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WO1998036056A1 - Enzyme a activite d'endo-1,3(4)-beta-glucanase - Google Patents

Enzyme a activite d'endo-1,3(4)-beta-glucanase Download PDF

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Publication number
WO1998036056A1
WO1998036056A1 PCT/DK1998/000057 DK9800057W WO9836056A1 WO 1998036056 A1 WO1998036056 A1 WO 1998036056A1 DK 9800057 W DK9800057 W DK 9800057W WO 9836056 A1 WO9836056 A1 WO 9836056A1
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WIPO (PCT)
Prior art keywords
enzyme
dna sequence
beta
glucanase
ser
Prior art date
Application number
PCT/DK1998/000057
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English (en)
Inventor
Marie-Louise Bang
Thomas Sandal
Original Assignee
Novo Nordisk A/S
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Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU58530/98A priority Critical patent/AU5853098A/en
Publication of WO1998036056A1 publication Critical patent/WO1998036056A1/fr

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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)
    • C12Y302/01006—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
    • C12N9/244—Endo-1,3(4)-beta-glucanase (3.2.1.6)

Definitions

  • the present invention relates to an enzyme with beta-glucanase activity, a cloned DNA sequence encoding the enzyme with beta- glucanase activity, a method of producing the enzyme, an enzyme composition comprising said enzyme with beta-glucanase activity, and the use of said enzyme and enzyme composition for a number of industrial applications.
  • an enzyme exhibiting beta-glucanase activity may be obtained from a strain of the genus Phaffia , more specifically Phaffia rhodozyma, and have succeeded in cloning a DNA sequence encoding said enzyme.
  • the invention relates to a cloned DNA sequence encoding an enzyme exhibiting beta- glucanase activity, which DNA sequence is selected from the group comprising of:
  • the invention relates to an isolated enzyme exhibiting beta-glucanase activity selected from the group consisting of:
  • the invention provides a recombinant expression vector, which enables heterologous recombinant production of an enzyme of the invention. Thereby it is possible to make a highly purified beta-glucanase composition, characterized in being free from homologous impurities. This is highly advantageous for a number of industrial applications.
  • the invention relates to an isolated substantially pure biological culture of the Escherichia coli strain DSM No.
  • 11342 harbouring a beta-glucanase encoding DNA sequence (the beta-glucanase encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in Escherichia coli DSM 11342) obtained from a strain of the filamentous fungus Phaffia rhodozyma , or any mutant of said E . coli strain having retained the beta-glucanase encoding capability; and to an isolated substantially pure biological culture of the filamentous fungus Phaffia rhodozyma CBS No. 6938, from which the DNA sequence presented as SEQ ID No. 1 has been derived.
  • a cloned DNA sequence refers to a DNA sequence cloned in accordance with standard cloning procedures used in genetic engineering to relocate a segment of DNA from its natural location to a different site where it will be reproduced. The cloning process involves excision and isolation of the desired DNA segment, insertion of the piece of DNA into the vector molecule and incorporation of the recombinant vector into a cell where multiple copies or clones of the DNA segment will be replicated. 5
  • the "cloned DNA sequence” of the invention may alternatively be termed "DNA construct” or "isolated DNA sequence” .
  • the term “obtained from” as used herein in connection with a l ⁇ specific microbial source means that the enzyme is produced by the specific source, or by a cell in which a gene from the source have been inserted.
  • An isolated polypeptide As defined herein the term, “an isolated polypeptide” or “isolated beta-glucanase”, as used
  • beta-glucanase of the invention is a beta-glucanase or beta-glucanase part which is essentially free of other non- beta-glucanase polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about
  • isolated polypeptide may alternatively be termed “purified polypeptide” .
  • impurities means any impurity (e.g. another polypeptide than the enzyme of the invention) which originate from the homologous cell where the enzyme of the invention is originally obtained from.
  • the homologous cell may e . g . be a strain of Phaffia rhodozyma .
  • beta-glucanase encoding part used in connection with a DNA sequence means the region of the DNA sequence which corresponds to the region which is translated into a polypeptide sequence.
  • the translated polypeptide may further, in addition to the mature sequence exhibiting beta-glucanase activity, comprise an N-terminal signal sequence.
  • the signal sequence generally guides the secretion of the polypeptide.
