U.S. patent application number 11/685939 was filed with the patent office on 2007-07-19 for deracemisation of amines.
This patent application is currently assigned to GLAXO GROUP LIMITED. Invention is credited to Marina Victorovna Alexeeva, Alexis Enright, Mahmoud Mahmoudian, Nicholas Turner.
Application Number | 20070166811 11/685939 |
Document ID | / |
Family ID | 9933245 |
Filed Date | 2007-07-19 |
United States Patent
Application |
20070166811 |
Kind Code |
A1 |
Alexeeva; Marina Victorovna ;
et al. |
July 19, 2007 |
Deracemisation of Amines
Abstract
Methods are described for the deracemisation or chiral inversion
of chiral amines by enzymatic treatment, such as by a
stereoselective enzymatic conversion and either a non-selective or
partially selective chemical or enzymatic conversion,
simultaneously or sequentially. Also described are methods for
selecting a suitable enzyme, particularly a suitable amine oxidase,
and for the generation of novel enzymes suitable for use in the
deracemisation method.
Inventors: |
Alexeeva; Marina Victorovna;
(Research Triangle Park, NC) ; Enright; Alexis;
(Research Triangle Park, NC) ; Mahmoudian; Mahmoud;
(Research Triangle Park, NC) ; Turner; Nicholas;
(Research Triangle Park, NC) |
Correspondence
Address: |
GLAXOSMITHKLINE;CORPORATE INTELLECTUAL PROPERTY, MAI B475
FIVE MOORE DR., PO BOX 13398
RESEARCH TRIANGLE PARK
NC
27709-3398
US
|
Assignee: |
GLAXO GROUP LIMITED
Glaxo Wellcome House Berkeley Avenue
Greenford
GB
UB6 0NN
|
Family ID: |
9933245 |
Appl. No.: |
11/685939 |
Filed: |
March 14, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10508356 |
Oct 4, 2005 |
7208302 |
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PCT/GB03/01198 |
Mar 19, 2003 |
|
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11685939 |
Mar 14, 2007 |
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Current U.S.
Class: |
435/183 |
Current CPC
Class: |
C12N 9/0022 20130101;
C12P 41/002 20130101 |
Class at
Publication: |
435/183 |
International
Class: |
C12N 9/00 20060101
C12N009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 19, 2002 |
GB |
0206415.2 |
Claims
1. A method for the enantiomeric conversion of amines comprising
treating a homochiral amine or a mixture of amine enantiomers with
an enzyme capable of catalysing oxidation of the amine in a
stereoselective manner and, subsequently or simultaneously,
treating with a reducing agent.
2. A method according to claim 1 which is a method of epimerisation
of one single amine enantiomer to the other.
3. A method according to claim 1 in which the mixture of amine
enantiomers is a racemic mixture.
4. A method according to claim 1 in which the reducing agent is
partially enantioselective.
5. A method according to claim 1 in which the reducing agent is a
chemical reducing agent and is non-enantioselective.
6. A method according to claim 5 in which the reducing agent is
selected from sodium borohydride, sodium cyanoborohydride, an
amine:borane complex or a transfer hydrogenation reagent.
7. A method according to claim 6 in which the transfer
hydrogenation reagent is ammonium formate with Pd/C.
8. A method according to claim 6 in which the amine:borane complex
is an ammonia:borane complex.
9. A method according to claim 1 comprising an oxidation-reduction
cycle in which the steps of treating with an enzyme capable of
catalysing oxidation of the amine and treating with a reducing
agent are performed sequentially, a plurality of times.
10. A method of improving the activity, substrate specificity, or
enantioselectivity of an amine oxidase enzyme by directed
evolution, the method comprising mutation of the enzyme and
selection of a mutant having improved activity against an
homochiral substrate.
11. A method of directing the evolution of an amino oxidase enzyme
comprising the steps of: a) mutating the enzyme to create at least
one enzyme variant; b) screening said enzyme variant for activity
against a homochiral substrate; and c) selecting one or more enzyme
variants which show greater activity toward the substrate than does
the originator enzyme.
12. A method according to claim 11, further comprising repeating
steps a), b) and c) one or more times, using the enzyme variant
selected in step c) as the enzyme to be mutated.
13. A method according to claim 11 further comprising performing
site directed mutagenesis on an enzyme selected in step c).
14. A method according to claim 11 in which the enzyme is a
monoamine oxidase.
15. A method according to claim 14 in which the enzyme is a
microbial monoamine oxidase.
16. A method according to claim 15 in which the enzyme is the
Aspergillus niger monoamine oxidase, or a variant thereof.
17. A method according to claim 16 in which the enzyme is a variant
of A. niger monoamine oxidase which differs from wild-type A. niger
monoamine oxidase by mutation in the region of amino acids
334-350.
18. A method according to claim 14 in which the enzyme is a variant
of A. niger monoamine oxidase which differs from wild type A. niger
monoamine oxidase by mutation of the amino acid at position number
336.
19. A method according to claim 14 in which the enzyme is a variant
of A. niger monoamine oxidase which differs from wild type A. niger
monoamine oxidase by incorporation of the mutation: N336S.
20.-23. (canceled)
24. A method according to claim 1 in which said enzyme is an
enantioselective monoamine oxidase (MAO) enzyme having the amino
acid sequence of SEQ ID NO:2.
25. A method according to claim 1 in which said enzyme is an
enantioselective monoamine oxidase enzyme which is a variant of
wild-type A. niger MAO, where said variant differs from wild-type
A. niger monoamine oxidase by incorporation of the mutation:
N336S.
26. A method according to claim 1 in which said enzyme is an
enantioselective monoamine oxidase enzyme which is a variant of
wild-type A. niger MAO, where said variant differs from wild-type
A. niger monoamine oxidase by incorporation of the mutations: N336S
and M348K.
27. A method according to claim 1 in which said enzyme is an
enantioselective monoamine oxidase enzyme which is a variant of
wild-type A. niger MAO, where said variant differs from wild-type
A. niger monoamine oxidase by incorporation of the mutations: N336S
and one or more of M348K, R259L and R260L.
28. (canceled)
Description
[0001] The present invention relates to a method for the
deracemisation or chiral inversion of chiral amines by enzymatic
treatment of a mixture of enantiomers. The method employs a
stereoselective enzymatic conversion and either a non-selective or
partially selective chemical or enzymatic conversion,
simultaneously or sequentially. The invention also provides a
method for selecting a suitable enzyme, particularly a suitable
amine oxidase, and for the generation of novel enzymes suitable for
use in the deracemisation method.
[0002] Enantiomerically pure chiral amines are valuable synthetic
intermediates, particularly for the preparation of pharmaceutical
target molecules. Traditionally, chiral amines have been obtained
by separation methods such as diastereomeric crystallisation using
a chiral acid to form a salt of one of the enantiomers, or by
kinetic resolution of a racemate using an enzyme to selectively
react one enantiomer allowing easier separation by physical methods
such as solvent partitioning or chromatography (1). Whilst such
methods can achieve high enantiomeric excess (e.e.), they can yield
only a maximum of 50% of the racemic starting material as the
required enantiomer. As with many chiral compounds, there is an
increasing desire to develop synthetic strategies for amines that
involve either asymmetric approaches or which combine resolution
with racemisation of the undesired enantiomer, both of which can in
principle deliver the product in 100% yield and 100% e.e.
Asymmetric methods suggested to date include the use of
transaminases for conversion of ketones to chiral amines (2, 3, 4).
Furthermore the kinetic resolution of amines using lipases such as
Burkholderia plantarii lipase (5, 6) or Candida antarctica lipase
(7) has been combined with racemisation of the unreacted amine
either by formation of an imine (5,6) or by transfer hydrogenation
with Pd/C as the catalyst (7).
