Note: Descriptions are shown in the official language in which they were submitted.
CA 02202~3 1997-04-11
-
"LIPASE, M~KuuKG~NISM ~UUU~L~G SAME, METHOD FOR PR$PARING SAID
LIPASE AMD USES T~R~"
The invention relates to a new lipase. The invention also
relates to a new strain of micro-organism producing this
llpase .
The invention also relates to processes to prepare this lipase,
to the uses thereof and to compositions comprising it.
It is known to include lipases in detergent compositions in
order to eliminate fatty deposits from the tissues (Enzyme
Microb. Technol., 1993 (3), pages 634-645). These fatty
deposits contain triglycerides contained, for example, in
sebum, yarious foodstuffs (oil, sauce, butter, fats), cosmetic
products. The lipases hydrolyze the triglycerides, forming
substances which are more readily soluble in water, mono- and
diglycerides, glycerol and free fatty acids.
Lipases which may be used in detergent compositions are known,
such as, in particular, the lipases originating from
Pseudomonas strains, for example the lipase produced by the
Pseudomonas stutzeri strain (British patent application
1372034), the lipase produced by Pseudomonas mendocina strain
(European patent application 0 571 982), the lipase produced
CA 02202~3 lss7-04-ll
-
by the Pseudomonas alcaliqenes strain (US patent 5,063,160),
and the lipase produced by the Pseudomonas pseudoalcaliqenes
strain (European patent application 0 218 272). Yet, despite
the properties of these lipases, the detergent compositions
containing these enzymes would appear not to be very effective.
Consequently, there is currently a need for a lipase which may
be used in the detergent field, which is highly stable and,
likewise, highly active in a broad pH and temperature range.
In addition, there is also a need for a lipase which is
particularly effective on grease stains, and this on a low
enzyme dose. In addition, there is also a need for a lipase
which is particularly effective on grease stains from the very
first washing cycle.
The object of the present invention is to provide a new lipase
which is active in a broad range of temperatures, active in a
broad alkaline pH range, and which is effective from the first
washing cycle.
A further object of the invention is to identify, isolate and
provide a strain, in particular a strain of Psçudomonas, which
produces said lipase naturally.
A further object of the invention is to prepare and provide a
composition and, in particular, a detergent composition which
contains said lipase.
CA 02202~3 1997-04-11
To this end, the present invention relates to a lipase,
isolated and purified, which originates from a strain of
Pseudomonas wisconsinensis. The present invention also relates
to a lipase, isolated and purified, which originates from a
derivative or a mutation of a strain of Pseudomonas
wisconsinensis which is capable of producing this lipase. The
lipase according to the invention is preferably derived from
the strain Pseudomonas wisconSinensis T 92.677/1 or from a
derivative or a mutation of this strain which is capable of
producing this lipase. The lipase of the invention is derived
from the strain Pseudomonas wisconsinensis T 92.677/1. The
lipase is classified in the international system under the
number E.C. 3.1.1.3; it is a glycerol ester hydrolase.
The lipase, isolated and purified, preferably has a relative
molecular mass of about 30 kDa. It is essentially
extracellular.
The N-terminal amino acid sequence (SEQ ID NO:l) of the lipase
according to the invention is as follows:
Asn Tyr Thr Lys Thr Lys Tyr Pro Ile Val Leu Val His Gly Val Thr
1 5 10 15
Gly Phe Asn Thr Ile Gly Gly Leu
The invention relates to a lipase which is derived from an
aerobic bacterium which is capable of producing the lipase in
an appropriate nutritive medium containing sources of carbon
.
CA 02202~3 1997-04-11
and nitrogen and mineral salts under aerobic conditions.
The invention relates to a lipase, isolated and purified, which
comprises the amino acid sequence from 1 to 286 amino acids
(SBQ ID NO:4) or a derived modified sequence thereof. The
amino acid sequence and the nucleotide sequence (SEQ ID NO:2)
coding for the mature lipase, as well as its translation into
amino acids (SEQ ID NO:3), is given in Figure 1 (Figures la
and lb).
The lipase according to the invention is synthesized in the
form of a precursor. The precursor contains 308 amino acids
(SEQ ID NO:7). The nucleotide sequence (SEQ ID NO:5) coding
for the precursor of the lipase is identified, together with
its translation into amino acids (SEQ ID NO:6). Figure 2
(Figures 2a and 2b) show the sequence of nucleotides (SEQ ID
NO:5) of the coding part of the lipase as well as its
translation into amino acids (SEQ ID NO:6).
The precursor contains the sequence of 286 amino acids (SEQ
ID NO:4) of the mature lipase and the sequence of 22 amino
acids (SEQ ID NO:10) of the presequence.
The sequence of the mature lipase is preceded by a presequence.
This is an additional sequence of 22 amino acids (SEQ ID
NO:10). The corresponding sequence o~ nucleotides (SEQ ID
NO:8), as well as its translation into amino acids (SEQ ID
NO:9), is identified. This presequence codes for the peptide
CA 02202~3 1997-04-11
signal of the lipase according to the invention.
In a preferred manner, said lipase has an estimated iso-
electric point of between about 9.8 and about 10.1. In a
particularly preferred manner, said lipase has an estimated
iso-electric point of about 9.95.
The lipase according to the invention is active in a broad
temperature range. The isolated and purified lipase according
to the invention develops an optimal enzymatic activity,
measured at a pH of 9.5, in a temperature range above about 40
C. The isolated and purified lipase according to the invention
develops an optimal enzymatic activity, measured at a pH of
9.5, in a temperature range below about 60 C. More
particularly, the lipase according to the invention develops
an optimal enzymatic activity, measured at a pH of 9.5, in a
temperature range of between about 40 C and about 60 C. In
a particularly preferred manner, the lipase according to the
invention develops an optimal enzymatic activity, measured at
a pH of about 9.5, at a temperature of about 55 C.
The isolated and purifi.ed lipase according to the invention
develops an enzymatic activity in excess of 50 ~ of the maximum
enzymatic activity in a temperature range of between about 40
C and about 60 C, at a pH of about 9.5, the maximum enzymatic
activity being measured at a temperature of 55 C and at a pH
of 9.5.
CA 02202~3 lss7-04-ll
The lipase according to the invention is active in a broad
alkaline pH range. The isolated and purified lipase according
to the invention usually develops an optimal enzymatic
activity, measured at a temperature of about 30 C, in a pH
range of more than or equal to about 8. The isolated and
purified lipase according to the invention develops an optimal
enzymatic activity, measured at a temperature of about 30 C,
in a pH range of less than or e~ual to about 10. More
particularly, the lipase according to the invention develops
an optimal enzymatic activity, measured at a temperature of
about 30 C, in a pH range of between about 8 and about 10.
In a particularly preferred manner, the lipase according to the
invention develops an optimal enzymatic activity, measured at
a temperature of about 30 C, in a pH range of between about
8 and about 9.5.
The isolated and purified lipase according to the invention
develops an enzymatic activity of more than 85 ~ of the maximum
enzymatic activity in a pH range of between about 8 and about
10 at a temperature of about 30 C, the maximum enzymatic
activity being measured at a temperature of 30 C and at a pH
of 9.5.
The lipase according to the invention is thermostable at an
alkaline pH. Indeed, the isolated and purified lipase
according to the invention displays a relative enzymatic
activity of at least 55 ~ measured after an incubation of 160
minutes at a temperature of 55 C and at a pH of 10 in a buffer
CA 02202~3 1997-04-11
solution with a hardness of 15. It displays a relative
enzymatic activity of at least 70 ~ measured after an
incubation of 80 minutes under the same conditions.
The term 'relative enzymatic activity' is understood to mean
the relationship between the enzymatic activity, measured
during a test under the given conditions regarding pH,
temperature, substrate and duration, and the maximum enzymatic
activity measured during the same test, the enzymatic activity
being measured after the hydrolysis of the triolein and the
maximum enzymatic activity being fixed arbitrarily at a value
of 100.
The invention also relates to a modified lipase, i.e. an
enzyme, the amino acid sequence of which differs from that of
the crude enzyme by at least one aminated acid. It is possible
for these modifications to be obtained by known DNA mutagenesis
processes, such as the exposure to ultraviolet rays, to
chemical substances, such as ethyl methane sulphonate (EMS),
N-methyl-N-nitro-N-nitrosoguanidine (MNNG), sodium nitrite or
O-methylhydroxyl amine, or by genetic engineering techniques,
such as, for example, controlled mutagenesis or random
mutagenesis. These techniques are known to the person skilled
in the art and are described, in particular, in MOLECULAR
CLONING - a laboratory manual - SAMBROOK, FRITSCH, MANIATIS -
Second Edition, 1989, Chapter 15.
The invention also relates to a mutated lipase obtained by
CA 02202~3 l997-04-ll
modification of the nucleotide sequence of the gene which codes
for the lipase. The techniques to obtain such mutated lipases
are known to the person skilled in the art and are described,
in particular, in MOLECULAR CLONING - a laboratorY manual -
SAMBROOK, FRITSCH, MANIATIS - Second Edition, 1989, Chapter 15.
The invention also relates to a lipase which has immuno-
chemical properties identical or partially identical to the
lipase obtained from the Pseudomonas wisconsinensis T 92.677/1
strain. The immuno-chemical properties may be determined in
an immunological manner by identity tests, in particular by
using specific polyclonal or monoclonal antibodies. Identity
tests, such as, in particular, the Ouchterlony immunodiffusion
method, or the immuno-electrophoresis method, are known to the
person skilled in the art. Examples of such methods are
described by Axelsen N.H. ~Handbook of Immunoprecipitation Gel
Techniques", Blackwell Scientific Publications, 1983, Chapters
5 and 14, the terms "antigenic identity~ and ~partial antigenic
identity" being described in that document in Chapters 5, 19
and 20. A serum containing the specific antibody is prepared
according to the described method by immunizing animals (for
example mice, rabbits or goats) with a preparation of purified
lipase. This preparation may be mixed with an additive, such
as the Freund adjuvant, and the mixture obtained is injected
into the animals. The polyclonal antibody is obtained after
one or more immunizations. An example involves injecting,
subcutaneously at two-weekly intervals, four parts each
containing 150 micrograms of purified lipase, the immunization
CA 02202 j j3 1997 - 04 -11
'
thus extending over 8 weeks. The serum is taken after the
immunization period and the immunoglobulin may be isolated
according to the method described by Axelsen N.H. (1983).
The present invention also relates to the isolation, the
identification and the supplying of a new bacteria which
produces the lipase. This aerobic bacteria is isolated and
purified. It generally belongs to the Pseudomonadaceae family.
It preferably belongs to the Pseudomonas genus. In a
particularly preferred manner, it is a strain of Pseudomonas
wisconsinensis. Good results were obtained with a strain of
Pseudomonas wisconsinensis T 92.677/1 or a derivative or
mutation of this strain.
