FIELD OF THE INVENTION

The present invention relates to a process for simulta¬ neously desizing and bleaching of a fabric comprising starch or starch-derivatives as well as to the use of an oxidation stable α-amylase for said process .

BACKGROUND OF THE INVENTION

In the textile processing industry, α-amylases are tradi¬ tionally used as auxiliaries in the desizing process to facilitate the removal of starch-containing size which has served as a protective coating on yarns during weaving.

Complete removal of the size coating after weaving is important to ensure optimum results in the subsequent processes, in which the fabric is generally scoured, bleached and dyed. Enzymatic starch break-down is pre¬ ferred because it does not involve any harmful effect on the fibre material.

In order to reduce processing cost and increase mill throughput, the 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 tra- ditional α-amylases are not very compatible with high pH levels and bleaching agents. Alternatively, unrealistic high amounts of α-amylases, optionally in protected form, have to be used for such combined processes. The non-enzy¬ matic breakdown of the starch size does lead to some fibre damage because of the rather aggressive chemicals used.

Accordingly, it would be desirable to use α-amylase enzymes having an improved resistance towards or being

compatible with oxidation (bleaching) agents at elevated pH, in order to retain the advantages of enzymatic size break down in a time-saving and environmentally desirable simultaneous desizing and bleaching process.

US 4,643,736 discloses a process for desizing and bleaching performed in a single operation, in which sodium chlorite is used in combination with a strong base, a surface active agent, an activator, and an amylolytic enzyme. However, the use of sodium chlorite is undesirable from an environmental point of view.

EP 119 920 discloses a process for simultaneous desizing and bleaching, in which sodium tetraborate decahydrate is used as buffer in a bath containing hydrogen peroxide, a sequestering agent, an amylase and a surfactant.

In both of the processes described in the above patent publications a relatively high amount of α-amylase is used, presumably in order to compensate for the low oxidation stability of the α-amylases used.

PCT/DK93/00230 discloses α-amylase mutants having improved oxidation stability. The mutants are indicated to be useful for desizing, but their use in a combined desizing and bleaching process is not mentioned.

BRIEF DISCLOSURE OF THE INVENTION

The fact that oxidation stable α-amylases now, for the first time, are available makes it possible to perform a combined enzymatic desizing and bleaching treatment of starch-containing fabrics. Accordingly, in a first aspect the invention relates to a process for simultaneously desizing and bleaching of a sized fabric containing starch or starch derivatives, which process comprises treating

the fabric with a bleaching composition and an oxidation stable α-amylase.

It is contemplated that the use of oxidation stable α- amylases in the above process constitute an environmentally desirable alternative to non-enzymatic alkali or oxidation agents used today for desizing and bleaching. Furthermore, the oxidation stable α-amylase may be used in amounts corresponding to the amounts of α-amylase used in today's desizing processes.

In the present context, the term "oxidation stable" is intended to indicate that under conditions prevailing during the combined process of the invention, the oxidation stable α-amylase performs better than the B . licheniformis α-amylase, commercially available from the Applicant under the trade na .-i Termamyl®. Termamyl® is presently considered to be highly useful for desizing, but is less suitable for a combined process due to a relatively low tolerance towards bleaching agents normally used for bleaching. The better performance may, e.g., be measured as described in the section entitled "Determination of oxidation stability" hereinafter.

The term "desizing" is intended to be understood in a conventional manner, i.e. the removal of size from the fabric, the term "scouring" the removal of non-cellulosic materials such as grease, wax, protein, hemi-cellvlosic material, pectin, ash, dirt and oil, and term "b2 ching" the bleaching of coloured impurities associated with the fibers of the fabric.

The term "simultaneously" is intended to indicate that the desizing and bleaching are carried out in a single operation. This has the obvious advantage that the washing and other treatments normally performed between separately conducted desizing and bleaching steps are no longer required. Thereby, the water and energy demand as well as

the demand to different equipment to be used for each of the processes are considerably reduced. Furthermore, depending on the type of fabric to be treated and the nature of impurities present thereon, a scouring effect may be obtained during the performance of the process of the invention. Thus, in such cases, no additional scouring treatment need to be performed.

The term "fabric containing starch or starch derivatives" is intended to indicate any type of fabric, in particular woven fabric prepared from a cellulose-containing material, containing starch or starch derivatives. The fabric is normally made of cotton, viscose, flax and the like. The main part of the starch or starch derivatives present on the fabric is normally size with which the yarns, normally warp yarns, have been coated prior to weaving. In the present context, the term "fabric" is also intended to include garments and other types of processed fabrics.

In a second aspect the invention relates to a composition to be used in a simultaneous desizing and bleaching process, which composition comprises an oxidation stable α-amylase in combination with at least one further component selected from the group consisting of wetting agents, dispersing agents, sequestering agents and emulsifying agents.

In a final aspect the invention relates to the use of an oxidation stable α-amylase for a simultaneous desizing and bleaching process.

DETAILED DISCLOSURE OF THE INVENTION

The oxidation stable α-amylase

A preferred example of an α-amylase to be used in the process of the invention is one which has been prepared

from a parent α-amylase by replacing one or more methionine residues of the parent α-amylase with any amino acid residue different from Cys or Met. Thus, according to the invention the amino acid residues to replace the methionine amino acid residue are the following: Ala, Arg, Asn, Asp, Gin, Glu, Gly, His, lie. Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

It has surprisingly been found that the mutant α-amylases prepared as described above exhibit a better activity level and a better stability in the presence of oxidizing agents than prior art mutant amylases.