[0003] An alternative approach, which has been termed
deracemisation, involves the stereoinversion of one enantiomer to
the other e.g. using a cyclic oxidation-reduction sequence. To date
it has been shown that such a system can be applied to the
preparation of L-.alpha.-amino acids by the use of an
enantioselective D-amino acid oxidase in combination with a
non-selective reducing agent. The original work (8) reported the
stereoinversion of D- to L-alanine, albeit in low yield, using
sodium borohydride as the reducing agent. The instability of sodium
borohydride at pH 7 precludes its use on a practical scale, and
recently we have shown that deracemisation of amino acids can be
made more efficient by the use of more suitable reducing agents
including sodium cyanoborohydride (13), ammonium formate with Pd/C
and also borohydride complexes or amine:boranes (14). However,
no-one to date has successfully applied a deracemisation method to
amines.
SUMMARY OF THE INVENTION
[0004] The present invention provides a method for the
deracemisation or chiral inversion (generally referred to herein as
enantiomeric conversion) of chiral amines by treatment of a mixture
of amine enantiomers with an enzyme capable of catalysing oxidation
of the amine in a stereoselective manner and, subsequently or
simultaneously, treating the mixture with a reducing agent. The
method is applicable to mixtures of enantiomers in varying
proportions, including racemic mixtures, and to conversion
(epimerisation) of one single enantiomer to the other. For example
the method is applicable to mixtures of R and S forms of an amine
in a ratio of 1:1, 1:2, 1:5, 1:10, 2:1, 5:1, 10:1, 100:1 or other
ratios. The product of the enantiomeric conversion is enriched in
the desired enantiomer over the starting material i.e. the desired
enantiomer is in enantiomeric excess. Preferably the product
comprises a substantially pure single enantiomer. Thus, in
preferred embodiments the enantiomeric conversion process of the
invention is employed to convert a mixture of amine enantiomers
into a composition consisting essentially of a single enantiomer,
or is employed to convert one substantially pure amine enantiomer
into the other, again in enantiomerically pure form.
[0005] The reducing agent may be partially stereoselective or
non-stereoselective and may be a chemical reducing agent.
Alternatively the reduction may be enzymatically catalysed. If a
chemical reducing agent is to be employed, this may advantageously
be selected from sodium borohydride, sodium cyanoborohydride,
amine:borane complexes or a transfer hydrogenation reagent such as
ammonium formate with Pd/C. If the stereoselective oxidation and
non-stereoselective (or partially selective) reduction are
performed sequentially, in an oxidation-reduction cycle, the cycle
may be performed a plurality of times until the desired
enantiomeric excess is achieved.
[0006] The enzyme capable of catalysing oxidation of the amine in a
stereoselective manner may be a monoamine oxidase (MAO),
particularly a microbial monoamine oxidase, but any amine oxidase
enzyme may be employed. One MAO which may advantageously be
employed in the method of the present invention is the Aspergillus
niger monoamine oxidase or a variant thereof, for example a variant
in which the enzyme differs from wild-type A. niger MAO by
incorporation of one or more mutations, especially in the region of
amino acids 25-265 and 334-350, particularly prefere4d are enzymes
having a mutation at one or more of amino acids 259, 260, 336 and
348, more particularly the mutation N336S or the double mutation
N336S, M348K.
[0007] The present invention also provides a method of directing
the evolution of an originator enzyme by: a) mutating the
originator enzyme to create at least one enzyme variant; b)
screening said enzyme variant for activity against a homochiral
substrate; and c) selecting one or more enzyme variants which show
greater activity toward the homochiral substrate than does the
originator enzyme. Optionally steps a), b) and c) may be repeated,
using the enzyme variant selected in step c) as an originator
enzyme. At appropriate stages, the enzyme variant(s) may be assayed
against the opposite enantiomer of the substrate, or against a
mixture of the substrate enantiomers, to confirm
enantioselectivity. In particular embodiments, the originator
enzyme is an oxidase which shows activity against amines, for
example an amine oxidase, especially a monoamine oxidase. The
substrate may be any chiral amine which can be oxidised to an
imine, including cyclic secondary amines, for example amines of
Formula I: ##STR1## In which: a) R is H or C.sub.1-4alkyl; R1 and
R2 are independently selected from substituted or unsubstituted
C.sub.1-10alkyl, C.sub.1-10alkenyl, C.sub.1-10cycloalkyl,
C.sub.1-10heterocycle, C.sub.1-10aryl, C.sub.1-10heteroaryl,
C.sub.1-4alkyl-aryl, C.sub.1-4alkyl-heteroaryl,
C.sub.1-4alkyl-C.sub.1-6cycloalkyl and C.sub.1-4alkyl-C.sub.1-6
heterocycle; or b) R is H or C.sub.1-4alkyl, R1 and R2 together
form a substituted or unsubstituted C.sub.1-10cycloalkyl ring
system or C.sub.1-10aryl ring containing one or more heteroatoms;
or c) R and R1 together form a substituted or unsubstituted
C.sub.1-10cycloalkyl or C.sub.1-10aryl ring system which may
contain one or more heteroatoms and R2 is defined as in a)
above.
[0008] As used herein, the terms "halo" or "halogen" refer to
fluorine, chlorine, bromine and iodine.
[0009] As used herein, the term "alkyl" refers to a straight or
branched hydrocarbon chain containing the specified number of
carbon atoms. For example, C.sub.1-C.sub.3alkyl means a straight or
branched hydrocarbon chain containing at least 1 and at most 3
carbon atoms. Examples of alkyl as used herein include, but are not
limited to; methyl, ethyl, n-propyl, i-propyl.
[0010] As used herein, the term "cycloalkyl" refers to a fully
saturated hydrocarbon ring containing the specified number of
carbon atoms. The term "cycloalkyl" encompasses single and bicyclic
ring structures. Examples of cycloalkyl as used herein include, but
are not limited to cyclohexyl, cyclopropyl.
[0011] As used herein, the term "aryl" refers to an unsaturated
which may be saturated or unsaturated hydrocarbon ring containing
the specified number of carbon atoms. The term "aryl" encompasses
single and bicyclic ring structures. Examples of cycloalkyl as used
herein include, but are not limited to phenyl, naphthyl.
[0012] Where cycloalkyl or aryl ring systems contain one or more
heteroatoms, these are selected from N, S or O, preferably N. Thus,
the terms "heterocyclic" and "heteroaryl" refer to cycloalkyl and
aryl groups, respectively, which contain up to three heteroatoms
selected from N, S or O, preferably N.
[0013] Where one or more of R, R1 and R2, or a ring formed
therebetween, are substituted, one to three substituents may be
present and are selected from halogen, hydroxy, C.sub.1-3alkyl,
C.sub.1-3alkenyl, C.sub.1-3alkoxy, nitro, nitrile and
CONH.sub.2.
[0014] Conveniently, the target substrate can be used for evolution
of enzymes with improved activity and enantioselectivity for that
particular substrate.
[0015] The invention therefore provides a method of improving the
catalytic activity, and optionally also the enantioselectivity, of
an amine oxidase, especially a monoamine oxidase, by directed
evolution comprising mutation of the enzyme and selection of a
mutant having improved activity against a homochiral substrate. The
use of an enzyme variant, selected by such a method, in a method
for the deracemisation or epimerisation of amines is also
provided.
[0016] The wild-type amino acid sequence of A. niger MAO is set out
in SEQ. ID No. 1. In one embodiment the invention provides a
variant of the A. niger MAO in which the enzyme differs from
wild-type A. niger MAO in the region of amino acids 334-350,
particularly amino acid 336 and/or amino acid 348, more
particularly by incorporation of the mutation N336S or the double
mutation N336S, M348K. The invention thus provides a variant of the
A. niger MAO having the amino acid sequence set out in SEQ. ID No.
2 and, in a further embodiment, a variant of the A. niger MAO
having the amino acid sequence set out in SEQ. ID No. 3.
