Classifications

• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
  • C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
• • A—HUMAN NECESSITIES
  • A21—BAKING; EDIBLE DOUGHS
  • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
  • A21D8/00—Methods for preparing or baking dough
  • A21D8/02—Methods for preparing dough; Treating dough prior to baking
  • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
  • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
• • 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)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
  • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

• the present invention relates to a polynucleotide encoding a lipolytic enzyme and to a lipolytic enzyme.
• the invention also relates to methods of producing and using the lipolytic enzyme.
• Lipolytic enzymes are capable of hydrolyzing carboxylic ester bonds in a substrate to release carboxylic acids. They are known to be useful, e.g., in baking and detergents. Various species of the related genera Acremonium and Fusanum are known to produce lipolytic enzymes. Thus, Roberts et al., Mycologia, 79, 265-273, 1987. Satyanarayana & John " , Current Science, 50, 680-682, 1981 state that some isolates oi Acremonium produce lipase.
• WO 98/26057 discloses a polypeptide having lipase and phospholipase activity (GenBank Ace. No. A85215) obtained from Fusanum oxysporum. A lipase from Fusarium heterosporum and- its sequence are known. Journal of Fermentation and Bioengineering, 75 (5), p 349-352, 1993. Journal of Biochemistry (Tokyo), 116 (3), p 536-540, 1994. A lipolytic enzyme from Fusarium culmorum CBS 513.94 and its N-terminal sequence are disclosed in US 5830736. A lipase/phospholipase from Fusarium oxysporum and its sequence are disclosed in WO 98/26057. US 5990069 discloses a lipase from a strain of Fusarium solani var. minus.
• one aspect of the invention provides a polynucleotide encoding a lipolytic enzyme which comprises: a) the DNA sequence encoding a mature lipolytic enzyme cloned into a plasmid present in Escherichia coli DSM 13539, b) the mature polypeptide-encoding DNA sequence shown in SEQ ID NO: 1 , 3, 5, 7 or 9, c) an analogue of the DNA sequence defined in a) or b) which i) has at least 80 % identity with said DNA sequence, or ii) hybridizes at high stringency with said DNA sequence, its complementary strand or a subsequence thereof.
• Another aspect of the invention also provides a lipolytic enzyme which may be a polypeptide having an amino acid sequence as the mature peptide shown in
• the lipolytic enzyme of the invention may be a polypeptide encoded by the lipolytic enzyme encoding part of the DNA sequence cloned into a plasmid present in Escherichia coli deposit number DSM 13539.
• the lipolytic enzyme may also be an analogue of the polypeptide defined above which: i) has at least 85 % identity with said polypeptide, ii) is immunologically reactive with an antibody raised against said polypeptide in purified form, iii) is an allelic variant of said polypeptide,
• the lipolytic enzyme of the invention may be a polypeptide which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with a complementary strand of the mature polypeptide-encoding nucleic acid sequence of SEQ ID NO: 1 , 3, 5, 7 or 9 or a subsequence thereof having at least 100 nucleotides.
• a further aspect of the invention provides a nucleic acid sequence comprising a nucleic acid sequence which encodes the lipolytic enzyme described above.
• aspects of the invention provide a recombinant expression vector comprising the DNA sequence, and a cell transformed with the DNA sequence or the recombinant expression vector.
• the DNA sequences of the invention have the following identities to known DNA sequences:
• a lipolytic enzyme of the invention may be derived from a strain of Fusanum or Acremonium, particularly F. venenatum, F. sulphureum, A. berkeleyanum, F. culmorum or F. solani, using probes designed on the basis of the DNA sequences in this specification, or from a Plasmid contained in an E. coli strain identified below.
• the Fusarium venenatum cell may be Fusarium venenatum A3/5, which was originally deposited as Fusarium graminearum ATCC 20334 and recently reclassified as Fusarium venenatum by Yoder and Christianson, 1998, Fungal Genetics and Biology 23: 62-80 and O'Donnell et al, 1998, Fungal Genetics and Biology 23: 57-67.
• the Fusarium venenatum cell may be a morphological mutant of Fusarium venenatum A3/5 or Fusarium venenatum ATCC 20334, as disclosed in WO 97/26330.
• ATCC 20334 is available on commercial terms from American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209 ("ATCC").
• strains are A. berkeleyanum CBS 301.38, F. culmorum CBS 513.94 and F. solani MUCL 38667.
• the two CBS strains are available on commercial terms from Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands (P.O.Box 85167, 3508 AD Utrecht, the Netherlands).
• MUCL 38667 is available on commercial terms from Mycotheque de I'Universite Catholique de Louvain, Place Croix du Sud 3, B-1348 Louvain-la-Neuve, Belgium by referring to US 5990069.
• DSM indicates a deposit made at DSMZ - Deutshe Sammmlung von Microorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig DE, Germany.
• the deposits DSM 13537, DSM 13538 and DSM 13539 were made by Novo Nordisk A/S and were later assigned to Novozymes A S.
• the lipolytic enzyme of the invention may be a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10 or it may be an amino acid variant derived from any of these sequences by substitution, deletion and/or insertion of one or more amino acid residues.
• the variant may be constructed in analogy with known variants of other lipases from Fusarium, e.g. from F. oxysporum and F. heterosporum as disclosed in WO 200032758 and T. Nagao, Journal of Bioscience and Bioengineering, 89 (5), 446-450 (2000).
• the lipolytic enzyme may be C-terminally processed, e.g. by KEX-2 hydrolysis of the bond after Arg and Lys corresponding to K274 and R275 of SEQ ID NO: 6 to remove a peptide at the C-terminal.
• the C-terminally processed polypeptide tends to have a lower thermostability than the full-length polypeptide.
• the variant may have substitutions of amino acids Arg and/or Lys corresponding to K274 and/or R275 of SEQ ID NO: 6, e.g. R275A and/or K274A. Such variants may be protected against C-terminal processing by KEX-2 hydrolysis to preserve the full-length polypeptide with higher thermostability.
• a polynucleotide sequence encoding any of the polypeptides described above may be constructed by principles known in the art.
• the lipolytic enzyme is able to hydrolyze carboxylic ester bonds and is classified as EC 3.1.1 according to Enzyme Nomenclature 1992, Academic Press,
• the enzyme has lipase (triacylglycerol lipase) activity (EC 3.1.1.3) and may also have other activities such as phospholipase activity, particularly phospholipase A1
• the expression vector of the invention typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a selectable marker, a transcription terminator, a repressor gene or various activator genes.
• the vector may be an autonomously replicating vector, or it may be integrated into the host cell genome.