  • beta-glucanase is defined according to standard enzyme EC-classification as EC 3.2.1.6.
  • the invention relates to a cloned DNA sequence encoding an enzyme exhibiting beta-glucanase activity, which DNA sequence comprises:
  • beta-glucanase encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in DSM 11342 such reference is also intended to include the beta-glucanase encoding part of the DNA sequence presented in SEQ ID NO 1.
  • the terms "the beta-glucanase encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in DSM 11342" and "the beta-glucanase encoding part of the DNA sequence presented in SEQ ID NO 1" may be used interchangeably.
  • the DNA sequence may be of genomic, cDNA, or synthetic origin or any combination thereof.
  • the present invention also encompasses a cloned DNA sequence which encodes an enzyme exhibiting beta-glucanase activity having the amino acid sequence set forth as the mature part of SEQ ID NO 2 , which differ from SEQ ID NO 1 by virtue of the degeneracy of the genetic code.
  • the DNA sequence shown in SEQ ID NO 1 and/or an analogue DNA sequence of the invention may be cloned from a strain of the filamentous fungus Phaffia rhodozyma producing the enzyme with beta-glucanase activity, or another or related organism as further described below (See section "Microbial sources").
  • the analogous sequence may be constructed on the basis of the DNA sequence presented as the beta- glucanase encoding part of SEQ ID No. 1, e . g .
  • nucleotide substitutions which do not give rise to another amino acid sequence of the beta-glucanase encoded by the DNA sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence (i.e. a variant of the beta-glucanase of the invention) .
  • amino acid changes are preferably of a minor nature, i.e. conservative amino acid substitutions that do not significantly affect the folding or activity of the protein, small deletions, typically of one to about 30 amino acids; small amino- or carboxyl- terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification, such as a poly-histidine tract; an antigenic epitope or a binding domain.
  • conservative substitutions are within the group of basic amino acids, such as arginine, lysine, histi- dine; acidic amino acids, such as glutamic acid and aspartic acid; polar amino acids, such as glutamine and asparagine; hydrophobic amino acids, such as leucine, isoleucine, valine; aromatic amino acids, such as phenylalanine, tryptophan, ty- rosine; and small amino acids, such as glycine, alanine, se- rine, threonine, methionine.
  • basic amino acids such as arginine, lysine, histi- dine
  • acidic amino acids such as glutamic acid and aspartic acid
  • polar amino acids such as glutamine and asparagine
  • hydrophobic amino acids such as leucine, isoleucine, valine
  • aromatic amino acids such as phenylalanine, tryptophan, ty- rosine
  • small amino acids such as g
  • beta-glucanase activity to identify amino acid residues that are critical to the activity of the molecule.
  • Sites of substrate-enzyme interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photo affinity labelling (cf. e.g. de Vos et al., (1992), Science 255, 306-312; Smith et al., (1992), J. Mol. Biol. 224, 899-904; Wlodaver et al., (1992), FEBS Lett. 309, 59-64) .
  • Polypeptides of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof.
  • a fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding another polypeptide to a nucleic acid sequence (or a portion thereof) of the present invention.
  • Techniques for producing fusion polypeptides are known in the art, and include, ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter (s) and terminator.
  • the DNA sequence of the invention can be cloned from the strain Escherichia coli DSM No. 11342 using standard cloning techniques e . g . as described by Sambrook et al., (1989), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab.; Cold Spring Harbor, NY.
  • the DNA sequence of the invention may also be cloned from an organism producing said enzyme, e.g. by purifying the enzyme, amino acid sequencing, and preparing a suitable probe or PCR primer based on this amino acid sequence.
  • DNA sequence of the invention can also be cloned by any general method involving
  • the DNA encoding a beta-glucanase of the invention may, in accordance .with well-known procedures, conveniently be cloned from a suitable source, such as any of organisms mentioned in the section "Microbial Sources", by use of synthetic oligonucleotide probes prepared on the basis of a DNA sequence disclosed herein.
  • a suitable oligonucleotide probe may be prepared on the basis of the beta- glucanase encoding part of the nucleotide sequences presented as SEQ ID No. 1 or any suitable subsequence thereof, or the basis of the amino acid sequence SEQ ID NO 2.
  • the DNA sequence homology referred to above is determined as the degree of identity between two sequences indicating a derivation of the first sequence from the second.
