U.S. patent application number 16/219454 was filed with the patent office on 2019-05-23 for enzymatic hydroxylation of aliphatic hydrocarbon.
This patent application is currently assigned to Novozymes A/S. The applicant listed for this patent is Novozymes A/S. Invention is credited to Martin Hofrichter, Lisbeth Kalum, Matthias Kinne, Henrik Lund, Sebastian Peter, Katrin Scheibner, Rene Ullrich.
Application Number | 20190153480 16/219454 |
Document ID | / |
Family ID | 43996790 |
Filed Date | 2019-05-23 |
United States Patent
Application |
20190153480 |
Kind Code |
A1 |
Hofrichter; Martin ; et
al. |
May 23, 2019 |
Enzymatic Hydroxylation of Aliphatic Hydrocarbon
Abstract
The invention relates to enzymatic methods for hydroxylation in
position 2 or 3 of substituted or unsubstituted, linear or branched
aliphatic hydrocarbons.
Inventors: |
Hofrichter; Martin;
(Dresden, DE) ; Scheibner; Katrin; (Jena, DE)
; Ullrich; Rene; (Zittau, DE) ; Kinne;
Matthias; (Gorlitz, DE) ; Peter; Sebastian;
(Zittau, DE) ; Lund; Henrik; (Vaerloese, DK)
; Kalum; Lisbeth; (Vaerloese, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novozymes A/S |
Bagsvaerd |
|
DK |
|
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
43996790 |
Appl. No.: |
16/219454 |
Filed: |
December 13, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15887636 |
Feb 2, 2018 |
10174342 |
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16219454 |
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15349024 |
Nov 11, 2016 |
9909147 |
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15887636 |
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14842953 |
Sep 2, 2015 |
9534238 |
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15349024 |
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13637716 |
Sep 27, 2012 |
9222109 |
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PCT/EP2011/054761 |
Mar 28, 2011 |
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14842953 |
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61318582 |
Mar 29, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 7/02 20130101; C12Y
114/14001 20130101; C12N 9/0071 20130101; C12P 7/00 20130101; C12P
7/04 20130101; C12P 7/42 20130101; C12Y 111/02001 20130101 |
International
Class: |
C12P 7/04 20060101
C12P007/04; C12P 7/02 20060101 C12P007/02; C12P 7/42 20060101
C12P007/42 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 28, 2010 |
EP |
10158092.6 |
Mar 28, 2010 |
EP |
10158093.4 |
Claims
1-17. (canceled)
18: A method for hydroxylation in position 2 or 3 of either end of
a substituted or unsubstituted, linear or branched, aliphatic
hydrocarbon having at least 3 carbons and having a hydrogen
attached to the carbon in position 2 or 3, comprising contacting
the aliphatic hydrocarbon with hydrogen peroxide and a polypeptide
having peroxygenase activity; wherein the polypeptide has at least
90% sequence identity to SEQ ID NO: 1.
19: The method of claim 18, wherein the carbon in position 2 or 3,
which is hydroxylated, is unsubstituted until it is contacted with
the peroxygenase.
20: The method of claim 18, wherein the amino acid sequence of the
polypeptide comprises the motif of SEQ ID NO: 9.
21: The method of claim 18, wherein the amino acid sequence of the
polypeptide comprises the motif of SEQ ID NO: 10.
22: The method of claim 18, wherein the amino acid sequence of the
polypeptide comprises the motif of SEQ ID NO: 11.
23: The method of claim 18, wherein the amino acid sequence of the
polypeptide comprises the motif of SEQ ID NO: 12.
24: The method of claim 18, wherein the amino acid sequence of the
polypeptide comprises the motif of SEQ ID NO: 13.
25: The method of claim 18, wherein the amino acid sequence of the
polypeptide comprises the motif of SEQ ID NO: 14.
26: The method of claim 18, wherein the polypeptide has at least
95% sequence identity to SEQ ID NO: 1.
27: The method of claim 18, wherein the polypeptide comprises or
consists of the amino acid sequence of SEQ ID NO: 1.
28: The method of claim 18, wherein the polypeptide is a fragment
of SEQ ID NO: 1 having peroxygenase activity.
29: The method of claim 18, wherein the substituents of the
aliphatic hydrocarbon are selected from the group consisting of
halogen, hydroxyl, carboxyl, amino, nitro, cyano, thiol, sulphonyl,
formyl, acetyl, methoxy, ethoxy, phenyl, benzyl, xylyl, carbamoyl
and sulfamoyl.
30: The method of claim 18, wherein the substituents are selected
from the group consisting of chloro, hydroxyl, carboxyl and
sulphonyl; in particular chloro and carboxyl.
31: The method of claim 18, wherein the aliphatic hydrocarbon is
unsubstituted.
32: The method of claim 18, wherein the aliphatic hydrocarbon is
linear.
33: The method of claim 18, wherein the aliphatic hydrocarbon is an
alkane.
34: The method of claim 33, wherein the alkane is propane, butane,
pentane, hexane, heptane, octane, nonane or decane, or isomers
thereof.
35: The method of claim 18, wherein the aliphatic hydrocarbon is
part of a fatty acid.
36: A method for hydroxylation in position 2 or 3 of the terminal
end of an acyl group of a lipid, comprising contacting the lipid
with hydrogen peroxide and a polypeptide having peroxygenase
activity; wherein the polypeptide has at least 90% sequence
identity to SEQ ID NO: 1.
37: The method of claim 36, wherein the polypeptide has at least
95% identity to SEQ ID NO: 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 15/887,636 filed on Feb. 2, 2018, now pending, which is a
divisional of U.S. application Ser. No. 15/349,024 filed on Nov.
11, 2016, now U.S. Pat. No. 9,909,147, which is a divisional of
U.S. application Ser. No. 14/842,953 filed on Sep. 2, 2015, now
U.S. Pat. No. 9,534,238, which is a divisional of U.S. application
Ser. No. 13/637,716, now U.S. Pat. No. 9,222,109, which is a 35
U.S.C. 371 national application of PCT/EP2011/054761 filed Mar. 28,
2011, which claims priority or the benefit under 35 U.S.C. 119 of
European application nos. 10158092.6 and 10158093.4, each filed on
Mar. 28, 2010, and U.S. provisional application No. 61/318,582
filed on Mar. 29, 2010. The content of each application is fully
incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING
[0002] This application contains a Sequence Listing in computer
readable form, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The present invention relates to the use of polypeptides
having peroxygenase activity for site specific hydroxylation of
aliphatic hydrocarbons.
Background
[0004] A peroxygenase denoted AaP from the agaric basidiomycete
strain Agrocybe aegerita (strain TM-A1) was found to oxidize aryl
alcohols and aldehydes. The AaP peroxygenase was purified from A.
aegerita TM A1 by several steps of ion chromatography and SDS-PAGE,
the molecular weight was determined and the N-terminal 14 amino
acid sequence was determined after 2-D electrophoresis but the
encoding gene was not isolated (Ullrich et al., 2004, Appl. Env.
Microbiol. 70(8): 4575-4581).
[0005] WO 2006/034702 discloses methods for the enzymatic
hydroxylation of non-activated hydrocarbons, such as, naphtalene,
toluol and cyclohexane, using the AaP peroxygenase enzyme of
Agrocybe aegerita TM A1. This is also described in Ullrich and
Hofrichter, 2005, FEBS Letters 579: 6247-6250.
[0006] WO 2008/119780 discloses eight different peroxygenases from
Agrocybe aegerita, Coprinopsis cinerea, Laccaria bicolor and
Coprinus radians; also shown as SEQ ID NOs: 1-8 in the present
application.
[0007] DE 103 32 065 A1 discloses methods for the enzymatic
preparation of acids from alcohols through the intermediary
formation of aldehydes by using the AaP peroxygenase enzyme of
Agrocybe aegerita TM A1.
[0008] A method was reported for the rapid and selective
spectrophotometric direct detection of aromatic hydroxylation by
the AaP peroxygenase (Kluge et al., 2007, Appl. Microbiol.
Biotechnol. 75: 1473-1478).
[0009] It is well-known that a direct regioselective introduction
of oxygen functions (oxygenation) into organic molecules
constitutes a problem in chemical synthesis. It is particularly
difficult to catalyse the selective hydroxylation of aliphatic
carbohydrates. The products may be used as important intermediates
in a wide variety of different syntheses.
[0010] In particular the chemical hydroxylation of alkanes is
relatively complex, requires aggressive/toxic chemicals/catalysts
and leads to a series of undesired by-products.
[0011] It is known that an intracellular enzyme, methane
monooxygenase (MMO, EC 14.13.25), oxygenates/hydroxylates the
terminal carbon of some hydrocarbons. The MMO enzyme consists of
several protein components and is formed by methylotrophic bacteria
(e.g. Methylococcus capsulatus); it requires complex electron
donors such as NADH or NADPH, auxiliary proteins (flavin
reductases, regulator protein) and molecular oxygen (O.sub.2). The
natural substrate of MMO is methane, which is oxidized to methanol.