• the lipolytic enzyme of the invention may be produced by transforming a suitable host cell with a DNA sequence encoding the lipolytic enzyme, cultivating the transformed organism under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
• the host organism may particularly be a eukaryotic cell, in particular a fungal
• a yeast cell such as a yeast cell or a filamentous fungal cell, e.g. a strain of Aspergillus, Fusarium, T choderma or Saccharomyces, particularly A. niger, A. oryzae, F. graminearum, F. sambucinum, F. cerealis or S. cerevisiae.
• the production of the lipolytic enzyme in such host organisms may be done by the general methods described in EP 238,023 (Novo Nordisk), WO 96/00787 (Novo Nordisk) or EP
• the invention provides production of a lipolytic enzyme by shuffling of at least two polynucleotides, expressing the shuffled polynucleotides to form recombinant polypeptides, screening the polypeptides to select a polypeptide having 20 lipolytic enzyme activity, and producing the selected polypeptide.
• the polynucleotides to be shuffled include a first polynucleotide comprising the and a second different polynucleotide encoding a lipolytic enzyme and having at least 50 % identity to the first polynucleotide.
• the second polynucleotide may comprise a second mature polypeptide-coding sequence of SEQ ID NO: 1 , 3, 5, 7 or 25 9 or may comprise a polynucleotide encoding a lipolytic enzyme from F. oxysporum (WO 98/26057) or from F. heterosporum.
• starting-point polynucleotides may involve fragmenting the polynucleotides and recombining the fragments, to obtain output polynucleotides (i.e. polynucleotides
• DNA recombination or shuffling may be a (partially) random process in which a library of chimeric genes is generated from two or more starting genes.
• a number of known formats can be used to carry out this shuffling or recombination process.
• the process may involve random fragmentation of parental DNA followed by reassembly by PCR to new full length genes, e.g. as presented in US5605793, US5811238, US5830721 , US6117679 .
• In-vitro recombination of genes may be carried out, e.g. as described in US6159687, WO98/41623, US6159688, US5965408, US6153510.
• the recombination process may take place in vivo in a living cell, e.g. as described in WO 97/07205 and WO 98/28416.
• the parental DNA may be fragmented by DNA'se I treatment or by restriction endonuclease digests as described by Kikuchi et al (Gene 236:159-167, 2000).
• Shuffling of two parents may be done by shuffling single stranded parental DNA of the two parents as described in Kikuchi et al (Gene 243:133-137, 2000).
• Hybridization The hybridization is used to indicate that a given DNA sequence is analogous to a nucleotide probe corresponding to a DNA sequence of the invention.
• the hybridization conditions are described in detail below.
• Suitable conditions for determining hybridization between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5 x SSC (standard saline citrate) for 10 min, and prehybridization of the filter in a solution of 5 x SSC (Sambrook et al. 1989), 5 x Denhardt's solution (Sambrook et al. 1989), 0.5 % SDS and 100 ⁇ g/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a random-primed (Feinberg, A. P. and Vogelstein, B.
• 5 x SSC standard saline citrate
• the lipolytic enzyme and the nucleotide sequence of the invention may have homologies to the disclosed sequences of at least 85 %, particularly at least 90 % or at least 95 %, e.g. at least 98 %.
• alignments of sequences and calculation of identity scores were done using a Needleman-Wunsch alignment (i.e. global alignment), useful for both protein and DNA alignments.
• the default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively.
• the penalty for the first residue in a gap is -12 for proteins and -16 for DNA, while the penalty for additional residues in a gap is -2 for proteins and -4 for DNA. Alignment is from the FASTA package version v20u6 (W.
• a substrate for lipase is prepared an emulsion of 5 % by volume of tributyrin
• the lipolytic enzyme of the invention can be used in various industrial application of lipolytic enzymes, e.g. in baking or detergents as described below. It can also be used for diglyceride synthesis (EP 307154), acidolysis, interesterification (WO 8802775), ester hydrolysis, oil degumming (JP-A 2-153997, US 5264367), production of lysolecithin (JP patent 2794574, JP-B 6-087751) and in a cheese-making process (WO 0054601 ). Use in baking
• the lipolytic enzyme of the invention can be used in the preparation of dough, bread and cakes, e.g. to improve the elasticity of the bread or cake.
• the lipolytic enzyme can be used in a process for making bread, comprising adding the lipolytic enzyme to the ingredients of a dough, kneading the dough and baking the dough to make the bread. This can be done in analogy with WO 9404035 and EP 585988.
• the lipolytic enzyme is typically added in an amount corresponding to 0.01-100 mg enzyme protein per kg of flour, particularly 0.1-25 mg enzyme protein per kg of flour, more particularly 0.1-10 mg enzyme protein per kg of flour, and most particularly 0.5-5 mg enzyme protein per kg of flour.
• the lipolytic enzyme may be used as a detergent additive, e.g. at a concentration (expressed as pure enzyme protein) of 0.001-10 (e.g. 0.01-1) mg per gram of detergent or 0.001 -100 (e.g. 0.01 -10) mg per liter of wash liquor.
• the detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations.
• the lipolytic enzyme may be effective for the removal of fatty stains, for whiteness maintenance and for dingy cleanup.
• a laundry detergent composition may be formulated as described in WO 97/04079, WO 97/07202, WO 97/41212, PCT/DK WO 98/08939 and WO 97/43375.
• the detergent composition of the invention may particularly be formulated for hand or machine dishwashing operations, e.g. as described in GB 2,247,025 (Unilever) or WO 99/01531 (Procter & Gamble).
• the lipolytic enzyme may be effective for removal of greasy/oily stains, for prevention of the staining /discoloration of the dishware and plastic components of the dishwasher by highly colored components and the avoidance of lime soap deposits on the dishware.
• Enzymes Enzymes for DNA manipulations are obtainable from New England Biolabs, Inc. and were used according to the manufacturer's instructions.
• Plasmids/vectors pT7Blue (Invitrogen, Netherlands) pCaHj483 is described in WO 9704079 and WO 9942566.
• LA PCRTM in vitro Cloning Kit (TaKaRa) was used for cloning and was used according to the manufacturer's instructions. Microbial strains
• Fusarium venenatum WTY700 3.8d a spore-purified r/5-minus, s1 -minus strain, was used as the recipient strain for transformation experiments.
• Fusarium venenatum WTY700 3.8d is a morphological mutant of Fusarium venenatum strain ATCC 20334 (Wiebe et al, 1991 , Mycol Research 95: 1284-1288), E. coli JM109 (TOYOBO, Japan)
• A. oryzae BECh-2 is described in Danish patent application PA 1999 01726. It is a mutant of JaL 228 (described in WO 98/12300) which is a mutant of IFO 4177.
