NZ241453A

NZ241453A - Xylanase and bacillus strains for its production.

Info

Publication number: NZ241453A
Application number: NZ24145392A
Authority: NZ
Inventors: Eric Samain; Philippe Debeire; Michele Debeire-Gosselin; Jean-Pierre Touzel
Original assignee: Agronomique Inst Nat Rech
Priority date: 1992-01-30
Filing date: 1992-01-30
Publication date: 1994-02-25

Description

<div class="application article clearfix" id="description"> Patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 U.Z. PATENT OFFICE 30 JAN 1992 COMPLETE SPECIFICATION RCCE!V""> XYLANASE, BACILLUS STRAINS PRODUCTIVE OF XYLANASE AND THEIR USES WE, INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE (INRA), a French company of 147, rue de 1'Universite, 75341 Paris Cedex 07, FRANCE hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: - 1 - (followed by page la) - la- The present invention relates to a xylanase, as well as to Bacillus strains productive of this enzyme. It also relates to the use of this enzyme and of these bacteria in the bleaching of paper pulp and the 5 preparation of xylose or of xylo-oligosaccharides from plant raw materials, in particular. Varied uses have been proposed for xylanases in the biotechnology field, especially in the foodstuffs field (Biely, Trends Biotechnol 3 (11): 286-290, 1985), 10 in the paper industry (Mora et al., J. Wood Chem. Technol, 6 : 147-165, 1986) or in the production of chemical compounds from hemicellulose (Reilly P.J., 1981, Xylanases: structure and function in trends in the biology of fermentations for fuels and chemicals. 15 A.J. Hollaender (Ed), Plenum, New York). The technical feasibility of such applications has been assessed chiefly using enzymes produced by mesophilic fungi. However, such applications could be facilitated by the use of fungi possessing better tem-20 perature stability. Various bacteria and enzymes are known for the production of xylanases (see, in particular, Wong et al. , Microbiological Reviews, 52, No. 3, 305-317, 1988). Hitherto, the highest yields of enzymes have been 25 obtained from fungi (Yu et al. Enzymo Microb. Technol. 9:16-24, 1987). However, hyperproductive strains of Bacillus have already been described (Okazaki et al. Appl. Microbiol. Biotechnology, 19: 335-340, 1984; Okazaki et al., Agric. Biol. Chem. 49, 2033-2039, 1985). 30 Such thermophilic species of Bacillus which degrade xylan can be good candidates for the industrial production of xylanases on account of their high growth rate and of their genetics being well known. The xylanases isolated by Okazaki et al. emanate 35 from two Bacillus strains referred to as W1 and W2 by the authors. In each of these strains, two components of the xylanase activity, referred to as I and II, have been demonstrated. The components I degrade xylan to xylobiose and to oligomers having a higher degree of (followed by page 2) - 2 - polymerisation, while the components II produce xylose in addition to the above compounds. The components I (referred to as Wl.I and W2.1) have respective molecular weights of 21.5 kDa and 5 22.5 kDa, as well as isoelectric points of 8.5 and 8.3. The components II (Wl.II and W2.II) have, for their part, respective molecular weights of 49.5 kDa and 50 kDa. The two components I and II are inhibited by Hg2+ ions and, to a lesser extent, by Cu2+. 10 Many other xylanases have been isolated from various species of Bacillus, Clostridium, Aspergillus, Streptomyces or Trichoderma, inter alia (Wong et al., 1988, cited above). Thus, the resume of Japanese Patent JP 130 96 84 15 (RIKAGAKU KENKYSHO) relates to a type WII xylanase having a molecular weight of 50 kD or 42 kD. No isoelectric point is mentioned for this xylanase. A paper by RAJARAM et al., (Applied Microbiology and Biotechnology, Vol. 34, No. 1, October 1990, pages 20 141-144) relates to a Bacillus strain isolated in the natural environment and which produces a xylanase having optimal activity at between 60°C and 70°C and at a pH of between 6 and 7. This enzyme is characterised neither by its molecular weight nor by its isoelectric point. This 25 strain produces, in addition, other enzymes such as cellulases. Another resume of a Japanese patent in the name of RIKAGAKU KENKYUSHO (JP-85 118 644) describes a xylanase having optimal activity at a pH of between 6 and 30 7. This enzyme is considered to have a molecular weight, determined by ultrafiltration, of between 50 and 100 kD. No isoelectric point is mentioned in this resume. A paper by GRUNINGER et al. (Enzyme Microbiology and Technology, Vol. 8, May 1986, pages 