As a particularly unspecific biocatalyst, MMO
oxygenates/hydroxylates, as well as methane, a series of further
substrates such as n-alkanes and their derivatives, cycloalkanes,
aromatics, carbon monoxide and heterocycles. Utilization of the
enzyme in biotechnology is currently not possible, since it is
difficult to isolate, like most intracellular enzymes, it is of low
stability, and the cosubstrates required are relatively
expensive.
SUMMARY OF THE INVENTION
[0012] In a first aspect, the inventors of the present invention
have provided a method for hydroxylation in position 2 or 3 of
either end of a substituted or unsubstituted, linear or branched,
aliphatic hydrocarbon having at least 3 carbons and having a
hydrogen attached to the carbon in position 2 or 3, comprising
contacting the aliphatic hydrocarbon with hydrogen peroxide and a
polypeptide having peroxygenase activity; wherein the polypeptide
comprises:
[0013] a) an amino acid sequence which has at least 50% identity to
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8; and
[0014] b) an amino acid sequence represented by one or more of the
following motifs:
TABLE-US-00001 Motif I: (SEQ ID NO: 9)
[FL]XX[YF]S[AN]X[FHY]G[GN]GX[YF]N; Motif II: (SEQ ID NO: 10)
G[GN]GX[YF]NXN[VA]AX[EH][LF]R; Motif III: (SEQ ID NO: 11)
RXXRI[EQ][DEQ]S[IM]ATN; Motif IV: (SEQ ID NO: 12)
S[IM]ATN[PG][EQN][FM][SDN][FL]; Motif V: (SEQ ID NO: 13)
P[PDK][DG]F[HFW]R[AP]; Motif VI: (SEQ ID NO: 14)
[TI]XXXLYPNP[TK][GV].
[0015] In an embodiment, the aliphatic hydrocarbon is a fatty
acid.
[0016] In another aspect is provided a method for hydroxylation in
position 2 or 3 of the terminal end of an acyl group of a lipid,
comprising contacting the lipid with hydrogen peroxide and a
polypeptide having peroxygenase activity; wherein the polypeptide
comprises:
[0017] a) an amino acid sequence which has at least 50% identity to
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8; and
[0018] b) an amino acid sequence represented by one or more of the
following motifs:
TABLE-US-00002 Motif I: (SEQ ID NO: 9)
[FL]XX[YF]S[AN]X[FHY]G[GN]GX[YF]N; Motif II: (SEQ ID NO: 10)
G[GN]GX[YF]NXN[VA]AX[EH][LF]R; Motif III: (SEQ ID NO: 11)
RXXRI[EQ][DEQ]S[IM]ATN; Motif IV: (SEQ ID NO: 12)
S[IM]ATN[PG][EQN][FM][SDN][FL]; Motif V: (SEQ ID NO: 13)
P[PDK][DG]F[HFW]R[AP]; Motif VI: (SEQ ID NO: 14)
[TI]XXXLYPNP[TK][GV].
[0019] In further aspects, the invention provides uses of
polypeptides having peroxygenase activity for removal of lipid
containing stains from laundry; and for reducing unpleasant odor
from laundry.
Definitions
[0020] Peroxygenase activity: The term "peroxygenase activity" is
defined herein as the capability to hydroxylate naphtalene using
hydrogen peroxide, also referred to as "unspecific peroxygenase",
EC 1.11.2.1. This is a heme-thiolate protein. Enzymes of this type
include glycoproteins secreted by agaric basidiomycetes. They
catalyse the insertion of an oxygen atom from H.sub.2O.sub.2 into a
wide variety of substrates, including aromatic rings such as
naphthalene, toluene, phenanthrene, pyrene and p-nitrophenol,
recalcitrant heterocycles such as pyridine, dibenzofuran, various
ethers (resulting in O-dealkylation) and alkanes such as propane,
hexane and cyclohexane. Additional reactions which may be catalysed
by peroxygenases include hydroxylation, epoxidation, N-oxidation,
sulfooxidation, O- and N-dealkylation, bromination and one-electron
oxidations. They have little or no activity toward chloride.
[0021] For purposes of the present invention, peroxygenase activity
is determined according to the spectrophotometric procedure
described by Kluge et al. (2007, Appl. Microbiol. Biotechnol. 75:
1473-1478).
[0022] The polypeptides of the present invention have at least 20%,
preferably at least 40%, more preferably at least 50%, more
preferably at least 60%, more preferably at least 70%, more
preferably at least 80%, even more preferably at least 90%, most
preferably at least 95%, and even most preferably at least 100% of
the peroxygenase activity of the mature polypeptide of SEQ ID NO:
1, 2, 3, 4, 5, 6, 7 or 8.
[0023] Isolated polypeptide: The term "isolated polypeptide" as
used herein refers to a polypeptide that is isolated from a source.
In a preferred aspect, the polypeptide is at least 1% pure,
preferably at least 5% pure, more preferably at least 10% pure,
more preferably at least 20% pure, more preferably at least 40%
pure, more preferably at least 60% pure, even more preferably at
least 80% pure, and most preferably at least 90% pure, as
determined by SDS-PAGE.
[0024] Substantially pure polypeptide: The term "substantially pure
polypeptide" denotes herein a polypeptide preparation that contains
at most 10%, preferably at most 8%, more preferably at most 6%,
more preferably at most 5%, more preferably at most 4%, more
preferably at most 3%, even more preferably at most 2%, most
preferably at most 1%, and even most preferably at most 0.5% by
weight of other polypeptide material with which it is natively or
recombinantly associated. It is, therefore, preferred that the
substantially pure polypeptide is at least 92% pure, preferably at
least 94% pure, more preferably at least 95% pure, more preferably
at least 96% pure, more preferably at least 96% pure, more
preferably at least 97% pure, more preferably at least 98% pure,
even more preferably at least 99%, most preferably at least 99.5%
pure, and even most preferably 100% pure by weight of the total
polypeptide material present in the preparation. The polypeptides
of the present invention are preferably in a substantially pure
form, i.e., that the polypeptide preparation is essentially free of
other polypeptide material with which it is natively or
recombinantly associated. This can be accomplished, for example, by
preparing the polypeptide by well-known recombinant methods or by
classical purification methods.
[0025] Mature polypeptide: The term "mature polypeptide" is defined
herein as a polypeptide having peroxygenase activity that is in its
final form following translation and any post-translational
modifications, such as N-terminal processing, C-terminal
truncation, glycosylation, phosphorylation, etc. In a preferred
aspect, the mature polypeptide has the amino acid sequence shown in
positions 1 to 330 of SEQ ID NO:1 based on the N-terminal peptide
sequencing data (Ullrich et al., 2004, Appl. Env. Microbiol. 70(8):
4575-4581), elucidating the start of the mature protein of AaP
peroxygenase enzyme. In another preferred aspect, the mature
polypeptide has the amino acid sequence shown in positions 1 to 328
of SEQ ID NO:2.
[0026] Identity: The relatedness between two amino acid sequences
or between two nucleotide sequences is described by the parameter
"identity".
[0027] For purposes of the present invention, the degree of
identity between two amino acid sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of the
EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277;
emboss.org), preferably version 3.0.0 or later. The optional
parameters used are gap open penalty of 10, gap extension penalty
of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution
matrix. The output of Needle labeled "longest identity" (obtained
using the--nobrief option) is used as the percent identity and is
calculated as follows:
(Identical Residues.times.100)/(Length of Alignment-Total Number of
Gaps in Alignment)
[0028] For purposes of the present invention, the degree of
identity between two deoxyribonucleotide sequences is determined
using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970,
supra) as implemented in the Needle program of the EMBOSS package
(EMBOSS: The European Molecular Biology Open Software Suite, Rice
et al., 2000, supra; emboss.org), preferably version 3.0.0 or
later. The optional parameters used are gap open penalty of 10, gap
extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI
NUC4.4) substitution matrix. The output of Needle labeled "longest
identity" (obtained using the--nobrief option) is used as the
percent identity and is calculated as follows:
(Identical Deoxyribonucleotides.times.100)/(Length of
Alignment-Total Number of Gaps in Alignment).
[0029] Modification: The term "modification" means herein any
chemical modification of the polypeptide consisting of the mature
polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8; or a homologous
sequence thereof; as well as genetic manipulation of the DNA
encoding such a polypeptide. The modification can be a
substitution, a deletion and/or an insertion of one or more
(several) amino acids as well as replacements of one or more
(several) amino acid side chains.
DETAILED DESCRIPTION OF THE INVENTION
Polypeptides Having Peroxygenase Activity (Peroxygenases)
[0030] The present invention relates to uses of an isolated
polypeptide, which is preferably recombinantly produced, having
peroxygenase activity, which comprises an amino acid sequence
having at least 50% identity, preferably at least 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 97%, or 98% identity to the
polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.