• Cove-2 30 g/L Sucrose, 20 ml/L COVE salt solution, 10mM, Acetamide, 30 g/L noble agar.
• Cove salt solution per liter 26 g KCI, 26 g MgSO4-7aq, 76 g KH2PO4, 50ml Cove trace metals.
• Cove trace metals per liter 0.04 g NaB4O7-10aq, 0.4 g CuSO4-5aq, 1.2 g FeSO4-7aq, 0.7 g MnSO4-aq, 0.7 g Na2MoO2-2aq, 0.7 g ZnSO4-7aq.
• AMG trace metals per liter 14.3 g ZnSO4-7aq, 2.5 g CuSO4-5aq, 0.5 g NiCl2, 13.8 g FeSO4, 8.5 g MnSO4, 3.0 g citric acid.
• YPG 4 g/L Yeast extract, 1 g/L KH2PO4, 0.5 g/L MgSO4-7aq, 5 g/L
• Cove top agarose 342.3 g/L Sucrose, 20 ml/L COVE salt solution, 10mM Acetamide, 10 g/L low melt agarose.
• MS-9 per liter 30 g soybean powder, 20 g glycerol, pH 6.0.
• MDU-pH5 per liter 45 g maltose-1aq, 7 g yeast extract, 12 g KH2PO4, 1 g MgSO4-7aq, 2 g K2SO4, 0.5 ml AMG trace metal solution and 25 g 2- morpholinoethanesulfonic acid, pH 5.0. Measurement of dough and breads properties
• the crumb firmness was measured using a texture analyzer TA-XT2 from Stable Micro Systems.
• Texture was measured according to a modified ACCA method (American Cereal Chemists' Association).
• Elasticity is a measure of the degree to which a dough can be stretched without tearing. It is evaluated by rolling a piece of dough (about 30 g) to a size of about 10 cm, and having a test panel carefully pulling at opposite ends to judge the resistance and elasticity. The results are expressed on a scale from 0 (low/weak elasticity) to 10 (high/strong elasticity) with the control (dough without enzyme addition) taken as 5.
• Crumb structure is determined as the property of a baked product with finer and/or thinner cell walls in the crumb and/or more uniform/homogenous distribution of cells in the crumb and is usually evaluated empirically by the skilled test baker.
• the shape factor is taken as the ratio between the height and diameter of rolls after baking (average of 10 rolls).
• the specific volume is calculated as volume ml per g bread, taking the mean value of the volumes of 4 loaves measured using the traditional rape seed method.
• the specific volume of the control (without enzyme) is defined as 100.
• the relative specific volume index is the percentage of the specific volume of 4 loaves per the specific volume of 4 control loaves.
• Example 1 Fermentation and mycelial tissue from F. venenatum
• Fusarium venenatum WTY700 3.8d was grown in a two-liter lab-scale fermentor using a fed-batch fermentation scheme with NUTRIOSETM (Roquette
• Mycelial samples were harvested at 2, 4, 6, and 8 days post-inoculum and quick-frozen in liquid nitrogen. The samples were stored at -80°C until they were disrupted for RNA extraction.
• Double-stranded cDNA was synthesized using approximately 5 ⁇ g of poly(A)+ mRNA according to the method of Gubler and Hoffman (1983, Gene 25: 263-269) except a ⁇ /ofl-(dT)18 primer (Pharmacia Biotech, Inc., Piscataway, NJ) was used to initiate first strand synthesis.
• the cDNA was treated with mung bean nuclease (Boehringer Mannheim Corporation, Indianapolis, IN) and the ends were made blunt with T4 DNA polymerase (New England Biolabs, Beverly, MA).
• the cDNA was digested with Notl, size selected by agarose gel electrophoresis (ca. 0.7-4.5 kb), and ligated with pZErO-2.1 (Invitrogen Corporation, Carlsbad, CA) which had been cleaved with Notl plus EcoRV and dephosphorylated with calf-intestine alkaline phosphatase (Boehringer Mannheim Corporation, Indianapolis, IN).
• the ligation mixture was used to transform competent E. coli TOP10 cells (Invitrogen Corporation, Carlsbad, CA). Transformants were selected on 2YT agar plates (Miller, 1992, A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor Press, Cold Spring Harbor, New York) which contained kanamycin at a final concentration of 50 ⁇ g/ml.
• the primary microtiter plates were stored frozen at -80°C.
• the secondary deep-dish plates were incubated at 37°C overnight with vigorous agitation (300 rpm) on rotary shaker.
• each secondary culture plate was covered with a polypropylene pad (Advanced Genetic Technologies Corporation, Gaithersburg, MD) and a plastic microtiter dish cover.
• DNA was isolated from each well using the 96-well Miniprep Kit protocol of Advanced Genetic Technologies Corporation (Gaithersburg, MD) as modified by Utterback et al (1995, Genome Sci. Technol 1 : 1-8).
• Nucleotide sequence data were scrutinized for quality, and samples giving improper spacing or ambiguity levels exceeding 3% were discarded or re-run. Vector sequences and ambiguous base calls at the ends of the DNA sequences were trimmed with assistance of FACTURATM software (Perkin-Elmer Applied Biosystems, Inc., Foster City, CA. All sequences were compared to each other to determine multiplicity using AutoAssemblerTM software (Perkin-Elmer Applied Biosystems, Inc., Foster City, CA).
• NRDB non-redundant database
• GeneAssistTM software Perkin- Elmer Applied Biosystems, Inc., Foster City, CA
• Smith-Waterman algorithm using the BLOSUM 62 matrix with a threshold score of 70.
• the NRDB was assembled from Genpept, Swiss-Prot, and PIR databases.
• Putative lipase clones were identified by comparing the deduced amino acid sequence of the ESTs to protein sequences deposited in publicly available databases such as Swissprot, Genpept, and PIR using a modified Smith-Waterman search algorithm (Perkin-Elmer Applied Biosystems, Foster City, CA). Tentative identification was based on amino acid sequence similarity to numerous fungal lipases.
• One clone, Fusarium venenatum EST FA0726 was selected for nucleotide sequence analysis which revealed that the cDNA clone was truncated at its 5 prime end.
• Example 6 Fusarium venenatum genomic DNA extraction Fusarium venenatum WTY700 was grown for 24 hours at 28°C and 150 rpm in 25 ml of YEG medium composed per liter of 5 g of yeast extract and 20 g of glucose. Mycelia were then collected by filtration through Miracloth (Calbiochem, La Jolla, CA) and washed once with 25 ml of 10 mM Tris-1 mM EDTA (TE) buffer. Excess buffer was drained from the mycelia which were subsequently frozen in liquid nitrogen.
