SK118594A3 - Delignifying preparation exhibiting alpha-l-arabinofuranosidase activity production and application thereof - Google Patents

Abstract

2132335 9320192 PCTABScor01 A preparation exhibiting enzymatic activity comprising a substantial portion of .alpha.-L-arabinofuranosidase activity having the capability of delignifying wood pulp at a pH of 8 - 9 and a temperature of 65 ·C, is disclosed. Said preparation is obtainable by aerobic fermentation of a Bacillus stearothermophilus strain selected from the deposited strain NCIMB 40494 and mutants and variants thereof. The deposited strain and mutants and variants thereof are comprised by the invention. A method of producing said preparation, a process comprising treatment of wood pulp with said preparation, and wood pulp that has been treated with said preparation, and also said preparation in association with a preparation exhibiting enzymatic activity produced by another microbial strain and having the capability of delignifying wood pulp at a temperature of at least 65 ·C and a pH of at least 9, are disclosed. Use of an enzyme expressed by the deposited strain and .alpha.-L-arabinofuranosidase which comprises one or two specified partial sequences and homologues thereof, are also included.

Description

Technical field

The invention relates to a preparation exhibiting enzymatic activity comprising, as a major component, α-L-arabinofuranosidase activity with the ability to delignify wood pulp at pH 8-9 and a temperature of 65 θθ. It further relates to a process for the preparation of this preparation by aerobic fermentation of a selected strain of Bacillus stearothermophilia. The invention also relates to this selected strain. In addition, it relates to a method for treating wood pulp with the composition of the invention and also to pulp treated with said composition as such. In addition, it relates to the use of an enzyme produced by a deposited strain of Bacillus stearothermophilia NCIMB 40494 in pulp processing. It also relates to α-L-arabinofuranosidase having an amino acid sequence comprising one or two specific amino acid partial sequences or homologues thereof.

BACKGROUND OF THE INVENTION

Given the environmental impact, pulp and paper industries are interested in bleaching processes leading to delignification and bleaching of pulp without the use of chlorine-containing compounds.

For this purpose, a number of patent applications have been filed recently describing enzymes for delignifying wood pulp. One of these applications is our WO 91/10724 (Swedish priority application for which a patent was issued on December 19, 1991) which claims, inter alia, a preparation exhibiting an enzymatic activity having the ability to delignify pulp at a temperature of at least 65 θθ and a minimum pH of 9. can be obtained by aerobic fermentation of a Baci11us stearothermophilia strain with the ability to produce this preparation as one of two strains deposited in the collection of microorganisms under NCIMB 40221 or NCIMB 40222. Purification of enzymatic activity yielded xylanase with MW / MW / 41,000-42,000 Da.

To date, efforts have been made to isolate another strain of Bacillus stearothermophilia which could be used to purify other delignification activities, such as xylanase activity. The aim was, for example, that α-L-arabinofuranosidase, which has the ability to delignify wood pulp at high temperature and pH, is also a useful tool in developing a bleaching operation without any bleaching chemicals containing chlorine.

Recently, WO 91/18976 (filed by Novo Nordisk A / S) has been published. Its claim No. 14 is directed to the arabinofuranosidase enzyme produced by Baci 11 µs stearothermophi 1 µs. On page 9, lines 11 and 12, there is no reference in the literature to arabinofuranosidase derived from thermophilic / thermophilic / Bacillus. Although WO 91/18976 claims the enzyme arabinofuranosidase produced by Bacillus stearothermophilus, it does not disclose a method for its preparation.

Three strains, two isolated and one mutant, were placed in the microorganism collection. However, none of them has been described as arabinofuranosidase producing strain in said application. .

The only strain described as arabinofuranosidase producing strain is the unsupported mutant strain designated BPS-3-H-17-4. It should further be noted that the preparation experiments described in the application (center above on page 17) were carried out with fermentation products obtained from this unsaved strain. On page 8, r.12-16 it is reported that this strain is derived from one of the deposited strains after mutagenesis with ethyl methanesulfonate. This mutagen is very non-specific and no conditions are described for obtaining arabinofuranosidase-producing strain. Thus, in the prior art, a way to obtain arabinofuranosidase from a thermophilic - thermophilic - Baci11us strain has not been described.

SUMMARY OF THE INVENTION

The present invention relates to a preparation exhibiting enzymatic activity in which a substantial part of the activity of α-L-arabinofuranosidase occupies the ability to delignify wood pulp at pH 8-9 and a temperature of 60 θθ.

In this specification and the appended claims, the term wood pulp in the broader sense is to be construed to include all types of 1 ignocellulose materials.

One object of the invention is directed to a preparation exhibiting enzymatic activity obtainable by aerobic fermentation in a suitable environment of a Baci11us stearotermophilus strain selected from NCIMB 40494 strain and mutants and variants thereof having substantially the same ability to produce said preparation as NCIMB 40494 strain, wherein, in the enzymatic activity of said preparation, a substantial part occupies α-L-arabinofuranosidase activity with the ability to delign wood pulp at pH 8-9 and a temperature of 65 ° C.

