SUBTILISIN BPN' VARIANTS HAVING DECREASED ADSORPTION AND INCREASED HYDROLYSIS

Abstract: The present invention relates to subtilisin BPN' variants having a modified amino acid sequence of wild-type BPN' amino 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 amino acid sequence comprises different amino acids than that occurring in wild-type subtilisin BPN' (i.e., substitution) at specifically identified positions in one or more of the loop regions whereby the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type subtilisin BPN'. The present invention also relates to the genes encoding such subtilisin BPN' variants. The present invention also relates to compositions comprising such subtilisin BPN' variants for cleaning a variety of surfaces.

Description: Subtilisin BPN' variants having decreased adsorption and Increased hydrolysis TECHNICAL FIELD 0 The present invention relates to novel enzyme variants useful in a variety of cleaning compositions, and the genes encoding such enzyme variants. BACKGROUND Enzymes make up the largest class of naturally occurring proteins. Each class of enzyme generally catalyzes (accelerates a reaction without being s 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 0 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. Unfortunately, the efficacy level of these proteins in their natural, bacterial environment, frequently does not translate into the relatively unnatural wash 5 environment. Specifically, 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 amino acid sequence of the protease determines the characteristics of the protease. A change of the amino acid sequence of the protease may alter 0 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 amino acid sequence. Several approaches have been taken to alter the wild-type amino acid sequence of proteases in an attempt to improve their properties, with the goal of increasing the efficacy of the protease in the wash 5 environment. These approaches include altering the amino acid sequence to enhance thermal stability and to improve oxidation stability under quite diverse conditions. Despite the variety of approaches described in the art, there is a continuing need for new effective variants of proteases useful for cleaning a variety of surfaces. Objects of the Present Invention It is an object of the present invention to provide subtilisin enzyme variants having improved hydrolysis versus the wild-type of the enzyme. It is also an object of the present invention to provide cleaning compositions comprising these subtilisin enzyme variants. SUMMARY The present invention relates to subtilisin BPN' variants having a modified amino acid sequence of wild-type BPN' amino 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 amino acid sequence comprises different amino acids than that occurring in wild-type subtilisin BPN' (i.e., substitution) at specifically identified positions in one or more of the loop regions whereby the BPN * variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type subtilisin BPN'. The present invention also relates to the genes encoding such subtilisin BPN" variants. The present invention also relates to compositions comprising such subtilisin BPN' variants for cleaning a variety of surfaces. DESCRIPTION I. Subtilisin Variants This invention pertains to subtilisin enzymes, in particular BPN', 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, "BPN 1 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 the mutant genes encoding for such BPN' variants. The 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 serine protease. A serine protease is distinguished by the fact that there is an essential serine residue at the active site. The observation that an enzyme's rate of hydrolysis of soluble substrates increases with enzyme concentration is well documented. It would therefore seem plausible that for surface bound substrates, such as is encountered in many cleaning applications, the rate of hydrolysis would increase with increasing surface concentration. This has been shown to be the case. (Brode, P.F. Ill and D. S. Rauch, LANG UIR, "Subtilisin BPN': Activity on an Immobilized Substrate", Vol. 8, pp. 1325-1329 (1992)). In fact, a linear dependence of rate upon surface concentration was found for insoluble substrates when the surface concentration of the enzyme was varied. (Rubingh, D. N. and M. D. Bauer, "Catalysis of Hydrolysis by Proteases at the Protein-Solution Interface," in POLYMER SOLUTIONS, BLENDS AND INTERFACES, Ed. by I. Noda and D. N. Rubingh, Elsevier, p. 464 (1992)). Surprisingly, when seeking to apply this principle in the search for variant proteases which give better cleaning performance, we did not find that enzymes which adsorb more give better performance. In fact, we surprisingly determined the opposite to be the case: decreased adsorption by an enzyme to a substrate resulted in increased hydrolysis of the substrate (i.e., better cleaning performance). While not wishing to be bound by theory, it is believed that improved performance, when comparing one variant to another, is a result of the fact that enzymes which adsorb less are also less tightly bound and therefore more highly mobile on the surface from which the insoluble protein substrate is to be removed. At comparable enzyme solution concentrations, this increased mobility is sufficient to outweigh any advantage that is conferred by delivering a higher concentration of enzyme to the surface. The mutations described herein are designed to change (i.e., decrease) the adsorption of the enzyme to surface-bound soils. In BPN', certain amino acids form exterior loops on the enzyme molecule. For purposes of discussion, these loops shall be referred to as first, second, third, fourth and fifth loop regions. Specifically, positions 59-66 form the first loop region; positions 95- 107 form the second loop region; positions 126-133 form the third loop region; positions 154-167 form the fourth loop region; positions 187-191 form the fifth loop region; and positioins 199-220 form the sixth loop region (position numbering analagous to positions in the amino acid sequence for wild-type subtilisin BPN' (SEQ ID NO: 1 )). It believed that these 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 BPN 1 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 BPN' molecule gives them a role in the catalytically productive adsorption of the enzyme to surface-bound substrates (peptides/protein soils). As used herein, "variant" means an enzyme having an amino acid sequence which differs from that of wild-type. As used herein, "mutant BPN' gene" means a gene coding for a BPN' variant. As used herein, "wild-type subtilisin BPN'" refers to a subtilisin enzyme represented by SEQ ID NO:1. The amino acid sequence for subtilisin BPN' is further described by Wells, J. A., E. Ferrari, D. J. Henner, D. A. Estell and E. Y. Chen, NUCLEIC ACIDS RESEARCH, Vol. II, 7911-7925 (1983), incorporated herein by reference. As used herein, the term "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 59-66, 95-107, 126-133, 154-167, 187- 191 and 199-220. As used herein, "more hydrophilic amino acid" refers to any other amino acid having greater hydrophilicity than a subject amino acid with reference to the hydrophilicity table below. The following hydrophilicity table (Table 1 ) 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, and the remaining amino acids are neutral. In a preferred embodiment of the present invention, the substituting amino acid is either neutral or negatively charged, more preferably negatively charged (i.e., Glu or Asp). Therefore, for example, 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. Likewise, 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. In one embodiment of the present invention, the BPN' variant has a modified amino acid sequence of wild-type amino acid sequence, wherein the modified amino acid sequence comprises a substitution at one or more positions in one or more of the first, second, third, fourth or fifth loop regions; whereby the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type subtilisin BPN'. In another embodiment of the present invention, the BPN' variant further comprises one or more substitutions to the sixth loop region. In a preferred embodiment of the present invention, the substituting amino 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 hydrophylic, preferably more hydrophylic, than the amino acid at the subject position in the wild-type amino acid sequence. A. Substitutions in the First Loop Region When a substitution occurs in the first loop region, the substitution occurs at one or more of positions 59, 60, 61 , 62, 63, 65 or 66. When a substitution occurs at position 59, the substituting amino acid is Asn, Asp, Glu or Ser. When a substitution occurs at position 60, the substituting amino acid is Glu. When a substitution occurs at position 61 , the substituting amino acid is Asp, Gin, Glu or Ser. When a substitution occurs at position 62, the substituting amino acid is Asp, Gin, Glu or Ser. When a substitution occurs at position 63, the substituting amino acid is Asp or Glu. When a substitution occurs at position 65, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser. When a substitution occurs at position 66, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser. B. Substitutions in the Second Loop Region When a substitution occurs in the second loop region, the substitution occurs at one or more of positions 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106 or 107. When a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Met, Pro, Ser or Thr. When a substitution occurs at position 96, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Met, Pro, Ser, Thr or Val. When a substitution occurs at position 97, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser. When a substitution occurs at position 98, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr. When a substitution occurs at position 99, the substituting amino acid is Glu. When a substitution occurs at position 100, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser. When a substitution occurs at position 101 , the substituting amino acid is Asp or Glu. When a substitution occurs at position 102, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser. When a substitution occurs at position 103, the substituting amino acid is Asn, Asp, Glu or Ser. When a substitution occurs at position 104, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr or Val. When a substitution occurs at position 105, the substituting amino acid is Asp or Glu. When a substitution occurs at position 106, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr, Tyr or Val. When a substitution occurs at position 107, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val. C. Substitutions in the Third Loop Region When a substitution occurs in the third loop region, the substitution occurs at one or more of positions 126, 127, 128, 129, 130, 131 , 132 or 133. When a substitution occurs at position 126, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Met, Pro, Ser, Thr or Val. When a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser. When a substitution occurs at position 128, the substituting amino acid is Asn, Asp, Gin, Glu, Gly or Ser. When a substitution occurs at position 129, the substituting amino acid is Asn, Asp, Gin, Glu, Gly or Ser. When a substitution occurs at position 130, the substituting amino acid is Asp or Glu. When a substitution occurs at position 131 , the substituting amino acid is Asn, Asp, Gin, Glu, Gly or Ser. When a substitution occurs at position 132, the substituting amino acid is Asp or Glu. When a substitution occurs at position 133, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr. D. Substitutions in the Fourth LOOP Region When a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166 or 167. When a substitution occurs at position 154, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser. When a substitution occurs at position 155, the substituting amino acid is Asp, Gin, Glu or Ser. When a substitution occurs at position 156, the substituting amino acid is Asp. When a substitution occurs at position 157, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser. When a substitution occurs at position 158, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser. When a substitution occurs at position 159, the substituting amino acid is Asp or Glu. When a substitution occurs at position 160, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser. When a substitution occurs at position 161 , the substituting amino acid is Asp or Glu. When a substitution occurs at position 162, the substituting amino acid is Asp or Glu. When a substitution occurs at position 163, the substituting amino acid is Asp or Glu. When a substitution occurs at position 164, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser. When a substitution occurs at position 165, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Met, Pro, Ser or Thr. When a substitution occurs at position 166, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser. When a substitution occurs at position 167, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr or Val. E. Substitutions in the Fifth LOOP Region When a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 187, 188, 189, 190 or 191. When a substitution occurs at position 187, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser and Thr. When a substitution occurs at position 188, the substituting amino acid is Asp or Glu. When a substitution occurs at position 189, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr, Tyr or Val. When a substitution occurs at position 190, the substituting amino acid is Asp or Glu. When a substitution occurs at position 191, the substituting amino acid is Asp or Glu. F. Substitutions in the Sixth Loop Region When a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 21 1 , 212, 213, 214, 215, 216, 217, 218, 219 or 220. When a substitution occurs at position 199, the substituting amino acid for position 199 is Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 200, the substituting amino acid for position 200 is His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 201 , the substituting amino acid for position 201 is Gly, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 202, the substituting amino acid for position 202 is Pro, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 203, the substituting amino acid for position 203 is Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 204, the substituting amino acid for position 204 is Asp, or Glu. When a substitution occurs at position 205, the substituting amino acid for position 205 is Leu, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 206, the substituting amino acid for position 206 is Pro, Asn, Ser, Asp, or Glu. When a substitution occurs at position 207, the substituting amino acid for position 207 is Asp or Glu. When a substitution occurs at position 208, the substituting amino acid for position 208 is Pro, Gly, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 209, the substituting amino acid for position 209 is He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 210, the substituting amino acid for position 210 is Ala, Gly, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 211 , the substituting amino acid for position 211 is Ala, Pro, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 212, the substituting amino acid for position 212 is Gin, Ser, Asp or Glu. When a substitution occurs at position 213, the substituting amino acid for position 213 is Trp, Phe, Tyr, Leu, lie, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 214, the substituting amino acid for position 214 is Phe, Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 215, the substituting amino acid for position 215 is Thr, Pro, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 216, the substituting amino acid for position 216 is His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 217, the substituting amino acid for position 217 is Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 218, the substituting amino acid for position 218 is Gin, Ser, Asp or Glu. When a substitution occurs at position 219, the substituting amino acid for position 219 is Pro, Gin, Asn, Ser, Asp or Glu. When a substitution occurs at position 220, the substituting amino acid for position 220 is Pro, Gly, Gin, Asn, Ser Asp or Glu. G. Preparation of enzyme variants Example 1 Mutant BPN' Genes A phagemid (pSS-5) containing the wild type subtilisin BPN' gene (Mitchinson, C. and J. A. Wells, (1989), "Protein Engineering of Disulfide Bonds in Subtilisin BPN', BIOCHEMISTRY, Vol. 28, pp. 4807-4815) is transformed into Escherichia coli ung-strain CJ236 and a single stranded uracil-containing DNA template is produced using the VCSM13 helper phage (Kunkel, T.A., J.D. Roberts and R.A. Zakour, "Rapid and efficient site-specific mutagenesis without phenotypic selection", METHODS IN ENZYMOLOGY, Vol. 154, pp. 367-382, (1987); as modified by Yuckenberg, P.D., F. Witney, J. Geisselsoder and J. McClary, "Site-directed in vitro mutagenesis using uracil-containing DNA and phagemid vectors", DIRECTED MUTAGENESIS - A PRACTICAL APPROACH, ed. M.J. McPherson, pp. 27-48, (1991); both of which are incorporated herein by reference). A single primer site-directed mutagenesis modification of the method of Zoller and Smith (Zoller, M.J., and M. Smith, "Oligonucleotide-directed mutagenesis using M13- derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA", NUCLEIC ACIDS RESEARCH, Vol. 10, pp. 6487- 6500, (1982), incorporated herein by reference) is used to produce all mutants (basically as presented by Yuckenberg, et al., 1991 , above). 