It is preferred that the oxidation stable α-amylase to be used in the present process is of microbial origin. More particularly, it is preferred that the α-amylase is derivable from a strain of Bacillus . Thus, Bacillus α- amylases exhibit in themselves a high heat stability, and by being mutated as describee ?bove, the mutants may exhibit an even better stabi y, especially in the presence of oxidizing agents.

In the present context the term "derivable" is intended not only to indicate an α-amylase produced by a strain of the organism in question, but also an α-amylase encoded by a DNA sequence isolated from such strain and produced in a host organism transformed with said DNA sequence. Furthermore, the term is intended to indicate an α-amylase which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the α-amylase in question.

Examples of parent Bacillus α-amylases useful for the present purpose are those derivable from a strain of B . licheniformis , a strain of B . amyloliquefaciens, or a strain of B . stearothermophilus.

The amino acid sequence for a B . licheniformis α-amylase useful for the present purpose is apparent from SEQ ID No. 2 (the corresponding DNA sequence is shown in SEQ ID No. 1). G. L. Gray et al., J. Bacteriol. 166, 635-643, 1986, FR 2665178 and EP 410 498 disclose variants of said α- amylase. The methionine numbers of the B . licheniformis α- a ylase are: 8, 15, 197, 256, 304, 366, and 438.

The amino acid sequence for a B . amyloliquefaciens α- amylase useful for the present purpose is apparent from SEQ ID No. 4 (the corresponding DNA sequence is shown in SEQ ID No. 3). Takkinen et al., J. Biol. Chem. 258. 1007- 1013, 1983 discloses a variant of said α-amylase. The methionine numbers of these B . amyloli-quefaciens α- amylases are: 6, 197, 256, 304, 366, and 438.

The amino acid sequence for a B. stearothermophilus α- amylase useful for the present purpose is apparent from SEQ ID No. 6 (the corresponding DNA sequence is shown in SEQ ID No. 5). G.L. Gray et al., J. Bacteriol. 166, 635- 643, 1986 disclose a variant of said α-amylase. The methionine numbers of these B. stearothermophilus α- amylases are: 8, 9, 97, 200, 206, 284, 307, 311, 316, and 437.

Furthermore, a parent α-amylase of fungal origin may be used, e.g. an α-amylase derivable from a strain of the fungal genus Aspergillus . For instance, the parent α- amylase may be derivable from a strain of the fungal species A. oryzae or A. niger. These α-amylases are all well characterized and their entire amino acid sequence is described.

The amino acid sequence for the Asp. oryzae α-amylase (sold commercially as FUNGAMYL®, by Novo Nordisk A/S) is shown in SEQ ID No. 7. The amino acid sequence of an A. niger α-amylase is shown in DK 5126/87.

In a preferred embodiment the parent α-amylase is selected from the group consisting of a B . licheniformis , B . amyloliquefaciens , B . stearothermophilus , A. oryzae and A. niger α-amylase, or is a functional analogue of any of 5 said parent α-amylases which

i) comprises an amino acid sequence being at least 60% homologous with the amino acid sequence of the parent α- amylase, IT ii) reacts with an antibody raised against the parent α- amylase, and/or

iii) is encoded by a DNA sequence which hybridizes with 15 the same probe as a DNA sequence encoding the parent α- amylase.

Property i) of the analogue is intended to indicate the degree of identity between the analogue and the parent α-

20 amylase indicating a derivation of the first sequence from the second. In particular, a polypeptide is considered to be homologous to the parent α-amylase if a comparison of the respective amino acid sequences reveals an identity of greater than about 60%, such as above 70%, 80%, 85%, 90%

25 or even 95%. Sequence comparisons can be performed via known algorithms, such at the one described by Lipman and Pearson (1985) .

The homologous α-amylase may be a genetically engineered 30 α-amylase, e.g. prepared in order to improve one or more properties such as ther ostability, acid/alkaline stability, temperature or pH optimum and the like.

The additional properties ii) and iii) of the analogue of 35 the parent α-amylase may be determined as follows:

Property ii) , i.e. the immunological cross reactivity, may be assayed using an antibody raised against or reactive

with at least one epitope of the parent α-amylase. The antibody, which may either be monoclonal or polyclonal, may be produced by methods known in the art, e.g. as described by Hudson et al., 1989. The immunological cross- reactivity may be determined using assays known in the art, examples of which are Western Blotting or radial immunodiffusion assay, e.g. as described by Hudson et al., 1989. In this respect, immunological cross-reactivity between parent B. licheniformis , B . amyloliquefaciens and B. stearothermophilus α-amylases having the amino acid sequences SEQ ID Nos. 2 , 4 and 6, respectively, have been found.

The oligonucleotide probe used in the characterization of the analogue in accordance with property iii) defined above, may suitably be prepared on the basis of the full or partial nucleotide or amino acid sequence of the parent α-amylase. The hybridization may be carried out under any suitable conditions allowing the DNA sequences to hybrid- ize. For instance, such conditions are hybridization under specified conditions, e.g. involving presoaking in 5xSSC and prehybridizing for lh at -40°C in a solution of 20% formamide, 5xDenhardt ^, s solution, 50mM sodium phosphate, pH 6.8, and 50μg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with lOOμM ATP for 18h at -40°C, or other methods described by e.g. Sambrook et al., 1989.

Specific examples of analogues of the B. licheniformis α- amylase comprising the amino acid sequence shown in SEQ ID No. 2 are Termamyl® (available from Novo Nordisk A/S) , Optither ® and Takather ® (available from Solvay) , Maxamyl® (available from Gist-Brocades) , Spezym AA® (available from Genencor) , and Keistase® (available from Daiwa) .

