MXPA99004822A - Chemically modified enzymes - Google Patents

Abstract

Modified enzymes are provided in which at least one amino acid, such as asparagine, leucine, methionine or serine, of an enzyme is replaced with a cysteine and the thiol hydrogen is replaced with a substituent group providing a thiol side chain selected from the group consisting of:a) -SR 1R 2, wherein R 1 is an alkyl and R 2 is a charged or polar moiety;b) -SR 3, wherein R 3 is a substituted or unsubstituted phenyl;c) -SR 4, wherein R 4 is substituted or unsubstituted cyclohexyl;d) -SR 5, wherein R 5 isC 10-C 15 alkyl;and e) -SR 6 wherein R 6 is a C 1-6 alkyl. Also, methods of producing the modified enzymes are provided, as well as detergent and feed additives and a composition for the treatment of a textile. A method for using the modified enzymes in organic synthesis is additionally provided. Further, modified enzymes having improved activity, altered pH profile and/or wash performance are provided.

Description

CHEMICALLY MODIFIED ENZYMES Background of the Invention Modification of enzyme properties by site-directed mutagenesis has been limited to replacements of natural amino acids, although molecular biological strategies to overcome this restriction have been derived recently (Cornish, VW et al (1995) Angew, Chem. , Int. Ed. Engl. 34: 621). However, these latter procedures are generally not easy to apply in most laboratories. In contrast, the controlled chemical modification of the enzymes offers a wide potential for the easy and flexible modification of the structure of the enzyme, which opens up extensive possibilities for the controlled adaptation of the specificity of the enzyme. The change of the properties of the enzyme by chemical modification has been previously explored, with the first report - which is from 1966 by Bender's groups (Polgar, L. et al. (1966) J. Am. Chem. Soc. 88: 3153) and Koshland (Neet, KE et al. (1966) Proc. Nati, Acad. Sci. USA 56: 1606), who created a thiolsubtilisin Rf0.030419 by chemical transformation (CH2OH - > CH2SH) Serine residue from the active site of the BPN 'of subtilisin for cysteine. Interest in artificially produced chemical enzymes, including some with synthetic potential, was renewed by Wu, Z.P. et al. (1989) J. Am. Chem. Soc. 111: 4514; Bell, I.M. et al. (1993) Biochemistry 32: 3754 and Peterson, E.B. et al. (1995) Biochemistry istry 34: 6616, and more recently by Suckling, C.J. et al. (1993) Bioorg. Med. Chem. Lett. 3: 531. Enzymes are now widely accepted as useful catalysts in organic synthesis. However, wild-type, natural enzymes can never be expected to accept all structures of synthetic chemical interest, nor do they always transform them stereospecifically into the enantiomerically pure materials necessary for synthesis. This limitation of potential on the synthetic capacities of enzymes has been recognized, and some progress has been made in not altering their specificities in a controlled manner using the techniques of site-directed mutagenesis and random engineering or protein design. . However, modification of the properties of the enzyme by the design of the proteins is limited to manufacturing replacements of natural amino acids, and the molecular biological methods contemplated to overcome this restriction are not easily feasible for the routine application or synthesis to a large extent. scale. The generation of new specificities or activities obtained by the chemical modification of enzymes has intrigued chemists for many years, and continues to do so. The inventors have adopted the strategy of chemical modification-site-directed mutagenesis, combined, since it offers virtually unlimited possibilities for the creation of new structural environments at any location of the amino acids. The '5,208,158 patent discloses chemically modified detergent enzymes wherein one or more methionines have been mutated into cysteines. The cysteines are subsequently modified to confer on the enzyme an improved stability towards the oxidizing agents. The chemical modification claimed is the replacement of the hydrogen of the thiol with an alkyl having 1 to 6 carbon atoms. Although US Pat. No. 5,208,158 describes the alteration of the oxidative stability of an enzyme, it may also be desirable to develop one or more enzymes with altered properties such as activity, nucleophile specificity, substrate specificity, stereoselectivity, thermal stability, the profile of the pH activity and the surface binding properties for use in, for example, detergents or organic synthesis.