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. Henner, (1984), "Cloning of the Neutral Protease Gene of Bacillus subtillis and the Use of the Cloned Gene to Create an In Vitro-der ' weό Deletion Mutation", JOURNAL OF BACTERIOLOGY, Vol. 160, pp. 15-21 ). For some of the mutants a modified pSS-5 with a frameshift-stop codon mutation at amino acid 217 is used to produce the uracil template. Oligonucleotides are designed to restore the proper reading frame at position 217 and also encoded for random substitutions at positions 59, 60, 61, 62, 63, 64, 65, 66; 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107; 126, 127, 128, 129, 130, 131 , 132, 133; 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167; 187, 188, 189, 190, 191 ; 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219 and 220 (equimolar and/or variable mixtures of all four nucleotides for all three bases at these codons). Mutations that correct for the frameshift-stop and produce a functional enzyme are identified by their ability to digest casein. The random substitutions are determined by DNA sequencing. Example 2 Fermentation The Bacillus subtilis cells (BE2036) 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 Agoo °f about 60 and harvested. Example 3 Purification The fermentation broth is taken through the following steps to obtain pure enzyme. The broth is cleared of Bacillus subtilis cells by centrifugation, 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-(Λ/- morpholino)ethanesulfonic acid). The enzyme is further purified by loading it onto either a cation exchange chromatography column or an affinity adsorption chromatography column and eluting it from the column with a NaCI or a propylene glycol gradient (see Scopes, R. K., PROTEIN PURIFICATION PRINCIPLES AND PRACTICE, Springer-Verlag, New York (1984), incorporated herein by reference). The pNA assay (DelMar, E.G., C. Largman, J.W. Brodrick 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- nitroaniline is released as the enzyme hydrolyzes the soluble synthetic substrate, succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (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. To avoid autolysis of the enzyme during storage, an equal weight of propylene glycol is added to the pooled fractions obtained from the chromatography column. Upon completion of the purification procedure the purity of the stock enzyme solution is checked with SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the absolute enzyme concentration is determined via an active site titration method using trypsin inhibitor type ll-T: turkey egg white purchased from Sigma Chemical Company (St. Louis, Missouri). The measured conversion factors will show which changes made in the enzyme molecule at the various positions result in the enzyme variant having increased activity over the wild-type, against the soluble substrate pNA. In preparation for use, 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 Example 4 Model Surface Preparation Aminopropyl controlled pore glass (CPG) purchased from CPG Inc. (Fairfield, New Jersey) is used as a support for covalently attaching the sAAPF- pNA substrate purchased from Bachem, Inc. (Torrence, California). The reaction is carried out in dimethyl sulfoxide and (1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide hydrochloride) (EDC) is used as a coupling agent. Upon completion (monitored by pNA assay), the excess solvent is removed, and the CPG:sAAPF-pNA is rinsed with dimethyl sulfoxide (DMSO) and doubly-distilled water. This is followed by oven drying with a N2 purge at about 70 C C. The reaction scheme and preparation of the immobilized substrate are conducted as described by Brode, P.F. Ill, and D.S. Rauch, "Subtilisin BPN': Activity on an Immobilized Substrate," LANGMUIR, Vol. 8, p. 1325-1329, (1992), incorporated herein by reference. The CPG surface will have 62,000 ± 7,000 pNA molecules/μm 2 . The surface area will remain unchanged from the value of 50.0m 2 /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). Example 5 Surface Hydrolysis Assay 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. Both the experimental procedure and the calculation of the adsorption and hydrolysis are conducted as described by Brode et al., 1992, above. All enzymes are monitored for stability against autolysis and should show no appreciable autolytic loss over the time course of this experiment. Therefore, 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. Example 6 Soluble Substrate Kinetic Analysis 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 μM 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 LOTUS™ spreadsheet (Lotus Development Corporation, Cambridge, Massachusetts). Analysis for kinetic parameters is conducted by the standard Lineweaver Burk analysis in which the data in the initial part of the run (generally the first minute) are fit to a linear regression curve to give v 0 . The v 0 and s 0 data are plotted in the standard inverse fashion to give KM and k ca t- I. Example BPN' variants BPN' variants of the present invention which have decreased adsorption to and increased hydrolysis of surface bound substrates are exemplified in Tables 2-25, below. In describing the specific mutations, the original amino acid occurring in wild-type is given first, the position number second, and the substituted amino acid third. TABLE 2 Loop 1 - Single Mutation Variants Gln59Asn Gln59Asp Gln59Glu Gln59Ser AspβOGlu Asn61Asp AsnβlGln AsnβlGlu AsnβlSer Asn62Asp Asn62Gln Asn62Glu Asn62Ser Ser63Asp Ser63Glu Gly65Asn Gly65Asp Gly65Gln GlyβδGlu Gly65Pro Glyβ5Ser ThrββAsn ThrββAsp ThrββGln ThrββGlu ThrββGly ThrββPro ThrββSer TABLE 3 Loop 1 - Double Mutation Variants Gln59Ser + Asn62Glu AspβOGlu + AsnβlSer AsnβlGlu + Asn62Ser Gln59Ser + GlyβδGln AsnβlGln + Gly65Asn AsnβlSer + Asn62Asp 10 Gln59Glu + AsnβlGln AspβOGlu + Gly65Gln Gln59Asp + Gly65Pro Asn61Asp + Gly65Asn Gln59Ser + Asn62Asp 15 Gln59Asn + GlyβδGln Asn62Asp + ThrββGly Gln59Asn + Asn62Glu AsnβlSer + Ser63Glu Gln59Ser + AspβOGlu 20 Asp60Glu + ThrββGln AsnβlGlu + ThrββGly Asp60Glu + Asn62Gln Asn62Gln + Gly65Pro Asn61Ser + ThrββSer 25 Asp60Glu + Gly65Pro Ser63Glu + GlyβδPro Asp60Glu + ThrββSer Gln59Ser + AsnβlGlu Asn62Asp + Gly65Gln 30 AsnβlGln + Ser63Asp Gln59Asp + Gly65Asn Ser63Asp + ThrββPro Ser63Glu + ThrββAsn Asn62Glu + ThrββAsn 35 Asn61Asp + Gly65Ser Gly65Pro + ThrββSer Gln59Ser + Asn62Ser Asp60Glu + Gly65Ser Ser63Asp + Gly65Ser 40 AsnβlGln + Ser63Glu AsnβlAsp + Asn62Ser Gln59Glu + Gly65Pro Gln59Ser + AsnβlAsp Gln59Asp + Asn62Ser 45 Gln59Asn + GlyβδSer Ser63Glu + ThrββSer AsnβlSer + Ser63Asp Asn62Ser + Gly65Pro TABLE 4 Leu96Met Leu96Pro Leu96Ser Leu96Thr 5 Leu96Val Gly97Asn Gly97Asp Gly97Gln Gly97Glu 10 Gly97Pro Gly97Ser Ala98Asn Ala98Asp Ala98Gln 15 Ala98Glu Ala98Gly Ala98His Ala98Pro Ala98Ser 20 Ala98Thr Asp99Glu GlylOOAsn GlylOOAsp GlylOOGln 25 GlylOOGlu GlylOOPro GlylOOSer SerlOlAsp SerlOlGlu 30 Glyl02Asn Glyl02Asp Glyl02Gln Glyl02Glu Glyl02Pro 35 Glyl02Ser Glnl03Asn Glnl03Asp Glnl03Glu Glnl03Ser 40 Tyrl04Ala Tyrl04Asn Tyrl04Asp Tyrl04Cys Tyrl04Gln 45 Tyrl04Glu Tyrl04Gly Tyrl04His Tyrl04Ile Tyrl04Leu 50 Tyrl04Met Tyrl04Pro Tyrl04Ser Tyrl04Thr Tyrl04Val 5 SerlOδAsp SerlOSGlu TrplOβAla TrplOβAsn TrplOβAsp 10 TrplOβCys TrplOβGln TrplOβGlu TrplO β Gly TrplOβHis 15 TrplOβlle TrplOβLeu TrplOβMet TrplOβPhe TrplOβPro 20 TrplOβSer TrplOβThr TrplOβTyr TrplOβVal Ilel07Ala 25 Ilel07Asn Ilel07Asp Ilel07Cys Ilel07Gln Ilel07Glu 30 Ilel07Gly Ilel07His Ilel07Leu Ilel07Met Ilel07Pro 35 Ilel07Ser Ilel07Thr Ilel07Val TABLE7 40 Loop 2 - Double Mutation Variants 45 Leu 96Asn + Asp 99Glu Glyl02Gln + TrplOβAsp Tyrl04Leu + TrplOβGlu Tyrl04Pro + Ilel07Asp Gly 97Ser + SerlOlAsp GlylOOPro + SerlOlGlu Val 9δAsn + Ala 98Asp Val 9δMet + Ilel07Gly Asp 99Glu + TrplOβCys 10 GlylOOAsn + TrplOβThr Glnl03Ser + TrplOβPro Glyl02Asp + Glnl03Ser Glyl02Ser + TrplOβGln SerlOlAsp + Glyl02Pro 15 Leu 96Cys + TrplOβAsp Asp 99Glu + Glyl02Ser Glyl02Asp + TrplOβVal Gly 97Ser + TrplOβPhe Glnl03Asp + Tyrl04Thr 20 Ala 98His + GlylOOGln SerlOSGlu + TrplOβLeu Leu 96His + Tyrl04Thr Gly 97Pro + SerlOlGlu Val 9δThr + TrplOβlle 25 GlylOOAsp + Tyrl04Ile Val 9δPro + Glnl03Asn Glnl03Asn + TrplOβlle Ala 98His + Glyl02Pro TrplOβAsn + Ilel07His 30 Val 9δGln + Leu 96Asp Gly 97Asp + Ala 98Gln GlylOOSer + SerlOlGlu Val 9 Asp + Tyrl04Gly Tyrl04Ala + SerlOδAsp 35 GlylOOPro + SerlOδGlu Leu 96Cys + Tyrl04Leu Val 9δGly + GlylOOSer Glyl02Gln + Tyrl04Ser Ala 98Gly + TrplOβPhe 40 GlylOOAsp + TrplOβPhe Val 9δGlu + Ala 98Gln SerlOlGlu + Tyrl04Asn Leu 96Val + SerlOlAsp Glyl02Glu + Glnl03Asn 45 Glyl02Glu + TrplOβGly Ala 98Gln + GlylOOAsp GlylOOGln + Glnl03Ser Gly 97Glu + Tyrl04Leu SerlOlAsp + Glyl02Ser 50 Ala 98His + SerlOlAsp Gly 97Asp + Glnl 03Asn TABLE 8 Loop 2 - Triple Mutation Variants 10 15 20 25 30 35 40 45 Glyl27Glu Glyl27Pro Glyl27Ser Glyl28Asn 5 Glyl28Asp Glyl28Gln Glyl28Glu Glyl28Pro Glyl28Ser 10 Prol29Asn Prol29Asp Prol29Gln Prol29Glu Prol29Gly 15 Prol29Ser Serl30Asp Serl30Glu Glyl31Asn Glyl31Asp 20 Glyl31Gln Glyl31Glu Glyl31Pro Glyl31Ser Serl32Asp 25 Serl32Glu Alal33Asn Alal33Asp Alal33Gln Alal33Glu 30 Alal33Gly Alal33His Alal33Pro Alal33Ser Alal33Thr 35 TABLE 11 Loop 3 - Double Mutation Variants Leul26Gln + Serl30Glu Glyl31Gln + Alal33Asn 40 Prol29Asp + Glyl31Gln Glyl28Ser + Serl30Glu Leul26Pro + Alal33Gly Glyl27Asp + Alal33Gly Leul26Asp + Prol29Gln 45 Glyl31Asn + Alal33Gln Glyl27Pro + Glyl31Glu Glyl28Asn + Glyl31Asp Prol29Gln + Serl30Glu Glyl28Pro + Serl30Asp Glyl28Gln + Prol29Ser Glyl28Asn + Prol29Gly Leul26Val + Serl30Asp Leul26Val + Prol29Ser Leul26Cys + Prol29Glu Glyl27Asp + Alal33Thr Glyl28Pro + Prol29Glu Glyl27Ser + Glyl31Asp Leul26His + Prol29Asp 10 Glyl31Pro + Alal33Glu Glyl27Ser + Glyl28Ser Prol29Asn + Glyl31Glu Leul26Val + Prol29Asp Prol29Gly + Alal33Asp 15 Leul26Val + Serl30Glu Prol29Glu + Alal33Pro Prol29Gly + Serl30Asp Leul26His + Glyl28Glu Glyl28Asn + Serl32Glu 20 Glyl27Pro + Serl32Asp Glyl27Gln + Prol29Gln Glyl28Pro + Prol29Asp Glyl28Asn + Serl30Glu Leul26Cys + Prol29Asn 25 Prol29Asn + Serl32Glu Leul26Ser + Serl32Asp Glyl28Glu + Glyl31Ser Prol29Asn + Serl30Asp Leul26Ser + Serl32Glu 30 Prol29Gln + Glyl31Pro Glyl27Asp + Glyl28Gln Glyl28Gln + Prol29Glu Glyl27Pro + Prol29Gly Prol29Gln + Alal33Gln 35 Leul26Val + Glyl28Asp Glyl28Ser + Serl32Glu Leul26Asn + Prol29Gly Leul26Ile + Alal33Gly Glyl28Ser + Glyl31Gln 40 Glyl27Ser + Serl30Asp Leul26Cys + Serl32Asp Glyl27Pro + Serl30Glu Leul26His + Alal33Asp Glyl31Ser + Alal33Glu 45 Glyl31Pro + Alal33Gln Glyl31Asp + Alal33Ser Leul26Asp + Alal33Asn Leul26Glu + Prol29Gln TABLE 12 Loop 3 - Triple Mutation Variants Leul26His + Prol29Glu + Alal33Asn Leul26Asp + Glyl28Ser + Glyl31Gln Prol29Asn + Glyl31Ser + Serl32Glu Glyl28Pro + Prol29Asn + Serl30Glu Glyl28Gln + Serl30Glu + Alal33Ser Glyl3lGln + Serl32Glu + Alal33Gln 10 Glyl28Asp + Glyl31Ser + Alal33Asn Glyl3lSer + Serl32Asp + Alal33Pro Prol29Ser + Glyl31Gln + Alal33Glu Glyl28Asn Serl30Glu + Glyl31Gln Leul26Gly Glyl27Gln + Glyl31Pro 15 Leul26Pro Glyl27Glu + Glyl28Pro Leul26Ser Prol29Ser + Serl32Asp Glyl28Ser Serl32Glu + Alal33Asn Leul26Val Serl32Glu + Alal33Gln Prol29Gly Serl30Glu + Glyl31Pro 20 Leul26Thr Glyl27Pro + Alal33Asn Leul26His Serl30Asp + Alal33Pro Leul26Cys Glyl27Ser + Prol29Ser Leul26Gly Serl32Asp + Alal33Ser Glyl28Gln + Prol29Gln + Glyl31Asn 25 Glyl28Asp + Glyl31Asn + Alal33His Leul26Cys Serl30Glu + Alal33Gly Glyl27Ser Serl30Asp + Alal33Gly Leul26His Prol29Asn + Serl30Asp Leul26Asn Glyl31Asp + Alal33Gln 30 Leul26Met Glyl28Asn + Serl32Asp Leul26Glu Glyl27Gln + Alal33His Leul26Met Serl32Asp + Alal33His Serl30Glu Glyl31Gln + Alal33Gln Glyl27Pro Glyl28Ser + Alal33Ser 35 Le l26Ala Prol29Gly + Serl32Glu Glyl31Asn Serl32Asp + Alal33Asn Leul26Val Glyl31Asp + Alal33Ser Leul26Ser Glyl27Asn + Alal33Gln Prol29Gln Serl30Glu + Alal33His 40 Leul26 et Glyl27Ser + Serl30Asp Leul26Cys Prol29Asn + Glyl31Asp Prol29Ser Serl30Asp + Alal33Asn Leul26Ser Prol29Gly + Serl32Glu Glyl27Ser Prol29Gln + Serl32Asp 45 Glyl27Pro + Glyl28Asn + Prol29Gln Leul26His + Serl32Asp + Alal33Asn Glyl28Pro + Prol29Glu + Alal33Thr Prol29Ser + Glyl31Glu + Alal33Pro Leul26His + Glyl28Pro + Prol29Gln Leul26Met + Glyl27Asp + Glyl28Asp Glyl28Pro + Glyl31Glu + Serl32Asp Glyl31Asp + Serl32Glu + Alal33Pro Glyl28Glu + Prol29Glu + Alal33Asn Prol29Ser + Serl32Glu + Alal33Glu Leul26Asn + Serl30Glu + Glyl31Asp Prol29Asn + Serl30Glu + Glyl31Asp Leul26His + Serl30Glu + Glyl31Glu Prol29Glu + Serl30Asp + Glyl31Asn Glyl27Ser + Prol29Asp + Serl30Asp Serl30Asp + Glyl31Asp + Serl32Asp Glyl28Asp + Serl30Glu + Glyl31Asn Leul26Met + Glyl28Glu + Serl30Asp Glyl28Asp + Prol29Asn + Serl30Glu TABLE 13 Loop 3 - Quadruple Mutation Variants Leul26Ser + Prol29Asn + Serl30Asp Alal 33His Leul26Met + Prol29Ser + Serl32Glu Alal33Asn Glyl27Ser + Glyl31Gln + Serl32Glu Alal33Gln Leul26Asn + Glyl27Pro + Glyl28Glu Prol29Gly Leul26Asn + Prol29Gly + Glyl31Asp Alal33Gly Leul26Gly + Prol29Gly + Serl32Glu Alal33Pro Leul26Gly + Glyl27Asp + Prol29Gly Glyl31Pro Glyl27Asn + Prol29Gln + Glyl31Asp Alal33Gly Leul26Pro + Glyl27Ser + Glyl28Gln Serl30G1U Leul26Ala + Glyl27Gln + Prol29Asn Serl30Glu Leul26Asn + Glyl27Ser + Serl30Glu Alal33Thr Glyl28Gln + Prol29Gln + Serl30Asp Glyl31Ser Leul26His + Glyl28Ser + Glyl31Ser Serl32Asp Leul26Gln + Prol29Ser + Serl30Asp Alal33His Leul26Val + Glyl28Pro + Prol29Asn Alal33Asp Leul26Val + Prol29Gly + Serl30Glu Alal33Thr Leul26Thr + Glyl27Pro + Serl32Glu Alal33Thr Glyl28Asp + Prol29Gly + Glyl31Pro Alal33Ser Leul26Asn + Glyl28Glu + Prol29Gln Glyl31Pro Leul26Pro + Glyl27Pro + Prol29Ser Serl30Asp Glyl27Pro + Glyl28Gln + Glyl31Glu Serl32Glu Leul26Ile + Glyl27Gln + Glyl31Asp Serl32Glu Leul26Val + Glyl31Asp + Serl32Asp Alal33Pro Glyl28Asp + Prol29Asp + Glyl31Asn Alal33Pro Prol29Asn + Glyl31Ser + Serl32Asp Alal33Asp Leul26Gln + Glyl31Pro + Serl32Asp Alal33Asp Glyl27Pro + Serl30Glu + Glyl31Glu Alal33His Leul26Gln + Prol29Gln + Serl30Asp Glyl31Glu Glyl27Ser + Serl30Asp + Glyl31Glu Alal33Gln Leul26Ser + Glyl27Pro + Prol29Glu Serl30Glu Serl30Glu + Glyl31Glu + Serl32Glu Alal33Ser Glyl27Gln + Serl30Glu + Glyl31Asp Serl32Asp ThrlδδAsp ThrlδδGln ThrlδβGlu ThrlδδGly 5 ThrlδδPro ThrlδβSer Serlδ9Asp Serlδ9Glu GlylβOAsn 10 GlylβOAsp GlylβOGln GlylβOGlu GlylβOPro GlylβOSer 15 SerlβlAsp SerlβlGlu Serl62Asp Serl62Glu Serl63Asp 20 Serl63Glu Thrl64Asn Thrl64Asp Thrl64Gln Thrl64Glu 25 Thrl64Gly Thrl64Pro Thrl64Ser VallβδAla VallβSAsn 30 VallβδAsp VallβδCys VallβδGln VallβδGlu VallβδGly 35 VallβδHis VallβδMet VallβδPro VallβδSer VallβδThr 40 GlylββAsn GlylββAsp GlylββGln GlylββGlu GlylββPro 45 GlylββSer Tyrl67Ala Tyrl67Asn Tyrl67Asp Tyrl67Cys 50 Tyrl67Gln Tyrl67Glu Tyrl67Gly Tyrl67His Tyrl67Ile Tyrl67Leu Tyrl67Met Tyrl67Pro Tyrl67Ser Tyrl67Thr 10 Tyrl67Val TABLE 15 Loop 4 - Double Mutation Variants AsnlδδSer + GlulδβAsp 15 Glylδ4Ser + Tyrl67Gln Glylδ4Glu + VailβδAla AsnlδδGlu + Thrl64Pro Glylδ7Pro + Serlδ9Asp Glylδ4Ser + Serl61Asp 20 SerlβlGlu + VailβδPro Glylδ4Gln + SerlβlGlu Asnl δAsp + ThrlδδPro Thrl64Asn + GlylββGln AsnlδδGlu + Tyrl67His 25 GlulδβAsp + ThrlδδGly Glylδ4Pro + Glylδ7Glu AsnlδδSer + Tyrl67Asp ThrlδδPro + GlylββAsp Thrl64Gln + Tyrl67Glu 30 GlylS7Gln + ThrlδδGlu ThrlδδAsn + Serl62Asp GlylS4Asn + Tyrl67Glu Glylδ7Gln + Serl61Asp Thrl64Asp + Tyrl67Ala 35 GlylβOAsp + VailβδHis GlylS4Glu + Glylδ7Ser GlulδβAsp + Tyrl67Ile AsnlδδSer + ThrlδδAsp GlylS7Gln + Thrl64Pro 40 Thrl64Ser + Tyrl67Ile Serlδ9Glu + Tyrl67Thr Thrl64Glu + VailβδGln ThrlδδGly + GlylβOSer SerlβlAsp + GlylββPro 45 Glylδ4Glu + GlylββSer GlylβOAsp + VailβδAsn Serl62Glu + VailβδGln Glylδ7Asn + Serlδ9Glu SerlβlAsp + VailβδAsn AsnlδδAsp + VallδδPro Glul56Asp + Glyl66Ser Glylδ4Pro + Serlδ9Asp Glylδ4Ser + Tyrl67Cys Glyl60Pro + Thrl64Asp SerlβlGlu + Vall β δGly Serl62Glu + Tyrl67Asn Glylδ4Asn + GlylββGlu SerlβlGlu + Tyrl67Ala 10 GlylβOGln + VallβδPro Glylδ4Glu + VallβδGly GlylβOSer + Serl63Asp Glylδ7Glu + ThrlδδAsn GlylβOAsp + VallβδPro 15 GlylβOAsn + Serl62Asp Thrl64Gln + GlylββGln AsnlδδSer + ThrlδδGln SerlβlGlu + Tyrl67Gly Serl62Asp + GlylββSer 20 Glylδ4Glu + ThrlδδGly Glylδ4Ser + ThrlδδSer Glylδ7Asp + GlylβOPro Serl63Glu + VallβδHis Glylδ4Pro + Glyl66Asp 25 TABLE 16 Loop 4 - Triple Mutation Variants Glylδ4Gln + AsnlδδSer Glulδ6Asp Glylδ4Ser + GlylβOAsp Tyrl67Gln 30 AsnlSSGlu + Glylδ7Ser Thrl64Pro Glylδ7Asn + Serlδ9Asp GlylβOSer Glulδ6Asp + GlylβOSer Vail65Thr GlylδOPro + Serl62Glu Thrl64Asn Glylδ4Ser + GlulδβAsp Thrl58Gln 35 Glyl60Asn + Serl62Glu GlylββSer GlylδOSer + VailβδGly GlylββGln ThrlδδGln + Serl62Asp Tyrl67Val Glylδ7Gln + Serl62Glu Tyrl67Leu Serl62Glu + Thrl64Gln Vail65Cys 40 Glylδ7Ser + VailβδMet Glyl66Glu Glylδ4Ser + Glulδ6Asp Glyl66Pro ThrlδδSer + Serl61Asp Thrl64Gly GlulδβAsp + Glylδ7Ser Glyl60Asn Glyl54Gln + AsnlδδAsp Glyl66Ser 45 Serl63Glu + Vail6δThr Tyrl67Pro GlylS7Asp + Thrlδ8Gln Vail65Ser Glylδ7Asn + Serlδ9Asp Glyl66Ser GlylβOGln + Serl63Glu Vail65Met Glylδ4Asn + AsnlδδAsp Glyl57Pro GlulδβAsp + ThrlδδPro Thrl64Gln VailβδPro AsnlδδGln + GlulδβAsp Thrl64Gly VailβδThr ThrlδδGly + GlylβOSer Serl63Asp Tyrl67Asn Serlδ9Asp + GlylβOGln GlylββSer Tyrl67Pro Glylδ4Pro + Thrl64Gln VallβδGly GlylββAsp Glylδ4Asn + GlylβOPro SerlβlGlu GlylββPro AsnlδδSer + Glylδ7Asn Thrl64Gln Tyrl67Asp Glylδ7Asn + ThrlδδAsn Serl63Glu VailβδGln GlylβOGlu + SerlβlAsp VallβδMet Tyrl67Pro AsnlδδGlu + GlulδβAsp ThrlδδGln GlylββPro AsnlδδAsp + GlulδβAsp VallβδAsn Glyl66Asn AsnlδδAsp + GlulδβAsp GlylβOSer Thrl64Asn Glylδ4Ser + ThrlδδGln Serl62Glu Serl63Glu Glylδ4Asn + AsnlδδGln Serl63Glu Thrl64Glu GlulδβAsp + Glylδ7Glu GlylβOGln Thrl64Gly GlulδβAsp + Glylδ7Glu ThrlδδSer VailβδCys Glylδ4Pro + Glylδ7Pro ThrlδδAsp Serlδ9Asp Glylδ4Ser + Glylδ7Asn ThrlδδGlu Serlδ9Glu Glylδ7Pro + GlylβOPro GlylββAsp Tyrlβ7Glu Glylδ4Asn + SerlβlGlu Serl62Glu Tyrl67Asn Glylδ4Asp + AsnlδδAsp Thrl64Gln GlylββAsn Glylδ4Gln + Serlδ9Glu GlylβOGlu Serl61Asp ThrlδδSer + Serlδ9Asp GlylβOAsp Serl61Asp AsnlδδSer + GlulδβAsp Glyl57Asp ThrlδδGlu Glylδ7Asn + Serlδ9Asp SerlβlGlu Serl62Glu Glylδ4Asn + GlulδβAsp Glyl57Glu Thrl64Glu Glylδ7Gln + GlylβOAsp Serl62Asp Vail6δThr GlylβOGlu + Serl62Asp Thrl64Asn GlylββGln Glylδ4Asp + AsnlδδSer GlulδβAsp Thrl64Ser Glylδ4Asp + GlulδβAsp Glylδ7Glu ThrlδδGly Glylδ4Gln + Glyl57Pro Serlδ9Asp SerlβlAsp Serlδ9Glu + SerlβlAsp GlylββSer Tyrl67His Serlδ9Asp + SerlβlAsp GlylββPro Tyrl67Ser GlulδβAsp + Thrl58Glu Vallβ Ala GlylββGln GlulδβAsp + ThrlδδAsp GlylββPro Tyrl67Ala AsnlδδGln + ThrlδδAsp Thrl64Asp Tyrl67Val Serl63Glu + Thrl64Asp VallβδMet GlylββGlu SerlβlAsp + Serl63Asp VallβδThr Tyrl67His SerlβlAsp + Serl63Glu Thrl64Gln GlylββAsn Glylδ7Pro + Serlδ9Glu SerlβlAsp Serl63Glu Glylδ4Pro + GlulδβAsp Serl63Asp Thrl64Glu AsnlδδAsp + GlulδβAsp ThrlδδAsp Thrl64Asn GlulδβAsp + Serlδ9Asp Thrl6 Asp VailβδAla ThrlδδGln + Serlδ9Asp Serl63Glu VailβδCys Glylδ4Gln + Serlδ9Asp Serl63Asp GlylββPro AsnlδδSer + GlylβOAsp Serl62Glu Thrl64Asp Glyl54Gln + GlylβOAsp Serl62Glu Thrl64Glu Glul56Asp + GlylβOPro VallβδPro GlylββGlu GlylβOGlu + Serl63Asp Thrl64Gly Tyrl67Leu GlylβOGlu + Serl63Glu Thrl64Pro GlylββGln AsnlδδAsp + Thrlδ8Pro + Serl63Glu + Thrl64Asp AsnlδδSer + GlulδβAsp + Serl63Asp + GlylββGlu TABLE 18 Loop 5 - Single Mutation Variants Alal87Asn Alalδ7Asp Alal87Gln Alal87Glu Alalδ7Gly Alalδ7His Alalδ7Pro Alal67Ser Alalδ7Thr SerlδδAsp SerlδδGlu Phelδ9Ala Phel69Asn Phel69Asp Phel89Cys Phelδ9Gln Phelδ9Glu Phelδ9Gly Phel69His Phel89Ile Phel89Leu Phel89Met Phelδ9Pro Phelδ9Ser Phel69Thr Phelδ9Tyr Phelδ9Val Serl90Asp Serl90Glu Serl91Asp Serl91Glu TABLE 19 Loop 5 - Double Mutation Variants Alalδ7Asp + Phel89Gln Alal67Ser + SerlδδAsp SerlδδGlu + Phel69Pro Alal87Asp + Phel69His Alal67Asn + Serl91Glu Alalδ7Gln + Serl91Asp Alal67Glu + Phel69Pro Alal67Pro + Phel89Asp SerlδδAsp + Phel69Cys Phelδ9His + Serl91Asp Serl86Glu + Phelδ9Ala Alalδ7His + Serl88Asp Alal67Asn + SerlδδGlu SerlδδGlu + Phel69Gln Alal87Asp + Phelδ9Ser SerlδδAsp + Phel89Val Alalδ7Gln + SerlδδGlu 10 Alal67Ser + SerlδδGlu Alal67Pro + Serl91Asp SerlδδGlu + Phelδ9Val Phel89Ser + Serl91Glu Alal67Gly + Serl91Glu 15 Alalδ7Asn + Serl91Asp Alal67Thr + Serl91Asp Alalδ7His + SerlδδGlu SerlδδGlu + Phel69Gly Alal67Ser + Phelδ9Ile 20 SerlδδGlu + Phel69Met Phel69Asn + Serl91Asp Alal67Gln + Phel89Tyr Alal67Gln + Serl91Glu Alal87Ser + Phelδ9Ala 25 Phel69Val + Serl91Asp SerlδδGlu + Phelδ9Leu Alal67Pro + SerlδδGlu Phelδ9Asn + Serl91Glu Phel69Ile + Serl91Asp 30 Alalδ7Glu + Phelδ9Met Alal87His + Serl91Glu SerlδδAsp + Phel69Tyr Alalδ7Gly + Phelδ9Val SerlδδAsp + Phel69Gln 35 Alalδ7Gly + Phelδ9Tyr Alalδ7Gln + Phelδ9Asp Phel89Tyr + Serl91Glu Alal67Ser + Serl91Asp Alalδ7Thr + SerlδδGlu 40 Alal87Asn + SerlδδAsp Alalδ7Gly + SerlδδAsp Alalδ7Gly + Phelδ9Cys Phel69Cys + Serl91Glu Alal67Asp + Phelδ9Gly 45 SerlδδAsp + Phelδ9Leu SerlδδAsp + Phelδ9Gly Alalδ7Asn + Phelδ9Asp Alalδ7Pro + Serl91Glu Phel69Met + Serl91Asp 50 Alal67Thr + SerlδδAsp Alalδ7Pro + Phel 69Cys Serl91Glu Alal67Thr + Phelδ9Tyr Serl91Glu Alalδ7Ser + SerlδδGlu Phelδ9Ser Alalδ7Gln + Phel69Asn Serl91Glu Alal67Gln + SerlδδAsp Phel69His Alal67Gln + SerlδδGlu Phel69His Alal87Gly + Serl88Asp Phel89Met Alal87Gly + SerlδδAsp Phel69Cys Alalδ7Pro + Phelδ9His Serl91Glu Alal67Pro + Phel69Gln Serl91Glu Alalδ7Asn + SerlδδAsp Phelδ9Asn Alalδ7Gly + SerlδδGlu Phelδ9Ser Alalδ7Gln + Phelδ9Met Serl91Asp Alal67Gly + SerlδδAsp Phel69Pro Alal67Thr + Phelδ9His Serl91Asp Alal67Asn + SerlδδGlu Phel89Cys Alalδ7Gln + Phel 69Val Serl91Glu Alalδ7Pro + Phel 89Met Serl91Glu Alal67Ser + Serl 88Glu Phel89His Alal87Ser + Phelδ9Gln Serl91Asp Alal87Gln + SerlδδAsp Phel69Pro Alal87Gly + SerlδδAsp + Phelδ9Gly Alalδ7His + Phel 69Gln + Serl91Glu Alalδ7Thr + SerlδδGlu + Phelδ9Ile Alalδ7Pro + Phel 69Gly + Serl91Glu Alal87Thr + Phel 69Met + Serl91Glu Alal67Gly + Phelδ9Thr + Serl91Glu Alal67Gln + Phelδ9Leu Serl91Glu Alalδ7Thr + Phel 69Thr Serl91Asp Alalδ7Gln + Serl 88Asp Phel89Met Alal67Pro + Phel 89Ser Serl91Glu Alalδ7Asp + SerlδδGlu Phelδ9Val Alalδ7Glu + SerlδδGlu Phel69Ser Alalδ7Asp + Serl 66Glu Phel89Met Alalδ7Asp + SerlδδAsp Phel89Gln Alalδ7Asp + SerlδδGlu Phelδ9Cys Alal67Asp + SerlδδGlu Phelδ9Tyr Alal67Glu + Serl 68Glu Phelδ9Tyr Alalδ7Asp + SerlδδAsp Phelδ9Gly Alal87Glu + SerlδδGlu Phel89Leu Alalδ7Asp + SerlδδGlu Phel89Ser Alalδ7Glu + SerlδδAsp Phelδ9Gly Alalδ7Asp + SerlδδAsp Phel69Pro Alal87Asp + SerlδδGlu Phelδ9His Alal87Glu + SerlδδGlu Phel89Thr TABLE 21 Loop 5 - Quadruple Mutation Variants SerlOSGlu + Phel69Val Tyrl67Asp + Alal67Thr SerlδlGlu + Ala216Thr Ser 63Asp + Glnl03Ser Leu 96Gln + Prol29Glu Ala 98Gly + Tyr214Glu Leu 96Asn + Asn212Ser Ser 63Asp + Phelδ9Leu ThrlδδGln + Lys213Glu 10 Leul26Gln + GlylδOAsp Serlδ9Asp + Tyr217Gln SerlOlAsp + Val203Ala GlylOOAsn + Gly21δGlu Gin δ9Asp + Glyl31Gln 15 Glylδ7Glu + Leu209Pro Trpl06Pro + Tyr217Ile Ala216Ser + Gly219Asp Thr ββGln + Leul26Asn Glyl02Gln + Gly219Asp 20 Asn212Ser + Lys213Asp Gln206Ser + Lys213Glu Tyrl04Glu + AsnlδδGln Val 9 Asp + Leul26Ser Tyrl04Asp + Glyl66Gln 25 Thr ββPro + Ser204Glu Asn βlGlu + Phelδ9Pro Asp βOGlu + Tyrl67Ala Prol29Gln + Gln206Asp GlylβOAsp + Ala216Asn 30 SerlβlGlu + GlylδβAsn Leu 96Pro + GlylOOAsp TrplOβAsn + Val203Asn SerlOlAsp + Glyl27Ser Alal33Gln + Val203Asp 35 SerlOlAsp + Gly202Ser Ilel07Ala + GlylβOAsn Alal33Thr + Tyr214Ile Phelδ9Ser + Ser204Asp Gly 97Asp + Trpl06Phe 40 Gin δ9Asn + GlulδδAsp Pro201Ser + Lys213Glu Serl62Glu + Gly202Gln Gly βδSer + Gln206Asp Lys213Asp + Ala216Pro 45 Val203Ala + Lys213Asp Ala216Thr + Tyr217Pro Glyl31Asn + Asn216Glu Tyrl04Glu + Glyl31Pro Glyl27Ser + ThrlδδAsp 50 Trpl06Gly + Serl32Asp Asn 62Ser + Alal67Ser Serl63Asp + Phelδ9Ser Pro201Gln + Gly21δGlu GlylOOGln + Tyr217Thr Serl30Glu + Glylδ4Asn Asp 60Glu + Tyr214Thr AsnlδδGlu + Tyr217Gln Ala 9δGln + Glyl02Asn Pro201Asn + Gly219Asp 10 Thr 66Ser + Glyl27Gln Leul26Glu + Ala216Thr Asn 61Ser + AsnlδδGlu Thr 66Ser + Glylδ7Asp Prol29Ser + Thrl64Gln 15 Ala216Asp + Tyr217Val Serl30Glu + Tyr217Leu Asn 62Asp + Tyr214Leu Val 9δSer + Phelδ9Val GlylOOPro + Serlδ9Asp 20 Asnl55Gln + Ser204Glu Prol29Asp + Val203Ser SerlOlGlu + ThrlδδAsn Alal87Pro + Asn218Asp Val 9δGly + SerlβlAsp 25 Gly202Pro + Ala216Gln Gly 97Ser + Gly21δAsp Tyrl67Asp + Gln206Ser Thr 66Ser + Asn212Glu Ala216Thr + Tyr217Gln 30 Ala200Asn + Tyr217Ala Asp βOGlu + Val203Pro Val 9δThr + Tyr217Met Val203Asn + Lys213Glu Glyl02Asp + Val203Gly 35 Serl30Asp + Alal33Thr Tyrl04Ala + GlylββSer Leu 96Met + Tyr217Asp SerlOlAsp + Glyl02Pro SerlOlAsp + Thr220Pro 40 Val 9δAsn *+ Ala216Pro Tyrl04Asn + Prol29Asp Gly202Asn + Gln206Asp Gin δ9Glu + Ilel07Cys Thr ββGlu + Tyrl04Pro 45 Val 9δMet + Asp 99Glu Ser204Glu + Gly211Pro Pro210Glu + Gly219Ser Leul26Pro + Ser204Glu Prol29Asp + Ala200His 50 Ilel07Gly + Gly21δPro Thr ββGlu + Gln206Asn AsnlδδAsp + Leu209His Gly211Asp + Tyr217Val Ala216Asp + Thr220Gln ThrlδδGly + Ser204Asp GlylOOGlu + Ilel07Ser Ala 9δSer + Glylδ4Asn Glnl03Asn + Ala216Glu Glylδ4Gln + Pro210Gln 10 Leul26Pro + Ala216His Ala216His + Tyr217Leu Glylδ4Glu + Tyr217Ser Gly 97Ser + Tyrl67Thr Trpl06Ile + Ala216Gly 15 Glyl02Ser + Phelδ9Gly Glylδ4Glu + Gly219Asn Lys213Glu + Ala216Pro Asn 62Asp + Leul26Ser Thr 66Gly + Gln206Glu 20 Glylδ7Pro + Val203Cys Gin S9Asp + Tyr214Ser Leu 96Met + GlylOOSer Ala 96Gly + Lys213Asp Asn 62Gln + Leu 96Asp 25 Glyl27Asn + Gln206Glu Glyl60Pro + Gly219Asn Leu 96Thr + Tyr217Ala TrplOβPhe + Tyr217Thr Glyl31Pro + Lys213Glu 30 Gly βδGln + Asp 99Glu Glyl27Asn + Glyl26Gln Alal33Asn + Glylδ4Asn Ser204Glu + Gly21δSer GlulδβAsp + Pro210Ser 35 Asp βOGlu + Gln206Ser Asn βlGln + Ala216Asn Pro210Asn + Asn212Asp Alal33Asp + Val203Asn Gly219Ser + Thr220Gly 40 Serl91Asp + Val203Thr GlylβOGlu + Ala216Thr Serl62Glu + Ala216Gln Ala 98Gln + Tyr217Asn Val 9δAsp + Gln206Asn 45 Tyrl04Ser + Ser204Asp GlylOOPro + Phel69Gln Gly 97Asp + Tyr217His Gln206Ser + Gly211Asn Alal87Asn + SerlδδAsp 50 Ala 96Gly + Asp 99Glu Thrl64Asn + Phelδ9Cys Val203Gln + Gln206Ser Trpl06Cys + Glylδ7Ser ThrlδδSer + Glyl60Ser SerlδδAsp + Tyr217Gly Glylδ7Asn + Phelδ9Met SerlδδAsp + Ala216Asn Glyl26Asn + Glyl66Ser Leul26Asn + Ala216Ser 10 Glyl27Asp + Gln206Asn Gin δ9Glu + Leu 96His Serl32Asp + Tyr217Ala Glyl66Ser + Gly219Glu Serl63Glu + Val203Met 15 Ala 96His + Tyr217Met Ala 9δPro + Serl30Asp Glyl60Asn + Ser204Glu Gln206Asn + Gly21δAsp Glnl03Ser + Serl30Asp 20 Alal33Gly + Thr220Gly Serl32Glu + Ala216Gln Asn 61Gln + Ilel07His Leul26Ala + Glyl31Glu Gln206Asp + Thr220Gly 25 Gln206Glu + Tyr217Cys Glylδ7Ser + Pro210Asp Glyl66Glu + Tyr214Gln SerlδδGlu + Ala216His Thr 66Glu + GlylββGln 30 Glyl02Pro + GlylββGlu Val 9δGln + Tyrl04Ile Serl91Glu + Gly219Ser Asp 99Glu + Asn216Gln GlylOOAsn + SerlOδGlu 35 Glyl66Pro + Pro210Asn Gin δ9Asn + Thrl64Ser Leul26His + Tyr214Ala Thr 66Pro + Lys213Asp Trpl06His + Gly211Ser 40 Tyrl67Leu + Ser204Glu Val 9δThr + Alal33Gly Ilel07Ser + Gln206Glu Phelδ9Tyr + Lys213Asp Gly 6δAsn + Asn21δAsp 45 Tyrl67Val + Lys213Glu Gly 97Gln + Serl32Glu Asp 99Glu + Glyl02Pro Leul26Cys + Ala216Asp Leul26Cys + Glyl27Ser 50 Serl91Asp + Ala216Asn GlylOOGln + Glylδ4Asp Asn 61Asp + Gly211Ser SerlβlAsp + Phelδ9Leu Ile205Gln + Ala216Glu Asn 62Gln + Tyr217Leu Ilel07Met + SerlβlAsp Leul26Ile + Tyr217Ser Ala 9δHis + Serl62Asp Asn 61Asp + Glyl26Ser 10 AsnlδδGlu + Gly21δGln AsnlδδGln + Ser204Asp AsnlδδGlu + Thr220Gln Lys213Asp + Tyr217His Glyl27Pro + Ser204Glu 15 Ser204Asp + Tyr217Ala Glul 6Asp + Val203Gly Glyl27Glu + Alal33His GlylOOAsn + Glyl31Ser Gly211Gln + Lys213Asp 20 Alalδ7Asp + Phelδ9Leu Ala216Glu + Tyr217Cys Ser204Asp + Ala216Thr Glyl31Ser + ThrlδδAsp GlylOOAsn + Gln206Asn 25 SerlOδAsp + Glyl31Gln Ser204Asp + Tyr214Val Tyr214Met + Tyr217Ile Ser 63Glu + Thrl64Asn Ilel07Cys + Ala216Pro 30 TrplOβGly + Gln206Asp Glyl02Asp + Thrl64Pro Asp 99Glu + Ala216Gln Lys213Glu + Ala216Gln Alal33Ser + Pro210Glu 35 Asp βOGlu + Tyrl04Asn Asn 62Gln + Ilel07Cys Tyrl67Ala + Gly211Asp GlulδβAsp + Tyr217Ile Glyl31Pro + Leu209Pro 40 Lys213Glu + Asn21δGln GlylβOSer + Val203Glu AsnlδδSer + Tyrl67Ala Asp βOGlu + Phelδ9Gly Thrl64Gln + Gly219Ser 45 Serl62Asp + Gln206Asn GlylOOGlu + Tyrl04Asn GlylβOPro + Gln206Ser Thr ββGly + Ala216Gly Tyrl04Ile + Gly21δPro 50 Pro201Gln + Ala21βThr 10 15 20 25 30 35 40 45 50 Leu 96Gln + Lys213Glu Gln206Glu + Ala216Thr Glylδ4Ser + AsnlδδGlu Serl32Asp + Tyr214Asn Prol29Gln + Alal33Pro Ala 96Asn + Glyl27Asp Gly211Gln + Asn216Asp TrplOβCys + Serl63Asp Leu 96His + Ala216Gly 10 Gly 97Asn + Ser204Asp Asn βlSer + Glylδ7Asp Pro210Asn + Tyr217His Asp βOGlu + Tyrl04Ala Thrl64Asn + Ala200Gly 15 Tyr214Val + Ala216Asp Leul26His + Ala216Ser Glyl26Gln + Asn212Asp Serl62Glu + Gln206Ser Gln206Glu + Ala216Ser 20 Thrl64Pro + Thr220Asp Val203Ser + Gly219Asp Gln206Asn + Gly219Asp Ser 63Asp + Ilel07Gln Glyl02Gln + Val203Ala 25 SerlOlGlu + VallβδGln Gin δ9Ser + GlylββGlu SerlOlGlu + Tyr217Ser Glyl31Asn + Alalδ7Glu Glyl02Ser + Tyr214Gly 30 ThrlδδSer + Thr220Glu Asp 99Glu + Gly21δGln Val 9δGly + Thr220Asp Ala200Ser + Tyr214Val SerlδδGlu + Ala216Asn 35 Tyr214His + Ala216Asp ThrlδδGlu + Phelδ9Asn AsnlδδGln + Serl91Asp Thr ββSer + Leul26Ser Thr ββGly + Gln206Asp 40 SerlOδAsp + Tyr214Thr Glyl02Pro + Thrl64Gln TrplOβGly + Pro210Gly AsnlδδAsp + Thr220Gln 45 TABLE23 Multi-loop Triple Mutation Variants Gin δ9Ser + Leu 96Gly + Ser204Glu Asn 62Ser + Val 9δGln + Asn21δAsp Tyrl04Cys + Glyl27Gln + Lys213Glu SerlδδGlu + Gly21δAsn + Ala216Pro Gly 97Gln + Ilel07Ala + Glylδ7Glu Serl62Glu + Pro210Gln + Gly21δAsn Thr ββPro + Val203Cys + Tyr217Ser SerlOδGlu + Alal33Gly + Phelδ9Val Leu 96Asn + Asn212Ser + Tyr214Glu Gin S9Asp + Glyl31Gln + Leu209Pro TrplOβPro + Glylδ7Glu + Tyr217Ile Thr ββGln + Leul26Asn + SerlδδGlu Asn212Ser + Lys213Asp + Gly219Gln Val 9δAsp + Leul26Ser + AsnlδδGln Asn βlGlu + Thr ββPro + Phelδ9Pro GlylβOAsp + GlylββAsn + Ala216Asn TrplOβAsn + Glyl27Ser + Val203Asn SerlOlAsp + Ilel07Ala + Gly202Ser Alal33Thr + Phel69Ser + Tyr214Ile Gin δ9Asn + Gly 97Asp + TrplOβPhe Glylδ7Pro + Pro210Gly + Ala216Glu GlylβOSer + Asn212Ser + Tyr217Thr Asn 62Gln + Gln206Asn + Ala216Ser Prol29Ser + Gly21δGlu + Tyr217Pro Ala 9δAsn + Tyr217His + Thr220Gly Val203Gly + Gly211Glu + Ala216Asn Glyl27Glu + Tyr214Asn + Ala216His TrplOβPro + Alal33Pro + Gln206Asp Val 9δSer + Glyl2δGlu + Tyr217Cys Serlδ9Asp + GlylββGln + Gly219Gln Leu 96Val + GlulδβAsp + Glylδ7Pro Alal33Gly + Thr208Pro + Tyr214Pro TrplOβAsn + Glyl28Pro + Val203Met Gly βδSer + Glyl02Asn + Alalδ7His Ala200Gln + Gln206Glu + Tyr217His Glnl03Ser + GlulδβAsp + Ala216Ser Gin δ9Asn + Ala216Thr + Gly219Pro Glyl02Ser + Pro210Asp + Tyr217Ile GlylOOGlu + Ilel07Ser + ThrlδδGly Ala 9δGlu + Glylδ4Gln + Pro210Gln Glnl03Glu + Leul26Pro + Ala216His Lys213Glu + Ala216His + Tyr217Leu Glylδ4Glu + Tyrl67Thr + Tyr217Ser Gly 97Ser + TrplOβlle + Ala216Gly Glyl02Ser + Phel69Gly + Gly219Asn Glylδ7Pro + GlylβOAsp + Val203Cys Leu 96Met + Ala 96Gly + GlylOOSer Glyl27Asn + GlylβOPro + Gln206Glu Leu 96Thr + Tyr217Ala + Gly219Asn TrplOβPhe + Lys213Glu + Tyr217Thr Glyl02Glu + Glyl27Asn + Glyl26Gln Alal33Asn + Glylδ4Asn + SerlβlAsp Asn βlGln + Gln206Ser + Ala216Asn Ser204Asp + Gly219Ser + Thr220Gly Ala 96Gln + Serlδ9Glu + Tyr217Asn Gly 97Asp + GlylOOPro + Phel69Gln Gln206Ser + Gly211Asn + Tyr217His Ala 9δGly + Alalδ7Asn + SerlδδAsp Asp 99Glu + Thrl64Asn + Phel69Cys TrplOβCys + Glylδ7Ser + Gln206Ser Glylδ7Asn + SerlδδAsp + Tyr217Gly GlylββSer + SerlδδAsp + Ala216Asn 10 Leul26Asn + Glyl28Asn + Ala216Ser Leu 96His + Serl32Asp + Tyr217Ala Ala 9δHis + Lys213Glu + Tyr217Met Ala 9δPro + Serl30Asp + GlylβOAsn Serl30Asp + Alal33Gly + Thr220Gly 15 Asn βlGln + Ilel07His + Asn216Glu Gln206Glu + Tyr217Cys + Thr220Gly Glylδ7Ser + Pro210Asp + Tyr214Gln Val 9δGln + Glyl02Pro + GlylββGlu Tyrl04Ile + Serl91Glu + Gly219Ser 20 Asp 99Glu + GlylOOAsn + Asn21δGln Glyl31Glu + GlylββPro + Pro210Asn Leul26His + Thrl64Ser + Tyr214Ala Thr ββPro + Gly211Ser + Lys213Asp TrplOβHis + Tyrl67Leu + Ser204Glu 25 Val 9δThr + Alal33Gly + Gln206Glu Gly 97Gln + Glyl02Pro + Serl32Glu Leul26Cys + Serl91Asp + Ala216Asn GlylOOGln + Glylδ4Asp + Gly211Ser Asn 62Gln + Ala216Glu + Tyr217Leu 30 Leul26Ile + SerlβlAsp + Tyr217Ser Prol29Glu + AsnlδδGln + ThrlδδGln Glyl27Glu + Alal33His + Val203Gly Glyl31Ser + Gly211Gln + Lys213Asp Glyl31Ser + ThrlδδAsp + Ala216Thr 35 GlylOOAsn + SerlOδAsp + Gln206Asn Gly 97Glu + GlylβOGln + Thrl64Asn Ilel07Cys + Lys213Asp + Ala216Pro TrplOβGly + Gln206Asp + Ala216His Alal33Ser + Lys213Glu + Ala216Gln 40 Asn 62Gln + Ilel07Cys + Thrl64Asp Glyl31Pro + Leu209Pro + Tyr217Ile AsnlδδSer + Tyrl67Ala + Phel69Gly Asp βOGlu + Thrl64Gln + Gly219Ser GlylβOPro + Ser204Glu + Gln206Ser 45 Thr ββGly + GlylOOAsp + Ala21βGly Tyrl04Ile + Gly21δPro + Ala216Thr Glyl27Ser + Lys213Asp + Tyr217Ser SerlδδGlu + Gln206Asn + Leu209His Asn 62Ser + Gln206Asn + Pro210Glu 50 Ala 96Glu + Leul26Met + Val203Gly Glylδ4Ser + SerlβlGlu + Ala216His Pro201Gly + Gly211Glu + Ala216Thr SerlβlAsp + Gly219Gln + Thr220Asn Asn 62Glu + ThrlδδSer + Gly21δSer Glyl02Asn + Leul26Glu + Ala216Pro Glyl27Gln + Ser204Glu + Tyr217Thr Alal33Gln + Phelδ9Ile + Lys213Asp Serl30Asp + Asn212Ser + Tyr217Thr Leul26Ile + Glylδ4Asn + Gln206Asp 10 Thr ββPro + Glnl03Asn + Lys213Asp Leul26Asn + Glylδ4Gln + Pro210Gly Leul26Val + Gly21δGlu + Ala216Pro Gln206Ser + Lys213Asp + Tyr217His Leu 96Asn + Leul26Pro + Ala216Ser 15 Ser 63Asp + TrplOβAsn + Gln206Asn Glyl27Ser + SerlβlGlu + Gly219Asn Val 9δThr + Thr206Gly + Lys213Glu Gly 97Pro + TrplOβTyr + Asn216Glu Leu 96Gln + Phel69Ile + Val203His 20 Serl32Asp + Alal33Pro + Tyr214Asn Ala 96Asn + Glyl27Asp + Gly211Gln Leu 96His + Gly 97Asn + Ala216Gly Pro210Asn + Gly21δGlu + Tyr217His Asp βOGlu + TrplOβTyr + Prol29Gln 25 Glylδ7Asn + Phelδ9Val + Asn21δAsp GlylOOAsp + Thrl64Asn + Ala200Gly Leul26His + Gln206Asp + Ala216Ser Ser 63Asp + Ilel07Gln + Val203Ala SerlOlGlu + Glyl02Gln + VallβδGln 30 Asp 99Glu + ThrlδδSer + Gly21δGln Ala200Ser + Ser204Glu + Tyr214Val AsnlδδGln + ThrlδδGlu + Phel69Asn Thr ββGly + SerlOδAsp + Tyr214Thr Glyl02Pro + Thrl64Gln + Pro210Gly 35 TrplOβGly + AsnlδδAsp + Thr220Gln ThrlδδGly + Alal67Gln + Ser204Glu Glylδ4Gln + Tyrl67Cys + Ser204Glu Asp βOGlu + Ala 96His + Glyl02Pro Glyl31Ser + Ile20δVal + Ala216Asp 40 Glyl26Gln + VallβδCys + Gly211Gln Gly 97Asn + Ilel07Gln + GlylββGln GlylβOAsp + GlylββPro + Tyr214Ile Gin S9Asp + Glylδ4Ser + Asn216Gln Glylδ4Ser + VallβδHis + Ser204Glu 45 Ser 63Glu + Prol29Ser + Tyr217Gly Glylδ7Pro + ThrlδδSer + Lys213Glu Thrl64Glu + Gly21SSer + Ala216Asn Thr ββPro + Asp 99Glu + Tyr217Cys TrplOβMet + Alal87Ser + Tyr217Ile 50 Ilel07Thr + GlulδβAsp + Tyr217Cys 10 15 20 25 30 35 40 45 50 Leu 96Cys + Thrl64Ser + Ser204Asp Glyl31Gln + Phel69Ile + Val203Asp Asp βOGlu + Gly βδGln + Thr 66Asn Glyl02Glu + Glyl26Ser + Ala216Gln Asn 62Gln + Val 9δGly + Gln206Asn Gly 97Pro + Glylδ4Asp + Asn21δGln Thr ββPro + Leu 96Val + Ala216Pro Gly 97Asn + AsnlδδGlu + Tyr214Val Tyrl04Ala + Tyrl67Glu + Ala216Pro 10 Glylδ7Asn + Asn21δGlu + Thr220Gly Alal33His + Thrl64Gln + Glyl66Ser Leul26Gln + Serlδ9Glu + GlylβOAsp Asn 61Asp + Asn 62Asp + Glyl26Ser Thr ββPro + GlylOOGlu + SerlOlGlu 15 Ser204Glu + Ile205Gln + Ala216Glu Ser204Asp + Ala216Glu + Tyr217Cys Ser204Asp + Ala216Asp + Thr220Gln Glnl03Asn + Ser204Glu + Ala216Glu Gly202Gln + Ser204Glu + Asn21δAsp 20 Ser204Glu + Gln206Asp + Ala216Asp Ser204Asp + Gln206Glu + Ala216Asp Tyrl67Ala + Ser204Asp + Tyr217Asp Gly211Asp + Lys213Glu + Ala216Thr Gly211Asp + Lys213Glu + Tyr217Pro 25 Tyrl67Val + Gly211Asp + Lys213Glu Asp βOGlu + Asn 62Asp + Tyr217Leu GlylβOGlu + Serl62Glu + Ala216Thr Ser204Glu + Gln206Asp + Tyr217Leu Ser204Glu + Gln206Glu + Ala216Thr 30 Ilel07Cys + Ser204Glu + Gln20βGlu Ser204Glu + Gln206Glu + 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Φ_ φ_ _ _ .. t_o- - tO M Φ -fi >ι tO Φ Φ M Φ to φ xi φ φ -β φ M Φ M M Φ >1 Φ to to -fi tD co co co co cu iaJ co iaJ CD co EH EH fal co co cD co ft r EH CO CO H i-H CD CO CD iaJ cO l-H CO taJ f-^ CD CD EH CD CD CD o o o £0 IΛ C\ o i •Λ o IΛ o CM « o o " ■ I- • < Glnl03Ser + Glyl66Asn + Tyr214Ile + Gly21δPro Asn 61Asp + Tyrl04Ser + Leul26His + Tyr214His Gly 6δGln + Glyl31Gln + Phelδ9Ile + Val203Asp Asn 62Gln + Thr 66Asp + Val 9δGly + Gln206Asn Thr 66Pro + Gly 97Pro + Glyl 4Asp + Ala216Pro Val 9δPro + Tyrl04Gly + Glyl27Ser + Gly21δAsp Asp 99Glu + Trpl06Ala + Pro201Gln + Ala216Gly Asn 61Gln + Val 9δAsp + Glyl02Asn + Alalδ7Asn Ilel07Gln + Val203Ser + Ser204Asp + Gly21δSer Val 9δThr + Gly202Gln + Ser204Asp + Ala216Asn ThrlδδPro + Val203Gly + Lys213Glu + Tyr217Ser Trpl06Pro + AsnlδδAsp + Gln206Ser + Tyr214Ala Glyl02Asn + Glylδ7Ser + Tyrl67Ala + Ala216Asn Glyl60Asn + Val203Thr + Pro210Glu + Asn216Gln Ilel07Ser + Glyl2δAsn + AsnlδδGlu + Ala216Gly Glnl03Asn + Prol29Gly + Glyl66Gln + Thr220Gly Asn 61Ser + Ser 63Asp + Thr 66Gly + Glylδ4Ser Tyrl04Gly + Prol29Ser + Gln206Ser + Gly219Ser Glyl02Pro + Glyl31Asp + AsnlδδSer + Tyr217His Asn 61Ser + Val 9δGln + Ser204Asp + Ala216Gln ThrlδδAsn + Alal67Gly + Tyr217Ala + Gly219Asp Gly 6δGln + Gly 97Pro + Serl30Glu + Pro210Asn Glyl26Asn + Serlδ9Glu + Pro201Ser + Tyr217Val Leul26Asn + AsnlδδGln + Gly202Gln + Asn212Ser Thr 66Ser + Tyrl04Val + Glylδ4Glu + Gly21δAsn Glyl02Asn + Glyl2δGln + SerlβlGlu + Tyr217Met Serl32Glu + ThrlδδGln + Thrl64Asn + Gln206Asn Asn 62Glu + Leu 96Ile + Gly211Ser + Gly219Ser Thr206Pro + Pro210Gly + Ala216Thr + Tyr217Met GlylOOGln + GlylβOAsn + Pro201Gly + Asn212Asp Tyrl04Asp + Glylδ4Pro + Alal67Asn + Val203Ser Leu 96Gln + Leul26Thr + Serl62Glu + Tyr217Val Glyl2δAsn + Alal87Pro + Pro201Gly + Ser204Glu Glnl03Ser + Glylδ7Glu + ThrlδδGln + Ala216Gln Leul26Ser + Thrl64Glu + Val203Pro + Gly211Gln Thrl64Gly + Val203Met + Ala216Asp + Tyr217Gln Serlδ9Asp + Val203Asn + Ile20δAsn + Pro210Ser Gly 6δAsn + Gln206Asp + Ala216Gly + Tyr217His Glnl03Asn + Ilel07Cys + Thrl64Asp + Val203Thr Glyl28Glu + AsnlδδGln + ThrlδδSer + GlylβOSer Ala 98His + Serl62Glu + Gln206Asn + Tyr217Gly Glyl26Ser + Thrl64Asn + Ser204Glu + Tyr217Gly Glyl27Gln + Glylδ7Ser + Serlδ9Asp + Tyr217Val Glylδ7Asn + Gln206Asn + Tyr217Val + Gly219Pro Thr 66Ser + Alal33Thr + Serl63Asp + Thr206Gln Leu 96Thr + Glyl31Asp + Gln206Asn + Ala216Gly Asn 61Ser + Serl32Glu + Gly211Ser + Asn21δGln GlylOOSer + Tyrl04Ala + Ser204Asp + Gly211Gln Leu 96His + Ala 98Glu + Prol29Gln + Alal33Asn Asn 62Glu + Glyl26Gln + Alal67Asn + Gly21δSer Leu 96Ile + Glylδ7Ser + Val203Ala + Ala216Ser Asn 61Gln + Val 9δThr + GlylδOAsp + Ala216His Leu 96Cys + Glyl26Pro + Serl91Glu + Thr20δAsn Trpl06Ala + Glyl31Gln + Val203Ala + Tyr214Gln Asn 61Ser + Ala216Gln + Tyr217Leu + Gly219Asn Tyrl04Gly + SerlOδGlu + ThrlδδSer + Leu209Thr Alal33Ser + Phel69Thr + Asn212Glu + Tyr217Thr Tyrl04Ser + ThrlδδGly + Thrl64Glu + Ala216Pro Gin δ9Asn + Thr 66Asn + Thrl64Gly + Alalδ7Pro Ilel07His + Glylδ7Ser + Lys213Glu + Tyr217Asn Glyl27Ser + Gln206Asp + Gly21δGln + Tyr217Leu Leul26Gly + Glyl31Glu + Tyrl67Met + Thr220Gln ThrlδδGln + Lys213Glu + Gly21δSer + Tyr217Gly Asn 61Gln + Leul26Gly + Thrl64Ser + Asn21δAsp Asn 62Asp + Prol29Gly + Gln206Ser + Ala216His Asp 60Glu + Val 9δGln + Leul26Pro + Val203Thr Glnl03Glu + Ilel07Val + Phel89Asn + Ala216Thr Ilel07Thr + Prol29Gln + Lys213Glu + Tyr217Thr Tyrl04His + Glylδ4Gln + Glylδ7Asp + Tyr217Ser Gin δ9Asn + Trpl06Cys + Ala200Thr + Ala216Gln Thr 66Gln + Gly 97Ser + Glyl27Pro + Tyr217Asp GlylOOAsn + Ser204Asp + Pro210Ser + Tyr214Gly Asn 62Ser + Ilel07Gly + Leul26Cys + Thr220Gly Leul26His + Glylδ4Asp + Asn216Gln + Thr220Asn SerlOlGlu + Glylδ7Gln + Tyr214Pro + Ala216His Asn 62Gln + Serl62Glu + Val203Ser + Ala216Thr Tyrl04Gln + Trpl06Gly + Leul26Asp + Asn212Gln Gin δ9Ser + Val 9δPro + Gly202Asn + Tyr217Ser Leu 96Pro + Glyl60Asp + SerlδlGlu + Glyl66Asn Serlδ9Glu + Glyl60Asp + Tyrl67Gly + Phelδ9Val Asn212Glu + Lys213Glu + Ala216Ser + Tyr217Gln ThrlδδAsp + Serlδ9Asp + Gly21δAsn + Ala216Thr Ala 96Asp + Asp 99Glu + Thrl64Gln + Alalδ7Ser Gly 97Pro + Glyl31Pro + Glylδ4Asp + AsnlδδAsp Glyl02Ser + Trpl06Gln + Glylδ7Glu + Phelδ9Asp GlylOOGln + Ser204Glu + Tyr214Ile + Ala216Glu Val 9δPro + Ser204Glu + Ala216Gly + Asn21δGlu Ser204Glu + Ile205Gln + Pro210Gly + Asn216Asp Gly 97Ser + Glylδ4Asn + Gln206Asp + Gly21δAsp Gly 97Asp + Ala 96Gln + Asp 99Glu + Glylδ4Ser ThrlδδGln + VallδδMet + Gly211Glu + Lys213Glu Glyl60Glu + Serl62Asp + Tyrl67Ile + Gly219Ser Asn 61Ser + Thr 66Ser + AsnlδδGlu + Glylδ7Asp ThrlδδAsp + Serlδ9Asp + Thrl64Asp + Gly211Asn Val 9 Asp + Glyl02Glu + Alal87Pro + Tyr217Pro Asn 62Glu + GlylOOAsp + Thr208Asn + Tyr217His Ser204Asp + Gln206Glu + Gly211Gln + Ala216His Glylδ4Asn + Ser204Glu + Gln206Asp + Tyr217Thr Thr 66Gln + Serl30Glu + Serl32Asp + ThrlδδPro Asp 60Glu + Gly 6 Asn + Thr 66Glu + Tyr214Ser Asp βOGlu + Gln206Ser + Pro210Glu + Gly219Ser ThrlδδAsp + Serl63Glu + Serl91Glu + Ile2OδGly Ser204Asp + Gly21δGln + Ala216Glu + Gly219Asp ThrlδδAsp + Alalδ7Asp + Phel69Glu + Tyr217Met Glyl2δGln + Prol29Asn + Val203Asp + Ala216Asp Gly 97Asn + Ilel07Gln + Ser204Glu + Gly219Glu TrplOβAsn + Glylδ7Gln + Ser204Asp + Gly219Asp Glyl27Asp + Glyl2δAsn + Serl30Asp + Gly219Gln Val 9δSer + Prol29Gly + AsnlδδGlu + SerlδδGlu AsnlδδAsp + SerlδδAsp + Phelδ9Asn + Ala216Gly TrplOβPhe + Ser204Asp + Gln206Asp + Tyr214Asp Asn 62Asp + Gly 97Gln + Pro210Asp + Gly2UGlu Val 9δAsp + Tyrl04Asp + Leul26Ser + AsnlδδGln GlylOOAsn + Gln206Asp + Lys213Glu + Ala216Asp Gln206Asp + Lys213Asp + Ala216Glu + Tyr217Asn Glyl02Gln + AsnlδδGlu + Val203Glu + Asn2lδAsp Gin δ9Glu + Thr ββGlu + Glyl02Pro + Glyl66Gln Leul26Cys + Glylδ7Asp + Serl63Asp + Ala216His Thr ββAsp + Gln206Asp + Ala216Asp + Gly219Pro Asn 62Asp + Ser 63Glu + Glyl31Asn + Lys213Glu Leul26Asn + Prol29Asn + Serl91Asp + Gly219Glu Thr ββAsp + GlylOOAsn + Glyl27Ser + Lys213G1U Ilel07Val + Phelδ9Asp + Val203Glu + Ala216Gln Ser 63Asp + Val 9δSer + Lys213Asp + Ala216Ser Ilel07His + Val203Cys + Tyr214Glu + Tyr217Asp Asn 62Ser + SerlOδAsp + TrplOβGly + Serl32Asp Ser 63Glu + Leu 96Cys + Pro210Glu + Ala216Glu Alalδ7Gly + Gly21δAsp + Tyr217Thr + Asn2lδGlu GlylβOSer + Gln206Glu + Lys213Glu + Ala216Ser Glyl31Pro + Phelδ9Leu + Gln206Glu + Lys213Glu Prol29Asn + Alal33Gln + Gln206Glu + Lys213Glu Ala 96His + Glylδ4Glu + Serl63Asp + Tyr217Met Val203His + Gln206Glu + Gly211Glu + Lys213Asp Leul26Ala + Ser204Glu + Gln206Asp + Lys213Glu Ilel07Leu + Glylδ7Glu + Val203His + Gly219G1U Ala 9δGlu + Glyl02Asp + SerlOδGlu + Leu209Thr Thr ββGln + Lys213Glu + Ala216Glu + Asn2lδGlu Ser204Glu + Gln206Asn + Pro210Glu + Gly2lδAsp Glyl27Asp + Serl32Asp + Glylδ4Asp + Vail6δGln Ser 63Glu + Val203His + Asn212Glu + Tyr217Leu Gln206Glu + Lys213Glu + Tyr217Ala + Asn2lδGlu Gln206Asp + Lys213Glu + Ala216Asn + Asn2lδAsp Glylδ7Pro + SerlδδGlu + Ser204Glu + Ala216Asp Gin δ9Glu + Thr 66Asp + GlylOOGln + Gly2lδGlu TrplOβSer + Alalδ7Asp + Gln206Glu + Tyr217Asp Serlδ9Glu + Asn212Gln + Gly21δAsp + Ala216Glu GlylβOAsp + SerlδlAsp + Gln206Asp + Tyr214Asn Thr ββGlu + Tyrl67Gln + Gln206Glu + Gly211Pro Prol29Asn + Serl63Glu + Tyr217Glu + Asn218Glu AsnlδδGlu + GlulδδAsp + Ser204Glu + Tyr214Thr Gin δ9Asp + Serl62Asp + Serl63Glu + Ala216Thr Leul26Pro + Serl62Glu + Serl63Glu + Tyr217Glu GlylOOGlu + Val203Cys + Asn212Asp + Lys213Glu SerlOδGlu + Alalδ7Ser + Val203Glu + Ser204Asp Glnl03Asp + Serl63Glu + Thrl64Glu + Pro201Gln Val 9δGln + GlulδβAsp + Glylδ7Asp + Lys213Glu Serl62Glu + Thrl64Gln + Ala216Asp + Tyr217Glu Asp 99Glu + GlylOOGlu + Serlδ9Glu + Ala216Thr Ala 98Glu + Asp 99Glu + Trpl06Gly + Glylδ4Asp Asn 62Glu + Ser 63Glu + Prol29Ser + AsnlδδAsp Asn 61Glu + Gln206Glu + Ala216Glu + Tyr217Cys Thr 66Pro + Glnl03Asp + Glulδ6Asp + Serl91Asp Asp 60Glu + Ser204Asp + Ala216Asp + Tyr217Ile SerlOδAsp + Ser204Asp + Gln206Ser + Ala216Glu Thrlδ8Asn + Serl62Asp + Ser204Asp + Asn2lδAsp Gin δ9Asp + Glylδ7Ser + Ser204Asp + Asn2lδAsp Gly 97Ser + Glyl26Glu + Gln206Glu + Gly2lδAsp Trpl06Asp + Val203Cys + Ser204Glu + Tyr217Glu SerlOδGlu + Alalδ7Thr + Ser204Glu + Tyr217Glu Gly 97Asn + AsnlδδGlu + Serl63Glu + Tyr214Val Val 9δAsp + TrplOβGlu + Alal67Pro + Val203Asp Glnl03Asp + TrplOβGlu + Glyl2δAsn + Serl62Asp Glyl28Glu + Serl30Asp + SerlδδGlu + Ala216Gln Glnl03Asp + SerlOδGlu + Glylδ4Glu + Ala216Thr Serlδ9Glu + Gly211Glu + Lys213Asp + Tyr217Gly Gin δ9Asn + SerlδδAsp + Gly211Glu + Lys213Glu Ilel07Glu + Gly211Glu + Lys213Asp Tyr217Gln Serlδ9Asp + Serl62Glu + Pro210Glu Ala216Asn Asp 60Glu + Asn 62Asp + Serl91Asp Tyr217Leu Asp 60Glu + Ser 63Asp + Ilel07Asn Phel69Glu Leu 96Cys + GlylββAsp + Pro210Asp Lys213Asp Val 9δGlu + Ala 9δAsn + Glyl02Glu Serl62Glu Ser 63Asp + Tyrl67His + Ala216Glu Gly219Glu Tyrl04Asp + ThrlδδAsp + Serl91Glu Asn218Ser Glylδ4Pro + Serlδ9Glu + Ser204Asp Gln206Asp Glyl02Glu + Ser204Asp + Gln206Glu Tyr217His AsnlδδGln + Serl63Asp + Ser204Glu Gln206Glu Glyl31Asp + ThrlδδGln + Ser204Asp Gln206Asp Tyrl67Asp + Ser204Glu + Gln206Glu + Tyr217Asn Gly 97Asp + Alal33Gly + Ser204Asp + Gln206Asp Glyl27Asp + Ser204Glu + Gln206Glu Tyr214Asn Glyl02Glu + Glyl27Gln + AsnlδδAsp Thr220Asp Gly 97Glu + Serl30Glu + Tyrl67Asp Tyr217Val Asn 62Glu + Alal87Gly + Pro210Asp Ala216Glu SerlOlAsp + SerlOδAsp + Ala216His + Tyr217His Serl30Asp + Serl32Glu + Asn212Glu + Ala216Gln Serl30Glu + Serl32Glu + GlylδOAsp + Thr220Gly GlylOOGlu + Tyrl04Thr + Serl30Asp + Serl32Asp Gin δ9Ser + GlylβOAsp + Gln206Glu + Tyr217Asp Glyl27Asp + Prol29Glu + SerlδδAsp + Gln206Asn Serlδ9Asp + Thrl64Glu + Phelδ9His + Lys213Glu Asn 61Asp + Gly 97Asp + Serlδ9Glu + Thr220Ser Serlδ9Glu + Serl63Glu + Ser204Glu + Tyr217Ser Thrl δAsp + Serl62Glu + Alalδ7Pro + Ala216Glu Leu 96Val + ThrlδδGlu + Serl62Asp + Gly219Asp Asp 99Glu + ThrlδδAsp + Serl62Asp + Val203Met Val 9 Asp + Glyl31Asn + Serl63Asp + Serl91Glu Asn βlGlu + Asp 99Glu + Ser204Asp + Tyr217Gly Asn 62Asp + GlylββSer + Ser204Asp + Gly21δGlu Glyl02Asp + SerlOδAsp + Tyrl67Ala + Gly211Glu SerlδδAsp + Serl91Glu + Ala216Gly + Tyr217Glu Asp βOGlu + Gly 97Asp + TrplOβAsn + Serlδ9Glu Thr ββAsp + Leu 96Glu + Phelδ9Gly + Gly21δAsp Asn 62Glu + Thr ββAsp + Tyrl04Pro + GlylββAsp Asn βlSer + Ala 9δAsp + AsnlδδAsp + SerlδδGlu GlylOOGlu + Tyrl04Glu + Serl30Glu + AsnlδδGln Asp βOGlu + Leul26Asn + Gln206Glu + Lys213Asp Ala 96Glu + Glylδ4Pro + GlulδβAsp + SerlδδGlu Glyl2δGln + Alal33Glu + Alal87Glu + Serl91Asp SerlOlGlu + Glylδ4Asn + Gly211Glu + Tyr214Glu Serl32Asp + AsnlδδAsp + ThrlδδGlu + Ala216Thr Asn βlGlu + AsnlδδAsp + Alalδ7Asp + Asn212Gln Glnl03Glu + GlylβOAsn + Gln206Glu + Asn216Glu Gin δ9Glu + GlylOOGlu + Thrl64Pro + Gly211Asp Ser 63Glu + SerlOlAsp + Glyl31Ser + Val203Pro Gin δ9Asp + Thr ββAsp + Tyrl04Val + Alal33Asp Ser 63Glu + SerlOlGlu + Alal33His + Ala216Glu Asp βOGlu + Val 9δAla + Lys213Glu + Tyr217Ala TrplOβMet + Serl91Glu + Lys213Glu + Gly219Glu Ser 63Asp + GlylβOAsp + Lys213Asp + Ala216His Glyl02Asp + Glylδ7Asn + Serl62Glu + Serl91Glu Gin δ9Ser + SerlOδAsp + Serl62Asp + Serl91Asp Glyl27Pro + Serl62Glu + Serl91Glu + Asn212Asp Ser 63Asp + SerlOδAsp + Serl32Asp + Ala216His Thr ββGln + Glyl2δGlu + GlulδβAsp + Ala216Asp Glyl2δAsp + Glylδ7Asn + Pro210Gln + Thr220Glu GlulδβAsp + Gln206Glu + Lys213Glu + Ala216Asn Asp 99Glu + Glylδ7Pro + Gln206Asp + Lys213Glu Serl63Asp + Gln206Asp + Lys213Glu + Tyr217Ala Glylδ4Glu + Serl63Glu + Pro210Gln + Tyr217Asp Glylδ4Asp + Glylδ7Asn + Serl63Asp + Ser204Glu Glylδ4Ser + Glylδ7Asp + Lys213Glu + Ala216Glu Glylδ7Ser + ThrlδδGlu + Lys213Asp + Ala216Asp SerlOlGlu + Glylδ4Pro + Lys213Asp + Ala216Glu GlylOOAsp + Lys213Glu + Ala216Asp + Tyr217Leu Asn 62Ser + ThrlδδGlu + Ser204Asp + Thr220Asp Thr ββAsn + Ilel07Val + Lys213Asp + Tyr217Asp Glylδ7Asn + Pro201Gln + Lys213Glu + Tyr217Asp Glyl27Glu + ThrlδδPro + Alalδ7Asp + Ser204Glu Asp 99Glu + Alal33Gly + SerlδδGlu + Thr220Glu Asp βOGlu + SerlδδGlu + Gln206Ser + Asn218Glu Gin δ9Asp + Leu 96Glu + Glyl31Gln + Serl32Asp SerlOlGlu + Prol29Asp + ThrlδδAsn + Val203Ser Ser 63Glu + Serl63Asp + Ala216Asp + Tyr217Gln Glyl02Gln + GlylβOGlu + Serl91Glu + Lys213Glu Val 9δGlu + Asp 99Glu + Gly21δGlu + Asn218Gln SerlOδGlu + Alal33Glu + Val203Glu + Asn21δGln Glnl03Asp + Serl32Glu + Serl62Glu + Gln206Ser Asp βOGlu + SerlOlAsp + Thrl64Gly + Lys213Asp Gin δ9Asp + Asp 99Glu + Glnl03Asn + Alal67Pro Asp βOGlu + Serlδ9Asp + Tyrl67Leu + SerlδδAsp Asn 62Glu + Serl63Glu + Gly211Glu + Ala216His Asn 62Glu + Serl32Asp + Pro210Gly + Gly211Glu Glyl02Asn + Serl62Asp + Gln206Asp + Gly219Asp SerlδδAsp + Ser204Asp + Tyr217Leu + Thr220Gln Ser 63Glu + Glyl66Gln + Ala216Thr + Asn218Glu Glnl03Glu + Glyl31Glu + Tyr217Thr + Thr220Glu Asp 60Glu + Phel69His + Asn212Glu + Ala216Asp AsnlδδGln + Gly21δGlu + Tyr217Pro + Gly219Asp Glyl02Asn + Leul26Glu + Serl30Glu + Lys213Asp Ala 9δAsp + Glyl66Glu + Pro210Asp + Tyr214Gln Asn 62Glu + AsnlδδSer + Lys213Asp + Tyr217Leu Asp 60Glu + SerlOδGlu + Lys213Glu + Thr220Gln Asp 60Glu + Gln206Ser + Lys213Asp + Asn218Asp Ser 63Glu + Gly 97Gln + Glnl03Asp + Gln206Asp Ser 63Glu + Val 9δAla + Serl30Asp + Gln206Asp Ser 63Asp + Ilel07Met + Serl91Asp + Gln206Asp Prol29Asn + Serl30Asp + Lys213Glu + Tyr217Glu Prol29Asn + Serl91Glu + Lys213Asp + Tyr217Glu Gly 97Gln + Glyl02Asp + Prol29Glu + Phel69Gln Gin δ9Asn + Serl62Glu + Phelδ9Asp + Ser204Asp Glyl27Pro + Glyl28Glu + Phelδ9Glu + Ser204Asp Leu 96Pro + SerlOδAsp + Serl30Glu + Alal33Gly Tyrl67His + Serl91Glu + Asn212Glu + Asn2lδAsp Asn 61Glu + ThrlδδGln + Lys213Asp + Tyr217Asn Gin δ9Asp + Glylδ7Asp + Gln206Ser + Asn2lδAsp Glylδ4Ser + Serl63Glu + SerlδδGlu + Ser204Asp Leu 96Asn + Serl30Asp + SerlδδAsp + Ser204Glu Ilel07Asp + SerlδδAsp + Ser204Asp + Gln206Asn Gln206Glu + Ala216Gly + Tyr217Leu + Thr220Asp Glyl02Glu + Leul26Cys + Serl30Glu + Tyr214Asp Asn 62Glu + GlylβOAsp + Lys213Glu + Ala216Gly SerlOlAsp + Trpl06Met + Glylδ4Asp + Serl62Asp Asp 60Glu + Glyl02Glu + Gln206Asn + Ala216Asp Glulδ6Asp + Gln206Ser + Pro210Asp + Tyr217Asp Prol29Glu + Serlδ9Asp + Gln206Glu + Tyr217Pro Prol29Asp + Serlδ9Glu + Lys213Asp + Tyr217His SerlOδGlu + TrplOβLeu + Glyl27Glu + Serl63Glu SerlOlAsp + Alal33Gln + Serl91Asp + Val203Asp Ser 63Glu + Serl30Asp + Tyr217Gln + Gly219Asp Glyl31Asp Serl63Asp + Glyl66Asn + Ser204Asp Ilel07Asp Gln206Ser + Asn212Glu + Ala2lβAsp Leul26Gly Serl30Asp + Glylδ4Asn + Asn2lδAsp Gin δ9Asp SerlOδAsp + Glyl66Gln + Ser204Asp Asn βlAsp SerlOδGlu + Alal67Gln + Ala2lβGly SerlOδAsp Phel89Ile + Lys213Glu + Gly219Gln Ser 63Glu Glyl31Gln + Ser204Glu + Gly219Asn Glylδ7Pro Thrl64Glu + Gln206Asn + Lys213Asp Leu 96Ile SerlOlAsp + Gln206Glu + Tyr214Ala Thr ββGln Leu 96Met + Tyrl67Glu + SerlδδGlu Tyrl04Cys Glyl60Asp + Ile2OδPro + Ala2lβGlu Asp βOGlu Serl30Asp + Pro2OlGln + Ala2lβGly Ilel07Asp Serl91Asp + Gln2OβAsp + Ala2lβThr Gin δ9Asp Val 9δAsn + SerlOlGlu + Serl63Glu Val 9δGln Tyrl04Cys + Lys213Glu + Asn2lδAsp Asn 62Asp Gly 97Asn + Ala 9δSer + Serl62Glu Glnl03Glu Ser204Asp + Gln2OβAsn + Ala2lβPro SerlOlAsp Serl62Asp + GlylββSer + Tyr217Thr Leul26Ile Glyl2δAsp + Pro2lOSer + Asn218Glu GlylOOGlu Glyl60Ser + GlylββGlu + Ala2lβThr Glnl03Asn Serl32Asp + Serl63Glu + SerlδδAsp TABLE25 Multi-loop Quintuple Mutation Variants Val 9δGln + Tyrl04Cys + Glyl27Gln Lys213Glu + Ala216Pro Asn βlSer + Leu 96His + Glylδ7Pro Val203Asp + Ala216Gly Leu 96Gln + Glyl27Gln + GlulδβAsp Tyr214Ala + Thr220Asn GlylOOGln + Tyrl67Cys + SerlδδGlu Val203Gln + Ala216His Asn 62Ser + TrplOβGly + Serl32Asp Alal67Ser + Phel69Ser Thr ββSer + Glyl27Gln + Pro201Asn Ala2lβThr + Gly219Asp Gly 97Asn Glylδ4Pro + Gln2OβAsn Pro210Glu + Gly211Pro Prol29Gly Serl32Glu + ThrlδδAsn VallβδThr + Gln2OβAsn Gly βδSer Gly 97Gln + Glyl28Ser Lys213Asp + Gly219Gln Leu 96Met Glnl03Asn + Alal33Ser Glylδ4Pro + Gly219Pro Asn βlGln + TrplOβAla + Gly211Pro Asn2lδAsp + Gly219Asn Thr ββGly + Tyrl04Ile + Gly211Glu Gly21δPro + Ala216Gly Leul26Ile Serl30Asp + Glylδ4Asn Asn212Ser + Tyr217Thr Leul26Val Gln206Ser + Pro210Gly Gly21δGlu + Ala216Pro Leu 96Asn Leul26Pro + Lys213Asp Ala216Ser + Tyr217His TrplOβAsn Glyl27Ser + SerlβlGlu Gln2OβAsn + Gly219Asn SerlOlGlu Glyl02Gln + Ilel07Gln VallβδGln + Val203Ala Asp βOGlu Ala 96Gly + Ilel07Ser Glylδ7Ser + Thrl64Ser Prol29Glu GlylβOPro + GlylββAsn Alalδ7Pro + Gly202Ser Leu 96Ile Tyrl67Thr + SerlδδAsp Val203His + Gln206Ser Asn βlGln Val 9δAsp + Glyl02Asn Glyl31Asn + Alalδ7Asn GlylβOAsn Val203Thr + Pro210Glu Asn216Gln + Thr220Gln Glyl2δAsn AsnlδδGlu + GlylββGln Ala216Gly + Thr220Gly Gly βδSer Val 9δMet + GlylOOAsn Glyl31Asp + Tyr214Gly Tyrl04Gly Prol29Ser + Serl63Glu Gln206Ser + Gly219Ser Asn βlSer + Val 9δGln + Ser204Asp + Pro2lOGly Ala216Gln Gly βδGln + Gly 97Pro + Serl30Glu + Glylδ4Ser Pro210Asn TrplOβSer + Glyl2δAsn + Serlδ9Glu + Pro2OlSer Tyr217Val Leu 96Met + Leul26Asn + AsnlδδGln + SerlδδGlu Gly202Gln GlylOOGlu + ThrlδδGln + Thrl64Asn + Gln2OβAsn Ala216Thr Asn 62Glu + Leu 96Ile + Gly 97Ser + Gly2USer Gly219Ser Glyl02Asp + Tyrl67Ala + Pro210Gly + Ala2lβThr Tyr217Met Serl32Glu + ThrlδδPro + Phelδ9Thr + Ala2OOGln Tyr214Ala Ala 98Pro + TrplOβPro + GlylβOPro + Ala2lβAsn Tyr217Asp Glyl27Pro + Alal33Asn + Thrl64Glu + Gly2UGln Tyr214Thr GlylOOAsn + TrplOβPro + Glyl27Ser + Lys213Glu Tyr214Ala Glylδ7Asn + Ser204Asp + Gln2OβAsn + Tyr217Val Gly219Pro Leu 96Thr + Glyl31Asp + Alal33Thr + Gln2OβAsn Ala216Gly GlylOOSer + Tyrl04Ala + Thrl64Asp + Gly2UGln Thr220Ser SerlOlAsp + Prol29Ser + Phel69Val + Pro2OlAsn + Ala216Ser Thr ββGly + Glyl02Asn + Tyrl04His + Trpl OβThr Alal87Asn Thr ββAsn + Glyl02Glu + TrplOβGly + GlylββSer Ala216Thr Glyl2δGln + Glylδ4Asn + Tyrl67Gly + Tyr217Leu Asn2lδGlu Alal33Ser + Glylδ7Ser + Phel69Thr + Gly202Asn Asn212Glu Tyrl04Ser + ThrlδδGly + Thrl64Glu + Gln2OβAsn Ala216Pro Gin δ9Asn + Glnl03Asn + Thrl64Gly + Alalδ7Pro Thr220Asp Gly 97Gln + Glyl02Asp + Glyl27Ser + Phel89Gln Tyr217Leu Thr ββAsn + Gln206Glu + Tyr214Ile + Ala2lβThr Tyr217Cys Asp βOGlu + Thr ββGly + Leu 9βGly + Ala2lβHis Tyr217Asn Ilel07Asp + GlylβOAsn + Val203Pro + Gly2UPro Gly219Asn Val 9δSer + TrplOβCys + VallβδGln + Pro2lOGln Tyr217Glu TrplOβThr + ThrlδδSer + Thrl64Pro + Ser204Glu Thr220Pro Glyl2δPro + Alal67Ser + Gln2OβAsn + Asn212Ser Gly2lδAsp TrplOβGln + Leul26Gly + Thrl64Ser + Val203Gln Asn21δAsp Asp βOGlu + Val 9δGln + Leul26Pro + Glylδ7Asn Val203Thr Gin δ9Asn + TrplOβCys + Ala200Thr + Gly2UGln Ala216Gln Asn 62Ser + Ilel07Gly + Leul26Cys + Pro2lOGlu Thr220Gly Asn 62Gln + ThrlδδGlu + Val203Ser + Gly2lδSer Ala2lβThr Gin δ9Asn + Asp βOGlu + TrplOβPhe + Glylδ4Gln Thr206Pro Thr ββSer + AsnlδδGln + Val203Gln + Gln2OβGlu Tyr217His Glyl26Pro + Phelδ9Met + Val203Gly + Ser204Glu Ala216Glu Gin δ9Ser + Asn 62Ser + Leu 96Gly + Ser204Glu Asn2lδAsp Glnl03Ser + Glyl26Gln + Ser204Glu + Gly2HAsn Asn21δGlu Gly 97Pro + Prol29Gln + Glylδ7Asn + Ser204Asp Asn2lδGlu Leul26Asn + ThrlδδGln + VallβδMet + Gly2UGlu Lys213Glu Glylδ7Ser + Ser204Glu + Gln206Asp + Tyr2 17Cys + Thr220Gly Alal33Thr + Phelδ9Ser + Ser204Asp + Gln2 OβAsp Tyr214Ile GlylOOGln + Glylδ4Asn + Ser204Glu + Gln2 OβAsp Tyr217Thr Glyl27Asp + Glyl26Glu + Glylδ4Glu + Glylδ7Asn Phelδ9Ser GlylOOGln + TrplOβThr + Serl30Asp + Tyrl 67Glu Tyr217Thr GlulδβAsp + ThrlδδAsp + Tyrl67Gly + Pro2 OlGln Gly21δSer Glylδ7Gln + Val203Asp + Ser204Asp + Ala2lβPro Gly219Asp Leul26Gly + Prol29Glu + Glyl31Glu + Tyrl 67Met Thr220Gln Leu 96Ser + Serl30Asp + GlylββGlu + Ala2lβGln Tyr217lle AsnlδδGlu + GlylβOAsn + GlylββGlu + Tyr2 17Cys Thr220Asp Asn 62Asp Gly 97Gln + TrplOβGly + Pro210Asp + Asn212Gln Val 9δAsp Tyrl04Glu + Leul26Ser + AsnlδδGln + Gln206Ser Glylδ4Glu ThrlδδAsp + Phelδ9Glu + Gly21δAsn + Tyr217Met Ilel07Leu Glylδ4Asp + Glylδ7Glu + Val203His + Gly219Glu TrplOβlle AsnlδδSer + Serlδ9Asp + Serl91Glu + Ala2lβThr GlylOOAsp Leul26Asp + Glyl27Ser + Prol29Gln + Thr220Ser Alal33Pro Gln206Glu + Tyr214Ala + Asn21δGlu + Gly219Ser Thr. ββGly SerlOlGlu + Glyl02Asn + Leul26Glu + Ala216Pro GlylOOGlu Glyl02Glu + Tyrl04Glu + AsnlδδGln + Val203Ala Leul26His Alal67Glu + Val203Glu + Gln2OβAsp + Asn2lδGlu Asp βOGlu Leu 96Asn + Prol29Gln + Gly211Glu + Tyr217Met Leu 96Cys Ilel07Ala + Alal33Pro + Glylδ7Asp + GlylβOAsp Ser 63Asp ThrlδδGly + Gln2OβAsp + Tyr214Asp + Tyr217Asp Gin δ9Asp + Asn 62Asp + GlylOOGlu + Phel69Tyr + Tyr214Met SerlOlGlu + Glyl27Glu + Alal67Gln + Gln2OβAsn + Tyr217Ile Asn 62Asp + Ser 63Glu + GlylOOAsp + Glyl31Asn + Lys213Glu Asp βOGlu + Gly 97Asp + Ala 96Glu + Phel69His + Gly211Glu Asp βOGlu + Val 9δGlu + Asp 99Glu + SerlOlAsp + VallβδThr Glyl02Gln + Glylδ4Glu + AsnlδδGlu + Serl91Asp + Gln2OβAsp Asn βlSer + Thr ββSer + Leul26Glu + AsnlδδGlu + Glylδ7Asp Prol29Asn + Alal33Gln + Phel69Ile + Gln206Glu + Lys213Glu Asn βlSer Gln2OβAsp + Lys213Glu + Tyr217Ala + Gly219Asn Gin δ9Asn Glyl26Asn + Ala200Thr + Gln206Glu + Lys213Glu Phel89Gln Val203Gly + Gln2OβAsp + Lys213Asp + Tyr217Pro Ala 98His Glylδ4Glu + Serl63Asp + Val203Met + Tyr217Met Leu 96Met Prol29Gly + Glylδ4Glu + Serl63Glu + Tyr217Ser Gly 97Pro Ser204Glu + Lys213Asp + Ala216Glu + Gly219Ser VallβδSer Lys213Glu + Tyr214Cys + Ala216Glu + Tyr217Pro Serl91Glu Ser204Glu + Gln2OβAsp + Tyr214His + Ala216Asp Glyl02Pro AsnlδδAsp + Ala216Glu + Tyr217His + Asn216Glu AsnlδδAsp + Gly21δPro + Ala216Glu + Tyr217Ser + Asn216Glu GlylβOSer + Ser204Glu + Gln206Glu + Lys213Glu + Ala216Ser Ala 98Thr + Alalδ7Ser + Ser204Glu + Gln206Glu + Lys213Asp Glyl27Pro + Ser204Glu + Gln206Glu + Lys213Glu Tyr217Ala Leul26Met + Prol29Glu + Serlβ3Glu + Phelδ9Thr Asn21δSer SerlOlAsp + Ser204Asp + Gln206Glu + Ala216Asn Tyr217Glu Val 9δAla Tyrl67Asp + Ser204Glu + Gln206Glu Tyr217Glu AsnlδδGlu GlulδβAsp + Thrl64Asp + Ser204Glu Tyr214Thr TrplOβPro Glyl27Asp + Serl30Asp + AsnlδδAsp Gly219Gln Prol29Ser Ser204Asp + Gln20βGlu + Pro210Asp Asn21δGlu Tyrl04Val Leul26Asp + Glylδ7Asp + Serl63Asp Thrl64Asp Leu 96Asp Gly 97Asp + Glnl03Asp + Tyr217Cys Gly219Asp Serlδ9Glu Asn212Gln + Lys213Glu + Gly2lδAsp + Ala216Glu Gin δ9Asp + Asn 62Glu + Ser 63Glu + Prol29Ser + AsnlδδAsp Glnl03Ser + Tyrl04Ala + Val203Asp + Gln2OβAsp Lys213Glu Val 9δGlu + GlulδβAsp + Glylδ7Asp + Tyr214Gly Thr220Asp Val 9δGlu + Gly21δGlu + Ala216Glu + Tyr217Leu Gly219Ser Ser 63Asp + GlylβOAsp + SerlβlGlu + Val203Ser Tyr217Cys GlylβOAsp + SerlβlAsp + Tyrl67Met + Ser204Asp + Tyr217Ala Leu 96His + TrplOβAsp + Gln2OβAsn + Asn2lδAsp + Gly219Asp GlylOOGlu SerlOlAsp TrplOβMet Serl62Asp + Thrl64Pro SerlOδGlu Alal67Ser Val203Glu Ser204Asp + Ala216Gly Asp βOGlu TrplOβAsn Val203Glu Ser204Glu + Ala216Gln Glnl03Asp Serl63Glu Thrl64Glu Pro201Gln + Ala216Pro Val 9δGln GlylOOAsn GlulδβAsp Glylδ7Asp + Lys213Glu ThrlδδAsp Serlδ9Asp Ser204Glu Gly21δAsn + Tyr217Cys SerlOδAsp TrplOβGlu Thrl64Asn Ala216Asp + Gly219Ser Gin δ9Glu Asp βOGlu Tyrl04Asn Serl91Glu + Pro201Gln Glnl03Asp SerlβlGlu Serl62Asp Gln206Ser + Tyr217His Ala 96Asp Asp 99Glu SerlOδGlu Thrl64Gln + Alal87Ser Glylδ4Asp AsnlδδAsp Ser204Glu Ala216Gln + Tyr217Ala Asn βlGlu Tyrl04Ser Gln206Glu Ala216Glu + Tyr217Cys Glylδ7Ser ThrlδδGlu Gln2OβAsp Lys213Asp + Ala216Asp Val 9δThr Glylδ7Glu SerlδδGlu Ser204Glu + Ala216Asp Tyrl04His AsnlδδGlu Glylδ7Asn Tyrl67Glu + Gly202Ser Glyl26Asp Glylδ7Asn Pro210Gln Asn216Glu + Thr220Glu Asn 62Glu Val 9δAla GlylOOAsp Lys213Glu + Tyr217His GlylββAsp Gln206Ser Gly21δPro Tyr217Asp + Gly219Asp Serl30Asp Serl63Asp Tyrl67Ser Serl91Asp + Tyr217Met Gly 97Pro Serl32Asp ThrlδδGly Ser204Glu + Ala216Asp Glylδ4Asp Serl91Asp Lys213Asp Tyr214Ala + Tyr217Asn Asn βlGln Ilel07His Ser204Glu Lys213Glu + Asn218Glu Gin δ9Asp Ala 9δGlu Glyl02Asp SerlOδGlu + Leu209Thr Alal33Gly Glylδ4Asp Gln206Glu Gly21δGlu + Thr220Gln Glylδ4Asn GlylβOSer GlylββGlu Gln2OβAsp + Gly2lδAsp Leu 96Glu Ala 96Asn Tyrl67Asn Gln206Glu + Gly21δGlu Serl62Glu Thrl64Glu Thr208Gln Ala216Asp + Tyr217Glu Val 9δAsp Ilel07Asp Tyrl67His SerlδδGlu + Thr220Asn Glylδ4Glu GlylββAsp Lys213Asp Ala216Ser + Tyr217Cys Gly 97Glu Asp 99Glu GlulδβAsp Tyrl67Ala + Ala216Pro Thr ββGly Glnl03Asp TrplOβGlu Glyl26Asn + Serl62Asp Glnl03Glu SerlOδGlu ThrlδδSer Leu209Thr + Lys213Glu Thr ββGln Thrl64Asp Val203His Gly211Glu + Lys213Glu Prol29Asn Glyl31Gln Thrl64Glu Gly211Glu + Lys213Asp Serlδ9Asp Serlβ2Glu Gln20βSer Pro210Glu + Tyr214Ala Asp 99Glu SerlOlAsp Glyl31Asn Lys213Glu + Gly21δSer Glnl03Glu Tyrl04Gly Thrl64Pro Pro210Asp + Asn212Glu Asn 62Ser Serl32Asp GlylβOGlu Serl62Glu + Ala216His GlylβOGlu Serl62Asp Tyrl6711e Ser204Glu + Gly219Ser Asp βOGlu Ser 63Asp Serl30Glu Gly202Gln + Gly21δSer Glylδ4Glu GlulδβAsp Pro210Glu Lys213Asp + Asn21δGln SerlOδAsp TrplOβGly Glyl27Asp Glylδ4Asp + VallβδGln Asn 62Glu GlylOOGlu Glylδ7Asn GlylββGlu + Tyr217Leu Asn 62Asp Prol29Gly Alal33Gly Ser204Asp + Gln2OβAsp Asp βOGlu GlylOOAsn Ser204Asp Gln206Glu + Pro210Ser Serl62Glu Thrl64Glu Val203Thr Ser204Asp + Asn212Ser Gly 97Glu Serl30Glu Tyrl67Asp Tyr217Val + Gly219Ser Glyl26Glu Serl63Glu GlylββGlu Gln206Glu + Ala216Ser Asp βOGlu Asn βlGlu Alal67Gly Lys213Glu + Ala216Glu Gly 97Asp SerlOlAsp Tyrl04Glu SerlβlGlu + Tyr217Val Ser 63Glu + Ilel07Gln + Gln206Asp + Ala216Asp + Thr220Glu Serl30Glu + Serl32Glu + Glyl60Asp + Ala216Gln + Thr220Gly Val 9δGlu + Serl30Asp + Serl32Glu + Ala200Gly + Tyr217His Thr ββGly + GlylOOGlu + Glnl03Asp + Serl32Asp + Tyr217Asn Asp βOGlu + Glyl28Glu + Gln206Asn + Pro210Glu + Ala216Gln Leul26Val + ThrlδδGlu + Val203Met + Lys213Asp + Gly21δGlu Asp 99Glu + Serlδ9Glu + Thrl64Glu + Tyrl67Leu + Gln206Ser Val 9δAsp + Prol29Asn + Thrl64Gln + Ala216Glu + Asn2lδGlu Glylδ4Asp + Alal67Gly + Gly2lδAsp + Tyr217Thr + Asn2lδGlu Asn 62Glu + Gly 97Asp + GlylOOAsn + Ser204Glu + Tyr217Cys Asn 62Glu + Gly 97Asp + Glulδ6Asp + Val203Cys + Ala216Gly Asn 62Asp + Gly 97Asp + Ser204Asp + Tyr214Leu + Tyr217Leu GlulδβAsp + Serl63Asp + Gln206Ser + Gly2lδAsp + Ala216Asp Serlδ9Glu + Serl63Glu + Phelδ9His + Ser204Glu + Tyr217Ser GlylOOPro + AsnlδδGln + Serlδ9Asp + Serlβ3Asp + Ser204Glu Glyl02Asp + Alal87Asp + SerlδδAsp + Val203His + Ser204Asp Asp 99Glu + ThrlδδAsp + Serl62Asp + Val203Met + Ala216Thr Val 9δCys + Gly 97Pro + Serl63Glu + Serl91Asp + Ser204Asp Leu 96Glu + Asp 99Glu + Serlδ9Glu + Gln2OβAsn + Ala2lβThr Glyl27Pro + Serl62Glu + Serl91Glu + Gly211Glu + Asn212Asp Ser 63Glu + Serl91Asp + Gln206Asp + Ala216Asp + Tyr217Gln Ser 63Glu + Phel69Ile + Val203Met + Gln2OβAsp + Gly211Glu TrplOβTyr + Phel89Asp + Pro210Asp + Lys213Glu + Asn2lδGlu Serl91Glu + Gln206Glu + Ala216Gly + Tyr217Leu + Thr220Asp Val 9δGly + Thrl δAsp + Serl61Asp + Alalδ7Pro + Asn2lδAsp Thr ββGlu + GlylββGlu + Phelδ9Val + Serl91Glu + Gly219Ser Asp βOGlu + Asp 99Glu + Gln206Glu + Gly211Pro + Ala216Glu Asn 61Asp + Ser 63Asp + Glnl03Glu + Lys213Asp + Tyr217Pro Tyrl04Glu + Glyl26Gln + Serl32Glu + Asn212Asp + Ala216Ser Asn 62Asp + Ser204Asp + Gly21δGlu + Ala216Gln + Tyr217Leu Asn 61Asp + GlylOOAsp + Trpl06Ala + Asn212Gln + Lys213Asp Glyl27Glu + Glylδ7Gln + Ser204Asp + Lys213Asp + Ala216Glu Leu 96Glu + Gly 97Ser + GlylOOGlu + Gln2OβAsp + Lys213Asp Asp 60Glu + Leu 96Cys + Gly 97Glu + Ser204Glu + Gly21δAsn Tyrl67Pro + Ser204Asp + Lys213Glu + Ala216His + Gly219Glu Gly 