U.S. patent application number 15/535277 was filed with the patent office on 2017-11-30 for stabilized enzyme compositions.
This patent application is currently assigned to NOVOZYMES A/S. The applicant listed for this patent is NOVOZYMES A/S. Invention is credited to Lars Lehmann Hylling Christensen, Esben Peter Friis, Lise Munch Mikkelsen, Frank Winther Rasmussen, Miguel Duarte Guilherme Toscano.
Application Number | 20170342349 15/535277 |
Document ID | / |
Family ID | 52282468 |
Filed Date | 2017-11-30 |
United States Patent
Application |
20170342349 |
Kind Code |
A1 |
Toscano; Miguel Duarte Guilherme ;
et al. |
November 30, 2017 |
STABILIZED ENZYME COMPOSITIONS
Abstract
The present invention relates to methods for obtaining protease
variants with improved properties. The present invention also
relates to protease variants, compositions comprising the protease
variants and methods of using the protease variants.
Inventors: |
Toscano; Miguel Duarte
Guilherme; (Greve, DK) ; Mikkelsen; Lise Munch;
(Roedovre, DK) ; Friis; Esben Peter; (Herlev,
DK) ; Christensen; Lars Lehmann Hylling; (Alleroed,
DK) ; Rasmussen; Frank Winther; (Roskilde,
DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVOZYMES A/S |
Bagsvaerd |
|
DK |
|
|
Assignee: |
NOVOZYMES A/S
Bagsvaerd
DK
|
Family ID: |
52282468 |
Appl. No.: |
15/535277 |
Filed: |
December 21, 2015 |
PCT Filed: |
December 21, 2015 |
PCT NO: |
PCT/EP2015/080765 |
371 Date: |
June 12, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/54 20130101; C12N
9/6408 20130101; C12N 9/96 20130101; C12Y 304/21014 20130101; C11D
3/00 20130101; C12Y 304/21062 20130101; C11D 3/386 20130101; C12N
9/00 20130101; C12N 9/50 20130101; C11D 3/38663 20130101; C12Q 1/37
20130101 |
International
Class: |
C11D 3/386 20060101
C11D003/386; C12N 9/96 20060101 C12N009/96; C12N 9/54 20060101
C12N009/54; C12Q 1/37 20060101 C12Q001/37; C12N 9/64 20060101
C12N009/64 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 19, 2014 |
EP |
14199399.8 |
Dec 18, 2015 |
EP |
PCT/EP2015/080610 |
Claims
1. The present invention relates to a method for improve binding of
a protease to an inhibitor of the protease, comprising the steps
of: a) identify the amino acid residues exposed to the inhibitor
when the inhibitor binds to the protease, b) modify the protease by
modifying the amino acid residues identified in step a) c)
determine the binding constant Ki d) select modified protease
generated in step b)
2. The method according to claim 1, wherein the improvement factor
of the modified protease selected in step d) is above 1.0.
3. The method of claim 1, wherein the subtilisin inhibitor is a
peptide aldehyde, or a hydrosulfite adduct thereof; or a phenyl
boronic acid, or a derivative thereof, such as 4-FPBA.
4. The method of claim 1, wherein the inhibitor is a peptide
aldehyde or ketone having the formula
P-(A).sub.y-L-(B).sub.x--B.sup.0--R* or a hydrosulfite adduct of
such aldehyde, wherein: a) R* is H (hydrogen), CH.sub.3, CX.sub.3,
CHX.sub.2, or CH.sub.2X; b) X is a halogen atom; c) B.sup.0 is a
single amino acid residue with L- or D-configuration of the formula
--NH--CH(R)--C(.dbd.O)--; d) x is 1, 2 or 3; e) B.sub.x is
independently a single amino acid residue, each connected to the
next B or to B.sup.0 via its C-terminal; f) L is absent or
independently a linker group of the formula --C(.dbd.O)--,
--C(.dbd.O)--C(.dbd.O)--, --C(.dbd.S)--, --C(.dbd.S)--C(.dbd.S)--
or --C(.dbd.S)--C(.dbd.O)--; g) A is absent if L is absent or is
independently a single amino acid residue connected to L via the
N-terminal of the amino acid; h) P is selected from the group
consisting of hydrogen or if L is absent an N-terminal protection
group; i) y is 0, 1, or 2, j) R is independently selected from the
group consisting of C.sub.1-6 alkyl, C.sub.6-10 aryl or C.sub.7-10
arylalkyl optionally substituted with one or more, identical or
different, substituent's R'; k) R' is independently selected from
the group consisting of halogen, --OH, --OR'', --SH, --SR'',
--NH.sub.2, --NHR'', --NR''.sub.2, --CO.sub.2H, --CONH.sub.2,
--CONHR'', --CONR''.sub.2, --NHC(.dbd.N)NH.sub.2; and l) R'' is a
C.sub.1-6 alkyl group. m) x may be 1, 2 or 3.
5. The method of claim 4, wherein the inhibitor is an aldehyde
having the formula P--B.sup.2--B.sup.1--B.sup.0--H or an adduct
having the formula P--B.sup.2--B.sup.1--N(H)--CHR--CHOH--SO.sub.3M,
wherein a) H is hydrogen; b) B.sup.0 is a single amino acid residue
with L- or D-configuration of the formula --NH--CH(R)--C(.dbd.O)--;
c) B.sup.1 and B.sup.2 are independently single amino acid
residues; d) R is independently selected from the group consisting
of C.sub.1-6 alkyl, C.sub.6-10 aryl or C.sub.7-10 arylalkyl
optionally substituted with one or more, identical or different,
substituent's R'; e) R' is independently selected from the group
consisting of halogen, --OH, --OR'', --SH, --SR'', --NH.sub.2,
--NHR'', --NR''.sub.2, --CO.sub.2H, --CONH.sub.2, --CONHR'',
--CONR''.sub.2, --NHC(.dbd.N)NH.sub.2; f) R'' is a C.sub.1-6 alkyl
group; and g) P is an N-terminal protection group.
6. The method of claim 4, wherein R is such that
B.sup.0=--NH--CH(R)--C(.dbd.O)-- is Phe, Tyr or Leu; and B.sup.1 is
Ala, Gly or Val; and B.sup.2 is Arg, Phe, Tyr or Trp.
7. The method of claim 4, wherein x=2, L is absent, A is absent,
and P is p-methoxycarbonyl (Moc) or benzyloxycarbonyl (Cbz).
8. The method of claim 4, wherein the inhibitor is
Cbz-Arg-Ala-Tyr-H, Ac-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-H,
Cbz-Gly-Ala-Tyr-CF.sub.3, Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H,
Cbz-Val-Ala-Leu-CF.sub.3, Moc-Val-Ala-Leu-CF.sub.3,
Cbz-Gly-Ala-Phe-H, Cbz-Gly-Ala-Phe-CF.sub.3, Cbz-Gly-Ala-Val-H,
Cbz-Gly-Gly-Tyr-H, Cbz-Gly-Gly-Phe-H, Cbz-Arg-Val-Tyr-H,
Cbz-Leu-Val-Tyr-H, Ac-Leu-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Tyr-H,
Ac-Tyr-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Leu-H, Ac-Phe-Gly-Ala-Phe-H,
Ac-Phe-Gly-Val-Tyr-H, Ac-Phe-Gly-Ala-Met-H, Ac-Trp-Leu-Val-Tyr-H,
MeO--CO-Val-Ala-Leu-H, MeNCO-Val-Ala-Leu-H,
MeO--CO-Phe-Gly-Ala-Leu-H, MeO--CO-Phe-Gly-Ala-Phe-H,
MeSO.sub.2-Phe-Gly-Ala-Leu-H, MeSO.sub.2-Val-Ala-Leu-H,
PhCH.sub.2O--P(OH)(O)-Val-Ala-Leu-H, EtSO.sub.2-Phe-Gly-Ala-Leu-H,
PhCH.sub.2SO.sub.2-Val-Ala-Leu-H,
PhCH.sub.2O--P(OH)(O)-Leu-Ala-Leu-H,
PhCH.sub.2O--P(OH)(O)-Phe-Ala-Leu-H, or
MeO--P(OH)(O)-Leu-Gly-Ala-Leu-H or a hydrosulfite adduct of any of
these, wherein Cbz is benzyloxycarbonyl and Moc is
methoxycarbonyl.
9. The method of claim 4, wherein the inhibitor is
Cbz-Gly-Ala-Tyr-H or Moc-Val-Ala-Leu-H, or a hydrosulfite adduct
thereof, wherein Cbz is benzyloxycarbonyl and Moc is
methoxycarbonyl.
10. The method according to claim 1 wherein the protease is
selected from a protease: a. having the amino acid sequence shown
in SEQ ID NO: 1 or a protease having at least 60%, preferably at
least 70%, preferably at least 80% or at least 90% sequence
identity hereto, or b. having the amino acid sequence shown in SEQ
ID NO: 2 or a protease having at least 60%, preferably at least
70%, preferably at least 80% or at least 90% sequence identity
hereto, or c. having the amino acid sequence shown in SEQ ID NO: 3
or a protease having at least 60%, preferably at least 70%,
preferably at least 80% or at least 90% sequence identity hereto,
or d. having the amino acid sequence shown in SEQ ID NO: 4 or a
protease having at least 60%, preferably at least 70%, preferably
at least 80% or at least 90% sequence identity hereto, or e. having
the amino acid sequence shown in SEQ ID NO: 5 or a protease having
at least 60%, preferably at least 70%, preferably at least 80% or
at least 90% sequence identity hereto, or f. protease having the
amino acid sequence shown in SEQ ID NO: 6 or a protease comprising
compared to SEQ ID NO 1 an insertion of glutamic acid at the
position corresponding to position 97 of SEQ ID NO 1 and having at
least 60%, preferably at least 70%, preferably at least 80% or at
least 90% sequence identity to SEQ ID NO 1.
11. A protease obtained by the method of claim 1.
12. The method according to claim 1 wherein the modified protease
variant has improved wash performance.
Description
REFERENCE TO A SEQUENCE LISTING
[0001] This application contains a Sequence Listing in computer
readable form, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention relates to method for improve binding
of an enzyme to an inhibitor of the enzyme. The enzyme variants
generated by the method of the invention are suitable for use in
cleaning processes and detergent compositions, such as laundry
compositions and dish wash compositions, including hand wash and
automatic dish wash compositions.
Description of the Related Art
[0003] Enzymes have been used within the detergent industry as part
of detergent formulations for many decades. Proteases are from a
commercial perspective the most relevant enzyme in such
formulations, but other enzymes including lipases, amylases,
cellulases, hemicellulases or mixtures of enzymes are also commonly
used. One family of proteases, which are widely used in detergents,
are the subtilases. A generally encountered problem using proteases
with or without other enzymes in particular liquid detergent
compositions, is that its needed to stabilize the protease in order
to minimize loss due to proteolysis or auto-proteolysis during
storage in the detergent in order not to get reduced enzyme wash
performance when finally washed. The storage stability of enzymes
in liquid cleaning compositions has been improved for example by
adding various protease inhibitors or stabilizers. The use of a
reversible peptide aldehyde protease inhibitor to stabilize liquid
detergent compositions is described in WO2007/141736, WO2007/145963
and WO2007/145964 (Procter and Gamble Co.) and compositions
containing enzymes stabilized with inhibitors is described in
US2003/157088 (Procter and Gamble Co.). WO96/41638 (Sanofi Winthrop
Inc.) and WO2005/105826 (Zaidan Hojin Biseibutsu) disclose peptide
aldehydes and ketones. Boric acid and boronic acids are known to
reversibly inhibit proteases and are described e.g. in WO1996041859
(Novozymes NS). The use of peptide aldehydes or hydrosulfite
derivatives thereof for stabilizing certain proteases in detergents
is described in e.g. WO2009/118375, WO2013/004636 and WO11036153
(Novozymes NS). An enzyme and its inhibitor interact in a specific
manner and the inhibitors are designed to fit the enzyme similar to
a key and a lock. Consequently, not all inhibitors will bind
equally strong to all enzymes and a specific protease inhibitor may
interact with optimal efficiency to a limited number of proteases
which will be relatively similar in the binding affinity to the
specific inhibitor. An optimal interaction of protease and protease
inhibitor is necessary for obtaining stabilized compositions where
the enzymes of the compositions are stabilized and do not adversely
affect each other. Enzymes formulated in cleaning compositions are
often modified to increase their cleaning performance i.e.
proteases are modified by introducing specific alterations in the
back bone (protease parent) and tested for performance in a
relevant assay. The optimized protease is subsequently formulated
in a detergent and in particular, when the protease formulated in
compositions comprising additional enzymes it may be necessary to
stabilize the protease to maintain optimal effect of the cleaning
composition until finally used in the wash. The optimized protease
may vary in the three dimensional structure from the parent
protease and thus may vary in the interaction with a specific
inhibitor for the parent protease. Further, proteases used in the
detergent industry are very different in structure and sequences.
Thus there is a need for ways to optimize the enzyme inhibitor
interaction in particular for methods to improve proteases toward
stronger binding to specific protease inhibitors.
SUMMARY OF THE INVENTION
[0004] The present invention relates to a method for improve
binding of a protease to an inhibitor of the protease, comprising
the steps of: [0005] a) identify the amino acid residues exposed to
the inhibitor when the inhibitor binds to the protease, [0006] b)
modify the protease by modifying the amino acid residues identified
in step a) [0007] c) determine the binding constant Ki [0008] d)
select modified protease generated in step b)
[0009] The invention further relates to a protease obtained by the
method of the invention.
Overview of Sequences Listing
SEQ ID NO: 1 Savinase
SEQ ID NO: 2 BPN'
SEQ ID NO: 3 TY145
SEQ ID NO: 4 BLAP
SEQ ID NO: 5 Protease P
SEQ ID NO: 6 Protease C
Definitions
[0010] The term "protease" is defined herein as an enzyme that
hydrolyses peptide bonds. It includes any enzyme belonging to the
EC 3.4 enzyme group (including each of the thirteen subclasses
thereof). The EC number refers to Enzyme Nomenclature 1992 from
NC-IUBMB, Academic Press, San Diego, Calif., including supplements
1-5 published in Eur. J. Biochem. 1994, 223, 1-5; Eur. J. Biochem.
1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem.
1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650;
respectively. The term "subtilases" refer to a sub-group of serine
protease according to Siezen et al., Protein Engng. 4 (1991)
719-737 and Siezen et al. Protein Science 6 (1997) 501-523. Serine
proteases or serine peptidases is a subgroup of proteases
characterized by having a serine in the active site, which forms a
covalent adduct with the substrate. Further, the subtilases (and
the serine proteases) are characterized by having two active site
amino acid residues apart from the serine, namely a histidine and
an aspartic acid residue. The subtilases may be divided into 6
sub-divisions, i.e. the Subtilisin family, the Thermitase family,
the Proteinase K family, the Lantibiotic peptidase family, the
Kexin family and the Pyrolysin family. The term "protease activity"
means a proteolytic activity (EC 3.4). Proteases of the invention
are endopeptidases (EC 3.4.21). For purposes of the present
invention, protease activity is determined according to the
procedure described in "Materials and Methods" below.
[0011] The term "parent", "protease parent" or "precursor protease"
means a protease in which an alteration is made to produce protease
variant. The parent may be a naturally occurring (wild-type)
polypeptide. In a particular embodiment the parent is a protease
with at least 60%, at least 62%, at least 63%, at least 64%, at
least 65%, at least 70%, at least 72%, at least 73%, at least 74%,
at least 75%, at least 80%, at least 81%, at least 82%, at least
83%, at least 84%, at least 85%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99% or 100% identity to a
polypeptide with the amino acid sequence shown in SEQ ID NO 1, 2,
3, 4, 5 or 6.
[0012] The term "protease variant" means a protease having protease
activity comprising an alteration, i.e., a substitution, insertion,
and/or deletion, preferably substitution, at one or more (or one or
several) positions compared to its parent which is a protease
having the identical amino acid sequence of said variant but not
having the alterations at one or more of said specified positions.
A substitution means a replacement of an amino acid occupying a
position with a different amino acid; a deletion means removal of
an amino acid occupying a position; and an insertion means adding
amino acids e.g. 1 to 10 amino acids, preferably 1-3 amino acids
adjacent to an amino acid occupying a position. Preferably, the
variant is modified by the hand of man.
[0013] The term "wild-type protease" means a protease expressed by
a naturally occurring organism, such as a bacterium, archaea,
yeast, fungus, plant or animal found in nature.
[0014] The term "mature polypeptide" means a polypeptide in its
final form following translation and any post-translational
modifications, such as N-terminal processing, C-terminal
truncation, glycosylation, phosphorylation, etc.
