Production of fatty acid alkyl esters using alkali treatment

Abstract

A method for producing fatty acid alkyl esters comprises providing a system comprising an oil phase / hydrophobic phase and a hydrophilic phase, and reacting a fatty acid raw material present in the oil phase / hydrophobic phase with an alcohol in the presence of water and one or more esterases.

Description

[0001] This application is a divisional application of the invention patent application with application date of May 28, 2015, application number 201580027195.1, and invention name "Production of fatty acid alkyl esters by alkali treatment".

[0002] References to sequence listings

[0003] This application contains a sequence listing in computer readable form. The computer readable form is incorporated herein by reference. Field of the Invention

[0004] The present invention relates to a method for producing fatty acid alkyl esters from fatty acid raw materials. The method according to the present invention comprises a polishing reaction, in which an alkaline reagent or a base is directly mixed into the holoenzyme reaction mixture. Background Art

[0005] Fatty acid alkyl esters can be used as a fuel, biodiesel, in a standard diesel engine. Biodiesel can be used alone or mixed with petroleum diesel. Biodiesel has become more attractive recently because of its environmental benefits.

[0006] Although biodiesel is currently mainly produced chemically (using, for example, NaOH and / or sodium methoxide as catalysts), there are several associated problems that limit its development, such as pre-processing of oils due to high content of free fatty acids, removal of chemical catalysts from the ester and glycerol phases for the need for a high alcohol surplus in the reaction, and removal of inorganic salts in the glycerol recovery process.

[0007] By using esterifying enzymes as catalysts, these disadvantages caused by chemical catalysts are largely avoided, and the use of lipases in transesterification for the production of biodiesel has attracted interest in recent years.

[0008] Biodiesel produced by enzymatic bioconversion (compared to chemical conversion) is more environmentally friendly. However, with very few exceptions, enzymatic technology is not currently used for commercial-scale biodiesel production.

[0009] Enzymatic production of fatty acid alkyl esters using liquid enzymes is described in, for example, WO 2006 / 072256, Lv et al., Process Biochemistry 45 (2010) 446-450) and WO 2012 / 098114.

[0010] In the method for producing fatty acid alkyl ester or biodiesel, fatty acid feedstock and alcohol (typically methanol) are reacted to produce fatty acid alkyl ester and glycerine. These methods comprise other steps, for example fatty acid alkyl ester is carried out alkali treatment to reduce the amount of free fatty acids. Before these processing steps, the oil phase / hydrophobic phase comprising fatty acid alkyl ester, free fatty acids etc. is separated from the hydrophilic phase of a part comprising for example water, glycerine and excessive alcohol. Before alkali treatment, the separation of oil phase / hydrophobic phase and hydrophilic phase is considered to be necessary, mainly because, it is believed that, before alkali treatment, there is no phase separation, and the glycerine formed as the byproduct of producing fatty acid alkyl ester will comprise a large amount of salts. This is undesirable, because glycerine itself is exactly a valuable byproduct, and wherein this glycerine is processed into technical grade glycerine.

[0011] Additionally, separation of the oily / hydrophobic phase and the hydrophilic phase prior to the alkaline treatment is considered advantageous in order to reduce evaporation of the alcohol, since the alkaline treatment is performed at elevated temperatures, which are close to the boiling point of the alcohol.

[0012] Finally, when producing fatty acid alkyl esters / biodiesel by enzymatic bioconversion, phase separation prior to alkaline treatment makes it possible to reuse large amounts of the enzyme which would otherwise be at least partially denatured during the alkaline treatment.

[0013] However, during the separation of the oil phase / hydrophobic phase and the hydrophilic phase, a third emulsion phase is formed, which also contains some fatty acid alkyl esters or biodiesel. This third phase is not collected with the oil phase / hydrophobic phase. Therefore, in addition to the technical challenges, this separation step also results in the loss of fatty acid alkyl esters or biodiesel.

[0014] Therefore, there is a need for more efficient methods of producing fatty acid alkyl esters or biodiesel. SUMMARY OF THE INVENTION

[0015] The present invention provides a process for producing fatty acid alkyl esters by reacting a fatty acid feedstock with an alcohol in a system comprising an oil phase / hydrophobic phase and a hydrophilic phase, wherein soaps / salts are formed from free fatty acids by treatment with one or more alkaline agents in the presence of the alcohol / the hydrophilic phase.

[0016] The present invention also provides a method for producing fatty acid alkyl esters, comprising

[0017] i) providing a system comprising an oil phase / hydrophobic phase and a hydrophilic phase, and

[0018] ii) reacting the fatty acid feedstock present in the oil phase / hydrophobic phase with an alcohol in the presence of water and one or more esterifying enzymes;

[0019] The total amount of the one or more esterases is in the range of 100 to 500 enzyme units / g oil phase / hydrophobic phase.

[0020] An advantage of the process of the present invention is that very little product (ie fatty acid esters / biodiesel) is lost in the process, which provides economic benefits and increases sustainability.Another advantage is that the process operations have been greatly simplified.

[0021] In a further aspect, the invention provides a composition comprising at least 90% (w / w) fatty acid alkyl esters, from 300 to 400 ppm soap, less than 0.25% (w / w) free fatty acids and less than 0.23% (w / w) glycerides. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : A schematic diagram showing a method according to the present invention, including the preparation of an oil phase / hydrophobic phase with a high content of fatty acid esters (FAME phase), which is separated from a hydrophilic phase of glycerol, alcohol and soap / salts containing residual fatty acids (glycerol / soap phase); acidification of the soap / fatty acid salts to produce free fatty acids, which can be recycled and used as fatty acid raw materials in enzyme reactions; and the production of high-grade / industrial-grade glycerol.

[0023] Figure 2 : shows an embodiment of the method of the present invention. The figure shows a process flow diagram of a batch operation device. In a specific embodiment, the reaction system includes 1% water, 10% glycerol, 1.4 equivalents of MeOH and 0.3% enzyme (lipase), which corresponds to 300 lipase units / g oil phase / hydrophobic phase. The device can also be constructed as a continuous stirred tank reactor system.

[0024] These drawings have been included for illustrative purposes only and should not be construed as limiting the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0025] definition

[0026] 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 biodiesels because they can be used as additives or replacements for petroleum diesel.

[0027] alcohol: The alcohol used in the process of the present invention is preferably an alcohol having 1 to 5 carbon atoms (C 1、 C2, C3, C4, or C5) short chain alcohols.

[0028] Fatty acid raw materials:The term "fatty acid feedstock" is defined herein as a substrate comprising triglycerides, diglycerides, monoglycerides, free fatty acids or any combination thereof. In principle, any oils and fats of plant or animal origin comprising fatty acids can be used as substrates in the method of the present invention to produce fatty acid alkyl esters.

[0029] Lipolytic enzymes

[0030] The one or more esterases used in the method of the present invention are selected from lipase, phospholipase, cutinase, acyltransferase or one or more mixtures of lipase, phospholipase, cutinase and acyltransferase. The one or more esterases are selected from enzymes in EC 3.1.1, EC 3.1.4 and EC 2.3. The one or more esterases can also be a mixture of one or more lipases. The one or more esterases can include lipase and phospholipase. The one or more esterases include lipases of EC 3.1.1.3. The one or more esterases include lipases active on tri-, di- and monoglycerides.

[0031] LipaseSuitable esterases may be polypeptides having lipase activity, for example, selected from Candida antarctica lipase A (CALA) as disclosed in WO 88 / 02775, C. antarctica lipase B (CALB) as disclosed in WO 88 / 02775 and shown in SEQ ID NO: 1 of WO 2008065060, Thermomyces lanuginosus (formerly Humicola lanuginosus) lipase disclosed in EP 258 068, Thermomyces lanuginosus variants disclosed in WO 2000 / 60063 or WO 1995 / 22615 (particularly, SEQ ID NO: 2003 of WO 95 / 22615). lipase at position 1-269 of ID NO: 2, Hyphozyma sp. lipase (WO 98 / 018912), and Rhizomucor manhei (Rhizomucor manhei) lipase (SEQ ID NO: 5 in WO 2004 / 099400)), from Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes (EP 218272), Pseudomonas cepacia (EP 331 376), Pseudomonas stutzeri (GB 1,372,034), Pseudomonas fluorescens, Pseudomonas sp. strain SD 705 (WO 95 / 06720 and WO 96 / 27002), Pseudomonas wisconsinensis (WO 96 / 12012); Bacillus lipase, for example from Bacillus subtilis (Dartois et al., (1993), Biochemica et Biophysica Acta, 1131, 253-360), Bacillus stearothermophilus (JP 64 / 744992) or Bacillus pumilus (WO 91 / 16422). Also preferred are lipases from any one of the following organisms: Fusarium oxysporum, Absidiareflexa, Absidia flexus, Rhizomucor manhei, Rhizopus oryzae, Aspergillus niger, Aspergillus tubingensis, Fusarium heterosporum, Aspergillus oryzae, Aspergillus camembert, Aspergillus foetidus, Aspergillus niger, Aspergillus oryzae and Thermomyces lanuginosus, e.g., a lipase selected from any one of SEQ ID NOs: 1 to 15 in WO 2004 / 099400.

[0032] Useful lipases relevant to the present invention are lipases having at least 60%, e.g., 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%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity to the mature polypeptide of SEQ ID NO: 2, the polypeptide at positions 1-269 of SEQ ID NO: 2 shown in WO 95 / 22615, or the polypeptide of SEQ ID NO: 1 shown in WO 2008 / 065060.

[0033] Commercial lipase preparations suitable for use in the method of the present invention include LIPOZYME CALB L, LIPOZYME (R) TL 100L and CALLERA TM TRANS (both available from Novozymes A / S).

[0034] Particularly useful lipases may be selected from the group consisting of

[0035] (a) a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2;

[0036] (b) a polypeptide that is a subsequence of the amino acid sequence set forth in SEQ ID NO: 1 or 2;

[0037] (c) a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 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% sequence identity to any of the polypeptides defined in (a) and (b).

[0038] The lipase set forth in (c) may be a variant of the amino acid sequence set forth in SEQ ID NO: 1, wherein the polypeptide comprises the following substitutions T231R and N233R.

[0039] The lipase as described in item (c) may have an amino acid sequence that differs from the polypeptide of SEQ ID NO: 1 or 2 in up to 40 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

[0040] The lipase can be a variant of a parent lipase, which has lipase activity and has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1, and includes a substitution at a position corresponding to T231R+N233R and at least one or more (e.g., several) of D96E, D111A, D254S, G163K, P256T, G91T, G38A, D27R, and N33Q of SEQ ID NO: 2.

