U.S. patent application number 15/568535 was filed with the patent office on 2018-12-06 for process for isolating and purifying ambrox.
The applicant listed for this patent is Givaudan SA. Invention is credited to Eric EICHHORN.
Application Number | 20180346434 15/568535 |
Document ID | / |
Family ID | 53488739 |
Filed Date | 2018-12-06 |
United States Patent
Application |
20180346434 |
Kind Code |
A9 |
EICHHORN; Eric |
December 6, 2018 |
PROCESS FOR ISOLATING AND PURIFYING AMBROX
Abstract
A method of isolating and purifying (-)-Ambrox from a reaction
mixture comprising (-)-Ambrox and one or more of the compounds
(II), (III) and (IV) ##STR00001##
Inventors: |
EICHHORN; Eric; (Zurich,
CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Givaudan SA |
Vernier |
|
CH |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20180134678 A1 |
May 17, 2018 |
|
|
Family ID: |
53488739 |
Appl. No.: |
15/568535 |
Filed: |
April 22, 2016 |
PCT Filed: |
April 22, 2016 |
PCT NO: |
PCT/EP2016/058997 PCKC 00 |
371 Date: |
October 23, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07B 2200/09 20130101;
C07D 307/92 20130101; C12P 17/04 20130101; C12Y 504/99017 20130101;
B01D 9/005 20130101; C11B 9/0076 20130101; C07B 63/00 20130101 |
International
Class: |
C07D 307/92 20060101
C07D307/92; C07B 63/00 20060101 C07B063/00; B01D 9/00 20060101
B01D009/00; C11B 9/00 20060101 C11B009/00; C12P 17/04 20060101
C12P017/04 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 24, 2015 |
GB |
1507170.7 |
Claims
1. A method of isolating and purifying (-)-Ambrox from a reaction
mixture, comprising one or more of the compounds (II), (III) and
(IV) ##STR00005##
2. The method according to claim 1 comprising the step of
selectively crystallizing (-)-Ambrox from a mixture comprising one
or more of the compounds (II), (III) or (IV).
3. A method of improving or enhancing the odour of (-)-Ambrox
comprising the step of separating (-)-Ambrox from a mixture
comprising one or more of the compounds (II), (III) or (IV)
##STR00006## by selective crystallization of (-)-Ambrox from the
mixture, such that after the step of separating, (-)-Ambrox
contains none, or only olfactory acceptable amounts, of the
compounds (II), (III) or (IV).
4. The method according to claim 1, wherein the reaction mixture is
free, or is substantially free, of homofarnesol.
5. The method according to claim 2, wherein the crystallizing
solvent is selected from the group consisting of water, methanol,
acetone, petroleum ether, hexane, t-butyl methyl ether, THF and
ethyl acetate ethanol, toluene and mixtures thereof.
6. The method according to claim 5, wherein the crystallizing
solvent is an ethanol water mixture.
7. The method according to claim 1, wherein the reaction mixture is
formed as a result of an enzyme-catalyzed cyclization of
homofarnesol comprising a mixture of 7E,3E and 7E,3Z homofarnesol
geometric isomers of homofarnesol, wherein the reaction is carried
out in the presence of a recombinant microorganism expressing the
gene encoding the enzyme.
8. The method according to claim 7, wherein the reaction mixture of
7E,3E and 7E,3Z homofarnesol is enriched in the 7E,3E geometric
isomer.
9. The method according to claim 7, wherein the reaction mixture of
7E,3E and 7E,3Z homofarnesol consists of 7E,3E and 7E,3Z
homofarnesol and no other geometric isomers of homofarnesol.
10. The method according to claim 7, wherein the weight ratio of
the 7E,3E isomer to 7E,3Z isomer is at least 80:20.
11. The method according to claim 7, wherein the enzyme is a
wild-type squalene hopene cyclase or a variant of the wild-type
squalene hopene cyclase.
12. A perfume ingredient consisting of (-)-Ambrox and olfactory
acceptable amounts of one or more of the compounds (II), (III) or
(IV) ##STR00007##
13. The perfume ingredient according to claim 12, comprising
crystalline (-)-Ambrox
14. A perfume composition comprising (-)-Ambrox and at least one
other perfume ingredient, wherein said perfume composition contains
olfactory acceptable amounts of one or more of the compounds (II),
(III) or (IV) ##STR00008##
15. The method according to claim 3, wherein the reaction mixture
is free, or is substantially free, of homofarnesol.
16. The method according to claim 3, wherein the crystallizing
solvent is selected from the group consisting of water, methanol,
acetone, petroleum ether, hexane, t-butyl methyl ether, THF and
ethyl acetate ethanol, toluene and mixtures thereof.
17. The method according to claim 16, wherein the crystallizing
solvent is an ethanol water mixture.
18. The method according to claim 3, wherein the mixture is formed
as a result of an enzyme-catalyzed cyclization of homofarnesol
comprising a mixture of 7E,3E and 7E,3Z homofarnesol geometric
isomers of homofarnesol, wherein the reaction is carried out in the
presence of a recombinant microorganism expressing the gene
encoding the enzyme.
19. The method according to claim 18, wherein the reaction mixture
of 7E,3E and 7E,3Z homofarnesol is enriched in the 7E,3E geometric
isomer.
20. The method according to claim 18, wherein the reaction mixture
of 7E,3E and 7E,3Z homofarnesol consists of 7E,3E and 7E,3Z
homofarnesol and no other geometric isomers of homofarnesol.
21. The method according to claim 18, wherein the weight ratio of
the 7E,3E isomer to 7E,3Z isomer is at least 80:20.
22. The method according to claim 18, wherein the enzyme is a
wild-type squalene hopene cyclase or a variant of the wild-type
squalene hopene cyclase.
23. The method according to claim 10, wherein the weight ratio of
the 7E,3E isomer to 7E,3Z isomer is at least 90:10.
24. The method according to claim 10, wherein the weight ratio of
the 7E,3E isomer to 7E,3Z isomer is at least 95:5.
Description
[0001] The present invention is concerned with a method of
preparing, isolating and purifying the isomer (-)-Ambrox. More
particularly, the invention is concerned with a method of preparing
(-)-Ambrox by means of a bioconversion, as well as to a method of
its recovery and purification from a reaction mixture.
[0002] AMBROFIX.TM. is the proprietary Givaudan trade name of the
enantiomerically pure compound (-)-Ambrox, which has the general
formula (I).
##STR00002##
[0003] AMBROFIX.TM. is a very important molecule in the perfumers'
palette of ingredients. It delivers a highly powerful, highly
substantive and highly stable ambery note for use in all perfumery
applications. AMBROFIX.TM., available from Givaudan, is the most
suitable material for obtaining an authentic ambergris odour
note.
[0004] Currently, AMBROFIX.TM. is produced synthetically from
starting materials of natural origin. The availability and quality
of certain starting materials are dependent on climatic conditions,
as well as socio-economic factors. Furthermore, since starting
materials may be extracted from natural resources, with modest
yields, they are available at prices that will, in all likelihood,
increasingly render their use uneconomical on an industrial scale.
Accordingly, if commercial industrial supplies of AMBROFIX.TM. are
to continue to be available at a reasonable cost, there is a need
for a more cost-effective process of production and purification,
which is capable of industrialization.
[0005] An industrially scalable biotechnological route into
AMBROFIX.TM. would be attractive because it is potentially less
complex and less polluting than fully synthetic procedures.
[0006] A potentially useful substrate on which to attempt a
bioconversion to provide (-)-Ambrox is homofarnesol. In their
seminal paper, Neumann et al (Biol. Chem. Hoppe-Seyler Vol. 367 pp
723-726 (1986)) discussed the feasibility of converting
homofarnesol to (-)-Ambrox under enzymatic catalysis, employing the
enzyme Squalene Hopene Cyclase (SHC). The homofarnesol employed was
a mixture of the four geometric isomers of this molecule. Of the
four isomers, only the 7E,3E geometric isomer (using conventional
nomenclature) could be cyclized, and then only with very low yield
of the desired (-)-Ambrox.
[0007] JP 2009-60799 (Kao) discloses a synthesis whereby SHC acts
on a homofarnesol substrate to produce (-)-Ambrox. The substrate is
a mixture of all four geometric isomers (3Z,7Z; 3E,7Z; 3Z,7E; and
3E,7E). The document only discloses the preparation of (-)-Ambrox
from homofarnesol extracts containing SHC. The homofarnesol mixture
is converted to (-)-Ambrox and its 9-epi stereoisomer, and
purification can be carried out by distillation or by column
chromatography. Kao does not describe a process whereby
homofarnesol is converted into (-)-Ambrox using intact
microorganisms producing SHC, and furthermore, it does not provide
any technical teaching related to the downstream processing of
complex reaction mixtures obtained by such processes that can yield
(-)-Ambrox in olfactively pure form.
[0008] To the applicant's knowledge, the prior art does not
describe any viable, industrially scalable processes, involving the
SHC-catalyzed bioconversion of homofarnesol, to provide (-)-Ambrox
in olfactively pure form.
[0009] Furthermore, if bioconversion of homofarnesol is to be
realized on an industrial scale, cost-efficient sources of highly
pure, 3E,7E-homfarnesol should be available. However, although
synthetic routes into homofarnesol have been described in the
literature (see for example US 2013/0273619), to the applicant's
knowledge there are no cost-effective, industrial-scale sources of
pure 7E,3E-homofarnesol currently available.
[0010] There remains a need to provide an economically feasible and
industrially scalable route into the valuable fragrance ingredient
(-)-Ambrox.
[0011] In co-pending patent applications PCT/EP2014/072891
(published as WO2015/059293) and PCT2014/EP/072882 (published as
WO2015/059290), the applicant describes an efficient method of
preparing 7E,3E/Z-homofarnesol mixture that is enriched in the
7E,3E geometric isomer. The 7E,3E/Z-homofarnesol mixture is
prepared from beta-farnesene, and the isomeric information
contained in this starting material is preserved, such that the
homofarnesol double bond at the 7-position is fixed in the
E-configuration. However, even 30 this elegant chemistry still
results in a 3E/Z isomer mixture. Pure 7E,3E-homofarnesol remains
synthetically challenging, and might only be achieved by means of
economically disadvantageous purification of isomeric mixtures.
[0012] Surprisingly, the applicant has found that
7E,3E/Z-homofarnesol mixtures can undergo a bioconversion process,
whereby the homofarnesol mixture is enzymatically cyclized in the
presence of a recombinant microorganism expressing an enzyme, in
particular a Squalene Hopene Cyclase (SHC) biocatalyst capable of
bioconverting homofarnesol to (-)-Ambrox, to yield a reaction
mixture from which (-)-Ambrox can be isolated in olfactively pure
form with surprisingly facile downstream processing.
[0013] In one aspect of the invention there is provided the
enzyme-catalyzed cyclisation of homofarnesol to provide a reaction
mixture comprising (-)-Ambrox, wherein the homofarnesol comprises a
mixture of 7E,3E/Z-geometric isomers of homofarnesol, and wherein
the reaction is carried out in the presence of a recombinant
microorganism producing the enzyme, more particularly an intact
recombinant microorganism producing the enzyme.
[0014] In an embodiment of the invention, the cyclization reaction
is carried out in the presence of an SHC biocatalyst capable of
bioconverting homofarnesol to (-)-Ambrox.
[0015] The SHC biocatalyst is a wild-type or a variant enzyme or is
a microorganism expressing a gene encoding the SHC enzyme,
preferably a recombinant E. coli microorganism. The SHC biocatalyst
can be used in any form such as but not limited to a purified SHC
enzyme, a crude extract containing an SHC enzyme or an immobilised
SHC enzyme (e.g. on a carrier), or the biocatalyst can be a
microorganism having produced or producing the SHC enzyme, such as
an intact recombinant whole cell and/or fragmented cell or a
membrane fraction containing the SHC enzyme.
[0016] In a particular embodiment of the present invention, the
homofarnesol mixture is enriched in the 7E,3E-geometric isomer.
[0017] In a more particular embodiment, the homofarnesol mixture is
at least 55/45 by weight 7E,3E/7E,3Z.
