WO1991019794A1

WO1991019794A1 - Activation of polysaccharide hydrolase

Info

Publication number: WO1991019794A1
Application number: PCT/DK1991/000161
Authority: WO
Inventors: Hans Adam Hagen; Thomas Videbaek; Tom Pickard; Lars Overvad Asferg; Henry Alexander Hamilton; Pedro Luiz Fernandes
Original assignee: Novo Nordisk A/S
Priority date: 1990-06-14
Filing date: 1991-06-14
Publication date: 1991-12-26
Also published as: AU8076791A; KR100237970B1; DK145090D0

Abstract

Addition of ethoxylated C12-C20 alcohol or alkyl-phenol with 10-100 ethoxy groups increases the effect of polysaccharide hydrolases. Practical applications include textile treatment with cellulase and desizing of textile with α-amylase.

Description

ACTIVATION OF POLYSACCHARIDE HYDROLASE

TECHNICAL FIELD

This invention relates to a method of increasing the effect of a polysaccharide hydrolase.

BACKGROUND ART

Polysaccharide hydrolases are enzymes classified in group EC 3.2.1 and having a polysaccharide as substrate. Examples are endo-glucanases such as Q.-amylase and cellulase, which promote endo-hydrolysis of the glucans starch and cellulose, respectively. Many industrial uses of polysaccharide hydrolases are known. Thus, it is known to use α-amylase for desizing, i.e. removal of starch size applied during textile production.

It is known to use cellulase to treat cellulosic textiles such as cotton to obtain such effects as softening and cleaning (see US 4,435,307), colour clarification (see EP 220,016), increasing water absorption (JP-B 52-48236) or formation of localized variation of colour to create "stone-washed" appearance in denim (EP 307,564). It is also known to use cellulase in the paper and pulp industry for deinking of waste paper (JP-A 59-9299, JP-A 63-59494) or for improvement of the draining properties of pulp (EP 262,040, EP 351,655).

STATEMENT OF THE INVENTION

We have now surprisingly found that the activity of polysaccharide hydrolases can be enhanced by incorporation of ethoxylated C^-C^ alcohol or alkyl- phenol with 10-100 ethoxy groups. Such ethoxylates are commonly used as dispersants, in contrast to similar ethoxylates with fewer ethoxy groups commonly used as non-ionic surfactants. It is known to use ethoxylated dispersant as a waxy material for prilling of enzymes (DE 2,060,095) or for coating of enzyme granules (CA 974,907), but an activating effect of this compound on the enzyme has not been reported.

Accordingly, the invention provides a method of increasing the effect of a polysaccharide hydrolase, characterized by incorporating as an enzyme activator a compound of the general formula:

R - O - (CH₂ CH₂ 0)_n H whereby R is an aliphatic alkyl or alkyl-phenyl with 12-20 carbon atoms, and n is 10- 100 into the reaction system in dissolved form. The invention also provides polysaccharide hydrolase preparations comprising said ethoxylated compound, a detergent composition comprising said enzyme and activator and processes using the enzyme and activator in combination.

DETAILED DESCRIPTION OF THE INVENTION

Polysaccharide hydrolase The invention is applicable to activation of a polysaccharide hydrolase, i.e. an enzyme in group EC 3.2.1 according to Enzyme Nomenclature having poly¬ saccharide as substrate. It is particularly applicable to activation of an endo- glucanase, i.e. an enzyme that effects endo-hydrolysis of a glucan (a polymer of glucose units), e.g. a cellulase or an α-amylase. For reasons of economy, microbial enzymes are generally preferred, either of fungal or microbial origin. Enzymes with either acidic, near-neutral or alkaline pH optimum may be chosen, depending on the intended conditions of use.

Cellulase (EC 3.2.1.4) may be derived from Aspergillus (particularly A niger), Trichoderma (particularly T. viride, T. reesei and T. koningii), Humicola (particularly H. insolens, see US 4,435,307) and alkalophilic Bacillus (US 3,844,890). Cellulase may also be prepared .according to PCT/DK 91/00123. Examples of commercially available cellulases are Denimax™ and Cellusoft™ 1.5 L, products of Novo Nordisk A/S. α-amylase (EC 3.2.1.1) may be derived from Bacillus, e.g. B. amyloliquefaciens, B. licheniformis or B. stearothermophilus.