[0041] In another embodiment, the lipase is a variant having lipase activity and having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1, and comprising substitutions at positions corresponding to T231R+N233R and at least one or more (e.g., several) of D96E, D111A, D254S, G163K, P256T, G91T, G38A, D27R, and N33Q of SEQ ID NO: 2, selected from the group consisting of:

[0042] a)D96E T231R N233R;

[0043] b) N33Q D96E T231R N233R;

[0044] c)N33Q T231R N233R

[0045] d) N33Q D111A T231R N233R;

[0046] e) N33Q T231R N233R P256T;

[0047] f)N33Q G38A G91T G163K T231R N233R D254S;

[0048] g)N33Q G38A G91T D96E D111A G163K T231R N233R D254S P256T;

[0049] h)D27R N33Q G38A D96E D111A G163K T231R N233R D254S P256T;

[0050] i)D27R N33Q G38A G91T D96E D111A G163K T231R N233R P256T;

[0051] j)D27R N33Q G38A G91T D96E D111A G163K T231R N233R D254S;

[0052] k)D27R G38A G91T D96E D111A G163K T231R N233R D254S P256T;

[0053] l)D96E T231R N233R D254S;

[0054] m)T231R N233R D254S P256T;

[0055] n)G163K T231R N233R D254S;

[0056] o)D27R N33Q G38A G91T D96E G163K T231R N233R D254S P256T;

[0057] p)D27R G91T D96E D111A G163K T231R N233R D254S P256T;

[0058] q)D96E G163K T231R N233R D254S;

[0059] r)D27R G163K T231R N233R D254S;

[0060] s)D27R G38A G91T D96E D111A G163K T231R N233R D254S;

[0061] t)D27R G38A G91T D96E G163K T231R N233R D254S P256T;

[0062] u)D27R G38A D96E D111A G163K T231R N233R D254S P256T:

[0063] v)D27R D96E G163K T231R N233R D254S;

[0064] w)D27R D96E D111A G163K T231R N233R D254S P256T;

[0065] x)D27R G38A D96E G163K T231R N233R D254S P256T.

[0066] Useful variants of these parent lipases are provided, for example, in WO 2015 / 049370.

[0067] Lipase activity:

[0068] In the context of the present invention, the esterification activity can be determined as lipase units (LU) using tributyrate as substrate. The method is based on the hydrolysis of tributyrate under the action of the enzyme, and the consumption of base to maintain a constant pH during the hydrolysis is recorded as a function of time.

[0069]

[0070] According to the present invention, one lipase unit (LU) can be defined as the amount of enzyme that releases 1 micromole of titratable butyrate per minute under standard conditions (i.e. at 30°C; pH 7.0; with 0.1% (w / v) gum arabic as emulsifier and 0.16 M tributyric acid as substrate).

[0071] Alternatively, esterase activity can be determined as long-chain lipase units (LCLU), Using the substrate pNP-Palmitate (C:16), when incubated at pH 8.0 at 30°C, the lipase hydrolyzes the ester bond and releases pNP, which is yellow and can be detected at 405 nm.

[0072]

[0073] Phospholipase :

[0074] The one or more esterases may include polypeptides with phospholipase activity, preferably phospholipase A1, phospholipase A2, phospholipase B, phospholipase C, phospholipase D, lyso-phospholipase activity, and / or any combination thereof. In the method of the present invention, the one or more esterases may be phospholipases, e.g., single phospholipases, such as A1, A2, B, C, or D; two or more phospholipases, such as, two phospholipases (including but not limited to, both A-type and B-type, both A1-type and A2-type, both A1-type and B-type, both A2-type and B-type, both A1-type and C-type, both A2-type and C-type) or two or more phospholipases of the same type.

[0075] The one or more esterases may be polypeptides having phospholipase activity and acyltransferase activity, for example, selected from the group consisting of polypeptides disclosed in WO 2003 / 100044, WO 2004 / 064537, WO 2005 / 066347, WO 2008 / 019069, WO 2009 / 002480, and WO 2009 / 081094. Acyltransferase activity may for example be determined by the assay described in WO 2004 / 064537.

[0076] The phospholipase may be selected from the polypeptides disclosed in WO 2008 / 036863 and WO 20003 / 2758. A suitable phospholipase preparation is PURIFINE (R) (available from Verenium) and LECITASE (R) ULTRA (available from Novozymes). Enzymes with acyltransferase activity and commercial enzyme preparations LYSOMAX (R) OIL (available from Danisco A / S) is also available.

[0077] Cutinase: The one or more esterifying enzymes may comprise a polypeptide having cutinase activity. For example the cutinase may be selected from the polypeptides disclosed in WO 2001 / 92502, in particular the Humicola insolens cutinase variant disclosed in Example 2.

[0078] Preferably, the one or more esterases are enzymes having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% identity to any of the previously mentioned lipases, phospholipases, cutinases, and acyltransferases.

[0079] In one embodiment, the one or more esterases have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least or even at least 99% identity to the amino acid sequence shown at positions 1-269 of SEQ ID NO:2 of WO 95 / 22615.

[0080] Enzyme sources and preparations: One or more esterases used in the method of the present invention can be derived from or obtainable from any of the sources mentioned herein. The term "derived from" in this context means that the enzyme can be separated from the organism in which the enzyme is naturally present, i.e., the identity of the amino acid sequence of the enzyme is the same as that of the natural enzyme. The term "derived from" also means that the enzyme can be recombinantly produced in a host organism, wherein the recombinantly produced enzyme has the same identity as the natural enzyme, or has a modified amino acid sequence, for example, deletion, insertion and / or substitution of one or more amino acids, i.e., the recombinantly produced enzyme is a mutant and / or fragment of the natural amino acid sequence. Natural variants are included in the meaning of the natural enzyme. In addition, the term "derived from" includes enzymes synthesized by, for example, peptide synthesis. The term "derived from" also includes enzymes that have been modified in vivo or in vitro by, for example, glycosylation, phosphorylation, etc. The term "obtainable" in this context means that the enzyme has the same amino acid sequence as the natural enzyme. The term covers enzymes that have been separated from an organism (in which the enzyme exists naturally, or in which the enzyme is recombinantly expressed in the same type or other type of organism), or enzymes synthesized by, for example, peptide synthesis. For recombinantly produced enzymes, the terms "obtainable" and "derived from" refer to the identity of the enzyme and not to the identity of the host organism in which the enzyme is recombinantly produced.

[0081] Accordingly, the one or more esterases can be obtained from microorganisms by using any suitable technology. For example, enzyme preparation can be obtained by fermenting suitable microorganisms and then by methods well known in the art from the fermented liquid or microorganism separation enzyme preparation of gained. The enzyme can also be obtained by using recombinant DNA technology. This method generally includes a host cell transformed with a recombinant DNA vector for cultivation, the vector comprising a DNA sequence encoding the enzyme in question, and the DNA sequence is operably connected with a suitable expression signal so that it can express the enzyme in the culture medium under the conditions allowing the enzyme to be expressed, and reclaim the enzyme from the culture. The DNA sequence can also be incorporated into the genome of the host cell. The DNA sequence can be a genome, a cDNA or a synthetic source, or any combination thereof, and can be separated or synthesized according to methods well known in the art.

[0082] The one or more esterolytic enzymes can be applied in any suitable formulation, for example, as a lyophilized powder or in an aqueous solution.

[0083] Sequence consistency:

[0084] The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0085] For purposes of the present invention, use Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) to determine the sequence identity between two amino acid sequences, this algorithm is as EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite (The European Molecular Biology Open Software Suite), Rice et al., 2000, Genetics Trend (Trends Genet.) 16:276-277) (preferred 5.0.0 version or updated version) Needle program is implemented.The parameter used is gap opening penalty 10, gap extension penalty 0.5, and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.The Needle output (obtained using-nobrief option) marked as "longest identity" is used as percentage identity and is calculated as follows:

[0086] (number of identical residues x 100) / (length of alignment - total number of gaps in alignment)

[0087] Method design

[0088] The invention provides, in the system / reaction system comprising oil phase / hydrophobic phase and hydrophilic phase, in the presence of water and one or more esterases, by reacting fatty acid raw material with alcohol to produce the method for fatty acid alkyl ester. The reaction of the fatty acid raw material (comprising any triglyceride, diglyceride, monoglyceride, free fatty acid or any combination thereof contained in the fatty acid raw material) and the alcohol produces fatty acid alkyl ester / biodiesel and glycerine. Along with reaction, fatty acid alkyl ester is accumulated in the oil phase / hydrophobic phase.

[0089] The inventors of the present invention have observed that in a process for the production of fatty acid alkyl esters / biodiesel by enzymatic bioconversion of a fatty acid feedstock, even if an alkali treatment is carried out without effectively separating the oil phase / hydrophobic phase from the hydrophilic phase, the resulting glycerol has unexpectedly low salt content and has a sufficiently low content of salts to be easily processed into high quality or industrial grade glycerol, for example by neutralization or salification.

[0090] Therefore, in method according to the present invention, before fatty acid ester / biodiesel is carried out any further processing (for example, handle with alkaline reagent to remove free fatty acids), fatty acid alkyl ester / biodiesel is to produce under the following conditions, and the oil phase / hydrophobic phase that will comprise fatty acid ester / biodiesel can be avoided from the separation of hydrophilic phase under this condition.This provides a large amount of remarkable benefits: at first, it is possible to design the method for producing fatty acid and finally being refined product (comprising biodiesel and glycerine), and this method step is less, step cost is lower, method time is shorter and has higher capacity.Also, because phase separation occurs after alkali treatment, it is possible to use the temperature that rises, and this temperature can make these phases separate faster.

[0091] The inventors of the present invention have also discovered that, after alkaline treatment, the process according to the present invention produces a fatty acid alkyl ester / biodiesel phase or composition having a reduced content of soaps or salts of free fatty acids, which makes the subsequent subsequent washing steps less costly.

[0092] Finally, the process according to the invention gives higher yields since very little fatty acid alkyl esters are trapped in the soap emulsion and no fatty acid alkyl esters are trapped in the enzyme emulsion phase.

[0093] In a main aspect of the present invention, in the presence of the alcohol / the hydrophobic phase, the formation of soap / salt free fatty acids in the oil phase / hydrophobic phase can be achieved by treating with one or more alkaline agents. Therefore, the method comprises contacting fatty acid alkyl esters, free fatty acids, water, alcohol and glycerol and one or more esterases with one or more alkaline agents under conditions that allow soap / salt formation of free fatty acids.

[0094] According to this aspect, the present invention provides a method for producing fatty acid alkyl esters, the method comprising

[0095] i) reacting a fatty acid feedstock with an alcohol to produce fatty acid alkyl esters and glycerol in a reaction system comprising an oil phase / hydrophobic phase and a hydrophilic phase in the presence of water and one or more esterifying enzymes; and

[0096] j) before separating the oily / hydrophobic phase from the hydrophilic phase, removing or reducing the amount of free fatty acids by treating with one or more alkaline agents to form soaps / salts of free fatty acids; for example when there are still oily / hydrophobic phases and hydrophilic phases in the reaction system.

[0097] The method according to the present invention may further comprise

[0098] i) providing a system, such as a reaction system, comprising the oil phase / hydrophobic phase and the hydrophilic phase,

[0099] ii) reacting the fatty acid feedstock present in the oil phase / hydrophobic phase with an alcohol in the presence of water and one or more esterifying enzymes to produce the fatty acid alkyl esters, free fatty acids and glycerol.

[0100] According to some embodiments, the method comprises separating the soap / salt of the free fatty acid produced in step ii) from the fatty acid esters and one or more esterolytic enzymes, for example by separating the fatty acid alkyl esters from the hydrophilic phase comprising the soap / salt and the one or more esterolytic enzymes.

[0101] Accordingly, the present invention provides a method for producing fatty acid alkyl esters, the method comprising

[0102] i) providing a system, such as a reaction system, comprising an oil phase / hydrophobic phase and a hydrophilic phase,

[0103] ii) reacting the fatty acid feedstock present in the oil phase / hydrophobic phase with an alcohol in the presence of water and one or more esterifying enzymes to produce the fatty acid alkyl esters;

[0104] iii) reacting free fatty acids in the oil phase / hydrophobic phase with one or more alkaline reagents; and

[0105] iv) separating the fatty acid alkyl ester from the hydrophilic phase

[0106] In another embodiment, the method according to the present invention comprises

[0107] i) providing a reaction system having an oil phase / hydrophobic phase comprising a fatty acid feedstock, and a hydrophilic phase comprising an alcohol, water and one or more esterifying enzymes;

[0108] ii) reacting a fatty acid feedstock with an alcohol in the presence of said water and said one or more esterifying enzymes to produce free fatty acids, glycerol, and fatty acid alkyl esters;

[0109] iii) adding one or more alkaline reagents to the reaction system to allow the formation of soaps / salts of the free fatty acids produced in step ii); and

[0110] iv) separating the fatty acid alkyl esters from the soaps / salts of the fatty acids and one or more esterifying enzymes.

