FI85986B - Enzyme product and method for production thereof - Google Patents

Description

85986

Enzyme preparation and method for its production

The present invention relates to an enzyme preparation according to the preamble of claim 1.

The invention also relates to a method for producing it according to the preamble of claim 3.

With regard to the state of the art, reference is made to the following publications: 1. Honda, H. et al., System. Appl. Microbiol. 8 (1986), 152-157 2. Honda, H. et al., Can. J. Microbiol. 31 (1985), 538-542 3. Esteban et al., Can. J. Microbiol. 28 (1982), 733-739 4. U.S. Patent 3,826,715 (1974), Horikoshi et al.

5. Poutanen, K. and Puls, J., Appl. Microb. Biotechnol. 28 (1988), 425-432

By alkaline xylanase is meant a xylan-degrading enzyme that functions in the alkaline pH range. Such enzymes can be used to facilitate the removal of residual lignin from alkaline cellulosic pulp.

It is known that alkaline bacterial strains, such as those belonging to the genus Bacillus, can produce xylanases that operate in the alkaline pH range. In general, however, the xylanase activity produced by these bacteria is so low that they are inefficient and expensive to produce the enzyme. The first two references [1, 2] describe the production of xylanase by the strain Bacillus sp. Well. C-125. According to the data given in the publications, the xylanase activity produced was 0.8 to 1 U / ml, which corresponds to an activity of 90 to 100 nkat / ml, calculated as catalysts. Similarly, Esteba and colleagues [3] have produced xylanase with Bacillus drculans strain WL-12 at xylanase activity of about 2 to 20 nkat / ml, with an optimum pH of 5.5 ___ 7.

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The object of the present invention is to obviate the drawbacks associated with the prior art and to provide an enzyme preparation having a high xylanase activity.

The invention is based on the surprising finding that by growing a certain Bacillus circulans strain, namely the Bacillus circulans strain deposited under ATCC No. 21783, on a xylan-containing medium at alkaline pH, a culture medium with high xylanase activity is obtained. Comparing the results reported in Honda et al., The invention achieves up to 50-fold activities. The difference in activity is even greater, i.e., £ 200-fold, relative to the xylanase produced by another B. circulans strain (Esteban et al., [3]).

B. circulans ATCC 21783 has previously been used to produce amylase [4], the main parameters of said bacterial strain are also described in detail in the reference. However, no data are available in the literature on its suitability for β-xylanase production.

More specifically, the solution according to the invention is mainly characterized by what is set forth in the characterizing part of claim 1.

The method according to the invention, in turn, is characterized by what is set forth in the characterizing part of claim 3.

As used in this application, the term "enzyme preparation" means any product that contains at least one enzyme. Thus, the enzyme preparation may be, for example, a culture medium containing the enzyme or enzymes, an isolated enzyme or a mixture of two or more enzymes.

By culturing the microorganism Bacillus circulans strain ATCC 21783 under temporal conditions, a culture medium containing at least two β-xylanase enzymes with isoelectric points (pI) of about 8.8 and about 3,85986 8.4-8.5 is obtained. The molecular weights of the enzymes corresponding to these isoelectric points have been estimated by electrophoresis to be 28,000 and 28,000 or 25,000, respectively. The enzyme preparation according to the invention contains at least one of the above-mentioned enzymes. Preferably, it comprises a culture medium containing both enzymes.

The β-xylanase activity of the culture medium prepared by the method is preferably at least about 2000 nkat / ml, preferably at least about 3000. As can be seen from the following working examples, we have provided growth solutions having an activity of up to 4500-5000 nkat / ml.

In addition to the high xylanase activity, the culture medium of the B. drculans strain we use also contains other enzymes, mainly xylan side chains or enzymes which hydrolyze other hemicellulose components. In addition to the xylanase in the solution, the activities of the main enzymes are as follows: β-xylosidase 0 to 100 nkat / ml, α-arabinosidase 0 to 50 nkat / ml, acetylxylan esterase 0 to 10 nkat / ml, α-glucuronidase 0 to 20 nkat / ml and β-mannanase 0 to 50 nkat / ml.

