U.S. patent application number 12/799639 was filed with the patent office on 2013-03-14 for novel fungal protease and use thereof.
This patent application is currently assigned to AB Enzymes Oy. The applicant listed for this patent is Kari Juntunen, Jarno Kallio, Susanna Makinen, Pentti Ojapalo, Marja Paloheimo, Leena Valtakari, Jari Vehmaanpera. Invention is credited to Kari Juntunen, Jarno Kallio, Susanna Makinen, Pentti Ojapalo, Marja Paloheimo, Leena Valtakari, Jari Vehmaanpera.
Application Number | 20130065810 12/799639 |
Document ID | / |
Family ID | 40590374 |
Filed Date | 2013-03-14 |
United States Patent
Application |
20130065810 |
Kind Code |
A2 |
Juntunen; Kari ; et
al. |
March 14, 2013 |
Novel Fungal Protease and Use Thereof
Abstract
The present invention is related to a fungal serine protease
enzyme, which comprises an amino acid sequence of the mature
Fe_RF6318 enzyme having an amino acid sequence of SEQ ID NO: 15.
The serine protease is obtainable from Fusarium equiseti, more
preferably from the deposited strain CBS 119568. Also disclosed are
nucleic acid sequences encoding said protease, such as plasmid
pALK2521 comprising the nucleotide sequence SEQ ID NO:9 deposited
in E. coli RF7664 under accession number DSM 22171 and plasmid
pALK2529 comprising the full-length gene SEQ ID NO: 10 deposited in
E. coli RF7800 under accession number DSM 22172. Said protease is
useful as an enzyme preparation applicable in detergent
compositions and for treating fibers, for treating wool, for
treating hair, for treating leather, for treating food or feed, or
for any applications involving modification, degradation or removal
of proteinaceous material.
Inventors: |
Juntunen; Kari; (Espoo,
FI) ; Valtakari; Leena; (Rajamaki, FI) ;
Makinen; Susanna; (Layliainen, FI) ; Kallio;
Jarno; (Jarvenpaa, FI) ; Vehmaanpera; Jari;
(Klaukkala, FI) ; Ojapalo; Pentti; (Tuusula,
FI) ; Paloheimo; Marja; (Vantaa, FI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Juntunen; Kari
Valtakari; Leena
Makinen; Susanna
Kallio; Jarno
Vehmaanpera; Jari
Ojapalo; Pentti
Paloheimo; Marja |
Espoo
Rajamaki
Layliainen
Jarvenpaa
Klaukkala
Tuusula
Vantaa |
|
FI
FI
FI
FI
FI
FI
FI |
|
|
Assignee: |
AB Enzymes Oy
Rajamaki
FI
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20110003729 A1 |
January 6, 2011 |
|
|
Family ID: |
40590374 |
Appl. No.: |
12/799639 |
Filed: |
April 28, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61214975 |
Apr 30, 2009 |
|
|
|
Current U.S.
Class: |
510/321 ;
435/223; 435/225; 435/254.1; 435/256.1; 435/320.1; 435/71.1;
510/320; 536/22.1 |
Current CPC
Class: |
C11D 3/38681 20130101;
C12N 9/58 20130101; C11D 3/386 20130101 |
Class at
Publication: |
510/321 ;
435/223; 435/225; 536/22.1; 435/320.1; 435/254.1; 435/71.1;
435/256.1; 510/320 |
International
Class: |
C11D 7/42 20060101
C11D007/42; C12N 9/62 20060101 C12N009/62; A61K 36/06 20060101
A61K036/06; C12N 15/57 20060101 C12N015/57; C12P 21/02 20060101
C12P021/02; C12N 9/58 20060101 C12N009/58; C07H 21/00 20060101
C07H021/00 |
Claims
1. A fungal serine protease enzyme, characterized in that said
enzyme has serine protease activity and comprises an amino acid
sequence of the mature Fe_RF6318 enzyme as defined in SEQ ID NO:15
or an amino acid sequence having at least 86% identity to the amino
acid sequence of the mature Fe_RF6318 enzyme defined in SEQ ID
NO:15.
2. The fungal serine protease enzyme according to claim 1,
characterized in that said enzyme is obtainable from Fusarium
equiseti.
3. The fungal serine protease enzyme according to claim 1 or 2,
characterized in that said enzyme has serine protease activity and
comprises the amino acid sequence SEQ ID NO:15 .
4. The fungal serine protease enzyme according to any of claims 1
to 3, characterized in that a mature form of said enzyme has a
molecular mass between 20 and 35 kDa.
5. The fungal serine protease enzyme according to any of claims 1
to 4, characterized in that the optimal temperature of said enzyme
at pH 9 using 15 min reaction time and casein as a substrate is
from 30.degree. C. to 70.degree. C.
6. The fungal serine protease enzyme according to any of claims 1
to 5, characterized in that said enzyme has pH optimum at
50.degree. C. using 15 min reaction time and casein as a substrate
at pH range from at least pH 6 to pH 11.
7. The fungal serine protease enzyme according to any of claims 1
to 6, characterized in that said enzyme is capable in degrading and
removing proteinaceous stains in the presence of detergent between
10.degree. C. and 60.degree. C.
8. The fungal serine protease enzyme according to any of claims 1
to 7, characterized in that said enzyme is encoded by an isolated
polynucleotide sequence, which hybridizes under stringent
conditions with a polynucleotide sequence included in plasmid
pALK2521 comprising the nucleotide sequence SEQ ID NO:9 deposited
in Escherichia coli RF7664 under accession number DSM 22171.
9. The fungal serine protease enzyme according to any of claims 1
to 8, characterized in that said enzyme is encoded by an isolated
polynucleotide sequence, which encodes a polypeptide comprising an
amino acid sequence of the mature Fe_RF6318 enzyme as defined in
SEQ ID NO:15 or an amino acid sequence having at least 86% identity
to the amino acid sequence of the mature Fe_RF6318 defined in SEQ
ID NO:15.
10. The fungal serine protease enzyme according to any of claims 1
to 9, characterized in that said enzyme is encoded by an isolated
nucleic acid molecule, which encodes a polypeptide comprising the
amino acid sequence characterized in SEQ ID NO:15.
11. The fungal serine protease enzyme according to any of claims 1
to 10, characterized in that said enzyme is encoded by an isolated
nucleic acid molecule comprising the nucleotide sequence SEQ ID
NO:14.
12. The fungal serine protease enzyme according to any of claims 1
to 11, characterized in that said enzyme is encoded by the
polynucleotide sequence included in pALK2529 deposited in
Escherichia coli RF7800 under accession number DSM 22172.
13. The fungal serine protease enzyme according to any of claims 1
to 12, characterized in that said enzyme is produced from a
recombinant expression vector comprising the nucleic acid molecule
encoding a fungal serine protease according to any of claims 1 to
12 operably linked to regulatory sequences capable of directing the
expression of the serine protease encoding gene in a suitable
host.
14. The fungal serine protease enzyme according to any of claims 1
to 13, characterized in that said enzyme is produced in a
heterologous host.
15. The fungal serine protease enzyme according to any of claims 1
to 14, characterized in that said enzyme is produced in a microbial
host.
16. The fungal serine protease enzyme according to any of claims 1
to 15, characterized in that said enzyme is produced in a host of
the genus Trichoderma, Aspergillus, Fusarium, Humicola,
Chrysosporium, Neurospora, Rhizopus, Penicillium and
Mortiriella.
17. The fungal serine protease enzyme according to claim 16,
characterized in that said enzyme is produced in Trichoderma or
Aspergillus.
18. The fungal serine protease enzyme according to claim 17,
characterized in that said enzyme is produced in T. reesei.
19. An isolated nucleic acid molecule encoding a fungal serine
protease enzyme selected from the group consisting of: (a) a
nucleic acid molecule encoding a polypeptide having serine protease
activity and comprising the amino acid sequence as depicted in SEQ
ID NO:15; (b) a nucleic acid molecule encoding a polypeptide having
serine protease activity and at least 86% identity to the amino
acid sequence of SEQ ID NO:15; (c) a nucleic acid molecule
comprising the coding sequence of the nucleotide sequence as
depicted in SEQ ID NO: 10; (d) a nucleic acid molecule comprising
the coding sequence of the polynucleotide sequence contained in DSM
22171 or DSM 22172; (e) a nucleic acid molecule the coding sequence
of which differs from the coding sequence of a nucleic acid
molecule of any one of (c) to (d) due to the degeneracy of the
genetic code; and (g) a nucleic acid molecule hybridizing under
stringent conditions with a nucleic acid molecule contained in DSM
22171, and encoding a polypeptide having serine protease activity
and an amino acid sequence which shows at least 86% identity to the
amino acid sequence as depicted in SEQ ID NO:15.
20. A recombinant expression vector comprising the nucleotide
sequence according to claim 19 operably linked to regulatory
sequences capable of directing the expression of said serine
protease encoding gene in a suitable host.
21. A host cell comprising the recombinant expression vector
according to claim 20.
22. The host cell according to claim 21, characterized in that said
host is a microbial host.
23. The host cell according to claim 21 or 22, characterized in
that said host is a filamentous fungus.
24. The host cell according to any of claims 21 to 23,
characterized in that said host is of a genus Trichoderma,
Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora,
Rhizopus, Penicillium and Mortiriella.
25. The host cell according to claim 24, characterized in that said
host is Trichoderma or Aspergillus.
26. The host cell according to claim 25, characterized in that said
host is T. reesei.
27. A process of producing a polypeptide having serine protease
activity, said process comprising the steps of culturing the host
cell according to any of claims 21 to 26 and recovering the
polypeptide.
28. A polypeptide having serine protease activity encoded by the
nucleic acid sequence according to claim 19 and which is obtainable
by the process according to claim 27.
29. A process for obtaining an enzyme preparation comprising the
steps of culturing a host cell according to any one of claims 21 to
26 and either recovering the polypeptide from the cells or
separating the cells from the culture medium and obtaining the
supernatant.
30. An enzyme preparation obtainable by the process according to
claim 29.
31. An enzyme preparation, which comprises the serine protease
enzyme according to any of claims 1 to 18.
32. The enzyme preparation according to claim 30 or 31,
characterized in that said preparation comprises other enzymes
selected from the group of protease, amylase, cellulase, lipase,
xylanase, mannanase, cutinase, pectinase or oxidase with or without
a mediator.
33. The enzyme preparation according to any of claims 30 to 32,
characterized in that said preparation comprises a suitable
additive selected from the group of stabilizers, buffers,
surfactants, builders, bleaching agents, mediators, anti-corrosion
agents, antiredeposition agents, caustics, abrasives, optical
brighteners, dyes, pigments, and preservatives.
34. The enzyme preparation according to any one of claims 30 to 33,
characterized in that said enzyme preparation is in the form of
liquid, powder or granulate.
35. Use of the serine protease enzyme according to any of claims 1
to 18 or the enzyme preparation according to any of claims 30 to 34
for detergents, for treating fibers, for treating wool, for
treating hair, for treating leather, for treating food or feed, or
for any applications involving modification, degradation or removal
of proteinaceous material.
36. Use of the serine protease enzyme according to any of claims 1
to 18 or the enzyme preparation according to any of claims 30 to 34
as a detergent additive.
37. Use according to claim 36 in detergent liquids.
38. Use according to claim 36 in detergent powders.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a fungal serine protease
enzyme useful in various applications, particularly in laundry and
dish-washing detergents. The invention relates to a nucleic acid
molecule encoding said enzyme, a recombinant vector, a host cell
for producing said enzyme, an enzyme composition comprising said
enzyme as well as a process for preparing such composition. This
invention relates also to various uses of said enzyme or
compositions comprising said enzyme.
BACKGROUND
[0002] Microbial proteases are among the most important hydrolytic
enzymes and find applications in various industrial sectors, such
as detergents, food, leather, pharmaceuticals, diagnostics, waste
management and silver recovery. Microbial extracellular proteases
account for a major part, more than one third, of the total
worldwide industrial enzyme sales (Cherry and Fidantsef, 2003).
Approximately 90% of the commercial proteases are detergent enzymes
(Gupta et al., 2002). Most commercial proteases, mainly neutral and
alkaline are produced by organisms belonging to the genus
Bacillus.
[0003] Serine proteases of the subtilisin family or subtilisins
produced by Bacillus species form the largest subgroup of
industrial proteases. These enzymes are commercially important as
protein degrading component or additive of washing detergents. The
commercial detergent preparations currently in use comprise the
naturally occurring alkaline serine proteases originating from
Bacillus species or are recombinant protease preparations (Maurer,
2004). Variants of the natural enzymes with improved catalytic
efficiency and/or better stability towards temperature, oxidizing
agents and changing washing conditions have been developed through
site-directed and/or random mutagenesis. Examples of commercial
proteases are such as subtilisin Carlsberg (Alcalase.RTM.,
Novozymes, D K), subtilisin 309 (Savinase.RTM., Novozymes, D K),
Subtilisin 147 (Esperase.RTM., Novozymes, DK), Kannase.RTM.
(Novozymes, D K), Purafect.RTM. (Genencor Inc., USA), Purafect.RTM.
Ox, Properase.RTM. (Genencor Inc., USA) and the BLAP S and X series
(Henkel, D E).
[0004] Several alkaline serine proteases (EC 3.4.21) and genes
encoding these enzymes have also been isolated from eukaryotic
organisms, including yeast and filamentous fungi. U.S. Pat. No.
3,652,399 and EP 519229 (Takeda Chemical Industries, Ltd., JP)
disclose an alkaline protease from the genus Fusarium (asexual
state, teleomorph) or Gibberella, (sexual state, anamorph)
particularly from Fusarium sp. S-19-5 (ATCC 20192, IFO 8884), F.
oxysporum f. sp. lini (IFO 5880) or G. saubinetti (ATCC 20193,
IFO6608), useful in the formulation of detergent and other cleanser
compositions. WO 88/03946 and WO 89/04361 (Novo Industri A/S, DK)
disclose an enzymatic detergent additive and a detergent
composition comprising a protease and a lipase, wherein the fungal
protease is derived from Fusarium, particularly F. oxysporum or F.
solani. A detergent additive comprising protease with specificity
for peptide bonds adjacent to only one or two specific amino acids
is disclosed in WO89/06270. WO1994025583 (NovoNordisk A/S, DK)
discloses an active trypsin-like protease enzyme derivable from a
Fusarium species, in particular a strain of F. oxysporum (DSM
2672), and the DNA sequence encoding the same.The amino acid
sequence of a novel protease deriving from Fusarium sp. BLB (FERM
BP-10493) is disclosed in WO 2006101140 (SODX Co. Ltd, Nakamura).
Also, alkaline proteases from fungal species such as Tritirachium
and Conidiobolus have been reported (reviewed in Anwar and
Saleemuddin, 1998).
[0005] Use of fungal serine proteases in different applications is
also known from several patent applications. For example,
combination of a cellulase and a protease, particularly a
trypsin-like protease from Fusarium sp. DSM 2672 as a detergent
additive or composition is disclosed in WO 1992018599 (NovoNordisk
A/S). Such detergent compositions may further comprise reversible
protease inhibitors for stabilizing the enzyme(s) as disclosed in
WO 1992003529 and WO 1992005239 (NovoNordisk A/S). Process for
removal or bleaching of soiling or stains from cellulosic fabrics
with an enzyme hybrid comprising a catalytically active amino acid
sequence such protease linked to an amino acid sequence comprising
a cellulose binding domain is disclosed in WO 1997028243
(NovoNordisk A/S). WO 1997002753 (NovoNordisk A/S) discloses a
method for gentle cleaning of soiled process equipment using a
lipase and a protease being preferably a serine protease obtainable
from Fusarium. Use of F. equiseti and other fungi in reducing
organic matter in waste waters is disclosed in the EP 1464626
patent application (Biovitis S .A., F R).
[0006] The socioeconomic challenges and governmental regulations
have forced detergent industry to take in consideration many
environmental aspects including not only the use of more lenient
chemicals, which can be used in minor amounts and therefore leave
less environmental waste trails, but also the need of energy
saving. Detergent enzymes, particularly proteases, are important
ingredient in detergent compositions. The need to save energy by
decreasing the washing temperatures and the increased use of
synthetic fibers which cannot tolerate high temperatures and
current lifestyle have changed customer habits towards low washing
temperatures and has created a demand for new enzymes, which are
effective in low temperatures.
[0007] Despite the fact that numerous patent publications, reviews
and articles have been published, in which serine proteases from
various microorganisms, for example, the low temperature alkaline
proteases from actinomycete (Nocardiopsis dassonvillei) and fungal
(Paecilomyces marquandii) microorganisms are disclosed, e.g. in EP
0290567 and EP 0290569 (Novo Nordisk A/S, DK), there is still a
great need for alternative serine proteases, which are suitable for
and effective in modifying, degrading and removing proteinaceous
materials particularly in low or moderate temperature ranges and
which are stable in the presence of detergents with highly varying
properties.
[0008] Detergent industry is making great advances in adapting its
new products to customers' habits and needs, the properties of new
textile products and new washing machines. It is evident that when
developing new detergents, particularly laundry and dish wash
compositions, a wide range of varying and rapidly changing demands
have to be satisfied. In order to fulfill all varying demands of
detergent industry and governmental regulations, new serine
protease ingredients for detergent compositions should not only be
able to accomplish their tasks in wide pH and temperature ranges
and remain stable in variety of conditions, including mechanical
and chemical interventions in combination with a variety of
different detergents, it is also desirable that the serine protease
can be produced in high amounts, which can be cost-effectively
down-stream processed, by easy separation from fermentation broth
and mycelia.
SUMMARY OF THE INVENTION
[0009] The aim of the present invention is to provide a serine
protease of fungal origin which shows broad substrate specificity,
is active at broad pH ranges and has a broad temperature optimum,
i.e. functions both at low and moderate temperatures. The serine
proteases for laundry and dish detergents have to be stable also in
the presence of detergents or to be compatible with detergents.
Particularly, the object of the invention is to provide a serine
protease, which is capable of removing proteinaceous material,
including stains in washing laundry and dishes, at lower
temperatures than the present commercial enzyme preparations,
thereby saving energy. The fungal serine protease can be produced
in high-yielding fungal hosts and its down-stream processing, e.g.
separation of fermentation broth and mycelia is easy to
perform.
[0010] The present invention relates to a fungal serine protease
enzyme, which has serine protease activity and comprises an amino
acid sequence of the mature Fe_RF6318 enzyme as defined in SEQ ID
NO:15 or an amino acid sequence having at least 86% identity to the
amino acid sequence of the mature Fe_RF6318 enzyme defined in SEQ
ID NO:15.
