VIIKARI LIISA (FI)
KANTELINEN ANNE (FI)
LINKO MATTI (FI)
RANUA MARJATTA (FI)
SUNDQUIST JORMA (FI)
| US4725544A | 1988-02-16 |
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol. 55, No. 5, May 1989, ROBERT C.A. YANG et al.: "Hyperexpression of a Bacillus Circulans Xylanase Gene in Escherichia Coli and Characterization of the Gene Product", see page 1192 - page 1195.
SYSTEM. APPL. MICROBIOL., Vol. 8, 1986, HIROSHI HONDA et al.: "Production of Extracellular Alkaline Xylanase of Alkalophilic Bacillus Sp. C-125, by Escherichia Coli Carrying pCX 311", see page 152 - page 157.
CANADIAN JOURNAL OF MICROBIOLOGY, Vol. 31, 1985, H. HONDA et al.: "Two Types of Xylanases of Alkalophilic Bacillus Sp. No. C-125", see page 538 - page 542.
APPL. MICROBIOL. BIOTECHNOL., Vol. 19, 1984, WATARU OKAZAKI et al.: "Production and Properties of Two Types of Xylanases from Alkalophilic Thermophilic Bacillus Spp.", see page 335 - page 340.
PATENT ABSTRACTS OF JAPAN, Vol. 94, No. 188, C295; & JP,A,58 166 008, 04-04-1985, RIKAGAKU KENKYUSHO.
BIOTECHNOLOGY AND BIOENGINEERING, Vol. 32, 1988, M.G. PAICE et al.: "Viscosity-Enhancing Bleaching of Hardwood Kraft Pulp with Xylanase from a Cloned Gene", see page 235 - page 239.
BIOMASS, Vol. 15, 1988, L. JURASEK et al.: "Biological Treatments of Pulps", see page 104 - page 106.
| 1. | Enzyme preparation, c h a r a t e r i z e d in that it has been preparared by cultivation of the strain ATCC of the microorganism Bacillus circulans, or a mutant thereof, and it contains at least one of two βxylanases having molecular weights which, determined by electrophoresis, are about 28,000 and about 41,000, respectively, and whose isoelectric points are in the range from about 8 to about 9. |
| 2. | 2 The enzyme preparation according to claim 1, c h a r a c t e r i z e d in that it comprises a fermentation broth containing both of said βxylanases enzymes. |
| 3. | Process for producing an enzyme preparationen, c h a r a c t e i z e d by cultivating the strain ATCC 217S3 of the microorganism Bacillus circulans, or a mutant thereof, for about 12 to about 96 hours on a cultivation medium containing a carbon source, a nitrogen source and mineral salts and having a pH of about 7 to about 11 and a temperature between about 20°C and about 50°C and, if desired, separating the enzyme(s) having xylanase activity. |
| 4. | The process according to claim 3, c h a r a c t e r i z e d by maintaining the pH of the cultivation medium in the range from about 8 to about 9.5. |
| 5. | The process according to claim 3, c h a r a c t e r i z e d by using as a carbon source material of plant origin containing xylan or an isolated xylan, such as a softwood xylan. |
| 6. | The process according to any one of claims 3 to 5, c h a r a c t e r i z e d by using as a nitrogen source corn steep liquor or distiller's spent grain. |
| 7. | The process according to any one of claims 3 to 6, c h a r a c t e r i z e d in that the fermentation medium contains ammonium sulphate, calcium chloride, magnesium sulphate, manganese sulphate, iron sulphate and sodium chloride. |
| 8. | The use of the enzyme preparation according to claim 1 in the bleaching of pulp. |
| 9. | The use according to claim 8, c h a r a c t e r i z e d in that it is carried out in the alkaline pH region. |
The present invention concerns an enzyme preparation in accordance with the preamble of claim 1.
The invention also relates to a process according to the preamble of claim 3 for the preparation thereof.
Further, the invention concerns the use of the enzyme preparation.