309-314) relates 35 to another Bacillus strains isolated from mud and which produces a heat-stable xylanase. The enzyme is characterised as having optimal activity at 78°C and at a pH value of 7.5. This enzyme is characterised neither by its molecular weight nor by its isoelectric pH. - 3 - The industrial production of xylanases is impeded by the simultaneous presence of contaminant activities such as cellulases, leading to additional purification costs. Furthermore, the use of genetically modified 5 microorganisms can present problems such as instability of the plasmids carrying the genes coding for the xylanases. As far as the Applicant is aware, the best productivity with respect to endoxylanase obtained with 10 a microorganism not producing cellulase has been obtained from a Streptomyces lividans mutant devoid of cellulase activity after introduction of a plasmid carrying genes coding for xylanases A and B. Productivities of the order of 6000 to 10,000 IU.l/h were observed in the culture 15 media. It should nevertheless be noted that, in this case, problems linked to the failure of xylanase A to hydrolyse insoluble xylan, and of thermal stability in the case of xylanase B, were encountered (Kluepfel et al. Biochem. J. 267, 47-50, 1990). 20 None of the enzymes described in the prior art hence possessed, as far as the Applicant is aware, features making an industrial application possible, that is to say good thermal stability, a large capacity for degradation of substrates and a means of production by 25 hyperproductive strains. The Applicant hence showed, surprisingly, that certain Bacillus strains produced heat-stable xylanases possessing high degradative activity. Still more surprisingly, the Applicant showed 30 that these Bacillus strains excreted large amounts of such enzymes without producing significant amounts of cellulases. The subject of the present invention is hence Bacillus strains hyperproductive of xylanase, and espe-35 cially the strains registered under Nos. 1-1017 and 1-1018 with the Collection Nationale de Cultures des Microorganismes (National Collection of Microorganism Cultures) of the Pasteur Institute. These strains were isolated from dung. - 4 - The bacteria occur as short, straight rods forming spores and averaging 0.5 ^ by 2.5 p in size. They are generally on their own or paired and display gram-positive staining, and the spores are oval and central. 5 These strains are strictly aerobic, no growth being observed in the absence of oxygen, and nitrate is not used as an electron acceptor. The optimal pH for growth and for production of xylanase is 7.8, while the limiting pH values for growth 10 lie between 6.5 and 8.5 and the maximal temperature is 63 °C. Apart from xylan, these strains utilise xylose, glucose, sucrose, maltose and also starch. Fructose, arabinose, cellulose, pectin and also chitin are not 15 utilised. Furthermore, growth is inhibited by a 5% concentration of sodium chloride. The G+C content of the DNA of these strains, determined by thermal denaturation, is 57.5 mol%. These strains are well differentiated from other 20 strains productive of xylanases, as shown in Table I, on the basis of the different culture times and of their different productivity with respect to xylanase. The strain 1-1018 was obtained by mutagenesis of the strain 1-1017. The strain 1-1018 possesses the same 25 general features as the strain 1-1017, but displays a hyperproduction of xylanase. The subject of the present invention is, moreover, a thermophilic xylanase, characterised in that it possesses a molecular mass of approximately 22 kDa and 30 an isoelectric point of approximately 7.7. This enzyme advantageously displays, moreover, great stability at 60°C for at least 24 hours, and a pH of optimal activity within the range extending from 4.8 to 7, and preferably approximately 6. 35 It should be noted that the pHi of this enzyme is fairly high but nevertheless lower than the pHi of the xylanases of similar molecular mass produced by some bacilli, in particular those described by Okazaki et al. (1985, publication cited above). P - 5 - pH 6 corresponds to an optimal pH, but the activity remains greater than 80% in the range between 4.8 and 7. The xylanase according to the invention can 5 possess the following N-terminal amino acid sequence: Asn-Thr-Tyr-Trp-Gln-Tyr-Trp-Thr-Asp-Gly-Ile-Gly-Tyr-Val-Asn-Ala-Thr-Asn-Gly-Gln. Advantageously, it comprises in its sequence 15 alanines, 6 arginines, 29 aspartates and asparagines, 19 10 glutamates and glutamines, 31 glycines, 3 histidines, 6 isoleucines, 