[0018] In a more particular embodiment, the homofarnesol mixture is
at least 70/30 by weight 7E,3E/7E,3Z.
[0019] In a still more particular embodiment, the homofarnesol
mixture is at least 80/20 by weight 7E,3E/7E,3Z
[0020] In a still more particular embodiment, the homofarnesol
mixture is at least 90/10 by weight 7E,3E/7E,3Z.
[0021] In a still more particular embodiment, the homofarnesol
mixture is at least 95/5 by weight 7E,3E/7E,3Z.
[0022] In a particular embodiment of the present invention, the
homofarnesol mixture consists of 7E,3E/Z-geometric isomers and no
other geometric isomers of homofarnesol.
[0023] The skilled person understands that the term 7E, 7Z, 3E or
3Z used in connection with homofarnesol refers respectively to the
orientation of the double bond at the 7-position and 3-position of
homofarnesol. The 7E,3E-homofarnesol compound has the CAS No.
459-89-2, whereas the 7E,3Z-homofarnesol compound has the CAS No.
138152-06-4. The use of the term 7E, 3E/Z-homofarnesol refers to a
mixture of the compounds.
[0024] Methods of obtaining homofarnesol mixtures useful as a
substrate in the cyclisation reaction in accordance with the method
of the present invention are set forth in the co-pending
applications PCT/EP2014/072891 (published as WO2015/059293) and
PCT2014/EP/072882 (published as WO2015/059290) referred to above,
which are hereby incorporated by reference in their entirety. In
general terms, they describe a synthesis of homofarnesol mixtures
that proceeds by converting farnesene, more particularly
alpha-farnesene and/or beta-farnesene, to its corresponding
cyclopropanated farnesene derivative, using an organic solution of
an N-alkyl-N-nitroso urea. The cyclopropanated derivative then
undergoes ring-opening and rearrangement reactions in the presence
of a Bronsted acid to afford the homofarnesol mixture, which is
selective for the 7E,3E geometric isomer. Using farnesene, as a
starting material is particularly preferred because it ensures that
the E-configuration of the double bond at the 7 position of
homofarnesol is fixed.
[0025] Specific reaction conditions, which form particular
embodiments of the present invention, are set forth in the
co-pending applications, as well as the examples hereinbelow, and
do not require more elaboration here.
[0026] The cyclization of homofarnesol to provide a reaction
mixture containing (-)-Ambrox may be catalysed by Squalene Hopene
Cyclase (SHC). SHC may be a wild type enzyme (e.g. SEQ ID No. 1),
or a variant thereof (e.g. SEQ ID No. 2, or SEQ ID No. 4). SHC can
be obtained from Alicyclobacillus acidocaldarius (Bacillus
acidocaldarius), Zymomonas mobilis or Bradyrhizobium japonicum (as
set forth in Example 3b of US20120135477A1).
[0027] However, the enzyme can also be produced by recombinant
means, using techniques that are generally known in the art.
[0028] The term "recombinant" as used with respect to the
production of enzymes shall refer to enzymes produced by
recombinant DNA techniques, i.e., produced from cells transformed
by an exogenous DNA construct encoding the desired enzyme. The term
"recombinant DNA" therefore includes a recombinant DNA incorporated
into a vector into an autonomously replicating plasmid or virus, or
into the genomic DNA of a prokaryote or eukaryote (or the genome of
a homologous cell, at a position other than the natural chromosomal
location).
[0029] Nucleic acid molecule(s) is/are operatively linked to
expression control sequences allowing expression in prokaryotic
and/or eukaryotic host cells. As used herein, "operatively linked"
means incorporated into a genetic construct so that expression
control sequences effectively control expression of a coding
sequence of interest. The transcriptional/translational regulatory
elements referred to above include but are not limited to inducible
and non-inducible, constitutive, cell cycle regulated,
metabolically regulated promoters, enhancers, operators, silencers,
repressors and other elements that are known to those skilled in
the art and that drive or otherwise regulate gene expression. Such
regulatory elements include but are not limited to regulatory
elements directing constitutive expression or which allow inducible
expression like, for example, CUP-1 promoter, the tet-repressor as
employed, for example, in the tet-on or tet-off systems, the lac
system, the trp system regulatory elements. By way of example,
Isopropyl .beta.-D-1-thiogalactopyranoside (IPTG) is an effective
inducer of protein expression in the concentration range of 100
.mu.M to 1.0 mM. This compound is a molecular mimic of allolactose,
a lactose metabolite that triggers transcription of the lac operon,
and it is therefore used to induce protein expression where the
gene is under the control of the lac operator.
[0030] Similarly, nucleic acid molecule(s) can form part of a
hybrid gene encoding additional polypeptide sequences, for example,
a sequence that functions as a marker or reporter. Examples of
marker and reporter genes including beta-lactamase, chloramphenicol
acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside
phosphotransferase dihydrofolate reductase (DHFR),
hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ
(encoding beta-galactosidase), and xanthine guanine
phosphoribosyltransferase (XGPRT). As with many of the standard
procedures associated with the practice of the disclosure, skilled
artisans will be aware of additional useful reagents, for example,
additional sequences that can serve the function of a marker or
reporter.
[0031] Recombinant polynucleotides can encode SHC enzymes such as
the wild type SHC or a variant thereof, which may be inserted into
a vector for expression and optional purification. One type of
vector is a plasmid representing a circular double stranded DNA
loop into which additional DNA segments are ligated. Certain
vectors can control the expression of genes to which they are
functionally linked. These vectors are called "expression vectors".
Usually expression vectors suitable for DNA recombination
techniques are of the plasmid type. Typically, an expression vector
comprises a gene such as the wild type SHC or a variant thereof. In
the present description, the terms "plasmid" and "vector" are used
interchangeably since the plasmid is the vector type most often
used.
[0032] Such vectors can include DNA sequences which include but are
not limited to DNA sequences that are not naturally present in the
host cell, DNA sequences that are not normally transcribed into RNA
or translated into a protein ("expressed") and other genes or DNA
sequences which one desires to introduce into the non-recombinant
host. It will be appreciated that typically the genome of a
recombinant host is augmented through the stable introduction of
one or more recombinant genes. However, autonomous or replicative
plasmids or vectors can also be used within the scope of this
disclosure. Moreover, the present disclosure can be practiced using
a low copy number, e.g., a single copy, or high copy number plasmid
or vector.
[0033] In a preferred embodiment the vector of the present
disclosure comprises plasmids, phagemids, phages, cosmids,
artificial bacterial and artificial yeast chromosomes, knock-out or
knock-in constructs. Synthetic nucleic acid sequences or cassettes
and subsets may be produced in the form of linear polynucleotides,
plasmids, megaplasmids, synthetic or artificial chromosomes, such
as plant, bacterial, mammalian or yeast artificial chromosomes.
[0034] It is preferred that the proteins encoded by the introduced
polynucleotide are produced within the cell upon introduction of
the vector. The diverse gene substrates may be incorporated into
plasmids. The plasmids are often standard cloning vectors, e.g.,
bacterial multicopy plasmids. The substrates can be incorporated
into the same or different plasmids. Often at least two different
types of plasmid having different types of selectable markers are
used to allow selection for cells containing at least two types of
vectors.
[0035] Typically bacterial or yeast cells may be transformed with
any one or more of the following nucleotide sequences as is well
known in the art. For in vivo recombination, the gene to be
recombined with the genome or other genes is used to transform the
host using standard transforming techniques. In a suitable
embodiment DNA providing an origin of replication is included in
the construct. The origin of replication may be suitably selected
by the skilled person. Depending on the nature of the genes, a
supplemental origin of replication may not be required if sequences
are already present with the genes or genome that are operable as
origins of replication themselves.
[0036] A bacterial or yeast cell may be transformed by exogenous or
heterologous DNA when such DNA has been introduced inside the cell.
The transforming DNA may or may not be integrated, i.e. covalently
linked into the genome of the cell. In prokaryotes, and yeast, for
example, the transforming DNA may be maintained on an episomal
element such as a plasmid. With respect to eukaryotic cells, a
stably transformed cell is one in which the transfected DNA has
become integrated into a chromosome so that it is inherited by
daughter cells through chromosome replication. This stability is
demonstrated by the ability of the eukaryotic cell to establish
cell lines or clones comprised of a population of daughter cells
containing the transforming DNA.
[0037] Generally, the introduced DNA is not originally resident in
the host that is the recipient of the DNA, but it is within the
scope of the disclosure to isolate a DNA segment from a given host,
and to subsequently introduce one or more additional copies of that
DNA into the same host, e.g., to enhance production of the product
of a gene or alter the expression pattern of a gene. In some
instances, the introduced DNA will modify or even replace an
endogenous gene or DNA sequence by, e.g., homologous recombination
or site-directed mutagenesis. Suitable recombinant hosts include
microorganisms, plant cells, and plants.
[0038] The present disclosure also features recombinant hosts. The
term "recombinant host", also referred to as a "genetically
modified host cell" or a "transgenic cell" denotes a host cell that
comprises a heterologous nucleic acid or the genome of which has
been augmented by at least one incorporated DNA sequence. A host
cell of the present disclosure may be genetically engineered with
the polynucleotide or the vector as outlined above.
[0039] The host cells that may be used for purposes of the
disclosure include but are not limited to prokaryotic cells such as
bacteria (for example, E. coli and B. subtilis), which can be
transformed with, for example, recombinant bacteriophage DNA,
plasmid DNA, bacterial artificial chromosome, or cosmid DNA
expression vectors containing the polynucleotide molecules of the
disclosure; simple eukaryotic cells like yeast (for example,
Saccharomyces and Pichia), which can be transformed with, for
example, recombinant yeast expression vectors containing the
polynucleotide molecule of the disclosure. Depending on the host
cell and the respective vector used to introduce the polynucleotide
of the disclosure the polynucleotide can integrate, for example,
into the chromosome or the mitochondrial DNA or can be maintained
extrachromosomally like, for example, episomally or can be only
transiently comprised in the cells.
[0040] The term "cell" as used herein in particular with reference
to genetic engineering and introducing one or more genes or an
assembled cluster of genes into a cell, or a production cell is
understood to refer to any prokaryotic or eukaryotic cell.
Prokaryotic and eukaryotic host cells are both contemplated for use
according to the disclosure, including bacterial host cells like E.
coli or Bacillus sp, yeast host cells, such as S. cerevisiae,
insect host cells, such as Spodoptora frugiperda or human host
cells, such as HeLa and Jurkat.
[0041] Specifically, the cell is a eukaryotic cell, preferably a
fungal, mammalian or plant cell, or prokaryotic cell. Suitable
eucaryotic cells include, for example, without limitation,
mammalian cells, yeast cells, or insect cells (including Sf9),
amphibian cells (including melanophore cells), or worm cells
including cells of Caenorhabditis (including Caenorhabditis
elegans). Suitable mammalian cells include, for example, without
limitation, COS cells (including Cos-1 and Cos-7), CHO cells,
HEK293 cells, HEK293T cells, HEK293 T-Rex.TM. cells, or other
transfectable eucaryotic cell lines. Suitable bacterial cells
include without limitation E. coli.
[0042] Preferably prokaryotes, such as E. coli, Bacillus,
Streptomyces, or mammalian cells, like HeLa cells or Jurkat cells,
or plant cells, like Arabidopsis, may be used.
[0043] Preferably the cell is an Aspergillus sp or a fungal cell,
preferably, it can be selected from the group consisting of the
genera Saccharomyces, Candida, Kluyveromyces, Hansenula,
Schizosaccharomyces, Yarrowia, Pichia and Aspergillus.
[0044] Preferably the E. coli host cell is an E. coli host cell
which is recognized by the industry and regulatory authorities
(including but not limited to an E. coli K12 host cell or as
demonstrated in the Examples, an E. coli BL21 host cell).
[0045] One preferred host cell to use with the present disclosure
is E. coli, which may be recombinantly prepared as described
herein. Thus, the recombinant host may be a recombinant E. coli
host cell. There are libraries of mutants, plasmids, detailed
computer models of metabolism and other information available for
E. coli, allowing for rational design of various modules to enhance
product yield. Methods similar to those described above for
Saccharomyces can be used to make recombinant E. coli
microorganisms.
