Note: Descriptions are shown in the official language in which they were submitted.
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ALKALINE BACILLUS AMYLASE
FIELD OF INVENTION
The present invention relates to amylases having improved
washing performance in an alkaline detergent solution at low
s temperature.
BACKGROUND OF THE INVENTION
For a number of years a-amylase enzymes have been used for
a variety of different purposes, the most important of which are
starch liquefaction, textile desizing, starch modification in
io the paper and pulp industry, and for brewing and baking. A
further use of a-amylases, which is becoming increasingly impor-
tant is the removal of starchy stains during washing with a
detergent at alkaline pH.
Examples of commercial a-amylase products are Termamyl°,
i5 BAN° and Fungamyl°, all available from Novo Nordisk A/S,
Denmark. These and similar products from other commercial sour
ces have an acidic to a neutral pH optimum, typically in the
range of from pH 5 to pH 7.5, and they do not display optimal
activity in detergent solutions at alkaline pH.
2o WO 95/26397 discloses an a-amylase from a Bacillus strain
which has optimum activity at pH 8. WO 96/23873 describes
variants of Bacillus amylases with improved performance under
washing conditions.
US 5,147,796 describes an alkaline pullulanase having
is alpha-amylase activity. Fig. 2b of the document shows optimum
amylase activity at pH 8-8.5.
M. Takagi et al., J. Ferment. Bioeng., vol 81, No. 6, 557-
559 (1996) describe an alkaliphilic alpha-amylase-pullulanase
from Bacillus sp. The enzyme has optimum amylase activity at pH
30 9, but the activity drops rapidly at higher pH, and the activity
at pH 10 is lower than at pH 7.
It is an object of the present invention to provide novel
a-amylases with improved performance in alkaline solutions,
especially in alkaline detergent solutions at pH around 9-11.
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SZJMMARY OF THE INVENTION
~r:e present invention provides alpha-amylase whic:~. is:
a) a polypeptide produced by Bacillus sp. NCINB 46916,
or
s b) a polypeptide having an amino acid sequence as shown
in positions 32-532 of SEQ ID NO: 2, or
c) - a polypeptide encoded by the alpha-amylase encoding
part of the DNA sequence cloned into a plasmid present in
Escherichia coli DSM 12662, or
io d) an analogue of the polypeptide defined in (a) or (b)
which:
i) is at least 60 % homologous with said
polypeptide, or
ii) is derived from said polypeptide by
is substitution, deletion and/or insertion of one or
several amino acids.
In another aspect, the invention provides an a-amylase
having one or more of the following characteristics:
~an activity at pH 10.5 which is at least two times
Zo higher than the activity at pH 7.3 when measured at 37°C.
~an activity at pH 9.5 which is at least 4 times higher
than the activity at pH 7.3 when measured at 37°C.
~an optimum pH of about 9.5 when measured at 37°C.
~a thermostability such that it retains more than 90 % of
Zs its activity after 20 minutes incubation at 25°C in a solution
of 3 g/1 of a test detergent containing 20% STPP, 25% Na2S04,
15% Na2C03, 20% LAS, 5% C12-C15 alcohol ethoxylate, 5%
Na2Si205, 0.3% NaCl at pH 10.5 and 6 degrees German hardness,
and retains less than 90 % of its activity after 20 minutes
3o incubation at 30°C in the same solution.
~a molecular weight of about 55 kDa as determined by SDS-
PAGE.
~an iso-electric point of about S as determined by
isoelectric focusing.
as The invention also provides an isolated DNA sequence
which encodes an alpha-amylase, wherein the alpha-amylase is
that described above, or wherein the DNA sequence comprises:
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a) the DNA sequence showy. in positions 94-1596 of SEQ ID
NO: ', or
b) an analogue of the DNA sequence defined in a) which
i) is at least 60 % homologous with said DNA
= sequence, or
ii) hybridizes with said DNA sequence at least 55°C.
Other aspects of the invention provide a recombinant
expression vector comprising the DNA sequence, and a cell
transformed with the Dh'~ sequence or the recombinant expression
io vector. .
The invention also provides a method for producing an
alpha-amylase by cultivating the cell and a detergent
composition comprising said alpha-amylase.
BRIEF DESCRIPTION OF DRAWINGS
is The present invention is further illustrated with
reference to the accompanying drawings, in which:
Fig. 1 shows a pH activity profile of the amylase from
NCIMB 40916 compared to two prior-art amylases (SP722 and
Termamyl).
zo Fig. 2 shows a temperature activity profile of the amylase
from NCIMB 40916.
Fig. 3 shows the stability of the amylase from NCIMB
40916 after incubation at various temperatures.
Fig. 4 shows the cloning vector pSJ1678 described in
~s Example 1.
Figs. 5 and 6 show results of washing tests described in
Example 4.
DETAILED DESCRIPTION OF THE INVENTION
Microbial source
3o The alpha-amylase of the invention may be derived from a
strain of Bacillus. A preferred strain is Bacillus sp. NCIMB
40916. This strain was deposited on 28 January 1998 by the
inventors under the terms of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for
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the Purposes ef Patent Procedure at the National Collections o
Industrial and Marine Bacteria Limited (NCIMB), 23 St. Machar
Drive, Aberdeen AB2 1RY, Scotland, United Kingdom. A Escheri-
chia coli strain termed JA388 containing the alpha amylase gene
s cloned in plasmid nJA388 has also been deposited on 3 February
1999 under the terms of the Budapest Treaty with the Deutshe
Sammmlung von Microorganismen and Zellkulturen GmbH (DSMZ),
Mascheroder Weg 1b, D-38124 Braunschweig DE, and given the
accession number DSM 12662.
io Production of alpha-amylase
The alpha-amylase of the invention can be produced by
cultivating a suitable amylase-producing strain of Bacillus or
the transformed host cell of the invention in a suitable nu
trient medium, and recovering the alpha-amylase from the cul
i5 ture medium.
