Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01032—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/189—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
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • 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)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2477—Hemicellulases not provided in a preceding group
  • C12N9/248—Xylanases
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
• • 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
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/07—Bacillus
  • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/832—Bacillus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/832—Bacillus
  • Y10S435/839—Bacillus subtilis

Definitions

• This invention relates to proteins with xylanase activity derived from bacteria, and in particular to xylanases which are free of any significant cellulase activity and which are active at high temperature and at neutral to alkaline pH. Xylanases having these characteristics are particularly useful in the bleaching of wood pulps, such as kraft pulps.
• Enzymes are proteins present in all living cells, where apart from controlling metabolic processes, they break down food materials into simpler compounds.
• the enzymes are catalysts which speed up processes which would otherwise proceed very slowly, or not at all.
• enzymes are very specific, breaking down only one type of compound.
• Xylan is a polysaccharide found in most plant cell walls, consisting of D-xylose units linked by ⁇ -1-4 glycosidic bonds. It occurs with another polysaccharide, cellulose and an amorphous binding polymer, lignin. Xylan forms a major component of plant hemicelluloses, and varies in the nature of substituents on the sugar groups, depending on the origin.
• xylans derived from hardwoods typically consist of a backbone of O-acetyl-4-O- methylglucuronoxylan, in which about 10% of the xylose units carry 4-O-methylglucuronic acid side chains linked via ⁇ -1,2 bonds, and 70% of the xylose residues are acetylated at C-2 or C-3.
• xylans derived from softwoods are usually arabino-4-O-methylglucuronoxylans in which over 10% of the xylose sub-units carry arabinofuranose residues linked via ⁇ -1,3 bonds.
• xylanases Enzymes which are able to degrade xylan are called xylanases (endo- 1,4- ⁇ -D-xylanases; International enzyme nomenclature EC 3.2.1.8) .
• the mashing process for the production of juices can be made to produce higher yields emd better processing with the application of cell wall degrading enzymes, which include xylanase.
• the primary source of cellulose for paper manufacture is wood, and may be either hardwood or softwood.
• the initial step in paper manufacture is the reduction of wood to the fibre state, which may be achieved by mechanical or chemical pulping methods.
• Chemical pulping involves the "cooking" of woodchips with chemical reagents in order to separate the cellulose fibres from the other wood components, and to break down the lignin and other extraneous compounds so that the cellulose is left in tact in its fibrous form.
• the most common process is the kraft or sulphate process, which can be applied to almost any timber species.
• the active ingredients are sodium hydroxide and sodium sulphide in a strongly alkaline solution.
• xylanase acts as a bleaching aid (bleach booster) by releasing some trapped residual lignin within the pulp matrix and giving better access to bleaching chemicals. It is widely believed that xylanase breaks down reprecipitated xylan which forms a coating on the pulp, thus releasing trapped residual lignin from within the pulp matrix, and allowing better access of bleaching chemicals to this matrix. Thus xylanase acts as a bleaching aid or bleach booster.
• the pulp In the kraft process, the pulp is typically handled at high temperatures and neutral to alkaline pH.
• Commercial xylanases typically have a temperature optimum of about 50°C and a pH optimum of about 5, and are thus subject to rapid denaturation under process conditions.
• xylanases which are able to act optimally on the kraft pulp without any requirement to adjust the temperature or pH.
• the xylanase In order to be useful as a bleaching aid, the xylanase must also be free of any significant cellulase activity, since cellulase would cause an undesirable loss of cellulose fibre.
• bacterium is a strain of Bacillus Subtilis which we have designated B230.
• the invention provides a bacterium, isolatable from wood compost, having the following characteristics:
• the bacterium is isolated such that a biologically pure culture exits.
• xylanase production is enhanced by growth in the presence of xylan or of lignocellulose substrates, or degradation products, including xylose and xylitol, derived from such substrates.
• the xylanase has at least one characteristic selected from the group consisting of activity at about pH between 4.5 and 9.5, a thermal activity range up to 70°C, and high thermal stability up to 65°C.
• the xylanase produced by the bacterium is effective on both soluble and insoluble xylans.
• the bacterium has the characteristics of the bacterial isolate designated B230, as deposited under the provisions of the Budapest Treaty in the Australian Government Analytical Laboratories, PO Box 385, Pymble, New South Wales 2073, Australia, on 6 September 1994, under Accession No. N94/41262, or a mutant or derivative thereof having the ability to produce a xylanase as described above.
• the term "mutant or derivative” thereof includes naturally occurring and artificially induced mutants which retain their ability to digest xylans. Production of such mutants or derivatives will be well known by those skilled in the art.
• the invention provides a process for producing at least one protein with xylanase activity said process comprising cultivating a bacterium under conditions and for a time sufficient to produce said protein and collecting culture medium wherein said bacterium has the following characteristics: A. Ability to grow at a temperature between 20 and 45°;
• the bacterium used is strain B320 or a mutant, variant or derivative thereof.
• the bacterium is grown under optimal conditions for extracellular production of said at least one protein. Still more preferably the production of said at least one protein is induced by the addition of xylitol to the culture medium.
