Title of Invention

A METHOD OF PRODUCING A LIPASE HAVING AN AMINO ACID SEQUENCE

Abstract

The invention relates to a method of producing a lipase having an amino acid sequence which: a) has at least 90% identity with the wild-type lipase derived from humicola lanuginosa strain DSM 4109; b) compared to said wild-type lipase, comprises a substitution of an electrically neutral or negatively charged amino acid at the surface of the three-dimensional structure within 15 A of E1 or Q249 with a positively charged amino acid; and c) comprises a peptide addtiion at the C-terminal; and/or d) meets the following limitations: ii) comprises a negatively charged amino acid in the region corresponding to positions 90-101 os said wild-type lipase; and iii) comprises a netural or negative amino acid at a position corresponding to N94 of said wild-type lipase and/or has negative or netural net electric charge in the region corresponding to positions 90-101 of said wild-type lipase, said method comprises the step of culturing a transformed host cell selected from Humicola lanuginosa strain DSM 4109, containing the DNA sequence encoding the said lipase under known conditions for the production of the said lipase and recovering the lipase from the resulting broth in a known manner.

Full Text

The present invention relates to a method of producing a lipase having an amino acid sequence. For a number of years, lipases have been used as detergent enzymes to remove lipid or fatty stains from clothes and other textiles, particularly a lipase derived from Humicola lanuginosa (EP 258 068 and EP 305 216) sold under the tradename Lipolase ® (product of Novo Nordisk A/S). WO 92/05249, WO 94/25577 WO 95/22615, WO 97/04079 and WO 97/07202 disclose variants of the H. lanuginosa lipase having improved properties for detergent purposes. Thus, WO 97/04079 discloses variants having a peptide addition (extension) at the N-terminal and/or the C-terminal. WO 97/07202 discloses lipase variants with "first wash performance" which are capable of removing substantial amounts of lard from a lard stained swatch in a one-cycle wash. There is an ever existing need for providing novel lipases with improved washing properties in a variety of commercial detergents. The present invention relates to such novel lipases. The inventors have found that certain variants of Lipolase (wild-type Humicola lanuginosa lipase) have a particularly good first-wash performance in a detergent solution. The lipases may flirter provide additional benefits, such as whiteness maintenance and dingy cleanup. The inventors found that the variants should comprise one or more substitutions with positive amino acids near the N-terminal in the three-dimensional structure. The variants should further comprise a peptide addition at the C-terminal and/or should meet certain limitations on electrically charged amino acids at positions 90-101 and 210. Accordingly, the present invention provides a method of producing a lipase having an amino acid sequence which: a) has at least 90 % identity with the wild-type lipase derived from Humicola lanuginosa strain DSM 4109; b) compared to said wild-type lipase. comprises a substitution of an electrically neutral or negatively charged amino acid at the surface of the three-dimensional structure within 15 A of El or Q249 with a positively charged amino acid; and c) comprises a peptide addition at the C-terminal; and/of d) meets the following limitations: i) comprises a negative amino acid in position E210 of said wild-type lipase; ii) comprises a negatively charged amino acid in the region corresponding to positions 90-101 of said wild-type lipase; and iii) comprises a neutral or negative amino acid at a position corresponding to N94 of said wild-type lipase and/or has a negative or neutral net electric charge in the region corresponding to positions 90-101 of said wild-type lipase, said method comprises the step of culturing a transformed host cell selected form Humicola lanuginosa strain DSM 4109, containing the DNA sequence encoding the said lipase under known conditions for the production of the said lipase and recovering the lipase from the resulting broth in a known manner. Humicola lanuginosa lipase The reference lipase used in this invention is the wild-type lipase derived from Humicola lanuginosa strain DSM 4109. It is described in EP 258 068 and EP 305216 and has the amino acid sequence shown in positions 1-269 of SEQ ID NO: 2 of US 5,869,438. In this specification, the reference lipase is also referred to as Lipolase. Substitution