U.S. patent application number 11/813121 was filed with the patent office on 2008-05-15 for production of fatty acid alkyl esters by use of two lipolytic enzymes.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Masanobu Abo, Morten Wurtz Christensen.
Application Number | 20080113419 11/813121 |
Document ID | / |
Family ID | 36572405 |
Filed Date | 2008-05-15 |
United States Patent
Application |
20080113419 |
Kind Code |
A1 |
Abo; Masanobu ; et
al. |
May 15, 2008 |
Production of Fatty Acid Alkyl Esters by Use of Two Lipolytic
Enzymes
Abstract
A method for producing fatty acid alkyl esters, wherein a
solution comprising triglyceride and alcohol is contacted with a
first lipolytic enzyme having a relatively higher activity on free
fatty acids than on triglyceride and a second lipolytic enzyme
having a relatively higher activity on triglyceride than on free
fatty acids.
Inventors: |
Abo; Masanobu; (Chiba-ken,
JP) ; Christensen; Morten Wurtz; (Raleigh,
NC) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE, SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
36572405 |
Appl. No.: |
11/813121 |
Filed: |
January 10, 2006 |
PCT Filed: |
January 10, 2006 |
PCT NO: |
PCT/DK06/00016 |
371 Date: |
June 29, 2007 |
Current U.S.
Class: |
435/134 |
Current CPC
Class: |
Y02E 50/10 20130101;
Y02E 50/13 20130101; C12P 7/649 20130101 |
Class at
Publication: |
435/134 |
International
Class: |
C12P 7/64 20060101
C12P007/64 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 10, 2005 |
DK |
PA 2005 00041 |
Claims
1-15. (canceled)
16. A method for producing fatty acid alkyl esters, comprising
making a reaction mixture by contacting a solution comprising a
substrate and an alcohol, which substrate comprises triglyceride,
with a first lipolytic enzyme having a ratio of activity on
triglyceride to activity on FFA below 0.2 and a second lipolytic
enzyme having a ratio of activity on triglyceride to activity on
FFA above 0.5.
17. The method of claim 16, wherein the substrate further comprises
free fatty acids in the range of 0.01-95% per weight free fatty
acids.
18. The method of claim 16, wherein the triglyceride is derived
from one or more of vegetable oil feedstock, rapeseed oil, soybean
oil, mustard oil, sunflower oil, canola oil, coconut oil, hemp oil,
palm oil, tall oil, animal fats including tallow, lard, poultry and
fish oil.
19. The method of claim 16, wherein the molar ratio between alcohol
and fatty acid residues is least 0.1 and maximum 10.
20. The method of claim 16, wherein the molar ratio between alcohol
and fatty acid residues is in the range 0.3-5.
21. The method of claim 16, wherein the molar ratio between alcohol
and fatty acid residues is in the range 0.4-2.
22. The method of claim 16, wherein the alcohol is methanol or
ethanol.
23. The method of claim 16, wherein the reaction mixture further
comprises water.
24. The method of claim 16, wherein the first lipolytic enzyme has
a ratio of activity on triglyceride to activity on FFA in the range
of 0.01-0.2 and the second lipolytic enzyme has a ratio of activity
on triglyceride to activity on FFA in the range of 0.5-20.
25. The method of claim 16, wherein the first lipolytic enzyme is
60% identical with a lipolytic enzyme selected from the group
consisting of Lipase B from Candida antarctica, Hyphozyma sp.
lipase and Candida parapsilosis lipase.
26. The method of claim 235 wherein the first lipolytic enzyme is
Lipase B from Candida antarctica, Hyphozyma sp. lipase or Candida
parapsilosis lipase.
27. The method of claim 16, wherein the second lipolytic enzyme is
60% identical with a lipolytic enzyme selected from the group
consisting of T. lanuginosus lipase, H. insolens cutinase, C.
antarctica lipase A, lipases from Candida rugosa, Pseudomonas
cepacia, Geotricum candidum, Rhizomucor miehei, Crytococcus spp.
S-2, Candida parapsilosis and Humicola lanuginosus.
28. The method of claim 27, wherein the second lipolytic enzyme is
one of T. lanuginosus lipase, H. insolens cutinase, C. antarctica
lipase A, lipases from Candida rugosa, Pseudomonas cepacia,
Geotricum candidum, Rhizomucor miehei, Cytococcus spp. S-2, Candida
parapsilosis and Humicola lanuginosus.
29. The method of claim 16, wherein the process is proceeding in a
batch mode.
