Note: Descriptions are shown in the official language in which they were submitted.
~ 94/14964 PCT~3/035~1
` 2151~38
MODIFIED CIJTINASES, 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-organisms by means of
rDNA t~c-hniques, especially the lipase from Thermom~ces
lanuginosus/Humicola lanuqinosa. EP-A-331 376 (Amano)
describes lipases and their production by rDNA techn;ques,
and their use, including an amino acid sequence of lipase
from Pseudomonas cePacia. Further examples of lipases
produced by rDNA t~chnlque are given in WO 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
35 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
W094/14964 21~ ~ O ~ 8 PCT~3/03551 -
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
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 conformational change in
10 the 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
andtor 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
hydrophilic amino acid residues in the lipid contact zone, or
substituting these residues by hydrophobic amino acids.
Cutinases are a sub-class of enzymes (EC 3.1.1.50),
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
094/14964 21510 3 8 PCT~3/03551
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
f;ndings appear to confirm the present theory about the
m~ch~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 general
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
W094/14964 ~CT~3103551 ~
2 ~ 3 8 4
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 therefore differ from classical lipases in that
lO they 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 l990, 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 CUti n~ , in particular the
cutinase from Fusarium solani pisi, exhibit a clear in-the-
wash effect. However, there is still a need for Cutinase
30 variants having improved in-the-wash lipolytic activity in
anionic-rich detergent compositions, and for methods for
producing such enzymes.
In accordance with the present invention, there are
provided Cutinase variants wherein the amino acid sequence
35 has been modified in such way that the compatibility to
anionic surfactants has been improved. More in particular, it
was found that the lipolytic activity of eukaryotic
Cutinases, more in particular of Cutinases from Fusarium
21~ 1 0 3 8 PCT~3/03~1
solani pisi, Colletotrichum capsici, Colletotrichum
qloeosporiodes and Maqnaporthe qrisea, in anionic-rich
detergent compositions may be improved by reducing the
binding of anionic surfactants to the enzyme.
DEFINITION OF THE INVENTION
A Cutinase variant of a parent Cutinase, wherein
the amino acid sequence has been modified in such way that
lO the compatibility to anionic surfactants has been improved,
in particular by reducing the binding of anionic surfactants
to the enzyme.
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 8S 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 Cutinases appears
25 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 p~-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.
W094/14964 PCT~3/03~51 ~
2~ 3~ 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) 9
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 ca~sici, Colletotrichum
qloeos~oriodes and Maqna~orthe qrisea. In Figure ll the
20 partial amino acid sequences of these Cutinases are shown and
it can be seen that there is a high degree of homology.
Alternative to the improvement of Fusarium solani
isi 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 ca~sici,
Colletotrichum qloeosPoriodes and MaqnaPorthe qrisea cDNA
encoding (pro)cutinase and probes recognizing conserved
sequences in other Cutinases and if necessary, using these
30 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
W0-A-92/05249). After cloning and expression the thus
obtained Cutinases encoding genes in E. coli according
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
~ 094/149~ 21~1 0 3 8 PCT~3103551
7 f
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 "in-the-wash" activity of lipolytic enzymes
and careful inspection of the 3D structure of Fusarium solani
Pisi cutinase (Martinez et al. (1992) Nature 356, 615-618)
and inspection of the 3D structure of Fusarium solani pisi
lO cutinase we have found a number of possibilities how to
improve the compatibility of this cutinase and Cutinases in
general to anionic surfactants by means of recombinant DNA
techniques.
Starting from the known 3D structure of the
15 Fusarium solani Pisi cutinase, the 3D-structure of the
cutinase from Colletotrichum ~loeosPoriodes 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.
20 Louis, Missouri). The obtained model of the Colletotrichum
qloeosPoriodes cutinase was refined by applying energy
minimization (EM) and molecular dynamics (MD) t~chniques as
implemented in the BIOSYM molecular modelling software
package (BIOSYM, San Diego, California). During EM and MD
25 refinement of the model a knowledge-based approach was
applied. The model was simultaneously optimized for the
detailed energy terms of the potential energy ~unction and
known structural criteria. Model ~uality was assessed by
criteria such as number and quality of hydrogen bonds,
30 hydrogen bonding patterns in the secondary structure
elements, the orientation of peptide units, the values of and
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,
35 extremely exposed hydrophobic residues and energetically
unfavourable positions of disulphide bridges. Relevant side-
chain rotamers were selected from the Ponder & Richards
rotamer library (Ponder et al. (1987) J.Mol.Biol. 193, 775-
WO94/149~ ~ PCT~3/03551
21 ~ 038 8
791). The final choice of a particular side-chain rotamer
from this library was based on structural criteria
evaluations as mentioned above. MD was used to anneal the
side-chain atoms into position. A similar approach was used
5 to obtain the 3D-structure of the cutinase from MaqnaPorthe
qrisea.
The present invention shows that Cutinases can be
modified in such a way that the interaction with anionic
surfactants can be reduced without changing the "in-the-wash"
lO performance of the modified Cutinase.
This may be achieved in a number of ways. First,
the binding of anionic surfactants to the enzyme may reduced
by reducing the electrostatic interaction between the anionic
surfactant and the enzyme. For instance, by replacing one or
15 more positively charged arginine residues which are located
close to a hydrophobic patch capable of binding the apolair
tail of an anionic surfactant, by lysine residues. It is also
possible to reduce the electrostatic interaction between the
anionic surfactant and the enzyme shielding the positive
20 charge of such an arginine residue by introducing within a
distance of about 6 A from said arginine a negative charge,
e.g. an glutamic acid residue. Alternatively, the
electrostatic interaction between the anionic surfactant and
the enzyme may be reduced by replacing one or more positively
25 charged arginine residues which are located close to a
hydrophobic patch capable of binding the apolair tail of the
anionic surfactant, by uncharged amino acid residues.