[0011] The enzyme of the invention is obtainable from Fusarium
equiseti, more preferably from the deposited strain CBS 119568.
[0012] The enzyme has a molecular mass between 25 and 35 kDa. The
enzyme has optimal temperature at the range from 30.degree. C. to
70.degree. C. at pH 9. Said enzyme has pH optimum at the pH range
of at least pH 6 to pH 11 at 50.degree. C. The temperature and pH
optima were determined using 15 min reaction time and casein as a
substrate. The serine protease of the invention is capable in
degrading or removing proteinaceous stains in the presence of
detergent between 10.degree. C. and 60.degree. C.
[0013] The fungal serine protease enzyme of the invention is
encoded by an isolated polynucleotide sequence, which hybridizes
under stringent conditions with a polynucleotide sequence included
in plasmid pALK2521 comprising the nucleotide sequence SEQ ID NO:9
deposited in E. coli RF7664 under accession number DSM 22171.
[0014] Said enzyme is encoded by an isolated polynucleotide
sequence, which encodes a polypeptide comprising an amino acid
sequence of the mature Fe_RF6318 enzyme as defined in SEQ ID NO:15
or an amino acid sequence having at least 86% identity to the amino
acid sequence of the mature Fe_RF6318 defined in SEQ ID NO:15.
Preferably, said enzyme is encoded by an isolated nucleic acid
molecule comprising the nucleotide sequence SEQ ID NO:14.
[0015] The full-length fungal serine protease enzyme of the
invention is encoded by the polynucleotide sequence included in
pALK2529 deposited in Escherichia coli RF7800 under accession
number DSM 22172.
[0016] The fungal serine protease enzyme is produced from a
recombinant expression vector comprising the nucleic acid molecule
encoding a fungal serine protease of the invention operably linked
to regulatory sequences capable of directing the expression of the
serine protease encoding gene in a suitable host. Suitable hosts
include heterologous hosts, preferably microbial hosts of the genus
Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium,
Neurospora, Rhizopus, Penicillium and Mortiriella.
[0017] Preferably said enzyme is produced in Trichoderma or
Aspergillus, most preferably in T. reesei.
[0018] The present invention relates also to an isolated nucleic
acid molecule encoding a fungal serine protease enzyme selected
from the group consisting of: [0019] (a) a nucleic acid molecule
encoding a polypeptide having serine protease activity and
comprising the amino acid sequence as depicted in SEQ ID NO:15;
[0020] (b) a nucleic acid molecule encoding a polypeptide having
serine protease activity and at least 86% identity to the amino
acid sequence of SEQ ID NO:15; [0021] (c) a nucleic acid molecule
comprising the coding sequence of the nucleotide sequence as
depicted in SEQ ID NO: 10; [0022] (d) a nucleic acid molecule
comprising the coding sequence of the polynucleotide sequence
contained in DSM 22171 or DSM 22172; [0023] (e) a nucleic acid
molecule the coding sequence of which differs from the coding
sequence of a nucleic acid molecule of any one of (c) to (d) due to
the degeneracy of the genetic code; and [0024] (f) a nucleic acid
molecule hybridizing under stringent conditions to a nucleic acid
molecule contained in DSM 22171, and encoding a polypeptide having
serine protease activity and an amino acid sequence which shows at
least 86% identity to the amino acid sequence as depicted in SEQ ID
NO:15.
[0025] The invention further relates to a recombinant expression
vector comprising the nucleotide sequence of the invention operably
linked to regulatory sequences capable of directing expression of
said serine protease encoding gene in a suitable host. Suitable
hosts include heterologous hosts, preferably microbial hosts of the
genus Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium,
Neurospora, Rhizopus, Penicillium and Mortiriella. Preferably said
enzyme is produced in Trichoderma or Aspergillus, most preferably
in T. reesei.
[0026] The invention relates also to a host cell comprising the
recombinant expression vector as described above. Preferably, the
host cell is a microbial host, such as a filamentous fungus.
Preferred hosts are of a genus Trichoderma, Aspergillus, Fusarium,
Humicola, Chrysosporium, Neurospora, Rhizopus, Penicillium and
Mortiriella. More preferably the host is Trichoderma or
Aspergillus, most preferably a filamentous fungus T. reesei.
[0027] The present invention relates to a process of producing a
polypeptide having serine protease activity, said process
comprising the steps of culturing the host cell of the invention
and recovering the polypeptide. Also within the invention is a
polypeptide having serine protease activity encoded by the nucleic
acid sequence of the invention and which is obtainable by the
process described above.
[0028] The invention relates to a process for obtaining an enzyme
preparation comprising the steps of culturing a host cell of the
invention and either recovering the polypeptide from the cells or
separating the cells from the culture medium and obtaining the
supernatant. Within the invention is also an enzyme preparation
obtainable by the process described above.
[0029] The invention relates to an enzyme preparation, which
comprises the serine protease enzyme of the invention.
[0030] The enzyme preparation of the invention may further comprise
other enzymes selected from the group of protease, amylase,
cellulase, lipase, xylanase, mannanase, cutinase, pectinase or
oxidase with or without a mediator as well as suitable additives
selected from the group of stabilizers, buffers, surfactants,
bleaching agents, mediators, anti-corrosion agents, builders,
antiredeposition agents, optical brighteners, dyes, pigments,
caustics, abrasives and preservatives, etc.
[0031] The spent culture medium of the production host can be used
as such, or the host cells may be removed, and/or it may be
concentrated, filtrated or fractionated. It may also be dried. The
enzyme preparation of the invention may be in the form of liquid,
powder or granulate.
[0032] Also within the invention is the use of the serine protease
enzyme or the enzyme preparation of the invention for detergents,
for treating fibers, for treating wool, for treating hair, for
treating leather, for treating food or feed, or for any
applications involving modification, degradation or removal of
proteinaceous material. Particularly, the enzyme or enzyme
preparation is useful as a detergent additive in detergent liquids
and detergent powders.
BRIEF DESCRIPTION OF DRAWINGS
[0033] FIG. 1 shows the nucleotide sequence of the Fusarium
equiseti RF6318 Fe prtS8A gene and the deduced amino acid sequence.
The putative signal peptide, analyzed by SignalP V3.0 program is in
lower case letters and underlined. The pro sequence and the deduced
amino acids of the pro sequence are in lower case letters. The
mature nucleotide and peptide sequences are in capital letters
(N-terminal sequence determined from the purified wild type
Fe_RF6318 protein). The location of the putative intron sequence is
in lower case, italic letters and marked by a dotted line below the
nucleotide sequence. The stop codon is shown by an asterisk below
the sequence. The N-terminal sequence and peptide sequences
obtained from the wild type Fe_RF6318 protein are highlighted with
gray background.
[0034] FIG. 1A shows the nucleotide sequence of Fe prt8A gene from
the ATG start codon to the CCT codon (nucleotides 898 to 900), the
sequence region encoding the amino acid sequence from Met 1 to
Va1278 of the Fe_RF6318 protein.
[0035] FIG. 1B shows the nucleotide sequence of Fe prt8A gene from
CTC codon (nucleotides 901 to 903) to the TAA stop codon, the
sequence region encoding the amino acid sequence from Leu279 to
Ala412 of the Fe_RF6318 protein.
[0036] FIG. 2 schematically shows the cassette used for expressing
the Fe prtS8A gene in Trichoderma reesei.
[0037] FIG. 3 shows the partially purified recombinant Fe_RF6318
protein analysed on 12% SDS PAGE gel. Lane 1. Sample of the
partially purified Fe_RF6318, Lane 2. MW marker (Bench Mark Protein
Ladder, Invitrogen).
[0038] FIG. 4A describes the temperature profile of recombinant
protein Fe_RF6318 assayed at pH 9 using 15 min reaction time and
casein as a substrate. The data points are averages of three
separate measurements.
[0039] FIG. 4B describes the effect of pH on the activity of
recombinant Fe_RF6318 protein. The buffer used was 40 mM
Britton-Robinson buffer, casein was used as a substrate, reaction
time was 15 min and reaction temperature was 50.degree. C. The data
points are averages of three separate measurements.
[0040] FIG. 5 describes the performance of recombinant Fe_RF6318
protein with blood/milk/ink stain (Art 116, EMPA) at 30.degree. C.,
pH 9, 60 min. Commercial preparations Savinase Ultra.RTM. 16 L
(Novozymes A/S, DK) and Purafect.RTM. 4000 L (Genencor Inc., USA)
were used for comparison. .DELTA.L* (deltaL*)=lightness value L* of
enzyme treated fabric-lightness value L* of fabric treated with
buffer only (enzyme blank).
[0041] FIG. 6 describes the performance of recombinant Fe_RF6318
protein with blood/milk/ink stain (Art. 116, EMPA) at 50.degree.
C., pH 9, 60 min. Commercial preparations Savinase.RTM. Ultra 16 L
and Purafect.RTM. 4000L were used for comparison. .DELTA.L*
(deltaL*) =lightness value L* of enzyme treated fabric-lightness
value L* of fabric treated with buffer only (enzyme blank).
[0042] FIG. 7A describes the performance of recombinant Fe_RF6318
protein with blood/milk/ink stain (Art 117, EMPA) at 40.degree. C.,
approximately at pH 10, 60 min in the presence of detergent powder
(Art. 601, EMPA). Commercial preparation Purafect.RTM. 4000L was
used for comparison. .DELTA.L* (deltaL*)=lightness value L* of
enzyme treated fabric-lightness value L* of fabric treated with
buffer only (enzyme blank).
[0043] FIG. 7B describes the performance of recombinant Fe_RF6318
protein with blood/milk/ink stain (Art 117, EMPA) at 40.degree. C.,
approx. at pH 10, 60 min in the presence of detergent powder and
bleaching agents (Art. 604 and 606, EMPA). Commercial preparation
Purafect.RTM. 4000L was used for comparison. .DELTA.L*
(deltaL*)=lightness value L* of enzyme treated fabric-lightness
value L* of fabric treated with buffer only (enzyme blank).
[0044] FIG. 8A describes the performance of recombinant Fe_RF6318
protein with blood/milk/ink stain (Art 117, EMPA) at 50.degree. C.,
approx. at pH 10, 60 min in the presence of detergent powder (Art.
601, EMPA). Commercial preparation Purafect.RTM. 4000L was used for
comparison. .DELTA.L* (deltaL*)=lightness value L* of enzyme
treated fabric-lightness value L* of fabric treated with buffer
only (enzyme blank).
[0045] FIG. 8B describes the performance of recombinant Fe_RF6318
protein with blood/milk/ink stain (Art 117, EMPA) at 50.degree. C.,
approx. at pH 10, 60 min in the presence of detergent powder and
bleaching agents (Art. 604 and 606, EMPA). Commercial preparation
Purafect.RTM. 4000L was used for comparison. .DELTA.L*
(deltaL*)=lightness value L* of enzyme treated fabric-lightness
value L* of fabric treated with buffer only (enzyme blank).
[0046] FIG. 9 shows the performance of recombinant Fe_RF6318
protein with blood/milk/ink stain (Art 117, EMPA) and liquid
detergent Ariel Sensitive at 40.degree. C., approx. at pH 7.9, 60
min. Commercial preparations Savinase.RTM. Ultra 16 L and
Purafect.RTM. 4000 L were used for comparison. Shown at x-axis
enzyme dosage (activity units/ml), at y-axis .DELTA.L*
(deltaL*)=lightness value L* of enzyme treated fabric-lightness
value L* of fabric treated with buffer only (enzyme blank).
[0047] FIG. 10 shows the performance of recombinant Fe_RF6318
protein with blood/milk/ink stain (Art 117, EMPA) with different
concentrations of liquid base detergent for coloured fabrics at
30.degree. C. Commercial preparations Purafect.RTM. 4000L and
Savinase.RTM. Ultra 16 L were used for comparison. .DELTA.L*
(deltaL*)=lightness value L* of enzyme treated fabric-lightness
value L* of fabric treated with buffer only (enzyme blank).
[0048] FIG. 10A shows performance with detergent concentration of 5
g/l and pH 7.5.
[0049] FIG. 10B shows performance with detergent concentration of 5
g/l (enzyme dosage calculated as protein).
[0050] FIG. 10C shows performance with detergent concentration of
3.3 g/l and pH 7.4.
[0051] FIG. 10D shows performance with detergent concentration of 1
g/l and pH 7.3.
[0052] FIG. 11 shows the performance of recombinant protein
Fe_RF6318 with blood/milk/ink stain (Art 117, EMPA) with different
concentrations of Ariel Sensitive (without enzymes) on fabrics at
30.degree. C. Commercial preparations Purafect.RTM. 4000L and
Savinase.RTM. Ultra 16 L were used for comparison. .DELTA.L*
(deltaL*)=lightness value L* of enzyme treated fabric-lightness
value L* of fabric treated with buffer only (enzyme blank).
[0053] FIG. 11A shows performance with detergent concentration of 5
g/l and pH 8.
[0054] FIG. 11B shows performance with detergent concentration of 5
g/l (enzyme dosage calculated as protein).
[0055] FIG. 11C shows performance with detergent concentration of
3.3 g/l and pH 7.9
[0056] FIG. 11D shows performance with detergent concentration of 1
g/l and pH 7.6.
[0057] FIG. 12 shows the performance of recombinant protein
Fe_RF6318 on different stains in Launder Ometer tests with liquid
detergent Ariel Sensitive (without enzymes) at 30.degree. C.
Commercial preparation Savinase Ultra 16 L was used for comparison.
.DELTA.L* (deltaL*)=lightness value L* of enzyme treated
fabric-lightness value L* of fabric treated with buffer only
(enzyme blank).
[0058] FIG. 12A shows performance on blood/milk/ink/PE-cotton (Art.
117, EMPA).
[0059] FIG. 12B shows performance on blood/milk/ink/Cotton (Art.
116, EMPA)
[0060] FIG. 12C shows performance on grass (Art. 164, EMPA).
[0061] FIG. 13 shows the performance of recombinant protein
Fe_RF6318 on different stains in Launder Ometer tests with liquid
Base detergent for coloured fabrics at 30.degree. C. Commercial
preparations Savinase.RTM. Ultra 16 L and Purafect.RTM. 4000 L were
used for comparison. .DELTA.L* (deltaL*)=lightness value L* of
enzyme treated fabric-lightness value L* of fabric treated with
buffer only (enzyme blank).
[0062] FIG. 13A shows performance on blood/milk/ink/PE-cotton (Art.
117, EMPA).
[0063] FIG. 13B shows performance on blood/milk/ink/Cotton (Art.
116, EMPA).
[0064] FIG. 13C shows performance on grass (Art. 164, EMPA).
[0065] FIG. 13D shows performance on Cocoa (Art. 112, EMPA).
[0066] FIG. 14 describes total stain removal efficiency (delta %
SR) of Fe_RF6318 enzyme preparation on eight different protease
sensitive stains (Table 5) in full-scale washing trials. Commercial
preparations Savinase.RTM. Ultra 16 L and Purafect.RTM. 4000L were
used for comparison.
[0067] FIG. 14A describes total stain removal efficiency when
protease preparations were dosed according to the activity.
[0068] FIG. 14B describes total stain removal efficiency when
protease preparations were dosed according to the amount of
protein.
[0069] FIG. 15 describes the stain removal effect with liquid
detergent base for coloured fabrics in full scale trial at
30.degree. C.
[0070] FIG. 15A describes stain removal on blood/milk/ink/Cotton
(C-05-014/CFT).
[0071] FIG. 15B describes stain removal on blood/milk/ink/PE-Cotton
(C-05-014/CFT).
[0072] FIG. 15C describes stain removal on chocolate
milk/pigment/Cotton (C-03-030/CFT).
[0073] FIG. 15D describes stain removal on groundnut
oil/milk/Cotton (C-05-014/CFT).
[0074] FIG. 15E describes stain removal on egg yolk/pigment/Cotton
(CS-38-010/CFT).
[0075] FIG. 16 describes the performance of recombinant Fe_RF6318
protein with blood/milk/ink stain (Art 117, EMPA) at temperatures
from 10.degree. C. to 60.degree. C., pH 9, 60 min. Commercial
preparations Savinase Ultra.RTM. 16 L (Novozymes A/S, DK),
Purafect.RTM. 4000L (Genencor Inc., USA) and Properase.RTM. 4000E
(Genencor Inc., USA) were used for comparison. .DELTA.L*
(deltaL*)=lightness value L* of enzyme treated fabric-lightness
value L* of fabric treated with buffer only (enzyme blank).
[0076] FIG. 16A shows the performance of recombinant protein
Fe_RF6318 and commercial protease preparations at 10.degree. C.
[0077] FIG. 16B shows the performance of recombinant protein
Fe_RF6318 and commercial protease preparations at 20.degree. C.
[0078] FIG. 16C shows the performance of recombinant protein
Fe_RF6318 and commercial protease preparations at 30.degree. C.
[0079] FIG. 16D shows the performance of recombinant protein
Fe_RF6318 and commercial protease preparations at 40.degree. C.
[0080] FIG. 16E shows the performance of recombinant protein
Fe_RF6318 and commercial protease preparations at 50.degree. C.
[0081] FIG. 16F shows the performance of recombinant protein
Fe_RF6318 and commercial protease preparations at 60.degree. C.
[0082] FIG. 17 shows the performance of recombinant Fe_RF6318
protein with blood/milk/ink stain (Art 117, EMPA) and Liquid Base
concentration of 3.3 g/l at 10.degree. C. and 20.degree. C.
Commercial preparations Savinase.RTM. Ultra 16 L, Purafect.RTM.
4000L and Properase.RTM. 4000 E were used for comparison. .DELTA.L*
(deltaL*)=lightness value L* of enzyme treated fabric-lightness
value L* of fabric treated with buffer only (enzyme blank).
[0083] FIG. 17A shows performance at 10.degree. C.
[0084] FIG. 17B shows performance at 20.degree. C.
SEQUENCE LISTING
[0085] SEQ ID NO:1 Sequence of an aminoterminal peptide #3792 from
Fusarium equiseti RF6318 protease.
[0086] SEQ ID NO:2 Sequence of a tryptic peptide 1246.673 from
Fusarium equiseti RF6318 protease.
[0087] SEQ ID NO:3 Sequence of a tryptic peptide 3341.633 from
Fusarium equiseti RF6318 protease.
[0088] SEQ ID NO:4 Sequence of a tryptic peptide 1503.799 from
Fusarium equiseti RF6318 protease.
[0089] SEQ ID NO:5 The sequence of the oligonucleotide primer PRO87
derived from the aminoterminal peptide SEQ ID NO:1.