30. The method of claim 16, wherein the process is proceeding in a
continuous mode.
31. The method of claim 16, further comprising mixing solution
phases in the reaction mixture using a high shear mixer.
32. The method of claim 16, wherein the process is conducted in a
counter-current mode.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for producing
fatty acid alkyl esters from triglyceride by use of a first
lipolytic enzyme which favours the conversion of triglyceride to
fatty acid alkyl esters and a second lipolytic enzyme which favours
the conversion of free fatty acids to fatty acid alkyl esters.
BACKGROUND ART
[0002] Biodiesel, generally classified as mono-alkyl esters of fats
and oils, has become more attractive recently because of its
environmental benefits. Although biodiesel is at present
successfully produced chemically (using e.g. NaOH and/or sodium
methoxide as catalyst), there are several associated problems to
restrict its development, such as pre-processing of oil due to high
contents of free fatty acids, removal of chemical catalyst from
ester and glycerol phase and removal of inorganic salts during
glycerol recovery.
[0003] The disadvantages caused by chemical catalysts are largely
prevented by using lipolytic enzymes as the catalysts and in recent
years interest has developed in the use of lipases with or without
immobilization in transesterification for the production of
biodiesel.
[0004] Fungal esterases may be used in the enzymatic production of
esters, where they may replace catalysts like mineral acid (e.g.
sulphuric acid, hydrogen chloride, and chlorosulfonic acid),
amphoteric hydroxides of metals of groups I, II, III, and IV, and
others. The use of enzymes for ester synthesis has been described
in the prior art, in particular enzymes classified in EC 3.1.1
Carboxylic ester hydrolases according to Enzyme Nomenclature
(Recommendations of the Nomenclature Committee of the International
Union of Biochemistry and Molecular Biology, 1992 or later).
[0005] WO 88/02775 discloses lipases A and B from Candida
antarctica. It states that C. antarctica lipase B (CALB) is more
effective for ester synthesis.
[0006] Cutinases are lipolytic enzymes capable of hydrolyzing the
substrate cutin. Cutinases are known from various fungi (P. E.
Kolattukudy in "Lipases", Ed. B. Borgstrom and H. L. Brockman,
Elsevier 1984, 471-504). The amino acid sequence of a cutinase from
Humicola insolens has been published (U.S. Pat. No. 5,827,719).
[0007] Many researchers have reported that a high yield of alkyl
esters could be reached in the presence of organic solvents, but
because of the toxicity and flammability of organic solvents
lipase-catalysed alcoholysis in a solvent-free medium is more
desirable. Methanolysis catalysed by lipases has been shown to take
place in a water-containing system free of organic solvents. In
such systems lipases which are less sensitive to methanol is
advantageous (Kaieda et al. J. Biosci. Bioeng. 2001, 91:12-15). It
is well known that excessive short-chain alcohols such as methanol
might inactivate lipase seriously. However, at least three molar
equivalents of methanol are required for the complete conversion of
the oil to its corresponding methyl ester. Du et al. (Biotechnol.
Appl. Biochem. 2003, 38:103-106) studied the effect of molar ratio
of oil/methanol comparatively during non-continuous batch and
continuous batch operation.
[0008] To avoid inactivation of the lipases the methanol
concentration has been kept low by step-wise addition of methanol
throughout the reaction (Shimada et al. J Mol. Catalysis Enzymatic,
2002, 17:133-142; Xu et al. 2004, Biocat. Biotransform.
22:45-48).
[0009] Boutur et al. (J. Biotechnol. 1995, 42:23-33) reported a
lipase from Candida deformans which were able to catalyse both
alcoholysis of triglyceride (TG) and esterification of free fatty
acids (FFA), but not under the same reaction conditions. Under the
conditions described by Boutur et al. only the esterification was
catalysed.
[0010] In order to obtain a more economic production of fatty acid
ethyl esters for biodiesel, there is a need for a faster conversion
of fats and oils to their corresponding methyl esters and a higher
yield in said conversion.
SUMMARY OF THE INVENTION
[0011] The present invention relates to a method for producing
fatty acid alkyl esters, such as fatty acid methyl esters (FAME)
and fatty acid ethyl esters. Such esters are also called biodiesel,
because they are used as an additive to mineral diesel to result in
a sulphur-free, higher-cetane-number fuel, which is partly based on
renewable resources.