[0015] The term "mature polypeptide coding sequence" means a
polynucleotide that encodes a mature polypeptide having protease
activity.
[0016] The term "cDNA" means a DNA molecule that can be prepared by
reverse transcription from a mature, spliced, mRNA molecule
obtained from a prokaryotic or eukaryotic cell. A cDNA lacks intron
sequences that may be present in the corresponding genomic DNA. The
initial, primary RNA transcript is a precursor to mRNA that is
processed through a series of steps, including splicing, before
appearing as mature spliced mRNA.
[0017] The relatedness between two amino acid sequences or between
two nucleotide sequences is described by the parameter "sequence
identity". For purposes of the present invention, the degree of
sequence 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
[0018] Biology Open Software Suite, Rice et al., 2000, Trends
Genet. 16: 276-277), 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)
[0019] The term "improved property" means a characteristic
associated with an enzyme that is improved compared to a reference
enzyme one example is a protease variants which is improved
compared to the parent protease. Such improved properties include,
but are not limited to, wash performance, enzyme activity, thermal
activity profile, thermostability, pH activity profile, pH
stability, substrate/cofactor specificity, surface properties,
substrate specificity, product specificity, increased stability
and/or inhibitor binding.
[0020] The term "improved protease activity" is defined herein as
an altered protease activity e.g. by increased protein conversion
of a protease displaying an alteration of the activity relative (or
compared) to the activity of a reference protease, e.g. altered
activity of a protease variant compared to the parent protease, by
increased protein conversion.
[0021] The term "stability" includes storage stability and
stability during use, e.g. during a wash process and reflects the
stability of an enzyme, such as a protease as a function of time
e.g. how much activity is retained when the protease variant is
kept in solution in particular in a detergent solution or in a wash
solution. The stability is influenced by many factors e.g. pH,
temperature, detergent composition e.g. amount of builder,
surfactants, other stabilizers etc.
[0022] The terms "improved stability" and "increased stability"
includes but are not limited to: improved stability under storage
conditions, improved performance after storage conditions, improved
tolerance to ingredients present in the detergent composition,
increased binding to a specific inhibitor, improved detergent
stability, improved in-wash stability, improved auto-proteolytic
stability (including reduced auto-proteolysis) and/or improved
chemical stability "improved chemical stability", "detergent
stability".
[0023] The term "improved chemical stability" is defined herein as
an enzyme, e.g. a protease variant displaying retention of
enzymatic activity after a period of incubation in the presence of
a chemical or chemicals, either naturally occurring or synthetic,
which reduces the enzymatic activity of the reference enzyme, e.g.
protease parent. Likewise, the term "improved detergent stability"
is defined herein as an enzyme e.g. protease variant displaying
retention of enzymatic activity after a period of incubation in the
presence of a detergent component such as bleach, surfactants,
builder and chelators, which reduces the enzymatic activity of the
reference enzyme e.g. parent protease.
[0024] The term "improved wash performance" or improved "cleaning
performance" is defined herein as e.g. a protease variant
displaying an improved wash performance relative to the wash
performance of the parent protease, or relative to a reference
protease when measured in a relevant assay such as AMSA. The term
"wash performance" includes wash performance in laundry but also
hand wash and dish wash.
[0025] The terms "cleaning composition" or "detergent composition",
includes unless otherwise indicated, granular or powder-form
all-purpose or heavy-duty washing agents, especially cleaning
detergents; liquid, gel or paste-form all-purpose washing agents,
especially the so-called heavy-duty liquid (HDL) types; unit
dose/liquid capsules formats, liquid fine-fabric detergents; hand
dishwashing agents or light duty dishwashing agents, especially
those of the high-foaming type;
[0026] machine dishwashing agents, including the various tablet,
granular, liquid, unit dose/liquid or solid capsules and rinse-aid
types for household and institutional use; liquid cleaning and
disinfecting agents, including antibacterial hand-wash types,
cleaning bars, soap bars, mouthwashes, denture cleaners, car or
carpet shampoos, bathroom cleaners; hair shampoos and hair-rinses;
shower gels, foam baths; metal cleaners; as well as cleaning
auxiliaries such as bleach additives and "stain-stick" or pre-treat
types. The terms "detergent composition" and "detergent
formulation" are used in reference to mixtures which are intended
for use in a wash medium for the cleaning of soiled objects. In
some embodiments, the term is used in reference to laundering
fabrics and/or garments (e.g., "laundry detergents"). In
alternative embodiments, the term refers to other detergents, such
as those used to clean dishes, cutlery, etc. (e.g., "dishwashing
detergents"). The terms "cleaning composition" and "detergent
composition" are not intended to be limited to compositions that
contain surfactants. It is intended that the term encompasses
detergents that may contain, surfactants, builders, chelators or
chelating agents, bleach system or bleach components, polymers,
fabric conditioners, foam boosters, suds suppressors, dyes,
perfume, tannish inhibitors, optical brighteners, bactericides,
fungicides, soil suspending agents, anti corrosion agents, dye
transfer inhibitors, acids or bases; buffering agents, salts,
enzymes and enzyme inhibitors or stabilizers, enzyme activators,
bluing agents and fluorescent dyes, antioxidants, or solubilizers
and other functional or visually impacting ingredients (ex
opacifiers, pearlescences).
[0027] The term "effective amount of enzyme" refers to the quantity
of enzyme necessary to achieve the enzymatic activity required in
the specific application, e.g., in a defined detergent composition.
Such effective amounts are readily ascertained by one of ordinary
skill in the art and are based on many factors, such as the
particular enzyme used, the cleaning application, the specific
composition of the detergent composition, and whether a liquid or
dry (e.g., granular, bar) composition is required, and the
like.
[0028] The term "water hardness" or "degree of hardness" or "dH" or
".degree. dH" as used herein refers to German degrees of hardness.
One degree is defined as 10 milligrams of calcium oxide per liter
of water.
[0029] The term "stain removing enzyme" as used herein, describes
an enzyme that aids the removal of a stain or soil from a fabric or
a hard surface. Stain removing enzymes act on specific substrates,
e.g., protease on protein, amylase on starch, lipase and cutinase
on lipids (fats and oils), pectinase on pectin; mannanases on
mannan/processed foods and hemicellulases on hemicellulose. Stains
are often depositions of complex mixtures of different components
which either results in a local discoloration of the material by
itself or which leaves a sticky surface on the object which may
attract soils dissolved in the washing liquor thereby resulting in
discoloration of the stained area. When an enzyme acts on its
specific substrate present in a stain the enzyme degrades or
partially degrades its substrate thereby aiding the removal of
soils and stain components associated with the substrate during the
washing process. For example, when a protease acts on a blood stain
it degrades the protein components in the blood.
[0030] The term "reduced amount" means in this context that the
amount of the component is smaller than the amount which would be
used in a reference process under otherwise the same
conditions.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The invention relates to a method of modifying enzymes in
order to optimize their binding to a suitable inhibitor. Enzymes
have long been applied in cleaning compositions. However, a
generally encountered problem using proteases with or without other
enzymes in particular liquid detergent compositions, is that its
needed to stabilize the protease in order to minimize loss due to
proteolysis or auto-proteolysis during storage in the detergent in
order not to get reduced enzyme wash performance when finally
washed. This may lead to reduced stability of the proteases and
other enzymes in the composition and an overall reduced performance
of the enzymes in the cleaning compositions. The storage stability
of enzymes in cleaning compositions may be improved by adding
protease inhibitors or stabilizers. The interaction between an
enzyme and an inhibitor suitable for the enzyme is preferably
reversible thus the effect of the inhibitor is reversed by removing
the inhibitor. The inhibitor constant, Ki, is an indication of the
potency of an inhibitor to a specific protease and it is the
inhibitor concentration required to decrease the maximal rate of
the reaction to half of the uninhibited value. The Ki is a measure
of the effectiveness of the inhibition of a specific function,
preferably the effectiveness of inhibition or stabilizing of a
protease. The present invention relates to a method of producing
enzyme variants, in particular protease variants which are improved
compared to the parent enzyme or proteases by being more
efficiently inhibited or stabilized by a specific inhibitor than
the corresponding parent protease. Therefore, Ki is lower for the
enzyme or protease variants than Ki for the corresponding parent.
With a lower Ki, either higher stability (more inhibition during
storage) or less inhibitor (more economically feasible) is needed
to obtain the same degree of inhibition. Preferably, the inhibitor
is a reversible inhibitor of enzyme/protease activity, e.g., serine
protease activity and the dilution into the wash liqueur leads to
dissociation. It is advantageous to stabilize proteases by protease
inhibitors, preferably, by a reversible protease inhibitor.
Preferred inhibitors include peptide aldehydes or hydrosulfite
derivatives inhibitors as described in WO 2009/118375; and
WO13004636 and WO11036153 (Novozymes NS), peptide aldehyde
inhibitors as described in WO2007/141736, WO2007/145963 and
WO2007/145964 (Procter and Gamble Co.) and boric acid and boronic
acids inhibitors as described e.g. in WO1996041859 (Novozymes
A/S).
[0032] As mentioned the interaction between an enzyme such as a
protease with its inhibitor is specific and depends on the
inhibitor but also of the enzyme e.g. the protease. The inhibitors
described above are designed to fit to specific enzymes to obtained
optimal inhibition. However, an optimal inhibitor interaction also
depend on the structure of the enzyme as such e.g. the protease.
Preferably, the enzymes to be inhibited are proteases in particular
serine proteases, preferably subtilases which may be of the
subtilisin-type. Many commercially available proteases are
performance optimized by introducing specific mutations into the
back bone. The mutations may influence the way the protease
interacts with the inhibitor. Preferably, the protease variant
produced by the method of the invention has improved binding to a
specific inhibitor compared to the binding of the protease parent
to the same inhibitor. Even more preferably the protease is a
protease which has improved binding to a specific inhibitor
compared to the binding of the protease parent to the same
inhibitor and has improved or retained cleaning performance such as
wash performance in laundry compared to the parent protease.
Several commercially available detergent proteases have been
developed and many of these are formulated in cleaning compositions
with various inhibitors. To obtain an optimal cleaning composition
with good cleaning properties, also after storage, the proteases
comprised in the compositions usually needs to be stabilized by an
inhibitor.
[0033] One embodiment of the invention is providing a method for
modifying enzymes such as proteases for optimal binding to an
inhibitor. There are numerous ways of modifying a protease one way
of modifying a protease is by protein engineering i.e. introducing
alterations in the primary sequence. The method of the invention
describes how the enzymes such as protease could be modified to
increase the binding to an inhibitor. Thus one embodiment of the
invention relates to a method for improve binding of an enzyme to
an inhibitor of the enzyme, comprising the steps of [0034] a)
identify the amino acid residues exposed to the inhibitor when the
inhibitor binds to the enzyme, [0035] b) modify the enzyme by
modifying the amino acid residues identified in step a)
[0036] One embodiment of the invention relates to a method for
improve binding of an enzyme to an inhibitor of the enzyme,
comprising the steps of [0037] a) identify the amino acid residues
exposed to the inhibitor when the inhibitor binds to the enzyme,
[0038] b) modify the enzyme by modifying the amino acid residues
identified in step a) [0039] c) optionally determine the binding
constant Ki [0040] d) select modified enzyme generated in step b)
optionally with improvement factor>1.0
[0041] Calculation of Improvement Factor (IF)
[0042] Having the Ki for each enzyme, it is possible to calculate
the improvement factor e.g. the effect of a single mutation, as
described in Materials and Methods. The relative Ki for the
mutation (Improvement Factor, or IF) is calculated:
Improvement Factor ( IF ) = Ki enzyme without mutation Ki enzyme
variant with mutation ##EQU00001##
[0043] A lower K.sub.i is desirable, as a satisfactory inhibition
can be obtained at a lower inhibitor concentration, so beneficial
mutations have an IF>1.0.
[0044] Preferably the improvement factor is above 1.0 and below 50,
preferably above 2.0 and below 40, preferably above 2.0 and below
30, preferably above 5.0 and below 20, preferably above 5.0 and
below 10. Preferably the improvement factor is above 1.0,
preferably above 2.0, preferably above 5.0, preferably above 10.0,
preferably above 15.0, preferably above 20.
[0045] In one embodiment the enzyme is a protease, preferred
proteases includes those of bacterial, fungal, plant, viral or
animal origin e.g. vegetable or microbial origin, preferably
microbial origin. In one aspect of the invention the enzyme is an
alkaline protease, preferably a serine protease and preferably of
the S1 family, such as trypsin, or the S8 family such as
subtilisin. In a preferred aspect the protease is a serine protease
such as a subtilase and/or a subtilisin. The term "subtilases"
refers to a sub-group of serine protease according to Siezen et
al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein
Science 6 (1997) 501-523. Examples of subtilases are those derived
from Bacillus such as Bacillus lentus, B. alkalophilus, B.
subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus
gibsonii described in; U.S. Pat. No. 7,262,042 and WO09/021867, and
subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus
licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and
subtilisin 168 described in WO89/06279 and protease PD138 described
in (WO93/18140). Other useful proteases may be those described in
WO92/175177, WO01/016285, WO02/026024 and WO02/016547. A further
preferred protease is the alkaline protease from Bacillus lentus
DSM 5483, as described for example in WO95/23221, and variants
thereof which are described in WO92/21760, WO95/23221, EP1921147
and EP1921148. In a preferred embodiment the protease is a protease
having an amino acid sequence which has at least 60%, at least 62%,
at least 63%, at least 64%, at least 65%, at least 70%, at least
72%, at least 73%, at least 74%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% or 100% sequence identity to a polypeptide having the
amino acid sequence of SEQ ID NO 1. In a preferred embodiment the
protease is a protease having an amino acid sequence which has at
least 60%, at least 62%, at least 63%, at least 64%, at least 65%,
at least 70%, at least 72%, at least 73%, at least 74%, at least
75%, at least 80%, at least 81%, at least 82%, at least 83%, at
least 84%, at least 85%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99% or 100% sequence identity to a
polypeptide having the amino acid sequence of SEQ ID NO 2. In a
preferred embodiment the protease is a protease having an amino
acid sequence which has at least 60%, at least 62%, at least 63%,
at least 64%, at least 65%, at least 70%, at least 72%, at least
73%, at least 74%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity to a polypeptide having the amino acid sequence
of SEQ ID NO 3.
[0046] In a preferred embodiment the protease is a protease having
an amino acid sequence which has at least 60%, at least 62%, at
least 63%, at least 64%, at least 65%, at least 70%, at least 72%,
at least 73%, at least 74%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% or 100% sequence identity to a polypeptide having the amino
acid sequence of SEQ ID NO 4.
[0047] In a preferred embodiment the protease is a protease having
an amino acid sequence which has at least 60%, at least 62%, at
least 63%, at least 64%, at least 65%, at least 70%, at least 72%,
at least 73%, at least 74%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% or 100% sequence identity to a polypeptide having the amino
acid sequence of SEQ ID NO 5.
[0048] In a preferred embodiment the protease is a protease having
an amino acid sequence which has at least 60%, at least 62%, at
least 63%, at least 64%, at least 65%, at least 70%, at least 72%,
at least 73%, at least 74%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% or 100% sequence identity to a polypeptide having the amino
acid sequence of SEQ ID NO 6.
[0049] The invention further relates to a method of generating
modified protease having improved binding to an inhibitor. In one
embodiment the amino acid identified in a) binds the inhibitor
through hydrogen binding or e.g. through hydrophobic attraction,
which allows modifying the binding pockets to better accommodate
the inhibitor and/or modifying side chains filling up the binding
pockets.
