Process for reducing free fatty acids

Abstract

The present invention relates to the process of enzymatic (trans)esterification / esterification of free fatty acids and glycerides. In particular, the present invention relates to this process by continuously using a drying operation for removing water from the enzyme reaction mixture, or separating the glycerol phase from the reaction mixture, then drying the glycerol phase and recycling it to reform the reaction mixture, specifically in biodiesel applications, thereby promoting the reduction of FFA in biodiesel.

Description

Technical Field

[0001] The present invention relates to a process for reducing the level of free fatty acids in biodiesel / fatty acid alkyl esters by an enzymatic combined (trans)esterification / esterification reaction. In particular, the present invention relates to this process using a low water content and glycerol recirculation in the combined (trans)esterification / esterification reaction.

Background Art

[0002] CO generated from fossil fuels 2 causes environmental problems and there is a strong need to search for sustainable solutions worldwide.

[0003] Currently, conventional diesel fuels derived from fossil carbon sources are the main cause of CO 2 emissions, and developing a sustainable and renewable fuel that can drive existing diesel motors is a viable way to reduce CO 2 emissions related to diesel. Such fuels are, for example, fatty acid methyl esters also called biodiesel, and can be obtained by esterifying fatty acids derived from oils and fats consisting of glycerides and free fatty acids (FFA).

[0004] Biomass raw materials for biodiesel production mainly consist of glycerides, but inexpensive and low-quality raw materials often hold large amounts of free fatty acids (FFA). However, since FFA can be converted to biodiesel by a (trans)esterification reaction, it is also a potential source of biodiesel.

[0005] Conventionally, the production of biodiesel has been carried out by chemical catalysts. The production of biodiesel with an alkali catalyst by the transesterification of glycerides is the most commonly used method, but it has drawbacks. Importantly, when the concentration of FFA is high, chemical alkali catalysts cannot cope with the quality of low-quality and variable raw materials. To utilize such raw materials, large-scale pretreatment is required. Currently, a typical pretreatment is the esterification of FFA with a sulfuric acid catalyst.

[0006] The need for large-scale preprocesses is a problem because it significantly reduces the profitability of the process. From the perspective of using low-quality raw materials, an enzymatic catalyst solution has been provided to achieve a more environmentally friendly, more profitable, and more robust process.

[0007] Enzymatic catalysts are not very affected by the quality of raw materials. This catalyst requires operating conditions that are quite close to the ambient environment, has high selectivity, and requires a small amount of additives, resulting in a smaller scale, cleaner, and more easily disposable waste treatment flow. In addition, enzymes are renewable catalysts, and processes using enzymes tend to be more environmentally friendly than chemical processes.

[0008] The enzymatic biodiesel process requires only a small amount of preprocessing compared to chemical processes and is capable of converting both FFA and glycerides into biodiesel.

[0009] However, the enzymatic process has some limitations. The enzymatic esterification reaction that converts free fatty acids into biodiesel and water with short-chain alcohols is reversible. Therefore, the presence of water promotes the reaction in an unfavorable direction for biodiesel production. At the same time, removing water is favorable for biodiesel formation, but it has the adverse effect of reducing the stability of the enzyme.

[0010] The current enzymatic transesterification process, while achieving high conversion rates, cannot avoid equilibrium constraints, so the product still contains 1 - 6% by weight of FFA (regardless of the raw materials). One solution could be to use corrosive washing to convert and remove the residual FFA, but this generates soap, leading to a large amount of wastewater as a by - product, thus requiring an additional process.

Summary of the Invention

Problems to be Solved by the Invention

[0011] Therefore, there is still a need to develop an enzymatic process for producing biodiesel, especially to improve the effectiveness of the enzymatic process.

Means for Solving the Problems

[0012] The present invention relates to a process for reducing the level of free fatty acids in biodiesel / fatty acid alkyl esters, comprising the steps of: (i) providing a fatty acid feedstock substrate comprising triglycerides, diglycerides, monoglycerides, free fatty acids, fatty acid esters, or any combination thereof; (ii) reacting the fatty acid feedstock substrate with an alcohol in the presence of one or more esterases and added glycerol to produce a fatty acid alkyl ester; (iii) separating the reaction mixture of step (ii) into a light phase comprising fatty acid methyl ester (FAME) and a heavy phase comprising esterase, glycerol, short - chain alcohol, and water; (iv) drying the mixture of step (ii) and then separating the light and heavy phases; and / or (v) drying the heavy phase of step (iii) in the presence of esterase, wherein the glycerol and esterase of the heavy phase are recycled and added back to step (ii), and the level of the obtained free fatty acid (FFA) concentration in the resulting biodiesel (FAME) product is less than 1% (weight / weight).

[0013] These and still other objects and advantages of the present invention will become apparent from the following description. In the following detailed description, preferred embodiments of the present invention are described in connection with 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, reference should be made to the claims of this specification to interpret the scope of the present invention.

[0014] The main improvements brought about by the present invention are to partially or completely eliminate corrosive polishing and the corresponding yield loss, salt side streams, and wastewater. This is also a further step towards substituting currently dominant chemical processes that are not very environmentally friendly.

Brief Description of the Drawings

[0015]

Figure 1

Modes for Carrying Out the Invention

[0016] The figures are included for illustrative purposes only and should not be construed as limiting the present invention.

[0017] Definitions Before disclosing and describing specific embodiments of the present invention, it should be understood that the present invention is not limited to the specific processes and materials disclosed herein and may therefore vary to some extent. It should also be understood that the terms used herein are used only for the purpose of describing specific embodiments and are not intended to be limiting, since the scope of the present invention is defined only by the appended claims and their equivalents.

[0018] In the description and claims of the present invention, the following terms are used.

[0019] The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, references to "a step" include references to one or more such steps.

[0020] As used herein, "substantially" when used in reference to an amount or quality of a material or to a particular one of those characteristics thereof, refers to an amount sufficient to achieve the effect intended to be provided by the material or characteristic. The exact degree of allowable variation may depend upon the particular context. Similarly, "substantially free of" refers to the identified element or agent being absent from the composition. In particular, an element identified as "substantially free of" is either completely absent from the composition or is present in such a small amount as to have no detrimental effect on the composition.

[0021] As used herein, a reference to "about" a numerical value or parameter includes embodiments that are about the numerical value or parameter itself. For example, a recitation of "about X" includes the embodiment "X". When used in combination with a measured value, "about" includes the range that encompasses at least the uncertainty associated with the method of measuring the particular value, and may include the range of plus or minus two standard deviations surrounding the recited value.

[0022] Similarly, a reference to a gene or polypeptide "derived from" another gene or polypeptide X includes the gene or polypeptide X.

[0023] It is to be understood that the embodiments described herein include embodiments "consisting of" and / or embodiments "consisting essentially of". As used herein, the terms "comprise", "comprises", "comprising", and the like, unless the context dictates otherwise by explicit language or necessary implication, are used in a comprehensive sense, i.e., to specify the presence of the stated features, but do not preclude the presence or addition of further features in various embodiments.

[0024] In this specification, concentrations, amounts, and other numerical data may be expressed in a range format. Such a range format is used merely for convenience and brevity and is to be construed flexibly as including not only the numerical values explicitly recited as limiting the range but also all individual numerical values or sub-ranges subsumed within the range as if each such numerical value and sub-range were explicitly recited. For example, a weight range of from about 1 percent to about 20 percent includes not only the explicitly recited concentration limits of from 1 percent to about 20 percent but also individual concentrations such as 2 percent, 3 percent, 4 percent, etc. and sub-ranges such as from 5 percent to 15 percent, from 10 percent to 20 percent, etc.

[0025] Lipid: The term "lipid" 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 nonpolar lipids, and their derivatives.

[0026] Esterification: As used herein, the term "esterification" refers to a reaction for combining an organic acid such as a fatty acid with any alcohol or polyol such as glycerol.

[0027] Hydrolysis: As used herein, the term "hydrolysis" refers to reacting water with an ester to produce an acid and an alcohol.

[0028] Alcoholysis: As used herein, the term "alcoholysis" refers to reacting an ester with a monohydric alcohol such as ethanol or butanol or a polyhydric alcohol such as glycerol to produce an ester having a different alkyl group.

[0029] Acidolysis: As used herein, the term "acidolysis" refers to a reaction of an ester with an acid that results in an exchange of acyl groups.

[0030] Intermolecular esterification: As used herein, the term "intermolecular esterification" refers to the reaction of a first ester and a second ester that results in a mixture between an acyl moiety and an alcohol moiety.

[0031] Transesterification: As used herein, the term "transesterification" refers to any of the following reactions: alcoholysis, acidolysis, and intermolecular esterification.

[0032] Synthesis: As used herein, the term "synthesis" or "synthesis of fatty acids" preferably refers to a one-step reaction selected from any one of the following reactions: esterification, intermolecular esterification, alcoholysis, acidolysis, transesterification, by which a fatty acid is covalently bonded at the sn-2 position of a glyceride.

[0033] The term "alkyl" or "alkyl group" is to be construed in its broadest sense for describing a monovalent aliphatic compound containing hydrocarbons.

[0034] The terms "glycerol derivative" and "glyceride" are used interchangeably herein to describe esters, ethers, and other derivatives of glycerol in which at least one hydrogen of any of the hydroxyl groups bonded to C1, C2, or C3 carbon is substituted. Examples of glycerol derivatives are tristearoyl glycerol (or tri-O-stearoyl glycerol or glycerol tristearate or glyceryl tristearate); 1,3-benzylidene glycerol (or 1,3-O-benzylidene glycerol); and especially glycerol 2-phosphate (or 2-phosphoglycerol). When the substitution is on a carbon atom rather than on the oxygen of the hydroxyl group, the compound may be regarded as a derivative of glycerol (e.g., 1,2,3-nonadecanetriol of C16H33CHOH-CHOH-CH2OH, which may also be regarded as 1-C-hexadecyl glycerol). The term "glycerol" as used herein is intended to include glycerol derivatives.