Brief Description of the Invention There is a need for enzymes such as proteases that have altered properties. As such, the present invention provides modified enzymes that have one or more amino acid residues replaced by cysteine residues. The cysteine residues are modified by replacing the hydrogen in the thiol with a substituent group that provides a thiol side chain selected from the group consisting of: a) -Si ^ R2, wherein R1 is an alkyl and R2 is a charged or polar portion; b) -SR3, wherein R3 is a substituted or unsubstituted phenyl; c) -SR4, wherein R4 is substituted or unsubstituted cyclohexyl; and d) -SR5, wherein R5 is alkyl with 10 to 15 carbon atoms. In preferred embodiments, the side chain groups -SR3 and -SR4 above also comprise an alkyl group, R, which is placed before either R3 or R4 to form the -SRR3 or -SRR4. R is preferably an alkyl with 1 to 10 carbon atoms. With respect to the group -SR -'- R2 of the thiol side chain, R2 can be positively or negatively charged. Preferably, R2 is S03", C00" or NH3 +.

In addition, R1 is preferably an alkyl with 1 to 10 carbon atoms. Preferably, the enzyme is a protease. More preferably, the enzyme is a Bacillus subtilisin. Also, preferably, the amino acids therein replaced by cysteines are selected from the group consisting of asparagine, leucine, methionine and serine. More preferably, the amino acid to be replaced is located in a subsite of the protease, preferably the subsites Si, Si 'or S2. Even more preferably, the amino acids to be replaced are N62, L217, M222, S156 and S166 wherein the numbered position corresponds to the subtilisin that is naturally present in Bacillus amyloliquefaciens or to equivalent amino acid residues in other subtilisins, such as as Bacillus lentus subtilisin. In a particularly preferred embodiment, the enzyme is a subtilisin of Bacillus lentus. In the most preferred embodiments, the amino acid to be replaced by the cysteine is N62 and the thiol side chain group is selected from the group: -S1R2 wherein R1 is CH2 and R2 is CH2S03 ~; -SRR3 wherein R is CH2 and R3 is C6H5; -SRR4 wherein R is CH2 and R4 is c-C6Hn; -SR5 where R5 is n-C? OH2 ?; or the amino acid to be replaced by the cysteine is L217 and the group of the thiol side chain is -SR5 where R5 is n-C? 0H2 ?. The present invention further provides modified enzymes that have one or more amino acid residues replaced by the cysteine residues.

The cysteine residues are modified by replacing the thiol hydrogen with a substituent group that provides an SR6 of the thiol side chain where R6 is an alkyl with 1 to 6 carbon atoms and the amino acid residues that are to be replaced by the cysteine are selected from the group consisting of asparagine, leucine, and serine. Preferably, the enzyme is a protease. More preferably, the enzyme is a subtilisin of Bacillus. More preferably, the amino acid is located in a subsite of the protease, preferably the subsites Si, Si 'or S2. Even more preferably, the amino acids that are to be replaced are N62, L217, M222, S156 and S166.

Preferably, the enzyme is a subtilisin of B. lentus, the amino acid to be replaced by a cysteine is N62 or L217 and the group of the thiol side chain is -SR6 wherein R6 is CH2C (CH3) 3 or C5Hn. The present invention provides a method for producing a modified enzyme, which includes providing an enzyme wherein one or more amino acids have been replaced with cysteine residues and replacing the thiol hydrogen of the cysteine residue with a substituent group that provides a side chain of thiol of the group consisting of: a) -SR1R2, wherein R1 is an alkyl and R2 is a charged or polar portion; b) -SR3, wherein R3 is a substituted or unsubstituted phenyl; c) -SR4, wherein R4 is substituted or unsubstituted cyclohexyl; and d) -SR5, wherein R5 is alkyl with 10 to 15 carbon atoms. In the preferred embodiments, the groups -SR3 and -SR4 of the thiol side chain above, further comprise an alkyl group, R, which is placed before either R3 or R4 to form -SRR3 or -SRR4. R is preferably an alkyl with 1 to 10 carbon atoms.

With respect to the group -SR1R2 of the thiol side chain, R2 may be positively or negatively charged. Preferably, R2 is S? 3 ~, C00 ~ or NH3 +.

