Classifications

• • 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
• • 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

Definitions

• the present invention relates to novel lipase enzyme variants with improved properties, DNA constructs coding for the ex ⁇ pression of said variants, host cells capable of expressing the variants from the DNA constructs, as well as a method of producing the variants by cultivating said host cells.
• lipases seem to have certain structural features in common, but that,on the other hand, major structural variations also exist among the lipases.
• WO 92/05249 discloses lipase variants with improved properties, in which certain characteristics of wild-type lipase enzymes have been changed by specific modifications of their amino acid sequences. For instance, the electrostatic charge and/or hydro- phobicity of a so-called lipid contact zone of the wild-type lipase enzymes have been changed, and the accessibility of lipid substrate to the active site has been improved, mainly by substituting or deleting amino acid residues present in the native lipase molecule.
• the present invention relates to a lipase variant of a parent lipase comprising a trypsin-like catalytic triad including an active serine located in a pre ⁇ dominantly hydrophobic, elongated binding pocket of the lipase molecule, in which a non-aromatic amino acid residue of a lipid contact zone comprising residues located within the part of the lipase structure containing the active serine residue, which residues may participate in the interaction with the substrate at or during hydrolysis, has been substituted with an aromatic amino acid residue.
• this type of lipase variant is termed lipase variant I.
• trypsin-like is intended to indicate that the parent lipase comprises a catalytic triad at the active site corresponding to that of trypsin, i.e. the ami- no acids Ser, His and one of Asp, Glu, Asn or Gin.
• Some lipases may also comprise a surface loop structure which covers the ac- tive serine when the lipase is in inactive form (an example of such a lipase is described by Brady et al.. Nature 343. 1990, pp. 767-770) .
• the loop structure When the lipase is activated, the loop structure is shifted to expose the active site residues, creating a sur- face with increased surface hydrophobicity which interacts with the lipid substrate at or during hydrolysis.
• this surface is termed the "lipid contact zone", in ⁇ tended to include amino acid residues located within or forming part of this surface (or a corresponding surface of lipases which do not comprise such a loop structure) .
• the amino acid residues optionally in the form of loop structures, may par ⁇ ticipate in lipase interaction with the substrate at or during hydrolysis where the lipase hydrolyses triglycerides from the lipid phase when activated by contact with the lipid surface.
• fatty acids and mono- and di-glycerides are formed in varying amounts.
• the lipid contact zone of the Humicola lanuginosa lipase dis- cussed in detail in the present application is defined by amino acid residues 21-25, 36-38, 56-62, 81-98, 110-116, 144-147, 172-174, 199-213 and 248-269. These residues have been ident ⁇ ified on the basis of computer model simulations of the inter ⁇ action between the lipase and a lipid substrate.
• an aromatic amino acid residue is intended to mean a residue of tyrosine, tryptophan or pheny- lalanine
• non-aromatic amino acid residue is intended to include a residue of an amino acid different from tyrosine, tryptophan and phenylalanine.
• the invention relates to specific lipase variants in which one or more amino acid residues in specific positions of the Humicola lanuginosa lipase disclosed in WO 92/05249, the cDNA and amino acid sequence of which are shown in SEQ ID Nos. 1 and 2, or in similar positions of lipases of other origins has/have been substituted with other amino acid residues.
• the present invention also relates to a DNA construct compris ⁇ ing a DNA sequence encoding a lipase variant as indicated a- bove, a recombinant expression vector carrying said DNA con ⁇ struct, a cell transformed with the DNA construct or the ex- pression vector, as well as a method of producing a lipase variant of the invention by culturing said cell under condi ⁇ tions conducive to the production of the lipase variant, after which the lipase variant is recovered from the culture.
• the invention further relates to a detergent additive compris ⁇ ing a lipase variant of the invention, optionally in the form of a non-dusting granulate, stabilised liquid or protected enzyme, as well as to a detergent composition comprising lipase variant of the invention.