[0050] In one aspect of the invention the protease to be modified
by the method of the invention is a protease which is optimized for
a specific purpose such as wash performance or stability. Thus the
protease may be a variant (i.e. comprising one or more alterations)
of protease e.g. any of the proteases listed above. In one aspect
the protease to be modified according to the method of the
invention is a protease having a polypeptide sequence which has at
least 60% sequence identity to SEQ ID NO 1, 2, 3, 4, 5 or 6. In one
aspect the protease to be modified according to the method of the
invention is a protease comprising one or more of the following
mutations compared to SEQ ID NO 1: S9E, S9R, A15T, S24G, S24R,
K27R, N42R, G52S, S55P, T56P, G59D, G59E, N60D, N60E, V66A, N74D,
S76N, S85N, S85R, S97A, S97E, S97D, S99E, S99D, S99G, S99N, S99H,
S99M, S101A, V102I, V102N, G116V, G116R, S126A, S126L, S154D,
A156E, G157S, G157D, G157P, S158E, Y161A, R164S, Q176E, N179E,
S182E, Q185N, A188P, N198D, V199I, Q200L, Y203W, S206G, S210V,
L211D, L211Q, L211E, N212D, N212E, N212S, M216S, M216A, N232H,
Q239R, N242D, S250D, S253D, N255W, N255D, N255E, S256E, S256D or
R269H, wherein the protease has at least 60%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the polypeptide having the amino acid sequence of SEQ
ID NO 1. In another aspect of the invention the protease to be
modified in the method according to the invention comprises the
following mutations compared to SEQ ID NO 1: Y161A+R164S+A188P,
S97AD, S97SE, V66A+S104A, S9R+A15T+V66A+N212D+Q239R,
S9R+A15T+G59E+V66A+A188P+V199I+Q239R+N255D, S9E+N42R+N74D,
N74D+Q200L, N74D+N198D, Q200L+Y203W, S85N+S99G+V102N,
K27R+V102Y+N115S+T268A, N74D+S101A+V102I,
S99G+S101A+V102I+G157D+A226V+Q230H+Q239R+N242D+N246K,
S99G+S101A+V102I+G157D+A226V+Q230H+Q239R+N242D+N246K,
S24G+S52G+S76N+S99N+G126A+Y211Q, S24G+S52G+S76N+S99N+G126S+Y211Q,
S85N+G116V+S126L+P127Q+S129A, S85N+G116V+S99M+S126L+P127Q+S129A,
N74D+S85R+G116R+S126L+P127Q+S129A, S97D+S99R+S101A+V102I+G157S,
S3T+V4I+S97D+S99R+S101A+V102I+G157S+A188P+V193M+V199I+L211D,
S3T+V4I+S97D+S99R+S101A+V102I+G157S+V193M+V205I+L217D,
S3T+V4I+S97D+S99R+S101A+V102I+G157S+V199I,
S97D+S99E+S101A+V102I+G157S,
S3T+V4I+S97D+S99E+S101A+V102I+G157S+V199I,
S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L+Y203W+S250D+S253D+N255W+L256E-
, S9E+N42R+N74D+A188P+V199I+Q200L+Y203W+S253D+N255W+L256E,
S9E+N42R+N74D+Q176E+A188P+V199I+Q200L+Y203W+S250D+S253D+N255W+L256E,
S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L+Y203W+S250D+N255W+L256E+*269a
H+*269bH, or
S3V+N74D+H118V+Q176E+N179E+S182E+V199I+Q200L+Y203W+S210V+S250D+N255W+L256-
E, wherein the protease has at least 60%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99% sequence
identity to the polypeptide having the amino acid sequence of SEQ
ID NO 1.
[0051] In one aspect the protease to be modified according to the
method of the invention is a protease comprising one or more of the
following mutations compared to SEQ ID NO 4: S9E, S9R, A15T, S24G,
S24R, K27R, N42R, G52S, S55P, T56P, G59D, G59E, N60D, N60E, V66A,
N74D, S76N, S85N, S85R, S97A, S97E, S99E, S99D, S99G, S99N, S99H,
S99M, V102N, G116V, G116R, S126A, S126L, S154D, A156E, G157D,
G157P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N, A188P,
N198D, V199I, Q200L, Y203W, S206G, S210V, L211D, L211Q, L211E,
N212D, N212E, N212S, M216S, M216A, N232H, Q239R, N242D, S250D,
S253D, N255W, N255D, N255E, S256E, S256D or R269H, wherein the
protease has at least 60%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99% sequence identity to the
polypeptide having the amino acid sequence of SEQ ID NO 4. In
another aspect of the invention the protease to be modified in the
method according to the invention comprises the following mutations
compared to SEQ ID NO 4: S3T+V4I, S3T+V4I+A188P+V193M+V199I+L211D,
S3T+V4I+V193M+V199I+L211D, S3T+V4I+V193M+V199I+L211D,
S3T+V4I+V199I, S3T+V4I+S99E+V199I, S99E, wherein the protease has
at least 60%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% sequence identity to the polypeptide having
the amino acid sequence of SEQ ID NO 4.
[0052] In one aspect of the invention the protease to be modified
in the method according to the invention comprises one or more of
the following mutations compared to SEQ ID NO 2: S9E, S9R, A15T,
S24G, S24R, K27R, K43R, S53G, F58P, N61D, N61E, N62D, N62E, V68A,
N76D, S78N, S87N, S87R, D99A, D99E, S101E, S101D, S101G, S101N,
S101H, S101M, Q103A, Y104I, Y104N, N118V, N118R, G128A, G128L,
E156D, E156S, G160S, G160D, G160P, S161E, Y167A, K1705, S182E,
Q185E, S188E, S191N, S204D, Q206L, L209W, N212G, A216V, Y217D,
L217Q, L217E, N218D, N218E, N218S, M222S, M222A, Q245R, S248D,
K256D, F261W, F261D, F261E, Y262E, Y262D or Q275R. In another
aspect of the invention the protease comprises one or more
following mutations compared to SEQ ID NO 2: S9E+K43R+N76D,
N76D+Q206L, N76D+S204D, Q206L+L209W, S87N+S101G+Y104N, K27R+N117S,
N76D+Q103A+Y104I,
S101G+Q103A+Y104I+G160D+A232V+S236H+Q245R+S248D+N252K,
S101G+Q103A+Y104I+G160D+A232V+S236H+Q239R+S248D+N252K,
S24G+S53G+S78N+S101N+G128A+Y217Q, S24G+S53G+S78N+S101N+G128S+Y217Q,
S87N+G118V+G128L+P129Q+S130A, S87N+G118V+S101M+G128L+P129Q+S130A,
N76D+S87R+G118R+G128L+P129Q+S130A, S101R+Q103A+Y104I+G160S,
S3T+V4I+S101R+Q103A+Y104I+G160S, Y217L, Y217D, Y217E or S101E
wherein the protease has at least 60%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99% sequence identity to
the polypeptide having the amino acid sequence of SEQ ID NO 2.
[0053] In one aspect the protease to be modified according to the
method of the invention is a protease which comprise a substitution
at one or more positions corresponding to positions 171, 173, 175,
179, or 180 of SEQ ID NO: 3. In one aspect the protease to be
modified according to the method of the invention is a protease
comprise one or more of the following mutations compared to SEQ ID
NO 3, S173P, S175P, F180Y, S175P+F180Y, S173P+F180Y, S173P+S175P,
S173P+S175P+F180Y, wherein the protease has at least 60%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
sequence identity to the polypeptide having the amino acid sequence
of SEQ ID NO 3.
[0054] In one aspect of the invention the protease to be modified
is a commercially available protease. Examples of suitable
commercially available protease include those sold under the trade
names Alcalase.RTM., Duralase.TM., Durazym.TM., Relase.RTM.,
Savinase.RTM., Primase.RTM., Polarzyme.RTM., Kannase.RTM.,
Liquanase.RTM., Blaze.RTM., Ovozyme.RTM., Coronase.RTM.,
Neutrase.RTM., Everlase.RTM. and Esperase.RTM. (Novozymes NS),
those sold under the tradename Maxatase.RTM., Maxacal.RTM.,
Maxapem.RTM., Purafect.RTM., Purafect Prime.RTM., Purafect MA.RTM.,
Purafect Ox.RTM., Purafect OxP.RTM., Puramax.RTM., Purafast.RTM.,
Properase.RTM., FN2.RTM., FN3.RTM., FN4.RTM., Excellase.RTM.,
Eraser.RTM., Ultimase.RTM., Opticlean.RTM., Optimase.RTM.,
ExcellenzP.RTM., Preferenz.RTM. and Effectenz.RTM.
(Danisco/DuPont), Axapem.TM. (Gist-Brocases N.V.), BLAP (sequence
shown in FIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof
(Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from
Kao.
[0055] The invention relates to a method for improve binding of a
protease to an inhibitor of the protease, comprising the steps of:
[0056] a) identify the amino acid residues exposed to the inhibitor
when the inhibitor binds to the protease, [0057] b) modify the
protease by modifying the amino acid residues identified in step a)
[0058] c) optionally determine the binding constant Ki [0059] d)
select modified protease generated in step b) which has improved
binding to the protease inhibitor.
[0060] In step a) amino acid residues exposed to the inhibitor when
the inhibitor binds to the protease are identified. To identify the
amino acids binding the inhibitor the three dimensional structure
of the protease may be consulted.
Protease TY145 (SEQ ID NO 3)
[0061] In one embodiment the amino acids binding the inhibitor is
determined in the protease TY145, having the polypeptide sequence
shown in SEQ ID NO 3.
[0062] The structure of TY145 (SEQ ID NO: 3) in complex with the
CI2 inhibitor is disclosed in WO2004067737A2 (Novozymes NS). CI2 is
Chymotrypsin Inhibitor 2 from Barley, which is known to inhibit
chymotrypsin and subtilisin family proteases (Svendsen, I.,
Jonassen, I., Hejgaard, J., & Boisen, S. (1980) Carlsberg Res.
Commun. 45, 389-395).
[0063] From this structure, where TY145 (SEQ ID NO:3) is chain A
and the CI2 inhibitor is chain B, the CI2 inhibitor residues
occupying the binding sub-sites S1-S4 in TY145 (SEQ ID NO 3) can be
identified as:
[0064] P4--Chain B, residue Ile 353
[0065] P3--Chain B, residue Val 354
[0066] P2--Chain B, residue Thr 355
[0067] P1--Chain B, residue Met 356
An initial selection of residues to test for altered inhibitor
binding is identified by selecting all residues in chain A having
their alpha-carbon within 16 angstroms of the alpha-carbon of Ile
353 of chain B. From this set, all residues having their
alpha-carbon within 10 angstroms of Met 356 are removed, leaving
residues with alpha carbon closer than 16 .ANG. to Ile 353 but at
least 10 .ANG. away from Met 356. The residues selected in this way
are: 33, 34, 35, 36, 37, 54, 55, 56, 57, 70, 73, 103, 104, 105,
106, 107, 108, 109, 111, 112, 113, 114, 115, 116, 117, 118, 119,
120, 121, 138, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,
154, 177, 178, 180, 181, 182, 183 and 184.
[0068] In one embodiment of the invention the protease selected in
step d) is a protease having at least 60% sequence identity to SEQ
ID NO 3; and the amino acid residues identified in step a) is
selected from the group consisting of 33, 34, 35, 36, 37, 54, 55,
56, 57, 70, 73, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 121, 138, 144, 145, 146, 147,
148, 149, 150, 151, 152, 153, 154, 177, 178, 180, 181, 182, 183 and
184. One embodiment relates to a method for improve binding of a
protease comprising the amino acid sequence of SEQ ID NO 3 or a
polypeptide having at least 60% sequence identity hereto to an
inhibitor of the protease, comprising the steps of: [0069] a)
identify the amino acid residues exposed to the inhibitor when the
inhibitor binds to the protease, [0070] b) modify the protease by
modifying the amino acid residues identified in step a), wherein
the amino acids identified are selected from the group consisting
of 33, 34, 35, 36, 37, 54, 55, 56, 57, 70, 73, 103, 104, 105, 106,
107, 108, 109, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,
121, 138, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,
177, 178, 180, 181, 182, 183 and 184 corresponding to the positions
of SEQ ID NO 3 and/or c) determine the binding constant Ki and/or
d) selecting the modified protease generated in step b) with
improved inhibitor binding.
[0071] Example 5 shows variants of SEQ ID NO 3 generated by the
method according to the invention, which have improved inhibitor
binding. Using the above method it will be possible to identify
specific mutations which improve the inhibitor binding of the
targeted protease. One embodiment relates to a method for improving
binding of a protease comprising the amino acid sequence of SEQ ID
NO 3 or a polypeptide having at least 60% sequence identity hereto
to an inhibitor of the protease, comprising the steps of: [0072] a)
identify the amino acid residues exposed to the inhibitor when the
inhibitor binds to the protease, [0073] b) modify the protease by
modifying the amino acid residues identified in step a), wherein
the modifications are selected from the group consisting of: Q70F,
Q70A, Q70N, Q70H, Q70Y, S111R, S111E, S111D, S111I, S114A, S114Q,
S114V, S114F, S144R, A145E, K146T, K146S, K146W, K146N, K146F,
K146A, K146R, I150N, I150S, I150I, I150T, I150A, I150V, N176 I178Y,
I178F, I178P, G182A, G182S, L184F, L184Y, L184W, L184D and
L184H.
Protease P (SEQ ID NO 5)
[0074] The structure of the protease P with the amino acid sequence
shown in SEQ ID NO 5 may be obtained by standard X-ray
crystallographic methods or by standard homology modelling known in
the art, see e.g. Emilio Xavier Esposito, Dror Tobi and Jeffry D.
Madura: Comparative Protein Modeling, in Reviews in Computational
Chemistry, Vol. 22, edited by Kenny B. Lipkowitz, Thomas R.
Cundari, and Valerie J. Gillet. 2006 Wiley-VCH, John Wiley &
Sons, Inc. A particular implementation of a homology modelling tool
is described in Z. Xiang, 2006, Curr. Protein Pept. Sci. 7(3):
217-227.
[0075] The structure of the protease with SEQ ID NO 5 can be
aligned to the TY145 structure (SEQ ID NO 3) of the TY145/CI2
inhibitor complex described above using standard superposition
methods, see eg. Marc A. Marti-Renom, Emidio Capriotti, Ilya N.
Shindyalov, and Philip E. Bourne, in Structural Bioinformatics,
2.sup.nd. Ed, by Jenny Gu and Philip E. Bourne, (2009) John Wiley
& Sons, Inc. A particular implementation of molecular
superposition is found in The PyMOL Molecular Graphics System,
Version 1.6, Schrodinger, LLC. In this way, a model of the Protease
P/CI2 inhibitor complex is obtained and used for selection of
Protease P residues to test for altered inhibitor binding. The
residues are identified by selecting all residues in Protease P
having their alpha-carbon within 16 angstroms of the alpha-carbon
of Ile353 of the C12 inhibitor. From this set, all residues having
their alpha-carbon within 10 angstroms of Met356 are removed,
leaving residues with alpha carbon closer than 16 .ANG. to Ile353
but at least 10A away from Met356. The residues selected in this
way are: 31, 32, 33, 34, 35, 54, 55, 56, 64, 67, 92, 93, 94, 95,
96, 97, 98, 100, 101, 102, 103, 103, 104, 105, 106, 107, 108, 109,
110, 122, 128, 129, 130, 131, 132, 133, 134, 135, 137, 138, 149,
160, 160, 161, 161, 162, 163, 164, 165 and 166. In one embodiment
of the invention the protease selected in step d) is a protease
having at least 60% sequence identity to SEQ ID NO 5; and the amino
acid residues identified in step a) is selected from the group
consisting of: 31, 32, 33, 34, 35, 54, 55, 56, 64, 67, 92, 93, 94,
95, 96, 97, 98, 100, 101, 102, 103, 103, 104, 105, 106, 107, 108,
109, 110, 122, 128, 129, 130, 131, 132, 133, 134, 135, 137, 138,
149, 160, 160, 161, 161, 162, 163, 164, 165 and 166. One embodiment
relates to a method for improve binding of a protease comprising
the amino acid sequence of SEQ ID NO 5 or a polypeptide having at
least 60% sequence identity hereto to an inhibitor of the protease,
comprising the steps of: [0076] a) identify the amino acid residues
exposed to the inhibitor when the inhibitor binds to the protease,
[0077] b) modify the protease by modifying the amino acid residues
identified in step a), wherein the amino acids identified are
selected from the group consisting of 31, 32, 33, 34, 35, 54, 55,
56, 64, 67, 92, 93, 94, 95, 96, 97, 98, 100, 101, 102, 103, 103,
104, 105, 106, 107, 108, 109, 110, 122, 128, 129, 130, 131, 132,
133, 134, 135, 137, 138, 149, 160, 160, 161, 161, 162, 163, 164,
165 and 166 corresponding to the positions of SEQ ID NO 5 and/or c)
determine the binding constant Ki and/or d) selecting the modified
protease generated in step b) with improved inhibitor binding.
[0078] In example 6 is shown variants of SEQ ID NO 5 generated by
the method according to the invention which have improved inhibitor
binding. Using the above method it will be possible to identify
specific mutations which improve the inhibitor binding of the
targeted protease. One embodiment relates to a method for improve
binding of a protease comprising the amino acid sequence of SEQ ID
NO 5 or a polypeptide having at least 60% sequence identity hereto
to an inhibitor of the protease, comprising the steps of: [0079] a)
identify the amino acid residues exposed to the inhibitor when the
inhibitor binds to the protease, [0080] b) modify the protease by
modifying the amino acid residues identified in step a), wherein
the modifications are selected from the group consisting of: Q64N,
Q64F, M103S, M103A, G128P and
G127S+G128P+G129S+Y130P+D131S+M134L.