Specific examples of analogues of the B. amyloliquefaciens α-amylase comprising the amino acid sequence shown in SEQ

ID No. 4 are BAN® (available from Novo Nordisk A/S) , Op- tiamyl® (available from Solvay) , Dexlo® and Rapidase® (available from Gist-Brocades) , Kazuzase® (a mixed α- amylase and protease product available from Showa Denko) .

Specific examples of analogues of the B. stearother¬ mophilus α-amylase comprising the amino acid sequence shown in SEQ ID No. 6 are Liquozyme 280L® (available from Novo Nordisk A/S) and G-zyme 995® (available from Enzyme BioSystems) .

In a preferred embodiment of the process of the invention the oxidation stable α-amylase is prepared from a parent α-amylase by replacing one or more of the methionine amino acid residues with a Leu, Thr, Ala, Gly, Ser, lie, Asn, or Asp amino acid residue, preferably a Leu, Thr, Ala, or Gly amino acid residue.

I :.he present context, a mutant α-amylase of particular ii. ;erest is one, in which the methionine amino acid residue in -?-sition 197 in B. licheniformis α-amylase or the methioii ne amino acid residue in homologous positions in other α-amylases is exchanged. The concept of homologous positions or sequence homology of α-amylases has been explained e.g. in Nakajima, R. et al.. 1986, Appl. Microbiol. Biotechnol. .23., 355-360 and Liisa Holm et al.. 1990, Protein Engineering 3_, 181-191. Sequence homology of Bacillus α-amylases from B. licheniformis , B . stearothermophilus and B. amyloliquefaciens are about 60%. This makes it possible to align the sequences in order to compare residues at homologous positions in the sequence. By such alignment of α-amylase sequences the number in each α-amylase sequence of the homologous residues can be found. The homologous positions will probably spatially be in the same position in a three dimensional structure (Greer, J. , 1981, J. Mol. Biol. 151, 1027-1042), thus having analogous impact on specific functions of the enzyme in question. In relation to position 197 in B. licheniformis α-a ylase the homologous positions in B.

stearothermophilus α-amylase are positions 200 and 206, and the homologous position in B. amyloliquefaciens α- amylase is position 197. Experimentally it has been found that these mutants exhibit both an improved activity level and an improved stability in the presence of oxidizing agents.

Accordingly, another type of oxidation stable α-amylases of interest for the present purpose, is an oxidation stable α-amylase prepared by replacing one or both of the methionine amino acid residues in positions 200 and 206 in a parent B. stearothermophilus α-amylase or the methionine amino acid residues in homologous positions in other α- amylases with other amino acid residues as explained above.

The mutant α-amylases disclosed above may be constructed in accordance with established methods, e.g. by use of site-directed mutagenesis.

Process conditions

It will be understood that the combined process of the invention may be performed in accordance with any suitable desizing or bleaching process known in the art, e.g. as described by Olson, E.S. "Textile Wet Processes, Vol. I, Noyes Publication, Park Ridge, New Jersey, USA (1983) , M. Peter und H.K Rouette, Grundlagen der Textilveredlung, Deutsche Fachverlag GmbH, Frankfurt am Main, Germany (1988) . Thus, the process conditions to be used in performing the present invention may be selected so as to match a particular equipment or a particular type of process which it is desirable to use. Preferred examples of process types to be used in connection with the present invention include Jigger/Winch, Pad-Roll and Pad-Steam types. These types are dealt with in further detail below.

The combined process of the invention may be carried out as a batch, semi-continuous or continuous process using

steam or the principles of cold-bleaching. As an example the process may comprise the following steps:

(a) Impregnating the fabric in a desizing and bleaching bath containing (as a minimum) an oxidation stable α- amylase and a bleaching agent followed by squeezing out excessive liquid so as to maintain the quantity of liquor necessary for the reaction to be carried out (normally between 60 and 120% of the weight of the dry fabric) ,

(b) subjecting the impregnated fabric to steaming so as to bring the fabric to the desired reaction temperature, gen¬ erally between 20° and 120°C, and

(c) holding by rolling up or pleating the cloth in a J- Box, U-Box, carpet machine or the like for a sufficient period of time (normally between a few minutes and several hours) to allow the desizing and bleaching to occur.

As mentioned above, scouring may be an inherent result obtained when performing the combined process of the invention. However, for certain types of fabric it may be advantageous and/or necessary to subject the fabric to a scouring treatment in order to obtain a final product of a desired quality. In such cases, oxidation stable α- amylases disclosed herein may be employed in a combined desizing and scouring process, in particular oxidation stable α-amylases which are sufficiently stable at the high pH values, at which scouring is normally performed. Typically, a combined desizing and scouring process is carried out using a sufficient amount of an oxidation stable α-amylase and a strong alkali, such as NaOH, under conditions known in the art for desizing and scouring to be performed. Subsequently, the fabric resulting from a combined desizing and scouring treatment may be subjected to bleaching.

Normally, the oxidation stable α-amylase and the bleaching composition are added separately to the equipment in which the combined process is to take place. However, the oxidation stable α-amylase may also be mixed with the bleaching composition immediately prior to the combined treatment to be performed.

Although any type of bleaching agent may be used (such as sodium chlorite, sodium hypo chlorit and hydrogen peroxide) in the process of the invention it is preferred that a hydrogen peroxide based bleaching composition is used. Hydrogen peroxide constitutes the most gentle and environmentally friendly bleaching agent available today. The hydrogen peroxide is normally used in the form of a 35% solution and in an amount of 1-50 g/1 of bleaching bath liquid, such as in an amount of 5-40 g/1, 5-30 g/1, 10-30 g/1 or 30-40 g/1 depending on the type of process to be used.