[0035] Esterase: The terms "esterase", "lipase", "lipase enzyme", "lipolytic enzyme", "lipid esterase", "lipolytic polypeptide", and "lipolytic protein" mean hydrolase enzymes that break down esters into acids and alcohols by a chemical reaction with water called hydrolysis. This term also refers to enzymes called carboxylic ester hydrolases that act on ester bonds and includes enzymes classified as EC 3.1.1 carboxylic ester hydrolases according to the Enzyme Nomenclature (available at http: / / www.chem.qmw.ac.uk / iubmb / enzyme or from Enzyme Nomenclature 1992, Academic Press, San Diego, California, with Supplement 1 (1993), Supplement 2 (1994), Supplement 3 (1995), Supplement 4 (1997) and Supplement 5, in Eur. J. Biochem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250; 1-6 and Eur. J. Biochem. 1999, 264, 610-650 respectively). Non-limiting examples of esterases include carboxylesterase, arylesterase, triacylglycerol lipase, acetyl esterase, acetylcholinesterase, cholinesterase, tropine esterase, pectin esterase, sterol esterase, chlorophyllase, L-arabinonolactonase, gluconolactonase, uronolactonase, tannase, retinyl palmitate esterase, hydroxybutyric acid dimer hydrolase, acylglycerol lipase, 3-oxoadipic acid enol lactonase, 1,4-lactonase, galactolipase, 4-pyridoxolactonase, acylcarnitine hydrolase, aminoacyl tRNA hydrolase, D-arabinonolactonase, 6-phosphogluconolactonase, phospholipase A1, 6-acetylglucose deacetylase, lipoprotein lipase, dihydrocoumarin lipase, limonin D-ring lactonase, steroid lactonase, triacetate lactonase, actinomycin lactonase, orsellinate depside hydrolase, cephalosporin C deacetylase, chlorogenic acid hydrolase, α-amino acid esterase, 4-methyloxaloacetate esterase, carboxymethylene butenolide hydrolase, deoxylimonate A-ring lactonase, 2-acetyl-1-alkylglycerophosphocholine esterase, fusarinine C ornithine esterase, sinapine esterase, wax ester hydrolase, phorbol diester hydrolase, phosphatidylinositol deacylase, sialic acid O-acetyl esterase, acetoxybutynylthiophene deacetylase, acetylsalicylic acid deacetylase, methylumbelliferyl acetate deacetylase, 2-pyrone-4,6-dicarboxylic acid lactonase, N-acetylgalactosaminoglycan deacetylase, juvenile hormone esterase, bis(2-ethylhexyl) phthalate esterase, protein glutamate methyl esterase, 11-cis-retinyl palmitate hydrolase, all-trans-retinyl palmitate hydrolase, L-rhamnonol-1,4-lactonase, 5-(3,4-diacetoxybut-1-ynyl)-2,2'-bithiophene deacetylase, fatty acyl ethyl ester synthase, xylonono-1,Examples include 4-lactonase, N-acetylglucosaminylphosphatidylinositol deacetylase, cetraxate benzyl esterase, acetylalkylglycerol acetylhydrolase and acetylxylan esterase. Non-limiting examples of esterases include carboxylic acid ester hydrolases classified from EC 3.1.1.1 to EC 3.1.1.85 according to the Enzyme Nomenclature (available on the website at the address www.chem.qmw.ac.uk / iubmb / enzyme). Esterases have broad specificity and can also hydrolyze vitamin A esters. Esterases can also arise from microsomes that catalyze the reactions of EC 3.1.1.2, EC 3.1.1.5, EC 3.1.1.6, EC 3.1.1.23, EC 3.1.1.28, EC 3.1.2.2, EC 3.5.1.4 and EC 3.5.1.13. This esterase can 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., the hydrolytic activity of lipase) can be determined by a pNP assay using substrates having various chain lengths.,

[0036] Parent or parent esterase: The term "parent" or "parent esterase" means an esterase that has been modified to give an enzyme variant. The parent esterase can be a naturally occurring (wild-type) polypeptide, but can also be its variant and / or fragment.

[0037] Sequence identity: The relationship between two amino acid sequences is represented by the parameter "sequence identity".

[0038] For the purposes of the present invention, the sequence identity between two amino acid sequences is preferably determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48; 443-453) as implemented in the Needle program of the EMBOSS package version 5.0.0 or later (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277). 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 Needle, displayed as "longest identity" (obtained using the -nobrief option), is used as percent identity and is calculated as follows. (Number of identical residues × 100) / (Length of alignment - Total number of gaps in alignment)

[0039] Substrate: Suitable substrates for producing fatty acid alkyl esters according to the present invention are a wide variety of vegetable oils and fats. Most commonly, rapeseed oil and soybean oil are used, but other crops such as mustard oil, sunflower oil, canola oil, coconut oil, hemp oil, palm oil, and even algae are used. The substrate can be of crude quality or further processed (refined, decolorized, and deodorized). Not only animal fats including tallow, lard, poultry oil, and fish oil, but also discarded vegetable and animal fats and oils (commonly known as yellow grease and brown grease) can be used. Suitable fats and oils can be pure triglycerides or mixtures of triglycerides and free fatty acids commonly found in discarded vegetable oils and animal fats. The substrate can also be obtained from the deodorized distillate of vegetable oils. The types of fatty acids in the substrate include those that occur naturally as glycerides in vegetable and animal fats and oils. These include, for example, oleic acid, linoleic acid, linolenic acid, palmitic acid, stearic acid, and lauric acid. Trace 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 free or esterified fatty acids such as those in triglycerides, diglycerides, monoglycerides, or fatty acid alkyl esters.

[0040] Biodiesel: Fatty acid alkyl esters (FAAE) of short-chain alcohols, such as fatty acid methyl esters (FAME) and fatty acid ethyl esters (FAEE), are also called biodiesel because they are used as additives or substitutes for fossil fuels. Since biodiesel is produced from renewable resources, it has become even more important as an additive or substitute for diesel fuels based on fossil fuels.

[0041] Alcohol: The alcohol used in the process of the present invention is preferably a short-chain, branched-chain or straight-chain alcohol having 1 to 5 carbon atoms (C 1 , C 2 , C 3 , C 4 or C5 is a “lower alcohol”) or a mixture thereof. Preferred lower alcohols are 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 fatty acids bound to glycerides). The alcohol can be added to the reaction mixture stepwise (such as in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more steps) and / or continuously. The addition can be carried out in one, some or all of the reactors. When two or more reactors are used in series and / or in parallel, the addition can be carried out evenly at the same dosage at all dosing times or unevenly by varying the dosing rate.

[0042] The term “equilibrium” can be defined herein as the point at which the free fatty acids in the reaction mixture no longer further decrease or the point at which the decrease in FFA is so small that it is not worthwhile to continue the reaction in, for example, the transesterification vessel.

[0043] Added glycerol: The term “added glycerol” is the glycerol added to the reactants at any point in time. This added glycerol may already be present as free glycerol in the feedstock oil. Bound glycerol that is released as a by-product by the reaction of glycerides is not taken into account.

[0044] Fatty acid raw material: The term "fatty acid raw material", or "oil and / or fat", or "vegetable oil raw material" is defined herein as a substrate containing fatty acid derivatives. The substrate may include fatty acid alkyl esters, triglycerides, diglycerides, monoglycerides, free fatty acids, or any combination thereof. Any oil and fat derived from vegetables or animals containing fatty acids can be used as a substrate for producing fatty acid alkyl esters in the process of the present invention. A fatty acid raw material consisting essentially of fatty acid alkyl esters is also suitable as a raw material (biodiesel raw material) of the present invention. Preferably, the free fatty acid content of the fatty acid raw material is more than 0.25%, more than 0.30%, more than 0.35%, more than 0.50%, more than 0.75%, more than 1.0%, more than 5.0%, more than 10.0%, more than 15.0%, more than 20.0%, more than 25.0%, more than 30.0%, more than 40% or even more than 50.0%. The fatty acid raw material can be an oil selected from the group consisting of microbial oil, algal oil, canola oil, coconut oil, castor oil, palm oil (copra oil), corn oil, cottonseed oil, linseed 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 oil derived from halophytes, sea buckthorn oil, camelina oil, jojoba oil, coriander seed oil, meadowfoam oil, coastal zinnia oil or any combination thereof.

[0045] The fatty acid raw material can be a fat selected from the group consisting of animal fats including lard from pigs, beef and sheep, chicken fat, fish oil or any combination thereof.

[0046] The fatty acid raw material can be crude, refined, decolorized, deodorized, degummed or any combination thereof.

[0047] Food quality oils and fats are expensive, so waste and by-products from their processes, as well as non-food grade oils and fats, are becoming more attractive raw materials for producing fatty acid alkyl esters. Soda oil sludge is a fraction of oil obtained at an oil refinery by treating oil with a base to convert free fatty acids into soap (e.g., sodium soap). Soda oil sludge usually contains not only soap but also a fraction of glycerides. Acid oil is a by-product from an essential oil refinery produced by acidifying soda oil sludge to solubilize the soap. Acid oil mainly contains free fatty acids (FFA) and acyl glycerols. Distillates such as palm fatty acid distillate (PFAD) are by-products of refined oils obtained from distillation processes used to remove free fatty acids from oils.

[0048] The term fatty acid feedstock is used interchangeably herein with the term biodiesel feedstock.

[0049] A fatty acid feedstock is crude oil, refined oil, or used / waste oil or a mixture thereof. The feedstock can be an intermediate product of oil, waste, or by-product or a refined oil and fat selected from the group consisting of: soda oil sludge; acid oil; fatty acid distillates such as PFAD, soybean fatty acid distillate, rapeseed fatty acid distillate, rice bran fatty acid distillate, poultry fatty acid distillate, tallow fatty acid distillate, etc.; gums resulting from degumming; by-products from products of omega-3 fatty acid derivatives derived from fish oil; fat trap oils and fats; yellow oils and brown oils, free fatty acids such as oleic acid; or fractions of oil obtained by physical separation; or any combination thereof.

[0050] The present invention relates to a process for reducing the level of free fatty acids in biodiesel / fatty acid alkyl esters.

[0051] In one aspect of the present invention, a process for reducing the level of free fatty acids in biodiesel / fatty acid alkyl esters, comprising: (i) providing a fatty acid feedstock substrate comprising triglycerides, diglycerides, monoglycerides, free fatty acids, fatty acid esters, or any combination thereof; (ii) reacting the fatty acid raw material substrate with an alcohol in the presence of one or more esterases and added glycerol to produce a fatty acid alkyl ester; (iii) separating the reaction mixture of step (ii) into a light phase containing fatty acid methyl ester (FAME) and a heavy phase containing esterase, glycerol, short-chain alcohol and water; (iv) drying the mixture of step (ii) and then separating the light phase and the heavy phase; and / or (v) a step of drying the heavy phase of step (iii) in the presence of an esterase, wherein the glycerol and esterase of the heavy phase are recycled and added back to step (ii), the step comprising (vi) the level of the obtained free fatty acid (FFA) concentration in the obtained biodiesel (FAME) product is less than 1% (weight / weight).

[0052] In one aspect of the present invention, the fatty acid raw material substrate comprises triglyceride, diglyceride, monoglyceride, free fatty acid, fatty acid ester or any combination thereof.

[0053] In one aspect of the present invention, the fatty acid raw material substrate is derived from one or more of algal oil, canola oil, coconut oil, castor oil, palm oil, copra oil, corn oil, distilled corn oil, cottonseed oil, linseed 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 derived from halophytes and / or animal fats such as lard, tallow, chicken fat, fish oil, palm oil free fatty acid distillate, soybean oil free fatty acid distillate, soda oil sludge fatty acid material, yellow grease, used cooking oil, palm oil waste liquid and brown grease or any combination thereof.

[0054] Some fatty acid feedstocks have high levels of free fatty acids. For example, fatty acid distillates, acid oils, animal fats or similar feedstocks derived from palm or soybeans can contain 10 - 90% FFA. The fatty acid feedstock can be crude biodiesel from another enzymatically catalyzed transesterification reaction with the residual FFA that remains unconverted. Such crude biodiesel retains FFA in the range of 0.25 - 8 wt%, for example 0.5 - 6 wt% or further 0.6 - 5 wt%, and the remainder is FAME and 0.25 - 5 wt% of unconverted mono-, di-, triglycerides. Such substrates can retain significant amounts of mono-, di- and triglycerides, including various amounts of FAME and FFA.

[0055] In another aspect of the invention, the substrate is crude FAME distilled from the light phase separated from the heavy phase that retains mainly FAME and FFA and contains trace amounts of glycerides. In such cases, the concentration of FFA is approximately the same as the concentration originally present in the crude FAME, but the quality of the feedstock is significantly improved, resulting in a better process in terms of reaction rate and product quality. Such distillation can be carried out at a temperature in the range of 140 °C - 240 °C under a vacuum of 0.5 - 100 mbara.