[0090] SEQ ID NO:6 The sequence of the oligonucleotide primer PRO88
derived from the aminoterminal peptide SEQ ID NO:1.
[0091] SEQ ID NO:7 The sequence of the oligonucleotide primer PRO89
derived from peptide SEQ ID NO:4.
[0092] SEQ ID NO:8 The sequence of the oligonucleotide primer PRO90
derived from peptide SEQ ID NO:4.
[0093] SEQ ID NO:9 The sequence of the PCR fragment obtained using
the primers PRO88 (SEQ ID NO:6) and PRO89 (SEQ ID NO:7) and
Fusarium equiseti RF6318 genomic DNA as a template.
[0094] SEQ ID NO:10 The nucleotide sequence of the full-length
Fusarium equiseti RF6318 protease gene (Fe prtS8A).
[0095] SEQ ID NO:11 The deduced amino acid sequence of the
full-length Fusarium equiseti RF6318 protease (Fe_RF6318) including
amino acids from Metl to Ala412.
[0096] SEQ ID NO:12 The nucleotide sequence encoding the amino acid
sequence of the proenzyme form of Fusarium equiseti RF6318
protease.
[0097] SEQ ID NO:13 The amino acid sequence of the proenzyme form
of Fusarium equiseti RF6318 protease including amino acids Ala21 to
Ala 412 of the full length protease.
[0098] SEQ ID NO: 14 The nucleotide sequence encoding the amino
acid sequence of the mature form of Fusarium equiseti RF6318
protease.
[0099] SEQ ID NO:15 The amino acid sequence of the mature form of
Fusarium equiseti RF6318 protease including amino acids Ala124 to
Ala412 of the full length enzyme. DEPOSITIONS
[0100] Fusarium equiseti RF6318 was deposited at the Centraalbureau
Voor Schimmelcultures at Uppsalalaan 8, 3508 AD, Utrecht, the
Netherlands on 7 Apr. 2006 and assigned accession number CBS
119568.
[0101] The E. coli strain RF7664 including the plasmid pALK2521 was
deposited at the Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH (DSMZ), Inhoffenstrasse 7 b, D-38124
Braunschweig, Germany on 14 Jan. 2009 and assigned accession number
DSM 22171.
[0102] The Ecoli strain RF7800 including the plasmid pALK2529 was
deposited at the Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH (DSMZ), Inhoffenstrasse 7 b, D-38124
Braunschweig, Germany on 14 Jan. 2009 and assigned accession number
DSM 22172.
DETAILED DESCRIPTION
[0103] The present invention provides a serine protease of fungal
origin, which protease shows broad substrate specificity, is stable
at high pH ranges and has a broad temperature optimum, i.e. good
performance both at low and moderate temperatures. The enzyme is
ideal for detergent applications, withstanding oxidizing and
chelating agents and being effective at low enzyme levels in
detergent solutions. Particularly, the serine protease is active at
temperatures as low as 10.degree. C., the preferred range being
from 10.degree. C. to 60.degree. C. Thus, the present invention
provides an alternative serine protease for use in detergent and
other applications. The fungal serine protease can be produced in
high-yielding fungal hosts and its down-stream processing, e.g.
separation of fermentation broth and mycelia is easy to
perform.
[0104] By "serine protease" or "serine endopeptidase" or "serine
endoproteinase" is in connection to this invention meant an enzyme
classified as EC 3.4.21 by the
[0105] Nomenclature of the International Union of Biochemistry and
Molecular Biology. Serine proteases are found in both single-cell
and complex organisms. Based on their structural similarities,
serine proteases have been grouped into at least six clans (SA, SB,
SC, SE, SF and SG; S denoting serine protease), which have been
further subgrouped into families with similar amino acid sequences
and three-dimensional structures (see, for example the Serine
protease home page at
http://www.biochem.wustl.edu/.about.protease/, Department of
Biochemistry and Molecular Biophysics, Washington University of
Medicine, St. Louis, Mo., USA). These protein hydrolyzing or
degrading enzymes are characterized by the presence of a
nucleophilic serine group in their active site, and the proteases
of clan SA and clan SB are also distinguished by having essential
aspartate and histidine residues, which along with the serine, form
a catalytic triad.
[0106] The major clans include the "chymotrypsin-like", including
chymotrypsin, trypsin and elastase (clan SA) and "subtilisins-like"
(clan SB) serine proteases. The enzymes target different regions of
the polypeptide chain, based upon the side chains of the amino acid
residues surrounding the site of cleavage. The serine protease of
the present invention belongs to clan SB.
[0107] The characterized "subtilisin-like serine proteases" or
"subtilases" are generally bacterial in origin. This class of
proteases, represented by various Bacillus, like B.
amyloliquifaciens, B. licheniformis and B. subtilis (Rao et al.,
1998), is specific for aromatic or hydrophobic residues, such as
tyrosine, phenylalanine and leucine.
[0108] By the term "serine protease activity" as used in the
invention is meant hydrolytic activity on protein containing
substrates, e.g. casein, haemoglobin, keratin and BSA. The methods
for analysing proteolytic activity are well-known in the literature
and are referred e.g. in Gupta et al. (2002).
[0109] Proteases can be classified using group specific inhibitors.
The diverse group of "serine protease inhibitors", includes
synthetic chemical inhibitors and natural proteinaceous inhibitors.
One group of natural inhibitors are serpins (abbreviated from
serine protease inhibitors), such as antithrombin and alpha
1-antitrypsin. Artificial synthetic inhibitors include
3,4-dichloroisocoumarin (3,4-DCI), diisopropylfluorophosphate
(DFP), phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-lysine
chloromethyl ketone (TLCK). Some of the serine proteases are
inhibited by thiol reagents such as p-chloromercuribenzoate (PCMB)
due to the presence of a cysteine residue near the active site.
Thus, the serine protease activity can be determined in an assay
based on cleavage of a specific substrate or in an assay using any
protein containing substrate with or without a specific inhibitor
of serine proteases under suitable conditions.
[0110] Serine proteases are generally active at neutral or alkaline
pH, with an optimum between pH 7 and 11, and have broad substrate
specificity. The "alkaline serine proteases" mean enzymes that are
active and stable at pH 9 to pH 11 or even at pH 10 to 12.5
(Shimogaki et al., 1991) and have isoelectric point around pH 9.
Those represent the largest subgroup of commercial serine
proteases. The molecular masses of alkaline serine proteases range
between 15 and 35 kDa. The temperature optima of the natural serine
proteases are around 60.degree. C. (Rao et al., 1998).
[0111] Microorganism strains capable of producing protease activity
can be screened and the activity on different substrates can be
determined. Chosen strains can be cultivated on a suitable medium.
After a sufficient amount of an interesting serine protease has
been produced, the enzyme can be isolated or purified and its
properties can be more thoroughly characterized. Alternatively,
genes encoding serine proteases in various organisms can be
isolated and the amino acid sequence encoded by the genes can be
compared with the amino acid sequences of the serine protease
isolated and characterized in the Examples here.
[0112] The produced protease enzymes, particularly the serine
proteases can be purified by using conventional methods of enzyme
chemistry, such as salt preparation, ultrafiltration, ion exchange
chromatography, affinity chromatography, gel filtration and
hydrophobic interaction chromatography. Purification can be
monitored by protein determination, enzyme activity assays and by
SDS polyacrylamide gel electrophoresis. The enzyme activity and
stability of the purified enzyme at various temperature and pH
values as well as the molecular mass and the isoelectric point can
be determined.
[0113] The purification of a preferred serine protease of the
present invention has been demonstrated in Example lb. The
filtrated culture supernatant was applied to a Q Sepharose FF
column. The flow through fraction was applied to phenyl Sepharose
HP column and proteins were eluted with a linear decreasing salt
gradient. Fractions showing protease activity were pooled,
concentrated and applied to a Superdex 75 10/300 GL column.
Purification was followed by activity assays on resorufin-labeled
casein as described in Example lb. Naturally, it is possible to
separate the enzyme of the present invention by using other known
purification methods instead, or in addition to the methods
described herein. The recombinant serine protease was purified as
described in Example 5 and used for characterization of pH and
temperature profiles.
[0114] The molecular mass of the purified serine protease can be
determined by mass spectrometry or on SDS-PAGE according to Laemmli
(1970). The molecular mass can also be predicted from the amino
acid sequence of the enzyme. The mature serine protease or mature
serine protease enzyme typically has a molecular mass between 20 to
35 kDa, typically around 25 to 30 kDa (Rao et al., 1998).
[0115] The serine proteases are synthesized as inactive "zymogenic
precursors" or "zymogens" in the form of a preproenzyme, which are
activated by removal of the signal sequence (secretion signal
peptide or prepeptide) and the prosequence (propeptide) to yield an
active mature form of the enzyme (Chen and Inouye, 2008). This
activation process involves action of proteases and may result from
limited self-digestive or autocatalytic processing of the serine
protease. The prosequence may be cleaved for example during
posttranslational phases of the production or in the spent culture
medium or during the storage of the culture medium or enzyme
preparation. Activation of the proenzyme may also be achieved by
adding a proteolytic enzyme capable of converting the inactive
proenzyme into active mature enzyme into the culture medium where
the host organism is cultivated or adding the proteolytic enzyme to
the culture supernatant after cultivation process. The shortening
of the enzyme can also be achieved e.g. by truncating the gene
encoding the polypeptide prior to transforming it to the production
host.
[0116] The term "mature" means the form of enzyme which after
removal of the signal sequence and propeptide comprises the
essential amino acids for enzymatic or catalytic activity. In
filamentous fungi it is the native form secreted into the culture
medium.
[0117] The temperature optimum of the serine protease can be
determined in a suitable buffer at different temperatures by using
casein as a substrate as described in Example 1c and 5 or by using
other substrates and buffer systems described in the literature
(Gupta et al., 2002). Determination of the pH optimum can be
carried out in a suitable buffer at different pH values by
following the activity on a protein substrate.
[0118] Protease activity is generally based on degradation of
soluble substrates. In detergent application proteases have to work
on substances which are at least partly insoluble. Thus an
important parameter for a detergent protease is the ability to
adsorb to and hydrolyse these insoluble fragments.
[0119] Another important parameter for selection of detergent
proteases is its isoelectric point or pI value. The detergent
proteases perform best when the pH value of the detergent solution
in which it works is approximately the same as the pI value for the
enzyme. pI can be determined by isoelectric focusing on an
immobilized pH gradient gel composed of polyacrylamide, starch or
agarose or by estimating the pI from the amino acid sequence, for
example by using the pI/MW tool at ExPASy server
(http://expasy.org/tools/pi_tool.html; Gasteiger et al., 2003).
[0120] The N-terminus of the purified protease as well as internal
peptides can be sequenced according to Edman degradation chemistry
(Edman and Begg, 1967) as described in Example 2 or by other
methods described in the literature.
[0121] The serine protease enzyme of the invention may derive from
any organism including bacteria, archaea, fungi, yeasts and even
higher eukaryote, such as plants. Preferably said enzyme originates
from a fungus, including filamentous fungi and yeasts, for example
from a genus selected from the group comprising Fusarium. Fungal
alkaline proteases are advantageous to the bacterial proteases due
to the ease of down-stream processing to produce a microbe-free
enzyme or enzyme composition. Mycelium can be easily removed
through filtration techniques prior to the purification of the
enzyme.
[0122] The present invention relates to fungal serine protease,
which has a good performance in the presence of detergents with
highly varying properties, at broad, i.e. from low to moderate
temperature ranges, such as 10.degree. C. to 60.degree. C.
[0123] In the present invention good performance in presence of
detergent means that the enzyme, in this case the fungal serine
protease of the invention, functions at lower temperature ranges
than many commercial subtilisins presently for sale. In other
words, good performance means that the enzyme is capable of
degrading or removing proteinaceous stains or material at low to
moderate temperature ranges, but especially at lower temperature
ranges than the present commercial products, for example the
commercial enzyme product Purafect.RTM. 4000L (Genencor Inc.,
USA).
[0124] The fungal serine protease of the invention functions at low
temperature ranges. For example, by modifying pH, selecting
detergents with suitable properties, including enzyme protecting
agents and by controlling washing conditions the activity of the
serine protease of the invention may be maintained at temperatures
as low as 10.degree. C. Therefore, the serine protease of the
invention depending on the washing conditions and auxiliary
ingredients and additives in detergents is useful particularly in
temperatures at or below 50.degree. C. The enzyme functions also at
temperatures at or below 45.degree. C., at or below 40.degree. C.,
at or below 35.degree. C., or at or below 30.degree. C.
[0125] In the presence of a detergent, the fungal serine protease
of the invention functions as defined above between 10.degree. C.
and 60.degree. C. In Examples 6 to 13, comparative experiments are
described, and from FIGS. 7 to 17 it is evident that the
performance of the fungal serine protease Fe_RF6318 in varying
conditions and exposed to varying treatments, on multitude of
different stains on different textile material, measured as deltaL*
or delta% SR, is by far better than the performance of the
commercial products, Savinase.RTM. Ultra 16L (Novozymes AIS, DK),
Purafect.RTM. 4000L (Genencor Inc, USA) and Properase.RTM. 4000E
(Genencor Inc., USA). Particularly, the stain removal effect of
said fungal serine protease Fe_RF6318 in low to moderate
temperature ranges such as from 10.degree. C. to 60.degree. C. is
remarkably higher than with Savinase.RTM. Ultra 16L and
Purafect.RTM. 4000L. It also has higher stain removal capacity at a
range from 30.degree. C. to 60.degree. C. when compared to
Properase.RTM. 4000E.
[0126] From said experimental results it can be concluded that the
fungal serine protease of the invention is capable of satisfying
the greatly varying demands of detergent customers and detergent
industry and industry providing washing machinery and is well
suited to the requirements of future regulations and customer
habits.
[0127] According to a preferred embodiment of the invention the
fungal serine protease enzyme is a polypeptide having serine
protease activity and comprising the mature enzyme of Fe_RF6318
having the amino acid sequence SEQ ID NO:15 or an amino acid
sequence having at least 86% identity to the amino acid sequence
SEQ ID NO:15 or at least 86% to the amino acid sequence SEQ ID
NO:11. Preferred enzymes show at least 86%, preferably at least
87%, more preferably at least 88%, even more preferably at least
90% identity. Still more preferably the amino acid sequences show
at least 92% or at least 94% or 96%, more preferably at least 98%,
most preferably 99% identity to the amino acid sequence of SEQ ID
NO:15. The identities of the two enzymes are compared within the
corresponding sequence regions, e.g. within the mature or
full-length region of the serine protease.
[0128] The serine protease of the present invention is marked
Fe_RF6318, an isolated serine protease originating from Fusarium
equiseti and is a member of clan SB, family 8 of serine
endoproteinases.
[0129] By the term "identity" is here meant the identity between
two amino acid sequences compared to each other within the
corresponding sequence region having approximately the same amount
of amino acids. For example, the identity of a full-length or a
mature sequence of the two amino acid sequences may be compared.
The amino acid sequences of the two molecules to be compared may
differ in one or more positions, which however does not alter the
biological function or structure of the molecules. Such variation
may occur naturally because of different host organisms or
mutations in the amino acid sequence or they may be achieved by
specific mutagenesis. The variation may result from deletion,
substitution, insertion, addition or combination of one or more
positions in the amino acid sequence. The identity of the sequences
is measured by using ClustalW alignment (e.g. in
www.ebi.ac.uk/Tools/Clustalw). The matrix used is as follows:
BLOSUM, Gap open:10, Gap extension: 0.5.
[0130] Preferably, the fungal serine protease is obtainable from
Fusarium, more preferably from Fusarium equiseti. According to the
most preferred embodiment the serine protease of the invention is
obtainable from the strain deposited at Centraalbureau voor
Schimmelcultures under accession number CBS 119568.
[0131] One preferred embodiment of the invention is a fungal serine
protease enzyme having serine protease activity and an amino acid
sequence of the mature Fe_RF6318 enzyme as defined in SEQ ID NO:15.
The mature enzyme lacks the signal sequence or prepeptide and the
prosequence or propeptide. The mature serine protease of the
invention includes amino acids Ala124 to Ala412 of the full length
protease characterized in SEQ ID NO:11. Thus, within the scope of
the invention is also the full-length Fe_RF6318 enzyme having SEQ
ID NO:11 including the signal sequence (prepeptide) and propeptide
and the mature enzyme as well as the proenzyme form lacking the
signal sequence (prepeptide) thus having SEQ ID NO:13.
[0132] The present invention relates to a fungal serine protease
enzyme, the mature form of which has a molecular mass or molecular
weight between 20 and 35 kDa, preferably between 25 and 33 kDa,
more preferably between 28 and 30 kDa. The most preferred MW is the
predicted molecular mass of Fe_RF6318 being 29 kDa for the mature
polypeptide obtained by using the Compute pI/MW tool at ExPASy
server (Gasteiger et al., 2003).
[0133] The enzyme of the invention is effective in degrading
proteinaceous material at a broad temperature range. The optimal
temperature of the enzyme is from 30.degree. C. to 70.degree. C.
(about 20% of the maximum activity), preferably from 40.degree. C.
to 60.degree. C. (at least about 40% of the maximum activity), and
more preferably between 50.degree. C. and 60.degree. C. (at least
70% of the maximum activity), most preferably at 60.degree. C. (the
maximum activity, Fe_RF6318) when measured at pH 9 using 15 min
reaction time and casein as a substrate as described in Example
5.
[0134] According to one preferred embodiment of the invention the
fungal serine protease enzyme has pH optimum at a pH range from at
least pH 6 to pH 11, showing over 40% of the maximum activity at pH
10 at 50.degree. C. using 15 min reaction time and casein as a
substrate as described in Example 5. In particular, the pH optimum
is between pH 6 and pH 10 (about 60% of the maximum activity), and
more preferably between pH 9 and pH 10 (about 80% of the maximum
activity), and most preferably at pH 10 at 50.degree. C.
[0135] The fungal serine protease of the invention has "good
performance in the presence of detergent", i.e. is capable of
degrading or removing proteinaceous stains or material in the
presence of detergent at low temperature ranges, specifically at
lower temperature ranges than the present commercial products, for
example the commercial enzyme product Purafect.RTM. 4000L (Genencor
Inc., USA). In the presence of a detergent the enzyme of the
invention functions between 10.degree. C. and 60.degree. C.,
preferably at or below 50.degree. C. The Fe_RF6318 enzyme functions
also in temperatures at or below 45.degree. C., at or below
40.degree. C., at or below 35.degree. C., or at or below 30.degree.
C.