Further components required for the process to be performed are typically added separately. Examples of such components include a stabilizer and a wetting agent. The stabilizer may be an agent stabilizing the hydrogen peroxide (such as water glass (Na20:Si02) and a Magnesium salt) so as to control the reactivity of the hydrogen peroxide.

The wetting agent serves to improve the wettability of the fibre whereby a rapid and even desizing and bleaching may be obtained. The wetting agent is preferably of an oxidation stable type.

In a preferred embodiment of the process of the invention, the oxidation stable α-amylase is used in an amount exceeding 1 g/1, preferably in an amount of 1-20 g/1, such as 1-10 g/1, 1-5 g/1 or 1-3 g/1. It will be understod that the amount of α-amylase to be used depend on the formulation of the α-amylase product in question.

Irrespective of the particular type of process to be used for the combined desizing and bleaching of the invention, the combined process is normally performed at a temperature in the range of 30-100°C, such as 50-100°C, 80-100°C, 90-100°C or 90-95°C and a pH in the range of 6.5-11, such as 9-10.8 or 10.0-10.8 . However, the actual process conditions may vary widely within these ranges as will be apparent from the following disclosure.

Preferred examples of the process conditions to be used in connection with the present invention include:

A batch type process, e.g. of the Jigger/Winch type, using

1-5 g/1 of an oxidation stable α-amylase,

6-25 g/1 of hydrogen peroxide (35%) ,

7-14 g/1 of stabilizer, e.g. water glass,

0.25-5 g/1 of a wetting agent, e.g. Arbyl R, available from Grϋnau, Germany, the process being performed at a pH in the range of 10-11

(obtained by addition of NaOH) and a temperature in the range of 90-95°C (obtained by steaming) , typically for 1-2 hours.

A semi-continuous process, e.g. of the Pad-Roll type, using

1-5 g/1 of an oxidation stable α-amylase,

30-40 g/1 of hydrogen peroxide (35%) ,

12-30 g/1 of stabilizer, e.g. water glass, 0.25-5 g/1 of a wetting agent, e.g. Arbyl R, the process being performed at a pH in the range of 10-11

(obtained by addition of NaOH) and a temperature in the range of 20-40°C, typically for 12-24 hours.

A continuous process, e.g. of the Pad-Steam type, using 1-5 g/1 of an oxidation stable α-amylase, 8-25 g/1 of hydrogen peroxide (35%) , 5-20 g/1 of stabilizer, e.g. water glass,

0.25 - 5 g/1 of a wetting agent, e.g. Arbyl R, the process being performed at a pH in the range of 10-11 (obtained by addition of NaOH) and a temperature in the range of 98-140°C (steaming) , the temperature above 115 preferably being maintained for a few seconds only and the process time typically being 0.5-3 min.

It will be understood that the combined process may be performed in any equipment sufficiently tolerant towards the conditions of the process.

Furthermore, it will be evident that in addition to the oxidation stability of the α-amylase to be used in the process of the invention this amylase should preferably be one which is active at a pH of above 6.5, such as above 9.0. Preferably the oxidation stable α-amylase has a high activity in the pH range of 10-10.8.

Composition of the invention Although the oxidation stable α-amylase may be added as such it is preferred that it is formulated into a suitable composition. Thus, the oxidation stable α-amylase may be used in the form of a granulate, preferably a non-dusting granulate, a liquid, in particular a stabilized liquid, a slurry, or in a protected form. Dust free granulates may be produced, e.g. as disclosed in US 4,106,991 and US 4,661,452 (both to Novo Nordisk A/S) and may optionally be coated by methods known in the art.

Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as e.g. propylene glycol, a sugar or sugar alcohol or acetic acid, according to established methods. Other enzyme stabilizers are well known in the art. Protected enzymes may be prepared according to the method disclosed in EP 238 216.

In principle the composition of the invention comprising an oxidation stable α-amylase may contain any other agent

to be used in the combined process of the invention. However, it is preferred that the composition is free from the bleaching agent and other highly oxidizing agents.

The composition of the invention comprises at least one further component selected from the group consisting of wetting agents, dispersing agents, sequestering agents and emulsifying agents. Examples of suitable wetting agents are disclosed above. The emulsifying agent serves to emulsify hydrophobic impurities present on the fabric. The dispersing agent serves to prevent that extracted impurities redeposit on the fabric. The sequestering agent serve to remove ions such as Ca, Mg and Fe, which may have a negative impact on the process and preferred examples include caustic soda (sodium hydroxide) and soda ash (sodium carbonate) .

Determination of oxidation stability

The amylase preparation is diluted to an amylase activity of 100 NU/ml (the NU (or KNU which is 1000 NU) amylase activity assay is defined in AF 207/1, which is available on request from Novo Nordisk A/S, and the unit is defined as follows: 1 KNU is the amount of enzyme which, per hour, under standard conditions, dextrinized 5.26 g starch dry substance Merch Amylum solubile, cat. no. 1253) in 50 mM of a Britton-Robinson buffer at pH 6-10 and incubated at 40-90°C. Subsequently H ₂ 0 ₂ (35%) is added to a concentration of 1-50 g/1, and the pH value is readjusted to the desired value, the activity is now measured after 15 seconds and after 5, 15, and 30 minutes, the activities are determined as described in AF 207/1 but with a standard curve constructed at the chosen pH and temperature instead of at pH 7.3, 37°C. the results are compared to measurements of a preparation of Termamyl® (available from Novo Nordisk A/S, Denmark) under the same conditions.

The following non-limiting examples illustrates the inven¬ tion.

