, N233C, K237C, D254S, and P256T, wherein the numbering is according to SEQ ID NO:1.

[0167] 21. The composition according to any one of paragraphs 18 to 20, wherein the sn-1,3 lipase variant further comprises one of the substitutions selected from the group consisting of S54T, S83T, G91A, A150G, I255A, and E239C, and the numbering is according to SEQ ID NO:1.

[0168] 22. The composition according to any one of paragraphs 18 to 21, wherein the sn-1,3 lipase variant comprises the substitutions E1C+N233C and one or more additional substitutions, the numbering being according to SEQ ID NO:1.

[0169] 23. The composition according to any one of paragraphs 18 to 22, wherein the sn-1,3 lipase has lipase activity and has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% but less than 100% sequence identity to SEQ ID NO:1 and comprises or consists of substitutions corresponding to one of the following sets of substitutions using SEQ ID NO:1 for numbering:

[0170] [Table 4]

[0171] [Table 5]

[0172] [Table 6]

[0173] 24. sn-1,3 lipase, i) a lipase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO:2; ii) variants having lipase activity with at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the lipase set forth in SEQ ID NO:2; iii) A fragment of lipase of (i) or (ii) having lipase activity. 24. The composition according to any one of paragraphs 12 to 23, wherein

[0174] 25. sn-2 lipase, i) a lipase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO:3; ii) variants having lipase activity having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the lipase set forth in SEQ ID NO:3; iii) A fragment of lipase of (i) or (ii) having lipase activity. 13. The composition according to paragraph 12, wherein

[0175] 26. The composition according to any one of paragraphs 12 to 25, wherein the total amount of the enzyme composition is in the range of 0.01 to 1.0 g enzyme protein (EP) / kg substrate.

[0176] 27. Use of the enzyme composition according to any one of paragraphs 12 to 26 for producing fatty acid alkyl esters. EXAMPLES

[0177] Example 1: Testing the combination of Eversa Transform (ET) 2.0 (SEQ ID NO: 1) in combination with Novocor ADL (CalA formulation) (SEQ ID NO: 3). Both enzymes are from Novozymes, Denmark. 30 g crude palm oil (CPO) or crude palm kernel oil (CPKO), NaOH (10 ppm for CPO, 50 ppm for CPKO), 2.5% water, 1.7 eq. MeOH, 0.3 eq. at the start and 1.4 eq. over 20 h.

[0178] Enzyme composition: 1:0,1% ET2.0HS, 2: 0.08% ET2.0HS + CalA equivalent ~ 0.02% ET2.0, 3: 0.06% ET2.0HS + CalA equivalent ~ 0.04% ET2.0HS, NB is ET2.0HS with a low enzyme dose of 0.1%.

[0179] Samples are made in doublets. 40°C, shaking incubator 250 rpm. Samples taken at 1, 3, 20 and 24 hours respectively. DG, analysis of relative glyceride composition, analyzed on Eurofins QTA by calibration B-100 (method according to AOCS method CK2-09) (24 hour sample) and FFA% of 24 hour sample measured by titration. Samples are dried in speed vacuum. ET HS 3.9% active enzyme-protein (39mg / mL) Novocor (CalA) ADL 1.65% active enzyme-protein. 0.243% Novocor(CalA)ADL=0.1% ET2.0HS

[0180] [Table 7]

[0181] [Table 8]

[0182] The results showed that in the case of CPO, better conversion with increasing CalA substitution, and in the case of CPKO, only 20% substitution showed good conversion, while 40% substitution was similar to pure ET.

[0183] Example 2: Various feedstocks with emphasis on total glycerides at the end of the reaction: 30 g of fatty acid feedstocks of various qualities. POME and UCO are typically low quality feedstocks with high initial FFA levels, whereas CPKO and CPO are relatively high quality triglyceride oils.

[0184] Higher initial FFA translates to more water in the endpoint reaction mixture and therefore higher FFA at equilibrium.

[0185] 2% water is added to the enzyme mixture. The total enzyme protein in the mixture is similar in all cases and corresponds to the amount of enzyme dosed with 0.2% ET2.0.

[0186] Concentration measurement using NIR (QTA, Eurofins, B100 method) at t=30 hours For CPKO and CPO, a low MeOH dose (1.6 equivalents) and a slow 20 hour dosing rate were optimal and are reported below. For POME and UCO, a high MeOH dose (2.2 equivalents) and a fast 10 hour dosing rate were optimal and are reported below.

[0187] [Table 9]

[0188] [Table 10]

[0189] [Table 11]

[0190] [Table 12]

[0191] In general, total glyceride levels are reduced when using ET / CALA mixtures.

[0192] Example 3: Comparison of SEQ ID NO:4 vs. SEQ ID NO:3: Standard biodiesel reaction with 2% water, 50 ppm sodium hydroxide. 1.7 equivalents methanol dosed over 20 hours. 30 g CPO in 100 mL square blue cap flasks incubated at 35C, 250 rpm shaking incubator. 0.2% of SEQ ID NO:1 enzyme as normalized enzyme protein mass dose is 2.34 mg enzyme protein total for all tests. 80 / 20 SEQ ID NO:1 / SEQ ID NO:3 (protein mass basis), or SEQ ID NO:1 / SEQ ID NO:4 (protein mass basis). Samples run in duplicate.

