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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
  • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C9/00—After-treatment of cellulose pulp, e.g. of wood pulp, or cotton linters ; Treatment of dilute or dewatered pulp or process improvement taking place after obtaining the raw cellulosic material and not provided for elsewhere
  • D21C9/10—Bleaching ; Apparatus therefor
  • D21C9/1026—Other features in bleaching processes
  • D21C9/1036—Use of compounds accelerating or improving the efficiency of the processes

Definitions

• the invention relates to a preparation exhibiting enzymatic activity comprising a substantial portion of ⁇ -L-arabinofurano-
• sidase activity having the capability of delignifying wood pulp at a pH of 8 - 9 and a temperature of 65°C. Further, it relates to a method of producing said preparation by aerobic fermenta ⁇ tion of a selected Bacillus stearothermophilus strain. The invention also comprises said selected strain. Moreover, it
• 15 relates to a process comprising treatment of wood pulp with a preparation of the invention and also wood pulp that has been treated with said preparation. Additionally, it relates to the use of an enzyme expressed by the deposited Bacillus stearother ⁇ mophilus strain NCIMB 40494 in the treatment of wood pulp. Also,
• ⁇ -L-arabinofuranosidase having an amino acid sequence which comprises one or two specified partial amino acid sequences or homologues thereof.
• the only strain shown to produce an arabinofuranosidase is a non-deposited mutant strain designated BPS-3-H-17-4. It should further be mentioned that the preparations used in the experiments shown in the application (cf. top of page 17) have been conducted with fermentation products obtained from said non-deposited strain. On page 8, lines 12-16 is disclosed that said strain derives from one of the deposited strains after mutagenesis with ethylmethansulfonate. This mutagen is very unspecific and no conditions of how to arrive at an arabinofura- nosidase-producing strain is given. Thus, there is still not disclosed in the art how to obtain an arabinofuranosidase from a thermophilic Bacillus.
• the present invention provides a preparation exhibiting enzymatic activity comprising a substantial portion of ⁇ -L- arabinofuranosidase activity having the capabality of deligni ⁇ fying wood pulp at a pH of 8 - 9 and a temperature of 65°C.
• wood pulp is to be interpreted broadly, and thus it is intended to comprise all kinds of lignocellulosic materials.
• One aspect of the invention is directed to a preparation exhibiting enzymatic activity, which is obtainable by aerobic fermentation, in a suitable medium, of a Bacillus stearother ⁇ mophilus strain selected from the strain NCIMB 40494 and mutants and variants thereof having substantially the same capability of producing said preparation as said strain NCIMB 40494, the enzymatic activity of said preparation comprising a substantial portion of ⁇ -L-arabinofuranosidase activity having the capabilitiesi ⁇ ty of delignifying wood pulp at a pH of 8 - 9 and a temperature of 65°C.
• the prepara ⁇ tion is a clarified culture broth.
• the preparation is a concentrated fraction of said culture broth exhibiting ⁇ -L-arabinofuranosida ⁇ se activity in a wood pulp medium.
• the ⁇ -L-arabinofuranosidase activity derives from an ⁇ -L-arabinofuranosidase which is composed of two sub-units having the approximative molecular weights of 52 500 Da and 57 500 Da, respectively.
• the preparation according to the above disclosed aspect of the invention is in association with a preparation exhibiting enzymatic activity produced by another microbial strain and having the capability of deligni- fying wood pulp at a temperature of at least 65°C and a pH of at least 9.
• the preparation comprises an ⁇ -L-arabinofuranosidase produced by the Bacillus stearothermophilus strain NCIMB 40494 and a xylanase produced by the Bacillus stearothermophilus strain NCIMB 40222.
• One additional aspect of the invention is directed to an ⁇ -L- arabinofuranosidase having an amino acid sequence which comprises the N-terminal partial amino acid sequence SEQ ID NO: 1:
• Another additional aspect of the invention is directed to an ⁇ -L-arabinofuranosidase having an amino acid sequence which comprises the N-terminal partial amino acid sequence SEQ ID NO: 1
• such a homologue may e.g. have a sequence which has 80% or more amino acids in common with the sequence SEQ ID NO: 1 and /or the sequence SEQ ID NO: 2.
• sequence SEQ ID NO: 3 An example of a homologue of the sequence SEQ ID NO: 2 is the sequence SEQ ID NO: 3:
• ⁇ -L-arabinofuranosidase which is the N-terminal sequence of an ⁇ -L-arabinofuranosidase produced by the Bacillus stearothermophilus strain NCIMB 40222.
• Said ⁇ -L-arabinofuranosidase is composed of two identical sub- units having an approximate molecular weight of 64 000 Da each (the native enzyme, 128 000 Da as judged by SDS gel).