The medium used to cultivate the cells may be any conven-
tional medium suitable for growing the host cell in question
and obtaining expression of the a-amylase of the invention.
Suitable media are available from commercial suppliers o~ :y
Zo be prepared according to published recipes (e.g. as desc~ d
in catalogues of the American Type Culture Collection).
The a-amylase secreted from the host cells may ~n-
veniently be recovered from the culture medium by well-known
procedures, including separating the cells from the medium by
is centrifugation or filtration, and precipitating proteinaceous
components of the medium by means of a salt such as ammonium
sulphate, followed by the use of chromatographic procedures
such as ion exchange chromatography, affinity chromatography,
or the like.
3o Properties of alpha-amylase
A preferred alpha-amylase is derived from Bacillus sp.
NCIMB 40916. It can be produced as described in the examples.
The amino acid sequence of the amylase and of the DNA encoding
it are shown in SEQ ID N0: 1 and 2. The following
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characteristics were found for an amylase of the invention
(purified alpha-amylase from NCIMB 40916):
A molecular weight of approximately 55 kDa as determined
by SDS-PAGE using a Novex, 4-25% gradient gel.
s A pI of approximately 5 was determined by isoelectric
focusing (Ampholine PAG, pH 3.5-9.5).
A pH-activity curve is shown in Fig. 1, taking the
activity at pH 7.3 as 100%. It was determined using the
Phadebas assay using 50 mM Britten-Robinson buffer adjusted to
io various pH-values. For reference, the pH profiles of two prior-
art Bacillus amylases (Termamyl derived from B. licheniformis
and SP722 produced according to WO 95/26397) were measured
under the same conditions are also shown in Figure 1. Figure 1
shows that the amylase of the invention has about 10 times
is higher activity than the two prior-art amylases at pH 9.5. Fig.
1 also shows that the optimum activity is about pH 9.5.
A temperature-activity curve was measured using the
Phadebas assay at various temperatures with 50 mM Britten-
Robinson buffer adjusted to pH 9.5. The results are shown in
zo Fig. 2. It is seen from Fig. 2 that the amylase of the
invention has optimum activity at about 55°C.
Stability of the amylase was measured at pH 10.5 (50 mM
CAPS) at 22, 37, 45, 55 and 65 °C after 30 minutes incubation.
The enzyme was diluted to 40 NU/ml in buffer and 25 microlitre
zs sample was incubated at the respective temperatures. After 30
minutes, the samples was chilled on ice and residual activity
was determined. A stability profile of the amylase of the
invention and a prior-art amylase (SP722) is shown in Figure 3.
Stability was tested by incubating 40 NU/ml of amylase in
ao a solution of 3 g/1 of the A/P model detergent described above,
at pH 10.5 and 6°dH (German hardness, Ca:Mg 2:1). After the
incubation, the residual activity was measured by Phadebas at
pH 7.3. The results were 95 % residual activity after
incubation at 25°C, and 87 % at 30°C.
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Homology of polypeptide and DNA sequence
The amino acid sequence hcmology may be determined as the
degree of identity between the two sequences indicating a
derivation of the first sequence from the second. The homology
s may suitably be determined by means of computer programs known
in the art. Thus, FASTA provided in GCG version 8 (Needleman,
S.B. and Wunsch, C.D., (1970), Journal of Molecular Biology,
48, 443-453) may be used with the following settings: Scoring
matrix: GenRunData:blosum50.cmp, Variable pamfactor used Gap
io creation penalty: 12, Gap extension penalty: 2. Alternatively,
Gap from GCG version 9 may be used with a translated version 8
peptide scoring matrix, a gap creation penalty of 30, a gap
extension penalty of 1 using ntol's matrix
(http://plasmid/-bioweb/matrix/) without end gap penalty.
is The amino acid sequence exhibits a degree of identity
preferably of at least 60%, preferably at least 70%, more
preferably at Least 80%, especially at least 90% or at least
95%, with the amino acid sequence shown in positions 32-532 of
SEQ ID NO: 2.
zo The DNA sequence homology may be determined as the degree
of identity between the two sequences indicating a derivation
of the first sequence from the second. The homology may
suitably be determined by means of computer programs known in
the art such as GAP provided in the GCG program package
is (described above). Thus, Gap GCGv8 may be used with the fol-
lowing default parameters: GAP creation penalty of 5.0 and GAP
extension penalty of 0.3, default scoring matrix . GAP uses the
method of Needleman/Wunsch/Sellers to make alignments.
The DNA construct of the present invention comprises a
ao DNA sequence exhibiting a degree of identity preferably of at
least 60%, preferably at least 70%, more preferably at least
80%, especially at least 90% or at least 95%, with the nucleic
acid sequence shown in positions 94-1596 of SEQ ID NO: 1.
A homology search of known sequences showed homologies
3s for the sequences of the invention with a number of Bacillus
amylases in the range 31-34 % on amino acid basis and 46-48 %
on DNA basis, determined as described above.
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Hybridization
The hybridization is used to indicate that a given DNA
sequence is analogous to a nucleotide probe corresponding to
SEQ ID N0: 1. The hybridization conditions are described in
s detail below.
Suitable conditions for determining hybridization between
a nucleotide probe and a homologous DNA or RNA sequence
involves presoaking of the filter containing the DNA fragments
or RNA to hybridize in 5 x SSC (standard saline citrate) for 10
io min, and prehybridization of the filter in a solution of 5 x
SSC (Sambrook et al. 1989), 5 x Denhardt's solution (Sambrook
et al. 1989), 0.5 % SDS and 100 ~Cg/ml of denatured sonicated
salmon sperm DNA (Sambrook et al. 1989), followed by
hybridization in the same solution containing a random-primed
is (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem.