with positive amino acid The lipase of the invention comprises one or more (e.g. 2-4, particularly two) substitutions of an electrically neutral or negatively charged amino acid near El or 25 Q249 with a positively charged amino acid, preferably R. The substitution is at the surface of the three-dimensional structure within 15 A of El or Q249, e.g. at any of positions 1-11, 90, 95, 169, 171-175, 192-211,213- 226, 228-258, 260-262. The substitution may be within 10 A of El or Q249, e.g. at any of positions 1-7,10, 175, 195, 197-202,204-206,209,215,219-224,230-239,242-254. The substitution may be within 15 A of El, e.g. at any of positions 1-11, 169, 171, 192-199, 217-225, 228-240, 243-247, 249, 261-262. The substitution is most preferably within 10 A of El, e.g. at any of positions 1-7, 10, 219-224 and 230-239. Thus, some preferred substitutions are S3R, S224R, P229R, T231R, N233R, D234RandT244R. Peptide addition at C-tenninal The lipase may comprise a peptide addition attached to C-terminal L269. The peptide addition improves the first-wash performance in a variety of detergents. The peptide addition preferably consists of 1-5 amino acids, e.g. 2, 3 or 4 amino acids. The amino acids of the peptide addition will be numbered 270, 271, etc. The peptide addition may consist of electrically neutral {e.g. hydrophobic) amino acids, e.g. PGL or PG. In an alternative embodiment, the lipase peptide addition consists of neutral (e.g. hydrophobic) amino acids and the amino acid C, and the lipase comprises substitution of an amino acid with C at a suitable location so as to form a disulfide bridge with the C of the peptide addition. Examples are: 270C linked to G23C or T37C 271C iinl 272C linked to D27C, T35C, E56C, T64C or R81C. Amino acids at positions 90-101 and 210 The lipase of the invention preferably meets certain limitations on electrically charged amino acids at positions 90-101 and 210. Lipases meeting the charge limitations are particularly effective in a detergent with high content of anionic. Thus, amino acid 210 may be negative. E210 may be unchanged or it may have the substitution E210D/C/Y, particularly E210D. The lipase may comprise a negatively charged amino acid at any of positions 90-101 (particularly 94-101), e.g. at position D96 and/or E99. Further, the lipase may comprise a neutral or negative amino acid at position N94, i.e. N94(neutral or negative), e.g. N94N/D/E. Also, the lipase may have a negative or neutral net electric charge in the region 90-101 (particularly 94-101), i.e. the number of negative amino acids is equal to or greater than the number of positive amino acids. Thus, the region may be unchanged from Lipolase, having two negative amino acids (D96 and E99) and one positive (K9B), and having a neutral amino acid at position 94 {N94), or the region may be modified by one or more substitutions. Alternatively, two of the three amino acids N94, N96 and E99 may have a negative or unchanged electric charge. Thus, all three amino acids may be unchanged or may be changed by a conservative or negative substitution, i.e. N94(neutrai or negative), D(negative) and E99(negative). Examples are N94D/E and D96E. Also, one of the three may be substituted so as to increase the electric charge, i.e. N94(positive), D96(neutral or positive) or E99 (neutral or positive). Examples are N94K/R, D96I/L/N/S/W or E99N/Q/K/R/H. The substitution of a neutral with a negative amino acid (N94D/E), may improve the perfomnance in an anionic detergent. The substitution of a neutral amino acid with a positive amino acid (N94K/R) may provide a variant lipase with good performance both in an anionic detergent and in an anionic/non-ionic detergent (a detergent with e.g. 40-70 % anionic out of total surfactant). Amino acids at other positions The inventors have found that a substitution Q249R/K/H may improve the performance both in anionic and in anionic/non-ionic detergent, and that a substitution of R209 with a neutral or negative amino acid (e.g. R209P/S) may improve the performance in anionic detergent. The lipase may optionally comprise the substitution G91A. The lipase may optionally comprise substitutions of one or more additional amino acids. Such substitutions may, e.g., be made according to principles l^nown in the ar^, e.g. substitutions described in WO 92/05249, WO 94/25577, WO 95/22615, WO 97/04079 and WO 97/07202. Combinations of substitutions A lipase variant with good first-wash performance may be obtained by modifying Lipolase as follows. Substitutions in parentheses are optional. T231R+N233R