[0012] The method of the invention includes a solution comprising
alcohol, triglyceride and/or free fatty acids, which solution is
contacted with a first lipolytic enzyme and a second lipolytic
enzyme of different specificity, wherein the lipolytic enzymes
catalyse the conversion of triglyceride or free fatty acids or a
mixture of both to fatty acid alkyl esters. The first lipolytic
enzyme is characterised in that it exhibits higher activity against
triglyceride than free fatty acids, whereas the second lipolytic
enzyme exhibits higher activity against free fatty acids than
triglyceride. The activity of the first and second lipolytic
enzymes is determined by use of the methods described in Example 1
and 2 below.
[0013] The first lipolytic enzyme is defined as one having a ratio
of activity on TG/activity on FFA below 0.2. The second lipolytic
enzyme is defined as one having a ratio of activity on TG/activity
on FFA above 0.5.
[0014] The combination of a first lipolytic enzyme and a second
lipolytic enzyme according to the present invention results in a
synergistic effect on the conversion of triglyceride and
triglyceride in combination with free fatty acids to fatty acid
alkyl esters, whereby a higher percentage of conversion is obtained
in a shorter period of time.
[0015] Further, the invention relates to a batch process or a
continuous, staged process to produce fatty acid alkyl esters using
a first and a second lipolytic enzyme as described above, wherein
the alcohol is added continuously or stepwise, and wherein the
enzymes are recycled or used only once.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention relates to a method for producing
fatty acid alkyl esters. The method of the invention includes a
solution comprising alcohol, and a substrate, which comprises
triglyceride and/or free fatty acids. The solution is contacted
with a first lipolytic enzyme and a second lipolytic enzyme of
different specificity, wherein the lipolytic enzymes catalyse the
conversion of triglyceride or free fatty acids or a mixture of both
to fatty acid alkyl esters.
[0017] Substrates Suitable substrates for production of fatty acid
alkyl esters in accordance with the present invention are a broad
variety of vegetable oils and fats; rapeseed and soybean oils are
most commonly used, though other crops such as mustard, sunflower,
canola, coconut, hemp, palm oil and even algae show promise. The
substrate can be of crude quality or further processed (refined,
bleached and deodorized). Also animal fats including tallow, lard,
poultry, marine oil as well as waste vegetable and animal fats and
oil, commonly known as yellow and brown grease can be used. The
suitable fats and oils may be pure triglyceride or a mixture of
triglyceride and free fatty acids, commonly seen in waste vegetable
oil and animal fats. The substrate may also be obtained from
vegetable oil deodorizer distillates. The type of fatty acids in
the substrate comprises those naturally occurring as glycerides in
vegetable and animal fats and oils. These include oleic acid,
linoleic acid, linolenic acid, palmetic acid and lauric acid to
name a few. Minor constituents in crude vegetable oils are
typically phospholipids, free fatty acids and partial glycerides
i.e. mono- and diglycerides. When used herein the phrase "fatty
acid residues" refers to fatty acids, either free or esterified as
in triglycerides, diglycerides, monoglycerides or fatty acid alkyl
esters.
[0018] Biodiesel Fatty acid alkyl esters, such as fatty acid methyl
esters (FAME) and fatty acid ethyl esters are also called
biodiesel, because they are used as an additive to fossil diesel.
Biodiesel constitutes an increasingly important additive or
substitute for diesel fuels based on fossil oil because it is
produced from renewable resources.
[0019] Alcohol The alcohol used in the method of the invention is
preferably a lower alcohol having 1 to 5 carbon atoms
(C.sub.1-C.sub.5). Preferred alcohols are methanol and ethanol.
[0020] Lipolytic enzyme The first lipolytic enzyme according to the
present invention is characterised in that it exhibits higher
activity against triglyceride than free fatty acids, whereas the
second lipolytic enzyme exhibits higher activity against free fatty
acids than triglyceride. The activity of the lipolytic enzymes
against triglycerides and free fatty acid is determined as
described in Example 1 and Example 2, respectively.
[0021] According to the present invention, the first lipolytic
enzyme is defined as one having a ratio of activity on triglyceride
(measured as conversion of triglyceride to fatty acid alkyl esters)
to activity on FFA (measured as conversion of FFA to fatty acid
alkyl esters) below 0.2. The second lipolytic enzyme is defined as
one having a ratio of activity on triglyceride (measured as
conversion of triglyceride to fatty acid alkyl esters) to activity
on FFA (measured as conversion of FFA to fatty acid alkyl esters)
above 0.5.