[0111] The one or more esterases used in the method of the present invention may in particular be selected from the group consisting of lipases, phospholipases, cutinases and mixtures thereof.

[0112] It is particularly preferred that at least one of the one or more esterases is a lipase, and optionally, one or more lipases are used in combination with one or more phospholipases and / or one or more cutinases. As will be appreciated by the skilled person, when the fatty acid feedstock contains impurities in the form of phosphate esters, it is meaningful to use a phospholipase in combination with other esterases (e.g., lipases and cutinases). The combined use of one or more lipases with one or more phospholipases results in a combined transesterification and a reduction (degumming) of phosphate esters and thus produces fatty acid alkyl esters with reduced phosphorus.

[0113] Such a method for producing phosphorus-reduced fatty acid alkyl esters is disclosed in WO 2006 / 133698, which comprises mixing an alcohol, a substrate comprising triglycerides and / or fatty acids with one or more esterases (including lipases, cutinases and acetyltransferases), and one or more phospholipases and water, the contents of which are incorporated herein by reference in their entirety.

[0114] In order to minimize the amount of enzyme (which is lost in the alkali treatment), the inventors of the present invention have also been able to reduce the amount of enzyme used in the method for producing fatty acid alkyl esters. Therefore, the amount of the one or more esterases is preferably in the range of 100 to 500 enzyme units / g oil phase / hydrophobic phase, for example, in the range of 100 to 490 enzyme units / g oil phase / hydrophobic phase, 100 to 480 enzyme units / g oil phase / hydrophobic phase, 100 to 475 enzyme units / g oil phase / hydrophobic phase, 100 to 450 enzyme units / g oil phase / hydrophobic phase, 100 to 425 enzyme units / g oil phase / hydrophobic phase, 100 to 400 enzyme units / g oil phase / hydrophobic phase, 100 to 450 enzyme units / g oil phase / hydrophobic phase, 100 to 460 enzyme units / g oil phase / hydrophobic phase, 100 to 480 enzyme units / g oil phase / hydrophobic phase, 100 to 49 ... 0 to 375 enzyme units / g oil phase / hydrophobic phase, 100 to 350 enzyme units / g oil phase / hydrophobic phase, 100 to 325 enzyme units / g oil phase / hydrophobic phase, 100 to 300 enzyme units / g oil phase / hydrophobic phase, 150 to 500 enzyme units / g oil phase / hydrophobic phase, 150 to 490 enzyme units / g oil phase / hydrophobic phase, 150 to 480 enzyme units / g oil phase / hydrophobic phase, 150 to 475 enzyme units / g oil phase / hydrophobic phase, 150 to 450 enzyme units / g 150 to 425 enzyme units / g oil phase / hydrophobic phase, 150 to 400 enzyme units / g oil phase / hydrophobic phase, 150 to 375 enzyme units / g oil phase / hydrophobic phase, 150 to 350 enzyme units / g oil phase / hydrophobic phase, 150 to 325 enzyme units / g oil phase / hydrophobic phase, 150 to 300 enzyme units / g oil phase / hydrophobic phase, 200 to 490 enzyme units / g oil phase / hydrophobic phase, 200 to 480 enzyme units / g oil phase / hydrophobic phase The amount of enzyme units / g oil phase / hydrophobic phase can be in the range of 200 to 475 enzyme units / g oil phase / hydrophobic phase, 200 to 450 enzyme units / g oil phase / hydrophobic phase, 200 to 425 enzyme units / g oil phase / hydrophobic phase, 200 to 400 enzyme units / g oil phase / hydrophobic phase, 200 to 375 enzyme units / g oil phase / hydrophobic phase, 200 to 350 enzyme units / g oil phase / hydrophobic phase, 200 to 325 enzyme units / g oil phase / hydrophobic phase, or for example, 200 to 300 enzyme units / g oil phase / hydrophobic phase.

[0115] Accordingly, the method of the present invention may include

[0116] i) providing a system comprising an oil phase / hydrophobic phase and a hydrophilic phase, and

[0117] ii) reacting the fatty acid feedstock present in the oil phase / hydrophobic phase with an alcohol in the presence of water and one or more esterifying enzymes;

[0118] Wherein, the total amount of the one or more esterases is in the range of 100 to 500 enzyme units / g oil phase / hydrophobic phase, for example, 100 to 490 enzyme units / g oil phase / hydrophobic phase, 100 to 480 enzyme units / g oil phase / hydrophobic phase, 100 to 475 enzyme units / g oil phase / hydrophobic phase, 100 to 450 enzyme units / g oil phase / hydrophobic phase, 100 to 425 enzyme units / g oil phase / hydrophobic phase, 100 to 400 enzyme units / g oil phase / hydrophobic phase, 100 to 375 enzyme units / g oil phase / hydrophobic phase, 100 to 350 enzyme units / g oil phase / hydrophobic phase, 100 to 325 enzyme units / g oil phase / hydrophobic phase, 100 to 300 enzyme units / g oil phase / hydrophobic phase, 150 to 500 enzyme units / g oil phase / hydrophobic phase, 150 to 490 enzyme units / g oil phase / hydrophobic phase, 150 to 480 enzyme units / g oil phase / hydrophobic phase, 150 to 475 enzyme units / g oil phase / hydrophobic phase, 150 to 450 enzyme units / g oil phase / hydrophobic phase, 150 to 425 enzyme units / g oil phase / hydrophobic phase, 150 to 400 enzyme units / g oil phase / hydrophobic phase, 150 to 375 enzyme units / g oil phase / hydrophobic phase, 150 to 350 enzyme units / g oil phase / hydrophobic phase, 150 to 325 enzyme units / g oil phase / hydrophobic phase, 150 to 300 enzyme units / g oil phase / hydrophobic phase, 200 to 490 enzyme units / g oil phase / hydrophobic phase, 200 to 480 enzyme units / g oil phase / hydrophobic phase The amount of enzyme units / g oil phase / hydrophobic phase can be in the range of 200 to 475 enzyme units / g oil phase / hydrophobic phase, 200 to 450 enzyme units / g oil phase / hydrophobic phase, 200 to 425 enzyme units / g oil phase / hydrophobic phase, 200 to 400 enzyme units / g oil phase / hydrophobic phase, 200 to 375 enzyme units / g oil phase / hydrophobic phase, 200 to 350 enzyme units / g oil phase / hydrophobic phase, 200 to 325 enzyme units / g oil phase / hydrophobic phase, or for example, 200 to 300 enzyme units / g oil phase / hydrophobic phase.

[0119] According to some embodiments of the present invention, wherein the one or more esterases are selected from lipases, such as the lipases set forth in SEQ ID NO: 1, it should be understood that the amount of the enzyme is provided in lipase units. Therefore, when lipases are preferred, the amount of the esterase is preferably in the range of 100 to 500 lipase units / g oil phase / hydrophobic phase, for example, 100 to 490 lipase units / g oil phase / hydrophobic phase, 100 to 480 lipase units / g oil phase / hydrophobic phase, 100 to 475 lipase units / g oil phase / hydrophobic phase, 100 to 450 lipase units / g oil phase / hydrophobic phase, 100 to 425 lipase units / g oil phase / hydrophobic phase, 100 to 400 lipase units / g oil phase / hydrophobic phase, 100 to 375 lipase units / g oil phase / hydrophobic phase, 100 to 350 lipase units / g oil phase / hydrophobic phase, 100 to 325 lipase units / g oil phase / hydrophobic phase, 100 to 300 lipase units / g oil phase / hydrophobic phase, 150 to 500 lipase units / g oil phase / hydrophobic phase, 150 to 490 lipase units / g oil phase / hydrophobic phase, 150 to 480 lipase units / g oil phase / hydrophobic phase, 150 to 475 lipase units / g oil phase / hydrophobic phase, 150 to 450 lipase units / g oil phase / hydrophobic phase 150 to 300 lipase units / g oil phase / hydrophobic phase, 200 to 490 lipase units / g oil phase / hydrophobic phase, 200 to 480 lipase units / g oil phase / hydrophobic phase. In the range of 200 to 300 lipase units / g oil phase / hydrophobic phase, 200 to 475 lipase units / g oil phase / hydrophobic phase, 200 to 450 lipase units / g oil phase / hydrophobic phase, 200 to 425 lipase units / g oil phase / hydrophobic phase, 200 to 400 lipase units / g oil phase / hydrophobic phase, 200 to 375 lipase units / g oil phase / hydrophobic phase, 200 to 350 lipase units / g oil phase / hydrophobic phase, 200 to 325 lipase units / g oil phase / hydrophobic phase, or for example, 200 to 300 lipase units / g oil phase / hydrophobic phase.

[0120] According to other embodiments of the present invention, wherein the one or more esterases are selected from lipases, such as the lipases set forth in SEQ ID NO: 2 and subsequences and variants thereof as disclosed above, the amount of the enzyme is provided in long-chain lipase units (LCLU). In such embodiments, the amount of the esterase is preferably in the range of 100 to 500 lipase units / g oil phase / hydrophobic phase, for example, in the range of 100 to 490 LCLU / g oil phase / hydrophobic phase, 100 to 480 LCLU / g oil phase / hydrophobic phase, 100 to 475 LCLU / g oil phase / hydrophobic phase, 100 to 450 LCLU / g oil phase / hydrophobic phase, 100 to 425 LCLU / g oil phase / hydrophobic phase, 100 to 400 LCLU / g oil phase / hydrophobic phase, 100 to 415 LCLU / g oil phase / hydrophobic phase, 100 to 425 LCLU / g oil phase / hydrophobic phase, 100 to 430 LCLU / g oil phase / hydrophobic phase, 100 to 440 LCLU / g oil phase / hydrophobic phase, 100 to 450 LCLU / g oil phase / hydrophobic phase, 100 to 460 LCLU / g oil phase / hydrophobic phase, 100 to 475 LCLU / g oil phase / hydrophobic phase, 100 to 480 LCLU / g oil phase / hydrophobic phase, 100 to 490 LCLU / g oil phase / hydrophobic phase, 100 to 490 LCLU / g oil phase / hydrophobic phase, 100 to 480 LCLU / g oil phase / hydroph 0 to 375 LCLU / g oil phase / hydrophobic phase, 100 to 350 LCLU / g oil phase / hydrophobic phase, 100 to 325 LCLU / g oil phase / hydrophobic phase, 100 to 300 LCLU / g oil phase / hydrophobic phase, 150 to 500 LCLU / g oil phase / hydrophobic phase, 150 to 490 LCLU / g oil phase / hydrophobic phase, 150 to 480 LCLU / g oil phase / hydrophobic phase, 150 to 475 LCLU / g oil phase / hydrophobic phase, 150 to 450 LCLU / g LCLU / g oil phase / hydrophobic phase, 150 to 425 LCLU / g oil phase / hydrophobic phase, 150 to 400 LCLU / g oil phase / hydrophobic phase, 150 to 375 LCLU / g oil phase / hydrophobic phase, 150 to 350 LCLU / g oil phase / hydrophobic phase, 150 to 325 LCLU / g oil phase / hydrophobic phase, 150 to 300 LCLU / g oil phase / hydrophobic phase, 200 to 490 LCLU / g oil phase / hydrophobic phase, 200 to 480 LCLU / g oil phase / hydrophobic phase The present invention relates to a novel aqueous phase comprising: a first phase, a second phase, a second phase, a third phase, a fourth phase, a fifth phase, a sixth phase, a fifth phase, a sixth phase, a sixth phase, a fifth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a sixth phase, a

[0121] In another embodiment of the present invention, the amount of the one or more esterases is preferably in the range of 0.005-5 g enzyme protein (EP) / kg oil phase / hydrophobic phase or fatty acid raw material, for example, 0.005-2.5 g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-1 g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.75 g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.5 g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.25 g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.1 g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.075 g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.05 g EP / kg oil phase / hydrophobic phase or fatty acid raw material. EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.025g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.01g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.01-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.02-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.03-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.04-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.05-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.06-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.07-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.08-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.09-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.1-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.2-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.3-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.4-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.5-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.6-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.7-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.8-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.9-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 1-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 2-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 3-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 4-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.01-4g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.02-3g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.03-2g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.0.04-1g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.05-0.9g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.06-0.8g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.07-0.7g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.08-0.6g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.09-0.5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.1-0.4g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.1-0.3g EP / kg oil phase / hydrophobic phase or fatty acid raw material, or for example in the range of 0.1-0.25g EP / kg oil phase / hydrophobic phase or fatty acid raw material. .

