Note: Descriptions are shown in the official language in which they were submitted.
W094/14963 2 1 ~ PCT~3/03550
MODIFIED CUTINASES, DNA, VECTOR AND HOST
TECHNICAL FIELD
The present invention generally relates to the
field of lipolytic enzymes. More in particular, the invention
is concerned with lipolytic enzymes which have been modified
by means of recombinant DNA techniques, with methods for
their production and with their use, particularly in
enzymatic detergent compositions.
BACKGROUND AND PRIOR ART
Lipolytic enzymes are enzymes which are capable of
hydrolysing triglycerides into free fatty acids and
diglycerides, monoglycerides and eventually glycerol. They
15 can also split more complex esters such as cutin layers in
plants or sebum of the skin. Lipolytic enzymes are used in
industry for various enzymatic processes such as the inter-
and trans-esterification of triglycerides and the synthesis
of esters. They are also used in detergent compositions with
20 the aim to improve the fat-removing properties of the
detergent product.
The most widely used lipolytic enzymes are lipases
(EC 3.1.1.3). For example, EP-A-258 068 and EP-A-305 216
(both Novo Nordisk) both describe production of fungal
lipases via heterologous host micro-orgAni~m~ by means of
rDNA techniques, especially the lipase from Thermomyces
lanuqinosus/Humicola lanuqinosa. EP-A-331 376 (Amano)
describes lipases and their production by rDNA t~chn; ques,
and their use, including an amino acid sequence of lipase
30 from Pseudomonas cepacia. Further examples of lipases
produced by rDNA technique are given in W0-A-89/09263 and EP-
A-218 272 (both Gist-Brocades). In spite of the large number
of publications on lipases and their modifications, only the
lipase from Humicola lanuqinosa has so far found wide-spread
commercial application as additive for detergent products
under the trade name Lipolase (TM).
A characteristic feature of lipases is that they
exhibit interfacial activation. This means that the enzyme
activity is much higher on a substrate which has formed
WO94/14963 PCT~3/03~50 ~
215~8~7 2
interfaces or micelles, than on fully dissolved substrate.
Interface activation is reflected in a sudden increase in
lipolytic activity when the substrate concentration is raised
above the critical micel concentration (CMC) of the
5 substrate, and interfaces are formed. Experimentally this
phenomenon can be observed as a discontinuity in the graph of
enzyme activity versus substrate concentration.
The mechanism of interfacial activation in lipases
has been interpreted in terms of a conformation change in the
lO protein structure of the lipase molecule. In the free,
unbound state, a helical lid covers the catalytic binding
site. Upon binding to the lipid substrate, the lid is
displaced and the catalytic site is exposed. The helical lid
is also believed to interact with the lipid interface, thus
15 allowing the enzyme to remain bound to the interface.
WO-A-92/05249 (Novo Nordisk) discloses genetically
modified lipases, in particular the lipase from Humicola
lanuqinosa, which have been modified at the lipid contact
zone. The lipid contact zone is defined in the application as
20 the surface which in the active form is covered by the
helical lid. The modifications involve deletion or
substitution of one or more amino acid residues in the lipid
contact zone, so as to increase the electrostatic charge
and/or decrease the hydrophobicity of the lipid contact zone,
25 or so as to change the surface conformation of the lipid
contact zone. This is achieved by deleting one or more
negatively charged amino acid residues in the lipid contact
zone, or substituting these residues by neutral or more
positively charged amino acids, and/or by substituting one or
30 more neutral amino acid residues in the lipid contact zone by
positively charged amino acids, and/or deleting one or more
hydrophillic amino acid residues in the lipid contact zone,
or substituting these residues by hydrophobic amino acids.
Cutinases are a sub-class of enzymes (EC 3.l.1.50),
35 the wax ester hydrolases. These enzymes are capable of
degrading cutin, a network of esterified long-chain fatty
acids and fatty alcohols which occurs in plants as a
protective coating on leaves and stems. In addition, they
~ 215~837
W094/14963 ~ PCT~3/03550
possess some lipolytic activity, i.e. they are capable of
hydrolysing triglycerides. Thus they can be regarded as a
special kind of lipases. Contrary to lipases, however,
cutinases do not exhibit any substantial interfacial
5 activation.
Cutinases can be obtained from a number of sources,
such as plants (e.g. pollen), bacteria and fungi. Because of
their fat degrading properties, cutinases have been proposed
as ingredients for enzymatic detergent compositions. For
10 example, WO-A-88/09367 (Genencor) suggests combinations of a
surfactant and a substantially pure bacterial cutinase enzyme
to formulate effective cleaning compositions. Disclosed are
detergent compositions comprising a cutinase obtained from
the Gram negative bacterium Pseudomonas putida ATCC 53552.
15 However, in the more recent European patent application EP-A-
476 915 (Clorox), it is disclosed that the same enzyme -
which is then referred to as a lipase - is no more effective
than other lipases in removing oil stains from fabrics, when
used by conventional methods.
Recently, the three-dimensional structure has been
determined of a cutinase from Fusarium solani Pisi (Martinez
et al. (1992) Nature 356, 615-618). It was found that this
cutinase does not possess a helical lid to cover the
catalytic binding site. Instead, the active site serine
25 residue appears to be accessible to the solvent. These
findings appear to confirm the present theory about the
mer-h~n;~m of interfacial activation in lipases.
The cutinase gene from Fusarium solani Pisi has
been cloned and sequenced (Ettinger et al., (1987)
30 Biochemistry 26, 7883-7892). WO-A-90/09446 (Plant Genetics
Systems) describes the cloning and production of this gene in
E. coli. The cutinase can efficiently catalyse the hydrolysis
and the synthesis of esters in aqueous and non-aqueous media,
both in the absence and the presence of and interface between
35 the cutinase and the substrate. On the basis of its qeneral
stability, it is suggested that this cutinase could be used
to produce cleaning agents such as laundry detergents and
other specialized fat dissolving preparations such as
WO94/14963 21~ ~ 8 3 7 PCT~W3/03550
cosmetic compositions and shampoos. A way to produce the
enzyme in an economic feasible way is not disclosed, neither
are specific enzymatic detergent compositions containing the
cutinase.
Because of this characteristic feature, i.e. the
absence of interfacial activation, we define for the purpose
of this patent application Cutinases as lipolytic enzymes
which exhibit substantially no interfacial activation.
Cutinases therefor differ from classical lipases in that they
lO do not possess a helical lid covering the catalytic binding
site.
As mentioned above, only the lipase derived from
Humicola lanuqinosa has so far found wide-spread commercial
application as additive for detergent products under the
15 trade name Lipolase (TM). In his article in Chemistry and
Industry 1990, pages 183-186, Henrik Malmos notes that it is
known that generally the activity of lipases during the
washing process is low, and Lipolase (TM) is no exception.
During the drying process, when the water content of the
20 fabric is reduced, the enzyme regains its activity and the
fatty stains are hydrolysed. During the following wash cycle
the hydrolysed material is removed. This also explains why
the effect of lipases is low after the first washing cycle,
but significant in the following cycles. Thus, there is still
25 a need for lipolytic enzymes which exhibit any significant
activity during the washing process.
We have found that Cutinases, in particular the
cutinase from Fusarium solani Pisi, exhibit a clear in-the-
wash effect. However, there is still a need for Cutinases
30 having improved in-the-wash lipolytic activity and for
methods for producing such enzymes.
The purpose of the present invention is to provide
Cutinases, which have been modified so as to improve their
performance, especially their in-the-wash lipolytic activity.
We have now surprisingly found that the lipolytic
activity of eukaryotic Cutinase enzymes, more in particular
of Cutinases from Fusarium solani pisi, Colletotrichum
capsici, Colletotrichum qloeosPoriodes and Maqna~orthe
~ 215~837
WO94/14963 PCT~3/03550
qrisea, may be improved by modifying the amino acid sequence
in such way that the hydrophobicity at the surface of the
enzyme has been increased.
DEFINITION OF THE INVENTION
A Cutinase variant of a parent Cutinase, wherein
the amino acid sequence has been modified in such way that
the hydrophobicity at the surface of the enzyme has been
increased. Preferably, the hydrophobicity at the surface of
the enzyme has been increased so as to form an enlarged lipid
contact zone.
15 DESCRIPTION OF THE INVENTION
The invention relates to variants of Cutinase
enzymes. As discussed above, Cutinases can be obtained from a
number of sources, such as plants (e.g. pollen), bacteria and
fungi. The Cutinase to be used as parent Cutinase or starting
20 material in the present invention for the modification by
means of recombinant DNA techniques, is chosen from the group
of eukaryotic Cutinases. Eukaryotic Cutinases can be obtained
from various sources, such as plants (e.g. pollen), or fungi.
The group of (eukaryotic) fungal Cut;n~ appears
2S to comprise two families with different specificities, leaf-
specificity and stem-specificity. Cutinases with leaf-
specificity tend to have an acidic or neutral pH-optimum,
whereas Cutinases with stem-specificity tend to have an
alkaline pH-optimum. Cutinases having an alkaline pH-optimum
30 are more suitable for use in alkaline built detergent
compositions such as heavy duty fabric washing powders and
liquids. Cutinase having an acidic to neutral pH-optimum are
more suitable for light duty products or rinse conditioners,
but also for industrial cleaning products.
In the following Table I, four different Cutinases
with stem-specificity are listed, together with their pH-
optima.
WO94/14963 PCT~3/035~0
2l5~8~ 6
TABLE I
ExamPles of cutinases with stem-sPecificity pH-oPtimum
Fusarium solani pisi 9
Fusarium roseum culmorum lO
5 Rhizoctonia solani 8.5
Alternaria brassicicola (PNBase I) g
Especially preferred in the present invention are
Cutinases which can be derived from wild type Fusarium solani
lO Pisi (Ettinger et al. 1987). When used in certain detergent
compositions, this Cutinase exhibits clear "in-the-wash"
effects.
Also suitable as parent Cutinase or starting
material in the present invention for the modification by
15 means of recombinant DNA techniques, are Cutinases having a
high degree of homology of their amino acid sequence to the
Cutinase from Fusarium solani Pisi. Examples are the
Cutinases from Colletotrichum capsici, Colletotrichum
qloeosPoriodes and MaqnaPorthe qrisea. In Figure 12 the
20 partial amino acid sequences of these Cut;n~ are shown and
it can be seen that there is a high dergee of homology.
