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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
  • A61K8/66—Enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
• • 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
  • C11D3/0005—Other compounding ingredients characterised by their effect
  • C11D3/0078—Compositions for cleaning contact lenses, spectacles or lenses
• • 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)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea

Definitions

• the present invention relates to novel enzyme variants useful in a variety of cleaning compositions, and DNA sequences encoding such enzyme variants
• Enzymes make up the largest class of naturally occurring proteins Each class of enzyme generally catalyzes (accelerates a reaction without being consumed) a different kind of chemical reaction
• One class of enzymes known as proteases are known for their ability to hydrolyze (break down a compound into two or more simpler compounds with the uptake of the H and OH parts of a water molecule on either side of the chemical bond cleaved) other proteins This ability to hydrolyze proteins has been taken advantage of by incorporating naturally occurring and protein engineered proteases as an additive to laundry detergent preparations Many stains on clothes are proteinaceous and wide-specificity proteases can substantially improve removal of such stains
• protease characteristics such as thermal stability, pH stability, oxidative stability and substrate specificity are not necessarily optimized for utilization outside the natural environment of the enzyme
• the ammo acid sequence of the protease determines the characteristics of the protease
• a change of the ammo acid sequence of the protease may alter the properties of the enzyme to varying degrees, or may even inactivate the enzyme, depending upon the location, nature and/or magnitude of the change in the ammo acid sequence
• Several approaches have been taken to alter the wild-type ammo acid sequence of proteases in an attempt to improve their properties, with the goal of increasing the efficacy of the protease in the wash environment
• These approaches include altering the am o acid sequence to enhance thermal stability and to improve oxidation stability under quite diverse conditions
• the present invention relates to Thermitase variants having a modified ammo acid sequence of wild-type Thermitase am o acid sequence, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region and a fifth loop region, wherein the modified am o acid sequence comprises different ammo acids than that occurring in wild-type Thermitase (i.e , substitution) at specifically identified positions in one or more of the loop regions whereby the Thermitase variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type Thermitase
• the present invention also relates to DNA sequences encoding such Thermitase variants
• the present invention also relates to compositions comprising such Thermitase variants for cleaning a variety of surfaces.
• This invention pertains to subtilisin enzymes, in particular Thermitase, that have been modified by mutating the various nucleotide sequences that code for the enzyme, thereby modifying the amino acid sequence of the enzyme.
• the modified subtilisin enzymes (hereinafter, "Thermitase variants") of the present invention have decreased adsorption to and increased hydrolysis of an insoluble substrate as compared to the wild-type subtilisin.
• the present invention also pertains to DNA sequences encoding for such Thermitase variants.
• subtilisin enzymes of this invention belong to a class of enzymes known as proteases.
• a protease is a catalyst for the cleavage of peptide bonds
• One type of protease is a se ⁇ ne protease.
• a serme protease is distinguished by the fact that there is an essential serme residue at the active site
• the mutations described herein are designed to change (i.e., decrease) the adsorption of the enzyme to surface-bound soils
• certain amino acids form exterior loops on the enzyme molecule.
• these loops shall be referred to as first, second, third, fourth and fifth loop regions.
• positions 66- 73 form the first loop region
• positions 103-115 form the second loop region
• positions 134-141 form the third loop region
• positions 162-171 form the fourth loop region
• positions 191-195 form the fifth loop region
• positions 204-224 form the sixth loop region (position numbering analogous to positions in the amino acid sequence for wild-type subtilisin Thermitase (SEQ ID N0.1 )).
• loop regions play a significant role in the adsorption of the enzyme molecule to a surface-bound peptide, and specific mutations in one or more of these loop regions will have a significant effect on this adsorption. While not wishing to be bound by theory, it is believed that the loop regions are important to the adsorption of the Thermitase molecule for at least two reasons. First, the amino acids which comprise the loop regions can make close contacts with any surfaces to which the molecule is exposed. Second, the proximity of the loop regions to the active-site and binding pocket of the Thermitase molecule gives them a role in the catalytically productive adsorption of the enzyme to surface-bound substrates (peptides/protein soils).
• variant means an enzyme having an amino acid sequence which differs from that of wild-type.