In one embodiment of this aspect of the invention, the composition is a purified culture medium. In another embodiment of this part of the invention, the composition is a concentrated fraction of said purified culture medium exhibiting α-L-arabinofuranosidase activity in a wood pulp environment. In yet another embodiment of the present invention, α-L-arabinofuranosidase activity is derived from α-L-arabinofuranosidase, which is composed of two subunits with an approximate molecular weight of 52,500 Da and 57,500 Da, respectively.

In a further aspect of the invention, the composition according to the aforementioned object of the invention comprises a composition exhibiting enzymatic activity produced by another microbial strain and having the ability to delignify wood pulp at a temperature of at least 65 and a pH of at least 9. In a specific embodiment arabinofuranosidase produced by NCIMB strain 40494 of Bacillus stearothermophilus and xylanase produced by NCIMB strain 40222 of Bacillus stearothermophilus.

Another subject of the invention is aimed on the α-L-Arabinofuranozi dase with ou sequence and y acidify n, which it contains an N-terminal member u sequence no acidic i n SEQ Ident.č.1: Met? Gin Pro Tyr Arg Pro? Glu Glu Leu or a homologue thereof. Another subject of the invention is aimed on the

α-L-arabinofuranosidase having an amino acid sequence comprising the N-terminal amino acid partial sequence of SEQ ID NO: 2:

Ser Met Lys Lys Ala Thr Met Goals Glu Lys Asp Phe Lys Goes A1 and Glu Goes Asp Lys Arg Goes Tyr or homologue thereof.

In connection with α-L-arabinofuranosidase having an amino acid sequence comprising the N-terminal amino acid partial sequence of SEQ ID NO. 1:

Met? Gin Pro Tyr Arg Pro? Glu Glu Leu, or a homologue thereof, and / or α-L-arabinofuranosidase having an amino acid sequence comprising an N-terminal amino acid partial sequence of SEQ ID NO: 2

Ser Met Lys Lys Ala Thr Met Goals Glu Lys Asp Phe Lys Goals Ala Glu Goals Asp Lys Arg Goes Tyr or a homolog thereof, such a homolog is an intended homolog with an amino acid sequence which, in relation to SEQ ID NO: 1 and / or SEQ ID NO: 2 has some substituents, extended or deleted portions of amino acids that do not cause the elimination of the α-L-arabinofuranosidase activity of the entire enzyme in, for example, wood pulp and preferably at 65 ° C and pH 8-9. Further, such a homolog may have, for example, a sequence having 80% or more of the common amino acids of SEQ ID NO. 1 and / or SEQ ID NO.

An example of a homology to SEQ ID NO: 2 is SEQ ID NO: 2. 3:

Al a Thr Lys Lys Al a Thr Met Ile Ile Glu Lys Asp Phe Lys Ile Al a Glu Ile Asp Lys Arg Ile Tyr Gly Ser Phe Ile Glu Hi s Leu Gly Arg Al a wall Tyr Gly Gly Ile Tyr Glu for Gly Hi s for gin Al a Asp Glu

Asn Gly, which is the M-terminal sequence of α-L-arabinofuranosidase produced by Bacillus stearothermophilia NCIMB 40222. Said α-L-arabinofuranosidase is composed of two identical subunits, each with an approximate molecular weight of 64,000 Da (natural enzyme, 128,000 Da, by SDS gel).

Thus, a homolog of SEQ ID NO: 1 and / or SEQ ID NO: 2 is any amino acid sequence that is sufficiently homologous at the nucleotide level to be recognized by any DNA or RNA probe derived from said sequence (s).

Another subject of the invention is a preparation which exhibits Bacillus stearothermophilus mutants and variants in producing the preparation both as an aerobic fermentation and in the activity of this preparation and the L-arabinofuranosyase delignification of the wood cell is directed to a method of producing enzymatic activity. with the same ability, strain NCIMB 40494 is subjected to a suitable environment and the enzymatic enzyme contains a substantial portion of the activity, with the ability to otherwise be at a pH of 8-9 and a temperature of 65 ° C.

Yet another object of the invention is directed to an isolated strain of NCIMB 44 Bacillus stearothermophilis and mutants and variants thereof, wherein the mutants and variants have substantially the same ability to produce a preparation exhibiting the enzymatic activity of said strain NCIMB 44, occupying a substantial fraction of this enzymatic activity. α-L-arabinofuranosidase activity with the ability to delignify wood pulp at pH 8-9 and 65 ° C.

A further object of the invention is to use an enzyme produced by Bacillus stearothermophilia strain NCIMB 40494 for the processing of wood pulp. As can be seen from Table 8 of the specification, this strain produces several types of enzymatic activities degrading extra-cellular carbohydrates, which are derived from the production of various genes contained in the genome of this bacterium.

Although these different activities have not yet been individually investigated, it is believed that different activities, separately or in different combinations, will be useful for processing wood pulp.

