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)
  • C12Y302/01039—Glucan endo-1,3-beta-D-glucosidase (3.2.1.39)
• • 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
  • 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
  • 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)
  • C12Y302/01058—Glucan 1,3-beta-glucosidase (3.2.1.58)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

• the present invention relates to a novel enzyme exhibiting ⁇ - 1,3-glucanase activity. More specifically a DNA construct encoding the novel enzyme, an expression vector comprising said DNA construct, a cell comprising said DNA construct or said recombinant expression vector, a method of producing said novel enzyme, an enzyme preparation comprising said novel enzyme, and use of the enzyme for degradation or modification of ⁇ -glucan containing material.
• fungi A large diverse group of nonmotile, heterotrophic, eukaryotic organisms are collectively referred to as fungi. Most fungi are saprophytes, i.e. securing their food from dead organic material. Because of their heterotrophic properties, i.e. using organic material as carbon source, many fungi produce metabolites of industrial interest. Certain species are further useful as sources for food, whereas others are responsible for spoiling almost any organic material in which they come in contact.
• Fungal cells range in size from microscopic unicellular organ ⁇ isms to macroscopic as e.g. mushrooms.
• True fungi are in general terrestrial and includes Zygomycetes , such as Rhizopus , Basidiomycetes such as Puccinxa graminis , Ascomycetes , such as Neurospora and Saccharomyces and _->e ⁇ -te--O-r.ycetes, such as Penici Ilium and Aspergillus .
• Fungal microorganisms include multicellular as well as unicel ⁇ lular organisms and are in general considered to consist of yeasts and molds. Unicellular fungi are primarily yeasts, while the term mold is used for fungi that are predominantly mycelial.
• Fungal cells have a quite complex structure, constituting of a cytoplasm, comprising nucleus, mitochondria, microbodies etc. encapsulated by a cytoplasmic membrane.
• a cytoplasmic membrane Chemically and structurally the cytoplasmic membrane consist of a bilayer of phospholipids with different proteins inserted into it.
• the cytoplasmic membrane is surrounded by the rigid cell wall.
• the cell walls of most true fungal microorganisms contain a network of glucan, which gives the cell wall strength. Further major fungal cell walls constituents are mannoprotein and chitin.
• Glucan and chitin is far more resistant to microbial degrada ⁇ tion than cellulose, which is the major constituent of the cell wall of many fungi-like organisms, such as Oomycetes .
• Glucan is predominantly 0-1,3-linked with some branching via 1,6-linkage (Manners et al., Biotechnol. Bioeng, 38, p. 977, 1973) , and is known to be degradable by certain 3-1,3-glucanase systems.
• /8-1,3-glucanase includes the group of endo-jS-1,3-glucanases also called laminarinases (E.C. 3.2.1.39 and E.C. 3.2.1.6, Enzyme Nomenclature, Academic Press, Inc, 1992) .
• the unit operation of cell disruption appears as an essential first step for intracellular products separation and downstream processing of valuable intracellular products.
• SCP Selective Cell Permeabilization
• SPR Selective Protein Recovery
• SCP and SPR involves the use of pure preparations of cell-wall- degrading 3-glucanases to increase fungal cell wall porosity (with very limited cell lysis) and facilitate the release of intracellular proteins.
• SCP gives primary separ ⁇ ation of the target product from some of its major contaminants.
• a major limitation to this approach is the relatively low level of expression of yeast lytic enzymes presently obtained in the bacteria used for the production of these enzymes (e.g. Oerskovia xanthineolytica, Andrews and Asenjo, Biotech. Bioeng, 30, p. 628, 1987).
• Today Oerskovia xanthineolytica are sometimes referred to as Cellulomonas cellulans . However, the name Oerskovia xanthineolytica will be used below.
• a number of commercial enzyme compositions useful in the enzymatic lysis of fungal cells are available. Such products normally comprise multiple enzymatic activities, e . g. including 0-1,3- and 0-1,6-glucanase, protease, chitinase, mannase and other enzymes capable of degrading cell wall components.
• the lvtic system of Oerskovia xanthineolytica LLG109 The lytic enzyme system of Oerskovia xanthineolytica LLG109 has partially been isolated and purified and some of the glucanase and protease components have been characterised (Ventom and Asenjo, Enzyme Microb. Technol., 13, p. 71, 1991).
• the specific activity of the enzyme was 11.1 U/mg.
• the K ⁇ for 0-1,3-glucanase activity on yeast glucan was 2.5 mg/ml for laminarin (a soluble 0-1,3-glucan) 0.95 mg/ml.
• the pH optimum for 0-1,3-glucanase was 8.0 on yeast glucan and 6.0 on laminarin substrate.
• the lytic 0-1,3-glucanase caused only limited lytic activity on viable yeast (Saccharomyces cerevisiae) cells (Ventom and Asenjo, supra , 1991) , but this was stimulated synergistically by the lytic protease component.