• YEG medium composed per liter of 5 g of yeast extract and 20 g of glucose.
• Mycelia were then collected by filtration through Miracloth (Calbiochem, La Jolla, CA) and washed once with 25 ml of 10 mM Tris-1 mM EDTA (TE) buffer. Excess buffer was drained from the mycelia which were subsequently frozen in liquid nitrogen.
• the frozen mycelia were ground to a fine powder in an electric coffee grinder, and the powder was added to 20 ml of TE buffer and 5 ml of 20% w/v sodium dodecylsulfate (SDS) in a disposable plastic centrifuge tube. The mixture was gently inverted several times to ensure mixing, and extracted twice with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1 v/v/v). Sodium acetate (3 M solution) was added to give a final concentration of 0.3 M and the nucleic acids were precipitated with 2.5 volumes of ice cold ethanol.
• SDS sodium dodecylsulfate
• the tube was centrifuged at 15,000 x g for 30 minutes and the pellet was allowed to air dry for 30 minutes before resuspension in 0.5 ml of TE buffer.
• DNase-free ribonuclease A was added to a concentration of 100 ⁇ g/ml and the mixture was incubated at 37°C for 30 minutes.
• Proteinase K 200 ⁇ g/ml was then added and the mixture was incubated an additional hour at 37°C.
• the mixture was extracted twice with phenol: chloroform: isoamyl alcohol (25:24:1 v/v/v) before precipitating the DNA with sodium acetate and ethanol according to standard procedures.
• the DNA pellet was dried under vacuum, resuspended in TE buffer, and stored at 4°C.
• Genomic libraries of Fusarium venenatum WTY700 were constructed in ⁇ ZipLox according to the manufacturer's instructions (Life Technologies, Gaithersburg, MD). Fusarium venenatum genomic DNA was partially digested with Tsp509l and size-fractionated on 1 % agarose gels. DNA fragments migrating in the size range 3-7 kb were excised and eluted from the agarose gel slices using Prep-a- Gene reagents (BioRad, Hercules, CA).
• the eluted DNA fragments were ligated with EcoRI-cleaved and dephosphorylated ⁇ ZipLox vector arms (Life Technologies, Gaithersburg, MD), and the ligation mixtures were packaged using commercial packaging extracts (Stratagene, La Jolla, CA). The packaged DNA libraries were plated and amplified in E. co//Y1090ZL cells.
• the cDNA from Fusarium venenatum clone FA0726 was excised from the vector plasmid by digestion with EcoRI and Not ⁇ yielding an approximately 900 bp fragment. The fragment was purified by gel electrophoresis, and radiolabeled with ⁇ [ 32 P] dCTP using a Prime-it Random Primer Labeling Kit (Stratagene, La Jolla, CA).
• Plasmid DNA was isolated from the clone by passage through E. coli DH10B cells (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. This clone was designated E. coli DH10B - pFvLipasel .
• Example 8 Characterization of the Fusarium venenatum genomic clone encoding lipase
• DNA sequencing was performed on an Perkin-Elmer Biosystems Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke etal, 1992, Journal of Virology Methods 38: 47-60). Contig sequences were generated using a transposon insertion strategy (Primer Island Transposition Kit, Perkin-Elmer/Applied Biosystems, Inc., Foster City, CA). The 2.94 kb genomic fragment was sequenced to an average redundancy of 4.8.
• the nucleotide sequence and deduced amino acid sequence are shown as SEQ ID NO: 1-2.
• the insert contains an open reading frame of 1.153 kb encoding a polypeptide of 350 amino acids.
• SignalP software program (Nielsen et al, 1997, Protein Engineering 10: 1-6)
• a signal peptide of 15 residues was predicted.
• the predicted signal peptide is followed by a 15 residue propeptide ending with a concanical propeptide Glu/Arg cleavage site.
• N-terminal sequencing of the lipase protein supports this propeptide cleavage site prediction.
• the open reading frame is interrupted by two introns of 49 bp and 58 bp.
• the mature Fusarium venenatum lipase comprises 319 amino acids and a predicted molecular weight of 33.6 kDa.
• Example 9 Construction of plasmid pSheBI
• the Fusarium venenatum expression vector pSheBI ( Figure 1 ) was generated by modification of pDM181 (WO 98/20136). The modifications included
• the lipase-expression vector, pEJG60 was constructed as follows.
• the lipase coding region was amplified from pFvlipasel using the following pair of primers: Primer 990658 and 990661 (SEQ ID NO: 17 and 18)
• the forward primer introduces a Sph ⁇ site which contains the ATG
• the reverse primer introduces a Pac ⁇ site after the stop codon.
• the amplification reaction (100 ⁇ l) contained the following components: 0.5 ⁇ g of genomic clone pFvLipasel , 50 pmol of the forward primer, 50 pmol of the reverse primer, 10 mM dNTPs (dATP, dCTP, dGTP, and dTTP), 1X Pwo DNA polymerase buffer, and 2.5 units of Pwo DNA polymerase (Boehringer Mannheim, Indianapolis, IN).
• the reactions were incubated in a Perkin-Elmer Model 480 Thermal Cycler programmed for 1 cycles at 95°C for 2 minutes; 10 cycles at 94°C for 45 seconds, 55°C for 45 seconds, and 72°C for 2 minutes; 17 cycles at 94°C for 45 seconds, 55°C for 45 seconds, and 72°C for 2 minutes with an extension of 20 seconds per cycle; 1 cycle at 72°C for 10 minutes; and a soak cycle at 4 °C.
• the reaction products were isolated on a 1% agarose gel where a 1.15 kb product band was excised from the gel and purified using Qiaquik Gel Extraction Kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions.
• the generated fragment was digested with Sph ⁇ , blunted with Klenow, digested with Pac ⁇ , and purified by agarose gel electrophoresis and Qiaquik Gel Extraction Kit (Qiagen, Chatsworth, CA).
• the purified DNA segment was ligated into pSheBI ( Figure 1) which was previously ⁇ /col digested, treated with DNA polymerase I (Klenow fragment), and digested with Pac ⁇ .
• the treatment of the ⁇ /col- digested vector with Klenow fragment resulted in a filling in of the ⁇ /col cohesive end, thereby making it blunt and compatible with the blunt site of the lipase DNA 5 segment.
• the resulting expression plasmid was designated pEJG60 ( Figure 2).