[0046] In one embodiment, the recombinant E. coli microorganism
comprises nucleotide sequences encoding SHC genes or functional
equivalents/homologies thereof including but not limited to
variants, homologues mutants, derivatives or fragments thereof.
[0047] Another preferred host cell to use with the present
disclosure is S. cerevisiae which is a widely used chassis organism
in synthetic biology. Thus, the recombinant host may be S.
cerevisiae. There are libraries of mutants, plasmids, detailed
computer models of metabolism and other information available for
S. cerevisiae, allowing for rational design of various modules to
enhance product yield. Methods are known for making recombinant S.
cerevisiae microorganisms.
[0048] Culturing of cells is performed, in a conventional manner.
The culture medium contains a carbon source, at least one nitrogen
source and inorganic salts, and vitamins are added to it. The
constituents of this medium can be the ones which are
conventionally used for culturing the species of microorganism in
question.
[0049] Carbon sources of use in the instant method include any
molecule that can be metabolized by the recombinant host cell to
facilitate growth and/or production of (-)-Ambrox. Examples of
suitable carbon sources include, but are not limited to, sucrose
(e.g., as found in molasses), fructose, xylose, glycerol, glucose,
cellulose, starch, cellobiose or other glucose containing
polymer.
[0050] In embodiments employing yeast as a host, for example,
carbon sources such as sucrose, fructose, xylose, ethanol,
glycerol, and glucose are suitable. The carbon source can be
provided to the host organism throughout the cultivation period or
alternatively, the organism can be grown for a period of time in
the presence of another energy source, e.g., protein, and then
provided with a source of carbon only during the fed-batch
phase.
[0051] The suitability of a recombinant host cell microorganism for
use in the methods of the present disclosure may be determined by
simple test procedures using well known methods. For example, the
microorganism to be tested may be propagated in a rich medium
(e.g., LB-medium, Bacto-tryptone yeast extract medium, nutrient
medium and the like) at a pH, temperature and under aeration
conditions commonly used for propagation of the microorganism. Once
recombinant microorganisms (i.e. recombinant host cells) are
selected that produce the desired products of bioconversion, the
products are typically produced by a production host cell line on
the large scale by suitable expression systems and fermentations,
e.g. by microbial production in cell culture.
[0052] In one embodiment of the present disclosure, a defined
minimal medium such as M9A is used for cell cultivation. The
components of M9A medium comprise: 14 g/L KH.sub.2PO.sub.4, 16 g/L
K.sub.2HPO.sub.4, 1 g/L Na.sub.3Citrate.2H.sub.2O, 7.5 g/L
(NH.sub.4).sub.2SO.sub.4, 0.25 g/L MgSO.sub.4.7H.sub.2O, 0.015 g/L
CaCl.sub.2.2H.sub.2O, 5 g/L of glucose and 1.25 g/L yeast
extract).
[0053] In another embodiment of the present disclosure, nutrient
rich medium such as LB (Luria-Bertani) was used. The components of
LB comprise: 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl).
[0054] Other examples of Mineral Medium and M9 Mineral Medium are
disclosed, for example, in U.S. Pat. No. 6,524,831B2 and US
2003/0092143A1.
[0055] The recombinant microorganism may be grown in a batch, fed
batch or continuous process or combinations thereof. Typically, the
recombinant microorganism is grown in a fermentor at a defined
temperature in the presence of a suitable nutrient source, e.g., a
carbon source, for a desired period of time to bioconvert
homofarnesol to (-)-Ambrox in a desired amount.
[0056] The recombinant host cells may be cultivated in any suitable
manner, for example by batch cultivation or fed-batch cultivation.
As used herein, the term "batch cultivation" is a cultivation
method in which culture medium is neither added nor withdrawn
during the cultivation. As used herein, the term "fed-batch" means
a cultivation method in which culture medium is added during the
cultivation but no culture medium is withdrawn.
[0057] One embodiment of the present disclosure provides a method
of producing (-)-Ambrox in a cellular system comprising producing
wild type SHC or variants thereof under suitable conditions in a
cellular system, feeding homofarnesol to the cellular system,
converting the homofarnesol to (-)-Ambrox using the SHC or variants
produced using the cellular system, collecting (-)-Ambrox from
cellular system and isolating the (-)-Ambrox from the system.
Expression of other nucleotide sequences may serve to enhance the
method. The bioconversion method can include the additional
expression of other nucleotide sequences in the cellular system.
The expression of other nucleotide sequences may enhance the
bioconversion pathway for making (-)-Ambrox.
[0058] A further embodiment of the present disclosure is a
bioconversion method of making (-)-Ambrox comprising growing host
cells comprising wild type SHC or variant genes, producing wild
type SHC or variant enzymes in the host cells, feeding homofarnesol
(e.g. EEH) to the host cells, incubating the host cells under
conditions of pH, temperature and solubilizing agent suitable to
promote the conversion of homofarnesol to Ambrox and collecting
(-)-Ambrox. The production of the wild type SHC or variant enzymes
in the host cells provides a method of making (-)-Ambrox when
homofarnesol is added to the host cells under suitable reaction
conditions. Achieved conversion may be enhanced by adding more
biocatalyst and SDS to the reaction mixture.
[0059] The recombinant host cell microorganism may be cultured in a
number of ways in order to provide cells in suitable amounts
expressing the wild type SHC or variant enzymes for the subsequent
bioconversion step. Since the microorganisms applicable for the
bioconversion step vary broadly (e.g. yeasts, bacteria and fungi),
culturing conditions are, of course, adjusted to the specific
requirements of each species and these conditions are well known
and documented. Any of the art known methods for growing cells of
recombinant host cell microorganisms may be used to produce the
cells utilizable in the subsequent bioconversion step of the
present disclosure. Typically the cells are grown to a particular
density (measurably as optical density (OD)) to produce a
sufficient biomass for the bioconversion reaction. The cultivation
conditions chosen influence not only the amount of cells obtained
(the biomass) but the quality of the cultivation conditions also
influences how the biomass becomes a biocatalyst. The recombinant
host cell microorganism expressing the wild type SHC or variant
genes and producing the wild type SHC or variant enzymes is termed
a biocatalyst which is suitable for use in a bioconversion
reaction. In some embodiments the biocatalyst is a recombinant
whole cell producing wild type SHC or variant enzymes or it may be
in suspension or an immobilized format.
[0060] In one embodiment, the biocatalyst is produced in sufficient
amounts (to create a sufficient biomass), harvested and washed (and
optionally stored (e.g. frozen or lyophilized)) before the
bioconversion step.
[0061] In a further embodiment, the cells are produced in
sufficient amounts (to create a sufficient biocatalyst) and the
reaction conditions are then adjusted without the need to harvest
and wash the biocatalyst for the bioconversion reaction. This one
step (or "one pot") method is advantageous as it simplifies the
process while reducing costs. The culture medium used to grow the
cells is also suitable for use in the bioconversion reaction
provided that the reaction conditions are adjusted to facilitate
the bioconversion reaction.
[0062] The bioconversion methods of the present disclosure are
carried out under conditions of time, temperature, pH and
solubilizing agent to provide for conversion of the homofarnesol
feedstock to (-)-Ambrox The pH of the reaction mixture may be in
the range of 4-8, preferably, 5 to 6.5, more preferably 4.8-6.0 for
the SHC variant enzymes and in the range of from about pH 5.0 to
about pH 7.0 for the wild type SHC enzyme and can be maintained by
the addition of buffers to the reaction mixture. An exemplary
buffer for this purpose is a citric acid buffer. The preferred
temperature is between from about 15.degree. C. and about
45.degree. C., preferably about 20.degree. C. and about 40.degree.
C. although it can be higher, up to 55.degree. C. for thermophilic
organisms especially if the wild type enzyme from a thermophilic
microorganism is used. The temperature can be kept constant or can
be altered during the bioconversion process.
[0063] The Applicant has demonstrated that it may be useful to
include a solubilizing agent (e.g. a surfactant, detergent,
solubility enhancer, water miscible organic solvent and the like)
in the bioconversion reaction. Examples of surfactants include but
are not limited to Triton X-100, Tween 80, taurodeoxycholate,
Sodium taurodeoxycholate, Sodium dodecyl sulfate (SDS), and/or
sodium lauryl sulfate (SLS).
[0064] The Applicant has selected and identified SDS as a
particularly useful solubilizing agent from a long list of other
less useful solubilizing agents. In particular, the Applicant
identified SDS as a remarkably better solubilizing agent than e.g.
Triton X-100 in terms of reaction velocity and yield for the
homofarnesol to (-)-Ambrox bioconversion reaction.
[0065] Without wishing to be bound by theory, the use of SDS with
recombinant microbial host cells may be advantageous as the SDS may
interact advantageously with the host cell membrane in order to
make the SHC enzyme (which is a membrane bound enzyme) more
accessible to the homofarnesol substrate. In addition, the
inclusion of SDS at a suitable level in the reaction mixture may
improve the properties of the emulsion (homofarnesol in water)
and/or improve the access of the homofarnesol substrate to the SHC
enzyme within the host cell while at the same time preventing the
disruption (e.g. denaturation/inactivation of the wild type SHC or
variant enzyme).
[0066] The concentration of the solubilising agent (e.g. SDS) used
in the bioconversion reaction is influenced by the biomass amount
and the substrate (EEH) concentration. That is, there is a degree
of interdependency between the solubilising agent (e.g. SDS)
concentration, the biomass amount and the substrate (EEH)
concentration. By way of example, as the concentration of
homofarnesol substrate increases, sufficient amounts of biocatalyst
and solubilising agent (e.g. SDS) are required for an efficient
bioconversion reaction to take place. If, for example, the
solubilising agent (e.g. SDS) concentration is too low, a
suboptimal homofarnesol conversion may be observed. On the other
hand, if, for example, the solubilising agent (e.g. SDS)
concentration is too high, then there may be a risk that the
biocatalyst is affected through either the disruption of the intact
microbial cell and/or an denaturation/inactivation of the SHC/HAC
enzyme.
[0067] The selection of a suitable concentration of SDS in the
context of the biomass amount and substrate (EEH) concentration is
within the knowledge of the Skilled Person. By way of example, a
predictive model is available to the Skilled Person to determine
the suitable SDS, substrate (EEH) and biomass concentrations.
[0068] The temperature of the bioconversion reaction for a wild
type SHC enzyme is from about 45-60.degree. C., preferably
55.degree. C.
[0069] The pH range of the bioconversion reaction for a wild type
SHC enzyme is from about 5.0 to 7.0, more preferably from about 5.6
to about 6.2, even more preferably about 6.0.
[0070] The temperature of the bioconversion reaction for a SHC
variant enzyme is about 34.degree. C. to about 50.degree. C.,
preferably about 35.degree. C.
[0071] The pH of the bioconversion reaction for a SHC variant
enzyme is about 4.8-6.4, preferably about 5.2-6.0.
[0072] Preferably the solubilising agent used in the bioconversion
reaction is SDS.
[0073] The [SDS]/[cells] ratio is in the range of about, 10:1-20:1,
preferably about 15:1-18:1, preferably about 16:1 when the ratio of
biocatalyst to EEH homofarnesol is about 2:1
[0074] The SDS concentration in the bioconversion reaction for a
SHC variant enzyme is in the range of about 1-2%, preferably in the
range of about 1.4-1.7%, even more preferably about 1.5% when the
homofarnesol concentration is about 125 g/l EEH and the biocatalyst
concentration is 250 g/l (corresponding to an OD of about 175 (650
nm)).
[0075] The ratio of biocatalyst to EEH homofarnesol substrate is in
the range of about 0.5:1-2:1, in some embodiments 2:1, preferably
about 1:1 or 0.5:1.
[0076] In some embodiments, (-)-Ambrox is produced using a
biocatalyst to which the homofarnesol substrate is added. It is
possible to add the substrate by feeding using known means (e.g.
peristaltic pump, infusion syringe and the like). Homofarnesol is
an oil soluble compound and is provided in an oil format. Given
that the biocatalyst is present in an aqueous phase, the
bioconversion reaction may be regarded as a two phase system when
homofarnesol is added to the bioconversion reaction mixture. This
is the case even when a solubilizing agent (e.g. SDS) is
present.