The term "derivative of this strain" is understood to mean any
naturally modified bacteria, i.e. modified by natural
selection. The term "mutation of this strain" is understood
to mean any artificially modified bacteria. The mutations of
this strain may be obtained by known modification techniques,
such as ultraviolet radiation, X-rays, mutagenic agents or
genetic engineering. These techniques are known to the person
skilled in the art and are described, in particular, in
SAMBROOK et al., 1989, Chapter 15. Examples of mutagenic
agents are described, in particular, by R. Scriban,
Biotechnoloqie (Technique et Documentation Lavoisier), 1982,
pp 365-368.
The strain of Pseudomonas wisconsinensis T 92.677/1 was
CA 02202~3 1997-04-11
,, .
submitted to the collection entitled BELGIA`N COORDINATED
COLLECTIONS OF MICROORGANISMS (LMG culture collection,
Université de Gand, Laboratoire de Microbiologie - K.L.
Ledeganckstraat 35, B-9000 Gand, Belgium) in accordance with
the Budapest Treaty under number LMG P-15151 on 12th October,
1994). The invention relates to an isolated and purified
culture of the Pseudomonas wisconsinensis strain and a culture
derived or mutated therefrom. More particularly, the invention
relates to an isolated and purified culture of the strain of
Pseudomonas wisconsinensis T 92.677/1 and a culture derived or
mutated therefrom.
The strain of the present invention was identified by its
biochemical characteristics. It is an aerobic Gram-negative
bacteria. It does not develop in anaerobiosis. No spores are
formed. The oxidase test is positive in the presence of 1
(p/v) of tetramethyl-1,4-phenylene diammonium dichloride. This
bacteria is not thermophilic. It does not produce gas from
glucose.
The invention also relates to the isolation and supplying of
a DNA molecule comprising the nucleotide sequence (SEQ ID NO:2)
which codes for the mature lipase of Pseudomonas wisconsinensis
T 92.677/1 or a derived modified sequence thereof.
The term 'derived modified sequence of the DNA molecule' is
understood to mean any DNA molecule obtained by modification
of one or more nucleotides of the gene which codes for the
CA 02202~3 1997-04-11
- 11
lipase according to the invention. The techniques to obtain
such sequences are known to the person skilled in the art and
are described, in particular, in MOLECULAR CLONING - a
laboratory manual - SAMBROOK, FRITSCH, MANIATIS - Second
Edition, 1989, Chapter 15. The derived modified sequence of
the DNA molecule usually comprises at least 70 ~ homology with
the sequence of nucleotides (SBQ ID NO:2) of the gene which
codes for the lipase according to the invention, i.e. at least
70 ~ identical nucleotides and having the same position in the
sequence. The derived modified sequence of the DNA molecule
preferably comprises at least 80 ~ homology with the nucleotide
sequence (SEQ ID NO:2) of the gene which codes for the lipase
according to the invention. In a particularly p~eferred
manner, the derived modified sequence of the DNA molecule
comprises at least 90 ~ homology with the nucleotide sequence
(SEQ ID NO:2) of the gene which codes for the lipase according
to the invention.
According to the invention, the DNA molecule usually comprises
at least the nucleotide sequence (SEQ ID NO:5) which codes for
the precursor of the lipase or a derived modified sequence
thereof. This nucleotide sequence (SEQ ID NO:5) comprises the
nucleotide sequence (SEQ ID NO:2) which codes for the mature
lipase of Pseudomonas wisconsinensis T 92.677/1 and its signal
sequence (presequence) (SEQ ID NO:8). This DNA molecule
preferably comprises the entire gene of the Pseudomonas
wisconsinensis T 92.677/l lipase.
CA 02202jj3 1997-04-11
12
The present invention also relates to a process for the
production of a lipase. This process comprises the culture of
an aerobic bacteria which is capable of producing the lipase
in an appropriate nutritive medium containing sources of carbon
and nitrogen and mineral salts under aerobic conditions, and
the collection of the lipase thus obtained. This culture
medium may be a solid or a liquid. The culture medium is
preferably a liquid medium. The aerobic bacteria is usually
a strain of Pseudomonas or a derivative or mutation of this
strain which is capable of producing the lipase. More
particularly, the aerobic bacteria is a strain of Pseudomonas
wisconsinensis or a derivative or mutation of this strain
capable of producing the lipase. The aerobic bacteria is
preferably a strain of Pseudomonas wisconsinensis T 92.677/1
or a derivative or mutation of this strain capable of producing
the lipase.
The culture conditions for these bacteria, which will permit
obtaining the lipase according to the invention, such as the
components of the nutritive medium, culture parameters,
temperature pH, ventilation, agitation, are well known to the
person skilled in the art Examples of the culture conditions
are described, in particular, in European patent application
0 571 982.
The techniques for the collection of the lipase are well known
to the person skilled in the art and are selected in terms of
the proposed use of the lipase. Usually, centrifugation,
CA 02202~3 l997-04-ll
13
filtration, ultrafiltration, evaporation, microfiltration,
crystallization or a combination of the one or the other of
these techniques, such as centrifugation followed by
ultrafiltration, are used. Examples of such techniques are
described, in particular, by R. Scriban, Biotechnoloqie,
(Technique et Documentation Lavoisier), 1982, pp 267-276.
It is then possible for the lipase to be purified, if
necessary, and depending on the proposed uses. The techniques
for the purification of enzymes are well known to the person
skilled in the art, such as precipitation with the aid of a
salt, such as ammonium sulphate, or with the aid of a solvent,
such as acetone or an alcohol. Examples of such techniques are
described, in particular, by R. Scriban, Biotechnoloqie,
(Technique et Documentation Lavoisier), 1982, pp 267-276. It
is also possible for the lipase to be dried by atomization or
lyophilization. Examples of such techniques are described, in
particular, by R. Scriban, Biotechnoloqie. (Techniuue et
Documentation Lavoisier), 1982, pp 267-276. The present
invention also relates to enzymatic compositions which comprise
the lipase according to the invention and at least one
additive. Depending on the proposed uses, the enzymatic
compositions which comprise the lipase according to the present
invention may be in solid or in liquid form.
The additives which are contained in the composition according
to the invention are known to the person skilled in the art and
are selected in terms of the proposed use of the composition.
CA 02202~3 1997-04-11
14
They must be compatible with the lipase and must not affect the
enzymatic activity of the lipase. These additives are usually
enzyme stabilizers, preservatives and formulation agents.
Bxamples of additives are described, in particular, in European
patent application 0 218 272. The following may be mentioned
as examples of additives: ethylene glycol, glycerine, 1,2-
propane diol, starch, a sugar such as glucose and sorbitol, a
salt such as sodium chloride, calcium chloride, potassium
sorbate and sodium benzoate, or a mixture of two or more of
these substances. Good results were obtained with 1,2-propane
diol. Good results were also obtained with sorbitol.
.
There are numerous openings in various industries, such as, for
example, the foodstuffs lndustry, the pharmaceutical industry
or the chemical industry, for a lipase according to the
invention.
The lipase may, in particular, be used for cleaning. The
present invention also relates to the use of the lipase, as
defined above, for cleaning. An example of a use of this kind
is described, in particular, in British patent application
1372034 and in Buropean patent application o 218 272. Within
this framework, it forms part of the detergent compositions.
The present invention thus also relates to detergent
compositions which contain the lipase. The components of
detergent compositions are known to the person skilled in the
art and are adapted depending on the proposed use of the
composition. Such compounds are, in particular, enzymes such
CA 02202~3 lss7-04-ll
as, for example, proteases, amylases and/or cellulases;
fillers, such as sodium tripolyphosphate; bleaching agents,
such as perborate; formulation additives; surface-active
agents. The detergent compositions according to the invention
may be used, depending on their formulation, in powder form,
in granular form or as a washing liquid for household washing;
as a stain-removing substance to remove stains or grease from
items or to remove stains from washing prior to cleaning; and
in powder form, granular form or liquid form to wash dishes.
The lipase may be used, in particular, for the treatment of
waste paper in order to remove oil-based inks. An example of
such use is described, in particular, in the summary Chemical
Abstract 113/154607.
The lipase may be used, in particular, during the processing
of paper pulp to prevent sticky deposits which are known as
"pitch". An example of such use is described, in particular,
in the document Enzyme Microb. Technol., 1993 (3), pages 634-
645.
The lipase may be used, in particular, in the foodstuffs
industry in order to develop the aroma of certain food
products, such as cheeses; and during the production of
special margarines. An example of such use is described, in
particular, in the document Enzyme Microb. Technol., 1993 (3),
pages 634-645.
CA 02202~3 1997-04-11
16
The present invention is illustrated by the following Examples.
Figure 1 (Figures la and lb) shows the amino acid sequence and
the nucleotide sequence (SEQ ID NO:2) coding for the mature
lipase, as well as its translation into amino acids (SEQ ID
NO:3).
Figure 2 (Figures 2a and 2b) shows the nucleotide sequence (SEQ
ID NO:5) of the coding part of the lipase as well as its
translation into amino acids (SEQ ID NO:6).
Example 1
Isolation and characterization of the Pseudomonas
wisconsinensis T 92.677/l strain
The Pseudomonas wisconsinensis T 92.677/1 strain was isolated
from a soil sample taken in the United States in the State of
Wisconsin.
1 g of soil is placed in suspension in 10 ml of demineralized
water which contains 9 g/l of NaCl. This suspension is diluted
10 times with demineralized water containing 9 g/1 of NaCl.
1 ml of the suspension of diluted soil is spread out on an
agar-agar nutritive medium A.
i
The medium A contains 10 g/l of tryptone (Difco), 5 g/l of
yeast extract, 5 g/1 of NaCl, 20 g/1 of agar, 2.5 g/l of
CA 02202~3 1997-04-11
17
NaHCO3, 7.5 g/l of Na2CO3, 10 g/l of olive oil, 1 g/l of
polyvinyl alcohol (25/140) and 0.01 g/l of rhodamine B (Sigma
6626).
The medium A is prepared as follows.
An olive oil emulsion is first prepared as follows. 50 ml of
distilled water is heated to 80 C. 1 g of polyvinyl alcohol
is added in small quantities to this heated water. Then 10
of olive oil is added to the polyvinyl alcohol suspension.
An emulsion is then provided by means of an Ultra-turax mixer
operating at 13500 revolutions per minute (shaft 18 GM). The
emulsion obtained is sterilized at 121 C for 30 minutes.
.
A gelose is then prepared as follows. 10 g of tryptone, 5 g
of yeast extract, S g of NaCl, 20 g of agar-agar are added to
850 ml of distilled water. The gelose suspension obtained is
sterilized at 121 C for 30 minutes.
1 l of carbonate buffer (pH 9.5), containing 25 g/l of NaHCO3
and 75 g/l of Na2CO3, was prepared, then sterilized at 121 C
for 30 minutes.
Then, 1 ml of an aqueous solution of [0.01 ~ (p/v)] rhodamine
B (Sigma 6626) is prepared. This solution is sterilized by
filtration through a 0.45 ~ sterilizing membrane (MILLIPORE).
The sterilized olive oil emulsion and the sterilized gelose
CA 02202~3 1997-04-11
are cooled to 60 C, then mixed under sterile conditions. The
sterilized rhodamine solution is then added. Then, 100 ml of
sterilized carbonate buffer is added in a manner so as to
obtain a pH of 9.5. The suspension thus obtained is then
emulsified by means of an Ultra-turax mixer at 13500
revolutions per minute (shaft 18 GM).