[0017] Throughout the specification and the claims which follow,
unless the context requires otherwise, the word `comprise`, and
variations such as `comprises` and `comprising`, will be understood
to imply the inclusion of a stated integer or step or group of
integers but not to the exclusion of any other integer or step or
group of integers or steps.
[0018] A chiral compound will have two or more enantiomers which
are stereochemically dissimilar. A composition which contains more
than one enantiomer of a chiral compound is referred to as a
"racemic mixture" if it contains the enantiomers in equal or
substantially equal amounts. By contrast a composition is
"homochiral", "enantiomerically pure" or a "substantially pure
single enantiomer" if it contains a single enantiomer,
substantially free of the corresponding enantiomer, or consists
essentially of one enantiomer in the absence of the other. By
`substantially free` is meant no more than about 5% w/w of the
corresponding enantiomer, particularly no more than about 3% w/w,
and more particularly less than about 1% w/w is present.
"Enantioselective" and "stereoselective" are used herein
interchangeably and refer to the tendency of a reaction to favour
one enantiomer of a chiral compound over the other. "Partially
stereoselective" (or "partially enantioselective"),
"non-enantioselective" etc. shall be understood accordingly.
[0019] The invention will now be described in more detail, with
reference to the accompanying drawings and sequence listings, in
which:
[0020] FIG. 1 shows the reaction scheme for the oxidation-reduction
cycle which results in deracemisation;
[0021] FIG. 2 shows diagrammatically the assay used to detect
enzymes having the desired enantioselective amine oxidase
activity;
[0022] FIG. 3 shows the results of 27 enzymes selected from the
detection assay of FIG. 2, assayed against L-AMBA and D-AMBA;
[0023] FIG. 4 shows the substrate specificity of the N336S, M348K
mutant A. niger monoamine oxidase;
[0024] FIG. 5 shows the enantioselectivity of the N336S, M348K
mutant A. niger monoamine oxidase;
[0025] SEQ ID NO:1 shows the amino acid sequence of the wild-type
Aspergillus niger monoamine oxidase enzyme (NB we found the
sequence to differ by 4 amino acids from that reported by Schilling
& Lerch (10,11). These changes most likely represent errors in
the original DNA sequencing; two of these differences are also
noted by Sablin (12).);
[0026] SEQ ID NO:2 shows the amino acid sequence of a variant
monoamine oxidase enzyme generated by directed evolution in the
following Examples. The mutation N336S is shown in bold face type
and underlined;
[0027] SEQ ID NO: 3 shows the amino acid sequence of a variant
monoamine oxidase enzyme generated by site directed mutagenesis of
the N336S mutant enzyme of SEQ ID NO: 2. The mutations N336S and
M348K are shown in bold face type and underlined.
[0028] Herein we report a significant new extension of
deracemisation, by applying the method for the first time to the
deracemisation of chiral amines. The reaction follows the general
scheme shown in FIG. 1. One enantiomer is converted to the other
because the enzyme reacts preferentially with one of the two chiral
forms, leaving the other unreacted. The enzyme reaction produces
the achiral imine, which does not have a chiral centre at the
nitrogen-bearing carbon. Non-selective reduction of the imine
results in the creation of a 1:1 mixture of amine enantiomers (if a
partially selective reducing agent were employed, an unequal
mixture of amine enantiomers would be formed). As this process is
allowed to undergo a number of cycles, the amine approaches
enantiomeric purity (assuming a sufficiently enantioselective amine
oxidase). The yield can approach 100% If the imine can be
efficiently reduced before undergoing hydrolysis to the ketone.
[0029] Amine oxidases have been classified into two groups, namely
Type I (Cu/TOPA dependent) and Type II (flavin dependent) (15). In
the catalytic cycle of the Type I enzyme, the intermediate imine
remains covalently bound to the protein, which mitigates against
intervention at this stage in the reaction to reduce the imine back
to the amine. The Type II enzymes have been extensively studied
from mammalian sources (9), however microbial sources of Type II
enzymes are poorly documented and indeed at the outset of our work
there were no reports of enantioselective transformations.
Schilling et al., (10, 11) reported the cloning and expression of a
Type II monoamine oxidase from Aspergillus niger (MAO-N) and
subsequently Sablin et al., (12) purified the enzyme to homogeneity
and carried out substrate specificity and kinetic studies. The
enzyme was reported to have high activity towards simple aliphatic
amines (e.g. amylamine, butylamine) but was also active, albeit at
a lower rate, towards benzylamine.
[0030] Our studies have revealed that the A. niger MAO-N enzyme
possesses very low, but measurable activity towards
L-.alpha.-methylbenzylamine with even slower oxidation of the
D-enantiomer. Thus the enzyme is partially enantioselective. We
have carried out in vitro evolution (16, 17) to generate new amine
oxidase enzymes having improved catalytic activity and
enantioselectivity over the wild-type A. niger MAO-N enzyme. We
have also demonstrated the applicability of the evolved mutants in
deracemisation reactions.
[0031] To generate a large library of enzyme variants we used a
mutator strain for random mutation of a plasmid containing an
insert encoding the wild-type A. niger amine oxidase enzyme. The E.
coli XL1-Red mutator strain (Stratagene) has been employed
previously for in vitro evolution experiments and has the advantage
that all parts of the plasmid are subject to mutation (cf. error
prone PCR where only the gene of interest is mutated) which can
result in improved production of enzyme as well as changes to
enzyme activity and/or specificity. By carrying out multiple cycles
with the mutator strain followed by transformation of the plasmid
library into E. coli BL21 cells, we were able to generate a library
of around 10.sup.6 variants.
[0032] Having generated the mutant enzymes, we assayed them for
amine oxidase activity. Amine oxidases are typical of all members
of the oxidase family in that they evolve hydrogen peroxide as a
by-product. Many of the reported assays for oxidase activity
exploit the production of hydrogen peroxide, especially coupled
with peroxidase in the presence of a substrate that upon oxidation
yields a highly coloured product. We first used
aminoantipyrine/tribromohydroxybenzoic acid as the substrate for
the peroxidase, which yielded distinct pinkish-red colonies which
could be easily visualised. However the coloured product is
relatively soluble and hence colour from the active colonies
rapidly diffused giving high background intensity. This problem was
overcome by switching to 3,3'-diaminobenzadine (DAB) as the
substrate which gave rise to a dark pink, insoluble product
resulting in both very high definition and contrast of the active
colonies. It should be noted that this high-throughput screen
should be generally applicable to other oxidase enzymes in addition
to amine oxidases.
[0033] The library was plated out directly onto nitro-cellulose
filters on agar-plates (around 3,000 colonies per plate) and a
sub-set of the plates taken through the screening protocol (see
FIG. 2). Each filter was transferred to a petri-dish and stored at
-20.degree. C. for 24-72 h in order to partially lyse the cells.
Thereafter, each plate was treated with a cocktail containing both
the assay mixture and also 2% agarose at 60.degree. C. The plates
were incubated at 37.degree. C. for 24-48 h after which they was
inspected for positive clones which were then removed and plated at
higher dilution to isolate pure colonies.
[0034] The result of screening a subset (ca. 150,000 clones) of the
initial library led to the identification of 35 clones with
improved activity towards L-AMBA compared to the wild-type enzyme.
Each of these clones was grown on a small scale and assayed against
both L- and D-AMBA as cell-free extracts resulting in the best 27
clones which were selected for further study. Each of these 27 were
assayed against L-AMBA and D-AMBA, the results of which are
presented in FIG. 3. Finally, the mutant enzyme clone which showed
greatest improvement over the wild-type enzyme in terms of its
selectivity, and activity, towards L-versus D-AMBA was selected for
more detailed examination. Further mutation was then introduced to
boost expression.
