WO2002012511A1 - Nouvelle methode de surproduction de lichenases dans des bacteries par isolement des genes directement d'echantillons naturels (lichenaturases) - Google Patents

Nouvelle methode de surproduction de lichenases dans des bacteries par isolement des genes directement d'echantillons naturels (lichenaturases) Download PDF

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WO2002012511A1
WO2002012511A1 PCT/ES2001/000303 ES0100303W WO0212511A1 WO 2002012511 A1 WO2002012511 A1 WO 2002012511A1 ES 0100303 W ES0100303 W ES 0100303W WO 0212511 A1 WO0212511 A1 WO 0212511A1
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PCT/ES2001/000303
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Vicente RODRÍGUEZ MUÑOZ
Rafael PÉREZ MELLADO
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Consejo Superior De Investigaciones Científicas
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Priority to AU2001282141A priority Critical patent/AU2001282141A1/en
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    • 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)
    • C12Y302/01073—Licheninase (3.2.1.73)
    • 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)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/244—Endo-1,3(4)-beta-glucanase (3.2.1.6)
    • 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)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2448—Licheninase (3.2.1.73)
    • 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)
    • C12Y302/01006—Endo-1,3(4)-beta-glucanase (3.2.1.6)

Definitions

  • Biotechnology Genetic engineering of microorganisms. Biodiversity Degenerate oligonucleotides Production of polysaccharide degrading enzymes. Liquenases
  • Hemicellulose which includes glucans, xylans and mannan
  • the 1, 3- 1, 4- ⁇ -glucans mixture forms the bulk of the cell wall of cereals such as wheat, oats and barley.
  • the enzyme endo-1, 3-1, 4- ⁇ -D-glucanhydrosilase (lichenase, EC, no. 3.2.1.73) cuts the ⁇ -1, 4 junctions adjacent to the ⁇ -1, 3 in glucans.
  • the ⁇ -glucanase enzymes are especially interesting in the beer industry because ⁇ -glucans cause problems in the filtration processes, and they have also been added to poultry feed to improve their digestibility.
  • the present invention describes two degenerated primers and a method for accessing new gene sequences encoding bacterial lichens of the genus Bacillus directly from the soil.
  • lichenurae The name of lichenurase comes to indicate the direct isolation of these genes from the soil, hence the "natural", It should be noted that you can not logically indicate the organism that originally encodes them by the procedure by which they have been identified. However, the lichen activity of these new proteins encoded by these new genes is demonstrated as indicated in the present invention. Furthermore, the present invention describes a method for cloning and overproduction of said lichens by using a vector designed for the overproduction of extracellular enzymes in Bacillus subtilis, which includes the promoter and signal peptide of the Bacillus subtilis chitosanase. The cloning of the lichens isolated from the soil in this vector allows overexpression, processing and secretion of said enzymes to the outside environment.
  • oligonucleotide sequences SEQ ID NO 1 and SEQ ID NO 2 and their use for amplification of nucleotide sequences encoding proteins with lichen activity, as well as variants thereof that allow improvements in the subsequent procedure of identification, cloning and overproduction;
  • the soils are a mixture of minerals, decomposing organic matter and numerous organisms and microorganisms. As such a mixture, it contains numerous possible sources of DNA as well as compounds of organic origin, such as humic acids, which can interfere with the specific binding of the primers and with the activity of the polymerase, making amplification by PCR difficult.
  • a wide variety of methods have been described to solve these problems, but the impossibility of handling a large number of samples and the slowness of the processes have generally been a remora.
  • the method used in this invention ["Fast DNA Spin Kit for Soil, Bio 101] was designed for rapid extraction of clean and valid DNA for PCR amplifications from soils. However, an additional purification step by fragmentation became necessary.
  • oligonucleotides were designated as Lich2F [5'CGGGATCCCAAAC (A / G) GG (C / T) GG (A / G) TCGTTTT (T / A) TG3 '] (SEQ ID NO 1) and Lich2R [5'CGGGATCCTTAT (T / C) TTTTT (G / C) TATA (G / A) CGCA (T / C) CC3 '] (SEQ ID NO 2) and form part of the present invention.
  • the oligonucleotides have at their ends target sequences for the BamHI restriction enzyme, necessary for cloning by cohesive ends in a vector cut with said enzyme.
  • the oligonucleotides can be designed to introduce modifications at the 5 'end that allow the identification of these sequences by methods well known in the state of the art, among others, coding of specific amino acids, dioxigenin mapping, biotin mapping, marking by radioactive isotopes and additional nucleotides, and are part of the present invention.
  • DNA fragments obtained from two different types of land by PCR amplification were ligated to the shuttle plasmid pNSP15.
  • This vector contains the kanamycin resistance gene and the origin of plasmid pPCT2 replication for Bacillus subtilis, the cat gene, promoter reference encoding the chloramphenicol acetyl transferase enzyme, the origin of plasmid replication pRMI cat from E. coli and the promoter and signal peptide of the chitosanase gene of Bacillus subtilis oriented in the direction of the cat gene [RP Mellado, M. Moreno, L. Rivas and V. Parro: New vector for the overproduction of proteins in Bacillus subtilis. Patent application No.