Enzvme activator

The compound used in the invention to increase enzyme activity may be a highly ethoxylated fatty alcohol (HEFA); the alcohol is preferably a linear fatty alcohol, e.g. with 16-18 carbon atoms. In the following, HEFA-n indicates HEFA with ^16-18 alcohol with n ethoxy groups.

Alternatively, the activator may be an alkylphenol ethoxylate; the alkyl group has 6-14 carbon atoms, e.g. 8-10, and is preferably linear. An example is nonyl phenol ethoxylate (NPE or NPE-n, where n is the number of ethoxy groups).

Some examples of commercial products are: Berol Wash (HEFA-12),

Berol 08 (HEFA-80), products of Berol Nobel, Sweden. Lutensol AT 25, AT 50 and

AT 80 (HEFA-25, -50 and -80, respectively), products of BASF. Synperonic NP 30

(NPE-30), product of ICI. The concentration of the activator during use is 0.01-4 g/l. In the case of cellulase it is preferably above 0.01 g/l, most preferably above 0.1 g/l, and is preferably below 1 g/l, more preferably below 0.5 g/l, e.g. below 0.3 g/l. In the case of α-amylase it is preferably 0.5-4 g/l.

The amount of the activator preferably corresponds to 0.5-30 g per kg of the polysaccharide-containing material (e.g. textile) to be treated. In an enzymatic detergent composition according to the invention, the amount of activator is preferably 2-10%.

The amount of activator in an enzyme preparation according to the invention is generally 5-40%. The ratio of activator to enzyme activity is preferably 0.2-2.0 g per 1000 EGU (unit defined below) of cellulase activity or 1-20 g per 1000 KNU of α-amylase activity (1 KNU = 1000 NU, see EP 252,730).

The enzyme may be further activated by using polyvinyl pyrrolidone (PVP) together with the ethoxylated compound, the ratio between the two preferably being 0.5-2. The enzyme activator may be incorporated into an enzyme preparation, or it may be added to the process separately from the enzyme. Enzyme preparation

The enzyme preparation of the invention may be solid or liquid, particularly a non-dusting granulate or a stabilized liquid.

Enzyme granulates may be made by known methods, e.g. by extrusion or drum granulation according to CA 974,907, US 4,106,991, US 4,435,307 or US 4,661 ,452. The amount of the activator is preferably above 1% by weight, preferably above 2%, particularly above 5%, and generally below 40%.

Alternatively, the activator may be incorporated by coating a preformed enzyme granulate. The amount of the activator in the coating should make up at least 1% by weight of the coated particles. The coating may be applied by known methods e.g. as described in British Patent No. 1 ,362,365, page 1 , line 82 to page 2, line 34, and British Patent application No. 34973/73 and 10842/74 corresponding to Belgium patent No. 146,802. The coating material may comprise, in addition to the ethoxylated compound, other waxy coating materials and particulate material, e.g. magnesium silicate, alumino silicates, CaSOψ CaCOs, bentonite, zeolite or TiO^

A liquid enzyme preparation according to the invention may be stabilized against microbial infection by incorporation of a stabilizing agent known in the art. Examples are inorganic salts (such as NaCI), sugars (such as sucrose and glucose), polyols (such as glycerol, propylene glycol and sorbitol) and alcohols (such as ethanol and iso-propanol). These are generally effective in amounts above 10%, especially above 20%. Another example is organic acids (such as benzoic, sorbic, propionic, lactic and formic), which are generally effective in amounts of 0.01 - 2% at low pH (below 5). Other examples of stabilizing agents are antioxidants (such as sulphur dioxide), 1 ,2-benz-iso-thiazolin-3-one (BIT) and parabens. Some of these may also serve to improve enzyme stability. Further additives may be incorporated to improve enzyme stability or physical stability of the enzyme preparation. The choice depends on the type of enzyme. Examples are CaC_2 (e.g. 0.1-0.5%), a protease inhibitor (such as borate and formate) or 0.5-5% of lower alcohol (such as ethanol or iso-propanol, preferably_used together with a larger amount of a polyol). The amount of the dispersant in the liquid enzyme preparation is preferably 1-10% by weight. A cellulase preparation of the invention will typically have a cellulase activity in the range 50-5000 EGU/g. In the treatment of textiles it will typically be used at a dosage of 2-200 g/kg of textile. An α-amylase preparation according to the invention typically has activity in the range 20-200 KNU/g.