  • the homology may suitably be determined by means of computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453).
  • the coding region of the DNA sequence exhibits a degree of identity preferably of at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97% with the beta-glucanase encoding part of the DNA sequence shown in SEQ ID No. 1.
  • hybridization referred to above is intended to comprise an analogous DNA sequence which hybridizes to a double-stranded nucleotide probe corresponding to the beta-glucanase encoding part of the DNA sequence shown in SEQ ID NO 1, i.e. nucleotides 1-1275, under at least low stringency conditions as described in detail below.
  • Suitable experimental conditions for determining hybridization at low, medium, or high stringency between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5 x SSC (Sodium chloride/Sodium citrate, Sambrook et al. 1989) for 10 min, and prehybridization of the filter in a solution of 5 x SSC, 5 x Denhardt ' s solution (Sambrook et al. 1989), 0.5 % SDS and 100 ⁇ g/ml of denatured sonicated salmon sperm DNA (Sambrook et al.
  • the filter is then washed twice for 30 minutes in 2 x SSC, 0.5 % SDS at least 55 °C (low stringency) , more preferably at least 60°C (medium stringency) , still more preferably at least 65 °C (medium/high stringency) , even more preferably at least 70 °C (high stringency) , even more preferably at least 75 °C (very high stringency) .
  • Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using a x-ray film.
  • the polypeptide homology referred to above is determined as the degree of identity between two sequences indicating a derivation of the first sequence from the second.
  • the homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453.
  • the mature part of a polypeptide encoded by an analogous DNA sequence exhibits a degree of identity preferably of at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, especially at least 97% with the mature part of the amino acid sequence shown in SEQ ID NO 2.
  • the present invention is also directed to Beta- glucanase variants which have an amino acid sequence which differs by no more than three amino acids, preferably by no more than two amino acids, and more preferably by no more than one amino acid from the mature part of the amino acid sequence set forth in SEQ ID NO 2.
  • Antibodies to be used in determining immunological cross- reactivity may be prepared by using a purified beta-glucanase. More specifically, antiserum against the beta-glucanase of the invention may be raised by immunizing rabbits (or other rodents) according to the procedure described by N. Axelsen et al. in A Manual of Quantitative Immunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter 23, or A. Johnstone and R. Thorpe, I munochemistry in Practice, Blackwell Scientific Publications, 1982 (more specifically p. 27-31) .
  • Purified immunoglobulins may be obtained from the antiserum obtained, for example by salt precipitation ((NH 4 ) S0 ) , followed by dialysis and ion exchange chromatography, e . g . on DEAE-Sephadex. Immunochemical characterization of proteins may be performed either by Outcherlony double-diffusion analysis
  • the beta-glucanase of and the corresponding cloned DNA sequence of the invention may be obtained from any fungal strain.
  • a preferred genus is Phaffia , wherein a preferred strain is Phaffia rhodozyma .
  • Phaffia rhodozyma CBS No. 6938 An isolate of a strain of Phaffia rhodozyma from which an beta-glucanase of the invention can be obtained is Phaffia rhodozyma CBS No. 6938, which has been deposited according to the Budapest Treaty on the International Recognition of the
  • CBS Calarn, The Netherlands
  • the expression plasmid pYES 2.0 comprising the full length cDNA sequence encoding the beta-glucanase of the invention has been transformed into a strain of the Escherichia coli which was deposited according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH. , Masheroder Weg lb, D- 38124 Braunschweig, Federal Republic of Germany, (DSM) . Deposit date : 18 of December 96
  • the expression vector of the invention may be any expression vector that is conveniently subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which the vector it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome (s) into which it has been integrated.
  • the DNA sequence encoding the beta-glucanase should be operably connected to a suitable promoter and terminator sequence.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins which are either homologous or heterologous to the host cell.
  • the procedures used to ligate the DNA sequences coding for the beta-glucanase, the promoter and the terminator and to insert them into suitable vectors are well known to persons skilled in the art (cf. e . g . Sambrook et al., (1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY) .