Alternative to the improvement of Fusarium solani
pisi cutinase by modification of its gene, genetic
information encoding Cutinases from other eukaryotic
25 organisms can be isolated using 5'- and 3'- DNA probes
derived from Fusarium solani ~isi, Colletotrichum caPsici,
Colletotrichum qloeosPoriodes and Maqna~orthe qrisea cDNA
encoding (pro)cutinase and probes recognizing conserved
sequences in other lipolytic enzymes and if necessary, using
30 these probes to multiply cDNA's derived from messenger RNA's
(mRNA's) of Cutinase producing eukaryotic cells using the
Polymerase Chain Reaction or PCR technology (see, for example
WO-A-92/05249). After cloning and expression the thus
obtained Cutinases encoding genes in E. coli according
35 standard procedures, the Cutinases are tested on their
performance in (fatty) soil removal under appropriate
conditions. In this way a number of natural occurring
variants of the above mentioned Cutinases can be obtained
WO94/14963 ~15~ 8 3 7 PCT~3/03550
with improved in-the-wash performance. Moreover, the
sequences of these natural occurring Cutinases provide an
excellent basis for further protein engineering of Fusarium
solani pisi cutinase.
On the basis of new ideas about the factors
determining the activity of "in-the-wash" lipolytic enzymes
and careful inspection of the 3D structure of Fusarium solani
~isi cutinase we have found a number of possibilities how to
improve the performance of this cutinase and Cutinases in
10 general by means of recombinant DNA techni~ues.
As Cutinases differ fundamentally from lipases like
Lipolase (TM), the interaction between Cutinase the substrate
(the lipid interface) will be based on principles different
from lid opening and exposure of a hydrophobic area that can
15 bind the substrate (Brzozowski et al. (1991) Nature 351, 491-
494).
The present invention shows that Cutinases can be
modified in such a way that the interaction with the
substrate can be improved without forming such large
20 hydrophobic areas on the surface of the modified Cutinase
that the Cutinase molecules start to aggregate. The
enlargement of the hydrophobic surface can be obtained by
introducing hydrophobic amino acids like alanine, valine,
leucine, isoleucine, proline, phenylalanine, tryptophan and
25 tyrosine, and methionine and to a lesser degree glutamic
acid, glutamine and histidine, provided that the hydrophobic
side ~-hA; n.c of these amino acids are not buried in the
hydrophobic core of the Cutinase. Methionine is not normally
considered to be a hydrophobic amino acid. However, when
integrated at certain positions, methionine can effectively
contribute to the increased hydrophobicity at the surface of
the Cutinase molecule.
In some cases it was found to be beneficia to
introduce beside the hydrophobic amino acids also charged
amino acids to avoid aggregation of the enzyme. Surprisinqly,
we have found that when integrated at certain positions in
the Cutinase molecule, positively charged amino acids like
lysine and arginine can also give enlargement of the
W094l14963 PCT~3/035~0
215~8~ 8
hydrophobic surface area. This is limited to those positions
in the Cutinase molecule where the methylene groups present
in lysine and arginine can not be buried in the molecule. The
advantage of using lysine or arginine is that the amino- or
imido groups increase the probability that the methylene
groups will be exposed and therefor will be able to interact
with the lipid phase.
Lysine and Arginine are not normally considered to
be hydrophobic amino acids. However, the atoms forming the
side chains of these residues contain a large number of
hydrophobic atoms (in the methylene moieties) which may
interact with the lipid phase. In fact, the size of the
hydrophobic part of a lysine residue is comparable to that of
a valine residue.
If other intrinsic properties of the Cutinase are
negatively affected by the introduction of the positive
charge, this may be compensated by the introduction of a
compensating negative charge or deletion of a positive charge
at the surface in or near that part of the Cutinase molecule
20 which interacts with the lipid phase.
Inspection of the hydrophobicity of the surface o~
~usarium solani Pisi cutinase around the active site shows
that the hydrophobicity is not optimal. To improve this
certain amino acids in this area residues should be replaced
25 by more bulky hydrophobic residues.
In order to get a better underst~n~;ng of the
relationship between structure and function in lipolytic
enzymes, we have carefully studied the three-dimensional (3D)
structures of a number of such enzymes. When these structures
30 had not been published, we derived the structures by means of
molecular modelling techniques.
The 3D structure of the Rhizomucor miehei lipase
has been determined by X-ray crystallographic methods (Brady
et al. (199O) Nature 343, 767-770, Brzozowski et al. (1991)
35 Nature 351, 491-494, Derewenda et al. (1992) Biochemistry 31,
1532-1541). The active-site Ser 144, belonging to a Ser-His-
Asp protease-like catalytic triad, is buried under a short
helical lid (residues 85-91). The structure in which the
wo 94~14963 21~ 0 8 3 7 PCT~3103550
active-site ser is buried is referred to below as the
"closed" conformation of the enzyme. It is ~elieved that the
adsorption at the oil-water interface is associated by a
movement of the helical lid. As a consequence of this
5 movement the active-site Serine becomes exposed and the
hydrophobic area around the active-site increases. It is
believed that the "open" conformation corresponds to the
activated enzyme adsorbed at the oil-water interface.
The C~-coordinates of the "closed" form of the
fungus Rhizomucor miehei lipase have been deposited in the
Protein Data Bank at Brookhaven. Elaborate computational
methods were used for generating full protein coordinates
(backbone and side-chains) of the Rhizomucor miehei lipase. A
crude starting model of the Rhizomucor miehei lipase was
15 generated by applying the computational procedures described
in S. Wodak et al. (1989) Protein Engineering 2, 335-345.
This method is implemented in the SYBYL molecular modelling
software package (TRIPOS associates, Inc. St. Louis,
M;~c~uri). Subsequently, the model was refined by applying
20 energy minimization (EM) and mole~~ r dynamics (MD)
t~chn;ques as implemented in the BIOSYM molecular modelling
software package tBIOSYM, San Diego, California). During EM
and MD refinement of the model a knowledge-based approach was
applied. The model was simultaneously optimized for the
25 detailed energy terms of the potential energy function and
known structural criteria. Model quality was A~c~ced by
criteria such as number and quality of hydrogen bonds,
hydrogen bonding patterns in the secondary structure
elements, the orientation of peptide units, the values of and
30 main chain dihedral angles, the angle of interaction of
aromatic groups and the sizes of cavities. Moreover, the
model was checked for inappropriately buried charges,
extremely exposed hydrophobic residues and energetically
unfavourable positions of disulphide bridges. Relevant side-
chain rotamers were selected from the Ponder & Richardsrotamer library (Ponder et al. (1987) J.Mol.Biol. 193, 775-
791). The final choice of a particular side-chain rotamer
from this library was based on structural criteria
WO94/14963 PCT~3/03550
2~8~7 lo
evaluations as mentioned above. MD was used to anneal the
side-chain atoms into position. Elaborate ~mi nation of the
model structure for consistency with known structural
properties and EM and MD calculations to optimize structural
5 characteristics allow to generate a reliable full atom model
of the Rhizomucor miehei lipase. The "open" conformation of
Rhizomucor miehei lipase was obtained by applying an MD
computer simulation in which a C1O-triglyceride was docked
into the active site of the lipase. Elaborate comparison to
10 the published conformational characteristics of the open
structure (Derewenda et al. Biochemistry (1992) 31, 1532-
1541) showed that the computer model of the "open"
conformation which was obtained in this way, is essentially
the same.
Starting from the known 3D structure of the fungus
Rhizomucor miehei lipase, the "open" and "closed" 3D-
structures of Humicola lanqinosa lipase were obtained by
applying rule-based comparative modelling t~chn; ques as
implemented in the COMPOSER module of the SYBYL mol~c~
20 modelling software package (TRIPOS associates, Inc. St.
Louis, Missouri). The obtained model of Humicola lanqinosa
lipase was refined by the same computational procedures as
mentioned above.
The part of the lipase molecule which is involved
in the adsorption of the substrate onto the enzyme was
identified by comparing the three-dimensional (3D) structures
of the fungus Rhizomucor miehei lipase and the Humicola
lanqinosa lipase.
Starting from the known 3D structure of the
30 Fusarium solani Pisi cutinase, the 3D-structure of the
cutinase from Colletotrichum qloeosPoriodes was obtained by
applying rule-based comparative modelling techniques as
implemented in the COMPOSER module of the SYBYL molecular
modelling software package (TRIPOS associates, Inc. St.
35 Louis, Missouri). The obtained model of the Colletotrichum
qloeosporiodes cutinase was refined by the same computational
procedures as mentioned above.
W094/14963 2 1 ~ 0 8 3 7 PCT~3/03550
11
From the three-dimensional structures of the
lipolytic enzymes listed below in Table II, it is was
unexpectedly observed that one can define a particular vector
which is the least-square fit through the C~-atoms of
5 residues 116 to 120 of the Fusarium solani pisi cutinase.
This vector is essentially perpendicular to the surface where
the interaction with the substrate occurs.
From the following Table II it follows that when
the primary sequences of a number of lipolytic enzymes from
10 different sources are compared, the amino acid residues 116
to 120 of the Cutinase ex Fusarium solani Pisi appear to be
located in an area having a large extent of functional
homology. The alignment can be guided by the use of the
consensus sequence Gly-(His/Tyr)-Ser-X-Gly for lipolytic
15 enzymes.
TABLE II
Humicola lanuginosa lipase ~Kvv~lGHSLGGALATVAGADLRGNGY
Mucor miehei lipase YKVAVTGHSLG~.~AT.T~T.GT.YQREE
human pancreas lipase ~Nvnv~ T~ A~GRRTNGTIG
20 Fusarium s.p. cutinase ATLIAGGYSQ~.AAT.AA~IEDLDSAIR
C. gloeosporiodes cutinase AAIVSGGYSQGTAVMAGSISGLSTTIK
Therefor we have used the vector through the amino
acid residues 116 to 120 in the Fusarium solani pisi cutinase
molecule to define the part of the Cutinase molecule in which
25 the amino acid modifications should be made in order to
obtain a Cutinase having improved in-the-wash activity. The
following Table III gives the structure of the neighbouring
amino acids for the lipolytic enzymes shown in Table II.
TABLE III
strand strand helix act.site
H. lanuginosa lip. 138-141 142 Ser 146 147-159 his258
Mucor miehei lipase 136-139 (fit:140-Ser 144) 145-157 his257
human pancreas lip. 144-147 (fit:148-Ser*152) 153-165 his263
35 F.s.pisi cutinase 112-115 (fit:116-Ser 120) 121-133 hisl88
C. gloe. cutinase 112-115 (fit:116-Ser*120) 121-133 hisl88
Ser* = active site Serine
W094/14963 21~ 0 8 3 7 PCT~3/03550
12
The invention in one of its aspects provides a
modified Cutinase enzyme having improved in-the-wash
lipolytic activity wherein the amino acid sequence has been
modified in such way that the hydrophobicity at the surface
5 of the enzyme has been increased. Preferably, the
hydrophobicity at the surface of the enzyme adjacent to the
lipid contact zone has been increased so as to form an
enlarged lipid contact zone.