In addition, R1 is preferably an alkyl with 1 to 10 carbon atoms. Preferably, the enzyme is a protease. More preferably, the enzyme is a Bacillus subtilisin. Also, preferably, the amino acids therein replaced by cysteines are selected from the group consisting of asparagine, leucine, methionine or serine. More preferably, the amino acid to be replaced is located in a subsite of the protease, preferably the subsites Si, Si 'or S2. Even more preferably, the amino acids to be replaced are N62, L217, M222, S156 and S166 where the numbered position corresponds to the subtilisin that is naturally present in Bacillus amyloliquefaciens or equivalent amino acid residues in other subtilisins, such as Bacillus subtilisin lentus In a preferred embodiment, the enzyme is a subtilisin of Bacillus lentus. In the most preferred embodiments, the amino acid to be replaced by cysteine is N62 and the thiol side chain group is selected from the group: -SXR2 wherein R1 is CH2 and R2 is CH2S03 ~; -SRR3 wherein R is CH and R3 is C6H5; -SRR4 wherein R is CH2 and R4 is c-C6Hu; -SR5 wherein R5 is n-C? 0H2 ?; or the amino acid to be replaced by the cysteine is L217 and the thiol side chain group is -SR5 wherein R5 is nC? or H2? - The present invention also provides modified enzymes having one or more amino acid residues replaced by cysteine residues. The cysteine residues are modified by replacing the hydrogen of the thiol with a substituent group that provides an -SR6 of the thiol side chain wherein R6 is an alkyl with C? _6 and the amino acid residues that are to be replaced by cysteine are selected of the group consisting of asparagine, leucine, and serine. Preferably, the enzyme is a protease. More preferably, the enzyme is a subtilisin of Bacillus. Even more preferably, the amino acid is located in a subsite of the protease, preferably the subsites of Si, Si 'or S2. More preferably, the amino acids to be replaced are N62, L217, M222, S156 and S166. Preferably, the enzyme is a subtilisin of B. lentus, the amino acid to be replaced by a cysteine is N62 or L217 and the group of the thiol side chain is -SR6 wherein R6 is CH2C (CH3) 3 or C5HU. Detergent additives including modified enzymes are also provided. Food additives are provided that include modified enzymes. Methods of using the modified enzymes in a detergent formulation are provided. Methods of using the modified enzymes in the treatment of tissues are provided. Methods of using the modified enzymes are provided in the preparation of a feed additive. Modified enzymes are provided that have an increased activity. Modified enzymes are provided that have altered pH profiles. Modified enzymes are provided that have improved washing performance. Methods of using the modified enzymes in organic synthesis are provided.

Brief Description of the Drawings Figure 1 is a bar graph of the results obtained after probing modified S156C mutants with boronic acid inhibitors at pH 8.6. Figure 2 is a bar graph of the results obtained after probing the S166C mutants modified with the boronic acid inhibitors at pH 8.6. Figure 3 is a graph of the pH profiles of wild type Bacillus lentus subtilisin (SBL-WT, squares) and a modified N62C mutant (N62C-Scy; circles). The points were made in duplicate.

Detailed description of the invention In one embodiment of the invention, a modified enzyme and a method for providing such an enzyme are provided, having one or more amino acid residues of a subtilin replaced by cysteine residues. The cysteine residues are then modified by replacing the thiol hydrogen with a substituent group that provides a thiol side chain selected from the group consisting of: a) -SR1R2, wherein R1 is an alkyl and R2 is a charged or polar portion; b) -SR3, wherein R3 is a substituted or unsubstituted phenyl; c) -SR4, wherein R4 is substituted or unsubstituted cyclohexyl; and d) -SR5, wherein R5 is alkyl with 10 to 15 carbon atoms. In the preferred embodiments, the side chain groups -SR3 and -SR4 above also comprise an alkyl group, R, which is placed before either R3 or R4 to form the -SRR3 or -SRR4. R is preferably an alkyl with 1 to 10 carbon atoms. With respect to the group -SR ^ 'R2 of the thiol side chain, R2 can be positively or negatively charged. Preferably, R2 is S03 ~, COO ~ or NH3 +.

In addition, R1 is preferably an alkyl with 1 to 10 carbon atoms. Preferably, the enzyme is a protease. More preferably, the enzyme is a subtilisin of Bacillus. Also, preferably, the amino acids therein replaced by cysteines are selected from the group consisting of asparagine, leucine, methionine and serine.