[0122] In some embodiments of the above aspects of the invention, the fatty acid feedstock or any triglyceride, diglyceride, monoglyceride, free fatty acid, or any combination thereof contained therein, is reacted with the alcohol so that the oil phase / hydrophobic phase contains or is essentially composed of fatty acid alkyl esters and free fatty acids.

[0123] The method of the present invention preferably comprises reacting the fatty acid raw material with the alcohol until at least 60% (w / w) (e.g., at least 65% (w / w), at least 70% (w / w), at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), at least 90% (w / w), or for example, at least 95% (w / w)) of the fatty acid acetyl groups and / or free fatty acids in the fatty acid raw material have been converted to fatty acid alkyl esters.

[0124] In a specific embodiment of the invention, the amount of water corresponds to 0.5% to 5.0% (w / w) of the oil phase / hydrophobic phase, for example 0.5% to 4.0% (w / w) of the oil phase / hydrophobic phase, 0.5% to 3.75%, 0.5% to 3.5%, 0.5% to 3.25%, 0.5% to 3.0%, 0.5% to 2.75%, 0.5% to 2.5%, 0.5% to 2.25%, %, 0.5% to 2.0%, 0.5% to 1.9%, 0.5% to 1.8.%, 0.5% to 1.75%, 0.75% to 2.0%, 0.75% to 1.8%, 0.75% to 1.75%, 0.75% to 1.5%, 1.0% to 2.0%, 1.0% to 1.9%, 1.0% to 1.8%, or for example 1.0% to 1.5% (w / w) of the oil phase / hydrophobic phase. As the skilled person will appreciate, the need to add water, respectively, in the process depends on the amount of water in the fatty acid feedstock.

[0125] In further embodiments, the process comprises increasing the amount of glycerol in the hydrophilic phase to 2% (w / w) to 30% (w / w), preferably from 2% (w / w) to 20% (w / w), for example from 2% (w / w) to 20% (w / w) relative to the oil phase / hydrophobic phase, by reaction of the fatty acid feedstock and optionally by further addition of glycerol.

[0126] In still further embodiments, the alcohol is added to achieve an amount in the range of 12% (w / w) to 34% (w / w) relative to the oil phase / hydrophobic phase, such as in the range of 17% (w / w) to 34% (w / w) relative to the oil phase / hydrophobic phase, or such as in the range of 12% (w / w) to 24% (w / w) relative to the oil phase / hydrophobic phase. When the alcohol is methanol, it is preferably in the range of 12% (w / w) to 24% (w / w) relative to the oil phase / hydrophobic phase, but when the alcohol is ethanol, it is preferably in the range of 17% (w / w) to 34% (w / w) relative to the oil phase / hydrophobic phase.

[0127] The fatty acid feedstock may be reacted with the alcohol at a temperature ranging from 32° C. to 45° C., preferably from 32° C. to 40° C. As will be appreciated by the skilled artisan, the upper temperature limit depends on the thermoaffinity of the esterolytic enzyme(s) employed in the process.

[0128] The method of the present invention may comprise reacting the fatty acid feedstock with the alcohol for 16-50 hours, for example for 24-50 hours, for example for 30-50 hours, for 30-45 hours, for 35-50 hours, for 35-45 hours, for 38-42 hours, preferably for 35-40 hours.

[0129] As will be appreciated by the skilled person, the process of the present invention can be carried out in batch mode or in continuous mode. In a continuous mode process, the two phases, the oil phase / hydrophobic phase and the hydrophilic phase, can be processed separately in countercurrent. Kosugi et al. (1990), Biotechnology & Bioengineering, Vol. 36, 617-622, describe a continuous countercurrent process for hydrolyzing vegetable oils by immobilized lipase.

[0130] The alcohol used in the process may be a C1-C5 alcohol, preferably ethanol or methanol. Methanol is currently the most preferred.

[0131] The alcohol may be added dropwise and / or continuously. The alcohol may be added dropwise in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more steps.

[0132] It will be appreciated that at least one of the one or more esterolytic enzymes used in the method of the invention may be a liquid enzyme.

[0133] In particular, the one or more esterases may be selected from lipases, phospholipases, cutinases and mixtures thereof.

[0134] According to some embodiments, it is preferred that the one or more esterases are one or more lipases. According to other embodiments, a mixture of lipase and phospholipase is used. If it is required to degumming the fatty acid raw material, this combination can be particularly preferred.

[0135] Specifically, the fatty acid feedstock can be derived from one or more of the following: algae oil, canola oil, coconut oil, castor oil, coconut oil, copra oil, corn oil, corn oil, corn oil, corn oil free fatty acid distillate, cottonseed oil, linseed oil, fish oil, grapeseed oil, hemp oil, jatropha oil, jojoba oil, mustard oil, canola oil, palm oil, palm stearin, palm olein, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, soybean oil, sunflower oil, tall oil, oils from halophytes and / or animal fats (including fats from pigs, cattle and sheep, lard, chicken fat, fish oil, palm oil free fatty acid distillate, soybean oil free fatty acid distillate, soap stock fatty acid material, yellow grease, and brown grease or any combination thereof).

[0136] According to other embodiments, the fatty acid raw material can be selected from the group consisting of algae oil, castor oil, coconut oil (coconut dry oil), 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, and oil from halophytes, or any combination thereof.

[0137] The fatty acid feedstock may be selected from the group consisting of animal fats, including tallow from pigs, cattle and sheep, lard, chicken fat, fish oil, or any combination thereof.

[0138] The fatty acid feedstock may be crude, refined, bleached, deodorized, degummed, or any combination thereof.

[0139] Food quality oil and fat are expensive, and therefore waste and byproduct and non-food grade oil and fat from their processing have become the raw materials with increasing attraction for fatty acid alkyl esters.Soap stock is the fraction of the oil obtained in the refinery, by converting free fatty acids to soap (for example, sodium soap) with alkali treatment of this oil.Except these soaps, this soap stock also comprises the fraction of glyceride usually.Acidic oil is the byproduct from the refinery, is to dissolve these soaps by acidifying the soap stock and produce.Acidic oil mainly comprises free fatty acids (FFA) and acylglycerols.Distillate (such as palm fatty acid distillate (PFAD)) is the byproduct of oil refining, from the distillation method for eliminating free fatty acids from this oil.

[0140] The raw material can be an intermediate, waste or by-product of oil or fat refining, the raw material being selected from the group consisting of: soap raw materials; acidic oils; fatty acid distillates, such as PFAD, soybean fatty acid distillates, rapeseed fatty acid distillates, rice bran fatty acid distillates, poultry fatty acid distillates, tallow fatty acid distillates, etc.; gums from degumming; by-products from the production of fish oil omega-3 fatty acid derivatives; fat trap grease; yellow grease, and brown grease, free fatty acids (such as oleic acid); or fractions of oils obtained by physical separation; or any combination thereof.

[0141] In embodiments, the solution phases in the system (eg, the oil phase / hydrophobic phase and the hydrophilic phase) are mixed using a high shear mixer or a cavitator.

[0142] In other embodiments, prior to adding the one or more esterases, an alkaline agent or base, such as NaOH and / or KOH, is added to the system. Specifically, an alkaline agent or base is added in an amount corresponding to 200 ppm or less, preferably in an amount within the range of 10-100 ppm. The main purpose of adding an alkaline agent or base at this stage in the method is to neutralize trace mineral acids in the oil.

[0143] In further embodiments, the oil phase / hydrophobic phase comprises fatty acid alkyl esters, free fatty acids, and optionally unreacted fatty acid starting material; and the hydrophilic phase comprises glycerol, alcohol, water, and the one or more esterolytic enzymes.

[0144] In other embodiments, the hydrophilic phase constitutes from 5% to 50%, 10% to 50%, 20% to 50%, 20% to 45%, or even 20% to 40% of the reaction system (reactant mixture) (w / w), and glycerol constitutes 30% to 85% (w / w), 40% to 85% (w / w), 45% to 85% (w / w), 50% to 85% (w / w) or even 60% to 80% (w / w) of the hydrophilic phase.

[0145] In other embodiments, glycerol constitutes 30% to 70% (w / w), 35% to 70% (w / w), 40% to 70% (w / w), or even 45% to 70% (w / w) of the hydrophilic phase.

[0146] As the method is carried out, in this glycerol-water phase, the amount of glycerol can be increased to exceed the ideal level. Therefore, it can be advantageous to separate the part of this hydrophilic phase from the oil phase / hydrophobic phase that comprises fatty acid alkyl esters, and for example reuse the separated part in the method for the present invention. As the technician will recognize, in order to reuse these enzymes, the part of this hydrophilic phase can be separated from this fat phase by a decanter, a condenser, a cyclone separator or an eddy current separator, a settler or by centrifugation. Reusing this part of the hydrophilic phase provides other advantages, because this will also cause the recycling of at least part of these esterases. When this part of this hydrophilic phase is returned in this reactor, fresh liquid lipase can be added so that lipase activity is maintained at the ideal level and alcohol (for example methanol) can be added.

[0147] Therefore, in some embodiments, the method according to the present invention includes

[0148] i) removing at least a portion of the glycerol / hydrophilic phase from the system during the reaction of the fatty acid feedstock with the alcohol or when the reaction has terminated; and

[0149] ii) In a process as defined in any one of the preceding claims, the process is combined with an additional fatty acid feedstock, wherein the process is the same or different from the process of removal of the glycerol / said hydrophilic phase.

[0150] According to some embodiments, 30% to 80% (w / w) of the glycerol / the hydrophilic phase is removed, such as 30% to 70% (w / w), such as 40% to 60% (w / w) or 45% to 55% (w / w) of the glycerol / the hydrophilic phase.

[0151] In still further embodiments, the oil phase / hydrophobic phase and the hydrophilic phase are subjected to a base treatment to form soaps / salts of the free fatty acids.