The increase in surface hydrophobicity of the
10 Cutinase can be achieved by replacing one or more amino acid
residues by amino acid residues selected from the group
consisting of alanine, valine, leucine, isoleucine, proline,
phenylalanine, tryptophan and tyrosine, methionine, glutamic
acid, glutamine and histidine. Preferred are valine, leucine,
isoleucine, phenylalanine, tryptophan and methionine.
It was found to be advantageous to modify the amino
acid sequence in such way that in addition to the increase in
hydrophobicity at the surface, one or more positive charges
have been introduced by introduction of one or more lysine or
20 arginine residues.
Preferably, the modified residues are located in
that part of the molecule which is defined by the vector
which is the least-square fit through the C~-atoms of
residues 116 to 120 of the Fusarium solani Pisi cutinase, or
25 the corresponding Ca-atoms of a different Cutinase, and the
plane perpendicular to said vector and cont~;ning the C~-atom
of residue 117, or the corresponding C~-atom of a different
Cutinase.
As said before, for the three-dimensional structure
30 of the Cutinase from Fusarium solani pisi has been published.
In that case it will be clear which modification will lead to
modifications within the scope of this invention. In case the
thee-dimensional structure of a particular Cutinase is not
yet known, it will nevertheless be possible by alignment of
35 the amino acid sequence with a known sequence (see Fig.12),
guided by the consensus sequence Gly-(His/Tyr)-Ser-X-Gly for
lipolytic enzymes, to arrive at suitable modifications within
~ WO94tl4963 215 0 8 3 7 PCT~3/03550
the scope of the present invention. Preferably, molecular
modelling techniques are also used in this process.
The Cutinases variants produced according to the
invention can bring advantage in enzyme activity, when used
as part of detergent or cleaning compositions. In particular,
they were found to possess an improved in-the-wash perfor-
mance during the main cycle of a wash process. By in-the-wash
performance during the main cycle of a wash process, it is
meant that a detergent composition containing the enzyme is
10 capable of removing a significant amount of oily soil from a
soiled fabric in a single wash process in a European type of
automatic washing machine, using normal washing conditions as
far as concentration, water hardness, temperature, are
concerned. It should be born in mind that under the same
15 conditions, the conventional commercially available lipolytic
enzyme Lipolase (TM) ex Novo Nordisk does not appear to have
any significant in-the-wash effect on oily soil.
The in-the-wash effect of an enzyme on oily soil
can be assessed using the following assay. New polyester test
20 having a cotton content of less than 10% are prewashed using
an enzyme-free detergent product such as the one given below,
and are subsequently thoroughly rinsed. Such unsoiled cloths
are then soiled with olive oil or another suitable,
hydrolysable oily stain. Each tests cloth (weighing approxi-
25 mately 1 g) is incubated in 30 ml wash liquor in a lO0 mlpolystyrene bottle. The wash liquor contains the detergent
product given below at a dosage of 1 g per litre. The bottles
are agitated for 30 minutes in a Miele TMT washing machine
filled with water and using a normal 30C main wash
30 programme. The Cutinase variant is preadded to the wash
liquor at 3 LU/ml. The control does not contain any enzyme.
The washing powder has the following composition (in % by
weight):
Ethoxylated alcohol nonionic surfactant 9.5
35 Sodium sulphate 38.6
Sodium carbonate 40.4
Sodium silicate (Na2O:Si2O = 2.4) 7.3
Water 4.2
WO94/14963 2 130 8 3 7 PCT~3/035~0
14
As nonionic surfactant we used C12-C15 ethoxylated
alcohol 10.5-13 E0, but the nature of the ethoxylated alcohol
nonionic can vary within wide limits.
After washing, the cloths are thoroughly rinsed
5 with cold water and dried in a tumble dryer with cold air,
and the amount of residual fat is assessed. This can be done
in several ways. The common method is to extract the test
cloth with petroleum ether in a Soxhlet extraction apparatus,
distilling off the solvent and determining the percentage
10 residual fatty material as a fraction of the initial amount
of fat on the cloth by weighing.
According to a second, more sensitive method,
brominated olive oil is used to soil the test cloths
(Richards, S., Morris, M.A. and Arklay, T.H. (1968), Textile
15 Research Journal 38, 105-107). Each test cloth is then
incubated in 30 ml wash liquor in a 100 ml polystyrene
bottle. A series of bottles is then agitated in a washing
machine filled with water and using a normal 30C main wash
~G~Lamme. After the main wash, the test cloths are carefully
20 rinsed in cold water during 5 seconds. Immediately after the
rinse, the test cloths dried in a dryer with cold air. After
drying the amount of residual fat can be determined by
measuring the bromine content of the cloth by means of X-ray
fluorescence spectrometry. The fat removal can be determined
25 as a percentage of the amount which was initially present on
the test cloth, as follows:
% Soil removal = Brominebl - Bromine~w * 100 %
Brominebw
wherein: Brominebw denotes the percentage bromine on the
30 cloth before the wash and Bromineaw the percentage bromine
after the wash.
A further method of assessing the enzymatic
activity is by measuring the reflectance at 460 nm according
to standard techniques.
In the context of this invention, a modified,
mutated or mutant enzyme or a variant of an enzyme means an
enzyme that has been produced by a mutant organism which is
expressing a mutant gene. A mutant gene (other than one
WO94/14963 215 0 ~ 3 7 PCT~3/035~0
containing only silent mutations) means a gene encoding an
enzyme having an amino acid sequence which has been derived
directly or indirectly, and which in one or more locations is
different, from the sequence of a corresponding parent
5 enzyme. The parent enzyme means the gene product of the
corresponding unaltered gene. A silent mutation in a gene
means a change or difference produced in the polynucleotide
sequence of the gene which (owing to the redundancy in the
codon-amino acid relationships) leads to no change in the
10 amino acid sequence of the enzyme encoded by that gene.
A mutant or mutated micro-organism means a micro-
organism that is, or is descended from, a parent micro-
organism subjected to mutation in respect of its gene for the
enzyme. Such mutation of the organism may be carried out
15 either (a) by mutation of a corresponding gene (parent gene)
already present in the parent micro-orgAniC~ or (b) by the
transfer (introduction) of a corresponding gene obtained
directly or indirectly from another source, and then
introduced (including the mutation of the gene) into the
20 micro-organism which is to become the mutant micro-organism.
A host micro-organism is a micro-organism of which a mutant
gene, or a transferred gene of other origin, forms part. In
general it may be of the same or different strain or species
origin or descent as the parent micro-organism.
In particular, the invention provides mutant forms
of the Fusarium solani Pisi cutinase disclosed in WO-A-
90/09446 (Plant Genetics Systems), and of the Cutinases from
Colletotrichum caPsici~ Colletotrichum qloeosPoriodes and
Maqnaporthe qrisea. These Cutinase variants can be produced
30 by a rDNA modified micro-organism containing a gene obtained
or made by means of rDNA techniques.
Once the amino acid residues have been identified
which are located in that part of the molecule which is
defined by the vector which is the least-square fit through
35 the C~-atoms of residues 116 to 120 of the Fusarium solani
E~ cutinase, or the corresponding C~-atoms of a different
Cutinase, and the plane perpendicular to said vector and
containing the C~-atom of residue 117, or the corresponding
WO94/14963 PCT~3/03550 -
2150~37 16
C~-atom of a different Cutinase, one can attempt to modify
the amino acid sequence by introduction of suitable amino
acids at one or more of the identified positions, for e~ample
mutation N712K relative to the sequence of Fusarium solani
isi cutinase or a homologue thereof.
It will be clear to the skilled man that such
modifications will affect the structure of the CutinaseO
Obviously, modifications are preferred which do not affect
the electrostaic charge around the active site too much.The
inventors have developed the necessary level of understanding
of the balance between the inevitable distortion of the
conformation of the enzyme and the benefit in increased
enzyme activity, which makes is possible to predict and
produce successful Cutinase variants with a high rate of
15 success.
In the following Table IV and elsewhere in this
specification, amino-acids and amino acid residues in peptide
sequences are indicated by one-letter and three-letter
abbreviations as follows:
TABLE IV
A = Ala = Alanine V = Val = Valine
L = Leu = Leucine I = Ile = Isoleucine
P = Pro = Proline F = Phe = Phenylalanine
W = Trp = Tryptophan M = Met = Methionine
25 G = Gly = Glycine S = Ser = Serine
T = Thr = Threonine C = Cys = Cysteine
Y = Tyr = Tyrosine N = Asn = Asparagine
Q = Gln = Glutamine D = Asp = Aspartic Acid
E = Glu = Glutamic Acid K = Lys = Lysine
30 R = Arg = Arginine H = His = Histidine
In this specification, a mutation present in khe
amino acid sequence of a protein, and hence the mutant
protein itself, may be described by the position and nature
35 of the mutation in the following abbreviated way: by the
identity of an original amino acid residue affected by the
mutation; the site (by sequence number) of the mutation; and
by the identity of the amino acid residue substituted there
~ 21~ 7
W094/149~ ~CT~W3103550
17 l
in place of the original. If there is an insertion of an
extra amino acid into the sequence, its position is indicated
by one or more sukscript letters attached to the number of
the last preceding member of the regular sequence or
5 reference sequence.
For example, a mutant characterised by substitution
of Asparagine by Lysine in position 172 is designated as:
Asnl72Lys or N172K. A (hypothetical) insertion of an
additional amino acid residue such as proline after the
10 Asparagine would be indicated as Asnl72AsnPro or N172NP,
alternatively as *172aP, with the inserted residue designated
as position number 172a. A (hypothetical) deletion of
Asparagine in the same position would be indicated by Asnl72*
or N172*. The asterisk stands either for a deletion or for a
15 missing amino acid residue in the position designated,
whether it is reckoned as missing by actual deletion or
merely by comparison or homology with another or a reference
sequence having a residue in that position.
Multiple mutations are separated by plus signs,
20 e.g. N172K+S54I+A128F designates a mutant protein carrying
three mutations by substitution, as indicated for each of the
three mentioned positions in the amino acid sequence. The
mutations given in the following table may be combined if
desired.
The Table V given below shows certain useful
examples of Cutinase variants according to the invention,
based on the sequence of Cutinase from Fusarium solani Pisi.
TABLE V
Variants of Fusarium solani Pisi Cutinase.