More preferably, the amino acid to be replaced is located in a subsite of the protease, preferably the subsites Si, Si 'or S2. Even more preferably, the amino acids to be replaced are N62, L217, M222, S156 and S166 wherein the numbered position corresponds to subtilisin which is naturally present in Bacillus amyloliquefaciens or to equivalent amino acid residues in other subtilisins, such as subtilisin of Bacillus lentus. In a particularly preferred embodiment, the enzyme is a subtilisin of Bacillus lentus. In the most preferred embodiments, the amino acid to be replaced by the cysteine is N62 and the thiol side chain group is selected from the group: -S ^ -R2 wherein R1 is CH2 and R2 is CH2S03"; SRR3 wherein R is CH2 and R3 is C6H5; -SRR4 wherein R is CH2 and R4 is c-C6Hn; -SR5 wherein R5 is nC? 0H2 ?; or the amino acid to be replaced by the cysteine is L217 and the thiol side chain group is -SR5 wherein R5 is nC? 0H2 - The present invention also provides modified enzymes that have one or more amino acid residues replaced by the cysteine residues.The cysteine residues are modified by replacing the hydrogen of the thiol with a substituent group that provides an -SR6 of the thiol side chain wherein R6 is an alkyl with 1 to 6 carbon atoms and the amino acid residues that are to be replaced by the cysteine are selected from the group consisting of of asparagine, leucine, and serine. The enzyme is a protease. More preferably, the enzyme is a Bacillus subtilisin. Even more preferably, the amino acid is located in a subsite of the protease, preferably, the subsites Si, Sx 'or S2. More preferably, the amino acids to be replaced are N62, L217, M222, S156 and S166. Preferably, the enzyme is a subtilisin of B. lentus, the amino acid to be replaced by a cysteine is N62 or L217 and the group of the thiol side chain is -SR6 where R6 is CH2C (CH3) 3 or C5Hu. A "modified enzyme" is an enzyme that has been changed by replacing an amino acid residue such as an asparagine, serine, methionine or leucine, with a cysteine residue and then replacing the hydrogen of the thiol. cysteine with a substituent group that provides a thiol side chain, i.e., a group such as an alkyl with C? -6 or an alkyl with C10-15 or a group that includes a phenyl group, a cyclohexyl group or a charged portion or polar. After modification, the properties of the enzyme, ie the activity or specificity of the substrate, can be altered. Preferably, the activity of the enzyme is increased. The term "enzyme" includes proteins that are capable of catalyzing chemical changes in other substances without being changed by themselves. The enzymes can be wild-type enzymes or variant enzymes. Enzymes within the scope of the present invention include pullulanases, proteases, amylases and isomerases, lipases, oxidases and reductases. The enzyme can be a wild-type or mutant protease. Wild type proteases can be isolated from, for example, Bacillus lentus or Bacillus amyloliquefaciens (also referred to as LBP '). The mutant proteases can be made according to the teachings of, for example, PCT Publication Nos. WO 95/10615 and WO 91/06637. Various types of portions can be used to replace the thiol hydrogen of the ciestein residue. These include -SR ^ 2, -SR3, -SR4, -SR5 or -SR6. R and R1 are independently defined as an alkyl with C? -? Or substituted or unsubstituted. R2 is a charged or polar group. R3 is a substituted or unsubstituted phenyl group. R 4 is a substituted or unsubstituted cyclohexyl group. R5 is alkyl with C10-15. R6 is an alkyl with C1-5. R1, R5 or R6 can be a straight chain or a branched and / or substituted or unsubstituted chain. A charged group is one or more atoms that together form a charged molecule, that is, S03", C00 ~ or NH3". The terms "thiol side chain group", "substituent group that provides a thiol side chain", "thiol containing group", and "thiol side chain" are terms which can be used interchangeably and include groups that They are used to replace the hydrogen in the thiol of the cysteine used to replace one of the amino acids in a subtilisin. Commonly, the thiol side chain group includes a sulfur through which the Rx groups defined above are fixed to the sulfur of the thiol of the cysteine. The term "substituted" refers to a group from which one hydrogen in the group has been replaced with another atom or molecule. For example, a hydrogen can be substituted, for example, with a methyl group, a fluorine atom or a hydroxyl group. In the present invention, the alkyl groups, the cyclohexyl group and the phenyl group may be substituted, that is, they have substitutions of one or more hydrogen atoms with another atom or molecule.