  • suitable promoters for use in filamentous fungus host cells are, e.g. the ADH3 promoter (McKnight et al . , The EMBO J . (1985) , 2093 - 2099) or the tpiA promoter.
  • Examples of other useful promoters are those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral a- amylase, Aspergillus niger acid stable a-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (gluA) , Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase or Aspergillus nidulans acetamidase.
  • Host Cells Host Cells
  • the present invention also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides.
  • host cell encompasses any progeny of a parent cell which is not identical to the parent cell due to mutations that occur during replication.
  • the cell is preferably transformed with a vector comprising a nucleic acid sequence of the invention followed by integration of the vector into the host chromosome.
  • Transformation means introducing a vector comprising a nucleic acid sequence of the present invention into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. Integration is generally considered to be an advantage as the nucleic acid sequence is more likely to be stably maintained in the cell. Integration of the vector into the host chromosome may occur by homologous or non-homologous recombination as described above. The choice of host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
  • the host cell may be a unicellular microorganism, e.g. a prokaryote, or a non- unicellular microorganism, e.g. a eukaryote.
  • the host cell is a fungal cell.
  • "Fungi” as used herein includes the phyla Ascomycota,
  • Basidiomycota, Chytridiomycota , and Zygo ycota as defined by Hawksworth et al . , In , Ainsworth and Bisby' s Dictionary of The Fungi , 8th edition, 1995, CAB International, University Press, Cambridge, UK
  • Oomycota as cited in Hawksworth et al . , 1995, supra , page 171
  • all mitosporic fungi Hawksworth et al . , 1995, supra
  • Basidiomycota include mushrooms, rusts, and smuts.
  • Representative groups of Chytridiomycota include, e.g., Allomyces , Blastocladiella , Coelomomyces , and aquatic fungi.
  • Representative groups of Oomycota include, e . g . , Saproleg- niomycetous aquatic fungi (water molds) such as Achlya . Examples of mitosporic fungi include Aspergillus , Penicillium , Candida , and Alternaria .
  • Representative groups of Zygomycota include, e . g. , Rhizopus and Mucor.
  • the fungal host cell is a filamentous fungal cell.
  • "Filamentous fungi” include all fila- 5 mentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al . , 1995, supra) .
  • the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium , Aspergillus , Fusarium, Humicola , Mucor, Myceliophthora , Neurospora ,
  • Penicillium 10 Penicillium , Thielavia , Tolypocladium , and Trichoderma or a teleomorph or synonym thereof.
  • Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se .
  • Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al . , 1983, Journal of Bacteriology
  • Mammalian cells may be transformed by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology 52:546) .
  • the present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence 35 encoding the enzyme, is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
  • a suitable host cell which has been transformed with a DNA sequence 35 encoding the enzyme
  • the resulting enzyme is recovered from the culture.
  • the homologous host cell may be a strain of Phaffia rhodozyma .
  • the medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question.
  • the expressed beta-glucanase may conveniently be secreted into the culture medium and may be recovered therefrom by well-known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography , or the like.
  • beta-glucanase of the invention Activity of beta-glucanase of the invention:
  • the beta-glucanase of the invention has ENDO-1, 3 (4) -BETA- GLUCANASE (EC 3.2.1.6) enzyme activity.
  • beta-glucanase of the invention may advantageously be used in any of the general known applications for a beta-glucanase.
  • the dosage of the enzyme either used as a single enzyme or in an composition, said composition comprising either other enzyme activities, or other ingredients according to the art, of the invention and other conditions under which the enzyme or composition is used may be determined on the basis of methods known in the art.
  • Examples are use of the beta-glucanase of the invention in the brewing industry, as the enzymes degrades the barley b- glucan and thereby reduces the viscosity and improves the filterability of the wort.
  • beta-glucanse according to the invention is preferably used as an agent for degradation or modification of b- glucan containing material such as microbial cell walls.
  • the enzyme preparation of the invention may be used for rupturing or lysing cell walls of microorganisms thereby enabling recovery of desirable products produced by the microorganism.