[0035] It is known that the codons used in the DNA and RNA to
encode amino acids show a certain redundancy. Thus, a given amino
acid may be coded for by more than one 3-base codon. However, a
given organism may more commonly use one of the alternative codons
than the other(s) for a given amino acid. This has implications if
a heterogeneous nucleic acid is introduced into a host organism for
expression. Sometimes the codons used by the originator organism
are the "less preferred" codons for the host, which can result in
difficulties in expression such as a reduced level of expression.
Similarly, certain amino acids may be less commonly represented in
the proteome of one organism when compared to another. It may
sometimes possible to boost the levels of heterologous expression
in a host cell by using an alternative nucleic acid sequence in
which codons and/or amino acids which are less preferred by the
host are exchanged for more preferred alternatives. The present
invention provides an isolated nucleic acid which encodes a
monoamine oxidase enzyme which is a variant of the A. niger MAO in
which the codons for R259 and R260 are optimised for expression in
a heterogeneous host cell. More particularly, when the expression
of the MAO is intended to take place in E. Coli, the arginine amino
acids at these positions are replaced by alternative amino acids of
a similar charge and size. These mutations may in addition to
mutations which alter the catalytic activity and/or
enantioselectivity of the mutant enzyme. In a further embodiment,
we provide an enantioselective monoamine oxidase enzyme which is a
variant of the A. niger MAO and which differs from wild-type A.
niger monoamine oxidase by incorporation of the mutations N336S and
one or more of M348K, R259L and R260L.
[0036] The mutant N336S identified below may be taken as the basis
for "hotspot" mutation in which further mutations are introduced in
the amino acids surrounding position 336. Mutations may be made by
site-directed mutagenesis, by the construction of chimeric
recombinant enzymes using a "cut and paste" methodology employing
restriction enzymes, or by other means well known to workers
skilled in the art. This method is used to identify further mutants
with enhanced activity over the N336S mutant, such as the N336S,
M348K double mutant identified herein.
EXAMPLE 1--PREPARATION OF MUTATED PLASMID DNA LIBRARY BY E. COLI
XL1-RED MUTATOR STRAIN
[0037] The MAO gene from Aspergillus niger cloned in pET3a was
obtained from B. Schilling (11). The gene was amplified by pfu
Turbo DNA polymerase (Stratagene) using primers designed according
to E. coli codon usage. The PCR product was subcloned into the
pET16b vector (Novagen). This construct (MAOpET16b) was submitted
for mutagenesis by the E. coli XL1-Red mutator strain.
[0038] E. coli XL1-Red mutator strain competent cells were obtained
from Stratagene. Competent cells were transformed as described in
the Stratagene protocol. 700 .mu.l of transformed cell suspension
(Transformation 1) was inoculated in 20 ml of LB medium (tryptone,
10 g/L; yeast extract, 5 g/L; NaCl, 10 g/L; pH 7) with ampicillin
(100 .mu.g/ml; LB Amp) and grown for 18 h in a 50 ml Falcon tube in
an incubator shaker at 37.degree. C. These growing conditions were
used throughout the experiment.
[0039] 20 .mu.l of growing culture was inoculated in 10 ml of fresh
LB Amp and grown for 24 h. The plasmid was purified (Qiagen Plasmid
DNA Miniprep Kit) from 1 ml culture (pMAO2) and used for the second
transformation (Transformation II) of the mutator strain. The total
transformed cell suspension was inoculated in 10 ml of LB Amp and
grown for 24 h. The plasmid was purified from 1 ml of culture
(pMAOretr1.1). 100 .mu.l of Transformation II growing culture was
used to inoculate 10 ml LB Amp. The culture was grown for 24 h, the
plasmid purified (pMAOretr1.2) and used for Transformation III.
Total transformed cell suspension (1 ml) was inoculated in 10 ml LB
Amp, grown for 24 h and the plasmid purified (pMAOretr2.1). 100
.mu.l of Transformation III growing culture was used to inoculate
the next 10 ml of LB Amp, grown for 24 h and the plasmid purified
from 1 ml culture (pMAOretr2.2) and used for Transformation IV.
Total suspension of transformed cells (1 ml) was used to inoculate
10 ml of LB Amp, grown for 24 h and the plasmid purified from 1 ml
culture (pMAOretr3.1). 100 .mu.l of Transformation IV growing
culture was used to inoculate fresh 10 ml LB Amp, grown for 24 h
and the plasmid purified (pMAOretr3.2).
[0040] Collected pools of mutated plasmid DNA (pMAOretr2.2,
pMAOretr3.1, pMAOretr3.2) were used to transform E. coli BL21 (DE3)
to express mutated MAO genes and detect activity towards
L-.alpha.-methylbenzylamine (L-AMBA) oxidation.
EXAMPLE 2--SCREENING FOR MAO MUTANTS
[0041] The plate assay method (FIG. 2) was used to identify MAO
mutants with activity towards L-AMBA. More specifically, E. coli
BL21 (DE3) transformants (2500 colonies per plate) were plated on
HiBond-C Extra (Amersham Pharmacia) membrane placed on an LB Amp
agar plates and incubated for 24 h at 37.degree. C. Membranes
containing the colonies were pulled from the plates, kept at
-20.degree. C. for 24 h and incubated with assay mixture at room
temperature for 24 h.
Assay Mixture:
[0042] 1 tablet of DAB (3,3'-diaminobenzidine, Sigma, D-4418)
[0043] 1 ml of K phosphate buffer (1M, pH 7.6) [0044] 30 .mu.l
L-AMBA (10 mM) [0045] 30 .mu.l horseradish peroxidase (Sigma) 1
mg/ml [0046] 10 ml 2% agarose (Bio-Rad) [0047] Water up to 20
ml
[0048] Positive clones were subjected to a second round of
screening (100-200 colonies per plate) to confirm activity.
EXAMPLE 3--ACTIVITY STUDIES
a) Clone Selection
[0049] 27 positive clones identified in the plate assay as having
improved activity towards L-AMBA compared to the wild-type enzyme
were grown on a small scale and assayed against both L- and D-AMBA.
10 ml LB Amp was inoculated with a single colony of E. coli BL21
(DE3) transformed with the protein expression vector pET16b
harbouring the gene of interest and cultured in a 50 ml Falcon tube
with agitation for 24 h. At the end of incubation 1 ml of cell
culture was centrifuged, the pellet was resuspended in 1 ml of 25
mM potassium phosphate buffer pH 7.6 and 0.1 ml was used to perform
a hydrogen peroxide formation assay using both L- and D-AMBA as
substrates.
Assay Mixture:
[0050] (Manfred Braun et al, Applied Microbiology and Biotechnology
(1992) 37:594-598) [0051] 5 ml phosphate buffer (1M, pH 7.6) [0052]
500 .mu.l 2,4,6-tribromohydrobenzoic acid (2% in DMSO), [0053] 37.5
.mu.l 4-aminoantipurine (1M) [0054] 32.5 .mu.l L or D-AMBA (final
concn 5 mM) [0055] Water up to 50 ml To 895 ul Assay Mixture was
Added: [0056] 5 .mu.l HRP (Sigma, P6782) (1 mg/ml)
[0057] Sample (100 .mu.l) was added and absorbance at 510 nm
measured after 24 hours against a control without sample. The
absorbance results are shown in FIG. 3.
[0058] Several of these 27 clones appear to be expression mutants,
as they demonstrate increased protein expression when visualised on
polyacrylamide gels (data not shown). The plasmid of the best
mutant identified, which appeared not to have increased expression
but which showed greatest improvement over the wild-type enzyme in
terms of its selectivity, and activity, towards L-versus D-AMBA,
was grown on a larger scale and the enzyme purified. The wild-type
enzyme was also purified by the same protocol and the two enzymes
compared for substrate specificity and enantioselectivity.