Protease C (SEQ ID NO 6)
[0081] The structure of the protease with the amino acid sequence
shown in SEQ ID NO 6 may be obtained by standard X-ray
crystallographic methods or by standard homology modelling known in
the art, see e.g. Emilio Xavier Esposito, Dror Tobi and Jeffry D.
Madura: Comparative Protein Modeling, in Reviews in Computational
Chemistry, Vol. 22, edited by Kenny B. Lipkowitz, Thomas R.
Cundari, and Valerie J. Gillet. 2006 Wiley-VCH, John Wiley &
Sons, Inc. A particular implementation of a homology modelling tool
is described in Z. Xiang, 2006, Curr. Protein Pept. Sci. 7(3):
217-227.
[0082] The structure of the protease with SEQ ID NO 6 can be
aligned to the TY145 structure (SEQ ID NO 3) of the TY145/CI2
inhibitor complex described above using standard superposition
methods, see e.g. Marc A. Marti-Renom, Emidio Capriotti, Ilya N.
Shindyalov, and Philip E. Bourne, in Structural Bioinformatics,
2.sup.nd. Ed, by Jenny Gu and Philip E. Bourne, (2009) John Wiley
& Sons, Inc. A particular implementation of molecular
superposition is found in The PyMOL Molecular Graphics System,
Version 1.6, Schrodinger, LLC. In this way, a model of the SEQ ID
NO 6/CI2 inhibitor complex is obtained and used for selection of
residues in the protease with the amino acid sequence shown in SEQ
ID NO 6 to test for altered inhibitor binding. The residues are
identified by selecting all residues in the protease with SEQ ID NO
6 having their alpha-carbon within 16 angstroms of the alpha-carbon
of Ile353 of the inhibitor. From this set, all residues having
their alpha-carbon within 10 angstroms of Met356 are removed,
leaving residues with alpha carbon closer than 16 .ANG. to Ile353
but at least 10A away from Met356. The residues selected in this
way are: 30, 31, 32, 33, 34, 48, 49, 50, 51, 60, 63, 91, 92, 93,
94, 95, 96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109,
110, 122, 128, 129, 130, 131, 132, 133, 134, 135, 137, 138, 149,
160, 162, 163, 164, 165, 166 and 169.
[0083] In one embodiment of the invention the protease by selected
in step d) is a protease having at least 60% sequence identity to
SEQ ID NO 1 and comprise, compared to SEQ ID NO 1, one insertion of
the amino acid Glutamic acid (Glu) in a position corresponding to
position 97 of SEQ ID
[0084] NO 6, in short S97SE; and the amino acid residues identified
in step a) is selected from the group consisting of 30, 31, 32, 33,
34, 48, 49, 50, 51, 60, 63, 91, 92, 93, 94, 95, 96, 97, 98, 101,
102, 103, 104, 105, 106, 107, 108, 109, 110, 122, 128, 129, 130,
131, 132, 133, 134, 135, 137, 138, 149, 160, 162, 163, 164, 165,
166 and 169 and 166. One embodiment relates to a method for improve
binding of a protease comprising the amino acid sequence of SEQ ID
NO 6 or a polypeptide having at least 60% sequence identity hereto
to an inhibitor of the protease, comprising the steps of: [0085] a)
identify the amino acid residues exposed to the inhibitor when the
inhibitor binds to the protease, [0086] b) modify the protease by
modifying the amino acid residues identified in step a), wherein
the amino acids identified are selected from the group consisting
of 30, 31, 32, 33, 34, 48, 49, 50, 51, 60, 63, 91, 92, 93, 94, 95,
96, 97, 98, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 122,
128, 129, 130, 131, 132, 133, 134, 135, 137, 138, 149, 160, 162,
163, 164, 165, 166 and 169 and 166 corresponding to the positions
of SEQ ID NO 6 and/or c) determine the binding constant Ki and/or
d) selecting the modified protease generated in step b) with
improved inhibitor binding.
[0087] Example 7 shows variants of SEQ ID NO 6 generated by the
method according to the invention, which have improved inhibitor
binding. Using the above method it will be possible to identify
specific mutations which improve the inhibitor binding of the
targeted protease. One embodiment relates to a method for improve
binding of a protease comprising the amino acid sequence of SEQ ID
NO 6 or a polypeptide comprising, compared to SEQ ID NO 1, one
insertion of the amino acid Glutamic acid (Glu) in a position
corresponding to position 97 of SEQ ID NO 1, in short S97SE and
having at least 60% sequence identity to SEQ ID NO 6, to an
inhibitor of the protease, comprising the steps of:
[0088] a) identify the amino acid residues exposed to the inhibitor
when the inhibitor binds to the protease, [0089] b) modify the
protease by modifying the amino acid residues identified in step
a), wherein the modifications are selected from the group
consisting of: N60A, N60F, N60H, S100I, V103A, L134V, A164S, Y166F,
N60H+S100I, N60F+S100I, S100I+L134V and S100I+Y166F.
Savinase (SEQ ID NO 1)
[0090] The structure of Savinase is known from several published
x-ray crystallography structures, e.g. C. Betzel, S. Klupsch, G.
Papendorf, S. Hastrup, S. Branner, K. S. Wilson, 1992, J. Mol. Biol
(223), p 427. The structure of Savinase can be aligned to the
TY-145 (SEQ ID NO 3) structure of the TY145/CI2 inhibitor complex
described above using standard superposition methods, see e.g. Marc
A. Marti-Renom, Emidio Capriotti, Ilya N. Shindyalov, and Philip E.
Bourne, in Structural Bioinformatics, 2.sup.nd. Ed, by Jenny Gu and
Philip E. Bourne, (2009) John Wiley & Sons, Inc. A particular
implementation of molecular superposition is found in The PyMOL
Molecular Graphics System, Version 1.6, Schrodinger, LLC. In this
way, a model of the Savinase/CI2 inhibitor complex is obtained and
used for selection of Savinase residues to test for altered
inhibitor binding. The residues are identified by selecting all
residues in Savinase having their alpha-carbon within 16 angstroms
of the alpha-carbon of Ile353 of the CI2 inhibitor. From this set,
all residues having their alpha-carbon within 10 angstroms of
Met356 are removed, leaving residues with alpha carbon closer than
16 .ANG. to Ile353 but at least 10A away from Met356. The residues
selected in this way are: 30, 31, 32, 33, 34, 48, 49, 50, 51, 60,
63, 91, 92, 93, 94, 95, 96, 97, 100, 101, 102, 103, 104, 105, 106,
107, 108, 109, 121, 127, 128, 129, 130, 131, 132, 133, 134, 136,
137, 148, 159, 161, 162, 163, 164, 165 and 168. In one embodiment
of the invention the protease selected in step d) is a protease
having at least 60% sequence identity to SEQ ID NO 1; and the amino
acid residues identified in step a) is selected from the group
consisting of: 30, 31, 32, 33, 34, 48, 49, 50, 51, 60, 63, 91, 92,
93, 94, 95, 96, 97, 100, 101, 102, 103, 104, 105, 106, 107, 108,
109, 121, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 148,
159, 161, 162, 163, 164, 165 and 168. One embodiment relates to a
method for improve binding of a protease comprising the amino acid
sequence of SEQ ID NO 1 or a polypeptide having at least 60%
sequence identity hereto to an inhibitor of the protease,
comprising the steps of: [0091] a) identify the amino acid residues
exposed to the inhibitor when the inhibitor binds to the protease,
[0092] b) modify the protease by modifying the amino acid residues
identified in step a), wherein the amino acids identified are
selected from the group consisting of 30, 31, 32, 33, 34, 48, 49,
50, 51, 60, 63, 91, 92, 93, 94, 95, 96, 97, 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 121, 127, 128, 129, 130, 131, 132,
133, 134, 136, 137, 148, 159, 161, 162, 163, 164, 165 and 168
corresponding to the positions of SEQ ID NO 1 and/or c) determine
the binding constant Ki and/or d) selecting the modified protease
generated in step b) with improved inhibitor binding.
[0093] BPN' (SEQ ID NO 2)
[0094] The structure of BPN' is known from several published x-ray
crystallography structures, e.g. R. Bott, M. Ultsch, A. Kossiakoff,
T. Graycar, B. Katz, S. Power (1988) J. Biol. Chem. (263), p. 7895.
The structure of BPN' can be aligned to the TY145 structure of the
TY145/C12 inhibitor complex described above using standard
superposition methods, see eg. Marc A. Marti-Renom, Emidio
Capriotti, Ilya N. Shindyalov, and Philip E. Bourne, in Structural
Bioinformatics, 2.sup.nd. Ed, by Jenny Gu and Philip E. Bourne,
(2009) John Wiley & Sons, Inc. A particular implementation of
molecular superposition is found in The PyMOL Molecular Graphics
System, Version 1.6, Schrodinger, LLC. In this way, a model of the
BPN'/inhibitor complex is obtained and used for selection of BPN'
residues to test for altered inhibitor binding. The residues are
identified by selecting all residues in BPN' having their
alpha-carbon within 16 angstroms of the alpha-carbon of Ile353 of
the CI2 inhibitor. From this set, all residues having their
alpha-carbon within 10 angstroms of Met356 are removed, leaving
residues with alpha carbon closer than 16 .ANG. to Ile353 but at
least 10A away from Met356. The residues selected in this way are:
30, 31, 32, 33, 34, 49, 50, 51, 52, 62, 65, 93, 94, 95, 96, 97, 98,
99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 123,
129, 130, 131, 132, 133, 134, 135, 136, 138, 139, 150, 164, 165,
167, 168, 169, 170 and 171. In one embodiment of the invention the
protease selected in step d) is a protease having at least 60%
sequence identity to SEQ ID NO 2; and the amino acid residues
identified in step a) is selected from the group consisting of: 30,
31, 32, 33, 34, 49, 50, 51, 52, 62, 65, 93, 94, 95, 96, 97, 98, 99,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 123, 129,
130, 131, 132, 133, 134, 135, 136, 138, 139, 150, 164, 165, 167,
168, 169, 170. One embodiment relates to a method for improve
binding of a protease comprising the amino acid sequence of SEQ ID
NO 2 or a polypeptide having at least 60% sequence identity hereto
to an inhibitor of the protease, comprising the steps of: [0095] a)
identify the amino acid residues exposed to the inhibitor when the
inhibitor binds to the protease, [0096] b) modify the protease by
modifying the amino acid residues identified in step a), wherein
the amino acids identified are selected from the group consisting
of 30, 31, 32, 33, 34, 49, 50, 51, 52, 62, 65, 93, 94, 95, 96, 97,
98, 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 123,
129, 130, 131, 132, 133, 134, 135, 136, 138, 139, 150, 164, 165,
167, 168, 169, 170 corresponding to the positions of SEQ ID NO 2
and/or c) determine the binding constant Ki and/or d) selecting the
modified protease generated in step b) with improved inhibitor
binding. Bacillus Lentus protease BLAP (SEQ ID NO 4)
[0097] The structure of BLAP is known from several published x-ray
crystallography structures, e.g. D. W. Goddette, C. Paech, S. S.
Yang, J. R. Mielenz, C. Bystroff, M. E. Wilke, R. J. Fletterick
(1992), J. Mol. Biol. (228), p 580. The structure of BLAP can be
aligned to the TY145 (SEQ ID NO 3) structure of the TY145/inhibitor
complex described above using standard superposition methods, see
eg. Marc A. Marti-Renom, Emidio Capriotti, Ilya N. Shindyalov, and
Philip E. Bourne, in Structural Bioinformatics, 2.sup.nd. Ed, by
Jenny Gu and Philip E. Bourne, (2009) John Wiley & Sons, Inc. A
particular implementation of molecular superposition is found in
The PyMOL Molecular Graphics System, Version 1.6, Schrodinger, LLC.
In this way, a model of the BLAP/CI2 inhibitor complex is obtained
and used for selection of BLAP residues to test for altered
inhibitor binding. The residues are identified by selecting all
residues in BLAP having their alpha-carbon within 16 angstroms of
the alpha-carbon of Ile353 of the CI2 inhibitor. From this set, all
residues having their alpha-carbon within 10 angstroms of Met356
are removed, leaving residues with alpha carbon closer than 16
.ANG. to Ile353 but at least 10A away from Met356. The residues
selected in this way are: 30, 31, 32, 33, 34, 48, 49, 50, 51, 60,
63, 91, 92, 93, 94, 95, 96, 97, 99, 100, 101, 102, 103, 104, 105,
106, 107, 108, 109, 121, 127, 128, 129, 130, 131, 132, 133, 134,
136, 137, 148, 159, 160, 161, 162, 163, 164 and 165. In one
embodiment of the invention the protease selected in step d) is a
protease having at least 60% sequence identity to SEQ ID NO 4; and
the amino acid residues identified in step a) is selected from the
group consisting of: 30, 31, 32, 33, 34, 48, 49, 50, 51, 60, 63,
91, 92, 93, 94, 95, 96, 97, 99, 100, 101, 102, 103, 104, 105, 106,
107, 108, 109, 121, 127, 128, 129, 130, 131, 132, 133, 134, 136,
137, 148, 159, 160, 161, 162, 163, 164 and 165. One embodiment
relates to a method for improve binding of a protease comprising
the amino acid sequence of SEQ ID NO 4 or a polypeptide having at
least 60% sequence identity hereto to an inhibitor of the protease,
comprising the steps of: [0098] a) identify the amino acid residues
exposed to the inhibitor when the inhibitor binds to the protease,
[0099] b) modify the protease by modifying the amino acid residues
identified in step a), wherein the amino acids identified are
selected from the group consisting of 30, 31, 32, 33, 34, 48, 49,
50, 51, 60, 63, 91, 92, 93, 94, 95, 96, 97, 99, 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 121, 127, 128, 129, 130, 131, 132,
133, 134, 136, 137, 148, 159, 160, 161, 162, 163, 164 and 165
corresponding to the positions of SEQ ID NO 4 and/or c) determine
the binding constant Ki and/or d) selecting the modified protease
generated in step b) with improved inhibitor binding.
[0100] The protease variants obtained by the method of the
invention are tested as described in examples 5 to 8. In one
embodiment the modified enzymes e.g. protease variants generated by
the method according to the invention have improved inhibitor
binding compared to the non-modified enzyme e.g. the reference or
parent protease. In one particular embodiment the protease variants
generated by the method of the invention have improved inhibition
by inhibitor compared to the parent protease or compared to a
reference protease. In another particular embodiment the protease
variants generated by the method of the present invention have
increased inhibition by a protease inhibitor compared to the
inhibition of a protease with SEQ ID NO 1, 2, 3, 4, 5 or 6 by the
same inhibitor.
[0101] The protease variants obtained by a method according to the
invention may be formulated into cleaning compositions such as
detergent compositions comprising a protease variant produced by
the method of the invention. In one embodiment the cleaning
composition is a liquid or powder laundry detergent, suitable for
e.g. washing at high temperature and/or pH, such as at or above
40.degree. C. and/or at or above pH 8. In one embodiment the
cleaning composition is a liquid or powder laundry detergent,
suitable for e.g. washing at low temperature and/or pH, such as at
or below 20.degree. C. and/or pH 6. The detergent may also be
formulated as a unit dose detergent and/or compact detergent
optionally with minimum or no water. The detergent may also be a
dish wash detergent which is preferably phosphate-free. The
cleaning composition may further comprise at least one additional
enzyme, such as carbohydrate-active enzymes like carbohydrase,
pectinase, mannanase, amylase, cellulase, arabinase, galactanase,
xylanase, or additional proteases such as metalloproteases, lipase,
a, cutinase, oxidase, e.g., a laccase, and/or peroxidase.
[0102] Inhibitors
[0103] Several protease inhibitors have been described in the art
and include protease inhibitors such as peptide aldehyde,
hydrosulfite adducts of peptide aldehydes; boric acid, or a boronic
acid; or a derivative of any of these. Preferably, the protease
inhibitor is a (reversible) subtilisin protease inhibitor. In
particular, the protease inhibitor may be a peptide aldehyde, boric
acid, or a boronic acid; or a derivative of any of these.