The binding site of an enzyme consists of a series of subsites through the surface of the enzyme. The substrate residues that correspond to the subsites are labeled with P and the subsites are labeled with S. By convention, the subsites are labeled Si, S2, S3, S4, Si 'and S2'. A description of the subsites can be found in Siezen et. to the. (1991) Protein Engineering 4: 719-737 and Fersht, A.E. (1985) Enzyme Structure and Mechanism 2 / a. ed., Freeman (New Yok) pp. 29-30. The preferred subsites are Si, Sx 'and S2. The amino acid residues of the present invention can be replaced with cysteine residues using site-directed mutagenesis methods or other methods well known in the art. (See, for example, PCT Publication No. WO 95/10615). A method for modifying the thiol hydrogen of the cysteine residue can be found in Example 4 below. In one aspect of the invention, the modified proteases have altered proteolytic activity when compared to the precursor protease, since the increase in such activity (numerically larger) makes it possible to use the enzyme to act more efficiently on a substrate. target or target Also of interest are modified enzymes having altered activity, nucleophile specificity, substrate specificity, stereo selectivity, thermal stability, pH activity profile and surface binding properties when compared to the precursor. Surprisingly, the modified proteases of the present invention can have altered pKas and therefore the pH profiles that are displaced from those of the parent protease (see Example 7) without changing the surface charge of the protease molecule. The modified enzymes of the invention can be formulated in known liquid and powdered detergents having a pH between 6.5 and 12.0 at levels of from about 0.01 to about 5% (preferably 0.1% up to 0.5%) by weight. These detergent or additive cleaning compositions may also include other enzymes such as known proteases, amylases, cellulases, lipases or endoglycosidases, as well as formers and stabilizers. The modified enzymes of the invention, especially the subtilisins, are useful in the formulation of various detergent compositions. Several of the known compounds are suitable surfactants useful in the compositions comprising the modified enzymes of the invention. These include non-ionic, anionic, cationic or zwitterionic detergents, as described in US 4,404,128 by Barry J. Anderson and US 4,261,868 by Jiri Flora et al. A suitable detergent formulation is that described in Example 7 of U.S. Pat. No. 5,204,015. The technique is familiar with the different formulations which can be used as cleaning compositions. In addition to typical cleaning compositions, it is readily understood that the modified enzymes of the present invention can be used for any purpose that wild type or natural type enzymes are used. Accordingly, these modified enzymes can be used, for example, in liquid soap or bar applications, formulations for dishwashing machines, solutions or products for cleaning contact lenses, synthesis of peptides, feeding applications such as feed additives. feed or preparation of food additives, waste treatment, textile applications such as the treatment of tissues, as enzymes for fusion cleavage in the production of proteins, etc. The modified enzymes of the present invention may comprise improved wash performance in a detergent composition (when compared to the precursor). When used here, improved washing performance in a detergent is defined as increasing the clearance of certain spots sensitive to enzymes such as grass or blood., as determined by the evaluation of light reflectance after a standard wash cycle. The addition of the modified enzymes of the invention to conventional cleaning compositions does not create any special use limitations. In other words, any temperature and pH suitable for the detergent are also suitable for the present compositions since the pH is within the above range and the temperature is below the denaturing temperature of the described modified enzyme. In addition, the modified enzymes of the invention can be used in a cleaning composition without detergents, again either alone or in combination with formers and stabilizers. In another aspect of the invention, the modified enzyme is used in the preparation of an animal feed, for example, a cereal-based feed. The cereal can be at least one of wheat, barley, corn, sorghum, rye, oats, triticale (hybrid wheat and rye cereal) and rice. Although the cereal component of a cereal-based food constitutes a protein source, it is usually necessary to include sources of supplemental protein in the food such as those derived from fishmeal, meatmeal or vegetables. Vegetable protein sources include at least one soybean with a full fat content, naba seeds, cañola (a variety of naba seeds with reduced levels of erucic acid), soybean meal, naba seed flour, and canola flour . The inclusion of a modified enzyme of the present invention in an animal feed may make it possible for the raw protein value and / or the digestibility and / or the amino acid content and / or the digestibility coefficients of the food to be increased, so that which allows a reduction in the amounts of alternative protein sources and / or amino acid supplements which have previously been necessary feed ingredients for animals. The feed provided by the present invention may also include other enzyme supplements such as one or more of β-glucanase, glucoarnilase, mannase, α-galactosidase, phytase, lipase, α-arabinofuranosidase, xylanase, α-amylase, esterase, oxidase, oxide-reductase and pectinase. It is particularly preferred to include a xylanase as an additional enzyme supplement such as a subtilisin derived from the genus Bacillus. Such xylanase is described in detail for example in PCT patent application WO 97/20920. One aspect of the invention is a composition for the treatment of a textile material that includes MP. The composition can be used to treat for example silk or wool as described in publications such as RD 216,034; EP 134,267; US 4,533,359; and EP 344,259. The modified enzymes of the present invention can be used in organic synthesis for example, to catalyze a desired reaction and / or to favor a certain stereoselectivity. See, for example, Noritomi et al. Biotech Bioeng. 51: 95-99 (1996); Dabulis et al. Biotech Bioeng. 41: 566-571 (1993); Fitzpatrick et al. J. Am. Chem. Soc. 113: 3166-3171 (1991). The following is presented by way of example and is not to be construed as limiting the scope of the claims.