[0059] The mutated gene was also sequenced and the sequence is
shown in SEQ ID NO: 2. There was a single amino acid change from
the wild-type enzyme with serine replacing asparagine at position
336.
b) Growth and Comparison of Mutant and Wild-Type Enzymes
Growth and Purification of MAO Mutant Expressed in E. coli BL21
(DE3)
[0060] LB medium (6.times.300 ml) containing ampicillin (100
.mu.gml.sup.-1) in 1 L baffled flasks was inoculated with a single
colony of monoamine oxidase mutant from an LB agar plate. Cultures
were incubated at 30.degree. C. for 22 hours (OD.sub.600.about.3.6)
then harvested and washed with phosphate buffer (50 mM, pH 8) to
yield a yellow-brown pellet (11.2 g).
[0061] The pellet was resuspended in Tris/HCl buffer (25 mM, pH
7.8, 30 ml) and sonicated on ice (30 s on, 30 s off for 10
minutes). The suspension was then centrifuged (20,000 rpm,
4.degree. C.) until clear supernatant was obtained and the
supernatant dialysed against Tris/HCl buffer (25 mM, pH 7.8). The
cell free extract was filtered through 0.45 .mu.m sterile membrane
and chromatographed on a QSepharose anion exchange column.
Fractions were assayed using colorimetric hydrogen peroxide based
assay and active fractions were stored at -80.degree. C. The active
fraction has a protein content of 1 mgml.sup.-1, and a specific
activity of 0.193 Umg.sup.-1 against amylamine.
Chromatography Conditions:
[0062] Column=HiFlow QSepharose 26/10 [0063] Buffer A=Tris/HCl (25
mM, pH 7.8) [0064] Buffer B=Tris/HCl (25 mM, pH 7.8)+1 M NaCl
[0065] Flow rate=4 mlmin.sup.-1 [0066] Fraction collect=10 ml
[0067] Column wash=2 CV 100% buffer A [0068] Elution=10 CV 100%
buffer A to 100% buffer B [0069] Column clean=4 CV 100% buffer B
Assay Mixture: [0070] 5 ml phosphate buffer (1 M, pH 7.6) [0071]
500 .mu.l 2,4,6-tribromo-3-hydroxybenzoic acid (2% in DMSO) [0072]
37.5 .mu.l 4-aminoantipyrine (1.5 M) [0073] 30 .mu.l amine
substrate (final concentration 0.015-5 mM) [0074] 44.4 ml water
Assay Conditions: [0075] 990 .mu.l assay mixture [0076] 5 .mu.l
horse radish peroxidase (1 mgml.sup.-1) [0077] 10 .mu.l enzyme
[0078] The spectrophotometer was blanked against assay mixture and
HRP. Enzyme was added and the absorbance at 500 nm measured at 3 s
intervals for a 10 minute period.
[0079] Results: TABLE-US-00001 Wild-type Enzyme Mutant Enzyme
Substrate K.sub.m mM k.sub.cat min.sup.-1 K.sub.m mM k.sub.cat
min.sup.-1 L-AMBA ND 0.17 0.4 8.0 D-AMBA ND 0.01 ND 0.08
benzylamine ND 371 ND 196 amylamine -- 1000 0.4 116
[0080] The K.sub.m values were calculated using `KaleidaGraph for
Windows` (Synergy Software). For calculation of the k.sub.cat, the
active enzyme concentration was determined by estimating the FAD
content from the absorbance at 458 nm using an extinction
coefficient of 11 mM.sup.-1 cm.sup.-1 (see ref 12).
[0081] The data reveal that the activity of the mutant amine
oxidase towards L-AMBA (kcat=8.0 min-1) is 47 fold higher than the
wild-type (kcat=0.17 min-1). Moreover the selectivity of the mutant
for the L-enantiomer versus D-AMBA (ca. 100:1) has also been
increased relative to the wild type enzyme (ca. 17:1). Thus the
outcome of the in vitro evolution experiments has been to
simultaneously improve both the enantioselectivity and catalytic
activity of the enzyme. For comparison, the activity towards the
best substrate for the wild-type enzyme, namely amylamine, and also
benzylamine, is presented. The substantial improvement in activity
and selectivity of the mutant was confirmed by chiral HPLC
(Chiralcel CrownPak CR+) in which after 24 h complete oxidation of
the L-enantiomer was apparent whereas there was no detectable
conversion of the D-enantiomer.
EXAMPLE 4--DERACEMISATION REACTION
[0082] Using DL-AMBA as the substrate, in the presence of the
mutant MAO-N, a range of reducing agents were screened (sodium
borohydride, catalytic transfer hydrogenation, amine:boranes). This
screen identified ammonia:borane as the optimal reagent which gave
a 77% yield of D-AMBA with an enantiomeric excess=93%. Greater
optical purities of the product (up to 99% e.e.) could be achieved
although at the expense of yield.
[0083] More specifically, MAO mutant N336S (100 .mu.l of 0.193
Uml.sup.-1=0.02 U) was added to a solution of DL-AMBA (0.13 .mu.l,
final concentration=0.8 mM) and ammonia-borane complex (10 .mu.l of
4M solution, final concentration=80 mM, 100 eq) in phosphate buffer
(400 .mu.l, 20 mM, pH 8). A 10 .mu.l aliquot was diluted in 990
.mu.l perchloric acid, pH 1.5 and analysed by HPLC. The reaction
mixture was incubated at 30.degree. C. and the reaction monitored
by HPLC at regular intervals until no further reaction was
observed.
[0084] Yield: D-AMBA=77%, e.e.=93%
Analytical Conditions:
[0085] Column=Chiralcel CrownPak CR+ [0086] Mobile phase=100%
perchloric acid, pH 1.5 [0087] Flow rate=0.8 mlmin.sup.-1 [0088]
Detection=200 nm [0089] Temperature=25.degree. C. [0090] r.t.
(L-AMBA)=12.8 min [0091] r.t. (D-AMBA)=16.5 min
EXAMPLE 5--STEREOINVERSION REACTION
[0092] We also carried out the stereoinversion of L- to D-AMBA (18%
yield, 99% e.e.) and showed that under identical conditions there
was no conversion of D- to L-AMBA.
[0093] More Specifically, MAO mutant N336S (100 .mu.l of 0.193
Uml.sup.-1=0.02 U) was added to a solution of L-AMBA (0.13 .mu.l,
final concentration=0.4 mM) and ammonia-borane complex (10 .mu.l of
4M solution, final concentration=80 mM, 200 eq) in phosphate buffer
(400 .mu.l, 20 mM, pH 8). A 10 .mu.l aliquot was diluted in 990
.mu.l perchloric acid, pH 1.5 and analysed by HPLC. The reaction
mixture was incubated at 30.degree. C. and the reaction monitored
by HPLC at regular intervals until no further reaction was
observed.
[0094] Yield D-AMBA=18%, e.e. >99%:
[0095] NB: no reaction was observed after 24 hours when D-AMBA was
used as substrate under identical reaction conditions.
Analytical Conditions:
[0096] Column=Chiralcel CrownPak CR+ [0097] Mobile phase=100%
perchloric acid, pH 1.5 [0098] Flow rate=0.8 mlmin.sup.-1 [0099]
Detection=200 nm [0100] Temperature=25.degree. C. [0101] r.t.
(L-AMBA)=12.8 min [0102] r.t. (D-AMBA)=16.5 min
[0103] Analysis of the HPLC chromatograms suggests that the yield
in the deracemisation reactions is prevented from reaching 100% due
to the formation a by-product with longer retention time as the
reaction progresses. We are currently optimising the deracemisation
protocol to achieve the exquisite levels of yield and selectivity
previously demonstrated for the deracemisation of .alpha.-amino
acids.
Abbreviations:
MAO--monoamine oxidase.