N94K+ D96L+ T231R+ N233R+ Q249R+ 270P+ 271G+ 272L D96L+T231R+N233R G91A+ E99K+ T231R+ N233R+ Q249R CN33Q) +D96L +T231R +N233R +Q249R +270 PGL R209P+T231R+N233R (N33Q) +E99N +N101S +T231R +N233R +Q249R +270 PGL K24C +(N33Q) +D96S +T231R +N233R +Q249R +270 PCL (N33Q) +G91A+E99K+T231R +N233R +Q249R +270 PGL El A-KN33Q) +G91A +E99K +T231R +N233R +Q249R+270PGL (N33Q) +G91A +E99K +G255R +T231R +N233R +Q249R +270 PGL (N33Q) +G91A +E99K +T231R +N233R +T244R +Q249R+270PGL G91A +E99K +T231R +N233R +Q249R E87K +G91D +D96L +G225P +T231R +N233R +Q249R +N251D G91A+E99K +T231R +N233R +Q249R +270AGVF G91A+E99K+T189G +T231R +N233R +Q249R D102G +T231R +N233R +Q249R T231R +N233R +Q249R +270AGVF R209P+T231R+N233R N33Q +N94K +D96L +T231R +N233R +Q249R +270PGLPFKRV N33Q +N94K +D98L +T231R +N233R +Q249R N33Q +D96S +T231R +N233R +Q249R N33Q +D96S +V2281 + +T231R +N233R +Q249R E1A+N33Q+G91A +E99K +T231R +N233R +Q249R +270PGLPFKRV N33Q +S83T +E87K +G91A + E99K +T231R +N233R +Q249R +270PGLPFKRV N33Q +G91A + E99K +T231R +N233R +Q249R +270PGLPFKRV T231R+N233R+270CP T231R+N233R+270RE N33Q+E99N +N101S +T231R +N233R +Q249R +270PGLPFKRV D62A +S83T + G91A +E99K +T231R +N233R +Q249R E99N +N101S +T231R +N233R +Q249R R84W ■»-G91A +E99K +T231R +N233R +Q249R G91A+E99K+T231R +N233R +Q249R +270SPG G91A +E99K +T231R +N233R +Q249R +270VWP G91A+E99K+T231R+N233R+Q249R+270LI_ASSGRGGHR G91A +E99K +T231R +N233R +Q249R +270VTT G91A+E99K+T231R +N233R +Q249R +270VLQ G91A +E99K +T231R +N233R +Q249R +270TST G91A +E99K +T231R +N233R +Q249R +270LRI V60G +D62E +091A +E99K +T231R +N233R +Q249R G91A +D96W +E99K +T231R +N233R +G263Q +L264A +I265T +G266S +T267A +L269N +270AGGFS Nomenclature for amino acid modifications The nomenclature used herein for defining mutations is essentially as described in WO 92/05249. Thus, T231R indicates a substitution of T in position 231 with R. PGL or 270P+ 271G+ 272L indicates the peptide addition PGL attached to the C-terminal {L269). Amino acid grouping In this specification, amino acids are classified as negatively charged, positively charged or electrically neutral according to their electric charge at pH 10, which is typical of the detergent of the invention. Thus, negative amino acids are E, D, C (cysteine) and Y, particularly E and D. Positive amino acids are R, K and H, particularly R and K. Neutral amino acids are G, A, V, L, I, P, F, W, S, T, M, N, Q and C when forming part of a disulfide bridge. A substitution with another amino acid in the same group (negative, positive or neutral) is termed a conservative substitution. The neutral amino acids may be divided into hydrophobic (G, A, V, L, I, P, F, W and C as part of a disulfide bridge) and hydrophilic (S, T, M, N, Q). Amino acid identity The lipase variant of the of the invention has an amino acid identity of at least 90 % (preferably more than 95 % or more than 98 %) with Lipoiase. The degree of identity may be suitably determined by means of computer programs known in the art. such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-45), using GAP with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1. DNA sequence, Expression vector, Host cell, Production of lipase The invention provides a DNA sequence encoding the lipase of the invention, an expression vector harboring the DNA sequence, and a transformed host cell containing the DNA sequence or the expression vector. These may be obtained by methods known in the art. The invention also provides a method of producing the lipase by culturing the transformed host cell under conditons conducive for the production of the lipase and recovering the lipase from the resulting broth. The method may be practiced according to principles known in the art. Detergent additive According to the invention, the lipase may typically be used as an additive in a detergent composition. This additive is conveniently formulated as a non-dusting granulate, a stabilized liquid, a slurry or a protected enzyme. The additive may be prepared by methods known in the art. DETERGENT COMPOSITION The detergent compositions of the invention may for example, be formulated as hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use in the pretreatment of stained fabrics, rinse added fabric softener compositions, and compositions for use in general household hard surface cleaning operations and dishwashing operations. The detergent composition of the invention comprises the lipase of the invention and a surfactant. Additionally, it may optionally comprise a builder, another enzyme, a suds suppresser, a softening agent, a dye-transfer