[0104] The protease inhibitors may be a phenyl boronic acid, or a
derivative thereof, of the following formula:
##STR00001##
wherein R is selected from the group consisting of hydrogen,
hydroxy, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkenyl and
substituted C1-C6 alkenyl. Preferably, R is hydrogen, CH3, CH3CH2
or CH3CH2CH2. Preferably the protease inhibitor (phenyl boronic
acid derivative) is 4-formyl-phenyl-boronic acid (4-FPBA). The
protease inhibitor is selected from the group consisting of:
[0105] thiophene-2 boronic acid, thiophene-3 boronic acid,
acetamidophenyl boronic acid, benzofuran-2 boronic acid,
naphtalene-1 boronic acid, naphtalene-2 boronic acid, 2-FPBA,
3-FBPA, 4-FPBA, 1-thianthrene boronic acid, 4-dibenzofuran boronic
acid, 5-methylthiophene-2 boronic, acid, thionaphtrene boronic
acid, furan-2 boronic acid, furan-3 boronic acid, 4,4
biphenyl-diborinic acid, 6-hydroxy-2-naphtalene, 4-(methylthio)
phenyl boronic acid, 4 (trimethyl-silyl)phenyl boronic acid,
3-bromothiophene boronic acid, 4-methylthiophene boronic acid,
2-naphtyl boronic acid, 5-bromothiphene boronic acid,
5-chlorothiophene boronic acid, dimethylthiophene boronic acid,
2-bromophenyl boronic acid, 3-chlorophenyl boronic acid,
3-methoxy-2-thiophene, p-methyl-phenylethyl boronic acid,
2-thianthrene boronic acid, di-benzothiophene boronic acid,
4-carboxyphenyl boronic acid, 9-anthryl boronic acid, 3,5
dichlorophenyl boronic, acid, diphenyl boronic acidanhydride,
o-chlorophenyl boronic acid, p-chlorophenyl boronic acid,
m-bromophenyl boronic acid, p-bromophenyl boronic acid,
p-flourophenyl boronic acid, p-tolyl boronic acid, o-tolyl boronic
acid, octyl boronic acid, 1,3,5 trimethylphenyl boronic acid,
3-chloro-4-flourophenyl boronic acid, 3-aminophenyl boronic acid,
3,5-bis-(triflouromethyl) phenyl boronic acid, 2,4 dichlorophenyl
boronic acid, 4-methoxyphenyl boronic acid.
[0106] Further boronic acid derivatives suitable as protease
inhibitors in the cleaning composition are described in U.S. Pat.
No. 4,963,655, U.S. Pat. No. 5,159,060, WO 95/12655, WO 95/29223,
WO 92/19707, WO 94/04653, WO 94/04654, U.S. Pat. No. 5,442,100,
U.S. Pat. No. 5,488,157 and U.S. Pat. No. 5,472,628.
[0107] The protease inhibitor may also be a peptide aldehyde having
the formula X--B.sup.1--B.sup.0--H, wherein the groups have the
following meaning: [0108] a) H is hydrogen; [0109] b) B.sup.0 is a
single amino acid residue with L- or D-configuration and with the
formula: NH--CHR'--CO; [0110] c) B.sup.1 is a single amino acid
residue; and [0111] d) X consists of one or more amino acid
residues (preferably one or two), optionally comprising an
N-terminal protection group.
[0112] NH--CHR'--CO (B.sup.0) is an L or D-amino acid residue,
where R' may be an aliphatic or aromatic side chain, e.g.,
alkylaryl, such as benzyl, where R' may be optionally substituted.
More particularly, the B.sup.0 residue may be bulky, neutral,
polar, hydrophobic and/or aromatic. Examples are the D- or L-form
of Tyr (p-tyrosine), m-tyrosine, 3,4-dihydroxyphenylalanine, Phe,
Val, Met, norvaline (Nva), Leu, Ile or norleucine (Nle).
[0113] In the above formula, X--B.sup.1--B.sup.0--H, the B.sup.1
residue may particularly be small, aliphatic, hydrophobic and/or
neutral. Examples are alanine (Ala), cysteine (Cys), glycine (Gly),
proline (Pro), serine (Ser), threonine (Thr), valine (Val),
norvaline (Nva) and norleucine (Nle), particularly alanine,
glycine, or valine.
X may in particular be one or two amino acid residues with an
optional N-terminal protection group (i.e. the compound is a tri-
or tetrapeptide aldehyde with or without a protection group). Thus,
X may be B.sup.2, B.sup.3--B.sup.2, Z--B.sup.2, or
Z--B.sup.3--B.sup.2 where B.sup.3 and B.sup.2 each represents one
amino acid residue, and Z is an N-terminal protection group. The
B.sup.2 residue may in particular be small, aliphatic and/or
neutral, e.g., Ala, Gly, Thr, Arg, Leu, Phe or Val. The B.sup.3
residue may in particular be bulky, hydrophobic, neutral and/or
aromatic, e.g., Phe, Tyr, Trp, Phenylglycine, Leu, Val, Nva, Nle or
Ile. The N-terminal protection group Z (if present) may be selected
from formyl, acetyl, benzoyl, trifluoroacetyl, fluoromethoxy
carbonyl, methoxysuccinyl, aromatic and aliphatic urethane
protecting groups, benzyloxycarbonyl (Cbz), t-butyloxycarbonyl,
adamantyloxycarbonyl, p-methoxybenzyl carbonyl (MOZ), benzyl (Bn),
p-methoxybenzyl (PMB) or p-methoxyphenyl (PMP), methoxycarbonyl
(Moc); methoxyacetyl (Mac); methyl carbamate or a methylamino
carbonyl/methyl urea group. In the case of a tetrapeptide aldehyde
with a protection group (i.e. X=Z--B3-B2 X=Z--B2), Z is preferably
a small aliphatic group, e.g., formyl, acetyl, fluoromethoxy
carbonyl, t-butyloxycarbonyl, methoxycarbonyl (Moc); methoxyacetyl
(Mac); methyl carbamate or a Methylamino carbonyl/methyl urea
group. In the case of a tripeptide aldehyde with a protection group
(i.e. X=Z--B.sup.2), Z is preferably a bulky aromatic group such as
benzoyl, benzyloxycarbonyl, p-methoxybenzyl carbonyl (MOZ), benzyl
(Bn), p-methoxybenzyl (PMB) or p-methoxyphenyl (PMP).
[0114] In the case of a tripeptide aldehyde with a protection group
(i.e. X=Z--B.sup.2), Z is preferably a bulky aromatic group such as
benzoyl, benzyloxycarbonyl, p-methoxybenzyl carbonyl (MOZ), benzyl
(Bn), p-methoxybenzyl (PMB) or p-methoxyphenyl (PMP) or Z is
preferably a small aliphatic group, e.g., formyl, acetyl,
fluoromethoxy carbonyl, t-butyloxycarbonyl, methoxycarbonyl (Moc);
methoxyacetyl (Mac); methyl carbamate or a Methylamino
carbonyl/methyl urea group. In the case of a tetrapeptide aldehyde
with a protection group (i.e. X=Z--B.sup.3--B2 X=Z--B.sup.2), Z is
preferably a small aliphatic group, e.g., formyl, acetyl,
fluoromethoxy carbonyl, t-butyloxycarbonyl, methoxycarbonyl (Moc);
methoxyacetyl (Mac); methyl carbamate or a Methylamino
carbonyl/methyl urea group.
[0115] Suitable peptide aldehydes are described in WO 94/04651, WO
95/25791, WO 98/13458, WO 98/13459, WO 98/13460, WO 98/13461, WO
98/13461, WO 98/13462, WO 2007/141736, 2007/145963, WO 2009/118375,
WO 2010/055052 and WO 2011/036153. More particularly, the peptide
aldehyde may be Cbz-RAY-H, Ac-GAY-H, Cbz-GAY-H, Cbz-GAL-H,
Cbz-VAL-H, Cbz-GAF-H, Cbz-GAV-H, Cbz-GGY-H, Cbz-GGF-H, Cbz-RVY-H,
Cbz-LVY-H, Ac-LGAY-H, Ac-FGAY-H, Ac-YGAY-H, Ac-FGAL-H, Ac-FGAF-H,
Ac-FGVY-H, Ac-FGAM-H, Ac-WLVY-H, MeO--CO-VAL-H, MeNCO-VAL-H,
MeO--CO-FGAL-H, MeO--CO-FGAF-H, MeSO2-FGAL-H, MeSO2-VAL-H,
PhCH2O(OH)(O)P-VAL-H, EtSO2-FGAL-H, PhCH2SO2-VAL-H,
PhCH2O(OH)(O)P-LAL-H, PhCH2O(OH)(O)P-FAL-H, or MeO(OH)(O)P-LGAL-H.
Here, Cbz is benzyloxycarbonyl, Me is methyl, Et is ethyl, Ac is
acetyl, H is hydrogen, and the other letters represent amino acid
residues denoted by standard single letter notification (e.g.,
F=Phe, Y=Tyr, L=Leu). Alternatively, the peptide aldehyde may have
the formula as described in WO 2011/036153:
P--O-(A.sub.i-X').sub.n-A.sub.n+1-Q
[0116] wherein Q is hydrogen, CH.sub.3, CX''.sub.3, CHX''.sub.2, or
CH.sub.2X'', wherein X'' is a halogen atom;
[0117] wherein one X is the "double N-capping group" CO, CO--CO,
CS, CS--CS or CS--CO, most preferred urido (CO), and the other X'
are nothing,
[0118] wherein n=1-10, preferably 2-5, most preferably 2,
[0119] wherein each of A.sub.i and A.sub.n+1 is an amino acid
residue having the structure:
[0120] --NH--CR''--CO-- for a residue to the right of X'=--CO--,
or
[0121] --CO--CR''--NH-- for a residue to the left of X'=--CO--
[0122] wherein R'' is H-- or an optionally substituted alkyl or
alkylaryl group which may optionally include a hetero atom and may
optionally be linked to the N atom, and wherein P is hydrogen or
any C-terminal protection group.
[0123] Examples of such peptide aldehydes include .alpha.-MAPI,
.beta.-MAPI, F-urea-RVY-H, F-urea-GGY-H, F-urea-GAF-H,
F-urea-GAY-H, F-urea-GAL-H, F-urea-GA-Nva-H, F-urea-GA-Nle-H,
Y-urea-RVY-H, Y-urea-GAY-H, F--CS-RVF-H, F--CS-RVY-H, F--CS-GAY-H,
Antipain, GE20372A, GE20372B, Chymostatin A, Chymostatin B, and
Chymostatin C. Further examples of peptide aldehydes are disclosed
in WO 2010/055052 and WO 2009/118375, WO 94/04651, WO 98/13459, WO
98/13461, WO 98/13462, WO 2007/145963, hereby incorporated by
reference.
[0124] Alternatively to a peptide aldehyde, the protease inhibitor
may be a hydrosulfite adduct having the formula
X--B.sup.1--NH--CHR'--CHOH-SO.sub.3M, wherein X, B.sup.1 and R' are
defined as above, and M is H or an alkali metal, preferably Na or
K. Peptide aldehyde hydrosulfite adducts are described in WO
2013/004636.
[0125] The peptide aldehyde may be converted into a water-soluble
hydrosulfite adduct by reaction with sodium bisulfite, as described
in textbooks, e.g., March, J. Advanced Organic Chemistry, fourth
edition, Wiley-Interscience, US 1992, p 895.
[0126] An aqueous solution of the bisulfite adduct may be prepared
by reacting the corresponding peptide aldehyde with an aqueous
solution of sodium bisulfite (sodium hydrogen sulfite, NaHSO3);
potassium bisulfite (KHSO3) by known methods, e.g., as described in
WO 98/47523; U.S. Pat. No. 6,500,802; U.S. Pat. No. 5,436,229; J.
Am. Chem. Soc. (1978) 100, 1228; Org. Synth., Coll. vol. 7:
361.
[0127] All the mentioned peptide aldehydes described here could be
either a peptide aldehyde or a hydrosulfite adduct thereof.
[0128] The molar ratio of the above-mentioned peptide aldehydes (or
hydrosulfite adducts) to the protease may be at least 1:1 or 1.5:1
and it may be less than 1000:1, more preferred less than 500:1,
even more preferred from 100:1 to 2:1 or from 20:1 to 2:1, or most
preferred, the molar ratio is from 10:1 to 2:1.
[0129] Formate salts (e.g., sodium formate) and formic acid have
also shown good effects as inhibitor of protease activity. Formate
can be used synergistically with the above-mentioned protease
inhibitors, as shown in WO2013/004635 (Novozymes NS). The formate
salts may be present in the detergent composition in an amount of
at least 0.1% w/w or 0.5% w/w, e.g., at least 1.0%, at least 1.2%
or at least 1.5%. The amount of the salt is typically below 5% w/w,
below 4% or below 3%.
[0130] WO2007/141736, WO2007/145963 and WO2007/145964 (Proctor and
Gamble Co.)
[0131] disclose the use of a reversible peptide protease inhibitor
to stabilize liquid detergent compositions. US2003/157088 (Proctor
and Gamble Co.) describes compositions containing enzymes
stabilized with inhibitors.
The invention is further described in the following
embodiments.
Embodiment 1
[0132] a method for improve binding of a protease to an inhibitor
of the protease, comprising the steps of: [0133] a) identify the
amino acid residues exposed to the inhibitor when the inhibitor
binds to the protease, [0134] b) modify the protease by modifying
the amino acid residues identified in step a) [0135] c) determine
the binding constant Ki [0136] d) select modified protease
generated in step b) optionally with improvement factor>1.0.
Embodiment 2
[0137] The method according to embodiment 1, wherein the he
improvement factor of the protease selected in step d) is above 1.0
and below 50, preferably above 2.0 and below 40, preferably above
2.0 and below 30, preferably above 5.0 and below 20, preferably
above 5.0 and below 10. Preferably the improvement factor is above
1.0, preferably above 2.0, preferably above 5.0, preferably above
10.0, preferably above 15.0, preferably above 20.
Embodiment 3
[0138] Method of embodiment 1 wherein at least one of the amino
acids identified in step a) bind to the inhibitor via hydrogen
binding.
Embodiment 4
[0139] The method according to any of embodiment 1 to 3 wherein the
structure of a targeted protease is aligned to the TY-145 structure
(SEQ ID NO 3) of the TY145/CI2 inhibitor complex as disclosed in
WO2004067737A2.
Embodiment 5
[0140] The method according to any of embodiment 4, wherein the at
least one binding amino acids are identified by selecting all
residues in the protease chain A having their alpha-carbon within
16 angstroms of the alpha-carbon of Ile 353 of the CI2 inhibitor
chain B.
Embodiment 6
[0141] The method of embodiment 5, wherein all residues having
their alpha-carbon within 10 angstroms of Met 356 of the CI2
inhibitor chain B are removed, Embodiment 7: The method according
to any of the proceeding embodiments wherein the amino acids
identified in step a) have alpha carbon closer than 16 .ANG. to the
isoleucine amino acid (Ile) at position 353 of chain B of the CI2
inhibitor but at least 10A away from methionine amino acid (Met) at
position 356 of chain B of the CI2 inhibitor.
Embodiment 8
[0142] The method of any one of embodiments 1-7, wherein the
subtilisin inhibitor is a peptide aldehyde, or a hydrosulfite
adduct thereof; or a phenyl boronic acid, or a derivative thereof,
such as 4-FPBA.
Embodiment 9
[0143] The method of any one of embodiments 1-8, wherein the
inhibitor is a peptide aldehyde or ketone having the formula
P-(A).sub.y-L-(B).sub.x--B.sup.0--R* or a hydrosulfite adduct of
such aldehyde, wherein: [0144] a) R* is H (hydrogen), CH.sub.3,
CX.sub.3, CHX.sub.2, or CH.sub.2X; [0145] b) X is a halogen atom;
[0146] c) B.sup.0 is a single amino acid residue with L- or
D-configuration of the formula --NH--CH(R)--C(.dbd.O)--; [0147] d)
x is 1, 2 or 3; [0148] e) B.sub.x is independently a single amino
acid residue, each connected to the next B or to B.sup.0 via its
C-terminal; [0149] f) L is absent or independently a linker group
of the formula --C(.dbd.O)--, --C(.dbd.O)--C(.dbd.O)--,
--C(.dbd.S)--C(.dbd.S)--C(.dbd.S)-- or --C(=S)--C(.dbd.O)--; [0150]
g) A is absent if L is absent or is independently a single amino
acid residue connected to L via the N-terminal of the amino acid;
[0151] h) P is selected from the group consisting of hydrogen or if
L is absent an N-terminal protection group; [0152] i) y is 0, 1, or
2, [0153] j) R is independently selected from the group consisting
of C.sub.1-6 alkyl, C.sub.6-10 aryl or C.sub.7-10 arylalkyl
optionally substituted with one or more, identical or different,
substituent's R';
[0154] k) R' is independently selected from the group consisting of
halogen, --OH, --OR'', --SH, --SR'', --NH.sub.2, --NHR'',
--NR''.sub.2, --CO.sub.2H, --CONH.sub.2, --CONHR'', --CONR''.sub.2,
--NHC(.dbd.N)NH.sub.2; and [0155] l) R'' is a C.sub.1-6 alkyl
group. [0156] m) x may be 1, 2 or 3.