[0136] The serine protease enzyme of the invention has pI, which as
predicted from the deduced amino acid sequence is between pI 9 and
pI 9.5, preferably between pI 9.1 and pI 9.4. The predicted pI of
Fe_RF6318 enzyme of the invention is pI 9.3.
[0137] Oligonucleotides synthesized on the amino acid sequence of
N-terminal or tryptic peptides of the purified enzyme or a PCR
product obtained by using the above oligonucleotides can be used as
probes in isolation of cDNA or a genomic gene encoding the serine
protease of the invention. The probe may be designed also based on
the known nucleotide or amino acid sequences of homologous serine
proteases. The serine protease clones may also be screened based on
activity on plates containing a specific substrate for the enzyme
or by using antibodies specific for a serine protease.
[0138] According to a preferred embodiment of the invention the
fungal serine protease enzyme is encoded by an isolated
polynucleotide sequence which hybridizes under stringent conditions
with a polynucleotide or probe sequence included in plasmid
pALK2521 comprising the nucleotide sequence SEQ ID NO:9 in E. coli
RF7664, deposited at the Deutsche Sammlung von Mikroorganismen and
Zellkulturen (DSMZ) under accession number DSM 22171.
[0139] In the present invention the Fe prt8A gene was isolated with
a probe prepared by PCR using stringent hybridization as described
in Example 3d. Standard molecular biology methods can be used in
isolation of cDNA or a genomic DNA of the host organism, e.g. the
methods described in the molecular biology handbooks, such as
Sambrook and Russell, 2001.
[0140] Hybridization with a DNA probe, such as for example SEQ ID
NO:9 consisting of more than 100-200 nucleotides, is usually
performed at "high stringency" conditions, i.e. hybridization at a
temperature, which is 20-25.degree. C. below the calculated melting
temperature (Tm) of a perfect hybrid, the Tm calculated according
to Bolton and McCarthy (1962). Usually prehybridization and
hybridization are performed at least at 65.degree. C. in
6.times.SSC (or 6.times.SSPE), SxDenhardt's reagent, 0.5% (w/v)
SDS, 100 .mu.g/ml denatured, fragmented salmon sperm DNA. Addition
of 50% formamide lowers the prehybridization and hybridization
temperatures to 42.degree. C. Washes are performed in low salt
concentration, e.g. in 2.times.SSC-0.5% SDS (w/v) for 15 minutes at
room temperature (RT), followed in 2.times.SSC-0.1% SDS (w/v) at
RT, and finally in 0.1.times.SSC-0.1% SDS (w/v) at least at
65.degree. C.
[0141] According to one preferred embodiment the fungal serine
protease enzyme of the invention is encoded by an isolated nucleic
acid molecule, which encodes a polypeptide comprising the amino
acid sequence characterized in SEQ ID NO:15, or a polypeptide
having at least 86% to the amino acid sequence SEQ ID NO:15 or at
least 86% to the amino acid sequence SEQ ID NO:11. Preferred
enzymes show at least 86%, preferably at least 87%, more preferably
at least 88%, even more preferably at least 90% identity. Still
more preferably the amino acid sequences show at least 92% or at
least 94% or 96%, more preferably at least 98%, most preferably 99%
identity to the amino acid sequence of SEQ ID NO:15. The identities
of the two enzymes are compared within the corresponding sequence
regions, e.g. within the mature or full-length region of the serine
protease.
[0142] Thus, within the scope of the invention is a polypeptide
sequence, which is encoded by a nucleic acid molecule encoding the
amino acid sequence of the full-length serine protease of the
invention including the prepeptide (signal sequence) and the
propeptide in addition to the mature form of the enzyme, and which
amino acid sequence is characterized in SEQ ID NO:11.
[0143] Also, within the scope of the invention is a polypeptide
sequence, which is encoded by a nucleic acid molecule encoding the
propeptide of serine protease enzyme of the invention including the
propeptide in addition to the mature form of the enzyme, and which
amino acid sequence is characterized in SEQ ID NO:13.
[0144] One preferred embodiment of the invention is the fungal
serine protease enzyme encoded by an isolated nucleic acid
molecule, which comprises the nucleotide sequence encoding the
mature form of the Fe_RF6318 serine protease having SEQ ID
NO:15.
[0145] According to one preferred embodiment the fungal serine
protease enzyme of the invention is encoded by an isolated nucleic
acid molecule comprising the nucleotide sequence SEQ ID NO:14
encoding the mature form of the Fe_RF6318 enzyme (SEQ ID
NO:15).
[0146] Thus, within the scope of the invention is the polypeptide
encoded by the nucleic acid molecule having the nucleotide sequence
SEQ ID NO:10 comprising the "coding sequence" for the enzyme. The
expression "coding sequence" means the nucleotide sequence which
initiates from the translation start codon (ATG) and stops at the
translation stop codon (TAA, TAG or TGA). The translated
full-length polypeptide starts usually with methionine and
comprises intron regions.
[0147] Also, within the scope of the invention is a fungal serine
protease enzyme encoded by a nucleic acid molecule comprising the
nucleotide sequence SEQ ID NO:12, which encodes the Fe_RF6318
proenzyme form.
[0148] According to another preferred embodiment of the invention
the fungal serine protease is encoded by the polynucleotide
sequence included in pALK2529 deposited in E. coli RF7800 under
accession number DSM 22172.
[0149] One embodiment of the invention is the serine protease
enzyme produced from a recombinant expression vector comprising the
nucleic acid molecule, which encodes the fungal serine protease
enzyme as characterized above operably linked to regulatory
sequences capable of directing the expression of said serine
protease encoding gene in a suitable host. Construction of said
recombinant expression vector and use of said vector is described
in more detail in Example 4.
[0150] Suitable hosts for production of the fungal serine protease
enzyme are homologous or heterologous hosts, such as the microbial
hosts including bacteria, yeasts and fungi. Filamentous fungi, such
as Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium
Neurospora, Rhizopus, Penicillium and Mortiriella, are preferred
production hosts due to the ease of down-stream processing and
recovery of the enzyme product. Suitable hosts include species such
as T. reesei, A. niger, A oryzae, A. sojae, A. awamori or A.
japonicus type of strains, F. venenatum or F. oxysporum, H.
insolens or H. lanuginosa, N. crassa and C. lucknowense, some of
which are listed as enzyme production host organisms in e.g. AMFEP
2007 list of commercial enzymes (http://www.amfep org/list.html).
More preferably, the enzyme is produced in a filamentous fungal
host of the genus Trichoderma or Aspergillus, such as T. reesei or
A. niger, A. oryzae or A. awamori. According the most preferred
embodiment of the invention the fungal serine protease enzyme is
produced in T. reesei.
[0151] The present invention relates also to an isolated nucleic
acid molecule encoding the fungal serine protease enzyme selected
from the group consisting of: [0152] (a) a nucleic acid molecule
encoding a polypeptide having serine protease activity and
comprising the amino acid sequence as depicted in SEQ ID NO:15;
[0153] (b) a nucleic acid molecule encoding a polypeptide having
serine protease activity and at least 86% to SEQ ID NO:15; [0154]
(c) a nucleic acid molecule comprising the coding sequence of the
nucleotide sequence as depicted in SEQ ID NO: 10; [0155] (d) a
nucleic acid molecule comprising the coding sequence of the
polynucleotide sequence contained in DSM 22171 or DSM 22172; [0156]
(e) a nucleic acid molecule the coding sequence of which differs
from the coding sequence of a nucleic acid molecule of any one of
(c) to (d) due to the degeneracy of the genetic code; and [0157]
(f) a nucleic acid molecule hybridizing under stringent conditions
with a nucleic acid molecule contained in DSM 22171, and encoding a
polypeptide having serine protease activity and an amino acid
sequence which shows at least 86% identity to the amino acid
sequence as depicted in SEQ ID NO:15.
[0158] The nucleic acid molecule of the invention may be RNA or
DNA, wherein the DNA may constitute of the genomic DNA or cDNA.
[0159] Standard molecular biology methods can be used in isolation
and enzyme treatments of the polynucleotide sequence encoding the
fungal serine protease of the invention, including isolation of
genomic and plasmid DNA, digestion of DNA to produce DNA fragments,
sequencing, E. coli transformations etc. The basic methods are
described in the standard molecular biology handbooks, e.g.
Sambrook and Russell, 2001.
[0160] Isolation of the Fe prtS8A gene encoding the Fe_RF6318
polypeptide is described in Example 3. Briefly, the 866 by PCR
fragment obtained by using the sequences of the degenerate
oligonucleotide primers (SEQ ID NO: 6 and SEQ ID NO: 7) was used to
isolate the Fe prt8A from Fusarium equiseti RF6318 in pBluescript
II KS+ vector. The full-length Fusarium equiseti Fe prtS8A gene was
included in the plasmid pALK2529 deposited in E. coli to the DSMZ
culture collection under accession number DSM 22172. The deduced
amino acid sequence of the serine protease was analyzed from the
DNA sequence.
[0161] The nucleotide sequence of Fusarium equiseti serine protease
Fe prtS8A (SEQ ID NO: 10) and the deduced sequence (SEQ ID NO:11)
are presented in FIG. 1A-B. The length of the gene is 1303 by
(including the stop codon). One putative intron was found having
the length of 64 bps. The deduced protein sequence consists of 412
amino acids including a predicted signal sequence of 20 amino acids
(SignalP V3.0; Nielsen et al., 1997 and Nielsen and Krogh, 1998)
and a propeptide from Ala21 to Arg123. The peptides purified from
the wild type Fe_RF6318 matched the deduced amino acid sequence
indicating that the gene cloned encodes the protease purified from
the Fusarium equiseti host RF6318 deposited to the CBS culture
collection under accession number CBS 119568. The predicted
molecular mass was 29 kDa for the mature polypeptide and the
predicted pI was 9.30. These predictions were made using the
Compute pI/MW tool at ExPASy server (Gasteiger et al., 2003). The
deduced amino acid sequence contained two possible N-glycosylation
sites (Asn77 and Asn255), but according to CBS Server NetNGlyc V1.0
only one site, Asn77 (located in the pro sequence) is probable. The
homologies to the published protease sequences were searched using
the BLAST program, version 2.2.9 at NCBI (National Center for
Biotechnology Information) (Altschul et al., 1990). The identity
values of the mature Fe_RF6318 sequence to the corresponding
regions of homologous sequences were obtained by using ClustalW
alignment (Matrix: BLOSUM, Gap open: 10, Gap extension: 0.5 (e.g.
in www.ebi.ac.uk/Tools/Clustalw) are shown in Table 3.
[0162] The serine protease Fe_RF6318 of the present invention
showed highest homology to Gibberella zeae hypothetical protein
PH-1, locus tag FG03315.1 (EMBL accession no. XP .sub.--383491,
unpublished), to T. harzianum CECT 2413 serine endopeptidase (EMBL
accession no. CAL25508, Suarez et al., 2007) and to T. atroviride
alkaline proteinase precursor 508.066, ALP (EMBL accession no.
M87516, Geremia et al. 1993), the latter disclosed as an amino acid
sequence SEQ ID NO:313 in US 60/818,910 (Catalyst Bioscience Inc.).
The identity to G. zeae hypothetical protein was within the
full-length enzyme 85%. When the mature polypeptides lacking the
signal sequence and propeptide were aligned, the identity was 85%.
Identity to T. harzianum CECT 2413 serine endopeptidase was 70%
(full-length enzyme) and 75% (mature enzyme). The identity to T.
atroviride ALP was 69% (full-length enzyme) and 74% (mature
enzyme).
[0163] Thus, within the scope of the invention is an isolated
polynucleotide sequence or isolated nucleic acid molecule, which
encodes a fungal serine protease enzyme or polypeptide comprising
the amino acid sequence of the mature form of the Fe_RF6318 enzyme
characterized in SEQ ID NO: 15, i e amino acids Ala124 to Ala412 of
the full length serine protease of SEQ ID NO:11.
[0164] Further, within the scope of the present invention are
nucleic acid molecules which encode a fragment of a fungal serine
protease polypeptide, wherein the fragment has serine protease
activity and has at least 86% identity to the amino acid sequence
SEQ ID No:15 or at least 86% to the amino acid sequence SEQ ID
NO:11. Preferred enzymes show at least 86%, preferably at least
87%, more preferably at least 88%, even more preferably at least
90% identity. Still more preferably the amino acid sequences show
at least 92% or at least 94% or 96%, more preferably at least 98%,
most preferably 99% identity to the amino acid sequence of SEQ ID
NO:15. The identities of the two enzymes are compared within the
corresponding sequence regions, e.g. within the full-length or
mature region of the serine protease.
[0165] The nucleic acid molecule is preferably a molecule
comprising the coding sequence as depicted in SEQ ID NO:10, which
encodes the full length form of the fungal serine protease enzyme
of this invention.
[0166] The isolated nucleic acid molecule of the invention may be a
molecule comprising the coding sequence of the polynucleotide
sequence contained in DSM 22171 or DSM 22172. DSM 22171 carries the
nucleotide sequence of the PCR fragment (SEQ ID NO:9) used in
cloning the full length Fe prtS8A gene. DSM 22172 carries the
nucleotide sequence of the full length Fe prtS8A gene (SEQ ID
NO:10).
[0167] The nucleic acid molecule of the invention may also be an
analogue of the nucleotide sequence characterized in above. The
"degeneracy" means analogues of the nucleotide sequence, which
differ in one or more nucleotides or codons, but which encode the
recombinant protease of the invention.
[0168] The nucleic acid molecule may also be a nucleic acid
molecule hybridizing under stringent conditions to a PCR probe
contained in plasmid pALK2521 deposited in E.coli under the
accession number DSM 22171 and encoding a polypeptide having serine
protease activity and an amino acid sequence which within the
corresponding sequene region shows at least 86% identity to the
amino acid sequence as depicted in SEQ ID NO:15. The hybridizing
DNA may originate from a fungus belonging to species Fusarium or it
may originate from other fungal species.
[0169] Thus, within the scope of the invention is an isolated
nucleic acid molecule comprising a nucleotide sequence as depicted
in SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14 and analogues
thereof.
[0170] The present invention relates also to a recombinant
expression vector or recombinant expression construct, which can be
used to propagate or express the nucleic acid sequence encoding the
chosen serine protease in a suitable prokaryotic or eukaryotic
host. The recombinant expression vector comprises DNA or nucleic
acid sequences which facilitate or direct expression and secretion
of the serine protease encoding sequence in a suitable host, such
as promoters, enhancers, terminators (including transcription and
translation termination signals) and signal sequences operably
linked the polynucleotide sequence encoding said serine protease.
The expression vector may further comprise marker genes for
selection of the transformant strains or the selection marker may
be introduced to the host in another vector construct by
co-transformation. Said regulatory sequences may be homologous or
heterologous to the production organism or they may originate from
the organism, from which the gene encoding the serine protease is
isolated.
[0171] Examples of promoters for expressing the serine protease of
the invention in filamentous fungal hosts are the promoters of A.
oryzae TAKA amylase, alkaline protease ALP and triose phosphate
isomerase, Rhizopus miehei lipase, Aspergillus niger or A. awamori
glucoamylase (glaA), Fusarium oxysporum trypsin-like protease,
Chrysosporium lucknowense cellobiohydrolase I promoter, Trichoderma
reesei cellobiohydrolase I (Ce17A) etc.
[0172] In yeast, for example promoters of S. cerevisiae enolase
(ENO-1), galactokinase (GAL1), alcohol dehydrogenase (ADH2) and
3-phosphoglycerate kinase can be used to provide expression.
[0173] Examples of promoter sequences for directing the
transcription of the serine protease of the invention in a
bacterial host are the promoter of lac operon of Escherichia coli,
the Streptomyces coelicolor agarase dagA promoter, the promoter of
the B. licheniformis alpha-amylase gene (amyL), the promoter of the
B. stearothermophilus maltogenic amylase gene (amyM), the promoters
of the B. sublitis xylA and xylB genes, etc.
[0174] Suitable terminators include those of the above mentioned
genes or any other characterized terminator sequences.
[0175] Suitable transformation or selection markers include those
which complement a defect in the host, for example the dal genes
from B. subtilis or B. licheniformis or Aspergillus amdS and niaD.
The selection may be based also on a marker conferring antibiotic
resistance, such as ampicillin, kanamycin, chloramphenicol,
tetracycline, phleomycin or hygromycin resistance.
[0176] Extracellular secretion of the serine protease of the
invention is preferable. Thus, the recombinant vector comprises
sequences facilitating secretion in the selected host. The signal
sequence of the serine protease of the invention or the presequence
or prepeptide may be included in the recombinant expression vector
or the natural signal sequence may be replaced with another signal
sequence capable of facilitating secretion in the selected host.
Thus, the chosen signal sequence may be homologous or heterologous
to the expression host.
[0177] Examples of suitable signal sequences are those of the
fungal or yeast organisms, e.g. signal sequences from well
expressed genes. Such signal sequences are well known from the
literature.
[0178] The recombinant vector may further comprise sequences
facilitating integration of the vector into the host chromosomal
DNA to obtain stable expression.
[0179] The Fe_RF6318 protease of the invention was expressed with
its own signal sequence from the T. reesei cbhl (cel7A) promoter as
described in Example 4. The expression construct used to transform
the T. reesei host included also cbhl terminator and amdS marker
for selecting the transformants from the untrasformed cells.
[0180] The present invention relates also to host cells comprising
the recombinant expression vector as described above. Suitable
hosts for production of the fungal serine protease enzyme are
homologous or heterologous hosts, such as the microbial hosts
including bacteria, yeasts and fungi. Production systems in plant
or mammalian cells are also possible.
[0181] Filamentous fungi, such Trichoderma, Aspergillus, Fusarium,
Humicola, Chrysosporium, Neurospora, Rhizopus, Penicillium and
Mortiriella, are preferred production hosts due to the ease of
down-stream processing and recovery of the enzyme product. Suitable
expression and production host systems are for example the
production system developed for the filamentous fungus host
Trichoderma reesei (EP 244234), or Aspergillus production systems,
such as A. oryzae or A. niger (WO 9708325, U.S. Pat. No. 5,843,745,
U.S. Pat. No. 5,770,418), A. awamori, A. sojae and A.
japonicus-type strains, or the production system developed for
Fusarium, such as F. oxysporum (Malardier et al., 1989) or F.
venenatum, and for Neurospora crassa, Rhizopus miehei, Mortiriella
alpinis, H. lanuginosa or H. insolens or for Chrysosporium
lucknowense (U.S. Pat. No. 6,573,086). Suitable production systems
developed for yeasts are systems developed for Saccharomyces,
Schizosaccharomyces or Pichia pastoris. Suitable production systems
developed for bacteria are a production system developed for
Bacillus, for example for B. subtilis, B. licheniformis, B.
amyloliquefaciens, for E. coli, or for the actinomycete
Streptomyces. Preferably the serine protease of the invention is
produced in a filamentous fungal host of the genus Trichoderma or
Aspergillus, such as T. reesei, or A. niger, A oryzae, A. sojae, A.
awamori or A. japonicus-type strains. According the most preferred
embodiment of the invention the fungal serine protease enzyme is
produced in T. reesei.