[0056] In another aspect of the invention, the fatty acid feedstock contains fatty acids in an amount in the range of 0.25 - 10 wt%, for example 1 - 8 wt%, 1 - 7 wt%, 1 - 5 wt% or 1 - 4 wt%.

[0057] In one aspect of the invention, the fatty acid feedstock is reacted with an alcohol in the presence of one or more esterases.

[0058] In one aspect of the invention, the fatty acid feedstock is reacted with an alcohol in the presence of one or more esterases and glycerol to produce fatty acid alkyl esters.

[0059] In another aspect of the invention, a process is used to convert the FFA level while leaving a significant concentration of unconverted glycerides.

[0060] In one aspect of the present invention, the process is a single enzymatic conversion of a fatty acid feedstock substrate to biodiesel.

[0061] The process is divided into several processing steps by any intermediate unit operation. Such intermediate unit operations can include, but are not limited to, transportation (e.g., to other parts of the factory or other factories or storage facilities), filtration, distillation, decolorization, washing, and storage. Dividing the process into two or more parts in this way can be beneficial when the first reaction step that causes a partial conversion cannot achieve a complete reaction due to, for example, equipment limitations, but is possible in some parts of an existing factory. In such cases, it can be beneficial to utilize the capabilities of such a factory before transporting the partially converted oil to other parts of the same factory or other factories where final conversion can be achieved. Another beneficial reason for dividing the process into parts by intermediate unit operations is that the efficiency of removing existing impurities may be improved after partial conversion to alkyl esters. This also applies when using waste oils rich in metals such as POME. Generate low-boiling alkyl esters while reducing high-boiling glycerides, and then distillatively remove the partially (or completely) converted alkyl esters and FFA while including impurities in the distillation bottoms.

[0062] In another aspect of the present invention, the process can be viewed as a two-step enzymatic conversion of a fatty acid feedstock substrate to biodiesel.

[0063] In another aspect of the present invention, the process is (i) providing a fatty acid feedstock substrate comprising triglyceride, diglyceride, monoglyceride, free fatty acid, fatty acid ester, or any combination thereof; (ii) reacting the fatty acid feedstock substrate with an alcohol in the presence of one or more esterases and added glycerol to produce a fatty acid alkyl ester; (iii) Separating the reaction mixture of step (ii) into a light phase containing fatty acid methyl ester and a heavy phase containing esterase, glycerol, short-chain alcohol and water; (iv) A step of drying the heavy phase of step (iii) in the presence of esterase, wherein the glycerol and esterase of the heavy phase are reused and added back to step (ii); The obtained free fatty acid (FFA) concentration level is less than 1% (weight / weight).

[0064] In the said process, the first step is enzymatic transesterification by esterase, and the second step is esterification of free fatty acid to biodiesel by esterase. The esterase used for transesterification and esterification can be the same esterase or another esterase. In this case, the substrate of step (i) can be present in the light phase of the reaction mixture from enzymatic transesterification.

[0065] Alternatively, the substrate of step (i) can be the complete reaction mixture from the transesterification reaction and is then dried to promote the esterification process. This can also be the complete reaction mixture from transesterification, which is isolated by separating the heavy phase and dried continuously or in a stepwise manner during the reaction at any starting and ending points and then recycled to the transesterification reaction.

[0066] The mixture of step (ii) is then incubated in an enzyme reactor. The enzyme reactor can be any container suitable for incubating the enzyme, and the incubation parameters are selected to promote the enzyme reaction. Those skilled in the art are aware of these parameters and can select, for example, the temperature, mixing, and container that assist this enzyme reaction.

[0067] In one aspect of the invention, the enzyme reaction mixture, i.e., the mixture provided in step (ii) and containing the enzyme reaction product and the residue of the substrate mixed together, is subjected to drying. Such drying can be carried out continuously during the reaction, for example, by directly applying a vacuum to the reaction vessel while continuously adding methanol to replace the evaporated portion. Such drying can also be carried out in an external vessel that loops the reaction mixture and feeds the dried mixture back into the reaction vessel. Drying can also be carried out by stripping using a suitable gas. Such drying can be stepwise and / or can be carried out stepwise between successive reaction vessels.

[0068] In another aspect of the invention, the reaction mixture is dried using step iv and then separated into a light phase and a heavy phase. The dried heavy phase is then recycled back to step (ii).

[0069] In one aspect, the reacted mixture of step (ii) is separated. Separation of the reacted mixture is typically carried out using conventional methods known in the art.

[0070] The enzyme reaction mixture can be subjected to separation based on gravity such as centrifugation and / or decantation, thereby producing a light phase and a heavy phase. The light phase contains FAME, i.e., biodiesel, residual FFA, and glycerides. This can optionally be dried by conventional drying at the temperatures and conditions normally used to dry biodiesel, i.e., in a drying step. Optionally, distillation may be carried out, which is preferred if the quality of the raw material is not recognized as biodiesel compliant with legally approved standards. The heavy phase contains glycerol, esterase, water, and short-chain alcohols and undergoes step v.

[0071] In one aspect of the invention, the water added in step (ii) is less than 2% weight / weight of the fatty acid raw material.

[0072] In one aspect of the invention, the added glycerol added in step (ii) is at least 2% weight / weight of the fatty acid raw material.

[0073] In one aspect of the present invention, the process is continuous and / or batch and / or fed-batch.

[0074] Those skilled in the art will recognize that the present invention is suitable not only independently or as an additional process with dedicated equipment, but also as an additional process in the stepwise processing after the main transesterification reaction, or as a process carried out in parallel with the transesterification reaction and can be implemented with existing equipment. Those skilled in the art will also recognize that such optimal design and operation depend on whether the process is batch or continuous and will be designed accordingly. In such cases, mainly a drying configuration is added, thus reducing the amount of additional equipment.

[0075] In one aspect of the present invention, even under mild conditions where partial enzyme activity is possible, it can be beneficial when the enzyme reaction mixture is sufficiently dried in a dryer. The drying in step (ii) is carried out under these mild conditions.

[0076] The mild drying conditions used in step ii maintain the esterase in an active state while significantly reducing water and short-chain alcohols. The reduction of water sufficiently promotes esterification and reduces the FFA level in the biodiesel when reused in step ii.

[0077] Therefore, the heavy phase can be reused in whole or in part during the process. The heavy phase can be resupplied to step (ii) of the process when glycerol, esterase, and short-chain alcohols are added to the substrate and mixed. In some embodiments, the reused heavy phase can be replenished by adding fresh glycerol, short-chain alcohols, and / or esterase.

[0078] In another aspect of the present invention, the fatty acid raw material optionally also includes a heavy phase mainly containing glycerol, water and alcohol. Optionally, this heavy phase may also contain enzymes from previous reaction steps and enzymes reused from downstream process steps.

[0079] In another aspect of the present invention, the substrate contains free fatty acids in an amount in the range of more than 10% by weight, for example in the range of 10 - 90% by weight, for example 10 - 85% by weight, 10 - 80% by weight, 10 - 75% by weight, 10 - 70% by weight, 10 - 65% by weight, 10 - 60% by weight, 10 - 55% by weight, 10 - 50% by weight, 10 - 45% by weight, 10 - 40% by weight, 10 - 35% by weight, 10 - 30% by weight, 10 - 25% by weight, 10 - 20% by weight, 10 - 15% by weight of free fatty acids; or for example 10 - 85% by weight, 15 - 80% by weight, 20 - 75% by weight, 20 - 65% by weight, 20 - 55% by weight of free fatty acids.

[0080] In one aspect of the present invention, the alcohol is one or more of C1 - C5 alcohols, preferably ethanol, propanol, methanol or a mixture thereof.

[0081] In another aspect of the present invention, one or more high - molecular alcohols with a molecular weight of more than 500 g / mol are used. For example, myristyl alcohol has a molar mass of 214.4 g / mol.

[0082] In one aspect of the present invention, the alcohol is administered such that a constant concentration of alcohol in the heavy phase of step ii) is obtained, for example 1 - 50% by weight, for example 2 - 40% by weight, for example 3 - 30% by weight. The target concentration of alcohol in the heavy phase depends on the stability of the enzyme, temperature and the composition of the target product.

[0083] In another aspect of the present invention, the alcohol is administered step - by - step or continuously without considering the amount of alcohol in the heavy phase. When several reactors are used in series or in parallel, the alcohol can be administered in various proportions in one, several or all of the reactors.

[0084] In another aspect of the present invention, the total amount of alcohol administered depends on the composition of the raw materials and the target quality. For example, in the case of substantially pure triglyceride oil, up to 3 molar excess, preferably 2.5 or less, is used based on the convertible fatty acids. For example, regardless of the conversion of glycerides, while converting 4.4% FFA, less than 0.25% FFA can be achieved by using only 0.2 molar equivalents based on the total fatty acids in the raw material oil.

[0085] Drying is a conventional technique for drying biodiesel based on the difference in boiling points between the heavy and light components of the biodiesel reactant. In the present invention, similar equipment is used for drying the heavy phase isolated from the light phase.

[0086] Since it can be difficult to grasp the temperature inside the drying chamber, the temperature at the inlet point can be measured.

[0087] In one aspect of the present invention, drying in step iv is carried out under conditions such that the esterase of dried step (ii) maintains at least 40%, preferably 60%, and most preferably 80% of its activity.

[0088] In another aspect of the present invention, the drying process is optionally carried out under optimal drying conditions without considering the stability of the enzyme. In this case, the denatured and inactivated enzyme is reused together with the dried glycerol.

[0089] In another aspect of the present invention, the fact that the enzyme tends to form an emulsified layer between the light and heavy phases is utilized. The enzyme-rich emulsified layer is separated from the light and heavy phases and can be reused regardless of the drying process. For example, in the case of reusing 2 / 3 of the heavy phase and thus removing 1 / 3 of it on the one hand, according to this principle, more than 2 / 3 of the enzyme activity can be reused, but 1 / 3 of the enzyme is lost together with the removed 1 / 3 of the heavy phase. This makes it possible to efficiently high-temperature dry 2 / 3 of the reused glycerol phase in some cases because many enzymes are maintained unchanged by avoiding severe drying conditions.

[0090] In one embodiment of the present invention, glycerol is dried, recycled, and accumulated until the heavy phase before the combined glycerol is released from the glyceride occupies 2 to 40%, for example 5 to 40%, preferably 10 to 40% of the reactor volume.

[0091] In one embodiment of the present invention, all or part of the heavy phase is collected and processed as a whole until a certain degree of drying is achieved, and then it or a part thereof is recycled to step ii.

[0092] In another embodiment of the present invention, a continuous stream of the glycerol phase is continuously dried. While a part of the added glycerol is continuously dried, another part can be dried discontinuously, such as in a batch drying unit.

[0093] The concentrations of water and methanol in the heavy phase depend on the accumulation of the dried glycerol in the heavy phase by dilution. The FFA in the light phase at equilibrium mainly depends on the ratio of water to methanol in the glycerol phase. As a result of the accumulation of glycerol, the concentration and activity of water decrease significantly, which is the main cause of chemically reducing FFA.