  • the specific composition of the enzyme preparation to be used should be adapted to the composition of the cell wall to be ruptured or lysed.
  • yeast cell walls have been found to comprise two main layers, an outer layer of protein-mannan complex and an inner glucan layer.
  • the enzyme preparation comprises at least protease, mannanase and b-glucanase activity.
  • the extract recovered after rupture of the microbial cell walls normally comprises a number of different components, such as pigments, vitamins, colorants and flavourants. Extracts obtained from rupture of yeast, i.e. yeast extracts, are used as such, e.g. for food or feed applications - or components thereof may be recovered and optionally further processed.
  • Such products include vitamins, colorants (e.g. carotenoids, Q-10 and astaxanthin) , enzymes, proteins and flavour components or flavour enhancers (e.g. MSG, 5 ' -GMP and 5' -IMP).
  • vitamins, colorants e.g. carotenoids, Q-10 and astaxanthin
  • enzymes e.g. enzymes, proteins and flavour components or flavour enhancers (e.g. MSG, 5 ' -GMP and 5' -IMP).
  • flavour enhancers e.g. MSG, 5 ' -GMP and 5' -IMP
  • the enzyme preparation of the invention may be used in the production of protoplasts from yeasts (e.g. of Saccharomyces sp. or Schizosaccharomyces sp.) or from fungi (e.g. Aspergillus sp. or Penicillium sp.). Preparation and regeneration of protoplast from such organisms are important in fusion, transformation and cloning studies.
  • the production of protoplasts may be performed in accordance with methods known in the art.
  • the invention may also be used for improving fungal transformation.
  • the enzyme or enzyme preparation according to the invention may be used in the preparation of pharmaceuticals, especially products entrapped inside the cells in the cytoplasmic membrane, the periplasmic space and/or the cell wall.
  • the enzyme preparation of the invention may be used in the modification of b-glucans such as curdlan and laminarin.
  • an preferred use of an beta-glucanase of the invention is for use in the process of wine-making, in particular for removing of beta-glucans before filtration of the wine.
  • Phaffia rhodozyma CBS No. 6938 comprises the beta- glucanase encoding DNA sequence of the invention.
  • Yeast strain The Saccharomyces cerevisiae strain used was W3124 (MATa; ura 3-52; leu 2-3, 112; his 3-D200; pep 4-1137; prcl::HIS3; prbl:: LEU2 ; cir+) .
  • E. coli strain DH10B (Life Technologies)
  • the Aspergillus expression vector pHD414 is a derivative of the plasmid p775 (described in EP 238 023) .
  • the construction of pHD414 is further described in WO 93/11249.
  • Fermentation procedure of Phaffia rhodozyma CBS No. 6938 for mRNA isolation Fermentation of Phaffia rhodozyma CBS No. 6938 was performed in shake-flasks with BPX media (described below) at 20 °C for 2-3 days.
  • Identification of positive yeast clones i.e. clones which comprise a gene encoding for beta-glucanase activity was done as described below.
  • the yeast transformants is plated on SC agar containing 0.1% AZCL beta-glucan (Megazyme, Australia) and 2% galactose and incubated for 3-5 days at 30°C
  • Beta-glucanase positive colonies is identified as colonies surrounded by a blue halo.
  • a beta-glucanase-producing yeast colony is inoculated into 20 ml YPD broth in a 50 ml glass test tube. The tube is shaken for 2 days at 30°C The cells are harvested by centrifugation for 10 min. at 3000 rpm. DNA is isolated according to WO 94/14953 and dissolved in 50 ml water. The DNA is transformed into E . coli by standard procedures. Plasmid DNA is isolated from E . coli using standard procedures, and analyzed by restriction enzyme analysis. The cDNA insert is excised using appropriate restriction enzymes and ligated into an Aspergillus expression vector.
  • Transformation of Aspergillus oryzae or Aspergillus niger Protoplasts may be prepared as described in WO 95/02043, p. 16, line 21 - page 17, line 12, which is hereby incorporated by reference.
  • Protoplasts are mixed with pA2BG171 (See example 1) . The mixture is left at room temperature for 25 minutes.
  • 0.2 ml of 60% PEG 4000 (BDH 29576), 10 mM CaCl and 10 mM Tris-HCl, pH 7.5 is added and carefully mixed (twice) and finally 0.85 ml of the same solution is added and carefully mixed.
  • the mixture is left at room temperature for 25 minutes, spun at 2500 g for 15 minutes and the pellet is resuspended in 2 ml of 1.2 M sorbitol. After one more sedimentation the protoplasts are spread on minimal plates (Cove, Biochem. Biophys. Acta 113 (1966) 51-56) containing 1.0 M sucrose, pH 7.0 , 10 mM acetamide as nitrogen source and 20 mM CsCl to inhibit background growth. After incubation for 4-7 days at 37 °C spores are picked and spread for single colonies. This procedure is repeated and spores of a single colony after the second reisolation is stored as a defined transformant .