[0031] In a preferred embodiment, the polypeptide comprises an
amino acid sequence represented by one or more of the following
motifs, preferably comprising two or more, three or more, four or
more, five or six of the following motifs:
TABLE-US-00003 Motif I: (SEQ ID NO: 9)
[FL]XX[YF]S[AN]X[FHY]G[GN]GX[YF]N; Motif II: (SEQ ID NO: 10)
G[GN]GX[YF]NXN[VA]AX[EH][LF]R; Motif III: (SEQ ID NO: 11)
RXXRI[EQ][DEQ]S[IM]ATN; Motif IV: (SEQ ID NO: 12)
S[IM]ATN[PG][EQN][FM][SDN][FL]; Motif V: (SEQ ID NO: 13)
P[PDK][DG]F[HFW]R[AP]; Motif VI: (SEQ ID NO: 14)
[TI]XXXLYPNP[TK][GV].
[0032] In another embodiment, the polypeptide comprises an amino
acid sequence having a substitution, deletion, and/or insertion of
one or several amino acids of the mature polypeptide of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, or SEQ ID NO:8.
[0033] In yet another embodiment, the polypeptide of the first
aspect comprises or consists of the amino acid sequence of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, or SEQ ID NO:8; or a fragment thereof having
peroxygenase activity; preferably the polypeptide comprises or
consists of the mature polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ
ID NO:8.
[0034] Preferably, amino acid changes are of a minor nature, that
is conservative amino acid substitutions or insertions that do not
significantly affect the folding and/or activity of the protein;
small deletions, typically of one to about 30 amino acids; small
amino- or carboxyl-terminal extensions, such as an amino-terminal
methionine residue; a small linker peptide of up to about 20-25
residues; or a small extension that facilitates purification by
changing net charge or another function, such as a poly-histidine
tract, an antigenic epitope or a binding domain.
[0035] Examples of conservative substitutions are within the group
of basic amino acids (arginine, lysine and histidine), acidic amino
acids (glutamic acid and aspartic acid), polar amino acids
(glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine and valine), aromatic amino acids (phenylalanine,
tryptophan and tyrosine), and small amino acids (glycine, alanine,
serine, threonine and methionine). Amino acid substitutions that do
not generally alter specific activity are known in the art and are
described, for example, by H. Neurath and R. L. Hill, 1979, In, The
Proteins, Academic Press, New York. The most commonly occurring
exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,
Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,
Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
[0036] In addition to the 20 standard amino acids, non-standard
amino acids (such as 4-hydroxyproline, 6-N-methyl lysine,
2-aminoisobutyric acid, isovaline, and alpha-methyl serine) may be
substituted for amino acid residues of a wild-type polypeptide. A
limited number of non-conservative amino acids, amino acids that
are not encoded by the genetic code, and unnatural amino acids may
be substituted for amino acid residues. "Unnatural amino acids"
have been modified after protein synthesis, and/or have a chemical
structure in their side chain(s) different from that of the
standard amino acids. Unnatural amino acids can be chemically
synthesized, and preferably, are commercially available, and
include pipecolic acid, thiazolidine carboxylic acid,
dehydroproline, 3- and 4-methylproline, and
3,3-dimethylproline.
[0037] Alternatively, the amino acid changes are of such a nature
that the physico-chemical properties of the polypeptides are
altered. For example, amino acid changes may improve the thermal
stability of the polypeptide, alter the substrate specificity,
change the pH optimum, and the like.
[0038] Essential amino acids in the parent polypeptide can be
identified according to procedures known in the art, such as
site-directed mutagenesis or alanine-scanning mutagenesis
(Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter
technique, single alanine mutations are introduced at every residue
in the molecule, and the resultant mutant molecules are tested for
biological activity (i.e., peroxygenase activity) to identify amino
acid residues that are critical to the activity of the molecule.
See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The
active site of the enzyme or other biological interaction can also
be determined by physical analysis of structure, as determined by
such techniques as nuclear magnetic resonance, crystallography,
electron diffraction, or photoaffinity labeling, in conjunction
with mutation of putative contact site amino acids. See, for
example, de Vos et al., 1992, Science 255: 306-312; Smith et al.,
1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett.
309: 59-64. The identities of essential amino acids can also be
inferred from analysis of identities with polypeptides that are
related to a polypeptide according to the invention.
[0039] Single or multiple amino acid substitutions, deletions,
and/or insertions can be made and tested using known methods of
mutagenesis, recombination, and/or shuffling, followed by a
relevant screening procedure, such as those disclosed by
Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and
Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413;
or WO 95/22625. Other methods that can be used include error-prone
PCR, phage display (e.g., Lowman et al., 1991, Biochem. 30:
10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and
region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145;
Ner et al., 1988, DNA 7: 127).
[0040] Mutagenesis/shuffling methods can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides expressed by host cells (Ness et
al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA
molecules that encode active polypeptides can be recovered from the
host cells and rapidly sequenced using standard methods in the art.
These methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide of interest, and
can be applied to polypeptides of unknown structure.
[0041] The total number of amino acid substitutions, deletions
and/or insertions of the mature polypeptide of SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7 or SEQ ID NO:8 is 10, preferably 9, more preferably 8, more
preferably 7, more preferably at most 6, more preferably 5, more
preferably 4, even more preferably 3, most preferably 2, and even
most preferably 1.
[0042] It is preferable that the polypeptide of the first aspect is
encoded by the polynucleotide contained in the plasmid which is
contained in E. coli NN049991 deposited 14 Mar. 2008 under the
terms of the Budapest Treaty with the DSMZ under accession number
DSM 21289; or which is encoded by the polynucleotide contained in
the plasmid which is contained in E. coli NN049992 deposited 14
Mar. 2008 under the terms of the Budapest Treaty with the DSMZ
under accession number DSM 21290.
[0043] Another preferred embodiment relates to the polypeptide of
the first aspect of the invention, wherein the mature polypeptide
is amino acids 1 to 330 of SEQ ID NO:1.
[0044] Yet another preferred embodiment relates to the polypeptide
of the first aspect of the invention, wherein the mature
polypeptide is amino acids 1 to 328 of SEQ ID NO:2.
[0045] Yet another preferred embodiment relates to the polypeptide
of the first aspect of the invention, wherein the mature
polypeptide is amino acids 1 to 344 of SEQ ID NO:4.
Hydrogen Peroxide
[0046] The hydrogen peroxide required by the peroxygenase may be
provided as an aqueous solution of hydrogen peroxide or a hydrogen
peroxide precursor for in situ production of hydrogen peroxide. Any
solid entity which liberates upon dissolution a peroxide which is
useable by peroxygenase can serve as a source of hydrogen peroxide.
Compounds which yield hydrogen peroxide upon dissolution in water
or an appropriate aqueous based medium include but are not limited
to metal peroxides, percarbonates, persulphates, perphosphates,
peroxyacids, alkyperoxides, acylperoxides, peroxyesters, urea
peroxide, perborates and peroxycarboxylic acids or salts
thereof.
[0047] Another source of hydrogen peroxide is a hydrogen peroxide
generating enzyme system, such as an oxidase together with a
substrate for the oxidase. Examples of combinations of oxidase and
substrate comprise, but are not limited to, amino acid oxidase (see
e.g. U.S. Pat. No. 6,248,575) and a suitable amino acid, glucose
oxidase (see e.g. WO 95/29996) and glucose, lactate oxidase and
lactate, galactose oxidase (see e.g. WO 00/50606) and galactose,
and aldose oxidase (see e.g. WO 99/31990) and a suitable
aldose.
[0048] By studying EC 1.1.3._, EC 1.2.3._, EC 1.4.3._, and EC
1.5.3..sub.-- or similar classes (under the International Union of
Biochemistry), other examples of such combinations of oxidases and
substrates are easily recognized by one skilled in the art.
[0049] Hydrogen peroxide or a source of hydrogen peroxide may be
added at the beginning of or during the method of the invention,
e.g. as one or more separate additions of hydrogen peroxide; or
continously as fed-batch addition. Typical amounts of hydrogen
peroxide correspond to levels of from 0.001 mM to 25 mM, preferably
to levels of from 0.005 mM to 5 mM, and particularly to levels of
from 0.01 to 1 mM hydrogen peroxide. Hydrogen peroxide may also be
used in an amount corresponding to levels of from 0.1 mM to 25 mM,
preferably to levels of from 0.5 mM to 15 mM, more preferably to
levels of from 1 mM to 10 mM, and most preferably to levels of from
2 mM to 8 mM hydrogen peroxide.