• xylitol is added in an amount of 0.01 to 2% of the culture medium which is preferably a broth.
• the invention provides a protein with xylanase activity said protein having an associated cellulase activity of less than 0.1% and a molecular weight of about 28kD as determined by SDS- PAGE.
• the protein has at least one characteristic selected from the group consisting of activity at about pH between 4.5 and 9.5, a thermal activity range up to 70°C and high thermal stability up to 65°C.
• the protein is effective in digesting both soluble and insoluble xylans.
• the protein with xylanase activity is isolatable from the bacterium described above. More preferably the protein is isolated from the bacterial strain B230.
• the protein with xylanase activity is an isolated preparation meaning that it has undergone some purification away from other proteins and/or non- proteinatious material.
• the purity of the preparation may be represented as at least 40% protein with xylanase activity, preferably at least 60% protein, more preferably at least 75% protein with xylanase activity, still more preferably at least 80% protein with xylanase activity or greater, as determined by weight, activity, amino acid composition or similarity, antibody reactivity or any other convenient means.
• the invention provides a composition
• a composition comprising said protein with xylanase activity as an active ingredient together with an industrially acceptable stabiliser.
• the composition may be used as a bleaching aid or bleaching booster or in paper deinking.
• industrially acceptable stabilisers which may be used such as glycerol, sorbitol or other polyalcohols.
• composition described above is for use in bleaching kraft pulp or deinking paper. Accordingly, in a fifth aspect the present invention provides a method of bleaching wood or paper pulp comprising administering a bleaching aid or bleaching booster effective amount of the composition to said pulp, for a time and under conditions sufficient to achieve the desired bleaching of the pulp.
• the protein of the present invention may also be used in the preparation of animal feed and in preparation of dough for bread-making.
• the bacterium B230 when grown under suitable fermentation conditions, will produce xylanase which accumulates in the extracellular fermentation broth.
• the xylanase from such a broth has a thermal activity range from ambient up to 70°C and a useful pH range from 5 to 9, with optimal activity at pH 6 - 6.5.
• the xylanase has very high thermal stability, retaining 100% activity after 3 hrs and 90% activity after 22 hrs at 60°C. Cellulase activity associated with the xylanase is minimal ( ⁇ 0.1%).
• the crude preparation may be used however partially purified xylanase may also be used.
• Figure 1 shows the variation of activity of xylanase from bacterium B230 with pH compared with that from bacterium B698,
• Figure 2 illustrates the variation in activity of xylanase from bacterium B230 with temperature, compared with that from bacterium B698.
• Figure 3 is a photograph of a SDS-PAGE gel of the purified enzyme having an approximate molecular weight of
• Figure 4 is a photograph of a SDS-PAGE gel of fermenter broth proteins including xylitol induced xylanase. Compared with proteins from non-induced cultures, the xylanase protein can be identified as having an approximate molecular weight of 28kD.
• Figure 5 illustrates the colour units release by xylanase from bacterium B230 at a range of pH and temperatures.
• a bacterium which we have designated B230 was isolated from a sample of white-rotted karri wood; this sample was collected from near Walpole, Western Australia, in May 1993.
• the composition of Luria-Bertani medium is: tryptone lOg yeast extract 5g sodium chloride lOg deionised water 1L
• LB Luria-Bertani
• All media were sterilised by autoclaving at 121°C for 20 minutes. The organism was isolated in pure culture, and a sample was deposited under the Budapest Treaty in the Australian Government Analytical Laboratories as described above.
• the bacterium has the following taxonomic characteristics: rod-shaped bacterium with a centrally-located spore
• the bacterium is not fastidious, and can be grown on a range of media, including LB broth.
• the requirements are: 1. a source of carbon, most conveniently a carbohydrate such as dextrose,
• the bacterium can be grown within the temperature range 20 to 45°C and within the pH range 5 to 9.5.
• the bacterium can be grown successfully under different fermentation conditions, including solid state or submerged culture; fermentation continues under aerobic conditions with or without agitation.
• bacterium B230 When grown under the conditions described in Example 2, bacterium B230 synthesises xylanase, and releases the enzyme into the extracellular medium. While xylanase is produced constitutively, addition of xylan to the culture medium as an additional carbon source further enhances the level of xylanase production.
• the added xylan may be in the form of isolated wood xylan, or may be a component of lignocellulosic material such as wheat bran.
• Xylanase was assayed using the following conditions:
• Buffer 50mM sodium phosphate/citric acid, pH 6.
• Incubation temperature 50°C
• the enzyme reaction was stopped with 3,5- dinitrosalicylic acid (DNS) reagent which measures, using xylose standards, the amount of reducing sugar produced in 20 minutes. Enzyme units are expressed in nanokatals (nkats), where 1 nkat is the amount of xylanase which will produce 1 nmole of xylose per second under the defined conditions.
• NDS 3,5- dinitrosalicylic acid
• Xylanase from B230 can conveniently be prepared by submerged fermentation.