EXAMPLE 1 Comparison of the pH-profile of oxidation stable α-amylase and conventional amylase

Materials and methods :

Textile:

100% pure cotton, unbleached, 5cm x 5cm swatches.

Enzymes:

A: Bacillus licheniformis oxidation stable α- amylase (methionin 197 replaced with leucin, activity 6.2 KNU _p /g, prepared by the Applicant, batch A943043K) .

B: Bacillus licheniformis α-amylase (Termamyl 120L, activity 142 KNU _p /g, commercially available from the Applicant, batch AXR 4025 94-3) .

Dosage: 30 KNU _p enzyme/100ml buffer.

Buffer: Britton-Robinson, pH 7 to 11 Dosage: 100ml buffer/shake flask

100 ml buffer was added to a shake flask and placed in a heated water bath at 85°C. When the buffer had reached the temperature of 85°C, the enzyme preparation (A or B, respectively) was added together with one swatch. The swatches were treated in a shaking waterbath for 20 minutes at 85°C.

After the enzyme treatment the swatches were washed at

95°C in 100 ml water containing 2g of Kieralon CD/litre (a surfactant) , followed by rinsing 4 times, each time in 100 ml cold deionized water. Then, the swatches were dried at

105°C for 10 minutes. After drying the swatches were tested using the conventional TEGEWA method (Method and standard scales obtainable from Verband TEGEWA, Karlstrasse 21, Frankfurt a.M., Germany).

TEGEWA method:

The samples of desized fabric were impregnated in a satu¬ rated iodine solution (5 ml/100 ml water) , rinsed with cold water, and wiped off with filter paper before compar- ing the (blue) colour intensity with the TEGEWA violet standard scale.

The TEGEWA standard scale ranges from 1 to 9, where l is the sized fabric while 9 is the totally desized fabric. A rating above 6 usually corresponds to an acceptable desizing.

The results from the pH experiments are shown in table 1 below..

Table l pH Reference Enzyme B Enzyme A

(conventiona1) (ox. stable)

7 1 6 7

8 1 5 7

9 1 4 7

10 1 2 5

The results show that the oxidation stable A-amylase is more pH-stable than the conventional A-amylase (Termamyl)

EXAMPLE 2

The oxidation stable α-amylase was tested and compared with a conventional amylase (Termamyl) in a combined process of desizing/bleaching.

Materials and methods:

As described in example 1, and further: H ₂ 0 ₂ concentration: 4, 6, 8, 10, 24 g/1, respectively NaOH cone: 0.5, 1.0 g/1, respectively Stabilizer: 2 g/1 (Product No. 1136 from the com¬ pany Harald Pedersen, Kastanievej 7, 1878 Frb.C, Denmark) Process temp.: 80°C, 85°C, 95°C, respectively

Process time: 60 minutes (20 min. for temp. 85°C and

95°C)

The desizing/bleaching solution was preheated to 80°C (85°C, 95°C) in a shake flask, the swatches were added to the solution, and the shake flask was placed in a shaking water bath for 1 hour. The concentration of H ₂ 0 ₂ in the solution was monitored by titration immediately before use. The washing/rinsing and evaluation of the treated swatches was as described in example 1.

The results from the desizing/bleaching experiments are shown in table 2 below.

Table 2

H ₂ 0 ₂ Temp. NaOH PH Reference Enzyme B Enzyme A g/i °C g/i score score score

4 80 0.5 10.1 2 5 7

6 80 0.5 9.9 1 3 6

6 80 1.0 10.1 2 3 5

8 80 1.0 10.2 2 3 5

10 85 0.5 9.6 2 4 5

10 95 0.5 9.5 3 5 6

24 85 0.5 9.1 3 4 6

24 95 0.5 8.7 4 5 7

10 95 1.0 10.0 4 5

The results demonstrate that the . ation stable A- amylase is more stable at. high pH nd at high H ₂ 0 ₂ concen¬ trations than the conventional amylase (Termamyl) .

REFERENCES CITED IN THE SPECIFICATION

Gray, G.L. et al. , J. Bacteriol. 166. 635-643, 1986;

Takkinen et al., J. Biol. Chem. 258. 1007-1013, 1983;

Lipman and Pearson, Science 227, 1435 (1985);

Hudson, L. , and Hay, F. , Practical Immunology, Third edition (1989) , Blackwell Scientific Publications;

Sambrook et al..Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor, 1989;

Nakajima, R. et al.. 1986, Appl. Microbiol. Biotechnol. 23. 355-360;

Liisa Holm et al.. 1990, Protein Engineering 1, 181-191;

Greer, J. , 1981, J. Mol. Biol. 153. 1027-1042;

Olson, E.S. "Textile Wet Processes, Vol. I, Noyes Publica¬ tion, Park Ridge, New Jersey, USA (1983) ;

M. Peter und H.K Rouette, Grundlagen der Textilveredlung, Deutsche Fachverlag GmbH, Frankfurt am Main, Germany (1988) ;

SEQUENCE LISTING

In the following SEQ ID Nos. 1, 3, 5 the 5', coding sequence and 3' sequence of the relevant α-amylase genes are illustrated. The 5' sequence is the first separate part of the sequence written with lower case letters, the coding sequence is the intermediate part of the sequence, where the signal sequence is written with lower case letters and the sequence encoding the mature α-amylase is written with upper case letters, and the 3' sequence is the third separate part of the sequence written with lower case letters.