[0077] Further details of a suitable bioconversion process are
disclosed in the examples, set forth herein below.
[0078] The bioconversion process according to the present invention
produces a reaction mixture containing the desired (-)-Ambrox, and
also a number of by-products. More particularly, the reaction
mixture contains, in addition to (-)-Ambrox a complex mixture of
by-products, including a novel constitutional isomer of (-)-Ambrox
according to the formula (II), as well as known stereo isomers of
(-)-Ambrox according to the formulae (III) and (IV)
##STR00003##
[0079] The applicant believes, although does not intend to be bound
by any particular theory, that the compound of formula (II) is
formed by the cyclization of the 7E,3Z-geometric isomer of
homofarnesol. It has been described as practically odourless, with
a detection threshold of >500 ng/l.
[0080] As stated above, the applicant believes that the compound of
formula (II) is a novel molecule, and as such, this compound forms
a further aspect of the present invention. Perfume ingredients and
perfume compositions consisting of or comprising the compound (II),
as well as perfumed articles containing same, form additional
aspects of the invention.
[0081] The use of the compound of formula (II) as a perfume
ingredient in perfumery applications, such as fine perfumes or
functional perfume compositions such as personal care, household
care and fabric care compositions, forms further additional aspects
of the invention.
[0082] Mixtures of (-)-Ambrox and an olfactory acceptable amount of
compound (II) forms still another aspect of the present
invention.
[0083] The term "olfactory acceptable amount" as used herein in
relation to the compound of formula (II), or any of the other
by-products (III) or (IV), or indeed, any material that may be
present as an impurity in (-)-Ambrox formed in accordance with a
method of the present invention, is understood to mean that the
compound or material is present in a mixture with (-)-Ambrox in an
amount below its odour detection threshold, or in an amount at
which it will not contribute its olfactory characteristics in a way
that will affect the olfactory character of (-)-Ambrox. (-)-Ambrox
containing an olfactory acceptable amount of any such compound or
material would be identifiable to a skilled perfumer as possessing
the odour character of commercial grades of (-)-Ambrox, such as
AMBROFIX.TM. obtained by a synthetic procedure ex-sclareol, and
available from Givaudan.
[0084] In preferred embodiments of the present invention, the
reaction mixture contains no, or substantially no, unreacted
homofarnesol.
[0085] The applicant discovered that homofarnesol was a powerful
solvent for (-)-Ambrox as well as for the aforementioned
by-products of the bioconversion process. As such, in the presence
of appreciable amounts of homofarnesol, (-)-Ambrox and the
by-products remain dissolved together in a crude, intractable
mixture, from which it is difficult, protracted and costly to
separate and ultimately isolate (-)-Ambrox in olfactively pure
form. Reducing the level of unreacted homofarnesol in admixture
with (-)-Ambrox and the compounds (II), (III) and (IV) was found to
considerably facilitate downstream processing and
isolation/purification of (-)-Ambrox.
[0086] Downstream processing, as will be appreciated by persons
skilled in the art, is a critical operation in the manufacture of
useful compounds formed by bioconversion processes. As part of the
synthesis of a compound, it can affect the compound's physical
properties. In the case of the preparation of perfume ingredients
by biotech methods, it is desirable that a target compound can be
separated from a reaction mixture in olfactively pure form in order
that the desired odour characteristics of the target compound are
not distorted by odour contributions of the complex mixture of
contaminants and by-products that may be present in the
fermentation medium or the biocatalyst.
[0087] Accordingly, the invention provides in another of its
aspects a method of isolating and purifying (-)-Ambrox from a
reaction mixture, comprising one or more of the compounds (II),
(III) and (IV).
[0088] In yet another aspect of the present invention there is
provided a method of improving or enhancing the odour of
(-)-Ambrox, comprising the steps of separation and purification of
(-)-Ambrox from a reaction mixture containing one or more of the
compounds (II), (III) and (IV).
[0089] In an isolated and purified form, (-)-Ambrox either does not
contain any of the compounds (II), (III) or (IV), or if it does
contain any of said compounds, then each should be present in an
olfactory acceptable amount.
[0090] The reaction mixture obtained from the bioconversion
process, such as a process as described herein above, generally
comprises a solid phase containing crude (-)-Ambrox and one or more
of the by-products (II), (III) and (IV), as well as cellular
material and/or debris thereof; and a liquid phase or liquid phases
comprising water and/or any unreacted homofarnesol.
[0091] The solid phase may be separated from the liquid phase or
phases by filtration or centrifugation. Furthermore, by selecting a
filter with an appropriate pore size, it is also possible to effect
separation of the crude (-)-Ambrox, from the cellular material
and/or debris. Once the crude (-)-Ambrox is separated from cellular
material and/or debris thereof, it may be washed, before being
subjected to further work-up procedures to isolate (-)-Ambrox from
compounds (II), (III) and (IV).
[0092] Alternatively, instead of filtration or centrifugation, the
reaction mixture can be warmed to a temperature above the melting
point of (-)-Ambrox, whereupon (-)-Ambrox forms an oil phase above
an aqueous phase containing the cellular material and debris.
Optionally, and in order to ensure a complete recovery of
(-)-Ambrox, the aqueous and cellular material can be washed with a
water-immiscible organic solvent (such as toluene) to remove any
residual (-)-Ambrox that may have been entrained in the aqueous
phase, and these washings can be combined with the oil phase. The
oil phase can thereafter be concentrated by evaporation to provide
a crude mixture comprising (-)-Ambrox and one or more of the
compounds (II), (III) and (IV),which mixture can then subjected to
further work-up procedures to isolate and purify (-)-Ambrox.
[0093] In another embodiment, instead of warming the reaction
mixture to form a (-)-Ambrox-containing oil phase, the reaction
mixture can be extracted with a suitable water-immiscible organic
solvent (such as toluene) to form an organic phase containing
(-)-Ambrox and one or more of the compounds (II), (III) and (IV),
which can be separated from an aqueous phase containing the
cellular material and debris. The organic phase can be concentrated
by evaporation to provide a crude mixture comprising (-)-Ambrox and
one or more of the compounds (II), (III) and (IV), which can be
subjected to further work-up procedures to isolate and purify
(-)-Ambrox.
[0094] In yet another alternative method, the reaction mixture can
be steam distilled to remove the distillate from the cellular
material and debris. The distillate can be collected as a biphasic
mixture, before the oil phase of the biphasic mixture comprising a
mixture of (-)-Ambrox and one or more of the compounds (II), (III)
and (IV) is separated from the aqueous phase, and then subjected to
further work-up procedures to isolate and purify (-)-Ambrox.
[0095] In a particular embodiment of the present invention, said
method of isolating and purifying (-)-Ambrox comprises the step of
selectively crystallizing (-)-Ambrox from a mixture containing one
or more of the compounds (II), (III) or (IV).
[0096] The phrase "selectively crystallizing" refers to a process
step whereby (-)-Ambrox is caused to crystallize from a solvent,
whilst the compounds (II), (III) and (IV) remain dissolved in the
crystallizing solvent, to such an extent that isolated crystalline
material contains only (-)-Ambrox, or if it contains any of the
compounds (II), (III) or (IV), then they are present only in
olfactory acceptable amounts.
[0097] Crystallization may be carried out in a suitable organic
solvent. The choice of solvent is based on considerations, such as
solubility differences at room temperature and at high
temperatures, or in boiling solvent; and for the need of an
abundance of crystals recoverable in cool solvent. Usually, a
compound to be separated is dissolved in a relatively polar solvent
and then a relatively less polar solvent can be added to bring the
dissolved compound to its solubility limit, whereupon it will start
to crystallize. Also, in an industrial process, issues of cost as
well as safety of handling are relevant. Suitable solvents include,
but are not limited to methanol, acetone, petroleum ether, hexane,
t-butyl methyl ether, THF and ethyl acetate. Preferred solvents
include toluene or ethyl alcohol. Pairs of solvents may also be
employed.
[0098] In a particularly preferred embodiment of the present
invention, selective crystallization is undertaken by dissolving
the mixture containing (-)-Ambrox and one or more of the compounds
(II), (III) and (IV) in warm ethanol and allowing (-)-Ambrox to
selectively crystallize by slowly adding a non-solvent, such as
water, to the cooling ethanolic solution.
[0099] Considering the close structural relationship of (-)-Ambrox
and the by-product compounds (II), (III) and (IV), which are
respectively a constitutional isomer and two stereoisomers of
(-)-Ambrox, it was remarkable that (-)-Ambrox could be selectively
crystallized from such a mixture, to provide (-)-Ambrox in
olfactively pure form and in high yields. The skilled person would
reasonably anticipate that the compounds would co-crystallize with
(-)-Ambrox, rendering downstream processing far more complex,
time-consuming and expensive than was found to be the case.
[0100] The surprisingly facile manner in which (-)-Ambrox could be
separated from a mixture containing compound (II), (III) and/or
(IV) by crystallization represents a particular advantage of the
present invention.
[0101] The ease with which (-)-Ambrox could be separated by
crystallization could be contrasted with the observation that
(-)-Ambrox could not be recovered in such a facile manner and in
such high yield from a mixture containing (II), (III) and/or (IV)
by other purification techniques, such as by distillation, owing to
the very similar boiling points of (-)-Ambrox and the by-products
(II), (III) and (IV).
[0102] The term "olfactively pure" as it is used in relation to
(-)-Ambrox, is intended to mean that (-)-Ambrox is free of
compounds (II), (III) or (IV), or any other material found in the
reaction mixture, or that if such compounds or materials should be
present, they are present in olfactory acceptable amounts, as that
term is defined herein.
[0103] In an embodiment of the invention (-)-Ambrox in olfactively
pure form contains less than 5% by weight of any of the compounds
(II), (III) or (IV).
[0104] In more particular embodiments, (-)-Ambrox in olfactively
pure form contains less than 4%, less than 3%, less than 2%, less
than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than
0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than
0.2%, less than 0.1%, or less than 0.05% by weight of each of the
compounds (II), (III) or (IV).
[0105] The quality of separation of (-)-Ambrox from the mixture of
the compounds (II), (III) and/or (IV) by selective crystallization
may be influenced by the composition of the mixture from which it
is separated. More particularly, the quality of the separation of
(-)-Ambrox from a mixture of compounds (II), (III) and/or (IV) by
crystallization was improved when the weight ratio of (-)-Ambrox to
the other compounds (II), (III) and (IV) in the mixture was greater
than 70:30, more particularly 80:20, more particularly 90:10, still
more particularly 95:5, and more particularly still 97:3.
[0106] Furthermore, the quality of the separation of (-)-Ambrox by
crystallization may be influenced by the amount of unreacted
homofarnesol present in the mixture from which it is separated.
More particularly, the quality of separation is improved when the
level of unreacted homofarnesol is less than 30 wt % by weight,
more particularly less than 20 wt %, more particularly less than
10% by weight, more particularly still less than 5 wt % and still
more particularly less than 3% by weight, still more particularly
less than 2% by weight, and more particularly still less than 1% by
weight, based on the weight of the mixture from which (-)-Ambrox is
crystallized.
[0107] Preferably, the reagents and reaction conditions employed in
the bioconversion process of the present invention are such that
the reaction proceeds with 100% conversion of homofarnesol, or
substantially so, thus leaving no unreacted homofarnesol in the
reaction mixture. However, if unreacted homofarnesol is present,
although economically disadvantageous, it can be separated from
(-)-Ambrox and other by-products by distillation, for example.
[0108] Accordingly, in a particular embodiment of the invention,
there is provided a method of isolating and purifying (-)-Ambrox
from a mixture comprising one or more of the compounds (II), (III)
and (IV), which mixture is free, or substantially free, of
homofarnesol.
[0109] In a more particular embodiment, the isolation and
purification of (-)-Ambrox from a mixture comprising one or more of
the compounds (II), (III) and (IV), and free or substantially free
of homofarnesol, is achieved by the selective crystallization of
(-)-Ambrox.