Surfactants
[0050] The method of the invention may include application of a
surfactant (for example, as part of a detergent formulation or as a
wetting agent). Surfactants suitable for being applied may be
non-ionic (including semi-polar), anionic, cationic and/or
zwitterionic; preferably the surfactant is anionic (such as linear
alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty
alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,
alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid
or soap) or non-ionic (such as alcohol ethoxylate, nonylphenol
ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,
ethoxylated fatty acid monoethanolamide, fatty acid
monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl
N-alkyl derivatives of glucosamine ("glucamides")), or a mixture
thereof.
[0051] When included in the method of the invention, the
concentration of the surfactant will usually be from about 0.01% to
about 10%, preferably about 0.05% to about 5%, and more preferably
about 0.1% to about 1% by weight.
Aliphatic Hydrocarbons
[0052] The hydrocarbons, which are hydroxylated in the method of
the invention, are aliphatic hydrocarbons having a chain of at
least 3 carbons, and having a hydrogen attached to the carbon in
position 2 or 3. Preferably, the aliphatic hydrocarbon is an alkane
or an alkene; more preferably, the aliphatic hydrocarbon is an
alkane, such as propane, butane, pentane, hexane, heptane, octane,
nonane or decane, or isomers thereof.
[0053] The aliphatic hydrocarbons are linear or branched, but not
cyclic, as site specific hydroxylation is not possible with cyclic
hydrocarbons. Branched hydrocarbons correspond to isomers of linear
hydrocarbons.
[0054] The aliphatic hydrocarbons are substituted or unsubstituted.
Preferably, the aliphatic hydrocarbons are unsubstituted, such as
non-activated hydrocarbons.
[0055] When the aliphatic hydrocarbons are substituted (functional
groups attached), the preferred substituents are halogen, hydroxyl,
carboxyl, amino, nitro, cyano, thiol, sulphonyl, formyl, acetyl,
methoxy, ethoxy, phenyl, benzyl, xylyl, carbamoyl and sulfamoyl;
more preferred substituents are chloro, hydroxyl, carboxyl and
sulphonyl; and most preferred substituents are chloro and
carboxyl.
[0056] The aliphatic hydrocarbons may be substituted by up to 10
substituents, up to 8 substituents, up to 6 substituents, up to 4
substituents, up to 2 substituents, or by up to one
substituent.
[0057] In a preferred embodiment, the aliphatic hydrocarbon is a
fatty acid (the substituent is a carboxyl group). Examples of fatty
acids include, but are not limited to, butanoic acid (butyric
acid), pentanoic acid (valeric acid), hexanoic acid (caproic acid),
heptanoic acid (enanthic acid), octanoic acid (caprylic acid),
nonanoic acid (pelargonic acid), decanoic acid (capric acid),
dodecanoic acid (lauric acid), tetradecanoic acid (myristic acid),
hexadecanoic acid (palmitic acid), octadecanoic acid (stearic
acid), eicosanoic acid (arachidic acid), linoleic acid, linolenic
acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic
acid.
[0058] In a second aspect, the aliphatic hydrocarbon is an acyl
group of a lipid, such as a monoglyceride, diglyceride,
triglyceride, phospholipid or sphingolipid; and the hydroxylation
takes place in position 2 or position 3 of the terminal end of the
acyl group. The acyl group must have at least one hydrogen attached
to the carbon in position 2 or 3 of the terminal end. The acyl
group may be saturated or unsaturated, and optionally functional
groups (substituents) may be attached. Examples of acyl groups
include, but are not limited to, the acyl forms of butanoic acid
(butyric acid), pentanoic acid (valeric acid), hexanoic acid
(caproic acid), heptanoic acid (enanthic acid), octanoic acid
(caprylic acid), nonanoic acid (pelargonic acid), decanoic acid
(capric acid), dodecanoic acid (lauric acid), tetradecanoic acid
(myristic acid), hexadecanoic acid (palmitic acid), octadecanoic
acid (stearic acid), eicosanoic acid (arachidic acid), linoleic
acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, and
docosahexaenoic acid.
Methods and Uses
[0059] The present invention provides a method for site specific
hydroxylation in position 2 or position 3 of an aliphatic
hydrocarbon using a peroxygenase and hydrogen peroxide. The
aliphatic hydrocarbon must include a chain of at least 3 carbons,
and either (one or more) end of the aliphatic hydrocarbon may be
used as the starting point to determine which carbon is in position
2 or 3. The aliphatic hydrocarbon must have at least one hydrogen
attached to the carbon (which is hydroxylated) in position 2 or 3.
In a preferred embodiment, the carbon in position 2 or 3, which is
hydroxylated with the peroxygenase, is unsubstituted (before the
hydroxylation is carried out).
[0060] Accordingly, in a first aspect, the present invention
provides a method for hydroxylation in position 2 or 3 of either
end (one or more ends) of a substituted or unsubstituted, linear or
branched, aliphatic hydrocarbon having at least 3 carbons and
having a hydrogen attached to the carbon in position 2 or 3,
comprising contacting the aliphatic hydrocarbon with hydrogen
peroxide and a polypeptide having peroxygenase activity; wherein
the polypeptide comprises:
[0061] a) an amino acid sequence which has at least 50% identity to
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8; and
[0062] b) an amino acid sequence represented by one or more of the
following motifs:
TABLE-US-00004 Motif I: (SEQ ID NO: 9)
[FL]XX[YF]S[AN]X[FHY]G[GN]GX[YF]N; Motif II: (SEQ ID NO: 10)
G[GN]GX[YF]NXN[VA]AX[EH][LF]R; Motif III: (SEQ ID NO: 11)
RXXRI[EQ][DEQ]S[IM]ATN; Motif IV: (SEQ ID NO: 12)
S[IM]ATN[PG][EQN][FM][SDN][FL]; Motif V: (SEQ ID NO: 13)
P[PDK][DG]F[HFW]R[AP]; Motif VI: (SEQ ID NO: 14)
[TI]XXXLYPNP[TK][GV].
[0063] The method of the invention may be used for a variety of
purposes, like bulk chemical synthesis (biocatalysis), increasing
aqueous solubility of aliphatic hydrocarbons, bioremediation, and
modification of the characteristics of food products.
[0064] The method of the invention may also be used for a number of
industrial processes in which said hydroxylation reactions are
beneficial. An example of such use is in the manufacture of pulp
and paper products where alkanes and other relevant aliphatic
hydrocarbons that are present in the wood (resin) can result in
depositioning problems in the pulp and paper manufacturing process.
These hydrophobic compounds are the precursors of the so-called
pitch deposits within the pulp and paper manufacturing processes.
Pitch deposition results in low quality pulp, and can cause the
shutdown of pulp mill operations. Specific issues related to pulps
with high extractives content include runnability problems, spots
and holes in the paper, and sheet breaks. Treatment with
peroxygenase can increase the solubility of said compounds and
thereby mitigate problems.
[0065] Yet another use of the method of the invention is in i.e.
oil or coal refineries where the peroxygenase catalyzed
hydroxylation can be used to modify the solubility, viscosity
and/or combustion characteristics of hydrocarbons. Specifically the
treatment can lead to changes in the smoke point, the kindling
point, the fire point and the boiling point of the hydrocarbons
subjected to the treatment.
[0066] In the synthesis of bulk chemicals, agro chemicals (incl.
pesticides), specialty chemicals and pharmaceuticals the method of
the invention may obviously be relevant in terms of selectively
introducing hydroxy groups in the substrates thereby affecting the
solubility of the modified compound. Furthermore, the selective
hydroxylation provides a site for further modification by methods
known in the art of organic chemical synthesis and chemo-enzymatic
synthesis.
[0067] Natural gas is extensively processed to remove higher
alkanes. Hydroxylation of such higher alkanes may be used to
improve water solubility, and thus facilitate removal of the higher
alkanes by washing the natural gas stream. Removal may be performed
at the well or during refining.
[0068] Hydroxylation of oil waste will significantly improve
biodegradability and will be applicable both in connection with
waste water treatment from refineries and bioremediation of
contaminated ground or water
[0069] In a second aspect, the present invention provides a method
for hydroxylation in position 2 or 3 of the terminal end of an acyl
group of a lipid, comprising contacting the lipid with hydrogen
peroxide and a polypeptide having peroxygenase activity; wherein
the polypeptide comprises:
[0070] a) an amino acid sequence which has at least 50% identity to
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8; and
[0071] b) an amino acid sequence represented by one or more of the
following motifs:
TABLE-US-00005 Motif I: (SEQ ID NO: 9)
[FL]XX[YF]S[AN]X[FHY]G[GN]GX[YF]N; Motif II: (SEQ ID NO: 10)
G[GN]GX[YF]NXN[VA]AX[EH][LF]R; Motif III: (SEQ ID NO: 11)
RXXRI[EQ][DEQ]S[IM]ATN; Motif IV: (SEQ ID NO: 12)
S[IM]ATN[PG][EQN][FM][SDN][FL]; Motif V: (SEQ ID NO: 13)
P[PDK][DG]F[HFW]R[AP]; Motif VI: (SEQ ID NO: 14)
[TI]XXXLYPNP[TK][GV].