[0094] In another embodiment of the present invention, a part of the heavy phase in step iii is removed either before and / or after drying, and the removed fraction is optionally reused in an earlier continuous reaction step in step ii or in an upstream reaction such as a transesterification reaction that produces an optional crude biodiesel feedstock.

[0095] In another embodiment of the present invention, the glycerol in step ii is added to a process outside the existing process.

[0096] Glycerol can be produced in various qualities as long as any contaminants do not significantly inhibit the enzyme.

[0097] In another aspect of the invention, the glycerol of step ii results from a transesterification process in which an optional crude biodiesel feedstock is produced. Optionally, such glycerol is dried and / or purified before entering the process.

[0098] Methanol tolerance refers to the decrease in stability (measurable, for example, by DSC as thermal stability) exhibited by most esterases in the presence of methanol.

[0099] In another aspect of the invention, optionally, the single or combined esterases used in step ii do not include the same enzymes as those used in the preparation of the crude biodiesel fatty acid feedstock of the preceding process step. Such new esterases are preferably more thermostable and / or more methanol-tolerant and / or active at a lower water activity level than the enzymes used to produce crude biodiesel.

[0100] In another aspect of the invention, additional (combined) esterases are added to the mixture at any point in step ii in addition to or subsequent to separating the existing enzymes of the previous reaction steps. Such additional esterases are preferably more thermostable and / or more methanol-tolerant and / or active at a lower water activity level than the enzymes used to produce crude biodiesel.

[0101] In one aspect of the invention, the esterase is preferably in a liquid formulation, a granule formulation, a dry formulation and / or a powder formulation. Although less preferred, the enzyme can be used in an immobilized form.

[0102] The process according to any of the preceding claims is carried out at a temperature of 20 to 90 °C, such as 25 to 85 °C, preferably 30 to 80 °C.

[0103] In one aspect of the invention, the process proceeds in batch mode or continuous mode.

[0104] In one aspect of the present invention, the total duration of the process is 1 to 72 hours, for example 2 to 48 hours, for example 4 to 40 hours in a batch process.

[0105] In one aspect of the present invention, the total duration of the process is 1 to 72 hours, for example 2 to 48 hours, for example 4 to 40 hours in a continuous process.

[0106] In another aspect of the present invention, the reaction is carried out, for example, in a storage container by the controlled addition of methanol selected such that any desired biodiesel quality can be obtained with the initial addition of water and glycerol and an equilibration time and the corresponding long reaction time. In this case, the reaction is carried out without considering the reaction time, and the addition of the enzyme and the drying of glycerol are suppressed by utilizing the slow administration of methanol and the reaction time until the biodiesel quality is reached. This is useful, for example, when a (storage) container that has not been used and has received ATEX certification is in a state of not being used in another way for a fairly long time, which is economically beneficial.

[0107] In one aspect of the present invention, the process includes one or more reactors used in series or in parallel.

[0108] In another aspect of the present invention, the process includes two or more reactors, and the heavy phase of each or some of the reactors is separated and dried separately before entering the next reactor.

[0109] In another aspect of the present invention, two or more reactors are used, and the heavy phase is separated and dried only between selected reactors, for example, only between reactor 1 and 2, 2 and 3, for example, between reactor "n" and "n + 1".

[0110] Optionally, some or all of the heavy phase of reactor "n" can be separated, optionally dried, and optionally returned to any previous reactor, for example from reactor 4 to reactor 1, or from 3 to 1, or from 8 to 5 for reuse. Optionally, the heavy phase of reactor n can be split and only a portion of the heavy phase dried and then introduced into a different reactor, for example the contents of reactor 4 can be separated into two parts x and y, part x dried and added to reactor 3 while the undried part y is added to reactor 1. There are numerous combinations, and the most preferred combination is one that results in the driest composition in the final reactor of any number of continuous and parallel reactors that is economically attractive while minimizing the FFA at equilibrium in the final product.

[0111] In another aspect of the invention, optionally, a portion of the light phase of step (iii) is recycled directly back to step (ii). Optionally, a portion of the FAME phase of any reactor n is recycled to a previous reactor such as from reactor 4 to reactor 2. Optionally, the FAME phase is dried regardless of the presence or absence of glycerol during drying.

[0112] In one aspect of the invention, the total amount of said esterase enzyme is in the range of 0.01 to 8 g of enzyme protein (EP) per kg of substrate.

[0113] In one aspect of the invention, the amount of FFA is reduced or maintained at less than 5 wt%, preferably less than 2 wt%, more preferably less than 1 wt%, for example 0.5 wt%, and most preferably less than 0.25 wt%.

[0114] In one aspect of the invention, when using a refined oil that retains an initial FFA value, for example less than 1% or even closer to zero, the present invention does not result in an increase in FFA beyond the FFA levels claimed in the present invention.

[0115] In one aspect of the invention, drying is effected by drying carried out at a pressure of less than 250 mbara, for example less than 150 mbara, preferably less than 100 mbara.

[0116] In one embodiment of the present invention, the dried heavy phase mixture of step (iii) obtained in step iv or v has a moisture content in the range of 0 to 10% by weight, for example 0.05 to 8% by weight, preferably 0.1 to 5% by weight, and most preferably 0.1 to 4% by weight.

[0117] Esterase The esterase provided in step (ii) is one or more esterases, for example one or more enzymes classified as carboxylesterases of EC 3.1.1 according to the Enzyme Nomenclature (recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, since 1992).

[0118] In one embodiment, the esterase catalyzes the transesterification reaction.

[0119] In another embodiment, the esterase catalyzes the esterification reaction.

[0120] In one embodiment, the process of the present disclosure comprises one or more esterases. Non-limiting examples of suitable esterases include polypeptides having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 having esterase activity.

[0121] In one aspect of the present disclosure, the esterase comprises an amino acid sequence having a degree of sequence identity of at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% with the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 having esterase activity. In embodiments, a suitable esterase according to the present disclosure comprises the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 having esterase activity. In embodiments, a suitable esterase according to the present disclosure comprises a combination of two or more of the polypeptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

[0122] In one aspect, the esterase of the present disclosure is an artificial mutant comprising substitution, deletion and / or insertion of one or more (or some) amino acids of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 or their homologous sequences.

[0123] In one aspect, the one or more esterases may be selected from the group consisting of C. antarctica lipase A as described in WO 88 / 02775, Thermomyces lanuginosus lipase as exemplified in WO 00 / 60063, Thermomyces lanuginosus (formerly Humicola lanuginosus) lipase variants, Humicola insolens cutinase variants as disclosed in Example 2 of WO 01 / 92502, lipases derived from Humicola lanuginosus (EP 258068), Chromobacterium Viscosum, Candida rugosa, Pseudomonas cepacia, Geotricum candidum, Rhizomucor miehei, Cryptococcus spp. S-2, Candida parapsilosis, Eversa Transform (Novozymes A / S), LIPOZYME CALB L, NS88007 and Callera Trans (Novozymes A / S).

[0124] Preferably, the amino acid changes are of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1 to about 30 amino acids; small amino-terminal or carboxyl-terminal extensions such as amino-terminal methionine residues; small linker peptides up to about 20 - 25 residues; or small extensions that facilitate purification by altering net charge or another function, such as polyhistidine sequences, antigenic epitopes or binding domains.

[0125] Examples of conservative substitutions are those made within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and amino acids with small molecular weights (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that generally do not alter a particular activity are known in the art and are described, for example, in H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.

[0126] Alternatively, the amino acid changes are of such a nature that the physicochemical properties of the polypeptide are altered. For example, the amino acid changes may be such that they improve the thermal stability of the polypeptide, change the substrate specificity, change the optimal pH, etc.

[0127] The important amino acids in the parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, to identify amino acid residues that are extremely important for the activity of the molecule, a single alanine mutation is introduced into every residue within the molecule, and the resulting mutant molecules are examined for cellulolytic activity-promoting activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site or other biological interactions of the enzyme can also be determined by physically analyzing in combination with mutations of the amino acids at the presumptive contact sites of the structure as measured by techniques such as nuclear magnetic resonance, crystal structure analysis, electron diffraction, or photoaffinity labeling. See, for example, de Vos et al., 1992, Science 255;306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identification of important amino acids can also be inferred from the analysis of identification by polypeptides related to the parent polypeptide.

[0128] Substitutions, deletions and / or insertions of one or more amino acids can be made and tested using known mutagenesis, recombination and / or shuffling methods, followed by the related screening procedures as disclosed in Reidhaar - Olson and Sauer, 1988, Science 241:53 - 57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86; 2152 - 2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error - prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832 - 10837; U.S. Pat. No. 5,223,409; WO 92 / 06204) and site - specific mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0129] By combining mutagenesis / shuffling methods with high - throughput automated screening methods, the activity of a cloned and mutagenized polypeptide expressed by a host cell can be detected (Ness et al., 1999, Nature Biotechnology 17; 893 - 896). The mutagenized DNA molecule encoding the active polypeptide can be recovered from the host cell and easily sequenced using standard methods in the art. These methods enable the rapid determination of individual important amino acid residues within the polypeptide.

[0130] In an embodiment, the total number of amino acid substitutions, deletions and / or insertions in the polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 is 10 or less, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0131] In an embodiment, a suitable esterase according to the present disclosure comprises or consists of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 having esterase activity.

[0132] One or more (some) components of the esterase according to the present disclosure can be a wild-type protein, a recombinant protein, or a combination of a wild-type protein and a recombinant protein. For example, one or more (some) components can be a natural protein of a cell and used as a host cell for recombinantly expressing one or more (some) other components of the esterase composition. One or more (some) components of the esterase composition can be produced as single components and then combined to form an enzyme composition. The enzyme composition can be a combination of a multi-component protein preparation and a single-component protein preparation.

[0133] The esterase used in the process of the present disclosure can be in any form suitable for use, such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cell debris, a semi-purified or purified enzyme preparation, or a host cell as a source of the esterase. The esterase composition can be a dry powder or granule, a non-dusty granule, a liquid, a stabilized liquid, or a stabilized and protected enzyme. The liquid esterase preparation can be stabilized, for example, by adding a stabilizer such as a sugar, a sugar alcohol, or another polyol and / or lactic acid or another organic acid by an established process.

[0134] The esterase can be derived or obtained from any suitable origin including bacterial, fungal, yeast, plant or mammalian origin. The term "obtained" as used herein means that the esterase can be isolated from an organism that naturally produces the esterase as a native enzyme. The term "obtained" as used herein means that the enzyme can be recombinantly produced in a host organism using the methods described herein, and this recombinantly produced esterase enzyme is either undenatured or foreign to the host organism, or has a modified amino acid sequence having, for example, one or more (several) amino acids deleted, inserted and / or substituted, i.e., a recombinantly produced enzyme that is a variant and / or a fragment of the native amino acid sequence or an enzyme produced by a nucleic acid shuffling process known in the art. Native variants are included within the meaning of the native enzyme, and variants obtained by recombination such as site-directed mutagenesis or shuffling methods are included within the meaning of the foreign enzyme.

[0135] Esterase preparation The term "esterase" as used herein is used to refer to an enzyme that catalyzes the esterification of FFA to fatty acid methyl esters.

[0136] The esterase can be provided in any suitable formulation, such as a lyophilized powder, immobilized or in an aqueous / liquid solution.

[0137] In one embodiment, the esterase is provided as a spray-dried or granulated dry formulation. When the esterase is provided as a dry formulation, the esterase is dissolved / suspended in an aqueous / liquid solution.