30 T19V, G41A, T45K, T45P, *I49a, S54I, N58R, G75R, A76P, G82A,
D83S, A85F, A85V, S92R, A93V, L99K, GlOOR, A127L, A128F,
N172K, T173I, T179F, I183F, V184I, A185L, L189F, A19OL,
D194R, G197V, E201K
Preferred variants according to the invention are
A85F, N172K and E201K of Fusarium solani Pisi cutinase, and
the corresponding variants of other Cutinases. An example of
such a corresponding Cutinase variant is the variant
WO94/14963 PCT~3/03550
2~83~ 18
Aspl72Lys or Dl72K derived from cutinase ex Colletotrichum
qloeosporiodes.
According to a further aspect of the invention,
there is provided a process for producing the Cutinase
5 variants of the invention. Naturally occurring Cutinase
producing micro-organisms are usually plant pathogens and
these micro-organisms are not very suitable to act as host
cell for modified Cutinases genes. Consequently, the genes
coding for modified (pro)Cutinases were integrated in rDNA
10 vectors that can be transferred into the preferred host
micro-organism for rDNA technology. For this purpose rDNA
vectors essentially similar to the rDNA vector described in
WO-A-90/09446 can be used.
Naturally occurring Cutinase producing micro-
15 organisms are not very suitable for fermentation processes.To improve the yield of the fermentation process a gene
coding for improved Cutinases should be transferred into
micro-org~ni~ that can growth fast on cheap medium an~ are
capable to synthesize and secrete large amounts of Cutinase.
20 Such suitable rDNA modified (host micro-organisms) according
to the present invention are bacteria, among others, Bacilli,
Corynebacteria, StaphYlococci and Stre~tomYces, or lower
eukaryotes such as Saccharomvces cerevisiae and related
species, KluYveromyces marxianus and related species,
25 Hansenula ~olYmorpha and related species, and species of the
genus AsPerqillus. Preferred host micro-organisms are the
lower eukaryotes, because these microorganisms are producing
and secreting enzymes very well in fermentation processes and
are able to glycolysate the Cutinase molecule. Glycosylation
30 can contribute to the stability of the Cutinase in detergent
systems.
The invention also provides genetic material
derived from the introduction of modified eukaryotic Cutinase
genes, e.g. the gene ~rom Fusarium solani pisi, into cloning
35 rDNA vectors, and the use of these to transform new host
cells and to express the genes of the Cutinase variants in
the new host cells.
WO94/14963 ! '~ ~ = ` PCT~W3/03550
19
Also provided by the invention are polynucleotides
made or modified by rDNA technique, which encode such
Cutinase variants, rDNA vectors containing such
polynucleotides, and rDNA modified microorganisms containing
5 such polynucleotides and/or such rDNA vectors. The invention
also provides corresponding polynucleotides encoding the
modified eukaryotic Cutinases, e.g. a polynucleotide having a
base sequence that encodes a mature Cutinase variant, in
which polynucleotide the final translated codon is followed
10 by a stop codon and optionally having nucleotide sequences
coding for the prepro- or pro-sequence of this Cutinase
variant directly upstream of the nucleotide sequences coding
for the mature Cutinase variant.
In such a polynucleotide, the Cutinase-encoding
15 nucleotide sequence derived from the organism of origin can
be modified in such a way that at least one codon, and
preferably as many codons as possible, are made the subject
of 'silent' mutations to form codons encoding equivalent
amino acid residues and being codons preferred by a new host,
20 thereby to provide in use within the cells of such host a
messenger-RNA for the introduced gene of improved stability.
Upstream of the nucleotide se~uences coding for the
pro-or mature Cutinases, there can be located a nucleotide
sequence that codes for a signal or secretion sequence
25 suitable for the chosen host. Thus an embodiment of the
invention relates to a rDNA vector into which a nucleotide
sequence coding for a Cutinase variant or a precursor thereof
has been inserted.
The nucleotide sequence can be derived for example
from:
(a) a naturally occurring nucleotide sequence (e.g. encoding
the original amino acid sequence of the propre- or pro-
cutinase produced by Fusarium solani Pisi);
(b) chemically synthesized nucleotide sequences consisting of
35 codons that are preferred by the new host and a nucleotide
sequence resulting in stable messenger RNA in the new host,
still encoding the original amino acid sequence;
WO94/14963 2 ~ 5 ~ 8 3 7 PCT~W3/03550
(c) genetically engineered nucleotide sequences derived from
one of the nucleotide sequences mentioned in preceding
paragraphs a or b coding for a Fusarium solani pisi Cutinase
with a different amino acid sequence but having superior
5 stability and/or activity in detergent systems.
Summarizing, rDNA vectors able to direct the
expression of the nucleotide sequence encoding a Cutinase
gene as described above in one of the preferred hosts
preferably comprise the following components:
(a) Double-stranded (ds) DNA coding for mature Cutinase or
precutinase or a corresponding precutinase in which at least
part of the presequence has been removed directly down stream
of a secretion signal (preferred for the selected host cell).
In cases where the part of the gene that should be translated
15 does not start with the codon ATG, an ATG codon should be
placed in front. The translated part of the gene should
always end with an appropriate stop codon;
(b) An expression regulon (suitable for the selected host
organism) situated upstream of the plus strand of the ds DNA
20 encoding the Cutinase (component (a));
(c) A terminator sequence (suitable for the selected host
organism) situated down stream of the plus strand of the ds
DNA encoding the Cutinase (component (a);
(dl) Nucleotide sequences which facilitate integration, of
25 the ds DNA into the genome of the selected host or,
(d2) an origin of replication suitable for the selected host;
(el) Optionally a (auxoLl~hic) selection marker. The
auxotrophic marker can consist of a coding region of the
auxotrophic marker and a defective promoter;
(e2) Optionally a ds DNA sequence encoding proteins involved
in the maturation and/or secretion of one of the precursor
forms of the Cutinase in the host selected.
Such a rDNA vector can also carry, upstream and/or
downstream of the polynucleotide as earlier defined, further
35 sequences facilitative of functional expression of the
cutinase. The auxotrophic marker can consist of a coding
region of the auxotrophic marker and a defective promoter
region.
~ WO94/14963 215 0 8 3 7 PCT~3/03550
Another embodiment of this invention is the
fermentative production of one of the various Cutinase
variants described above. Such a fermentation can either be a
normal batch fermentation, fed-batch fermentation or
continuous fermentation. The selection of a process to be
used depends on the host strain and the preferred down stream
processing method (known per se). Thus, the invention also
provides a process for producing a Cutinase variant as
specified herein, which comprises the steps of fermentatively
cultivating an rDNA modified micro-organism containing a gene
made by rDNA technique which carries at least one mutation
affecting the amino acid sequence of the Cutinase thereby to
confer upon the Cutinase improved activity by comparison with
the corresponding parent enzyme, making a preparation of the
Cutinase variant by separating the Cutinase produced by the
micro-organism either from the fermentation broth, or by
separating the cells of the micro-organism from the
fermentation broth, disintegrating the separated cells and
concentrating or part purifying the Cutinase variant either
20 from said broth or from said cells by physical or chemical
concentration or purification methods. Preferably conditions
are chosen such that the Cutinase variant is secreted by the
micro-organism into the fermentation broth, the enzyme being
recovered from the broth after removal of the cells either by
25 filtration or centrifugation. Optionally, the Cutinase
variant can then be concentrated and purified to a desired
extent. These fermentation processes in themselves apart from
the special nature of the micro-organisms can be based on
known fermentation techniques and commonly used fermentation
30 and down stream processing equipment.
Also provided by the invention is a method for the
production of ~ modified micro-organism capable of producing
a Cutinase variant by means of rDNA techniques, characterized
in that the gene coding for the Cutinase variant that i5
introduced into the micro-organism is fused at its 5'-end to
a gene fragment encoding a (modified) pre-sequence functional
as a signal- or secretion-sequence for the host organism.
WO94/14963 PCT~3/03S50
21~083~ 22
According to a further aspect of the invention,
there are provided rDNA modified micro-organisms containing a
Cutinase variant gene and able to produce the Cutinase
variant encoded by said gene. In an rDNA modified micro-
organism, a gene (if originally present) encoding the nativeCutinase is preferably removed, e.g. replaced by another
structural gene.
According to a further aspect of the present
invention, there are provided enzymatic detergent
10 compositions comprising the Cutinase variants of the
invention. Such compositions are combinations of the
Cutinases variants and other ingredients which are commonly
used in detergent systems, including additives for detergent
compositions and fully-formulated detergent and cleaning
15 compositions, e.g. of the kinds known per se and described
for example in EP-A-258 068. More specifically, they may
comprise from 5 - 60, preferably from 20 - 50% by weight of a
detergency builder and from 0.1 - 50 % by weight of an active
system, which in turn comprises 0 - 95 % by weight of one or
20 more anionic surfactants and 5 - 100 % by weight of one or
more nonionic surfactants.
The other components of such detergent compositions
can be of any of many known kinds, for example as described
in GB-A-1 372 034 (Unilever), US-A-3 950 277, US-A-4 011 169,
25 EP-A-179 533 (Procter & Gamble), EP-A-205 208 and EP-A-206
390 (Unilever), JP-A-63-078000 (1988), and Research
Disclosure 29056 of June 1988, together with each of the
several specifications mentioned therein, all of which are
hereby incorporated herein by reference.
The Cutinase variants of the present invention can
usefully be added to the detergent composition in any
suitable form, i.e. the form of a granular composition, a
solution or a slurry of the enzyme, or with carrier material
(e.g. as in EP-A-258 068 and the Savinase(TM) and
35 Lipolase(TM) products of Novo Nordisk).
The added amount of Cutinase variant can be chosen
within wide limits, for example from 10 - 20,000 LU per gram,
and preferably 50 -2,000 LU per gram of the detergent
W094/l4963 23 PCT~3/~3550
composition. In this specification LU or lipase units are
defined as they are in EP-A-258 068 (Novo Nordisk).
Similar considerations apply mutatis mutandis in
the case of other enzymes, which may also be present. See
5 also European patent application EP-A-407 225.
Advantage may be gained in such detergent
compositions, where protease is present together with the
cutinase, by selecting such protease from those having pI
lower than 10. EP-A-271 154 (Unilever) describes a number of
such proteases. Proteases for use together with Cutinase
variants can in certain circumstances include subtilisin of
for example BPN' type or of many of the types of subtilisin
disclosed in the literature, some of which have already been
proposed for detergents use, e.g. mutant proteases as
15 described in for example EP-A-130 756 or EP-A-251 446 (both
Genentech); US-A-4 760 025 (Genencor); EP-A-214 435 (Henkel);
WO-A-87/04661 (Amgen); WO-A-87/05050 (Genex); Thomas et al.