[0022] Accordingly, the present invention relates to a method for
producing fatty acid alkyl esters, characterised in that a solution
comprising triglyceride and alcohol is contacted with a first
lipolytic enzyme having a ratio of activity on triglyceride to
activity on FFA below 0.2 and a second lipolytic enzyme having a
ratio of activity on triglyceride to activity on FFA above 0.5.
[0023] The first lipolytic enzyme preferably has a ratio of
activity on triglyceride to activity on FFA in the range of
0.01-0.2, more preferably in the range of 0.01-0.1, more preferably
in the range of 0.0125-0.05, more preferably in the range of
0.015-0.025, even more preferably in the range of 0.02-0.024. The
second lipolytic enzyme preferably has a ratio of activity on
triglyceride to activity on FFA in the range of 0.5-20, more
preferably in the range of 0.6-10, more preferably in the range of
0.7-5, even more preferably in the range of 0.8-1.5.
[0024] As stated above, the activity of the lipolytic enzymes
against triglycerides and free fatty acid is determined as
described in Example 1 and Example 2, respectively. Below, the
ratio of activity on triglyceride (abbreviated TG) as measured in
Example 1 to activity on free fatty acids (abbreviated FFA) as
measured in Example 2, has been calculated for the tested lipolytic
enzymes:
[0025] CALB: TG/FFA=0.55/26.41=0.021
[0026] H. insolens cutinase: TG/FFA=12.13/10=1.213
[0027] T. lanuginosus lipase: TG/FFA=13.22/16.25=0.814.
[0028] The combination of a first lipolytic enzyme and a second
lipolytic enzyme according to the present invention results in a
synergistic effect on the conversion of triglyceride and/or free
fatty acids to fatty acid alkyl esters, whereby a higher percentage
of conversion is obtained in a shorter period of time.
[0029] In a preferred embodiment of the method of the present
invention a first lipolytic enzyme of the present invention is
lipase B from Candida antarctica (CALB) as disclosed in WO
88/02775, whereas the second lipolytic enzyme is one of the
Thermomyces lanuginosus (previously Humicola lanuginosus) lipase
variants exemplified in WO 00/60063 and the Humicola insolens
cutinase variants disclosed in Example 2 of WO 01/92502,
hereinafter referred to as T. lanuginosus lipase and H. insolens
cutinase respectively. In a second preferred embodiment a first
lipolytic enzyme includes Hyphozyma sp. lipase and Candida
parapsilosis lipase, whereas a second lipolytic enzyme of the
present invention includes C. antarctica lipase A as disclosed in
WO 88/02775 and lipases from Humicola lanuginosus (EP 258 068),
Candida rugosa, Pseudomonas cepacia, Geotricum candidum, Rhizomucor
miehei, Ctytococcus spp. S-2 and Candida parapsilosis.
[0030] In a third embodiment the first lipolytic enzyme is
homologous with CALB, Hyphozyma sp. lipase or Candida parapsilosis
lipase, whereas the second lipolytic enzyme is homologous with T.
lanuginosus lipase, H. insolens cutinase or any of the lipases from
Humicola lanuginosus (EP 258 068), Candida rugosa, Pseudomonas
cepacia, Geotricum candidum, Rhizomucor miehei, Crytococcus spp.
S-2 and Candida parapsilosis.
[0031] Preferably, the first lipolytic enzyme according to the
method of the present invention is 60% identical with CALB, whereas
the second lipolytic enzyme is 60% identical with the T.
lanuginosus lipase, the H. insolens cutinase. More preferably the
first lipolytic enzyme is 70% identical with CALB, even more
preferably the first lipolytic enzyme is 75%, 80%, 85%, 88%, 90%,
92%, 94%, 95%, 96%, 97%, 98% or even 99% identical with CALB.
Similarly, the second lipolytic enzyme is preferably 70% identical
with T. lanuginosus lipase and H. insolens cutinase, more
preferably the second lipolytic enzyme is 75%, 80%, 85%, 88%, 90%,
92%, 94%, 95%, 96%, 97%, 98% or even 99% identical with T.
lanuginosus lipase or H. insolens cutinase.
[0032] The enzymes may be applied as lyophilised powder,
immobilised or in aqueous solution.
[0033] For purposes of the present invention, the degree of
identity may be suitably determined according to the method
described in Needleman, S. B. and Wunsch, C. D., (1970), Journal of
Molecular Biology, 48, 443-45, with the following settings for
polypeptide sequence comparison: GAP creation penalty of 3.0 and
GAP extension penalty of 0.1. The determination may be done by
means of a computer program known such as GAP provided in the GCG
program package (Program Manual for the Wisconsin Package, Version
8, August 1994, Genetics Computer Group, 575 Science Drive,
Madison, Wis., USA 53711).