SEQ ID No. 1 cggaagattggaagtacaaaaataagcaaaagattgtcaatcatgtcatgagccatgc gggagacggaaaaatcgtctta atgcacgatattt- atgcaacgttcgcagatgctgctgaagagattattaaaaagctgaaa- gcaaaaggctatcaattggt aactgtatctcagcttgaagaagtgaagaag- cagagaggctattgaataaatgagtagaagcgccatatcggcgcttttc tttt- ggaagaaaatatagggaaaatggtacttgttaaaaattcggaatatttatac- aacatcatatgtttcacattgaaa ggggaggagaatc

atgaaacaacaaaaacggctttacgcccgattgctgacgctgttatttgcgctcatc t tcttgctgc ctcattctgcagcagcggcgGCAAATCTTAATGGGACG- CTGATGCAGTATTTTGAATGGTACATGCCCAATGACGGCCAA CAT- TGGAGGCGTTTGCAAAACGACTCGGCATATTTGGCTGAACAC ,GTAT- TACTGCCGTCTGGATTCCCCCGGCATATAA GGGAACGAG-

CCAAGCGGATGTGGGCTACGGTGCTTACGACCTTTATGATTTAGGGGAGTTTCATCA A AAAGGGACGGTTC GGACAAAGTACGGCACAAAAGGAGAGCTGCAATCTGCGATCAAAAGT-

CTTCATTCCCGCGACATTAACGTTTACGGGGAT GTGGTCATCAACCACAAAGGCGGCGC-

TGATGCGACCGAAGATGTAACCGCGGTTGAAGTCGATCCCGCTGACCGCAACCG CGTAATTTCAGGAGAACACCTAATTAAAGCCTGGACACATTT- TCATTTTCCGGGGCGCGGCAGCACATACAGCGATTTTA AATGGCAT- TGGTACCATTTTGACGGAACCGATTGGGACGAGTCCCGAAAGCTGAAC- CGCATCTATAAGTTTCAAGGAAAG GCTTGGGATTGGGAAGTTTCCAATGAA- AACGGCAACTATGATTATTTGATGTATGCCGACATCGATTATGACCATCCTGA

TGTCGCAGCAGAAATTAAGAGATGGGGCACTTGGTATGCCAATGAACTGCAAT- TGGACGGTTTCCGTCTTGATGCTGTCA AACACATTAAATTTTCTTTTTTGCGGGATTGGGTTAATC-

ATGTCAGGGAAAAAACGGGGAAGGAAATGTTTACGGTAGCT GAATATTGGCAGAAT- GACTTGGGCGCGCTGGAAAACTATTTGAACAAAACAAATTTTAATCATTCAGTGTTTG ACGTGCC GCTTCATTATCAGTTCCATGCTGCATCGACACAG- GGAGGCGGCTATGATATGAGGAAATTGCTGAACGGTACGGTCGTTT CCAAG- CATCCGTTGAAATCGGTTACATTTGTCGATAACCATGATACACAGCCGGGGCAA- TCGCTTGAGTCGACTGTCCAA ACATGGTTTAAGCCGCTTGCTTACGCTTTTAT- TCTCACAAGGGAATCTGGATACCCTCAGGTTTTCTACGGGGATATGTA CGGGA- CGAAAGGAGACTCCCAGCGCGAAATTCCTGCCTTGAAACACAAAATTGAACCGA- TCTTAAAAGCGAGAAAACAGT ATGCGTACGGAGCACAGCATGATTATTTCGAC- CACCATGACATTGTCGGCTGGACAAGGGAAGGCGACAGCTCGGTTGCA AATTCAGG- TTTGGCGGCATTAATAACAGACGGACCCGGTGGGGCAAAGCGAATGTATGTCGGCCGG CAAAACGCCGGTGA GACATGGCATGAC-

ATTACCGGAAACCGTTCGGAGCCGGTTGTCATCAATTCGGAAG- GCTGGGGAGAGTTTCACGTAAACG GCGGGTCGGTTTCAATTTATGTTCAAAGATAG

aagagcagagaggacggatttcctgaaggaaatccgtttttttatttt

SEQ ID No. 2

ANLNGTLMQYFEWYMPNDGQHWRRLQNDSAYLAEHGITAV WIPPAYKGTSQADVGYGAYDLYDLGEFHQKGTVRTKYGTK GELQSAIKSLHSRDINVYGDWINHKGGADATEDVTAVEV DPADRNRVISGEHLIKAWTHFHFPGRGSTYSDFKWHWYHF

DGTDWDESRKLNRIYKFQGKAWDWEVSNENGNYDYLMYAD IDYDHPDVAAEIKRWGTWYANELQLDGFRLDAVKHIKFSF LRDWVNHVREKTGKEMFTVAEYWQNDLGALENYLNKTNFN HSVFDVPLHYQFHAASTQGGGYDMRKLLNGTWSKHPLKS VTFVDNHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQ

VFYGDMYGTKGDSQREIPALKHKIEPILKARKQYAYGAQH DYFDHHDIVGWTREGDSSVANSGLAALITDGPGGAKRMYV GRQNAGETWHDITGNRSEPWINSEGWGEFHVNGGSVSIY VQR