  • Plasmid pNSP15 was propagated in a culture of competent E. coli MC1061 cells. The strain was inoculated in 10 ml of LB medium with 100 mg / ml of kanamycin and incubated at 37 ° C for 12 h. The culture was centrifuged and the resulting pellet was used to extract the plasmid with the Promega Wizard plus SV minipreps kit. The plasmid obtained was digested with the restriction enzyme BamHI and the PCR fragments obtained were ligated with the degenerated oligonucleotides. The ligaments were transformed into E.
  • coli MC1061 cells and the transformants obtained in LB plates with 100 mg / ml kanamycin were selected by PCR, using an oligonucleotide complementary to the sequence of the chitosanase signal peptide of B. subtilis, 701 B (5 ' cgcggatccAGGGGAAGTGAAATGAAAATCAG3 ' ) (SEQ ID NO 3) and oligonucleotide Lich2R used as reverse in amplification of the gene fragments for land lichen. Only the clones with the fragment in the proper orientation (in the transcription direction of the chitosanase promoter) produced a 778 bp fragment.
  • each of the possible new lichenurae is individualized in a plasmid. Sequence of one of the clones of the transformations of the fragments from earth n ° 1 (p5L2-6, SEQ ID NO 4) and another from those from earth n ° 2 (p16L2-4, SEQ) were sequenced ID NO 5) which were used in the following comparative tests of the lichen activity (endo 1, 3-1, 4- ⁇ -glucanase).
  • Plasmids with the insert were extracted from the E. coli clones in the manner described above and transformed into competent Bacillus subtilis 168 cells. The selection of positive transformants was performed by replicates of the Bacillus clones that grew in kanamycin in LB plates with lichen and congo red for the detection of halos of degradation of the polysaccharide significantly greater than those produced by the lichenase itself of B. subtilis. Subsequently, the clones of B. subt ⁇ llis with inserts p5L2-6 and p16L2-4 were selected to determine the lichen activity of the supernatants of their cultures and compared with that obtained for the wild strain B. subtilis 168 [pNSP15] and B. subtilis 168 transformed with plasmids pBSL and pHML.
  • the endo 1, 3-1, 4- ⁇ -glucanase activity was measured by determining the concentration of reducing sugars released following the method of Wolf et al [Wolf, M., Geczi, A., Simón, O. and Borriss, R. 1995. Genes encoding xylan and beta-glucan hydrolysing enzyme in B. subtilis: characterization, mapping and construction of strains deficient in lichenase, cellulase and xylanase. Microbiology, 141: 281-290]. The maximum activity was observed in the stationary phase of the culture. Enzymatic activities were higher in the clones with the p5L2-6 and p16L2-4 inserts compared to those obtained with the control strains.
  • degenerate oligonucleotides described in the present invention allows the identification of new nucleotide sequences (SEQ ID NO 4 and SEQ ID NO 6 are examples of 2 of them) that encode proteins with lichenose activity (liquenaturases) from samples of soil (SEQ ID NO 5 and SEQ ID N 7) and all of them form part of the present invention.
  • SEQ ID NO 4 and SEQ ID NO 6 are examples of 2 of them
  • proteins with lichenose activity liquenaturases
  • an expression vector which allows the recombinant protein to be secreted outside the cell, which represents an advantage when obtaining these proteins from the culture media, and forms part of the present invention.
  • the state of the art of genetic engineering allows the use of multiple expression vectors and host cells for this same purpose and form part of the present invention.
  • Example 1 Extraction and purification of DNA from soil samples.
  • Land No. 1 comes from the jungle of the island of Bali, Indonesia, and Land No. 2 comes from Avila, Spain.
  • the DNA was extracted from the soil using the "fastDNA SPIN kit for soil" Bio101 kit.
  • the DNA solution resulting from the extraction was used for partial amplification of bacterial ribosomal genes with highly conserved oligonucleotides to check the viability of the DNA for use in PCR. No fragment was obtained, so an extra purification step was necessary.
  • the DNA was loaded on an agarose gel and subjected to electrophoresis. In this way the possible impurities of the high molecular size DNA extracted from the earth were separated.
  • the DNA was extracted from the gel by removing the agarose with phenol and precipitating the aqueous phase to recover the DNA.
  • Example 2 Amplification of new lichens directly from natural samples.
  • the Lich2F oligonucleotide (SEQ ID NO 1) was derived from the DNA region of the lichenase genes corresponding to the first 22 nucleotides of the mature part of the gene.
  • the oligonucleotide Lich2R (SEQ ID NO 2) was derived from the last 23, including the termination codon of the translation of the genes analyzed. Both oligonucleotides have at their 5 ' ends the target sequences for cutting with the restriction enzyme BamHI.
  • the reaction mixture for the PCR contained 280 ng of each of the oligonucleotides (Lich2F and Lich2R), the DNA extracted from the soil, (approximately 50 ng, estimated amount by gel comparison), 10 ml of enzyme buffer, dNTPs 1 mM of final concentration, CI2Mg 1 mM of final concentration and 1 U of DNA polymerase (Ecogen), completing the final volume at 100 ml.
  • the amplification process consisted of: 1 cycle at 95 ° C (5 min), 29 cycles of three stages: 1 min at 95 ° C, 1 min at 50 ° C and 1 min at 72 ° C, and a final cycle of 10 min at 72 ° C.
  • the reactions were carried out in a PTC 100 model thermal cycler from MJ Research Inc. Two positive reactions were arranged with the genomic DNA of B. subtilis and B. amiloliquefaciens, which produced bands of the expected size of 646 bp. As a negative control, two reactions without template DNA were arranged to verify that possible contamination of the reagents were not responsible for the bands obtained using the earth's DNA as a substrate.