132:6-13), 'ZP-dCTP-labeled (specific activity > 1 x 10" cpm/ug
probe for 12 hours at approx. 45°C. The filter is then washed
two times for 30 minutes in 2 x SSC, 0.5 % SDS at a temperature
of at least 55°C, more preferably at least 60°C, more
so preferably at least 65°C, even more preferably at least 70°C,
especially at least 75°C.
Molecules to which the oligonucleotide probe hybridizes
under these conditions are detected using a x-ray film.
Recombinant expression vector
zs The expression vector of the invention typically includes
control sequences encoding a promoter, operator, ribosome
binding site, translation initiation signal, and, optionally, a
repressor gene or various activator genes.
The recombinant expression vector carrying the DNA
3o sequence encoding the a-amylase of the invention may be any
vector which may conveniently be subjected to recombinant DNA
procedures, and the choice of vector will often depend on the
host cell into which it is to be introduced. Thus, the vector
may be an autonomously replicating vector, i.e. a vector which
35 exists as an extrachromosomal entity, the replication of which
is independent of chromosomal replication, e.g. a plasmid, a
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bacteriophage or an extrachromosomal element, minichromosome or
an artificial chromosome. Alternatively, the vector may be one
which, when introduced into a host cell, is integrated into the
host cell genome and replicated together with the chromosomes)
into which it has been integrated.
In the vector, the DNA sequence should be operably '
connected to a suitable promoter sequence. The promoter may be
any DNA sequence which shows transcriptional activity in the
host cell of choice and may be derived from genes encoding pro
o teins either homologous or heterologous to the host cell.
Examples of suitable promoters for directing the transcription
of the DNA sequence encoding an a-amylase of the invention,
especially in a bacterial host, are the promoter of the Iac
operon of E.coli, the Streptomyces coelicolor agarase gene dagA
is promoters, the promoters of the Bacillus licheniformis a-
amylase gene (amyl), the promoters of the Bacillus
stearothermophilus maltogenic amylase gene (amyM), the promo-
ters of the Bacillus Amyloliquefaciens a-amylase (amyQ), the
promoters of the Bacillus subtilis xylA and xylB genes etc. For
zo transcription in a fungal host, examples of useful promoters
are those derived from the gene encoding A. oryzae TAKA
amylase, Rhizomucor miehei aspartic proteinase, A. niger neu-
tral a-amylase, A. niger acid stable a-amylase, A. niger glu-
coamylase, Rhizomucor miehei lipase, A. oryzae alkaline
zs protease, A. oryzae triose phosphate isomerase or A. nidulans
acetamidase.
The expression vector of the invention may also comprise
a suitable transcription terminator and, in eukaryotes, poly-
adenylation sequences operably connected to the DNA sequence
3o encoding the a-amylase of the invention. Termination and poly-
adenylation sequences may suitably be derived from the same
sources as the promoter. -
The vector may further comprise a DNA sequence enabling
the vector to replicate in the host cell in question. Examples
35 Of such sequences are the origins of replication of plasmids
pUCl9, pACYC177, pUB110, pE194, pAMBl and pIJ702.
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The vector may also comprise a selectable marker, e.g. a
gene the product of which complements a defect in the host
cell, such as the dal genes from B. subtilis or B. lichenifor-
mis, or one which confers antibiotic resistance such as ampi-
cillin, kanamycin, chloramphenicol or tetracyclin resistance.
Furthermore, the vector may comprise Aspergillus selection
markers such as amdS, argB, niaD and sC, a marker giving rise
to hygromycin resistance, or the selection may be accomplished
by co-transformation, e.g. as described in WO 91/17243.
io While intracellular expression may be advantageous in
some respects, e.g. when using certain bacteria as host cells,
it is generally preferred that the expression is extracellular.
Procedures suitable for constructing vectors of the
invention encoding an a-amylase , and containing the promoter,
i5 terminator and other elements, respectively, are well known to
persons skilled in the art icf., for instance, Sambrook et al.
(1989) ] .
Host cells
The cell of the invention, either comprising a DNA con
Zo struct or an expression vector of the invention as defined
above, is advantageously used as a host cell in the recombinant
production of the a-amylase of the invention. The cell may be
transformed with the DNA construct of the invention encoding
the amylase, conveniently by integrating the DNA construct (in
is one or more copies) in the host chromosome. This integration is
generally considered to be an advantage as the DNA sequence is
more likely to be stably maintained in the cell. Integration of
the DNA constructs into the host chromosome may be performed
according to conventional methods, e.g. by homologous or
3o heterologous recombination. Alternatively, the cell may be
transformed with an expression vector as described above in
connection with the different types of host cells.
The cell of the invention may be a cell of a higher
organism such as a mammal or an insect, but is preferably a
35 microbial cell, e.g. a bacterial or a fungal (including yeast)
cell.
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Examples of bacterial host cells which, on cultivation,
are capable of producing the enzyme of the invention are
gram-positive bacteria such as strains of Bacillus, such as
strains of B. subtilis, B. Iicheniformis, B. lentus, B.
s clausii, B. brevis, B. stearothermophilus, B. alkalophilus, B.
amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B.
megaterium or B. thuringiensis, or strains of Streptomyces,
such as S. lividans or S. murinus, or gram-negative bacteria
such as Escherichia coli. The transformation of the bacteria
io may be effected by protoplast transformation, electroporation,
conjugation, or by using competent cells in a manner known per
se (cf. Sambrook et al., supra).
The yeast organism may favourably be selected from a
species of Saccharomyces or Schizosaccharomyces, e.g.
is Saccharomyces cerevisiae. The filamentous fungus may advan
tageously belong to a species of Aspergillus, e.g. Aspergillus
oryzae or Aspergillus niger. Fungal cells may be transformed by
a process involving protoplast formation and transformation of
the protoplasts followed by regeneration of the cell wall in a
zo manner known per se. A suitable procedure for transformatic: of
Aspergillus host cells is described in EP 238 023.