• the PCR-amplified lipase gene segment was re-sequenced to verify the absence of any errors.
• Example 11 Transformation of Fusarium venenatum and analysis of Fusarium venenatum transformants
• Protoplasts were prepared by inoculating 100 ml of YEPG medium with 4 X 10 7 spores of Fusarium venenatum WTY700 and incubating for 16 hours at 24°C
• the protoplasts were stored at -80°C, after control led-rate freezing in a Nalgene Cryo 1°C Freezing Container (VWR Scientific, Inc., San Francisco, CA).
• Frozen protoplasts of Fusarium venenatum WTY700 were thawed on ice.
• 35 protoplasts was added, mixed gently, and incubated on ice for 30 minutes.
• One ml of SPTC was added and incubated 20 minutes at room temperature.
• 25 ml of 40°C COVE top agarose the mixture was poured onto an empty 150 mm diameter plate and incubated overnight at room temperature.
• an additional 25 ml of 40°C COVE top agarose containing 10 mg of BASTATM per ml was poured on top of the plate and incubated at room temperature for up to 14 days.
• the active ingredient in the herbicide BASTATM is phosphinothricin.
• BASTATM was obtained from AgrEvo (Hoechst Schering, Rodovre, Denmark) and was extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1), and once with chloroform: isoamyl alcohol (24:1) before use.
• Lipase activity was determined as follows: 100 ⁇ l of substrate (3.92 ml of 100 mM MOPS pH 7.5, 4 mM CaCI 2 , 990 ⁇ l of DMSO, 80 ⁇ l of 1% AOS, and 20 ⁇ l of p- nitrophenyl butyrate) was added to 100 ⁇ l of diluted sample. The samples were diluted accordingly in 100 mM MOPS pH 7.5, 4 mM CaCI 2 . The absorbance at 405 nm was monitored for 3 minutes at room temperature in a 96-well microtiter plate using a Molecular Devices Thermomax Microplate Reader.
• the lipase assay results indicated that at both 5 and 7 days, most of the transformants produced lipase activity well above that of the untransformed control.
• a prominent polypeptide at a apparent molecular weight of 32-33 kD was observed at both time points and for each transformant harboring pEJG60.
• Example 12 Cloning and expression of lipase gene from Fusarium sulphureum
• Aspergillus oryzae strain BECh-2 was inoculated to 100 ml of YPG medium and incubated for16 hrs at 32°C at 120 rpm. Pellets were collected and washed with 0.6 M KCI, and resuspended 20 ml 0.6 M KCl containing a commercial ⁇ -glucanase product (Glucanex, product of Novo Nordisk A/S) at the concentration of 30 ⁇ l/ml. Cultures were incubated at 32°C at 60 rpm until protoplasts formed, then washed with STC buffer twice.
• the protoplasts were counted with a hematometer and resuspended in an 8:2:0.1 solution of STC:STPC:DMSO to a final concentration of 2.5x10e7 protoplasts/ml.
• About 3 ⁇ g of DNA was added to 100 ⁇ l of protoplasts solution, mixed gently and incubated on ice for 30 min.
• One ml of SPTC was added and incubated 30 min at 37°C.
• 10 ml of 50°C Cove top agarose the reaction was poured onto Cove agar plate. Transformation plates were incubated at 32°C for 5 days.
• a strain of Fusarium sulphureum was used as a genomic DNA supplier.
• PCR reactions on Fusarium sulphureum genomic DNA was done with two following primer sets: Iip3 / Iip15 (SEQ ID NO: 20/23) and Iip10 / Iip17 (SEQ ID NO: 21/24) designed based upon the alignment of 3 lipases from Fusarium.
• Reaction components (2.6 ng / ⁇ l of genomic DNA, 250 mM dNTP each, primer 250 nM each, 0.1 U/ ⁇ l of Taq polymerase in 1X buffer (Roche Diagnostics, Japan)) were mixed and submitted for PCR under the following conditions.
• Steps 1 to 3 were repeated 30 times.
• 450bp of fragment and 280 bp of fragment were amplified from primer sets of Iip3/lip15 (SEQ ID NO: 20/23) and Iip10/lip17 (SEQ ID NO: 21/24), respectively. They were gel-purified with GFXTM PCR DNA and Gel Band Purification kit (amersham pharmacia biotech) and ligated into a pT7Blue vector with ligation high (TOYOBO, Japan) . The ligation mixtures were transformed into E. coli JM109.
• adaptor PCR was done.
• a cassette was made by mixing of adaptor L (SEQ ID NO: 26) and adaptor S:: acctgccc.
• 3' and 5' of adaptor S are dephosphorylated and amidated, respectively.
• Obtained fragments were purified by GFXTM PCR DNA and Gel Band Purification kit (Amersham Pharmacia Biotech) and sequenced with each primers which amplified the fragment.
• the missing C-terminal part was cloned with LA PCRTM in vitro Cloning Kit (TaKaRa) following to the manufacturer's instructions.
• 350 bp of DNA fragment corresponding to C-terminal region was obtained from Xho I digested genome ligated to Sal I cassette of the kit with 27C2 primer (SEQ ID NO: 29). Obtained fragments were purified by GFXTM PCR DNA and Gel Band Purification kit (Amersham Pharmacia Biotech) and sequenced with 27C2 primer.
• Reaction components (6 ng / ⁇ l of genomic DNA, 250 mM dNTP each, primer 250 nM each, 0.05 U7 ⁇ l of Expand high fidelity polymerase in 1X buffer (Roche Diagnostics, Japan)) were mixed and submitted for PCR under the following conditions.
• Amplified DNA fragment was gel-purified with GFXTM PCR DNA and Gel Band Purification kit (amersham pharmacia biotech) and ligated into a pT7Blue vector with ligation high (TOYOBO, Japan) .
• the ligation mixtures were transformed into E. coli JM109.
• pT27w-3 has no PCR error and it is defined as Fusarium sulphureum lipase nucleotide sequence.
• the lipase gene was digested from pT27w-3 with BamH I and Sal I and ligated into the BamH I and Xhol sites in the Aspergillus expression cassette pCaHj483 which has Aspergillus niger neutral amylase promoter, Aspergillus nidulans TPI leader sequences, Aspergillus niger glucoamylase terminator and Aspergillus nidulans amdS gene as a marker.
• the resultant plasmid was pNL27w-8.
• pNL27w-8 was transformed into Aspergillus oryzae BECh-2.
• the selected transformants were inoculated in 100 ml of MS-9 media and cultivated at 30°C for 1 day. 3 ml of grown cell in MS-9 medium was inoculated to 100 ml of MDU-2BP medium and cultivated at 32°C for 3 days. The supernatant was obtained by centrifugation.