Detergent

Detergent according to the invention may be prepared by incorporating the enzyme activator into known enzymatic detergent compositions. See e.g. US

4,435,307 and WO 89/04862. The detergent will typically contain 10-1000 EGU/g of cellulase; it may be in particulate or liquid form, and will generally contain 1-60% of anionic and/or non-ionic detergent.

Use of polysaccharide hydrolase

Polysaccharide hydrolase activated according to the invention can be used in any process involving treatment of material that contains the polysaccharide on which the enzyme is active. Thμs, α-amylase is used to treat starch-containing material, e.g. in desizing of starch-sized textile, preferably with 1 -4 g/l of the activator. Cellulase is used to treat cellulose-containing material, typically at a concentration corresponding to an activity of 20-5000 EGU/I, in the case of textile treatment generally 50-2000 EGU/I.

The cellulose-containing material may be a textile, such as cotton, denim, flax or polyester/cotton, typically using 0.05-3 g activator/kg textile. Thus, the ethoxylated dispersant may be incorporated into known processes to enhance the cellulase activity, e.g. for softening and cleaning of textiles (US 4,435,307), colour clarification (EP 220,016), increasing water absorption (JP-B 52-48236) or formation of localized variation of colour, e.g. in blue denim, to create "stone-washed" appearance (EP 307,564).

Finishing of blue denim may be made at slightly acid conditions, e.g. at liquor/fabric ratio 5-10 l/kg, pH 4.5-5.5, 45-55°C for 45-90 minutes, using Trichoderma cellulase at a dosage of 2,000-4,000 EGU/kg of fabric (EGU activity unit, see below). Alternatively, the finishing of blue denim may be made at near-neutral conditions, e.g. at liquor/fabric ratio about 10 l/kg, pH 6-8, 50-60°C for 45-120 minutes, using Humicola cellulase at a dosage of 1 ,000-3,000 EGU/kg of fabric.

Other cellulose-containing materials can also be treated. Thus, the process of the invention may be used in the paper and pulp industry for deinking of waste paper or for improvement of pulp drainage.

Determination of cellulase activity (EGU)

A substrate solution is prepared, containing 34.0 g/l CMC (Hercules 7 LFD) in 0.1 M phosphate buffer at pH 6.0. The enzyme sample to be analyzed is dissolved in the same buffer. 5 ml substrate solution and 0.15 ml enzyme solution are mixed and transferred to a vibration viscosimeter (e.g. MIVI 3000 from Sofraser, France), thermostated at 40°C. One Endo-Glucanase Unit (EGU) is defined as the amount of enzyme that reduces the viscosity to one half under these conditions. The amount of enzyme sample should be adjusted to provide 0.01-0.02 EGU/ml in the reaction mixture.

EXAMPLE 1

Hydrolysis of carboxymethyl cellulose bv cellulase

The EGU method described above for cellulase activity was applied to cellulases derived from Humicola insolens and Trichoderma reesei except that HEFA-80 (Berol 08) and PVP Albigen A, product of BASF) were added to the buffers used to dilute the enzyme samples, as indicated below. The below results indicate the concentration of HEFA or PVP in the reaction mixture and the measured cellulase activity in percent of the reference.

It is seen that both cellulases are activated by addition of as little as 0.01 g/l of HEFA. Maximum activation is obtained with about 0.5 g/l of HEFA. The activity is further increased by adding PVP together with HEFA.

EXAMPLE 2

Effect of activator concentration in washing with cellulase

Pre-washed, desized denim fabric was cut into swatches of the size 5x6 cm and 4x5 cm, then folded and sewn together. Using a laboratory washing machine (Laundrometer, Atlas LP1) with 20 cups, 7.5 g of the sewn denim and five metal balls were added to each cup. Two white cotton/polyester (50/50%) swatches (8x8 cm) were added in order Jo measure the redeposition of the indigo colour on the fabric (backstaining). 150 ml ofbuffer containing 300-700 EGU/I of cellulase and dispersant as indicated was then added to each cup. Cellulase from Trichoderma reesei was used with 60 mM acetate buffer (pH 5), and Humicola insolens cellulase was used with 60 mM phosphate buffer (pH 6). The cups were then tumbled at 55°C for 90 minutes. Each sample was run in triplicate.