AMBA--.alpha.-methylbenzylamine
TBHBA--2,4,6-tribromo-3-hydroxybenzoic acid
AAP--4-aminoantipyrine
HRP--horse radish peroxidase type VI from bovine liver
LB--Lubria Bertani
r.t.--retention time
EXAMPLE 6--SUBSTRATE SPECIFICITY OF THE N336S MUTANT ENZYME
[0104] The activity of the mutant enzyme described above was
studied in relation to a variety of amine substrates. Assay
conditions were essentially as set out in Example 3. The substrates
tested were: ##STR2##
[0105] Results: TABLE-US-00002 Relative Sample Rate (S)-.alpha.
Methylbenzylamine 1 (R)-.alpha. Methylbenzylamine * (rac)-.alpha.
Methylbenzylamine * (S)-4-Methylphenylethylamine 0.28
(R)-4-Methylphenylethylamine *
(S)-.alpha.-Methyl-4-nitrobenzylamine 0.91
(R)-.alpha.-Methyl-4-nitrobenzylamine 0.06
(S)-4-Bromo-.alpha.-phenylethylamine 0.24
(R)-4-Bromo-.alpha.-phenylethylamine *
(rac)-4-Bromo-a-phenylethylamine * (S)-1-4-Methoxyphenylethylamine
0.81 (R)-1-4-Methoxyphenylethylamine 0.13 (S)-MTQ 0.67 (R)-MTQ *
(rac)-MTQ 0.13 (S)-3-Methyl-2-butylamine 5.4
(R)-3-Methyl-2-butylamine * (S)-3-3-Dimethyl-2-butylamine 1.4
(R)-3-3-Dimethyl-2-butylamine * 2-Methylcyclohexylamine 0.43
N-heptylamine 12.1 N-amylamine 25.2 Hexylamine 18.6
Bis(.alpha.-methyl)benzylamine * 1,3-Dimethylbutylamine 1.8
1,2,3,4-Tetrahydro-1-naphthylamine 0.13 * No measurable rate
EXAMPLE 7--CREATION OF A FURTHER MUTANT CLONE
[0106] The first published sequence of wild-type A. niger monoamine
oxidase showed a lysine at position 348. Some of the mutant enzymes
generated above and initially tested in Example 3(a) were thought
to be double mutants because they had a methionine at this
position. However, when the wild-type sequence was checked
(resequenced), the wild-type amino acid at position 348 was shown
to be methionine (as set out in SEQ ID NO:1). In order to elucidate
the impact (if any) of a mutation at this site, site-directed
mutagenesis was performed on the wild-type enzyme to introduce the
M348K mutation. It was noted that the identity of the amino acid at
this position influenced the efficiency of expression, with lysine
giving rise to increased expression without altering the catalytic
activity of the enzyme (absolute activity in U/ml is greater than
wild type, but correction for the amount of protein gives a value
of kt which is the same as for the wild-type enzyme). Site directed
mutagenesis was then performed upon the N336S mutant described
above to generate a second mutation (M348K), thus combining the
catalytic enhancement of the N336S mutation with an increase in
expression. The amino acid sequence of the double mutant is set out
in SEQ ID NO: 3.
EXAMPLE 8--SUBSTRATE SPECIFICITY OF THE N336S, M348K MUTANT
ENZYME
[0107] The activity of the double mutant enzyme described above was
studied in relation to a variety of amine substrates. Assay
conditions were essentially as set out in Example 3. The substrates
tested and the relative rates of conversion (compared to L-AMBA set
at 100) are shown in FIG. 4. For some substrates, both enantiomers
were tested as substrates. The enzyme appears to demonstrate
enantioselectivity in every case, with activity against the
opposite enantiomer being undetectable in several cases (the
enantioselectivity data is shown in FIG. 5).
EXAMPLE 9--APPLICATION OF EXPRESSION MUTANT R260K
[0108] The mutation R260K was found to be in low occurrence codon
for arginine. The sequencing results revealed that all of the
"expression" mutants had the same mutation at position 260 wherein
arginine was replaced by lysine.
[0109] The wild type MAO-N amino acid sequence has two arginines at
position 259 and 260 both encoded by the codon (AGG), which has a
low occurrence in E. coli genes (4%). Lysine and arginine are both
basic amino acids and hence replacement of one by the other should
not affect the charge of the protein, however the replacement of a
low frequency codon for Arg AGG (4%) by a high frequency codon for
Lys AAG (22%) results in improved protein expression in E. coli.
Thus we decided to replace the codons of both Arg 259 and Arg 260
with the codon CGT (38%) by site directed mutagenesis to evaluate
the effect upon the expression level of MAO-N. An alternative
approach would be to create a "silent" mutation by altering the AGG
codon to a different codon which still encodes arginine (AGA, CGT,
CGC, CGA, CGG).
[0110] MAO WT and MAOArg259/260 were partially purified from
recombinant E. coli BL21(DE3) harbouring a pET16b carrying the
corresponding mao-n gene. Cell free extracts were prepared by
sonication and the specific activities of MAO WT and MAOArg259/260
towards M were measured for both the soluble and insoluble
fractions. (Table 3) TABLE-US-00003 TABLE 3 Specific activities
towards AA in partially purified MAO WT and MAOArg259/260 towards
amylamine. Protein Soluble Insoluble Total concentration fraction
fraction activity Enzyme mg ml.sup.-1 U mg.sup.-1 U mg.sup.-1 U MAO
WT 1.18 0.23 0.33 0.57 MAOArg259/260 1.30 0.46 0.56 1.2
[0111] To confirm the increased level of the R260K mutant
expression, Cell free extracts of MAO WT, mutant 4 (R260K) and
MAOArg259/260 were obtained and assayed towards L-AMBA. This was
achieved by incubating 50 .mu.l of each sample for 240 minutes and
specific activities were determined. (Table 4) TABLE-US-00004 TABLE
4 Soluble .DELTA.Abs fraction Enzyme 510 nm U mg.sup.-1 MAO WT 0.06
3.6 .times. 10.sup.-5 MAOArg259/260 0.12 7.0 .times. 10.sup.-5 MAO
mutant 0.16 5.7 .times. 10.sup.-5 (R260K)
[0112] In conclusion, we have significantly extended the
deracemisation strategy by applying the method for the first time
to the deracemisation of chiral amines. In so doing we have
successfully achieved the `directed evolution` of an enzyme in
order to meet the specific requirements of a novel
biotransformation. Another interesting aspect of the present work
is the identification of a highly enantioselective mutant by using
a single enantiomer substrate in the screen (L-AMBA). There has
been much discussion concerning the need for truly enantioselective
screens in which racemates are used, thereby mimicking the
real-life situation found in a kinetic resolution in which the two
enantiomers compete for the enzyme. It may be that if one is able
to screen truly large and diverse libraries of variant genes (e.g.
10.sup.6) then it is possible to select for enantioselectivity in
the manner described herein, using inherently simpler screens.
[0113] The application of which this description and claims form
part may be used as a basis for priority in respect of any
subsequent application. The claims of such subsequent application
may be directed to any feature or combination of features described
herein. They may take the form of product, composition, process or
use claims and may include, by way of example and without
limitation, one or more of the following claims:
REFERENCES
[0114] (1) L. E. Iglesias, V. M. Sanchez, F. Rebolledo and V.
Gotor, Tetrahedron: Asymmetry (1997) 8, 2675-2677. [0115] (2) J. S.
Shin, B. G. Kim, A. Liese and C. Wandrey, Biotechnol. Bioeng.
(2001) 73 179-187; [0116] (3) G. Matcham, M. Bhatia, W. Lang, C.
Lewis, R. Nelson, A. Wang and W. Wu, Chimia (1999) 53, 584-589;
[0117] (4) J. S. Shin, B. G. Kim, Biotechnol. Bioeng., (1999) 65,
206-211. [0118] (5) G. Hieber and K. Ditrich, Chimica Oggi (2001)
19, 16-20; [0119] (6) F. Balkenhohl, K. Ditrich, B. Hauer and W.