[0138] In another embodiment, the esterase is provided in a liquid formulation. That is, the esterase is not immobilized on a carrier, but is present in an aqueous formulation or a glycerol formulation and / or a sorbitol formulation. This indicates that the cost can be reduced as a liquid formulation enzyme, the production becomes easier, and thus it becomes cheaper.

[0139] Immobilized enzymes such as Novozym 435 are bound to solid particles, making them easy to recover from the reaction mixture and reusable in the process. However, recovering and reusing immobilized enzymes requires additional process steps such as filtration. In addition, small particles can cause fouling or damage to the equipment that reduces the profitability of the process.

[0140] Furthermore, the immobilized enzyme itself is expensive to immobilize.

[0141] The present invention presents a surprising process for reusing liquid formulation enzymes, resulting in cost reduction / improvement of process efficiency in the use of liquid formulation enzymes.

[0142] There are advantages to using esterase in liquid formulations, but immobilized esterase can still be used if necessary.

[0143] A particular embodiment relates to a process according to the invention in which the esterase comprises or consists of CALB in a liquid formulation.

[0144] Providing the esterase as a liquid has the problem that water is introduced into the system (which shifts the equilibrium away from biodiesel production).

[0145] However, the inventors have surprisingly discovered that glycerol significantly shifts the equilibrium in the direction of increasing the conversion of FFA and reduces the effect of water introduction.

[0146] In addition, the enzyme can be recovered by isolating the heavy glycerol phase in which the enzyme is present.

[0147] Advantages The present invention provides at least the following advantages: - The drying process can further promote the enzyme reaction in the direction of generating more FAME and improve the yield of FAME. - Surprisingly, when using water-dependent enzymes in another way, water can be completely replaced with glycerol. This makes it possible to use enzymes that are faster and better than the CALB enzyme, which is the only enzyme described so far for this problem. CALB cannot efficiently convert triglycerides and thus cannot be used for transesterification, but the present invention enables the efficient use of other enzymes, thereby enabling the transesterification of triglycerides into biodiesel. - Reusing the reaction mixture, for example, reusing the enzyme and recycling glycerol, provides an economic benefit to the process. - This economic improvement results in the ability to consider FFA feedstocks that could not be used before. - By drying the entire mixture, energy costs are saved. - Since only the heavy phase (glycerol phase) instead of the entire reaction mixture is processed, the drying unit operation is significantly reduced. - Optionally, when using step v, by not drying the FAME, the tank dimensions, which are materials passing through heat and vacuum, are reduced, further improving the operating cost / capital cost.

[0148] A further advantage is that the process layout is simplified based on conventional engineering unit operations, thereby enabling the use of liquid lipase formulations at a lower cost.

[0149] The present invention is further described in the following paragraphs.

[0150] Paragraph 1. A process for reducing the level of free fatty acids in biodiesel / fatty acid alkyl esters, comprising: (i) providing a fatty acid feedstock substrate comprising triglycerides, diglycerides, monoglycerides, free fatty acids, fatty acid esters, or any combination thereof; (ii) reacting the fatty acid raw material substrate with an alcohol in the presence of one or more esterases and added glycerol to produce a fatty acid alkyl ester; (iii) separating the reaction mixture of step (ii) into a light phase containing fatty acid methyl ester (FAME) and a heavy phase containing esterase, glycerol, short-chain alcohol and water; (iv) drying the mixture of step (ii) and then separating the light phase and the heavy phase; and / or (v) a step of drying the heavy phase of step (iii) in the presence of an esterase, wherein the glycerol and esterase of the heavy phase are recycled and added back to step (ii), comprising, wherein the level of the resulting free fatty acid (FFA) concentration in the obtained biodiesel (FAME) product is less than 1% (weight / weight), a process.

[0151] Paragraph 2. The esterase of step (ii) is not immobilized, the process according to paragraph 1.

[0152] Paragraph 3. The esterase of step (ii) is added as a liquid, granule and / or powder, the process according to paragraph 1.

[0153] Paragraph 4. The water added in step (ii) is less than 2% weight / weight of the fatty acid raw material, the process according to any one of paragraphs 1 to 3.

[0154] Paragraph 5. The added glycerol of step (ii) is at least 2% weight / weight of the fatty acid raw material, the process according to any one of paragraphs 1 to 4.

[0155] Paragraph 6. The esterase can transesterify monoglyceride (MG), diglyceride (Dg) and triglyceride (TG), the process according to any one of paragraphs 1 to 5.

[0156] Paragraph 7. The process according to paragraph 6, wherein the esterase is an esterase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% or at least 100% sequence identity with the esterase of SEQ ID NO: 1 or and the polypeptide of SEQ ID NO: 1.

[0157] Paragraph 8. (v) providing a fatty acid feedstock substrate comprising triglyceride, diglyceride, monoglyceride, free fatty acid, fatty acid ester or any combination thereof; (vi) reacting the fatty acid feedstock substrate with an alcohol in the presence of one or more esterases and added glycerol to produce a fatty acid alkyl ester; (vii) separating the reaction mixture of step (ii) into a light phase comprising fatty acid methyl ester and a heavy phase comprising esterase, glycerol, short-chain alcohol and water; (viii) a step of drying the heavy phase of step (iii) in the presence of an esterase, wherein the glycerol and esterase of the heavy phase are recycled and added back to step (ii), the step comprising, wherein the level of the resulting free fatty acid (FFA) concentration is less than 1 (weight / weight)%, the process according to paragraph 1.

[0158] Paragraph 9. The water added in step ii) is 2% weight / weight or more of the fatty acid feedstock, and the biodiesel product according to claim 1 reacts with a further esterase in a second esterification step to result in an FFA concentration of less than 1%, less than 0.5%, for example less than 0.3%, the process according to paragraph 1.

[0159] Paragraph 10. The process according to paragraph 9, wherein the further esterase is an esterase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% or at least 100% sequence identity with the esterase of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 3.

[0160] Paragraph 11. The process according to any one of paragraphs 1 to 10, which is continuous or batch / fed-batch.

[0161] Paragraph 12. The process according to any one of paragraphs 1 to 11, wherein the drying in step v) is carried out until the FFA concentration reaches less than 1% by weight.

[0162] Paragraph 13. The process according to any one of paragraphs 1 to 12, wherein the alcohol is a C1-C5 alcohol.

[0163] Paragraph 14. The process according to any one of paragraphs 1 to 13, wherein the fatty acid alkyl ester is a methyl ester or an ethyl ester.

[0164] Paragraph 15. The process according to any one of paragraphs 1 to 14, wherein the added glycerol is dried, optionally before being recycled, to contain less than 20% by weight of water, preferably less than 10% by weight of water, and most preferably less than 2% by weight of water.

[0165] Paragraph 16. The drying in step iv) and / or step v) is carried out under conditions such that the dried esterase of step ii) maintains at least 40%, preferably 60%, most preferably 80% of its activity, in the process according to any one of paragraphs 1 to 15.

[0166] Paragraph 17. The drying is carried out in the range of 30°C to 100°C, such as 40°C to 90°C, preferably 45°C to 85°C, most preferably 50°C to 80°C, in the process according to any one of paragraphs 1 to 16.

[0167] Paragraph 18. The reaction in step ii) is carried out at a temperature of 20 to 90°C, such as 25 to 85°C, preferably 30 to 80°C, in the process according to any one of paragraphs 1 to 17.

[0168] Paragraph 19. The substrate is derived from one or more of algal oil, canola oil, coconut oil, castor oil, palm oil, copra oil, corn oil, distilled corn oil, cottonseed oil, linseed 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 derived from halophytes and / or animal fats such as lard, tallow, chicken fat, fish oil, palm oil free fatty acid distillate, soybean oil free fatty acid distillate, soda oil sludge fatty acid material, yellow grease, used cooking oil, palm oil waste liquid and brown grease, or any combination thereof, in the process according to any one of paragraphs 1 to 18.

[0169] Paragraph 20. The total duration of the process is 1 to 72 hours, such as 2 to 48 hours, such as 4 to 40 hours in a batch process, and the total duration of the process is 1 to 72 hours, such as 2 to 48 hours, such as 4 to 40 hours in a continuous process, in the process according to any one of paragraphs 1 to 19.

[0170] Paragraph 21. The total amount of the esterase is within the range of 5 to 8000 ppm (weight of enzyme protein / weight of substrate), the process according to any one of Paragraphs 1 to 20.

[0171] Paragraph 22. The total amount of the non-immobilized esterase is within the range of 5 to 1000 ppm (weight of enzyme protein / weight of substrate), the process according to any one of Paragraphs 1 to 21.

[0172] Paragraph 23. The amount of FFA decreases to less than 5% by weight, preferably less than 2% by weight, more preferably less than 1% by weight, for example 0.5% by weight, and most preferably less than 0.25% by weight, the process according to any one of Paragraphs 1 to 22.

[0173] Paragraph 24. The esterase is Aspergillus lipase; Aspergillus niger lipase; Thermomyces lanuginosa lipase; Candida Antarctica lipase A; Candida Antarctica lipase B; Candida cylindracae lipase; Candida deformans lipase; Candida lipolytica lipase; Candida parapsilosis lipase; Mucor miehei, Chromobacterium; Candida rugosa lipase; Corynebacterium acnes lipase; Humicola lanuginose, Cryptococcus spp.)S-2 lipase; lipase from Fusarium culmorum; lipase from Fusarium heterosporum; lipase from Fusarium oxysporum; lipase from Mucor javanicus; lipase from Rhizomucor miehei; lipase from Rhizomucor delemar; lipase from Burkholderia (Pseudomonas) cepacia; lipase from Pseudomonas sp, ATCC 21808, Pseudomonas camembertii; lipase from Pseudomonas fluorescens; lipase from Rhizopus; lipase from Rhizopus arrhizus; lipase from Staphylococcus aureus; lipase from Geotrichium candidum; lipase from Hyphozyma sp.; lipase from Klebsiella oxytoca; and wild-type orthologs and homologs thereof; and variants thereof, a process according to any one of paragraphs 1 to 23 selected from the group consisting of.

[0174] Paragraph 25. The process according to any one of paragraphs 1 to 24, further comprising the separation of the fatty acid alkyl ester.

[0175] Paragraph 26. The process according to any one of paragraphs 1 to 25, wherein the biodiesel product contains bound glycerol at a concentration of less than 1% by weight, preferably less than 0.5% by weight, most preferably less than 0.3% by weight.

[0176] Paragraph 27. The process according to paragraph 1, wherein the light phase contains monoglycerides at a concentration of less than 0.8% by weight.

[0177] Paragraph 28. The light phase is the process according to paragraph 1, comprising bound glycerol with a value of less than 0.24% by weight.

[0178] Paragraph 29. The light phase is the process according to paragraph 1, comprising FFA at a concentration of less than 0.5% by weight, preferably 0.25% by weight.

[0179] Paragraph 30. The light phase is the process according to paragraph 1, which is further subjected to a corrosive cleaning treatment.

[0180] Paragraph 31. The light phase is the process according to paragraph 1 or 30, which is subjected to further treatments such as distillation, washing, drying, decolorization, or a combination thereof.

[0181] Paragraph 32. The process according to any one of paragraphs 1 to 31, which is carried out in any number of consecutive and / or parallel reactors, and the reactions can be partial reactions or complete reactions respectively.

