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  • 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
  • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
  • C12N9/2457—Pullulanase (3.2.1.41)
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  • 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
  • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
  • C12N9/2411—Amylases
  • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
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  • 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
  • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
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  • 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
  • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
  • C12N9/246—Isoamylase (3.2.1.68)
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  • 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)
  • C12Y302/01001—Alpha-amylase (3.2.1.1)
• • 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)
  • C12Y302/01041—Pullulanase (3.2.1.41)
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  • 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)
  • C12Y302/01068—Isoamylase (3.2.1.68)
• • C—CHEMISTRY; METALLURGY
  • C13—SUGAR INDUSTRY
  • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
  • C13K1/00—Glucose; Glucose-containing syrups
  • C13K1/06—Glucose; Glucose-containing syrups obtained by saccharification of starch or raw materials containing starch
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates, inter alia , to the use of a 5 hybrid between a carbohydrate-binding domain ("CBD”) and an enzyme of a type employed in industrial starch processing [notably starch processing for the production (vide infra) of sweeteners, particularly glucose- and/or fructose-containing syrups] , especially an amylolytic enzyme, such as an ⁇ -amylase 0 employed in a so-called “starch liquefaction" process (vide infra) in which starch is degraded (often termed “dextrinized”) to smaller oligo- and/or polysaccharide fragments, or a debranching enzyme (such as an isoamylase or a pullulanase) employed to debranch amylopectin-derived starch fragments in 5 connection with the so-called “saccharification” process (vide infra) which is normally carried out after the liquefaction stage.
• the invention also relates to hybrid
• the present invention is of particular value in the field of starch processing (starch conversion) .
• starch conversion starch conversion
• Conditions for conventional starch conversion processes and for liquefaction and/or saccharification processes are described in, 5 e.g., US 3,912,590 and in EP 0,252,730 and EP 0,063,909.
• a "traditional" process for the production of glucose- and fructose-containing syrups from starch normally consists of three 0 consecutive enzymatic processes, viz. a liquefaction process followed by a saccharification process and (for production of fructose-containing syrups) an isomerization process.
• a liquefaction process followed by a saccharification process and (for production of fructose-containing syrups) an isomerization process.
• starch initially in the form starch suspension in aqueous medium
• dextrins oligo- and 5 polysaccharide fragments of starch
• TermamylTM (Bacillus licheniformis ⁇ -amylase) , available from Novo Nordisk A/S, Bagsvaerd, Denmark], typically at pH values between 5.5 and 6.2 and at temperatures of 95-160°C for a period of approximately 2 hours.
• approximately lmM of calcium (ca. 40 ppm free calcium ions) is typically added to the starch suspension.
• the dextrins are converted into dextrose (D-glucose) by addition of a glucoamylase (amyloglucosidase, EC 3.2.1.3; e.g. AMGTM, from Novo Nordisk A/S) and, typically, a debranching enzyme, such as an isoamylase (EC 3.2.1.68) or a pullulanase (EC 3.2.1.41; e.g. PromozymeTM, from Novo Nordisk A/S) .
• glucoamylase e.g. AMGTM, from Novo Nordisk A/S
• a debranching enzyme such as an isoamylase (EC 3.2.1.68) or a pullulanase (EC 3.2.1.41; e.g. PromozymeTM, from Novo Nordisk A/S) .
• the pH of the medium is normally reduced to a value below 4.5 (e.g pH 4.3), maintaining the high temperature (above 95°C) , and the liquefying ⁇ -amylase activity is thereby denatured.
• the temperature is then normally lowered to 60°C, and glucoamylase and debranching enzyme are added.
• the saccharification process is normally allowed to proceed for 24-72 hours.
• the pH of the medium is increased to a value in the range of 6-8, preferably pH 7.5, and calcium ions are removed by ion exchange.
• the resulting syrup may then be converted into high fructose syrup using, e.g., an immobilized "glucose isomerase” (xylose isomerase, EC 5.3.1.5; e.g. SweetzymeTM, from Novo Nordisk A/S).
• an immobilized "glucose isomerase” xylose isomerase, EC 5.3.1.5; e.g. SweetzymeTM, from Novo Nordisk A/S.
• an ⁇ -amylolytic enzyme which is stable and highly active at low concentrations of free calcium ( ⁇ 40 ppm) is required.
• Such an enzyme should preferably have a pH optimum at a pH in the range of 4.5-6.5, more preferably in the range of 4.5- 5.5.
• Achievement of the above-mentioned goal requires the availability of an ⁇ -amylolytic enzyme which is stable at a pH in the range of 4.5-5.5, and which preferably maintains a high specific activity.
• TermamylTM 120 L generates "limit dextrins" (which are poor substrates for B . acidopullulyticus pullulanase) by hydrolysing 1,4-alpha-glucosidic linkages close to and on both sides of the branching points in amylopectin. Hydrolysis of these limit dextrins by glucoamylase leads to a build-up of the trisaccharide panose, which is only slowly hydrolysed by glucoamylase.
• thermostable ⁇ -amylolytic enzyme which does not suffer from this disadvantage would be a significant process improvement, as no separate inactivation step would be required.
• One object of the present invention is to achieve improved performance of ⁇ -amylolytic enzymes in relation to starch liquefaction processes - e.g. by achieving one or more or the above-outlined improvements - by changing the affinity of the enzyme for the starch substrate, whereby the modified enzyme comes into more intimate contact with the substrate.
• One aspect of the invention relates to an improved enzymatic process for liquefying starch employing a modified form of a liquefying ⁇ -amylase, wherein the ⁇ -amylase in question is linked to an amino acid sequence comprising a carbohydrate-binding domain (vide infra) .
• the invention also relates to an improved enzymatic process for liquefying starch which besides a modified ⁇ -amylase also is treated with a debranching enzyme.
• the debranching enzyme may be modified by linkage to an amino acid sequence comprising a carbohydrate-binding domain.
• an analogously modified (i.e. CBD- derivatized) form of a debranching enzyme such as an isoamylase or a pullulanase
• a debranching enzyme such as an isoamylase or a pullulanase
• the present invention thus relates to a method for liquefying starch, wherein a starch substrate is treated in aqueous medium with a modified enzyme (enzyme hybrid) which comprises an amino acid sequence of an ⁇ -amylase linked (i.e. covalently bound) to an amino acid sequence comprising a carbohydrate-binding domain (CBD) .
• a modified enzyme enzyme hybrid
• the invention also relates to an improved enzymatic process for liquefying starch which besides a modified ⁇ -amylase also is treated with a debranching enzyme.
• the debranching enzyme may be modified by linkage to an amino acid sequence comprising a carbohydrate-binding domain.
• a further aspect of the present invention relates to a method for saccharifying starch which has been subjected to a liquefaction process, wherein the reaction mixture after liquefaction is treated with a modified enzyme (enzyme hybrid) which comprises an amino acid sequence of an amylopectin- debranching enzyme (e.g. an isoamylase or a pullulanase) linked (i.e. covalently bound) to an amino acid sequence comprising a carbohydrate-binding domain (CBD) .