• the lipase productivity of selected transformants was determined as LU activity.
• the productivity of the best transformant TNL27-75 was 130 LU/ml and BECh2 has no lipase activity.
• Example 13 Immunological characterization of lipolytic enzyme from Fusarium sulphureum
• a purified lipolytic enzyme sample having the amino acid sequence shown as amino acids 1-319 of SEQ ID NO: 4 was tested by immunodiffusion against a polyclonal antibody raised against the Fusarium oxysporum lipase. No immunological cross-reaction was found.
• Transformation in Aspergillus strain Aspergillus oryzae strain BECh-2 was inoculated to 100 ml of YPG medium and incubated for16 hrs at 32°C at 120 rpm. Pellets were collected and washed with 0.6 M KCI, and resuspended 20 ml 0.6 M KCI containing a commercial ⁇ -glucanase product (Glucanex, product of Novo Nordisk A/S) at the concentration of 30 ⁇ l/ml. Cultures were incubated at 32°C at 60 rpm until protoplasts formed, then washed with STC buffer twice.
• Glucanex product of Novo Nordisk A/S
• the protoplasts were counted with a hematometer and resuspended in an 8:2:0.1 solution of STC:STPC:DMSO to a final concentration of 2.5x10e7 protoplasts/ml.
• About 3 ⁇ g of DNA was added to 100 ⁇ l of protoplasts solution, mixed gently and incubated on ice for 30 min.
• One ml of SPTC was added and incubated 30 min at 37°C.
• 10 ml of 50°C Cove top agarose the reaction was poured onto Cove agar plate. Transformation plates were incubated at 32°C for 5 days.
• a strain of Acremonium berkeleyanum was used as a genomic DNA supplier. PCR reactions on Acremonium berkeleyanum genomic DNA was done with two following primer sets: lip3 / Iip11 (SEQ ID NO: 20/22) and Iip10 / Iip21 (SEQ ID NO: 21/25) designed based upon the alignment 3 lipases from Fusarium.
• Reaction components 2.5 ng / ⁇ l of genomic DNA, 250 mM dNTP each, primer 250 nM each, 0.1 U/ ⁇ l in Taq polymerase in 1X buffer (Roche Diagnostics, Japan) were mixed and submitted for PCR under the following conditions.
• Steps 1 to 3 were repeated 30 times.
• 340 bp of fragment and 330 bp of fragment were amplified from primer sets of Iip3/lip11 and Iip10/lip21, respectively. They were gel-purified with GFXTM PCR DNA and Gel Band Purification kit (amersham pharmacia biotech) and ligated into a pT7Blue vector with ligation high (TOYOBO, Japan) . The ligation mixtures were transformed into E. coli M109. The resultant plasmids, pTAc-0310 and pTAc-1021 , were sequenced and compared to the Fusarium oxysporum lipase, showing that a clone encodes the internal part of the lipase.
• LA PCR ⁇ T"M 1 " in vitro Cloning Kit (TaKaRa) was used for genome walking.
• One kb of DNA fragment corresponding to N-terimal region was obtained from Sal I digested genome ligated to Sal I cassette of the kit with AcN3 primer (SEQ ID NO: 32).
• One kb of DNA fragment corresponding to C-terminal region was obtained from Hind III digested genome ligated to Hind III cassette of the kit with AcC3 primer (SEQ ID NO: 33).
• Obtained fragments were purified by GFXTM PCR DNA and Gel Band Purification kit (amersham pharmacia biotech) and sequenced with each primer which amplified the fragment. The sequences were compared to Fusarium oxysporum lipase, showing that an amplified DNA fragments encodes the whole part of lipase.
• the fidelity of taq polymerase is not good so in order to get the right sequence whole gene was amplified the primers 152-N (Bel) and 152-C(Xho) (SEQ ID NO: 34 and 35).
• Reaction components (6 ng / ⁇ l of genomic DNA, 250 mM dNTP each, primer 250 nM each, 0.05 U/ ⁇ l of Expand high fidelity polymerase in 1X buffer (Roche Diagnostics, Japan) were mixed and submitted for PCR under the following conditions.
• Amplified DNA fragment was gel-purified with GFXTM PCR DNA and Gel
• pT152-1 and pT152-2 were sequenced and they are identical.
• pT152-1 sequence is defined as Acremonium berkeleyanum lipase nucleotide sequence.
• Aspergillus oryzae expression plasmid pCaHj 483 (described in WO 98/00529) consists of an expression cassette based on the Aspergillus niger neutral amylase II promoter fused to the Aspergillus nidulans triose phosphate isomerase non translated leader sequence (Pna2/tpi) and the Aspergillus niger amyloglycosidase terminater (Tamg). Also present on the plasmid is the Aspergillus selective marker amdS from Aspergillus nidulans enabling growth on acetamide as sole nitrogen source. These elements were cloned into the E. coli vector pUC19.
• the ampicillin resistance marker enabling selection in E coli of this plasmid was replaced with the URA3 marker of Saccharomyces cerevisiae that can complement a pyrF mutation in E. coli in the following way:
• the pUC19 origin of replication was PCR amplified from pCaHj483 with the primers 142779 and 142780 (SEQ ID NO: 40 and 41).
• the primer 142780 introduces a Bbu I site in the PCR fragment.
• the URA3 gene was amplified from the general S. cerevisiae cloning vector pYES2 (Invitrogen corporation, Carlsbad, Ca, USA) using the primers 140288 and 142778 (SEQ ID NO: 36 and 39).
• the primer 140288 introduces an EcoR I site in the PCR fragment.
• the two PCR fragments were fused by mixing them and amplifying using the primers 142780 and 140288 using the splicing by overlap method (Horton et al (1989) Gene, 77, 61-68).
• the resulting fragment was digested with EcoR I and Bbu I and ligated to the largest fragment of pCaHj 483 digested with the same enzymes.
• the ligation mixture was used to transform the pyrF E. coli strain DB6507 (ATCC 35673) made competent by the method of Mandel and Higa (Mandel, M. and A. Higa (1970) J. Mol. Biol. 45, 154). Transformants were selected on solid M9 medium (Sambrook et. al (1989) Molecular cloning, a laboratory manual, 2. edition, Cold Spring Harbor Laboratory Press) supplemented with 1 g/l casaminoacids, 500 ⁇ g/l thiamine and 10 mg/l kanamycin.
• pCaHj 527 A plasmid from such a transformant was termed pCaHj 527.