Ladner, J. Prakt. Chem. (1997) 339, 381. [0120] (7) M. T. Reetz and
K. Schimossek, Chimia, (1996), 50, 668-669. [0121] (8) E. W. Hafner
and D. Wellner, Proc. Nat. Acad. Sci. (1971) 68, 987. [0122] (9) R.
B. Silverman, J. M. Cesarone and X. Liu, J. Am. Chem. Soc. (1993)
115, 4955. [0123] (10) B. Schilling and K. Lerch, Biochim. Biophys.
Acta. (1995), 1243, 529. [0124] (11) B. Schilling and K. Lerch,
Mol. Gen. Genet. (1995) 247, 430. [0125] (12) S. O. Sablin, V.
Yankovskaya, S. Bernard, C. N. Cronin and T. P. Singer, Eur. J.
Biochem. (1998) 253, 270. [0126] (13) T Beard and N J Turner Chem.
Commun. 2002 in press. [0127] (14) F R Alexandre, D P Pantaleone, P
P Taylor, I G Fotheringham, D J Ager and N J Turner (2002)
Tetrahedron Lett. 43, 707-710. [0128] (15) P L Dostert, M S
Benedetti and K F Tipton (1989) Medicinal Research Reviews 9,
45-89. [0129] (16) J D Sutherland (2000) Curr. Opinion. Chem. Biol.
263. [0130] (17) F H Arnold (1998) Acc. Chem. Res. 31, 125.
Sequence CWU 1
1
9 1 495 PRT Aspergillus niger 1 Met Thr Ser Arg Asp Gly Tyr Gln Trp
Thr Pro Glu Thr Gly Leu Thr 1 5 10 15 Gln Gly Val Pro Ser Leu Gly
Val Ile Ser Pro Pro Thr Asn Ile Glu 20 25 30 Asp Thr Asp Lys Asp
Gly Pro Trp Asp Val Ile Val Ile Gly Gly Gly 35 40 45 Tyr Cys Gly
Leu Thr Ala Thr Arg Asp Leu Thr Val Ala Gly Phe Lys 50 55 60 Thr
Leu Leu Leu Glu Ala Arg Asp Arg Ile Gly Gly Arg Ser Trp Ser 65 70
75 80 Ser Asn Ile Asp Gly Tyr Pro Tyr Glu Met Gly Gly Thr Trp Val
His 85 90 95 Trp His Gln Ser His Val Trp Arg Glu Ile Thr Arg Tyr
Lys Met His 100 105 110 Asn Ala Leu Ser Pro Ser Phe Asn Phe Ser Arg
Gly Val Asn His Phe 115 120 125 Gln Leu Arg Thr Asn Pro Thr Thr Ser
Thr Tyr Met Thr His Glu Ala 130 135 140 Glu Asp Glu Leu Leu Arg Ser
Ala Leu His Lys Phe Thr Asn Val Asp 145 150 155 160 Gly Thr Asn Gly
Arg Thr Val Leu Pro Phe Pro His Asp Met Phe Tyr 165 170 175 Val Pro
Glu Phe Arg Lys Tyr Asp Glu Met Ser Tyr Ser Glu Arg Ile 180 185 190
Asp Gln Ile Arg Asp Glu Leu Ser Leu Asn Glu Arg Ser Ser Leu Glu 195
200 205 Ala Phe Ile Leu Leu Cys Ser Gly Gly Thr Leu Glu Asn Ser Ser
Phe 210 215 220 Gly Glu Phe Leu His Trp Trp Ala Met Ser Gly Tyr Thr
Tyr Gln Gly 225 230 235 240 Cys Met Asp Cys Leu Ile Ser Tyr Lys Phe
Lys Asp Gly Gln Ser Ala 245 250 255 Phe Ala Arg Arg Phe Trp Glu Glu
Ala Ala Gly Thr Gly Arg Leu Gly 260 265 270 Tyr Val Phe Gly Cys Pro
Val Arg Ser Val Val Asn Glu Arg Asp Ala 275 280 285 Ala Arg Val Thr
Ala Arg Asp Gly Arg Glu Phe Val Ala Lys Arg Val 290 295 300 Val Cys
Thr Ile Pro Leu Asn Val Leu Ser Thr Ile Gln Phe Ser Pro 305 310 315
320 Ala Leu Ser Thr Glu Arg Ile Ser Ala Met Gln Ala Gly His Val Asn
325 330 335 Met Cys Thr Lys Val His Ala Glu Val Asp Asn Met Asp Met
Arg Ser 340 345 350 Trp Thr Gly Ile Ala Tyr Pro Phe Asn Lys Leu Cys
Tyr Ala Ile Gly 355 360 365 Asp Gly Thr Thr Pro Ala Gly Asn Thr His
Leu Val Cys Phe Gly Thr 370 375 380 Asp Ala Asn His Ile Gln Pro Asp
Glu Asp Val Arg Glu Thr Leu Lys 385 390 395 400 Ala Val Gly Gln Leu
Ala Pro Gly Thr Phe Gly Val Lys Arg Leu Val 405 410 415 Phe His Asn
Trp Val Lys Asp Glu Phe Ala Lys Gly Ala Trp Phe Phe 420 425 430 Ser
Arg Pro Gly Met Val Ser Glu Cys Leu Gln Gly Leu Arg Glu Lys 435 440
445 His Gly Gly Val Val Phe Ala Asn Ser Asp Trp Ala Leu Gly Trp Arg
450 455 460 Ser Phe Ile Asp Gly Ala Ile Glu Glu Gly Thr Arg Ala Ala
Arg Val 465 470 475 480 Val Leu Glu Glu Leu Gly Thr Lys Arg Glu Val
Lys Ala Arg Leu 485 490 495 2 495 PRT Artificial sequence Variant
monoamine oxidase enzyme generated by direct evolution 2 Met Thr
Ser Arg Asp Gly Tyr Gln Trp Thr Pro Glu Thr Gly Leu Thr 1 5 10 15
Gln Gly Val Pro Ser Leu Gly Val Ile Ser Pro Pro Thr Asn Ile Glu 20
25 30 Asp Thr Asp Lys Asp Gly Pro Trp Asp Val Ile Val Ile Gly Gly
Gly 35 40 45 Tyr Cys Gly Leu Thr Ala Thr Arg Asp Leu Thr Val Ala
Gly Phe Lys 50 55 60 Thr Leu Leu Leu Glu Ala Arg Asp Arg Ile Gly
Gly Arg Ser Trp Ser 65 70 75 80 Ser Asn Ile Asp Gly Tyr Pro Tyr Glu
Met Gly Gly Thr Trp Val His 85 90 95 Trp His Gln Ser His Val Trp
Arg Glu Ile Thr Arg Tyr Lys Met His 100 105 110 Asn Ala Leu Ser Pro
Ser Phe Asn Phe Ser Arg Gly Val Asn His Phe 115 120 125 Gln Leu Arg
Thr Asn Pro Thr Thr Ser Thr Tyr Met Thr His Glu Ala 130 135 140 Glu
Asp Glu Leu Leu Arg Ser Ala Leu His Lys Phe Thr Asn Val Asp 145 150
155 160 Gly Thr Asn Gly Arg Thr Val Leu Pro Phe Pro His Asp Met Phe
Tyr 165 170 175 Val Pro Glu Phe Arg Lys Tyr Asp Glu Met Ser Tyr Ser
Glu Arg Ile 180 185 190 Asp Gln Ile Arg Asp Glu Leu Ser Leu Asn Glu
Arg Ser Ser Leu Glu 195 200 205 Ala Phe Ile Leu Leu Cys Ser Gly Gly