  • Each of the A . oryzae transformants are inoculated in 10 ml of YPM (cf. below) and propagated. After 2-5 days of incubation at 30°C, the supernatant is removed.
  • the beta-glucanase activity is identified by applying 20 ⁇ l supernatant to 4 mm diameter holes punched out in agar plates containing 0.2% AZCL beta- glucan (Megazyme, Australia) . Beta-glucanase activity is then identified as a blue halo.
  • Fed batch fermentation was performed in a medium comprising maltodextrin as a carbon source, urea as a nitrogen source and yeast extract.
  • the fed batch fermentation was performed by inoculating a shake flask culture of A. oryzae host cells in question into a medium comprising 3.5% of the carbon source and 0.5% of the nitrogen source. After 24 hours of cultivation at pH 7.0 and 34 °C the continuous supply of additional carbon and nitrogen sources were initiated. The carbon source was kept as the limiting factor and it was secured that oxygen was present in excess. The fed batch cultivation was continued for 4 days.
  • Fermentation of A . oryzae was performed in shake-flasks with DAP-2C-1 media (described below) .
  • the inoculation was performed by cultivation of A . oryzae in cove tubes containing 1,2 M sorbitol, 1 % glucose and 0,01 M urea at 37 °C for 4-5 days.
  • the spores from cove tubes was dissolved in sterile water added 0,1 % tween and used to inoculate the shake-flasks.
  • the fermentation was continued at 30 °C for 3 days.
  • the beta-glucanase encoding part of the DNA sequence shown in SEQ ID No. 1 coding for the beta-glucanase of the invention can be obtained from the deposited organism Escherichia coli DSM 11342 by extraction of plasmid DNA by methods known in the art (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY).
  • YPD 10 g yeast extract, 20 g peptone, H 0 to 900 ml. Autoclaved, 100 ml 20% glucose (sterile filtered) added.
  • YPM 10 g yeast extract, 20 g peptone, H 2 0 to 900 ml. Autoclaved, 100 ml 20% maltodextrin (sterile filtered) added.
  • 10 x Basal salt 75 g yeast nitrogen base, 113 g succinic acid, 68 g NaOH, H 2 0 ad 1000 ml, sterile filtered.
  • SC-URA 100 ml 10 x Basal salt, 28 ml 20% casamino acids without vitamins, 10 ml 1% tryptophan, H 0 ad 900 ml, autoclaved, 3.6 ml 5% threonine and 100 ml 20% glucose or 20% galactose added.
  • SC-agar SC-URA, 20 g/1 agar added.
  • SC-variant agar 20 g agar, 20 ml 10 x Basal salt, H 2 0 ad 900 ml, autoclaved
  • BPX media 50 g potato flour, 25 g barley flour, 0,05 g BAN 800 MG, H 2 0 ad 800 mL. Inkubated at 60-85 °C for 30 min. 5 g Na- Caseinate at 60 °C added and dissolve. Thereafter 10 g soy grit, 4,5 g Na 2 HP0 4 12H 2 0, 0,1 L Pluronic added, H 2 0 ad 1000 mL. pH adjusted to 7,4-7,5 with 4 N NaOH. Autoclaved.
  • DAP-2C-1 media 11 g MgSO 4 7H 2 0, 1 g KH 2 P0 4 , 2 g citric acid, 30 g maltodextrin, 6 g K 3 PO 3H 2 0, 0,5 g yeast extract (Difco) , 1 mL pluronic, 0,5 mL KU-6 (see below) , H 2 0 ad 1000 mL. 1 CaC0 3 tablet per 250 mg added. Autoclaved, 3,5 mL 50 % (NH 4 ) HPO 4 and 5 mL 20 % lactic acid (sterile filtered) added. pH is 5,2.
  • Beta-glucanase-positive colonies were identified and isolated on SC-agar plates as described above (vide supra) .
  • cDNA inserts were amplified directly from the yeast colonies and characterized as described in the Materials and Methods section above.
  • the DNA sequence of the cDNA encoding the beta- glucanase is shown in SEQ ID No. 1 and the corresponding amino acid sequence is shown in SEQ ID No. 2.
  • SEQ ID No. 1 DNA nucleotides from No 1 to No. 1275 define the beta-glucanase encoding region.
  • the cDNA is obtainable from the plasmid in DSM 11342.
  • Total DNA was isolated from a yeast colony and plasmid DNA was rescued by transformation of E . coli as described above.
  • the DNA was digested with appropriate restriction enzymes, size fractionated on gel, and a fragment corresponding to the beta- glucanase gene was purified.
  • the gene was subsequently ligated to pHD414, digested with appropriate restriction enzymes, resulting in the plasmid pA2BG171. After amplification of the DNA in E . coli the plasmid was transformed into Aspergillus oryzae as described above.
  • Substrates 0.1 w/v% AZCL- ⁇ -Glucan; 0.1 w/v% AZCL-curdlan; 0.1 w/v% AZCL-He-Cellulose. (all from MegaZyme, Australia) .
  • the pH was adjusted to 5.0 before mixing with 800 ml of 2- methoxyethanol .
  • the substrate AZCL-He-Cellulose comprises substantially only 1,4-bindings in ⁇ -D-glycanes, where the other two comprise 1,3- bindings in ⁇ -D-glycanes.
  • SEQ ID No. 1 shows a cloned DNA sequence of the invention, comprising a DNA sequence encoding an enzyme exhibiting beta-glucanase activity.
  • ORGANISM Phaffia rhodozyma
  • GCT TCA AAT TCT TCC TCC GAG CAC GAG ACG AAC CGA ATC GCT AGC
  • GCT 192 Ala Ser Asn Ser Ser Ser Glu His Glu Thr Asn Arg lie Ala Ser Ala 50 55 60
  • SEQ ID No. 2 shows the amino acid sequence of a beta-glucanase of the invention.
  • Lys Gly Glu lie Asp lie Leu Glu Gly Thr His Ser Trp Asp Arg Asn 225 230 235 240