The medium A, on which the soil suspension was spread out, is
incubated at 30 C for 48 hours. The micro-organisms which
produce lipase are detected by means of an ultraviolet light,
they are encircled by a fluorescent halo.
The micro-organisms detected as producing lipase are cultured
on a gelose nutritive medium B.
The medium B contains 10 g/l of tryptone (Difco), 5 g/l of
yeast extract, 5 g/l of NaCl, 20 g/l of agar-agar, 2.5 g/l of
NaHCO3, 7.5 g/l of Na2CO3. The tryptone, yeast extract, NaCl,
agar-agar, which form the medium B, are mixed with 900 ml of
distilled water, then sterilized at 121 C for 30 minutes.
The pH is adjusted to 9.5 by the addition of 100 ml of
previously sterilized carbonate buffer (containing 25 g/l of
NaHCO3 and 75 g/l of Na2CO3).
The micro-organism was identified by its biochemical
characteristics: Gram-negative bacteria, aerobic. No spores
are formed.
CA 02202~3 1997-04-11
19
The dimensions of the vegetative cells are 0.5-0.7 ~m x 1.5-
4.0 ~m. The mobility of the vegetative cells is positive. The
lysis test by 3 ~ (m/v) of KOH is positive. The catalase test
is positive in the presence of 10 ~ (v/v) hydrogen peroxide.
The oxidase test is positive in the presence of 1 ~ (m/v) of
tetramethyl-1,4-phenylene-diammonium dichloride. The urease
test is negative. The test for the reduction in nitrate is
positive. Comparable tests have been described, in particular,
in European patent application 0 218 272.
This strain is aerobic, i.e. it develops in aerobiosis. It
does not develop in anaerobiosis, i.e. in an atmosphere of 84
~ (v/v) of N2, 8 ~ (v/v) of CO2, 8 ~ (v/v) of H2 at 37 C.
The abbreviation '~ (v/v)' represents a percentage expressed
in terms of volume per volume. The abbreviation '~ (v/m)'
represents a percentage expressed in terms of volume by mass.
The abbreviation '~ (m/v)' represents a percentage expressed
in terms of mass per volume. The abbreviation '~ (m/m)~
represents a percentage expressed in terms of mass by mass.
This strain is not thermophilic. It exhibits a normal
development after incubation on the gelose medium B at 20 C,
30 C, 37 C and 41 C.
The strain does not produce gas from glucose.
The strain uses azelate, caprate, citrate, glucose, gluconate,
CA 02202~3 1997-04-11
L-arginine, L-histidine, betaine and geraniol. The strain does
not use adipate, phenylacetate, L-arabinose and maltose. It
does not hydrolyze gelatine, starch and esculin.
The strain belongs to the genus Pseudomonas and to the RNA-I
group.
The biochemical characteristics clearly differentiate the
Pseudomonas wisconsinensis strain and, in particular, the
Pseudomonas wisconsinensis T 92.677/1 strain, from a
Pseudomonas mendocina strain, a Pseudomonas ~seudoalcaliqenes
strain, a Pseudomonas alcaliqenes strain and from a Pseudomonas
stutzeri strain. This will be clearly demonstrated in Table
1 which sets out the main biochemical characteristics of these
S strains.
CA 02202~3 1997-04-11
21
Table 1
Characteristics Pseudomonas
wisconsinensis stutzeri mendo- alcali- ~seudoalcali-
T 92.677/1 cina aenes qenes
Dimensions ~m x 0.5-0.7 0.7-0.8 0.7-0.8 0.5 0.7-0.8
~m 1.5-4.0 1.4-2.8 1.4-2.8 2.0-3.0 1.2-2.5
Yellow pigment + - + d
Starch hydrolysis - +
Arginine dehydrolase - - + + d
Use of glucose + + +
Use of gluconate + d + - d
Use of geraniol + - +
Use of L-histidine + - + d d
Use of L-arginine + - + + +
Use of betaine + - + - +
+ = test positive in respect of 90 % or more of the strains
- = test negative in respect of 90 % or more of the strains
d = test positive in respect of more than 10 % but less than 90 % of the strains
The isolated bacteria thus belongs to the genus Pseudomonas; no
known species could be determined.
The strain of Pseudomonas wisconsinensis T 92.677/1 was submitted to
the collection entitled BELGIAN COORDINATED COLLECTIONS OF
MICROORGANISMS (LMG culture collection) under number LMG P-15151 on
12th October, 1994.
CA 02202~3 1997-04-11
22
Example 2
Production of the lipase by the Pseudomonas wisconsinensis T
92.677/1 strain
The Pseudomonas wisconsinensis T 92.677/1 strain was cultured
at 30 C for 24 hours in a Petri dish containing the gelose
medium B.
Then, from this culture, a culture is prepared in 25 ml of a
liquid medium C. The medium C contains 10 g/l of tryptone
(Difco), 5 g/l of yeast extract, 10 g/l of NaCl, the pH of the
medium is adjusted to 7.0 with NaOH 0.lN, the medium is
sterilized at 121 C for 30 minutes. The culture is produced
at 30 C under orbital agitation at a rate of 200 revolutions
per minute, with an amplitude of about 2.54 cm.
After an incubation of 16 hours, this culture is introduced
into a 20 litre capacity fermentation vessel which contains 13
lites of the sterillzed liquid medium D.
The medium D contains 2.5 g/l of K2HPO~, 2.5 g/l of KH2 PO~
1 g/l of MgSO4.7H2O, 2 g/l of (NH~)2SO~, 2 g/l of (NH2)2CO,
g/l CaCl2, 20 g/l of soya bean meal, 2 g/l of yeast extract,
20 g/l of glucose, 5 g/l anti-foam oil (Mazuol, from Mazes
Chemicals). The pH is adjusted to 7.4 (using ordinary
phosphoric acid and ordinary caustic soda) prior to and after
sterilization in the fermentation vessel (30 minutes at 121
CA 02202~53 Iss7-04-
23
C). The glucose is sterilized separately at pH 4.0 (pH
adjusted with ordinary phosphoric acid) at 121 C for 30
minutes. The medium is sterilized in the fermentation vessel
at 121 C for 30 minutes.
The culture in the fermentation vessel is produced at a
temperature of 23 C, at a pressure of 0.1S x 105 Pa (Pa =
Pascal) (0.15 bar), with ventilation at 0.3 VVM (volume of air
per volume of culture medium per minute), with an axial
agitation of 200 revolutions per minute, the control of the
dissolved oxygen being fixed at 10 ~ (v/v) by controlling the
agitation rate.
After 24 hours of fermentation, the enzymatic activity of the
culture thus obtained is measured, using the following method.
The hydrolysis of the triolein is quantified by neutralizing
the fatty acids freed by the action of the lipase. This step
is carried out with the aid of an automatic titrating
apparatus, an apparatus which maintains the pH at a constant
at a set value by adding NaOH 0.01 N.
A lipase unit (LU) is defined as the quantity of enzyme which
catalyses the freeing of one micromol of fatty acid per minute
under the standard test conditions described above.
10 g of triolein (Roth 5423.1) and 10 g of gum arabic (Fluka
51200) are mixed in 100 ml of distilled water. This mixture
CA 02202~3 1997-04-11
24
is emulsified by means of a Ultra-Turrax mixer at 13500
revolutions per minute (axial agitation) three times for 5
minutes, maintaining the mixture under nitrogen and in an ice
bath.
A dilution buffer, which contains 2.34 g/l of NaCl, 2.94 g/l
of CaCl2.2H2O and 0.61 g/l of tri(2-amino-2-hydroxymethyl-1,3-
propane diol), is prepared.
An automatic titrating apparatus, which is equipped with a
burette containing NaOH 0.01N, a temperature sensor and a pH
probe and with a thermostat-controlled reactor, is used.
A small magnetic agitation bar, 10 ml of triolein emulsion and
20 ml of dilution buffer are introduced into the thermostat-
controlled reactor. The pH of the solution thus obtained is
adjusted to 9.5 with NaOH 0.lN. Then, 0.5 ml of the sample
to be tested and containing the lipase are introduced, the
sample possibly having been diluted such that it contains only
a maximum of 5 LU. The pH is controlled with NaOH 0.01N for
the first two minutes. Subsequently, the consumption of soda
between 2 and 4 minutes is recorded, while maintaining the pH
constant (volume of soda consumed between 2 and 4 minutes = Vl
in ~l).
Subsequently, the same test is carried out, but the sample
containing the lipase is replaced by 0.5 ml of dilution buffer
(volume of soda consumed between 2 and 4 minutes = V2 in ~l).
CA 02202~3 lss7-04-ll
One lipase unit (LU) is determined as follows:
1 LU/ml = (V1-V2) x possible dilution of the sample x 10
According to this method, a lipase activity is detected in the
culture.
Example 3
Preparation of a concentrated lipase solution
The pH of the culture, as obtained upon completion of the
fermentation of Example 2, is adjusted to a pH of 8, using the
concentrated caustic soda 10 N.
Then, 1 ~ (v/v) of Triton X-114 (SERVA 37214) is added to this
culture. The mixture is stirred gently at 15 C for 2 hours.
Subsequently, 1 ~ (v/v) of Optifloc FC205 (SOLVAY) in the form
of a 10 ~ (v/v) solution is added to the mixture. The mixture
is stirred gently at 15 C for 1 hour.
The mixture is centrifuged for 15 minutes at 9000 revolutions
per minute (BECKMAN J21, rotor JA10) at a temperature of 4 C.
The supernatant from the centrifugation is retained.
The centrifugation supernatant is heated to 40 C for 5
minutes. A separation of the phases is observed. The top
phase is removed. 35 ~ (v/v) of acetone at 4 C is added to
CA 02202~3 1997-04-11
26
the bottom phase. The suspension is incubated at 4 C for 15
minutes with moderate stirring.
The suspension is then centrifuged at 9000 revolutions per
minute (BECKMAN J21, rotor JA10) for 15 minutes at a
temperature of 4 C. The supernatant from the centrifugation
is retained.
Acetone is added to the centrifugation supernatant at 4 C
until an acetone concentration of 65 ~ (v/v) is obtained. The
mixture is incubated at 4 C for 16 hours.
Subsequently, the mixture is centrifuged at 9000 revolutions
per minute (Beckman J21, rotor JA10) for lS minutes at a
temperature of 4 C. The precipitate from the centrifugation
is retained and is suspended in 150 ml of a buffer (pH 7) which
contains 5 mM of Brij 58 (ICI), 25 mN of CaCl2 and 20 mM of
Tris.
The suspension, which contains the precipitate, is then
centrifuged at 9000 revolutions per minute (Beckman J21, rotor
JA10) at 4 C for 15 minutes. The supernatant from the
centrifugation which forms a concentrated lipase solution is
retained.
CA 02202~3 1997-04-11
27
Example 4
Purification of the lipase
In order to purify the concentrated lipase solution as obtained
in Example 3, the purification method which employs a
hydrophobic interaction chromatography is used, followed by the
purification method which employs a molecular filtering
chromatography.