97Ser + SerlOδAsp + AsnlδδGlu + GlylββAsp + Val203Asn Glyl02Asn + GlylβOAsn + Thrl64Glu + Gln2OβAsn + Thr220Asp Asn 61Ser + Ala 98Asp + AsnlδδAsp + SerlδδGlu + Val203Ser GlulδβAsp + Ser204Asp + Gln206Glu + Lys213Glu + Ala216Pro Asp 99Glu + Glylδ7Pro + Ser204Glu + Gln2OβAsp + Lys213Glu Serl30Asp + GlylβOAsn + Ser204Glu + Gln2OβAsn + Gly2lδAsp Glyl27Glu + GlulδβAsp + Ser204Glu + Gln2OβAsp + Tyr214Pro Ala 96Glu + Asp 99Glu + Trpl06Gly + Glylδ4Asp + Asn21δGlu Gin δ9Ser + Val 9δGlu + Ala 98Asn + SerlOδGlu + Gln206Glu Gly 97Pro + Glyl28Glu + Lys213Asp + Ala216Glu + Asn21δGlu Glnl03Asp + Ilel07Asp + Glylδ7Pro + Tyrl67Glu + Ala216Glu Asp 60Glu + Gln206Glu + Lys213Asp + Gly21δPro + Asn2lδGlu Serl30Glu + Thrl64Glu + Val203Met + Ser204Asp + Gln2OβAsp Asp βOGlu + Ser 63Glu + Glylδ4Asp + GlylββSer + SerlδδAsp Leu 96His + Serl30Glu + GlulδβAsp + Tyrl67Glu + Lys213Glu Gin δ9Ser + GlulδβAsp + GlylδOGlu + Gly211Glu + Lys213Glu Glyl27Glu + AsnlδδAsp + Alal67His + Ala216Glu + Tyr217His Glnl03Glu + GlylβOAsn + Gln206Glu + Tyr214Gly + Asn2lδGlu Ser 63Asp + Gly202Pro + Lys213Asp + Gly21δGln + Asn218Asp Asp βOGlu + Leu 96Glu + Thrlδ8Gln + GlylδβPro + Gln2OβAsp Gly 97Asp + Glnl03Asp + Phel89Ala + Gln206Ser + Lys213Asp Asn 62Asp + Thr 66Glu + Tyrl04Pro + Serl32Asp + Asn212Asp Ala 98Pro + Prol29Asp + Serl30Asp + Lys213Glu + Tyr217Glu Ser 63Asp + GlulδδAsp + Gln206Glu + Lys213Glu + Ala216Pro Asp βOGlu + Glyl02Gln + SerlOδGlu + Thrl64Gln + Gly211Glu Asp βOGlu + ThrlδδGln + Lys213Glu + Ala216Gln + Tyr217Val Ilel07Asp + Glyl31Asp + Ala216Asp + Tyr217His + Asn2lδAsp Ser 63Asp + GlylOOGlu + Glnl03Asp + Gln206Asn + Gly219Asp AsnlδδGlu + Glylδ7Glu + Gln206Asn + Pro210Asp + Ala216Glu Ser 63Asp + Ilel07Met + Prol29Asn + Serl91Asp + Gly219Glu Ser 63Asp + Val 9δAla + Asp 99Glu + Leul26Thr + Serl63Asp Thr ββGlu + GlylOOGln + Glnl03Asp + Lys213Asp + Ala216Asn ThrlδδAsp + SerlδlAsp + Gln206Asp + Tyr217Cys + Gly219Asp Ser 63Glu + Serl62Asp + Alalδ7Gln + Gly211Asn + Lys213Asp Gly 97Ser + SerlOlAsp + Val203Cys + Tyr214Glu + Tyr217Asp Val 9δGlu + Asp 99Glu + Ser204Asp + Gly21δGlu + Asn216Gln Gin δ9Glu + Thr 66Asp + Serl63Asp + Pro201Gln + Gly21δGlu Ala 9δHis + SerlOlGlu + Glyl66Gln + SerlδδAsp + Val203Asp Ser 63Asp + GlylδOAsp + Val203Ala + Ser204Asp + Gln206Glu Glyl27Glu + Serl62Glu + Serl63Glu + Lys213Asp + Ala216His Serl62Asp + Alal87Glu + Pro201Gln + Gln206Asp + Tyr217Glu Glylδ7Glu + Phel89Tyr + Val203Glu + Ser204Glu + Lys213Glu GlylβOGlu + SerlδlAsp + Tyrl67Glu + Gly202Asn + Gln206Glu Asp βOGlu + Serlδ9Asp + Thrl64Glu + Phelδ9His + Lys213Glu Tyrl04Cys + Serl62Glu + Lys213Glu + Asn2lδAsp + Gly219Glu Tyrl04Asp + Glyl28Asn + Serl30Asp + Glylδ7Ser + Ser204Glu Serl32Glu + Glylδ7Ser + Serl63Asp + Asn212Asp + Lys213Glu Gly 97Asp + Ala 98Asp + Prol29Glu + Tyrl67Leu + Gln206Asp SerlOlGlu + ThrlδδGln + Alalδ7Glu + SerlδδGlu + Gln206Glu Asp 99Glu + GlylOOAsp + AsnlδδAsp + GlylββGln + Ser204Glu Serl30Glu + Serl61Glu + Serl62Asp + Thrl64Asn + Gly211Asp Gin δ9Asn + Tyrl04Asp + ThrlδδAsp + Serl91Glu + Asn21δGlu Asp βOGlu + SerlOlGlu + Ser204Glu + Gln206Ser + Pro210Asp Serl30Asp + Serlδ9Asp + Serl63Glu + Pro210Gln + Tyr217Asp Asn 61Asp + GlylOOAsp + Trpl06Pro + Glyl26Glu + Tyr217Asp Glyl02Pro + Glyl31Asp + SerlδδAsp + Ser204Glu + Gln206Glu GlulδβAsp + Ser204Asp + Gln206Asp + Asn212Asp + Ala216His Thr ββPro + Glnl03Asp + Glul 6Asp + Serl91Glu + Gln206Asp Glyl31Pro + Phel69Leu + Serl91Glu + Gln206Glu + Lys213Glu Ala 96Glu + Glylδ7Ser + Gln206Asp + Lys213Asp + Gly21δGln Tyrl04Leu + ThrlδδGlu + Gly202Ser + Gln206Glu + Lys213Glu Ser 63Glu + Ala 96Gln + Glyl02Asn + Serl30Asp + Tyr217Glu ThrlδδGlu + Glyl66Asn + Pro210Glu + Lys213Glu + Thr220Glu TrplOβThr + Glylδ4Ser + Glylδ7Asp + Lys213Glu + Ala216Glu Ala 9δSer + Alal67Glu + Lys213Asp + Gly21δGln + Ala216Asp Tyrl04Pro + Serlδ9Asp + Gly202Asn + Lys213Glu + Ala216Asp Leul26Asn + AsnlδδGlu + Thrl64Asn + Lys213Asp + Ala216Glu SerlβlAsp + Val203His + Ser204Asp + Gly211Asp + Tyr217Asp Asn 61Asp + Serl63Asp + Val203His + Ser204Glu + Tyr217Asp Val 9δAsp + TrplOβGlu + SerlβlGlu + Alalδ7Pro + Ser204Asp Leu 96Glu + GlylOOAsp + TrplOβCys + SerlδδGlu + Gln2OβAsp SerlOlGlu + Ser204Asp + Gly211Glu + Lys213Asp + Gly21δAsn Asp 99Glu + Serlδ9Glu + Serl62Glu + Ser204Asp + Gly219Asn Leu 96Ala + Glnl03Asp + Leul26Val + Glyl26Asp + Ser204Asp Ala 9δGlu + SerlOδGlu + Glylδ4Glu + GlulδβAsp + Phel89Pro Asn βlGlu + Serlδ9Glu + Gln206Ser + Pro210Glu + Ala216Glu Gly 97Asp + SerlOlAsp + Alal33Glu + Gln206Glu + Gly219Pro Leul26Ala + Glyl31Glu + Ser204Glu + Pro210Asp + Lys213Glu Val 9δGlu + Ala 98Asn + Glyl02Glu + Serl62Asp + Ser204Glu Asn βlGlu + GlylOOAsn + Prol29Asp + Serl63Glu + Asn218Ser Glyl02Asp + Glyl27Ser + ThrlδδAsp + GlylβOGlu + Lys213Glu Serl30Asp + AsnlδδGln + ThrlδδGlu + Serl91Asp + Gly21δGlu Alal33Asp + Serlδ9Glu + SerlβlAsp + Ser204Asp + Ala216Gln Serl32Glu + Thrl64Asp + Ser204Asp + Gln206Glu + Tyr217Pro Glylδ7Glu + Tyrl67Asp + Ser204Glu + Gln206Glu + Ala216Asn Thr ββSer + Serl30Glu + ThrlδδGlu + Ser204Glu + Gln206Glu Asp 99Glu + Serlδ9Glu + Ser204Glu + Gln206Glu + Tyr217Pro Thr ββSer + SerlOδAsp + Serlδ9Glu + Ser204Glu + Gln2OβAsp Asp βOGlu + Glyl27Asp + Ser204Glu + Gln206Glu + Tyr214Asn Ser 63Glu + Serl30Asp + Gln2OβAsp + Ala216Gly + Asn2lδAsp Prol29Gly + Serlδ9Glu + SerlδδGlu + Phelδ9Cys + Ser204Asp Glyl31Asp + GlulδβAsp + Serl62Glu + Alal67Pro + Tyr214Gly Glyl02Asp + TrplOβGlu + Serlδ9Glu + Pro210Gln + Thr220Asp Glyl31Asp + SerlβlAsp + Serl63Asp + GlylββAsn + Ser204Asp Gin δ9Asn + SerlδδAsp + Gln2OβAsp + Gly211Glu + Tyr217Glu Ala 9δGlu + Glylδ7Asp + Thrl64Asp + Phel89Thr + Lys213Asp Glnl03Asp + TrplOβTyr + GlylβOAsp + Lys213Glu + Gly2lδAsp Val 9 Asp + Glyl31Gln + Serlδ9Asp + Ala216Asp + Asn2lδAsp SerlOlAsp + Glnl03Glu + SerlβlGlu + Gln206Glu + Ala216His Thr ββGlu + Glyl2δPro + Glylδ4Asp + Thrl64Asp + Ser204Glu Val 9δAsp + Glyl31Glu + Serl63Asp + Serl91Glu + Gln2OβAsn Val 9δSer + Ala 96Glu + SerlOlAsp + Glyl31Asp + Phelδ9Asp Asn 62Asp + Leul26His + Glyl31Pro + Lys213Glu + Tyr217Asp Ser 63Asp + Serl30Glu + ThrlδδPro + Ala216Glu + Tyr217Ile Gin δ9Asp + Glylδ7Asp + Gln206Glu + Tyr214Val + Asn2lδAsp Val 9δGlu + Asp 99Glu + Gly2lδAsp + Ala216Asn + Tyr217Ile Serl32Glu + Glylδ4Gln + Glylδ7Glu + SerlβlAsp + Tyr214Ser SerlOlAsp + Glyl31Pro + SerlδδAsp + Serl91Glu + Gln206Glu Thr ββAsp + Leu 96Glu + GlulδβAsp + Val203His + Gly2lδAsp Asn 62Glu + GlylββGln + SerlδδGlu + Gly211Glu + Ala216His Ilel07Asp + Alalδ7Asp + Serl91Asp + Gln2OβAsp + Ala216Thr SerlOδAsp + Serlδ9Glu + Serl91Asp + Lys213Asp + Ala2lβThr Asnl δAsp + Serl63Asp + VallβδAsn + Gln206Ser + Lys213Glu SerlOlGlu + Glyl31Asn + AsnlδδGlu + Alal87Glu + Lys213Asp Gin δ9Glu + GlylβOAsp + SerlδδAsp + Val203Glu + Tyr217Ile In another embodiment of the present invention, an effective amount of one or more of the enzyme variants are included in compositions useful for cleaning a variety of surfaces in need of proteinaceous stain removal. Such cleaning compositions include detergent compositions for cleaning hard surfaces, unlimited in form (e.g., liquid and granular); detergent compositions for cleaning fabrics, unlimited in form (e.g., granular, liquid and bar formulations); dishwashing compositions (unlimited in form); oral cleaning compositions, unlimited in form (e.g., dentifrice, toothpaste and mouthwash formulations); denture cleaning compositions, unlimited in form (e.g., liquid, tablet); and contact lens cleaning compositions, unlimited in form (e.g., liquid, tablet). The cleaning compositions also comprise, in additin to the BPN' variants described hereinbefore, one or more cleaning composition materials compatible with the protease enzyme, the term "cleaning composition material", as used herein, means any liquid, solid or gaseous material selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, granule, bar, spray, stick, paste, gel), which materials are also compatible with the BPN' variant used in the composition, the specific selection of cleaning composition materials are readily made by considering the surface material to be cleaned, the desired form of the composition for the cleaning condition during use (e.g., through the wash detergent use). The term "compatible", as used herein, means the cleaning composition materials do not reduce the proteolytic activity of the BPN' variant to such an extent that the protease is not effective as desired during normal use situations. Specific cleaning composition materials are exemplified in detail hereinafter. As used herein, "effective amount of enzyme variant" refers to the quantity of enzyme variant necessary to achieve the enzymatic activity necessary in the specific cleaning composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and is based on many factors, such as the particular enzyme variant used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) composition is required, and the like. Preferably the cleaning compositions comprise from about 0.0001% to about 10% of one or more enzyme variants of the present invention, more preferably from about 0.001 % to about 1%, more preferably still from about 0.01% to about 0.1%. Several examples of various cleaning compositions wherein the enzyme variants may be employed are discussed in further detail below. All parts, percentages and ratios used herein are by weight unless otherwise specified. As used herein, "non-fabric cleaning compositions" include hard surface cleaning compositions, dishwashing compositions, oral cleaning compositions, denture cleaning compositions and contact lens cleaning compositions. A. Cleaning Compositions for Hard Surfaces. Dishes and Fabrics The enzyme variants of the present invention can be used in a variety of detergent compositions where high sudsing and good insoluble substrate removal are desired. Thus the enzyme variants can be used with various conventional ingredients to provide fully-formulated hard-surface cleaners, dishwashing compositions, fabric laundering compositions and the like. Such compositions can be in the form of liquids, granules, bars and the like. Such compositions can be formulated as modern "concentrated" detergents which contain as much as 30%-60% by weight of surfactants. The cleaning compositions herein can optionally, and preferably, contain various anionic, nonionic, zwitterionic, etc., surfactants. Such surfactants are typically present at levels of from about 5% to about 35% of the compositions. Nonlimiting examples of surfactants useful herein include the conventional C-j 1-C-J8 a -ky* benzene sulfonates and primary and random alkyl sulfates, the C10-C18 secondary (2,3) alkyl sulfates of the formulas CH 3 (CH 2 )x(CHOSO 3 )-M + )CH 3 and CH 3 (CH 2 )y(CHOSO 3 -M + ) CH 2 CH 3 wherein x and (y+1 ) are integers of at least about 7, preferably at least about 9, and M is a water-solubilizing cation, especially sodium, the C-|o-C-|8 a ** y* alkoxy sulfates (especially EO 1-5 ethoxy sulfates), C-|o-C-|8 alkyl alkoxy carboxylates (especially the EO 1-5 ethoxycarboxylates), the C10-OJ8 alkyl polyglycosides, and their corresponding sulfated polyglycosides, C12-C18 alpha-sulfonated fatty acid esters, C12-C18 a " < y* anc * a " < y* phenol alkoxylates (especially ethoxylates and mixed ethoxy/propoxy), C12-C-18 betaines and sulfobetaines ("sultaines"), C-jo-C-18 a * ne oxides, and the like. The alkyl alkoxy sulfates (AES) and alkyl alkoxy carboxylates (AEC) are preferred herein. (Use of such surfactants in combination with the aforesaid amine oxide and/or betaine or sultaine surfactants is also preferred, depending on the desires of the formulator.) Other conventional useful surfactants are listed in standard texts. Particularly useful surfactants include the C10-C-18 N-methyl glucamides disclosed in US Patent 5, 194,639, Connor et al., issued March 16, 1993, incorporated herein by reference. A wide variety of other ingredients useful in detergent cleaning compositions can be included in the compositions herein, including other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, solvents for liquid formulations, etc. If an additional increment of sudsing is desired, suds boosters such as the C-10-C16 alkolamides can be incorporated into the compositions, typically at- about 1 % to about 10% levels. The C-10-C1 monoethanol and diethanol amides illustrate a typical class of such suds boosters. Use of such suds boosters with high sudsing adjunct surfactants such as the amine oxides, betaines and sultaines noted above is also advantageous. If desired, soluble magnesium salts such as MgCl2, MgSO , and the like, can be added at levels of, typically, from about 0.1 % to about 2%, to provide additionally sudsing. The liquid detergent compositions herein can contain water and other solvents as carriers. Low molecular weight primary or secondary alcohols exemplified by methanol, ethanol, propanol, and isopropanol are suitable. Monohydric alcohols are preferred for solubilizing surfactants, but polyols such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (e.g., 1 ,3-propanediol, ethylene glycol, glycerine, and 1 ,2-propanediol) can also be used. The compositions may contain from about 5% to about 90%, typically from about 10% to about 50% of such carriers. The detergent compositions herein will preferably be formulated such that during use in aqueous cleaning operations, the wash water will have a pH between about 6.8 and about 11.0. Finished products thus are typically formulated at this range. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art. When formulating the hard surface cleaning compositions and fabric cleaning compositions of the present invention, the formulator may wish to employ various builders at levels from about 5% to about 50% by weight. Typical builders include the 1-10 micron zeolites, polycarboxylates such as citrate and oxydisuccinates, layered silicates, phosphates, and the like. Other conventional builders are listed in standard formularies. Likewise, the formulator may wish to employ various additional enzymes, such as cellulases, lipases, amylases and proteases in such compositions, typically at levels of from about 0.001% to about 1% by weight. Various detersive and fabric care enzymes are well-known in the laundry detergent art. Various bleaching compounds, such as the percarbonates, perborates and the like, can be used in such compositions, typically at levels from about 1 % to about 15% by weight. If desired, such compositions can also contain bleach activators such as tetraacetyl ethylenediamine, nonanoyloxybenzene sulfonate, and the like, which are also known in the art. Usage levels typically range from about 1 % to about 10% by weight. Various soil release agents, especially of the anionic oligoester type, various chelating agents, especially the aminophosphonates and ethylenediaminedisuccinates, various clay soil removal agents, especially ethoxylated tetraethylene pentamine, various dispersing agents, especially polyacrylates and polyasparatates, various brighteners, especially anionic brighteners, various suds suppressors, especially silicones and secondary alcohols, various fabric softeners, especially smectite clays, and the like can all be used in such compositions at levels ranging from about 1 % to about 35% by weight. Standard formularies and published patents contain multiple, detailed descriptions of such conventional materials. Enzyme stabilizers may also be used in the cleaning compositions. Such enzyme stabilizers include propylene glycol (preferably from about 1 % to about 10%), sodium formate (preferably from about 0.1 % to about 1 %) and calcium formate (preferably from about 0.1 % to about 1 %). 