Furthermore, the electrostatic interaction between the
anionic surfactant and the enzyme may reduced by replacing
30 one or more positively charged arginine residues which are
located close to a hydrophobic patch capable of binding the
apolair tail of the anionic surfactant, by negatively charged
amino acid residues.
Another approach to reduce the binding between an
35 anionic surfactant and the enzyme is to replace one or more
amino acid residues which are located in a hydrophobic patch
capable of binding the apolair tail of the anionic
surfactant, by less hydrophobic amino acid residues. These
~ 094/149~ 21~ 1 0 3 8 PCT~3/03551
less hydrophobic amino acid residues are preferably selected
from the group consisting of glycine, serine, alanine,
aspartic acid and threonine.
Due to their improved anionics compatibility, the
5 Cutinases variants produced according to the invention can
bring advantage in enzyme activity, when used as part of an
anionic-rich detergent or cleaning compositions. In the
context of this invention, anionic-rich means that the
detergent or cleaning composition contains a surfactant
lO system which consists for more than 5%, generally more than
lO~, and in particular more than 20% of anionic surfactants.
The Cutinase variants of the present invention were
found to possess an improved in-the-wash performance during
the main cycle of a wash process. By in-the-wash performance
15 during the main cycle of a wash process, it is meant that a
detergent composition containing the enzyme is 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
20 far as concentration, water hardness, temperature, are
concerned. It should be born in mind that under the same
conditions, the conventional commercially available lipolytic
enzyme Lipolase (TM) ex Novo Nordisk does not appear to have
any significant in-the-wash effect at all on oily soil.
The in-the-wash effect of an enzyme on oily soil
can be assessed using the following assay. New polyester test
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
30 are then soiled with olive oil or another suitable,
hydrolysable oily stain. Each tests cloth (weighing
approximately l g) is incubated in 30 ml wash liquor in a lOo
ml polystyrene bottle. The wash liquor contains the detergent
product given below at a dosage of l g per litre. The bottles
35 are agitated for 30 minutes in a Miele TMT washing machine
filled with water and using a normal 30C main wash
programme. The Cutinase variant is preadded to the wash
liquor at 3 LU/ml. The control does not contain any enzyme.
W094/149~ PCT~3/03551
~ S1~3~ lo
The washing powder has the following composition (in % by
weight):
LAS 6.9
Soap 2.0
5 Nonionic surfactant lO.o
Zeolite 27.0
Sodium carbonate 10.2
Sodium sulphate 13.0
After washing, the cloths are thoroughly rinsed
10 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
testcloth with petroleum ether in a Soxhlet extraction
apparatus, distilling off the solvent and determining the
lS percentage 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
20 Research Journal 38, 105-107). Each test cloth is then
incubated in 30 ml wash liquor in a 100 ml poly~yLene
bottle. A series of bottles is then agitated in a washing
machine filled with water and using a normal 30C main wash
programme. After the main wash, the test cloths are carefully
25 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
30 as a percentage of the amount which was initially present on
the test cloth, as fo'lows:
% Soil removal = Bromineb~ - Bromine~w * 100 %
Brominebw
35 wherein: Brominebw denotes the percentage bromine on the
cloth before the wash and Bromineaw the percentage bromine
after the wash.
~ 094/14964 21~1 Q 3 ~ PCT~3103551
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,
5 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
containing only silent mutations) means a gene encoding an
enzyme having an amino acid sequence which has been derived
lO directly or indirectly, and which in one or more locations is
different, from the sequence of a corresponding parent
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
15 sequence of the gene which (owing to the redundancy in the
codon-amino acid relationships) leads to no change in the
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-
20 organism subjected to mutation in respect of its gene for theenzyme. Such mutation of the organism may be carried out
either (a) by mutation of a corresponding gene (parent gene)
already present in the parent micro-organism, or (b) by the
transfer (introduction) of a corresponding gene obtained
25 directly or indirectly from another source, and then
introduced (including the mutation of the gene) into the
micro-organism which is to become the mutant micro-org~n; ~m.
A host micro-organism is a micro-organism of which a mutant
gene, or a transferred gene of other origin, forms part. In
30 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
35 Colletotrichum capsici, Colletotrichum qloeosporiodes and
Maqnaporthe qrisea. These Cutinase variants can be produced
by a rDNA modified micro-organism containing a gene obtained
or made by means of rDNA techniques.
W094/14964 21 S 10 ~ ~ PCT~3/035~1 -
12
Once the amino acid residues have been identified
that should be replaced by another amino acid residue, for
example mutation R17E relative to the sequence of Fusarium
solani Pisi cutinase or a homologue thereof.
S It will be clear to the skilled man that such
modifications will affect the structure of the Cutinase.