While following the direction for use as specified by the
supplier (Pharmacia) with regard to the hydrophobic interaction
chromatography column, a 16/10 Phenyl-Sepharose Pharmacia
Hiload column (Ref. 17-10~5-01) is charged with 140 ml of the
concentrated lipase solution as obtained in Example 3.
As the equilibrium buffer, a 20 mM phosphate buffer at a pH of
7.2 is used; as the elution buffer, a 20 mM phosphate buffer
at a pH of 7.2 and containing 30 ~ (v/v) of isopropanol is
used. The flow rate is fixed at 1.5 ml per minute.
The lipase with the eluted fraction is collected with the
phosphate buffer containing the isopropanol.
The enzymatic activity of the fraction is measured according
to the method described in Example 2.
The eluted fraction containing the lipase is diafiltered in
CA 02202~3 lss7-04-ll
28
an Amicon cell provided with a YM10 membrane, with 10 volumes
of a buffer (pH 7) containing 25 mM CaCl2 and 20 mM Tris.
The diafiltered fraction is then concentrated to 0.5 ml by
ultrafiltration by means of the same Amicon cell which is
provided with a YM10 membrane.
Subsequently, the concentrated fraction (0.5 ml) is injected
into a molecular filtering chromatography column (Pharmacia
Superdex 75 HR 10/30 column, Ref. 17-1047-01). The separation
is initiated by an input of 0.5 ml per minute of a buffer (pH
7) containing 25 mM CaCl2 and 20 mM Tris.
Three absorption peaks at 280 nm are separated. The lipase
corresponds to the first absorption peak at 280 nm. The
corresponding fraction, which contains the purified lipase, is
retained.
Example 5
Determination of the N-terminal sequence
The method descri~ed by Vandekkerhove J. et al., Eur. J.
Biochemistry, 152 9 (1985) is used to determine the N-terminal
sequence of the lipase.
The fraction containing the purified lipase as obtained in
Example 4 is used.
CA 02202~3 1997-04-11
29
The N-terminal sequence (SEQ ID NO:1) is the following:
Asn Tyr Thr Lys Thr Lys Tyr Pro Ile Val Leu Val His Gly Val Thr
1 5 10 15
Gly Phe Asn Thr Ile Gly Gly Leu
This sequence differs from the N-terminal sequences of the
other lipases secreted by the other strains of Pseudomonas,
which sequences are published, in particular, in Enzyme Microb.
Technol., 1993 (3), pages 634-645.
.
Bxample 6
Amino acid sequence
The amino acid sequence of the lipase according to the present
invention is determined indirectly from nucleotide sequence
(SEQ ID NO:5) of the gene which codes for said lipase, the
~ethod whereby it is obtained being described in Example 17.
This is carried out by means of the computer program
IntelliGenetics Suite Software for Molecular Biology (Release
No. 5.4) from IntelliGenetics, Inc., USA.
Figure 2 (Figures 2a and 2b) show the nucleotide sequence (SEQ
ID NO:5) of the coding part of the lipase as well as its
translation into amino acids (SEQ ID NO:6).
The lipase is synthesized in the form of a precursor. The
precursor of the lipase contains 308 amino acids (SEQ ID NO:7).
CA 02202~3 1997-04-11
The nucleotide sequence (SEQ ID NO:5) coding for the precursor
of the lipase, as well as its translation into amino acids ~SEQ
ID NO:6) is identified.
The presequence of the synthesized lipase is identified in the
form of a precursor. It is a sequence of 22 amino acids (SEQ
ID NO:10) which constitutes the signal peptide. The
corresponding nucleotide sequence (SEQ ID NO:8) is identified.
Subsequently, the amino acid se~uence of the mature lipase is
identified. The mature lipase contains 286 amino acids (SEQ
ID NO:4).
Figure 1 (Figure la and Figure lb) represents the nucleotide
sequence (SEQ ID NO:2) coding for the mature lipase, as well
as its translation into amino acids (SEQ ID NO:3).
Example 7
Amino acid distribution
The distribution of amino acids in the mature lipase,
determined from the amino acid sequence (SEQ ID NO:4) of the
lipase (Example 6) is set out in Table 2.
CA 02202553 1997-04-11
31
TABLE 2
Symbol Amino Acids Number % mol
(relative molecular mass~
A alanine 28 9.790
B aspartic acid 0 0
C cysteine 2 0.699
D aspartic acid 10 3.497
E glutamic acid 7 2.448
F phenyl alanine 7 2.448
G glycine 35 12.238
H histidine 10 3.497
isoleucine 14 4.895
K Iysine 9 3.147
L leucine 22 7.692
M methionine 1 0.350
N asparagine 25 8.741
P pr~line 11 3.846
Q glutamine 6 2.098
R arginine 13 4.545
S serine 24 8.392
T threonine 18 6.294
V valine 29 10.140
W tryptophan 5 1.748
X unknown o o
Y tyrosine 10 3.497
Z glutamine 0 0
glutamic acid
Example 8
Calculation of relative molecular mass
The relative molecular mass of the lipase is estimated, by
calculation, from the amino acid sequence of the mature form
of the lipase and from the amino acid sequence of the lipase
CA 02202~3 1997-04-11
32
including the signal peptide, as described in Bxample 6.
From the calculation, a relative molecular mass of 30093
Daltons is deduced in respect of the mature form, and a
relative molecular mass of 32365 Daltons in respect of the form
comprising the signal peptide.
Example 9
Determination of the relative molecular mass of the lipase bv
SDS-PAGE analysis
.
A polyacrylamide gel electrophoresis under denaturing
conditions (SDS-PAGE) is carried out on the fraction which
contains the purified lipase as obtained in Example 4. The gel
system used is the system known as PHASTSYSTEM of PHARMACIA LKB
BIOTECHNOLOGY (File of use No. 110), with the gels containing
a polyacrylamide gradient of 10-15 ~ (v/v). The conditions of
the electrophoresis are those specified by the supplier. As
reference, the molecular mass markers PHARMACIA LMW (Low
Molecular Weight), reference 17-0446-01 are used. The markers
used are phosphorylase b (94 kD), albumin (67 kD), ovalbumin
(43 kD), carboanhydrase (30 kD), trypsin inhibitor (20.1 kD~
and alpha-lactalbumin (14.4 kD).
The gel thus obtained shows that the fraction which contains
the purified lipase as obtained in Example 4 is pure.
A Coomassie blue staining (Fast Coomassie staining, Pharmacia,
CA 02202~3 1997-04-11
33
file of use No. 200) of the gel shows a polypeptide having a
relative molecular mass of about 30 (+/- 0.5) kD.
Example 10
Bstimation of the iso-electric point
The iso-electric point of the lipase is calculated on the basis
of the amino acid sequence of the mature form of the lipase
and on the basis of the amino acid sequence of the lipase,
including the signal peptide, as described in Example 6.
An iso-electric point of 9.95 is deduced for the mature form
and 10.12 for the form comprising the signal peptide.
Exam~le 11
Determination of the optimal pH of the li~ase
The enzymatic activity of the lipase at different pH values is
measured according to the method described in Example 2. Thus,
the hydrolysis of the substratum (triolein emulsion) is
determined after the action of the lipase at different pH
values, all the other conditions being identical to the
standard conditions, as described in Example 2, i.e. at a
temperature of 30 C and a duration of two minutes.
The fraction containing the purified lipase as obtained in
Example 4 is used.
CA 02202~3 l997-04-ll
34
The results are set out in Table 3.
Table 3
Temperature Relative Activity
C
18 19
24 26
41
34 45
56
52
49 91
100
54
76 43
During the test, the maximum enzymatic activity was measured
for the specimen set at a pH o~ about 9.5 and at a temperature
of about 55 C. By that very fact, a relative enzymatic
activity of 100 ~ was thus allocated to that sample.
This Example shows that the lipase according to the invention
has an optimal enzymatic activity measured at a pH of 9.5
within a temperature range of between about 40 and about 60C.
CA 02202~3 l997-04-1l
This Example also shows that the lipase according to the
invention develops an optimal enzymatic activity, measured at
a pH of 9.S, at a temperature of about 55 C.
The lipase according to the invention develops an enzymatic
activity of more than 50 ~ of the maximum enzymatic activity
in a temperature range of about 40 to 60 C, for a pH of about
9.5.
Exam~le 12
Determination of the oP~imal pH of the lipase
The enzymatic activity of the lipase is measured at different
pH values according to the method described in Example 2.
Thus, the hydrolysis of the substratum (triolein emulsion) is
determined after the action of the lipase at different pH
values, all the other conditions being identical to the
standard conditions, as described in Example 2, i.e. at a
temperature o~ 30 C and a duration of two minutes.
The fraction containing the purified lipase as obtained in
Example 4 is used.
The results are set out in Table 4.
CA 02202~3 1997-04-11
Table 4
pH Relative Activity
7.0 17
~.0 100
9,O 100
9.5 100
10.O 91
10.5 71
11.0 72
12.0 47
This Example shows that the lipase according to the invention
develops an optimal enzymatic activity, measured at a
temperature of about 30 C, in a pH range of between about 8
and 10.
During the test, the maximum enzymatic activity was measured
for the sample set at a pH of about 9.5 and a temperature of
about 30 C. By this very fact, a relative enzymatic activity
of 100 ~ was thus allocated to this sample.
The lipase according to the invention develops an enzymatic
activity of more than about 90 ~ of the maximum enzymatic
activity in a pH range between about 8 and about 10, at a
temperature of about 30 C.
CA 02202~3 1997-04-11
37
The lipase according to the invention develops an en~ymatic
activity of more than about 70 ~ of the maximum enzymatic
activity in a pH range of between about 8 and about 11, at a
temperature of about 30 C.
Example 13
Stability of the lipase with respect to ~he temperature
The part of the fraction which contains the purified lipase,
as obtained in Example 4, at a pH of 10 and at 55 C, is
incubated in an aqueous buffer with a hardness of 15 (the
buffer containing calcium chloride 1.98 mM, magnesium chlbride
0.69 mM and sodium bicarbonate 2.5 mM).
At regular intervals, as shown in detail in Table 4 (incubation
time in minutes), a sample is taken and its enzymatic activity
is measured according to the method described in Example 2.
The results are set out in Table 5.
CA 02202~3 1997-04-11
38
Table 5
Incubation time - minutes Relative Activity -
o 100
84
81
79
93
86
72
100 59
120 59
130 60
140 61
160 61
260 39
1140 25
The deactivation constant (k) over the period 0-260 minutes is
0.000333 min-l. [The deactivation constant k is obtained
according to the definition ln (At/Ao) = -k.t, t being the
incubation time, Ao the activity relative to the incubation
time 0 and At being the activity relative to the incubation
time t.]
During this test, the maximum enzymatic activity was measured
for the sample at the time 0. By this very fact, a relative
CA 02202~3 1997-04-11
39
enzymatic activity of 100 ~ was thus allocated to this sample.
It is concluded, from this Bxample, that the lipase according
to the invention shows a relative enzymatic activity of less
than 55 ~ measured after an incubation of 160 minutes at a
temperature of 55 C and at a pH of 10 in a buffer solution
having a hardness of 15. It shows a relative enzymatic
activity of at least 70 ~ measured after an incubation of 80
minutes under the same conditions.