After the tumbling, 80 ml of the liquid was poured off and filtered through a weighed Whatman G glass fibre filter. The filter was left in the funnel for 2 hours to drain off liquid. It was dried for 1 hour at 90°C and then weighed. The amount of lint trapped on the filter from the washing liquid was then measured as the weight gain of the filter.

Blanks, with no enzyme, were included. After deducting the blank lint formation, the washing activity of the enzyme was expressed as lint formation per EGU of cellulase activity (mg lint/EGU).

Similarly, the washing activity of the enzyme was determined without any dispersant present; this was taken as 100%, and the washing performance with dispersant was expressed as the washing activity with dispersant in percent of the activity without dispersant. A series of experiments was made to illustrate the effect of dispersant concentration, using HEFA-80:

The results indicate that the maximum activation of washing effect is obtained with approximately 0.25 g HEFA/I. The washing performance of the Trichoderma enzyme is generally more enhanced than the Humicola enzyme. The results show that it is possible to increase the performance of Trichoderma cellulase by 69% by adding 0.25 g/l of dispersant compared to 36% for Humicola cellulase. The trial with only dispersant present (no enzyme) shows that the dispersant itself gives a negligible contribution to the washing performance. EXAMPLE 3

Effect of activator type in washing with cellulase

A series of experiments was made as in Example 2, using different types of dispersant, as indicated.

The results indicate no significant difference in washing effect performance between the different dispersants. Comparison with Example 2 shows that the concentration dependent action of the other types of HEFA is similar to HEFA-80.

The combination of 0.1 g/l HEFA-80 and 0.1 g/l PVP is seen to be particularly effective in increasing the washing performance of Humicola cellulase. EXAMPLE 4

Granulated cellulase preparation

A cellulase granulate with HEFA in the core is prepared by the procedure of Example XVIII of US 4,106,991 , replacing ALCALASE^® concentrate with cellulase concentrate, and replacing BEROL^® 067 with dispersant according to the invention.

EXAMPLE 5

Coated cellulase preparation

Cellulase granulate is coated with dispersant as in Example XXII of US 4,106,991, replacing PEG 1500 with dispersant according to the invention.

EXAMPLE 6

Stabilized liquid cellulase preparation

A liquid is formulated as follows:

The preparation has cellulase activity 100-120 EGU/ml and pH 4.5 - 7.5. EXAMPLE 7

Cellulase-containino detergent

10% of HEFA-80 is added to composition A or B (cellulase-containing detergent) of the Experimental Part of WO 89/04862.

EXAMPLE 8

Desizing with α-amylase

Desizing test were carried out using α-amylase (Aquazym 30 L, derived from S. amyloliquefaciens, activity 30 KNU/g, product of Novo Nordisk A/S) and ethoxylated alcohol (Lutensol AT 80). The procedure was as follows. Enzyme solution: 10 g Aquazym 30 L, 3 g NaCI, 1 g wetting agent (Umectol DSS) and Lutensol (as indicated below). Complete to

1000 ml.

Desizing: Cut standard sized raw cotton cloth into strips of approx. 5 g. Weigh cloth strips and place in numbered beakers (250 ml). Add 10 ml enzyme solution for each g of cloth in beaker. Place in

70°C water bath during minimum 30 min and maximum 60 min with occasional mixing. Remove all samples at the same time elapsed and wash in water at 90°C. Dry strips and press with iron.

Iodine test "Tegewa": Place fabric for 1 min in iodine solution (10 g Kl, 0.65 l₂, 800 ml H₂0, 95% ethanol to 1000 ml).Rinse in cold water.