Another object of the invention is directed to a method comprising treating wood pulp, wherein the wood pulp is treated in at least one operation with a composition of the invention. In one embodiment of the present invention, the wood pulp to be treated is kraft pulp. In another embodiment, the kraft pulp to be treated is a partially delignified kraft pulp. In yet another embodiment of the present invention, the treated partially delignified kraft pulp is oxygen delignified kraft pulp.

Another object of the invention is directed to wood pulp that has been treated in at least one operation with a composition of the invention.

Yet another object of the invention is directed to a DNA or RNA probe that recognizes a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 1:

Met? Gin Pro Tyr Arg Pro? Glu Glu Leu and / or amino acid sequence SEQ ID NO: 2:

Ser Met Lys Lys Ala Thr Met Goal Glu Lys Asp Phe Lys Goal Ala Glu Goal Asp Lys Arg Go Tyr.

The latter object is useful for detecting α-L-arabinofuranosidase with the ability to delignify wood pulp at a temperature of at least 65 ° C and a pH of at least 8-9.

Storage of microorganisms:

The Baci11us stearothermophi1us L1 strain was deposited under the Treaty of Budapest in the National Collections of Industrial and Marine Bacteria Ltd (NCIMB) / National Collection of Industrial and Marine Bacteria spol. / - (NCIMB) Limited in Aberdeen on March 24, 1992 and received NCIMB registration number 40494.

The strain Bacillus stearothermophilus T-6 received NCIMB registration number 40222 in the same collection of microorganisms and was deposited earlier in the context of priority application WO 91/10724.

DETAILED DESCRIPTION OF THE INVENTION

Isolation of a thermostable α-L-arabinofuranosidase producing a Bacillus stearothermophilia L1 strain.

A. Preparation of alkaline extracted pulp (AKP)

A suspension of K15, oxygen-bleached fine wood kraft pulp (6-10% fiber by weight in dry matter) in 0.02 N NaOH (pH 11.7) was incubated at 60-62 θθ for 18-20 h. While still hot, the fluid was separated from the fibers by suction at

The resulting solution was a sintered glass funnel.

neutralized to pH 7 and concentrated by ultrafiltration (removal of 10,000 unit molecular weight).

The retained amount (AKP-1) contained 5.2 mg / ml lignin,

1.85 mg / ml carbohydrates (phenol sulfuric acid) and 0.83 mg / ml pentose (orcinol = 5-methyl-1,3-benzenediol).

B. Enriching culture

Salts (0.1 MgSO 4 .7H 2 O, 10 mM K 2 HPO 4 0.1% urea and trace elements) were added to AKP-1 and inoculated samples of sludge and water collected from the Korsnäs treatment tanks. The flasks were incubated by shaking at 65 ° C and pH 9.0 for 48 hours. The fraction of the cloudy culture was then inoculated into a tube containing as medium 15 ml of 0.2% gal of actomanan (acacia resin, Sigma). After incubation for 24 hours. at 65 ° C and pH 9.0, the culture was seeded in LB agar. After incubation, approximately 20 different types of colonies were isolated and recovered in pure culture. After preliminary testing, one of the strains was studied in detail: strain L1, which contained the thermophilic α-L-arabinofuranosidase.

Strain L1 was a Gram-positive, thermophilic bacterium; it grew well at 60-65 θθ, but did not grow below 40 θθ. It was rod-shaped with terminal spores, aerobic and oxydase positive. The L1 strain grew on the following carbon sources: D-glucose, D-xylose, L-arabinose, D-mannose, and xylan. It did not grow on carbon sources such as melobiose and galactose. Based on these properties, strain L1 was classified as a strain of a heterogeneous group, Bacillus stearothermophi 1 us.

Initial experiments (with K15 pulp)

C. Thermophilic α-L-arabinofuranosidase from the L1 strain selected for flowing on the medium was further studied because the crude culture grown far away was absorbed at the time of absorbance at the digestion point sensitive.

350 nm evaluation as enzyme preparation test. Values measured on supernatant (supernatant) percentage of strain L1

Strain L1 was extracellular galactomannan KI5 pulp. To extract spectrophotometer CKY ^{(Α 35θ) bo1i} samples grown and evolved

The tribe grew and casas new and galactose observed as an activity.

the measured values are presented as lignin. Growth and delignification activity on different carbon sources are summed up to some acidity or at least 5.1% at a tax rate of 7.6% for xylose cultures.

was and extract at which

At most 1 yeast rates without addition of another carbon source delignification. Additional growth was of all possible sugars except hampered the formation of delicative activity was obtained on arabinose, 7.1%, respectively.

The extracellular enzymatic activities of strain L1 grown in xylose and arabininose are summarized in Table 2. Xylanase activity was determined by incubating fresh xylan solution with extracellular supernatant fluid and assay for enhancing sugar reduction by the ferricyanide method (Spiro, R.G. 1966 Methods of Enzymology 8: 7-9). The assay buffer was 50 mM Tris.CL, pH 7.0 and 0.5% xylan (non-cultivated oat, Sigma). Units of activity for xylanase are umol of reduced sugar formed at 65 θθ.