[0182] Paragraph 33. The alcohol is added stepwise and / or continuously, and the stepwise addition of alcohol can be in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more steps. The process according to any one of paragraphs 1 to 32.

[0183] Paragraph 34. The process according to paragraphs 1 to 33, in which two or more reactors are used in series, and a partial amount equal to or not equal to the total amount of alcohol is added to some or all of the reactors.

[0184] Paragraph 35. The process according to any one of paragraphs 1 to 34, further comprising the separation of the fatty acid alkyl ester.

[0185] Paragraph 36. The glycerol in the reaction mixture dried in step ii) is partially or completely reused. The process according to paragraph 1.

[0186] Paragraph 37. The process according to paragraph 1, wherein the heavy phase in step iii) is dried in the presence of an esterase, and part or all of the glycerol and esterase in the heavy phase are reused and added back to step ii) as part or all of the added glycerol.

[0187] Paragraph 38. The process according to paragraph 1, wherein one or more esterases have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% or at least 100% sequence identity with the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

[0188] Paragraph 39. The process according to paragraph 38, wherein one or more esterases comprise or consist of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

[0189] Paragraph 40. The process according to paragraph 13, wherein the alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol or mixtures thereof.

[0190] Paragraph 41. The process according to paragraph 40, wherein the alcohol is methanol.

[0191] Paragraph 42. The process according to paragraphs 1 to 41, wherein the FFA decreases along with the conversion of triglycerides and is less than 30%, preferably less than 20%, most preferably less than 10%.

[0192] Paragraph 43. The process according to paragraphs 1 to 42, wherein crude oil containing less than 1% by weight of FFA is reacted to reduce the FFA level to less than 1% by weight, preferably less than 0.5% by weight, most preferably less than 0.25% by weight.

[0193] Paragraph 44. The process according to paragraphs 1 to 43, wherein the esterase is selected from polypeptides having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% or at least 100% sequence identity with the polypeptide of SEQ ID NO: 1.

[0194] The present invention will be further illustrated by the following examples, which should not be construed as limiting the scope of the present invention.

Examples

[0195] Example 1: Crude palm oil (CPO) containing 4.4% by weight of free fatty acid (FFA) and 6.1% by weight of diacylglycerol (DAG) was melted at 60 °C and used without prior drying. Then, 30 g of CPO was weighed into a 100 mL Erlenmeyer flask, and then 20% (weight / weight of oil) of dried industrial grade glycerol was added. The mixture was adjusted in a shaking incubator oven at 35 °C while mixing at 250 rpm for 30 minutes to ensure that the temperature was reached. 100 ppm of sodium hydroxide was added as a 50% aqueous solution.

[0196] The dosages (liquid and immobilized) of various enzymes of SEQ ID NO: 1 or 2 were added at the 0-hour time point. The reaction was carried out at 35 °C in a shaking incubator at 250 rpm while continuously linearly administering 1.7 molar equivalents of methanol based on free fatty acid and bound fatty acid over 40 hours. After completing the administration of methanol at the time point indicating 40 hours, the reaction was continued until the time point indicating 48 hours, and the results were measured. A 2 mL sample was pipetted into a 2 mL Eppendorf tube. The sample was incubated at 99 °C for 10 minutes to inactivate the enzyme and then centrifuged at 2000 rpm for 1 minute. Finally, the sample was dried under vacuum at 80 °C for 2 hours to remove methanol.

[0197] Analysis method: FAME, mono, di and triglycerides by AOCS official method Ck 2-09 using the provided Quality Trait Analysis (QTA) NIR method.

[0198]

Table 1

[0199] From the results, it is shown that the immobilized enzyme does not function equivalently to the liquid enzyme and requires a much higher dosage to obtain similar results. It is also worth noting that the enzyme protein concentration of the immobilized preparation is 1.5 to 2 times higher than that of the liquid preparation. This is a comparison between two very similar enzymes, and any differences in the efficacy of the molecules are explained by the large difference in the dosage of the enzyme protein. Therefore, while it is clear that the liquid enzyme preparation is preferred, the immobilized enzyme can be administered to enable sufficient reaction, but it results in an unacceptable cost.

[0200] Example 2: Biodiesel reaction Crude palm oil (CPO) containing triglycerides, 4.4 wt% FFA, and 6.1 wt% DAG oil was melted at 60 °C. Next, 30 g of CPO was weighed into a 100 mL Erlenmeyer flask, and then 2% (oil weight / weight) water was added. The mixture was prepared in a shaking incubator oven at 35 °C while mixing at 250 rpm for 30 minutes to ensure that the temperature was reached. 100 ppm NaOH was added as a 50% aqueous solution to adjust the measured pH of the extract to around 5.5 (the measured pH of the extract was measured by mixing 10 g of oil with 10 g of 0.1 wt% KCL solution at 60 °C for 30 minutes and measuring the pH of the aqueous phase). The addition of NaOH was determined from a standard curve prepared previously with the raw material oil. 0.2% of the enzyme of SEQ ID NO: 1 was added at the 0-hour time point. The reaction was carried out at 35 °C in a shaking incubator at 250 rpm while continuously linearly dosing 1.7 molar equivalents of methanol based on free and bound fatty acids over 20 hours. After completing the methanol dosing at the time point indicating 20 hours, the reaction was continued until the time point indicating 48 hours, and after the reaction was considered to have reached equilibrium, the results were measured.

[0201] Collect 2 mL of the sample with a pipette into a 2 mL Eppendorf tube. Incubate the sample at 99 °C for 10 minutes to inactivate the enzyme, and then centrifuge at 2000 rpm for 1 minute. Finally, dry the sample under vacuum at 80 °C for 2 hours to remove the excess methanol dissolved in the FAME.

[0202] FFA by AOCS official method Ca 5a-40. Mono, di, and triglycerides by AOCS official method Ck 2-09 using the Quality Trait Analysis (QTA) NIR method provided by Eurofins.

[0203]

Table 2

[0204] From the above table, the remaining total reaching 100% is mainly FAME. The levels of glycerides are all within the range of the biodiesel standard specification levels, but the FFA is shown to be significantly above 0.25 wt%. At the end of the reaction, there is also a heavy phase containing many enzymes, the initially dosed 2% water, much of the dosed excess methanol, and 10 - 13% (oil weight / weight) glycerol liberated by the reaction. In today's processes, it is standard to add water, which is the reason for the high FFA levels relative to above the most preferred level of 0.25 wt%.

[0205] Example 3: Addition of Glycerol CPO (4.4 wt% FFA, 6.1 wt% DAG) oil was melted at 60 °C. Then, 30 g of CPO was weighed into a 100 mL Erlenmeyer flask, followed by the addition of 0.5 or 1% (oil weight / weight) water and 0.5 or 10% (oil weight / weight) of dried industrial grade glycerol. The mixture was adjusted in a shaking incubator oven at 35 °C while mixing at 250 rpm for 30 minutes to ensure reaching that temperature. 100 ppm of NaOH was added as a 50% aqueous solution. 0.2% (oil weight / weight) of the enzyme of SEQ ID NO: 1 was added at the 0 - hour time point. The reaction was carried out at 35 °C in a shaking incubator at 250 rpm while continuously linearly dosing 1.7 or 2.0 molar equivalents of methanol over 20 hours based on free and bound fatty acids. After completing the dosing of methanol at the time point indicating 20 hours, the reaction was continued until the time point indicating 48 hours and the results were measured after the reaction was considered to have reached equilibrium. 2 mL of the sample was pipetted into a 2 mL Eppendorf tube. The sample was incubated at 99 °C for 10 minutes to inactivate the enzyme and then centrifuged at 2000 rpm for 1 minute. Finally, the sample was dried under vacuum at 80 °C for 2 hours to remove methanol.

[0206] Analysis methods: FFA by AOCS official method Ca 5a - 40. Mono, di, and triglycerides by AOCS official method Ck 2 - 09 using the Quality Trait Analysis (QTA) NIR method provided by Eurofins.

[0207]

Table 3

[0208] The combination of 0% glycerol, 2% water, and 1.7 equivalents of methanol / dosing for 20 hours is a standard reaction as described in Example 1. Thus, considering the reaction without initially adding glycerol, it is clear that when less than 2% water was added, the reaction was significantly inhibited. The difference in glyceride conversion between 24 hours and 48 hours was small, suggesting that the reaction stalled due to enzyme inactivation and could not reach equilibrium. Thus, considering the results of the reaction with initially added glycerol, it is clear that glycerol brings about an improvement. By adding 5% glycerol together with only 1% water and 1.7 equivalents of methanol, not only is a significant improvement brought about compared to the reaction of Example 1 with an acceptable glyceride level, but the FFA level after 48 hours of the reaction also significantly decreases. By adding 10% glycerol, a further improvement in all parameters is brought about, but by adding only 0.5% water, an almost acceptable glyceride level is achieved. Further, by adding 10% (weight / weight of oil) glycerol, when most of the glyceride is converted, 20 - 23% glycerol is produced in the total mixture at equilibrium. Thus, the significant improvement brought about by glycerol is clearly demonstrated.

[0209] Example 4: Administration of Methanol CPO (4.4 wt% FFA, 6.1 wt% DAG) oil was melted at 60 °C. Next, 30 g of CPO was weighed into a 100 mL Erlenmeyer flask, and then 0.5 or 1% (oil weight / weight) of water and 0.5 or 10% (oil weight / weight) of dried industrial grade glycerol were added. The mixture was adjusted in a shaking incubator oven at 35 °C while mixing at 250 rpm for 30 minutes to ensure that the temperature was reached. 100 ppm of NaOH was added as a 50% aqueous solution. 0.2% (oil weight / weight) of the enzyme of SEQ ID NO: 1 was added at the 0-hour time point. The reaction was carried out at 35 °C in a shaking incubator at 250 rpm while continuously linearly dosing 1.7 or 2.0 molar equivalents of methanol based on free and bound fatty acids over 40 hours. After completion of the methanol dosing at the time point indicating 40 hours, the reaction was continued until the time point indicating 48 hours, and the results were measured. 2 mL of the sample was pipetted into a 2 mL Eppendorf tube. The sample was incubated at 99 °C for 10 minutes to inactivate the enzyme and then centrifuged at 2000 rpm for 1 minute. Finally, the sample was dried under vacuum at 80 °C for 2 hours to remove methanol.

[0210] Analysis method: FFA by AOCS official method Ca 5a-40. Mono, di and triglycerides by AOCS official method Ck 2-09 using the Quality Trait Analysis (QTA) NIR method provided by Eurofins.

[0211]

Table 4

[0212] The results show that, when compared with the results of Example 2, by reducing the methanol administration rate, obvious improvements have been achieved. The above table also shows, although it is obvious here, that when considering especially the glyceride level, reducing the amount of water added places a burden on the enzyme. These results indicate that, when compared with Example 2, a milder methanol administration schedule can mitigate the negative impact of reducing the amount of water added. From the results of this example, although it is good when there is little water and no glycerol, it is obvious that the conversion becomes less sufficient when the addition of glycerol is accompanied by a decrease in the water dosage.