• a homologue of the sequence SEQ ID NO: 1 and /or the sequence SEQ ID NO: 2 is any amino acid sequence which is sufficiently homologous on the nucleotide level to be recognized by any DNA or RNA probe derived from said sequence(s).
• Yet another aspect of the invention is directed to a method of producing a preparation exhibiting enzymatic activity, whereby a Bacillus stearothermophilus strain selected from the strain NCIMB 40494 and mutants and variants thereof having substantial ⁇ ly the same capability of producing said preparation as said strain NCIMB 40494, is subjected to aerobic fermentation, in a suitable medium, the enzymatic activity of said preparation comprising a substantial portion of ⁇ -L-arabinofuranosidase activity having the capability of delignifying wood pulp at a pH of 8 - 9 and a temperature of 65°C.
• Still another aspect of the invention is directed to the isolated Bacillus stearothermophilus strain NCIMB 40494 and mutants and variants thereof, said mutants and variants having substantially the same capability of producing a preparation exhibiting enzymatic activity as said strain NCIMB 40494, said enzymatic activity comprising a substantial portion of ⁇ -L- arabinofuranosidase activity having the capability of deligni ⁇ fying wood pulp at a pH of 8 - 9 and a temperature of 65°C.
• a further aspect of the invention is directed to the use of an enzyme expressed by the B_;_ stearothermophilus strain NCIMB 40494 for treatment of wood pulp.
• said strain produces several types of extra- cellular carbohydrate-degrading enzymatic activities which derive from the expression of different genes comprised by the genome of said bacterium.
• An additional aspect of the invention is directed to a process comprising treatment of wood pulp, whereby wood pulp is treated in at least one- step with a preparation according to the invention.
• the wood pulp to be treated is sulphate pulp.
• the sulphate pulp to be treated is a partially delignified sulphate pulp.
• the partially delignified sulphate pulp to be treated is an oxygen-delignified sulphate pulp.
• a further aspect of the invention is directed to wood pulp which has been treated in at least one step with a preparation ' according to the invention.
• Still another aspect of the invention is directed to a DNA or RNA probe which recognizes the nucleotide sequence coding for the amino acid sequence SEQ ID NO: 1: Met? Gin Pro Tyr Arg Pro? Glu Glu Leu and/or the amino acid sequence SEQ ID NO: 2:
• the last mentioned aspect of the invention is useful in the finding of ⁇ -L-arabinofuranosidases having the capability of delignifying wood pulp at a temperature of at least 65°C and a pH of at least 8 - 9.
• the Bacillus sterothermophilus strain LI was deposited under the Budapest Treaty at the National Collections of Industrial and Marine Bacteria Ltd (NCIMB), Aberdeen, on March 24, 1992 and was given the accession number NCIMB 40494.
• Bacillus stearothermophilus strain T-6 given the accession number NCIMB 40222 by the same depository, was deposited earlier in connection with the filing of the priority application for WO 91/10724.
• AKP-1 was supplemented with salts (0.1% MgS0 4 .7H 2 0, 10 mM K 2 HP0 4 , 0.1% urea and trace elements) and inoculated with mud and water samples taken from the water treatment pools at Korsnas.
• the flasks were incubated with shaking at 65°C and pH 9.0 for 48 h.
• a drop of turbid culture was then inoculated into a flask containing 15 ml of 0.2% galactomannan (Locust bean gum, Sigma) medium. After incubation for 24 hours at 65°C and pH 9.0, the culture was streaked onto LB agar. After incubation, approxima- tely 20 different colony types were isolated and obtained in pure culture.
• strain LI that contained a thermophilic ⁇ -L- arabinofuranosidase.
• Strain LI was a Gram-positive, thermophilic bacterium; it grew well at 60-65°C, but did not grow at less than 40°C. It was rod- shaped with a terminal spore, aerobic and oxidase-positive. Strain LI grew on the following carbon sources: D-glucose, D- xylose, L-arabinose, D-mannose and xylan. It did not grow on melobiose and galactose as carbon sources. Based on these properties LI was classified as a strain of the heterogenous group. Bacillus stearothermophilus.
• Strain LI was selected for further study because the crude extracellular fluid of culture grown on galactomannan medium was active on delignification of K15 pulp.
• a sensitive spectrophotometric assay was used for the estimation of how much lignin is made extractable by the enzymatic preparation.
• the absorbance values at 350 nm(A 350 ) were measured on the supernatant liquids of the samples, and measured values are presented as % lignin released.
• the growth and delignification activity of strain LI grown on different carbon sources is summarized in Table 1. The strain grew to some extent on the yeast extract and casamino acids without addition of another carbon source and yielded a net delignification of 5.1%.