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 succesful Cutinase variants with a high rate of
succes. In the following Table II 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 II
A = Ala = Alanine V = Val = Valine
20 L = Leu = Leucine I = Ile = Isoleucine
P = Pro = Proline F = Phe = Phenylalanine
W = Trp = Tryptophan M = Met = Methionine
G = Gly = Glycine S = Ser = Serine
T = Thr = Threonine C = Cys = Cysteine
25 Y = Tyr = Tyrosine N = Asn = Asparagine
Q = Gln = Glutamine D = Asp = Aspartic Acid
E = Glu = Glutamic Rcid K = Lys = Lysine
R = Arg = Arginine H = His = Histidine
In this specification, a mutation present in the
30 amino acid sequence of a protein, and hence the mutant
protein itself, may be described by the position and nature
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
35 by the identity of the amino acid residue substituted there
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 subscript letters attached to the number of
~ 094/14964 2 1 ~1 ~ 3 8 PCT~3/03551
13
the last preceding member of the regular sequence or
reference sequence.
For example, a mutant characterised by substitution
of Arginine by Glutamine in position 17 is designated as:
5 Argl7Glu or R17E. A (hypothetical) insertion of an additional
amino acid residue such as proline after the Arginine would
be indicated as Argl7ArgPro or R17RP, alternatively as *17aP,
with the inserted residue designated as position number 17a.
A (hypothetical) deletion of Arginine in the same position
10 would be indicated by Argl7* or R17*. The asteris~ stands
either for a deletion or for a 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
15 position.
Multiple mutations are separated by plus signs,
e.g. R17E+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
20 mutations given in the following Table may be co~bined if
desired.
The Table III given below shows certain useful
examples of Cutinase variants according to the invention,
based on the sequence of the Cutinases from Fusarium solani
isi, and Magnaporthe qrisea.
TABLE III
Variants of Fusarium solani Pisi cutinase:
R17L, R17K, R17E, L51A, L51S, R78L, T80D, R88E, R96N, R96Q,
R156L, A195S, R196A, R196K, R196E.
Variants of Magna~orthe qrisea cutinase:
A80D, A88E, R156L.
According to a further aspect of the invention,
there is provided a process for producing the Cutinase
variants of the invention. Naturally occurring Cutinase
35 producing micro-organisms are usuall~y 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
W094/14964 ~ PCT~3/03551
14
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-
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-organisms that can growth fast on cheap medium and are
10 capable to synthesize and secrete large amounts of Cutinase.
Such suitable rDNA modified (host micro-organisms) according
to the present invention are bacteria, among others, Bacilli,
Corynebacteria, StaPhYlococci and StrePtomYCes, or lower
eukaryotes such as SaccharomYces cerevisiae and related
species, KluYveromyces marxianus and related species,
Hansenula Polymorpha 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
20 are able to glycolysate the Cutinase molecule. Glycosylation
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
25 genes, e.g. the gene from Fusarium solani isi, into cloning
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.
Also provided by the invention are polynucleotides
30 made or modified by rDNA technique, which encode such
Cutinase variants, rDNA vectors containing such
polynucleotides, and rDNA modified microorganisms containing
such polynucleotides and/or such rDNA vectors. The invention
also provides corresponding polynucleotides encoding the
35 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
by a stop codon and optionally having nucleotide sequences
~ 094/14964 21510 3 8 PCT~3/03551
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
5 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
aminoacid residues and being codons preferred by a new host,
lO 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 sequences coding for the
pro-or mature Cutinases, there can be located a nucleotide
sequence that codes for a signal or secretion sequence
15 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
20 from:
(a) a naturally occurring nucleotide sequence (e.g. encoding
the original amino acid sequence of the prepro- or pro-
cutinase produced by Fusarium solani ~isi);
(b) chemically synthesized nucleotide sequences consisting of
25 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;
(c) genetically engineered nucleotide sequences derived from
one of the nucleotide sequences mentioned in preceding
30 paragraphs a or b coding for a Fusarium solani Pisi Cutinase
with a different amino acid sequence but having superior
stability and/or activity in detergent systems.
Summarizing, rDNA vectors able to direct the
expression of the nucleotide sequence encoding a Cutinase
35 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
W094tl4964 I ; PCT~3/03551
2~ 16
-
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
does not start with the codon ATG, an ATG codon should be
5 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
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
the ds DNA into the genome of the selected host or,
(d2) an origin of replication suitable for the selected host;
(el) Optionally a (auxotrophic) 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
sequences facilitative of functional expression of the
25 cutinase. The auxotrophic marker can consist of a coding
region of the auxotrophic marker and a defective promoter
region.
Another embodiment of this invention is the
fermentative production of one of the various Cutinase
30 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
35 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
~ 094/149~ 2 1 ~ 1 0 3 8 ~ PCT~3/035Sl
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
5 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 varaint either
from said broth or from said cells by physical or chemical
lO 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
filtration or centrifugation. Optionally, the Cutinase
15 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
and down stream processing equipment.
Also provided by the invention is a method for the
production of a modified micro-organism capable of producing
a Cutinase variant by means of rDNA t~chn;ques, characterized
in that the gene coding for the Cutinase variant that is
introduced into the micro-org~n;~ is fused at its 5'-end to
25 a gene fragment encoding a (modified) pre-sequence functional
as a signal- or secretion-sequence for the host orgAn;~.
According to a further aspect of the invention,
there are provided rDNA modified micro-organisms containing a
Cutinase varaint gene and able to produce the Cutinase
30 variant encoded by said gene. In an rDNA modified micro-
organism, a gene (if originally present) encoding the native
Cutinase is preferably removed, e.g. replaced by another
structural gene.
According to a further aspect of the present
3~ invention, there are provided enzymatic detergent
compositions comprising the Cutinase variants of the
invention. Such compositions are combinations of the
Cutinases variants and other ingredients which are commonly
W094/14964 ~ 3 g PCT~3/03551
used in detergent systems, including additives for detergent
compositions and fully-formulated detergent and cleaning
compositions, e.g. of the kinds known per se and described
for example in EP-A-258 068.