Embodiment 10
[0157] The method of embodiment 9, wherein the inhibitor is an
aldehyde having the formula P--B.sup.2--B.sup.1--B.sup.0--H or an
adduct having the formula
P--B.sup.2--B.sup.1--N(H)--CHR--CHOH--SO.sub.3M, wherein [0158] a)
H is hydrogen; [0159] b) B.sup.0 is a single amino acid residue
with L- or D-configuration of the formula --NH--CH(R)--C(.dbd.O)--;
[0160] c) B.sup.1 and B.sup.2 are independently single amino acid
residues; [0161] d) R is independently selected from the group
consisting of C.sub.1-6 alkyl, C.sub.6-10 aryl or C.sub.7-10
arylalkyl optionally substituted with one or more, identical or
different, substituent's R'; [0162] e) R' is independently selected
from the group consisting of halogen, --OH, --OR'', --SH, --SR'',
--NH.sub.2, --NHR'', --NR''.sub.2, --CO.sub.2H, --CONH.sub.2,
--CONHR'', --CONR''.sub.2, --NHC(.dbd.N)NH.sub.2; [0163] f) R'' is
a C.sub.1-6 alkyl group; and [0164] g) P is an N-terminal
protection group.
Embodiment 11
[0165] The method of embodiment 9 or 10, wherein R is such that
B.sup.0=--NH--CH(R)--C(.dbd.O)-- is Phe, Tyr or Leu; and B.sup.1 is
Ala, Gly or Val; and B.sup.2 is Arg, Phe, Tyr or Trp.
Embodiment 12
[0166] The method of any one of embodiments 9-11, wherein x=2, L is
absent, A is absent, and P is p-methoxycarbonyl (Moc) or
benzyloxycarbonyl (Cbz).
Embodiment 13
[0167] The method of any one of embodiments 9-12, wherein the
inhibitor is Cbz-Arg-Ala-Tyr-H, Ac-Gly-Ala-Tyr-H,
Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-CF.sub.3, Cbz-Gly-Ala-Leu-H,
Cbz-Val-Ala-Leu-H, Cbz-Val-Ala-Leu-CF.sub.3,
Moc-Val-Ala-Leu-CF.sub.3, Cbz-Gly-Ala-Phe-H,
Cbz-Gly-Ala-Phe-CF.sub.3, Cbz-Gly-Ala-Val-H, Cbz-Gly-Gly-Tyr-H,
Cbz-Gly-Gly-Phe-H, Cbz-Arg-Val-Tyr-H, Cbz-Leu-Val-Tyr-H,
Ac-Leu-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Tyr-H, Ac-Tyr-Gly-Ala-Tyr-H,
Ac-Phe-Gly-Ala-Leu-H, Ac-Phe-Gly-Ala-Phe-H, Ac-Phe-Gly-Val-Tyr-H,
Ac-Phe-Gly-Ala-Met-H, Ac-Trp-Leu-Val-Tyr-H, MeO--CO-Val-Ala-Leu-H,
MeNCO-Val-Ala-Leu-H, MeO--CO-Phe-Gly-Ala-Leu-H,
MeO--CO-Phe-Gly-Ala-Phe-H, MeSO.sub.2-Phe-Gly-Ala-Leu-H,
MeSO.sub.2-Val-Ala-Leu-H, PhCH.sub.2O--P(OH)(O)-Val-Ala-Leu-H,
EtSO.sub.2-Phe-Gly-Ala-Leu-H, PhCH.sub.2SO.sub.2-Val-Ala-Leu-H,
PhCH.sub.2O--P(OH)(O)-Leu-Ala-Leu-H,
PhCH.sub.2O--P(OH)(O)-Phe-Ala-Leu-H, or
MeO--P(OH)(O)-Leu-Gly-Ala-Leu-H or a hydrosulfite adduct of any of
these, wherein Cbz is benzyloxycarbonyl and Moc is
methoxycarbonyl.
Embodiment 14
[0168] The method of any one of embodiments 9-13, wherein the
inhibitor is Cbz-Gly-Ala-Tyr-H or Moc-Val-Ala-Leu-H, or a
hydrosulfite adduct thereof, wherein Cbz is benzyloxycarbonyl and
Moc is methoxycarbonyl.
Embodiment 15
[0169] The method according to any of the proceeding embodiments
wherein the protease is selected from a protease having the amino
acid sequence shown in SEQ ID NO: 1 or a protease having at least
60%, preferably at least 70%, preferably at least 80% or at least
90% sequence identity hereto.
Embodiment 16
[0170] The method according to embodiment 15 wherein the protease
is a variant of SEQ ID NO 1 comprising compared to SEQ ID NO 1 one
or more mutation selected from the list consisting of: S9E, S9R,
A15T, S24G, S24R, K27R, N42R, G52S, S55P, T56P, G59D, G59E, N60D,
N60E, V66A, N74D, S76N, S85N, S85R, S97A, S97E, S97D, S99E, S99D,
S99G, S99N, S99H, S99M, S101A, V102I, V102N, G116V, G116R, S126A,
S126L, S154D, A156E, G157S, G157D, G157P, S158E, Y161A, R164S,
Q176E, N179E, S182E, Q185N, A188P, N198D, V199I, Q200L, Y203W,
S206G, S210V, L211D, L211Q, L211E, N212D, N212E, N212S, M216S,
M216A, N232H, Q239R, N242D, S250D, S253D, N255W, N255D, N255E,
S256E, S256D and R269H, wherein the protease variant has at least
60%, at least 70%, at least 80%, at least 90% or at least 95%,
sequence identity to the polypeptide having the amino acid sequence
of SEQ ID NO 1.
Embodiment 17
[0171] The method according to embodiment 15 or 16, wherein the
protease is a variant of SEQ ID NO 1 comprising compared to SEQ ID
NO 1 the mutations selected from the list consisting of:
Y161A+R164S+A188P, S97AD, S97SE, V66A+S104A,
S9R+A15T+V66A+N212D+Q239R,
S9R+A15T+G59E+V66A+A188P+V199I+Q239R+N255D, S9E+N42R+N74D,
N74D+Q200L, N74D+N198D, Q200L+Y203W, S85N+S99G+V102N,
K27R+V102Y+N115S+T268A, N74D+S101A+V102I,
S99G+S101A+V102I+G157D+A226V+Q230H+Q239R+N242D+N246K,
S99G+S101A+V102I+G157D+A226V+Q230H+Q239R+N242D+N246K,
S24G+S52G+S76N+S99N+G126A+Y211Q, S24G+S52G+S76N+S99N+G126S+Y211Q,
S85N+G116V+S126L+P127Q+S129A, S85N+G116V+S99M+S126L+P127Q+S129A,
N74D+S85R+G116R+S126L+P127Q+S129A, S97D+S99R+S101A+V102I+G157S,
S3T+V4I+S97D+S99R+S101A+V102I+G157S+A188P+V193M+V199I+L211D,
S3T+V4I+S97D+S99R+S101A+V102I+G157S+V193M+V205I+L217D,
S3T+V4I+S97D+S99R+S101A+V102I+G157S+V199I,
S97D+S99E+S101A+V102I+G157S,
S3T+V4I+S97D+S99E+S101A+V102I+G157S+V199I,
S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L+Y203W+S250D+S253D+N255W+
[0172] L256E,
S9E+N42R+N74D+A188P+V199I+Q200L+Y203W+S253D+N255W+L256E,
S9E+N42R+N74D+Q176E+A188P+V199I+Q200L+Y203W+S250D+S253D+N255W+L256E,
S9E+N42R+N74D+H118V+Q176E+A188P+V199I+Q200L+Y203W+S250D+N255W+L256E+*269a-
H+*269bH and
S3V+N74D+H118V+Q176E+N179E+S182E+V199I+Q200L+Y203W+S210V+S250D+N255W+
[0173] L256E, wherein the protease variant has at least 60%, at
least 80%, at least 90%, at least 95% sequence identity to the
polypeptide having the amino acid sequence of SEQ ID NO 1.
Embodiment 18
[0174] The method according to any of embodiments 1 to 14, wherein
the protease is selected from a protease having the amino acid
sequence shown in SEQ ID NO: 2 or a protease having at least 60%,
preferably at least 70%, preferably at least 80% or at least 90%
sequence identity hereto.
Embodiment 19
[0175] The method according to embodiment 18 wherein the protease
is a variant of SEQ ID NO 2 comprising compared to SEQ ID NO 2 one
or more mutation selected from the list consisting of: S9E, S9R,
A15T, S24G, S24R, K27R, K43R, S53G, F58P, N61D, N61E, N62D, N62E,
V68A, N76D, S78N, S87N, S87R, D99A, D99E, S101E, S101D, S101G,
S101N, S101H, S101M, Q103A, Y104I, Y104N, N118V, N118R, G128A,
G128L, E156D, E156S, G160S, G160D, G160P, S161E, Y167A, K1705,
S182E, Q185E, S188E, S191N, S204D, Q206L, L209W, N212G, A216V,
Y217D, L217Q, L217E, N218D, N218E, N218S, M222S, M222A, Q245R,
S248D, K256D, F261W, F261D, F261E, Y262E, Y262D and Q275R, wherein
the protease variant has at least 60%, at least 70%, at least 80%,
at least 90% or at least 95%, sequence identity to the polypeptide
having the amino acid sequence of SEQ ID NO 2.
Embodiment 20
[0176] The method according to embodiment 18 or 19, wherein the
protease is a variant of SEQ ID NO 2 comprising compared to SEQ ID
NO 2 the mutations selected from the list consisting of:
S9E+K43R+N76D, N76D+Q206L, N76D+S204D, Q206L+L209W,
S87N+S101G+Y104N, K27R+N117S, N76D+Q103A+Y104I,
S101G+Q103A+Y104I+G160D+A232V+S236H+Q245R+S248D+N252K,
S101G+Q103A+Y104I+G160D+A232V+S236H+Q239R+S248D+N252K,
S24G+S53G+S78N+S101N+G128A+Y217Q, S24G+S53G+S78N+S101N+G128S+Y217Q,
S87N+G118V+G128L+P129Q+S130A, S87N+G118V+S101M+G128L+P129Q+S130A,
N76D+S87R+G118R+G128L+P129Q+S130A, S101R+Q103A+Y104I+G160S,
S3T+V4I+S101R+Q103A+Y104I+G160S, Y217L, Y217D, Y217E and S101E,
wherein the protease variant has at least 60%, at least 80%, at
least 90%, at least 95% sequence identity to the polypeptide having
the amino acid sequence of SEQ ID NO 2.
Embodiment 21
[0177] The method according to any of embodiments 1 to 14, wherein
the protease is selected from a protease having the amino acid
sequence shown in SEQ ID NO: 3 or a protease having at least 60%,
preferably at least 70%, preferably at least 80% or at least 90%
sequence identity hereto.
Embodiment 22
[0178] The method according to embodiment 21, wherein the protease
is a variant of SEQ ID NO 3 comprising compared to SEQ ID NO 3 one
or more mutation selected from the list consisting of: S173P,
S175P, F180Y, S175P+F180Y, S173P+F180Y, S173P+S175P,
S173P+S175P+F180Y, wherein the protease variant has at least 60%,
at least 70%, at least 80%, at least 90% or at least 95%, sequence
identity to the polypeptide having the amino acid sequence of SEQ
ID NO 3.
Embodiment 23
[0179] The method according to any of embodiments 1 to 14, wherein
the protease is selected from a protease having the amino acid
sequence shown in SEQ ID NO: 4 or a protease having at least 60%,
preferably at least 70%, preferably at least 80% or at least 90%
sequence identity hereto.
Embodiment 24
[0180] The method according to embodiment 23 wherein the protease
is a variant of SEQ ID NO 4 comprising compared to SEQ ID NO 4 one
or more mutation selected from the list consisting of: S9E, S9R,
A15T, S24G, S24R, K27R, N42R, G52S, S55P, T56P, G59D, G59E, N60D,
N60E, V66A, N74D, S76N, S85N, S85R, S97A, S97E, S99E, S99D, S99G,
S99N, S99H, S99M, V102N, G116V, G116R, S126A, S126L, S154D, A156E,
G157D, G157P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N,
A188P, N198D, V199I, Q200L, Y203W, S206G, S210V, L211D, L211Q,
L211E, N212D, N212E, N212S, M216S, M216A, N232H, Q239R, N242D,
S250D, S253D, N255W, N255D, N255E, S256E, S256D and R269H, wherein
the protease variant has at least 60%, at least 70%, at least 80%,
at least 90% or at least 95%, sequence identity to the polypeptide
having the amino acid sequence of SEQ ID NO 4.
Embodiment 25
[0181] The method according to embodiment 23 or 24, wherein the
protease is a variant of SEQ ID NO 4 comprising compared to SEQ ID
NO 4 the mutations selected from the list consisting of: S3T+V4I,
S3T+V4I+A188P+V193M+V199I+L211D, S3T+V4I+V193M+V199I+L211D,
S3T+V4I+V193M+V199I+L211D, S3T+V4I+V199I, S3T+V4I+S99E+V199I and
S99E, wherein the protease variant has at least 60%, at least 80%,
at least 90%, at least 95% sequence identity to the polypeptide
having the amino acid sequence of SEQ ID NO 4.
Embodiment 26
[0182] The method according to any of embodiments 1 to 14, wherein
the protease is selected from a protease having the amino acid
sequence shown in SEQ ID NO: 5 or a protease having at least 60%,
preferably at least 70%, preferably at least 80% or at least 90%
sequence identity hereto.
Embodiment 27
[0183] The method according to any of embodiments 1 to 14, wherein
the protease is a variant of SEQ ID NO 5 comprising compared to SEQ
ID NO 5 one or more mutation selected independently from
insertions, deletions and substitutions, wherein the protease
variant has at least 60%, at least 70%, at least 80%, at least 90%
or at least 95%, sequence identity to the polypeptide having the
amino acid sequence of SEQ ID NO 5.
Embodiment 28
[0184] The method according to any of embodiments 1 to 14, wherein
the protease is selected from a protease having the amino acid
sequence shown in SEQ ID NO: 6 or a protease comprising compared to
SEQ ID NO 1 an insertion of glutamic acid at the position
corresponding to position 97 of SEQ ID NO 1 and having at least
60%, preferably at least 70%, preferably at least 80% or at least
90% sequence identity to SEQ ID NO 1.
Embodiment 29
[0185] The method according to claim 28, wherein the protease is a
variant of SEQ ID
[0186] NO 6 comprising compared to SEQ ID NO 6 one or more mutation
selected independently from insertions, deletions and
substitutions, wherein the protease variant has at least 60%, at
least 70%, at least 80%, at least 90% or at least 95%, sequence
identity to the polypeptide having the amino acid sequence of SEQ
ID NO 6.
Embodiment 30
[0187] A protease obtained by the method of any of embodiments
1-29
Embodiment 31
[0188] The method according to any of the proceeding embodiments
wherein the modified protease variant has improved wash
performance.
Materials and Methods
Protease Activity Assay
1) Suc-AAPF-pNA Activity Assay:
[0189] The proteolytic activity can be determined by a method
employing the Suc-AAPF-pNA substrate. Suc-AAPF-pNA is an
abbreviation for
N-Succinyl-Alanine-Alanine-Proline-Phenylalanine-p-Nitroanilide,
and it is a blocked peptide which can be cleaved by endo-proteases.
Following cleavage a free pNA molecule is liberated and it has a
yellow colour and thus can be measured by visible spectrophotometry
at wavelength 405 nm. The Suc-AAPF-PNA substrate is manufactured by
Bachem (cat. no. L1400, dissolved in DMSO).
[0190] The protease sample to be analyzed was diluted in residual
activity buffer (100 mM Tris pH 8.6). The assay was performed by
transferring 30 .mu.l of diluted enzyme samples to 96 well
microtiter plate and adding 70 .mu.l substrate working solution
(0.72 mg/ml in 100 mM Tris pH8.6). The solution was mixed at room
temperature and absorption is measured every 20 sec. over 5 minutes
at OD 405 nm. The slope (absorbance per minute) of the time
dependent absorption-curve is directly proportional to the activity
of the protease in question under the given set of conditions. The
protease sample should be diluted to a level where the slope is
linear.