  • Example 3 Cloning of the amplification fragments in E. coli.
  • An E. coli recombinant containing plasmid pNSP15 was cultured in LB medium with 100 mg / ml ampicillin.
  • Plasmid DNA was obtained with the Promega Wizard plus SV kit following the manufacturer's instructions: the pellet obtained was resuspended by centrifugation of 5 ml of culture in 250 ml of resuspension buffer to which 250 ml of lysis buffer was added. The two solutions were mixed until a clear supernatant was obtained and 10 ml of alkaline protease was added. The samples were incubated at room temperature for 5 min, then adding 350 ml of neutralization solution.
  • the tubes were inverted and centrifuged at 13,000 rpm for 10 min.
  • the resulting supernatant was collected and purified on a Wizard plus SV minipreps spin column, which was washed with 750 ml of wash solution.
  • the columns were then centrifuged and 100 ml of nuclease-free water was added to extract the retained DNA.
  • the columns were centrifuged again at 13,000 rpm for 10 min and the solution was collected with the purified plasmid.
  • the PCR fragment obtained and the plasmid pNSP15 DNA were digested with the restriction endonuclease BamHI, using 100 ng of DNA in each case, to obtain cohesive ends.
  • the samples were digested in a final volume of 20 ml, adding 2 ml of enzyme buffer, 1 ml of enzyme and sterile distilled water to each tube until the final volume was completed. Both fragments were purified using the CONCERT Nucleic Acid Purification System, Life & Technologies, following the manufacturer's instructions.
  • the 5 'ends of the plasmid were dephosphorylated with alkaline phosphatase (Amersham) to prevent recirculation of the plasmid, in a final volume of 20 ml with 100 ng of plasmid, 2 ml of phosphatase buffer and 1 ml of alkaline phosphatase for 1 h at 37 ° C.
  • the 646 bp fragments were ligated to the plasmid, using 100 ng of plasmid DNA and a 5-fold molar excess of the respective inserts. The volume of water needed was added to ensure adequate proportion in a final volume of 10 ml. 1 ml of enzyme buffer and 1 ml of T4 DNA ligase was added. Each reaction was incubated between 4 and 16 h at 16 ° C. 5 ml of ligation was transformed into E. coli MC1061 and the cells were plated in LB medium with 100 mg / ml ampicillin to select the transformants.
  • SEQ ID oligonucleotides 701 B
  • Plasmid DNA was extracted from the clones in which the presence of the insert was checked in the proper orientation according to the method described above and competent Bacillus subtilis cells were transformed with said plasmids.
  • the transformed cells were plated on agar LB with kanamycin 7.5 mg / ml.
  • the transformants were replicated in LB agar with kanamycin 7.5 mg / ml, and with 0.2% lichenano (Sigma) and 0.1% congo red to select the transformants with the possible new lichenase genes by the presence of lichens lysis halos around colonies noticeably larger than those produced by the chromosomal copy of the ⁇ gene itself. subtilis Of the six positive clones in E.
  • the endo 1, 3-1, 4- ⁇ -glucanase activity was measured by determining the concentration of reducing sugars released following the method of Wolf et al [Wolf, M., Geczi, A., Simón, O. and Borriss, R. 1995. Genes encoding xylan and beta-glucan hydrolysing enzyme in B. subtilis: characterization, mapping and construction of strains deficient in lichenase, cellulase and xylanase. Microbiology, 141: 281-290].
  • the reaction mixture contained 500 ml of 2.5% lichen, 500 ml of 50 mM phosphate buffer and 50 ml of the supernatant of each culture, conveniently diluted; It was incubated at 37 ° C for 15 minutes. The reaction was stopped by adding 1 ml of 3,5-dinitrosalicylic acid reagent and boiling the samples for 10 min. The final volume was brought to 7 ml and the optical density was measured at 540 nm. The unit of enzymatic activity (UAE) was defined as the micromoles of reducing sugars obtained in the reaction per minute. Cultures of B.
  • subtilis 168 transformed with plasmids p5L2-6 and p16L2-4 grew in LB medium and the lichen activity obtained in different phases of the culture was compared with the obtained for the wild strain B. subtilis 168 [pNSP15] and B. subtilis 168 transformed with plasmids pBSL and pHML. The maximum activity was observed in the stationary phase of the culture. Enzymatic activities were 19 UAE / ml for clone p5L2-6, 29.8 UAE / ml for clone p16L2-4, 8.76 UAE / ml for clone pBSL, 17.2 UAE / ml for clone pHML and 0.57 UAE / ml for B.
  • Enzymatic activities per milligram of protein were determined by concentrating the total protein in the stationary phase supernatant by dialysis in ammonium sulfate, measuring the optical density units at 540 nm and correcting by amount of total protein. Said amount of protein was determined using the BIO-RAD (Protein Assay kit) kit for clones containing plasmids pBSL, pHML, p5L2-6 and p16L2-4, following the supplier's instructions.
  • Enzymatic activities were 2000 UAE / mg for the pBSL clone, 3512 UAE / mg for the pHML clone, 3000 UAE / mg for the p5L2-6 clone and 3341 UAE / mg for the p16L2-4 clone.
  • the presence of the respective mature proteins in the extracellular medium was verified by 15% PAGE-SDS electrophoresis gels, observing a majority band of 20 kDa of relative molecular mass and the amino-terminal end of the protein in the p5L2 clones was sequenced. 6 and p16L2-4 to check its correct processing in the cell.
  • the sequence obtained for clone p5L2-6 in its first residues was AGLN.
  • the first nine amino acids of clone p 6L2-4 were AGLNKGSQT.
  • the amino acids AGLNK belong to the first 5 amino acids of the mature chitosanase of B. subtilis and the following GSQT belong to the first amino acids of the mature lichenase.