[0034] Two given sequences can be aligned according to the method
described in Needleman (supra) using the same parameters. This may
be done by means of the GAP program (supra).
[0035] Further, the invention relates to a batch process and/or a
continuous, staged process to produce fatty acid alkyl esters using
a first and a second lipolytic enzyme as described above, wherein
the alcohol is added continuously or stepwise, and wherein the
enzymes are recycled or used only once. If the enzymes are in an
aqueous phase, this phase can be separated from the fatty phase by
a decanter, a settler or by centrifugation. In the continuously
process the two phases, oil and aqueous, respectively, can be
processed counter-currently. Kosugi, Y; Tanaka, H. and Tomizuka,
(1990), Biotechnology and Bioengineering, vol .36, 617-622,
describes a continuous, counter-current process to hydrolyse
vegetable oil by immobilized lipase.
General Description of Preparation of Fatty Acid Alkyl Esters
[0036] The substrate comprising triglyceride is mixed with alcohol,
preferably methanol or ethanol and heated to 30-60.degree. C.,
preferably 50.degree. C. on a reciprocal water shaking bath (200
rpm). Preferably water is added and the solution is mixed and
further heated to the desired temperature. The enzymes are added
and the solution is mixed vigorously and left on reciprocal water
shaking bath at the desired temperature, preferably 50.degree. C.
and 200 rpm to react. The phases of the reaction mixture can be
mixed by the use of high shear mixers, such as types from Silverson
or IKA Labortechnik, as used in enzymatic degumming of vegetable
oil (Clausen, K. (2001), European Journal of Lipid Science and
Technology, vol. 103, 333-340).
[0037] The [methanol]/[fatty acid residue] molar ratio should be at
least 0.1 and maximum 10, preferable in the range 0.3-5, more
preferable 0.4-2. The alcohol can be added stepwise to the reaction
over time. Water can be added separately or within an aqueous
enzyme solution. The final concentration of water in the reaction
mixture can be 0-50% (w/w), preferably 5-40%, more preferably
5-30%. The substrate comprises 1-99% (w/w) triglyceride, preferably
in the range of 70-95%. Further, the substrate may comprise free
fatty acids amounting to 0.01-95% (w/w), preferably in the range of
0.01-30%. Also, mono- and diglycerides and phospholipids may be
present.
[0038] The course of the reaction can be followed by withdrawing
samples from the reaction mixture after a certain period of
reaction time. The samples are centrifuged for 14 minutes at 14000
rpm. The upper layer consists of fatty material not soluble in the
water phase and this is analyzed by .sup.1H NMR (using CDCl.sub.3
as solvent). After the reaction has ended the glycerol phase is
removed either by decanting or centrifugation.
Cloning a DNA Sequence Encoding a Lipolytic Enzyme
[0039] The DNA sequence encoding a parent lipolytic enzyme may be
isolated from any cell or microorganism producing the lipolytic
enzyme in question, using various methods well known in the art.
First, a genomic DNA and/or cDNA library should be constructed
using chromosomal DNA or messenger RNA from the organism that
produces the lipolytic enzyme to be studied. Then, if the amino
acid sequence of the lipolytic enzyme is known, labelled
oligonucleotide probes may be synthesized and used to identify
lipolytic enzyme-encoding clones from a genomic library prepared
from the organism in question. Alternatively, a labelled
oligonucleotide probe containing sequences homologous to another
known lipolytic enzyme gene could be used as a probe to identify
lipolytic enzyme-encoding clones, using hybridization and washing
conditions of lower stringency.
[0040] Yet another method for identifying lipolytic enzyme-encoding
clones would involve inserting fragments of genomic DNA into an
expression vector, such as a plasmid, transforming
cutinase-negative bacteria with the resulting genomic DNA library,
and then plating the transformed bacteria onto agar containing a
substrate for lipolytic enzyme (i.e. triglyceride), thereby
allowing clones expressing the lipolytic enzyme to be
identified.
[0041] Alternatively, the DNA sequence encoding the enzyme may be
prepared synthetically by established standard methods, e.g. the
phosphoroamidite method described by S. L. Beaucage and M. H.
Caruthers, (1981), Tetrahedron Letters 22, p. 1859-1869, or the
method described by Matthes et al., (1984), EMBO J. 3, p. 801-805.