Example 14
Use of a deterqent composition containinq the lipase
A piece of white woven fabric (R. Hoppe GmbH), of cotton (65
~) and polyester (35 ~) and measuring 10 cm by 10 cm, is
prepared and impregnated with bacon fat and Sudan red dye. The
fabric is impregnated as follows.
A homogeneous solution of Sudan red 7B (SIGMA Cat. No. F 1000)
and bacon fat (Laru GmbH) is prepared by adding 0.1 ~ (m/m) of
Sudan red dye to the bacon fat The mixture is heated to 90
C until the Sudan red has dissolved completely. A roll of
fabric is immersed in the solution of Sudan red and bacon fat
maintained at 90 C and is continuously displaced in this
solution at a rate of 0.5 m/minute. Subsequently, the fabric
is dried by passing it through a roller mangle under a constant
linear pressure. The fabric thus impregnated contains 31.5 ~
(m/m) of fats. The impregnated fabric is kept at -18 C for
CA 02202~3 1997-04-11
22 hours until the fats are completely crystallized. The
fabric is cut into pieces of 10 cm by 10 cm. The pieces of
impregnated fabric are then stored at -18 C in the dark.
A liquid detergent composition containing 4 g/l of a solld
washing powder (Eurocompact powder from UNILEVER) and diverse
lipase concentrations of 500, 1000 and 2000 LU/l is prepared
in water having a hardness of 15 (water which contains calcium
chloride 1.98 mM, magnesium chloride 0.69 mM and sodium
bicarbonate 2.5 mM). The initial pH of the liquid detergent
composition is about 10. Use is made of the lipase, as
obtained in Example 4, and this is diluted with water having
a hardness of 15, in order to obtain the desired
concentrations.
The fabric impregnated with the bacon fat and the Sudan red dye
is then washed with 200 ml of the liquid detergent composition
in a 250 ml reactor. The washing takes place at 35 C for 45
minutes with an agitation of 40 r.p.m. (to and fro agitation
in 20-second cycles). After washing, the fabric is rinsed
three times with 200 ml of distilled water, then dried at 22
C between two sheets of paper for 24 hours.
Subsequently, the reflectance (RL) is determined at 460 nm of
the dried fabric by means of a colour measuring instrument
(Tricolor LFM 3).
.
This test is repeated three times, i.e. the washing cycle,
CA 02202~3 1997-04-11
41
using the liquid detergent composition which contains the
lipase, and the rinsing cycle are then repeated three times on
the same fabric sample without impregnating it with Sudan red
and bacon fat between cycles. Upon completion of each cycle,
the reflectance at 460 nm of the dried fabric is determined.
An exactly identical test is carried out with a detergent
composition which does not contain lipase. At the end of each
cycle, the reflectance (RO) at 460 nm of the dried fabric is
determined.
The washing performance is defined in ~ as the difference
between the reflectance (RL) obtained with washing in the
presence of lipase and the reflectance (RO) obtained with
washing in the absence of lipase, i.e. RL - RO expressed in ~.
The results of this Example show that the lipase according to
the invention is effective at a low dose of enzyme in the
detergent composition.
The results of this Example also show that the lipase according
to the invention is particularly effective starting with the
second washing cycle, and that it retains an enhanced
effectiveness after three and four washing cycles.
In particular, the lipase is particularly effective at the
concentration of 500 LU/l after three washing cycles.
CA 02202~3 1997-04-11
42
In addition, the lipase is found to be particularly effective
at the concentration of 1000 LU/l from the first washing cycle.
The lipase is found to be most particularly effective at the
concentration of 1000 LU/l during all the washing cycles.
Example 15
Cloning of the qene of Pseudomonas wisconsinensis T 92.677/1
gene
1. Extraction of the chromosomal DNA from the Pseudomonas
wisconsinensis T 92.677/1 strain
The DNA genome is prepared by following the method described
by WILSON, 1990, Current Protocols in Molecular Biology, Vol.
1 (Unit 2.4), with the modifications described below.
Using the culture as obtained in Example 2, a culture of 200
ml of the Pseudomonas wisconsinensis T 92.677/1 strain is
produce-d in an LB growth medium at 37 C for 16 hours. The
LB growth medium is the following: 10 g/l of TR~PTONE (DIFCO),
5 g/l of yeast extract, 10 g/l of NaCl.
The culture obtained is Gentrifuged !soRvALL R~ SC Plus
centrifuge, SS-34 rotor) at 2000 G for 15 minutes. The
centrifugation residue thus obtained is taken up in a solution
which contains 9.5 ml of the TE buffer at a pH of 8.0; 500 ~l
of a solution of SDS (sodium dodecyl sulphate) at 10 ~ (m/v);
and 50 ~l of a K proteinase solution (marketed by BOEHRINGER,
of Mannheim) at 20 mg/ml (prepared extemporaneously).
CA 02202~3 1997-04-11
43
The TE buffer (pH 8.0) is composed of 10 mM TRIS-HCl (TRIS-HCl
= tris(hydroxymethyl)amino methane)-HCl) and 1 mM EDTA
(ethylene diamine tetra-acetic acid).
The suspension thus obtained, containing the centrifugation
residue, is incubated at 65 C for 90 minutes.
Subsequently, 1.8 ml of a solution of NaCl 5 M and 15 ml of a
CTAB/NaCl solution at 10 ~ (m/v) are added to this suspension
(CTAB = cetyltrimethyl ammonium bromide, NaCl at 0.7 M). A
lysate is obtained.
Using this lysate, an extraction is carried out with 15 ml of
a mixture of chloroform/isoamylic alcohol (3-methyl-1-butanol)
24/1 under the conditions and according to the procedures
described in Molecular Cloninq - a laboratory manual
SAMBROOK, FRITSCH, MANIATIS - Second Edition, 1989, page E.3,
until an neat interface is obtained, as is described therein.
0.6 volumes (v/v) of isopropanol are added to the aqueous phase
recovered, and a viscous suspension is obtained.
The DNA contained in the viscous suspension is precipitated
according to the method described in SAMBROOK et al., 1989, p.
9.18. The precipitated DNA is wound around a Pasteur pipette,
then washed three times with 70 ~ (v/v) of ethanol. The washed
DNA is air-dried for 5 minutes at ambient temperature. The
CA 02202~3 1997-04-11
44
dried DNA is suspended in 2.5 ml of TE buffer at a pH of 8Ø
2. Construction of a ~enomic library of Pseudomonas
wisconsinensis T 92.677/1
Using the suspension obtained as above, the chromosomic DNA 915
~g) of Pseudomonas wisconsinenSis T 92.677/1 is cleaved
partially by the restriction enzyme Sau3AI. The method applied
is that of successive dilution of the restriction enzyme and
the restriction conditions are those described in SAMBROOK et
al., 1989, pages 5.28-5.32.
Cleavage is partially inhibited by the addition of 1 ~l of EDTA
0.5 M at a pH of 8.0 in the presence of ice.
Agarose gel electrophoresis is used to determine the fractions,
obtained after cleavage, which contain fragments having
dimensions of between 20 and 40 kpb.
All the fragments thus obtained are then subjected to a
precipitation process according to the method described in
SAMBROOK et al., 1989, p. 9.18.
The DNA fragments are then ligated according to the method
described by SAMBROOK et al., 1989, pages 1.68-1.69, with the
pRG930 plasmid (750 ng), first cut at the BamHI site, as
described by SAMBROOK et al., 1989, pages 1.60-1.61. A
collection of plasmids, which are named pRG930::WI, is
CA 02202 j j3 1997 - 04 -11
obtained.
The process to obtain the pRG930 plasmid is described in
Molecular Plant-Microbe Interactions, 1992, Vol. 5 (3) pages
228-234.
The ligation thus obtained is used for the transfection of the
cells of E. coli HB101 (PROMEGA), by using the kit sold under
the name of GIGAPACK II PACKAGING EXTRACT KIT (STRATAGENE) and
following the manufacturer's recommendations regarding the use
thereof.
The transfected cells of E. coli HB101 are cultured in a Petri
dish containing the gelose LB medium, 25 ~g/ml of streptomycin
and 50 ~g/ml of spectinomycin, for about 24 hours at 37 C.
A collection of transfected strains, which are referred to as
E. coli HB101 (pRG930::WI) is obtained.
Using 12 randomly selected colonies of E. coli HB101
(pRG930::WI), the DNA fragments are isolated according to the
method described in SAMBROOK et al., 1989, page 1.85.
A restriction analysis of these DNA fragments is carried out
(SAMBROOK et al., 1989, page 1.85) after cleavage with the
restriction enzymes EcoRI and PstI.
This analysis indicates that the dimensions of the inserted
fragments present in the pRG930::WI plasmids is between about
CA 02202~3 1997-04-11
46
20 and 30 kpb (kpb = 1000 pairs of bases) and that these
fragments differ from one another. This indicates that the
genomic library which has been set up is representative.
1500 colonies of E. coli HB101 (pRG930::WI) are then cultured
in a Petri dish containing the gelose LB medium, 25 ~g/ml of
streptomycin and 50 ~g/ml of spectinomycin, for 24 hours at 37
C. These 1500 colonies of E. coli HB101 (pRG930::WI)
constitute the genomic library.
3. Obtaininq a chromosomic fraqment containinq the qçne of the
Pseudomonas wisconsinensis T 92.677/1 lipase
In order to establish the nucleotide sequence of a probe
capable of screening the genomic library, the N-terminal
sequence of the Pseudomonas wisconsinensis T 92.677/1 lipase,
as obtained in Example 5, is taken as the initial reference
base.
In order to raise the ambiguity maximum in the translation of
the amino acids toward the nucleotides, on the one hand, the
nucleotide sequences of a plurality of lipases produced by
different strains of Pseudomonas and published in Gilbert J.
et al., Enzyme Microbioloqical Technoloqy, 1993, 15, pp. 634-
645, and, on the other hand, the property known by the name
"codon usage" of the Pseudomonas aeruqinos strain, published
in West S. et al., Nucleic Acid Research, 1988, 16, pp. 9323-
9335, are taken into account.
CA 02202 j j3 1997 - 04 -11
47
On the basis of these elements, the sequence of a synthetic
oligonucleotide of 72 pb (pb = base pairs) is established.
This synthetic oligonucleotide is prepared according to the
method described in BEAUCAGE et al. (1981), Tetrahedon Letters,
22, pp. 1859-1882, and using ~-cyanoethyl phosphoro-amidites
[sic.] in a CYCLONE SYNTHESIZER apparatus from BIOSEARCH.
The sequence of this synthetic oligonucleotide is the
following: SEQ ID NO:ll
5' - AACTACACCAAGACCAAATACCCCATCGTGCTGGTCCA -
- CGGCGTGACCGGCTTCAACACTATCGGCGGGCTC - 3'
This synthetic oligonucleotide is marked at its termination by
means of ~2p ATP with a kinase polynucleotide T4 enzyme,
following the method described in the SEQUITHERM CYCLE
SEQUENCING KIT (BYOZYME).