7 leucines, 3 lysines, 1 methionine, 4 phenyl-alanines, 8 prolines, 19 serines, 13 threonines, 22 tyrosines and 14 valines. This enzyme is, furthermore, inhibited by Hg2+, 15 but not by Ag2+ (1 mM) or by 0.1% SDS. The Michaelis constant (K^) with respect to birch xylan is 0.9 g/1. The neutral oligosaccharides produced by this enzyme from birch xylan (5 g/1 of birch xylan; 500 U/l, 2 0 6 0°C) are composed: - with very short enzyme incubation times (15 minutes), of xylotriose and xylo-oligosaccharides having higher degrees of polymerisation; - with long incubation times (24 to 72 hours), of 25 xylobiose, xylotriose and xylotetraose, the latter in very small amounts. This endoxylanase is, in particular, excreted by Bacillus, and especially by Bacillus strains 1-1017 and 1-1018 described above. 30 It is excreted into the culture medium. The culture medium for these strains is, in particular, characterised by an absence of endo-^-1,4-glucanase (cellulase) activity and by the presence of a very weak contaminant £-xylosidase activity 35 (approximately 0.06 IU/ml of culture supernatant for both strains). It should be noted that the culture supernatant may be lyophilised without significant loss of xylanase activity, and is stored frozen for at least one year without loss of activity. Endo-0-1,4-glucanase activities were determined by measurement of the reducing sugars liberated from 1% carboxymethylcellulose (CMC) or Avicel (1%) in 50 mM sodium acetate buffer, pH 6. ^-Xylosidase activity was determined by measurement of the liberation of p-nitrophenol from p-nitrophenyl-^-D-xyloside (0.1%) in 50 mM sodium acetate buffer, pH 6. One unit of xylanase activity (IU) is defined as one micromole of xylose equivalent reducing sugar liberated per minute at 60 °C. The concentration of reducing sugar is determined by the method of Kidby and Davidson (Anal. Biochem., 55; 321-325, 1973). The purified enzyme, for its part, is stored in frozen form in 20% ethylene glycol without significant loss of activity for one year. The subject of the present invention is also a process for obtaining the xylanase as described above, comprising the following steps: - concentration of the Bacillus culture supernatant , - passage through an ion exchange column such as a column of Q Sepharose Fast Flow (Pharmacia), passage through a hydrophobic interaction column such as a column of Phenyl-Sepharose (Pharmacia). Concentration of the supernatant can, in particular, be performed by ultrafiltration through a polysulphone membrane having an exclusion threshold above 10 kDa. This process enables a substantially pure xylanase preparation to be obtained. The present application relates, moreover, to a process for the production of the xylanase described above, comprising the steps: - of growth of the bacteria in a medium containing a growth substrate such as glucose, and - of production of xylanase induced by feeding the culture continuously with suitable amounts of xylo-oligosaccharides. - 7 - The subject of the present invention is also the use of the xylanase described above in the bleaching of paper pulp. An advantage of this xylanase lies in the fact that the degree of hydration of the paper pulp is of little importance. It is not obligatory to dilute the pulp greatly in order to obtain good enzymatic attack. The use of this xylanase as an auxiliary in the bleaching of paper pulp is all the more advantageous for the fact that the preparations are devoid of cellulase contaminants. This xylanase may also be used for the preparation of xylose or of xylo-oligosaccharides from raw materials of plant origin, which are inexpensive and renewable raw materials (for example maize cobs). Other uses of xylanases have been mentioned in the literature. The review by Zeikus et al. (Thermostable saccharidases New Sources uses and Biodesigns in "Enzymes in biomass conversion", Leatham and Himmel, ACS Washington D.C., 1991) lists the main uses of xylanases. They are mainly used in food manufacture, where their properties enable bread-making, the clarification of fruit juices and wines and the nutritional qualities of cereal fibres to be improved, and in the production of thickeners for foodstuffs. The second sphere of application relates to the paper pulp and fibre industries, where they are used for the bleaching of pulps, the manufacture of wood pulp and the purification of fibres for rayon manufacture. Uses are also noted in poultry feeding, in which uses xylanases are employed in order to decrease the viscosity of the feeds (Van Paridon et al. Xylans