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,
EP-A-179 S33 (Procter & Gamble), EP-A-205 208 and EP-A-206
390 (Unilever), JP-A-63-078000 (1988), and Research
10 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
Lipolase(TM) products of Novo Nordisk).
The added amount of Cutinase variant can be chosen
20 within wide limits, for example from 10 - 20,000 LU per gram,
and preferably 50 -2,000 LU per gram of the detergent
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
25 the case of other enzymes, such as proteA~ , amylases,
cellulases which may also be present. Advantage may be gained
in such detergent compositions, where protease is present
together with the Cutinase variant, 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 include
subtilisin of for example BPN' type or of many of the types
of subtilisin disclosed in the literature, e.g. mutant
proteases as described in for example EP-A-130 756 or EP-A-
35 251 446 (both Genentech); US-A-4 760 025 (Genencor); EP-A-214
435 (Henkel); WO-A-87jO4661 (Amgen); WO-A-87/05050 (Genex~; -
Thomas et al. J.Mol.Biol. (1987) 193, 803-813; Russel et al.
Nature (1987) 328, 496-500.
~ 094/l49~ 2 1 ~ 1 0 3 8 PCT~3/03551
19
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),
- 5 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 synthetic
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
~usarium 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. 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 transit~ons 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 the encoded phoA signal
W094/149~ ~ 8 PCT~3/03551
~ 20
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.
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.
15 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
Pisi 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 ~nCoA; ng region
with a plant a-galactosidase gene under the control
of the yeast gal7 promoter.
30 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 pAW14B obtained by insertion of a 5.3 kb
SalI fragment of AsPerqillus niqer var. awamori
genomic DNA in the SalI site of pUC19.
Fig. 9. Plasmid pUR7280 obtained by displacing the BspHI-
AflII fragment comprising the exlA open reading
094/14964 2 ~ 3 8 PCT~3/03551
frame in pAW14B with a BspHI-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.
Fig. 10. Plasmid pUR7281 obtained by introduction of both
the A. nidulans amdS and A. niqer var. awamori pyrG
selection markers in pUR7280.0 Fig. 11. Partial amino acid sequences of the cutinases from
Fusarium solani Pisi, Colletotrichum capsici,
Colletotrichum qloeosPoriodes and MaqnaPorthe
grisea, showing the location of the residues in the
3-D structure.5 Fig. 12. Compatibility of Fusarium solani pisi cutinase and
Cutinase variants to a LAS-based detergent
composition.
Fig. 13. Compatibility of Fusarium solani Pisi cutinase and
Cutinase variants to a PAS-based detergent
composition.
Fig. 14. Compatibility of Fusarium solani pisi cutinase and
Cutinase variants to a high-nonionic detergent
composition.
Fig. 15. Compatibility of Fusarium solani pisi cutinase and
Cutinase variants to SDS.
Fig. 16. In-the-wash effect for Fusarium solani Pisi
cutinase and Cutinase variant R17E.
REFERENCES0 Sambrook, J., Fritsch, E.F. and Maniatis,T. (1989). Molecular.t -
Cloning: a laboratory manual (2nd ed). Cold Spring Harbor
Lakoratory Press, Cold Spring Harbor, New York. ISNB 0-87969-
309-6.
Furste, J.P., Pansegrau, W., Frank, R., Blocker, H., Scholz,
35 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. 11983). J. Bacteriol 154, 366-
W094/14964 PCT~31035~1 ~
~ ~3~3~ 22
Tartof and Hobbs (1988). Gene 67, 169-182.
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
5 plants. Proc.Natl.Acad.Sci. USA 81, 3939-3943.
Noqi, Y. and Fukasawa, T. (1983). Nucleotide sequence of the
transcriptional initiation region of the yeast GAL7 gene.
Nucleic Acids Res. 11, 8555-8568.
Taussiq, R. and Carlsson, M. (1983). Nucleotide sequence of
10 the yeast SUC2 gene for invertase. Nucleic Acids Res. 11,
1943-1954.
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
15 yeast SaccharomYces cerevisiae. Ph.D. thesis. Rijks
Universiteit Utrecht, The Netherlands.
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.
20 Rotterdam, C. (1992). Xylanases and their application in
bakery. In: Visser, J., Beldman, G., Kusters-van Someren,
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.
25 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
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.
Ettinger, 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 I, 1-3.
21~1038
094/149~ - PCT~3/03551
23
De Graaff, L.H., H.W.J. van den Broek and J. Visser (198~).
Isolation and expression of the Aspergillus nidulans pyruvate
kinase gene. Curr. Genet. 13, 315-321.
EXAMPLE 1
~ Construction of a synthetic gene encoding Fusarium solani
pisi pre-pro-cutinase.
A synthetic gene encoding Fusarium solani pisi pre-
10 pro-cutinase was constructed essentially according to the
method described in EP-A-407 225 (Unilever). Based on
published nucleotide sequences of Fusarium solani pisi genes
(Soliday et al. (1984) and W0-A-90/09446, Plant Genetic
Systems), a completely synthetic DNA fragment was designed
15 which comprises a region encoding the Fusarium solani Pisi
pre-pro-cutinase polypeptide. Compared to the nucleotide
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
sequence of the entire synthetic cutinase gene is presented
in Fig. lD.