• ThePna2/tpi promoter present on pCaHj527 was subjected to site directed mutagenises by a simple PCR approach.
• Nucleotide 134 - 144 was altered from gtactaaaacc (SEQ ID NO: 57) to ccgttaaattt (SEQ ID NO: 58) using the mutagenic primer 141223 (SEQ ID NO: 38).
• Nucleotide 423 - 436 was altered from atgcaatttaaact (SEQ ID NO: 59) to cggcaatttaacgg (SEQ ID NO: 60) using the mutagenic primer 141222: (SEQ ID NO: 37). The resulting plasmid was termed pMT 2188.
• the plasmid pT152-1 was transformed to JM110 and non-methylated pT152-
• the lipase gene was digested from non-methylated pT152-1 with Bel I and Xho I and ligated into the BamH I and Xhol sites in the Aspergillus expression cassette pMT2188 which has Aspergillus niger neutral amylase promoter, Aspergillus nidulans TPI leader sequences, Aspergillus niger glucoamylase terminator and Aspergillus nidulans amdS gene as a marker and Saccharomyces cerevisiae URA3 gene as a marker for a plasmid construction.
• the ligation mixture was transformed E.coli 6507 by electroporation and the resultant plasmid was pNL152-3 (24).
• pNL152-3 (24) was transformed into Aspergillus oryzae BECh-2.
• the selected transformants were inoculated in 100 ml of MS-9 media and cultivated at
• the lipase productivity of selected transformants was determined as LU activity.
• Example 15 Cloning and expression of lipase gene from Fusarium culmorum Transformation in Aspergillus strain
• Aspergillus oryzae strain BECh-2 was inoculated to 100 ml of YPG medium and incubated for16 hrs at 32°C at 120 rpm. Pellets were collected and washed with 0.6 M KCl, and resuspended 20 ml 0.6 M KCI containing a commercial ⁇ -glucanase product (Glucanex, product of Novo Nordisk A/S) at the concentration of 30 ⁇ l/ml. Cultures were incubated at 32°C at 60 rpm until protoplasts formed, then washed with STC buffer twice.
• the protoplasts were counted with a hematometer and resuspended in an 8:2:0.1 solution of STC:STPC:DMSO to a final concentration of 2.5x10e7 protoplasts/ml.
• About 3 ⁇ g of DNA was added to 100 ⁇ l of protoplasts solution, mixed gently and incubated on ice for 30 min.
• One ml of SPTC was added and incubated 30 min at 37°C.
• 10 ml of 50°C Cove top agarose the reaction was poured onto Cove agar plate. Transformation plates were incubated at 32°C for 5 days.
• a strain of Fusarium culmorum was used as a genomic DNA supplier. PCR reactions on Fusarium culmorum genomic DNA was done with two following primer set: Iip2 / Iip21 (SEQ ID NO: 19/25) designed based upon the alignment 3 lipases from Fusarium.
• Reaction components (6 ng / ⁇ l of genomic DNA, 250 mM dNTP each, primer 250 nM each, 0.1 U/ ⁇ l in Taq polymerase in 1X buffer (Roche Diagnostics, Japan) were mixed and submitted for PCR under the following conditions.
• Steps 1 to 3 were repeated 30 times.
• LA PCR iT'MTM 1 in vitro
• Cloning Kit (TaKaRa) was used for genome walking.
• 0.5 kbp of DNA fragment corresponding to N-terimal region was obtained from BamH I digested genome ligated to Sau3A I cassette of the kit with 12N1 primer (SEQ ID NO: 42).
• 1.8 kb of DNA fragment corresponding to C-terminal region was obtained from Bgl II digested
• Obtained fragments were purified by GFXTM PCR DNA and Gel Band Purification kit (amersham pharmacia biotech) and sequenced with each primer which amplified the fragment. Their sequence were compared to the Fusarium oxysporum lipase, showing that the amplified DNA covered N-terminal and C-
• Reaction components (6 ng / ⁇ l of genomic DNA, 250 mM dNTP each, primer
• An amplified DNA fragment was gel-purified with GFXTM PCR DNA and Gel Band Purification kit (amersham pharmacia biotech) and ligated into a pT7Blue vector with ligation high (TOYOBO, Japan) .
• the ligation mixtures were transformed into E. coli JM109.
• the resultant plasmids, pT12-1 , pT12-2, pT12-3, and pT12-4, were sequenced and all of them are identical. The sequence is defined as Fusarium culmorum lipase DNA sequence.
• the plasmid pT12-6 was transformed to JM110 and non-methylated pT12-6 was extracted.
• the lipase gene was digested from non-methylated pT12-6 with Bel I and Sal I into T-vector and ligated into the BamH I and Xhol sites in the Aspergillus expression cassette pMT2188 which has Aspergillus niger neutral amylase promoter,
• Aspergillus nidulans TPI leader sequences Aspergillus niger glucoamylase terminator and Aspergillus nidulans amdS gene as a marker and Saccharomyces cerevisiae URA3 gene as a marker for a plasmid construction.
• the ligation mixture was transformed E.coli 6507 by electroporation and the resultant plasmid was pNL12-***.
• pNL12-*** was transformed into Aspergillus oryzae BECh-2.
• the selected transformants were inoculated in 100 ml of MS-9 media and cultivated at 30°C for 1 day.
• Example 16 Cloning of a phospholipase gene from the Fusarium solani strain MUCL 38667. A genomic DNA preparation of the strain MUCL 38667 was made as described in WO 00/24883.
• a PCR reaction (96°C 5 min, 30* (94°C 30 sec., 55°C 30 sec, 72°C 1 min), 72°C 5 min) was run using PWO polymerase in 2.5 mM MgSO 4 as recommended by the manufacturer (Boehringer Mannheim) with the MUCL 38667 genomic DNA as template, with oligo 161000J1 and 161000J2 (SEQ ID NO: 46 and 47). These oligo ' es were designed based conserved sequences in homologous phospholipases.
• a fragment of 180 bp was isolated from a 2 % gel. Because the amounts of DNA was very small, a new identical per was run, this time using the 180 bp fragment as template rather than MUCL 38667 genomic DNA.
• This fragment was cloned into pCR4 using TOPO-cloning as recommended by the manufacture (Invitrogen) and transformed into the E. coli strain TOPO10.
• DNA preparations where made using the Qiagen minispinprep kit and the clones where sequenced using M13 rev and M13 fwp primer supplied with the TOPO-cloning kit (Invitrogen). Sequence alignment was made to all available DNA sequence using SRS. The 180 bp fragment was identified as originating from a phospholipase gene.