• mutant Thermitase DNA means a DNA sequence coding for a Thermitase variant.
• wild-type Thermitase refers to an enzyme represented by SEQ ID NO:1.
• the amino acid sequence for Thermitase is further described by Meloun, B., Baudys, M., Kostka, V., Hausdorf, G., Frommel, C, and Hohne, W.E., FEBS LETT., Vol. 183, pp. 195-200 (1985), incorporated herein by reference.
• the term "Thermitase wild-type amino acid sequence” encompasses SEQ ID NO:1 as well as SEQ ID NO:1 having modifications to the amino acid sequence other than at any of positions 66, 67, 68, 69, 70, 72, 73, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 134, 135, 136, 137, 138, 139, 140, 141 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 191 , 192,193,194, 195, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223 or 224.
• hydrophilicity table lists amino acids in descending order of increasing hydrophilicity (see Hopp, T.P., and Woods, K.R., "Prediction of Protein Antigenic Determinants from Amino Acid Sequences", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE USA, Vol. 78, pp. 3824-3828, 1981 , incorporated herein by reference).
• Table 1 also indicates which amino acids carry a charge (this characteristic being based on a pH of from about 8-9).
• the positively charged amino acids are Arg and Lys
• the negatively charged amino acids are Glu and Asp
• the remaining amino acids are neutral.
• the substituting amino acid is either neutral or negatively charged, more preferably negatively charged (i.e., Glu or Asp).
• the statement “substitute Gin with an equally or more hydrophilic amino acid which is neutral or has a negative charge” means Gin would be substituted with Asn (which is equally hydrophilic to Gin), or Ser, Glu or Asp (which are more hydrophilic than Gin); each of which are neutral or have a negative charge, and have a greater hydrophilicity value as compared to Gin.
• the statement “substitute Pro with a more hydrophilic amino acid which is neutral or has a negative charge” means Pro would be substituted with Gin, Asn, Ser, Glu or Asp.
• the Thermitase variant has a modified amino acid sequence of Thermitase wild-type amino acid sequence, wherein the wild-type amino acid sequence comprises a substitution at one or more positions in one or more of the first loop region, the second loop region, the third loop region, the fourth loop region, the fifth loop region or the sixth loop region; whereby the Thermitase variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type Thermitase
• the substituting ammo acid for one or more of the positions in one or more of the loop regions is with reference to Table 1 neutral or negatively charged and equally or more hydrophilic, preferably more hydrophilic, than the ammo acid at the subject position in the wild-type ammo acid sequence A Substitutions in the First Loop Region
• substitution occurs at one or more of positions 66, 67, 68, 69, 70, 72 or 73
• the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser
• the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser
• the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser
• the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser B Substitutions in the Second LOOP Region
• substitution occurs at one or more of positions 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114 or 115
• the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Met, Pro, Ser or Thr.
• the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Met, Pro, Ser , Thr or Val
• the substituting ammo acid is Asp, Gin, Glu or Ser
• the substituting ammo acid is Asp or Glu.
• the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser.
• the substituting amino acid is Asp or Glu.
• the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser.
• the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser.
• the substituting amino acid is Ala. Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Met, Phe, Pro, Ser, Thr, Tyr or Val.
• the substituting amino acid is Asn, Asp, Gin. Glu, Gly, Pro or Ser.
• the substituting amino acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr.
• the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Met, Pro, Ser or Thr.
• substitution occurs at one or more of positions 134, 135, 136, 137, 138, 139, 140 or 141.
• the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Met, Pro, Ser, Thr or Val.
• the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser.
• the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser.
• the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser.
• the substituting amino acid is Ala, Asn, Asp, cys, Gin, Glu, Gly, His, Met, Pro, Ser or Thr.
• the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser.
• the substituting amino acid is Asp, gin, Glu or Ser.
• substitution occurs at one or more of positions 162, 163, 164, 165, 166, 167, 168, 169, 170 or 171
• the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser
• the substituting ammo acid is Asp, Gin, Glu or Ser.
• the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr.
• the substituting am o acid is Asn, Asp, Gin, Glu, Pro or Ser
• the substituting ammo acid is Asp, Gin, Glu or Ser
• the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser
• the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr.