[0182] The production host cell may be homologous or heterologous
to the serine protease of the invention. The host may be free of
homogenous proteases due to removal of proteases either by
inactivation or removal of one or more host proteases, e.g. by
deletion of the gene(s) encoding such homogenous or homologous
proteases.
[0183] The present invention relates also to a process for
producing a polypeptide having serine protease activity, said
process comprising the steps of culturing the natural or
recombinant host cell carrying the recombinant expression vector
for a serine protease of the invention under suitable conditions
and optionally isolating said enzyme. The production medium may be
a medium suitable for growing the host organism and containing
inducers for efficient expression. Suitable media are well-known
from the literature.
[0184] The invention relates to a polypeptide having serine
protease activity, said polypeptide being encoded by the nucleic
acid molecule of the invention and which is obtainable by the
process described above. Preferably, the polypeptide is a
recombinant protease enzyme obtained by culturing the host cell
carrying the recombinant expression vector for a serine protease of
the invention.
[0185] The invention further relates to a process for obtaining an
enzyme preparation comprising a polypeptide, which has serine
protease activity, said process comprising the steps of culturing a
host cell carrying the expression vector of the invention and
either recovering the polypeptide from the cells or separating the
cells from the culture medium and obtaining the supernatant having
serine protease activity.
[0186] The present invention relates also to an enzyme preparation,
which comprises the serine protease enzyme characterized above. The
enzyme preparation or composition has serine protease activity and
is obtainable by the process according to the invention.
[0187] Within the invention is an enzyme preparation which
comprises the fungal serine protease of the invention, preferably
the recombinant serine protease obtained by culturing a host cell,
which carries the recombinant expression vector of the
invention.
[0188] Said enzyme preparation may further comprise different types
of enzymes in addition to the serine protease of this invention,
for example another protease, an amylase, a lipase, a cellulase,
cutinase, a pectinase, a mannanase, a xylanase and/or an oxidase
such as a laccase or peroxidase with or without a mediator. These
enzymes are expected to enhance the performance of the serine
proteases of the invention by removing the carbohydrates and oils
or fats present in the material to be handled. Said enzymes may be
natural or recombinant enzymes produced by the host strain or may
be added to the culture supernatant after the production
process.
[0189] Said enzyme preparation may further comprise a suitable
additive selected from the group of surfactants or surface active
agent, buffers, anti-corrosion agents, stabilizers, bleaching
agents, mediators, builders, caustics, abrasives and preservatives,
optical brighteners, antiredeposition agents, dyes, pigments,
etc.
[0190] Surfactants are useful in emulsifying grease and wetting
surfaces. The surfactant may be a non-ionic including semi-polar
and/or anionic and/or cationic and/or zwitterionic.
[0191] Buffers may be added to the enzyme preparation to modify pH
or affect performance or stability of other ingredients.
[0192] Suitable stabilizers include polyols such as propylene
glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric
acid, or boric acid derivatives, peptides, etc.
[0193] Bleaching agent is used to oxidize and degrade organic
compounds. Examples of suitable chemical bleaching systems are
H.sub.2O.sub.2 sources, such as perborate or percarbonate with or
without peracid-forming bleach activators such as
tetraacetylethylenediamine, or alternatively peroxyacids, e.g.
amide, imide or sulfone type. Chemical oxidizers may be replaced
partially or completely by using oxidizing enzymes, such as
laccases or peroxidases. Many laccases do not function effectively
in the absence of mediators.
[0194] Builders or complexing agents include substances, such as
zeolite, diphosphate, triphosphate, carbonate, citrate, etc. The
enzyme preparation may further comprise one or more polymers, such
as carboxymethylcellulose, poly(ethylene glycol), poly(vinyl
alcohol), poly(vinylpyrrolidone), etc. Also, softeners, caustics,
preservatives for preventing spoilage of other ingredients,
abrasives and substances modifying the foaming and viscosity
properties can be added.
[0195] According to one preferred embodiment of the invention said
enzyme preparation is in the form of liquid, powder or
granulate.
[0196] The fungal serine protease of the present invention may like
other proteases, particularly alkaline proteases be used in the
detergent, protein, brewing, meat, photographic, leather, dairy and
pharmaceutical industries (Kalisz, 1988; Rao et al., 1998). For
example, it may be used as an alternative to chemicals to convert
fibrous protein waste (e.g. horn, feather, nails and hair) to
useful biomass, protein concentrate or amino acids (Anwar and
Saleemuddin, 1998). The use of fungal serine protease of the
present invention may like other enzymes prove successful in
improving leather quality and in reducing environmental pollution
and saving energy and it may like alkaline proteases be useful in
synthesis of peptides and resolution of the mixture of D,L-amino
acids. Subtilisin in combination with broad-spectrum antibiotics in
the treatment of burns and wounds is an example of the use of
serine proteases in pharmaceutical industry, therefore the fungal
serine protease of the present invention.may also find such use and
may also like alkaline proteases be applicable in removal of blood
on surgical equipments and cleaning contact lenses or dentures.
Like alkaline protease from Conidiobolus coronatus, the fungal
serine protease of the present invention may be used for replacing
trypsin in animal cell cultures. The proteases of the invention can
also be used in cleaning of membranes and destruction of biofilms.
In baking the proteases can be used e.g. in destruction of the
gluten network and in other food applications in hydrolysis of food
proteins, e.g proteins in milk. They can also be used e.g. in
treating yeast, rendering (extracting more protein from animal
bones), creating new flavours, reducing bitterness, changing
emulsifying properties, generating bioactive peptides and reducing
allergenicity of proteins. The substrates include animal, plant and
microbial proteins.
[0197] Detergent industry, particularly the laundry detergent
industry, has emerged as the single major consumer of proteases
active at high pH range (Anwar and Saleemuddin, 1998). The ideal
detergent protease should possess broad substrate specificity to
facilitate the removal of large variety of stains due to food,
grass, blood and other body secretions. It has to be active in the
pH and ionic strength of the detergent solution, the washing
temperature and pH, and tolerate mechanical handling as well as the
chelating and oxidizing agents added to detergents. The pI of the
protease must be near the pH of the detergent solution. Due to
present energy crisis and the awareness for energy conservation, it
is currently desirable to use the protease at lower
temperatures.
[0198] The present invention relates also to the use of the serine
protease enzyme or the enzyme preparation for detergents, treating
textile fibers, for treating wool, for treating hair, for treating
leather, for treating feed or food, or for any application
involving modification, degradation or removal of proteinaceous
material.
[0199] One preferred embodiment of the invention is therefore the
use of the serine protease enzyme as characterized above as a
detergent additive useful for laundry detergent and dish wash
compositions, including automatic dish washing compositions.
[0200] The expression "detergent" is used to mean substance or
material intended to assist cleaning or having cleaning properties.
The term "detergency" indicates presence or degree of cleaning
property. The degree of cleaning property can be tested on
different proteinaceous or protein containing substrate materials
or stains or stain mixtures bound to solid, water-insoluble
carrier, such as textile fibers or glass. Typical proteinaceous
material includes blood, milk, ink, egg, grass and sauces. For
testing purposes mixtures of proteinaceous stains are commercially
available. The function of the detergent enzyme is to degrade and
remove the protein-containing stains. Test results depend on the
type of stain, the composition of the detergent and the nature and
status of textiles used in the washing test (Maurer, 2004).
[0201] Typically, the protease or wash performance is measured as
"stain removal efficiency" or "stain removal effect" or "degree of
cleaning property", meaning a visible and measureable increase of
lightness or change in colour of the stained material, e.g. in
artificially soiled swatches or test cloths. Lightness or change in
colour values can be measured, for example by measuring the colour
as reflectance values with a spectrophotometer using L*a*b* colour
space coordinates as described in Examples 6 to 10. Fading or
removal of proteinaceous stain indicating of the protease
performance (stain removal efficiency) is calculated for example as
.DELTA.L*, which means lightness value L* of enzyme treated fabric
minus lightness value L* of fabric treated with buffer or washing
liquor without enzyme (enzyme blank or control). The presence of
detergent may improve the performance of the enzyme in removing the
stains.
[0202] The serine protease of the present invention degrades
various kinds of proteinaceous stains under conditions of neutral
and alkaline pH and even in the presence of detergents with
different compositions (as shown in Examples 6 to 13).
[0203] As shown in Example 6 the serine protease of the invention
removed the blood/milk/ink stain at 50.degree. C. and especially at
30.degree. C. in pH 9 buffer better than the commercial protease
preparations Savinase.RTM. Ultra 16L and Purafect.RTM. 4000L (FIGS.
5 and 6). The enzyme preparations were dosed as activity units. The
stain removal effect on blood/milk/ink stain was tested also at the
whole temperature range from 10.degree. C. to 60.degree. C. as
described in Example 12. Fe_RF6318 protease preparation showed
higher stain removal capacity compared to the commercial protease
preparation Savinase.RTM. Ultra 16L and Purafect.RTM. 4000L. It
also showed higher stain removal capacity at a range from
30.degree. C. to 60.degree. C. compared to Properase.RTM. 4000E
(FIG. 16).
[0204] The performance of the Fe_RF6318 protease was tested also in
detergent powder at 40.degree. C/50.degree. C. at pH 10 as
described in Example 7. The ability of the enzyme in removing
blood/milk/ink stain on polyester-cotton material was assayed. Each
enzyme preparation was dosed as activity units (.mu.mol
tyrosine/minute). As shown in FIGS. 7 and 8 the protease of the
invention is suitable also for powder detergents at very alkaline
conditions and its resistance for bleaching agents was slightly
higher than with commercial protease Purafect.RTM. 4000L.
[0205] The Fe_RF6318 protease removed blood/milk/ink standard stain
also in liquid base detergent and in Ariel Sensitive (Procter &
Gamble, UK) in the presence of the liquid base detergent and at
30.degree. C. (Example 9). The efficiency on blood/milk/ink stain
was considerably higher than with the commercial preparations
Savinase.RTM. Ultra 14L and Purafect.RTM. 4000L (FIGS. 10 and 11).
The enzyme preparations were dosed as activity units. The same
effect was observed also when the dosing was calculated as amount
of added protein (FIG. 10B and 11B). Washings performed with liquid
detergent concentration of 3.3. g/l and at 10.degree. C. and
20.degree. C. again showed superior performance over commercial
preparations Savinase.RTM. Ultra 14L, Purafect.RTM. 4000L and
Properase.RTM. 4000E (Example 13; FIG. 17).
[0206] In addition to the blood/milk/ink stains the Fe_RF6318
protease was effective in removing stains, such as grass and cocoa
when tested in liquid detergents at 30.degree. C. Treatments were
performed in ATLAS LP-2 Launder-Ometer. Results (FIGS. 12 and 13)
show that the Fe_RF6318 was effective on several stains at low
temperatures like 30.degree. C.
[0207] The performance of recombinant Fe_RF6318 enzyme preparation
produced in T. reesei was tested in the presence of liquid base
detergent in full scale in a washing machine at 30.degree. C.
(Example 11). Eight different protease sensitive tracers for
testing side effects are presented in Table 5 and the process
conditions in Table 6. Enzyme dosages used in the test trials were
calculated both as enzyme activities and as amount of enzyme
protein. Results presented in FIGS. 14A-B show that the sum of the
results obtained with the different stains was higher with the
Fe_RF6318 was higher than with the commercial protease preparations
Savinase.RTM. Ultra 16L and Purafect.RTM. 4000L when the enzyme was
dosed as amount of activity or as protein. The Fe_RF6318 was most
efficient on blood/milk/ink, chocolate/milk, groundnut oil/milk and
egg yolk stains (FIGS. 15A-E).
[0208] According to a preferred embodiment of the invention the
fungal serine protease of the invention is useful in detergent
liquids and detergent powders as shown in Examples 6 to 13. The
enzyme of enzyme preparation of the invention may be formulated for
use in a hand or machine laundry or may be formulated for use in
household hard surface cleaning or preferably in hand or machine
dishwashing operations.
EXAMPLE 1
Production and Purification of the Fusarium equiseti RF6318
Protease
[0209] (a) Cultivation of Fusarium equiseti RF6318
[0210] Fungal strain RF6318 was previously isolated as a
filamentous fungus producing cellulases. It was identified as
Fusarium cf. equiseti (Libert) Desmazieres (by Arthur de Cock,
Identification Services, Centralbureau Voor Schimmelcultures, P.O.
Box 85167, 3508 AD Utrecht, The Netherlands). F. equiseti RF6318
was shown to produce protease activity on agar plate assay
containing haemoglobin as a substrate. As the plate cultivations
were performed at about 10.degree. C. this result suggested that
RF6318 produces a protease or proteases acting at cold
temperatures. The F. equiseti RF6318 was grown, maintained and
sporulated on Potato Dextrose (PD) agar (Difco) at +4.degree. C.
For enzyme production spores from RF6318 slant were inoculated into
a culture medium which contained: 30 g/l Corn meal (finely ground),
5 g/l Corn steep powder, 4 g/l Soybean meal (defatted), 2 g/l
KH.sub.2PO.sub.4, 1 g/l NaCl and 1 g/l Paraffin oil. The pH of the
medium was adjusted before sterilization with NaOH to 8.5 and the
medium was autoclaved for 30 minutes at 121.degree. C. The microbe
was cultivated in 50 ml volume on a shaker (200 rpm) at 28.degree.
C. for 7 days. The spent culture supernatant was shown to contain
alkaline protease activity, when activity measurements were
performed (according to Example 1c) at different pH values (pH
7-10). Because of this alkaline activity, the RF6318 strain was
chosen as putative protease gene donor strain.
[0211] (b) Purification of Protease from the F. equiseti RF6318
Culture Medium
[0212] Cells and solids were removed from the spent culture medium
by centrifugation for 30 min, 50000 g at +4.degree. C. (Sorvall RC6
plus). 50 ml of the supernatant was used for purification of the
protease. After centrifugation, pH of supernatant was adjusted to
8.0 with addition of HCl. The supernatant was then filtered through
0.44 .mu.m filter (MILLEX HV Millipore) and applied to a 5 mL Q
Sepharose FF column (GE Healthcare) equilibrated in 20 mM Tris-HCl,
pH 8. Flow through fraction was collected and its pH was lowered to
7.5 by adding HCl. Solid ammonium sulfate was added to the flow
through fraction to obtain a final salt concentration of 1 M. The
flow through fraction was then filtered through 0.44 .mu.m filter
before applying to phenyl Sepharose HP (1 mL) column (GE
Healthcare) equilibrated in 20 mM Tris-HCl-1M ammonium sulfate pH
7.5. The proteins were eluted with a linear decreasing ammonium
sulfate gradient (from 1 to 0 M). Fractions of 1 ml were collected
and analyzed for protease activity on resorufin-labelled casein
(Boehringer Mannheim Biochemica) at pH 8.0 as instructed by the
manufacturer. Fractions with protease activity were pooled and
ultra filtrated with 10 k membrane (Amicon). The concentrated
filtrate was applied to a Superdex 75 10/300 GL column (GE
Healthcare) equilibrated with 20 mM Tris-HCl-200 mM NaCl, pH 7.5.
Proteins were eluted with the same buffer and 0.5 ml fractions were
collected. The protease activity from these fractions was analysed.
The fractions with protease activity were analyzed on sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The
fractions were shown to contain one major protein band having a
molecular mass of about 29 kDa. The chosen fractions were pooled.
The pooled fractions were used for preparation of peptides (Example
2). According to activity assay measurements, the purified protein
had its pH optimum at pH 10. This purified F. equiseti RF6318
protease is named as Fe_RF6318.
[0213] (c) Protease Activity Assay
[0214] Protease activity was assayed by the casein
Folin--Ciocalteau method using casein as a substrate. Rate of
casein degradation by a protease was measured by
spectrophotometrical monitoring of the release of acid-soluble
fragments as a function of time. Casein substrate used in the assay
was prepared as follows: 6 g of Casein Hammerstein Grade MP
Biomedicals, LLC (101289) was dissolved in 500 ml of 30 mM Tris,
2.0 mM CaCl.sub.2, 0.7 mM MgCl.sub.2, 2.5 mM NaHCO.sub.3. The pH of
the substrate solution was adjusted to 8.5. The enzyme reactions
were stopped using 0.11 M TCA solution. The Folin reagent used in
the assay was prepared by diluting 25 ml of 2 N Folin-Ciocalteu's
phenol reagent (SIGMA, F 9252) to 100 ml by distilled water. The
reaction was started by first incubating 2.5 ml of substrate
solution for 5 min at 50.degree. C. after which 0.5 ml of enzyme
solution was added and reaction was conducted for 30 min. After 30
min reaction 2.5 ml of reaction stop solution was added, the
contents were mixed and allowed to stand for 30 minutes at room
temperature. Tubes were centrifuged 4000 rpm for 10 minutes
(Hettich Rotanta 460). One ml of clear supernatant was mixed with
2.5 ml 0.5 M Na.sub.2CO.sub.3 and 0.5 ml diluted Folin reagent.
After waiting for at least 5 min (color development) the absorbance
of the mixture (colour) was measured at 660 nm against an enzyme
blank. The enzyme blank was prepared as follows: 0.5 ml enzyme
solution was mixed with 2.5 ml stopping solution and 2.5 ml
substrate, and the mixture was incubated for 30 min at 50.degree.
C. One unit of enzyme activity was defined as the enzyme quantity
that liberates the acid soluble protein hydrolysis product
corresponding to 1 .mu.g of tyrosine per ml (or g) of the reaction
mixture per min.
EXAMPLE 2
N-Terminal and Internal Amino Acid Sequencing of the Purified F.
Equiseti protease Fe_RF6318
[0215] For determination of internal sequences, the Coomassie
Brilliant Blue stained band was cut out of the polyacrylamide gel
and "in-gel" digested essentially as described by Shevchenko et al.
(1996). Proteins were reduced with dithiothreitol and alkylated
with iodoacetamide before digestion with trypsin (Sequencing Grade
Modified Trypsin, V5111, Promega).