Thr Leu Glu Asn Ser Ser Phe 210 215 220 Gly Glu Phe Leu His Trp Trp
Ala Met Ser Gly Tyr Thr Tyr Gln Gly 225 230 235 240 Cys Met Asp Cys
Leu Ile Ser Tyr Lys Phe Lys Asp Gly Gln Ser Ala 245 250 255 Phe Ala
Arg Arg Phe Trp Glu Glu Ala Ala Gly Thr Gly Arg Leu Gly 260 265 270
Tyr Val Phe Gly Cys Pro Val Arg Ser Val Val Asn Glu Arg Asp Ala 275
280 285 Ala Arg Val Thr Ala Arg Asp Gly Arg Glu Phe Val Ala Lys Arg
Val 290 295 300 Val Cys Thr Ile Pro Leu Asn Val Leu Ser Thr Ile Gln
Phe Ser Pro 305 310 315 320 Ala Leu Ser Thr Glu Arg Ile Ser Ala Met
Gln Ala Gly His Val Ser 325 330 335 Met Cys Thr Lys Val His Ala Glu
Val Asp Asn Met Asp Met Arg Ser 340 345 350 Trp Thr Gly Ile Ala Tyr
Pro Phe Asn Lys Leu Cys Tyr Ala Ile Gly 355 360 365 Asp Gly Thr Thr
Pro Ala Gly Asn Thr His Leu Val Cys Phe Gly Thr 370 375 380 Asp Ala
Asn His Ile Gln Pro Asp Glu Asp Val Arg Glu Thr Leu Lys 385 390 395
400 Ala Val Gly Gln Leu Ala Pro Gly Thr Phe Gly Val Lys Arg Leu Val
405 410 415 Phe His Asn Trp Val Lys Asp Glu Phe Ala Lys Gly Ala Trp
Phe Phe 420 425 430 Ser Arg Pro Gly Met Val Ser Glu Cys Leu Gln Gly
Leu Arg Glu Lys 435 440 445 His Gly Gly Val Val Phe Ala Asn Ser Asp
Trp Ala Leu Gly Trp Arg 450 455 460 Ser Phe Ile Asp Gly Ala Ile Glu
Glu Gly Thr Arg Ala Ala Arg Val 465 470 475 480 Val Leu Glu Glu Leu
Gly Thr Lys Arg Glu Val Lys Ala Arg Leu 485 490 495 3 495 PRT
Artificial sequence Variant monoamine oxidase enzyme generated by
site directed mutagenesis of the N336S mutant enzyme of SEQ ID NO 2
3 Met Thr Ser Arg Asp Gly Tyr Gln Trp Thr Pro Glu Thr Gly Leu Thr 1
5 10 15 Gln Gly Val Pro Ser Leu Gly Val Ile Ser Pro Pro Thr Asn Ile
Glu 20 25 30 Asp Thr Asp Lys Asp Gly Pro Trp Asp Val Ile Val Ile
Gly Gly Gly 35 40 45 Tyr Cys Gly Leu Thr Ala Thr Arg Asp Leu Thr
Val Ala Gly Phe Lys 50 55 60 Thr Leu Leu Leu Glu Ala Arg Asp Arg
Ile Gly Gly Arg Ser Trp Ser 65 70 75 80 Ser Asn Ile Asp Gly Tyr Pro
Tyr Glu Met Gly Gly Thr Trp Val His 85 90 95 Trp His Gln Ser His
Val Trp Arg Glu Ile Thr Arg Tyr Lys Met His 100 105 110 Asn Ala Leu
Ser Pro Ser Phe Asn Phe Ser Arg Gly Val Asn His Phe 115 120 125 Gln
Leu Arg Thr Asn Pro Thr Thr Ser Thr Tyr Met Thr His Glu Ala 130 135
140 Glu Asp Glu Leu Leu Arg Ser Ala Leu His Lys Phe Thr Asn Val Asp
145 150 155 160 Gly Thr Asn Gly Arg Thr Val Leu Pro Phe Pro His Asp
Met Phe Tyr 165 170 175 Val Pro Glu Phe Arg Lys Tyr Asp Glu Met Ser
Tyr Ser Glu Arg Ile 180 185 190 Asp Gln Ile Arg Asp Glu Leu Ser Leu
Asn Glu Arg Ser Ser Leu Glu 195 200 205 Ala Phe Ile Leu Leu Cys Ser
Gly Gly Thr Leu Glu Asn Ser Ser Phe 210 215 220 Gly Glu Phe Leu His
Trp Trp Ala Met Ser Gly Tyr Thr Tyr Gln Gly 225 230 235 240 Cys Met
Asp Cys Leu Ile Ser Tyr Lys Phe Lys Asp Gly Gln Ser Ala 245 250 255
Phe Ala Arg Arg Phe Trp Glu Glu Ala Ala Gly Thr Gly Arg Leu Gly 260
265 270 Tyr Val Phe Gly Cys Pro Val Arg Ser Val Val Asn Glu Arg Asp
Ala 275 280 285 Ala Arg Val Thr Ala Arg Asp Gly Arg Glu Phe Val Ala
Lys Arg Val 290 295 300 Val Cys Thr Ile Pro Leu Asn Val Leu Ser Thr
Ile Gln Phe Ser Pro 305 310 315 320 Ala Leu Ser Thr Glu Arg Ile Ser
Ala Met Gln Ala Gly His Val Ser 325 330 335 Met Cys Thr Lys Val His
Ala Glu Val Asp Asn Lys Asp Met Arg Ser 340 345 350 Trp Thr Gly Ile
Ala Tyr Pro Phe Asn Lys Leu Cys Tyr Ala Ile Gly 355 360 365 Asp Gly
Thr Thr Pro Ala Gly Asn Thr His Leu Val Cys Phe Gly Thr 370 375 380
Asp Ala Asn His Ile Gln Pro Asp Glu Asp Val Arg Glu Thr Leu Lys 385
390 395 400 Ala Val Gly Gln Leu Ala Pro Gly Thr Phe Gly Val Lys Arg
Leu Val 405 410 415 Phe His Asn Trp Val Lys Asp Glu Phe Ala Lys Gly
Ala Trp Phe Phe 420 425 430 Ser Arg Pro Gly Met Val Ser Glu Cys Leu
Gln Gly Leu Arg Glu Lys 435 440 445 His Gly Gly Val Val Phe Ala Asn
Ser Asp Trp Ala Leu Gly Trp Arg 450 455 460 Ser Phe Ile Asp Gly Ala
Ile Glu Glu Gly Thr Arg Ala Ala Arg Val 465 470 475 480 Val Leu Glu
Glu Leu Gly Thr Lys Arg Glu Val Lys Ala Arg Leu 485 490 495 4 8 PRT
Aspergillus niger 4 Leu Ile Lys Ala Ile Lys Gly Tyr 1 5 5 12 PRT
Aspergillus niger 5 Ile Ser Tyr Tyr Ile Gln His Asn Tyr Thr Cys Glu
1 5 10 6 32 PRT Aspergillus niger 6 Trp Gly His Ile Gln Ser Ile Ser
Ser Arg Cys Arg Trp Arg Cys Ser 1 5 10 15 Ser Arg Glu Gln Arg Ile
Ser Ala Pro Trp Arg Gln Cys Arg Thr Gln 20 25 30 7 35 PRT
Aspergillus niger 7 Arg His Ser Ser Gly Arg Cys Arg Pro Glu Cys Arg
Asp Leu Arg Arg 1 5 10 15 Cys Arg Tyr His Arg Gln Arg Tyr Arg Lys
Cys Arg Trp Cys Cys Pro 20 25 30 Arg Arg Arg 35 8 9 PRT Aspergillus
niger 8 Gln His Arg Gly Ser Cys Arg Gly Gly 1 5 9 9 PRT Aspergillus
niger 9 Cys Arg Arg Ser Phe Arg Trp Met Val 1 5
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