• a modified enzyme enzyme which comprises an amino acid sequence of an amylopectin- debranching enzyme (e.g. an isoamylase or a pullulanase) linked (i.e. covalently bound) to an amino acid sequence comprising a carbohydrate-binding domain (CBD) .
• an amylopectin- debranching enzyme e.g. an isoamylase or a pullulanase
• starch liquefaction processes as referred to in the context of the present invention do not embrace, for example, textile de-sizing processes wherein starch ("size") present in fabrics or textiles (normally cellulosic or cellulose-containing fabrics or textiles) is removed from the fabric or textile by an enzymatic process.
• a carbohydrate-binding domain is a polypeptide amino acid sequence which binds preferentially to a poly- or oligosaccharide (carbohydrate) , frequently - but not necessarily exclusively to a water-insoluble (including crystalline) form thereof.
• CBDs Although a number of types have been described in the patent and scientific literature, the majority thereof - many of which derive from cellulolytic enzymes (cellulases) - are commonly referred to as "cellulose-binding domains"; a typical cellulose-binding domain will thus be a CBD which occurs in a cellulase.
• CBDs which typically occur in chitinases
• CBDs which typically occur in chitinases
• ylan-binding domains CBDs which typically occur in xylanases
• mannan-binding domains CBDs which typically occur in mannanases
• starch-binding domains [CBDs which may occur in certain amylolytic enzymes, such as certain glucoamylases, or in enzymes such as cyclodextrin glucanotransferases (“CGTases”) ] , and others.
• CCTases cyclodextrin glucanotransferases
• CBDs are found as integral parts of large polypeptides or proteins consisting of two or more polypeptide amino acid sequence regions, especially in hydrolytic enzymes (hydrolases) which typically comprise a catalytic domain containing the active site for substrate hydrolysis and a carbohydrate-binding domain (CBD) for binding to the carbohydrate substrate in question.
• hydrolytic enzymes hydrolytic enzymes
• CBDs carbohydrate-binding domain
• Such enzymes can comprise more than one catalytic domain and one, two or three CBDs, and optionally further comprise one or more polypeptide amino acid sequence regions linking the CBD(s) with the catalytic domain (s) , a region of the latter type usually being denoted a "linker".
• hydrolytic enzymes comprising a CBD - some of which have already been mentioned above - are cellulases, xylanases, mannanases, arabinofuranosidases, acetylesterases and chitinases.
• CBDs have also been found in algae, e.g. in the red alga Porphyra purpurea in the form of a non-hydrolytic polysaccharide-binding protein [see P. Tomme et al. Cellulose- Binding Domains - Classification and Properties in Enzymatic Degradation of Insoluble Carbohydrates, John N. Saddler and Michael H. Penner (Eds.), ACS Symposium Series, No. 618 (1996)].
• most of the known CBDs [which are classified and referred to by P. Tomme et al. (op cit . ) as "cellulose- binding domains"] derive from cellulases and xylanases.
• cellulose-binding domain is intended to be understood in the same manner as in the latter reference (P. Tomme et al., op . cit) , and the abbreviation "CBD" as employed herein will thus often be interpretable either in the broader sense (carbohydrate-binding domain) or in the - in principle - narrower sense (cellulose- binding domain) .
• the P. Tomme et al. reference classifies more than 120 "cellulose-binding domains" into 10 families (I-X) which may have different functions or roles in connection with the mechanism of substrate binding. However, it is anticipated that new family representatives and additional CBD families will appear in the future.
• proteins/polypeptides in which CBDs occur e.g. enzymes, typically hydrolytic enzymes
• a CBD may be located at the N or C terminus or at an internal position.
• That part of a polypeptide or protein (e.g. hydrolytic enzyme) which constitutes a CBD per se typically consists of more than about 30 and less than about 250 amino acid residues.
• those CBDs listed and classified in Family I in accordance with P. Tomme et al. (op . cit . ) consist of 33-37 amino acid residues
• those listed and classified in Family Ila consist of 95-108 amino acid residues
• those listed and classified in Family VI consist of 85-92 amino acid residues
• one CBD derived from a cellulase from Clostridium thermocellum listed and classified in Family VII consists of 240 amino acid residues.
• the molecular weight of an amino acid sequence constituting a CBD per se will typically be in the range of from about 4kD to about 40kD, and usually below about 35kD.
• Enzyme classification numbers (EC numbers) referred to in the present specification with claims are in accordance with the
• modified enzymes as referred to herein include species comprising an amino acid sequence of an amylolytic enzyme [which in the context of the present invention may, e.g., be an ⁇ -amylase (EC 3.2.1.1), an isoamylase (EC 3.2.1.68) or a pullulanase (EC 3.2.1.41)] linked (i.e. covalently bound) to an amino acid sequence comprising a CBD.
• an amylolytic enzyme which in the context of the present invention may, e.g., be an ⁇ -amylase (EC 3.2.1.1), an isoamylase (EC 3.2.1.68) or a pullulanase (EC 3.2.1.41)] linked (i.e. covalently bound) to an amino acid sequence comprising a CBD.
• CBD-containing enzyme hybrids of interest in relation to degradation of starch include, e.g., hybrids comprising an amino acid sequence of a glucan 1, 4- ⁇ -maltohydrolase (EC 3.2.1.133), a ⁇ -amylase (EC 3.2.1.2), a glucoamylase (EC 3.2.1.3), or a neopullulanase (EC 3.2.1.135).
• CBD-containing enzyme hybrids as well as detailed descriptions of the preparation and purification thereof, are known in the art [see, e.g., WO 90/00609, WO 94/24158 and WO 95/16782, as well as Greenwood et al. , Biotechnology and Bioengineering 44 (1994) pp.
• fusion protein may be described by the following general formula:
• A-CBD is the N-terminal or the C- terminal region of an amino acid sequence comprising at least the carbohydrate-binding domain (CBD) per se .
• MR is the middle region (the "linker")
• X is the sequence of amino acid residues of a polypeptide encoded by a DNA sequence encodng the enzyme (or other protein) to which the CBD is to be linked.
• the moiety A may either be absent (such that A-CBD is a CBD per se , i.e. comprises no amino acid residues other than those constituting the CBD) or may be a sequence of one or more amino acid residues (functioning as a terminal extension of the CBD per se) .
• the linker may be a bond, or a short linking group l ⁇ comprising from about 2 to about 100 carbon atoms, in particular of from 2 to 40 carbon atoms.
• MR is preferably a sequence of from about 2 to about 100 amino acid residues, more preferably of from 2 to 40 amino acid residues, such as from 2 to 15 amino acid residues.
• the moiety X may constitute either the N-terminal or the C- terminal region of the overall enzyme hybrid.
• the CBD in an enzyme hybrid of the type in question may be positioned C- terminally, N-terminally or internally in the enzyme hybrid.
• cellulase refers to an enzyme which catalyses the degradation of cellulose
• Preferred cellulases i.e. cellulases comprising preferred CBDs
• microbial cellulases particularly bacterial or fungal cellulases.