(1986/5) Nature 316, 375-376 and (1987) J.Mol.Biol. 193,
803-813; Russel et al. (1987) Nature 328, 496-500.
The invention will now be further illustrated in
the following Examples. All techniques used for the
manipulation and analysis of nucleic acid materials were
performed essentially as described in Sambrook et al. (1989),
except where indicated otherwise.
In the accompanying drawings is:
Fig. lA. Nucleotide sequence of cassette 1 of the synthetic
Fusarium solani Pisi cutinase gene and of the
constituting oligo-nucleotides. Oligonucleotide
transitions are indicated in the cassette sequence.
Lower case letters refer to nucleotide positions
outside the open reading frame.
Fig. lB. Nucleotide sequence of cassette 2 of the syntheti_
Fusarium solani pisi cutinase gene and of the
constituting oligo-nucleotides. Oligonucleotide
transitions are indicated in the cassette sequence.
Fig. lC. Nucleotide sequence of cassette 3 of the synthetic
Fusarium solani Pisi cutinase gene and of the
WO94/14963 215 ~ ~ 3 ~ PCT~3/03550
24
constituting oligo-nucleotides. Oligonucleotide
transitions are indicated in the cassette sequence.
Lower case letters refer to nucleotide positions
outside the open reading frame.
5 Fig. lD. Nucleotide sequence of the synthetic cutinase gene
encoding Fusarium solani Pisi pre-pro-cutinase. The
cutinase pre-sequence, pro-sequence and mature
sequence are indicated. Also the sites used for
cloning and the oligonucleotide transitions are
indicated. Lower case letters refer to nucleotide
positions outside the open reading frame.
Fig. 2. Nucleotide sequence of a synthetic DNA fragment for
linking the Fusarium solani Pisi pro-cutinase
encoding sequence to a sequence encoding a
derivative of the E. coli phoA pre-sequence. The
ribosome binding site (RBS) and the restriction
enzyme sites used for cloning are indicated. Also
the amino acid sequences of ~he encoded phoA signal
sequence and part of the cutinase gene are
indicated using the one-letter code.
Fig. 3. Nucleotide sequence of cassette 8, a SacI-BclI
fragment which encodes the fusion point of the
coding sequences for the invertase pre-sequence and
mature Fusarium solani Pisi cutinase.
25 Fig. 4. Plasmid pUR2741 obtained by deletion of a 0.2 kb
SalI-NruI from pUR2740, is an E. coli-S. cerevisiae
shuttle vector comprising part of pBR322, an origin
of replication in yeast cells derived from the 2~m
plasmid, a yeast leu2D gene and a fusion of the
yeast invertase signal sequence encoding region
with a plant ~-galactosidase gene under the control
of the yeast gal7 promoter.
Fig. 5. Plasmid pUR7219 is an E. coli-S. cerevisiae shuttle
vector comprising part of pBR322, an origin of
replication in yeast cells derived from the 2~m
plasmid, a yeast leu2D gene and a fusion of the
yeast invertase signal sequence encoding region
with the region encoding the mature Fusarium solani
WO94/14963 ~1 5 08 ~ 7 . PCT~3/03550
E~ cutinase under the control of the yeast gal7
promoter.
Fig. 6. Plasmid pUR2740 is an E. coli-S. cerevisiae shuttle
vector comprising part of pBR322, an origin of
replication in yeast cells derived from the 2~m
- plasmid, a yeast leu2D gene and a fusion of the
yeast invertase signal sequence encoding region
with a plant ~-galactosidase gene under the control
of the yeast gal7 promoter.
lO Fig. 7. Nucleotide sequence of cassettes 5, 6 and 7,
comprising different types of connections of the
coding sequences of the exlA pre-sequence and
mature Fusarium solani pisi cutinase.
Fig. 8. Plasmid pAWl4B obtained by insertion of a 5.3 kb
SalI fragment of AsPerqillus niqer var. awamori
genomic DNA in the SalI site of pUCl9.
Fig. 9. Plasmid pUR7280 obtained by displacing the BspHI-
AflII fragment comprising the exlA open reading
frame in pAWl4B with a Bs~HI-AflII fragment
comprising the Fusarium solani pisi pre-pro-
cutinase coding sequence. Thus, plasmid pUR7280
comprises the Fusarium solani Pisi pre-pro-cutinase
gene under the control of the A. niqer var. awamori
promoter and terminator.
25 Fig. lO. Plasmid pUR7281 obtained by introduction of both
the A. nidulans amdS and A. ni~er var. awamori pyrG
selection markers in pUR7280.
Fig. ll. Schematical representation of the Fusarium solani
Pisi cutinase molecule.
30 Fig. 12. Partial amino acid sequences of the cutinases from
Fusarium solani pisi, Colletotrichum caPsici~
Colletotrichum qloeosporiodes and MaqnaPorthe
qrisea, showing the location of the residues in the
3-D structure.
Fig. 13. In-the-wash effect for Fusarium solani pisi
cutinase and the Cutinase variant Nl72K.
Fig. 14. In-the-wash effect for Fusarium solani pisi
cutinase and the Cutinase variant E20lK.
W094/14963 21~ ~ 8 ~ 7 PCT~3/03550
26
Fig. 15. In-the-wash effect for Fusarium solani Pisi
cutinase and the Cutinase variant A85F.
REFERENCES
5 Sambrook, J., Fritsch, E.F. and Maniatis,T. (1989). Molecular
Cloning: a laboratory manual (2nd ed). Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York. ISNB 0-87969-
309-6.
Furste, J.P., Pansegrau, W., Frank, R., Blocker, H., Scholz,
10 P. Bagdasarian, M. and Lanka, E. (1986). Molecular cloning of
the plasmid RP4 primase region in a multi-host-range tacP
expression vector. Gene 48, 119-131.
Michaelis et al. (1983). J. Bacteriol 154, 366-
Tartof and Hobbs (1988). Gene 67, 169-182.
15 Soliday, C.L., Flurkey, W.H, Okita, T.W. and Kolattukudy,
P.E. (1984). Cloning and structure determination of cDNA for
cutinase, an enzyme involved in fungal penetration of
plants. Proc.Natl.Acad.Sci. USA 81, 3939-3943.
~oai, Y. and Fukasawa, T. (1983). Nucleotide sequence of the
20 transcriptional initiation region of the yeast GAL7 gene.
Nucleic Acids Res. 11, 8555-8568.
Taussiq, R. and Carlsson, M. (1983). Nucleotide sequence of
the yeast SUC2 gene for invertase. Nucleic Acids Res. 11,
1943-1954.
25 Erhart, E and Hollenberg, C.P. (1981) Curing of Saccharomyces
cerevisiae 2-~m DNA by transformation. Curr. Genet. 3, 83-89.
Verbakel, J.A.M.V. (1991) Heterologous gene expression in the
yeast SaccharomYces cerevisiae. Ph.D. thesis. Rijks
Universiteit Utrecht, The Netherlands.
30 Maat, J., Roza, M., Verbakel, J., Stam, J., Santos da Silva,
M.J., Bosse, M., Egmond, M.R., Hagemans, M.L.D., v. Gorcom,
R.F.M., Hessing, J.G.M., v.d. Hondel, C.A.M.J.J. and v.
Rotterdam, C. (1992). Xylanases and their application in
bakery. In: Visser, J., Beldman, G., Kusters-van Someren,
35 M.A. and Voragen, A.G.J. (Eds), Xylans and Xylanases.
Progress in Biotechnology Volume 7, Elsevier Science
Publishers, Amsterdam. ISBN 0-444-89477-2.
WO94/14963 215 0 8 3 ~ PCT~3/03S50
27
de Graaff, L.H., van den Broek, H.C., van Ooijen, A.J.J. and
Visser, J. (1992). Structure and regulation of an AsPerqillus
xylanase gene. In: Visser, J., Beldman, G., Kusters-van
Someren, M.A. and Voragen, A.G.J. (Eds), Xylans and
5 Xylanases. Progress in Biotechnology Volume 7, Elsevier
Science Publishers, Amsterdam. ISBN 0-444-89477-2.
Hankin, L. and Kolattukudy, P.E. (1968). J. Gen. Microbiol.
51, 457-463.
Ettinqer, W.F., Thukral, S.K. and Kolattukudy, P.E. (1987).
Structure of cutinase gene, cDNA, and the derived amino acid
sequence from phytopathogenic fungi. Biochemistry 26, 7883-
7892.
Huse, W.D. and Hansen, C. (1988). Strategies 1, 1-3.
De Graaff, L.H., H.W.J. van den Broek and J. Visser (1988).
Isolation and expression of the Aspergillus nidulans pyruvate
kinase gene. Curr. Genet. 13, 315-321.
EXAMPLF 1
20 Construction of a synthetic gene encoding Fusarium solani
E~ pre-pro-cutinase.
A synthetic gene encoding Fusarium solani Pisi pre-
pro-cutinase was constructed essentially according to the
method described in EP-A-407 225 (Unilever). Based on
25 published nucleotide sequences of Fusarium solani Pisi genes
(Soliday et al. (1984) and WO-A-90/09446, Plant Genetic
Systems), a completely synthetic DNA fragment was designed
which comprises a region encoding the Fusarium solani ~isi
pre-pro-cutinase polypeptide. Compared to the nucleotide
30 sequence of the original Fusarium solani pisi gene, this
synthetic cutinase gene comprises several nucleotide changes
through which restriction enzyme recognition sites were
introduced at convenient positions within the gene without
affecting the encoded amino acid sequence. The nucleotide
35 sequence of the entire synthetic cutinase gene is presented
in Fig. lD.
Construction of the synthetic cutinase gene was
performed by assembly of three separate cassettes starting
W094/14963 PCT~3/035~0 -
21 SB8~
28
from synthetic DNA oligonucleotides. Each synthetic DNA
cassette is equipped with an EcoRI site at the start and a
HindIII site at the end. Oligonucleotides were synthesized
using an Applied Biosystems 380A DNA synthesizer and purified
5 by polyacrylamide gel electrophoresis. For the construction
of each of the cassettes the procedure outlined below was
followed. Equimolar amounts (50 pmol) of the oligonucleotides
constituting a given cassette were mixed, phosphorylated at
their 5'-end, annealed and ligated according to standard
10 techniques. The resulting mixture of double stranded DNA
molecules was cut with EcoRI and HindIII, size-fractionated
by agarose gel electrophoresis and recovered from the gel by
electro-elution. The resulting synthetic DNA cassette was
ligated with the 2.7 kb EcoRI-HindIII fragment of pUC9 and
15 transformed to Escherichia coli. The EcoRI-HindIII insert of
a number of clones was completely sequenced in both
directions using suitable oligonucleotide primers to verify
the sequence of the synthetic cassettes. Using this procedure
pUR7207 (comprising cassette l, Fig. lA), pUR7208 (comprising
20 cassette 2, Fig. lB) and pUR7209 (comprising cassette 3, Fig.
lC) were constructed. Finally, the synthetic cutinase gene
was assembled by combining the 2.9 kb EcoRI-~E~I fragment of
pUR7207 with the 0.2 kb ApaI-NheI fragment of pUR7208 and the
O.3 kb NheI-HindIII fragment of pUR7209, yielding pUR7210.