• B230 seed culture can be prepared overnight in LB broth at 37°C. This inoculum is added to an LB broth containing beechwood xylan (2% w/v) .
• the pH of the broth is increased to pH 7.8 by the addition of 2M sodium hydroxide, and the temperature adjusted to 37°C.
• the broth is stirred (1,000 rpm) and aerated with filtered sterile air (0.7 L of air/L of broth/min) .
• the seed inoculum is added to the broth and the above conditions of temperature, pH, agitation and aeration maintained. Samples of culture are taken at regular intervals to monitor the production of xylanase. Optimal levels of xylanase (11,000 nkat/mL) are obtained within 90 hours of fermentation.
• the crude enzyme preparation from the fermenter broth was characterised with respect to pH and temperature.
• the xylanase activity was determined as described above, with the exception that the buffer was changed to obtain a stable pH. The results are listed in Table 1 below. The data is further expressed in Figure 1. The optimal pH for xylanase activity was found to be pH 6-6.5. Table 1 pH Profile of B230 Xylanase
• the xylanase activity of B230 enzyme was determined as described above, except that the temperature was altered within the range from 40 to 80°C. Results are listed in Table 2 and further expressed in Figure 2. The optimal temperature for xylanase activity was found to be 60°C.
• B230 xylanase The stability of B230 xylanase was determined at pH 6 and 60°C, the optimal pH and temperature respectively for the enzyme system. Samples were tested for residual activity at regular intervals as described in the xylanase assay conditions above. After 3 hours, 100% xylanase activity was retained. Even after 22 hours, 90% of the xylanase activity was retained. Thus, xylanase from B230 is very thermally stable.
• the stability of B230 xylanase was determined at 4°C by storing it at that temperature. Samples were tested for activity at regular intervals under the conditions described in the xylanase assay conditions above. After 22 days, 100% of the original activity was retained.
• a fraction of xylanases was partially purified by conventional purification techniques involving DEAE Sepharose and size exclusion chromatography.
• the xylanase fraction had a single band on SDS-PAGE at 28kDa as shown in Figure 3 and a purity of > 80%.
• B230 seed culture was prepared overnight in LB broth at 37°. This inoculum was added equally to 2 flasks containing corn steep liquor (2%) and incubated at 37°C. To one flask, xylitol (to 0.1%) was added daily for 5 days. After 5 days both flask broths were centrifuged. The cell free broths were assayed for xylanase activity. Xylitol induces xylanase (2,OOOnkat/ml) compared with uninduced broth (50nkat/ml) . A sample of each broth was concentrated by ultrafiltration (5kDa membrane), and the retentate run on an SDS-PAGE gel. As shown in figure 4, a protein band at approximately 28kDa was induced by xylitol. This is consistent with the purified xylanase in Example 8, figure 3.
• X - xylanase treatment pH 5, 7 or 9, 60°C, 1 hr Kappa number is a measure of the amount of lignin in wood pulp. It is defined as the number of millilitres of 0.02M potassium permanganate solution which would be consumed by 1 gram of moisture-free pulp under AS 1301, APPITA P201 m-86, specified conditions.
• Example 11 Use of B230 as a Bleaching Aid - further example
• the crude xylanase system (167nkat/g of pulp) was mixed with unbleached kraft pulp (35g oven dried basis) at 8% consistency and adjusted to pH 5,6,7,8,9 or 10 with appropriate buffer.
• the mixture was incubated for 1 hr at either 50,60 or 70°C. After the set time, the pulp was filtered to obtain a filtrate sample.
• the filtrate sample was briefly centrifuged and the absorbance at 456 nm was measured in a spectrometer.
• Absorbance units were converted to Pt-Co colour units from a standard graph where 500 colour units was obtained by dissolving K 2 PtCl 6 (1.246g), CoCl 2 .6H 2 0 (l.OOg) and HC1 (lOOmL, 12M) in 1L of water.
• the colour units released from the pulp by the xylanase is a measure of the final bleach chemical savings.
• the optimal effective pH was found to be pH 7, independent of temperatures between 50 and 70°C (see figure 5) .
• PCT/AU95/00202 describes a xylanase-producing bacterium designated B698, which was isolated from wood compost, and which was deposited under the Budapest Treaty in the Australian Government Analytical Laboratories as Accession No. 94/7647.
• the temperature profile, pH profile, thermal stability at 60°C and 65°C at different pH values, and bleach boosting activity of xylanases for B230 and B698 were compared, using the methods described above, and the results are summarised in Tables 5 to 9.
• B698 xylanase retains more activity over a wider temperature range at pH 6.
• B6 8 xylanase is clearly more thermally stable at 65°C over the pH range 6-9 than B230 xylanase.
• B230 xylanase is more effective than B698 as a bleach xylanase boosting agent. This is a significant advantage.
• bacterium B230 expresses more xylanase than bacterium B698 (11,000 nkat/ml and 7,000 nkat/ml respectively.

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• 1994
  • 1994-10-26 AU AUPM9008A patent/AUPM900894A0/en not_active Abandoned
• 1995
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