[0213] Example 5: CPO (4.4 wt% FFA, 6.1 wt% DAG) oil was dried in a rotary evaporator at 80 °C under a vacuum of 5 mbar for 1 hour. Then, 30 g of pre-dried CPO was weighed into a 100 mL Erlenmeyer flask, and then 20% (oil weight / weight) of dried industrial grade glycerol was added. The mixture was adjusted in a shaking incubator oven at 35 °C while mixing at 250 rpm for 30 minutes to ensure that the temperature was reached. 100 ppm of NaOH was added as a 50% aqueous solution. 0.25 or 0.5% (oil weight / weight) of the enzyme of SEQ ID NO: 1 was added at the 0-hour time point. The reaction was carried out at 35 °C in a shaking incubator at 250 rpm while continuously linearly administering 2 molar equivalents of methanol based on free fatty acid and bound fatty acid over 40 hours. After completing the methanol administration at the time point indicating 40 hours, the reaction was continued until the time point indicating 72 hours, and the results were measured. A 2 mL sample was pipetted into a 2 mL Eppendorf tube. The sample was incubated at 99 °C for 10 minutes to inactivate the enzyme and then centrifuged at 2000 rpm for 1 minute. Finally, the sample was dried under vacuum at 80 °C for 2 hours to remove methanol.

[0214] Analysis method: FFA by AOCS official method Ca 5a-40. Monoglycerides, diglycerides and triglycerides by AOCS official method Ck 2-09 using the provided Quality Trait Analysis (QTA) NIR method.

[0215]

Table 5

[0216] The results show glycerides compliant with EN-14214, with the quality of standard palm oil, and the FFA level is achieved using the enzyme of SEQ ID NO: 1 in a single reaction step. When 0.25% of the enzyme is used, acceptable results are obtained in 72 hours, but when the enzyme dosage is doubled, similar results are obtained in 48 hours.

[0217] Example 6: 30 g of crude biodiesel containing 4.9 wt% FFA, 1 wt% monoglyceride, 0.3% diglyceride, and less than 0.1 wt% triglyceride. This crude biodiesel was prepared using crude palm oil (CPO) containing 2% added water (per weight of CPO) and 0.3% of SEQ ID NO: 3 (per weight of CPO), and 1.7 equivalents of methanol were dosed over 20 hours (relative to the number of moles of fatty acids in the raw material), and the final crude FAME product was completed after 24 hours. The crude FAME resulting from the reaction was then dried at 60 °C under 5 mbar vacuum for 16 hours before use in the reaction. Reactions were carried out with 30 g of crude biodiesel with the addition of 0.1% CALB solution, 5 or 20% industrial grade (over 99%) glycerol and varying amounts of methanol.

[0218] FFA was measured using AOCS official method Ca 5a-40. MG and BG were measured by Eurofins' QTA.

[0219]

Table 6

[0220]

Table 7

[0221] Glycerol has a great effect on both the rate and the influence of methanol on the activity of the enzyme. The measured values for 2 hours are sufficient to indicate the rate, and the enzyme is much more active with 4% methanol than with 10% methanol when 5% glycerol is added, whereas 8% methanol is optimal when 20% glycerol is added. More importantly, in a single lab batch experiment using 20% glycerol, FFA, MG, and BG within the range of the standard levels can be achieved. The standard levels at the time of writing this specification are that FFA is less than 0.25% by weight, MG is less than 0.8% by weight, and BG is less than 0.24% by weight. BG is bound glyceride and is the total value of MG, DG, and TG combined.

[0222] Example 7: Removal of FFA before producing chemical biodiesel When producing chemical biodiesel using methoxide, FFA cannot be present in the raw material at a concentration of less than 0.5% by weight because it leads to soap formation and degrades the catalyst. Attempts have been made and are currently being made to remove FFA by chemical reaction with glycerol or by non-catalytic reaction to produce glycerides or FAME. In addition, some producers remove FFA by a deodorization process carried out above 220°C. However, it has been shown that the method described in this application is also applicable to this treatment, and an example is shown in this specification. The reaction rate is an important point in this reaction, but this reaction is regarded as an industrial pretreatment and the available operating cost is low. Therefore, the key point of this example is to show that when using glycerol to stabilize while immediately administering the amount of methanol and mainly using TAG-inactivated CALB enzyme to esterify FFA while transesterifying DAG, the FFA that may be obtained in the crude oil can be rapidly reduced.

[0223] The raw material is CPO (4.4 wt% FFA, 6.1 wt% DAG), and the rest is mainly TAG. The CALB enzyme is adopted because it shows little reactivity to TAG, which is beneficial because it enables stoichiometric dosing only to the FFA, MAG, and DAG in methanol. Methanol is dosed based on the total fatty acids in the raw material oil and is reported in equivalents as follows. Melt the oil and weigh 30 g. Then add glycerol and methanol and preheat the mixture to 40°C. Finally, dose the enzyme at the zero-time point. In the following table, the maximum residue of 100% was triglyceride and a small amount of MG, which was not measured here.

[0224] Analysis method: FFA by AOCS official method Ca 5a-40. FAME was measured by a customized NMR method. DAG was measured by a customized HPLC method.

[0225]

Table 8

[0226]

Table 9

[0227] From Tables 8 and 9, the FFA levels are often significantly below 0.5% after only 2 hours of reaction, and in some cases even below 0.2%. After 2 hours of reaction time, the TAG has not been substantially converted. However, at 24 hours of reaction time, judging from the sum of the measured components in the table, CALB converts up to 7% of the TAG under specific conditions. This setting is competitive, for example, in the pretreatment of CPO for producing chemical biodiesel, due to the extremely fast reaction of only 2 hours and the relatively low methanol dosage compared to the esterification method using acid catalysts currently used industrially.

[0228] In addition, the amount of added glycerol is not consumed and may even increase by the transesterification of MG and DG. Since the CALB enzyme can be reused, the cost is further reduced compared to the currently adopted industrial method. This means that it may be sufficient with extremely low dosages to replace the currently adopted process with a more environmentally friendly and economical alternative.

[0229] Example 8: Ability of the esterase of SEQ ID NO: 4 CPO (4.4 wt% FFA, 6.1 wt% DAG) oil is melted at 60 °C. Then, 30 g of CPO is weighed into a 100 mL Erlenmeyer flask, and then 0 or 2% (wt / wt of oil) water and 0 or 5% (wt / wt of oil) dried industrial grade glycerol are added. The mixture is adjusted in a shaking incubator oven at 40 °C while mixing at 250 rpm for 30 minutes to ensure that the temperature is reached. 100 ppm of NaOH is added as a 50% aqueous solution.

[0230] An amount of the active enzyme protein of SEQ ID NO: 4 equal to 0.2% (wt / wt of oil) is added at the 0-hour time point. The dry powder of lipase is dissolved and added to 30 g of CPO as a 101 μL aqueous solution, which means that a small amount of water that cannot be avoided is added together with the lipase.

[0231] The reaction was carried out at 40 °C in a shaking incubator at 250 rpm while continuously linearly administering 1.7 molar equivalents of methanol based on free fatty acid and bound fatty acid over 10 hours or 20 hours. After completing the administration of methanol at the time point indicating 10 hours or 20 hours, the reaction was continued until the time point indicating 24 hours, and the results were measured.

[0232] 2 mL of the sample is pipetted into 2 mL Eppendorf tubes. These are immediately centrifuged at 2000 rpm for 1 minute and then incubated at 99 °C for 10 minutes to inactivate the enzyme. Finally, the sample is dried under vacuum at 80 °C for 2 hours to remove methanol.

[0233] Analysis method: - FFA by AOCS official method Ca 5a-40. - Monoglycerides, diglycerides and triglycerides by AOCS official method Ck 2-09 using the Quality Trait Analysis (QTA) NIR method provided by Eurofins.

[0234]

Table 10

[0235] Considering the results using a 20-hour dosing time comparable to the previous examples, these results show that by adding 5% glycerol instead of 2% water, not only the glyceride level but also the resulting FFA concentration is improved.

[0236] The methanol-tolerant lipase gives much better results at a 10-hour methanol dosing time than those obtained using SEQ ID NO: 1. When water is added, considering the glyceride concentration, the faster the methanol dosing, the faster the reaction. Furthermore, comparing the results of a 10-hour dosing time and a 20-hour dosing time using 5% glycerol instead of 2% water, the enzyme becomes significantly slower. Thus, even for the esterase of SEQ ID NO: 4, if too much is administered too quickly, it is clearly affected adversely by methanol.

[0237] Finally, comparing these results with the previous examples, no further quality improvement is brought about by this highly methanol-tolerant and rapid lipase, and the final FFA concentration is similar and highly correlated with the amount of water added. This is the case despite a large difference in the reaction rate and the ability to administer methanol quickly when using SEQ ID NO: 4 instead of SEQ ID NO: 1. Clearly, the reaction is a limited equilibrium, and when using a lipase with sufficient methanol tolerance, the choice of lipase only determines the reaction rate rather than the product quality.

[0238] Example 9: Influence of Glycerol Dosage in SEQ ID NO: 4 Melt CPO (4.4 wt% FFA, 6.1 wt% DAG) oil at 60°C. Then, weigh 30 g of CPO into a 100 mL Erlenmeyer flask, and add 5, 10, or 20% (oil weight / weight) of dried industrial grade glycerol. Adjust the mixture in a shaking incubator oven at 40°C while mixing at 250 rpm for 30 minutes to ensure that the temperature is reached.

[0239] Add an amount of active enzyme protein of SEQ ID NO: 4 equal to 0.2% (oil weight / weight) of SEQ ID NO: 1 at the 0-hour time point. Dissolve the dry powder of lipase and add it as a 101 μL aqueous solution to 30 g of CPO, which means that an unavoidable small amount of water is added together with the lipase.

[0240] Carry out the reaction at 40°C in a shaking incubator at 250 rpm while continuously linearly administering 1.7, 2, or 2.3 molar equivalents of methanol based on free fatty acid and bound fatty acid over 20 hours. After completing the administration of methanol at the time point indicating 20 hours, continue the reaction until the time point indicating 24 hours and measure the results.

[0241] Collect 2 mL of the sample with a pipette into 2 mL Eppendorf tubes. Centrifuge these immediately at 2000 rpm for 1 minute, then incubate at 99°C for 10 minutes to inactivate the enzyme. Finally, dry the sample under vacuum at 80°C for 2 hours to remove methanol.

[0242] Analysis method: - FFA by AOCS official method Ca 5a-40. - Mono, di, and triglycerides by AOCS official method Ck 2-09 using the Quality Trait Analysis (QTA) NIR method provided by Eurofins.

[0243]

Table 11

[0244] In particular, the performance at the FFA level generally improves as more glycerol is added. This is probably because the chemical activity of water in glycerol is significantly reduced, 1 which means that glycerol has an additional effect of effectively drying the reactants as its concentration increases.

[0245] Notably, by combining 2 equivalents of methanol with 20% glycerol, 0.25 wt% FFA, which is the target FFA level, is produced. The level of glycerides is generally higher than the standard level of biodiesel, but the reaction is carried out for only 24 hours, and further conversion of glycerides can be expected as the reaction time is lengthened.

[0246] The difference in results between 1.7 equivalents and 2.3 equivalents of methanol is small, suggesting that water and glycerol are much more influential parameters.