Construction of the synthetic cutinase gene was
25 performed by assembly of three separate cassettes starting
from synthetic DNA oligonucleotides. Each synthetic DNA
cassette is equipped with an ~_RI site at the start and a
HindIII site at the end. Oligonucleotides were synthesized
using an Applied Biosystems 380A DNA synthesizer and purified
30 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
35 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
WOg4/149~ 215~3~ PCT~3/03551
24
ligated with the 2.7 kb EcoRI-HindIII fragment of pUC9 and
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
5 the sequence of the synthetic cassettes. Using this procedure
pUR7207 (comprising cassette 1, Fig. lA), pUR7208 (comprising
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-APaI fragment of
lO pUR7207 with the 0.2 kb ApaI-NheI fragment of pUR7208 and the
O.3 kb NheI-HindIII fragment of pUR7209, yielding pUR7210.
This plasmid comprises an open reading frame encoding the
complete pre-pro-cutinase of Fusarium solani Pisi (Fig. lD).
15 EXAMPLE 2
Expression of Fusarium solani pisi (pro)cutinase in
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
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
25 signal sequence which provides a translational initiation
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
35 A1~-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
21510 ~ 8 PCT~3/03551
periplasmatic space (see WO-A-90/09446, Plant Genetic
Systems).
To obtain such a construct, the 69 bp EcoRI-SPeI
fragment comprising the cutinase pre-sequence and part of the
5 pro-sequence was removed from pUR7210 and replaced with the
synthetic DNA linker sequence (EcoRI-SpeI fragment) providing
the derivative of the E. coli phoA pre-sequence and the
alterated N-terminal amino acid residu 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
encoding region. This fragment was ligated with the 8.9 kb
BamHI-HindIII fragment of pMMB67EH (Furste et al., 1986) to
15 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
tac-promoter.
E. 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 K2HPO4
12 g/l Bacto-tryptone
25 24 g/l Bacto-yeast extract
0.4 % glycerol (vtv)
Cultures were grown overnight at 25C - 30C in the
presence of 100 ~g/ml ampicillin under vigorous ~h~k;ng (150
rpm) to an OD at 610 nm of 10-12. Then IPTG (isopropyl-~-D-
30 thiogalactopyranoside) was added to a final concentration of10 ~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 ~udged by the
analysis of samples withdrawn from the cultures, the cells
35 were harvested by centrifugation and resuspended in the
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
W094/149~ ~ 38 PCT~3/035~1
26
causing lysis of the cells through osmotic shock. Cell debris
was removed by centrifugation and the cell free extract was
acidified to pH 4.8 with acetic acid, left overnight at 4OC
and the resulting precipitate was removed. A better than 75
5 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.
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 concentatred alkaline solution
through a suitable volume of DEAE-cellulose (Whatman DE-52)
and direct application of the DEAE flow-through to a Q-
sepharose HP (Pharmacia) column. Elution with a salt gradient
15 yielded a homogenous cutinase preparation with a typical
overall yield of better than 75%.
EXAMPLE 3
Construction of genes encoding variants of Fusarium solani
20 Ei~i cutinase.
Using the synthetic gene for Fusarium solani Pisi
pre-pro-cutinase described in Example l, variant genes
comprising alterations in the encoded amino acid sequence
were constructed. For this construction essentially the same
25 approach was followed as described in Example l for the
construction of the three cassettes constituting the complete
synthetic gene. For example, a new version of cassette l was
assembled using the same oligonucleotides (oligos) as
described in Example l, except for the two oligos which cover
30 the coding triplet for the position which is to be mutated.
Instead, two new oligos were used, which comprise the mutant
sequence but are otherwise identical to the original oligos
which they are replacing.
ExamPle 3A
A gene coding for Fusarium solani Pisi cutinase
variant Rl7E was constructed using using a variant of
cassette l incorporating a variant of CUTIlC IG (containing
GAG instead of AGA) and a variant of CUTIlI IG (containing
~ 094/14964 21~10 38 PCT~3/03551
.. . .
27
CTC instead of TCT) instead of CUTIlC IG and CUTIlI IG (see
Fig. lA). The new cassette 1 was cloned and sequenced
essentially as described in Example 1 and the about 120 bp
EcoRI/NruI DNA fragment comprising the mutation R17E was
5 exchanged for the corresponding fragment in pUR7210, yielding
pUR7240 (R17E). The 0.6 kB SpeI-HindIII fragment from this
plasmids was used to replace the corresponding ~ragment in
pUR7220, yielding the E. coli expression plasmid pUR7222
(R17E). This E. coli expression plasmid was transformed to E.
10 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. Similarly
Arg 17 could be replaced by Lys or by Leu.
ExamPle 3B
A gene coding for Fusarium solani Pisi cutinase
variant R196E was constructed using using a variant of
cassette 3 incorporating a variant of CUTI3F MH (containing
GAG instead of CGG) and a variant of CUTI3M MH (con~;n;ng
CTC instead of CCG) instead of CUTI3F MH and CUTI3M MH (see
20 Fig. 3A). The new cassette 3 was cloned and sequenced
essentially as described in Example 1 and the about 120 bp
EcoRI/NruI DNA fragment comprising the mutation R196E was
exchanged for the corresponding fragment in pUR7210, yielding
pUR7241 (R196E). The 0.6 kB S~eI-~adIII fragment from this
25 plasmids was used to replace the corresponding fragment in
pUR7220, yielding the E. coli expression plasmid pUR7225
(R196E). 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
30 and purified essentially as described in Example 2. By the
same method Arg 196 was replaced by Lys (R196K), using a
variant of CUTI3F MH (containing A~G instead of CGG) and a
variant of CUTI3M MH (containing CTT instead of CCG) instead
of CUTI3F MH and CUTI3M MH. Similarly, Arg 196 was replaced
35 by Leu (R196L), using a variant of CUTI3F MH (containing CTT
instead of CGG) and a variant of CUTI3M MH (containing AAG
instead of CCG) instead of CUTI3F MH and CUTI3M MH. The same
method was used to replace Arg 196 by Ala (R196A).