• DD 226012 (Akad. Horshaft DDR) which concerns a method for production of a Bacillus 0-1,3-glucanase.
• JP 61040792 A (DOI K) describes a cell wall-cytolase 0-1,3-glucanase recombinant plasmid for removing the cell walls of yeast.
• the gene is derived from Arthrobacter and is trans ⁇ formed into Escherichia group bacteria.
• EP 440.304 concerns plants provided with improved resistance against pathogenic fungi transformed with at least one gene encoding an intracellular chitinase, or an intra- or extracellular 0-1,3-glucanase.
• the matching recombinant polynucleotides is also disclosed.
• WO 87/01388 describes a method for preparing cell lytic enzymes, such as 0-1,3-glucanases, which can be produced by Oerskovia .
• WO 92/03557 discloses a recombinant DNA expression vector comprising a 2.7 kb DNA sequence, derived from Oerskovia xanthineolytica , encoding a 0-1,3-glucanase. Said glucanase, expressed in E. coli, exhibits glucanase activity and no protease activity.
• E. coli has a number of deficiencies in connection with large scale industrial enzyme production. First of all the glucanase is expressed intercellular. Consequently the cells need to be opened to get access to the enzyme. This makes recovery of the enzyme cumbersome and expensive.
• a 0-1,3-glucanase substantially free of protease activity which is capable of opening the cell walls in a gentle way. This will facilitate the recovery and purification of the target protein. Further, it would be advantageous to express the gene encoding the target protein in a heterologous host cell capable of increasing the production yield.
• the invention relates to a DNA construct comprising a DNA sequence encoding a novel enzyme exhibiting 0-1,3-glucanase activity, which DNA sequence a) comprises the DNA sequence shown in SEQ ID No. l, or b) comprises an analogue of the DNA sequence shown in SEQ ID No. 1 , which i) hybridizes with the same oligonucleotide probe as the DNA sequence shown in SEQ ID No. 1, and ii) encodes a polypeptide which is homologous with the polypeptide encoded by a DNA sequence comprising the DNA sequence shown in SEQ ID No.
• the Oerskovia xanthineolytica LLG109 strain has been deposited by the inventors according to the Budapest Treaty at the Deutshe Sammlung von Mikroorganis en und Zellkulturen GmbH., (DSM) .
• analogue of the DNA sequence shown in SEQ ID No. 1 is intended to indicate any DNA sequence encoding an enzyme exhibiting 0-1,3-glucanase activity which has the properties i)-iii) above.
• the analog ⁇ ous DNA sequence is intended to indicate any DNA sequence encoding an enzyme exhibiting 0-1,3-glucanase activity which has the properties i)-iii) above.
• - is constructed on the basis of the DNA sequence shown in SEQ ID No. 1, e.g. by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the 0- 1,3-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 do give rise to a different amino acid sequence and therefore, possibly, a different protein structure which might give rise to a 0-1,3-glucanase mutant with different properties than the native enzyme.
• the analogous DNA sequence may be a subsequence of the DNA sequences shown in SEQ ID No. 1, for which the encoded protein exhibits 0-1,3- glucanase activity.
• hybridization referred to in i) above is intended to indicate that the analogous DNA sequence hybridizes to the same probe as the DNA sequence encoding the novel 26 kDa 0-1,3- glucanase enzyme under certain specified conditions which are described in detail in the Materials and Methods section hereinafter.
• the analogous DNA sequence is highly homologous to the DNA sequence such as at least 60% homologous to the sequence shown above encoding a 0-1,3-glucanase of the inven ⁇ tion, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or even at least 95% homologous to the sequence shown in SEQ ID no. 1 below.
• the degree of homology referred to in ii) above is determined as the degree of identity between the 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 (Needleman, S.B.
• the coding region of the DNA sequence exhibits a degree of identity preferably of at least 60%, such as at least 75% or 90% with the enzyme encoded by a DNA construct comprising the DNA sequence SEQ ID No. 1.
• the term "derived from” in connection with property iii) above is intended not only to indicate a 0-1,3-glucanase produced by Oerskovia xanthineolytica LLG109, but also a 0-1,3-glucanase encoded by a DNA sequence isolated from Oerskovia xanthineolytica LLG109 and produced in a host organism trans- formed with a vector comprising said DNA sequence.
• the immunological reactivity may be determined by the method described in the Materials and Methods section below.
• the invention relates to the construction of an expression vector harbouring a DNA construct of the invention, a cell comprising the DNA construct or expression vector, and a method of producing a novel enzyme exhibiting 0- 1,3-glucanase activity, which method comprises culturing said cell under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
• the invention relates to a novel enzyme exhibiting 0-1,3-glucanase activity, which enzyme a) is encoded by a DNA construct of the invention b) produced by the method of the invention, and/or c) is immunologically reactive with an antibody raised against a purified 0-1,3-glucanase encoded by the DNA sequence shown in SEQ ID No. 1 derived from Oerskovia xanthineolytica LLG109.