[0193] [Table 13]

[0194] Considering the 20 hour sample, which is the furthest from equilibrium, it is clear that SEQ ID NO:3 increases the reaction rate compared to SEQ ID NO:1 alone, with SEQ ID NO:3 providing an improvement over SEQ ID NO:4. Then, considering the 24 hour sample, SEQ ID NO:3 provides an improvement over SEQ ID NO:4 and SEQ ID NO:1 alone. SEQ ID NO:1 alone is better than SEQ ID NO:4 at the 24 hour reaction time, meaning it is closer to equilibrium, and the exceptional esterification ability of SEQ ID NO:4 may adversely affect the results. Therefore, SEQ ID NO:3 is preferred.

[0195] The present invention as described and claimed herein should not be limited in scope by the specific embodiments disclosed herein, since these embodiments are intended to be illustrative of some embodiments of the invention. Any equivalent embodiments are intended to be within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, the present disclosure, including definitions, will control.

Claims

1. A process for producing fatty acid alkyl esters, a) A step of providing a substrate comprising triglycerides, diglycerides, monoglycerides, free fatty acids, or any combination thereof, b) A step of producing a fatty acid alkyl ester by reacting the substrate with an enzyme composition comprising sn-1,3 lipase and sn-2 lipase, wherein the sn-1,3 lipase is a lipase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, and the sn-2 lipase is a lipase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity with SEQ ID NO: 3, and the lipase is in liquid form. A process that includes this.

2. The process according to claim 1, further comprising saponification and / or esterification in the presence of an enzyme or acid, followed by reduction of the free fatty acid in the fatty acid alkyl ester produced in step b) by separation and optionally recovery of glycerol.

3. The process according to claim 1, wherein alcohol and optionally water are added during step b).

4. The process according to claim 1, wherein the alcohol is a C1-C5 alcohol, preferably methanol, ethanol, propanol, or a mixture thereof.

5. The process according to claim 1, wherein the substrate is derived from one or more of the following: algal oil, canola oil, coconut oil, castor oil, coconut oil, copra oil, corn oil, distilled corn oil, cottonseed oil, flaxseed oil, fish oil, grape seed oil, hemp oil, jatropha oil, jojoba oil, mustard oil, canola oil, palm oil, palm stearin, palm olein, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, soybean oil, sunflower oil, tall oil, oils from salt-tolerant plants, and / or animal fats including animal fats derived from pigs, cattle, and sheep, lard, chicken fat, fish oil, palm oil free fatty acid distillates, soybean oil free fatty acid distillates, soapstock fatty acid materials, yellow grease, used cooking oil, palm oil factory wastewater, and brown grease, or any combination thereof.

6. The process according to claim 1, wherein at least 80%, at least 85%, at least 90%, or for example, at least 95% of the fatty acid acyl groups or free fatty acids in the substrate are converted to fatty acid alkyl esters.

7. An enzyme composition for producing a fatty acid alkyl ester comprising sn-1,3 lipase and sn-2 lipase, wherein the sn-1,3 lipase is a lipase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, and the sn-2 lipase is a lipase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity with SEQ ID NO: 3, and the lipase is in liquid form.

8. The composition according to claim 7, wherein the sn-1,3 lipase and the sn-2 lipase are of microbial origin, particularly fungal or bacterial origin.

9. The aforementioned sn-1,3 lipase and the aforementioned sn-2 lipase are Aspergillus lipase, Aspergillus niger lipase, Thermomyces lanuginosa lipase, Candida Antarctica lipase A, Candida Antarctica lipase B, Candida cylindracea lipase, Candida deformans lipase, Candida lipopolitica lipase of *Lipolitis*, lipase of *Candida* parapsilosis, lipase of *Mucor miehei*, lipase of *Candida* rugosa, lipase of *Corynebacterium* acnes, lipase of *Humicola* lanuginosa, S-2 lipase of *Cryptococcus* species, lipase of *Fusarium* culmorum, lipase of *Fusarium* heterosporum heterosporum lipase, Fusarium oxysporum lipase, Mucorjavanicus lipase, Rhizomucor miehei lipase, Rhizomucor delemar lipase, Burkholderia (Pseudomonas) cepacia lipase, Pseudomonas species ATCC 21808, Pseudomonas camembertii (cambertii) lipase, Pseudomonas fluorescein lipase, Rhizopus lipase, Rhizopus arrhizus lipase, Staphylococcus aureusThe composition according to claim 7, selected from the group consisting of aureus lipase, Geotrichum candidum lipase, Hyphozyma species lipase, and Klebsiella oxytoca lipase.

10. Use of the enzyme composition according to claim 7 for producing a fatty acid alkyl ester.