• lipase variant I is preferably one in which the non-aromatic amino acid residue to be substituted is a glutamic acid or an aspartic acid residue, and preferably one located in position 96 of the amino acid sequence of the mature H. lanuginosa lipase shown in SEQ ID No. 2, or in a similar position of a parent lipase of another origin as discussed in further detail below.
• Specific lipase variants of the invention prepared from said H. lanuginosa lipase comprises one or more amino acid residues substituted as follows:
• the lipase variant of the invention comprises more than one substi ⁇ tution, preferably two substitutions.
• the fol ⁇ lowing variants of the H . lanuginosa lipase have been found to be of interest:
• Lipase variants prepared from lipases of other origins by similar substitutions as those described above for the H . lanuginosa lipase are also considered to be within the scope of the present invention.
• the term "similar substitutions" is intended to indicate amino acid substitutions of other lipases, which are performed in similar positions to those identified above for the H . lanuginosa lipase. Similar positions may be identified on the basis of a comparison of the three-dimension ⁇ al structure of the lipase in question with that of the H. lanuginosa lipase.
• the three-dimensional structure of the H. lanuginosa lipase is shown in Fig. IA and IB of WO 92/05249, and the three-dimensional structure of the parent lipase to be modified may either be known or elucidated by conventional methods, e.g. involving X-ray analysis.
• amino acid residues to be substituted and the ones to be inserted preferably belong to the same type of amino acid (e.g. hydrophobic, hydrophillic, etc.), but need not be identical with the actual amino acid residue of the H . lanuginosa lipase.
• the parent lipase may be derived from a variety of sources such as mammalian lipases, e.g. pancreatic, gastric, hepatic or lipoprotein lipases, it is generally preferred that it is a microbial lipase.
• the parent lipase may be selected from yeast, e.g. Candida , lipases, bacterial, e.g. Pseudomonas. lipases or fungal, e.g. Humicola or Rhizomucor lipases. It is particularly preferred to select the parent lipase from a group of structurally homologous lipases.
• the parent lipase may be a Rhizomucor miehei lipase, in particular the lipase described in EP 238 023, or, as mentioned above, a H. lanuginosa lipase.
• H . lanuginosa lipase and the Rhizomucor miehei lipase belong to the same group of lipases. This implies that the overall three-dimensional structure of the two lipases is very similar and has been shown by X-ray crystallography to be highly homologous (a computer model of the H . lanuginosa and the Rh . miehei lipase is shown in Figs. IA and B and 2A and B, respectively, of WO 92/05249 from which the similarities between the lipid contact zones of the two lipases are clearly apparent) . It is therefore probable that modifications of the type indicated for the H. lanuginosa lipase will also be functional for the Rh . miehei lipase.
• the lipase variants of the invention may be prepared by isolating a DNA sequence encoding a parent lipase, suitable modifying said sequence, e.g. by site-directed mutagenesis, to encode for the variant in question and subsequently introducing the modified DNA sequence into a suitable host organism capable of expressing the variant in question.
• the DNA sequence of the DNA construct of the invention may be a cDNA, genomic DNA or 5 synthetic DNA sequence or any combination of such sequences obtained in accordance with conventional technology. Suitable techniques for cloning and mutating DNA sequences are disclosed in detail in WO 92/05249, the content of which is hereby incor ⁇ porated by reference. The techniques are further exemplified in 10 the following examples.
• lipase variants of the invention may be obtained as follows.
• an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, trans ⁇ lation initiation signal, and, optionally, a repressor gene or various activator genes.
• nucleotides encoding a "signal sequence" may be inserted prior to the lipase-coding sequence.
• a target gene to be treated according to the invention is operably linked to the control sequences in the proper reading frame.
• 25 sequences that can be incorporated into plasmid vectors, and which can support the transcription of the mutant lipase gene include but are not limited to the prokaryotic ⁇ -lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 7_5:3727-3731) and the tac promoter (DeBoer, et al.,
• a cell of the genus Bacillus such 35 as . licheniformi ⁇ , B . lentu ⁇ , or B . subtili ⁇ is transformed by an expression vector carrying the mutated DNA.