25 This plasmid comprises an open reading frame encoding the
complete pre-pro-cutinase of Fusarium solani PiSi (Fig. lD).
EXAMPLE 2
Expression of Fusarium solani pisi (pro)cutinase in
30 Escherichia coli.
With the synthetic cutinase gene an expression
vector for E. coli was constructed which is functionally
similar to the one described in WO-A-90/09446 (Plant Genetic
Systems). A construct was designed in which the part of the
35 synthetic gene encoding Fusarium solani pisi pro-cutinase is
preceded by proper E. coli expression signals, i.e. (i) an
inducible promoter, (ii) a ribosome binding site and (iii) a
signal sequence which provides a translational initiation
WO94/14963 21 ~ ~ 8 ~ ~ PCT~3/03550
29
codon and provides information required for the export of the
pro-cutinase across the cytoplasmic membrane.
A synthetic linker was designed (see Fig. 2) for
fusion of a derivative of the E. coli phoA signal sequence
(Michaelis et al., 1983) to the pro-sequence of the synthetic
cutinase gene. To optimize cleavage of the signal peptide and
secretion of pro-cutinase, the nucleotide sequence of this
linker was such that the three C-terminal amino acid residues
of the phoA signal sequence (Thr-Lys-Ala) were changed into
10 Ala-Asn-Ala and the N-terminal amino acid residu of the
cutinase pro-sequence (Leu 1, see Fig. lD) was changed into
Ala. This construction ensures secretion of cutinase into the
periplasmatic space (see W0-A-90/09446, Plant Genetic
Systems).
To obtain such a construct, the 69 bp EcoRI-SpeI
fragment comprising the cutinase pre-se~uence and part of the
pro-sequence was removed from pUR7210 and replaced with the
synthetic DNA linker se~uence (EcoRI-S~eI fragment) providing
the derivative of the E. coli phoA pre-se~uence and the
20 altered N-terminal amino acid residue of the cutinase pro-
sequence (Fig. 2). The resulting plasmid was named pUR7250
and was used for the isolation of a 0.7 kb BamHI-HindIII
fragment comprising a ribosome binding site and the pro-
cutinase encoding region fused to the phoA signal sequence
25 encoding region. This fragment was ligated with the 8.9 kb
BamHI-HindIII fragment of pMMB67~H (Furste et al., 1986) to
yield pUR7220. In this plasmid the synthetic gene encoding
pro-cutinase is fused to the altered version of the phoA
signal se~uence and placed under the control of the inducible
30 tac-promoter.
~ . coli strain WK6 harboring pUR7220 was grown in 2
litre shakeflasks containing 0.5 litre IXTB medium (Tartof
and Hobbs, 1988) consisting of:
0.017 M KH2PO4
0.017 M K2HP04
12 g/l Bacto-tryptone
24 g/l Bacto-yeast extract
0.4 ~ glycerol (v/v)
WO94/14963 21~ ~ 83 7 PCT~3/03550
Cultures were grown overnight at 25C - 30OC in the
presence of 100 ~g/ml ampicillin under vigorous shaking (150
rpm) to an OD at 610 nm of 10-12. Then IPTG (isopropyl-B-D-
thiogalactopyranoside) was added to a final concentration of
10 ~M and incubation continued for another 12-16 hours. When
no further significant increase in the amount of produced
lipolytic activity could be observed, as judged by the
analysis of samples withdrawn from the cultures, the cells
were harvested by centrifugation and resuspended in the
10 original culture volume of buffer containing 20% sucrose at
0C. The cells were collected by centrifugation and
resuspended in the original culture volume of icecold water
causing lysis of the cells through osmotic shock. Cell debris
was removed by centrifugation and the cell free extract was
15 acidified to pH 4.8 with acetic acid, left overnight at 4C
and the resulting precipitate was removed. A better than 75%
pure cutinase preparation essentially free of endogenous
lipases was obtained at this stage by means of ultra-
filtration and freeze drying of the cell free extract.
20 Alternatively, cutinase could be purified to homogeneity
(i.e. better than 95% pure) by loading the acidified cell
free extract onto SP-sephadex, eluting the enzyme with buffer
at pH 8.0, passage of the concentrated alkaline solution
through a suitable volume of DEAE-cellulose (Whatman DE-52)
25 and direct application of the DEAE flow-through to a Q-
sepharose HP (Pharmacia) column. Elution with a salt gradient
yielded a homogenous cutinase preparation with a typical
overall yield of better than 75%.
30 EXAMPLE 3
Construction of genes encoding variants of Fusarium solani
pisi cutinase.
Usir.g the synthetic gene for Fusarium solani pisi
pre-pro-cutinase described in Exampie 1, variant genes
35 comprising alterations in the encoded amino acid sequence
were constructed. For this construction essentially the same
approach was followed as described in Example 1 for the
construction of the three cassettes constituting the complete
WO94114963 PCT~3/035S0
21~Q8~
^31~
synthetic cutinase gene. For example, a gene coding for
Fusarium solani Pisi cutinase variant N172K was constructed
by assembling a new version of cassette 3 using the same
- oligonucleotides as described in Example l, except for the
5 two oligos which cover the coding triplet for the position
which is to be mutated, i.c. Asn 172 . Instead, two new
synthetic oligos were used, which comprise the mutant
sequence but are otherwise identical to the original oligos
which they are replacing. More specifically, a new cassette 3
lO comprising mutation N172K was assembled by incorporating a
variant of CUTI3D MH (containing AAG instead of AAT) and a
variant of CUTI3K MH (containing CTT instead of ATT) instead
of CUTI3D MH and CUTI3K MH (see Fig. lC). The new cassette 3
was cloned and sequenced essentially as described in Example
l and the 0. 3 kb NheI-HindIII DNA fragment comprising the
mutation was exchanged for the corresponding fragment in
pUR7210, yielding pUR72~7 (Nl72K). The 0.6 kb S~eI-HindIII
fragment from this plasmids was used to replace the
corresponding fragment in pUR7220, yielding the E. coli
20 expression plasmid pUR7224 (Nl72K). This E. coli expression
plasmid was transformed to E. coli strain WK6. Transformants
were grown as outlined in Example 2 and the variant pro-
cutinase enzyme was recovered and purified essentially as
described in Example 2.
A gene coding for Fusarium solani Pisi cutinase
variant E201K was constructed in an analogous way by
assembling a new version of cassette 3 incorporating a
variant of CUTI3F MH (containing AAG instead of GAG) and a
variant of CUTI3M MH (containing CTT instead of CTC) instead
30 of CUTI3F MH and CUTI3M MH (see Fig. lC).
A gene coding f or Fusarium solani Pisi cutinase
variant A85F was constructed in an analogous way by
assembling a new version of cassette 2 incorporating a
variant of CUTI2C MH (containing TTC instead of GCT) and a
35 variant of CUTI2I MH (containing GAA instead of AGC) instead
of CUTI2C MH and CUTI2I MH (see Fig. lB).
WO94/14963 32 PCT~3/03550
EXAMPLE 4 215 ~ 8 3 7
Expresslon of Fusarium solani Pisi cutinase in Saccharom~ces
cerevlslae.
For the expression of the synthetic Fusarium solani
5 Pisi cutinase gene in Saccharomyces cerevisiae an expression
vector was constructed in which a synthetic gene encoding the
mature cutinase is preceded by the pre-sequence of S.
cerevisiae invertase (Taussig and Carlsson, 1983) and the
strong, inducible gal7 promoter (Nogi and Fukasawa, 1983). To
10 prepare the synthetic cutinase gene for such a fusion, an
adaptor fragment was synthetized in which the coding sequence
for the invertase pre-sequence is fused to the sequence
encoding the N-terminus of mature cutinase. This fragment was
assembled as an EcoRI-HindIII cassette in pUC9 essentially as
15 described in Example 1 (cassette 8, see Fig. 3), yielding
pUR7217. Plasmids pUR7210 and pUR7217 were transformed to E.
coli JM110 (a strain lacking the dam methylase activity) and
the 2.8 kb BclI-HindIII fragment of pUR7217 was ligated with
the 0.6 kb ~clI-HindIII fragment of pUR7210, yielding pUR7218
in which the nucleotide sequence coding for the mature
cutinase polypeptide is fused with part of the S. cerevisiae
invertase pre-sequence coding region.
The expression vector pUR2741 (see Fig. 4) was
derived from pUR2740 (Verbakel, 1991, see Fig. 6) by
isolation of the 8.9 kb NruI-SalI fragment of pUR2740,
filling in the SalI protruding end with Klenow polymerase,
and recircularization of the fragment. The 7.3 kb SacI-
HindIII fragment of pUR2741 was ligated with the 0.7 kb SacI-
HindIII fragment of pUR7218, yielding pUR7219 (see Fig. 5).
30 Optionally, a S. cerevisiae polII terminator can be placed
behind the cutinase gene, in the HindIII site, which turned
out not to be essential for efficient expression of the
cutinase gene. The E. coli-S. cerevisiae shuttle plasmid
pUR7219 contains a origin for replication in S. cerevisiae
35 strains harboring the 2~ plasmid (cir+ strains), a promoter-
deficient version of the S. cerevisiae Leu2 gene permitting
selection of high copy number transformants in S. cerevisiae
leu2~ strains, and the synthetic gene encoding the mature
215~8~
WO 94/14963 PCT/EP93/03550
33
part of Fusarium solani Pisi cutinase operably linked to the
S. cerevisiae invertase pre-sequence under the regulation of
the strong, inducible S. cerevisiae gal7 promoter.
S. cerevisiae strain SU50 (a, cir, leu2, his4,
5 canl), which is identical to strain YT6-2-lL (Erhart and
Hollenberg, 1981), was co-transformed with an equimolar
mixture of the 2,~L S. cerevisiae plasmid and pUR7219 using a
standard protocol for electroporation of yeast cells.