[0110] (-)-Ambrox obtained according to a method of the present
invention is obtained in olfactively pure form. Olfactively pure
(-)-Ambrox forms another aspect of the present invention.
[0111] (-)-Ambrox in crystalline form forms yet another aspect of
the present invention.
[0112] (-)-Ambrox formed in accordance with the method of the
present invention may be mixed with one or more additional perfume
ingredients to form perfume compositions that find use in perfumery
applications, including use in fine perfumery, as well as use in
consumer products, such as personal care, fabric care and household
care applications.
[0113] Accordingly, the invention provides in another of its
aspects a perfume composition comprising (-)-Ambrox and at least
one other perfume ingredient, wherein said perfume composition
contains olfactory acceptable amounts of one or more of the
compounds (II), (III) or (IV).
[0114] The invention will be further illustrated with reference to
the following examples.
EXAMPLES
Example 1
[0115] Preparation of Homofarnesol
[0116] General Analytical Conditions:
[0117] Non-polar GC/MS: 50.degree. C./2 min, 20.degree. C./min
200.degree. C., 35.degree. C./min 270.degree. C. GC/MS Agilent
5975C MSD with HP 7890A Series GC system. Non-polar column: BPX5
from SGE, 5% phenyl 95% dimethylpolysiloxane 0.22 mm.times.0.25
mm.times.12 m. Carrier Gas: Helium. Injector temperature:
230.degree. C. Split 1:50. Flow: 1.0 ml/min. Transfer line:
250.degree. C. MS-quadrupol: 106.degree. C. MS-source: 230.degree.
C.
A) Preparation of MNU in THF
[0118] A solution of urea (175 g, 2.9 mol) and methylamine
hydrochloride (198 g, 2.9 mol) in water (400 ml) is heated at
reflux (105.degree. C.) for 3.5 h under stirring. At 40.degree. C.
NaNO2 (101 g, 1.45 mol) dissolved in water (200 ml) is added. After
15 min THF (1000 ml) is added which results in a transparent
2-phase mixture. Conc. H2SO4 (110 g, 1.1 mol) is added at
0-5.degree. C. and stirring within 1.5 h. After another 0.5 h at
0-5.degree. C. the two transparent phases are separated at
25.degree. C. The organic phase (A) (1065 ml, theoretically 1.35 M)
is stored for a few days at 0-5.degree. C. or forwarded immediately
to the cyclopropanation reactor.
[0119] After phase separation the water phase is extracted twice
with THF (2.times.1 l). This gives 1100 ml of phase B and 1075 of
phase C. Whereas phase A gives a 51% conversion of a terminal
alkene to a cyclopropane in a subsequent cyclopropanation reaction,
phase B gives <0.5% cyclopropane and phase C gives no detectable
conversion. We conclude that >99% MNU is extracted after the
first phase separation. Usually the water phase is therefore
discarded after the first phase separation (from organic phase A)
after treatment with conc. aqueous KOH and acetic acid.
B) Preparation of E-.DELTA.-Farnesene Using MNU in THF
##STR00004##
[0121] N-Methyl-N-nitroso urea 1.35 M in THF (136 ml, 184 mmol) is
added dropwise at 0.degree. C. to a rapidly stirred mixture of
E-beta-Farnesene (CAS 18794-84-8) (25 g, 122 mmol) and aqueous KOH
(50 ml, 40%) at 0-5.degree. C. After the addition of 4 ml of the
MNU solution, Pd(acac)2 (7.4 mg, 0.024 mmol, 0.02%) pre-dissolved
in 0.5 ml dichloromethane is added. The remaining MNU solution is
added over 4 h at 0-5.degree. C. A GC at this stage showed 28%
unconverted E-beta-Farnesene, 65% of the desired monocyclopropane
(shown above) and 3% of a biscyclopropanated compound 5. After 16 h
at 25.degree. C. acetic acid (100 ml) is added at 0-5.degree. C.,
then tert-butyl methyl ether (250 ml). After phase separation the
organic phase is washed with 2M HCl (250 ml) and the aqueous phase
extracted with tert-butyl methyl ether (250 ml). The combined
organic layers are washed with water (2.times.100 ml), aqueous 10%
NaOH (2.times.100 ml) and water (2.times.100 ml), dried over
MgSO.sub.4, filtered and concentrated to give 26.9 g of a slightly
yellow liquid which contains 9% E-beta-Farnesene, 82% of the
desired monocyclopropane compound and 6% of a biscyclopropanated
side product.
[0122] The desired compound could be further isolated by
distillative purification.
[0123] Addition of 1 g K.sub.2CO.sub.3 (1 g) and distillation over
a 30 cm steel coil column at 40-60 mbar gives 147 g
monocyclopropane compound (68% corr) at 135-145.degree. C. The
fractions are pooled to give 92 g monocyclopropane compound of 100%
purity.
[0124] Analytical data of E-.DELTA. Farnesene:
[0125] 1H-NMR (CDCl3, 400 MHz): 5.1 (2 m, 2 H), 4.6 (2 H), 2.2 (2
H), 2.1 (4 H), 2.0 (2 H), 1.7 (s, 3 H), 1.6 (2 s, 6 H), 1.3 (1 H),
0.6 (2 H), 0.45 (2 H) ppm. 13C-NMR (CDCl3, 400 MHz): 150.9 (s),
135.1 (s), 131.2 (s), 124.4 (d), 124.1 (d), 106.0 (t), 39.7 (t),
35.9 (t), 26.7 (t), 25.7 (q), 17.7 (q), 16.0 (d), 6.0 (t) ppm.
GC/MS: 218 (2%, M+), 203 (5%, [M-15]+), 175 (11%), 147 (31%), 134
(15%), 133 (20%), 121 (12%), 107 (55%), 95 (16%), 93 (30%), 91
(20%), 82 (11%), 81 (33%), 79 (42%), 69 (100%), 67 (22%), 55 (20%),
53 (21%), 41 (75%). IR (film): 3081 (w), 2967 (m), 2915 (m), 2854
(m), 1642 (m), 1439 (m), 1377 (m), 1107 (w), 1047 (w), 1018 (m),
875 (s), 819 (m), 629 (w). Anal. calcd. for C16H26: C, 88.00; H,
12.00. Found: C, 87.80; H, 12.01.
C) Preparation of (7E)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol
((7E)-homofarnesol)
[0126] A mixture of
(E)-(6,10-dimethylundeca-1,5,9-trien-2-yl)cyclopropane (E-.DELTA.
Farnesene) (1 g, 4.6 mmol), dodecane (0.2 g, 1.15 mmol, internal
standard) and L-(+)-tartaric acid (1 g, 6.9 mmol) in a pressure
tube is heated under stirring at 150.degree. C. After 18 h and
complete conversion (according to GC) the mixture is poured on
water (50 ml) and toluene (50 ml). The phases are separated and the
aqueous phase extracted with toluene (50 ml). The combined organic
layers are washed with conc. aqueous Na.sub.2CO.sub.3 (50 ml) and
conc. NaCl (2.times.50 ml), dried over MgSO.sub.4, filtered and
evaporated under reduced pressure to give a brownish resin (1.35 g)
which is mixed with 30% aqueous KOH (4.3 ml) and stirred at
25.degree. C. for 2 h. GC analysis reveals formation of 96%
(7E)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol according to the
internal standard. E/Z ratio 68:22. The analytical data of the
E-isomer are consistent with the ones from the literature, see for
example P. Kocienski, S. Wadman J. Org. Chem. 54, 1215 (1989).
Example 2
[0127] SHC Plasmid Preparation and Biocatalyst Production
[0128] SHC Plasmid Preparation
[0129] The gene encoding Alicyclobacillus acidocaldarius squalene
hopene cyclase (AacSHC) (GenBank M73834, Swissprot P33247) was
inserted into plasmid pET-28a(+), where it is under the control of
an IPTG inducible T7-promotor for protein production in Escherichia
coli. The plasmid was transformed into E. coli strain BL21(DE3)
using a standard heat-shock transformation protocol.
[0130] Erlenmeyer Flask Cultures
[0131] For protein production were used either rich medium (LB
medium) or minimal media. M9 is one example of minimal media, which
were successfully used.
[0132] Media Preparation
[0133] The minimal medium chosen as default was prepared as follows
for 350 ml culture: to 35 ml citric acid/phosphate stock (133 g/l
KH.sub.2PO.sub.4, 40 g/l (NH.sub.4).sub.2HPO.sub.4, 17 g/g citric
acid.H.sub.2O with pH adjusted to 6.3) was added 307 ml H.sub.2O,
the pH adjusted to 6.8 with 32% NaOH as required. After autoclaving
0.850 ml 50% MgSO.sub.4, 0.035 ml trace elements solution
(composition in next section) solution, 0.035 ml Thiamin solution
and 7 ml 20% glucose were added.
[0134] SHC Biocatalyst Production (Biocatalyst Production)
[0135] Small scale biocatalyst production (wild-type SHC or SHC
variants), 350 ml culture (medium supplemented with 50 .mu.g/ml
kanamycin) were inoculated from a pre-culture of the E. coli strain
BL21(DE3) containing the SHC production plasmid. Cells were grown
to an optical density of approximately 0.5 (OD.sub.650 nm) at
37.degree. C. with constant agitation (250 rpm).
[0136] Protein production was then induced by the addition of IPTG
to a concentration of 300 .mu.M followed by incubation for a
further 5-6 hours with constant shaking. The resulting biomass was
finally collected by centrifugation, washed with 50 mM Tris-HCl
buffer pH 7.5. The cells were stored as pellets at 4.degree. C. or
-20.degree. C. until further use. In general 2.5 to 4 grams of
cells (wet weight) were obtained from 1 litre of culture,
independently of the medium used.
[0137] The fermentation was prepared and run in 750 ml InforsHT
reactors. To the fermentation vessel was added 168 ml deionized
water. The reaction vessel was equipped with all required probes
(pO.sub.2, pH, sampling, antifoam), C+N feed and sodium hydroxide
bottles and autoclaved. After autoclaving, the following
ingredients are added to the reactor: [0138] 20 ml 10.times.
phosphate/citric acid buffer [0139] 14 ml 50% glucose [0140] 0.53
ml MgSO.sub.4 solution [0141] 2 ml (NH.sub.4).sub.2SO.sub.4
solution [0142] 0.020 ml trace elements solution [0143] 0.400 ml
thiamine solution [0144] 0.200 ml kanamycin stock
[0145] The reaction conditions are set as follows: pH=6.95,
pO.sub.2=40%, T=30.degree. C., Stirring at 300 rpm. Cascade: rpm
setpoint at 300, min 300, max 1000, flow l/min set point 0.1, min
0, max 0.6. Antifoam control: 1:9.
[0146] The fermenter was inoculated from a seed culture to an
OD.sub.650 nm of 0.4-0.5. This seed culture was grown in LB medium
(+Kanamycin) at 37.degree. C., 220 rpm for 8 h. The fermentation
was run first in batch mode for 11.5 h, where after was started the
C+ N feed with a feed solution (sterilized glucose solution (143 ml
H.sub.2O+ 35 g glucose) to which had been added after
sterilization: 17.5 ml (NH.sub.4).sub.2SO.sub.4solution, 1.8 ml
MgSO.sub.4 solution, 0.018 ml trace elements solution, 0.360 ml
Thiamine solution, 0.180 ml kanamycin stock. The feed was run at a
constant flow rate of approx. 4.2 ml/h. Glucose and NH.sub.4.sup.+
measurements were done externally to evaluate availability of the
C- and N-sources in the culture. Usually glucose levels stay very
low.
[0147] Cultures were grown for a total of approximately 25 hours,
where they reached typically and OD.sub.650 nm of 40-45. SHC
production was then started by adding IPTG to a final concentration
of approx. 1 mM in the fermenter (as IPTG pulse or over a period of
3-4 hours using an infusion syringe), setting the temperature to
40.degree. C. and pO.sub.2 to 20%. Induction of SHC production
lasted for 16 h at 40.degree. C. At the end of induction the cells
were collected by centrifugation, washed with 0.1 M citric
acid/sodium citrate buffer pH 5.4 and stored as pellets at
4.degree. C. or -20.degree. C. until further use.