[0152] In the method according to the present invention, it is preferred that the separation of the soap raw material fraction containing FFA is promoted, and before the treatment, the oil phase and the hydrophilic phase are rarely separated or not separated when the treatment with an alkaline agent or a base (preferably NaOH or KOH or a mixture thereof) occurs. Therefore, in these embodiments of the present invention, both the oil phase / hydrophobic phase and the hydrophilic phase are subjected to an alkaline treatment to form soap / fatty acid salts of at least a portion of the free fatty acids present in the oil phase / hydrophobic phase. However, this does not exclude that before the treatment with the alkaline agent, the hydrophilic phase is reduced, for example when part of the glycerol / hydrophilic phase is removed in order to be combined with another fatty acid raw material as disclosed above. Specifically, before the treatment with the alkaline agent, the hydrophilic phase can be reduced by 30% to 80% (w / w), for example, by 30% to 70% (w / w), for example, by 40% to 60% (w / w) or by 45% to 55% (w / w).

[0153] The alkaline agent / base can be added as a 1-6N solution in water, such as a 2-9N solution, a 3-9N solution, a 4-9N solution, a 5-9N solution, a 6-9N solution, a 7-9N solution, an 8-9N solution, such as a 3-6N solution, a 4-6N solution, or a 5-6N solution. In some embodiments, a 6-9N solution is preferred because this introduces very little additional water into the system.

[0154] In some embodiments, the amount of base is in the range of 1.0-2.0 molar equivalents of the amount of free fatty acids, such as in the range of 1.05-1,30 molar equivalents or such as 1.05-1.25 molar equivalents of the amount of free fatty acids.

[0155] The alkali treatment can be carried out at a temperature in the range of 35°C to 85°C, for example in the range of 35°C to 80°C, for example 35°C to 75°C, 35°C to 70°C, 35°C to 60°C, 35°C to 50°C, 35°C to 45°C, 35°C to 40°C, 40°C to 80°C, for example 40°C to 75°C, 40°C to 70°C, 40°C to 60°C, 40°C to 50°C, 40°C to 45°C, 45°C to 80°C, for example 45°C to 75°C, 45°C to 70°C, 45°C to 60°C, or for example 45°C to 50°C.

[0156] In general, it is advantageous to operate at as low a temperature as possible or well below the boiling point of the alcohol used, for example at 35° C. to 45° C., no or only little additional heating is required. Thus, the alkaline treatment is preferably carried out at a temperature in the range of 35° C. to 45° C., for example in the range of 36° C. to 45° C., for example in the range of 37° C. to 45° C., 38° C. to 45° C., 39° C. to 45° C. or 40° C. to 45° C. The skilled person will appreciate that the upper temperature limit is primarily defined by the boiling point of the alcohol and further depends on the amount of alcohol present in the system and the efficiency of the scrubbing system installed on the production plant.

[0157] The duration of the alkali treatment may be from 5 minutes to 2 hours, such as from 7 minutes to 2 hours, 10 minutes to 2 hours, 15 minutes to 2 hours, 30 minutes to 2 hours, such as from 30 minutes to 1,5 hours or such as from 30 minutes to 1 hour. In particular, when operating a continuous system, a skilled short duration, such as a few minutes, is possible.

[0158] In another embodiment of the present invention, the alkaline agent / base is added as a non-aqueous solution. Specifically, the alkaline agent / base is dissolved in an alcohol (eg methanol).

[0159] According to other embodiments, the alkaline agent is sodium methoxide or potassium methoxide or a mixture thereof.

[0160] The method according to the present invention may further comprise the step of separating the fatty acid alkyl ester from the soap / fatty acid salt. The separation of the fatty acid alkyl ester from the soap / fatty acid salt is particularly effective when carried out in the method according to the present invention. This is mainly because, before any further processing (e.g., treatment with an alkaline agent to remove free fatty acids) of the fatty acid ester / biodiesel, the separation of the oil phase / hydrophobic phase containing the fatty acid ester / biodiesel from the hydrophilic phase can be avoided. As a result, it is fast to separate the alkyl ester phase from the glycerol / soap phase and no or little formation of a third enzyme emulsion phase containing fatty acid ester / biodiesel, glycerol and enzyme is formed, and the product (fatty acid ester / biodiesel) is rarely lost in the method.

[0161] According to some embodiments of the present invention, additional advantages may be provided by using a large amount or volume of glycerol, which is effective in extracting soaps / fatty acid salts from fatty acid esters / biodiesel.

[0162] The fatty acid alkyl esters may be separated from the soaps / fatty acid salts by gravity settling, decantation and / or centrifugation.

[0163] In some embodiments of the invention, the treatment with the one or more alkaline agents is carried out under conditions and using an amount of alkaline agent that allows the amount of free fatty acids in the oil phase / hydrophobic phase to be reduced to less than 2% (w / w), for example less than 1.5% (w / w), less than 1% (w / w), less than 0.75% (w / w), less than 0.5% (w / w), or for example less than 0.25% (w / w).

[0164] According to a further embodiment of the invention, the method comprises separating a composition comprising fatty acyl esters from the soap / fatty acyl salts, wherein the content of free fatty acids in the composition is lower than 2% (w / w), such as lower than 1.5% (w / w), lower than 1% (w / w), lower than 0.75% (w / w), lower than 0.5% (w / w), or such as lower than 0.25% (w / w) and / or the content of glycerides (preferably expressed as "bound glycerol"; i.e. glycerol bound in glycerides) in the composition is lower than 2% (w / w), such as lower than 1.5% (w / w), lower than 1% (w / w), lower than 0.75% (w / w), lower than 0.5% (w / w), or such as lower than 0.23% (w / w).

[0165] Preferably, the process comprises separating a composition comprising fatty acyl esters from said soaps / fatty acyl salts, wherein the content of free fatty acids in said composition is lower than 0.25% (w / w) and / or the content of glycerides (preferably expressed as "bound glycerol"; i.e. glycerol bound in glycerides) in said composition is lower than 0.23% (w / w). Thus, according to these embodiments, the product of the invention is preferably a product that meets the requirements specified for biodiesel without the need for further refining.

[0166] In further embodiments, the method according to the present invention comprises separating the oily / hydrophobic phase from the hydrophilic phase to provide a composition comprising at least 90% (w / w) fatty acid alkyl esters, from 300 to 400 ppm soap, less than 0.25% (w / w) free fatty acids and less than 0.23% (w / w) glycerides (preferably expressed as "bound glycerol"; i.e., glycerol bound in glycerides).

[0167] The soap / fatty acid salts may be subjected to acidification to produce free fatty acids, for example by contacting the soap / fatty acid salts with HCl, H3PO4 and / or H2SO4. Currently, H2SO4 and possibly H3PO4 are preferred because the resulting salts precipitate easily and can therefore be easily separated from the glycerol to provide a valuable high-grade / industrial-grade glycerol product. A schematic diagram of a process according to the invention comprising the production of industrial-grade glycerol is shown in Figure 1 middle.

[0168] As the skilled person will appreciate, the free fatty acids produced by acidification of the soaps / fatty acid salts may be used as fatty acid feedstock in the process according to the invention as disclosed above.

[0169] The amount of free fatty acids produced by acidification of the soap / fatty acid salt may range from 0.5% to 3% (w / w) of the feedstock, for example from 1% to 2% (w / w) of the feedstock.

[0170] In a specific embodiment, the method according to any one of the present invention comprises the following steps

[0171] i) providing a reaction system comprising an oil phase / hydrophobic phase and a hydrophilic phase; for example, a reaction system wherein the oil phase / hydrophobic phase comprises a fatty acid feedstock (the fatty acid feedstock contains free and / or glyceride-bound fatty acids), and the hydrophilic phase comprises water, one or more esterases, and optionally glycerol;

[0172] ii) adding an alcohol, such as a C1-C5 alcohol, preferably ethanol or methanol, dropwise or continuously to the reaction system to reach an amount ranging from 14% to 24% (w / w) relative to the oil phase / hydrophobic phase;

[0173] iii) subjecting the fatty acid feedstock and the one or more esterifying enzymes to conditions which allow transesterification of the free and / or glyceride-bound fatty acids to provide a composition comprising fatty acid alkyl esters;

[0174] iv) contacting the composition with an alkaline agent / base under conditions which allow the formation of fatty acid salts / soaps from residual free fatty acids in the composition; and

[0175] V) separating the fatty acid alkyl esters from the fatty acid salts / soaps.

[0176] In another embodiment, the method according to the present invention comprises

[0177] i) providing a system comprising said oil phase / hydrophobic phase and said hydrophilic phase,

[0178] ii) reacting the fatty acid feedstock present in the oil phase / hydrophobic phase with an alcohol in the presence of water and one or more esterifying enzymes to produce the fatty acid alkyl esters and the free fatty acids.

[0179] iii) subjecting the oil phase / hydrophobic phase and the hydrophilic phase to an alkali treatment to form soaps / fatty acid salts of the free fatty acids present in the oil phase / hydrophobic phase

[0180] iv) separating the fatty acid alkyl esters from the hydrophilic phase comprising the soap / fatty acid salts

[0181] v) The hydrophilic phase is subjected to acidification, for example by adding H2SO4 or H3PO4.

[0182] In particular, the soap / fatty acid salt may be neutralized / subjected to acidification, for example by adding H2SO4 or H3PO4, to form a salt, such as Na2SO4 or K2SO4, which is precipitated in the glycerol.

[0183] In a further step, the process preferably comprises separating the precipitated salt from the glycerol, for example by filtration.

[0184] In order to provide high-grade or technical-grade glycerol, the process may further comprise drying the glycerol to remove, for example, water and alcohols, such as methanol, from the glycerol.

[0185] In particular, the glycerol can be purified, for example by drying and / or removing alcohol, and / or by filtering to produce a composition in which the content of glycerol is higher than 95% (w / w), for example higher than 97% (w / w), higher than 97.5% (w / w), higher than 98% (w / w), higher than 98.5% (w / w), higher than 99% (w / w), higher than 99.5% (w / w), higher than 99.75% (w / w), higher than 99.8% (w / w) or higher than 99.9% (w / w).

[0186] Additionally, according to these embodiments, the fatty acid raw material can be reacted with an alcohol in the presence of an amount of glycerol corresponding to 2% to 30% (w / w) or 10% to 12% (w / w) relative to the oil phase / hydrophobic phase, an amount of water corresponding to 1.5% to 5.0% (w / w) or 1.5% to 2.0% (w / w) relative to the oil phase / hydrophobic phase, and an amount of alcohol (e.g., methanol) in the range of 14% to 24% (w / w) relative to the oil phase / hydrophobic phase.

[0187] The alcohol may be added over a period of 1 to 30 hours, such as over a period of 1 to 20 hours, such as over a period of 16 hours.

[0188] In certain specific embodiments, the fatty acid feedstock comprises or consists essentially of corn oil or distillers corn oil.

[0189] In another aspect, the present invention provides a method for producing fatty acid alkyl esters, the method comprising contacting a fatty acid feedstock with an esterase; the esterase is selected from the group consisting of

[0190] (a) a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2;

[0191] (b) a polypeptide that is a subsequence of the amino acid sequence set forth in SEQ ID NO: 2;

[0192] (c) a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 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% sequence identity to any of the polypeptides defined in (a) and (b).

[0193] The lipase as described in item (c) may have an amino acid sequence that differs from the polypeptide of SEQ ID NO: 2 in up to 40 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

[0194] The lipase can be a variant of a parent lipase, which has lipase activity and has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1, and includes a substitution at a position corresponding to T231R+N233R and at least one or more (e.g., several) of D96E, D111A, D254S, G163K, P256T, G91T, G38A, D27R, and N33Q of SEQ ID NO: 2.