• Xylanase activity was determined by incubating a fresh solution of xylan with extracellular supernatant fluid and assaying for increase in reducing sugars by the ferricyanide method (Spiro, R.G. 1966, Methods in Enzymology 8 . : 7-9) .
• Assay buffer was 50 mM Tris. Cl, pH 7.0 and 0.5% xylan (oat spelts, Sigma). Units of activity for xylanase are ⁇ mol reducing sugar generated per minute at 65°C.
• the arabinofuranosidase assay is a modification of the standard assay for ⁇ -galactosidase (G. Tabolt and Syguson, Appl. & Enviro. Microbiol, vol 56, No-. 11, 1990).
• Test tube contains 0.5 ml of 40 mM Tris-buffer, pH 7.0. 0.4 ml of the enzyme sample and 0.1 ml 10 mM p-nitrophenyl- ⁇ -L-arabinofuranoside (Sigma Chemicals) was incubated at 65°C for 15 min. The reaction was terminated by putting the test tube in ice water bath. The release of p-nitrophenol is determined spectrophotometricaly at 401 nm. 10 micromole of p-nitro-phenol per ml has an absorbance of 18.4. A unit of enzyme acting is defined as micromole of p- nitrophenyl release per min.
• ⁇ -L-arabinofuranosidase (AF) activity was concentrated from a ten liter culture of LI grown on A-medium (Table 3). The activity was precipitated with 80% saturated ammonium sulfate, yielding a crude enzyme preparation with 11 units per ml AF and 0.96 units per ml of xylanase. The AF activity did not bind to carboxyl-methyl cellulose (CMC), but did adsorb completely to DEAE cellulose or DEAE-Sephacel.
• CMC carboxyl-methyl cellulose
• the concentrated AF demonstrated significant delignification activity (Table 4) . It should be pointed out that we have not yet optimized the conditions for using AF to delignify pulp. A potentially useful property of AF is that it should break the bond close to lignin, thereby leaving most of the hemi-cellulose with the cellulose fibers. This should give a higher yield of delignified pulp. In addition, it appears that the delignifica ⁇ tion activity of AF plus xylanase T-6 is more than additive.
• Tables 5-7 describe some of the enzymatic properties of the purified AF, using PNP- ⁇ -L-Ara as substrate.
• the enzyme is most active between pH 6.5-8.0, with an optimum at pH 7.0.
• the enzyme has low activity at pH 9.0 at 70°C.
• the temperature optimum was 70°C at both pH 7.0 and pH 8.0.
• the enzyme was most active at 20 mM Na 2 S0 4 , pH 7.5 in 10 mM phosphate buffer at 70°C.
• pH 7.0 the enzyme was very stable at 60°C, but lost about 50% of its activity at 70°C during 75 min and was completely inacti ⁇ vated at 80°C for 15 min. Extended experiments (with K20 pulp)
• Xylanase and two other endohemicellulases were assayed in 10 mM phosphate buffer, pH 8.0 at 60°C in 1 ml total assay volume.
• An appropriately diluted enzyme sample (0.1 ml) was added to 0.25 ml substrate, 0.1 ml 100 mM buffer and 0.55 ml water. The reaction was terminated by transferring to an ice water bath. Reducing sugar was determined by 3,5- dinitrosalicylic acid method as described by Miller (Miller, GL, (1959) Anal Chem 31:426-428).
• the substrates were 4% Oat splet xylan, 0.4% Locust Bean Gum-galactomannan, 4% arabinogalactan from Larchwood and 4% mannan from Saccharomyces cerevisiae.
• the enzymatic release of lignin from softwood pulp was determi ⁇ ned by adding 200 mg wet weight of K-20 pulp (from the Korsnas paper mill Gavle, Sweden) to 3 ml of enzyme solution, adjusting to pH 8.0 or 9.0 with a concentrated NaOH solution, and incubating at 65°C with shaking. After 2 h, 1.5 ml of the liquid was removed and centrifuged at 10,000 x g for 5 min in a minifuge to remove residual pulp fibers. The clear liquid (0.5 ml) was diluted with 1.0 ml of 0.1 N NaOH for determination of absorbance at 350 nm. The control for each assay was incubation of the pulp under the same conditions without enzyme. Since 1 mg lignin per ml had an A 350 of 9.1, and pulp used in these experiments had a lignin content of 1.3% (Klason, determined by Z. Zosim) , the
• % lignin released ⁇ A 350 x 3 x 100
• P x 0.013 where P is the pulp dry weight and ⁇ A 35Q is the absorbance after incubation minus absorbance before incubation. The net lignin released is the total minus the no enzyme control.