A screening of the genomic library is carried out by the method
referred to as "colony blot" (AMERSHAM), using the synthetic
oligonucleotide as prepared above as the probe.
The 1500 colonies of the genomic library (E. coli HB101
(pRG930::WI)) are cultured for 18 hours at 37 C on membranes
referred to as "hybond-N~" (AMERSHAM), following the method
given by the manufacturer.
The membranes (400 m2) are placed in plastic bags containing
45 ml of prehybridization solution.
CA 02202~3 Iss7-04-
48
The prehybridization solution contains 15 ml of 20X SSC (3 M
NaCl and 0.3 M o sodium citrate, pH of 7.0), 5 ml of Denhardt
solution and 500 ~g of the DNA of denatured and fragmented
salmon sperm (AMERSHAM).
500 ml of the Denhardt solution contain 5 g of FICOL~ of the
400 type (PHARMACIA), 5 g of polyvinyl pyrrolidone and 5 g of
bovine serum albumin.
The membranes placed in the plastic bags are incubated at 68
C in a water-bath under stirring (100 revolutions per minute,
amplitude of about 2.54 cm) for 4 hours.
The membranes are then incubated at 68 C with a hybridization
solution in a water-bath under stirring (100 revolutions per
minute, amplitude of about 2.54 cm) for 18 hours;
The hybridization solution is prepared by mixing 5 ml of the
prehybridization solution heated to 68 C and the marked
synthetic oligonucleotide, and, having been incubated in the
water-bath for 5 minutes, the final concentration of the
synthetic nucleotide is 0.3 pMol.
The membranes are subsequently collected. The membranes are
then washed with a solution which contains 100 ml of 2X SSC
(0.3 M NaCl and 0.03 M of sodium citrate, pH of 7.0) and 0.1
(m/v) of SDS for 5 minutes at ambient temperature.
-
CA 02202~3 1997-04-11
49
The washed membranes are then dried between two sheets of
absorbent paper. The dried membranes are covered with a
foodstuff-quality transparent plastics sheet. They are then
subjected to X-ray autoradiography (AMERSHAM).
The Pseudomonas wisconsinensis T 92.677/1 strain is used as
positive control and the E. çoli HB101 and E. çoli HB101
(pRG930) strains are used as negative control.
The E. coli HB101 (pRG930) strain was obtained by the
transformation method described in Moleçular Cloninq, a
laboratory Manual - MANIATIS et al., 1982, Cold Spring Harbor
Laboratory, pp. 150-151, using the E. coli HB101 strain and the
pRG930 plasmid.
A clear and strong signal is observed for the wild strain of
Pseudomonas wisconsinensis T 92.677/1 and no signal is observed
for the E. coli HB101 and E. coli HB101 (pRG930) strains. The
screening of the genomic library displays 80 colonies which
give a signal.
This is con~irmed by a fresh hybridization test.
A colony, which presents a strong signal, from the genomic
library is isolated and cultured in a Petri dish containing the
gelose LB medium, 25 ~g/ml of streptomycin and 50 ~g/ml of
spectinomycin, for 24 hours at 37 C. This colony is
CA 02202~3 1997-04-11
designated as E. coli HB101 (pRG930::WI12).
A hybridization test is carried out afresh with the E. coli
HB101 (pRG930::WI12) colony, in order to confirm the results
obtained.
Example 16
Analysis of the pRG930::WI12 plasmid present in the E. coli
HB101 (pRG930::WI12) strain - Analysis by ~he "Southern Blot"
method
The DNA is isolated from the E. coli HB101 (pRG930::WI12)
strain, obtained in Example 15, according to the method
described by SAMBROOK et al., 1989, pp. 1.25-1.28, and a
restriction analysis is carried out using the EcoRI restriction
enzyme. The DNA fragments obtained are separated by agarose
gel electrophoresis, according to the method described in
SAMBROOK et al., 1989, pp. 6.01-6.19.
This analysis demonstrates that the DNA fragment inserted in
the pRG930::WI12 plasmid has a dimension of about 27 kpb.
The DNA is then transferred to a membrane known as "hybond-N~"
(AMERSHAM) and hybridization is carried out according to the
method indicated by the manufacturer, as illustrated in Example
15. The pRG930 plasmid is used as the neyative control.
A single band is observed giving a hybridization signal with
CA 02202~3 lss7-04-ll
51
the synthetic oligonucleotide (SEQ ID NO:11) on the gel
electrophoresis. The fragment carried by this band has a
dimension of about 24.5 kpb, it is tied to the pRG930~vector.
Example 17
Nucleotide sequence of the çompletç qene which codes for the
Pseudomonas wisconsinensis T 92.677/1 lipase
1. Obtaininq the Pseudomonas wisconsinensis RC13 strain
A restriction analysis of the pRG930::WI12 plasmid is carried
out, using the EcoRI restriction enzyme. The DNA fragments
obtained are separated by agarose gel electrophoresis.
An analysis is carried out by the Southern Blot method, as
described in Example 16.
This shows that the fragment inserted in the pRG930::WI12
plasmid is formed, on the one hand, by 4 fragments which
together have a dimension of about 18.5 kpb and, on the other
hand, by a fragment attached to the pRG930 vector. This
fragment has a dimension of about 8.5 kpb.
The 8.5 kpb fragment, attached to the pRG930 vector, gives a
hybridization signal with the synthetic oliyonucleotide (SEQ
ID NO:ll), the 4 other fragments give no signal.
The 8.5 kpb fragment attached to the pRG930 vector is then
ligated on itself according to the method described by SAMBROOK
CA 02202~3 1997-04-11
et al., 1989, pp. 1.68-1.69. The plasmid pRG930::WI13 is
obtained.
The ligation thus obtained is used to transform the E. coli
DH5~ (GIBCO) cells, as described in SAMBROOK et al., 1989, pp.
1.82-1.84.
A colony of E, coli DH5~ (pRG930::WI13) is obtained and is
isolated and cultured in a Petri dish containing the gelose LB
medium, 25 ~g/ml of streptomycin and 50 ~g/ml of spectinomycin,
for about 24 hours at 37 C.
A map of the partial restriction of the pRG930::WI13 plasmid
is established, using the method described in Molecular
Cloninq, a laboratory Manual - MANIATIS et al., 1982, Cold
Spring Harbor Laboratory, pp. 374-379, and using the ClaI,
EcoRI, NcoI, SalI, HindIII, BglII, XhoI, BamHI, SmaI, SacI,
SacII, RpnI, SphI, XbaI, EcoRV and SpeI restriction enzymes.
2. Identification of the nucleotide se~uence of the lipase
A restriction analysis of the pPRG930::WIl3 [sic.~ plasmid is
carried out using the PstI restriction enzyme. The DNA
fragments obtained are separated by agarose gel
electrophoresis.
An analysis according to the Southern Blot method is carried
out, as described in Example 16.
CA 02202~3 1997-04-11
53
This shows that the fragment inserted in the pRG930::WI13
plasmid is formed, on the hand, by 5 fragments and, on the
other hand, by one small fragmen~. This small fragment has a
dimension of about 2.5 kpb.
The 2.5 kpb fragment gives a hybridization signal with the
synthetic oligonucleotide (SEQ ID N0:11), while the other 5
fragments do not give a signal.
The 2.5 kpb fragment is ligated with the pPRG930 [sic.] vector
according to the method described by SAMBROOK et al., 1989, pp.
1.68-1.69. The plasmid pRG930::WI14 is obtained.
From the pRG930:WI14 plasmid, the 2.5 kpb fragment is inserted
in the pBLUESCRIPT plasmid.
The pBLUESCRIPT plasmid is obtainable from the company
STRATAGENE.
The plasmid pBLUESCRIPT::WI14 is obtained.
The sequence of the 2.5 kpb fragment inserted in the
pBLUESCRIPT::WI14 plasmid is obtained using the SEQUITHERM
CYCLE SEQUENCING KIT (BIOZYM) and following the method
specified by the manufacturer.
In order to complete and verify the nucleotide sequence
CA 02202~3 1997-04-11
54
obtained, the above Example is repeated with the following
modification. The restriction analysis of the pPRG930::WI14
plasmid is carried out, successively using the SalI, KpnI or
SacII restriction enzymes, instead of the PstI restriction
enzyme.
The nucleotide sequence of the Pseudomonas wisconsinensis T
92.677/1 lipase is identified. The amino acid sequence and
the nucleotide sequence (SEQ ID N0:2) coding for the mature
lipase, as well as its translation into amino acids (SBQ ID
N0:3), are given in Figure 1 (Figures la and lb).
It is shown that the first amino acids of the sequence of the
lipase, thus identified, correspond to the N-terminal sequence
determined in Example 5.
The lipase is synthesized in the form of a precursor. The
precursor contains 308 amino acids (SEQ ID NO:7). The
nucleotlde sequence (SEQ ID N0:5) coding for the precursor of
the lipase is identified, as is its translation into amino
acids (SEQ ID NO:6).
The precursor contains the sequence of 286 amino acids (SEQ
ID N0:4) of the mature lipase and the sequence of 22 amino
acids (SEQ ID N0: 10) of the presequence.
The sequence of the mature lipase is preceded by a presequence.
It is an additional sequence of 22 amino acids (SEQ ID NO:10).
CA 02202553 1997-04-11
The corresponding nucleo~ide sequence (SEQ ID NO:8) is
identified, as is its translation into amino acids (SEQ ID
NO:9). This presequence codes for the signal peptide of the
Pseudomonas wisconsinensis T 92.677/1 lipase.