[0195] In another embodiment, the lipase is a variant having lipase activity and having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1, and comprising substitutions at positions corresponding to T231R+N233R and at least one or more (e.g., several) of D96E, D111A, D254S, G163K, P256T, G91T, G38A, D27R, and N33Q of SEQ ID NO: 2, selected from the group consisting of:

[0196] a)D96E T231R N233R; <h2 style=";text-align:left;direction:ltr">

[0197] <h2 style=";text-align:left;direction:ltr"> b)N33Q D96E T231R N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0198] <h2 style=";text-align:left;direction:ltr"> c)N33Q T231R N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0199] <h2 style=";text-align:left;direction:ltr"> d)N33Q D111A T231R N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0200] <h2 style=";text-align:left;direction:ltr"> e)N33Q T231R N233R P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0201] <h2 style=";text-align:left;direction:ltr"> f)N33Q G38A G91T G163K T231R N233R D254S;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0202] <h2 style=";text-align:left;direction:ltr"> g)N33Q G38A G91T D96E D111A G163K T231R N233R D254S P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0203] <h2 style=";text-align:left;direction:ltr"> h)D27R N33Q G38A D96E D111A G163K T231R N233R D254S P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0204] <h2 style=";text-align:left;direction:ltr"> i)D27R N33Q G38A G91T D96E D111A G163K T231R N233R P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0205] <h2 style=";text-align:left;direction:ltr"> j)D27R N33Q G38A G91T D96E D111A G163K T231R N233R D254S;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0206] <h2 style=";text-align:left;direction:ltr"> k)D27R G38A G91T D96E D111A G163K T231R N233R D254S P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0207] <h2 style=";text-align:left;direction:ltr"> l)D96E T231R N233R D254S;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0208] <h2 style=";text-align:left;direction:ltr"> m)T231R N233R D254S P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0209] <h2 style=";text-align:left;direction:ltr"> n)G163K T231R N233R D254S;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0210] <h2 style=";text-align:left;direction:ltr"> o)D27R N33Q G38A G91T D96E G163K T231R N233R D254S P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0211] <h2 style=";text-align:left;direction:ltr"> p)D27R G91T D96E D111A G163K T231R N233R D254S P256T;<h2 style=";text-align:left;direction:ltr">

[0212] q)D96E G163K T231R N233R D254S;

[0213] r)D27R G163K T231R N233R D254S;

[0214] s)D27R G38A G91T D96E D111A G163K T231R N233R D254S;

[0215] t)D27R G38A G91T D96E G163K T231R N233R D254S P256T;

[0216] u)D27R G38A D96E D111A G163K T231R N233R D254S P256T;

[0217] v)D27R D96E G163K T231R N233R D254S;

[0218] w)D27R D96E D111A G163K T231R N233R D254S P256T;

[0219] x)D27R G38A D96E G163K T231R N233R D254S P256T。

[0220] In a specific embodiment, the method comprises contacting the fatty acid raw material with the following amount of lipase, which amount is preferably in the range of 0.005-5g enzyme protein (EP) / kg fatty acid raw material, for example, 0.005-2.5g EP / kg fatty acid raw material, 0.005-1g EP / kg fatty acid raw material, 0.005-0.75g EP / kg fatty acid raw material, 0.005-0.5g EP / kg fatty acid raw material, 0.005-0.25g EP / kg fatty acid raw material, 0.005-0.1g EP / kg fatty acid raw material, 0.005-0.075g EP / kg fatty acid raw material, 0.005-0.05g EP / kg fatty acid raw material, 0.005-0.025g EP / kg fatty acid raw material, 0.005-0.01g EP / kg fatty acid raw material, 0.01-5g EP / kg fatty acid raw material, 0.02-5g EP / kg fatty acid raw material. EP / kg fatty acid raw material, 0.03-5g EP / kg fatty acid raw material, 0.04-5g EP / kg fatty acid raw material, 0.05-5g EP / kg fatty acid raw material, 0.06-5g EP / kg fatty acid raw material, 0.07-5g EP / kg fatty acid raw material, 0.08-5g EP / kg fatty acid raw material, 0.09-5g EP / kg fatty acid raw material, 0.1-5g EP / kg fatty acid raw material, 0.2-5g EP / kg fatty acid raw material, 0.3-5g EP / kg fatty acid raw material, 0.4-5g EP / kg fatty acid raw material, 0.5-5g EP / kg fatty acid raw material, 0.6-5g EP / kg fatty acid raw material, 0.7-5g EP / kg fatty acid raw material, 0.8-5g EP / kg fatty acid raw material, 0.9-5g EP / kg fatty acid raw material, 1-5g EP / kg fatty acid raw material, 2-5g EP / kg fatty acid raw material, 3-5g In the present invention, the present invention relates to a fatty acid raw material comprising: a fatty acid raw material, ...

[0221] Fatty acid alkyl ester composition and use thereof

[0222] Fatty acid alkyl esters are used in a wide range of products and as synthetic intermediates. Some of their industrial applications include being used as lubricants, plasticizers, rust inhibitors, drilling and cutting oils and as starting materials for synthesizing superamides and fatty alcohols. Some embodiments of the invention specifically relate to fuels. Due to the similarity with petroleum-based fuels in cetane number, energy content, viscosity and phase transition, the fatty acid alkyl esters of short-chain alcohols are nontoxic, biodegradable and are excellent all or part of substitutes for petroleum-based fuels.

[0223] Another aspect of the present invention relates to the following composition, which comprises at least 90% (w / w) fatty acid alkyl esters, from 300 to 400 ppm soap, less than 0.25% (w / w) free fatty acids and less than 0.23% (w / w) glycerides (preferably expressed as "bound glycerol"; that is, glycerol bound to glycerides). In terms of free fatty acids and glycerides, the product meets the specified requirements for biodiesel. For example, these specifications are provided by ASTM International (Specification for biodiesel (B100)-ASTM 6751) (available at http: / / www.astm.org / Standards / ). Compared with the soap content in the product from the previously known method, the soap content is 5-10x low; this content can be removed or reduced to an acceptable level by filtering. Specifically, the composition can be a composition obtainable by the procedure disclosed above.

[0224] In some embodiments, the composition according to the present invention is obtainable by a process wherein the fatty acid feedstock comprises corn oil or consists essentially of it, and the alkaline agent is KOH. The inventors unexpectedly discovered that when corn oil is used as a feedstock, the process provides a product that typically does not require further processing or purification and is nearly colorless.

[0225] In other aspects, the compositions according to the present invention can potentially be refined or purified by methods known in the art (e.g., distillation (including flash evaporation, stripping, and deodorization); phase separation; extraction; and drying). The purpose of such refining can be to remove or recover one or more of the above-mentioned components from the composition. Examples include, but are not limited to, drying for removing water. Thus, the crude reactant mixture (composition) can be used without further refining or by refining by one or more methods.

[0226] A further aspect of the invention provides a composition comprising glycerol, said composition being obtainable by a process according to the invention, wherein said soap / fatty acid salt is subjected to acidification to produce free fatty acids and salts, such as Na2SO4 or K2SO4, by contacting the soap / fatty acid salt with H3PO4 and / or H2SO4. As mentioned above, acidification of the soap / fatty acid salt with these acids results in the formation of salts which are easily precipitated and which can therefore be easily separated from the glycerol to provide a valuable high grade / technical grade glycerol product. As in Figure 1 As shown in the method for preparing high-grade / industrial-grade ethanol, the salt can be removed by filtering or other methods. The method may further include drying and / or reducing the amount of alcohol (e.g., methanol) in the glycerol. The composition obtainable according to the present invention can have a glycerol content higher than 97% (w / w), such as higher than 97.5% (w / w), higher than 98% (w / w), higher than 98.5% (w / w), higher than 99% (w / w), higher than 99.5% (w / w), higher than 99.75% (w / w), higher than 99.8% (w / w) or higher than 99.9% (w / w).

[0227] project

[0228] The present invention is further disclosed in and defined by any of the following items:

[0229] 1. A method for producing fatty acid alkyl esters, the method comprising: in a reaction system comprising an oil phase / hydrophobic phase and a hydrophilic phase, in the presence of one or more esterases, reacting a fatty acid raw material with an alcohol to produce fatty acid alkyl esters and glycerol, wherein in the presence of the alcohol / the hydrophilic phase, soaps / salts are formed from free fatty acids in the oil phase / hydrophobic phase by treating with one or more alkaline agents.

[0230] 2. A method for producing fatty acid alkyl esters, the method comprising: in a reaction system comprising an oil phase / hydrophobic phase and a hydrophilic phase, in the presence of water and one or more esterases, reacting a fatty acid raw material with an alcohol to produce fatty acid alkyl esters and glycerol; and before separating the oil phase / hydrophobic phase from the hydrophilic phase, removing or reducing the amount of free fatty acids by treating with one or more alkaline reagents to form soaps / salts of free fatty acids.

[0231] 3. The method according to item 1 or 2, comprising:

[0232] i) providing a system comprising said oil phase / hydrophobic phase and said hydrophilic phase,

[0233] ii) reacting the fatty acid feedstock present in the oil phase / hydrophobic phase with an alcohol in the presence of water and one or more esterifying enzymes to produce the fatty acid alkyl esters, free fatty acids and glycerol.

[0234] 4. The method according to any one of the above items, comprising separating the fatty acid alkyl esters from the hydrophilic phase comprising the soap / salt and the one or more esterolytic enzymes.

[0235] 5. The method according to any one of the above items, wherein the one or more esterases are selected from lipases, phospholipases, cutinases and mixtures thereof.

[0236] 6. The method according to any one of the above items, comprising:

[0237] i) providing a reaction system having an oil phase / hydrophobic phase comprising a fatty acid feedstock, and a hydrophilic phase comprising an alcohol, water and one or more esterifying enzymes;

[0238] ii) reacting a fatty acid feedstock with an alcohol in the presence of said water and said one or more esterifying enzymes to produce free fatty acids, glycerol, and fatty acid alkyl esters;

[0239] iii) adding one or more alkaline reagents to the reaction system to allow the formation of soaps / salts of the free fatty acids produced in step ii); and

[0240] iv) separating the fatty acid alkyl esters from the soaps / salts of the fatty acids and one or more esterifying enzymes.

[0241] 7. The method according to any of the above items, wherein the one or more esterases are one or more lipases, optionally in combination with one or more phospholipases and / or one or more cutinases.

[0242] 8. The method according to any one of the above items, wherein the total amount of the one or more esterases is in the range of 0.005-5 g enzyme protein (EP) / kg oil phase / hydrophobic phase or fatty acid raw material, for example, 0.005-2.5 g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-1 g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.75 g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.5 g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.25 g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.1 g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.075 g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.05 g EP / kg oil phase / hydrophobic phase or fatty acid raw material. EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.025g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.005-0.01g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.01-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.02-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.03-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.04-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.05-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.06-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.07-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.08-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.09-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.1-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.2-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.3-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.4-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.5-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.6-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.7-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.8-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.9-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 1-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 2-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 3-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 4-5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.01-4g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.02-3g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.03-2g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.0.04-1g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.05-0.9g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.06-0.8g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.07-0.7g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.08-0.6g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.09-0.5g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.1-0.4g EP / kg oil phase / hydrophobic phase or fatty acid raw material, 0.1-0.3g EP / kg oil phase / hydrophobic phase or fatty acid raw material, or for example in the range of 0.1-0.25g EP / kg oil phase / hydrophobic phase or fatty acid raw material. .