• a signal sequence may follow the translation initia- tion signal and precede the DNA sequence of interest. The signal sequence acts to transport the expression product to the cell wall where it is cleaved from the product upon secretion.
• control sequences as defined above is intended to include a signal sequence, when is present.
• a filamentous fungus is used as the host organism.
• the filamentous fungus host organism may conveniently be one which has previously been used as a host for producing recombinant proteins, e.g. a strain of Aspergillus sp. , such as A . niger, A. nidulans or A. oryzae .
• a strain of Aspergillus sp. such as A . niger, A. nidulans or A. oryzae .
• the use of A. oryzae in the production of recombinant proteins is extensively described in, e.g. EP 238 023.
• the DNA sequence coding for the lipase variant is preceded by a promoter.
• the promoter may be any DNA sequence exhibiting a strong transcriptional activity in Aspergillus and may be derived from a gene encoding an extracellular or intracellular protein such as an amylase, a glucoamylase, a protease, a lipase, a cellulase or a glycolytic enzyme.
• suitable promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral ⁇ -amylase, A . niger acid stable ⁇ - amylase, A . niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease or A. oryzae triose phosphate isomerase.
• a preferred promoter for use in the process of the present invention is the A. oryzae TAKA amylase promoter as it exhibits a strong transcriptional activity in A. oryzae .
• the sequence of the TAKA amylase promoter appears from EP 238 023.
• Termination and polyadenylation sequences may suitably be de ⁇ rived from the same sources as the promoter.
• the techniques used to transform a fungal host cell may suitab ⁇ ly be as described in EP 238 023.
• the DNA sequence encoding the lipase variant may be preceded by a signal sequence which may be a naturally occurring signal sequence or a functional part thereof or a synthetic sequence providing secretion of the protein from the cell.
• the signal sequence may be derived from a gene encoding an Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease, or a gene encoding a Humicola cellulase, xylanase or lipase.
• the signal sequence is preferably derived from the gene encoding A. oryzae TAKA amylase, A. niger neutral ⁇ -amylase, A . niger acid-stable ⁇ - amylase or A . niger glucoamylase.
• the medium used to culture the transformed host cells may be any conventional medium suitable for growing Aspergillus cells.
• the transformants are usually stable and may be cultured in the absence of selection pressure. However, if the transformants are found to be unstable, a selection marker introduced into the cells may be used for selection.
• the mature lipase protein secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
• the lipase variant may typically be a component of a detergent composition.
• it may be included in the detergent composition in the form of a non- dusting granulate, a stabilized liquid, or a protected enzyme.
• Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by methods known in the art.
• waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000, ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
• PEG poly(ethylene oxide) products
• PEG polyethyleneglycol
• Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
• a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
• Other enzyme stabilizers are well known in the art.
• Protected enzymes may be prepared according to the method disclosed in EP 238,216.
• the detergent composition of the invention may be in any con ⁇ venient form, e.g. as powder, granules, paste or liquid.
• a liquid detergent may be aqueous, typically containing up to 70 % water and 0-30 % organic solvent, or nonaqueous.
• the detergent composition comprises one or more surfactants, each of which may be anionic, nonionic, cationic, or zwit- terionic.
• the detergent will usually contain 0-50 % of anionic surfactant such as linear alkylbenzenesulfonate (LAS) , alpha- olefinsulfonate (AOS) , alkyl sulfate (fatty alcohol sulfate) (AS) , alcohol ethoxysulfate (AEOS or AES) , secondary alkane- sulfonates (SAS) , alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinic acid or soap.
• anionic surfactant such as linear alkylbenzenesulfonate (LAS) , alpha- olefinsulfonate (AOS) , alkyl sulfate (fatty alcohol sulfate) (AS) , alcohol ethoxysulfate (
• Nonionic surfactant such as alcohol ethoxylate (AEO or AE) , carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as described in WO 92/06154) .