• Endoglucanases EC
• bacterial cellulases are cellulases derived from or producible by bacteria from the group consisting of Pseudomonas , Bacillus, Cellulomonas, Clostridium, Microspora,
• the cellulase may be an acid, a neutral or an alkaline cellulase, i.e. exhibiting maximum cellulolytic activity in the acid, neutral or alkaline range, respectively.
• a useful cellulase is an acid cellulase, preferably a fungal acid cellulase, which is derived from or producible by fungi from the group of genera consisting of Trichoderma, Myrothecium, Aspergillus , Phanaerochaete , Neurospora, Neocallimastix and Botrytis.
• a preferred useful acid cellulase is one derived from or producible by fungi from the group of species consisting of Tri- choderma viride, Trichoderma reesei, Trichoderma longibrachiatum, Myrothecium verrucaria, Aspergillus niger, Aspergillus oryzae, Phanaerochaete chrysosporium, Neurospora crassa, Neocallimastix partriciarum and Botrytis cinerea .
• Another useful cellulase is a neutral or alkaline cellulase, preferably a fungal neutral or alkaline cellulase, which is derived from or producible by fungi from the group of genera consisting of Aspergillus, Penicillium, Myceliophthora, Humicola, Irpex, Fusarium, Stachybotrys , Scopulariopsis , Chaetomium, Myco- gone, Verticillium, Myrothecium, Papulospora, Gliocladium, Cepha- losporium and Acremonium .
• a preferred alkaline cellulase is one derived from or producible by fungi from the group of species consisting of Humicola insolens, Fusarium oxysporum, Myceliopthora thermophila, Penicillium janthinellum and Cephalosporium sp., preferably from the group of species consisting of Humicola insolens DSM 1800, Fusarium oxysporum DSM 2672, Myceliopthora thermophila CBS 117.65, and Cephal ospori urn sp. RYM-202.
• a preferred cellulase is an alkaline endoglucanase which is immunologically reactive with an antibody raised against a highly purified ⁇ 43kD endoglucanase derived from Humicola insolens DSM 1800, or which is a derivative of the latter ⁇ 43kD endoglucanase and exhibits cellulase activity.
• Other examples of useful cellulases are variants of parent cellulases of fungal or bacterial origin, e.g. a parent cellulase derivable from a strain of a species within one of the fungal genera Humicola , Trichoderma or Fusarium .
• CBDs examples include xylanases, mannanases, arabinofuranosidases, acetylesterases and chitinases.
• CBDs have also been found, for example, in certain algae, e.g. in the red alga Porphyra purpurea in the form of a non-hydrolytic polysaccharide-binding protein. Reference may be made to P. Tomme et al. (op cit . ) for further details concerning sources (organism genera and species) of such CBDs.
• Further CBDs of interest in relation to the present invention include CBDs deriving from glucoamylases (EC 3.2.1.3) or from CGTases (EC 2.4.1.19) .
• CBDs deriving from such sources will also be generally be suitable for use in the context of the invention.
• techniques suitable for isolating e.g., xylanase genes, mannanase genes, arabinofuranosidase genes, acetylesterase genes, chitinase genes (and other relevant genes) are well known in the art.
• a cellulose-binding domain of, e.g., a cellulase several genetic engineering approaches may be used.
• One method uses restriction enzymes to remove a portion of the gene and then to fuse the remaining gene-vector fragment in frame to obtain a mutated gene that encodes a protein truncated for a particular gene fragment.
• Another method involves the use of exonucleases such as Bal31 to systematically delete nucleotides either externally from the 5 ' and the 3 ' ends of the DNA or internally from a restricted gap within the gene.
• exonucleases such as Bal31 to systematically delete nucleotides either externally from the 5 ' and the 3 ' ends of the DNA or internally from a restricted gap within the gene.
• Appropriate substrates for evaluating the binding ability include cellulosic materials such as AvicelTM and cotton fibres.
• Other methods include the use of a selective or specific protease capable of cleaving a CBD, e.g. a terminal CBD, from the remainder of the polypeptide chain of the protein in question
• nucleotide sequence encoding the substrate-binding (carbohydrate-binding) region may then be manipulated in a variety of ways to fuse it to a DNA sequence encoding the enzyme of interest.
• the DNA fragment encoding the carbohydrate-binding amino acid sequence, and the DNA encoding the enzyme of interest are then ligated with or without a linker.
• the resulting ligated DNA may then be manipulated in a variety of ways to achieve expression.
• Preferred microbial expression hosts include certain Aspergillus species (e.g. A. niger or A. oryzae) , Bacillus species, and organisms such as Escherichia coli or Saccharomyces cerevisiae .
• Amylases (in particular ⁇ -amylases) which are appropriate as the basis for CBD/amylase hybrids of the types employed in the context of the present invention include those of bacterial or fungal origin. Chemically or genetically modified mutants of such amylases are included in this connection.
• Relevant ⁇ -amylases include, for example, ⁇ -amylase ⁇ obtainable from Bacillus species, in particular a special strain of B . licheniformis , described in more detail in GB 1296839.
• amylases include DuramylTM, TermamylTM, FungamylTM and BANTM (all available from Novo Nordisk A/S, Bagsvaerd, Denmark) , and RapidaseTM and Maxamyl PTM (available from Gist-Brocades, Holland) , and OptithermTM (available from Solvay) , and Spezy AATM and Spezyme Delta AA
• amylases which are appropriate as the basis for CBD/amylase hybrids of the types employed in the context of the present invention include a hybrid ⁇ -amylase consisting of 1-35 N-terminal amino acids of BAN (available from Novo Nordisk) and the C-terminal 36-483 C- terminal amino acids of Termamyl ⁇ (available from Novo Nordisk) with one or more of the following mutations H156Y, A181T, N190F A209V, Q264S; Termamyl with one or more of the following mutations I201E, D207H, E211Q, H205S; or MaxamylTM (available from Gist-brocades/Genencor) , with one or more of the following mutations H133Y, N188P,S.
• a hybrid ⁇ -amylase consisting of 1-35 N-terminal amino acids of BAN (available from Novo Nordisk) and the C-terminal 36-483 C- terminal amino acids of Termamyl ⁇ (available from Novo Nordisk) with
• Isoamylases isoamylases (EC 3.2.1.68) appropriate as the basis for CBD/isoamylase hybrids of the types employed in the context of the present invention include those of bacterial origin. Chemically or genetically modified mutants of such isoamylases are included in this connection.
• Relevant isoamylases include, for example, isoamylases obtainable from Pseudomonas species, (e.g. Pseudomonas sp. SMP1 or P. amyloderomosa SB15) , Bacillus species (e.g. B . amyloliquefaciens) , Flavobacterium species or Cytophaga (Lysobacter) species.
• Pullulanases pullulanases (EC 3.2.1.41) appropriate as the basis for CBD/pullulanase hybrids of the types employed in the context of the present invention include those of bacterial origin. Chemically or genetically modified mutants of such pullulanases are included in this connection. Relevant pullulanases include, for example, pullulanases obtainable from Bacillus species (e.g.