[0148] Results Ia
[0149] In general, with all other conditions unchanged the specific
activity of the produced biocatalyst was higher when a minimal
medium was used compared with a rich medium.
[0150] Induction was carried out successfully at 30 or 37.degree.
C. It was noted that when the induction was done at 40-43.degree.
C., a biocatalyst of higher specific activity was obtained.
[0151] Results Ib
[0152] The following Table 1 shows for two examples the culture
volume, optical density and amount of cells both at induction start
and induction end as well as the amount of biomass collected (wet
weight).
TABLE-US-00001 TABLE 1 cells cells Volume.sub.induction start
calculated Volume.sub.induction end collected (ml) OD.sub.650 nm
induction start (g) (ml) OD.sub.650 nm, .sub.induction end (g)
Example 1 273 40 10.9 342 55 28 Example 2 272 44 12.0 341 57 23
OD.sub.650 nm at inoculation: 0.45 (Example 1) and 0.40 (Example
2). Starting volumes: 205 ml.
TABLE-US-00002 Wild type SHCamino acid sequence (GenBank M73834,
Swissprot P33247) (SEQ ID No. 1)
MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQRE-
DGTWALY
PGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFL-
GKRMPLN
IYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPF-
RRAAEIR
ALDWLLERQAGDGSWGGIQPPWFYALTALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLA-
VLALRAA
GLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRD-
AMTKGFR
WIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQ-
KPDGSWF
GRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWAL-
MALIAGG
RAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR
Variant F601Y SHC amino acid sequence-variant with respect to SEQ
ID No. 1 (SEQ ID No. 2)
MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQRE-
DGTWALY
PGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFL-
GKRMPLN
IYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPF-
RRAAEIR
ALDWLLERQAGDGSWGGIQPPWFYALTALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLA-
VLALRAA
GLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRD-
AMTKGFR
WIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQ-
KPDGSWF
GRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWAL-
MALIAGG
RAESEAARRGVQYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR
Variant F605W SHC nucleotide sequence (SEQ ID No. 3)
ATGGCTGAGCAGTTGGTGGAAGCGCCGGCCTACGCGCGGACGCTGGATCGCGCGGTGGAGTATCTCCTCTCCTG-
CCAAAAG
GACGAAGGCTACTGGTGGGGGCCGCTTOTGAGCAACGTCACGATGGAAGCGGAGTACGTCCTCTTGTGCCACAT-
TCTCGAT
CGCGTCGATCGGGATCGCATGGAGAAGATCCGGCGGTACCTGTTGCACGAGCAGCGCGAGGACGGCACGTGGGC-
CCTGTAC
CCGGGTGGGCCGCCGGACCTCGACACGACCATCGAGGCGTACGTCGCGCTCAAGTATATCGGCATGTCGCGCGA-
CGAGGAG
CCGATGCAGAAGGCGCTCCGGTTCATTCAGAGCCAGGGCGGGATCGAGTCGTCGCGCGTGTTCACGCGGATGTG-
GCTGGCG
CTGGTGGGAGAATATCCGTGGGAGAAGGTGCCCATGGTCCCGCCGGAGATCATGTTCCTCGGCAAGCGCATGCC-
GCTCAAC
ATCTACGAGTTTGGCTCGTGGGCTCGGGCGACCGTCGTGGCGCTCTCGATTGTGATGAGCCGCCAGCCGGTGTT-
CCCGCTG
CCCGAGCGGGCGCGCGTGCCCGAGCTGTACGAGACCGACGTGCCTCCGCGCCGGCGCGGTGCCAAGGGAGGGGG-
TGGGTGG
ATCTTCGACGCGCTCGACCGGGCGCTGCACGGGTATCAGAAGCTGTCGGTGCACCCGTTCCGCCGCGCGGCCGA-
GATCCGC
GCCTTGGACTGGTTGCTCGAGCGCCAGGCCGGAGACGGCAGCTGGGGCGGGATTCAGCCGCCTTGGTTTTACGC-
GCTCATC
GCGCTCAAGATTCTCGACATGACGCAGCATCCGGCGTTCATCAAGGGCTGGGAAGGTCTAGAGCTGTACGGCGT-
GGAGCTG
GATTACGGAGGATGGATGTTTCAGGCTTCCATCTCGCCGGTGTGGGACACGGGCCTCGCCGTGCTCGCGCTGCG-
CGCTGCG
GGGCTTCCGGCCGATCACGACCGCTTGGTCAAGGCGGGCGAGTGGCTGTTGGACCGGCAGATCACGGTTCCGGG-
CGACTGG
GCGGTGAAGCGCCCGAACCTCAAGCCGGGCGGGTTCGCGTTCCAGTTCGACAACGTGTACTACCCGGACGTGGA-
CGACACG
GCCGTCGTGGTGTGGGCGCTCAACACCCTGCGCTTGCCGGACGAGCGCCGCAGGCGGGACGCCATGACGAAGGG-
ATTCCGC
TGGATTGTCGGCATGCAGAGCTCGAACGGCGGTTGGGGCGCCTACGACGTCGACAACACGAGCGATCTCCCGAA-
CCACATC
CCGTTCTGCGACTTCGGCGAAGTGACCGATCCGCCGTCAGAGGACGTCACCGCCCACGTGCTCGAGTGTTTCGG-
CAGCTTC
GGGTACGATGACGCCTGGAAGGTCATCCGGCGCGCGGTGGAATATCTCAAGCGGGAGCAGAAGCCGGACGGCAG-
CTGGTTC
GGTCGTTGGGGCGTCAATTACCTCTACGGCACGGGCGCGGTGGTGTCGGCGCTGAAGGCGGTCGGGATCGACAC-
GCGCGAG
CCGTACATTCAAAAGGCGCTCGACTGGGTCGAGCAGCATCAGAACCCGGACGGCGGCTGGGGCGAGGACTGCCG-
CTCGTAC
GAGGATCCGGCGTACGCGGGTAAGGGCGCGAGCACCCCGTCGCAGACGGCCTGGGCGCTGATGGCGCTCATCGC-
GGGCGGC
AGGGCGGAGTCCGAGGCCGCGCGCCGCGGCGTGCAATACCTCGTGGAGACGCAGCGCCCGGACGGCGGCTGGGA-
TGAGCCG
TACTACACCGGCACGGGCTTCCCAGGGGATTGGTACCTCGGCTACACCATGTACCGCCACGTGTTTCCGACGCT-
CGCGCTC GGCCGCTACAAGCAAGCCATCGAGCGCAGGTGA Variant F605W SHC amino
acid sequence-variant with respect to SEQ ID No. 1 (SEQ ID No. 4)
MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQRE-
DGTWALY
PGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFL-
GKRMPLN
IYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPF-
RRAAEIR
ALDWLLERQAGDGSWGGIQPPWFYALTALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLA-
VLALRAA
GLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRD-
AMTKGFR
WIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQ-
KPDGSWF
GRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWAL-
MALIAGG
RAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDWYLGYTMYRHVFPTLALGRYKQAIERR
Example 3
[0153] Bioconversion of 7E, 3E/Z-Homofarnesol Mixture
[0154] Bioconversion was undertaken using the following reaction
conditions:
[0155] The reaction (150.1 g total volume) run in 0.1 M citric
acid/sodium citrate buffer pH 5.4 in an InforsHT 750 ml fermenter
contained 146 g/l total homofarnesol using a homofarnesol
substrate, which was a mixture of 7E,3E:7E,3Z of 86:14, 250 g/l
cells (formed in accordance with the method of Example 2,
fermentation) and 1.55% SDS. The reaction was run at 35.degree. C.
with constant stirring (900 rpm), pH control was done using 10 to
40% citric acid in water.
[0156] The reaction mixture was subjected to isolation and
purification steps as set forth in Example 4, below.
Example 4
[0157] Downstream Processing Procedure
[0158] A reaction mixture formed from the bioconversion of 7E,
3E/Z-homofarnesol (86:14 3E:3Z) was subjected to steam
distillation. The distillate was collected as a biphasic mixture.
The organic phase was retained and the aqueous phase discarded. The
composition of the organic phase was analysed by GC and the results
shown in the Table 2 below (see "crude").
[0159] The organic phase was then concentrated to dryness. Ethanol
was then added to the crude, dried product and the mixture warmed
until the product was dissolved. At room temperature water is
slowly added and (-)-Ambrox crystallizes under occasional stirring
and cooling in an ice bath.
[0160] Table 1 shows the GC analytics results for the crystallized
product. The data show a strong enrichment of (-)-Ambrox, with
practically no by-products (a), (b) or (c) being found in the
crystallized sample.
[0161] It should be noted that in Table 2, "a", "b" and "c" refer
to compound (II), compound (IV) and compound (III) respectively.
"EZH" and "EEH" refer to 7E,3Z-homofarnesol and 7E,3E-homofarnesol
respectively.
TABLE-US-00003 TABLE 1 Peak area (GC) (--)-Ambrox a b c (--)-Ambrox
EZH EEH (%) Crude 215073 190376 588769 6751605 13429 14184 86.9
Crystallized 10088 8951 64625 9032941 0 0 99.1
Example 5
[0162] Extraction of the solid phase of the reaction broth:
[0163] Given that (-)-Ambrox is not soluble in water and is not
liquid at temperatures below approx. 75.degree. C., these
properties were taken as possible advantages to extract the product
from the solid phase of the biotransformation using either
water-miscible solvents (e.g. ethanol) and water-immiscible
solvents (e.g. toluene).
[0164] 200 ml reaction broth was centrifuged to separate the solid
from the liquid (aqueous) phase (Sorvall GS3, 5000 rpm, 10 min,
10.degree. C.). This separated approx. 80 ml solid pellet from
approx. an 120 ml liquid phase. Analysis (Gas chromatography) of
the aqueous phase after MTBE extraction showed that it contained
not more than approx. 0.3% of the (-)-Ambrox initially present in
the 200 ml reaction broth. Toluene and ethanol 99% were used for
extracting (-)-Ambrox from the solid phase.
[0165] Toluene Extraction:
[0166] 80 ml solid phase was extracted 6.times. with 45 ml toluene
(approx. 1/2 solid phase volume, vigorous shaking for 30 s,
centrifugation (Sorvall GS3, 5000 rpm, 10 min, 10.degree. C.). The
solvent phase was analyzed with GC for its (-)-Ambrox content. Over
99.5% of (-)-Ambrox initially present in the reaction broth was
extracted with 6 extractions representing a total toluene vol. of
1.35.times. the initial whole reaction broth volume (200 ml) or
3.4.times. the vol. of the solid phase.
[0167] Ethanol Extraction:
[0168] 80 ml solid phase was extracted (Infors Multifors HT,
35.degree. C., 1000 rpm, 30 min) with approx. 160 ml (2 vol.)
ethanol 99%, followed by centrifugation. (-)-Ambrox did not
crystallize during the extraction procedure. After 4 washes (total
640 ml ethanol, i.e. 3.2.times. the initial whole reaction broth
volume or 8.times. the volume of the solid phase), about 99% of
(-)-Ambrox initially present in the reaction broth was recovered.
Sufficient ethanol is required in the first extraction step to
prevent (-)-Ambrox crystallization (solubility in ethanol). When
only 1 or 1/2 vol of the solid phase was used in the first
extraction step, a sticky paste was obtained, which was difficult
to handle and (-)-Ambrox crystallized as needles on the pellet
during centrifugation. Temperature appeared as not being the factor
responsible for this crystallization (extraction and centrifugation
tested at room temperature and approx. 35.degree. C.-40.degree.
C.).
[0169] The (-)-Ambrox concentration in the ethanol phase as well as
the ethanol/water ratio of the liquid phase (residual moisture of
the solid phase) appeared to be responsible for crystal formation.
It was however noted that it was possible to reduce the volume of
ethanol to 1 vol of the solid phase.