Experimental stage Example 1 Production of Cis Mutants The gene for B. lentus subtilisin (SBL) was cloned into the bacteriophage vector M13mpl9 for mutagenesis (US Patent 5,185,258). The mutagenesis directed to the oligonucleotides was carried out as described in Zoller et al. (1993) Methods Enzymol. 100: 468-500. The mutated sequences were cloned, excised and reintroduced into the expression plasmid GG274 in the B. subtilis host. PEG (50%) was added as a stabilizer. The crude protein concentrate obtained was purified by first passing it through a Sephadex® G-25 desalting matrix with a buffer solution of pH 5.2 (20 mM sodium acetate, 5 mM CaCl2) to remove the small molecular weight contaminants. The pooled fractions for the desalting column were then applied to a strong cation exchange column (SE Sepharose® FF) in the sodium acetate buffer (above), and the SBL was eluted with a one-step gradient of 0- 200 mM of NaCl acetate buffer, pH 5.2. The powder of the salt-free enzyme was obtained after the dialysis of the eluent against the Millipore purified water, and the subsequent lyophilization. The purity of the wild-type and mutant enzymes, which have been denatured by incubation with 0.1M HCl at 0 ° C for 30 minutes, is determined by SDS-PAGE on homogeneous gels using the Phast® System from Pharmacia (Uppsala, Sweden ). The concentration of SBL was determined using the Bio-Rad staining or dye reagent kit which is based on the method of Bradford (1976) Analytical Biochemistry 72: 248-254. The specific activity of the enzymes was determined in a buffer solution of pH 8.6 using the method described below.

Example 2 Preparation of Certain Portions 3-methylbutyl metantiosulfonate The reaction mixture of l-bromo-3-methylbutane (1.7520 g, 0.0116 moles) and sodium metantiosulfonate (1554 g, 0.0116 moles) in dry DMF (5 ml) is heated at 50 ° C for 2 hours. At room temperature, water (15 ml) is added and the mixture is extracted with ether (3x30 ml).

The combined extracts are washed with brine, dried, concentrated. The residue is subjected to flash column chromatography on silica gel with EtOAc-hexanes (1: 4) The product was obtained as a colorless liquid (1.4777 g, 70%). IR (film): 3030 (w), 3011 (w), 2958 (st), 2932 (st), 2873 (st), 1468 (m), 1410 (w), 1388 (w), 1367 (w), 1319 (st), 1136 (st), 955 (st), 748 cm "1 (st) .XHR NMR (200 MHz, CDC13): d 3.33 (s, 3H, CH3S02S); 3.19 (t , J = 7.1 Hz, 2H, SCH2CH2), 1.70-1.58 (m, 3H, SCH2CH2CHMe2), 0. 95 (d, J = 5.3 Hz, 6H, CHMe2); 13 C NMR (50 MHz, CDC13): d 50.60, 38.19, 34.59, 27.40, 22.06.

Neopentyl metantiosulfonate The mixture of the reaction of neopentyl iodide (3.054 g, 0.0154 mol), sodium methansulfonate (2.272 g, 0.0170 mol) and dry DMF (4 ml) is heated at 90 ° C for 90 hours. The reaction vessel is wrapped with an aluminum foil to avoid direct sunlight on the reaction mixture, since the iodide is sensitive to sunlight. At the end of the heating, the reaction mixture turned a reddish-brown color. At room temperature, water (15 ml) was added and the mixture extracted with ether (3x30 ml). The combined ether extracts were washed twice with brine, dried, concentrated and the residue was subjected to column chromatography on silica gel with EtoAc-Hexanes (1: 2) to give a colorless oil which slowly solidified (1.2395 g, 44%). The product was recrystallized from 95% EtOH. P.f: 28.5-29.0 ° C; IR (cast CH2C12) 3021 (m), 2956 (m), 2868 (m), 1467 (m), 1433 (m), 1321 (st), 1310 (st), 1125 (st), 951 (m), 757 (m) and 724 cm "1 (m), XH NMR (200 MHz, CDC13): d 3.32 (s, 3H, CH3S02S), 3.13 (s, 2H, SCH2C), 1.05 (s, 9H, CMe3); 13 C NMR (50 MHz. CDC13): d 50.23, 50.09, 32.14, 28.79, MS (El): 182 (M +), 57 (base peak, CMe3 +).