Calculation of Ki
[0191] It is assumed that protease and inhibitor react according to
the equation:
E+IEI
[0192] Where E is the protease, I is the inhibitor and EI is the
protease-inhibitor complex. It is furthermore assumed that
equilibrium between protease, inhibitor and protease-inhibitor
complex is reached during the 1 hour incubation in Model B, and
that addition of substrate solution does not result in significant
alteration of this equilibrium during the measuring time used for
linear regression. The concentration of enzyme-inhibitor in the
detergent solution is then given by:
[ EI ] = ( ( [ E tot ] + [ I tot ] + K i - sqrt ( ( [ E tot ] + [ I
tot ] + K i ) 2 - 4 * [ E tot ] * [ I tot ] ) ) / 2 [ EI ] = [ E
tot ] + [ I tot ] + K i - ( [ E tot ] + [ I tot ] + K i ) 2 - 4 * [
E tot ] * [ I tot ] 2 ##EQU00002##
[0193] where E.sub.tot is the total protease concentration
([E.sub.tot]=[E]+[EI]), Itot is the total inhibitor concentration
([I.sub.tot]=[I]+[EI]), and K.sub.i is the equilibrium binding
constant for the reaction. The measured slopes V are given by:
V = V 0 * ( 1 - [ EI ] / [ E tot ] ) = V 0 * ( 1 - ( ( [ E tot ] +
[ I tot ] + K i - sqrt ( ( [ E tot ] + [ I tot ] + K i ) 2 - 4 * [
E tot ] * [ I tot ] ) ) / 2 [ E tot ] ) V = V 0 * ( 1 - [ EI ] [ E
tot ] ) = V 0 * ( 1 - [ E tot ] + [ I tot ] + K i - ( [ E tot ] + [
I tot ] + K i ) 2 - 4 * [ E tot ] * [ I tot ] 2 * [ E tot ] )
##EQU00003##
[0194] where V.sub.0 is the slope without inhibitor added. To
calculate apparent binding constant K.sub.i least square fitting of
measured slopes at various inhibitor concentrations to this
equation is performed with K.sub.i and V.sub.0 as variables.
[0195] The K.sub.i for the parent protease and for each variant was
calculated as described above
[0196] Having the Ki for each variant, it is possible to calculate
the effect of a single mutation, if data are available for at least
two variants that differ only by that mutation. In this case, the
relative Ki for the mutation (Improvement Factor IF) is
calculated:
Improvement Factor ( IF ) = Ki variant without mutation Ki variant
with mutation ##EQU00004##
A lower K.sub.i is desirable, as a satisfactory inhibition can be
obtained at a lower inhibitor concentration, so beneficial
mutations have an IF>1.0.
Detergent
TABLE-US-00001 [0197] TABLE 1 Model B composition Ingredient wt %
Laundry liquid (C10-C13)alkylbenzene sulfonic acid 7.2 Model B
sodium lauryl ether sulfate 10.6 detergent cocoa fatty acid 2.75
soy fatty acid 2.75 alcohol ethoxylate with 6.6 8 mol EO sodium
hydroxide 1.1 Ethanol 3 propane-1,2-diol 6 Glycerol 1.7
Triethanolamine 3.3 sodium formiate 1 sodium citrate 2
diethylenetriaminepentakis(methylene)- 0.5 pentakis(phosphonic
acid), heptasodium salt copoly(acrylic acid/maleic acid), sodium
salt 0.5 deionized water ad 100
Example 1: Construction of Variants by Site-Directed
Mutagenesis
[0198] Site-directed variants were constructed of the relevant back
bone e.g. SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6, comprising
specific substitutions according to the invention. The variants
were made by traditional cloning of 30 DNA fragments (Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring
Harbor, 1989) using PCR together with properly designed mutagenic
oligonucleotides that introduced the desired mutations in the
resulting sequence.
[0199] Mutagenic oligos were designed corresponding to the DNA
sequence flanking the desired site(s) of mutation, separated by the
DNA base pairs defining the insertions/deletions/substitutions, and
purchased from an oligo vendor such as Life Technologies. In this
manner, the variants listed in Table 2, Table 3 and Table 4 below
were constructed and produced.
[0200] In order to test the protease variants of the invention, the
mutated DNA comprising a variant of the invention were transformed
into a competent B. subtilis strain and fermented using standard
protocols (liquid media, 3-4 days, 30.degree. C.). The culture
broth was centrifuged (26000.times.g, 20 5 min) and the supernatant
was carefully decanted from the precipitate. The supernatant was
filtered through a Nalgene 0.2 .mu.m filtration unit in order to
remove the rest of the Bacillus host cells.
TABLE-US-00002 TABLE 2 Variants of SEQ ID NO 3 Mutations compared
to SEQ ID NO 3 Q70F Q70A Q70N S111R S111E S111D S114A S144R A145E
I150N I150S G182A L184F L184Y Y240R S173P + S175P Q70N + S173P +
S175P S114V + S173P + S175P I150L + S173P + S175P S114V + I150L +
S173P + S175P S173P + S175P + F180Y Q70H + S173P + S175P + F180Y
Q70Y + S173P + S175P + F180Y Q70F + S173P + S175P + F180Y I150T +
S173P + S175P + F180Y I150A + S173P + S175P + F180Y I150S + S173P +
S175P + F180Y I150N + S173P + S175P + F180Y I150V + S173P + S175P +
F180Y K146T + S173P + S175P + F180Y K146S + S173P + S175P + F180Y
K146N + S173P + S175P + F180Y K146W + S173P + S175P + F180Y K146F +
S173P + S175P + F180Y K146A + S173P + S175P + F180Y S114A + S173P +
S175P + F180Y S114Q + S173P + S175P + F180Y S114F + S173P + S175P +
F180Y Q70A + S173P + S175P + F180Y S173P + S175P + F180Y + L184F
S173P + S175P + F180Y + G182A S173P + S175P + I178Y + F180Y S173P +
S175P + I178F + F180Y S173P + S175P + I178P + F180Y K146R + S173P +
S175P + F180Y S173P + S175P + F180Y + L184F S173P + S175P + F180Y +
L184Y S173P + S175P + F180Y + G182A S173P + S175P + I178F + F180Y
S173P + S175P + F180Y + L184W S111I + S173P + S175P + F180Y S173P +
S175P + F180Y + G182S S173P + S175P + F180Y + L184D S173P + S175P +
F180Y + L184H
TABLE-US-00003 TABLE 3 Variants of SEQ ID NO 5 Mutations compared
to SEQ ID NO 5 Q64N Q64F M103S M103A G128P G127S + G128P + G129S +
Y130P + D131S + M134L
TABLE-US-00004 TABLE 4 Variants of SEQ ID NO 6 Mutations compared
to SEQ ID NO 6 N60A N60F N60H S100I V103A L134V A164S Y166F N60H +
S100I N60F + S100I S100I + L134V S100I + Y166F
Example 2: Purification Protease Variants of SEQ ID NO 3
[0201] The 0.2 .mu.m filtrate was mixed 1:1 with 3.0M (NH4)2SO4 and
the mixture was applied to a Phenyl-sepharose FF (high sub) column
(from GE Healthcare) equilibrated in 100 mM H3BO3, 10 mM MES/NaOH,
2 mM CaCl2, 1.5M (NH4)2SO4, pH 6.0. After washing the column with
the equilibration buffer, the protease was step-eluted with 100 mM
H3BO3, 10 mM MES, 2 mM CaCl2, pH 6.0. The eluted peak (containing
the protease activity) was collected and applied to a Bacitracin
agarose column (from Upfront chromatography) equilibrated in 100 mM
H3BO3, 10 mM MES, 2 mM CaCl2, pH 6.0. After washing the column
extensively with the equilibration buffer, the protease was eluted
with 100 mM H3BO3, 10 mM MES, 2 mM CaCl2, 1M NaCl, pH 6.0 with 25%
(v/v) 2-propanol. The elution peak (containing the protease
activity) was transferred to 20 mM MES, 2 mM CaCl2, pH 6.0 on a G25
sephadex column (from GE Healthcare). The G25 transferred peak was
the purified preparation and was used for further experiments.
Example 3: Purification of Protease Variants of SEQ ID NO 5
[0202] Solid ammonium sulphate was added to the 0.2 .mu.m filtrate
to a final ammonium sulfate concentration of 1.5M
(NH.sub.4).sub.2SO.sub.4. The solution became slightly turbid and
was again filtered through a Nalgene 0.2 .mu.m filtration unit. The
clear filtrate was applied to a Phenyl-sepharose FF (high sub)
column (from GE Healthcare) equilibrated in 20 mM HEPES, 2 mM
CaCl.sub.2, 1.5M (NH.sub.4).sub.2SO.sub.4, pH 7.0. After washing
the column extensively with the equilibration buffer, the protease
was eluted with a linear gradient over three column volumes between
the equilibration buffer and 20 mM HEPES, 2 mM CaCl.sub.2, pH 7.0
with 25% (v/v) 2-propanol. Fractions from the column were analysed
for protease activity (using the Suc-AAPF-pNA assay at pH 9). The
protease peak was pooled and the pool was transferred to 100 mM
H.sub.3BO.sub.3, 10 mM MES, 2 mM CaCl.sub.2, pH 6.0 on a G25
Sephadex column (from GE Healthcare). The G25 sephadex transferred
enzyme was applied to a SP-sepharose FF column (from GE Healthcare)
equilibrated in 100 mM H.sub.3BO.sub.3, 10 mM MES, 2 mM CaCl.sub.2,
pH 6.0. After washing the column extensively with the equilibration
buffer, the protease was eluted with a linear NaCl gradient
(0-->0.5M) in the same buffer over five column volumes.
Fractions from the column were analysed for protease activity
(using the Suc-AAPF-pNA assay at pH 9) and active fractions were
further analysed by SDS-PAGE. Fractions, where only one band was
seen on the coomassie stained SDS-PAGE gel, were pooled as the
purified preparation and was used for further characterization.
Example 4: Purification of Protease Variants of SEQ ID NO: 6
[0203] The pH in the 0.2 .mu.m filtrate was adjusted to pH 8 with 3
M Tris base and the pH adjusted filtrate was applied to a MEP
Hypercel column (Pall Corporation) equilibrated in 20 mM Tris/HCl,
1 mM CaCl2, pH 8.0. After washing the column with the equilibration
buffer, the column was step-eluted with 20 mM CH3COOH/NaOH, 1 mM
CaCl2, pH 4.5. Fractions from the column were analyzed for protease
activity using the Suc-AAPF-pNA assay at pH 9 and peak-fractions
were pooled. The pH of the pool from the MEP Hypercel column was
adjusted to pH 6 with 20% (v/v) CH3COOH or 3 M Tris base and the pH
adjusted pool was diluted with deionized water to the same
conductivity as 20 mM MES/NaOH, 2 mM CaCl2, pH 6.0. The diluted
pool was applied to a SP-Sepharose.RTM. Fast Flow column (GE
Healthcare) equilibrated in 20 mM MES/NaOH, 2 mM CaCl2, pH 6.0.
After washing the column with the equilibration buffer, the
protease variant was eluted with a linear NaCl gradient (0>0.5
M) in the same buffer over five column volumes. Fractions from the
column were analyzed for protease activity using the Suc-AAPF-pNA
assay at pH 9 and active fractions were analyzed by SDS-PAGE.
Fractions, where only one band was observed on the Coomassie
stained SDS-PAGE gel, were pooled as the purified preparation and
was used for further experiments.
Example 5: Inhibitor Binding Assay of SEQ ID NO 3 Variants
[0204] Purified protease variants are pre-diluted to approximately
0.2 mg/ml in dilution buffer. The experimental conditions are
described in Table 5. 40 .mu.l diluted protease is then mixed with
40 .mu.l inhibitor solution Inhibitor 1:
(Cbz-Gly-Ala-NHCH(CH.sub.2C.sub.6H.sub.4pOH)C(OH)(SO.sub.3Na)--H,
wherein Cbz is benzyloxycarbonyl) or 40 .mu.l Inhibitor 2: (4-FPBA)
solution in the well of a 96 well microtiter plate (Nunc F 96-MTP).
For each protease variant 8 concentrations of Inhibitor 1 solutions
(600 .mu.M, 200 .mu.M, 67 .mu.M, 22 .mu.M, 7.4 .mu.M, 2.47 .mu.M,
0.82 .mu.M and 0 .mu.M) and 8 concentrations of inhibitor 2 (120
mM, 40 mM, 13.3 mM, 4.44 mM, 1.48 mM, 0.494 mM, 0.164 mM and 0 mM)
are tested. After 10 min mixing of protease and inhibitor at room
temperature, 30 .mu.l of the mixture is transferred to a microtiter
plate (Nunc U96 PP 0.5 ml) with 270 .mu.l Model B detergent
(described in Table 1) in the wells resulting in inhibitor 1
concentrations of 0-30 .mu.M and inhibitor 2 concentrations of
0-6000 .mu.M. Protease, inhibitor and detergent is mixed for 1 hour
using magnetic bars for 1 hour at room temperature to reach
equilibrium. Then 20 .mu.l is transferred to a microtiter plate
(Nunc F 96-MTP). 100 .mu.l substrate solution is added and after 5
sec mixing absorbance at 405 nm is measured every 20 sec for 5 min
on a SpectraMax Plus reader. Slope from linear regression of
initial increase in absorbance at 405 nm is used for calculation of
apparent binding constants. Ki values were calculated according to
the "Calculation of Ki" method described in "Materials and
Methods"
TABLE-US-00005 TABLE 5 Experimental Conditions Dilution buffer
0.01% Triton X-100 Detergent Laundry liquid Model B detergent
(Table 1) Substrate buffer 100 mM Tris, pH 8.6 Substrate stock 100
mg/ml Suc-Ala-Ala-Pro-Phe-pNA (Bachem L-1400) in DMSO Substrate
solution 7 .mu.l/ml substrate stock solution in substrate buffer
Inhibitor 1 stock Inhibitor (414 mM) Inhibitor 1 working Inhibitor
1 stock diluted to 600 .mu.M, 200 .mu.M, solutions 67 .mu.M, 22
.mu.M, 7.4 .mu.M, 2.47 .mu.M, 0.82 .mu.M and 0 .mu.M with dilution
buffer Inhibitor 2 stock 4-FPBA (1801 mM) stock solution Inhibitor
2 working inhibitor 2 stock solution diluted to 120 mM, solutions
40 mM, 13.3 mM, 4.44 mM, 1.48 mM, 0.494 mM, 0.164 mM and 0 mM with
dilution buffer
[0205] Calculation of Improvement Factor (IF)
[0206] Having the Ki for each variant, it is possible to calculate
the effect of a single mutation, if data are available for at least
two variants that differ only by that mutation. In this case, the
relative Ki for the mutation (Improvement Factor, or IF) is
calculated:
Improvement Factor ( IF ) = Ki variant without mutation Ki variant
with mutation ##EQU00005##
[0207] A lower K.sub.i is desirable, as a satisfactory inhibition
can be obtained at a lower inhibitor concentration, so beneficial
mutations have an IF>1.0
TABLE-US-00006 TABLE 6 Inhibitor 1 binding constant improvement
factors (IF) in Model B relative to SEQ ID NO 3 Mutations compared
to IF SEQ ID NO 3 (inhibitor 1) SEQ ID NO 3 1.00 Y240R 1.19 S111R
1.11 Q70F 8.53 Q70A 2.54 Q70N 3.33 S114A 2.14 I150N 3.68 I150S 4.94
G182A 1.86 L184F 2.27 L184Y 4.47
TABLE-US-00007 TABLE 7 inhibitor 2 binding constant improvement
factors (IF) in Model B relative to SEQ ID NO 3 Mutations compared
to SEQ ID NO 3 IF Inhibitor 2 SEQ ID NO 3 1.00 S111E 1.23 S111D
1.53 A145E 1.03 S144R 2.03 Q70N 3.59 S114A 1.40 L184F 1.39
TABLE-US-00008 TABLE 8 Inhibitor 1 binding constant improvement
factors (IF) in Model B relative to SEQ ID NO 3 comprising S173P +
S175P. Mutations compared to SEQ ID NO 3 IF (inhibitor 1) S173P +
S175P 1.00 Q70N + S173P + S175P 4.48 S114V + S173P + S175P 1.83
I150L + S173P + S175P 2.05 S114V + I150L + S173P + S175P 6.14
TABLE-US-00009 TABLE 9 inhibitor 1 binding constant improvement
factors (IF) in Model B relative to SEQ ID NO 3 comprising S173P +
S175P + F180Y Mutations compared to IF SEQ ID NO 3 (inhibitor 1)
S173P + S175P + F180Y 1.00 Q70H + S173P + S175P + F180Y 3.78 Q70Y +
S173P + S175P + F180Y 7.44 Q70F + S173P + S175P + F180Y 11.76 I150T
+ S173P + S175P + F180Y 4.15 I150A + S173P + S175P + F180Y 2.52
I150S + S173P + S175P + F180Y 5.70 I150N + S173P + S175P + F180Y
3.58 I150V + S173P + S175P + F180Y 1.80 K146T + S173P + S175P +
F180Y 15.91 K146S + S173P + S175P + F180Y 1.13 K146N + S173P +
S175P + F180Y 1.36 K146W + S173P + S175P + F180Y 5.47 K146F + S173P
+ S175P + F180Y 5.17 K146A + S173P + S175P + F180Y 3.72 S114A +
S173P + S175P + F180Y 3.33 S114Q + S173P + S175P + F180Y 2.03 S114F
+ S173P + S175P + F180Y 1.41 Q70A + S173P + S175P + F180Y 4.13
S173P + S175P + F180Y + L184F 4.28 S173P + S175P + F180Y + G182A
2.49 S173P + S175P + I178Y + F180Y 2.15 S173P + S175P + I178F +
F180Y 2.34 S173P + S175P + I178P + F180Y 2.22 K146R + S173P + S175P
+ F180Y 1.31 S173P + S175P + F180Y + L184F 3.02 S173P + S175P +
F180Y + L184Y 3.91 S173P + S175P + F180Y + G182A 2.01 S173P + S175P
+ I178F + F180Y 1.83 S173P + S175P + F180Y + L184W 2.23 S111I +
S173P + S175P + F180Y 1.12 S173P + S175P + F180Y + G182S 1.69 S173P
+ S175P + F180Y + L184D 3.28 S173P + S175P + F180Y + L184H 2.50
Example 6: Inhibitor Binding Assay of SEQ ID NO 5 Variants
[0208] Purified protease variants are pre-diluted to approximately
0.2 mg/ml in dilution buffer. The experimental conditions are
described in Table 5. 40 .mu.l diluted protease is then mixed with
40 .mu.l inhibitor solution Inhibitor 1 or 40 .mu.l Inhibitor 2
solution in the well of a 96 well microtiter plate (Nunc F 96-MTP).