[0170] As (-)-Ambrox is not in the liquid phase at room
temperature, it separates with the biomass and can be extracted
with an organic solvent (e.g. a water-miscible solvent (e.g.
ethanol) or a water-immiscible solvent (e.g. toluene). The
centrifugation step that separates the (-)-Ambrox into the solid
phase of the reaction mixture is advantageous because it reduces
the amount of solvent required to extract (-)-Ambrox.
Example 6
[0171] Sensory Analysis
[0172] Purpose: to carry out a sensory analysis of (-)-Ambrox and
the compounds (II), (III) and (IV) formed in the crude material and
in the crystallised material.
[0173] Biotransformation of E,E-homofarnesol results in (-)-Ambrox,
and compound (IV).
[0174] Biotransformation of E,Z-homofarnesol results in the
macrocyclic ether compound (II) and epi-Ambrox compound (III).
[0175] A crude mixture of (-)-Ambrox comprises the desired
(-)-Ambrox, compound (II), (III) and (IV) present in an amount of
87.1 wt %, 2.8 wt %, 2.5 wt % and 7.6 wt % respectively.
[0176] When a crude mixture is selectively crystallised (lab
scale), the crystallised material has the same components as the
crude mixture, but they are present in an amount of 99.1 wt %, 0.1
wt %, 0.1 wt % and 0.7 wt % respectively.
[0177] The Sensory Analytical Results were as follows:
[0178] (-)-Ambrox: Odour Threshold 0.2 ng/l.
[0179] Compound (IV): weak, IsoE, woody, GC-detection threshold
5-10 ng.
[0180] Compound (II): "odorless" (GC-threshold >500 ng).
[0181] Compound (III): GC-threshol about 10.times. higher than
(-)-Ambrox (circa 2 ng).
[0182] The sensory analysis of the 3 by-products (compounds II, III
and IV) indicates a weaker odour than that from (-)-Ambrox. In
fact, the epi-ambrox (Compound III) odor is about 10 fold weaker
than (-)-Ambrox suggesting that it is essentially odorless.
Example 7
[0183] Ambrox Recovery by Steam Extraction
[0184] Resulting Purity of the Crude (Steam Extracted) and
Crystallized (-)-Ambrox
[0185] The biotransformation of EE:EZ-homofarnesol 86:14 provided a
reaction mixture that was steam extracted. The steam distillate was
collected as a biphasic mixture. The organic phase was retained and
the aqueous phase discarded. The composition of the organic phase
was analysed by GC and the results shown in the Table below (see
"crude"). The organic phase was then concentrated to dryness.
Ethanol was then added to the crude, dried product and the mixture
warmed until the product was dissolved. At room temperature water
is slowly added and (-)-Ambrox crystallizes under occasional
stirring and cooling in an ice bath.
[0186] The tabulated data also shows the GC analytics results for
products obtained after the steam extraction/distillation step
("crude") and the crystallized product ((-)-Ambrox). The references
in the Table to "EZH" and "EEH" refer to (3Z,7E)-homofarnesol and
7E,3E-homofarnesol respectively.
[0187] The tabulated data below indicates that the particular
starting material (EEH:EZH 86:14) produces the desired end product
(-)-Ambrox and a very specific mixture of by-products (II, IV and
III) using the WT SHC enzyme or a SHC derivative. The data for the
selective crystallization show a strong enrichment of (-) Ambrox,
with practically no by-products (II), (IV) or (III) being found in
the crystallized sample. Accordingly, this EE:EZ mixture provides
an olfactively pure (-)-Ambrox product, which is selectively
crystallised in a relatively straightforward and cost-effective
matter.
TABLE-US-00004 TABLE shows the GC analytics results for the
crystallized product. Peak area (GC) Ambrox (II) (IV) (III) Ambrox
EZH EEH (%) Crude 215073 190376 588769 6751605 13429 14184 86.9
Crystallized 10088 8951 64625 9032941 0 0 99.1
[0188] Steam extraction/filtration are environmentally friendly
methods for isolating (-)-Ambrox because it offers a convenient
solvent-free isolation of (-)-Ambrox with an associated
inactivation of the biocatalyst.
[0189] The (-)-Ambrox produced using the bioconversion reaction may
be extracted using solvent from the whole reaction mixture (e.g.
using a water-immiscible solvent or by steam
extraction/distillation or by filtration) or from the solid phase
(e.g. using a water miscible solvent) using methods which are known
to those skilled in the art.
Sequence CWU 1
1
41631PRTAlicyclobacillus acidocaldarius 1Met Ala Glu Gln Leu Val
Glu Ala Pro Ala Tyr Ala Arg Thr Leu Asp 1 5 10 15 Arg Ala Val Glu
Tyr Leu Leu Ser Cys Gln Lys Asp Glu Gly Tyr Trp 20 25 30 Trp Gly
Pro Leu Leu Ser Asn Val Thr Met Glu Ala Glu Tyr Val Leu 35 40 45
Leu Cys His Ile Leu Asp Arg Val Asp Arg Asp Arg Met Glu Lys Ile 50
55 60 Arg Arg Tyr Leu Leu His Glu Gln Arg Glu Asp Gly Thr Trp Ala
Leu 65 70 75 80 Tyr Pro Gly Gly Pro Pro Asp Leu Asp Thr Thr Ile Glu
Ala Tyr Val 85 90 95 Ala Leu Lys Tyr Ile Gly Met Ser Arg Asp Glu
Glu Pro Met Gln Lys 100 105 110 Ala Leu Arg Phe Ile Gln Ser Gln Gly
Gly Ile Glu Ser Ser Arg Val 115 120 125 Phe Thr Arg Met Trp Leu Ala
Leu Val Gly Glu Tyr Pro Trp Glu Lys 130 135 140 Val Pro Met Val Pro
Pro Glu Ile Met Phe Leu Gly Lys Arg Met Pro 145 150 155 160 Leu Asn
Ile Tyr Glu Phe Gly Ser Trp Ala Arg Ala Thr Val Val Ala 165 170 175
Leu Ser Ile Val Met Ser Arg Gln Pro Val Phe Pro Leu Pro Glu Arg 180
185 190 Ala Arg Val Pro Glu Leu Tyr Glu Thr Asp Val Pro Pro Arg Arg
Arg 195 200 205 Gly Ala Lys Gly Gly Gly Gly Trp Ile Phe Asp Ala Leu
Asp Arg Ala 210 215 220 Leu His Gly Tyr Gln Lys Leu Ser Val His Pro
Phe Arg Arg Ala Ala 225 230 235 240 Glu Ile Arg Ala Leu Asp Trp Leu
Leu Glu Arg Gln Ala Gly Asp Gly 245 250 255 Ser Trp Gly Gly Ile Gln
Pro Pro Trp Phe Tyr Ala Leu Ile Ala Leu 260 265 270 Lys Ile Leu Asp
Met Thr Gln His Pro Ala Phe Ile Lys Gly Trp Glu 275 280 285 Gly Leu
Glu Leu Tyr Gly Val Glu Leu Asp Tyr Gly Gly Trp Met Phe 290 295 300
Gln Ala Ser Ile Ser Pro Val Trp Asp Thr Gly Leu Ala Val Leu Ala 305
310 315 320 Leu Arg Ala Ala Gly Leu Pro Ala Asp His Asp Arg Leu Val
Lys Ala 325 330 335 Gly Glu Trp Leu Leu Asp Arg Gln Ile Thr Val Pro
Gly Asp Trp Ala 340 345 350 Val Lys Arg Pro Asn Leu Lys Pro Gly Gly
Phe Ala Phe Gln Phe Asp 355 360 365 Asn Val Tyr Tyr Pro Asp Val Asp
Asp Thr Ala Val Val Val Trp Ala 370 375 380 Leu Asn Thr Leu Arg Leu
Pro Asp Glu Arg Arg Arg Arg Asp Ala Met 385 390 395 400 Thr Lys Gly
Phe Arg Trp Ile Val Gly Met Gln Ser Ser Asn Gly Gly 405 410 415 Trp
Gly Ala Tyr Asp Val Asp Asn Thr Ser Asp Leu Pro Asn His Ile 420 425
430 Pro Phe Cys Asp Phe Gly Glu Val Thr Asp Pro Pro Ser Glu Asp Val
435 440 445 Thr Ala His Val Leu Glu Cys Phe Gly Ser Phe Gly Tyr Asp
Asp Ala 450 455 460 Trp Lys Val Ile Arg Arg Ala Val Glu Tyr Leu Lys
Arg Glu Gln Lys 465 470 475 480 Pro Asp Gly Ser Trp Phe Gly Arg Trp
Gly Val Asn Tyr Leu Tyr Gly 485 490 495 Thr Gly Ala Val Val Ser Ala
Leu Lys Ala Val Gly Ile Asp Thr Arg 500 505 510 Glu Pro Tyr Ile Gln
Lys Ala Leu Asp Trp Val Glu Gln His Gln Asn 515 520 525 Pro Asp Gly
Gly Trp Gly Glu Asp Cys Arg Ser Tyr Glu Asp Pro Ala 530 535 540 Tyr
Ala Gly Lys Gly Ala Ser Thr Pro Ser Gln Thr Ala Trp Ala Leu 545 550
555 560 Met Ala Leu Ile Ala Gly Gly Arg Ala Glu Ser Glu Ala Ala Arg
Arg 565 570 575 Gly Val Gln Tyr Leu Val Glu Thr Gln Arg Pro Asp Gly
Gly Trp Asp 580 585 590 Glu Pro Tyr Tyr Thr Gly Thr Gly Phe Pro Gly
Asp Phe Tyr Leu Gly 595 600 605 Tyr Thr Met Tyr Arg His Val Phe Pro
Thr Leu Ala Leu Gly Arg Tyr 610 615 620 Lys Gln Ala Ile Glu Arg Arg
625 630 2631PRTArtificial sequenceSynthetic AacSHC Derivative 2Met
Ala Glu Gln Leu Val Glu Ala Pro Ala Tyr Ala Arg Thr Leu Asp 1 5 10
15 Arg Ala Val Glu Tyr Leu Leu Ser Cys Gln Lys Asp Glu Gly Tyr Trp
20 25 30 Trp Gly Pro Leu Leu Ser Asn Val Thr Met Glu Ala Glu Tyr
Val Leu 35 40 45 Leu Cys His Ile Leu Asp Arg Val Asp Arg Asp Arg
Met Glu Lys Ile 50 55 60 Arg Arg Tyr Leu Leu His Glu Gln Arg Glu
Asp Gly Thr Trp Ala Leu 65 70 75 80 Tyr Pro Gly Gly Pro Pro Asp Leu
Asp Thr Thr Ile Glu Ala Tyr Val 85 90 95 Ala Leu Lys Tyr Ile Gly