CA 02202~53 1997-04-11
56
LIST OF SEQUENCES
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: SOLVAY (Société anonyme)
(B) STREET: rue du Prince Albert, 33
(C) TOWN: Brussels
(E) COUNTRY: Belgium
(F) POSTAL CODE: 1050
(ii) TITLE OF THE INVENTION: Lipase, micro-organism
producing it, process to prepare said lipase, and uses thereof
(iii) NUMBER OF SEQUENCES: 11
(iv) COMPUTER-LEGIBLE FORMAT:
(A) TYPE OF SUPPORT: Floppy disc
(B) COMPUTER: IBM PC compatible
(C) USER SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version
#1.30 (OEB)
(2) INFORMATION FOR THE SEQ ID NO: 1:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) NUMBER OF STRANDS:
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: peptide
(v) TYPE OF FRAGMENT: N-terminal
(vi) ORIGIN:
(A) ORGANISM: Pseudomonas
(B) STRAIN: Pseudomonas wisconsinensis
(C) INDIVIDUAL/ISOLATED: T 92.677/1
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
Asn Tyr Thr Lys Thr Lys Tyr Pro Ile Val Leu Val His Gly
1 5 10
Val Thr Gly Phe Asn Thr Ile Gly Gly Leu
(2) INFORMATION FOR THE SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 861 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF STRANDS: simple
(D) CONFIGURATION: linear
CA 02202553 1997-04-11
57
(ii) TYPE OF MOLECULE: DNA (genomic)
(v) TYPE OF FRAGMENT: internal
(vi) ORIGIN:
(A) ORGANISM: Pseudomonas
(B) STRAIN: Pseudomonas wisconsinensis
(C) INDIVIDUAL/ISOLATED: T 92.677/1
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
AACTACACCA AGACCAAGTA TCCGATCGTG CTGGTACACG GCGTGACCGG GTTCA~T~CC 60
ATCGGCGGGC TGGTCAATTA CTTCCATACC ATTCCCTGGA ACCTAGAGCG CGATGGCGCC 120
CGGGTGCACG TCGCCAGTGT CGCTGCCTTC AATGACAGCG AGCAGCGCGG CGCCGAGCTG 180
GCCCGGCAGA TCGTGCCCTG GGCCGCAG~C GGAGGCGGCA AGGTCAACCT GATCGGCCAC 240
AGTCAGGGCT CGCCGACCTC GCGCGTGGCG G~l-lC~ll~C GGCCGGATCT GGTGGCATCG 300
GTGACCTCGA TCAACGGCGT CAACAAGGGC TCCAAGGTCG CCGATGTGGT GCGCGG~l~ 360
CTGCCACCGG GTAGCGGTAT CGAAGGCGGC GCCAATGCCA TCGCCAACGC C~-lCG~l~CG 420
GTGATCAATC TG~l~l~l~G CTCAAGCAAC CCGCAAAACG GTATCAACGC GCTAGGCACC 480
CTGACCACCG CGG~CACCAG TGCGCTGAAC AGTCGCCACC C~l~GGGCGT CAACACCAGC 540
AGCTACTGCG CCAAGTCCAC CGAAGTGCAC AATGTGCGCG GTCACAGCAT CCGCTACTAC 600
TCCTGGACCG GTAATGCCGC CTATACCAAC GTGCTCGATG CGGCCG~TCC ~l-lC~-l~GCC 660
TTCACCGGCC l~l~ll~GG CAGCGAGAAG AACGACGGTC TGGTGGGCGT ATGTTCCACC 720
TATCTGGGGC AGGTGATCGA CGACAGCTAC AACATGAACC ACGTCGATGC GATCAACCAC 780
~l~llC~GCA TTCGTGGCTG GACCGAACCG GTGTCGCTGT ATCGCCAGCA CGCCAACCGC 840
CTGAAGAACA AGGGCGTCTG A a 61
(2) INFORMATION FOR THE SEQ ID NO: 3:
(i) CHARACTERISTICS OF THE SEOUENCE:
(A) LENGTH: 861 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF STRANDS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: DNA (genomic)
CA 02202553 1997-04-11
58
(ix) CHARACTERISTICS:
(A) NAME/K~Y: CDS
(B) LOCATION: 1..858
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3
AAC TAC ACC AAG ACC AAG TAT CCG ATC GTG CTG GTA CAC GGC GTG ACC 48
Asn Tyr Thr Ly~ Thr Lys Tyr Pro Ile Val Leu Val His Gly Val Thr
1 5 10 15
GGG TTC AAT ACC ATC GGC GGG CTG GTC AAT TAC TTC CAT ACC ATT CCC 96
Gly Phe Asn Thr Ile Gly Gly Leu Val Asn Tyr Phe His m r Ile Pro
20 25 30
TGG AAC CTA GAG CGC GAT GGC GCC CGG GTG CAC GTC GCC AGT GTC GCT 144
Trp Asn Leu Glu Arg Asp Gly Ala Arg Val His Val Ala Ser Val Ala
35 40 45
GCC TTC AAT GAC AGC GAG CAG CGC GGC GCC GAG CTG GCC CGG CAG ATC 192
Ala Phe Asn Asp Ser Glu Gln Arg Gly Ala Glu Leu Ala Arg Gln Ile
50 55 60
GTG CCC TGG GCC GCA GGC GGA GGC GGC AAG GTC AAC CTG ATC GGC CAC 240
Val Pro Trp Ala Ala Gly Gly Gly Gly Ly6 Val A~n Leu Ile Gly ~is
65 70 75 80
AGT CAG GGC TCG CCG ACC TCG CGC GTG GCG GCT TCG TTG CGG CCG GAT 288
Ser Gln Gly Ser Pro Thr Ser Arg Val Ala Al~ Ser Leu Arg Pro Asp
85 90 95
CTG GTG GCA TCG GTG ACC TCG ATC AAC GGC GTC AAC AAG GGC TCC AAG 336
Leu Val Ala Ser Val Thr Ser Ile Asn Gly Val Asn Lys Gly Ser Lys
100 105 110 ~
GTC GCC GAT GTG GTG CGC GGC GTG CTG CCA CCG GGT AGC GGT ATC GAA 384
Val Ala Asp Val Val Arg Gly Val Leu Pro Pro Gly Ser Gly Ile Glu
115 120 125
GGC GGC GCC AAT GCC ATC GCC AAC GCC CTC GGT GCG GTG ATC AAT CTG 432
Gly 61y Ala Asn Ala Ile Ala Asn Ala Leu Gly Ala Val Tl e Asn Leu
130 135 140
CTG TCT GGC TCA AGC AAC CCG CAA ~AC GGT ATC AAC GCG CTA GGC ACC 480
Leu Ser Gly Ser Ser Asn Pro Gln A~n Gly Ile Asn Ala Leu Gly Thr
145 150 155 160
CTG ACC ACC GCG GGC ACC AGT GCG CTG AAC AGT CGC CAC CCG TGG GGC 528
Leu Thr Thr Ala Gly Thr Ser Ala Leu Asn Ser Arg His Pro Trp Gly
165 170 175
GTC AAC ACC AGC AGC TAC TGC GCC AAG TCC ACC GAA GTG CAC AAT GTG 576
Val Asn Thr Ser Ser Tyr Cys Ala Lys Ser Thr Glu Val His Asn Val
180 185 190
CA 02202553 l997-04-ll
59
CGC GGT CAC AGC ATC CGC TAC TAC TCC TGG ACC G~T AAT GCC GCC TAT 624
Arg Gly His Ser Ile Arg Tyr Tyr Ser Trp Thr Gly Asn Ala Ala Tyr
195 200 205
ACC AAC GTG CTC GAT GCG GCC GAT CCC TTC CTG GCC TTC ACC GGC CTG 672
Thr Asn Val Leu Asp Ala Ala Asp Pro Phe Leu Ala Phe Thr Gly Leu
210 215 220
GTG TTC GGC AGC ~AG AAG AAC GAC GGT CTG GTG G~C GTA TGT TCC ACC 720
Val Phe Gly Ser Glu Lys Asn Asp Gly Leu Val Gly Val Cys Ser Thr
225 230 235 240
TAT CTG GGG CAG GTG ATC GAC GAC AGC TAC AAC ATG AAC CAC GTC GAT 768
Tyr Leu Gly Gln Val Ile Asp Asp Ser Tyr Asn Met Asn His Val Asp
245 250 2'5
GCG ATC AAC CAC CTG TTC GGC ATT CGT GGC TGG ACC GAA CCG GTG TCG 816
Ala Ile Asn His Leu Phe Gly Ile Arg Gly Trp Thr Glu Pro Val Ser
260 265 270
CTG TAT CGC CAG CAC GCC AAC CGC CTG AAG AAC AAG GGC GTC TGA 861
Leu Tyr Arg Gln His Ala Asn Arg Leu Lys Asn Lys Gly Val
275 280 285
(2) INFORMATION FOR THE SEQ ID NO: 4:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 286 amino acids
(B) TYPE: amino acids
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
Asn Tyr Thr Lys Thr Lys Tyr Pro Ile Val Leu Val His Gly Val Thr
1 5 lO 15
ly Phe Asn Thr Ile Gly Gly Leu Val Asn Tyr Phe His Thr Ile Pro
Trp Asn Leu Glu Arg Asp Gly Ala Arg Val His Val Ala ser Val Ala
~5
la Phe Asn A~p Ser Glu Gln Arg Gly Ala Glu Leu Ala Arg Gln Ile
. 55 60
Val Pro Trp Ala Ala Gly Gly Gly Gly ~ys Val Asn Leu Ile Gly His
er Gln Gly Ser Pro Thr Ser Arg Val Ala Ala Ser Leu Arg Pro Asp
._
CA 02202553 1997-04-11
~, .