[0216] Electrospray ionization quadrupole time-of-flight tandem
mass spectra for de novo sequencing were generated using a Q-TOF
instrument (Micromass, Manchester, UK) connected to an Ultimate
nano liquid chromatograph (LC-Packings, The Netherlands) essentally
as described previously (Poutanen et al., 2001) but using a 150
.mu.m.times.1.0 mm trapping column (3 .mu.m, 120 .ANG., #222403,
SGE Ltd UK) for peptide preconcentration.
[0217] For N-terminal sequence analysis SDS-PAGE/separated proteins
were transferred by electroblotting into a polyvinylidine
difluororide membrane (ProBlott; Perkin Elmer Applied Biosystems
Division, Foster City, Calif.). After being stained with Coomassie
brilliant blue, the protein bands of interest were removed and
subjected to N-terminal sequence analysis by Edman degradation on a
Procise 494A protein sequencer (Perkin Elmer Applied Biosystems
Division, Foster City, Calif.)
[0218] The N-terminal and internal peptide sequences determined
from the purified Fe_RF6318 protease are shown in Table 1. The
peptide sequences showed homology to published protease sequences
from Fusarium oxysporum serine proteases with EMBL Accession
numbers Q5R2N9 and 074236.
TABLE-US-00001 TABLE 1 N-terminal and internal peptide sequences
determined from Fe_RF6318 protease Peptide Sequence SEQ ID Comments
#3792 ALTTQSNAPWGLAAISRXTP NO: 1 N-terminal sequence X may be C, S,
T or R 1246.673 TVAAADSSWR NO: 2 3341.633 XTYGVAK NO: 3 1503.799
EA(L/I)TVGATTSADAK NO: 4 Third amino acid is not conclusive, can be
L or I
EXAMPLE 3
Cloning of the F. equiseti RF6318 Gene Encoding Fe_RF6318
Protease
[0219] (a) Isolation of DNA and Molecular Biology Methods used
[0220] Standard molecular biology methods were used in the
isolation and enzyme treatments of DNA (e.g. isolation of plasmid
DNA, digestion of DNA to produce DNA fragments), in E. coli
transformations, sequencing etc. The basic methods used were either
as described by the enzyme, reagent or kit manufacturer or as
described in the standard molecular biology handbooks, e.g.
Sambrook and Russell (2001). Isolation of genomic DNA from F.
equiseti RF6318 was done as described in detail by Raeder and Broda
(1985).
[0221] (b) Primers for Probe Preparation
[0222] The probe for cloning the gene encoding the Fe_RF6318
protein was synthesised by PCR. Degenerate oligos were planned
basing on the amino acid sequences of the peptides obtained from
the purified Fe_RF6318 (Table 1). The sequences of the primers are
shown in Table 2 (SEQ ID NOs: 5-8).
TABLE-US-00002 TABLE 2 Oligonucleotides (SEQ ID NOs: 5-8) used as
PCR primers in probe amplification. Oligos, SEQ ID NOs, oligo
lengths and degeneracies, oligonucleotides and the amino acids of
the peptide used in planning of the oligonucleotide sequence. SEQ
Length Oligo ID NO: (nts) Degeneracy Sequence.sup.(a Peptide.sup.(b
PRO87 5 20 256 CARTCNAAYGCNCCNTGGGG (s) #3792 PRO88 6 20 128
CARAGYAAYGCNCCNTGGGG (s) #3792 PRO89 7 20 2048 GCRTCNGCNGANGTNGTNGC
(as) 1503.799 PRO90 8 20 1024 GCRTCNGCRCTNGTNGTNGC (as) 1503.799
.sup.(aN = A , T, C or G; R = A or G, Y = T or C; "s" in the
parenthesis = sense strand, "as" in the parenthesis = antisense
strand. .sup.(bThe peptide sequences are included in Table 1.
[0223] (c) PCR Reactions and Selection of Probes for Cloning
[0224] F. equiseti RF6318 genomic DNA was used as a template for
probe synthesis. The PCR reaction mixtures contained 10 mM
Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl.sub.2,
0.2 mM dNTPs, 1 .mu.M each primer and 4 units of Dynazyme II DNA
polymerase (Finnzymes, Finland) and approximately 3 .mu.g of
genomic DNA per 100 .mu.l reaction volume. The conditions for the
PCR reactions were the following: 5 min initial denaturation at
96.degree. C., followed by 32 cycles of 1 min at 96.degree. C., 30
sek annealing at 55.5.degree. C., 1 min extension at 72.degree. C.
and a final extension at 72.degree. C. for 5 min. Primer
combination PRO88 (SEQ ID NO:6) and PRO89 (SEQ ID NO:7) produced a
specific DNA product having the expected size (according to
calculations basing on published fungal protease sequences). The
DNA product was isolated and purified from the PCR reaction mixture
and cloned to pCR.RTM.2.1-TOPO.RTM. vector according to the
manufacturers instructions (Invitrogen, USA). The 866 by DNA
fragment was sequenced from this plasmid (SEQ ID NO:9). The
pCR.RTM.2.1-TOPO.RTM. plasmid containing this PCR amplified DNA
fragment was named pALK2521. The E. coli strain RF7664 including
the plasmid pALK2521 was deposited to the DSM collection under the
accession number DSM 22171.
[0225] The deduced amino acid sequence of the PCR fragment included
the sequences of the internal Fe_RF6318 peptides 1246.673 (SEQ ID
NO:2) and 3341.633 (SEQ ID NO:3) (Table1). Also the C-terminal part
of the N-terminal peptide #3792 (SEQ ID NO:1) not included in the
primer was found from the deduced amino acid sequence (Table 1).
This confirms that the DNA fragment obtained from the PCR reaction
was part of the gene encoding the Fe_RF6318 protein and was thus
used as a probe for screening the full length gene from F. equiseti
genomic DNA.
[0226] (d) Cloning of the F. equiseti RF6318 Gene Encoding
Fe_RF6318 Protease F. equiseti genomic DNA was digested with
several restriction enzymes for Southern blot analysis. The
hybridization was performed with the 884 kb EcoRI fragment
including SEQ ID NO:9, cut from the plasmid pALK2521 as a probe
(Example 3c). The above probe was labeled by using digoxigenin
according to supplier's instructions (Roche, Germany).
Hybridisation was performed over night at 65.degree. C. After
hybridization the filters were washed 2.times.5 min at RT using
2.times.SSC-0.1% SDS followed by 2.times.15 min at 65.degree. C.
using 0.1.times.SSC-0.1% SDS.
[0227] Several hybridizing fragments in the F. equiseti genomic DNA
digests could be detected. Genomic Munl and BglII digests contained
hybridizing fragment with approximate sizes of 4.2 kb and 3.2 kb,
respectively. The single hybridizing fragment sizes were large
enough to contain the full length gene encoding the Fe_RF6318
protein according to calculations basing on published fungal
protease sequences. Genomic DNA fragments were isolated from the
RF6318 genomic Munl and BglII digests from the size range of the
hybridizing fragment (approximately 4 kb for Munl digestion and
approximately 3 kb for the BglII digestion). The genomic fragments
were isolated from agarose gel and were cloned to pBluescript II
KS+ (Stratagene, USA) vectors cleaved with EcoRI and BamHI.
Ligation mixtures were transformed to Escherichia coli XL10-Gold
cells (Stratagene) and plated on LB (Luria-Bertani) plates
containing 50-100 .mu.g/ml ampicillin. The E. coli colonies were
screened for positive clones using colonial hybridization with the
pALK2521 insert as a probe. Hybridisation was performed as
described for the RF6318 genomic DNA digests. Four positive BgIII
and eight positive Munl clones were collected from the plates. They
were shown by restriction digestion to contain inserts of expected
sizes. The insert from the Munl fragment ligated to the BamHI
vector was cut with Pstl-SalI double digestion. The insert from the
BglII fragment ligated to the EcoRI vector was cut with PstI-NotI
double digestion. A Southern blot was performed on inserts of the
collected clones (Southern blot hybridization at 68.degree. C. and
washed 2.times.5 min at RT using 2.times..times.SSC- 0.1% SDS
followed by 2.times.15 min at 68.degree. C. using
0.1.times.SSC-0.1% SDS). Three of the collected BglII clones and
six of the collected Munl clones contained the hybridizing fragment
in Southern blot. The full-length gene encoding the Fe_RF6318
protease (SEQ ID NO:10) was sequenced from the 3.2 kb BglII insert
and the plasmid containing this insert was named pALK2529. The E.
coli strain RF7800 including the plasmid pALK2529 was deposited to
the DSM collection under the accession number DSM 22172. The gene
encoding the Fe_RF6318 protein was named as Fe prtS8A.
[0228] (e) Characterisation of the Gene Encoding Fe_RF6318 Protease
and the Deduced Amino Acid Sequence
[0229] The Fe prtS8A sequence (SEQ ID NO:10) and the deduced amino
acid sequence (SEQ ID NO:11) are shown in FIG. 1A-B. The length of
the gene is 1303 by (including the stop codon). One putative intron
was found having the length of 64 bps (5' and 3' border sequences
according to those of fungal introns, according to Gurr et al.
(1987). The deduced protein sequence (SEQ ID NO:11) consists of 412
amino acids including a predicted signal sequence of 20 amino acids
(SignalP V3.0; Nielsen et al., 1997 and Nielsen and Krogh, 1998).
The whole N-terminal peptide #3792 (also the N-terminal part not
included in the probe sequence) was included in the deduced amino
acid sequence. The predicted molecular mass was 29141.09 Da for the
mature polypeptide and the predicted pI was 9.30. These predictions
were made using the Compute pI/MW tool at ExPASy server (Gasteiger
et al., 2003). The deduced amino acid sequence contained two
possible N-glycosylation sites at amino acid positions Asn77 and
Asn255 (FIG. 1), but according to CBS Server NetNGlyc V1.0 only the
site at position Asn77 (located in the pro sequence) is
probable.
[0230] (1) Homology, Identity and Alignment Studies
[0231] The homologies to the published protease sequences were
searched using the BLASTX program version 2.2.9 at NCBI (National
Center for Biotechnology Information) with default settings
(Altschul et al., 1990). The highest homologies were to a
hypothetical protein from Gibberella zeae Fusarium graminearum)
(EMBL accession number XP .sub.--383491) and Trichoderma harzianum
(Hypocrea lixii) serine endopeptidase (EMBL Accession number
CAL25508). Also homology was found to a sequence included in the
patent application US 60/818,910 (Catalyst Bioscience Inc.) as SEQ
ID NO:313. The Fe_RF6318 sequence was aligned with the above
homologous sequences. The identity values obtained by using
ClustalW alignment (www.ebi.ac.uk/Tools/clustalw; Matrix: BLOSUM,
Gap open:10, Gap extension: 0.5) are shown in Table 3.
TABLE-US-00003 TABLE 3 The identity values (%) obtained from
ClustalW alignment of the deduced protease amino acid sequences.
The mature amino acid sequences excluding the signal peptides and
propeptides were aligned. Matrix: BLOSUM, Gap open: 10, Gap
extension: 0.5, EMBL_ EBI. G. zeae, XP_ 383491; T. harzianum
CAL25508; U.S. Pat. No. 60/818,910, SEQ ID NO: 313 in the
application. U.S. Pat. No. Fe_RF6318 G. zeae T. harz. 60/818,910
Fe_RF6318 100 85 75 74 G. zeae 100 78 76 T. harz. 100 94 U.S. Pat.
No. 100 60/818,910
EXAMPLE 4
Production of the Recombinant Fe_RF6318 Protease in Trichoderma
Reesei
[0232] (a) Preparing the Production (Host) Vector
[0233] The expression plasmid pALK2533 was constructed for
production of recombinant Fe_RF6318 protease in Trichoderma reesei.
The Fe prtS8A gene with its own signal sequence was exactly fused
to the T. reesei cbhl (cel7A) promoter by PCR. The Fe prtS8A gene
fragment was excised from its 3'-end by BamHI (a site created after
stop codon in PCR). This leaves no original Fe prtS8A terminator in
the construct prior to the cbhl terminator sequence. An amdS marker
gene was added to the construction including the cbhl promoter and
cbhl terminator. The construction is analogous to that described in
Paloheimo et al. (2003) and the 8.7 kb linear expression cassette
is presented in FIG. 2. The expression cassette was isolated from
the vector backbone after EcoRI digestion and was used for
transforming T. reesei protoplasts. The host strain used does not
produce any of the four major T. reesei cellulases (CBHI, CBHII,
EGI, EGII). The transformations were performed as in Penttila et
al. (1987) with the modifications described in Karhunen et al.
(1993). The transformants were purified on selection plates through
single conidia prior to sporulating them on PD.
[0234] (b) Protease Production in Shake Flasks and Laboratory Scale
Bioreactor
[0235] The transformants were inoculated from the PD slants to
shake flasks containing 50 ml of complex lactose-based cellulase
inducing medium (Joutsjoki et al., 1993) buffered with 5%
KH.sub.2PO.sub.4 at pH 6.0. The protease production of the
transformants was analyzed from the culture supernatants after
growing them for 7 days at 30.degree. C., 250 rpm. In SDS-PAGE
gels, a major protein band of about 29 kDa corresponding to
recombinant Fe_RF6318 protease was detected from the spent culture
supernatants. The protease activity was assayed using casein as a
substrate as described in Example lc. Clearly increased activities
compared to host were measured from the culture supernatants The
integration of the expression cassette into the fungal genomes was
confirmed from chosen transformants by using Southern blot analysis
in which several genomic digests were included and the expression
cassette was used as a probe.
[0236] The T. reesei transformants producing the best protease
activities in the shake flask cultivations were chosen to be
cultivation in laboratory scale bioreactors. Cellulase inducing
complex medium was used in the cultivations. The spent culture
medium obtained from the cultivations was used in application tests
(Examples 6-11) and as starting material for purification and
further characterization of the recombinant Fe_RF6318 protease.
EXAMPLE 5
Purification and Characterization of the Recombinant Fe_RF6318
Protease
[0237] Cells and solids were removed from the spent culture medium
obtained from the fermentation (Example 4) by centrifugation for 30
min, 50000 g at +4.degree. C. (Sorvall RC6 plus). 15 ml of the
supernatant was used for purification of protease. All purification
steps were performed at cold room. After centrifugation, sample was
filtered through 0.44 .mu.m filter (MILLEX HV Millipore) before
applying to HiPrep 26/10 Desalting column (from GE Healthcare)
equilibrated in 20 mM Tris pH 8.8. Gel filtered sample was applied
to a 20 mL Q Sepharose FF column (from GE Healthcare) equilibrated
in 20 mM Tris pH 8.8. The flow through fraction was collected and
analysed on 12% SDS PAGE gel (FIG. 3). This enzyme sample was used
for characterization of pH and temperature profiles.
[0238] Temperature Profile
[0239] Temperature profile was obtained for Fe_RF6318 protease by
using the assay described in Example lc, except using 15 min
reaction time and pH 9.0. The result is shown in FIG. 4A. The
protease has an optimal temperature around 60.degree. C.
[0240] pH-Profile
[0241] The pH profile of the protease was determined at 50.degree.
C. using casein as a substrate as described in Example lc, except
that casein was dissolved in 40 mM Britton-Robinson buffer, the pH
of the reaction was adjusted to pH 6-12, the reaction time was 15
min and the enzyme reactions were stopped using a 0.11 M TCA
solution which contained 0.22 M sodium acetate and 0.33 M acetic
acid. The results are shown in FIG. 4B. The recombinant Fe_RF6318
protease exhibits relative activity over 60% from pH 6 to pH 10
with optimal activity around pH 10. The pH profile of the purified
recombinant Fe_RF6318 protease corresponded to the pH profile of
the wild type Fe_RF6318 protease purifed as described in Example
la.
EXAMPLE 6
Performance of Recombinant Fe_RF6318 Protease at pH 9 Buffer at
Different Temperatures
[0242] Recombinant protein Fe_RF6318 preparation produced in
Trichoderma (as described in Example 4), was tested for its ability
to remove blood/milk/ink standard stain (Art.116, 100% cotton, EMPA
Testmaterialen AG, Switzerland) at temperatures 30.degree. C. and
50.degree. C. Commercial protease preparations Savinase.RTM. Ultra
16 L (Novozymes) and Purafect.RTM. 4000L (Genencor International)
and treatment without enzyme (control) were used for comparison.
The stain fabric was first cut in to 1.5 cm x 1.5 cm swatches and
the pieces were made rounder by cutting the corners. Pieces were
placed in wells of microtiter plates (Nunc 150200). Into each well
having a diameter of 2 cm, 1.5 ml enzyme dilution in Glysine-NaOH
buffer pH 9 was added on top of the fabric. Each enzyme was dosed
0, 0.2, 0.4, 0.8, 1.6, 4, and 8 activity units (.mu.mol
tyrosine/min) per 1.5 ml buffer. Activity was measured using 30 min
reaction time as described in Example 1(c) using 10 min time for
color development after addition of diluted Folin reagent.
Microtiter plates with samples were incubated in a horizontal
shaker at 30.degree. C. and 50.degree. C. for 60 min with 125 rpm.
After that the swatches were carefully rinsed under running water
(appr. 45.degree. C.) and dried overnight at indoor air, on a grid,
protected against daylight.
[0243] The stain removal effect was evaluated by measuring the
colour as reflectance values Minolta CM 2500 spectrophotometer
using L*a*b* colour space coordinates (illuminant) D65/2.degree.).
The colour from both sides of the swatches was measured after the
treatment. Each value was the average of at least 2 parallel fabric
samples measured from both side of the fabric. Fading of
blood/milk/ink stain indicating of the protease performance (stain
removal efficiency) was calculated as .DELTA.L*, which means
lightness value L* of enzyme treated fabric minus lightness value
L* of fabric treated with washing liquor (buffer) without enzyme
(enzyme blank, control).
[0244] The results are shown in FIGS. 5 and 6. Fe_RF6318 protease
preparation showed considerably higher stain removal capacity at
50.degree. C. and especially at 30.degree. C. in pH 9 buffer
compared to commercial protease preparations Savinase.RTM. Ultra
16L and Purafect.RTM. 4000L.
EXAMPLE 7
Performance of Recombinant Protein Fe_RF6318 with Detergent Powder
at 40.degree. C/50.degree. C. and pH 10
[0245] Recombinant protein Fe_RF6318 preparation produced in
Trichoderma (as described in Example 4) was tested for its ability
to remove blood/milk/ink standard stain in the presence of
phosphate containing reference detergent with and without bleaching
agent at 40.degree. C. and 50.degree. C. (pH ca. 10). Standard
stain Art.117 (blood/ milk/ink, polyester+cotton, EMPA) was used as
test material. Commercial protease Purafect.RTM. 4000L and
treatment without enzyme (control) were used for comparison. Each
enzyme was dosed 0, 0.2, 0.4, 0.8, 1.6, 4, and 8 activity units
(.mu.mol tyrosine/min) per ml wash liquor. Activity was measured as
described in Example 6.