As far as the prior art is concerned, reference is made to the following publications:
1. Honda, H. et al., System. Appl. Microbiol. 8 (1986), pp. 152-157
2. Honda, H. et al., Can. J. Microbiol. 31 (1985), pp. 538-542
3. Esteban et al., Can. J. Microbiol. 28 (1982), pp. 733-739
4. US-patenttijulkaisu 3,826,715 (1974), Horikoshi et al.
5. Poutanen, K. and Puls, J. , Appl. Microb. Biotechnol. 23 (1988) .
The term "alkaline xylanases" denotes enzymes capable of breaking down xylane in the alkaline pH region. These enzymes can be used for facilitating the removal of residual lignin from pulp prepared by an alkaline pulping process.
It is known in the prior art that xylanases which are active in the alkaline pH region can be produced by alkalophilic bakterial strains, such as strains belonging to the Bacillus genus. Generally, the activity of the xylanases produced by these bacteria is so low that enzyme production is inefficient and expensive. The first two references [1, 2] describe the
production of xylanase by cultivation of the strain Bacillus sp. No. C-125. According to the data given in the publication, the activity of the xylanase was in the range from 0,8 to 1 U/ml, which calculated as katals corresponds to an activity of between 90 and 100 nkat/ml.
Esteban with his co-workers [3] has produced xylanase by using the strain Bacillus circulans WL-12, a xylanase activity in the range from 2 to 20 nkat/ml being obtained at a optimum pH value of 5.5 to 7.
The present invention aims at eliminating the drawbacks of the prior art while providing an enzyme preparation having a high xylanase activity.
The invention is based on the surprising observation that by cultivating a specific Bacillus circulans strain, namely the Bacillus circulans strain deposited under the ATCC number 21783, or a mutant thereof, on a cultivation medium containing xylan at an alkaline pH, it is possible to provide a cultivation liquor (in the following: fermentation broth) having a high xylanase activity. In comparison to the results disclosed in the publications by Honda et al., it is possible to attain up to 50- fold higher activities by means of the invention. The difference between the activity of the present invention preparation and the prior art is even larger, i.e. ^ 200, when the comparison is based on the xylanase produced by another B . circulans strain (Esteban et al, [3]).
The B . circulans strain ATCC 21783 has earlier been used for the preparation of amylase, cf reference [4]. That prior art publication also contains a detailed description of the most important parametres of the said bacterial strain. The prior art fails, however, to provide any references to the use of the strain for producing β-xylanase.
In particular, the preparation according to the invention is
mainly characterized by what is stated in the characterizing part of claim 1.
The process according to the invention is, again, characterized by what is stated in the characterizing part of claim 3.
The term "enzyme preparation" is used throughout this specification to designate any product containing at least one enzyme. Thus, the enzyme preparation may comprise, for instance, a fermentation broth containing one or several enzyme(s) , it may comprise an isolated enzyme of a mixture of two or several enzymes.
By cultivating the strain ATCC 21783 of the microorganism Bacillus circulans , or natural mutants or artificial mutants thereof which have been obtained by mutageniza ion, in alkaline conditions on a fermentation medium containing xylan, a fermentation broth is obtained which contains at least two β-xylanases whose molecular weights (determined by SDS-poly- acryl amidegelphoresis) are about 28 kD and about 41 kD, respectively. The isoelectric points (pi) of the enzymes are between about 8 and 9. The enzyme preparation according to the invention contains at least one of the two above-mentioned enzymes. Preferably, it comprises a fermentation broth containing both enzymes.
The composition of the product obtained by fermentation varies to some degree depending on the fermentation medium and the fermentation conditions. Thus, in addition to the two main xylanases, two xylanases whose pi's are about 5.5 to 7 have, in some cases, been found in the fermentation broths.
The activity of the β-xylanases in the fermentation broth prepared by the process according to the invention is preferably in excess of about 2000 nkat/ml, in particular at least about 3000 nkat/ml. As the following working examples will show, we have even been able to prepare fermentation broths having xylanas
acitivities in the range from 6000 to 7000 nkat/ml.
In addition to high xylanase activity, the fermentation broth of the B . circulans strain used contains several other enzymes. These enzymes are generally capable of hydrolyzing the side chains of xylan or other hemicellulose components. Apart from the xylanases, the most important enzymes and their activities are given below:
β-xylosidase 0 to 100 nkat/ml, α-arabinosidase 0 to 50 nkat/ml, acetyl xylanesterase 0 to 10 nkat/ml, α-glucuronidase 0 to 20 nkat/ml and β-mannanase 0 to 50 nkat/ml.