• the present invention also relates to an enzyme preparation useful for the degradation or modification of 0-glucan contain ⁇ ing materials, in particular icrobial cell wall material, said composition being enriched by an enzyme exhibiting 0-1,3,- glucanase activity, as described above.
• the final object of the invention is to provide a process for recovery of biological components from fungal cells, by subjecting the fungal cells in question to the novel 0-1,3- glucanase or an enzyme preparation thereof.
• Figure l shows the map of plasmid pPFFl.
• Figure 2 shows the map of plasmid pPF8A.
• Figure 3 shows the 1.5 kb BamHI-Kpnl fragment sequence accord ⁇ ing to the invention with compared with the predicted amino acid sequence and the partially determined amino acid sequence
• Figure 4 shows the map of plasmid pPF15BK
• Figure 5 shows the map of plasmid pPFUBgK.
• the present inventors have now surprisingly succeeded in providing a novel 0-1,3-glucanase substantially free of protease activity.
• the novel enzyme opens up the cells in a gently way, which involves making large and porous pores in the cell walls. This will further lead to a reduced amount of contaminants.
• the inventors have accomplished to express the gene encoding the novel 0-1,3-glucanase in a suitable heterologous host cell. Specifically in a strain of Bacillus subtil is , suitable for large scale industrial production. The yield of the novel 0-1,3-glucanase is increased in comparison to the parent cell from which the gene originates.
• DNA sequence of the invention encoding an enzyme exhibiting 0-1,3-glucanase activity may be isolated by a general method involving
• the DNA sequence coding for the enzyme may for instance be isolated from Oerskovia xanthineolytica strain LLG109 (Lechevalier, Int. J. Sys. Bacteriol., 22(4), p. 260, 1972), and selecting for clones expressing the appropriate enzyme activity (i.e. 0-1,3-glucanase activity as defined by the ability of the enzyme to hydrolyse 0-1,3-glucan bonds of a suitable substrate such as laminarin or AZCL-curdlan, cf. the Materials and Methods section hereinafter) .
• Oerskovia xanthineolytica strain LLG109 Lechevalier, Int. J. Sys. Bacteriol., 22(4), p. 260, 1972
• the appropriate enzyme activity i.e. 0-1,3-glucanase activity as defined by the ability of the enzyme to hydrolyse 0-1,3-glucan bonds of a suitable substrate such as laminarin or AZCL-curd
• the Oerskovia xanthineolytica LLG109 strain has been deposited by the inventors according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Deutshe Sam lung von Mikroorganismen und Zellkulturen GmbH. , (DSM) . Deposit date : 13.10.95
• the appropriate DNA sequence may then be isolated from the clone by standard procedures, e .g. as described in Materials and Methods section.
• the DNA construct comprises a DNA sequence, which a) comprises the DNA sequence shown in SEQ ID No. 1, or b) comprises an analogue of the DNA sequence shown in SEQ ID No. 1 , which i) hybridizes with the same oligonucleotide probe as the DNA sequence shown in SEQ ID No. 1, and ii) encodes a polypeptide which is homologous with the polypeptide encoded by a DNA sequence comprising the DNA sequence shown in SEQ ID No. 1, or iii) encodes a polypeptide which is immunologically reactive with an antibody raised against a purified 0-1,3-glucanase encoded by the DNA sequence shown in SEQ ID No. 1 derived from OersJtovia xanthineolytica LLG109.
• the DNA construct comprises the sequence shown in SEQ ID No. 1 or SEQ ID No. 3.
• polypeptide encoded by said DNA sequences shown in SEQ ID No. 1 and SEQ ID No. 3 are shown in SEQ ID No. 2 and SEQ ID No. 3.
• a preferred method of amplifying specific DNA sequences are by the use of polymerase chain reaction (PCR) using degenerate oligonucleotide probes synthesized.
• PCR polymerase chain reaction
• the PCR may be carried out using the techniques described in US Patent No. 4,683,202 or by R.K. Saiki et al. (1988), Science, 239, 487-491
• DNA sequence coding for a homologous enzyme i.e. an analogous DNA sequence
• the DNA sequence may be derived from another microorganism, in particular either a fungus or bacteria.
• Such DNA sequences may originates from fungi, comprising a strain of an Aspergillus sp. , in particular a strain of A. a ⁇ uleatus or A. niger, a strain of Trichoderma sp. , in particu ⁇ lar a strain of T. reesie , T. viride , T. longibrachiatum or T. koningii , T. harzianum or a strain of a Fusarium sp. , in particular a strain of F. oxysporum, or a strain of a Humicola sp. .
• DNA sequence encoding a homologous enzyme may be expected to derive from bacteria, such as another strain of a Oerskovia sp. , or a strain of an Arthrobacter sp. , Cytophaga sp. , --hodoti-er-nus sp. , in particular a strain of Rh . marinu ⁇ , or a strain of a Clostrium , in particular strains of Cl . thermocellum, or a strain of Bacillus sp. , in particular strains of B . lichenitormi ⁇ ., B . amyloliquefaciens, or B. circulans .