For each protease variant 8 concentrations of inhibitor 1 solutions
(600 .mu.M, 200 .mu.M, 67 .mu.M, 22 .mu.M, 7.4 .mu.M, 2.47 .mu.M,
0.82 .mu.M and 0 .mu.M) and 8 concentrations of Inhibitor 2 (120
mM, 40 mM, 13.3 mM, 4.44 mM, 1.48 mM, 0.494 mM, 0.164 mM and 0 mM)
are tested. After 10 min mixing of protease and inhibitor at room
temperature, 30 .mu.l of the mixture is transferred to a microtiter
plate (Nunc U96 PP 0.5 ml) with 270 .mu.l Model B detergent
(described in Table 1) in the wells resulting in inhibitor 1
concentrations of 0-30 .mu.M and inhibitor 2 concentrations of
0-6000 .mu.M. Protease, inhibitor and detergent is mixed for 1 hour
using magnetic bars for 1 hour at room temperature to reach
equilibrium. Then 20 .mu.l is transferred to a microtiter plate
(Nunc F 96-MTP). 100 .mu.l substrate solution is added and after 5
sec mixing absorbance at 405 nm is measured every 20 sec for 5 min
on a SpectraMax Plus reader. Slope from linear regression of
initial increase in absorbance at 405 nm is used for calculation of
apparent binding constants. Ki values were calculated according to
the "Calculation of Ki" method described in "Materials and
Methods"
[0209] Calculation of Improvement Factor (IF)
[0210] Having the Ki for each variant, it is possible to calculate
the effect of a single mutation, if data are available for at least
two variants that differ only by that mutation. In this case, the
relative Ki for the mutation (Improvement Factor, or IF) is
calculated:
Improvement Factor ( IF ) = Ki variant without mutation Ki variant
with mutation ##EQU00006##
[0211] A lower K.sub.i is desirable, as a satisfactory inhibition
can be obtained at a lower inhibitor concentration, so beneficial
mutations have an IF>1.0
TABLE-US-00010 TABLE 10 inhibitor 1 binding constant improvement
factors (IF) in Model B relative to SEQ ID NO 5 Mutations compared
to SEQ ID NO 5 IF (inhibitor 1) SEQ ID NO 5 1.00 Q64N 1.38 Q64F
3.44 M103S 1.73 M103A 2.31 G128P 1.19 G127S + G128P + G129S + 1.05
Y130P + D131S + M134L
TABLE-US-00011 TABLE 11 inhibitor 2 binding constant improvement
factors (IF) in Model B relative to SEQ ID NO 5 Mutations compared
to SEQ ID NO 5 IF (Inhibitor 2) SEQ ID NO 5 1.00 Q64N 1.24 G128P
1.54 G127S + G128P + G129S + 1.37 Y130P + D131S + M134L
Example 7: Inhibitor Binding Assay of SEQ ID NO 6 Variants
[0212] Purified protease variants are pre-diluted to approximately
0.2 mg/ml in dilution buffer. The experimental conditions are
described in Table 5 40 .mu.l diluted protease is then mixed with
40 .mu.l inhibitor solution Inhibitor 1 or 40 .mu.l inhibitor 2
solution in the well of a 96 well microtiter plate (Nunc F 96-MTP).
For each protease variant 8 concentrations of inhibitor 1 solutions
(600 .mu.M, 200 .mu.M, 67 .mu.M, 22 .mu.M, 7.4 .mu.M, 2.47 .mu.M,
0.82 .mu.M and 0 .mu.M) and 8 concentrations of inhibitor 2 (120
mM, 40 mM, 13.3 mM, 4.44 mM, 1.48 mM, 0.494 mM, 0.164 mM and 0 mM)
are tested. After 10 min mixing of protease and inhibitor at room
temperature, 30 .mu.l of the mixture is transferred to a microtiter
plate (Nunc U96 PP 0.5 ml) with 270 .mu.l Model B detergent
(described in Table 1) in the wells resulting in inhibitor 1
concentrations of 0-30 .mu.M and inhibitor 2 concentrations of
0-6000 .mu.M. Protease, inhibitor and detergent is mixed for 1 hour
using magnetic bars for 1 hour at room temperature to reach
equilibrium. Then 20 .mu.l is transferred to a microtiter plate
(Nunc F 96-MTP). 100 .mu.l substrate solution is added and after 5
sec mixing absorbance at 405 nm is measured every 20 sec for 5 min
on a SpectraMax Plus reader. Slope from linear regression of
initial increase in absorbance at 405 nm is used for calculation of
apparent binding constants. Ki values were calculated according to
the "Calculation of Ki" method described in "Materials and
Methods"
[0213] Calculation of Improvement Factor (IF)
[0214] Having the Ki for each variant, it is possible to calculate
the effect of a single mutation, if data are available for at least
two variants that differ only by that mutation. In this case, the
relative Ki for the mutation (Improvement Factor, or IF) is
calculated:
Improvement Factor ( IF ) = Ki variant without mutation Ki variant
with mutation ##EQU00007##
[0215] A lower K.sub.i is desirable, as a satisfactory inhibition
can be obtained at a lower inhibitor concentration, so beneficial
mutations have an IF>1.0
TABLE-US-00012 TABLE 12 inhibitor 1 binding constant improvement
factors (IF) in Model B relative to SEQ ID NO 6 Mutations compared
to IF SEQ ID NO 6 (inhibitor 1) SEQ ID NO 6 1.00 N60A 1.22 N60F
3.25 N60H 1.24 S100I 6.46 V103A 1.49 L134V 2.89 Y166F 1.31 N60H +
S100I 6.65 N60F + S100I 11.26 S100I + L134V 15.14 S100I + Y166F
11.26
TABLE-US-00013 TABLE 14 inhibitor 2 binding constant improvement
factors (IF) in Model B relative to SEQ ID NO 6 Mutations compared
to IF SEQ ID NO 6 (inhibitor 2) SEQ ID NO 6 1.00 N60A 1.74 V103A
1.86 A164S 1.84 Y166F 1.62 S100I + L134V 1.1 S100I + Y166F 2.31
Sequence CWU 1
1
61269PRTBacillus lentus 1Ala Gln Ser Val Pro Trp Gly Ile Ser Arg
Val Gln Ala Pro Ala Ala 1 5 10 15 His Asn Arg Gly Leu Thr Gly Ser
Gly Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Ser Thr His
Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 35 40 45 Phe Val Pro Gly
Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 50 55 60 His Val
Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 65 70 75 80
Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 85
90 95 Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp
Ala 100 105 110 Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly
Ser Pro Ser 115 120 125 Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser
Ala Thr Ser Arg Gly 130 135 140 Val Leu Val Val Ala Ala Ser Gly Asn
Ser Gly Ala Gly Ser Ile Ser 145 150 155 160 Tyr Pro Ala Arg Tyr Ala
Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175 Asn Asn Asn Arg
Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 180 185 190 Val Ala
Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 195 200 205
Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 210
215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln
Ile 225 230 235 240 Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly
Ser Thr Asn Leu 245 250 255 Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala
Ala Thr Arg 260 265 2275PRTBacillus amyloliquefaciens 2Ala Gln Ser
Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His
Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25
30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala
35 40 45 Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn
Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn
Asn Ser Ile Gly 65 70 75 80 Val Leu Gly Val Ala Pro Ser Ala Ser Leu
Tyr Ala Val Lys Val Leu 85 90 95 Gly Ala Asp Gly Ser Gly Gln Tyr
Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ala Asn Asn
Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro Ser Gly Ser
Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala 130 135 140 Ser Gly
Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly 145 150 155
160 Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala
165 170 175 Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser
Ser Val 180 185 190 Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser
Ile Gln Ser Thr 195 200 205 Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn
Gly Thr Ser Met Ala Ser 210 215 220 Pro His Val Ala Gly Ala Ala Ala
Leu Ile Leu Ser Lys His Pro Asn 225 230 235 240 Trp Thr Asn Thr Gln
Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys 245 250 255 Leu Gly Asp
Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala
Ala Gln 275 3311PRTBacillus sp. 3Ala Val Pro Ser Thr Gln Thr Pro
Trp Gly Ile Lys Ser Ile Tyr Asn 1 5 10 15 Asp Gln Ser Ile Thr Lys
Thr Thr Gly Gly Ser Gly Ile Lys Val Ala 20 25 30 Val Leu Asp Thr
Gly Val Tyr Thr Ser His Leu Asp Leu Ala Gly Ser 35 40 45 Ala Glu
Gln Cys Lys Asp Phe Thr Gln Ser Asn Pro Leu Val Asp Gly 50 55 60
Ser Cys Thr Asp Arg Gln Gly His Gly Thr His Val Ala Gly Thr Val 65
70 75 80 Leu Ala His Gly Gly Ser Asn Gly Gln Gly Val Tyr Gly Val
Ala Pro 85 90 95 Gln Ala Lys Leu Trp Ala Tyr Lys Val Leu Gly Asp
Asn Gly Ser Gly 100 105 110 Tyr Ser Asp Asp Ile Ala Ala Ala Ile Arg
His Val Ala Asp Glu Ala 115 120 125 Ser Arg Thr Gly Ser Lys Val Val
Ile Asn Met Ser Leu Gly Ser Ser 130 135 140 Ala Lys Asp Ser Leu Ile
Ala Ser Ala Val Asp Tyr Ala Tyr Gly Lys 145 150 155 160 Gly Val Leu
Ile Val Ala Ala Ala Gly Asn Ser Gly Ser Gly Ser Asn 165 170 175 Thr
Ile Gly Phe Pro Gly Gly Leu Val Asn Ala Val Ala Val Ala Ala 180 185
190 Leu Glu Asn Val Gln Gln Asn Gly Thr Tyr Arg Val Ala Asp Phe Ser
195 200 205 Ser Arg Gly Asn Pro Ala Thr Ala Gly Asp Tyr Ile Ile Gln
Glu Arg 210 215 220 Asp Ile Glu Val Ser Ala Pro Gly Ala Ser Val Glu
Ser Thr Trp Tyr 225 230 235 240 Thr Gly Gly Tyr Asn Thr Ile Ser Gly
Thr Ser Met Ala Thr Pro His 245 250 255 Val Ala Gly Leu Ala Ala Lys
Ile Trp Ser Ala Asn Thr Ser Leu Ser 260 265 270 His Ser Gln Leu Arg
Thr Glu Leu Gln Asn Arg Ala Lys Val Tyr Asp 275 280 285 Ile Lys Gly
Gly Ile Gly Ala Gly Thr Gly Asp Asp Tyr Ala Ser Gly 290 295 300 Phe
Gly Tyr Pro Arg Val Lys 305 310 4269PRTbacillius lentus 4Ala Gln
Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 1 5 10 15
His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 20
25 30 Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala
Ser 35 40 45 Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly
His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn
Ser Ile Gly Val Leu 65 70 75 80 Gly Val Ala Pro Ser Ala Glu Leu Tyr
Ala Val Lys Val Leu Gly Ala 85 90 95 Asp Gly Arg Gly Ala Ile Ser
Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 Gly Asn Asn Gly Met
His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125 Pro Ser Ala
Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 130 135 140 Val
Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Ser Ser Ile Ser 145 150
155 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp
Gln 165 170 175 Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly
Leu Asp Ile 180 185 190 Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr
Pro Gly Ser Thr Tyr 195 200 205 Ala Ser Leu Asn Gly Thr Ser Met Ala
Thr Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys
Asn Pro Ser Trp Ser Asn Val Gln Ile 225 230 235 240 Arg Asn His Leu
Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 245 250 255 Tyr Gly
Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265 5273PRTBacillus
sp. 5Met His Asn Asn Gln Arg Trp His Tyr Glu Met Ile Asn Ala Pro
Gln 1 5 10 15 Ala Trp Gly Ile Thr Thr Gly Ser Ser Asn Val Arg Ile
Ala Val Leu 20 25 30 Asp Thr Gly Ile Asp Ala Asn His Pro Asn Leu
Arg Asn Leu Val Asp 35 40 45 Thr Ser Leu Gly Arg Ser Phe Val Gly
Gly Gly Thr Gly Asp Val Gln 50 55 60 Gly His Gly Thr His Val Ala
Gly Thr Ile Ala Ser Tyr Gly Ser Val 65 70 75 80 Ser Gly Val Met Gln
Asn Ala Arg Leu Ile Pro Val Lys Val Leu Gly 85 90 95 Asp Asn Gly
Ser Gly Ser Met Tyr Gly Ile Gln Gln Gly Ile Leu Tyr 100 105 110 Ala
Ala Ser Ile Asn Ala Asp Val Ile Asn Met Ser Leu Gly Gly Gly 115 120
125 Gly Tyr Asp Ser Gly Met Asn Asn Ala Ile Asn Thr Ala Val Ser Ser
130 135 140 Gly Thr Leu Val Ile Ala Ala Ser Gly Asn Asp Gly Arg Gly
Ser Ile 145 150 155 160 Ser Tyr Pro Ala Ala Tyr Ser Asn Ala Ile Ala
Val Gly Ser Val Thr 165 170 175 Ser Asn Arg Thr Arg Ser Asn Phe Ser
Asn Tyr Gly Ser Gly Leu Glu 180 185 190 Leu Met Ala Pro Gly Ser Asn
Ile Tyr Ser Thr Tyr Pro Asn Gly Gln 195 200 205 Phe Arg Thr Leu Ser
Gly Thr Ser Met Ala Thr Pro His Val Ala Gly 210 215 220 Val Ala Gly
Leu Ile Lys Ser Ala Asn Pro Asn Leu Ser Val Thr Gln 225 230 235 240
Val Arg Asn Ile Leu Arg Asp Thr Ala Gln Tyr Ala Gly Ser Ser Asn 245
250 255 Gln Tyr Gly Tyr Gly Ile Val Asn Ala Tyr Ala Ala Val Gln Ala
Ala 260 265 270 Gly 6270PRTArtificialProtease C 6Ala Gln Ser Val
Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 1 5 10 15 His Asn
Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 20 25 30
Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 35
40 45 Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly
Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile
Gly Val Leu 65 70 75 80 Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val
Lys Val Leu Gly Ala 85 90 95 Ser Glu Gly Ser Gly Ser Val Ser Ser
Ile Ala Gln Gly Leu Glu Trp 100 105 110 Ala Gly Asn Asn Gly Met His
Val Ala Asn Leu Ser Leu Gly Ser Pro 115 120 125 Ser Pro Ser Ala Thr
Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg 130 135 140 Gly Val Leu
Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile 145 150 155 160
Ser Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp 165
170 175 Gln Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu
Asp 180 185 190 Ile Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro
Gly Ser Thr 195 200 205 Tyr Ala Ser Leu Asn Gly Thr Ser Met Ala Thr
Pro His Val Ala Gly 210 215 220 Ala Ala Ala Leu Val Lys Gln Lys Asn
Pro Ser Trp Ser Asn Val Gln 225 230 235 240 Ile Arg Asn His Leu Lys
Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn 245 250 255 Leu Tyr Gly Ser
Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265 270
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