• the detergent composition may additionally comprise one or more other enzymes, such as an amylase, a lipase, a cutinase, a pro ⁇ tease, a cellulase, a peroxidase or an oxidase.
• the detergent may contain 1-65 % of a detergent builder or com- plexing agent such as zeolite, diphosphate, triphosphate, phos ⁇ phonate, citrate, nitrilotriacetic acid (NTA) , ethylenediami- netetraacetic acid (EDTA) , diethylenetriaminepentaacetic acid (DTMPA) , alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst) .
• the detergent may also be unbuilt, i.e. essentially free of detergent builder.
• the detergent may comprise one or more polymers.
• examples are carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP) , polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA) , polycar- boxylates such as polyacrylates, maleic/acrylic acid copo- lymers and lauryl methacrylate/acrylic acid copolymers.
• CMC carboxymethylcellulose
• PVP poly(vinylpyrrolidone)
• PEG polyethyleneglycol
• PVA poly(vinyl alcohol)
• polycar- boxylates such as polyacrylates, maleic/acrylic acid copo- lymers and lauryl methacrylate/acrylic acid copolymers.
• the detergent may contain a bleaching system which may comprise a H 2 0 2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetra- acetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS) .
• TAED tetra- acetylethylenediamine
• NOBS nonanoyloxybenzenesulfonate
• the bleaching system may comprise per- oxyacids of e.g. the amide, imide, or sulfone type.
• the enzymes of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g. a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative as e.g. an aromatic borate ester, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.
• a polyol such as propylene glycol or glycerol
• a sugar or sugar alcohol lactic acid, boric acid, or a boric acid derivative as e.g. an aromatic borate ester
• the detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil- suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, or perfume.
• fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil- suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, or perfume.
• the pH (measured in aqueous solution at use concentration) will usually be neutral or alkaline, e.g. 7-11.
• a detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
• alkyl sulfate e.g. C 16 ., 8
• minor ingredients e.g. suds suppressors, perfume, optical brightener, photobleach
• a detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
• alcohol ethoxysulfate e.g. C 12 ., 8 alcohol, 1-2 EO
• alkyl sulfate e.g. C 16 . 18
• a detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
• minor ingredients e.g. suds suppressors, perfume
• An aqueous liquid detergent composition comprising
• An aqueous structured liquid detergent composition compris ⁇ ing
• alcohol ethoxylate e.g. C 12 . 15 alcohol, 7 EO or i 2 -i 5 alcohol, 5 EO
• soap as fatty acid e.g. oleic acid
• - anchoring polymers as e.g. lauryl metharylate/acrylic acid copolymer; molar ratio 25:1; MW 3800 0 - 3%
• a detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
• a detergent composition formulated as a granulate comprising
• polymers e.g. PVP, maleic/acrylic acid copolymer, PEG 1 - 5%
• minor ingredients e.g. suds suppressors, perfume
• a detergent composition formulated as a granulate comprising
• bleach activator e.g. NOBS or TAED 1 - 5% - carboxymethylcellulose 0 - 2%
• polymers e.g. polycarboxylate or PEG 1 - 5%
• An aqueous liquid detergent composition comprising
• hydrotrope e.g. sodium toluenesulfonate 2 - 6s-
• An aqueous liquid detergent composition comprising
• minor ingredients e.g. hydrotropes, dispersants, perfume, optical brighteners 0 - 5%
• a detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising - anionic surfactant (linear alkylbenzenesulfonate, alkyl sulfate, alpha- olefinsulfonate, alpha-sulfo fatty acid methyl esters, alkanesulfonates, soap) 25 - 40% - nonionic surfactant
• Detergent composition formulated as a nonaqueous detergent liquid comprising a liquid nonionic surfactant as e.g. linear alkoxylated primary alcohol, a builder system (e.g. phosphate) , enzyme and alkali.