• bacteria such as another strain of a Oerskovia sp. , or a strain of an Arthrobacter sp. , Cytophaga sp. , --hodoti-er-nus sp. , in particular a strain of Rh . marinu ⁇ , or a strain
• the DNA coding for a 0-1,3-glucanase of the invention may, in accordance with well-known procedures, conveniently be isolated from DNA from any of the above mentioned organisms 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 a partial nucleotide sequence of the sequence shown in SEQ ID No. 1.
• the DNA sequence may subsequently be inserted into a recombi ⁇ nant expression vector.
• This may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which 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 0-1,3-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 either homologous or heterologous to the host cell.
• a vector under control of the promoter for the maltogenic 0-1,3-amylase from Bacillus stearothermophilus and/or the signal of Bacillus stearothermophilus is preferred.
• the expression vector comprises the plasmid pPFFl shown in Figure 1.
• the host cell which is transformed with the DNA sequence encoding the enzyme of the invention may be either eukaryotic cells or prokaryotic cells.
• Suitable prokaryotic host cells are in particular bacterial cells.
• Examples of such bacterial host cells which, on cultivation, are capable of producing the novel enzyme of the invention are gram-positive bacteria such as strains of Bacillus , such as strains of B . subtilis, B . licheniformis, B . lentus, B . brevis, B . stearothermophilus, B . alkalophilus , B . amyloliquefaciens, B . coagulan ⁇ , B . circulans, B . lautus, B . megaterium or B . thuringiensis, or strains of Streptomyces, such as S . lividans or S. murinus , or gram-negative bacteria such as Escherichia coli .
• the transformation of the bacteria may be effected by protoplast transformation or by using competent cells in a manner known per se (cf. Sambrook et al., supra , 1989).
• the polypeptide When expressing the enzyme in bacteria such as E. coli , the polypeptide may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies) , or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the polypeptide is refolded by diluting the denaturing agent. In the latter case, the polypeptide may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the polypeptide.
• sonication or osmotic shock to release the contents of the periplasmic space and recovering the polypeptide.
• the bacterial host cell is a strain of Bacillus subtilis, especially the strain B . subtilis DN1885 or the protease deficient strain B. suJtilis TOC46.
• Suitable eukaryotic cells are in particular fungal cells such as yeasts or filamentous fungal cells.
• yeast cells include cells of Saccharomyces spp. , in particular strains of Saccharomyces cerevisiae , Saccharomyces kluyveri, Sacchromyces uvarum , or
• Schizosaccharo yces spp. such as Schizosaccharomyces pombe .
• the DNA sequence encoding the polypeptide of the invention may be preceded by a signal sequence and optionally a leader sequence , e .g. as described above.
• suitable yeast cells are strains of Kluyvero yces spp., such as K. lactis , or Hansenula spp., e . g. H . polymorpha , or Pichia spp., e .g. P. pastori ⁇ , Yarrowia spp., such as Yarrowia lipolytica (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; US patent no. 4,882,279).
• Examples of other fungal cells are cells of filamentous fungi, e . g. Aspergillus spp., Neuro ⁇ pora spp., Fu ⁇ arium spp. or Trichoderma spp., in particular strains of A. oryzae , A. nidulan ⁇ or A. niger.
• Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 238 023 and EP 184 438.
• the transformation of F. oxysporum may, for instance, be carried out as described by Malardier et al., Gene 78, p. 147-156, 1989.
• a filamentous fungus When a filamentous fungus is used as the host cell, it may be transformed with the DNA construct of the invention, conveni ⁇ ently by integrating the DNA construct in the host chromosome to obtain a recombinant host cell.
• This integration is gen ⁇ erally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e .g. by homologous or heterologous recombination.
• the present invention relates to a method of producing an enzyme according to the invention, wherein a suitable host cell transformed with a DNA sequence encoding the enzyme is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
• the medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection) .
• the present invention relates to a novel enzyme preparation useful for the modification or degradation of 0-glucan containing materials, said preparation being enriched in an enzyme exhibiting 0-1,3-glucanase activity as described above.
• the enzyme preparation having been enriched with the novel 0- 1,3-glucanase enzyme of the invention may e . g. be an enzyme preparation comprising multiple enzymatic activities, in particular an enzyme preparation comprising different enzyme activities required for the modification or degradation of microbial cell walls.
• Such enzyme preparations include lytic enzyme systems, in particular of microbial (fungal or bacterial) origin, e .g. derived from a strain of Trichoderma , such as Trichoderma harzianum, Trichoderma viride or Trichoderma reesie , a strain of Oerskovia sp. , such as Oerskovia xanthineolytica (sometimes called Cellulomonas cellelan ⁇ ) , a strain of Art ⁇ roJa ⁇ ter sp. such as Art roJa ⁇ ter luteu ⁇ , a strain of Rhizoctonia sp. or Cytophaga sp. , a strain of a Staphylococcu ⁇ sp. , or a strain of Streptomyce ⁇ sp..