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Abstract

La présente invention concerne l'isolement, l'amplification et la purification de gènes codant pour des lichenases directement d'échantillons de sols à l'aide d'oligonucléotides dégénérés (lichenaturases). L'invention concerne également une méthode de surproduction et de sécrétion des produits de ces gènes dans le milieu extérieur à l'aide de l'organisme hôte Bacillus subtilis 168.
PCT/ES2001/000303 2000-07-28 2001-07-27 Nouvelle methode de surproduction de lichenases dans des bacteries par isolement des genes directement d'echantillons naturels (lichenaturases) WO2002012511A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001282141A AU2001282141A1 (en) 2000-07-28 2001-07-27 Novel method for overproduction of lichenase in bacteria by isolating the genes directly from natural samples (lichenaturase)

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ESP200001922 2000-07-28
ES200001922A ES2164610B1 (es) 2000-07-28 2000-07-28 Nuevo metodo para la sobreproduccion de liquenasas en bacterias aislando los genes directamente de muestras naturales (liquenaturasas).

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990009436A1 (fr) * 1989-02-16 1990-08-23 Carlsberg A/S (1,3-1,4)-β-GLUCANASE THERMOSTABLE

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990009436A1 (fr) * 1989-02-16 1990-08-23 Carlsberg A/S (1,3-1,4)-β-GLUCANASE THERMOSTABLE

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BORRISS R.: "Structure of the beta-1, 3-1, 4-glucanase of bacillus macerans: homologies to other beta-glucanases", MOL. GEN. GENET., vol. 222, 1990, pages 278 - 283, XP002909653 *
HOFEMEISTER J.: "The beta-glucanase gene from bacillus amyloliquefaciens shows extensive homology with that of bacillus subtilis", GENE, vol. 49, no. 2, 1986, pages 177 - 187, XP002909652 *
LLOBERAS J.: "Molecular cloning, expression and nucleotide sequence of the endo-beta-1, 3-1, 4-D-glucanase gene from bacillus licheniformis, predictive structural analyses of the encoded polypeptide", EUR. J. BIOCHEM., vol. 197, 1991, pages 337 - 343, XP002947760 *
MURPHY N.: "The DNA sequence of the gene and genetic control sites for the excreted B. subtilis enzyme beta-glucanase", NUCLEIC ACIDS RES., vol. 12, 1984, pages 5355 - 5367, XP002909651 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

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