Hexyl methansulfonate The reaction mixture of 1-bromohexane (1.046 g, 0.00635 mole), sodium metantiosulfonate (0.850 g, 0.00635 mole) and dry DMF (6 mL) is heated at 60 ° C for 2 hours.- At room temperature, add water (15 ml) and the resulting mixture is extracted with ether (3x30 ml). The extracts are washed with brine, dried, concentrated and the residue is subjected to flash column chromatography on silica gel with EtOAc-Hexanes (1: 4) to give a colorless liquid (2.057 g, 82%). IR (cast CDC13): 3030 (w), 3010 (w), 2955 (st), 2930 (st), 2860 (st), 1460 (m), 1320 (st), 1133 (st), 955 (st) , 747 cm "1 (st); XH NMR (200 MHz, CDC13): d 3.33 (s, 3H, CH3S02S), 3.18 (t, J = 7.4 Hz, 2H, SCH2CH2), 1.77 (pseudo p, J = 7.2 Hz, 2H, SCH2CH2), 1.50-1.20 (m, 6H, CH2CH2CH2CH3), 0.90 (, 3H, CH2CH3); 13 C NMR (50 MHz, CDC13): d 50.64, 36.50, 31.13, 29.46, 28.26, 22.44, 13. 96 Cyclohexylmethyl methanediosulfonate The reaction mixture of bromomethylcyclohexane (1560 g, 0.00881 mol), sodium metantiosulfonate (1180 g, 0.00881 mol) and dry DMF (6 mL) is heated at 50 ° C for 24 hours. At room temperature, water (15 ml) is added and the mixture is extracted with ether (3x30 ml). The extracts are washed with brine, dried, concentrated and the residue is subjected to flash column chromatography on silica gel with EtOAc-hexanes (1: 4) to give a colorless oil (1.5033 g, 82%). IR (CDC13): 3030 (w), 3012 (w), 3012 (w), 2926 (st), 2853 (st), 1446 (m), 1410 (m), 1320 8st), 1134 (st), 955 (st), 746 cm "1 (st); XH NMR (200 MHz, CDC13): d 3.32 (s, 3H, CH3S02S), 3.07 (d, J = 6.9 Hz, 2H, SCH2CH), 1.95-1.55 (m , 6H), 1.40-0.90 (m, 5H); 13C NMR (50 MHz, CDC13): d 50.42, 43.30, 37.83, 32.43, 26.02, 25.82.

Decile metantiosulfonate The mixture of 1-bromodecane (2095 g, 0.00947 moles), sodium metantiosulfonate and dry DMF (6 ml) is heated at 60 ° C for 2 hours. At room temperature, water (15 ml) is added and the mixture is extracted with ether (3x30 ml). The ether extracts are washed with brine, dried, concentrated and the residue subjected to flash column chromatography on silica gel with EtOAc-hexanes (1: 4) to give a white solid (2.063 g, 94%). It is recrystallized from 95% EtOH. P.f; 28.0-29.5 ° C. IR (cast CDC13): 2954 (m), 2921 (st), 2852 (st), 1469 (m), 1305 (st), 1128 (st), 965 (m), 758 (m) and 720 cm "1 (m): X H NMR (200 MHz, CDCl 3): d 3.32 (s, 3 H, CH 3 S0 2 S), 3.17 (t, J = 7.4 Hz, 2 H, SCH 2 CH 2), 1.77 (m, 2 H, SCH 2 CH 2), 1.50-1.20 ( m, 14H, - (CH2) 7-), 0.88 (m, 3H, CH2CH3); 13C NMR (50 MHz, CDC13) d 50.64, 36.49, 31.84, 29.45 (two carbons), 29.37, 29.33, 28.94, 28.57, 22.64, 14.08.