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Abstract

La présente invention concerne une enzyme ayant une activité de β-glucanase, une séquence d'ADN clonée codant cette enzyme, un procédé de production de cette enzyme, une composition enzymatique renfermant ladite enzyme à activité de β-glucanase, ainsi que l'utilisation de ladite enzyme et de la composition enzymatique pour un grand nombre d'applications industrielles. Cette enzyme peut être obtenue à partir de la Phaffia rhodozyma.
PCT/DK1998/000057 1997-02-14 1998-02-13 Enzyme a activite d'endo-1,3(4)-beta-glucanase WO1998036056A1 (fr)

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AU58530/98A AU5853098A (en) 1997-02-14 1998-02-13 An enzyme with endo-1,3(4)-beta-glucanase activity

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DK0161/97 1997-02-14
DK16197 1997-02-14

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031533A1 (fr) * 1994-05-11 1995-11-23 Novo Nordisk A/S ENZYME PRESENTANT UNE ACTIVITE D'ENDO-1,3(4)-β-GLUCANASE

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031533A1 (fr) * 1994-05-11 1995-11-23 Novo Nordisk A/S ENZYME PRESENTANT UNE ACTIVITE D'ENDO-1,3(4)-β-GLUCANASE

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 5, Biosis, Dialog Accession No. 1970589, Biosis Accession No. 62060149, VILLA T.G. et al., "Method of Purifying Beta-1-3 Glucanase EC-3.2.1.6 from Candida-Utilis"; & APPL. ENVIRON. MICROBIOL., 32(1), 1976, 185-187. *

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