W094/14964 PCT~3/03551
~ 28
Example 3C
.
A gene coding for Fusarium solani Pisi cutinase
variant L51A was constructed using using a variant of
cassette 1 incorporating a variant of CUTIlF IG (containing
5 GCT instead of CTC) and a variant of CUTIlL IG (containing
AGC instead of GAG) instead of CUTIlF IG and CUTIlL IG (see
Fig. lA). The new cassette 1 was cloned and sequenced
essentially as described in Example 1 and the about 120 bp
EcoRI/NruI DNA fragment comprising the mutation L51A was
10 exchanged for the corresponding fragment in pUR7210, yielding
pUR7242 (L51A). The 0.6 kB SpeI-HindIII fragment from this
plasmid was used to replace the corresponding fragment in
pUR7220, yielding the E. coli expression plasmid pUR7245
(L51A). This E. coli expression plasmid was transformed to E.
15 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. Similarly
Leu 51 could be replaced by Ser.
Example 3D
Using the cassettes constructed in the examples 3A
and 3B, a Cutinase variant with two modifications can be
constructed. In example 3A the construction of pUR7240 (R17E)
has been described. In example 3B the construction of the
EaqI/HindlII DNA fragment comprising the mutation Rl96E has
25 been described. The A~aI/HindlII DNA fragment of pUR7240
(R17E) was replaced by the APaI/HindlII DNA fragment of
pUR7241, yielding pUR7243 (Rl7E+R196E). The 0.6 kB S~eI-
HindIII fragment from this plasmid was used to replace the
corresponding fragment in pUR7220, yielding the E. coli
30 expression plasmid pUR7226 (Rl7E+Rl96E). This E. coli
expression plasmid was used to transform 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.
35 Example 3E . .
Using the cassettes constructed in the examples 3A
and 3C, a Cutinase variant with two modifications can be
constructed. In example 3A the construction of pUR7240 (R17E)
~ 094/14964 21~ 1 0 3 8 PCT~3103~51
has been described. In example 3C the construction of the DNA
fragment comprising the mutation L51A has been described. The
BclI/ApaI fragment of pUR7242 was exchanged for the
corresponding fragment in pUR7240, yielding pUR7244
(R17E+L51A). The 0.6 kB S~eI-HindIII fragment from this
plasmids was used to replace the corresponding fragment in
pUR7220, yielding the E. coli expression plasmid pUR7246
(R17E+L51A). This E. coli expression plasmid was used to
transform E. coli strain WK6. Transformants were grown as
10 outlined in Example 2 and the variant pro-cutinase enzyme was
recovered and purified essentially as described in Example 2.
EXAMPLE 4
Expression of Fusarium solani Pisi cutinase in SaccharomYces
15 cerevisiae.
For the expression of the synthetic Fusarium solani
pisi cutinase gene in Saccharomvces cerevisiae an expression
vector was constructed in which a synthetic gene encoding the
mature cutinase is preceded by the pre-sequence of S.
20 cerevisiae invertase (Taussig and Carlsson, 1983) and the
strong, inducible gal7 promoter (Nogi and Fukasawa, 1983). To
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
25 encoding the N-terminus of mature cutinase. This fragment was
assembled as an EcoRI-HindIII cassette in pUC9 essentially as
described in Example 1 (cassette 8, see Fig. 3), yielding
pUR7217. Plasmids pUR?210 and pUR7217 were transformed to E.
coli JMllO (a strain lacking the dam methylase activity) and
30 the 2.8 kb BclI-HindIII fragment of pUR7217 was ligated with
the 0.6 kb BclI-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,
WO94/149~ ~ PCT~3/03551
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).
Optionally, a S. cerevisiae polII terminator can be placed
5 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
strains harboring the 2~ plasmid (cir+ strains), a promoter-
10 deficient version of the S. cerevisiae Leu2 gene permittingselection of high copy number transformants in S. cerevisiae
leu2~ strains, and the synthetic gene encoding the mature
part of Fusarium solani Pisi cutinase operably linked to the
S. cerevisiae invertase pre-sequence under the regulation of
15 the strong, inducible S. cerevisiae gal7 promoter.
S. cerevisiae strain SU50 (a, cir, leu2, his4,
canl), which is identical to strain YT6-2-lL (Erhart and
Hollenberg, 1981), was co-transformed with an equimolar
mixture of the 2~ S. cerevisiae plasmid and pUR7219 using a
20 standard protocol for ele~roporation of yeast cells.
Transformants were selected for leucine prototrophy and total
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
25 the leu2 gene contained on pUR7219 can only ~unctionally
complement leu2 deficient strains when present in high copy
numbers due to the simultaneous presence of the 2~ yeast
plasmid. One of the transformants was cured for the pUR7219
plasmid by cultivation on complete medium for more than 40
30 generations followed by replica-plating on selective and
complete solid media, yielding S. cerevisiae strain SU51 (a,
cir+, leu2, his4, canl).