[0246] An amount of 3.3 g of phosphate containing ECE reference
detergent 77 without optical brightener (Art. 601, EMPA) was
dissolved in lliter of tap water (water, dH .ltoreq.4), mixed well
with magnetic stirrer and tempered to 40.degree. C./50.degree. C.
Stain fabric was cut into pieces like described in Example 6.
Swatches were placed in well's of microtiter plates (Nunc 150200)
and 1.5 ml wash liquor containing detergent and enzyme dilution in
water (below 60 .mu.l) was added on top of the fabric. The plates
with samples were in incubated in horizontal shaker at 40.degree.
C./50.degree. C. for 60 min with 125 rpm. After that the swatches
were carefully/thoroughly rinsed under running water (appr.
45.degree. C.) and dried overnight at indoor air, on a grid,
protected from daylight. Another test series was made in a same way
except 0.81 g sodium perborate tetrahydrate (Art. 604, EMPA) and
0.16 g bleaching activator TAED tetraacetylethylendiamine (Art.
606, EMPA) was added in addition to detergent.
[0247] The colour of the swatches after treatment was measured with
Minolta CM 2500 spectrophotometer using L*a*b* colour space
coordinates and stain removal effect calculated as .DELTA.L* as
described in example 6.
[0248] The results shown in FIGS. 7A, 7B, 8A and 8B, showed that
protease Fe_RF6318 is also suitable with for powder detergents at
very alkaline conditions and its resistance for bleaching agents
was slightly higher than with commercial protease Purafect.RTM.
4000L.
EXAMPLE 8
Performance of Recombinant Protein Fe_RF6318 with Liquid Detergent
at 40.degree. C.
[0249] Recombinant protein Fe_RF6318 preparation produced in
Trichoderma (as described in Example 4) was tested for its ability
to remove blood/milk/ink standard stain in the presence of liquid
detergent Ariel Sensitive (Procter & Gamble, England), not
containing enzyme, at 40.degree. C. and pH ca. 7.9. Standard stain,
artificially soiled test cloth Art.117 (blood/milk/ink,
polyester+cotton, EMPA) was used as test material. Commercial
protease preparations Purafect.RTM. 4000L, Savinase.RTM. Ultra 16 L
and treatment without enzyme (control) were used for comparison.
Each enzyme was dosed 0, 0.2, 0.4, 0.8, 1.6, 4, and 8 activity
units (.mu.mol tyrosine/min) per ml wash liquor. Activity was
measured as described in Example 6.
[0250] An amount of 3.3 g of Ariel Sensitive was dissolved in 1
liter of tap water (dH .ltoreq.4), mixed well with magnetic stirrer
and tempered to 40.degree. C. Stain fabric was cut into pieces like
described in Example 6. Swatches were placed in well's of
microtiter plates (Nunc 150200) and 1.5 ml wash liquor containing
detergent and enzyme dilution in water (below 60 .mu.l) was added
on top of the fabric. The plates with samples were in incubated in
a horizontal shaker at 40.degree. C. for 60 min with 125 rpm. After
that the swatches were carefully rinsed under running water (appr.
45.degree. C.) and dried overnight at indoor air, on a grid,
protected against daylight.
[0251] The colour of the swatches after treatment was measured with
Minolta CM 2500 spectrophotometer using L*a*b* colour space
coordinates and stain removal effect calculated as .DELTA.L* as
described in example 6.
[0252] Based on the results shown in FIG. 9 Fe_RF6318 protease has
excellent performance with liquid detergent at 40.degree. C. The
efficiency of Fe_RF6318 on blood/milk/ink stain (polyester+cotton)
was considerably higher compared to commercial preparations
Purafect.RTM. 4000L and Savinase.RTM. Ultra 14L, when same amount
of protease activity was dosed. The dosage of 4-8 units of
commercial enzymes per ml of wash liquor was equal to dosage of
about 0.2-0.5% of enzyme preparation per weight of detergent, which
is a typical use level for detergent enzymes.
EXAMPLE 9
Performance of Recombinant Protein Fe_RF6318 with Different Liquid
Detergent Concentrations at 30.degree. C.
[0253] Recombinant protein Fe_RF6318 preparation produced in
Trichoderma (as described in Example 4) was tested for its ability
to remove blood/milk/ink standard stain with liquid detergent at
concentrations 1-5 g/l at 30.degree. C. Ariel Sensitive (Procter
& Gamble, England) containing no enzymes and liquid base
detergent for coloured fabric containing 25% washing active
substances, polyol and polymers (Table 4) were used as detergents
and standard stain Art.117 (blood/milk/ink, cotton+polyester, EMPA)
was used as test material. Commercial protease preparations
Purafect.RTM. 4000L, Savinase.RTM. Ultra 16 L and treatment without
enzyme (control) were used for comparison. Each enzyme was dosed 0,
0.2, 0.4, 0.8, 1.6, 4, and 8 activity units (.mu.mol tyrosine/min)
per ml wash liquor. Activity was measured as described in Example
6.
TABLE-US-00004 TABLE 4 Composition of Liquid Base detergent for
colored fabric Ingredients % NaLES (sodium lauryl ether sulphate)
4.9 Nonionic C12-15 7EO (ethylene oxide) 15 Na-Soap (Palm Kernel
FA) 4.4 Coco Glucoside 1 <Total Surfactant> <25.30>
Polyol (Glycerin) 5 Phosphonate (32%) (ThermPhos) 2 PVP-Sokalan HP
53 (BASF) 1 Sokalan PA 15 (BASF) 1.56 Sorez -100 (ISP) 0.4 Water up
to 100 %
[0254] Amounts of 1, 3.3 and 5 g of liquid detergent was dissolved
in 1 liter of tap water (dH <4), mixed well with magnetic
stirrer and tempered to 30.degree. C. The pH in the wash liquors
was ca. 7.3-7.5 with Base detergent or ca. 7.6-8.0 with Ariel,
depending on detergent concentration. Stain fabric was cut into
pieces like described in Example 6. Swatches were placed in wells
of microtiter plates (Nunc 150200) and 1.5 ml wash liquor
containing detergent and enzyme dilution in water (below 60 .mu.l)
was added on top of the fabric. The plates with samples were in
incubated in a horizontal shaker at 30.degree. C. for 60 min with
125 rpm. After that the swatches were carefully/thoroughly rinsed
under warm running water and dried overnight at indoor air, on a
grid, protected against daylight.
[0255] The colour of the swatches after treatment was measured with
Minolta CM 2500 spectrophotometer using L*a*b* colour space
coordinates and stain removal effect calculated as .DELTA.L* as
described in example 6.
[0256] Results obtained with base detergent for coloured fabrics
are shown in FIGS. 10 A-D and results obtained with Ariel Sensitive
are shown in FIGS. 11A-D. The efficiency of Fe_RF6318 on
blood/milk/ink stains was considerably higher compared to
commercial preparations Purafect.RTM. 4000L and Savinase.RTM. Ultra
14L with both detergents and at all detergent concentrations, when
same amount of activity was dosed. Also if dosing is calculated as
amount of added protein (FIGS. 10B and 11B), the stain removal
efficiency is highest with Fe_RF6318. The amount of protein from
the enzyme preparations was determined by Bio-Rad protein assay
(Bio-Rad Laboratories, Hercules, Calif.) using bovine gammaglobulin
(Bio-Rad) as standard. Results of these tests indicate that
Fe_RF6318 protease has excellent performance with liquid detergents
at low temperatures, like 30.degree. C.
EXAMPLE 10
Efficiency of Recombinant Protein Fe_RF6318 on Different Stains
with Liquid Detergents at 30.degree. C. (Launder Ometer)
[0257] Recombinant protein Fe_RF6318 preparation produced in
Trichoderma (as described in Example 4) was tested for its ability
to remove different stains with liquid detergent at 30.degree. C.
and compared to commercial protease preparations Purafect.RTM.
4000L and/or Savinase Ultra.RTM. 16 L. The following artificially
soiled test cloths from EMPA were used: blood/milk/ink (Art.117,
polyester+cotton), blood/milk/ink (Art.116, cotton), grass (Art.
164, cotton) and cocoa (Art.112, cotton). The fabric was cut in 9
cm x 12 cm swatches and the edges were neated by zig-zag stitches.
Two test series were performed: first with Ariel Sensitive without
enzymes and second later on with a liquid base detergent for
coloured fabric (Example 8). Different batches of stains were used
in the above two experiments.
[0258] Stain removal treatments were performed in LP-2 Launder
Ometer as follows. Launder Ometer was first preheated to 30.degree.
C. 450 ml of tempered wash liquor containing 5 g detergent per
litre tap water (dH .ltoreq.4) and enzyme dilution was added in
containers containing stains Art. 116 and Art. 117 or stains Art.
164 and Art. 112. Each enzyme was dosed 0, 2, 5, 10, and in some
tests 20 or 30 activity units (.mu.mol tyrosine/min) per ml wash
liquor. Activity was measured as described in Example 6. The
Launder Ometer was run at 30.degree. C. for 60 min and pH about 7.5
(base detergent) or 8 (Ariel). After that the swatches were
carefully rinsed under running water (ca. 20.degree. C.) and dried
overnight at indoor air, on a grid, protected against daylight.
[0259] The colour of the swatches after treatment was measured with
Minolta CM 2500 spectrophotometer using L*a*b* color space
coordinates and stain removal effect calculated as .DELTA.L* as
described in Example 6. The colour from both sides of the swatches
was measured after the treatment. Each value was the average of at
least 20 measurements per swatch. The measurements were avoided
from areas with crease marks formed during the treatment because of
the folding of the fabric (in cotton stains Art. 116 and Art.
112).
[0260] Results obtained with Ariel.RTM. Sensitive (FIG. 12 A-C)
show that efficiency on blood/milk/ink and grass stains was
considerably higher with Fe_RF6318 compared to commercial
preparation Savinase.RTM. Ultra 16L at 30.degree. C. when same
amount of protease activity was dosed. Also results obtained with
cocoa stain were better with Fe_RF6318 (data not shown).
[0261] Also results obtained with base detergent (5g/l) for
coloured fabrics (FIG. 13 A-D) showed that efficiency on
blood/milk/ink, grass and cocoa stains was considerably higher with
Fe_RF6318 compared to commercial preparations Savinase.RTM. Ultra
16L and Purafect.RTM. 4000L at 30.degree. C., when same amount of
protease activity was dosed. The dosage of 10 units of commercial
enzymes per ml of liquor was equal to dosage approximately 0.4% of
enzyme preparation per weight of detergent, which is in typical use
level for detergent enzymes. Results of these tests indicate that
Fe_RF6318 protease is efficient on several stains at low
temperatures like 30.degree. C.
EXAMPLE 11
Evaluation of the Performance of the Recombinant Protein Fe_RF6318
in Liquid Laundry Detergent in Full Scale Trials at 30.degree.
C.
[0262] The performance of recombinant protein Fe_RF6318 preparation
produced in Trichoderma (Example 4) was tested in liquid detergent
in full scale in a washing machine at 30.degree. C. and compared to
commercial protease preparations Purafect.RTM. 4000L and
Savinase.RTM. Ultra 16L and treatment with detergent without
enzyme. Liquid base detergent for coloured fabrics, as described in
Example 9, and 8 different protease sensitive tracers (Table 5)
were used. In addition two pieces of ballast soil (CFT-SBL) per
wash were placed in wash net to avoid contamination by contact with
the other swatches. Tracers were from CFT (Center For Testmaterials
BV, The Netherlands). Stain swatches 10 cm.times.10 cm were
stitched to kitchen towels. The process parameters and conditions
are described in Table 6. Enzyme dosages used in the trials were
calculated both as enzyme activities (ca. 0-14 activity units per
ml wash liquor) and as amount of protein (ca. 0-2.2 mg per litre
wash liquor). Protease activities and protein contents of the
preparations were measured as described in Examples 6 and 9.
TABLE-US-00005 TABLE 5 Protease sensitive tracers used in test. Nr.
Monitor/Swatch Substrate 1 CFT/CS-01-106 Blood (aged)/Cotton 2
CFT/C-03-030 Chocolate milk/pigment/Cotton 3 CFT/C-05-059b
Bood/milk/ink/Cotton 4 CFT/PC-05-014 Blood/Milk/Ink/PE-Cotton 5
CFT/CS-08-069 Grass/Cotton 6 CFT/C-10-186b Groundnut
oil/milk/Cotton 7 CFT/CS-25-016 Spinach/Cotton 8 CFT/CS-38-010 Egg
Yolk/Pigment/Cotton
TABLE-US-00006 TABLE 6 Process parameters and conditions Machine
Miele N-Tronic Frontstar Program 30.degree. C., short program
Hardness of water about 11.2.degree. dH with 13 liter intake
Ballast Load 2.5 kg bed sheets/bath towels,and kitchen towels
Detergent dosage 80 g/machine load Amount of each tracers 2 stain
trips 10 cm .times. 10 cm per machine Number of washes 2
[0263] Evaluation of stain removal effect was based on measurement
of reflectance (R 457 nm) with Datacolor- spectrophotometer with an
UV-filter and calculated as percentual stain removal effect (%
SR):
% SR = Reflectance after washing - Reflectance before washing
.times. 100 % Reflectance of unsoiled fabric - Reflectance before
washing ##EQU00001##
[0264] Results were calculated as A% SR (delta % SR), which means %
SR of enzyme treated fabric minus % SR of treatment without enzyme
(detergent only).
[0265] Two washes containing two swatches of each stain were
performed with each dosage of enzyme and three measurements were
measured of each stain swatch, so the values are based upon the 12
measurements per stain per product.
[0266] The results of total soil removal efficiencies (delta % SR)
are shown in FIGS. 14A-B and FIGS. 15 A-E. The total stain removal
effect based on the sum of the results obtained with the eight
different protease sensitive stains (Stains 1-8, table was higher
with Fe_RF6318 compared to commercial protease preparations
Purafect.RTM. 4000L and Savinase.RTM. Ultra 16L, when proteases
were dosed as amount of activity and as protein per volume of wash
liquor (FIGS. 14A-B). Fe_RF6318 was efficient especially on
blood/milk/ink, chocolate/milk, groundnut oil/milk and egg yolk
(FIGS. 15A-E).
[0267] A general detergency tracer (pigment/oil) was used as a
control in repeats in different machines. No differencies between
the various tests were observed.
EXAMPLE 12
Performance of Recombinant Protein Fe_RF6318 in pH 9 Buffer at
Temperatures from 10.degree. C. to 60.degree. C.
[0268] Recombinant protein Fe_RF6318 produced in Trichoderma (as
described in Example 4) was tested for its ability to remove
blood/milk/ink standard stain (Art.117, polyester+cotton, EMPA
Testmaterialen AG, Swizerland) at temperatures from 10 to
60.degree. C. Commercial protease preparations Savinase.RTM. Ultra
16 L, Purafect.RTM. 4000 L and Properase.RTM. 4000 E and treatment
without enzyme (control) were used for comparison. The tests were
performed at pH 9 buffer as described in Example 6, except the
incubation temperature range was broader, stain material was
different and the temperature of the rinsing water of the swatches
was similar to washing temperature. The colour of the swatches
after treatment was measured with Minolta CM 2500 spectrophotometer
using L*a*b* colour space coordinates and stain removal effect
calculated as.DELTA.L* as described in Example 6.
[0269] The results are shown in FIGS. 16A-F. Fe_RF6318 protease
preparation showed higher stain removal capacity at whole
temperature range from 10.degree. C. to 60.degree. C. in pH 9
buffer, compared to commercial protease preparations Savinase.RTM.
Ultra 16L and Purafect.RTM. 4000L. It also showed higher stain
removal capacity at range from 30.degree. C. to 60.degree. C.
compared to Properase.RTM. 4000E.
EXAMPLE 13
Performance of Recombinant Protein Fe_RF6318 with Liquid Detergent
at Cold Washing Temperatures 10.degree. C. and 20.degree. C.
[0270] Recombinant protein Fe_RF6318 produced in Trichoderma (as
described in Example 2) was tested for its ability to remove
blood/milk/ink standard stain (Art.117, cotton+polyester, EMPA)
with Liquid Base detergent at low temperatures. The testing method
was similar to Example 9, except only detergent concentration of
3.3 g/l and lower incubation temperatures, 10.degree. C. and
20.degree. C., were used. The temperature of the rinsing water of
the swatches was similar to washing temperature.
[0271] The colour of the swatches after treatment was measured with
Minolta CM 2500 spectrophotometer using L*a*b* colour space
coordinates and stain removal effect calculated as .DELTA.L* as
described in Example 6. For treatment without enzyme (enzyme
blank), detergent solution was used as washing liquor.
[0272] Results obtained with Liquid Base detergent concentration of
3.3 g/l at 10.degree. C. and 20.degree. C. are shown in FIGS. 17A
and B. The efficiency of Fe_RF6318 on blood/milk/ink stain was
considerably higher both at 10.degree. C. and 20.degree. C.
compared to commercial preparations Savinase.RTM. Ultra 16L,
Purafect.RTM. 4000L and Properase.RTM. 4000E, when same amount of
activity was dosed. Results of these tests indicate that Fe_RF6318
protease has excellent performance with liquid detergents at very
low washing temperatures.
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Sequence CWU 1
1
15120PRTFusarium equisetiMISC_FEATURESequence of an aminoterminal
peptide #3792 from Fusarium equiseti RF6318 protease. 1Ala Leu Thr
Thr Gln Ser Asn Ala Pro Trp Gly Leu Ala Ala Ile Ser1 5 10 15Arg Cys
Thr Pro 20210PRTFusarium equisetiMISC_FEATURESequence of a tryptic
peptide 1246.673 from Fusarium equiseti RF6318 protease. 2Thr Val
Ala Ala Ala Asp Ser Ser Trp Arg1 5 1037PRTFusarium
equisetiMISC_FEATURESequence of a tryptic peptide 3341.633 from
Fusarium equiseti RF6318 protease. 3Xaa Thr Tyr Gly Val Ala Lys1
5414PRTFusarium equisetiMISC_FEATURESequence of a tryptic peptide
1503.799 from Fusarium equiseti RF6318 protease 4Glu Ala Leu Thr
Val Gly Ala Thr Thr Ser Ala Asp Ala Lys1 5 10520DNAArtificial
SequenceThe sequence of the oligonucleotide primer PRO87 derived
from the aminoterminal peptide SEQ ID NO1. 5cartcnaayg cnccntgggg
20620DNAArtificial SequenceThe sequence of the oligonucleotide
primer PRO88 derived from the aminoterminal peptide SEQ ID NO1.
