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
• • 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)

Definitions

• the present invention relates to a xylanase and to a nucleotidic sequence encoding it. It also relates to the use of this enzyme in the bleaching of paper pulp and the preparation of xylose or of xylo-oligosaccha des 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), in the paper industry (Mora et al., J.
• the xylanases isolated by Okazaki et al. emanate 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 polymerization, while the components II produce xylose in addition to the above compounds.
• the components I (referred to as W1.I and W2.I) have respective molecular weights of 21.5 kDa and 22.5 kDa, as well as isoelectric points of 8.5 and 8.3.
• the components II have, for their part, respective molecular weights of 49.5 kDa and 50 kDa.
• the two components I and II are inhibited by Hg ' + ions and, to a lesser extent, by Cu "+ .
• RIKAGAKU KENKYSHO relates to a type Wll xylanase having a molecular weight of 5O kD or 42 kD. No isoelectric point is mentioned for this xylanase.
• Bacillus strain isolated in the natural environment and which produces a xylanase having optimal activity at between 6O°C and 7O°C and at a pH of between 6 and 7. This enzyme is characterized neither by its molecular weight nor by its isoelectric point. This strain produces, in addition, other enzymes such as cellulases.
• RIKAGAKU KENKYUSHO JP-85 118 6464 describes a xylanase having optimal activity at a pH of between 6 and 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.
• Applicant is aware, features making an industrial application possible, that is to say good thermal stability, a large capacity for degradation of substrates, a means of production by hyperproductive strains and possibilities to modify the aminoacids sequence.
• Another xylanase has been isolated from a Bacillus strain deposited at the CNCM Culture Collection under the number 1-1017. It has been described in EP 0.573.536 application filed under the name of the present applicant. This xylanase displays a temperature stability. However its sequence has not been determined and it can only be produced by growing the said Bacillus strain.
• the applicant has thus cloned the gene encoding this xylanase and has sequenced it.
• thermophilic xylanase having a sequence sharing an homology of at least 8O%, preferentially 90%, and more preferentially 95%, with the following one (SEQ ID N°2):
• the degree of homology can be determined using pairwise alignment methods such as the GAP and the BESTFIT programs of the Genetics Computer Group, Inc. Package (GCG)
• GAP Genetics Computer Group, Inc. Package
• FASTA Altschul et al.
• BLAST BLAST
• Xylanases falling under this definition are in particular the ones in which one or a few aminoacids have been changed, compared to the sequence SEQ ID N°2.
• Such a xylanase can possess a molecular mass of approximately 22 kDa, determined by SDS-PAGE, or 20.7 kDa determined by mass spectroscopy and an isoelectric point of approximately 7.7.
• This enzyme advantageously displays great stability at 6O°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.
• pHj of this enzyme is fairly high but nevertheless lower than the pHj of the xylanases of similar molecular mass produced by some bacilli, in particular those described by Okazaki et al. (1985, publication cited above).
• pH 6 corresponds to an optimal pH, but the activity remains greater than 80% in the range between 4.8 and 7.
• nucleotidic sequence coding for the said xylanase is a nucleotidic sequence coding for the said xylanase.
• This sequence can be DNA or RNA sequence and in particular c DNA, plasmidic DNA, genomic DNA or m RNA.
• nucleotidic sequence is the following one (SEQ ID N°1 ):
• the xylanase according to the present invention can thus also be produced by a microorganism strain, appropriately chosen, transformed by a vector coding for the said xylanase.
• the said microorganism is grown in an appropriate medium and thereafter the xylanase is isolated as described below.
• Such a microorganism is chosen in order to be able to produce and to excrete it.
• the vector coding for the xylanase is chosen in order to be expressed in the said microorganism. It can be a plasmid, such as pBluescript or preferentially pET..
• the pET E.coli expression system is one of the most widely used bacterial expression system (Studier et al., 1990, Meth. Enzymol.,
• the expression of recombinant xylanase can be achieved in particular as following.
• the DNA fragment encoding the mature xylanaxe i.e. the sequence SEQ ID N°1
• the PCR fragment is cloned blunt-ended into pBluescript (Stratagene Cloning Systems) before cloning as a Ndel/BamHI fragment into pET3a.
• the recombinant enzyme is expressed from pET3a in the E. coli strain BL21 (DE3) carrying pLysS. Cultures are grown in L-broth containing ampicillin (100 ⁇ g/ml) and chloramphenicol (25 ⁇ g/ml) until an A 600 °f 3 was reached, before induction with 0.1 mM isopropyl ⁇ -D- thiogalactoside (IPTG) for 3 hours. Large-scale cultures for protein purification are centrifuged and the cells are lysed in a buffer containing 5O mM Tris-HCI, pH 8.O, 1 mM EDTA by passage through a French press (10 MPa). The same process described in EP 0.573.536 for purifying the xylanase from the culture supernatant of the Bacillus can be used. One can expect about 1 mg of recombinant protein per ml of cell culture.
• An advantage of this way of production of the xylanase is that the nucleoditic sequence can be mutated before to be introduced in the microorganism. It is therefore easy to obtain various mutations corresponding to xylanases having various sequences.
• thermostable xylanases already described in the prior art, such as the one described by GRUNINGER et al. (previously cited), since their sequences were not known.