Dab with filter paper. Compare immediately with TEGEWA scale and note value (Violet scale for assessing the desizing degree of starch containing fabrics - Edition Oct 1981 , elaborated by: Verband der Textilhilfsmittel-, Lederhilfsmittel-, Gerbstoff- und Waschrohstoffindustrie e.V. Verband Tegewa, KarlstraBe 21 , D-6000 Frankfurt am Main).

The results were as follows (the higher Tegewa number indicates more complete desizing): % Lutensol Tegewa value

0 5

0.1% 5

0.2% 7

0.3% 6 0.4% 6

The results show that the addition of ethoxylated alcohol increases the desizing activity of α-amylase. The maximum effect is seen with 0.2% activator.

EXAMPLE 9

Desizing with α-amylase

A plant-scale test was done to try and find out if the synergistic effect of adding a small quantity of highly ethoxylated fatty alcohol (Lutensol AT 80) to Aquazym 30 is a constant effect. During a period in January and February 1991 desizing trials were carried out using the TEGEWA Method (Edition October 1981) and alternately a method of visual qualitative comparison, and the results were accumulated.

Results of tests with Tegewa ratings (the higher Tegewa number indicates more complete desizing):

6 Jan 18 Jan 21 Jan 22 Jan 23 Jan 20 Feb

Aq.30L

Aq.30L + 0.2% Lut. 6.5 ^* 6

The results of visual comparison were that Aquazym 30 L + 0.2% Lutensol had a better desizing effect than the Aquazym 30 L without Lutensol. The conclusion was that the addition of 0.2% Lutensol to α-amylase has a synergistic effect in textile desizing. In this specific case the desizing effect with 0.2% highly ethoxylated alcohol (Lutensol) was equivalent to α-amylase with 33% higher activity.

Claims

1. A method of increasing the effect of a polysaccharide hydrolase, characterized by incorporating a compound of the general formula:

R - O - (CH₂ CH₂ 0)_n H whereby R is an aliphatic alkyl or alkyl-phenyl with 12-20 carbon atoms, and n is 10- 100 into the reaction system in dissolved form.

2. A polysaccharide hydrolase preparation, characterized by comprising 5-40% of the ethoxylated compound defined in Claim 1.

3. An endo-glucanase preparation according to Claim 2, preferably a cellulase or α-amylase preparation.

4. A preparation according to Claim 3 of cellulase derived from Tricoderma or Humicola.

5. A preparation according to Claim 3 of α-amylase derived from Bacillus, preferably from B. amyloliquefaciens, B. licheniformis or β. stearothermophilus.

6. A cellulase preparation, characterized by comprising the ethoxylated compound defined in Claim 1, in an amount of 0.2-2.0 g per 1000 EGU of cellulase activity.

7. An α-amylase preparation, characterized by comprising the ethoxylated compound defined in Claim 1, preferably in an amount of 1-20 g per 1000 KNU of α-amylase activity.

8. A Trichoderma cellulase preparation, characterized by comprising at least 2% of the ethoxylated compound defined in Claim 1.

9. A liquid polysaccharide hydrolase preparation, characterized by comprising at least 2% of the ethoxylated compound defined in Claim 1.

10. A particulate polysaccharide hydrolase preparation, characterized by having a coating comprising the ethoxylated compound defined in Claim 1, the

5 amount of said compound in the coating being at least 5% by weight of the preparation.

11. A polysaccharide hydrolase granulate prepared by extrusion or drum granulation, characterized by comprising at least 5% of the ethoxylated compound defined in Claim 1.

10 12. A detergent composition containing polysaccharide hydrolase, characterized by further comprising 2-10% of the ethoxylated compound defined in Claim 1.

13. Use of a polysaccharide preparation according to any of claims 2-11 in a process involving treatment of material containing polysaccharide substrate.

15 14. A process involving treatment by a polysaccharide hydrolase of material containing polysaccharide substrate, characterized in that said enzyme is used in an aqueous solution containing 0.01-4 g/l of the ethoxylated compound defined in Claim 1.

15. A process according to Claim 13 or 14 for desizing of starch-sized 0 textile with α-amylase, whereby the concentration of the ethoxylated compound is preferably 0.5-4 g/l.

16. A process according to-Claim 13 or 14 involving treatment of cellulosic textile with cellulase, whereby the concentration of the ethoxylated compound is preferably 0.01-0.3 g/l.