SEQ ID No. 3 gccccgcacatacgaaaagactggctgaaaacattgagcctttgatga- ctgatgatttggctgaagaagtggatcgattg tttgagaaaagaaga-

agaccataaaaataccttgtctgtcatcagacagggtattttttatgctgt^ca- gactgtccgct gtgtaaaaataaggaataaaggggggttgttattattttact- gatatgtaaaatataatt tataagaaaatgagaggg agaggaaac

atgattcaaaaacgaaagcggacagtttcgttcagacttgtgcttatgtgcacg- ctgttatttgtcagttt gccgattacaaaaacatcagccGTAAATGGCACGC- TGATGCAGTATTTTGAATGGTATACGCCGAACGACGGCCAGCATT GGAAAC- GATTGCAGAATGATGCGGAACATTTATCGGATATCGGAATCACTGCCGTCTGGA- TTCCTCCCGCATACAAAGGA TTGAGCCAATCCGATAACGGATACGGACCTTAT- GATTTGTATGATTTAGGAGAATTCCAGCAAAAAGGGACGGTCAGAAC GAAATA- CGGCACAAAATCAGAGCTTCAAGATGCGATCGGCTCACTGCATTCCCGGAACGT- CCAAGTATACGGAGATGTGG TTTTGAATCATAAGGCTGGTGCTGATGCAACAG- AAGATGTAACTGCCGTCGAAGTCAATCCGGCCAATAGAAATCAGGAA ACTTCG- GAGGAATATCAAATCAAAGCGTGGACGGATTTTCGTTTTCCGGGCCGTGGAAAC- ACGTACAGTGATTTTAAATG GCATTGGTATCATTTCGACGGAGCGGACTGGGA- TGAATCCCGGAAGATCAGCCGCATCTTTAAGTTTCGTGGGGAAGG _^ A AAGCGT- GGGATTGGGAAGTATCAAGTGAAAACGGCAACTATGACTATTTAATGTATGCTG- ATGTTGACTACGACCACCCT GATGTCGTGGCAGAGACAAAAAAATGGGGTATC- TGGTATGCGAATGAACTGTCATTAGACGGCTTCCGTATTGATGCCGC CAAACA- TATTAAATTTTCAT ^m TCTGCGTGATTGGGTTCAGGCGGTCAGACAGGCGACGGG- AAAAGAAATGTTTACGGTTG CGGAGTATTGGCAGAATAATGCCGGGAAACTCG- AAAACTACTTGAATAAAACAAGCTTTAATCAATCCGTGTTTGATGTT CCGCTT- CATTTCAATTTACAGGCGGCTTCCTCACAAGGAGGCGGATATGATATGAGGCGT- TTGCTGGACGGTACCGTTGT GTCCAGGCATCCGGAAAAGGCGGTTACATTTGT- TGAAAATCATGACACACAGCCGGGACAGTCATTGGAATCGACAGTCC AAACTTGGT- TTAAACCGCTTGCATACGCCTTTATTTTGACAAGAGAATCCGGTTATCCTC- AGGTGTTCTATGGGGATATG TACGGGACAAAAGGGACATCGCCAAAGGAAATT- CCCTCACTGAAAGATAATATAGAGCCGATTTTAAAAGCGCGTAAGGA GTACGC- ATACGGGCCCCAGCACGATTATATTGACCACCCGGATGTGATCGGATGGACGAG- GGAAGGTGACAGCTCCGCCG CCAAATCAGGTTTGGCCGCTTTAATCACGGACGGAC- CCGGCGGATCAAAGC.GATGTATGCCGGCCTGAAAAATGCCGGC GAGACATGG- TATGACATAACGGGCAACCGTTCAGATACTGTAAAAATCGGATCTGACGGCTGG- GGAGAGTTTCATGTAAA CGATGGGTCCGTCTCCATTTATGTTCAGAAATAA

ggtaataaaaaaacacctccaagctgagtgcgggtatcagcttgga ggtgcgtt- tattttl".cagccgtatgacaaggtcggcatcaggtgtgacaaatacggtatgc- tggctgtcataggtgaca aatccgggttttgcgccgtttggctttttcacatg- tctgatttttgtataatcaacaggcacggagccggaatctttcgc cttggaaa-

aataagcggcgatcgtagctgcttccaatatggattgttcatcgggatcgctgc- ttttaatcacaacgtggg atcc

SEQ ID No. 4 VNGTLMQYFEWYTPNDGQHWKRLQNDAEHLSDIGITAVWI

PPAYKGLSQSDNGYGPYDLYDLGEFQQKGTVRTKYGTKSE

LQDAIGSLHSRNVQVYGDWLNHKAGADATEDVTAVEVNP

ANRNQETSEEYQIKAWTDFRFPGRGNTYSDFKWHWYHFDG

ADWDESRKISRIFKFRGEGKAWDWEVSSENGNYDYLMYAD VDYDHPDWAETKKWGIWYANELSLDGFRIDAAKHIKFSF

LRDWVQAVRQATGKEMFTVAEYWQNNAGKLENYLNKTSFN

QSVFDVPLHFNLQAASSQGGGYDMRRLLDGTWSRHPEKA

VTFVENHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQ

VFYGDMYGTKGTSPKEIPSLKDNIEPILKARKEYAYGPQH DYIDHPDVIGWTREGDSSAAKSGLAALITDGPGGSKRMYA

GLKNAGETWYDITGNRSDTVKIGSDGWGEFHVNDGSVSIY

SEQ ID No. 5 aaattcgatattgaaaacgattacaaataaaaattataataga- cgtaaacgttcgagggtttgctccctttttactcttt ttatgcaatcgtttcc- cttaattttttggaagccaaaccgtcgaatgtaacatttgattaagggggaag- ggcatt