[0243] 9. A method according to any of the above items, wherein step ii) comprises reacting the fatty acid raw material with the alcohol until at least 60% (w / w) (e.g., at least 65% (w / w), at least 70% (w / w), at least 75% (w / w), at least 80% (w / w), at least 85% (w / w), at least 90% (w / w), or for example, at least 95% (w / w)) of the fatty acid acyl groups or free fatty acids in the fatty acid raw material have been converted into fatty acid alkyl esters. 10. A method according to any of the above items, wherein the amount of water corresponds to 0.5% to 5.0% (w / w) of the oil phase / hydrophobic phase, for example 0.5% to 4.0% (w / w) of the oil phase / hydrophobic phase, 0.5% to 3.0% (w / w) of the oil phase / hydrophobic phase, 0.5% to 2.5% (w / w) of the oil phase / hydrophobic phase or for example 0.5% to 2.0% (w / w) of the oil phase / hydrophobic phase.

[0244] 11. A method according to any of the above items, comprising increasing the amount of glycerol in the hydrophilic phase to 2% to 30% (w / w), preferably from 2% to 20% (w / w), for example from 2% to 20% (w / w) relative to the oil phase / hydrophobic phase, by reaction of the fatty acid raw material and optionally by further addition of glycerol.

[0245] 12. A method according to any one of the above items, wherein the alcohol is added to reach an amount in the range of 12% to 34% (w / w) relative to the oil phase / hydrophobic phase, for example in the range of 17% to 34% (w / w) relative to the oil phase / hydrophobic phase, or for example in the range of 12% to 24% (w / w) relative to the oil phase / hydrophobic phase.

[0246] 13. The method according to any one of the above items, wherein the fatty acid raw material is reacted with the alcohol at a temperature in the range of 32°C to 45°C, preferably in the range of 32°C to 40°C.

[0247] 14. The method according to any one of the above items comprises reacting the fatty acid feedstock with the alcohol for 16-50 hours, for example for 24-50 hours, for example for 30-50 hours, for 30-45 hours, for 35-50 hours, for 35-45 hours, for 38-42 hours, preferably for 35-40 hours.

[0248] 15. The process according to any one of the above items, wherein the process is carried out in batch mode or in continuous mode.

[0249] 16. The method according to any one of the above items, wherein the alcohol is a C1-C5 alcohol, preferably ethanol or methanol.

[0250] 17. The method according to any one of the above items, wherein the alcohol is added dropwise or continuously.

[0251] 18. The method according to any of the above items, wherein at least one of the one or more esterases is a liquid enzyme.

[0252] 19. The method according to any one of the above items, wherein the fatty acid raw material is derived from one or more of the following: algae oil, canola oil, coconut oil, castor oil, coconut oil, copra oil, corn oil, corn oil, cottonseed oil, linseed oil, fish oil, grapeseed oil, hemp oil, jatropha oil, jojoba oil, mustard oil, canola oil, palm oil, palm stearin, palm olein, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, soybean oil, sunflower oil, tall oil, oil from halophytes and / or animal fats (including fats from pigs, cattle and sheep, lard, chicken fat, fish oil, palm oil free fatty acid distillate, soybean oil free fatty acid distillate, soap stock fatty acid material, yellow grease, and brown grease or any combination thereof).

[0253] 20. The method according to any of the preceding items, wherein the solution phase in the system / reaction system is mixed using a high shear mixer or a cavitator.

[0254] 21. The method according to any of the above items, wherein an alkaline agent or base, such as NaOH and / or KOH, is added to the system before adding the one or more esterolytic enzymes.

[0255] 22. The method according to item 21, wherein the alkaline agent or base is added in an amount corresponding to 200 ppm or less, preferably in an amount in the range of 10-100 ppm.

[0256] 23. A method according to any one of the above items, wherein

[0257] The oil phase / hydrophobic phase comprises fatty acid alkyl esters, free fatty acids and optionally unreacted fatty acid feedstock; and

[0258] The hydrophilic phase comprises glycerol, alcohol, water and the one or more esterifying enzymes.

[0259] 24. A method according to any one of the above items, wherein

[0260] The hydrophilic phase constitutes from 5% to 50%, 10% to 50%, 20% to 50%, 20% to 45%, or even 20% to 40% of the system / reaction system (w / w), and

[0261] Glycerol constitutes 30% to 85% (w / w), 40% to 85% (w / w), 45% to 85% (w / w), 50% to 85% (w / w) or even 60% to 80% (w / w) of the hydrophilic phase.

[0262] 25. The method according to any of the above items, wherein glycerol constitutes 30% to 80% (w / w), 30% to 70% (w / w), 35% to 70% (w / w), 40% to 70% (w / w) or even 45% to 70% (w / w) of the hydrophilic phase.

[0263] 26. The method according to any one of the above items, comprising

[0264] i) removing at least a portion of the glycerol / hydrophilic phase from the system / reaction system during the reaction of the fatty acid feedstock with the alcohol or when the reaction has terminated; and

[0265] ii) In a process as defined in any of the above items, the process is combined with an additional fatty acid feedstock, wherein the process is the same as or different from the process of removal of the glycerol / said hydrophilic phase.

[0266] 27. The method according to item 26, wherein 30% to 70% (w / w) of the glycerol / the hydrophilic phase is removed, for example 40% to 60% (w / w) or 45% to 55% (w / w) of the glycerol / the hydrophilic phase.

[0267] 28. The method according to any one of the above items, wherein the oil phase / hydrophobic phase and the hydrophilic phase are subjected to a base treatment to form soaps / salts of the free fatty acids.

[0268] 29. The method according to any of the preceding items, wherein the oil phase / hydrophobic phase and the hydrophilic phase are subjected to an alkali treatment to form soaps / fatty acid salts of at least a portion of the free fatty acids present in the oil phase / hydrophobic phase.

[0269] 30. The method according to any one of items 28 to 29, wherein the alkaline treatment comprises contacting the oil phase / hydrophobic phase and the hydrophilic phase with an alkaline agent or base selected from KOH or NaOH or a mixture thereof.

[0270] 31. A method according to any one of items 28 to 30, wherein the alkaline agent / base is added as a 1-6N solution in water, such as a 2-9N solution, a 3-9N solution, a 4-9N solution, a 5-9N solution, a 6-9N solution, a 7-9N solution, an 8-9N solution, such as a 3-6N solution, a 4-6N solution or a 5-6N solution.

[0271] 32. The process according to any one of items 28 to 31, wherein the amount of base is in the range of 1.0-2.0 molar equivalents of the amount of free fatty acids, such as in the range of 1.05-1,30 molar equivalents or such as 1.05-1.25 molar equivalents of the amount of free fatty acids.

[0272] 33. The method according to any one of items 28 to 32, wherein the alkaline treatment is carried out at a temperature in the range of 35°C to 85°C, such as in the range of 35°C to 80°C.

[0273] 34. A method according to any one of items 28 to 33, wherein the duration of the alkali treatment is from 30 seconds to 2 hours, for example from 30 seconds to 1 hour, from 30 seconds to 30 minutes, from 1 to 30 minutes, from 2-30 minutes, from 3-30 minutes, from 5 minutes to 2 hours, for example from 7 minutes to 2 hours, 10 minutes to 2 hours, 15 minutes to 2 hours, 30 minutes to 2 hours, for example from 30 minutes to 1.5 hours or for example from 30 minutes to 1 hour.

[0274] 35. The method according to any one of items 28 to 34, wherein the alkaline agent / base is added as a non-aqueous solution.

[0275] 36. A process according to any one of items 28 to 35, wherein the alkaline agent / base is dissolved in an alcohol, such as methanol.

[0276] 37. The method according to any one of items 28 to 34, wherein the alkaline agent is sodium methoxide or potassium methoxide or a mixture of the two.

[0277] 38. The process according to any one of items 28 to 37, wherein the fatty acid alkyl esters are separated from the soaps / fatty acid salts.

[0278] 39. The process according to any one of items 28 to 38, wherein the fatty acid alkyl esters are separated from the soaps / fatty acid salts by gravity settling, decantation and / or centrifugation.

[0279] 40. The method according to any one of items 28 to 39, wherein the treatment with the one or more alkaline agents is carried out under conditions and using an amount of alkaline agent that allows the amount of free fatty acids in the oil phase / hydrophobic phase to be reduced to less than 2% (w / w), such as less than 1.5% (w / w), less than 1% (w / w), less than 0.75% (w / w), less than 0.5% (w / w), or such as less than 0.25% (w / w). 41. The method according to any one of items 28 to 39, which comprises separating a composition comprising fatty acyl esters from the soap / fatty acyl salts, wherein the content of free fatty acids in the composition is less than 0.25% (w / w) and / or the content of glycerides in the composition is less than 0.23% (w / w).

[0280] 42. The method according to any one of the above items, which comprises separating a composition comprising fatty acyl esters from the soap / fatty acyl salts, wherein the content of free fatty acids in the composition is lower than 2% (w / w), such as lower than 1.5% (w / w), lower than 1% (w / w), lower than 0.75% (w / w), lower than 0.5% (w / w), or such as lower than 0.25% (w / w) and / or the content of glycerides (preferably expressed as "bound glycerol"; i.e. glycerol bound in glycerides) in the composition is lower than 2% (w / w), such as lower than 1.5% (w / w), lower than 1% (w / w), lower than 0.75% (w / w), lower than 0.5% (w / w), or such as lower than 0.23% (w / w).

[0281] 43. The method according to any of the preceding items, comprising separating a composition comprising fatty acyl esters from said soaps / fatty acyl salts, wherein the content of free fatty acids in said composition is lower than 0.25% (w / w) and / or the content of glycerides (preferably expressed as "bound glycerol"; i.e. glycerol bound in glycerides) in said composition is lower than 0.23% (w / w). Thus, according to these embodiments, the product of the invention is preferably a product that meets the requirements specified for biodiesel without the need for further refining.

[0282] 44. The method according to any one of the above items, comprising separating the oil phase / hydrophobic phase from the hydrophilic phase to provide a composition comprising at least 90% (w / w) fatty acid alkyl esters, from 300 to 400 ppm soap, less than 0.25% (w / w) free fatty acids and less than 0.23% (w / w) glycerides. 45. The method according to any one of items 28 to 44, wherein the soap / fatty acid salt is subjected to acidification to produce free fatty acids, for example by contacting the soap / fatty acid salt with HCl, H3PO4 and / or H2SO4.

[0283] 46. ​​The process according to any one of items 41 to 45, wherein free fatty acids produced by acidification of the soap / fatty acid salt can be used as fatty acid raw material in the process according to any one of the above items.

[0284] 47. A method according to any one of items 41 to 46, wherein the amount of free fatty acids produced by acidification of the soap / fatty acid salt is in the range of 0.5% to 3% (w / w) of the raw material, for example from 1% to 2% (w / w) of the raw material.

[0285] 48. The method according to any one of the above items, comprising the following steps

[0286] i) providing a reaction system comprising an oil phase / hydrophobic phase and a hydrophilic phase; for example, a reaction system wherein the oil phase / hydrophobic phase comprises a fatty acid feedstock (the fatty acid feedstock contains free and / or glyceride-bound fatty acids), and the hydrophilic phase comprises water, one or more esterases, and optionally glycerol;

[0287] ii) adding an alcohol, such as a C1-C5 alcohol, preferably ethanol or methanol, dropwise or continuously to the reaction system to reach an amount ranging from 14% to 24% (w / w) relative to the oil phase / hydrophobic phase;

[0288] iii) subjecting the fatty acid feedstock and the one or more esterifying enzymes to conditions which allow transesterification of the free and / or glyceride-bound fatty acids to provide a composition comprising fatty acid alkyl esters;

[0289] iv) contacting the composition with an alkaline agent / base under conditions which allow the formation of fatty acid salts / soaps from residual free fatty acids in the composition; and

[0290] V) separating the fatty acid alkyl esters from the fatty acid salts / soaps.