Industrial Applications
Owing to their activity at alkaline pH values, the a
amylases of the invention are well suited for use in a variety
zs of industrial processes, in particular the enzyme finds
potential applications as a component in detergents, e.g.
laundry and hard surface cleaning detergent compositions, but it
may also be useful in the production of sweeteners and ethanol
from starch. Thus, it may be used in conventional starch-
3o converting processes, such as liquefaction and saccharification
processes described in US Patent No. 3,912,590 and EP patent
publications Nos. 252,730 and 63,909.
The alkaline a-amylase of the invention may also be used
in the production of lignocellulosic materials, such as pulp,
35 paper and cardboard, from starch reinforced waste paper and
cardboard, especially where repulping occurs at pH above 7 and
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where amylases ca:: facilitate the disintegration of the waste
material through degradation of the reinforcing starch. The a-
amylase of the invention is especially useful in a process for
producing a papermaking pulp from starch-coated printed paper.
s The process may be performed as described in WO 95/14807,
comprising the following steps:
a) disintegrating the paper to produce a pulp,
b) treating with a starch-degrading enzyme before,
during or after step a), and
to c) separating ink particles from the pulp after
steps a) and b).
The a-amylases of the invention may also be very useful
in modifying starch where enzymatically modified starch is used
in papermaking together with alkaline fillers such as calcium
is carbonate, kaolin and clays. With the alkaline a-amylases of
the invention it becomes possible to modify the starch in the
presence of the filler thus allowing for a simpler integrated
process.
The a-amylase of the invention may also be very useful in~
zo textile desizing. In the textile processing industry, a-
amylases are traditionally used as auxiliaries in the desizing
process to facilitate the removal of starch-containing size
which has served as a protective coating on weft yarns during
weaving. Complete removal of the size coating after weaving is
zs important to ensure optimum results in the subsequent pro-
cesses, in which the fabric is scoured, bleached and dyed.
Enzymatic starch break-down is preferred because it does not
involve any harmful effect on the fiber material. In order to
reduce processing cost and increase mill throughput, the
3o desizing processing is sometimes combined with the scouring and
bleaching steps. In such cases, non-enzymatic auxiliaries such
as alkali or oxidation agents are typically used to break down
the starch, because traditional a-amylases are not very
compatible with high pH levels and bleaching agents. The non-
3s enzymatic breakdown of the starch size does lead to some fiber
darrtage because of the rather aggressive chemicals used.
Accordingly, it would be desirable to use the a-amylases of the
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invention as they have an improved performance in alkaline
solutions. The a-amylases may be used alone or in combination
with a cellulase when desizing cellulose-containing fabric or
textile.
s The a-amylases of the invention may also be very useful
in a beer-making process; the a-amylases will typically be
added during the mashing process.
Detergent Compositions
According to the invention, the a-amylase may typically be
io a component of a detergent composition, e.g., a laundry
detergent composition. As such, it may be included in the
detergent composition in the form of a non-dusting granulate, a
stabilized liquid, or a protected enzyme. Non-dusting granulates
may be produced, e.g., as disclosed in US 4,106,991 and
is 4,661,452 (both to Novo Industri A/S) and may optionally be
coated by methods known in the art. Examples of waxy coating
materials are polyethylene oxide) products (polyethyleneglycol,
PEG) with mean molecular weights of 1000 to 20000; ethoxylated
nonylphenols having from 16 to 50 ethylene oxide units;
2o ethoxylated fatty alcohols in which the alcohol contains from 12
to 20 carbon atoms and in which there are 15 to 80 ethylene
oxide units; fatty alcohols; fatty acids; and mono- and di- and
triglycerides of fatty acids. Examples of film-forming coating
materials suitable for application by fluid bed techniques are
is given in patent GS 1483591. Liquid enzyme preparations may, for
instance, be stabilized by adding a polyol such as propylene
glycol, a sugar or sugar alcohol, lactic acid or boric acid
according to established methods. Other enzyme stabilizers are
well known in the art. Protected enzymes may be prepared
ao according to the method disclosed in EP 238,216.
The properties of the alpha-amylase of the invention make
it particularly suitable for use in alkaline detergents, e.g.
at pH 9.5-10.5, and for washing at low temperatures, e.g. 20-
40°C.
35 The detergent composition of the invention may be in any
convenient form, e.g. as powder, granules, paste or liquid. A
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liquid detergent may be aqueous, typically containing up to 70%
water and 0-30% organic solvent, or non-aqueous.
The detergent composition comprises one or more surf
actants, each of which may be anionic, nonionic, cationic, or
s amphoteric (zwitterionic). The detergent will usually contain
0-50% of anionic surfactant such as linear alkylbenzene-
sulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate
(fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or
AES), secondary alkanesulfonates (SAS), alpha-sulfo fatty acid
io methyl esters, alkyl- or alkenylsuccinic acid, or soap. It may
also contain 0-40% of nonionic surfactant such as alcohol
ethoxylate (AEO or AE), alcohol propoxylate, carboxylated
alcohol ethoxylates, nonylphenol ethoxylate, alkylpolygly-
coside, alkyldimethylamine oxide, ethoxyiated fatty acid
is monoethanolamide, fatty acid monoethanolamide, or polyhydroxy
alkyl fatty acid amide (e. g. as described in WO 92/06154).
The detergent composition may additionally comprise one
or more other enzymes, such as pullulanase, esterase, lipase,
cutinase, protease, cellulase, peroxidase, or oxidase, e.g.,
ao laccase.
Normally the detergent contains 1-65% of a detergent
builder or complexing agent such as zeolite, diphosphate, tri-
phosphate, phosphonate, citrate, nitrilotriacetic acid (NTA),
ethylenediaminetetraacetic acid (EDTA), diethylenetri-
zs aminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinic acid,
soluble silicates or layered silicates (e. g. SKS-6 from
Hoechst).