[0072] Hydroxylation of the acyl group of a lipid generally
improves the aqueous solubility of the lipid. Accordingly, the
method of the invention may be used to remove or reduce oil or
lipid containing stains, like chocolate, from laundry, by
contacting the laundry with a peroxygenase and a source of hydrogen
peroxide, and optionally a surfactant.
[0073] The methods of the invention may be carried out with an
immobilized polypeptide having peroxygenase activity
(peroxygenase).
[0074] The methods of the invention may be carried out in an
aqueous solvent (reaction medium), various alcohols, ethers, other
polar or non-polar solvents, or mixtures thereof. By studying the
characteristics of the aliphatic hydrocarbon used in the methods of
the invention, suitable examples of solvents are easily recognized
by one skilled in the art. By raising or lowering the pressure at
which the hydroxylation is carried out, the solvent (reaction
medium) and the aliphatic hydrocarbon can be maintained in a liquid
phase at the reaction temperature.
[0075] The methods according to the invention may be carried out at
a temperature between 0 and 90.degree. C., preferably between 5 and
80.degree. C., more preferably between 10 and 70.degree. C., even
more preferably between 15 and 60.degree. C., most preferably
between 20 and 50.degree. C., and in particular between 20 and
40.degree. C.
[0076] The methods of the invention may employ a treatment time of
from 10 seconds to (at least) 24 hours, preferably from 1 minute to
(at least) 12 hours, more preferably from 5 minutes to (at least) 6
hours, most preferably from 5 minutes to (at least) 3 hours, and in
particular from 5 minutes to (at least) 1 hour.
[0077] In another aspect, the methods of the invention may be used
to reduce unpleasant odors from laundry by contacting the laundry
with a peroxygenase and a source of hydrogen peroxide, and
optionally a surfactant. The method of the invention results in
reduction of the amount of butanoic acid (butyric acid) in the
laundry. Butanoic acid is formed during washing of laundry when
certain animal fats and plant oils are hydrolyzed, e.g. by
detergent lipase, to yield free fatty acids, including butanoic
acid. Butanoic acid has an extremely unpleasant odor. The
peroxygenase hydroxylates the butanoic acid to 2-hydroxybutyric
acid (alpha-hydroxybutyric acid) or 3-hydroxybutyric acid
(beta-hydroxybutyric acid).
[0078] Unless otherwise specified, the nomenclature used is
standard IUPAC nomenclature.
[0079] The present invention is further described by the following
examples that should not be construed as limiting the scope of the
invention.
EXAMPLES
[0080] The polypeptide having peroxygenase activity from Agrocybe
aegerita, which is shown as SEQ ID NO:2, is referred to as AaeAPO
in the following examples.
Example 1
[0081] Hydroxylation of n-Hexane
[0082] Enzymatic hydroxylation of hexane was performed in the pure
substrate (n-hexane, >97%, Sigma Aldrich) containing 2 U
ml.sup.-1 (0.31 nmol) AaeAPO added as aqueous enzyme solution (10
.mu.l). H.sub.2O.sub.2 (4 mM) was added by syringe pumps over 1
hour. The experiment was done in 200 .mu.l scale (total volume) in
1 ml glass vials stirred with a magnetic stirrer. Products were
analyzed by GC-MS (Varian) by direct injection of the reaction
mixture. Controls were processed identically except that water (10
.mu.l) was added instead of enzyme solution.
[0083] The gas chromatogram and mass spectra of the sample with
active enzyme (AaeAPO) and n-hexane showed formation of high
amounts of 2-hexanol, and 3-hexanol; the control without enzyme did
not contain any of these peaks.
Example 2
[0084] Hydroxylation of n-Decane
[0085] Enzymatic conversion (in 200 .mu.l total volume) was done in
pure n-decane (>97%, Sigma Aldrich) supplemented with 2 U
ml.sup.-1 (0.31 nmol) AaeAPO. H.sub.2O.sub.2 (8 mM) was added by
syringe pumps over 2 hours and the sample was stirred with a
magnetic stirrer. Products were measured by GC-MS. Controls were
processed identically except that water (10 .mu.l) was added
instead of enzyme.
[0086] The gas chromatogram and mass spectra of the sample with
active enzyme (AaeAPO) and n-decane showed formation of high
amounts of two n-alkanols, 3-decanol and 2-decanol; the control
without enzyme did not contain these peaks.
Example 3
Enzymatic Hydroxylation of Lauric Acid
[0087] Enzymatic hydroxylation of lauric acid was performed using a
total reaction mixture of 4 ml containing 50 mM potassium phosphate
buffer, 40 v/v % acetronitrile, 1 mM lauric acid (>98% pure,
Aldrich W261408 was dissolved in acetonitrile), 0.01 mg
peroxygenase protein/ml (the peroxygenase shown as SEQ ID NO:4) and
2 mM ascorbic acid was added according to the table below.
[0088] The reaction was started by addition of hydrogen peroxide
corresponding to a concentration of 0.5 mM in the reaction mixture.
The reaction mixtures were incubated for 60 minutes at 35.degree.
C. using a heat block. A second addition of peroxide was added
after 30 minutes incubation to a total concentration of 1 mM. The
reactions were stopped by a heat treatment of 85.degree. C. in a
water bath for 5 minutes. Products were measured by GC-FID (Varian
3900) by injection at 100.degree. C. in split mode with ratio of
10:1 (helium was used as carrier gas at a constant flow of 25
ml/min). A temperature gradient were applied heating to 200.degree.
C. at a rate of 10.degree. C./min, then proceeding to 360.degree.
C. at a rate of 50.degree. C./min. The results were recorded as
peak area (see Table 1).
TABLE-US-00006 TABLE 1 Lauric acid Product Treatment (Area@3.2 min)
(Area @4.3 min) Peroxygenase + H.sub.2O.sub.2 No peak No peak
Lauric acid + H.sub.2O.sub.2 + ascorbic acid 16011 91 Lauric acid +
peroxygenase + 14406 395.5 H.sub.2O.sub.2 + ascorbic acid
[0089] A product peak appeared in the presence of the peroxygenase.
The elution time of the product was slightly shifted compared to
12-Hydroxydodecanoic acid, which is an iso-form of 2-hydroxy lauric
acid and 3-hydroxy lauric acid. Hence, the elution time was in
accordance to the expected product hydroxylated in the 2 or 3
position.
Example 4
Enzymatic Hydroxylation of Palmitic Acid
[0090] Enzymatic hydroxylation of palmitic acid was performed using
a total reaction mixture of 4 ml containing 50 mM potassium
phosphate buffer, 40 v/v % acetronitrile, 1 mM palmitic acid
(>99% pure, Sigma P0500) and 0.01 mg peroxygenase protein/ml
(the peroxygenase shown as SEQ ID NO:4).
[0091] The reaction was started by addition of hydrogen peroxide
corresponding to a concentration of 1 mM in the reaction mixture.
The reaction mixtures were incubated for 1, 2, 3 and 10 minutes at
35.degree. C. using a heat block. The reactions were stopped by a
heat treatment of 85.degree. C. in a water bath for 5 minutes.
Products were measured by GC-FID (Varian 3900) by injection at
100.degree. C. in split mode with ratio of 10:1 (helium was used as
carrier gas at a constant flow of 25 ml/min). A temperature
gradient were applied heating to 200.degree. C. at a rate of
10.degree. C./min, then proceeding to 360.degree. C. at a rate of
50.degree. C./min. The results were recorded as peak area (see
Table 2).
TABLE-US-00007 TABLE 2 Incubation time Palmitic acid Product (min)
(Area) (Area) 0 1799.5 No peak 1 1481.2 82.8 2 979.1 191.3
[0092] A product peak appeared already after 1 minutes of
incubation, and increased after two minutes incubation. The elution
profile was slightly shifted compared to 16-Hydroxyhexadecanoic
acid, which is an iso-form of 2-hydroxy palmitic and 3-hydroxy
palmitic. Hence, the elution time was in accordance to the expected
product hydroxylated in the 2 or 3 position.