The Arabinofuranosidase assay is a modification of the standard assay for β-galactosidase (G. Tabold and Syguson, Appl. Enviro. Microbiol. Vol. 56, No. 11, 1960). The test tube contains 40 mM Tris buffer, pH 7.0. An amount of 0.4 ml of the enzyme sample and 0.1 ml of 10 mM p-ni trophenyl-L-arabinofuranoside (Sigma chemicals) were incubated at 65 ° C for 15 min. The reaction was terminated by placing the test tube in an ice water bath.

The release of p-nitrophenol is determined spectrophotometrically at 401 nm. An amount of 10 micromoles of p-nitrophenol per ml has an absorbance of 18.4. The unit of active enzyme is defined as the amount of p-nitrophenyl micromoles released per minute.

Only two activities were observed in the xylose environment: 1.3 units per ml of xylanase and 0.004 units per ml of α-L-arabinofuranosidase. The activities of α-L-arabinofuranosidase, mannanase, α-D-galactosidase or α-L-mannosidase were not detected. Thus, the delignifying activity of the L1 strain was shown to be due to xylanase activity during cell growth in xylose environment. However, once the cells were grown in arabinose environment, α-L-arabinofuranosidase (0.5 units per ml) showed the main activity. Thus, α-L-arabinofuranosidase has been shown to be responsible for delignification in the A-environment.

Α-L-arabinofuranosidase (AF) activity was obtained by concentrating a 10-liter culture of the L1 strain grown in A-medium (Table 3). The activity was precipitated with 80% ammonium sulfate and the yield was a crude enzyme preparation with 11 units per ml AF and 0.96 units per ml xylanase. AF activity did not bind to carboxymethyl cellulose (CMC) but adsorbed completely to DEAE cellulose or DEAE-Sephacel.

Concentrated AF showed significant delignifying activity (Table 4). It should be emphasized that the optimum conditions for using AF for pulp delignification have not been investigated. The potentially useful property of AF is that it should break down the binding of lignin, leaving most of the hemicellulose with the cellulose fibers. This step should provide a higher yield of delignified pulp. In addition, it is apparent that the delignifying activity of AF along with the xylanase activity of T-6 is more than additional.

Tables 5-7 show some enzymatic properties of purified AF using ΡΝΡ-α-L-Ara as substrate. The enzyme is most active between pH 6.5 - 8.0, optimally at pH 7.0. The enzyme has low activity at pH 9.0 and 70 θθ. The optimum temperature was 70 θθ at both pH 7.0 and pH 8.0. The enzyme was most active at 20 mM Na ₂ SO ₄ , in 10 mM in phosphate buffer, pH

7.5 and 70 ° C, but lost about 50% of its activity at 70 ° C for 75 min and was completely inactive at 80 ° C for 15 min.

Extended experiments (with K20 pulp)

Extracellular Enzymatic Degradation Activities of Bacillus stearothermophilus - L1.

Specific extracellular carbohydrate degrading enzymatic activities were tested by monitoring growth in various environments (Table 8). Cells were grown on various carbon sources such as: p-nitrophenyl-β-D-manopyranoside, p-nitrophenyl-α-D-galactopyranoside, and p-nitrophenyl α-L-arabinopyranoside.

Xylanase and two and arabinase) were tested at pH 8.0 diluted further in 10 mM phosphate buffered total assay volume.

(0.1 ml) was added to the buffer and 0.55 and transferred to an ice water bath, sugar was detected by the 3,5-dinitrosalicylic endohemi cellulase method (solution manasse and 60 ° C sample in 1 ml)

Suitably the substrate, the reaction is

reduced

0.1 ml terminate 1 enzyme

100 mM

0.25 ml of water.

acids as described by Miller (Miller, GL, (1959) Anal. Chem 31: 426-428). The substrates were 4% non-cultivated oat xylan, 0.4% acacia grain-galactomannan resin, 4% arabinogal larch wood actan, and 4% mannan from Saccharomyces cerevisiae.

Enzymatic release of lignin from softwood pulp was determined by adding 200 mg of wet K-20 pulp (from Korsnäs paper mill in Gävle, Sweden) to 3 ml of enzyme solution, adjusting the pH to 8.0 or 9.0 with concentrated NaOH and incubating at θε with constant shaking. After 2 hours 1.5 ml of liquid was collected and centrifuged at 10,000 x g for 5 min. in a minifuge to remove residual pulp fibers. Pure liquid (0.5 mL) was diluted with 1.0 mL, 0.1 N NaOH to determine the absorbance at 350 nm. The control for each assay was incubation of the pulp under the same conditions without enzyme. Since 1 mg of lignin per ml showed A35Q = 9.1 and the pulp used in these experiments had a lignin content of 1.3% (Klason, determined by Z. Zosim), the delta A35Q x 3% lignin released = ------ --------- x 100

P x 0.013 where P is the weight of dry pulp and delta A35Q is the absorbance after incubation minus the absorbance before incubation. The amount of net lignin released is the total amount minus the amount found under enzyme-free conditions.

resin

Xylanase and achieved important activities namely xylanase Xylanase was found at low carbon in the culture medium was (LBG), D-glucose and L-arabinose. as a carbon source of activity.

was significant in L-arabinose, where mannase activity, α-L-arabinopyranose dase.