• xylanase was found in low concentration when the carbon sources in the growth media were locust bean gum (LBG) , D-glucose and L-arabinose.
• LBG locust bean gum
• the xylanase activity was amplified with D-xylose as the carbon source, reaching 1.23 U/ml.
• Mannose inhibited production of xylanase activity.
• ⁇ -L- arabinofuranosidase activity was low, but significant on D- xylose medium and high on L-arabinose medium, reaching 1.5 U/ml. No activities of mannase, ⁇ -D-galactosidase, ⁇ -D-mannosidase or ⁇ -L-arabinopyranosidase were found.
• the cell free extracellular fluid contained 1.2 U/ml of ⁇ -L-arabinofurano ⁇ sidase activity with a specific activity of 1.7 U/mg (Table 9).
• the purified enzyme was examined by FPLC Superose 12 gel filtration in 100 mM potassium phosphate buffer, 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. By SDS PAGE, the purified enzyme showed two bands with molecular weights of 57,500 and 52,500. Analysis of these two bands revealed, after blotting on PVDF membrane and sequencing on an Applied Biosystems model 475A gas phase sequencer, the two N-terminal sequences SEQ ID NO: 1: and SEQ ID NO: 2.
• the purified enzyme was completely stable at 60°C for at least 80 min, retained 50% of its maximum activity after incubation at 70°C for 75 min, and lost all its activity after 15 min at 80°C.
• the optimum pH for activity was 7.0; at pH 6.5 and pH 8.0, the activities were 55% and 50% of the optimum activity, respectively.
• the optimum temperature for activity was 70°C.
• the enzyme showed maximum activity in 20-50 mM Na 2 S0 4 ; at 100 mM and 150 mM Na 2 S0 4 , the activity decreased 10% and 50%, respectively.
• the kinetic parameters of the enzyme were measured using pNP- ⁇ - L-ara-f as the substrate.
• K m and V ma ⁇ were 2.2 x 10 " M and 101 ⁇ ol min ⁇ 1 mg -1 , respectively.
• the enzyme showed only low activity on high molecular weight substrates, such as arabinoxylan and arabinogalactan.
• Table 10 summarizes a typical experiment in which delignifica ⁇ tion activity was examined with pure ⁇ -L-arabinofuranosidase, pure xylanase and a mixture of the two enzymes.
• the mixture of 38 U/ml ⁇ -L-arabinofuranosidase and 5 U/ml xylanase T6 released a net of 19.2% of the lignin from the pulp, whereas the sum of each enzyme acting separately was only 16.5%.
• the mixture of enzymes released 18.4% lignin, compared to only 13.7% for the sum of the two activities acting separately.
• the two enzymes acted synergistically in releasing lignin from the pulp.
• the growth media consisted of carbon source (0.2%), 0.1% yeast extract, 0.5% casamino acids and E. salts (0.1% urea, 0.02% MgS0 4 '7H 2 0, 5mM KH 2 P0 4 , pH 9.0, 20 mM Tris ⁇ Cl, pH 9.0, 0.1% NaCl and standard trace elements mixture). All of the sugars were the D-configuration, except for L- arabinose; LB is locust bean galactomannane. Delignification of K15 was carried out with the extra ⁇ cellular supernatant (adjusted to pH 9.0), at 65°C for 2h.
• Xylan and LB-GM (locust bean galactomannan) degrading activities were determined by production of reducing su ⁇ gars and PNP-X activities were measured by liberation of PNP (p-nitrophenol) .
• Cells were grown for 24 hrs in either X-medium or A-medium (xylose or arabinose medium described in Table 1) and centrifuged to remove the cells.
• Xylanase T-6 is produced by NCIMB 40222, disclosed in WO
• 91/10724 is capable of delignifying wood pulp at a pH of at least 9 and a temperature of at least 65°C.
• LGB Locust Bean Gum, a galactomannan
• pNP is p-nitro- phenol.
• a 1.4 liter culture of strain LI was prepared as described in the specification. The culture was centrifuged for 30 min at 10,000 x g. The supernatant fluid was then passed through a 0.8 ⁇ m filter to remove residual cells. This crude supernatant was the starting material for the purification procedure.
• the conditions used were as follows: 10 mM each of Na 2 S0 4 and (NH 4 ) 2 S0 4 , 65°C, 2h at pH 8.0 or 9.0.
• NAME The Technion Research and Development Foundation Ltd
• NAME Ramot - University Authority for Applied Res.&Ind.De . Ltd
• ORGANISM Bacillus stearothermophilus
• ORGANISM Bacillus stearothermophilus
• ORGANISM Bacillus stearothermophilus

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