Sequence CWU 1
1
141330PRTAgrocybe aegeritamat_peptide(1)..(330) 1Glu Pro Thr Gln
Pro Pro Gly Pro Pro Glu Asp Thr Ser Ala Lys Leu1 5 10 15Val Asn Asp
Lys Asp His Pro Trp Lys Pro Leu Arg Pro Gly Asp Ile 20 25 30Arg Gly
Pro Cys Pro Gly Leu Asn Thr Leu Ala Ser His Gly Tyr Leu 35 40 45Pro
Arg Asn Gly Val Ala Thr Pro Ala Gln Ile Ile Asn Ala Val Gln 50 55
60Glu Gly Phe Asn Met Asp Asn Ser Val Ala Leu Phe Ala Thr Tyr Glu65
70 75 80Ala His Leu Met Val Gly Asn Leu Leu Thr Asp Leu Leu Ser Ile
Gly 85 90 95Arg Lys Thr Pro Leu Thr Gly Pro Asp Leu Pro Pro Pro Ala
Asn Ile 100 105 110Gly Gly Leu Ser Glu His Gly Leu Phe Glu Gly Asp
Ala Ser Met Thr 115 120 125Arg Gly Asp Ala Phe Phe Gly Asn Asn Asp
Glu Phe Asn Glu Glu Leu 130 135 140Phe Gln Gln Phe Ile Asp Tyr Ser
Asn Arg Phe Gly Gly Gly Tyr Tyr145 150 155 160Asn Leu Thr Val Ala
Val Glu Leu Arg Phe Lys Arg Ile Gln Asp Ser 165 170 175Ile Ala Thr
Asn Pro Glu Phe Asn Phe Val Ser Pro Arg Phe Phe Ala 180 185 190Ala
Tyr Gly Glu Ser Val Ala Pro Asn Asn Phe Phe Val Asp Gly Arg 195 200
205Lys Asp Asp Gly His Leu Asp Met Asp Ala Ala Arg Gly Phe Phe Gln
210 215 220Phe Gly Arg Met Pro Asp Gly Phe Phe Arg Pro Asn Gly Thr
Lys Gly225 230 235 240Asn Ala Gly Leu Asp Asp Val Val Arg Ala His
Pro Val Gln Pro Gly 245 250 255Arg Asn Leu Gly Arg Val Asn Ser Tyr
Thr His Asp Pro Thr Ser Ala 260 265 270Asp Phe Thr Thr Pro Cys Leu
Leu Tyr Glu Asn Phe Ala Asn Lys Thr 275 280 285Val Thr Ala Leu Tyr
Pro Asn Pro Lys Gly Gln Leu Arg Arg Ala Ile 290 295 300Lys Ala Asn
Leu His Phe Leu Phe Leu Ala Ile Asn Arg Thr Val Gly305 310 315
320Cys Ala Glu Val Phe Pro Tyr Gly Arg Asp 325 3302328PRTAgrocybe
aegeritamat_peptide(1)..(328) 2Glu Pro Gly Leu Pro Pro Gly Pro Leu
Glu Asn Ser Ser Ala Lys Leu1 5 10 15Val Asn Asp Glu Ala His Pro Trp
Lys Pro Leu Arg Pro Gly Asp Ile 20 25 30Arg Gly Pro Cys Pro Gly Leu
Asn Thr Leu Ala Ser His Gly Tyr Leu 35 40 45Pro Arg Asn Gly Val Ala
Thr Pro Val Gln Ile Ile Asn Ala Val Gln 50 55 60Glu Gly Leu Asn Phe
Asp Asn Gln Ala Ala Val Phe Ala Thr Tyr Ala65 70 75 80Ala His Leu
Val Asp Gly Asn Leu Ile Thr Asp Leu Leu Ser Ile Gly 85 90 95Arg Lys
Thr Arg Leu Thr Gly Pro Asp Pro Pro Pro Pro Ala Ser Val 100 105
110Gly Gly Leu Asn Glu His Gly Thr Phe Glu Gly Asp Ala Ser Met Thr
115 120 125Arg Gly Asp Ala Phe Phe Gly Asn Asn His Asp Phe Asn Glu
Thr Leu 130 135 140Phe Glu Gln Leu Val Asp Tyr Ser Asn Arg Phe Gly
Gly Gly Lys Tyr145 150 155 160Asn Leu Thr Val Ala Gly Glu Leu Arg
Phe Lys Arg Ile Gln Asp Ser 165 170 175Ile Ala Thr Asn Pro Asn Phe
Ser Phe Val Asp Phe Arg Phe Phe Thr 180 185 190Ala Tyr Gly Glu Thr
Thr Phe Pro Ala Asn Leu Phe Val Asp Gly Arg 195 200 205Arg Asp Asp
Gly Gln Leu Asp Met Asp Ala Ala Arg Ser Phe Phe Gln 210 215 220Phe
Ser Arg Met Pro Asp Asp Phe Phe Arg Ala Pro Ser Pro Arg Ser225 230
235 240Gly Thr Gly Val Glu Val Val Ile Gln Ala His Pro Met Gln Pro
Gly 245 250 255Arg Asn Val Gly Lys Ile Asn Ser Tyr Thr Val Asp Pro
Thr Ser Ser 260 265 270Asp Phe Ser Thr Pro Cys Leu Met Tyr Glu Lys
Phe Val Asn Ile Thr 275 280 285Val Lys Ser Leu Tyr Pro Asn Pro Thr
Val His Val Arg Lys Ala Leu 290 295 300Asn Thr Asn Leu Asp Phe Phe
Phe Gln Gly Val Ala Ala Gly Cys Thr305 310 315 320Gln Val Phe Pro
Tyr Gly Arg Asp 3253377PRTLaccaria bicolor 3Met Ala Arg Leu Thr Phe
Leu Ala Ala Ile Ala Leu Ala Leu Ser Ser1 5 10 15Thr Thr Val Leu Ala
Phe Pro Ser Tyr Gly Ser Leu Ala Gly Leu Ser 20 25 30Glu Ala Glu Leu
Asp Arg Ile Ile Pro Leu Leu Glu Ala Arg Asn Ala 35 40 45Gly Pro Pro
Pro Gly Pro Leu Lys Asn Thr Ser Thr Lys Leu Val Asn 50 55 60Asp Lys
Asn His Pro Trp Lys Pro Leu Gly Tyr Gly Asp Ile Arg Gly65 70 75
80Pro Cys Pro Gly Leu Asn Thr Leu Ala Ser His Gly Trp Leu Pro Arg
85 90 95Asn Gly Ile Ala Thr Pro Ala Gln Ile Val Asn Ala Val Gln Glu
Gly 100 105 110Phe Asn Met Gly Asn Asp Leu Ala Val Phe Val Thr Tyr
Ala Ala His 115 120 125Leu Val Asp Gly Asn Gln Val Thr Asp Leu Leu
Ser Ile Gly Gly Lys 130 135 140Thr Pro Gln Thr Gly Pro Asp Pro Pro
Ala Pro Ala Ile Val Gly Gly145 150 155 160Leu Asn Thr His Ala Val
Phe Glu Gly Asp Ala Ser Met Thr Arg Gly 165 170 175Asp Ala Phe Phe
Gly Asp Asn His Ser Phe Asn Glu Thr Gln Phe Asp 180 185 190Glu Phe
Ser Ala Phe Ser Asn Lys Phe Gly Gly Gly Tyr Tyr Asn Leu 195 200
205Ser Val Ala Ala Glu Phe Arg Trp Gln Arg Ile Gln Glu Ser Ile Ala
210 215 220Thr Asn Pro Asn Phe Ser Leu Ile Ser Pro Arg Tyr Phe Thr
Ala Tyr225 230 235 240Ala Glu Ser Val Phe Pro Leu Val Phe Phe Val
Asp Gly Arg Val Ser 245 250 255Asp Gly Arg Leu Ser Leu Pro Asn Ala
Arg Gly Phe Phe Gln Asn Ser 260 265 270Gln Met Pro Lys Asp Phe Phe
Arg Pro Asn Gln Ser Ile Gly Leu Asn 275 280 285Glu Ile Gly Asp Gly
Ile Ser Ala Ile Ala Ser Ala His Pro Ile Ala 290 295 300Pro Gly Lys
Asn Glu Gly Val Gly Asn Tyr Val Leu Asp Pro Thr Ser305 310 315
320Ala Asp Phe Asp His Phe Cys Leu Leu Tyr Ile Asn Phe Val Asn Gln
325 330 335Thr Val Lys Ser Leu Tyr Pro Asn Pro Lys Gly Val Leu Leu
Asp Ala 340 345 350Leu Lys Arg Asn Leu Asn Asn Phe Tyr Gly Pro Leu
Asn Gly Ser Asp 355 360 365Cys Glu Gln Ile Phe Pro Tyr Gly Lys 370
3754344PRTCoprinopsis cinereamat_peptide(1)..(344) 4Thr Ser Lys Leu
Pro Ile Val Phe Pro Pro Pro Pro Pro Glu Pro Ile1 5 10 15Lys Asp Pro
Trp Leu Lys Leu Val Asn Asp Arg Ala His Pro Trp Arg 20 25 30Pro Leu
Arg Arg Gly Asp Val Arg Gly Pro Cys Pro Gly Leu Asn Thr 35 40 45Leu