• plasmids capable of expressing fusion proteins having the amino acid sequences derived from fragments of more than one polypeptide are well known in the art (see, e.g. WO 90/00609 and WO 95/16782) .
• the expression cassette may be included within a replication system for episomal maintenance in an appropriate cellular host or may be provided without a replication system, where it may become integrated into the host genome.
• the DNA may be introduced into the host in accordance with known techniques such as transformation, microinjection or the like.
• the host may be grown to express the fused gene. Normally it is desirable additionally to add a signal sequence which provides for secretion of the fused gene.
• Typical examples of useful fused genes are:
• pro-peptide sequence normally contains 5-25 amino acid residues.
• the recombinant product may be glycosylated or non- glycosylated.
• the ⁇ -amylolytic activity of an enzyme or enzyme hybrid may be determined using potato starch as substrate. This method is based on the break-down (hydrolysis) of modified potato starch, and the reaction is followed by mixing samples of the starch/enzyme or starch/hybrid enzyme solution with an iodine solution. Initially, a blackish-blue colour is formed, but during the break-down of the starch the blue colour becomes weaker and gradually turns to a reddish-brown. The resulting colour is compared with coloured glass calibration standards.
• KNU One Kilo Novo ⁇ -Amylase Unit
• Test conditions e.g. conditions of pH, temperature, calcium concentration etc.
• CBD/ ⁇ -amylase, CBD/isoamylase or CBD/pullulanase enzyme hybrids as described herein will suitably be conditions as already described above in connection with industrial starch conversion processes.
• Assay methods suitable for determining enzymatic activity under various conditions e.g. pH, temperature, calcium concentration etc., depending on the nature of the enzyme hybrid
• Assay methods suitable for determining enzymatic activity under various conditions e.g. pH, temperature, calcium concentration etc., depending on the nature of the enzyme hybrid
• the invention also relates to an isolated DNA sequence encoding a hybrid enzyme with amylolytic activity comprising:
• hybrid enzyme comprising an enzyme and a CDB connected via a linker that they are not very stable due to the linker.
• the inventors have found that when using the linker shown in SEQ ID NO. 21 or essential parts thereof the hybrids are very stable.
• the isolated DNA sequence of the invention typically encodes an enzyme with amylolytic activity, such as ⁇ -amylase activity, in particular a Bacillus ⁇ -amylase activity, especially the activity of Termamyl ⁇ or a variant thereof, or one of the amylolytic activities mentioned above in the section "Amylolytic enzymes".
• the CBD may be any CBD e.g the CBDs described above in the section "Carbohydrate-binding domains".
• the CBD is the CBD of the Bacillus agaradherens NCIMB No. 40482 alkaline cellulase Cel5A or the CBD-dimer of Clostridium stercorarium (NCIMB 11754) XynA..
• the isolated DNA sequence is the Termamyl
• the invention relates to a DNA construct comprising the isolated DNA sequence of the invention operably linked to one or more control sequences capable of directing the expression of the DNA sequence in a suitable expression host.
• 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.
• suitable promoters for directing the transcription of the DNA encoding the cellulytic enzyme of the invention in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha- a ylase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylanase or xylosidase gene, the phage Lambda P R or P promoters, or the E. coli lac, trp or tac promoters.
• promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al. (1980) J. Biol. Chem. 255:12073-12080; Alber and Kawasaki (1982) J. Mol. Appl. Gen. 1:419-434) or alcohol dehydrogenase genes (Young et al. (1982) in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York), or the TPI1 (US 4,599,311) or ADH2-4c (Russell et al. (1983) Nature 304:652-654) promoters.
• a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the expression vector.
• the secretory signal sequence is joined to the DNA sequence encoding the enzyme hybrid in the correct reading frame.
• Secretory signal sequences are commonly positioned 5 ' to the DNA sequence encoding the amylolytic enzyme.
• the secretory signal sequence may be that normally associated with the amylolytic enzyme or may be from a gene encoding another secreted protein.
• the expression vector of the invention may comprise a secretory signal sequence substantially identical to the secretory signal encoding sequence of the Bacillus licheniformis ⁇ -amylase gene, e.g. as described in WO 86/05812.
• measures for amplification of the expression may be taken, e.g. by tandem amplification techniques, involving single or double crossing-over, or by multicopy techniques, e.g. as described in US 4,959,316 or WO 91/09129.
• the expression vector may include a temperature sensitive origin of replication, e.g. as described in EP 283,075.
• the invention also relates to a recombinant expression vector comprising the DNA construct of the invention, a promoter, and transcriptional and translational stop signals.
• the host cell of the invention into which the DNA construct or the recombinant expression vector of the invention is to be introduced, may be any cell which is capable of producing the amylolytic enzyme and includes bacteria, yeast, fungi and higher eukaryotic cells.
• Examples of bacterial host cells which, on cultivation, are capable of producing the cellulytic enzyme of the invention are grampositive bacteria such as strains of Bacillus , in particular a strain of B . subtilis, B . licheniformis, B . lentus, B . brevis, B . stearothermophilus, B . alkalophilus, B . amyloliquefaciens, B . coagulans, B . circulan ⁇ , B . lautus , B . megatherium, B . pumilus, B . thuringiensis or B . agaradherens , or strains of Streptomyces , in particular a strain of S . lividan ⁇ or S .
• grampositive bacteria such as strains of Bacillus , in particular a strain of B . subtilis, B . licheniformis, B . lentus, B . brevis, B . stearothermophilus, B
• 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 . (1989) supra) .
• the enzyme When expressing the CBD/enzyme hybrid in bacteria such as E. coli , the enzyme 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.
• the cells are lysed and the granules are recovered and denatured after which the cellulytic enzyme is refolded by diluting the denaturing agent.
• the hybrid enzyme 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 hybrid enzyme.
• the transformed or transfected host cell described above is then cultured in a suitable nutrient medium under conditions permitting the expression of the cellulytic enzyme, after which the resulting cellulytic 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 cellulytic enzyme produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrif gation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g., ammonium sulphate, purification by a variety of chromatographic procedures, e.g., ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the type of cellulytic enzyme in question.
• a salt e.g., ammonium sulphate
• the present invention also relates to methods for producing a CBD/enzyme hybrid of the present invention comprising (a) cultivating a Bacillus strain to produce a supernatant comprising the polypeptide; and (b) recovering the polypeptide.
• the present invention also relates to methods for producing a hybrid enzyme of the present invention comprising (a) cultivating a host cell under conditions conducive to expression of the polypeptide; and (b) recovering the polypeptide.
• the cells are cultivated in a nutrient medium suitable for production of the hybrid enzyme using methods known in the art.
• the cell may be cultivated by shake flask cultivation, small-scale or large- scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.
• the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e.g. , references for bacteria and yeast; Bennett, J.W. and LaSure, L. , eds. (1991) More Gene Manipulations in Fungi, Academic Press, CA) .
• Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection) . If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it is recovered from cell lysates.
• the hybrid enzyme may be detected using methods known in the art that are specific for the hybrid enzymes. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the enzyme. Procedures for determining amylolytic activity are known in the art and are described below.
• the resulting hybrid enzyme may be recovered by methods known in the art.
• the hybrid enzyme may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
• the recovered hybrid enzyme may then be further purified by a variety of chromatographic procedures, e.g. , ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like.
• the hybrid enzyme of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion) , electrophoretic procedures (e.g., preparative isoelectric focusing (IEF) , differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification (Janson and Ryden, eds.), VCH Publishers, New York, 1989).
• chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
• electrophoretic procedures e.g., preparative isoelectric focusing (IEF)
• differential solubility e.g., ammonium sulfate precipitation
• extraction see, e.g., Protein Purification (Janson and Ryden, eds.), VCH Publishers, New York,
• the invention relates to an isolated and purified CBD/enzyme hybrid encoded by the isolated DNA sequence of the invention, in particular the hybrid shown in SEQ ID No. 20.
• -linker-CBDEGV Hybrid of Termamyl ⁇ and the fungal CBD EGV from Humicola insolens EGV. The construction of the hybrid is described in Example 9.
• Hybrid of the CBD CenA from Cellulomonas fimi endoglucanase A (CenA) and Termamyl ⁇ via a linker. The construction of the hybrid is described in Example 8.
• pBluescriptKSII- (Stratagene, USA) .
• pDN1981 P.L. J ⁇ rgensen, C.K.Hansen, G.B.Poulsen and
• Bacillus AC13 NCIMB 40482 (identical to Bacillus agaradherens DSM 8721) expressing the endoglucanase enzyme encoding DNA sequence of SEQ ID NO: 1. described in Example 1 below
• E. coli strain Cells of E . coli SJ2 (Diderichsen et al. (1990) J. Bacteriol. 172:4315-4321), which encodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis were prepared for and transformed by electroporation using a Gene PulserTM electroporator from BIO-RAD as described by the supplier.
• B . subtilis PL2306 was used as the transformation host strain. It is a cellulase-negative strain developed by introducing a disruption in the transcriptional unit of the known Bacillus subtilis cellulase gene in B . subtilis strain
• DN1885 (Diderichsen, B. , Wedsted, U. , Hedegaard, L. , Jensen, B. R. , Sj ⁇ holm, C. (1990) Cloning of aldB , which encodes alpha- acetolactate decarboxylase, an exoenzyme from Bacillus brevis . J. Bacteriol. 172:4315-4321). Not only was the cellulase gene of DN1885 disrupted but also two protease encoding genes where disrupted, namely the aprE (Stahl,M.L. and E.Ferrari 1984 Replacement of the Bacillus subtilis subtilisin structural gene with an In vitro-derived deletion mutation.
• the disruption was performed essentially as described in Bacillus subtilis and other Gram-Positive Bacteria; A.L. Sonenshein, J.A. Hoch and Richard Losick, Eds. American Society for Microbiology, 1993, p.618).
• Bacillus subtilis ToC46 (Diderichsen, B. , Wedsted, U. , Hedegaard, L. , Jensen, B. R. , Sj ⁇ holm, C. (1990) Cloning of aldB, which encodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis. J. Bacteriol., 172, 4315-4321) was used as a secondary expression host, competent cells and transformation was performed as described above.
• Buffer 0.05 M potassium phosphate, pH 7.5
• neocuproine assay Dygert, Li Floridana(1965) Anal. Biochem. No 368, .
• the principle of the neocuproine assay is that CuS0 4 is added to the sample, Cu ++ is reduced by the reducing sugar and the formed neocuproine complex is measured at 450 nm.
• Cellulytic activity may be measured in cellulase viscosity units (CEVU) , determined at pH 9.0 with carboxymethyl cellulose (CMC) as substrate.
• CEVU cellulase viscosity units
• Cellulase viscosity units are determined relatively to an enzyme standard ( ⁇ 1% water, kept in N 2 atmosphere at -20°C; arch standard at -80°C) .
• the standard used, 17-1187, is 4400 CEVU/g under standard incubation conditions, i.e., pH 9.0, Tris Buffer 0.1 M, CMC Hercules 7 LFD substrate 33.3 g/1, 40.0°C for 30 minutes.
• the strain NCIMB 40482 (identical to Bacillus agaradherens DSM 8721) was propagated in liquid medium as described in WO 94/01532. After 16 hours of incubation at 30°C and 300 rpm, the cells were harvested, and genomic DNA was isolated by the method described by Pitcher et al. (1989) Lett. Appl. Microbiol . 8 : 151-156) .
• Genomic DNA was partially digested with restriction enzyme Sau3A and size-fractionated by electrophoresis on a 0.7 % agarose gel. Fragments of between 2 and 7 kb in size were isolated by electrophoresis onto DEAE-cellulose paper (Dretzen et al. (1981) Anal. Biochem. 112:295-298). Isolated DNA fragments were ligated to BamHI digested, pSJ1678 plasmid DNA.
• the ligation mixture was used as DNA template in a PCR reaction containing 200 mM of each nucleotide (dATP, dCTP, dGTP and dTTP) , 2.5 mM MgCl 2 , Expand High Fidelity buffer, 2.0 units of Expand High Fidelity PCR system enzyme mix and 300 nM of each of the following primers:
• Primer 1 is a degenerated primer designed to match the amino acid sequence (Val-Val-Glu-Glu-His-Gly-Gln) (SEQ ID No. 4) of the N-terminal amino acid sequence presented in WO94/01532. The last amino acid is only presented by the first nucleotide of the codon namely C. C is the 3 ' -nucleotide of the primer.
• Primer 2 corresponds to a sequence present in the pSJ1678 vector.
• the PCR cycling was performed in a Hans Landgraf THERMOCYCLER (Hans Landgraf, Germany) , following the profile: 1 x (120 seconds at 94 °C) ;
• the PCR product was gel purified by gel eletrophoresis in a 0.7% agarose gel, and the relevant fragment (approx. 1.7 kb) was excised from the gel and purified using QIAquick Gel extraction Kit (Qiagen, USA) according to the manufacturer's instructions.
• the purified DNA was eluted in 50 ⁇ l of lO M Tris-HCl, pH 8.5.
• This DNA was used as a template for a PCR re-amplification using the same primers, mixture and cycle profile as above.
• the PCR product was gel purified by gel eletrophoresis in a 0.7% agarose gel, and the relevant fragment was excised from the gel and purified using QIAquick Gel extraction Kit.
• the purified DNA was eluted in 50 ⁇ l of 10 mM Tris-HCl, pH 8.5.
• the purified DNA was digested with Notl and Hindlll, gel purified as above, and ligated to the vector pBluescriptll KS- (Stratagene, USA) , also digested with Notl and Hindlll, and the ligation mixture was used to transform E . coli SJ2.