• microbial fungal or bacterial
• enzyme preparations which may conveni- ently be boosted with an enzyme of the invention includes Novozyme® 234 and Cereflo 200L, both available from Novo Nordisk A/S, Denmark, Cellulase (available from Merck) , Cellulase CP and Cellulase CT (both available from Sturge) , and/or Chitinase (available from Sigma) , Zymolase from Kirin Breweries.
• Novozyme® 234 and Cereflo 200L both available from Novo Nordisk A/S, Denmark
• Cellulase available from Merck
• Cellulase CP and Cellulase CT both available from Sturge
• Chitinase available from Sigma
• enriched is intended to indicate that the 0-1,3-glucanase activity of the enzyme preparation has been increased, e.g. with an enrichment factor of at least 1.1, conveniently due to addition of an enzyme of the invention prepared by the method described above.
• the enzyme preparation enriched in an enzyme exhibiting 0-1,3,-glucanase activity may be one which comprises an enzyme of the invention as the major enzymatic component, e .g. a mono-component enzyme composition.
• the enzyme preparation may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry preparation.
• the enzyme preparation may be in the form of a granulate or a microgranulate.
• the enzyme to be included in the preparation may be stabilized in accordance with methods known in the art.
• the enzyme preparation of the invention may, in addition to a 0-1,3-glucanase of the invention, contain one or more other cell wall degrading enzymes, for instance those with celluly- tic, mannanolytic, chitinolytic or proteolytic activities such as endo- and exo-glucanase, mannanase, endo- and exo- chitinase, protease, or - or 0-mannosidase.
• cell wall degrading enzymes for instance those with celluly- tic, mannanolytic, chitinolytic or proteolytic activities such as endo- and exo-glucanase, mannanase, endo- and exo- chitinase, protease, or - or 0-mannosidase.
• the additional enzyme(s) may be producible by means of a microorganism belonging to the genus Aspergillus , preferably Aspergillus niger, Aspergillus aculeatu ⁇ , Aspergillu ⁇ aw amor i or Aspergil ⁇ lus oryzae , or the genus Trichoderma , or the genus Oerskovia , the genus Cytophaga , or the genus Arthrobacter or any of the microorganisms mentioned above in connection with the commer ⁇ cially available enzyme preparations.
• a microorganism belonging to the genus Aspergillus preferably Aspergillus niger, Aspergillus aculeatu ⁇ , Aspergillu ⁇ aw amor i or Aspergil ⁇ lus oryzae , or the genus Trichoderma , or the genus Oerskovia , the genus Cytophaga , or the genus
• the dosage of the enzyme composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art.
• the enzyme preparation according to the invention is preferably used as an agent for degradation or modification of 0-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 composi ⁇ tion 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 0-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.
• Examples of 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) .
• the products to be recovered may be inherent products of the microorganism in question, or may be products which the microorganism has been constructed to produce, e . g . recombinant products.
• 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 transform ⁇ ation.
• the enzyme or enzyme preparation according to the 5 invention may be used in the preparation of pharmaceuticals, especially products entrapped inside the cells in the cyto ⁇ plasmic membrane, the periplasmic space and/or the cell wall.
• the enzyme preparation of the invention may be 10 used in the modification of 0-glucans such as curdlan and laminarin.
• Oerskovia xanthineolytica LLG109 (M. Lechevalier, Int. J. sys. Bacteriol, 22(4) , p. 260, 1972).
• Oerskovia xanthineolytica LLG109 strain has been deposited at 30 the Deutshe Sammlung von Mikroorganismen und Zellkulturen GmbH., (DSM).
• Bacillus subtili ⁇ DN1885 (Diderichsen et al., Journal of Bacteriology, vol. 172, p. 4315-4321, 1990)
• PF8A E. coli JM109 harbouring plasmid pPF8A PF15BK: B. subtilis DN1885 harbouring plasmid pPF15BK PFllBgK: B . subtilis DN1885 harbouring plasmid pPFUBgK PFF1: B. subtilis DN1885 harbouring plasmid pPFFl
• DS140 5' ttY gtN gaY ggN caR caR tt 3' DS142: 3' aaR caN ctR ccN gtY gtY aa 5'
• Hindlll Primers used for plasmid pDN1777 DNA amplification by PCR. Double underlined nucleotide sequences correspond to B . ⁇ tearothermophilu ⁇ , whereas the underlined nucleotide sequence correspond to O . Xanthineolytica LLG109
• Plas ids pPFFl: See figure 1 pPF8A: 2.7 Kb BamHI-BamHI fragment from O . xanthineolytica
• LLG109 see figure 2 .
• pDN520 (Diderichsen, B. and Christiansen, L. , FEMS Microb- 10 iology Letters 56, p. 53-60, 1988) .