Ala Ser His Gly Tyr Leu Pro Arg Asp Gly Val Ala Thr Pro Ala 50 55
60Gln Ile Ile Thr Ala Val Gln Glu Gly Phe Asn Met Glu Tyr Gly Ile65
70 75 80Ala Thr Phe Val Thr Tyr Ala Ala His Leu Val Asp Gly Asn Pro
Leu 85 90 95Thr Asn Leu Ile Ser Ile Gly Gly Lys Thr Arg Lys Thr Gly
Pro Asp 100 105 110Pro Pro Pro Pro Ala Ile Val Gly Gly Leu Asn Thr
His Ala Val Phe 115 120 125Glu Gly Asp Ala Ser Met Thr Arg Gly Asp
Phe His Leu Gly Asp Asn 130 135 140Phe Asn Phe Asn Gln Thr Leu Trp
Glu Gln Phe Lys Asp Tyr Ser Asn145 150 155 160Arg Tyr Gly Gly Gly
Arg Tyr Asn Leu Thr Ala Ala Ala Glu Leu Arg 165 170 175Trp Ala Arg
Ile Gln Gln Ser Met Ala Thr Asn Gly Gln Phe Asp Phe 180 185 190Thr
Ser Pro Arg Tyr Phe Thr Ala Tyr Ala Glu Ser Val Phe Pro Ile 195 200
205Asn Phe Phe Thr Asp Gly Arg Leu Phe Thr Ser Asn Thr Thr Ala Pro
210 215 220Gly Pro Asp Met Asp Ser Ala Leu Ser Phe Phe Arg Asp His
Arg Tyr225 230 235 240Pro Lys Asp Phe His Arg Ala Pro Val Pro Ser
Gly Ala Arg Gly Leu 245 250 255Asp Val Val Ala Ala Ala Tyr Pro Ile
Gln Pro Gly Tyr Asn Ala Asp 260 265 270Gly Lys Val Asn Asn Tyr Val
Leu Asp Pro Thr Ser Ala Asp Phe Thr 275 280 285Lys Phe Cys Leu Leu
Tyr Glu Asn Phe Val Leu Lys Thr Val Lys Gly 290 295 300Leu Tyr Pro
Asn Pro Lys Gly Phe Leu Arg Lys Ala Leu Glu Thr Asn305 310 315
320Leu Glu Tyr Phe Tyr Gln Ser Phe Pro Gly Ser Gly Gly Cys Pro Gln
325 330 335Val Phe Pro Trp Gly Lys Ser Asp 3405351PRTCoprinopsis
cinerea 5Met Val Ser Cys Lys Leu Pro Leu Pro Leu Leu Thr Leu Ala
Ile Ala1 5 10 15Leu Ala Asn Val Asn Ala Phe Pro Ala Tyr Gln Ser Leu
Gly Gly Leu 20 25 30Ser Lys Arg Gln Leu Glu Thr Ile Ile Pro Gly Leu
Pro Val Val Asn 35 40 45Pro Gly Pro Pro Pro Gly Pro Leu Ala Asp Ser
Thr Leu Lys Leu Val 50 55 60Asn Asp Ala Ala His Pro Tyr Gln Ala Pro
Arg Pro His Leu Asp His65 70 75 80Arg Gly Pro Cys Pro Gly Leu Asn
Thr Leu Ala Asn His Gly Tyr Leu 85 90 95Pro Arg Ser Gly Ile Ala Thr
Pro Ala Gln Ile Val Gln Ala Val Met 100 105 110Glu Gly Phe Asn Met
Glu Asn Thr Phe Ala Lys Phe Val Thr Tyr Ala 115 120 125Ala Phe Leu
Val Asp Gly Asn Pro Ile Thr Asn Leu Met Ser Ile Gly 130 135 140Gly
Lys Thr Trp Arg Thr Gly Ile Ile Glu Pro Pro Pro Pro Ala Ile145 150
155 160Val Gly Gly Leu Asn Thr His Ala Val Phe Glu Gly Asp Thr Ser
Met 165 170 175Thr Arg Gly Asp Phe His Phe Gly Asp Asn His Ser Phe
Asn Gln Thr 180 185 190Leu Phe Asp Gln Phe Val Glu Tyr Ser Asn Ile
His Gly Gly Gly Phe 195 200 205Tyr Asn Leu Thr Ala Ala Thr Glu Leu
Arg Tyr Gln Arg Ile Gln Gln 210 215 220Ser Ile Ala Thr Asn Pro Glu
Met Ser Phe Val Ser Pro Arg Trp Phe225 230 235 240Thr Ala Ile Leu
Leu Gln Asp Glu Lys Phe Pro Asp Asp Phe His Arg 245 250 255Ala Pro
Gly Pro Phe Ser Phe Glu Gly Leu Gly Tyr Leu Val Thr Arg 260 265
270Arg Pro Met Pro Pro Gly Arg Asn Val Gly Gly Val Asp Asn Tyr Val
275 280 285Pro Asp Pro Asn Ser Ala Asp Phe Asn Ser Phe Cys Lys Met
Tyr Glu 290 295 300Asp Phe Val Asn Asp Ile Val Val Ala Leu Tyr Pro
Asn Pro Thr Gly305 310 315 320Leu Leu Arg Arg Asn Leu Ile Lys Asn
Leu Glu Tyr Phe Trp Thr Gly 325 330 335Met Phe Asp Pro Ala Cys Thr
Glu Val Lys Pro Tyr Gly Thr Leu 340 345 3506386PRTCoprinopsis
cinerea 6Met Asn Gly Leu Phe Ala Thr Val Lys Leu Ala Leu Val Thr
Leu Leu1 5 10 15Ala Ser Gln Ser Gln Phe Ala Asn Ala Phe Pro Ala Trp
Gln Ser Leu 20 25 30Gly Gly Leu Ser Glu Arg Gln Leu Asp Glu Val Met
Pro Met Leu Lys 35 40 45His Arg Val Pro Pro Pro Pro Pro Gly Pro Pro
Ala Phe Thr Gly Ala 50 55 60Lys Leu Val Asn Asp Lys Ala His Pro Phe
Lys Pro Leu Lys Lys Gly65 70 75 80Asp Val Arg Gly Pro Cys Pro Gly
Leu Asn Thr Leu Ala Ser His Gly 85 90 95Tyr Leu Pro Arg Asn Gly Val
Ala Ser Pro Ser Gln Ile Ile Asp Ala 100 105 110Val Gln Glu Gly Phe
Asn Met Glu Asn Glu Leu Ala Arg Phe Thr Thr 115 120 125Tyr Val Ala
His Leu Val Asp Gly Asn Leu Val Thr Asp Leu Leu Ser 130 135 140Ile
Gly Glu Lys Thr Arg Lys Thr Gly Pro Asp Pro Pro Pro Pro Ala145 150
155 160Ile Val Gly Gly Leu Asn Asn His Gly Thr Phe Glu Gly Asp Ala
Ser 165 170 175Leu Thr Arg Gly Asp Ala Phe Phe Gly Asp Asn His Asn
Phe Asn Gln 180 185 190Glu Leu Phe Asp Gln Phe Lys Asn Phe Ser Ala
Val Tyr Gly Asn Gly 195 200 205Phe Phe Asn Met Thr Val Ala Gly Glu
Leu Arg Phe His Arg Ile Gln 210 215 220Gln Ser Ile Ala Thr Asn Pro
Glu Phe Ser Leu Val Gly Leu Arg His225 230 235 240Leu Thr Ala Tyr
Ala Glu Ala Ser Phe Pro Ser Leu Phe Phe Val Asp 245 250 255Gly Arg
Lys Thr Gly Ala Glu Ala Gly Gln Leu Asp Met Ala Thr Ala 260 265
270Glu Ser Phe Phe Arg Asp Met Met Tyr Pro Pro Asp Phe Phe Arg Pro
275 280 285Ala Ala Pro Val Ala Gly Asp Ala Gly Ala Ile Phe Leu Ala
His Pro 290 295 300Phe Gln Pro Gly Arg Asn Val Gly Gly Val Asn Asn
Phe Thr Val Asp305 310 315 320Asp Ser Leu Gly Ser Leu Leu Asp Phe
Cys Gly Phe Tyr Glu Asn Phe 325 330 335Val Asn Lys Thr Leu Lys Ala
Leu Tyr Pro Asn Pro Lys Gly Val Leu 340 345 350Arg Arg Asn Leu Asn
Ile Asn Leu Gln Phe Phe Phe Glu Ser Leu Pro 355 360 365Lys Asp Glu
Ser Gly Thr Pro Val Cys Thr Gln Val Phe Pro Tyr Gly 370 375 380Arg
Asn3857341PRTCoprinopsis cinerea 7Met Leu Lys Pro Arg Val Pro Pro
Pro Pro Pro Gly Pro Leu Ala Phe1 5 10 15Asn Gly Thr Lys Leu Val Asn
Asp Glu Asp His Pro Phe Met Pro Pro 20 25 30Arg Lys Gly Asp Ala Arg
Gly Pro Cys Pro Gly Leu Asn Thr Leu Ala 35 40 45Ser His Gly Tyr Leu
Pro Arg Asn Gly Ile Ala Thr Pro Ala Gln Ile 50 55 60Ile Asn Ala Val
Gln Glu Gly Phe Asn Met Glu Asn Glu Ile Ala Arg65 70 75 80Phe Thr