S. cerevisiae strain SU51 harboring pUR7219 was
grown-in 1 litre shakeflasks containing O.2 litre MM medium
35 consisting of: !
- yeast nitrogen base (YNB) without amino acids 6.7 g/l
- histidine 20 mg/l
- glucose 20 g/l
2151~38
~094/14964 PCT~3/035~1
31
Cultures were grown overnight at 30OC under
vigorous shaking (150 rpm) to an OD at 610 nm of 2-4. Cells
were collected by centrifugation and resuspended in l litre
of YPGAL medium 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
12-16 hours. At regular intervals samples were withdrawn from
10 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
sample between 100 and 200 ~l 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 ~aC12). 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
20 titrant against time was obtained. The amount of lipase
activity contained in the sample was calculated from the
~i um 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. .r
EXAMPLE 5
- Expression of variants of Fusarium solani pisi cutinase in S.
cerevisiae.
The 0.5 k~ ApaI-HindIII fragment of pUR7241 (R196E)
was used to replace the analogous fragment of p-~R7218,
yielding pUR7229 (R196E), in which the gene comprising the
mutation is operably fused to the sequence encoding the S.
I
WO94/14964 ~ 3 8 PCT~3/03551
32
cerevisiae signal sequence. The 7.0 kb SacI-HindIII fragment
of pUR2741 was ligated with the 0.7 kb SacI-HindIII fragment
of pUR7229 (R196E), yielding pUR7235 (R196E). This plasmid
was used to transform to S. cerevisiae strain SU51. The
5 resulting transformants were incubated as described in
Example 4 and the variant enzyme produced was recovered from
the culture broth as described in Examples 4 and 1.
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
15 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
20 deposited in an E. coli strain JM109 with the Centraalbureau
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
25 2.0 kb of 3'-flanking sequences (Fig.8). In pAW14B the exlA
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
30 facilitated the construction of pUR7280.
The construction was carried out as follows: pAW14B
(7.9 kb) was cut partially with BsPHI and the linearized
plasmid (7.9 kb) was isolated from an agarose gel.
Subsequently the isolated 7.9 kb fragment was cut with BsmI,
35 which cuts a few nucleotides downstream of the Bs~HI site of
interest, to remove plasmids linearized at other BsDHI sites.
The fragments were separated on an agarose gel and the 7.9 kb
BspHI-BsmI fragment was isolated. This was partially cut with
~ Og4/14g64 21510 3 8 PCT~3103551
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
solani pisi pre-pro-cutinase was ligated with the 7.2 kb
BspHI-AflII fragment of pAW14B, yielding pUR7280. The
constructed vector (pUR7280) can subsequently transferred to
moulds (for example Aspergillus niqer, AsPergillus 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 EcoRI 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. niger var. awamori pyrG gene together with
their own promoters and terminators were equiped with
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 ~usarium solani pisi cutinase gene in
AsPerqillus niqer 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. niger var. awamori exlA.
To prepare the synthetic cutinase gene for such
fusio~s, 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.
W094/149~ 2 ~ PCT~3/03551
34
In cassette 6 the exlA pre-sequence is fused with the N-
terminal residu 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
5 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-
S~eI 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 niqer
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 l litre shake flasks with 400 ml
35 synthetic media (pH 6.5) were inoculated with spores (final
concentration: lOE6/ml). The medium had the following
composition (AW Medium):
'0 94/14964 21~ 10 3 ~ PCT/EP93/03551
sucrose 10 g/l
NaN03 6.0 g/l
KCl 0.52 g/l
KH2P04 1.52 g/l
- 5 MgS04 7H20 0.49 g/l
Yeast extract 1.0 g/l
ZnS04 7H2O 22 mg/l
H3B03 11 mg/l
MnCl2 4H20 5 mg/l
FeS04 7H20 5 mg/l
CaCl2 6H2o 1.7 mg/l
CUs04- 5H20 1. 6 mg/l
NaH2Moo4 2H20 1.5 mg/l
Na2EDTA 50 mg/l
Incubation took place at 30C, 200 rpm for 24 hours
in a Mk X incubator shaker. After growth cells were collected
by filtration (0.45 ,um 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 con~;l;n;ng 50 ml salt solution to which xylose
has been added to a final concentration of lO 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 pUR7240 (P17E) or pUR7241
(R196E) or pUR724 (L51A) instead of pUR7210 for the
construction of fungal expression vectors, variants of
Fusarium solani pisi cutinase comprising the above mentioned
3S mutations were produced in AsPerqillus niqer var. awamori.
WO94114964 PCT~3/035~1 ~
33 36
EXAMPLE 8
Identification and isolation of genes related to the Fusarium
solani Pisi cutinase gene.
Genes encoding cutinases with a varying degree of
5 homology with ~usarium solani pisi cutinase were isolated
from different fungi. Fungal cultures were grown in 500 ml
shakeflasks containing 200 ml of the medium described by
Hankin and Kolattukudy (1968) supplemented with 0.25% glucose
and incubated for 4 days at 28C in a Mk X incubator shaker
(lO0 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.