• pUC18 (Yanish-Perron, C, Viera, J and Messing, J. ,
• pDN2801 (Diderichsen, B. , Wested, U. , Hedegaard, L. ,
• Chromosomal DNA from O . xanthineolytica LLG109 was partially 30 digested with BamHI.
• the digested DNA was fractionated by agarose gel electrophoresis. Fragments with sizes of about 1.6 to 4 kb were isolated and purified.
• the resulting BamHI fragments were mixed with BamHI-digested-Bacterial Alkaline dephosphorilated pUC18 plasmid vector (pUC18 BamHI/BAP from 35 Pharmacia) , and ligate with T4 ligase (BRL) . This ligation mixture was used to transform electrocompetent E.
• E coli JM109 cells by electroporation (Bio-Rad Gene Pulser; 125 ⁇ FD capaci ⁇ tance, 200 ⁇ Resistance) .
• E coli JM109 cells were made competent according to Sambrook et al., "A laboratory manual, Cold Spring Harbor Laboratory Press, New York,, 1989.
• the partial DNA library from strain LLG109 was plated in a total of 11 plates (with about 50 white (recombinant) colonies/plate) .
• the library was replicated on sterile nylon membranes (Hybond-N, Amersham) as described by Sambrook, supra ,
• the replica membranes were probed with the radiolabelled PCR product according to Sambrook et al., supra , 1989, under the following conditions: Hybridization temperature: 65°C, hybridization solution (100 ml) 6XSSC, 0.5% non-fat dried milk. Hybridization was carried out overnight (18 hours) . After
• filters were washed as follows: 10 min + 15 min in 200 ml of 2x SSC at 65°C and 5 min at 65°C in 0.2x SSC, 0.1% SDS. Filters were subjected to autoradiography (Fuji Film) overnight as -70°C to detect 32 P-labelled probe on filter.
• PCR amplification from O . xanthineolytica LLG109 The PCR reactions were carried out using 100 pmols of each degenerate primer per reaction. An amount of 100 ng of Oerskovia DNA was used per reaction. Taq DNA Polymerase from 25 Promega was used using the conditions recommended by the manufacturer. Cycling conditions were as followed: Hot start: 95°C for 1 minute 40 cycles.
• PCR product was cloned in pUCl ⁇ /Smal as described by 30 Kanungo and Pandey, Biotechniques, 14(6), p. 912, 1993, for subsequent sequencing.
• Radiolabelling of double-stranded DNA fragments Radiolabelling of double-stranded DNA fragments was done according to Sambrook et al., supra , 1989.
• Hybridization of Southern blots on nylon filters (Hybond-N, Amersham) with 32 P-labelled PCR probe were carried out following methods described by Sambrook et al., supra , 1989.
• the hybrid ⁇ ization conditions were as follows: Hybridization temperature: 65°C.
• Hybridization solution 6xSSC, 0,5% non-fat dried milk). The hybridization solution was carried out overnight.
• Competent cells were prepared and transformed as described by Yasbin, R.E., Wilson, G.A. and Young, F.E., J. Bacteriol. vol. 121, p. 296-304, 1975.
• DNA sequencing was done using the same SEQUENASE sequencing kit from USBI following the manufactures recommendations. DNA compressions were resolved by using the reagent kit for DNA sequencing using 7-deaza-dGTP and sequenase (also from USBI) . Isolation of plasmid DNA:
• E. coli The isolation of E. coli plasmid was performed according to Sambrook et al., ⁇ upra , 1989, using the method described in Promega Protocols and Application Guide)
• B . subtili ⁇ The isolation of B . ⁇ ubtili ⁇ was done as described by Kieser, T. , Plasmid, 12, p. 19-36, 1984.
• Antibodies to be used in determining immunological cross-reactivity may be prepared by use of a purified 0-1,3,-glucanase. More specifically, antiserum against the 0-1,3-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, Immunochemistry in Practice, Blackwell Scientific Publications, 1982 (more specifically p. 27-31) .
• Purified immunoglobulins may be obtained from the antisera, for example by salt precipi ⁇ tation ((NH 4 ) 2 S0 4 ) , followed by dialysis and ion exchange chromatography, e . g . on DEAE-Sephadex.
• Immunochemical charac ⁇ terization of proteins may be done either by Outcherlony double-diffusion analysis (O. Ouchterlony in: Handbook of Experimental Immunology (D.M. Weir, Ed.), Blackwell Scientific Publications, 1967, p. 655-706), by crossed immunoelectrophoresis (N. Axelsen et al., ⁇ upra , Chapters 3 and 4) , or by rocket immunoelectrophoresis (N. Axelsen et al.. Chapter 2) .
• the volume was adjusted to 1.0 litre with tap water.
• the pH was adjusted to 5.0 with H 3 P0 4 before sterilization in an autoclave at 121 ⁇ C for 60 minutes.