and Xylanases, International Symposium, Wageningen, 8-11 December 1991; Bedford and Classen H.L. Xylans and Xylanases, International Symposium, Wageningen, 8-11 December 1991). The use of xylanase in enhancing the value of byproducts of the paper pulp industry is more specifically mentioned in the paper by Biely (Trends in Biotechnology, W - 8 - ™ Vol. 3, No. 11, 1985). Mention may also be made of the two European Patents EP 228,732 and EP 227,159, which relate to the use of xylanases for improving the filterability of 5 glucose syrup and of beer, respectively. The possibility of using the xylanases for the production of chemical compounds from hemicellulose (Reilly, cited above) will also be noted. These various publications show that the 10 xylanases which are the subject of the present invention may be used in a large number of applications. The present invention is illustrated, without, however, being limited, by the application examples which follow. 15 Figure 1 shows the changes occurring in discon tinuous cultures of the strain 1-1017. Figure 2 shows the changes occurring in a continuous culture of the strain 1-1017 fed continuously with solutions of xylo-oligosaccharides at different 20 concentrations. Figure 3 shows the comparative xylanase production of the strains 1-1017 and 1-1018 in the presence of different concentrations of xylo-oligosaccharides. Figure 4 shows the liberation of xylo-oligosac-25 charides from unbleached kraft pulp with different concentrations of xylanase. Figure 5 shows the liberation of xylo-oligosaccharides from ground maize cobs. EXAMPLE 1: 30 Selection of the strain 1-1017 Various soil, compost and dung samples were used as an inoculum in order to enrich with xylanolytic strains. After 24 hours' incubation, the xylanase activities are tested in the culture supernatants, and 35 the three enriched preparations showing the largest activities are diluted and plated out on agar medium containing xylan. A base culture medium containing minerals and vitamins (Zeikus and Wolfe, J. Bacteriol. 109: 707-713, I 1972) and yeast extract (0.2%) was supplemented with agar (2%) and oat xylan (0.5%). After sterilisation, KHC03 (25 mM) is added from a sterile 1M stock solution to the mixed medium for isolation of the strains. The inoculated Petri dishes are incubated at 55°C in closed jars. The colonies forming clear zones are purified. The most productive isolates (strain 1-1017) are selected and characterised. The enrichment as well as the growth of the cultures are performed aerobically in an agitated water-bath in closed 125-ml bottles containing 10 ml of medium. The incubation temperature is 55 °C and the medium is buffered to pH 7 by adding 50 mM KHC03. EXAMPLE 2: Growth of the strain 1-1017 The kinetics of xylanase production are determined at 55°C in 2-litre fermenters in an initial volume of medium of 1.5 litres. After sterilisation, 50 ml of 1M KHC03 solution are added, and the pH is adjusted to 7.8 and maintained at this value. The concentration of dissolved oxygen is regulated and maintained at a saturation of 70%. Figure 1 shows the growth of the strain 1-1017 in discontinuous culture on 0.5% oat xylan. The strain 1-1017 produces up to 110 units of xylanase/ml. During the initial hours of culture, a transient accumulation of reducing sugars is observed. This accumulation is due to the presence in the inoculum of small amounts of xylanase which catalyse a rapid hydrolysis of the soluble xylan fraction to xylo-oligosaccharides. The bacterial growth then makes possible the consumption of the xylo-oligosaccharides and the synthesis of xylanase which begins immediately after their concentration becomes limiting for the growth of the bacteria. Xylanase production is broadly constant over 4 hours (30 IU.ml/h). Great variability is observed in xylanase production in accordance with the prior xylanase activity of - 10 - 4 1 4 § the inoculum, the size of the stationary phase and the physical properties of the xylan. b) Influence of the amount of xylo-oligosaccharides on the growth of the strain 1-1017. 5 In order to obtain reproducible culture conditions, xylanase production was tested in cultures fed with a solution in the presence of xylo-oligosaccharides. Figure 2 shows the influence of the concentration of xylo-oligosaccharides. 