• a liquid nonionic surfactant as e.g. linear alkoxylated primary alcohol, a builder system (e.g. phosphate) , enzyme and alkali.
• the detergent may also comprise anionic surfactant and/or a bleach system.
• the lipase variant of the invention may be incorporated in con ⁇ centrations conventionally employed in detergents. It is at present contemplated that, in the detergent composition of the invention, the lipase variant may be added in an amount cor ⁇ responding to 0.001-100 mg of enzyme per liter of wash liquor.
• the lipase variant may be used as an ingredient in dishwashing detergent composition.
• the dishwashing detergent composition comprises a surfactant which may be anionic, non- ionic, cationic, amphoteric or a mixture of these types.
• the detergent will contain 0-90% of non-ionic surfactant such as low- to non-foaming ethoxylated propoxylated straight-chain alcohols.
• the detergent composition may contain detergent builder salts of inorganic and/or organic types.
• the detergent builders may be subdivided into phosphorus-containing and non-phosphorus- containing types.
• the detergent composition usually contains 1- 90% of detergent builders.
• Examples of phosphorus-containing inorganic alkaline detergent builders when present, include the water-soluble salts espe ⁇ cially alkali metal pyrophosphates, orthophosphates , polyphos- phates, and phosphonates.
• Examples of non-phosphorus-containing inorganic builders when present, include water-soluble alkali metal carbonates, borates and silicates as well as the various types of water-insoluble crystalline or amorphous alumino silicates of which zeolites are the best-known representatives.
• suitable organic builders include the alkali metal, ammonium and substituted ammonium, citrates, succinates, malo ⁇ nates, fatty acid sulphonates, carboxymetoxy succinates, ammo ⁇ nium polyacetates, carboxylates, polycarboxylates, aminopoly- carboxylates, polyacetyl carboxylates and polyhydroxsulphona- tes.
• Suitable organic builders include the higher molecular weight polymers and co-polymers known to have builder prop ⁇ erties, for example appropriate polyacrylic acid, polymaleic and polyacrylic/poly aleic acid copolymers and their salts.
• the dishwashing detergent composition may contain bleaching agents of the chlorine/bromine-type or the oxygen-type.
• inorganic chlorine/bromine-type bleaches are li ⁇ thium, sodium or calcium hypochlorite and hypobromite as well as chlorinated trisodium phosphate.
• organic chlo ⁇ rine/bromine-type bleaches are heterocyclic N-bromo and N- chloro imides such as trichloroisocyanuric, tribromoiso- cyanuric, dibromoisocyanuric and dichloroisocyanuric acids, and salts thereof with water-solubilizing cations such as potassium and sodium.
• Hydantoin compounds are also suitable.
• oxygen bleaches are preferred, for example in the form of an inorganic persalt, preferably with a bleach precursor or as a peroxy acid compound.
• suitable peroxy bleach compounds are alkali metal perborates, both tetra- hydrates and monohydrates, alkali metal percarbonates, per- silicates and perphosphates.
• Preferred activator materials are TAED and glycerol triacetate.
• the dishwashing detergent composition of the invention may be stabilized using conventional stabilizing agents for the en ⁇ zyme(s) , e.g. a polyol such as e.g.propylene glycol, a sugar or a sugar alcohol, lactic acid, boric acid, or a boric acid de ⁇ rivative, e.g. an aromatic borate ester.
• a polyol such as e.g.propylene glycol, a sugar or a sugar alcohol, lactic acid, boric acid, or a boric acid de ⁇ rivative, e.g. an aromatic borate ester.
• the dishwashing detergent composition may also comprise other enzymes, in particular an amylase, a protease and/or a cellulase.
• the dishwashing detergent composition of the invention may also contain other conventional detergent ingredients, e.g. defloc- culant material, filler material, foam depressors, anti-cor- rosion agents, soil-suspending agents, sequestering agents, anti-soil redeposition agents, dehydrating agents, dyes, bac- tericides, fluorescers, thickeners and perfumes.