inhibiting agent and other components conventionally used in detergents such as soil-suspending agents, soil-releasing agents, optical brighteners, abrasives, bactericides, tarnish inhibitors, coloring agents, and/or encapsulated or non-encapsulated perfumes. The detergent composition according to the invention can be in liquid, paste, gels, bars or granular forms. The pH (measured in aqueous solution at use con¬centration) will usually be neutral or alkaline, e.g. in the range of 7-11, particularly 9-11. Granular compositions according to the present invention can also be in "compact form", i.e. they may have a relatively higher density than conventional granular detergents, i.e. form 550 to 950 g/l. The lipase of the invention, or optionally another enzyme incorporated in the detergent composition, is normally incorporated in the detergent composition at a level from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level from 0.01% to 0.2% of enzyme protein by weight of the composition. The detergent composition of the invention may comprise the lipase in an amount corresponding to 10-50,000 LU per gram of detergent, preferably 20-5,000 LU/g, e.g. 100-1000 LU/g. The detergent may be dissolved in water to produce a wash liquor containing lipolytic enzyme in an amount corresponding to 25-15,000 LU per liter of wash liquor, particularly 100 - 5000 LU/l, e.g. 300-2000 LU/l. The amount of lipase protein may be 0.001-10 mg per gram of detergent or 0.001-100 mg per liter of Vk^ash liquor More specifically, the lipase of the invention may be incorporated in the detergent compositions described in WO 97/04079, WO 97/07202. WO 97/41212, PCT/DK WO 98/03939 and WO 97/43375. Surfactant system The surfactant system may comprise nonionic, anionic, cationic, ampholytic, and/or zwitterionic surfactants. As described above, the lipase variants of the invention are particularly suited for detergents comprising of a combination of anionic and nonionic surfactant w\ih 70-100 % by weight of anionic surfactant and 0-30 % by weight of nonionic, particularly 80-100 % of anionic surfactant and 0-20 % nonionic. As further described, some preferred lipases of the invention are also suited for detergents comprising 40-70 % anionic and 30-60 % non-ionic surfactant. The surfactant is typically present at a level from 0.1% to 60% by weight, e.g. 1% to 40%, particularly 10-40 %. preferably from about 3% to about 20% by weight. Some examples of surfactants are described below. Anionic surfactants Preferred anionic surfactants include alkyl sulfate, alkyl ethoxy sulfate, linear alkyl benzene sulfonate and mixtures of these. The alkyl sulfate surfactants are water soluble salts or acids of the fomiula ROSOaM wherein R preferably is a C10-C24 hydrocarbyl, preferably an alkyl or hydroxyalkyi having a GIO-CJD alkyl component, more preferably a C12-C1B alkyl or hydroxyalkyi, and M is H or a cation, e.g., an alkali metal cation (e.g. sodium, potassium, lithium), or ammonium or substituted ammonium. Alkylbenzene sulfonates are suitable, especially linear (straight-chain) alkyl benzene sulfonates (LAS) wherein the alkyl group preferably contains from 10 to 18 carbon atoms. Suitable anionic surfactants include alkyl alkoxylated sulfates which are water soluble salts or acids of the formula R0(A)mS03M wherein R is an unsubstituted Cio-C-2-i alkyl or hydroxyalkyi group having a C10-C24 alkyl component, preferably a Cii-Cjo alkyl or hydroxyalkyi, more preferably CI;-GIB alkyl or hydroxyalkyi, A is an ethoxy or propoxy unit, m is greater than zero, typically between about 0.5 and about 6, more preferably between about 0.5 and about 3, and M is H or a cation which can be, for example, a metal cation (e.g., sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or substituted-ammonium cation. Alkyl ethoxylated sulfates as well as alkyl propoxylated sulfates are contemplated herein. Specific examples of substituted ammonium cations include methyl-, dimethyl, trimethyi-ammonium cations and quaternary ammonium cations such as tetramethyl-ammonium and dimethyl piperdinium cations and those derived from alkylamines 5 such as ethylamine, diethylamine, triethylamine, mixtures thereof, and the like. Other anionic surfactants include salts (including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono- di- and triethanolamine salts) of soap, C8-C22 primary or secondary alkanesulfonates, Ca-C;* olefinsulfonates, sulfonated polycartDoxylic acids prepared by sulfonation of the 1 pyrolyzed product of alkaline earth metal citrates. Nonionic surfactant