• Cells were plated on LB agar plates containing ampicillin (200 ⁇ g/ml) supplemented with X-gal (5-Bromo-4-chloro-3-indolyl alpha-D-Galactopyranoside, 50 ⁇ g/ml) .
• the transformed cells were plated on LB agar plates containing ampicillin (200 ⁇ g/ml) supplemented with X-gal (5- Bromo-4-chloro-3-indolyl alpha-D-Galactopyranoside, 50 ⁇ g/ml) , and incubated at 37 °C overnight. The next day white colonies were rescued by restreaking these onto fresh LB-ampicillin agar plates and incubated at 37 °C overnight. The day after, single colonies of each clone were transferred to liquid LB medium containing ampicillin (200 ⁇ g/ml) , and incubated overnight at 37 °C with shaking at 250 rpm.
• Plasmids were extracted from the liquid cultures using QIAgen Plasmid Purification mini kit. 5 ⁇ l samples of the plasmids are digested with Notl and Hindlll . The digestions were checked by gel electrophoresis on a 0.7 % agarose gel (NuSieve, FMC) . The appearance of a DNA fragment of approximately 1.0 kb indicated a positive clone.
• the DNA was sequenced using an Applied Biosystems 373A automated sequencer according to the manufacturers instructions. Analysis of the sequence data is performed according to Devereux et al. (1984) Nucleic Acids Res. 12:387-395).
• Genomic DNA was isolated as described above. 2 mg of pure genomic DNA was digested with EcoRI. The EcoRI was heat inactivated at 65 °C for 20 minutes, after which a phenol: chloroform extraction of DNA was performed. DNA was finally ethanol precipitated and resuspended in 20 ml TE.
• Primer 3 (#19719): 5 ' -TGACCCGTACGGTCCGTGGG-3 ' (SEQ ID No. 8), and
• Primer 4 (#19720): 5 • -GGCTCTTGATTTTGTGTCCACC-3 • (SEQ ID No.9).
• the PCR cycling was performed in a Hans Landgraf THERMOCYCLER (Hans Landgraf, Germany), following the profile: 1 x (120 seconds at 94 °C) ;
• the PCR product was gel purified by gel eletrophoresis in a 0.7% agarose gel, and the relevant fragment (approx. 4-5 kb) was excised from the gel and purified using QIAquick Gel extraction Kit.
• the purified DNA was eluted in 50 ⁇ l of lO M Tris-HCl , pH 8 . 5 .
• Qiagen purified DNA was sequenced with the Taq deoxy terminal cycle sequencing kit (Perkin Elmer, USA) , and the primer 1, 3 and 4 described above, using an Applied Biosystems 373A automated sequencer according to the manufacturers instructions. Analysis of the sequence data is performed according to Devereux et al. (1984) supra) . Based upon the obtained sequence two new primers were designed in order to clone the alkaline endoglucanase as presented as SEQ ID No. 12. The primers were #20887 (SEQ ID No. 10) and #100084 (SEQ ID NO. 14) as described below.
• the nucleotide sequence in SEQ ID No. 12 was cloned by PCR for introduction in an expression plasmid pDN1981.
• PCR was performed as described below on 500 ng of genomic DNA, using the following two primers containing Ndel and Kpnl
• restriction sites for introducing the endoglucanase encoding DNA sequence to pDN1981 for expression:
• the underlined nucleotides of Primer 5 corresponds to the Ndel site, and the underlined nucleotides in the Primer 7 is part of the Kpnl site present in the sequence.
• the PCR reactions was performed using a DNA Thermal Cycler (available from Landgraf, Germany) .
• One incubation at 94°C for 2 minutes followed by ten cycles of PCR performed using a cycle profile of denaturation at 94°C for 10 seconds, annealing at 55°C for 30 seconds, and extension at 68°C for 4 minutes.
• 25 cycles of PCR performed using a cycle profile of denaturation at 94°C for 10 seconds, annealing at 55°C for 30 seconds, and extension at 68°C for 3 minutes (this duration of extension is extended with 20 seconds for each of the 25 cycles) .
• the PCR fragment was purified using QIAquick PCR column Kit (Qiagen, USA) according to the manufacturer's instructions.
• the purified DNA was eluted in 50 ⁇ l of lOmM Tris-HCl, pH 8.5, digested with Ndel and Kpnl, and purified and ligated to digested pDN1981.
• the ligation mixture was used to transform B . subtilis PL2304.
• Competent cells were prepared and transformed as described by Yasbin et al . [ Yasbin R E, Wilson G A & Young F E; Transformation and transfection in lysogenic strains of Bacillus subtilis : evidence for selective induction of prophage in competent cells; J Bacteriol 1975 121 296-304].
• the transformed cells were plated on LB agar plates containing 10 mg/ml Kanamycin, 0.4% glucose, 10 mM KH2P04 and 0.1% AZCL HE-cellulose (Megazyme, Australia), and incubated at
• Each of the positive transformants were inoculated in 10 ml TY-medium containing 10 mg/ml Kanamycin. After 1 day of incubation at 37°C and stirring at 250 rpm, 50 ml supernatant was removed. The endoglucanase activity was identified by adding 50 ml supernatant to holes punched in the agar of LB agar plates containing 0.1 % AZCL HE-cellulose.
• the protein sequence derived from the cloned endoglucanase gene shows an endoglucanase of the following composition:
• Amino acid residues 1 to 26 correspond to a signal peptide; amino acid residues 27 to 326 constitute the actual endoglucanase (ho ologues to other family 5 glycosyl hydrolases) ; amino acid residues 327 to 354 correspond to a linker; amino acid residues 355 to 400 correspond to a cellulose binding domain (as described in Example 3) ; amino acid residues 401 to 416 correspond to a linker; and amino acid residues 417 to 462 constitute a second cellulose binding domain (highly homologues to the first one (at amino acid residues 355 to 400) ) .
• the molar extinction coefficient was determined as 146,370.
• the molecular weight was approximately 52 kD.
• the molar extinction coefficient was determined as 146.370.
• the molecular weight was approximately 49 kD.
• the enzyme has no cysteine, and the charged amino acids give a calculated pi of around 4.
• the ⁇ -amylase gene encoded on pDN1528 was PCR amplified for introduction of a BamHI site in the 3 ' -end of the coding region.
• the PCR and the cloning was done as follows. Approximately 10 to 20 ng of plasmid pDN1528 was PCR amplified in HiFidelity PCR buffer (Boehringer Mannheim, Germany) supplemented with 200 ⁇ M of each dNTP, 2.6 units of HiFidelity Expand enzyme mix, and 300 pmol of each primer: 5
• the PCR reactions was performed using a DNA thermal cycler
• Plasmids were extracted from the liquid cultures using QIAgen Plasmid Purification mini kit (Qiagen, USA) according to the manufacturer's instructions. 5 ⁇ l samples of the plasmids were digested with Pstl and BamHI. The digestions were checked by gelelectrophoresis on a 1.0% agarose gel (NuSieve, FMC). One positive clone, containing the Pstl-BamHI fragment containing part of the alfa-amylase gene, was designated pMB335. This plasmid was then used in the construction of ⁇ -amylase-CBD hybrids.