In the phosphoroamidite method, oligonucleotides are synthesized,
e.g. in an automatic DNA synthesizer, purified, annealed, ligated
and cloned in appropriate vectors.
[0042] Finally, the DNA sequence may be of mixed genomic and
synthetic origin, mixed synthetic and cDNA origin or mixed genomic
and cDNA origin, prepared by ligating fragments of synthetic,
genomic or cDNA origin (as appropriate, the fragments corresponding
to various parts of the entire DNA sequence), in accordance with
standard techniques. The DNA sequence may also be prepared by
polymerase chain reaction (PCR) using specific primers, for
instance as described in U.S. Pat. No. 4,683,202 or R. K. Saiki et
al., (1988), Science 239, 1988, pp. 487-491.
Expression Vector
[0043] The recombinant expression vector carrying the DNA sequence
encoding a lipolytic enzyme of the invention may be any vector
which may conveniently be subjected to recombinant DNA procedures,
and the choice of vector will often depend on the host cell into
which it is to be introduced. The vector may be one which, when
introduced into a host cell, is integrated into the host cell
genome and replicated together with the chromosome(s) into which it
has been integrated. Examples of suitable expression vectors
include pMT838.
[0044] The expression vector of the invention may also comprise a
suitable transcription terminator and, in eukaryotes,
polyadenylation sequences operably connected to the DNA sequence
encoding the lipolytic enzyme of the invention. Termination and
polyadenylation sequences may suitably be derived from the same
sources as the promoter.
[0045] The vector may further comprise a DNA sequence enabling the
vector to replicate in the host cell in question. Examples of such
sequences are the origins of replication of plasmids pUC19,
pACYC177, pUB110, pE194, pAMB1 and pIJ702.
[0046] The vector may also comprise a selectable marker, e.g. a
gene the product of which complements a defect in the host cell,
such as the dal genes from B. subtilis or B. licheniformis, or one
which confers antibiotic resistance such as ampicillin, kanamycin,
chloramphenicol or tetracyclin resistance. Furthermore, the vector
may comprise Aspergillus selection markers such as amdS, argB, niaD
and sC, a marker giving rise to hygromycin resistance, or the
selection may be accomplished by co-transformation, e.g. as
described in WO 91/17243.
[0047] The procedures used to ligate the DNA construct of the
invention encoding a cutinase variant, the promoter, terminator and
other elements, respectively, and to insert them into suitable
vectors containing the information necessary for replication, are
well known to persons skilled in the art (cf., for instance,
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,
Cold Spring Harbor, 1989).
Promoter
[0048] In the vector, the DNA sequence should be operably connected
to a suitable promoter sequence. The promoter may be any DNA
sequence which shows transcriptional activity in the host cell of
choice and may be derived from genes encoding proteins either
homologous or heterologous to the host cell.
[0049] Examples of suitable promoters for directing the
transcription of the DNA sequence encoding a lipolytic enzyme of
the invention, especially in a bacterial host, are the promoter of
the lac operon of E. coli, the Streptomyces coelicolor agarase gene
dagA promoters, the promoters of the Bacillus licheniformis
alfa-amylase gene (amyL), the promoters of the Bacillus
stearothermophilus maltogenic amylase gene (amyM), the promoters of
the Bacillus amyloliquefaciens alfa-amylase (amyQ), the promoters
of the Bacillus subtilis xylA and xylB genes etc. For transcription
in a fungal host, examples of useful promoters are those derived
from the gene encoding A. oryzae TAKA amylase, the TPI (triose
phosphate isomerase) promoter from S. cerevisiae (Alber et al.
(1982), J. Mol. Appl. Genet 1, p. 419-434, Rhizomucor miehei
aspartic proteinase, A. niger neutral alfa-amylase, A. niger acid
stable alfa-amylase, A. niger glucoamylase, Rhizomucor miehei
lipase, A. oryzae alkaline protease, A. oryzae triose phosphate
isomerase, or A. nidulans acetamidase.
Host Cells
[0050] The cell of the invention, either comprising a DNA construct
or an expression vector of the invention as defined above, is
advantageously used as a host cell in the recombinant production of
a lipolytic enzyme of the invention. The cell may be transformed
with the DNA construct of the invention encoding the lipolytic
enzyme, conveniently by integrating the DNA construct (in one or
more copies) in the host chromosome. This integration is generally
considered to be an advantage as the DNA sequence is more likely to
be stably maintained in the cell. Integration of the DNA constructs
into the host chromosome may be performed according to conventional
methods, e.g. by homologous or heterologous recombination.