The detergent builders may be subdivided into phosphorus
containing and non-phosphorous-containing types. Examples of
3o phosphorus-containing inorganic alkaline detergent builders
include the water-soluble salts, especially alkali metal
pyrophosphates, orthophosphates, polyphosphates and phospho-
nates. Examples of non-phosphorus-containing inorganic builders
include water-soluble alkali metal carbonates, borates and
3s silicates as well as layered disilicates and the various types
of water-insoluble crystalline or amorphous alumino silicates
of which zeolites is the best known representative.
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Examples of suitable organic builders include alkali
metal, ammonium or substituted ammonium salts of succinates,
malonates, fatty acid malonates, fatty acid sulphonates,
carboxymethoxy succinates, polyacetates, carboxylates, polycar-
s boxylates, aminopolycarboxylates and polyacetyl carboxylates.
The detergent may also be unbuilt, i.e. essentially free of
detergent builder.
The detergent may comprise one or more polymers. Examples
are carboxymethylcellulose (CMC), poly(viny.~-ayrrolidone) (PVP),
~o polyethyleneglycol (PEG), polyvinyl alc~_:ol) (PVA), poly
carboxylates such as polyacrylates, polymaleates,
maleic/acrylic acid copolymers and lauryl methacrylate/acrylic
acid copolymers.
The detergent composition may contain bleaching agents of
is the chlorine/bromine-type or the oxygen-type. The bleaching
agents may be coated or encapsulated.
Examples of inorganic chlorine/bromine-type bleaches are
lithium, sodium or calcium hypochlorite or hypobromite as well
as chlorinated trisodium phosphate. Examples of organic
zo chlorine/bromine-type bleaches are heterocyclic N-bromo and N-
chloro imides such as trichloroisocyanuric, tribromoiso-
cyanuric, dibromoisocyanuric and dichloroisocyanuric acids, and
salts thereof with water solubilizing cations such as potassium
and sodium. Hydantoin compounds are also suitable. The
Zs bleaching system may also comprise peroxyacids of, e.g., the
amide, imide, or sulfone type.
The oxygen-type bleach may be an inorganic persalt,
preferably with a bleach activator, or a peroxy acid compound.
Examples of inorganic persalts are alkali metal perborates,
3o both tetrahydrates and monohydrates, alkali metal
percarbonates, persilicates and perphosphates. The activator
may be tetraacetylethylenediamine (TAED) or nonanoyloxybenzene-
sulfonate (NOBS) .
The enzymes of the detergent composition of the invention
3s may be stabilized using conventional stabilizing agents, e.g. a
polyol such as propylene glycol or glycerol, a sugar or sugar
alcohol, lactic acid, boric acid, or a boric acid derivative
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such as, e.g., an aromatic borate ester, and she composition
may be formulated as described in, e.g., WO 92/19709 and WO
92/19708. The enzymes of the invention may also be stabilized
by adding reversible enzyme inhibitors, e.g., of the protein
s type as described in EP 0 544 777 B1.
The detergent may also contain other conventional deter-
gent ingredients such as, e.g., fabric conditioners including
clays, deflocculant material, foam boosters, foam depressors,
suds suppressors, anti-corrosion agents, soil-suspending
~o agents, anti-soil-redeposition agents, dyes, dehydrating
agents, bactericides, optical brighteners, or perfume.
The pH (measured in aqueous solution at use concen-
tration) will usually be neutral or alkaline, e.g. in the range
of 7-11.
i5 More specifically, the alpha-amylase of the invention may
be incorporated in any of the detergent formulations described
in WO 96/23873.
The a-amylases of the invention may be incorporated in
concentrations conventionally employed in detergents. It is at
Zo present contemplated that, in the detergent composition of the
invention, the a-amylase may be added in an amount correspon-
ding to 0.00001-1 mg (calculated as pure enzyme protein) of a-
amylase per liter of wash liquor.
The present invention is further illustrated in the
z5 following examples which are not intended to be in any way
limiting to the scope of the invention as claimed.
EXAMPLES
Host organism:
Escherichia coli SJ2 is described in Diderichsen, B.,
ao Wedsted, U., Hedegaard, L., Jensen, B.R., Sjoholm, C. (1990)
Journal of Bacteriology, Vo1.172, No. 8, p. 4315-4321.
Plasmids:
The gene bank vector was pSJ1678 which is further
disclosed in W094/19454 which is hereby incorporated by
3s reference.
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16
The gene bank vector pSJ1678 is further disclosed in WO
94/19454 which is hereby incorporated by reference.
Model deterqent:
A/P (Asia/Pacific) Model Detergent has the following
s composition: 20% STPP (sodium tripolyphosphate), 25% Na~SO"
15% Na~C03, 20% LAS (linear alkyl benzene sulfonate, Nansa 80S) ,
S% C1~-C,5 alcohol ethoxylate (Dobanol 25-7) , 5% Na2Siz05, 0.3%
NaCl.
Example 1
io Cloning of alpha-amylase into E. coli
DNA was isolated from Bacillus sp. NCIMB 40916 by the
method of Pitcher, D. G., Saunders,N. A., and Owen, R. J.
(1989) Lett. Appl. Microbiol. 8, 151-156. Chromosomal DNA was
partially digested with the restriction enzyme Sau3AI. The
is fragments were cloned into the BamHI site of the cloning vector
pSJ1678, as shown in Figure 4 and transformed into Escherichia
coli SJ2, thereby creating a gene library of Bacillus sp. NCIMB
40916.
This gene library was screened for alpha amylase activity
2o and the strain showing alpha amylase activity was termed JA388.