10 The initial concentration of sugar is 2 g/1. The substrate is consumed during the exponential growth phase, during which no xylanase production is detected. When the substrate concentration begins to become limiting for growth, an immediate synthesis of xylanase 15 is observed. The rate and also the duration of production of the enzyme are dependant on the supply of substrate, which varies from 0.26 g/h to 0.67 g/h with a flow rate of substrate of 24 ml/h and an initial volume of the 2 0 culture medium of 1.5 1. The arrow in Figure 2 corresponds to the addition of xylo-oligosaccharides. The largest production of enzymes (230 IU/ml) is obtained when the substrate is supplied at a rate of 25 0.36 g/h. Larger amounts of substrates lead to a shorten ing of the period of xylanase production. When glucose is used in place of xylo-oligosaccharides, xylanase production is considerably decreased (14 IU/ml and lasts less than 2 hours. 30 On the other hand, the use of glucose as an initial growth substrate does not modify the output of xylanase in comparison to the use of xylo-oligosaccharides. In order to prepare xylo-oligosaccharides as 35 substrates in continuous cultures, a 5% suspension of oat xylan is incubated for 8 hours at 60°C and pH 6 in the presence of 5 units of xylanase per ml. This suspension is then centrifuged, and the supernatant is recovered and thereafter autoclaved. - 11 - EXAMPLE 3: Purification of the xvlanase from a culture of the strain 1-1017 The process for purification of the xylanase 5 comprises the following steps: - concentration of the Bacillus culture supernatant by ultrafiltration through a polysulphone membrane having an exclusion threshold of 10 kDa; passage through a Sepharose Fast Flow 10 (Pharmacia) ion exchange column; - passage through a Phenyl-Sepharose (Pharmacia) hydrophobic interaction column. The results of this purification are collated in Table II. 15 This purification shows that the endoxylanase is the major protein among the proteins excreted into the culture medium (approximately 50% of the total excreted proteins). EXAMPLE 4: 20 Characterisation of the xvlanase 40 /*g of xylanase purified according to Example 3 were sequenced using an Applied Biosystems type 470 A gas phase sequencer. The phenylthiohydantoin (PTH) derivatives of each amino acid were identified by high 25 pressure liquid chromatography (HPLC) using a PTH analyser coupled to an Applied Biosystems 120 A apparatus. The N-terminal sequence of the first 20 amino acids which was obtained is as follows: 30 Asn-Thr-Tyr-Trp-Gln-Tyr-Trp-Thr-Asp-Gly-Ile-Gly-Tyr-Val- Asn-Ala-Thr-Asn-Gly-Gln. The overall amino acid composition was also determined after hydrolysis with 5.6 N hydrochloric acid at 100°C for 24 hours. 35 The results are shown in Table III, in which the molar percentage of each amino acid, the estimated number of residues of each type in the protein and, in comparison, the number of residues of each type in the N-terminal end of 20 amino acids are recorded. W - 12 - It will be noted that the asparagine and glutamine residues have been reckoned with the aspartate and glutamate residues, respectively, and that the tryptophan and cysteine residues were not measured. 5 EXAMPLE 5: Mutagenesis of the strain 1-1017 Cultures of the strain 1-1017 were prepared on a medium containing xylan and ethyl methanesulphonate (EMS). 10 The treated cells are washed twice, incubated overnight and plated out on an agar/xylan medium. The clones are cultured in glass microtubes (8 x 35 mm) filled with 100 microlitres of a culture medium supplemented with oat xylan (0.5%). The micro-15 tubes are closed using silicone stoppers in order to prevent evaporation and incubated for 24 hours at 55°C on a shaker. For the selection of mutants, 96 samples are tested in parallel; 20 pi of each microtube culture are 20 transferred using a multichannel pipette to polypropylene microtubes, where hydrolysis of the xylan is performed at room temperature. The reactions are initiated by adding 300 ^1 of substrate suspension to each tube, and stopped with 400 /j1 of 3,5-dinitrosalicylic acid. After boiling 25 for 15 minutes, 50 ^1 of each tube are transferred to a microtitration plate in order to read the optical density. The clones showing the largest xylanase activities are retested by five successive subculturings 30 and compared with the parent strain. Mutants having a significant enhancement of xylanase activity were selected from 500 clones isolated after mutagenesis with EMS. These mutants show an increase in xylanase 35 activity in discontinuous cultures on medium containing xylan. The xylanase productivity of the mutants is also determined in continuously fed cultures. With a flow rate of xylo-oligosaccharides of 0.36 g/h, the xylanase output 1 - 13 - of the mutant 1-1018 is not significantly increased (less than 10%) compared to that of the wild-type strain 1-1017, as shown in Figure 3, which shows the differential growth of the strains 1-1017 and 1-1018 at 5 two concentrations of xylo-oligosaccharides. When the flow rate of xylo-oligosaccharides is increased to 0.67 g/h, the strain 1-1018, for example, shows a twofold larger increase in xylanase production compared to the strain 1-1017 (parent strain). 