According to the invention, B. drculans ATCC 21783 is grown in a culture medium containing xylan compounds as a carbon source. The substrate is made alkaline, i.e. its pH is adjusted to about 7 to about 11. Preferably, its pH is adjusted to about 8 to about 9.5. Aqueous solutions of suitable alkaline compounds, preferably sodium carbonate, can be used to control the alkalinity of the medium. Of the xylan sources, hardwood decylans, especially birch and beech xylan, are particularly suitable. The cultivation can be carried out, for example, as a shaking culture or in a fermentor. Adjusting the pH of the culture medium is easier to perform in the fermentor.

In a preferred embodiment of the invention, birch xylan is used as the carbon source in the shaking culture.

The culture medium contains, for example, peptone or corn steepwater as a nitrogen source. Other alternative nitrogen sources include urea, meat extract, yeast extract and casein hydrolysates. In addition, the culture medium may contain nutrient salts and trace elements. Cations include calcium, magnesium, manganese, ammonium and iron ions, and anions such as phosphate and sulfate chloride ions.

The growth is normally performed at a temperature of about 20 ° C to about 50 ° C, with a growth time of about 12 to about 96 hours. However, these conditions may be waived if necessary.

The invention provides considerable advantages. Thus, we have surprisingly been able to find that the enzyme preparation according to the invention obtained has, in addition to its high activity, also a wide range of activity pH. Thus, it dissolves xylan in a still alkaline pH range of £ 9.

The xylanase growth solution according to the invention is very well suited for use specifically in pulp bleaching, e.g. to reduce the use of bleaching chemicals such as chlorine dioxide. At the same time, the residual chemical amount of the pulp can be effectively reduced. Because the cellulase activity of the culture medium is very low (£ 0.05 FPU), it can be used as such for pulp treatment without having a cellulose-degrading - and thus degrading - pulp quality effect. The β-xylosidase activity of the culture medium is reasonably high, which is why at pH 7 the main product of xylan hydrolysis is xylose. At higher pH values, less monosaccharide is formed, apparently due to the low pH stability of β-xylosidase.

If desired, the β-xylanase enzymes prepared can be isolated and purified by methods known per se.

The invention will now be examined in more detail by means of a few non-limiting examples. Examples 1-3 5 85986 describe the preparation of a culture medium containing β-xylanase and Example 4 describes the xylan-dissolving effect of the culture medium.

The xylanase activity of the prepared media was determined using the method described by Poutanen and Puis [5]. The substrate is 1% Larchwood xylan (supplied by Sigma), 50 mM sodium citrate buffer, pH 6.5.

Example 1. Culturing of Bacillus circulans strain ATCC 21783 as a shake crop

The strain of Bacillus circulans was intended to be shaken in a 50 ml solution volume in a 250 ml vessel. The growth temperature was 28®C. The medium contained birch xylan 10 g / l, yeast extract 5 g / l, peptone 2.5 g / l, K2HPO4 0.5 g / l, MgSO4-7H2O 0.125 g / l, (NH4) 2SO4 0.25 g / l, NaCl 0.125 g / l, CaCl 2 · 2H 2 O, 0.025 g / l, MnSO 4 H 2 O 0.0025 g / l, FeSO 4 - 7H 2 O 0.0025 g / l.

Separately sterilized 10% Na 2 CO 3 solution 2 ml / 50 ml was added to the autoclaved medium. Used as an inoculum-. . overnight Tryptic Soy.

After three days, the xylanase activity in the centrifuged medium was 3000 nkat / ml.

The initial pH was about 9.5 and dropped to about 7 during growth.