Met Ser Arg Asp Glu Glu Pro Met Gln Lys 100 105 110 Ala Leu Arg Phe
Ile Gln Ser Gln Gly Gly Ile Glu Ser Ser Arg Val 115 120 125 Phe Thr
Arg Met Trp Leu Ala Leu Val Gly Glu Tyr Pro Trp Glu Lys 130 135 140
Val Pro Met Val Pro Pro Glu Ile Met Phe Leu Gly Lys Arg Met Pro 145
150 155 160 Leu Asn Ile Tyr Glu Phe Gly Ser Trp Ala Arg Ala Thr Val
Val Ala 165 170 175 Leu Ser Ile Val Met Ser Arg Gln Pro Val Phe Pro
Leu Pro Glu Arg 180 185 190 Ala Arg Val Pro Glu Leu Tyr Glu Thr Asp
Val Pro Pro Arg Arg Arg 195 200 205 Gly Ala Lys Gly Gly Gly Gly Trp
Ile Phe Asp Ala Leu Asp Arg Ala 210 215 220 Leu His Gly Tyr Gln Lys
Leu Ser Val His Pro Phe Arg Arg Ala Ala 225 230 235 240 Glu Ile Arg
Ala Leu Asp Trp Leu Leu Glu Arg Gln Ala Gly Asp Gly 245 250 255 Ser
Trp Gly Gly Ile Gln Pro Pro Trp Phe Tyr Ala Leu Ile Ala Leu 260 265
270 Lys Ile Leu Asp Met Thr Gln His Pro Ala Phe Ile Lys Gly Trp Glu
275 280 285 Gly Leu Glu Leu Tyr Gly Val Glu Leu Asp Tyr Gly Gly Trp
Met Phe 290 295 300 Gln Ala Ser Ile Ser Pro Val Trp Asp Thr Gly Leu
Ala Val Leu Ala 305 310 315 320 Leu Arg Ala Ala Gly Leu Pro Ala Asp
His Asp Arg Leu Val Lys Ala 325 330 335 Gly Glu Trp Leu Leu Asp Arg
Gln Ile Thr Val Pro Gly Asp Trp Ala 340 345 350 Val Lys Arg Pro Asn
Leu Lys Pro Gly Gly Phe Ala Phe Gln Phe Asp 355 360 365 Asn Val Tyr
Tyr Pro Asp Val Asp Asp Thr Ala Val Val Val Trp Ala 370 375 380 Leu
Asn Thr Leu Arg Leu Pro Asp Glu Arg Arg Arg Arg Asp Ala Met 385 390
395 400 Thr Lys Gly Phe Arg Trp Ile Val Gly Met Gln Ser Ser Asn Gly
Gly 405 410 415 Trp Gly Ala Tyr Asp Val Asp Asn Thr Ser Asp Leu Pro
Asn His Ile 420 425 430 Pro Phe Cys Asp Phe Gly Glu Val Thr Asp Pro
Pro Ser Glu Asp Val 435 440 445 Thr Ala His Val Leu Glu Cys Phe Gly
Ser Phe Gly Tyr Asp Asp Ala 450 455 460 Trp Lys Val Ile Arg Arg Ala
Val Glu Tyr Leu Lys Arg Glu Gln Lys 465 470 475 480 Pro Asp Gly Ser
Trp Phe Gly Arg Trp Gly Val Asn Tyr Leu Tyr Gly 485 490 495 Thr Gly
Ala Val Val Ser Ala Leu Lys Ala Val Gly Ile Asp Thr Arg 500 505 510
Glu Pro Tyr Ile Gln Lys Ala Leu Asp Trp Val Glu Gln His Gln Asn 515
520 525 Pro Asp Gly Gly Trp Gly Glu Asp Cys Arg Ser Tyr Glu Asp Pro
Ala 530 535 540 Tyr Ala Gly Lys Gly Ala Ser Thr Pro Ser Gln Thr Ala
Trp Ala Leu 545 550 555 560 Met Ala Leu Ile Ala Gly Gly Arg Ala Glu
Ser Glu Ala Ala Arg Arg 565 570 575 Gly Val Gln Tyr Leu Val Glu Thr
Gln Arg Pro Asp Gly Gly Trp Asp 580 585 590 Glu Pro Tyr Tyr Thr Gly
Thr Gly Tyr Pro Gly Asp Phe Tyr Leu Gly 595 600 605 Tyr Thr Met Tyr
Arg His Val Phe Pro Thr Leu Ala Leu Gly Arg Tyr 610 615 620 Lys Gln
Ala Ile Glu Arg Arg 625 630 31896DNAArtificial sequenceSynthetic
AacSHC Derivative 3atggctgagc agttggtgga agcgccggcc tacgcgcgga
cgctggatcg cgcggtggag 60tatctcctct cctgccaaaa ggacgaaggc tactggtggg
ggccgcttct gagcaacgtc 120acgatggaag cggagtacgt cctcttgtgc
cacattctcg atcgcgtcga tcgggatcgc 180atggagaaga tccggcggta
cctgttgcac gagcagcgcg aggacggcac gtgggccctg 240tacccgggtg
ggccgccgga cctcgacacg accatcgagg cgtacgtcgc gctcaagtat
300atcggcatgt cgcgcgacga ggagccgatg cagaaggcgc tccggttcat
tcagagccag 360ggcgggatcg agtcgtcgcg cgtgttcacg cggatgtggc
tggcgctggt gggagaatat 420ccgtgggaga aggtgcccat ggtcccgccg
gagatcatgt tcctcggcaa gcgcatgccg 480ctcaacatct acgagtttgg
ctcgtgggct cgggcgaccg tcgtggcgct ctcgattgtg 540atgagccgcc
agccggtgtt cccgctgccc gagcgggcgc gcgtgcccga gctgtacgag
600accgacgtgc ctccgcgccg gcgcggtgcc aagggagggg gtgggtggat
cttcgacgcg 660ctcgaccggg cgctgcacgg gtatcagaag ctgtcggtgc
acccgttccg ccgcgcggcc 720gagatccgcg ccttggactg gttgctcgag
cgccaggccg gagacggcag ctggggcggg 780attcagccgc cttggtttta
cgcgctcatc gcgctcaaga ttctcgacat gacgcagcat 840ccggcgttca
tcaagggctg ggaaggtcta gagctgtacg gcgtggagct ggattacgga
900ggatggatgt ttcaggcttc catctcgccg gtgtgggaca cgggcctcgc
cgtgctcgcg 960ctgcgcgctg cggggcttcc ggccgatcac gaccgcttgg
tcaaggcggg cgagtggctg 1020ttggaccggc agatcacggt tccgggcgac
tgggcggtga agcgcccgaa cctcaagccg 1080ggcgggttcg cgttccagtt
cgacaacgtg tactacccgg acgtggacga cacggccgtc 1140gtggtgtggg
cgctcaacac cctgcgcttg ccggacgagc gccgcaggcg ggacgccatg
1200acgaagggat tccgctggat tgtcggcatg cagagctcga acggcggttg
gggcgcctac 1260gacgtcgaca acacgagcga tctcccgaac cacatcccgt
tctgcgactt cggcgaagtg 1320accgatccgc cgtcagagga cgtcaccgcc
cacgtgctcg agtgtttcgg cagcttcggg 1380tacgatgacg cctggaaggt
catccggcgc gcggtggaat atctcaagcg ggagcagaag 1440ccggacggca
gctggttcgg tcgttggggc gtcaattacc tctacggcac gggcgcggtg
1500gtgtcggcgc tgaaggcggt cgggatcgac acgcgcgagc cgtacattca
aaaggcgctc 1560gactgggtcg agcagcatca gaacccggac ggcggctggg
gcgaggactg ccgctcgtac 1620gaggatccgg cgtacgcggg taagggcgcg
agcaccccgt cgcagacggc ctgggcgctg 1680atggcgctca tcgcgggcgg
cagggcggag tccgaggccg cgcgccgcgg cgtgcaatac 1740ctcgtggaga
cgcagcgccc ggacggcggc tgggatgagc cgtactacac cggcacgggc
1800ttcccagggg attggtacct cggctacacc atgtaccgcc acgtgtttcc
gacgctcgcg 1860ctcggccgct acaagcaagc catcgagcgc aggtga
18964631PRTArtificial sequenceSynthetic AacSHC Derivative 4Met Ala
Glu Gln Leu Val Glu Ala Pro Ala Tyr Ala Arg Thr Leu Asp 1 5 10 15
Arg Ala Val Glu Tyr Leu Leu Ser Cys Gln Lys Asp Glu Gly Tyr Trp 20
25 30 Trp Gly Pro Leu Leu Ser Asn Val Thr Met Glu Ala Glu Tyr Val
Leu 35 40 45 Leu Cys His Ile Leu Asp Arg Val Asp Arg Asp Arg Met
Glu Lys Ile 50 55 60 Arg Arg Tyr Leu Leu His Glu Gln Arg Glu Asp
Gly Thr Trp Ala Leu 65 70 75 80 Tyr Pro Gly Gly Pro Pro Asp Leu Asp
Thr Thr Ile Glu Ala Tyr Val 85 90 95 Ala Leu Lys Tyr Ile Gly Met
Ser Arg Asp Glu Glu Pro Met Gln Lys 100 105 110 Ala Leu Arg Phe Ile
Gln Ser Gln Gly Gly Ile Glu Ser Ser Arg Val 115 120 125 Phe Thr Arg
Met Trp Leu Ala Leu Val Gly Glu Tyr Pro Trp Glu Lys 130 135 140 Val
Pro Met Val Pro Pro Glu Ile Met Phe Leu Gly Lys Arg Met Pro 145 150
155 160 Leu Asn Ile Tyr Glu Phe Gly Ser Trp Ala Arg Ala Thr Val Val
Ala 165 170 175 Leu Ser Ile Val Met Ser Arg Gln Pro Val Phe Pro Leu
Pro Glu Arg 180 185 190 Ala Arg Val Pro Glu Leu Tyr Glu Thr Asp Val
Pro Pro Arg Arg Arg 195 200 205 Gly Ala Lys Gly Gly Gly Gly Trp Ile
Phe Asp Ala Leu Asp Arg Ala 210 215 220 Leu His Gly Tyr Gln Lys Leu
Ser Val His Pro Phe Arg Arg Ala Ala 225 230 235 240 Glu Ile Arg Ala
Leu Asp Trp Leu Leu Glu Arg Gln Ala Gly Asp Gly 245 250 255 Ser Trp
Gly Gly Ile Gln Pro Pro Trp Phe Tyr Ala Leu Ile Ala Leu 260 265 270
Lys Ile Leu Asp Met Thr Gln His Pro Ala Phe Ile Lys Gly Trp Glu 275
280 285 Gly Leu Glu Leu Tyr Gly Val Glu Leu Asp Tyr Gly Gly Trp Met
Phe 290 295 300 Gln Ala Ser Ile Ser Pro Val Trp Asp Thr Gly Leu Ala
Val Leu Ala 305 310 315 320 Leu Arg Ala Ala Gly Leu Pro Ala Asp His
Asp Arg Leu Val Lys Ala 325 330 335 Gly Glu Trp Leu Leu Asp Arg Gln
Ile Thr Val Pro Gly Asp Trp Ala 340 345 350 Val Lys Arg Pro Asn Leu
Lys Pro Gly Gly Phe Ala Phe Gln Phe Asp 355 360 365 Asn Val Tyr Tyr
Pro Asp Val Asp Asp Thr Ala Val Val Val Trp Ala 370 375 380 Leu Asn
Thr Leu Arg Leu Pro Asp Glu Arg Arg Arg Arg Asp Ala Met 385 390 395
400 Thr Lys Gly Phe Arg Trp Ile Val Gly Met Gln Ser Ser Asn Gly Gly
405 410 415 Trp Gly Ala Tyr Asp Val Asp Asn Thr Ser Asp Leu Pro Asn
His Ile 420 425 430 Pro Phe Cys Asp Phe Gly Glu Val Thr Asp Pro Pro
Ser Glu Asp Val 435 440 445 Thr Ala His Val Leu Glu Cys Phe Gly Ser
Phe Gly Tyr Asp Asp Ala 450 455 460 Trp Lys Val Ile Arg Arg Ala Val
Glu Tyr Leu Lys Arg Glu Gln Lys 465 470 475 480 Pro Asp Gly Ser Trp
Phe Gly Arg Trp Gly Val Asn Tyr Leu Tyr Gly 485 490 495 Thr Gly Ala
Val Val Ser Ala Leu Lys Ala Val Gly Ile Asp Thr Arg 500 505 510 Glu
Pro Tyr Ile Gln Lys Ala Leu Asp Trp Val Glu Gln His Gln Asn 515 520
525 Pro Asp Gly Gly Trp Gly Glu Asp Cys Arg Ser Tyr Glu Asp Pro Ala
530 535 540 Tyr Ala Gly Lys Gly Ala Ser Thr Pro Ser Gln Thr Ala Trp
Ala Leu 545 550 555 560 Met Ala Leu Ile Ala Gly Gly Arg Ala Glu Ser
Glu Ala Ala Arg Arg 565 570 575 Gly Val Gln Tyr Leu Val Glu Thr Gln
Arg Pro Asp Gly Gly Trp Asp 580 585 590 Glu Pro Tyr Tyr Thr Gly Thr
Gly Phe Pro Gly Asp Trp Tyr Leu Gly 595 600 605 Tyr Thr Met Tyr Arg
His Val Phe Pro Thr Leu Ala Leu Gly Arg Tyr 610 615 620 Lys Gln Ala
Ile Glu Arg Arg 625 630
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