• CBD of Bacillus agaradherens NCIMB No. 40482 CBD of Bacillus agaradherens NCIMB No. 40482.
• the primers were designed to amplify the linker and most C- terminal CBD of the endoglucanase encoding gene of
• the PCR reaction was performed using a DNA thermal cycler 5 (Landgraf, Germany) .
• PCR polvmerase chain reaction
• Plasmids were extracted from the liquid cultures using QIAgen Plasmid Purification mini kit (Qiagen, USA) according to 5 the manufacturer's instructions. Five- ⁇ l samples of the plasmids were digested with BamHI and Notl. The digestions were checked by gelelectrophoresis on a 1.5% agarose gel (NuSieve, FMC) . The appearance of a DNA fragment of the same size as seen from the PCR amplification indicated a positive clone. 10 One positive clone, containing the fusion construct of the ⁇ -amylase gene and the CBD of Bacillus agaradherens NCIMB No. 40482 alkaline cellulase Cel5A, was designated MBamyC5ANewlink.
• the pDN1528 vector contains the amyL gene of B . licheniformis this gene is actively expressed in B . subtilis resulting in the production of active ⁇ -amylase appearing in the supernatant.
• the ligation mixture was used to transform competent cells of PL2306.
• Cells were plated on LB agar plates containing chloramphenicol (6 ⁇ g/ml), 0.4% glucose and lOmM potassium
• Plasmids were extracted from the liquid cultures using QIAgen Plasmid Purification mini kit (Qiagen, USA) according to the manufacturer's instructions, however the resuspension buffer was supplemented with 1 mg/ml of Chicken Egg White Lysozyme (SIGMA, USA) prior to lysing the cells at 37°C for 15 minutes. 5 ⁇ l samples of the plasmids were digested with BamHI and Notl. The digestions were checked by gelelectrophoresis on a 1.5% agarose gel (NuSieve, FMC). The appearance of a DNA fragment of the same size as seen from the PCR amplification indicated a positive clone. One positive clone was designated MB492.
• the clone MB492 (expressing Termamyl ⁇ fused to Bacillus agrara ⁇ "here.ns-Cel5A-linker-CBD) was incubated for 20 hours in SB-medium at 37°C and 250 rpm. 1 ml of cell-free supernatant was mixed with 200 ⁇ l of 10% Avicel. The mixture was left for 1 hour incubation at 0°C. After this binding of CBD to Avicel the Avicel with CBD was spun 5 minutes at 5000g.
• the pellet was re- suspended in 100 ⁇ l of SDS-page buffer, boiled at 95°C for 5 minutes, spun at 5000g for 5 minutes and 25 ⁇ l was loaded on a 4-20% Laemmli Tris-Glycine, SDS-PAGE NOVEX gel (Novex, USA) .
• the samples were electrophoresed in a Xcell Mini-Cell (NOVEX, USA) as recommended by the manufacturer, all subsequent handling of gels including staining with comassie, destaining and drying were performed as described by the manufacturer.
• -Linker-CBD fusion encoded on the plasmid pMB492 (SEQ ID No. 19) .
• the expression protein sequence of the fusion construction of pMB492 is shown in SEQ ID No. 20.
• the linker region of interest as described in this example is the specific sequence:
• Clostridium stercorarium NCIMB 11754 was grown anaerobically at 60°C in specified media as recommended by The National Collections of Industrial and Marine Bacteria Ltd. (Scotland) . Cells were harvested by centrifugation.
• Genomic DNA was isolated as described by Pitcher et al. (Pitcher, D. G. , Saunders, N. A., Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett. Appl. Microbiol., 8, 151-156).
• genomic DNA Approximately 100 to 200 ng of genomic DNA (isolated as described above) was PCR amplified in HiFidelity PCR buffer
• the primers were designed to amplify the DNA encoding the Cellulose Binding Domain of the XynA encoding gene of
• the PCR reaction was performed using a DNA thermal cycler (Landgraf, Germany) .
• PCR polymerase chain reaction
• PCR products generated as described above are purified using QIAquick PCR purification kit (Qiagen, USA) according to the manufacturer's instructions.
• the purified DNA is eluted in 50 ⁇ l of 10 mM Tris-HCl, pH 8.5. 25 ⁇ l of the purified PCR fragment is digested with BamHI and Eagl, electrophoresed in 1.0% low gelling temperature agarose (SeaPlaque GTG, FMC) gels, the relevant fragment is excised from the gels, and purified using QIAquick Gel extraction Kit (Qiagen, USA) according to the manufacturer's instructions. The isolated DNA fragment is then ligated to BamHI-Notl digested pMB335 and the ligation mixture is used to transform E . coli SJ2.
• CBD Cel5A -linker-Termamyl i.e. Bacillus agaradherens NCIMB 40482 endoglucanase C- terminal CBD linked to Termamyl ⁇ via the linker shown in SEQ ID No. 21 constructed as described in Example 4
• a shaking oil bath is heated to 105 °C.
• Two starch slurries (30% DS with 40 ppm Ca ++ ) are prepared, the pH is adjusted to 6.0 with NaOH.
• and Termamyl ⁇ , respectively, are well mixed into the slurries.
• the thermostat of the oil bath is adjusted to 95.4°C and 2 litre oil at room temperature are added to the oil bath.
• a clock is started and samples (1 flask of each slurry) are taken after 20, 40, 60, and 90 minutes. 2 drops of 1 N HC1 is added to each flask to inactivate the amylase.
• the DE-value is then determined as a function of time to compare the starch liquefaction per ⁇ g enzyme/g DS of CBD Cel5A - linker-Termamyl
• CBD Cen ⁇ expression vector pCBDTOOl The gene fragment encoding the 103 residue CBDcenA from Cellulomonas fimi endoglucanase A (CenA) was cloned in the high expression vector pTugE07K3. Appropriate restriction sites were introduced at the 5' and 3' ends of the CBD CenA gene by PCR. Each PCR mixture (50 ml total volume) contained 25 ng template DNA (pTZ18R-l.6ce.nA; Damude 1995 Doctoral thesis, University of British Columbia.
• Primer 14 5 ' -AGGTCTACTAGTCCCGGCTGCCGCGTCGAC-3 ' (SEQ ID No. 27)
• the plasmid pSJ3368 a derivative of pDN1528 (S. J ⁇ rgensen et al. (1991) Journal of Bacteriology, vol. 173, No., p-559-567.) containing the Termamyl ⁇ gene, was isolated from Bacillus by standard methods. Appropriate restriction sites for recloning the Termamyl ⁇ gene fragment in the E . coli vector pCBDTOOl and for the construction of the hybrids were introduced by PCR.
• Each PCR reaction mixture (50 ml total volume) contained 15 ng template DNA (pSJ3368) , 3 pmol primers (PAM1 and PAM2) , 2 mM MgS ⁇ 4, 10 % dimethyl sulfoxide, 0.4 mM 2 ' -deoxynucleotide 5'- triphosphates and 1U Vent DNA polymerase in "Thermopol" buffer (New England BioLabs) .