• Sucrose feed solution Content in 1 litre flask: MgCl 2 ,6H 2 0 8.08 gram Lic-tracemetals 34 ml Antifoam SB2121 1.3 ml Sucrose 241 gram Tapwater to 500 ml Also autoclaved at 121°C for 60 minutes,
• CAL 18-2 medium Yeast extract 40.0 gram MgS0 4 1.3 gram Maltodextrin 02 50.0 gram NaH 2 P0 4 20.0 gram
• the pH was adjusted to 6.0 with NaOH and autoclaved at 121°C for 60 minutes.
• AZCL-curdlan Megazyme, Sydney, Australia
• Laminarin Sigma
• PCR amplification of native genomic DNA from O . xanthineolytica LLG109 Native genomic DNA from O . xanthineolytica was prepared as described above and amplified using PCR technic.
• Primers DS96 and DS97 were synthesized for the 5' region of the yeast-lytic 57 kDa.0-1,3-glucanase gene from O . xanthineolytica LLG109 starting from the putative start codon and for the 3' region just downstream from the putative stop codon. Bases for sequences for EcoRI and BamHI were introduced at the 5' primer (DS96) and 3' primer (DS97) .
• PCR amplification from genomic DNA from strain O . xanthineolytica LLG109 using these primers gave a 1.7 kb DNA fragment.
• the PCR-fragment was checked by digesting it at various restriction sites internal to the gene.
• amino acid sequences were initially obtained from the N-terminal of the native 0-1,3-glucanase and from peptides generated by trypsin digestion of the native enzyme. Proteolytic digestion, peptide separation and amino acid sequencing were done according to standard procedures.
• N-Terminal native enzyme N-Ala-Pro-Gly-Asp-Leu-Leu-Trp-Xaa-Asp-Glu-Phe-
• Peptide 2 Phe-Val-Asp-Gly-Gln-Gln-Phe-Xaa-Arg-Val-
• Peptide 3 Val-Asp-Tyr-Val-Arg-Val-Tyr-Asp-
• O . xanthineolytica LLG109 chromosomal DNA amplification by PCR.
• Chromosomal DNA from O. xanthineolytica LLG109 was amplified by PCR using different combinations of the mixed oligonucleotides synthesized on the basis of reverse translation of amino acid sequence from 0-1,3-glucanase.
• the PCR probe strongly hybridised (under the same conditions) to either a 8 kb BamHI-BamHI band, a 8 kb Kpnl-Kpnl band or an about 5 kb BamHI-Kpnl band in Southern blot hybrid ⁇ ization analysis of chromosomal DNA from Strain 73/14.
• the pattern of bands, in the Southern blots from both Oer ⁇ kovia strains showing strong hybridization to the radiolabelled probe was different to that observed using the PCR product DS133/DS135.
• Plasmid pPF8A contained 2.7 kb BamHI insert. In the plasmid the probe was hybridizing to the 1.5 kb BamHI- Kpnl fragment contained in the 2.7 kb BamHI insert. This fact was consistent with the genomic Southern blot hybridization result using DS140/DS143 PCR probe.
• the nucleotide sequence of the 1.5 kb BamHI-Kpnl fragment from pPF8A was determined by the Dideoxi Chain Termination method.
• Double-stranded DNA was denaturated by alkali or heat treatment before using it as a template for sequencing reactions. Both strands of this region from pPF8A were sequences according to the sequence strategy shown.
• the nucleotide sequence contains an Open Reading Frame (ORF) of 921 nucleotides encoding a protein composed of 306 amino acids. Amino acid sequences identical or very similar to those existing in the native protein were present along the amino acid sequence predicted from the DNA sequence (amino acid residues different are discussed below) .
• the codon ATG is used as initiation codon in this OFR.
• a putative Ribosome Binding Site (RBS) is present 4 nucleotides upstream the initiation codon. Upstream of this ORF, there are two other in-frame ATG codons (nucleotides 321 and 351) .
• the 1.5 kb BamHI-Kpnl DNA fragment has a G+C content of 67%, and the G+C content of the ORF was, similar to that of Oer ⁇ kovia chromosomal DNA reported previous ⁇ ly (70-75% G+C) (Stackebrandt and Prauser, System. Appl. Microbiol., 14, p. 261-265, 1991) .
• Amino acid sequence of the precursor of the ⁇ -1.3 glucanase The ORF of the 0-1,3 glucanase gene encode a 306 amino acid protein.
• the NH 2 -terminal end of the deduced amino acid sequence exhibits signal sequence characteristics found in other secreted proteins (von Heine, Nucl. Acids Res., 14(11), p. 4683, 1986).
• Following the initiation codon there are three positive charged amino acid residues (histidine 5 and arginine 12 and 13) which are trailed by a long stretch of hydrophobic amino acid (Leu ,7 -Ala 23 and Ala 26 -Ala 35 ) .
• prokaryotic secretory signal sequence gave the following positions as best potential cleavage sites: i) potential cleavage site between position 35 and 36.
• the NH 2 -terminal sequence of the native mature form of 0-1,3-glucanase purified by Ventom and Asenjo, supra, 1991, from strain LLG109 fermenta ⁇ tion broth was determined as APGLLWSDEFDGAAGS.