1. Hard surface cleaning compositions As used herein "hard surface cleaning composition" refers to liquid and granular detergent compositions for cleaning hard surfaces such as floors, walls, bathroom tile, and the like. Hard surface cleaning compositions of the present invention comprise an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001 % to about 10%, more preferably from about .01 % to about 5%, more preferably still from about .05% to about 1 % by weight of active enzyme of the composition. In addition to comprising one or more of the enzyme variants, such hard surface cleaning compositions typically comprise a surfactant and a water-soluble sequestering builder. In certain specialized products such as spray window cleaners, however, the surfactants are sometimes not used since they may produce a filmy/streaky residue on the glass surface. The surfactant component, when present, may comprise as little as 0.1 % of the compositions herein, but typically the compositions will contain from about 0.25% to about 10%, more preferably from about 1 % to about 5% of surfactant. Typically the compositions will contain from about 0.5% to about 50% of a detergency builder, preferably from about 1 % to about 10%. Preferably the pH should be in the range of about 8 to 12. Conventional pH adjustment agents such as sodium hydroxide, sodium carbonate or hydrochloric acid can be used if adjustment is necessary. Solvents may be included in the compositions. Useful solvents include, but are not limited to, glycol ethers such as diethyleneglycol monohexyl ether, diethyleneglycol monobutyl ether, ethyleneglycol monobutyl ether, ethyleneglycol monohexyl ether, propyleneglycol monobutyl ether, dipropyleneglycol monobutyl ether, and diols such as 2,2,4-trimethyl-1 ,3- pentanediol and 2-ethyl-1,3-hexanediol. When used, such solvents are typically present at levels of from about 0.5% to about 15%, preferably from about 3% to about 11%. Additionally, highly volatile solvents such as isopropanol or ethanol can be used in the present compositions to facilitate faster evaporation of the composition from surfaces when the surface is not rinsed after "full strength" application of the composition to the surface. When used, volatile solvents are typically present at levels of from about 2% to about 12% in the compositions. The hard surface cleaning composition embodiment of the present invention is illustrated by the following examples. Examples 7-12 Oisodium N-diethyleneglycol-N,N-iminodiacetate **Na4 ethylenediamine diacetic acid 'Diethyleneglycol monohexyl ether *AII formulas adjusted to pH 7 In Examples 7-10, the BPN' variants recited in Tables 2-25, among others, are substituted for Ser105Glu, with substantially similar results. In Examples 11-12, any combination of the BPN' variants recited in Tables 2-25, among others, are substituted for Gly127Gln + Ala216Pro, with substantially similar results. Examples 13-18 Spray Compositions for Cleaning Hard Surfaces and Removing Household Mildew Product pH is about 7. In Examples 13-16, the BPN' variants recited in Tables 2-25, among others, are substituted for Tyr104lle + Gly215Pro, with substantially similar results. In Examples 17-18, any combination of the BPN' variants recited in Tables 2-25, among others, are substituted for Tyr104lle + Gly215Pro and Asp99Glu, with substantially similar results. 2. Dishwashing Compositions In another embodiment of the present invention, dishwashing compositions comprise one or more enzyme variants of the present invention. As used herein, "dishwashing composition" refers to all forms for compositions for cleaning dishes, including but not limited to, granular and liquid forms. The dishwashing composition embodiment of the present invention is illustrated by the following examples. Examples 19-24 Dishwashing Composition Product pH is adjusted to 7. In Examples 19-22, the BPN * variants recited in Tables 2-25, among others, are substituted for Gln59SSer + Leu96Gly + Ser204Glu, with substantially similar results. In Examples 23-24, any combination of the BPN' variants recited in Tables 2-25, among others, are substituted for Gln59SSer + Leu96Gly + Ser204Glu and Lys96Gly + Ser204Glu, with substantially similar results. 3. Fabric cleaning compositions In another embodiment of the present invention, fabric cleaning compositions comprise one or more enzyme variants of the present invention. As used herein, "fabric cleaning composition" refers to all forms for detergent compositions for cleaning fabrics, including but not limited to, granular, liquid and bar forms. Preferred fabric cleaning compositions are those in the liquid form, a. Granular fabric cleaning compositions The granular fabric cleaning compositions of the present invention contain an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001% to about 10%, more preferably from about 0.005% to about 5%, more preferably from about 0.01% to about 1% by weight of active enzyme of the composition. In addition to one or more enzyme variants, the granular fabric cleaning compositions typically comprise at least one surfactant, one or more builders, and, in some cases, a bleaching agent. The granular fabric cleaning composition embodiment of the present invention is illustrated by the following examples. Examples 25-28 Granular Fabric Cleaning Composition In Examples 25-26, the BPN' variants recited in Tables 2-25, among others, are substituted for SerlOlAsp, with substantially similar results. In Examples 27-28, any combination of the BPN' variants recited in Tables 2-25, among others, are substituted for SerlOlAsp and Thr66Glu, with substantially similar results. Examples 29-32 Granular Fabric Cleaning Composition In Examples 29-30, the BPN' variants recited in Tables 2-25, among others, are substituted for Val95Asp + Leu126Ser + Asn155Gln, with substantially similar results. In Examples 31-32, any combination of the BPN" variants recited in Tables 2-25, among others, are substituted for Val95Asp + Leu126Ser + Asn155Gln and Gly65Ser + Gly102Asn + Val203Glu, with substantially similar results. Examples 33-36 Granular Fabric Cleaning Composition In Examples 33-34, the BPN' variants recited in Tables 2-25, among others, are substituted for Ser63Glu, with substantially similar results. In Examples 35-36, any combination of the BPN' variants recited in Tables 2-25, among others, are substituted for Ser63Glu and Leu96Asn + Lys213Asp, with substantially similar results. Examples 37-40 Granular Fabric Cleaning Composition Example No. Component 37 38 39 40 Asn62Ser +Ser163Asp + Phe189Ser 0.10 0.20 0.03 0.05 + Ala216Glu Gly97Ser + Trp106lle + Tyr217Leu 0.02 0.05 C-|2 a 'ky' benzene sulfonate 12.00 12.00 12.00 12.00 Zeolite A (1-10 micrometer) 26.00 26.00 26.00 26.00 2-butyl octanoic acid 4.00 4.00 4.00 4.00 C-J2-C-14 secondary (2,3) alkyl sulfate, 5.00 5.00 5.00 5.00 Na salt Sodium citrate 5.00 5.00 5.00 5.00 Optical brightener 0.10 0.10 0.10 0.10 Sodium sulfate 17.00 17.00 17.00 17.00 Water and minors balance to 100% In Examples 37-38, the BPN' variants recited in Tables 2-25, among others, are substituted for Asn62Ser + Ser163Asp + Phe189Ser + Ala216Glu, with substantially similar results. In Examples 39-40, any combination of the BPN' variants recited in Tables 2-25, among others, are substituted for Asn62Ser + Ser163Asp + Phe189Ser + Ala216Glu and Gly97Ser + Trp106lle + Tyr217Leu, with substantially similar results. Examples 41-42 Granular Fabric Cleaning Composition Examples 43-44 Granular Fabric Cleaning Composition Alkyl Sulphate 8.0 Alkyl Ethoxy Sulphate 2.0 Mixture of C25 and C45 alcohol 3 and 7 times ethoxylated 6.0 Polyhydroxy fatty acid amide 2.5 Zeolite 17.0 Layered silicate/citrate 16.0 Carbonate 7.0 Maleic acid acrylic acid copolymer 5.0 Soil release polymer 0.4 Carboxymethyl cellulose 0.4 Poly (4-vinylpyridine) -N-oxide 0J Copolymer of vinylimidazole and vinylpyrrolidone 0.1 PEG2000 ' 0.2 Val95Gln + Tyrl 04Glu + Glyl 27Gln + Lys213Glu 0.5 + Ala216Asp Lipase 0.2 Example 47 Granular Fabric Cleaning Composition Component Weight % Liquid fabric cleaning compositions of the present invention comprise an effective amount of one or more enzyme variants of the present invention, preferably from about 0.005% to about 5%, more preferably from about 0.01% to about 1%, by weight of active enzyme of the composition. Such liquid fabric cleaning compositions typically additionally comprise an anionic surfactant, a fatty acid, a water-soluble detergency builder and water. The liquid fabric cleaning composition embodiment of the present invention is illustrated by the following examples. Examples 48-52 Liquid Fabric Cleaning Compositions In Examples 48-50 the BPN' variants recited in Tables 2-25, among others, are substituted for Ser161 Glu + Gly219Asn, with substantially similar results. In Examples 51-52, any combination of the BPN' variants recited in Tables 2-25, among others, are substituted for Ser161 Glu + Gly219Asn and Asn62Ser + lle107Ala + Glu206Asp + Tyr217Thr, with substantially similar results. Examples 53-57 Liquid Fabric Cleaning Compositions In Examples 53-55 the BPN' variants recited in Tables 2-25, among others, are substituted for SerlOlAsp + He 107Ala + Gly202Ser, with substantially similar results. In Examples 56-57, any combination of the BPN' variants recited in Tables 212, among others, are substituted for SerlOlAsp + He 107Ala + Gly202Ser and Val95Thr + Thr208Gly, with substantially similar results. Examples 58-59 ' Granular Fabric Cleaning Composition Example No. Component 58 59 C-12-14 alkenyl succinic acid 3.0 8.0 Citric acid monohydrate 10.0 15.0 Sodium C- j 2-15 a lkyl sulphate 8.0 8.0 Sodium sulfate of C-|2-15 βlcohol 2 times ethoxylated - 3.0 C-J2-15 alcohol 7 times ethoxylated - 8.0 C-i 2-15 alcohol 5 times ethoxylated 8.0 Diethylene triamine penta (methylene phosphonic acid)0.2 Oleic acid 1.8 Ethanol 4.0 4.0 Propanediol 2.0 2.0 AspδOGIu + Gln206Asn 0.2 0.2 Polyvinyl pyrrolidone 1.0 2.0 Suds suppressor 0J5 0.15 NaOH up to pH 7.5 Perborate 0.5 1 Phenol sulphonate 0.1 0.2 Peroxidase 0.4 0.1 Waters and minors up to 100 parts In each of Examples 58 and 59 herein, the BPN' variants recited in Tables 2-25, among others, are substituted for Asp60Glu + Gln206Asn, with substantially similar results. Examples 60-62 Liquid Fabric Cleaning Composition Bar fabric cleaning compositions of the present invention suitable for hand-washing soiled fabrics contain an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001 % to about 10%, more preferably from about 0.01 % to about 1 % by weight of the composition. The bar fabric cleaning composition embodiment of the present invention is illustrated by the following examples. Examples 63-66 Bar Fabric Cleaning Compositions *Can be selected from convenient materials such as CaCO 3 , talc, clay, silicates, and the like. In Examples 63-64 the BPN' variants recited in Tables 2-25, among others, are substituted for Gly97Glu + Thr164Pro, with substantially similar results. In Examples 65-66, any combination of the BPN' variants recited in Tables 2-25, among others, are substituted for Gly97Glu + Ghr164Pro and Ala98Ser + Gly154Asn, with substantially similar results. Examples 67-70 Bar Fabric Cleaning Compositions *Can be selected from convenient materials such as CaCO 3 , talc, clay, silicates, and the like. In Example 67, the BPN' variants recited in Tables 2-25, among others, are substituted for Val203Glu, with substantially similar results. In Example 68, the BPN' variants recited in Tables 2-25, among others, are substituted for GlylOOGlu + He107Ser, with substantially similar results. In Examples 69-70, any combination of the BPN' variants recited in Tables 2-25, among others, are substituted for Val203Glu and GlylOOGlu + lle107Ser, with substantially similar results. B. Additional Cleaning Compositions In addition to the hard surface cleaning, dishwashing and fabric cleaning compositions discussed above, one or more enzyme variants of the present invention may be incorporated into a variety of other cleaning compositions where hydrolysis of an insoluble substrate is desired. Such additional cleaning compositions include but are not limited to, oral cleaning compositions, denture cleaning compositions, and contact lens cleaning compositions. 1. Oral cleaning compositions In another embodiment of the present invention, a pharmaceutically- acceptable amount of one or more enzyme variants of the present invention are included in compositions useful for removing proteinaceous stains from teeth or dentures. As used herein, "oral cleaning compositions" refers to dentifrices, toothpastes, toothgels, toothpowders, mouthwashes, mouth sprays, mouth gels, chewing gums, lozenges, sachets, tablets, biogels, prophylaxis pastes, dental treatment solutions, and the like. Preferably, the oral cleaning compositions comprise from about 0.0001 % to about 20% of one or more enzyme variants of the present invention, more preferably from about 0.001% to about 10%, more preferably still from about 0.01% to about 5%, by weight of the composition, and a pharmaceutically-acceptable carrier. As used herein, "pharmaceutically- acceptable" means that drugs, medicaments or inert ingredients which the term describes are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, incompatibility, instability, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio. Typically, the pharmaceutically-acceptable oral cleaning carrier components of the oral cleaning components of the oral cleaning compositions will generally comprise from about 50% to about 99.99%, preferably from about 65% to about 99.99%, more preferably from about 65% to about 99%, by weight of the composition. The pharmaceutically-acceptable carrier components and optional components which may be included in the oral cleaning compositions of the present invention are well known to those skilled in the art. A wide variety of composition types, carrier components and optional components useful in the oral cleaning compositions are disclosed in U.S. Patent 5,096,700, Seibel, issued March 17, 1992; U.S. Patent 5,028,414, Sampathkumar, issued July 2, 1991 ; and U.S. Patent 5,028,415, Benedict, Bush and Sunberg, issued July 2, 1991; all of which are incorporated herein by reference. The oral cleaning composition embodiment of the present invention is illustrated by the following examples. Examples 71-74 Dentifrice Composition Example No. Component 71 72 73 74 Gln59Asp + Ala98Glu + Gly102Asp 2.000 3.500 1.500 2.000 +Ser105Glu + Leu109Thr Sorbitol (70% aqueous solution) 35.000 35.000 35.000 35.000 PEG-6* 1.000 1.000 1.000 1.000 Silica dental abrasive** 20.000 20.000 20.000 20.000 Sodium fluoride 0.243 0.243 0.243 0.243 Titanium dioxide 0.500 0.500 0.500 0.500 Sodium saccharin 0.286 0.286 0.286 0.286 Sodium alkyl sulfate (27.9% 4.000 4.000 4.000 4.000 aqueous solution) Flavor 1.040 1.040 1.040 1.040 Carboxyvinyl Polymer** * 0.300 0.300 0.300 0.300 Carrageenan* 0.800 0.800 0.800 0.800 Water balance to 100% *PEG-6 = Polyethylene glycol having a molecular weight of 600. **Precipitated silica identified as Zeodent 119 offered by J.M. Huber. ***Carbopol offered by B.F. Goodrich Chemical Company. ****lota Carrageenan offered by Hercules Chemical Company. In Examples 71-74 the BPN' variants recited in Tables 2-25, among others, are substituted for Gln59Asp + Ala98Glu + Gly102Asp + Ser105Glu + Leu209Thr, with substantially similar results. Examples 75-78 Mouthwash Composition In Examples 75-78, the BPN' variants recited in Tables 2-25, among others, are substituted for Leu96Thr + Gly128Asp + Ala133Glu+ Asn155Glu + Lys213Asp+ Ala216Asp, with substantially similar results. Examples 79-82 Lozenge Composition In Examples 79-82,' the BPN' variants recited in Tables 2-25, among others, are substituted for Ser132Asp + Tyr217Leu, with substantially similar results. Examples 83-86 Chewing Gum Composition • Supplied by L.A. Dreyfus Company. In Examples 83-86, the BPN' variants recited in Tables 2-25, among others, are substituted for Thr66Pro + Gln103Asn + Lys213Asp, with substantially similar results. 