Two α-L-arabinofuranosidases were detected. concentration where the source of acacia grain activity was increased by D-xylose 1.23 U / ml. Mannose inhibited α-L-arabinofuranosidase activity was high

Unlike the D-xylose medium, it reached 1.5 U / ml. α-D-galactosidase.

xylanase low, but no β-D-mannosidases in the environment, or

Purification of α-L-arabinofuranosidase from Bacillus stearothermophilia

After culturing Bacillus stearothermophilus L1 for 24 hours at 60 ° C in a medium containing L-arabinose, the free extracellular fluid without cells contained 1.2 U / ml α-L-arabinofuranosidase activity with a specific activity of 1.7 U / mg (Table 9). Preliminary experiments showed that the enzyme concentration could be accomplished by precipitation by saturation with 90% ammonium sulfate. However, only 68% activity was obtained and no significant increase in specific activity. Therefore, ammonium sulfate precipitation was not used and the crude enzyme was adsorbed directly onto a DEAE Sephacel column (Tab.9, anion exchange). After flushing the column with 10 mM phosphate-potassium buffer at pH 8.0, scrubbing was performed with a NaCl solution with a linear gradient from 0.1 to 0.9 M. The activity of α-β-arabinofuranosidase was shown as a sharp point between 0, 53 and 0.57 M NaCl. After subsequent concentration and desalting of the active fraction by dialysis against polyethylene glycol, the specific activity was increased 38-fold with 100% recovery of activity. This material was directly transferred to a Sephadex G-100 packed column. The active material eluted with a single sharp peak and an apparent molecular weight of 108,000. The total purification was 59-fold at a yield of 80%. The purified enzyme was checked by gel filtration on FPLC Superose in 100 mM phosphate-potassium buffer at pH 7.0 and 100 mM NaCl. Over 95% of the activity and protein eluted as a single sharp peak with an apparent molecular weight of 114,800. SDS PAGE showed purified enzyme as two bands of 57,500 and 52,500 respectively. After aspiration on a PVDF membrane and sequencing on the Applied Biosystem model 475A gas sequencer, analysis of these bands showed two N-terminal sequences of SEQ ID NO: 1 and SEQ ID NO. .č.2.

Characteristics of Bacillus stearothermophilus α-L-arabinofuranosidase L1

At pH 7.0, the purified enzyme was completely stable at 60 ° C for at least 80 min, retained 50% of its maximum activity after incubation at 70 θθ for 75 min, and lost all activity for 15 min at 80 θθ. At 70 ° C, pH 7.0 was optimal for activity; at pH 6.5 and pH 8.0, the respective activities made 55% and 50% optimal activity, respectively. At pH 7.0 and 8.0, the optimum temperature for the activity was 70 ° C. The enzyme showed maximal activity in 20-50 mM NagSO 4; at 100 mM and 150 mM Na ₂ SO ₄ , activity decreased by 10% and 10%, respectively. 50%.

The kinetic parameters of the enzyme were measured using ρΝΡ-α-L-ara-f as substrate. At pH 7.0 and 65 ° C, K _m and ^v _max were 2.2 x 10 ^-4 M and 101 gmol / min / mg. The enzyme showed only low activity on a high molecular weight substrate such as arabinoxylan and arabinogalactan.

Delignification activity of Bacillus stearothermophilius L1 α-L-arabinofuranosidase

During the purification of the enzyme (Table 9), eluents from the column were periodically examined for delignification activity using partially bleached pulp as substrate. The delignification activity was associated with a) the peak activity of α-L-arabinofuranosase,

b) with a peak of low molecular weight endoxylanase activity, c) with fractions containing a lower value of both activities. Since the highest specific delignification activity was manifested in the presence of both enzymes, it was decided to investigate the possibility of synergistic activity of α-L-arabinofuranosidase and the previously purified heat-stabilized T6 xylanase described in PCT application WO 91/10724.

Table 10 summarizes a typical experiment in which delignification activity was investigated using pure α-L-arabinofuranosidase, pure xylanase and a mixture of both enzymes. At pH 8.0 and 65 for 2 hours, pure 19.2% lignin was released from pulp from a mixture of 38 / ml aL-arabinofuranosidase and 5 U / ml T6 xylanase, yielding only 16.5% for each enzyme acting alone. . To obtain 16.5% of the released pure lignin using only T6 xylanase under these conditions, 50 U / ml enzyme was required. At pH 9.0 and 65 ° C for 2 hours, the enzyme mixture released 18.4% lignin compared to only 13.3% resulting when each activity acted separately. It is clear that both enzymes act synergistically in releasing lignin from the pulp.