• This amino acid sequence shows a high similarity to that predicted from the nucleotide sequence in positions Thr ⁇ -Ser 80 : TPESLAWSDEFDGAAGS ⁇
• Amino acid differences (see figure 3) observed between the predicted amino acid sequence and the sequence obtained from the native 0-1,3-glucanase (underlined amino acid residues) suggest that the ORF found in pPF8A may code for a different 0- 1,3-glucanase, an isoenzyme to the enzyme described by Ventom & Asenjo, supra , 1991.
• the amino acid residue Thr 64 forms a NH 2 -terminal end of the 0-1,3-glucanase isoenzyme.
• the peptide containing 63 amino acids should be considered as a prepro-region.
• the molecular weight of the mature 0-glucanase (Thr ⁇ -Gln 243 ) was calculated to be 26.278, which is similar to that previously calculated from SDS-PAGE by Ventom and Asenjo, ⁇ upra , 1991, for the characterized 0-1,3- glucanase.
• the predicted pi for this new enzyme is 3.77, which is so much lower than the previously reported (Ventom and Asenjo, supra , 1991) pi of 5.0, that no doubts can be left that a new hitherto unknown 0-glucanase was found.
• 0-glucanase gene was performed by replacing the native expression signals with those from the well-expressed Bacillu ⁇ stearothermophilus gene mentioned above.
• This final construct pPFFl was made as follows: A 0.2 kb Hindlll-Aval fragment from pPFUBgK was replaced by a 0.15 kb Hindlll-Aval fragment made by PCR.
• the PCR fragment con ⁇ tained the RBS and the signalpeptide coding regions of the B.stearothermophilus maltogenic ⁇ -amylase.
• the PCR fragment was obtained following pDN520 (Diderichsen et al., supra, 1988) DNA amplification using primers DK15 and DK16.
• Plasmid pPFFl was finally transformed into B.subtilis DN1885 (Yasbin et al, ⁇ upra , 1975). In this case 0-1,3-glucanase activity could be detected after 24 hours, 37°C on laminarin (0.04%) containing 5 LB agar plates after staining with congo red.
• the strain Bacillu ⁇ subtilis DN1885/pPFFl was grown in a 2 litre fermentation vessel supplied with a magnetic coupled
• the temperature was controlled at 34.0°C +/- 0.1°C, pH to 6.00 +/- 0.10 by addition of diluted ammonia in water or 5 v/v% H 3 P0 4 .
• the strain B . subtilis DN1885/pPFFl was grown on LB agar plates containing 10 ⁇ g/ml Chloramphenicol for 3 days at 30°C. A single colony was transferred to a 500 ml shakeflask containing 100 ml of CAL 18-2 medium and 1 mg of chloramphenicol. Growth was propagated at 30°C, on a rotating table at 250 RPM, for 3 days. Samples were taken every day and activity, pH, OD ⁇ oo and dry weight biomass determined.
• the activity determination was made as described in the section Methods and Materials, except that we used 0.05 ml supernatant + 0.25 ml curdlan + 0.2 ml water and stopped with 2 ml of stop reagent. Also the incuba- tion time was extended to 20 hours.
• N-terminal amino acid seguence determination and mass spec- trometry of purified recombinant 0-1.3-glucanase The fermentation broth containing the recombinant 0-1,3-gluca- nase was ultrafiltrated and washed with 20 mM Tris-HCl pH 8.5 in a Filtron concentrator equipped with a Minisette 10 kDa membrane. The sample was applied on a Q-Sepharose column equilibrated with 20 mM Tris-HCl pH 8.5 and the 0-1,3-glucanase was eluted with a linear gradient from 0 to 1 M NaCl in 10 column volumes. The fractions containing 0-1,3-glucanase activity were pooled.
• the pool was made 1.7 M with respect to ammonium sulfate and pH was adjusted to 6.5.
• the pool was further purified on a Phenyl-Sepharose column equilibrated with 30 mM MOPS containing 1.7 M ammonium sulfate pH 6.5. Elution of the 0-1,3-glucanase was performed with 10 mM Tris-HCl pH 8.5 using a linear gradient in 10 column volumes.
• the fractions containing 0-1,3-glucanase activity were pooled and dialyzed extensively against 10 mM sodium borate pH 9.0.
• the dialyzed sample was applied on a Mono Q column equilibrated with 10 mM sodium borate and the 0-1,3-glucanase was eluted with a linear gradient from 0 to 1 M NaCl in 40 column volumes.
• N-terminal amino acid sequence of the purified recombinant 0-1,3-glucanase is identical to the N-terminal sequence deduced from the SEQ ID No. 1.
• Matrix assisted laser desorption ionisation time-of-flight mass spectrometry of the purified recombinant 0-1,3-glucanase gave a mass of 26 462 Da which within the experimental error is in accordance with the mass calculated from the amino acid sequen ⁇ ce.
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