The surfactant may comprise polyalkylene oxide {e.g. polyethylene oxide) condensates of alkyl phenols. The alkyl group may contain from about 6 to about 14 carbon atoms, in a straight chain or branched-chain. The ethylene oxide may be . present in an amount equal to from about 2 to about 25 moles per mole of alkyl phenol. The surfactant may also comprise condensation products of primary and secondary aliphatic alcohols with about 1 to about 25 moles of ethylene oxide. The alkyl chain of the aliphatic alcohol can either be straight or branched, and generally contains from about 8 to about 22 carbon atoms. Further, the nonionic surfactant may comprise polyethylene oxide condensates of alkyl phenols, condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethylene oxide, alkylpolysaccharides, and mixtures hereof. Most preferred are Cs-Cu alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and CB-CIB alcohol ethoxyiates (preferably Cio avg,) having from 2 to 10 ethoxy groups, and mixtures thereof. Preferred nonionic surfactants are alcohol ethoxylate, alcohol phenol ethoxyiate, polyhydroxy fatty acid amide, alkyl polyglucoside and mixtures of these. EXAMPLE First-wash performance at various dosages in anionic detergent The following 5 variants according to the invention were prepared and tested with 3 types of soiled swatches, and the parent lipase was included for comparison: T231R+N233R. G91A +D96W +E99K +G263Q +L264A +1265T +G266D +T267A +L269N +270AGGFSWRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS R209P+T231R+N233R N33Q +D96S +T231R +N233R +Q249R E99N +N101S +T231 R +N233R +Q249R A commercial US detergent with a high content of anionic surfactant was heated to inactivate the enzymes already present, and was used at 1.4 g/l in a Tergot-o-meter ™ laboratory washing machine. The water hardness was 6"dH (Ca".Mg 2:1), and the washing conditions were 1 cycle at SO"C for 12 minutes, followed by over-night drying on filter paper. The lipase variant was added al dosages of 6400 and 12800 LU/I. Double determinations were made in two separate washes (same TOM). The following three types of textile swatches tested were: Lard/Sudan Red on cotton Style 400, freshly prepared; wfk 20-LS Lipstick on polyester/cotton; and wfK 1Q-LS lipstick on cotton. The soil removal was evaluated by measuring the remission at 460 nm af^er the first washing cycle, and the results were expressed as AR by subtracting the remission of a blank without enzyme. The results were that each variant at each dosage showed an improved first-wash performance on each type of soiled swatch (AR of 5 to 13), whereas the parent enzyme had essentially no effect {AR = 0 ± 2) under the same conditions. WE CLAIM: 1. A method of producing a lipase having an amino acid sequence which: a) has at least 90 % identity with the wild type lipase derived from Humicola lanuginosa strain DSM 4109; b) compared to said wild-type lipase, comprises a substitution of an electrically neutral or negatively charged amino acid at the surface of the three-dimensional structure within 15 A of E1 or Q249 with a positively charged amino acid; and c) comprises a peptide addition at the C-terminal; and/or d) meets the following limitations: i) comprises a negative amino acid in position E210 of said wild- type lipase; ii) comprises a negatively charged amino acid in the region corresponding to positions 90-101 of said wild-type lipase; and iii) comprises a neutral or negative amino acid at a position corresponding to N94 of said wild-type lipase ^id/or has a negative or neutral net electric charge in the region corresponding to positions 90-101 of said wild-type lipase, said method comprises the step of culturing a transformed host cell selected form Humicola lanuginosa strain DSM 4109, containing the DNA sequence encoding the said lipase under known conditions for the production of the said lipase and recovering the lipase from the resulting broth in a known manner. 2. A method of producing a lipase having an amino acid sequence substainlly as herein described and examplified.

Documents:

in-pct-2001-1300-che abstract.pdf

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in-pct-2001-1300-che claims.pdf

in-pct-2001-1300-che correspondence-others.pdf

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in-pct-2001-1300-che description(complete).pdf

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in-pct-2001-1300-che form-26.pdf

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in-pct-2001-1300-che pct.pdf

in-pct-2001-1300-che petition.pdf