• the xylanase as described above can be obtained through a process, comprising the following steps: - concentration of the microorganism culture supernatant,
• an ion exchange column such as a column of Q Sepharose Fast Flow (Pharmacia),
• Concentration of the supernatant can , in particular, be performed by ultrafiltration through a polysulphone membrane having an exclusion threshold above 10 kDa.
• the xylanase described above can be produced through a process comprising the steps:
• the subject of the present invention is also the use of the xylanase described above in the bleaching of paper pulp.
• 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).
• xylanases Other uses of xylanases have been mentioned in the literature.
• 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.
• Fig. 1 illustrates the homology degree between the xylanase according to the present invention (XYL2O) and other xylanases.
• Fig.2 represents HCA plots of four xylanases, including the one of the present invention.
• the strain 1-1017 was grown at 55°C in the liquid medium described in examples 1 and 2 of patent application EP-0.573.536.
• the nucleotide sequence of the forward primer (P1 ) (SEQ ID N°3) was AAYACNTAYTGGCARTAYTGGACNGAYGG (derived from the sequence NTYWQYWTDG in the N-terminus end of the XYL2O); that of the reverse primer (P2) (SEQ ID N°4) was YTGWCKNACRCTCCARTAYTG (corresponding to the sequence QYWSVRQ, a conserved region near the C-terminus of other xylanases from different Bacillus species).
• PCR was performed with chromosomal DNA as a template and the primers P1 and P2 on a thermocycler (Perkin-Elmer. France) with the following temperature profile: 1 min 94°C -1 min 5O°C - 2 min 72°C for 35 cycles.
• the PCR product was purified on a 1 % agarose gel and was ligated into EcoRV-digested pBluescript.
• the chimaeric plasmid (pBX2O) was used to transform SURE cells.
• Recombinant cells were selected on L-agar plates containing ampicillin (4O ⁇ g/ml), isopropyl- ⁇ -D-thiogalactoside (O.2 mM) and 5-bromo-4- chloro-3-indolyl- ⁇ -D-galactoside (4O ⁇ g/ml).
• ampicillin 4O ⁇ g/ml
• isopropyl- ⁇ -D-thiogalactoside O.2 mM
• 5-bromo-4- chloro-3-indolyl- ⁇ -D-galactoside (4O ⁇ g/ml).
• Chromosomal DNA was partially digested with Sau3AI and the resulting DNA fragments in the size range 1.5-8 kb were purified and ligated into BamHI-digested ZAP Express.
• the library was constructed using XL1-Blue cells as indicated by the manufacturer.
• pBX2O was digested with BamHI and Hindlll and the DNA insert was purified and labeled with digoxigenin (Boehringer Mannheim) following the instructions of the manufacturer. The labeled DNA was used to screen the genomic library. After the third screening, positive lambda plaques were isolated and the recombinant plasmid pBK-CMV inserted in the vector ZAP Express was excised using the filamentous phage ExAssist and then recovered by infecting the XLOLR cells in the presence of kanamycin (10 ⁇ g/ml).
• Plasmid preparations for sequence determination were performed using Qiagen tip 100 (Diagen, Coger, France). Double- stranded DNA sequencing was done by the dideoxy chain termination method of Sanger et al (Proc. Nat. Acad. Sci. USA, 1977, 74, 5463- 5467), using the SequenaseTM 2.0 DNA sequencing kit from United States Biochemical . Both universal and specific primers were used to sequence the sense and antisense strands of inserts in the plasmids.
• Hydrophobic Cluster Analysis is a method to compare amino acid sequence (Gaboriaud et al. FEBS Lett., 1987, 224, 149-155) which is derived from the theory of Lim (J. Mol. Biol, 1974, 88, 857-872). The method involves the drawing of the sequence of a theoretical A-helix where the hydrophobic residues form clusters.
• a part of the gene coding for the xylanase has been amplified by PCR using two degenerate primers, P1 and P2, corresponding to the N-terminus end of the xylanase and to a conserved region near the C- terminus, respectively.
• a 450 bp DNA fragment was obtained and cloned into the vector pBluescript.
• the sequence of the resultant plasmid pBX2O can be attributed without any doubt to the xylanase.
• a genomic library of B. sp 1-1017 was prepared in E. coli XL1-blue using the phage vector ZAP Express.
• This library was screened with the insert of the plasmid pBX2O.
• One positive plaque, designated pBX52A2 was shown to contain the complete gene of the xylanase.
• the nucleotidic sequence of this clone is indicated in the sequence list hereunder as SEQ ID N° 1.
• SEQ ID N°2 The complete protein sequence of the xylanase is shown as SEQ ID N°2 is the sequence list hereunder.
• the figure 3 summarizes the prediction of the occurrence of secondary structural elements which can be proposed for the xylanase according to the present invention on the basis of its primary structure and a thorough protein sequence analysis. These structural predictions can be translated into a putative three-dimensional model to be used in Molecular Isomorphism Replacement in view of solving the crystalline structure of this xylanase.
• MOLECULE TYPE DNA (genomic)
• HYPOTHETICAL NO
• ORGANISM Bacillus sp

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• 1996
  • 1996-10-15 HU HU9900738A patent/HUP9900738A2/hu unknown
  • 1996-10-15 CA CA002234998A patent/CA2234998A1/en not_active Abandoned
  • 1996-10-15 AU AU72932/96A patent/AU7293296A/en not_active Abandoned
  • 1996-10-15 EP EP96934697A patent/EP0857215A1/en not_active Withdrawn
  • 1996-10-15 JP JP9515518A patent/JPH11514235A/ja active Pending
  • 1996-10-15 WO PCT/EP1996/004485 patent/WO1997014803A1/en not_active Application Discontinuation
• 1998
  • 1998-04-16 NO NO981707A patent/NO981707L/no unknown

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