Transformants were selected for leucine prototrophy and total
10 DNA was isolated from a number of transformants. All
transformants appeared to contain both the 2~ plasmid and
pUR7219, exemplifying that the promoter-deficient version of
the leu2 gene contained on pUR7219 can only functionally
complement leu2 deficient strains when present in high copy
15 numbers due to the simultaneous presence of the 2~L yeast
plasmid. One of the transformants was cured for the pUR7219
plasmid by cultivation on complete medium for more than 40
generations followed by replica-plating on selective and
complete solid media, yielding S. cerevisiae strain SU51 (a,
20 cir+, leu2, his4, canl).
S. cerevisiae strain SU51 harboring pUR7219 was
grown in 1 litre shakeflasks containing O.2 litre MM medium
consisting of:
- yeast nitrogen base (YNB) without amino acids 6.7 g/l
25 - histidine 20 mg/l
- glucose 20 g/l
Cultures were grown overnight at 30C under vigorous sh~k; ng
(150 rpm) to an OD at 610 nm of 2-4. Cells were collected by
centrifugation and resuspended in 1 litre of YPGAL medium
30 consisting of:
- yeast extract 10 g/l
- bacto peptone 20 g/l
- galactose 50 g/l
in 2 litre shake flasks and incubation continued for another
35 12-16 hours. At regular intervals samples were withdrawn from
the culture and centrifugated to remove biomass. The
supernatant was analyzed for cutinase activity by a
titrimatic assay using olive oil as a substrate. For each
WO94/14963 PCT~3/03550
~15~37
sample between 100 and 200 ~1 of filtrate was added to a
stirred mixture of 5.0 ml lipase substrate (Sigma, containing
olive oil as a substrate for the lipase) and 25.0 ml of
buffer (5 mM Tris-HCl pH 9.0, 40 mM NaCl, 20 mM CaC12). The
assay was carried out at 30C and the release of fatty acids
was measured by automated titration with 0.05 M NaOH to pH
9.0 using a Mettler DL25 titrator. A curve of the amount of
titrant against time was obtained. The amount of lipase
activity contained in the sample was calculated from the
10 maximum slope of this curve. One unit of enzymatic activity
is defined as the amount of enzyme that releases 1 ~mol of
fatty acid from olive oil in one minute under the conditions
specified above. Such determinations are known to those
skilled in the art.
When the production of cutinase activity did no
longer increase, cells were removed by centrifugation and the
cell free extract was acidified to pH 4.8 with acetic acid
and cutinase was recovered as described in Example 1.
20 EXAMPLE 5
Expression of variants of Fusarium solani pisi cutinase in S.
cerevisiae.
Variant N176K of Fusarium solani Pisi cutinase was
expressed in S. cerevisiae in the following way. The 0.5 kb
25 ApaI-HindIII fragment of pUR7257 (N172K) was used to replace
the analogous fragment of pUR7218, yielding pUR7228 (N172K),
in which the gene comprising the mutation is operably fused
to the sequence encoding the S. cerevisiae signal se~uence.
The 7.0 kb SacI-HindIII fragment of pUR2741 was ligated with
30 the 0.7 kb SacI-HindIII fragment of pUR7228 (N172K), yielding
pUR7234 (N172K). This plasmid was transformed to S.
cerevisiae strain SU51. The resulting transformants were
incubated as described in Example 4 and the variant enzyme
produced was recovered from the culture broth as described in
35 Examples 4 and 1.
Variant E201K of Fusarium solani Pisi cutinase was
produced in S. cerevisiae in the same way 5 using a variant
W094/149~ ~15 0 ~ 3 7 PCT~3/03~0
of the Fusarium solani pisi cutinase gene coding for the
cutinase variant E201K, as described in Example 3.
Variant A85F of Fusarium solani pisi cutinase was
produced in S. cerevisiae in the same way using a variant of
5 the Fusarium solani pisi cutinase gene coding for the
- cutinase variant A85F, as described in Example 3.
EXAMPLE 6
Expression of Fusarium solani pisi cutinase in Aspergilli.
For the expression of the synthetic Fusarium solani
pisi cutinase gene in AsPerqillus niqer var. awamori an
expression vector was constructed in which the synthetic gene
encoding Fusarium solani Pisi pre-pro-cutinase was placed
under the control of the A. niqer var. awamori strong,
inducible exlA promoter (Maat et al.,1992, de Graaff et al.,
1992).
The pre-pro-cutinase expression plasmid (pUR7280)
was constructed starting from plasmid pAW14B, which was
deposited in an E. coli strain JM109 with the Centr~lh~reau
20 voor Schimmelcultures, Baarn, The Netherlands, under N CBS
237.90 on 31st May 1990, and contains a ca. 5.3 kb SalI
fragment on which the 0.7 kb endoxylanase II (exlA) gene is
located, together with 2.5 kb of 5'-flanking sequences and
2.0 kb of 3'-flanking sequences (Fig.8). In pAW14B the exlA
25 coding region was replaced by the pre-pro-cutinase coding
region. A Bs~HI site (5'-TCATGA-3') comprising the first
codon (ATG) of the exlA gene and an AflII site (5'-CTTAAG-
3'), comprising the stopcodon (TAA) of the exlA gene
facilitated the construction of pUR7280.
The construction was carried out as follows: pAW14B
(7.9 kb) was cut partially with ~HI and the linearized
plasmid (7.9 kb) was isolated from an agarose gel.
Subsequently, the isolated 7.9 kb fragment was cut with BsmI,
which cuts a few nucleotides downstream of the BspHI site of
35 interest, to remove plasmids linearized at other Bs~HI sites.
The fragments were separated on an agarose gel and the 7.9 kb
BspHI-BsmI fragment was isolated. This was partially cut with
W094/14963 ~ 5 PCT~3/03S50 -
36
AflII and the resulting 7.2 kb BspHI-AflII fragment was
isolated.
The 0.7 kb BsPHI-AflII fragment of pUR7210
comprising the entire open reading frame coding for Fusarium
5 solani pisi pre-pro-cutinase was ligated with the 7.2 kb
Bs~HI-AflII fragment of pAW14B, yielding pUR7280. The
constructed vector (pUR7280) can subsequently transferred to
moulds (for example Asperqillus niqer, AsPerqillus niqer var.
awamori, etc) by means of conventional co-transformation
10 techniques and the pre-pro-cutinase gene can then be
expressed via induction of the endoxylanaseII promoter. The
constructed rDNA vector can also be provided with
conventional selection markers (e.g. amdS or pyrG, hygromycin
etc.) and moulds can be transformed with the resulting rDNA
15 vector to produce the desired protein. As an example, the
amdS and pyrG selection markers were introduced in the
expression vector, yielding pUR7281 (Fig. 10). For this
purpose a NotI site was created by converting the ~_RI site
(present 1.2 kb upstream of the ATG codon of the pre-pro-
20 cutinase gene) into a NotI site using a syntheticoligonucleotide (5'-AATTGCGGCCGC-3'), yielding pUR7282.
Suitable DNA fragment comprising the entire A. nidulans amdS
gene and the A. niqer var. awamori pyrG gene together with
their own promoters and terminators were equiped with
25 flanking NotI sites and introduced in the NotI site of
pUR7282, yielding pUR7281 (Fig. 10).
As an alternative approach for the expression of
the synthetic Fusarium solani ~isi cutinase gene in
AsPerqillus niger var. awamori, expression vectors were
30 constructed in which a synthetic gene encoding the mature
cutinase is not preceded by its own pre-pro-sequence, but by
the pre-sequence of A. niqer var. awamori exlA.
To prepare the synthetic cutinase gene for such
fusions, several adaptor fragments were synthetized in which
35 the coding sequence for the exlA pre-sequence is connected to
the sequence encoding the N-terminus of mature cutinase in
different ways. In cassette 5 this connection is made by
fusing the exlA pre-sequence to the pro-sequence of cutinase.
~ WO94/149~ 21 5 Q~3 7 PCT~3/03550
In cassette 6 the exlA pre-sequence is fused with the N-
terminal residue of mature cutinase. Cassette 7 is identical
with cassette 6, but here the N-terminal residue of the
encoded mature cutinase polypeptide has been changed from the
original Glycine into a Serine residue in order to better fit
the requirements for cleavage of the signal peptide.
Cassettes 5, 6 and 7 were assembled from synthetic
oligonucleotides essentially as described in Example 1 (see
Fig. 7). Cassette 5 was used to displace the 0.1 kb EcoRI-
10 SPeI fragment of pUR7210, yielding pUR7287. Cassettes 6 and 7were used to displace the 0.1 kb EcoRI-BclI fragment of
pUR7210, yielding pUR7288 and pUR7289, respectively. For each
of the plasmids pUR7287, pUR7288 and pUR7289 the 0.7 kb
BspHI-AflII fragment was ligated with the 7.2 kb BspHI-AflII
fragment of pAW14B, yielding pUR7290, pUR7291 and pUR7292,
respectively.
The constructed rDNA vectors subsequently were
transferred to moulds (As~erqillus niqer, Asperqillus niger
var. awamori) by means of conventional co-transformation
20 techniques and the pre-(pro)-cutinase gene were expressed via
induction of the endoxylanaseII promoter. The constructed
rDNA vectors can also be provided with conventional selection
markers (e.g. amdS or pyrG, hygromycin) and the mould can be
transformed with the resulting rDNA vector to produce the
25 desired protein, as illustrated in this example for pUR7280
(see above).
Aspergillus strains transformed with either of the
expression vectors pUR7280, pUR7281, pUR7290, pUR7291,
pUR7292 (containing the Fusarium solani PiSi mature cutinase
30 encoding region with or without the corresponding pro-
sequence and either the cutinase signal sequence or the exlA
signal sequence under the control of A. niqer var. awamori
exlA promoter and terminator) were grown under the following
conditions: multiple 1 litre shake flasks with 400 ml
synthetic media (pH 6.5) were inoculated with spores (final
concentration: lOE6/ml). The medium had the following
composition (AW Medium):
WO94/14963 PCT~3/03550 -
837 38
sucrose lo g/ 1
NaN03 6.0 g
KCl 0.52 g/l
KH2P04 1.52 g/l
MgS04 7H2O 0.49 g/l
Yeast extract l.o g/l
ZnS04 7H2O 22 mg/l
H3B03 11 mg/l
MnC12 4H20 5 mg/l
FeS04 7H20 5 mg/l
CaCl2 6H2 1.7 mg/l
CuS04 5H20 1.6 mg/l
NaH2Moo4 2H2o 1.5 mg/l
Na2EDTA 50 mg/l
Incubation took place at 30C, at 200 rpm for 24
hours in a Mk X incubator shaker. After growth cells were
collected by filtration (0.45 ~m filter), washed twice with
AW Medium without sucrose and yeast extract (salt solution),
resuspended in 50 ml salt solution and transferred to 300 ml
20 shake flasks contA;n;ng 50 ml salt solution to which xylose
has been added to a final concentration of 10 g/l (induction
medium). Incubation under the same conditions as described
above was continued overnight. The resulting cultures were
filtered over miracloth to remove biomass and cutinase was
25 recovered essentially as described in Example 2.