This Escherichia coli strain JA388 which was deposited and
given the accession number DSM 12662, and which harbours the
plasmid termed pJA388 encoding the alpha amylase was used for
DNA sequencing.
is Example 2
Sequencing of DNA and alpha-amylase
The Alpha amylase gene cloned in plasmid pJA388 was
characterized by DNA sequencing by primer walking, using the
Taq deoxyterminal cycle sequencing kit (Perkin Elmer, USA),
3o fluorescent labeled terminators and appropriate
oligonucleotides as primers.
Analysis of the sequence data was performed according to
Devereux et al. (1984) Nucleic Acids Res. 12, 387-395. The
sequence corresponds to the DNA sequence shown in SEQ ID N0: 1.
35 The predicted protein sequence of the alpha amylase including
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77
the signal peptide and the mature alpha amylase are presented
in SEQ ID N0: 2. The deduced N-terminal sequence was verified
by sequencing 34 amino acids a~ the N-terminal of the protein.
Example 3
s Production of alpha-amylase
E, coli JA388 harboring plasmid pJA388 was cultivated
over night in LB-broth containing chloramphenicol 10 ~.g/ml, 37
°C, 250 rpm. Cells were harvested from 2.7 1 culture broth by
centrifugation at 6000 rpm for 15- minutes. The intracellular
io located amylase was released from the cells by using the
following osmotic shock procedure:
1) Cells were resuspended and washed in 500 ml 10 mM
Tris- HC1, pH 7.0 (EKV-buffer) followed by centrifugation.
2) Cells were resuspended in 75 ml 20% sucrose, 30 mM
is Tris-HC1 pH 8, 1 mM EDTA and added Lysozyme to a concentration
of 5 mg/ml.
3 ) The solution was incubated 15 min on ice followed by
centrifugation. The majority of the amylase was now contained
in the supernatant.
2o The supernatant was dialyzed overnight against 10 1 in
EKV-buffer with one buffer change to decrease the high
concentration of sucrose. The solution was filtered through a
0.45 y.m membrane vacuum filter.
The enzyme solution was applied on a Pharmacia Q
z5 Sepharose column FF previously equilibrated in EKV-buffer, pH
7, and the column was washed with EKV-buffer. Bound proteins
were eluted with a linear NaCl gradient from 0-500 mM over 10
column volumes. Amylase containing fractions were pooled and
dialyzed against EKV-buffer over night.
3o The solution was then applied on a Q Sepharose column FF
previously equilibrated in EKV-buffer, pH 8, the column was
washed with EKV-buffer, and the amylase was eluted with a
linear NaCl gradient from 0-500 mM over ZO column volumes.
Amylase containing fractions were pooled.
as A Phenyl Superose column previously equilibrated in EKV-
buffer, pH 8 containing 1 M ammonium sulfate was loaded with
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18
the enzyme solution added ammonium sulfate to a concentration
of 1 M. Unbound material was washed out with the ammonium
sulfate buffer and the column was eluted with a linear NaCl
gradient from 1-0 M ammonium sulfate over 20 column volumes.
Amylase containing fractions were pooled.
The purified amylase was analyzed by SDS-PAGE and only
one band was obtained after staining with Coomasie Blue.
Example 4
Washing test
o Washing performance was evaluated by washing soiled test
swatches for 15 minutes at 25°C in a detergent solution with
the alpha-amylase of the invention.
The detergent used was the A/P Model Detergent described
above at 3 g/1 having pH 10.5, or a commercial detergent from
i5 Malaysia (FAB Total from Colgate) at 3 g/1 having a pH of
approx. 9.7. The purified amylase of Example 3 was added to the
detergent solution at the concentration indicated below. The
test swatches were soiled with orange rice starch (CS-28
swatches available from CFT, Center for Test Material,
zo Holland) .
After washing, the swatches were evaluated by measuring
the remission at 460 nm. The results are expressed as DR -
remission of the swatch washed with the alpha-amylase minus the
remission of a swatch washed at the same conditions without the
alpha-amylase.
Alpha-amylase DR OR
concentration Model detergent Malaysian detergent
(mg enzyme protein
/ 1)
0 (reference) - 0 - 0
0.1 0.9 1
0.2 1.9 1.3
0.5 3 2.4
1.5 4.1 4.3
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19
The results are shown in rigures S and 6. The results
demonstrate that the alpha-amylase of the invention is
effective in both detergents at highly alkaline pH.
CA 02320759 2000-08-17
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SEQUENCE LISTING
<110> Novo Nordisk A!S
<120> Alkaline Bacillus Amylase
<130> 5942
<140>
<141>
<160> 2
<170> PatentIn Ver. 2.0
<210> 1
<211> 1599
<212> DNA
<213> Bacillus sp.