The obvious paradox is that the enzyme α-L-arabinofuranosidase showed less than 1% of its optimal activity at pH 9.0 using the model substrate p-nitrophenol α-L-arabinofuranosidase. However, in the biological bleaching process, the enzyme was almost as effective at pH 9.0 as at pH 8.0. It is possible that the pulp in some way protected and preserved the activity of the enzyme at higher pH values. These results are encouraging because the industrial delignification process is easier to implement at pH 9.0 and 65 ° C than at lower temperature and pH values.

Tab. 1

Growth and delignification activity of L1 strain on various media

source carbon ^and Growth ( ^Α 56θ) Deli gni fi caci a total (%) ^b Net any 0.73 7.1 5.1 0.2 LB 1.30 8.8 6.8 0.2 mannose 1.83 8.0 6.0 0.2 galactose 0.84 4.9 2.9 0.2 glucose 2.62 8.9 6.9 0.2 xy1óza 2.04 9.1 7.1 0.2 arabi nóza 1.81 10.5 7.6

The culture medium consisted of a carbon source (0.2%), 0.1% yeast extract, 0.5% casaminic acid, and E salt (0.1% urea, 0.02% MgSO ₄ .7H ₂ 0). , 5 mM KH ₂ PO ₄ , pH 9.0, 20 mM Tris HCl, pH 9.0, 0.1% NaCl and standard trace element mixture).

All sugars had a D-configuration except L-arabinose; LB is an acacia resin resin.

^b ) Delignification of K15 pulp was performed with extracellular supernatant (pH adjusted to 9.0) at 65 ° C, for 2 hours.

Tab. 2

Extracellular enzymatic activity of strain L1

X media um ^and A-Medium & substrate Acti v (U / ml) Akti v. (U / mL) xyl án 1.3 0.05 PNP-a-L-AF 0,004 0.5 PNP-a-Gal <0.001 PNP-a-Man <0.001 PNP-and-AP <0.001 LB-GM <0.001

^(a ) The degradation activities of xylan and LB-GM (acacia grain galactomannan) were determined from the production of reducing sugars and PNP-X activities were measured by released PNP (p-ni trofenol).

The cells were cultured for 24 hours on either X-medium or A-medium (xylose or arabinose medium described in Table 1) and the mixture centrifuged to remove cells.

Tab. 3

Preliminary cleaning α-L-arabinofuranosidase z Ll grown on the A-media u faction 1 1 1! 1. r- 1 -n E i 1 -Q * 1 1 O 1 1 1 1 1 Protei n ^and AF (U / ml) xylanase (U / mL) Crude supernatant 8100 1.11 0.82 0.03 (NH ₄₎ S0 ₄ ppt 400 13.3 11.0 0.96 DEAE-Sephacel 20 41.1 46.6 0.09

^and BioRad protein

Tab 4

Delignification of K15 with concentrated α-L-arabinofuranosidase

enzyme % released Pretty lignin ^and chi (20 U / ml) ^b 8.6 3.0 T-6 ^c xylanase (5 U / ml) 11.3 5.7 AF + T-6 xylanase 16.1 11.5

^(a ) 10 mM phosphate buffer, pH 8,0, 65 θθ 2 hr.

b) Concentrated by DEAE-Sepharel chromatography; contains <0,1 U / ml xylanase.

^(c ) T-6 xylanase produced by strain NCIMB 40222 described in Pr.

Tab. 5

Influence of temperature on AF activity at pH 7 and 8

Temperature ° C pH 7 AF (U / ml) ^a pH 8 AF (U / ml) ^b 40 2.1 1.0 50 5.0 2.4 60 8.3 5.2 65 10.1 7.6 70 10.3 9.6 75 9.9 7.5 80 4.2 1.0 90 0.8 0.3 100 0 0

^and in 20 mM Tri with buffer

Tab. 6

Effect of salt concentration on AF activity

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ^ 21 l 3 Q> 1 i 3 ro i 1 CO 1 1 O 1 1 -> 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 AF (U / ml) ^a 0 3.1 20 3.6 50 3.4 100 3.0 150 1.0

^a ) In 10 mM phosphate buffer, pH 7.5, at 70

Tab. 7

Stability of α-L-arabinofuranosidase (AF) activity

Reaction time (mi n) 60 ° C AF ^and activity (U / ml) 70 ° C AF ^and activity (U / ml) 80 ° C AF ^and activity (U / ml) 2 7.7 6.7 5.0 5 7.5 7.0 - 15 8.4 7.0 0 30 8.4 6.7 45 7.5 5.5 60 7.7 4.2 75 8.0 2.7

and

In 20 mM Tris buffer, at pH 7.0

Tab. 8

Extracellular enzymatic activities of Bacillus stearothermophilia

L1 ^and the degradation and carbohydrates

Carbohydrate substrates used for:

growth Acti welcomes the enzyme ^b Type < enzyme Aktivi ta (U / mL) LGB ^C LBG mannanase <0.01 LGB xyl án xylanase 0, 136 LGB ρΝΡ-α-D-Gal p α-D-galactose dase 0,008 D-glucose LBG mannanase <0.01 D-glucose xyl án xylanase 0,319 D-xylose xyl án xylanase 1.25 D-xylose LBG mannanase <0.01 X-xylose arabi nogalaktan galactanase / arabi cutidase <0.01 D-xylose ρΝΡ-α-D-Gal p α-D-galactose dase <0.001 D-xylose ρΝΡ-β-D-Man-p β-D-mannose dase <0.001 D-xylose pNP-a-L-ara-p α-L-arabino-pyranose dase <0.001 D-xylose pNP-a-L-ara-f α-L-arabino-furanozase 0.01 D-mannose mannan mannanase <0.01 D-mannose xyl án xylanase <0.01 D-mannose LBG mannanase / galactose dase <0.01 D-mannose ρΝΡ-β-D-Man-p β-D-mannose dase <0.001 L-arabiose arabi nogalaktan α-L-Arabi nosdase <0.01 L-arabiose xyl án xylanase 00:11 L-arabiose pNP-a-L-ara-f α-L-arabino-furanoziase 1.5

^(a ) The cells were cultured for 24 hours at 60 θθ and pH 8.0 in E-salts containing 0.1% yeast extract, 0.1% casaminic acids and 0.2% carbon source.

After incubation for 24 hours, the culture was centrifuged and extracellular carbohydrate degrading enzymes were tested as described in the specification.

^(c ) LGB is an acacia resin - galactomannan; pNP is p-ni trophenol

Tab. 9

Overview of the procedure for purification of ce-L-arabinofuranose g-L-arabinofuranose

Cleaning operations e Obj em (Ml) Proteí n (Mg / ml) (U / mL) Sp.akti v. (U / mg) yield Crude ^a 1400 0.70 1.21 1.71 Anion exchanger 4.5 5.8 377 65 100 Goal filtration ⁰ 18 0.74 75.6 101 80

^a ) 1.4 liters of L1 culture were prepared as described in the application. The culture was centrifuged for 30 min at 10,000 x g. The supernatant fluid was then filtered and on a 0.8 µm filter to remove cell debris. This crude supernatant was the starting material for the purification procedure.

b) Passage through a DEAE sephacel column followed by dialysis against polyethylene glycol.

^c) Sephadex G-100

Tab. 10

Delignification activity of α-L-arabinofuranosidase and T6 xylanase on partially bleached pulp

Enzyme ^a ) Deli gni fi caci a (%) ^b Whether or not PH Pretty no enzyme 8.0 5.3 no enzyme 9.0 5.2 α-L-Arabinofuranoziase 8.0 7.6 2.3 α-L-Arabinofuranoziase 9.0 7.3 2.1 xylanase 8.0 19.5 14.2 xylanase 9.0 16.8 11.6 α-L-arabinofuranosidase + xylanase 8.0 24.5 19.2 α-L-arabinofuranosidase + xylanase 9.0 24.6 18.4

^a ) Purified α-L-arabinofuranosidase (38 units / ml, specific activity 126 U / mg) and xylanase T6 (5 U / ml, 192 U / mg) were used. The enzymes were in 10 mM phosphate buffer.

The conditions used were: 10 mM Na ₂ SO ₄ and 10 nM (NH ₄ ) ₂ SO ₄ , 65 ° C at pH 8.0 or 9.0.

Industrial usability

The present invention can be advantageously used in the pulp industry for delignification and bleaching of all types of lignin-containing wood pulp, especially in processes in which the application of chlorine is not desired.

LIST OF SEQUENCES

(D General information and: (I) When voting: (A) First Name: Korsnas AB (B) Street: unlisted (C) The city: Gavl e (E) Country Sweden (F) Zip: S-801 81 (G) Phone: 026-151000 (H) Telef ax: 026-195253 (A) name: The Technion Research and Development Foundation Ltd. (B) U1i ca: Technion City (C) The city: Hai fa (E) Country: Israel (F) Zip: specified (A) name: Ramot - Universi ty Authority for Applied Res.S Ind.Dev.Ltd (B) U1i ca: 32 H Levanon Street (C) The city: Tel Aviv (E) Country: Israel (F) Zip: specified (II) Inv Name Disease: Del-igniting preparation exhibiting alpha-L-arabi-furanosidase activity, process for its production and use (III) Number se 3 (IV) i Mold on reading from computer: (A) Carrier type : F1 oppy disk (° C) Computer: IBM PC compatible l Operating System: PC-DOS / MS-DOS (D) software: Patentln Release # 1.0, Verses # 1.24 (EPO) (2) INFORMATION FOR SEQ ID NO (I) sequence Characteristics: (A) length: 9 amino acids (B) Type: ami nokyseli at (D) Topologies and: unknown

(II) Molecule type: proteí n (III) Hypoteti cký: not (IN) Fragment type: N-terminal (VI) Original source: (A) Organism: Bacillus stearothermophius

(XI) Sequence description: SEQ ID NO: 1:

Xaa Gin Pro Tyr Arg Xaa Glu Glu Leu 1 5 (2) INFORMATION FOR SEQ IDENT No.2 (I) Sequence Characterization:

(A) Length: 23 amino acids (B) Type: amino acid (D) Topology: unknown (II) Molecule type: protein (III) Hypothetical: no (V) Fragment type: N-terminal (VI) Original source:

(A) Organism: Bacillus stearothermophilus (XI) Sequence description: SEQ ID NO: 2:

Ser Met Lys Lys Ala Thr Met White Glu Lys Asp Phe Lys White Al 1 10 15

Glu clay Asp Lys Arg clay Tyr (2) INFORMATION FOR SEQ IDENT No.3 (I) Sequence characteristic:

(A) Length: 50 amino acids (B) Type: amino acid (D) Topology: unknown (II) Molecule type: protein (III) Hypothetical: no (V) Fragment type: N-terminal (VI) Original source:

(A) Organism: Bacillus stearothermophi1us (XI) Sequence description: SEQ ID NO: 3:

Al a Thr Lys Lys Al a Thr Met Ile Ile Glu Lys Asp Phe Lys Ile Al a 1 5 10 15 G1 u Ile Asp Lys Arg Ile Tyr Gly Ser Phe Ile G1 u H i s Leu Gly Arg 20 25 30 Ala wall Tyr Gly Gly Ile Tyr G1 u for Gly Hi s for gin Al a Asp G1 u 35 40 45

Asn Gly

Claims (17)

PATENT CLAIMS A preparation exhibiting enzymatic activity, characterized in that it is capable of being aerobically fermentable in a suitable medium of a strain of Baci11us stearothermophilus obtained by selection from strain NCIMB 40494 and its mutants and variants, which has substantially the same ability to produce said preparation49 as strain NCIMB 40. wherein the enzymatic activity of the formulation comprises a substantial fraction of the activity of αL-arabinofuranosidase with the ability to delignify wood pulp at pH 8-9 and a temperature of 65 ° C. Composition according to claim 1, characterized in that it consists of a purified culture medium. Composition according to claim 2, characterized in that it consists of a concentrated fraction of culture medium showing α-L-arabinofuranosidase activity in wood pulp environment. Composition according to claim 3, characterized in that the activity of α-L-arabinofuranosidase is derived from α-L-arabinofuranosidase, which is composed of two subunits with an approximate molecular weight of 52,000 Da and 57,500 Da. Composition according to any one of claims 1 to 4, characterized in that it is associated with a composition having enzymatic activity produced by another microbial strain and having the ability to delignify wood pulp at a temperature of at least 65 and a pH of at least 9. A composition according to claim 5, characterized in that it comprises α-L-arabinofuranosidase produced by NCIMB strain 40494 Bacillus stearothermophilus and xylanase produced by NCIMB strain 40222 Bacillus stearothermophilus. An α-L-arabinofuranosidase enzyme characterized by an amino acid sequence comprising an N-terminal amino acid partial sequence (SEQ ID NO: 1) Met? Gin Pro Tyr Arg Pro? G1u Glu Leu or a homologue thereof. 8) Enzyme α-L-arabinofuranosidase characterized by an amino acid sequence that contains an N-terminal amino acid partial sequence (SEQ ID NO: 2) Ser Met Lys Lys Al and Thr Met Goals Glu Lys Asp Phe Lys Goes Ala Glu Goes Asp Lys Arg Goes Tyr or homologue thereof. A method for producing a preparation exhibiting enzymatic activity, characterized in that the Bacillus stearothermophilia strain selected by selection from NCIMB 40494 strain, including mutants or variants thereof, having substantially the same ability to produce the preparation as NCIMB 40494 strain, is subjected to aerobic fermentation in a suitable fermentation. medium, wherein α-L-arabinofuranosidase activity with the ability to delignify wood pulp at pH 8-9 and a temperature of 65 ° C occupies a substantial part in the enzymatic activity of the preparation. An isolated strain of Bacillus stearothermophi 1 us NCIMB 040494, including mutants and variants thereof, having substantially the same ability to produce a preparation exhibiting enzymatic activity as strain NCIMB 40494, which occupies a substantial part of α-L-arabinofuranosidase activity in this enzymatic activity. wood pulp at pH 8-9 and 65 ° C. Use of an enzyme produced by the Baci11us stearothermophilia NCIMB 040494 strain in the processing of wood pulp. A method comprising treating wood pulp, characterized in that the wood pulp is treated in at least one operation with a preparation according to one of claims 1 to 6. The method of claim 12, wherein the wood pulp is kraft pulp. The method of claim 13, wherein the wood pulp is a partially delignified kraft cell. The method of claim 13, wherein the delignified kraft pulp is an oxygen delignified kraft pulp. Wood pulp, characterized in that it is treated in at least one operation with a preparation according to any one of claims 1 to 6. 17. A DNA or RNA probe recognizing a nucleotide sequence that encodes an amino acid sequence (SEQ ID NO: 1)