• other conventional detergent ingredients e.g. defloc- culant material, filler material, foam depressors, anti-cor- rosion agents, soil-suspending agents, sequestering agents, anti-soil redeposition agents, dehydrating agents, dyes, bac- tericides, fluorescers, thickeners and perfumes.
• the variant of the invention may be used in conventio- nal dishwashing detergents, e.g. any of the detergents de ⁇ scribed in any of the following patent publications: EP 551670, EP 533239, WO 9303129, EP 507404, US 5141664, GB 2247025, EP 414285, GB 2234980, EP 408278, GB 2228945, GB 2228944, EP 387063, EP 385521, EP 373851, EP 364260, EP 349314, EP 331370, EP 318279, EP 318204, GB 2204319, EP 266904, US 5213706, EP 530870, CA 2006687, EP 481547, EP 337760, WO 93/14183, US 5223179, WO 93/06202, WO 93/05132, WO 92/19707, WO 92/09680, WO 92/08777, WO 92/06161, WO 92/06
• lipase variants of the invention may be used in softening compositions:
• the lipase variant may be used in fabric softeners, e.g. as de ⁇ scribed in Surfactant and Consumer Products, Ed. by J. Falbe, 1987, pp 295-296; Tenside Surfactants Detergents, - (1993), 6, pp 394-399; JAOCS, Vol. j61 (1984), 2, pp 367-376; EP 517 762; EP 123 400; WO 92/19714; WO 93/19147; US 5,082,578; EP 494 769; EP 544 493; EP 543 562; US 5,235,082; EP 568 297; EP 570 237.
• fabric softeners e.g. as de ⁇ scribed in Surfactant and Consumer Products, Ed. by J. Falbe, 1987, pp 295-296; Tenside Surfactants Detergents, - (1993), 6, pp 394-399; JAOCS, Vol. j61 (1984), 2, pp 367-376; EP 517 762; EP 123
• Fig. 1 is a schematic representation of the preparation of plasmids encoding lipase variants by polymerase chain reaction (PCR) ;
• Fig. 2 is a schematic representation of the three-step mutagenesis by PCR
• H. lanuginosa lipase Cloning of H. lanuginosa lipase is described in EP 305,216. This patent application also describes expression and charac ⁇ terization of the lipase in Aspergillus oryzae .
• the expression plasmid used is termed p960.
• the expression plasmid used in this application is identical to p960, except for minor modifications immediately 3' to the li- pase coding region.
• the modification was made in the following way: p960 was digested with Nrul and BamHI restriction enzymes. Between these two sites the BamHI/Nhel fragment from plasmid pBR322, in which the Nhel fragment was filled in with Klenow polymerase, was cloned, thereby creating plasmid pAOl (shown in Fig. 5 of WO 92/05249) which contains unique BamHI and Nhel sites. Between these unique sites a BamHI/Xbal fragment from p960 was cloned to give pAHL (shown in Fig. 6 of WO 92/05249) .
• the approach used for introducing mutations into the lipase genes is described in Nelson & Long, Analytical Biochemistry, 180, 147-151 (1989) . It involves the 3-step generation of a PCR (polymerase chain reaction) fragment containing the desired mu- tation introduced by using a chemically synthesized DNA-strand as one of the primers in the PCR-reactions. From the PCR-gene- rated fragment, a DNA fragment carrying the mutation can be i- solated by cleavage with restriction enzymes and re-inserted into the expression plasmid. This method is thoroughly descri- bed in example 3. In figures 1 and 2 the method is further out ⁇ lined.
• the circular plasmid pAHL was linearized with the restriction enzyme Sphl in the following 50 ⁇ l reaction mixture: 50 mM NaCl, 10 mM Tris-HCl, pH 7.9, 10 mM MgCl 2 , 1 mM dithiothreitol, 1 ⁇ g plasmid and 2 units of Sphl.