Leu Val Ala Ser Val Thr Ser Ile Asn Gly Val Asn Ly6 Gly Ser Ly6
100 105 110
Val Ala A6p Val Val Arg Gly Val Leu Pro Pro Gly Ser Gly Ile Glu
115 120 125
Gly Gly Ala Asn Ala Ile Ala Asn Ala Leu Gly Ala Val Ile Asn Leu
130 135 140
Leu Ser Gly Ser Ser Asn Pro Gln A~n Gly Ile Asn Ala Leu Gly Thr
145 150 155 160
Leu Thr Thr Ala Gly Thr Ser Ala Leu Asn Ser Arg His Pro Trp Gly
165 170 175
Val Asn Thr Ser Ser Tyr Cy6 Ala Lys Ser Thr Glu Val His Asn Val
180 185 190
Arg Gly ~is Ser Ile Arg Tyr Tyr Ser Trp Thr Gly Asn Ala Ala Tyr
195 200 205
Thr Asn Val Leu Asp Ala Ala Asp Pro Phe Leu Ala Phe Thr Gly Leu
210 215 220
Val Phe Gly Ser Glu Ly6 Asn Asp Gly Leu Val Gly Val Cys Ser Thr
225 - 230 235 240
Tyr Leu Gly Gln Val Ile Asp Asp Ser Tyr Asn ~et A~n Hi6 Val A~p
245 250 255
Ala Ile Asn His Leu Phe Gly Ile Arg Gly Trp Thr Glu Pro Val Ser
260 265 270
Leu Tyr Arg Gln ~i6 Ala Asn Arg Leu Lys Asn Lys Gly Val
275 2B0 2BS
(2) INFORMATION FOR THE SEQ ID NO: 5:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 927 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF STRANDS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: DNA (genomic)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:
ATGCGTCGCG TCTATACCGC TGCCCTGGCA ACACTCGCTC TGCTTGGCGC CGTCGAGGCC 60
CAGGCCAACT ACACCAAGAC CAAGTATCCG ATCGTGCTGG TACACGGCGT GA~--GG~llC 120
AATACCATCG GCGGGCTGGT CAATTA-TTC CATACCATTC CCTGGAACCT AG~GCGCGhT 180
CA 02202553 l997-04-ll
GGCGCCCGGG TGCACGTCGC CAGTGTCGCT GCCTTCAATG ACAGCGAGCA GCGCGGCGCC 2 4 0
GAGCTGGCCC GGCAGATCGT GCC'L1GGGCC GCAGGCGGAG GCGGCAAGGT CAACCTGATC 3 0 0
GGCCACAGTC AGGGCTCGCC GACCTCGCGC GTGGCGGCTT L~1-1~CGGCC GGATCTGGTG 360
GCATCGGTGA CCTCGATCAA CGGCGTCAAC AAGGGCTCCA AGGTCGCCGA 1~1~1~CGC 420
GGCGTGCTGC CACCGG~1AG CGGTATCGAA GGCGGCGCCA ATGCCATCGC CAACGCCCTC 480
GGTGCGGTGA TCAATCTGCT GTCTGGCTCA AGCAACCCGC AAAACGGTAT CAACGCGCTA 540
GGCACCCTGA CCACCGCGGG CACCAGTGCG CTGAACAGTC GCCACCCGTG GGGCGTCAAC 600
ACCAGCAGCT ACTGCGCCAA GTCCACCGAA GTGCACAATG TGCGCGGTCA CAGCATCCGC 6 6 0
TACTACTCCT GGACCGGTAA TGCCGCCTAT ACCAACGTGC TCGATGCGGC CGATCCCTTC 720
CTGGCCTTCA CCGGCCTGGT GTTCGGCAGC GAGAAGAACG AC~ G1 GGGCGTATGT 7 8 0
TCCACCTATC TGGGGCAGGT GATCGACGAC AGCTACAACA TGAACCACGT CGATGCGATC 8 4 0
AACCACCTGT TCGGCATTCG TGGCTGGACC GAACCGGTGT CGCTGTATCG CCAGCACGCC 900
AACCGCCTGA AGAACAAGGG CGTCTGA 9 2 7
(2) INFORMATION FOR THE SEQ ID NO: 6:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 927 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF STRANDS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: DNA (genomic)
(ix) CHARACTERISTICS:
(A) NAME/KEY: sig peptide
(B) LOCATION: 1..66
(ix) CHARACTERISTICS:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 67..924
(ix) CHARACTERISTICS:
(A) NAME/KEY: CDS
(B) LOCATION: 1..924
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:
ATG CGT CGC GTC TAT ACC GCT GCC CTG GCA ACA CTC GC'T CTG CTT GGC 4 8
Met Arg Arg Val TYr Thr A1a A1a Leu A1a Thr Leu A1a Leu Leu G1Y
-22 -20 _ -15 -10
CA 02202~3 1997-04-11
62
GCC GTC GAG GCC CAG GCC AAC TAC ACC AAG ACC AAG TAT CCG ATC GTG 96
Ala Val Glu Ala Gln Ala Asn Tyr Thr Lys Thr Lys Tyr Pro Ile Val
-5 1 5 10
CTG GTA CAC GGC GTG ACC GGG TTC AAT ACC ATC GGC GGG CTG GTC AAT 144
Leu Val His Gly Val Thr Gly Phe Asn Thr Ile Gly Gly Leu Val Asn
15 20 25
TAC TTC CAT ACC ATT CCC TGG AAC CTA GAG CGC GAT GGC GCC CGG GTG 192
Tyr Phe His Thr Ile Pro Trp Asn Leu Glu Arg Asp Gly Ala Arg Val
30 35 40
CAÇ GTC GCC AGT GTC GCT GCC TTC AAT GAC AGC GAG CAG CGC GGC GCC 240
His Val Ala Se_ Val Ala Ala Phe Asn Asp Ser Glu Gln Arg Gly A1A
45 50 55
GAG CTG GCC CGG CAG ATC GTG CCC TGG GCC GCA GGC GGA GGC GGC AAG 288
Glu Leu Ala Arg Gln Ile Val Pro Trp Ala Ala Gly Gly Gly Gly Lys
60 65 70
GTC AAC CTG ATC GGC CAC AGT CAG GGC TCG CCG ACC TCG CGC GTG GCG 336
Val Asn Leu Ile Gly Hi6 Ser Gln Gly Ser Pro Thr Ser Arg Val Ala
~75 80 85 90
GCT TCG TTG CGG CCG GAT CTG GTG GCA TCG GTG ACC TCG ATC AAC GGC 384
Ala Ser Leu Arg Pro Asp Leu Val Ala Ser Val Thr Ser Ile Asn Gly
95 100 105
GTC AAC AAG GGC TCC AAG GTC GCC GAT GTG GTG CGC GGC GTG CTG CCA 432
Val Asn Lys Gly Ser Lys Val Ala Asp Val Val Arg Gly Val Leu Pro
110 115 120
CCG GGT AGC GGT ATC GAA GGC GGC GCC AAT GCC ATC GCC AAC GCC CTC 480
Pro Gly Ser Gly Ile Glu Gly Gly Ala Asn Ala Ile Ala Asn Ala Leu
125 130 135
GGT GCG GTG ATC AAT CTG CTG TCT GGC TCA AGC AAC CCG CAA AAC GGT 528
Gly Ala Val Ile Asn Leu Leu Ser Gly Ser Ser Asn Pro Gln Asn Gly
140 145 150
ATC AAC GCG CTA GGC ACC CTG ACC ACC GCG GGC ACC AGT GCG CTG AAC 576
Ile Asn Ala Leu Gly Thr Leu Thr Thr Ala Gly Thr Ser Ala Leu Asn
155 160 165 170
AGT CGC CAC CCG TGG GGC GTC AAC ACC AGC AGC TAC TGC GCC AAG TCC 624
Ser Arg His Pro Trp Gly Val Asn Thr Ser Ser Tyr Cy6 Ala Lys Se~
175 180 185
ACC GAA GTG CAC AAT GTG CGC GGT CAC AGC ATC CGC TAC TAC TCC TGG 672
Thr Glu Val His Asn Val Arg Gly His Ser Ile Arg Tyr Tyr Ser Trp
190 195 200
CA 02202553 1997-04-11
63
ACC GGT AAT GCC GCC TAT ACC AAC GTG CTC GAT GCG GCC GAT GCC TTC 720
Thr Gly Asn Ala Ala Tyr Thr A~n Val Leu Asp Ala Ala Asp Pro Phe
205 210 215
CTG GCC TTC ACC GGC CTG GTG TTC GGC AGC G~G AAG AAC GAC GGT CTG 768
Leu Ala Phe Thr Gly Leu Val Phe Gly Ser G1U Lys Asn Asp Gly Leu
220 225 230
GTG GGC GTA TGT TCC ACC TAT CTG GGG CAG GTG ATC GAC GAC AGC TAC 816
Val Gly Val Cys Ser Thr Tyr Leu Gly Gln Val Ile Asp Asp Ser Tyr
235 240 245 250
AAC ATG AAC CAC GTC GAT GCG ATC AAC CAC CTG TTC GGC ATT CGT GGC 864
Asn Mes Asn His Val Asp Ala Ile Asn His Leu Phe Gly Ile Arg Gly
255 260 265
TGG ACC GAA CCG GTG TCG CTG TAT CGC CAG CAC GCC AAC CGC CTG AAG 912
Trp Thr Glu Pro Val Ser Leu Tyr Arg Gln His Ala Asn Arg Leu Lys
270 275 2~0
AAC AAG G&C GTC TGA 927
Asn Lys Gly Val
285
(2) INFORMATION FOR THE SEQ ID NO: 7:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 308 amino acids
(B) TYPE: amino acids
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:
Met Arg Arg Val Tyr Thr Ala Ala Leu Ala Thr Leu Ala Leu Leu Gly
-22 -20 -15 -10
Ala Val Glu Ala Gln Ala Asn Tyr Thr Ly~ ~hr Lys Tyr Pro Ile Val
-5 1 5 10
eu Val His Gly Val Thr G~ Phe Asn Thr Ile Gly Gly Leu Val Asn
yr Phe His Thr Ile Pro Trp Asn Leu Glu Arg Asp Gly Ala Arg Val
His Val Ala Ser Val Ala Ala Phe Asn Asp Ser Glu Gln Ars Gly Ala
Glu Leu Ala Arg Gln Ile Val Pro Trp Ala Ala Gly Gly Gly Gly Lys
CA 02202~3 1997-04-11
>
64
Val Asn Leu Ile Gly His Ser Gln Gly Ser Pro Thr Ser Arg Val Ala
7S B0 85 90
Ala Ser Leu Arg Pro Asp Leu Val Ala Ser Val Thr Ser Ile Asn Gly
9S 100 lOS
Val Asn Lys Gly Ser Lys Val Ala Asp Val Val Arg Gly Val Leu Pro
110 115 120
Pro Gly Ser Gly Ile Glu Gly Gly Ala Asn Ala Ile Ala Asn Ala Leu
12S 130 135
Gly Ala Val Ile Asn Leu Leu Ser Gly Ser Ser Asn Pro Gln Asn Gly
140 145 150
Ile Asn Ala Leu Gly Thr Leu Thr Thr Ala Gly Thr Ser Ala Leu Asn
lSS 160 16S 170
Ser Arg His Pro Trp Gly Val Asn Thr Ser Ser Tyr Cys Ala Lys Ser
175 180 185
Thr Glu Val His Asn Val Arg Gly His Ser Ile Arg Tyr Tyr Ser Trp
190 l9S 200
Thr Gly.Asn Ala Ala Tyr Thr Asn Val Leu Asp Ala Ala Asp Pro Phe
20S 210 21S
Leu Ala Phe Thr Gly Leu Val Phe Gly Ser Glu Lys Asn Asp Gly Leu
220 225 230
Val Gly Val Cys Ser Thr Tyr Leu Gly Gln Val Ile Asp Asp Ser Tyr
235 240 245 2S0
Asn Met Asn His Val Asp Ala Ile Asn His Leu Phe Gly Ile Arg~Gly
255 260 265
Trp Thr Glu Pro V~l Ser Leu Tyr Arg Gln Hifi Ala Asn Arg Leu Lys
270 275 280
Asn Ly6 Gly Val
28S
(2) INFORMATION FOR THE SEQ ID NO: 8:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 66 base pairs
(B) TYPE: nucleotide
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: DNA (yenomic)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:
CA 02202553 1997-04-11
ATGC~l~GCG TCTATACCGC TGC'C~-l~GCA ACA~lCG~lC TGCTTGGCGC CGTCGAGGCC 60
CAGGCC 66
(2) INFORMATION FOR THE SEQ ID NO: 9-
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 66 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF STRANDS: simple
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: DNA (genomic)
(ix) CHARACTERISTICS:
(A) NAME/KEY: CDS
(B) LOCATION: 1..66
(ix) CHARACTERISTICS:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 1..66
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:
ATG CGT CGC GTC TAT ACC GCT GCC CTG GCA ACA CTC GCT CTG CTT GGC 48
Met Arg Arg Val Tyr Thr Ala Ala LRU Ala Thr Leu Ala Leu Leu Gly
1 5 10 15
GCC GTC GAG GCC CAG GCC 66
Ala Val Glu Ala Gln Ala
(2) INFORMATION FOR THE SEQ ID NO: 10:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acids
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10:
Met Arg Arg Val Tyr Thr Ala Ala Leu Ala Thr Leu Ala Leu Leu Gly
1 5 10 15
Ala Val Glu Ala Gln Ala
CA 02202553 1997-04-11
.,
66
(2) INFORMATION FOR THE SEQ ID NO: 11:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 72 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF STRANDS: sim,ple
(D) CONFIGURATION: linear .
(ii) TYPE OF MOLECULE: Further nucleic acid
(A) DESCRIPTION: /desc = "synthetic
oligonucleotide"
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:
AACTACACCA AGACCAAATA CCCCATCGTG CTGGTCCACG GCGTGACCGG CTTCAACACT 60
ATCGGCGGGC TC 72