• Thirty successive cycles were performed as follows: denaturation at 95°C for 1 min, annealing at 55°C for 1 min and primer extension at 72 °C for 1.54 min.
• a Nhel (underlined) and Ncol site were introduced at the 5' end of the gene with the oligonucleotide (PAM1)
• CBD C A -PTPTTP-Termamyl ⁇ production and purification Overnight cultures of E . coli JM101, harboring plasmid pNAMl.0, were diluted 500-fold in terrific broth (TB; 12 g tryptone, 24 g yeast extract, 9.8 g K2HPO4, 2.2 g KH2PO4 and 8 g (10 ml) glycerol in 11) (Sambrook et al . , 1989) (ref: Sambrook J. , Fritsch, E.F., & Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2nd ed.
• Proteins were recovered by centrifugation at 11,000 x g and the pellet was dissolved in 20 mM Tris-HCl, pH 8.0 (binding buffer). After further centrifugation at 15000 x g, the clarified supernatants was loaded onto a Ni + agarose column (Novagen, Markham, ON) . The column was washed with 40 mM imidazole, 200 mM NaCl, 20 mM Tris-HCl, pH 8.0 (wash buffer). Bound proteins were eluted with a gradient of imidazole (0-500 mM) in 20 mM Tris-HCl buffer containing 500 mM NaCl.
• EXAMPLE 8 Termamyl linker fungal CBD from Humicola insolens EGV. pNAMK6.1 (Termamyl
• Each PCR reaction mixture (50 ml total volume) contained 15 ng template DNA (pSJ3368) , 3 pmol primers (5Term2 and 3Term2) , 2 mM MgS ⁇ 4, 10 % dimethyl sulfoxide, 0.4 mM 2 ' -deoxynucleotide 5 ' -triphosphates and 1U Vent DNA polymerase in "Thermopol" buffer (New England BioLabs) .
• Thirty successive cycles were performed as follows: denaturation at 95 °C for 1 min, annealing at 55°C for 1 min and primer extension at 72 °C for 1.54 min.
• Nhel (underlined) and EcoRI (in bold) sites were introduced at the 5' end of the Termamyl gene with the oligonucleotide (5Term2) Primer 18
• primer The construct was verified by restricion digesting and by automated sequencing.
• the fungal CBD vector pCBDT006 was obtained by cloning the gene fragment encoding CBDEGV from Humicola insolens endoglucanase V (WO 91/17243) in pTugE07K3. Appropriate restriction sites were introduced at the 5' and 3' ends of the CBDEGV gene by PCR.
• Each PCR mixture (50 ml total volume) contained 25 ng template DNA 25-50 pmole primers (N137 and NlPTcs) , 10 % dimethyl sulfoxide, 0.4 mM 2 ' - deoxynucleotide 5 '-triphosphates, and 1U Vent DNA polymerase in "Thermopol" buffer (New England BioLabs) . Twenty successive cycles of denaturation at 96 °C for 45 seconds, followed by annealing at 50 °C for 60 seconds, and primer extension at 72 °C for 35 seconds were performed. The last cycle was followed by extension at 72 °C for 90 seconds.
• vector NAM2.0 was digested with Nhel and Stul and the resulting 1.48 kb fragment was gel purified using the
• the product has the following characterization MW 60863.
• a shaking oil bath was heated to 105°C.
• Three starch slurries (30% DS with 40 ppm Ca ++ ) were prepared, the pH was adjusted to 6.0 with NaOH.
• the enzyme was well mixed into the slurries according to the scheme:
• Slurry 1 Termamyl
• Slurry 2 Termamyl
• Slurry 3 Termamyl ⁇ 10.9 ⁇ g/g DS starch
• -linker- CBDEGV gives a improved liquefaction per ⁇ g enzyme/g DS compared to Termamyl ⁇ .
• a shaking oil bath was heated to 105°C.
• Two starch slurries (30% DS with 40 ppm Ca ++ ) were prepared, the pH was adjusted to 6.0 with NaOH.
• the enzyme was well mixed to the slurries according to the scheme:
• Slurry 1 CBD CenA -Termamyl 75NU/g DS starch
• Slurry 2 Termamyl ⁇ 75NU/g DS starch
• the flasks were placed in the oil bath for 8 minutes at 105°C and then 90 minutes at 95°C.
• ORGANISM Bacillus agaradherens
• Trp Tyr Gly Gin Phe Val Asn Tyr Glu Ser Met Lys Trp Leu Arg Asp 65 70 75 80
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid
• FEATURE FEATURE
• MOLECULE TYPE DNA (genomic)
• ORIGINAL SOURCE
• ORGANISM Bacillus agaradherens
• Trp Tyr Gly Gin Phe Val Asn Tyr Glu Ser Met Lys Trp Leu Arg Asp 65 70 75 80
• MOLECULE TYPE other nucleic acid
• FEATURE FEATURE
• GGT ATT ACT GCC GTC TGG ATT CCC CCG GCA TAT AAG GGA ACG AGC CAA 240 Gly He Thr Ala Val Trp He Pro Pro Ala Tyr Lys Gly Thr Ser Gin 65 70 75 80
• MOLECULE TYPE other nucleic acid
• GGT ATT ACT GCC GTC TGG ATT CCC CCG GCA TAT AAG GGA ACG AGC CAA 240 Gly He Thr Ala Val Trp He Pro Pro Ala Tyr Lys Gly Thr Ser Gin 65 70 75 80
• AAA AAT ATT GAT TTT GGT GAC GGC GCA ACG TCC GTA ACA GCA AGA GTA 2160 Lys Asn He Asp Phe Gly Asp Gly Ala Thr Ser Val Thr Ala Arg Val 705 710 715 720 GCT ACC CAG AAT GCT ACT ACC ATT CAG GTA AGA TTG GGA AGT CCA TCG 2208 Ala Thr Gin Asn Ala Thr Thr He Gin Val Arg Leu Gly Ser Pro Ser 725 730 735
• AAATTGCTGA ACGGTACGGT CGTTTCCAAG CATCCGTTGA AATCGGTTAC ATTTGTCGAT 1440
• CTTGCTTACG CTTTTATTCT CACAAGGGAA TCTGGATACC CTCAGGTTTT CTACGGGGAT 1560
• AAAACGCCAG CAACGCGGCC TTTTTACGGT TCCTGGCCTT TTGCTGGCCT TTTGCTCACA 4200
• MOLECULE TYPE other nucleic acid
• MOLECULE TYPE other nucleic acid

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• 1997
  • 1997-10-13 EP EP97943797A patent/EP0950093A2/de not_active Withdrawn
  • 1997-10-13 CN CN 97198640 patent/CN1233286A/zh active Pending
  • 1997-10-13 WO PCT/DK1997/000448 patent/WO1998016633A1/en not_active Application Discontinuation
  • 1997-10-13 AU AU45510/97A patent/AU4551097A/en not_active Abandoned

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