[0247] Example 10: Obtaining less than 0.4 wt% MG after reaction by washing CPO (4.4 wt% FFA, 6.1 wt% DAG) oil was used without pre-drying. Then, 300 g of CPO was weighed and added to a 1 L glass reactor stirred by a stirrer, and then 20% (weight / weight of oil) of dried industrial grade glycerol was added. The mixture was adjusted at 35 °C at a stirring speed of 400 rpm for 30 minutes to ensure that the temperature was reached. 0.5% (weight / weight of oil) of the enzyme of SEQ ID NO: 1 was added at the 0 hour point. The reaction was carried out at 35 °C in the reactor at 400 rpm while continuously linearly dosing 2 molar equivalents of methanol based on free fatty acid and bound fatty acid over 40 hours. After the dosing of methanol was completed at the time point indicating 40 hours, the reaction was continued until the time point indicating 72 hours, and the results were measured (0.4 wt% FFA, 0.72 wt% MAG, and 0.11 wt% DAG were measured). This material was then used for the following various washings.

[0248] Washing: 10 g of pre-prepared FAME close to the standard was added to a 25 mL plastic centrifuge tube. Subsequently, the selected washing mixture (see the results in the table below) was added. If applicable, the additive was premixed with the washing water. After initially mixing the mixture manually, an ultrasonic bath was performed at 60 °C for 2 minutes. Finally, the phases were separated by thorough centrifugation, and the washed oil phase was sampled in Table 12 with the obtained FFA, MAG, DAG, and TAG values.

[0249] For all samples, 2 mL of the sample was pipetted into a 2 mL Eppendorf tube. These were immediately centrifuged at 2000 rpm for 1 minute and then incubated at 99 °C for 10 minutes to inactivate the enzyme. Finally, the samples were dried under vacuum at 80 °C for 2 hours to remove methanol.

[0250] Analysis method: FFA by AOCS official method Ca 5a - 40. Mono, di, and triglycerides by AOCS official method Ck 2 - 09 using the provided Quality Trait Analysis (QTA) NIR method.

[0251] [Table 12]

[0252] The fact that the FFA level after the initial reaction is not less than 0.25 wt% but 0.4 wt% is due to insufficient drying of the raw material CPO. The FFA increased from 0.4 to 0.64 and 0.65 in the above two tests, suggesting that a small amount of active esterase is present in the raw material oil after separation and hydrolysis occurs due to the presence of moisture. However, the main result considered in this example is the change in the MAG level.

[0253] Since it is conceivable that soap may promote emulsification and thereby reduce MAG, the reduction of MAG due to the saponification of residual FFA was investigated. Since low pH might bring about surprising results, the effect of pH reduction by citric acid and chelation was investigated. Since it was possible that it had affected the attraction of the hydrophilic part of MAG to the aqueous phase, the effect of high ionic strength by the addition of salts was investigated. As a baseline, the effect of pure water (high purity, MilliQ) itself was also measured. Finally, silica particles with low hydrophilicity were tested as it was possible to adsorb MAG and then remove it by filtration.

[0254] The MAG levels were reasonably reduced to less than 0.4 wt% in all cases, suggesting that the oil-water emulsification properties inherent to MAG are generally useful for washing out this minor component when the MAG level exceeds the desired level.

[0255] Since soap is a by-product of this process and is washed away with water, washing the finished biodiesel is a standard method in today's existing chemical biodiesel plants. In today's state-of-the-art enzymatic biodiesel plants, an alkaline washing step is currently also used to saponify residual FFA. Therefore, a simple water washing step is not something new introduced to existing plants; rather, such a washing step is likely to produce cleaner waste liquor than existing processes, leading to a reduction in wastewater treatment costs. Although losses during washing are high in any existing process, the losses in this optional MAG removal washing step are small, and MAG is likely to be reusable after being separated from the wash water. Such separation can be achieved, for example, by setting a high temperature and optionally a low pH to break down the MAG-water emulsion, or by extracting a solvent (biodiesel). By accumulating MAG in the sedimentation tank of the wash water, the wash water can be made reusable, and then MAG can be reused from there to improve the overall process yield.

[0256] Example 11: Biodiesel reaction using two enzymes with moderate to poor methanol stability Crude palm oil (CPO) oil without the measured composition was melted at 60 °C. Then, 30 g of CPO was weighed into a 100 mL Erlenmeyer flask. 50 ppm of NaOH was added as a 50% aqueous solution. The addition of NaOH was determined from a standard curve prepared previously with this oil. The mixture was prepared in a shaking incubator oven at 35 °C while mixing at 250 rpm for 30 minutes to ensure that the temperature was reached. Enzyme was added at the 0 hour time point.

[0257] 2.9 mg of esterase (SEQ ID NO: 5 or SEQ ID NO: 6) was added as a pre-prepared esterase and glycerol mixture. SEQ ID NO: 5 and SEQ ID NO: 6 originally had concentrations of 1.01 and 0.50 mg / mL, respectively, when measured by the standard A280 method, which means that approximately 10% water was administered with the enzyme. Thus, the esterase was administered to the intended 20% (w / w) glycerol and dried at 5.1 mbar and room temperature for 10 hours to remove moisture.

[0258] Based on the assumption that there is 100% triglyceride in CPO, the reaction was carried out at 35 °C in a shaking incubator at 250 rpm while continuously linearly dosing 2.0 molar equivalents of methanol over 40 hours. This meant that the dosing amount relative to the true molar equivalent was slightly inaccurate when considering the concentrations of FFA, MG, and DG in the oil, which was unknown for this particular oil. After completing the methanol dosing at the time point indicating 40 hours, the reaction was continued until the time point indicating 120 hours, and the results were measured after the reaction reached equilibrium.

[0259] 2 mL of the sample was pipetted into a 2 mL Eppendorf tube. The sample was incubated at 99 °C for 10 minutes to inactivate the enzyme and then centrifuged at 2000 rpm for 1 minute. Finally, the sample was dried under vacuum at 80 °C for 2 hours to remove the excess methanol dissolved in the FAME.

[0260] FFA by AOCS official method Ca 5a-40. FAME, mono, di and triglycerides by AOCS official method Ck 2-09 using the Quality Trait Analysis (QTA) NIR method provided by Eurofins.

[0261]

Table 13

[0262] SEQ ID NO: 5 or SEQ ID NO: 6 functioned well during the first 24 hours of the reaction, at which point 1.2 equivalents of methanol were added to the reaction. The reaction stopped at the end of the 40-hour methanol dosing schedule with 2 equivalents of methanol added, and further poor conversion rates were obtained after further reaction time. This example shows the fact that enzymes that are not sufficiently stable, especially with respect to methanol tolerance in a low moisture environment, cannot make the most of the present invention because such enzymes are inactivated too quickly to achieve equilibrium with them. SEQ ID NOs: 1 and 4 are examples of enzymes with excellent methanol tolerance, and SEQ ID NO: 4 has particularly excellent methanol tolerance. Such sufficient alcohol tolerance of the selected enzyme is also a requirement for using the present invention with any selected alcohol.

Claims

1. A process for reducing the level of free fatty acids in biodiesel / fatty acid alkyl esters, comprising: I. providing a fatty acid feedstock substrate comprising triglycerides, diglycerides, monoglycerides, free fatty acids, fatty acid esters, or any combination thereof; II. reacting the fatty acid feedstock substrate with an alcohol in the presence of one or more esterases and added glycerol to produce a fatty acid alkyl ester; III. separating the reaction mixture of step (ii) into a light phase comprising fatty acid methyl ester (FAME) and a heavy phase comprising esterase, glycerol, short-chain alcohol, and water; IV. drying the mixture of step (ii) and then separating the light phase and the heavy phase; and / or V. drying the heavy phase of step (iii) in the presence of the esterase, wherein the glycerol and esterase of the heavy phase are recycled and added back to step (ii). The process, wherein the level of the obtained free fatty acid (FFA) concentration in the obtained biodiesel (FAME) product is less than 1% (weight / weight).

2. The process according to claim 1, wherein the free fatty acid (FFA) concentration is less than 1% by weight in the light phase of step (iii) after removing methanol from the light phase of step (iii).

3. The process according to claim 1, wherein the esterase in step (ii) is not immobilized.

4. The process according to claim 1, wherein the esterase in step (ii) is added as a liquid, granule, and / or powder.

5. The process according to any one of claims 1 to 4, wherein the water added in step (ii) is less than 2% weight / weight of the fatty acid feedstock.

6. The process according to any one of claims 1 to 5, wherein the esterase can transesterify monoglyceride (MG), diglyceride (Dg), and triglyceride (TG).

7. The process according to claim 6, wherein the esterase is an esterase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% or at least 100% sequence identity with the esterase of SEQ ID NO: 1 or and the polypeptide of SEQ ID NO:

1.

8. I. providing a fatty acid feedstock substrate comprising triglyceride, diglyceride, monoglyceride, free fatty acid, fatty acid ester or any combination thereof; II. reacting the fatty acid feedstock substrate with an alcohol in the presence of one or more esterases and added glycerol to produce a fatty acid alkyl ester; III. separating the reaction mixture of step (ii) into a light phase comprising fatty acid methyl ester and a heavy phase comprising esterase, glycerol, short-chain alcohol and water; IV. a step of drying the heavy phase of step (iii) in the presence of the esterase, wherein the glycerol and esterase of the heavy phase are recycled and added back to step (ii). The process according to claim 1, comprising, wherein the level of the resulting free fatty acid (FFA) concentration is less than 1% (weight / weight).

9. The process according to claim 1, wherein the alcohol is a C1-C5 alcohol.

10. The process according to any one of claims 1 to 9, which is carried out at a temperature of 20 to 90 °C, such as 25 to 85 °C, preferably 30 to 80 °C.

11. The substrate is derived from one or more of algal oil, canola oil, coconut oil, castor oil, palm 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, oil derived from halophytes and / or animal fats derived from pigs, beef, and sheep, lard, chicken fat, fish oil, palm oil free fatty acid distillate, soybean oil free fatty acid distillate, soda oil sludge fatty acid material, yellow grease, used cooking oil, palm oil waste liquid, and brown grease, or any combination thereof, for the process according to any one of claims 1 to 10.

12. The esterase is Aspergillus lipase; Aspergillus niger lipase; Thermomyces lanuginosa lipase; Candida Antarctica lipase A; Candida Antarctica lipase B; Candida cylindracae lipase; Candida deformans lipase; Candida lipolytica lipase; Candida parapsilosis lipase; Mucor miehei, Chromobacterium; Candida rugosa lipase; Corynebacterium acnes lipase; Humicola lanuginosa, Cryptococcus spp. S-2 lipase; Fusarium culmorum lipase; Fusarium heterosporum lipase; Fusarium oxysporum lipase; Mucor javanicus lipase; Rhizomucor miehei lipase; Rhizomucor delemar lipase; Burkholderia (Pseudomonas) cepacia lipase; Pseudomonas sp., ATCC 21808, Pseudomonas camembertii lipase; Pseudomonas fluorescens lipase; Rhizopus lipase; Rhizopus arrhizus lipase;A process according to any one of claims 1 to 11, selected from the group consisting of Staphylococcus aureus lipase; Geotrichium candidum lipase; Hypozyma sp. lipase; Klebsiella oxytoca lipase; and their wild-type orthologs and homologs; and their variants.

13. The FFA decreases along with the conversion of triglycerides and is less than 30%, preferably less than 20%, and most preferably less than 10%, for the process according to any one of claims 1 to 12.

14. One or more esterases have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or at least 100% sequence identity with the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, for the process according to any one of claims 1 to 13.

15. One or more esterases comprise or consist of the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, for the process according to any one of claims 1 to 14.

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