According to the invention, the B . circulans strain ATCC 21783 is grown on a fermentation medium containing material of plant origin as a carbon source. The medium is made alkaline, i.e. its pH is adjusted to a value in the range from about 7 to about 11. In particular, the pH of the medium is adjusted to about 8 - about 9.5. Aqueous solutions of suitable alkaline compounds, preferably of sodium carbonate or ammonium hydroxide, can be used for adjusting the pH of the medium. The carbon sources may comprise different materials of plant origin (based on wood or cereals) which contain xylan or they may consist of isolated xylans, such as softwood xylans, in particular birch or beech xylans. The concentration of the carbon source is from 10 to 100 g/1. The cultivation may be carried out as a shake flask cultivation or in a fermentor. It is easier to maintain the pH of the fermentation medium in the preferred range for the production of xylanase in a fermentor. Thus, according to one preferred embodiment of the invention, the cultivation is conducted in a fermentor in order to provide fermentation broths having high activities of the xylanases.
According to a further preferred embodiment of the invention, the carbon source comprises birch xylan.
The cultivation medium contains as a source of nitrogen for instance peptone, yeast extract, corn-steep liquor or distiller's spent grain. Other alternative nitrogen sources comprise urea, meat extract, yeast extract and casein hydrolysates. The cultivation medium may also contain nutrient salts and trace elements. The following cations may be mentioned: calcium, magnesium, manganese, ammonium and iron ions, and the following anions: phosphate, sulphate and chloride ions. As far as the growth of the B . circulans strain and the production of xylanases on some cultivation media are concerned, we have noticed that it is especially beneficial to add a solution of trace elements containing the following compounds: ammonium sulphate, calcium sulphate, magnesium sulphate, manganese sulphate, iron sulphate and sodium chloride. By selecting corn-steep liquor or distiller's spent grain as nitrogen sources, good fermentation results are obtained.
The cultivation is normally carried out at a temperature in the range from about 20 β C to about 50°C for a cultivation period of time extending from about 12 hours to about 96 hours. However, if necessary, it is possible to deviate from these conditions.
By means of the invention, considerable benefits can be obtained. Thus, it has surprisingly been found that the enzyme preparation provided by means of the invention does not only have a high activity but also a broad pH range of activity. Thus it is capable of solubilizing xylan even in the alkaline region ≥ 9.
The xylanase fermentation broth according to the invention is well-suited for use, in particular, in the bleaching of pulp, for instance for reducing the required amounts of bleaching chemicals, such as chlorine dioxide. At the same time, it is possible efficiently to decrease the residual amounts of chemicals contained in the pulp. Since the cellulase activity of the fermentation broth is extremely low ( -.0,05 FPU), the broth can be used as such for the treatment of pulp, without it having
a degrading effect on cellulose, which would impair the quality of the pulp. The β-xylosidase activity of the fermentation broth is fairly high. Because of this, the main product of xylane hydrolysis at pH 7 is xylose. At higher pH values, less mono- saccharides are formed, which obviously is caused by the low pH stability of the β-xylosidase.
If desired, the β-xylanase enzymes can be separated and purified by methods known per se .
In the following, the invention will be examined in more detail with the aid of non-limiting working examples. The examples 1 to 5 describe the preparation of fermentation broths containing β-xylanases, the example 6 elucidates the solubilizing effect of fermentation broths upon xylans and the examples 7 and 8 disclose the use of the enzyme preparation in the bleaching of pulp.
The xylanase activity of the prepared fermentation broths is assayed by the method described by Poutanen and Puls [5]. The substrate consists of 1 % Larchwood xylan (delivered by Sigma) , 50 M sodium citrate buffert, pH 6.5.
Example 1 - Shake flask cultivation of the Bacillus circulans strain ATCC 21783
The Bacillus circulans strain was cultured with shaking in a 50 ml solution volume in a 250 ml flask. The cultivation temperature was 28°C. The fermentation medium contained 10 g/1 birch xylan, 5 g/1 yeast extract, 2.5 g/1 peptone, 0.5 g/1 K 2 HP0 4 , 0.125 g/1 MgS0 4 .7H 2 0, 0.25 g/1 (NH 4 ) 2 S0 4 , 0.125 g/1, NaCl, 0.025 g/1 CaCl 2 .2H 2 0, 0.0025 g/1 MnS0 4 H 2 0, 0.0025 g/1 FeS0 4 .7H 2 0.