<220>
<221> CDS
<222> (1)..(1599)
<220>
<221> sig peptide
<222> (1)..(93)
<220>
<221> mat_peptide
<222> (94)..(1596)
<400> 1
atg caa aac aca gcg aaa aac tcc atc tgg cag agg gtg cgc cac agc 48
Met Gln Asn Thr Ala Lys Asn Ser Ile Trp Gln Arg Val Arg His Ser
-30 -25 -20
gcc att gcc tta tcc get ctc agt tta tcc ttt ggc ctg cag gcc agc 96
Ala Ile Ala Leu Ser Ala Leu Ser Leu Ser Phe Gly Leu Gln Ala Ser
-15 -10 -5 -1 1
gag tta cca caa att cca cca cag cag gtg aac aac acc atg tac cag 144
Glu Leu Pro Gln Ile Pro Pro Gln Gln Val Asn Asn Thr Met Tyr Gln
10 15
gca ttt tat tgg gat gcc tac cct ggc ctt tgg gcc aat tta ccg gcc 192
Ala Phe Tyr Trp Asp Ala Tyr Pro Gly Leu Trp Ala Asn Leu Pro Ala
20 25 30
1
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atg gcg gcc cct ttg gcc gag cgt ggc att acc tcg atg tgg ttg ccg 240
Met Ala Ala Pro Leu Ala Glu Arg Gly Ile Thr Ser Met Trp Leu Pro
35 40 45
ccc gcc gcc aaa ggc atg aat ggt act ttc agt gtc ggt tac gat gta 288
Pro Ala Aia Lys Gly Met Asn Gly Thr Phe Ser Val Gly Tyr Asp Val
50 55 60 65
tac gat ttc tgg gat ctg ggc gag ttt aac caa aaa ggc acc acc gcc 336
Tyr Asp Phe Trp Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Thr Ala
70 75 80
acc cgt tac ggt act cgt cag cag ctg caa caa gca ctg agt get ctg 389
Thr Arg Tyr Gly Thr Arg Gln Gln Leu Gln Gln Ala Leu Ser Ala Leu
85 90 95
gac caa ctg ggt att cag gcc tat ttt gat gtg gtg ttt aac cac cgc 432
Asp Gln Leu Gly Ile Gln Ala Tyr Phe Asp Val Val Phe Asn His Arg
100 105 110
atg ggc gcc gat gca cag gag aat att cct ggc ttt ggc ctg gcc tgg 480
Met Gly Ala Asp Ala Gln%Glu Asn Ile Pro Gly Phe Gly Leu Ala Trp
I15 120 125
acc gag tat cat ctg caa ggt cgt cag gcg cat tat acc cag caa aac 528
Thr Glu Tyr His Leu Gln Gly Arg Gln Ala His Tyr Thr Gln Gln Asn
130 135 140 145
tgg ggc tac ttg tgg cac gac ttt gac tgg aac tgg acc gcg ttt aat 576
Trp Gly Tyr Leu Trp His Asp Phe Asp Trp Asn Trp Thr Ala Phe Asn
150 155 160
ggc tcc gac aat cag ctc tac ccc ggc aaa tgg tgg ggc aat acc ttc 624
Gly Ser Asp Asn Gln Leu Tyr Pro Gly Lys Trp Trp Gly Asn Thr Phe
165 170 175
cac ttc cct tat ttg atg ggt gag gat gtc gat tac aac cgc ttt gaa 672
His Phe Pro Tyr Leu Met Gly Glu Asp Val Asp Tyr Asn Arg Phe Glu
180 185 190
gtg cag cag gaa atg aaa gcc tgg ggc gag tgg atc atc aac agc gtt 720
Val Gln Gln Glu Met Lys Ala Trp Gly Glu Trp Ile Ile Asn Ser Val
195 200 205
ggc ttt agc ggc ttt cgg atg gat gcc atc gcc cat gtc gat acc gat 768
Gly Phe Ser Gly Phe Arg Met Asp Ala Ile Ala His Val Asp Thr Asp
210 215 220 225
2
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WO 99/42567 PCT/DK99/00066
ttt acc cgt gac tgg atc aa~ cac gtg cag tgg gcc acc agt gag gat 816
Phe Thr Arg Asp Trp I1e Asn His Va1 Gln Trp Ala Thr Ser G_u Asp
230 235 240
gtg ttc ttt gtc get gaa gcc tgg gtc agt gat atc aac ggc tat ctg 864
Val Phe Phe Val Ala Glu Ala Trp Val Ser Asp Ile Asn Gly Tyr Leu
295 250 255
gat gca gtc aat acg ccg cat ttg cgc get ttt gat ttc aat ttg cgc 912
Asp Ala Val Asn Thr Pro His Leu Arg Ala Phe Asp Phe Asn Leu Arg
260 265 270
gaa gac ttc gtt get tta agc agc ggt agc aaa gac atg cgt tgg tgg 960
Glu Asp Phe Val Ala Leu Ser Ser Gly Ser Lys Asp Met Arg Trp Trp
275 280 285
ggc ggt ctg gtc aat agc cag cac cgt gat cgg gcg gtc act ttt gtc 1008
Giy Gly Leu Val Asn Ser Gln His Arg Asp Arg Ala Val Thr Phe Val
290 295 300 305
gat aac cac gat acc agc cgg gcc ggc aac cct tat ggc atg ccg cag 1056
Asp Asn His Asp Thr Ser Arg Ala Gly Asn Pro Tyr Gly Met Pro Gln
310 315 320
gtg atc aac tac aag aac cag gcc tac get tac att ctg ttg cgt gag 1109
Val Ile Asn Tyr Lys Asn Gln Ala Tyr Ala Tyr Ile Leu Leu Arg Glu
325 330 335
cat ggg gtg ccg act gtg ttt gcc cgc gat tac gac gaa ttt ggc atg 1152
His Gly Val Pro Thr Val Phe Ala Arg Asp Tyr Asp Glu Phe Gly Met
340 345 350
gcg cca acg ctg gat aaa ttg att gag gcg cgc cgc tac ttt get tat 1200
Ala Pro Thr Leu Asp Lys Leu Ile Glu Ala Arg Arg Tyr Phe Ala Tyr
355 360 365
ggt cct ggc cat gag tac tcc ggc aat acc gag gcc gtc tac gcc tat 1248
Gly Pro Gly His Glu Tyr Ser Gly Asn Thr Glu Ala Val Tyr Ala Tyr
370 375 380 385
gtg cgc gaa ggg ctt agc act gtg ccg ggt acc ggt ctg gtg atg ctg 1296
Val Arg Glu Gly Leu Ser Thr Val Pro Gly Thr Gly Leu Val Met Leu
390 395 400
ata tcg ggt cga aac tgg ggt ggt cag cag tcg ttc acc atc aac agc 1349
Ile Ser Gly Arg Asn Trp Gly Gly Gln Gln Ser Phe Thr Ile Asn Ser
405 410 915
3
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cac cag ccg aat acc acc t~~ tac gat tat acc ggc aat gtt agc ggc 1392
His Gln Pro Asn Thr Thr Phe Tyr Asp Tyr Thr Gly Asn Val Ser Gly
420 425 930
acg gtg acc acc aat gcg cag ggc tat ggc agc ttc ccg gtc act atg 1940
Thr Val Thr Thr Asn Ala Gln Gly Tyr Gly Ser Phe Pro Val Thr Met
435 440 495
acg gaa agt acc ggt tgg tca gtc ttg ggt acc aca atc caa tgg tgg 1488
Thr Glu Ser Thr Gly Trp Ser Val Leu Gly Thr Thr Ile Gln Trp Trp
950 455 460 465
cac tca gcc ggg atc cat tac cct gcg gat gac caa gga tgt tgg cta 1536
His Ser Ala Gly Ile His Tyr Pro Ala Asp Asp Gln Gly Cys Trp Leu
470 475 480
tgg ctt ttc gtt gtt ctt cac cgg cag cag tgc gga act gac caa ctg 1589
Trp Leu Phe Val Val Leu His Arg Gln G1n Cys Gly Thr Asp Gln Leu
485 490 495
ggg cgg cgg tat tga 1599
Gly Arg Arg Tyr
500
<210> 2
<211> 532
<212> PRT
<213> Bacillus sp.