(1987). At regular intervals samples were withdrawn from the
culture and analyzed for the presence of lipolytic activity
15 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 t~hni~ues. The mycelia were
washed, frozen in liquid nitrogen and lyophilized according
20 to standard techn;ques. Total cellular RNA preparations were
isolated using the guanidinium thiocyanate method and
purified by cesium chloride density gradient centrifugation,
essentially as described by Sambrook et al. (1989). PolyA(+)
mRNA fractions were isolated using a polyATtract mRNA
25 isolation kit (Promga). The polyA(+) mRNA fractions were used
in a Northern hybridization analysis using a cDNA fragment
from the ~usarium solani ~isi cutinase gene as a probe
according to standard techn;ques, to verify the expression of
cutinase-related genes. Preparations of mRNA comprising
30 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
supplier, yielding cDNA fragments with an XhoI cohesive end
flanking the poly-A region and an EcoRI adaptor at the other
35 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,
La Jolla), allowing expression of ~-galactosidase fusion
21sla3s
094/149~ PCT~W3/03551
37
proteins (Huse et al.,1988). These libraries were screened
using antiserum raised against Fusarium solani pisi cutinase.
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
lO preparations of cDNA 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
15 electroforesis and isolated from the gel.
As an alternative approach, the PCR screening
techique 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
20 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
25 electroforesis and isolated from the gel.
For strains which scored positive in either the
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
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
WO 94/14964 PCT/EP93103551 ~
21~103~ 38
genomic DNA was size-fractionated by gel electroforesis and
fragments of the appropriate size were isolated from the gel
and subcloned in pUCl9. These genomic libraries were screened
with the corresponding cDNA insert (expression library
5 approach) or the PCR fragment (PC~ screening approach),
yielding clones comprising the genomic copy of the cutinase
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.,
lO 1987). The N-terminal end of the mature cutinase polypeptide
was also deduced from such a comparison. Using standard PCR
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
15 Saccharomvces cerevisiae invertase gene (preceded by a SacI
site, compare cassette 8, Fig. 3) was fused to the sequences
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
20 pre-sequence were ligated with the 7.3 kb SacI-~dIII
fragment of pUR7241 (see Fig. 4) and transformed to S.
cerevisiae strain SU51. The fungal cutinases were expressed
and recovered from the culture broth essentially as described
in Example 4.
EXAMPLE 9
The compatibility of Fusarium solani Pisi Cutinase variants
Rl7E, Rl96E and Rl7E+Rl96E to various anionic surfactants.
The compatibility of Fusarium solani pisi cutinase
30 and of the Fusarium solani Pisi cutinase variants Rl7E, Rl96E
and Rl7E+Rl96E to various anionic surfactants was tested as
follows. Solutions of the enzyme in various c~etergent
products were prepared. The solutions were incubated at 40C
and at intervals samples were taken. Then the enzyme activity
35 was determined following the assay described in Example 4.
The following detergent products A-C were used:
~ 094/14964 21510 3 8 PCT~Pg3/03551
39
Product A
compound weight
Na-Linear alkyl benzene sulphonate 11.7
Nonionic surfactant 7E0 5.8
5 Nonionic surfactant 3E0 3.2
Zeolite 38.8
Sokolan CP7 4.8
Sodium CMC 0.8
Sodium carbonate 13.9
10 Sodium perborate 8.0
TAED 5.4
Sodium silicate 2.5
Product B
15 compound weight %
Sodium Primary Alkyl Sulphate 6.5
Nonionic surfactant 7E0 6.5
Nonionic surfactant 3E0 8.3
Soap 2.3
20 Zeolite 38.0
Sodium carbonate 15.9
Sodium perborate 8.0
TAED 5-4
Sodium silicate 2.5
WO94/14964 PCT~3/03551
3 8 40
Product C
compound weight %
Na-Linear alkyl benzene sulphonate 6.9
Nonionic surfactant 10.0
5 Soap 2.0
Zeolite 27.0
Sodium carbonate 10.2
The results for the compositions A-C are given in
Figure 12-14. It follows that, in particular in the anionic-
rich composition A, the Cutinase variants are more stable
than wildtype Fusarium solani Pisi cutinase.
EXAMPLE 10
15 The compatibility of Fusarium solani ~isi Cutinase variants
Rl96K and R196L to Sodium Dodecyl Sulphate (SDS).
The compatibility of Fusarium solani pisi cutinase
and of the Fusarium solani pisi cutinase variants R196K and
R196L to Sodium Dodecyl Sulphate (SDS) was tested as follows.
20 Solutions of the enzymes in 0.4 mM SDS and 10 mM Tris at 0FH
were prepared. The solutions were incubated at 40C and at
intervals samples were taken and the residual enzyme activity
was determined following the assay described in Example 4.
The results are shown in Figure 15. It can be seen that both
25 Cutinase variants are more stable to the anionic surfactant
Sodium Dodecyl Sulphate (SDS) than wildtype Fusarium solani
,Plsl cutinase.
EXAMPLE 11
30 Determining the In-the-wash activity of Fusarium solani pisi
Cuti~ase variant R17E.
Test cloths made of woven polyester/cotton were
soiled with pure olive oil. Each tests cloth was then
incubated in 30 ml wash liquor in a 100 ml polystyrene
35 bottle. The bottles were agitated in a Miele TMT washing
~ 094/14964 ~ PCT~3/03551
2 ~ 3 8
machine filled with water and using a normal 40C main wash
programme. The wash liquor consisted of 2 grams per litre (at
27FH) of washing powders A and B of Example 9.
The results are shown in Figure 16. The enhancement
of the in-the-wash performance (oily soil removal) of
Cutinase varaiant R17E relative to wild-type Fusarium solani
~isi cutinase under various wash conditions is evident. For
comparison, the same experiments were also carried out with
Lipolase (TM). Under all conditions, the Cutinase variant
10 R17E was superior.