Alternatively, the cell may be trans-formed with an expression
vector as described above in connection with the different types of
host cells.
[0051] The cell of the invention may be a cell of a higher organism
such as a mammal or an insect, particularly a microbial cell, e.g.
a bacterial or a fungal (including yeast) cell.
[0052] Examples of suitable bacteria are Gram positive bacteria
such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus,
Bacillus brevis, Bacillus stearothermophilus, Bacillus
alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans,
Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus
thuringiensis, or Streptomyces lividans or Streptomyces murinus, or
gram negative bacteria such as E. coli. The transformation of the
bacteria may, for instance, be effected by protoplast
transformation or by using competent cells in a manner known per
se.
[0053] The yeast organism may favorably be selected from a species
of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces
cerevisiae.
[0054] The host cell may also be a filamentous fungus e.g. a strain
belonging to a species of Aspergillus, particularly Aspergillus
oryzae or Aspergillus niger, or a strain of Fusarium, such as a
strain of Fusarium oxysporum, Fusarium graminearum (in the perfect
state named Gibberella zeae, previously Sphaeria zeae, synonym with
Gibberella roseum and Gibberella roseum f. sp. cerealis), or
Fusarium sulphureum (in the prefect state named Gibberella
puricaris, synonym with Fusarium trichothecioides, Fusarium
bactridioides, Fusarium sambucinum, Fusarium roseum, and Fusarium
roseum var. graminearum), Fusarium cerealis (synonym with Fusarium
crokkwellense), or Fusarium venenatum.
[0055] In a particular embodiment of the invention the host cell is
a protease deficient or protease minus strain. This may for
instance be the protease deficient strain Aspergillus oryzae JaL
125 having the alkaline protease gene named "alp" deleted. This
strain is described in WO 97/35956 (Novo Nordisk).
[0056] Filamentous fungi cells may be transformed by a process
involving protoplast formation and transformation of the
protoplasts followed by regeneration of the cell wall in a manner
known per se. The use of Aspergillus as a host microorganism is
described in EP 238 023 (Novo Nordisk A/S), the contents of which
are hereby incorporated by reference.
Production of Lipolytic Enzyme by Cultivation of Transformant
[0057] The invention relates, inter alia, to a method of producing
a lipolytic enzyme of the invention, which method comprises
cultivating a host cell under conditions conducive to the
production of the lipolytic enzyme and recovering the lipolytic
enzyme from the cells and/or culture medium.
[0058] The medium used to cultivate the cells may be any
conventional medium suitable for growing the host cell in question
and obtaining expression of the lipolytic enzyme of the invention.
Suitable media are available from commercial suppliers or may be
prepared according to published recipes (e.g. as described in
catalogues of the American Type Culture Collection).
[0059] The lipolytic enzyme secreted from the host cells may
conveniently be recovered from the culture medium by well-known
procedures, including separating the cells from the medium by
centrifugation or filtration, and precipitating proteinaceous
components of the medium by means of a salt such as ammonium
sulphate, followed by the use of chromatographic procedures such as
ion exchange chromatography, affinity chromatography, or the
like.
Materials and Methods
Lipase Activity on Tributyrin (LU)
[0060] A substrate for lipolytic enzymes is prepared by emulsifying
tributyrin (glycerin tributyrate) using gum Arabic as emulsifier.
The hydrolysis of tributyrin at 30.degree. C. at pH 7 is followed
in a pH-stat titration experiment. One unit of lipase activity (1
LU) equals the amount of enzyme capable of releasing 1 .mu.mol
butyric acidimin at the standard conditions.
Preparation of Fatty Acid Alkyl Ester
[0061] 8.00 gram of substrate is mixed with methanol (0.500
ml=>0.395 gram). The following types of substrates were used:
[0062] Example 1) 100% salad oil (refined, bleached and deodorized
soybean oil, RBD SBO); [0063] Example 2) 100% oleic acid; [0064]
Example 3) mixture of 20% w/w oleic acid in RBD SBO
[0065] The substrate-methanol mixture is heated to 50.degree. C. on
a reciprocal water shaking bath (200 rpm). Demineralised water is
added (volume depending on added enzyme volume; total amount of
water: 4.00 ml including water from enzyme addition), corresponding
to 32 w/w % of the total mixture. The mixture is heated to
50.degree. C. Then enzyme is added to the mixture and vigorously
mixed for 10 sec. and left on reciprocal water shaking bath at
50.degree. C. and 200 rpm. The phases of the reaction mixture can
be mixed by the use of high shear mixers, such as types from
Silverson Ltd. UK or IKA Kunkel.