• the digestion was carried out for 2 hours at 37°C.
• the reaction mixture was extracted with phenol (equilibrated with Tris-HCl, pH 7.5) and precipitated by adding 2 volumes of ice-cold 96% ethanol. After centrifugation and drying of the pellet, the linearized DNA was dissolved in 50 ⁇ l H 2 0 and the concentration estimated on an agarose gel.
• 3-step mutagenisation involves the use of four primers:
• step l 100 pmol primer A, 100 pmol primer B and l fmol linearized plasmid was added to a total of 100 ⁇ l reaction mixture and 15 cycles consisting of 2 minutes at 95°C, 2 minutes at 37°C and 3 minutes at 72°C were carried out.
• step 2 The concentration of the PCR product was estimated on an agarose gel. Then, step 2 was carried out. 0.6 pmol step 1 product and 1 fmol linearized plasmid was contained in a total of 100 ⁇ l of the . previously mentioned buffer and 1 cycle consisting of 5 minutes at 95°C, 2 minutes at 37°C and 10 minutes at 72°C was carried out.
• step 2 To the step 2 reaction mixture, 100 pmol primer C and 100 pmol primer D was added (1 ⁇ l of each) and 20 cycles consisting of 2 minutes at 95°C, 2 minutes at 37°C and 3 minutes at 72°C were carried out. This manipulation comprised step 3 in the mutage ⁇ nisation procedure.
• the product from step 3 was isolated from an agarose gel and re-dissolved in 20 ⁇ l H 2 0. Then, it was digested with the restriction enzymes BamHI and Bstxl in a total volume of 50 ⁇ l with the following composition: 100 mM NaCl, 50 mM Tris-HCl, pH 7.9, 10 mM MgCl 2 , 1 mM DTT and 10 units of each enzyme. Incubation was at 37°C for 2 hours. The 733 bp BamHI/BstxI fragments as isolated from an agarose gel.
• the expression plasmid pAHL was cleaved with BamHI and Bstxl under conditions indicated above and the large fragment was isolated from an agarose gel. To this vector, the mutated fragment isolated above was ligated and the ligation mix was used to transform E.coli. The presence and orientation of the fragment was verified by cleavage of a plasmid preparation from a transformant with restriction enzymes. Sequence analysis was carried out on the double-stranded plasmid using the di-deoxy chain termination procedure developed by Sanger. The plasmid was named pAHLD96W and is identical to pAHL, except for the altered codon.
• mutants were constructed using the same method as described in example 1, except that the restriction enzyme Xhol and Bstxl were used for digesting the PCR-product and the vec- tor used for recloning of the mutated fragments for D254K/L259I and L259I. Plasmid names and primers used for the modifications are listed below.
• YPD Garnier et al.. Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1981
• the mycelium was harvested by filtration through miracloth and washed with 200 ml of 0.6 M MgS0 4 .
• the suspension was cooled on ice and 1 ml of buffer containing 120 mg of Novozym * 234, batch 1687 was added.
• protoplasts were resuspended in 0.2 - 1 ml of STC.
• 100 ⁇ l of protoplast suspension was mixed with 5 - 25 ⁇ g of p3SR2 (an Aj t . nidulans amdS gene carrying plasmid described in Hynes et al., Mol. and Cel. Biol., Vol. 3, No. 8, 1430-1439, Aug. 1983) in 10 ⁇ l of STC. The mixture was left at room temperature for 25 min.
• lipase variant D96 in A. oryzae pAHLD96W was transformed into A ⁇ orvzae IFO 4177 by cotransfor- mation with p3SR2 containing the amdS gene from A ⁇ nidulans as described in example 3.
• Protoplasts prepared as described were incubated with a mixture of equal amounts of pAHLD96W and p3SR2, approximately 5 ⁇ g of each were used.
• 9 transformants which could use acetamide as sole nitrogen source were reiso- lated twice. After growth on YPD for three days, culture supernatants were analyzed using the assay for lipase activity described in example 5 (Purification of lipase variants of the invention) .