• the substituting ammo acid is Asn, Asp, Gin, Glu, Gly or Ser
• the substituting am o acid is Asp, Gin, Glu or Ser.
• the substituting am o acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr or Val.
• substitution occurs at one or more of positions 191 , 192, 193, 194 or 195.
• the substituting am o acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr, Tyr or Val.
• the substituting ammo acid is Asp or Glu.
• the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser.
• substitution occurs at one or more of positions 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218 or 219, 220, 221 , 223 or 224.
• the substituting amino acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr.
• the substituting amino acid is Asn, Asp, Gin, Glu, Gly or Ser.
• the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser.
• the substituting amino acid is Asp or Glu.
• the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr, Tyr or Val.
• the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val.
• the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr or Val.
• the substituting amino acid is Asp or Glu.
• the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser.
• the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr or Val.
• the substituting amino acid is Asn, Asp, Gin, Glu, Gly, or Ser.
• the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser.
• the substituting amino acid is Asp or Glu.
• the substituting amino acid is Asn Asp, Gin, Glu, Gly, Pro or Ser
• the substituting ammo acid is Ala Asn Asp, Cys Gin, Glu, Gly, His, lie, Leu, Met, Pro Ser, Thr or Val
• the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr
• the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Met, Pro, Ser, Thr or Val
• the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser
• the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser G Preparation of enzyme variants
• TP phagemid
• Thermitase gene is constructed The 2 8 Kbp Pvu II restriction enzyme fragment of plasmid pUC119, (Vieira, J and Messing, J , "Production of Single-Stranded Plasmid DNA", 153 METHODS IN ENZYMOLOGY 3-11 (1989)) is cloned into the Pvu II site of plasmid pUB110 (Bacillus Genetic Stock Center, Columbus OH 1 E9)
• the pUC119-pUB110 hybrid plasmid is named pJMA601
• Into the 8amH1 restriction site of PJMA601 is cloned the polymerase chain reaction-amplified Thermitase gene gene giving TP Phagemid TP is transformed into Escherichia coli Ung" strain CJ236 and a single stranded uracil-contaming DNA template is produced using the VCSM13 helper phage (Kunkel, T A ,
• Oligonucleotides are made using an Applied Biosystem Inc 380B DNA synthesizer Mutagenesis reaction products are transformed into Escherichia coli strain MM294 (American Type Culture Collection E. coli 33625) All mutants are confirmed by DNA sequencing and the isolated DNA is transformed into the Bacillus subtilis expression strain BG2036 (Yang, M Y., E Ferrari and D. J.
• Example 2 Fermentation The Bacillus subtilis cells (BG2036) containing a subtilisin mutant of interest are grown to mid-log phase in a one liter culture of LB-glucose broth and inoculated into a Biostat ED fermenter (B Braun Biotech, Inc., Allentown, Pennsylvania) in a total volume of 10 liters
• the fermentation media contains Yeast Extract, starch, antifoam, buffers and trace minerals (see FERMENTATION A PRACTICAL APPROACH, Ed. B McNeil and L M Harvey, 1990)
• the broth is kept at a constant pH of 7.0 during the fermentation run.