Example 2. Growth of Bacillus circulans strain ATCC 21783 in a fermentor

Bacillus circulans strains were grown in a laboratory fermentor. The medium contained 20 g / l of beech xylan, 20 ml / l of corn steep water, 0.54 g / l of K 2 HPO 4, 0.15 g / l of MgSO 4 * 7H 2 O. The growth temperature was 30 ° C and the pH was maintained between 8 and 8.4. The initial pH was adjusted with sodium carbonate solution. During the growth, the pH was automatically adjusted by the addition of ammonium hydroxide.

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After three days of culture, the xylanase activity in the culture medium was 4500 nkat / ml.

Example 3. Culturing of Bacillus circulans strain ATCC 21783

The Bacillus circulans strain was grown in a laboratory fermentor under the culture conditions of Example 2 in terms of temperature and pH. Beech xylan 20 g / l was used as the carbon source. The other components of the medium were the same as in Example 1. After three days of growth, the xylanase activity in the centrifuged medium was 4000 nkat / ml.

In addition to xylanase activity, the medium was analyzed for the activities of other enzymes: β-xylosidase 40 nkat / ml, α-arabinosidase 10 nkat / ml, acetylxylan esterase 2 nkat / ml, α-glucuronidase 5 nkat / ml and β-mannanase 10 nkat / ml ml.

Example 4. Hydrolysol of xylan

The deacetylated xylan was hydrolyzed with the enzyme preparation of Example 3. The pH of the hydrolysis solution was adjusted to 6, 7, 8, 9 and 10 with citrate-phosphate buffer. Xylanase was used at 100 nkat / g xylan. After the treatment, the carbohydrates had dissolved in the xylan as follows: pH dissolved carbohydrates (% of the original xylan) 6 15.5 7 18.6 8 14.1: 9 13.9 10 8.5

Claims (12)

Enzyme preparation according to Claim 1, characterized in that it is in the form of a culture medium which contains both β-xylanase enzymes. 3. A method for producing a β-xylanase-containing enzyme preparation which is substantially free of cellulase activity, characterized in that the strain of the microorganism Bacillus clrculans ATCC 21783 is grown for about 12 to about 96 hours in a medium containing a carbon source and a nitrogen source and minera The pH is about 7 to about 1 ° C and the temperature is about 20 ° C to about 50 ° C, and if desired, the enzyme (s) having xylanase activity is isolated. A method according to claim 3, characterized in that the pH of the culture medium is maintained at a value of about 8 to about 9.5. Method according to Claim 3, characterized in that hardwood silane is used as the carbon source. Process according to one of Claims 3 to 5, carried out as a shaking culture, characterized in that birch xylan is used as the carbon source. 8 85986 Use of an enzyme preparation produced according to claim 3 in the bleaching of cellulose pulp. 9 85986 1. β-xylanashaltig enzyme products, which are derived from the genome of the genome or atoms of the microorganism Bacillus drculans as an alkaline-derived medium, ca. 28,000 resp. ca. 25,000 ... ca. 28,000 and the stem isoelectric bunker is 8.8 respectful ca 8.4 ... 8.5, och - we are tired of the cellular activity. 2. An enzyme product according to claim 1, which comprises a form of a compound having the characteristic β-xylan enzyme. 3. A compound for the production of β-xylan acid enzyme products, which is preferably free of cellulosic activity, which is the subject of ATCC 21783, is a microorganism of Bacillus drculans, which is circulating l2 ... circular 96, The pH of the same mineral salt and stem is adjusted to about 7 ... about 11 and the temperature is about 20 "to about 50 ° C, about 50 ° C, with the presence of enzymes with xylan activity. 4. For the purposes of claim 3, the pH of the chewing medium is adjusted to about 8 to about 9.5. 5. The first patent claim 3, which relates to a third column, provides a method. 6. The first of these claims is set out in claims 1-5, the first of which is indicated by the invention. 10 85986 7. The preparation of an enzyme product in the form of a compound 3 according to cellulosic composition.