6caragyaayg cnccntgggg 20720DNAArtificial SequenceThe sequence of
the oligonucleotide primer PRO89 derived from peptide SEQ ID NO4.
7gcrtcngcng angtngtngc 20820DNAArtificial SequenceThe sequence of
the oligonucleotide primer PRO90 derived from peptide SEQ ID NO4.
8gcrtcngcrc tngtngtngc 209866DNAArtificial SequenceThe sequence of
the PCR fragment obtained using the primers PRO88 (SEQ ID NO6) and
PRO89 (SEQ ID NO7) and Fusarium equiseti RF6318 genomic DNA as a
template. 9aaagcaacgc accgtggggt cttgctgcca tctcccgccg aacccccggt
ggcagcacct 60acacctacga caccactgcc ggtgccggta cttacggtta cgtcgttgac
tctggtatca 120acaccgccca cactgacttt ggcggccgtg cttctctcgg
ttacaacgct gctggtggcg 180cccacactga tacccttggc cacggtaccc
acgttgctgg taccattgcc tccaacacct 240acggtgttgc caagcgtgta
agtacaatca taccccacat gagctacaac atgatctgaa 300ctttatactt
actattatta ggccaacgtc atctctgtca aggttttcgt cggtaaccaa
360gcttctacct ctgttatcct tgctggtttc aactgggctg tcaacgacat
cacctccaga 420accgtgctag cccgctctgt catcaacatg tctctcggtg
gtccctcttc tcagacctgg 480gctactgcca tcaacgctgc ctacagccaa
ggtgtcctct ccgttgttgc tgccggtaac 540ggtgattcca acggtcgtcc
tctccccgcc tctggccagt ctcctgccaa cgttcccaac 600gctatcaccg
ttgctgccgc cgactccagc tggcgaactg cctctttcac caactacggt
660cctgaggtcg atgtcttcgg tcctggtgtc aacatccagt ccacctggta
cacctccaac 720agcgctacca acaccatcag cggtacctcc atggcttgcc
ctcacgttgc tggtcttgct 780ctctacctcc aggctctcga gaacctcaat
acccctgctg ccgtcaccaa ccgcatcaag 840tctcttgcaa ctacctccgc tgacgc
866101303DNAFusarium equisetimisc_featureThe nucleotide sequence of
the full-length Fusarium equiseti RF6318 protease gene (Fe prtS8A)
. 10atgactagct tccgccgtat cgctcttggc cttgcagctc tgctgcccgc
agtcctcgcc 60gctcccaccg agaagcgaca ggagctcact gccgcgcctg acaagtacat
catcaccctc 120aagcccgagg ctgctgaggc caaggtcgag gctcacatgg
cctgggttac cgacgtccac 180cgccgcagcc tcggcaagcg tgacacttcc
ggtgttgaga agaagttcaa catcagcagc 240tggaacgcct actctggcga
gttcgacgat gctaccattg ctgagatcaa gaagagcccc 300gaggttgcct
tcgtcgagcc cgactacatt gtcaccctcg actacaaggt tgagcctctc
360tctgaccgtg ctctgaccac tcagagcaac gctccttggg gtcttgctgc
catctcccgc 420cgaacccccg gtggcagcac ctacacctac gacaccactg
ccggtgccgg tacttacggt 480tacgtcgttg actctggtat caacaccgcc
cacactgact ttggcggccg tgcttctctc 540ggttacaacg ctgctggtgg
cgcccacact gatacccttg gccacggtac ccacgttgct 600ggtaccattg
cctccaacac ctacggtgtt gccaagcgtg taagtacaat cataccccac
660atgagctaca acatgatctg aactttatac ttactattat taggccaacg
tcatctctgt 720caaggttttc gtcggtaacc aagcttctac ctctgttatc
cttgctggtt tcaactgggc 780tgtcaacgac atcacctcca agaaccgtgc
tagccgctct gtcatcaaca tgtctctcgg 840tggtccctct tctcagacct
gggctactgc catcaacgct gcctacagcc aaggtgtcct 900ctccgttgtt
gctgccggta acggtgattc caacggtcgt cctctccccg cctctggcca
960gtctcctgcc aacgttccca acgctatcac cgttgctgcc gccgactcca
gctggcgaac 1020tgcctctttc accaactacg gtcctgaggt cgatgtcttc
ggtcctggtg tcaacatcca 1080gtccacctgg tacacctcca acagcgctac
caacaccatc agcggtacct ccatggcttg 1140ccctcacgtt gctggtcttg
ctctctacct ccaggctctc gagaacctca atacccctgc 1200tgccgtcacc
aaccgcatca agtctcttgc cactaccggc cgcatcactg gcagcctcag
1260cggcagcccc aacgccatgg ctttcaacgg cgctactgct taa
130311412PRTFusarium equisetiMISC_FEATUREThe deduced amino acid
sequence of the full-length Fusarium equiseti RF6318 protease
(Fe_RF6318) including amino acids from Met1 to Ala412. 11Met Thr
Ser Phe Arg Arg Ile Ala Leu Gly Leu Ala Ala Leu Leu Pro1 5 10 15Ala
Val Leu Ala Ala Pro Thr Glu Lys Arg Gln Glu Leu Thr Ala Ala 20 25
30Pro Asp Lys Tyr Ile Ile Thr Leu Lys Pro Glu Ala Ala Glu Ala Lys
35 40 45Val Glu Ala His Met Ala Trp Val Thr Asp Val His Arg Arg Ser
Leu 50 55 60Gly Lys Arg Asp Thr Ser Gly Val Glu Lys Lys Phe Asn Ile
Ser Ser65 70 75 80Trp Asn Ala Tyr Ser Gly Glu Phe Asp Asp Ala Thr
Ile Ala Glu Ile 85 90 95Lys Lys Ser Pro Glu Val Ala Phe Val Glu Pro
Asp Tyr Ile Val Thr 100 105 110Leu Asp Tyr Lys Val Glu Pro Leu Ser
Asp Arg Ala Leu Thr Thr Gln 115 120 125Ser Asn Ala Pro Trp Gly Leu
Ala Ala Ile Ser Arg Arg Thr Pro Gly 130 135 140Gly Ser Thr Tyr Thr
Tyr Asp Thr Thr Ala Gly Ala Gly Thr Tyr Gly145 150 155 160Tyr Val
Val Asp Ser Gly Ile Asn Thr Ala His Thr Asp Phe Gly Gly 165 170
175Arg Ala Ser Leu Gly Tyr Asn Ala Ala Gly Gly Ala His Thr Asp Thr
180 185 190Leu Gly His Gly Thr His Val Ala Gly Thr Ile Ala Ser Asn
Thr Tyr 195 200 205Gly Val Ala Lys Arg Ala Asn Val Ile Ser Val Lys
Val Phe Val Gly 210 215 220Asn Gln Ala Ser Thr Ser Val Ile Leu Ala
Gly Phe Asn Trp Ala Val225 230 235 240Asn Asp Ile Thr Ser Lys Asn
Arg Ala Ser Arg Ser Val Ile Asn Met 245 250 255Ser Leu Gly Gly Pro
Ser Ser Gln Thr Trp Ala Thr Ala Ile Asn Ala 260 265 270Ala Tyr Ser
Gln Gly Val Leu Ser Val Val Ala Ala Gly Asn Gly Asp 275 280 285Ser
Asn Gly Arg Pro Leu Pro Ala Ser Gly Gln Ser Pro Ala Asn Val 290 295
300Pro Asn Ala Ile Thr Val Ala Ala Ala Asp Ser Ser Trp Arg Thr
Ala305 310 315 320Ser Phe Thr Asn Tyr Gly Pro Glu Val Asp Val Phe
Gly Pro Gly Val 325 330 335Asn Ile Gln Ser Thr Trp Tyr Thr Ser Asn
Ser Ala Thr Asn Thr Ile 340 345 350Ser Gly Thr Ser Met Ala Cys Pro
His Val Ala Gly Leu Ala Leu Tyr 355 360 365Leu Gln Ala Leu Glu Asn
Leu Asn Thr Pro Ala Ala Val Thr Asn Arg 370 375 380Ile Lys Ser Leu
Ala Thr Thr Gly Arg Ile Thr Gly Ser Leu Ser Gly385 390 395 400Ser
Pro Asn Ala Met Ala Phe Asn Gly Ala Thr Ala 405
410121243DNAFusarium equisetimisc_featureThe nucleotide sequence
encoding the amino acid sequence of the proenzyme form of Fusarium
equiseti RF6318 protease. 12gctcccaccg agaagcgaca ggagctcact
gccgcgcctg acaagtacat catcaccctc 60aagcccgagg ctgctgaggc caaggtcgag
gctcacatgg cctgggttac cgacgtccac 120cgccgcagcc tcggcaagcg
tgacacttcc ggtgttgaga agaagttcaa catcagcagc 180tggaacgcct
actctggcga gttcgacgat gctaccattg ctgagatcaa gaagagcccc
240gaggttgcct tcgtcgagcc cgactacatt gtcaccctcg actacaaggt
tgagcctctc 300tctgaccgtg ctctgaccac tcagagcaac gctccttggg
gtcttgctgc catctcccgc 360cgaacccccg gtggcagcac ctacacctac
gacaccactg ccggtgccgg tacttacggt 420tacgtcgttg actctggtat
caacaccgcc cacactgact ttggcggccg tgcttctctc 480ggttacaacg
ctgctggtgg cgcccacact gatacccttg gccacggtac ccacgttgct
540ggtaccattg cctccaacac ctacggtgtt gccaagcgtg taagtacaat
cataccccac 600atgagctaca acatgatctg aactttatac ttactattat
taggccaacg tcatctctgt 660caaggttttc gtcggtaacc aagcttctac
ctctgttatc cttgctggtt tcaactgggc 720tgtcaacgac atcacctcca
agaaccgtgc tagccgctct gtcatcaaca tgtctctcgg 780tggtccctct
tctcagacct gggctactgc catcaacgct gcctacagcc aaggtgtcct
840ctccgttgtt gctgccggta acggtgattc caacggtcgt cctctccccg
cctctggcca 900gtctcctgcc aacgttccca acgctatcac cgttgctgcc
gccgactcca gctggcgaac 960tgcctctttc accaactacg gtcctgaggt
cgatgtcttc ggtcctggtg tcaacatcca 1020gtccacctgg tacacctcca
acagcgctac caacaccatc agcggtacct ccatggcttg 1080ccctcacgtt
gctggtcttg ctctctacct ccaggctctc gagaacctca atacccctgc
1140tgccgtcacc aaccgcatca agtctcttgc cactaccggc cgcatcactg
gcagcctcag 1200cggcagcccc aacgccatgg ctttcaacgg cgctactgct taa
124313392PRTFusarium equisetiMISC_FEATUREThe amino acid sequence of
the proenzyme form of Fusarium equiseti RF6318 protease including
amino acids Ala21 to Ala 412 of the full length protease. 13Ala Pro
Thr Glu Lys Arg Gln Glu Leu Thr Ala Ala Pro Asp Lys Tyr1 5 10 15Ile
Ile Thr Leu Lys Pro Glu Ala Ala Glu Ala Lys Val Glu Ala His 20 25
30Met Ala Trp Val Thr Asp Val His Arg Arg Ser Leu Gly Lys Arg Asp
35 40 45Thr Ser Gly Val Glu Lys Lys Phe Asn Ile Ser Ser Trp Asn Ala
Tyr 50 55 60Ser Gly Glu Phe Asp Asp Ala Thr Ile Ala Glu Ile Lys Lys
Ser Pro65 70 75 80Glu Val Ala Phe Val Glu Pro Asp Tyr Ile Val Thr
Leu Asp Tyr Lys 85 90 95Val Glu Pro Leu Ser Asp Arg Ala Leu Thr Thr
Gln Ser Asn Ala Pro 100 105 110Trp Gly Leu Ala Ala Ile Ser Arg Arg
Thr Pro Gly Gly Ser Thr Tyr 115 120 125Thr Tyr Asp Thr Thr Ala Gly
Ala Gly Thr Tyr Gly Tyr Val Val Asp 130 135 140Ser Gly Ile Asn Thr
Ala His Thr Asp Phe Gly Gly Arg Ala Ser Leu145 150 155 160Gly Tyr
Asn Ala Ala Gly Gly Ala His Thr Asp Thr Leu Gly His Gly 165 170
175Thr His Val Ala Gly Thr Ile Ala Ser Asn Thr Tyr Gly Val Ala Lys
180 185 190Arg Ala Asn Val Ile Ser Val Lys Val Phe Val Gly Asn Gln
Ala Ser 195 200 205Thr Ser Val Ile Leu Ala Gly Phe Asn Trp Ala Val
Asn Asp Ile Thr 210 215 220Ser Lys Asn Arg Ala Ser Arg Ser Val Ile
Asn Met Ser Leu Gly Gly225 230 235 240Pro Ser Ser Gln Thr Trp Ala
Thr Ala Ile Asn Ala Ala Tyr Ser Gln 245 250 255Gly Val Leu Ser Val
Val Ala Ala Gly Asn Gly Asp Ser Asn Gly Arg 260 265 270Pro Leu Pro
Ala Ser Gly Gln Ser Pro Ala Asn Val Pro Asn Ala Ile 275 280 285Thr
Val Ala Ala Ala Asp Ser Ser Trp Arg Thr Ala Ser Phe Thr Asn 290 295
300Tyr Gly Pro Glu Val Asp Val Phe Gly Pro Gly Val Asn Ile Gln
Ser305 310 315 320Thr Trp Tyr Thr Ser Asn Ser Ala Thr Asn Thr Ile
Ser Gly Thr Ser 325 330 335Met Ala Cys Pro His Val Ala Gly Leu Ala
Leu Tyr Leu Gln Ala Leu 340 345 350Glu Asn Leu Asn Thr Pro Ala Ala
Val Thr Asn Arg Ile Lys Ser Leu 355 360 365Ala Thr Thr Gly Arg Ile
Thr Gly Ser Leu Ser Gly Ser Pro Asn Ala 370 375 380Met Ala Phe Asn
Gly Ala Thr Ala385 39014934DNAFusarium equisetimisc_featureThe
nucleotide sequence encoding the amino acid sequence of the mature
form of Fusarium equiseti RF6318 protease. 14gctctgacca ctcagagcaa
cgctccttgg ggtcttgctg ccatctcccg ccgaaccccc 60ggtggcagca cctacaccta
cgacaccact gccggtgccg gtacttacgg ttacgtcgtt 120gactctggta
tcaacaccgc ccacactgac tttggcggcc gtgcttctct cggttacaac
180gctgctggtg gcgcccacac tgataccctt ggccacggta cccacgttgc
tggtaccatt 240gcctccaaca cctacggtgt tgccaagcgt gtaagtacaa
tcatacccca catgagctac 300aacatgatct gaactttata cttactatta
ttaggccaac gtcatctctg tcaaggtttt 360cgtcggtaac caagcttcta
cctctgttat ccttgctggt ttcaactggg ctgtcaacga 420catcacctcc
aagaaccgtg ctagccgctc tgtcatcaac atgtctctcg gtggtccctc
480ttctcagacc tgggctactg ccatcaacgc tgcctacagc caaggtgtcc
tctccgttgt 540tgctgccggt aacggtgatt ccaacggtcg tcctctcccc
gcctctggcc agtctcctgc 600caacgttccc aacgctatca ccgttgctgc
cgccgactcc agctggcgaa ctgcctcttt 660caccaactac ggtcctgagg
tcgatgtctt cggtcctggt gtcaacatcc agtccacctg 720gtacacctcc
aacagcgcta ccaacaccat cagcggtacc tccatggctt gccctcacgt
780tgctggtctt gctctctacc tccaggctct cgagaacctc aatacccctg
ctgccgtcac 840caaccgcatc aagtctcttg ccactaccgg ccgcatcact
ggcagcctca gcggcagccc 900caacgccatg gctttcaacg gcgctactgc ttaa
93415289PRTFusarium equisetiMISC_FEATUREThe amino acid sequence of
the mature form of Fusarium equiseti RF6318 protease including
amino acids Ala124 to Ala412 of the full legth enzyme. 15Ala Leu
Thr Thr Gln Ser Asn Ala Pro Trp Gly Leu Ala Ala Ile Ser1 5 10 15Arg
Arg Thr Pro Gly Gly Ser Thr Tyr Thr Tyr Asp Thr Thr Ala Gly 20 25
30Ala Gly Thr Tyr Gly Tyr Val Val Asp Ser Gly Ile Asn Thr Ala His
35 40 45Thr Asp Phe Gly Gly Arg Ala Ser Leu Gly Tyr Asn Ala Ala Gly
Gly 50 55 60Ala His Thr Asp Thr Leu Gly His Gly Thr His Val Ala Gly
Thr Ile65 70 75 80Ala Ser Asn Thr Tyr Gly Val Ala Lys Arg Ala Asn
Val Ile Ser Val 85 90 95Lys Val Phe Val Gly Asn Gln Ala Ser Thr Ser
Val Ile Leu Ala Gly 100 105 110Phe Asn Trp Ala Val Asn Asp Ile Thr
Ser Lys Asn Arg Ala Ser Arg 115 120 125Ser Val Ile Asn Met Ser Leu
Gly Gly Pro Ser Ser Gln Thr Trp Ala 130 135 140Thr Ala Ile Asn Ala
Ala Tyr Ser Gln Gly Val Leu Ser Val Val Ala145 150 155 160Ala Gly
Asn Gly Asp Ser Asn Gly Arg Pro Leu Pro Ala Ser Gly Gln 165 170
175Ser Pro Ala Asn Val Pro Asn Ala Ile Thr Val Ala Ala Ala Asp Ser
180 185 190Ser Trp Arg Thr Ala Ser Phe Thr Asn Tyr Gly Pro Glu Val
Asp Val 195 200 205Phe Gly Pro Gly Val Asn Ile Gln Ser Thr Trp Tyr
Thr Ser Asn Ser 210 215 220Ala Thr Asn Thr Ile Ser Gly Thr Ser Met
Ala Cys Pro His Val Ala225 230 235 240Gly Leu Ala Leu Tyr Leu Gln
Ala Leu Glu Asn Leu Asn Thr Pro Ala 245 250 255Ala Val Thr Asn Arg
Ile Lys Ser Leu Ala Thr Thr Gly Arg Ile Thr 260 265 270Gly Ser Leu
Ser Gly Ser Pro Asn Ala Met Ala Phe Asn Gly Ala Thr 275 280
285Ala
* * * * *
References