• Chloramphenical is added for antibiotic selection of mutagenized plasmid
• the cells are grown overnight at 37°C to an AQQQ of about 60 and harvested
• the fermentation broth is taken through the following steps to obtain pure enzyme
• the broth is cleared of Bacillus subtilis cells by centnfugation, and clarified by removing fine particulates with a 100K cutoff membrane This is followed by concentration on a 10K cutoff membrane, and flow dialysis to reduce the ionic strength and adjust the pH to 5 5 using 0 025M MES buffer (2-( ⁇ -morphol ⁇ no)ethanesulfon ⁇ c acid)
• the enzyme is further purified by loading it onto either a cation exchange chromatography column or an affinity adsorption chromatography column and elutmg it from the column with a NaCI or a propylene glycol gradient (see Scopes, R K , PROTEIN PURIFICATION PRINCIPLES AND PRACTICE, Sp ⁇ nger-Verlag, New York (1984), incorporated herein by reference)
• the pNA assay (DelMar, E G , C Largman, J W Brod ⁇ ck and M C Geokas, ANAL BIOCHEM , Vol 99, pp 316-320, (1979), incorporated herein by reference) is used to determine the active enzyme concentration for fractions collected during gradient elution This assay measures the rate at which p-nitroani ne is released as the enzyme hydrolyzes the soluble synthetic substrate, succinyl-alanine-alanine-prolme-phenylalanine-p- nitroani de (sAAPF-pNA) The rate of production of yellow color from the hydrolysis reaction is measured at 410 nm on a spectrophotometer and is proportional to the active enzyme concentration In addition, absorbance measurements at 280 nm are used to determine the total protein concentration The active enzyme/total-protein ratio gives the enzyme purity, and is used to identify fractions to be pooled for the stock solution
• the enzyme stock solution is eluted through a Sephadex-G25 (Pharmacia, Piscataway, New Jersey) size exclusion column to remove the propylene glycol and exchange the buffer.
• the MES buffer in the enzyme stock solution is exchanged for 0.1 M Tris buffer (Tris(hydroxymethyl-aminomethane) containing 0.01 M CaCl2 and pH adjusted to 8.6 with HCI. All experiments are carried out at pH 8.6 in Tris buffer thermostated at 25°C. H. Characterization of enzyme variants
• CPG Aminopropyl controlled pore glass
• DMSO dimethyl sulfoxide
• the CPG surface will have 62,000 ⁇ 7,000 pNA molecules/ ⁇ m 2 .
• the surface area will remain unchanged from the value of 50.0m2/g reported by CPG Inc. for the CPG as received. This suggests that the procedure used to add sAAPF-pNA to CPG does not damage the porous structure (mean diameter is 486 A).
• CPG:sAAPF-pNA Using CPG:sAAPF-pNA, adsorption of an enzyme variant and hydrolysis of a CPG-bound peptide can be measured in a single experiment. A small volume of enzyme variant stock solution is added to a flask containing Tris buffer and CPG:sAAPF-pNA which has been degassed.
• the flask is shaken on a wrist-action shaker for a period of 90 minutes during which the shaker is stopped at various time intervals (for example, every 2 minutes during the early stages of adsorption hydrolysis - e.g., the first 20 minutes - and every 10 minutes towards the end of the experiment)
• the CPG:sAAPF-pNA is allowed to settle and the solution is sampled
• enzyme adsorption can be determined by measuring solution depletion The difference between the initial enzyme variant concentration and the concentration measured at each individual time point gives the amount of enzyme variant adsorbed
• the amount of pNA hydrolyzed from the surface is measured by taking an absorbance reading on an aliquot of the sample at 410 nm
• the total amount of pNA hydrolyzed is calculated by adding the amount sampled and the amount remaining in the flask This value is corrected by subtracting the amount of pNA that is hydrolyzed by Tris buffer at pH 8.6 when no enzyme is present. This base-hydrolysis ranges from 7-29% of the total hydrolysis depending on the efficiency of the enzyme
• the rates of hydrolysis of the soluble substrate sAAPF-pNA are monitored by measuring the adsorbance increase as a function of time at 410 nm on a DU-70 spectrophotometer.
• the enzyme concentration is held constant and is prepared to be in the range of 6-10 nanomolar while the substrate concentration is varied from 90-700 ⁇ M sAAPF-pNA for each kinetic determination.
• An adsorbance data point is taken each second over a period of 900 seconds and the data are transferred to a LOTUSTM spreadsheet (Lotus Development Corporation, Cambridge, Massachusetts).
• Thermitase variants of the present invention which have decreased adsorption to and increased hydrolysis of surface bound substrates are exemplified in Tables 2-36, below.
• Tables 2-36, below the original amino acid occurring in wild-type is given first, the position number second, and the substituted amino acid third.
• Tyr218Asp 4- Leu221Asn Thr212Pro + Tyr213Ser + Thr215Asp 4- Ser216Asp +
• Ser220Asp 4- Leu221Glu + Thr224Ser Trp208Met 4- Tyr210Thr + Thr212Asn -J- Ala219Gln +

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