• the generated PCR fragment of app. 1500 bp was cloned into pCR4 using TOPO-cloning as recommended by the manufacture (Invitrogen) and transformed into the E. coli strain TOPO10. DNA preparations where made using the Qiagen minispinprep kit and the clones where sequenced using M13 rev and M13 fwp primer supplied with the
• the MUCL 38667 genomic DNA (app. 1 ⁇ g) was cut with Hindlll in a volume of 10 ⁇ l and ligated in a volume of 500 ⁇ l. The DNA was precipitated in ethanol and
• the generated PCR fragment of app. 350 bp was cloned into pCR4 using TOPO-cloning as recommended by the manufacture (Invitrogen) and transformed 15 into the E. coli strain TOPO10.
• DNA preparations were made using the Qiagen minispinprep kit and the clones where sequenced using T3 and T7 primer supplied with the TOPO-cloning kit
• the 350 bp fragment was identified as originating from the 5 'end of a
• a PCR reaction (96°C 5 min, 30* (94°C 30 sec, 55°C 30 sec, 72°C 2 min), 25 72°C 5 min) was run using PWO polymerase in 2.5 mM MgSO 4 as recommended by manufacture (Boehringer Mannheim) with the MUCL38667 genomic DNA as template, with oligo 290101 J2 and 020301 J1 (SEQ ID NO: 53 and 54).
• the generated PCR fragment of app. 1100 bp were cloned into pCR4 using TOPO-cloning as recommended by the manufacture (Invitrogen) and transformed 30 into the E coli strain TOPO10.
• the cloned phospholipase gene in the TOPO vector, as well as pJVi9 (WO 97/47746) was cut with the restriction enzymes BamHI and Xhol.
• the pJVI9 vector and the phospholipase gene were purified from a 1 % agarose gel, and ligated o/n. The ligation was transformed into the E.coli strain DH10b, and transformants were isolated.
• YPD Yeast et al., (1981 ), Methods in Yeast Genetics, Cold Spring Harbor Laboratory
• the mycelium is harvested by filtration through miracloth and washed with 200 ml of 0.6 M MgSO 4 .
• the mycelium is suspended in 15 ml of 1.2 M MgSO , 10 mM NaH 2 PO 4 , pH 5.8. The suspension is cooled on ice and 1 ml of buffer containing 120 mg of Novozym ® 234 is added.
• the suspension is filtered through miracloth, the filtrate transferred to a sterile tube and overlayed with 5 ml of 0.6 M sorbitol, 100 mM Tris-HCl, pH 7.0. Centrifugation is performed for 15 min. at 1000 g and the protoplasts are collected from the top of the MgSO 4 cushion. 2 volumes of STC (1.2 M sorbitol, 10 mM Tris- HCl, pH 7.5, 10 mM CaCI 2 ) are added to the protoplast suspension and the mixture is centrifuged for 5 min. at 1000 g. The protoplast pellet is resuspended in 3 ml of STC and repelleted. This is repeated.
• the protoplasts are resuspended in 0.2-1 ml of STC.
• 100 ⁇ l of protoplast suspension are mixed with 5-25 ⁇ g of p3SR2 (an A. nidulans amdS gene carrying plasmid described in Hynes et al., Mol. and Cel. Biol., Vol. 3, No. 8, 1430-1439, Aug. 1983) and 5 ⁇ g of the pJVI9-phospholipase plasmid in 10 ⁇ l of STC.
• the mixture is left at room temperature for 25 min.
• A. oryzae JaL 125 (WO 97/35956) is derived from Aspergillus oryzae IFO 4177 available from Institute for Fermention, Osaka; 17-25 Juso Hammachi 2- Chome Yodogawa-ku, Osaka, Japan, having the alkaline protease gene named "alp” (described by Murakami K et al., (1991), Agric. Biol. Chem. 55, p. 2807-2811) deleted by a one step gene replacement method (described by G. May in "Applied Molecular Genetics of Filamentous Fungi” (1992), p. 1-25. Eds. J. R. Kinghom and G. Turner; Blackie Academic and Professional), using the A. oryzae pyrG gene as marker.
• R275A variant of Acremonium berkeleyanum lipase (SEQ ID NO: 6) was constructed and expressed in A. oryzae. Preliminary tests using crude culture broth confirmed the variant enzyme to be more heat stable than the wildtype. SDS-PAGE showed that around 60% of the molecules still were processed, compared to 100% for the wildtype. The temperature-stability curve of the crude variant broth was found to be biphasic, and the unprocessed form seemed to be around 10°C more stable than the processed one.
• Example 19 Determination of phospholipase and galactolipase activities
• the lipolytic enzymes from F. venenatum, F. sulphureum, A. berkeleyanum, F. culmorum and F. solani were tested for phospholipase and galactolipase activities by plate assay methods, as described in 15 WO 0032758.
• the lipolytic enzyme from F. oxysporum was included for comparison.
• Doughs were made using spiral mixers from 2 kg of Meneba flour (batch 25 941-2) according to the straight dough method (AACC Method 10-10B in Approved Methods of the American Association of Cereal Chemists, Ninth Edition, March 1995; AACC, St. Paul MN, USA).
• Lipase activity was determined using pH-stat titration and tributyrin as substrate at 30°C and pH 7. Enzymes were dosed according to the protocol below:
• Example 21 Dough and bread evaluation and dough stability measurements
• Each dough described in the previous Example was split into 15 rolls for 45 minute fermentation; 15 rolls for 70 minute fermentation. Two pan breads were evaluated for of the breads crumb structure; and 2 pan breads were evaluated for texture analysis.
• the results of Dough 5 showed that the effect of the Fusarium venenatum lipase dosed at 40 LU/kg flour was similar to that of the Fusarium oxysporum lipase dosed at 1000 LU/kg flour. Increased dosages of the Fusarium venenatum lipase resulted in softer, more sticky dough which had a lower extensibility and higher elasticity. At a dosage of 150 LU/kg flour, the Fusarium venenatum lipase yielded a dough which broke apart easily when stretched, similar to undermixed doughs. The effect of the Fusarium venenatum lipase on dough parameters was dosage dependent.
• DATEM di-acetylated-tartaric acid-esters of mono-and diglycerides of fatty acids
• the Fusarium venenatum lipase When dosed at 100 and 150 LU/kg flour, the Fusarium venenatum lipase yielded a better shape-factor, but lower volume after normal fermentation time than the Fusarium oxysporum lipase. After an extended fermentation time, 100 and 150
• the Fusarium venenatum lipase performed similarly to the Fusarium oxysporum lipase on initial softness and elasticity.

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