10 Mutant production and optimisation of the con ditions of continuously fed cultures permit an increase by a factor of 3 in xylanase production by comparison with discontinuous cultures of the wild-type strain on xylan. These high enzyme levels (up to 392 IU/ml) are 15 obtained in 7 hours of culture, and the xylanase productivity with 1-1018 is hence considerably greater than that of the best strains of fungi and bacteria (Bertrand et al., Biotechnol. Bioeng., 33:791-794, 1989) (Yu et al.,1987 cited above) productive of xylanase, 20 which necessitate several days before such enzyme levels are obtained. EXAMPLE 6: Treatment of paper pulps (unbleached kraft) with the xvlanase 25 8.5 g of wet pulp (corresponding to 1 g dry weight) originating from maritime pine and containing 60 mg of xylan were treated with 25 ml of buffer (containing various concentrations of xylanase) at 60°C for 24 hours. 30 The kinetics of liberation of xylo-oligosac- charides are shown in Figure 4. A concomitant liberation of lignin derivatives was shown (by measurement of the optical density at 280 nm). EXAMPLE 7: 35 Production of xvlo-oligosaccharides from ground maize cobs 1 g (dry weight) of maize cob powder (particle size > 100 mesh) were suspended in 100 ml of 50 mM sodium acetate buffer, pH 6, containing 1000 IU of xylanase, and - 14 - incubated at 60°C for 18 hours. Figure 5 shows that it is possible under these conditions to convert up to 38% of the xylan to oligosaccharides (xylo-oligosaccharides and arabino-xylo-5 oligosaccharides). These compounds may be used as they are or as precursors of xylose and of arabinose. o o TABLE I Comparison of xylanase production by the strains according to the invention and by strains of the prior art Strain Growth substrate Xylanase productivity (IU ml"1 h"1) Culture time (M References Clostridium thermolacticum Clostridium stercorarium Thermoascus auriantacus glucose xylan xylan 0.65 0.78 2.40 40 72 240 Brodel, B., E. Samain, and P. Debeire 1990. Biotechnol. Lett. 12(1);65-70 Berenger, J.F., C. Frixon J. C. Frixon J. Bigliardi, and N. Creuzet. 1985. Can. J. Microbiol. 31:635-642 Yu, E.K.C., L.U.L. Tan, M.K.H. Chan, L. Deschatelets and J.N. Saddler. 1987. Enzyme Microb. Technol. 9:16-24 Ul i Thielavia terrestris Streptomyces lividans A Solka-Floc xylan 1.04 16.76 18 72 Solka-Floc: purified cellulose containing 5 to 7% of hemicullulose Merchant, R., F. Merchant, and A. Margaritis. 1988 Biotechnol. Lett. 10(7):513-516 Bertrand, J.L., R. Morosoli F. Shareck, and D. Kluepfel. 1989. Biotechnol. Bioeng. 33(6): 791-794 c-ea& TABLE I (continued) Strain Comparison of xylanase production by the strains according to the invention and by strains of the prior art Growth substrate Xylanase productivity (IU ml"1 h"1) Culture time (h) References Streptomyces lividans B xylose 3.25 65 Kluepfel, D.S. Vats-Mehta F. Aumont, F. Shareck, and R. Morosoli 1990. Biochem. j. 267:45-50 Bacillus sp. alcalophile Bacillus sp. according to the invention xylose xylo- oligosaccharides 2.47 44 48 Okazaki W., T. Akiba, K. Horikoshi, and R. Akahoshi. 1984. Appl. Microbial. Biotechnol 19:335-340 (Tl I Nutant Bacillus sp. according to the invention xylo- oligosaccharides 78.4 o o • • TABLE II Step Fraction Volume Total proteins (mg) Total xylanase activity (IU) Specific activity (IU/mg) <%) Yield Culture supernatant 1500 216 232000 1071 100 I Concentration 150 197 225000 1142 97 II Q Sepharose Fast Flow 250 142 204250 1438 88 III Phenyl-Sepharose 205 72 143500 1993 62 TABLE III: Overall amino acid composition of the xylanase Amino acid Mol% Hydrolysis Sequencing Estimated number Number of residues (in of residues the partial sequence 1-20) Alanine 7.5 15 1 Arginine 3.0 6 0 Asparagine (a) (a) 3 Aspartic acid 14.5 29 1 Cysteine (c) (c) 0 Glutamic acid 9.5 19 0 Glutamine (b) (b) 2 Glycine 15.5 31 3 Histidine 1.5 3 0 Isoleucine 3.0 6 1 Leucine 3.5 7 0 Lysine 1.5 3 0 Methionine 0.5 1 0 Phenylalanine 2.0 4 0 Proline 4.0 8 0 Serine 9.5 19 0 Threonine 6.5 13 3 Tryptophan (c) (c) 2 Tyrosine 11.0 22 3 Valine 7.0 14 1 (a) (b) (c) Reckoned as aspartic acid Reckoned as glutamic acid Not measured </div>