• the best transformant was selected for further studies and grown in a 1 1 shake-flask on 200 ml FG4 medium (3% soy meal, 3% maltodextrin, 1% peptone, pH adjusted to 7.0 with
• Assay for lipase activity A substrate for lipase was prepared by emulsifying glycerine tributyrat (MERCK) using gum-arabic as emulsifier. Lipase activity was assayed at pH 7 using pH stat method. One unit of lipase activity (LU/mg) was defined as the amount needed to liberate one micromole fatty acid per minute.
• Step l Centrifuge the fermentation supernatant, discard the precipitate. Adjust the pH of the supernatant to 7 and add gradually an equal volume of cold 96 % ethanol. Allow the mixture to stand for 30 minutes in an ice bath. Centrifuge and discard the precipitate.
• Step 2 - Ion exchange chromatography. Filter the supernatant and apply on DEAE-fast flow (Pharmacia TM) column equilibrated with 50 mM tris-acetate buffer pH 7. Wash the column with the same buffer till absorption at 280 nm is lower than 0.05 OD. Elute the bound enzymatic activity with linear salt gradient in the same buffer (0 to 0.5 M NaCl ) using five column volumes. Pool the fractions containing enzymatic activity .
• Step 3 Hydrophobic chromatography. Adjust the molarity of the pool containing enzymatic activity to 0.8 M by adding solid Ammonium acetate. Apply the enzyme on TSK gel Butyl- Toyopearl 650 C column (available from Tosoh Corporation Japan) which was pre-equilibrated with 0.8 M ammonium acetate. Wash the unbound material with 0.8 M ammonium acetate and elute the bound material with distilled water.
• Step 4 Pool containing lipase activity is diluted with water to adjust conductance to 2 S and pH to 7. Apply the pool on High performance Q Sepharose (Pharmacia) column pre- equilibrated with 50 mM tris -acetate buffer pH 7. Elute the bound enzyme with linear salt gradient.
• H__. lanuginosa lipase variants of the invention was evaluated on the basis of the enzyme dosage in mg of protein per litre according to OD 280 compared to the wild-type H. lanuginosa lipase.
• wash trials were carried out in 150 ml beakers placed in a thermostated water bath. The beakers were stirred with triangu ⁇ lar magnetic rods.
• Wash liquor 100 ml per beaker Swatches: 6 swatches (3.5 x 3.5 cm) per beaker Fabric: 100% cotton.
• Test Fabrics style #400 Stain Lard coloured with Sudan red (0.75 mg dye/g of lard) . 6 ⁇ l of lard heated to 70°C was applied to the centre of each swatch. After application of the stain, the swatches were heated in an oven at 75°C for 30 minutes. The swatches were then stored overnight at room temperature prior to the first wash.
• Dose-response curves were compared for the lipase variants and the native H___ lanuginosa lipase.
• the dose-response curves were calculated by fitting the measured data to the following equation: c 0 - 5
• K is a constant expressing the maximum effect
• K 2 expresses the enzyme concentra ⁇ tion at which half of the maximum effect is obtained.
• ORGANISM Humicola lanuginosa
• GGG AAT CTT AAC TTC GAC TTG AAA GAA ATA AAT GAC ATT TGC TCC GGC 384 Gly Asn Leu Asn Phe Asp Leu Lys Glu lie Asn Asp lie Cys Ser Gly
• ACG TTA AGG CAG AAG GTG GAG GAT GCT GTG AGG GAG CAT CCC GAC TAT 480 Thr Leu Arg Gin Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr 125 130 135
• Glu Tyr Trp lie Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp
• ATC GTG AAG ATA GAA GGC ATC GAT GCC ACC GGC GGC AAT AAC CAG CCT 816 lie Val Lys He Glu Gly He Asp Ala Thr Gly Gly Asn Asn Gin Pro 235 240 245 250

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• 1993
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