2 ml of a sterilized 10 % Na 2 C0 3 solution was separately added to 50 ml of the autoclaved cultivation medium. The innoculum used
comprised a bacterial culture grown overnight on a Tryptic Soy medium. After three days, the activity of the xylanase in the centrifuged cultivation medium was 3000 nkat/ml.
The initial pH was about 9.5 and it dropped during the cultivation to about 7.
Example 2 - Cultivation of the Bacillus circulans strain ATCC 21783 in a fermentor
The Bacillus circulans strain was cultivated in a laboratory fermentor. The cultivation medium contained 20 g/1 of beech xylane, 20 ml/1 of corn-steep liquor, 0.54 g/1 of K 2 HP0 4 , 0.15 g/1 of MgS0 4 .7H 2 0. The cultivation temperature was 30°C and the pH was maintained at a value in the range from 8 to 8.5. The initial pH was adjusted by the addition of a sodium carbonate solution. During the fermentation, the pH was automatically adjusted by additions of ammonium hydroxide.
After three days, the xylanase activity of the fermentation broth was 4500 nkat/ml.
Example 3 - Cultivation of the Bacillus _circulans strain ATCC 21783 in a fermentor
The Bacillus circulans strain was cultivated in a laboratory fermentor in the same cultivation conditions as in example 2 as far as temperature and pH were concerned. The carbon source used comprised 20 g/1 of beech xylane. The other components of the fermentation medium were the same as in example 1. After three days of cultivation, the xylanase activity of the centrifugated cultivation medium was 4000 nkat/ml.
In addition to the activity of the xylanase, the fermentation broth was analysed for the activities of the other enzymes which were found to be as follows:
β-xylosidase 40 nkat/ml, α-arabinosidase 10 nkat/ml, acetyl esterase 2 nkat/ml, α-glucuronidase 5 nkat/ml and β-mannanase 10 nkat/ml.
Example 4 - Cultivation of the Bacillus circulans strain ATCC 21783 in a fermentor
The Bacillus cirulans strain was cultivated in a laboratory fermentor. The medium contained 20 g/1 of beech xylane, 20 ml/1 of corn-steep liquor, the other components of the medium were the same as in example l. After sterilisation 30 ml/1 of a 0.1 M sodium bicarbonate solution was separately added to the medium. The fermentation temperature was 30°C and the pH was kept in the range from 8 to 8.5. pH adjustment was effected by adding ammonium hydroxide. After 48 hours of cultivation the xylanase activity of the fermentation broth was 6900 nkat/ml.
Example 5 - Cultivation of the Bacillus circulans strain ATCC 21783 in a fermentor
The Bacillus circulans strain was cultivated in accordance with the example 4, while using 10 g/1 of distiller's spent grains (wheat) instead of the corn-steep liquor. After 76 hours of cultivation the xylanase activity of the fermentation broth was 6800 nkat/ml.
Example 6 - Hydrolysis of xylan
Deacetylated xylan was hydrolysed by the enzyme preparation of example 3. The pH of the hydrolysis solution was adjusted by means of citrate-phosphate buffer to 6, 7, 8 and 9, respectively. 100 nkat xylanase was used for each gram of xylan. After the treatment, carbohydrates had been solubilized from xylan in the following amounts:
pH solubilized carbohydrates
(% of original xylan)
6 15.5 7 18.6 8 14.1 9 13.9
Example 7 - Peroxide bleaching of pulp
A spruce kraft pulp was treated with an enzyme preparation produced from the Bacillus circulans strain ATCC 21783. For each gram of the pulp an amount of xylanase corresponding to an activity of 30 nkat was added. The enzyme treatment time was 24 h. The reference was treated as the sample with the exception that no enzyme was added. After the enzyme treatment the pulp was treated with peroxide in alkaline conditions. In comparison to the kappa levels (the lignin contents) of the pulps after pulping, the following reductions were attained at different pH values:
Kappa reduction, % brightness, ISO
Treatment pH
6
7 8 9
Example 8 - Chlorine bleaching of pulp
A spruce kraft pulp was bleached according to the previous example enzymatically in combination with a chlorine bleaching sequence (D50C50)ED-ED 2 . The intermediate kappa values, the final brigthness and the viscosity obtained were determined.