EXAMPLE 7
Expression of variants of Fusarium solani pisi cutinase in
Aspergilli.
By following essentially the route outlined in
Example 6, but now using plasmid pUR7257 (N172K) instead of
pUR7210 for the construction of fungal expression vectors, a
variant of Fusarium solani pisi cutinase comprising mutation
N172K was produced in Asperqillus niqer var. awamori.
EXAMPLE 8
Identification and isolation of genes related to the Fusarium
solani Pisi cutinase gene.
W094/14963 2 1 ~ ~ 8 3 7 J ~' e PCT~3/03550
39
Genes encoding cutinases with a varying degree of
homology with Fusarium solani pisi cutinase were isolated
from different fungi. Fungal cultures were grown in 500 ml
shakeflasks containing 200 ml of the medium described by
5 Hankin and Kolattukudy (1968) supplemented with 0.25% glucose
and incubated for 4 days at 28C in a Mk X incubator shaker
(100 rpm). At this time the glucose had been consumed and
cutinase production was induced by the addition of cutin
hydrolysate essentially as described by Ettinger et al.
lo (1987). At regular intervals samples were withdrawn from the
culture and analyzed for the presence of lipolytic activity
according to standard techniques (see example 4). Normally,
about two days after induction lipolytic activity could be
demonstrated and at that time the cells were harvested by
filtration using standard techniques. The mycelia were
washed, frozen in liquid nitrogen and lyophilized according
to standard techniques. Total cellular RNA preparations were
isolated using the guanidinium thiocyanate method and
purified by cesium chloride density gradient centrifugation,
20 essentially as described by Sambrook et al. (1989). PolyA(+)
mRNA fractions were isolated using a polyAT tract mRNA
isolation kit (Promga). The polyA(+) mRNA fractions were used
in a Northern hybridization analysis using a cDNA fragment
from the Fusarium solani ~isi cutinase gene as a probe
25 according to st~n~rd techniques, to verify the expression of
cutinase-related genes. Preparations of mRNA comprising
material capable of hybridizing with the probe were used for
the synthesis of cDNA using a ZAP cDNA synthesis kit
(Stratagene, La Jolla) according to the instructions of the
30 supplier, yielding cDNA fragments with an XhoI cohesive end
flanking the poly-A region and an EcoRI adaptor at the other
end. The obtained cDNA fragments were used for the
construction of expression libraries by directional cloning
in the sense orientation in lambda ZAPII vectors (Stratagene,
35 La Jolla), allowing expression of ~-galactosidase fusion
proteins (Huse et al.,1988). These libraries were screened
using antiserum raised against Fusarium solani pisi cutinase.
WO94/14963 PCT~3/03550
21~37
Alternatively, the synthesized cDNA fractions were
subjected to PCR-screening using cutinase specific primers
(see table 2). These primers were derived from comparison of
the amino acid sequence of several fungal Cutinase genes
(Ettinger et al., 1987). The conditions for the PCR reaction
were optimized for each set of primers, using cDNA from
Fusarium solani Pisi cutinase as a control. For those
preparations of cDNA with which a specific PCR fragment could
be generated with a length that is similar to (or greater
lO than) the length of the PCR fragment generated with the cDNA
from Fusarium solani Pisi cutinase under the same conditions,
the PCR fragment was purified by gel electroforesis and
isolated from the gel.
As an alternative approach, the PCR screening
15 technique using cutinase specific primers was also applied
directly to genomic DNA of some fungal strains, using genomic
DNA of Fusarium solani Pisi as a positive control. For those
preparations of fungal genomic DNA with which a specific PCR
fragment could be generated with a length that is similar to
(or greater than) the length of the PCR fragment generated
with the cDNA from Fusarium solani Pisi cutinase under
identical conditions, the PCR fragment was purified by gel
electrophoresis and isolated from the gel.
For strains which scored positive in either the
25 expression library approach or the PCR screening approach
(either with cDNA or genomic DNA) as well as a number of
other strains, high molecular weight genomic DNA was
isolated. Strains were grown essentially as described by
Ettinger et al. (1987), and genomic DNA was isolated as
30 described by de Graaff et al. (1988). Genomic DNA was
digested with various restriction enzymes and analyzed by
Southern hybridization using either the analogous cDNA insert
(expression library approach) or the PCR fragment (PCR
screening approach) or the Fusarium solani pisi cutinase gene
(other strains) as a probe, and a physical map of the
cutinase genes was constructed. An appropriate digest of
genomic DNA was size-fractionated by gel electrophoresis and
fragments of the appropriate size were isolated from the gel
W094/14963 4l PCT~W3/0355~
and subcloned in pUCl9. These genomic libraries were screened
with the corresponding cDNA insert (expression library
approach) or the PCR fragment (PCR screening approach),
yielding clones comprising the genomic copy of the cutinase
5 genes. These genes were sequenced in both directions. Introns
were identified by sequencing the corresponding cDNA or by
comparison with other cutinase sequences (Ettinger et al.,
1987). The N-terminal end of the mature cutinase polypeptide
was also deduced from such a comparison. Using standard PCR
lO techniques, the introns were removed, a HindIII site was
engineered immediately downstream of the open reading frames
and the coding sequence for the pre-sequence of the
SaccharomYces cerevisiae invertase gene (preceded by a SacI
site, compare cassette 8, Fig. 3) was fused to the sequences
15 encoding the N-terminus of the mature cutinases. The obtained
SacI-HindIII fragments comprising the cutinase genes operably
linked to the sequence encoding the S. cerevisiae invertase
pre-sequence were ligated with the 7.3 kb SacI-HindIII
fragment of pUR724l (see Fig. 4) and transformed to S.
20 cerevisiae strain SU51. The fungal cutinases were expressed
and recovered from the culture broth essentially as described
in Example 4.
EXAMPLE 9
25 The In-the-wash activity of Fusarium solani Pisi Cutinase
variant Nl72K.
The effect on fat removal of the Cutinase variant
Nl72K was compared to that of the wild type Fusarium solani
~isi Cutinase. In the test polyester test cloths soiled with
30 brominated olive oil were used as monitors. The amount of fat
on the test cloth was determined by measuring the amount of
bromine on the test cloth by means of X-ray fluorescence
spectrometry (as described above).
The amount of enzymatic soil removal of the wild
35 type Fusarium solani pisi Cutinase (WT) and Nl72K variant was
determined at a dosage of 3 LU/ml under several experimental
conditions:
W094/14963 PCT~W3/03550 -
2 ~ ~083 ~ 42
Soil Soil Temperature Detergent Water
removal removal (C) Product hardness
(%) WT (~) N172K (FH)
4.9 lO.1 40 A (1 g/l) 6
1.6 6.7 40 B (2 g/l) 6
20.2 24.6 40 C (2 g/l) 27
13.2 13.4 40 B (1 g/l) 27
30.8 40.6 30 C (2 g/l) 27
17.4 22.4 30 B (2 g/l) 27
The compositions (in % by weight) of the Detergent
Products A-C were as follows:
Product A
compound weight %
nonionic surfactant C12-C15alcohol 9.5
10.5-13EO
Sodium sulphate 38.6
Sodium Carbonate 40.4
Sodium silicate (Na2O:Si2O = 2.4) 7.3
20 Water 4.2
Product B
compound weight %
DOBS 6.4
25 Soap 1.7
Synperonic A7 3.0
Zeolite 43
Sokolan CP7 9.9
~ WO94/14963 21~ Q 8 3 7 - `
43
waterglass l.2
Sodium CMC 0.77
Sodium carbonate l0.16
NaOH 2.6
5 Water to l00
Product C
compound weight %
Coco-primary alkyl sulphate 5.2
Nonionic surfactant Cl2-Cl5 alcohol 5.2
7 EO
Nonionic surfactant Cl2-Cl5 alcohol 6.6
3 EO
Sodium silicate 0.45
15 Zeolite 4A 32.00
Sodium carbonate ll.52
Hardened Tallow soap 2.00
The enhancement of the in-the-wash performance
(oily soil removal) relative to wild-type Fusarium solani
Pisi cutinase under various wash conditions is evident.
Figure 13 shows the in-the-wash performance at various enzyme
concentrations using 2 g/l Detergent Product C in a 30 minute
wash at 30C at 27FH.
For comparison, the same experiments were also
carried out with Lipolase (TM). Under all conditions,
cutinase variant Nl72K was superior.
EXAMPLE l2
30 The In-the-wash activity of Fusarium solani PiSi Cutinase
variant E20lK.
WO94/14963 2 1 5 ~ ~ 3 7 PCT~W3/03550
The effect on fat removal of the Cutinase variant
E201K at various enzyme concentrations was compared to that
of the wild type Fusarium solani Pisi Cutinase using 2 g/l
Detergent Product C (see Example 11) in a 30 minute wash at
30C at 27FH. In the test polyester test cloths soiled with
brominated olive oil were used as monitors. The amount of fat
on the test cloth was determined by measuring the amount of
bromine on the test cloth by means of X-ray fluorescence
spectrometry (as described above).
The results are shown in Figure 14. The enhancement
of the in-the-wash performance (oily soil removal) relative
to wild-type Fusarium solani ~isi cutinase is evident. For
comparison, the same experiment was also carried out with
Lipolase (TM). It was found that the performance of the E201K
15 cutinase variant was clearly superior.
EXAMPLE 13
The In-the-wash activity of Fusarium solani ~isi Cutinase
variant A85F.
The effect on fat removal of the Cutinase variant
A85F at various enzyme concentrations was compared to that of
the wild type Fusarium solani pisi Cutinase using 2 g/l
Detergent Product C (see Example 11) in a 30 minute wash at
30C at 27FH. In the test polyester test cloths soiled with
25 brominated olive oil were used as monitors. The amount of fat
on the test cloth was determined by measuring the amount of
bromine on the test cloth by means of X-ray fluorescence
spectrometry (as described above).
The results are shown in Figure 15. The enhancement
30 of the in-the-wash performance (oily soil removal) relative
to wild-type Fusarium solani pisi cutinase is evident. For
comparison, the same experiment was also carried out with
Lipolase (TM). It was found that the performance of the A85F
cutinase variant was clearly superior.
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