Thr Tyr Thr Ala His Leu Met Asp Gly Asn Leu Val Thr Asp 85 90 95Leu
Leu Ser Ile Gly Pro Lys Thr Pro Lys Thr Gly Pro Asp Pro Pro 100 105
110Pro Pro Ala Ile Val Gly Gly Leu Asn Asn His Gly Thr Phe Glu Gly
115 120 125Asp Ala Ser Leu Ser Arg Ala Asp Ala Phe Phe Gly Asp Asn
His Ser 130 135 140Phe Asp Gln Glu Leu Phe Asp Gln Phe Arg Asn Phe
Ser Ala Ile Tyr145 150 155 160Gly Asn Gly Phe Phe Asn Met Thr Val
Ala Ala Glu Leu Arg Phe His 165 170 175Arg Ile Gln Gln Ser Ile Ala
Thr Asn Pro Glu Phe Ser Phe Ala Gly 180 185 190Leu Arg His Ile Thr
Ala Tyr Ala Glu Ala Ser Phe Pro Pro Ile Phe 195 200 205Phe Val Asp
Gly Arg Lys Thr Gly Ala Glu Ala Gly Gln Leu Asp Met 210 215 220Ala
Ala Ala Glu Ser Phe Phe Lys His Met Met Tyr Pro Pro Asp Phe225 230
235 240His Arg Pro Ala Glu Pro Val Asn Ser Asp Ala Gln Ala Val Phe
Glu 245 250 255Val His Pro Phe Gln Pro Gly Arg Asn Val Gly Gly Val
Asn Asn Tyr 260 265 270Thr Val Asp Glu Ser Leu Gly Gly Leu Leu Asp
Phe Cys Gly Phe Tyr 275 280 285Glu Asn Phe Val Asn Lys Thr Ile Lys
Gly Leu Tyr Pro Asn Pro Thr 290 295 300Gly Val Leu Lys Arg Asn Leu
Asn Ile Asn Leu Asp Phe Leu Phe Glu305 310 315 320Ala Leu Pro Lys
Ala Gly Asp Gly Ser Gln Pro Cys Thr Gln Val Phe
325 330 335Pro Tyr Gly His Asp 3408325PRTCoprinus radians 8Pro Pro
Pro Glu Tyr Val Gly Pro Lys Leu Val Asn Asp Ala Asp His1 5 10 15Pro
Trp Glu Pro Leu Arg Pro Gly Asp Ile Arg Gly Pro Cys Pro Gly 20 25
30Leu Asn Thr Leu Ala Ser His Gly Tyr Leu Pro Arg Asn Gly Val Ala
35 40 45Thr Pro Ala Gln Ile Ile Asn Ala Ile Val Glu Gly Phe Asn Phe
Asn 50 55 60Tyr Glu Gly Ala Val Phe Val Thr Tyr Phe Ala His Ile Val
Asp Gly65 70 75 80Asn Leu Val Thr Asp Leu Leu Ser Ile Gly Gly Lys
Thr Asn Leu Thr 85 90 95Gly Glu Asp Thr Gly Ala Pro Ala Ile Ile Gly
Gly Leu Asn Thr His 100 105 110Ser Val Phe Glu Gly Asp Ala Ser Met
Thr Arg Asp Asp Phe His Phe 115 120 125Gly Asp Asn His Ser Phe Asn
Gln Thr Leu Phe Asp Gln Phe Val Glu 130 135 140Tyr Ser Asn Thr Tyr
Gly Gly Gly Phe Tyr Asn Gln Glu Val Ala Gly145 150 155 160His Leu
Arg Arg Arg Arg Ile Glu Gln Ser Ile Ala Thr Asn Pro Glu 165 170
175Phe Asp Phe Thr Ser Pro Arg Phe Phe Thr Ala Phe Ala Glu Ser Ser
180 185 190Phe Pro Tyr Ser Phe Phe Val Asp Gly Arg Ile Thr Glu Arg
Pro Gly 195 200 205Gly Leu Ser Met Glu Asn Ala Thr Leu Phe Phe Arg
Asp His Lys Met 210 215 220Pro Asp Asp Phe Trp Arg Ala Pro Glu Pro
Thr Gly Gly Leu Asn Val225 230 235 240Leu Asp Ile Tyr Arg Ala Ser
Gly Ser Pro Pro Ala Gly Arg Asn Val 245 250 255Asn Gly Thr Asn Thr
Phe Thr Pro Asp Pro Asn Ser Ala Asp Phe Asp 260 265 270Asn Pro Cys
Glu Leu Tyr Tyr Asp Tyr Val Asn Arg Ile Val Lys Ser 275 280 285Leu
Tyr Pro Asn Pro Thr Gly Ile Leu Arg Asp Asn Leu Asn Ile Ala 290 295
300Leu Gly His Val Phe Asp Ser Met Asp Phe Gly Asp Cys Glu Gln
Leu305 310 315 320Phe Pro Tyr Gly Arg 325914PRTArtificial
sequencePeroxygenase motif IVARIANT(1)..(1)The amino acid may be
Phe or Leu.misc_feature(2)..(3)Xaa can be any naturally occurring
amino acidVARIANT(4)..(4)The amino acid may be Tyr or
Phe.VARIANT(6)..(6)The amino acid may be Ala or
Asn.misc_feature(7)..(7)Xaa can be any naturally occurring amino
acidVARIANT(8)..(8)The amino acid may be Phe, His or
Tyr.VARIANT(10)..(10)The amino acid may be Gly or
Asn.misc_feature(12)..(12)Xaa can be any naturally occurring amino
acidVARIANT(13)..(13)The amino acid may be Tyr or Phe. 9Xaa Xaa Xaa
Xaa Ser Xaa Xaa Xaa Gly Xaa Gly Xaa Xaa Asn1 5 101014PRTArtificial
sequencePeroxygenase motif IIVARIANT(2)..(2)The amino acid may be
Gly or Asn.misc_feature(4)..(4)Xaa can be any naturally occurring
amino acidVARIANT(5)..(5)The amino acid may be Tyr or
Phe.misc_feature(7)..(8)Xaa can be any naturally occurring amino
acidVARIANT(9)..(9)The amino acid may be Val or
Ala.misc_feature(11)..(11)Xaa can be any naturally occurring amino
acidVARIANT(12)..(12)The amino acid may be Glu or
His.VARIANT(13)..(13)The amino acid may be Leu or Phe. 10Gly Xaa
Gly Xaa Xaa Asn Xaa Xaa Xaa Ala Xaa Xaa Xaa Arg1 5
101112PRTArtificial sequencePeroxygenase motif
IIImisc_feature(2)..(3)Xaa can be any naturally occurring amino
acidVARIANT(6)..(6)The amino acid may be Gln or
Glu.VARIANT(7)..(7)The amino acid may be Asp, Glu or
Gln.VARIANT(9)..(9)The amino acid may be Ile or Met. 11Arg Xaa Xaa
Arg Ile Xaa Xaa Ser Xaa Ala Thr Asn1 5 101210PRTArtificial
sequencePeroxygenase motif IVVARIANT(2)..(2)The amino acid may be
Ile or Met.VARIANT(6)..(6)The amino acid may be Pro or
Gly.VARIANT(7)..(7)The amino acid may be Glu, Gln or
Asn.VARIANT(8)..(8)The amino acid may be Phe or
Met.VARIANT(9)..(9)The amino acid may be Ser, Asp or
Asn.VARIANT(10)..(10)The amino acid may be Phe or Leu. 12Ser Xaa
Ala Thr Asn Xaa Xaa Xaa Xaa Xaa1 5 10137PRTArtificial
sequencePeroxygenase motif VVARIANT(2)..(2)The amino acid may be
Pro, Asp or Lys.VARIANT(3)..(3)The amino acid may be Asp or
Gly.VARIANT(5)..(5)The amino acid may be His, Phe or
Trp.VARIANT(7)..(7)The amino acid may be Ala or Pro. 13Pro Xaa Xaa
Phe Xaa Arg Xaa1 51411PRTArtificial sequencePeroxygenase motif
VIVARIANT(1)..(1)The amino acid may be Thr or
Ile.misc_feature(2)..(4)Xaa can be any naturally occurring amino
acidVARIANT(10)..(10)The amino acid may be Thr or
Lys.VARIANT(11)..(11)The amino acid may be Gly or Val. 14Xaa Xaa
Xaa Xaa Leu Tyr Pro Asn Pro Xaa Xaa1 5 10
* * * * *