2. Denture cleaning compositions In another embodiment of the present invention, denture cleaning compositions for cleaning dentures outside of the oral cavity comprise one or more enzyme variants of the present invention. Such denture cleaning compositions comprise an effective amount of one or more of the enzyme variants, preferably from about 0.0001 % to about 50% of one or more of the enzyme variants, more preferably from about 0.001% to about 35%, more preferably still from about 0.01% to about 20%, by weight of the composition, and a denture cleansing carrier. Various denture cleansing composition formats such as effervescent tablets and the like are well known in the art (see for example U.S. Patent 5,055,305, Young, incorporated herein by reference), and are generally appropriate for incorporation of one or more of the enzyme variants for removing proteinaceous stains from dentures. The denture cleaning composition embodiment of the present invention is illustrated by the following examples. Examples 87-90 Two-layer Effervescent Denture Cleansing Tablet *Tetraacetylethylene diamine In Examples 87-90, the BPN' variants recited in Tables 2-25, among others, are substituted for Gln59Glu + Ser63Glu + Val95Met + Gly97Pro + Tyr217Ala, with substantially similar results. 3. Contact Lens Cleaning Compositions In another embodiment of the present invention, contact lens cleaning compositions comprise one or more enzyme variants of the present invention. Such contact lens cleaning compositions comprise an effective amount of one or more of the enzyme variants, preferably from about 0.01% to about 50% of one or more of the enzyme variants, more preferably from about 0.01% to about 20%, more preferably still from about 1% to about 5%, by weight of the composition, and a contact lens cleaning carrier. Various contact lens cleaning composition formats such as tablets, liquids and the like are well known in the art (see for example U.S. Patent 4,863,627, Davies, Meaken and Rees, issued September 5, 1989; U.S. Patent Re. 32,672, Huth, Lam and Kirai, reissued May 24, 1988; U.S. Patent 4,609,493, Schafer, issued September 2, 1986; U.S. Patent, 4,690,793, Ogunbiyi and Smith, issued September 1 , 1987; U.S. Patent 4,614,549, Ogunbiyi, Riedhammer and Smith, issued September 30, 1986; and U.S. Patent 4,285,738, Ogata, issued August 25, 1981 ; each of which are incorporated herein by reference), and are generally appropriate for incorporation of one or more enzyme variants of the present invention for removing proteinaceous stains from contact lens. The contact lens cleaning composition embodiment of the present invention is illustrated by the following examples. Examples 91-94 Enzymatic Contact Lens Cleaning Solution Example No. Component 91 92 93 94 Water balance to 100% In Examples 91-94, the BPN' variants recited in Tables 2-25, among others, are substituted for Ser191Glu + Gly219Ser, with substantially similar results. While particular embodiments of the subject invention have been described, it will be obvious to those skilled in the art that various changes and modifications of the subject invention can be made without departing from the spirit and scope of the invention. It is intended to cover, in the appended claims, all such modifications that are within the scope of the invention. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: BRODE, PHILIP F. et al. (ii) TITLE OF INVENTION: BPN * VARIANTS HAVING DECREASED ADSORPTION AND INCREASED HYDROLYSIS WHEREIN ONE OR MORE LOOP REGIONS ARE SUBSTITUTED (iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: THE PROCTER & GAMBLE COMPANY (B) STREET: 11810 EAST MIAMI RIVER ROAD (C) CITY: ROSS (D) STATE: OH (E) COUNTRY: USA (F) ZIP: 45061 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: CORSTANJE, BRAHM J. (B) REGISTRATION NUMBER: 34,804 (C) ATTORNEY DOCKET NO. 5597 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 513-627-2858 (B) TELEFAX: 513-627-0260 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 275 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Ala Gin Ser Val Pro Tyr Gly Val Ser Gin He Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gin Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val He Asp 20 25 30 Ser Gly He Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala 35 40 45 Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gin Asp Asn Asn Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser He Gly 65 70 75 80 Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Gly Ala Asp Gly Ser Gly Gin Tyr Ser Trp He He Asn Gly He Glu 100 105 110 Trp Ala He Ala Asn Asn Met Asp Val He Asn Met Ser Leu Gly Gly 115 120 125 Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala 130 135 140 Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly 145 150 155 160 Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val He Ala 165 170 175 Val Gly Ala Val Asp Ser Ser Asn Gin Arg Ala Ser Phe Ser Ser Val 180 185 190 Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser He Gin Ser Thr 195 200 205 Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu He Leu Ser Lys His Pro Asn 225 230 235 240 Trp Thr Asn Thr Gin Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys 245 250 255 Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu He Asn Val Gin Ala 260 265 270 Ala Ala Gin 275

Claims: What is claimed is: 1. A BPN' variant having a modified amino acid sequence of wild-type amino 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; characterized in that the modified amino acid sequence comprises a substitution at one or more positions in one or more of the loop regions; wherein A. when a substitution occurs in the first loop region, the substitution occurs at one or more of positions 59, 60, 61 , 62, 63, 65 or 66; wherein a. when a substitution occurs at position 59, the substituting amino acid is Asn, Asp, Glu or Ser; b. when a substitution occurs at position 60, the substituting amino acid is Glu; c. when a substitution occurs at position 61 , the substituting amino acid is Asp, Gin, Glu or Ser; d. when a substitution occurs at position 62, the substituting amino acid is Asp, Gin, Glu or Ser; e. when a substitution occurs at position 63, the substituting amino acid is Asp or Glu; f. when a substitution occurs at position 65, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser; and g. when a substitution occurs at position 66, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser; B. when a substitution occurs in the second loop region, the substitution occurs at one or more of positions 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106 or 107; wherein a. when a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Met, Pro, Ser or Thr; b. when a substitution occurs at position 96, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Met, Pro, Ser, Thr or Val; c. when a substitution occurs at position 97, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser; d. when a substitution occurs at position 98, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr; e. when a substitution occurs at position 99, the substituting amino acid is Glu; f. when a substitution occurs at position 100, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser; g. when a substitution occurs at position 101 , the substituting amino acid is Asp or Glu; h. when a substitution occurs at position 102, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser; i. when a substitution occurs at position 103, the substituting amino acid is Asn, Asp, Glu or Ser; j. when a substitution occurs at position 104, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr or Val; k. when a substitution occurs at position 105, the substituting amino acid is Asp or Glu; I. when a substitution occurs at position 106, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr, Tyr or Val; and m. when a substitution occurs at position 107, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; when a substitution occurs in the third loop region, the substitution occurs at one or more of positions 126, 127, 128, 129, 130, 131 , 132 or 133; wherein a. when a substitution occurs at position 126, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Met, Pro, Ser, Thr or Val; b. when a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser; c. when a substitution occurs at position 128, the substituting amino acid is Asn, Asp, Gin, Glu, Gly or Ser; d. when a substitution occurs at position 129, the substituting amino acid is Asn, Asp, Gin, Glu, Gly or Ser; e. when a substitution occurs at position 130, the substituting amino acid is Asp or Glu; f. when a substitution occurs at position 131 , the substituting amino acid is Asn, Asp, Gin, Glu, Gly or Ser; g. when a substitution occurs at position 132, the substituting amino acid is Asp or Glu; and h. when a substitution occurs at position 133, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr; when a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166 or 167; wherein a. when a substitution occurs at position 154, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser; b. when a substitution occurs at position 155, the substituting amino acid is Asp, Gin, Glu or Ser; c. when a substitution occurs at position 156, the substituting amino acid is Asp; d. when a substitution occurs at position 157, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser; e. when a substitution occurs at position 158, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser; f. when a substitution occurs at position 159, the substituting amino acid is Asp or Glu; g. when a substitution occurs at position 160, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser; h. when a substitution occurs at position 161 , the substituting amino acid is Asp or Glu; i. when a substitution occurs at position 162, the substituting amino acid is Asp or Glu; j. when a substitution occurs at position 163, the substituting amino acid is Asp or Glu; k. when a substitution occurs at position 164, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser; I. when a substitution occurs at position 165, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Met, Pro, Ser or Thr; m. when a substitution occurs at position 166, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser; and n. when a substitution occurs at position 167, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr or Val; and E. when a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 187, 188, 189, 190 or 191; wherein a. when a substitution occurs at position 187, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser and Thr; b. when a substitution occurs at position 188, the substituting amino acid is Asp or Glu; c. when a substitution occurs at position 189, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His,. He, Leu, Met, Pro, Ser, Thr, Tyr or Val; d. when a substitution occurs at position 190, the substituting amino acid is Asp or Glu; and e. when a substitution occurs at position 191 , the substituting amino acid is Asp or Glu; whereby the BPN' variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin BPN'. 2. The BPN' variant of Claim 1 , wherein one or more substitutions occur in the first loop region. 3. The BPN' variant of Claim 1 , wherein one or more substitutions occur in the second loop region. 4. The BPN' variant of Claim 1 , wherein one or more substitutions occur in the third loop region. 5. The BPN' variant of Claim 1 , wherein one or more substitutions occur in the fourth loop region. 6. The BPN' variant of Claim 1 , wherein one or more substitutions occur in the fifth loop region. 7. The BPN' variant of any of Claims 1-6, wherein the wild-type amino acid sequence further comprises a sixth loop region, characterized in that the modified amino acid sequence further comprises one or more substitutions in the sixth loop region; wherein the substitution(s) in the sixth loop region occurs at one or more of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219 or 220; wherein a. when a substitution occurs at position 199, the substituting amino acid for position 199 is Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu; b. when a substitution occurs at position 200, the substituting amino acid for position 200 is His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu; c. when a substitution occurs at position 201 , the substituting amino acid for position 201 is Gly, Gin, Asn, Ser, Asp or Glu; d. when a substitution occurs at position 202, the substituting amino acid for position 202 is Pro, Gin, Asn, Ser, Asp or Glu; e. when a substitution occurs at position 203, the substituting amino acid for position 203 is Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu; f. when a substitution occurs at position 204, the substituting amino acid for position 204 is Asp, or Glu; g. when a substitution occurs at position 205, the substituting amino acid for position 205 is Leu, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu; h. when a substitution occurs at position 206, the substituting amino acid for position 206 is Pro, Asn, Ser, Asp, or Glu; i. when a substitution occurs at position 207, the substituting amino acid for position 207 is Asp or Glu; j. when a substitution occurs at position 208, the substituting amino acid for position 208 is Pro, Gly, Gin, Asn, Ser, Asp or Glu; k. when a substitution occurs at position 209, the substituting amino acid for position 209 is He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu; I. when a substitution occurs at position 210, the substituting amino acid for position 210 is Ala, Gly, Gin, Asn, Ser, Asp or Glu; m. when a substitution occurs at position 211 , the substituting amino acid for position 211 is Ala, Pro, Gin, Asn, Ser, Asp or Glu; n. when a substitution occurs at position 212, the substituting amino acid for position 212 is Gin, Ser, Asp or Glu; o. when a substitution occurs at position 213, the substituting amino acid for position 213 is Trp, Phe, Tyr, Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu; p. when a substitution occurs at position 214, the substituting amino acid for position 214 is Phe, Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu; q. when a substitution occurs at position 215, the substituting amino acid for position 215 is Thr, Pro, Gin, Asn, Ser, Asp or Glu; r. when a substitution occurs at position 216, the substituting amino acid for position 216 is His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu; s. when a substitution occurs at position 217, the substituting amino acid for position 217 is Leu, He, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gin, Asn, Ser, Asp or Glu; t. when a substitution occurs at position 218, the substituting amino acid for position 218 is Gin, Ser, Asp or Glu; u. when a substitution occurs at position 219, the substituting amino acid for position 219 is Pro, Gin, Asn, Ser, Asp or Glu; and v. when a substitution occurs at position 220, the substituting amino acid for position 220 is Pro, Gly, Gin, Asn, Ser Asp or Glu. 8. A cleaning composition selected from the group consisting of a hard surface cleaning composition, a dishwashing composition, an oral cleaning composition, a denture cleansing composition, a contact lens cleaning composition and a fabric cleaning composition, characterized in that the cleaning composition comprises the BPN' variant of any of Claims 1-7 and a cleaning composition carrier. 9. The cleaning composition of Claim 8, wherein the cleaning composition is a hard surface cleaning composition. 10. The cleaning composition of Claim 8, wherein the cleaning composition is a fabric cleaning composition. 11. A mutant BPN' gene encoding the BPN' variant of any of Claims 1 -7.

Inventors: BARNETT BOBBY LEE, BRODE PHILIP FREDERICK III, GHOSH CHANCHAL KUMAR, RUBINGH DONN NELTON, BARNETT, BOBBY, LEE, BRODE, PHILIP, FREDERICK, III, GHOSH, CHANCHAL, KUMAR, RUBINGH, DONN, NELTON
Applicants: PROCTER & GAMBLE, THE PROCTER & GAMBLE COMPANY
Application year: 1995
Main classification: C12N15
IPC codes: A61K8, A61Q11, C11D3, C12N9, C12N15