<400> 2
Met Gln Asn Thr Ala Lys Asn Ser Ile Trp Gln Arg Val Arg His Ser
1 5 10 15
Ala Ile Ala Leu Ser Ala Leu Ser Leu Ser Phe Gly Leu Gln Ala Ser
20 25 30
Glu Leu Pro Gln Ile Pro Pro Gln Gln Val Asn Asn Thr Met Tyr Gln
35 40 45
Ala Phe Tyr Trp Asp Ala Tyr Pro Gly Leu Trp Ala Asn Leu Pro Ala
50 55 60
Met Ala Ala Pro Leu Ala Glu Arg Gly Ile Thr Ser Met Trp Leu Pro
65 70 75 80
Pro Ala Ala Lys Gly Met Asn Gly Thr Phe Ser Val Gly Tyr Asp Val
4
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WO 99/42567 PCT/DK99/00066
85 90 95
Tyr Asp Phe Trp Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Thr Ala
100 105 110
Thr Arg Tyr Gly Thr Arg Gln Gln Leu Gln Gln Ala Leu Ser Ala Leu
115 120 I25
Asp Gln Leu Gly Ile Gln Ala Tyr Phe Asp Val Val Phe Asn His Arg
130 135 140
Met Gly Ala Asp Ala Gln Glu Asn Ile Pro Gly Phe Gly Leu Ala Trp
145 150 155 160
Thr Glu Tyr His Leu Gln Gly Arg Gln Ala His Tyr Thr Gln Gln Asn
165 170 175
Trp Gly Tyr Leu Trp His Asp Phe Asp Trp Asn Trp Thr Ala Phe Asn
180 185 190
Gly Ser Asp Asn Gln Leu Tyr Pro Gly Lys Trp Trp Gly Asn Thr Phe
195 200 205
His Phe Pro Tyr Leu Met Gly Glu Asp Val Asp Tyr Asn Arg Phe Glu
210 215 220
Val Gln Gln Glu Met Lys Ala Trp Gly Glu Trp Ile Ile Asn Ser Val
225 230 235 240
Gly Phe Ser Gly Phe Arg Met Asp Ala Ile Ala His Val Asp Thr Asp
295 250 255
Phe Thr Arg Asp Trp Ile Asn His Val Gln Trp Ala Thr Ser Glu Asp
260 265 270
Val Phe Phe Val Ala Glu Ala Trp Val Ser Asp Ile Asn Gly Tyr Leu
275 280 285
Asp Ala Val Asn Thr Pro His Leu Arg Ala Phe Asp Phe Asn Leu Arg
290 295 300
Glu Asp Phe Val Ala Leu Ser Ser Gly Ser Lys Asp Met Arg Trp Trp
305 310 315 320
Gly Gly Leu Val Asn Ser Gln His Arg Asp Arg Ala Val Thr Phe Val
325 330 335
Asp Asn His Asp Thr Ser Arg Ala Gly Asn Pro Tyr Gly Met Pro Gln
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340 395 350
Val Ile Asn Tyr Lys Asn Gln Ala Tyr Ala Tyr Ile Leu Leu Arg Glu
355 360 365
His Gly Val Pro Thr Val Phe Ala Arg Asp Tyr Asp Glu Phe Gly Met
370 375 380
Ala Pro Thr Leu Asp Lys Leu Ile Glu Ala Arg Arg Tyr Phe Ala Tyr
385 390 395 400
Gly Pro Gly His Glu Tyr Ser Gly Asn Thr Glu Ala Val Tyr Ala Tyr
405 910 ' 915
Val Arg Glu Gly Leu Ser Thr Val Pro Gly Thr Gly Leu Val Met Leu
420 425 430
Ile Ser Gly Arg Asn Trp Gly Gly Gln Gln Ser Phe Thr Ile Asn Ser
435 490 945
His Gln Pro Asn Thr Thr Phe Tyr Asp Tyr Thr Gly Asn Val Ser Gly
450 955 460
Thr Val Thr Thr Asn Ala Gln Gly Tyr Gly Ser Phe Pro Val Thr Met
465 470 475 480
Thr Glu Ser Thr Gly Trp Ser Val Leu Gly Thr Thr Ile Gln Trp Trp
485 490 495
His Ser Ala Gly Ile His Tyr Pro Ala Asp Asp Gln Gly Cys Trp Leu
500 505 510
Trp Leu Phe Val Val Leu His Arg Gln Gln Cys Gly Thr Asp Gln Leu
515 520 525
Gly Arg Arg Tyr
530
6