gtgct aacgtttcaccgcatcattcgaaaaggatggatgttcctgctcgcgtt- tttgctcactgtctcgctgttctgcccaacag gacagcccgccaaggctGCCG- CACCGTTTAACGGCACCATGATGCAGTATTTTGAATGGTACTTGCCGGATGATG- GCACG TTATGGACCAAAGTGGCCAATGAAGCCAACAACTTATCCAG- CCTTGGCATCACCGCTCTTTGGCTGCCGCCCGCTTACAA AGGAACAAGCCGCA- GCGACGTAGGGTACGGAGTATACGACTTGTATGACCTCGGCGAATTCAATCAAA- AAGGGACCGTCC GCACAAAATACGGAACAAAAGCTCAATATCTTCAAGCCAT- TCAAGCCGCCCACGCCGCTGGAATGCAAGTGTACGCCGAT GTCGTGTTCGAC- CATAAAGGCGGCGCTGACGGCACGGAATGGGTGGACGCCGTCGAAGTCAATCCG- TCCGACCGCAACCA AGAAATCTCGGGCACCTATCAAATCCAAGCATGGACGAAATT- TGATTTTCCCGGGCGGGGCAACACCTACTCCAGCTTTA AGTGGCGCTGGTACCAT- TTTGACGGCGTTGATTGGGACGAAAGCCGAAAATTGAGCCGCATTTACAAAT-

TCCGCGGCATC GGCAAAGCGTGGGATTGGGAAGTAGACACGGAAAACGGAAAC- TATGACTACTTAATGTATGCCGACCTTGATATGGATCA TCCCGAAGTCGTGAC- CGAGCTGAAAAACTGGGGGAAATGGTATGTCAACACAACGAACATTGATGGGTT-

CCGGCTTGATG CCGTCAAGCATATTAAGTTCAGTTTTTTTCCTGATTGGTTG - CGTATGTGCGTTCTCAGACTGGCAAGCCGCTATTTACC GTCGGGGAATATTGG- AGCTATGACATCAACAAGTTGCACAATTACATTACGAAAACAGACGGAACGATG- TCTTTGTTTGA TGCCCCGTTACACAACAAATTTTATACCGCTTCCAAATCAGG- GGGCGCATTTGATATGCGCACGTTAATGACCAATACTC TCATGAAAGATCAAC- CGACATTGGCCGTCACCTTCGTTGATAATCATGACACCGAACCCGGCCAAGCGC- TGCAGTCATGG GTCGACCCATGGTTCAAACCGTTGGCTTACGCCTTTATTCTA- ACTCGGCAGGAAGGATACCCGTGCGTCTTTTATGGTGA CTATTATGGCATTCC- ACAATATAACATTCCTTCGCTGAAAAGCAAAATCGATCCGCTCCTCATCGCGCG- CAGGGATTATG CTTACGGAACGCAACATGATTATCTTGATCACTCCGACATCA- TCGGGTGGAC&AGGGAAGGGGGCACTGAAAAACCAGGA TCCGGACTGGCCGCA- CTGATCACC ..TGGGCCGGGAGGAAGCAAATGGATGTACGTTGGCAAACAACAC- GCTGGAAAAGT GTTCTATGACCTTACCGGCAACCGGAGTGACACCGTCACCAT- CAACAGTGATGGATGGGGGGAATTCAAAGTCAATGGCG GTTCGGTTTCGGTTT- GGGTTCCTAGAAAAACGACCGTTTCTACCATCGCTCGGCCGATCACAACCCGAC-

CGTGGACTGGT GAATTCGTCCGTTGGACCGAACCACGGTTGGTGGCATGGCCTTGA

tgcctgcga

SEQ ID No. 6

AAPFNGTMMQYFEWYLPDDGTLWTKVANEANNLSSLGITA LWLPPAYKGTSRSDVGYGVYDLYDLGEFNQKGTVRTKYGT KAQYLQAIQAAHAAGMQVYADWFDHKGGADGTEWVDAVE VNPSDRNQEISGTYQIQAWTKFDFPGRGNTYSSFKWRWYH FDGVDWDESRKLSRIYKFRGIGKAWDWEVDTENGNYDYLM

YADLDMDHPEWTELKNWGKWYVNTTNIDGFRLDAVKHIK FSFFPDWLSYVRSQTGKPLFTVGEYWSYDINKLHNYITKT DGTMSLFDAPLHNKFYTASKSGGAFDMRTLMTNTLMKDQP TLAVTFVDNHDTEPGQALQSWVDPWFKPLAYAFILTRQEG YPCVFYGDYYGIPQYNIPSLKSKIDPLLIARRDYAYGTQH

DYLDHSDIIGWTREGGTEKPGSGLAALITDGPGGSKWMYV GKQHAGKVFYDLTGNRSDTVTINSDGWGEFKVNGGSVSVW VPRKTTVSTIARPITTRPWTGEFVRWTEPRLVAW

SEQ ID No. 7

1 ATPADWRSQS IYFLLTDRFA RTDGSTTATC 31 NTADQKYCGG TWQGIIDKLD YIQGMGFTAl

WITPVTAQLP QTTAYGDAYH GYWQQDIYSL NENYGTADDL KALSSALHER GMYLMVDWA NHMGYDGAGS SVDYSVFKPF SSQDYFHPFC FIQNYEDQTQ VEDCWLGDNT VSLPDLDTTK DWKNEWYDW VGSLVSNYSI DGLRIDTVKH VQKDFWPGYN KAAGVYCIGE VLDGDPAYTC PYQNVMDGVL NYPIYYPLLN AFKSTSGSMD DLYNMINTVK SDCPDSTLLG TFVENHDNPR FASYTNDIAL AKNVAAFIIL NDGIPIIYAG QEQHYAGGND PANREATWLS GYPTDSELYK LIASANAIRN YAISKDTGFV TYKNWPIYKD DITIAMRKGT DGSQIVTILS NKGASGDSYT LSLSGAGYTA GQQLTEVIGC TTVTVGSDGN VPVPMAGGLP RVLYPTEKLA GSKICSSS