Claims

<div class="application article clearfix printTableText" id="claims"> 24 14 5 3 - 19 - WHAT WE CLAIM IS:

1. Bacillus strain productive of xylanase, registered under No. 1-1017 with the Collection Nationale de Cultures des Microorganismes (National Collection of 5 Microorganism Cultures) of the Pasteur Institute.

2. Bacillus strain productive of xylanase, registered under No. 1-1018 with the Collection Nationale de Cultures des Microorganismes (National Collection of Microorganism Cultures) of the Pasteur Institute. 10

3. A substantially pure thermophilic xylanase, characterised in that it possesses a molecular mass of substantially 22kDa and an isoelectric point of substantially 7.7.

4. Xylanase according to Claim 3, characterised in that it is stable at approximately 60°C. 15

5. Xylanase according to any one of claims 3 and 4, characterised in that its pH of optimal activity is within the range extending from 4.8 to 7.

6. Xylanase according to any one of claims 3 to 5, 2 0 characterised in that it possesses the following N-terminal amino acid sequence: Asn-Thr-Tyr-Trp-Gln-Tyr-Trp-Thr-Asp-Gly-Ile-Gly-Tyr-Val-Asn-Ala-Thr-Asn-Gly-Gln.

7. Xylanase according to any one of claims 3 to 6, 25 characterised in that it comprises in its sequence 15 alanines, 6 arginines, 29 aspartates and asparagines, 19 glutamates and glutamines, 31 glycines, 3 histidines, 6 isoleucines, 7 leucines, 3 lysines, 1 methionine, 4 phenylalanines, 8 prolines, 19 serines, 13 threonines, 22 30 tyrosines and 14 valines.

8. Xylanase according to any one of claims 3 to 7, characterised in that it is secreted by a Bacillus.

9. Xylanase according to Claim 8, characterised in that it is secreted by one of the Bacillus strains j^^ajtecording to any' one of claims 1 and 2. ',:^ffo\. Process for obtaining purified xylanase according to \ any one of claims 8 and 9, comprising the following steps: - concentration of a Bacillus culture supernatant of a Bacillus strain according to claim 1 or claim 2, .J - 20 - 24 14 5 3 - passage through an ion exchange column, and passage through a hydrophobic interaction column.

11. Process for the production of xylanase according 5 to one of Claims 8 and 9, comprising the steps: - of growth of a Bacillus strain according to claim 1 or claim 2 in a medium containing a growth substrate, and - of production of xylanase induced by feeding the culture continuously with suitable amounts of xylo- 10 oligosaccharides.

12. Use of the xylanase according to any one of claims 3 to 9 in the bleaching of paper pulp.

13. Use of the xylanase according to any one of claims 3 to 9 in the preparation of xylo-oligosaccharides from raw 15 materials of plant origin.

14. A xylanase according to claim 5 wherein the pH is substantially 6.

15. A process as claimed in claim 11 in which the growth substrate is glucose.

16. A bacillus strain as claimed in claim 1, substantially as herein described with reference to any example thereof.

17. Xylanase as claimed in any one of claims 7 to 9 inclusive, substantially as herein described with reference to any example thereof.

18. A process as claimed in claim 10, substantially as herein described with reference to any example thereof. INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE (INRA) By thei BALDWIN, attorneys SON & CAREY </div>