[0291] 49. The method according to any one of the above items, comprising:

[0292] i) providing a system comprising said oil phase / hydrophobic phase and said hydrophilic phase,

[0293] ii) reacting the fatty acid feedstock present in the oil phase / hydrophobic phase with an alcohol in the presence of water and one or more esterifying enzymes to produce the fatty acid alkyl esters and the free fatty acids.

[0294] iii) subjecting the oil phase / hydrophobic phase and the hydrophilic phase to an alkali treatment to form soaps / fatty acid salts of the free fatty acids present in the oil phase / hydrophobic phase

[0295] iv) separating the fatty acid alkyl esters from the hydrophilic phase comprising the soap / fatty acid salts

[0296] v) The hydrophilic phase is subjected to acidification, for example by adding H2SO4.

[0297] 50. The method according to any of the above items, wherein the soap / fatty acid salt is neutralized / subjected to acidification, such as by adding H2SO4 or H3PO4, to form a salt, such as Na2SO4 or K2SO4, which is precipitated in glycerol.

[0298] 51. A method according to claim 50, comprising separating the precipitated salt from the glycerol, for example by filtration.

[0299] 52. The method according to any of the preceding items, comprising drying the glycerol to remove, for example, water and alcohols, such as methanol, from the glycerol.

[0300] 53. A method according to any one of items 50 to 52, wherein the glycerol is purified, for example by drying and / or removing alcohol, and / or by filtering to produce a composition in which the content of glycerol is higher than 95% (w / w), for example higher than 97% (w / w), higher than 97.5% (w / w), higher than 98% (w / w), higher than 98.5% (w / w), higher than 99% (w / w), higher than 99.5% (w / w), higher than 99.75% (w / w), higher than 99.8% (w / w) or higher than 99.9% (w / w). 47. A method according to any one of the above items, wherein the fatty acid raw material is reacted with alcohol in the presence of an amount of glycerol corresponding to 2% to 30% (w / w) relative to the oil phase / hydrophobic phase, an amount of water corresponding to 1.5% to 5.0% (w / w) relative to the oil phase / hydrophobic phase, and an amount of alcohol (such as methanol) in the range of 14% to 24% (w / w) relative to the oil phase / hydrophobic phase.

[0301] 54. The method according to any of the above items, wherein the alcohol is added over a period of 1 to 30 hours, such as over a period of 1 to 20 hours, such as over a period of 16 hours.

[0302] 55. A method according to any one of the above items, wherein the fatty acid feedstock comprises or consists mainly of corn oil or distiller's corn oil.

[0303] 56. A composition comprising at least 90% (w / w) fatty acid alkyl esters, from 300 to 400 ppm soap, less than 0.25% (w / w) free fatty acids and less than 0.23% (w / w) glycerides.

[0304] 57. The composition according to item 50, which is obtainable by a method according to any one of items 1 to 55.

[0305] 58. The composition according to any one of items 55 to 57, which is obtainable by the following method, wherein the fatty acid feedstock comprises or mainly consists of corn oil, and the alkaline reagent is KOH.

[0306] 59. A composition comprising glycerol, said composition being obtainable by a process as defined in any one of items 1 to 46, wherein the soap / fatty acid salt is subjected to acidification to produce free fatty acids and salts, such as Na2SO4 or K2SO4, by contacting the soap / fatty acid salt with H3PO4 and / or H2SO4.

[0307] 60. The composition of claim 59, wherein the process comprises separating the salt from the glycerol, such as by filtration.

[0308] 61. A composition according to claim 59 or 60, wherein the process comprises drying and / or reducing the amount of alcohol, such as methanol, in the glycerol. 56. A composition according to item 55, wherein the glycerol content is higher than 95% (w / w), such as higher than 97% (w / w), higher than 97.5% (w / w), higher than 98% (w / w), higher than 98.5% (w / w), higher than 99% (w / w), higher than 99.5% (w / w), higher than 99.75% (w / w), higher than 99.8% (w / w) or higher than 99.9% (w / w).

[0309] The present invention is further described by the following examples, which should not be construed as limiting the scope of the present invention.

[0310] Example 1

[0311] Fatty acid methyl esters (FAME) were produced using transesterification of distillers corn oil using a low dosage of 300 LU of liquid lipase (SEQ ID NO: 1) per gram of oil.

[0312] In this experiment, glycerol and water concentrations were varied (see Table 1).

[0313] Table 1:

[0314]

[0315] *% w / w based on oil dosage

[0316] A 100 ml square glass flask was used as the reactor and the procedure used was as follows:

[0317] After addition of 32 g of oil, 100 ppm of NaOH dissolved in demineralized water are added. The mixture is mixed for 10 minutes at 35°C and 250 rpm, followed by addition of glycerol and liquid lipase. The reaction is started by addition of 0,70 g of methanol, followed by continuous dosing of 4,17 g of methanol over 16 hours.

[0318] The reaction was carried out in an Innova incubator at 35°C and 250 rpm. The reaction time per batch was 24 hours.

[0319] The yield of fatty acid methyl esters in the oil / hydrophobic phase was quantified by gas chromatography and the content of free fatty acids was measured by NaOH titration.

[0320] Table 2:

[0321] test %FAME@24h %FFA@24h Reference 84.8 2.2 Low Water 78.6 2.1 High Glycerin 89.1 1.3 High glycerin and low water 86.4 1.2 Medium water 81.4 2.1 Medium Glycerin 84.5 1.6

[0322] The highest yield expressed as total FAME and the lowest possible free fatty acid content was achieved by adding 10% glycerol and 1.5%-2.0% water (w / w of oil).

[0323] Example 2

[0324] Fatty acid methyl esters (FAME) were produced using transesterification of distillers corn oil using a low dosage of 300 LU liquid lipase per gram of oil. These enzyme reactions were performed in a 1 L stirred jacketed glass reactor at 35°C and 530 rpm for 24 hours.

[0325] The procedure used was as follows:

[0326] 700 g of oil was added to 1.7 g of 1 N NaOH and 8.75 g of demineralized water. The mixture was mixed at 35° C. and 530 rpm for 10 minutes, followed by the addition of 70.0 g of glycerol and 300 LU of liquid lipase (SEQ ID NO: 1) per gram of oil. The reaction was started by adding 19.2 g of methanol, followed by continuous dosing of 115.0 g of methanol over 16 hours.

[0327] The yield of fatty acid methyl esters and the content of bound glycerol in the oil / hydrophobic phase were quantified by gas chromatography and the content of free fatty acids was measured by NaOH titration.

[0328] Table 3:

[0329]

[0330] At 530 rpm, the mixture was heated to 60° C. and 28 ml of 3N KOH were added continuously over 3 minutes. The temperature was then raised to 82° C. for 2 hours. Samples of the mixture were taken out after 1 hour and 2 hours and centrifuged at 2000 rpm for 10 minutes and the oil phase / hydrophobic phase was analyzed for the content of fatty acid methyl esters, free fatty acids and bound glycerol.

[0331] Table 4:

[0332]

[0333] The heat and agitation were turned off and the mixture was allowed to stand for 1 hour before the levels of fatty acid methyl esters, free fatty acids, bound glycerol and soap were measured in the oil / hydrophobic phase.

[0334] Table 5:

[0335]

[0336] Example 3

[0337] The lipase of SEQ ID NO:2 was used in a large-scale trial to test the application for biodiesel. The lipase described in SEQ ID NO:1 was used at two levels of dosage and compared with standard production. Production was carried out in a 10,000 gal reactor and four batches were made for each series of tests. Batch number one, 30 gal of the formulated enzyme with 181 LCLU / g activity was added; corresponding to 17 mg enzyme protein / 100 g oil. No additional enzyme was added in the following batches B, C, and D. During the first 10 hours, methanol was added in an amount that maintained the concentration of methanol in the heavy phase at about 15%. This addition corresponds to 1.5 molar equivalents of fatty acids in batch A and is slightly reduced in successive batches (about 1.2 equivalents). After all batches, mixing was stopped and the heavy phase was left to stand at the bottom of the kettle for resuspension in the next batch. Only from batch B, about 650 gal of the heavy phase was discharged from the bottom of the kettle after the reaction and left to stand. Using the lipase of SEQ ID NO: 2, for these experiments a higher temperature was maintained to take advantage of the higher thermotolerance of this enzyme.

[0338] Table 6 Addition of the first 30 gal enzyme (SEQ ID NO: 2)

[0339]

[0340]

[0341] Table 7 Addition of 20 gal enzyme (SEQ ID NO: 2)

[0342]

[0343] Table 8 50 gal SEQ ID NO: 1 control, 5 gal added in batches B, C, and D

[0344]

Claims

1. A method for producing fatty acid alkyl esters, comprising the following steps: i) providing a reaction system comprising an oil phase / hydrophobic phase and a hydrophilic phase, ii) reacting a fatty acid feedstock with methanol in the oil phase / hydrophobic phase in the presence of water and one or more esterifying enzymes to produce the fatty acid alkyl esters and the fatty acids and glycerol, iii) subjecting the oil phase / hydrophobic phase and the hydrophilic phase to an alkali treatment to form soaps / fatty acid salts of the free fatty acids present in the oil phase / hydrophobic phase; iv) separating the fatty acid alkyl esters from the hydrophilic phase containing the soap / fatty acid salt; v) subjecting the soap / fatty acid salt to acidification by contacting the soap / fatty acid salt with H3PO4 and / or H2SO4 to form free fatty acids and salts, wherein the salts are precipitated in glycerol; vi) purifying said glycerol by drying to remove water and alcohol and by filtering to remove said salt formed in iii) to produce a composition wherein the glycerol content is greater than 95% by weight; and vii) separating the fatty acid alkyl esters from the hydrophilic phase comprising the soap / salt and the one or more esterifying enzymes.

2. The method according to claim 1, wherein the alkaline agent is added in an amount corresponding to 1.0-2.0 molar equivalents of free fatty acids.

3. The process of claim 1, comprising reacting a fatty acid feedstock with the methanol until at least 60% by weight of the fatty acid acyl groups or free fatty acids in the fatty acid feedstock have been converted to fatty acid alkyl esters.

4. The method according to any one of claims 1 to 3, wherein the total amount of the one or more esterases is in the range of 0.005-5 g enzyme protein / kg oil or fatty acid feedstock.

5. The method according to any one of claims 1 to 3, wherein the fatty acid feedstock is derived from one or more of the following: algae oil; canola oil; castor oil; coconut oil; corn oil; distillers corn oil; cottonseed oil; linseed oil; grapeseed oil; hemp oil; jatropha oil; jojoba oil; mustard 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; palm oil free fatty acid distillate; and soybean oil free fatty acid distillate or any combination thereof.

6. The process according to any one of claims 1 to 3, wherein the fatty acid feedstock is derived from oils from halophytes and / or animal fats.

7. The method according to claim 6, wherein the animal fat comprises grease from pigs, cattle and sheep, chicken fat, fish oil.

8. The method according to any one of claims 1 to 3, wherein the alkali treatment is carried out at a temperature in the range of 35°C to 45°C.

9. The process according to any one of claims 1 to 3, wherein the fatty acid raw material is reacted with an alcohol in the presence of glycerol in an amount corresponding to 2% to 30% by weight relative to the oil phase / hydrophobic phase, water in an amount corresponding to 1.5% to 5.0% by weight relative to the oil phase / hydrophobic phase, and methanol in an amount ranging from 14% to 24% by weight relative to the oil phase / hydrophobic phase.

10. The method according to any one of claims 1-3, wherein the fatty acid raw material is derived from a soap stock fatty acid material.

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