[0066] Samples are withdrawn from the reaction mixture after 3 hrs.
reaction time and centrifuged for 14 minutes at 14000 rpm. The
upper layer consists of fatty material not soluble in the water
phase and this is analyzed by .sup.1H NMR (using CDCl.sub.3 as
solvent) Varian 400 MHz spectrometer (Varian Inc. CA, USA). The
conversion of the fatty acids residues into fatty acid methyl ester
is determined by the ratio of the methyl signals from the fatty
acid methyl esters, --COOCH.sub.3 (3.70 ppm) and CH.sub.3CH.sub.2--
(1.0-0.9 ppm) from the fatty acid residues.
[0067] The enzyme dose is based on a total 0.4 mg protein/8.00 gram
substrate. For testing a synergistic effect of two enzymes
combined; 0.2 mg of each enzyme were added to 8 gram of substrate
and compared to the each of the single enzymes at a dose of 0.4
mg/8 gram of substrate. To relate the amount of protein to an
enzyme activity, a standard enzyme activity assay can be applied,
in this case the LU-assay as described above (lipase activity on
tributyrine). The following enzyme preparations were tested:
[0068] 1. T. lanuginosus lipase (TLL, specific activity 7000 LU/mg
protein)
[0069] 2. C. antarctica lipase B (CALB, specific activity 500 LU/mg
protein)
[0070] 3. H. insolens cutinase (cutinase, specific activity 1800
LU/mg protein)
Enzyme dose and additional water volumes for experiments with
single enzymes:
[0071] 1. TLL: 0.700 ml of a 4000 LU/ml enzyme solution+3.30 ml
water
[0072] 2. CALB: 1.680 ml of a 119 LU/ml enzyme solution+2.32 ml
water
[0073] 3. Cutinase: 0.450 ml of a 1600 LU/ml enzyme solution+3.55
ml water
Enzyme dose and additional water volumes for experiments with
combination of enzymes:
[0074] 1. TLL+CALB: (0.350 ml of a 4000 LU/ml TLL solution+0.840 ml
of a 119 LU/ml CALB solution+2.810 ml water)
[0075] 2. Cutinase+CALB: (0.225 ml of a 1600 LU/ml cutinase
solution+0.840 ml of a 119 LU/ml CALB solution+2.935 ml water).
EXAMPLES
Example 1
Preparation of Fatty Acid Alkyl Esters from Triglycerides
[0076] Refined, bleached and deodorized soybean oil (RBD SBO, salad
oil) was used as substrate according to the general method
described above.
The conversions (%) of fatty acid residues into FAME after 3 hours
reaction time using different lipolytic enzymes are shown in Table
1, whereas the conversion (%) achieved with a combination of CALB
and TLL is shown in Table 2. Coefficient of Variation in % (CV %)
of four identical experiments was determined to be 2.2%.
TABLE-US-00001 TABLE 1 Single enzymes, % conversion of fatty acid
residues into FAME CALB 0.55 Cutinase 12.13 TLL 13.22
TABLE-US-00002 TABLE 2 Combination of enzymes, % conversion of
fatty acid residues into FAME. CALB + TLL 16.21
Example 2
Preparation of Fatty Acid Alkyl Esters from Oleic Acid
[0077] Oleic acid was used as substrate according to the general
method described above. The conversions (%) of fatty acid residues
into FAME after 3 hours reaction time using different lipolytic
enzymes are shown in Table 3.
TABLE-US-00003 TABLE 3 Single enzymes, % conversion of fatty acid
residues into FAME. CALB 26.41 Cutinase 10 TLL 16.25
Example 3
Preparation of Fatty Acid Alkyl Esters from Triglyceride Containing
Free Fatty Acids
[0078] A mixture of 20% w/w oleic acid in RBD SBO was used as
substrate according to the general method described above. The
conversions (%) of fatty acid residues into FAME after 3 hours
reaction time using different lipolytic enzymes and combinations of
said enzymes are shown in Table 4 and 5.
TABLE-US-00004 TABLE 4 Single enzymes, % conversion of fatty acid
residues into FAME. CALB 16.58 Cutinase 11.22 TLL 14.09
TABLE-US-00005 TABLE 5 Combination of enzymes, % conversion of
fatty acid residues into FAME. CALB + Cutinase 18.82 CALB + TLL
20
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