Sodium metantiosulfonate Mesyl chloride (46.6 ml, 0.602 mol) is added dropwise to a solution of Na2S "9H20 (142.2 g, 0.592 mol) in water (150 ml) at 80 ° C. After the addition, the reaction mixture is heated under reflux and returns from a faint yellow to yellow in 15 hours. During this time, some yellow precipitates are also formed. The reaction mixture is cooled to room temperature and the water is evaporated. After the solid residue is ground with a mortar and pestle and the powder is further dried at 50 ° C and 1 torr. The absolute ethanol (700 ml) is used to crush the powder in 4 portions and the ethanol filtrate is concentrated and cooled with an ice bath to obtain a precipitate which is collected by vacuum filtration. The filtrate is further concentrated to obtain a second crop of precipitates. After repeated concentration and filtration (4 X), the final volume of the filtrate was about 10 ml. The combined precipitates are redissolved in absolute ethanol at room temperature and filtered to remove trace amounts of sodium chloride and sodium sulfide. The filtrate is concentrated and cooled and the solids are collected by vacuum filtration. Again, the process of concentration, cooling and filtration is repeated 3 times to give crystals in the form of flakes, white, which are dried additionally to 1 torr. all night. (24.51 g, 31%) IR (KBr): 3004, 2916, 1420, 1326, 1203, 1095, 980, 772 cm. "1 XH NMR (200 MHz, D20): d 3.23 (s). MHz, D20, with DMSO-d6 as an internal standard): d 39.72 ppm.

Benzyl metantiosulfonate The benzyl bromide (9.07 g, 0.053 mole) is added slowly to a suspension of sodium metantiosulfonate (7.10 g, 0.0530 mole) in absolute EtOH (100 mL) and the reaction mixture is heated to reflux overnight. The reaction mixture is cooled with an ice bath and the solid (sodium bromide and sodium metantiosulfonate) is removed by filtration. The filtrate is concentrated to give a crude product which was mainly the desired product. The pure product was obtained by flash chromatography on silica gel with EtOAc-hexanes (1: 6) (7.92 g, 74%). The product was further purified by recrystallization from absolute ethanol. P.f. 39.5-40.2 ° C (bed 40-42.5 ° C) IR (KBr): 3089, 3068, 3017, 3000, 2981, 2936, 2918, 1602, 1582, 1496, 1305, 1131, 960, 771, 741, 702 cm "1. XH NMR (200 MHz, CDC13): d 7.38 (m, 5H, phenyl), 4.38 (s, 2H, SCH2), 2.91 (s, 3H, CH3S02). 13C NMR (50 MHz, CDC13): d 135.12, 129.14, 129.03, 128.26, 51.09, 40.79 Reagents CH3S02-SCH2CH2S03"Na + and CH3S02-SCH2CH2NH3 + Br" were purchased from Toronto Research Chemicals (Toronto, Ontario).

Example 3 Modification of the Cis Mast tants The following is exemplary for the method used to modify the Cis mutants, i.e., N62C.

Modification of M222C To a solution of the cis mutant, M222C, B. lentus (25.1 mg, 0.94 μmoles) in buffer (250 ml, 70 mM CHES, 5 mM MES, 2 mM CaCl2, pH 9.5) in a polypropylene test tube which it has been pre-coated with an aqueous solution of polyethylene glycol 10,000 (0.1% w / v), a solution of methyl metantiosulfonate described in Example 2 in 95% EtOH (100 μl, 92.4 μmol) is added. The solution is swirled and swirled and allowed to rotate slowly on a rotating end over end device at room temperature (22 ° C). A model solution containing ethanol instead of the reagent solution is run in parallel. The modification is monitored by activity measurements on 10 μl extraction samples and determined according to the method described above. The reaction is terminated after 2.5 hours when the addition of another aliquot of the reagent to the reaction did not change the activity of the protease. The solution (2.5 ml) is purified on a disposable desalting column (Pharmacia Biotech PD-10®, Sephadex® G-25M). The column was equilibrated with buffer (25 ml, 5 mM MES, 2 mM CaCl 2, pH 6.5) and the sample was loaded on top. The first 2.5 ml collected are discarded. The protein eluted is buffer-MES (3.5 ml) and collected in three fractions. All the fractions appeared as a single band when verified on a gel (SDS-PAGE, Pharmacia Phast-System®) and could not be differentiated from the Cys mutant or from the wild type which both run as references. The three fractions were mixed and dialyzed against deionized water (3 x 1 1) at 0 ° C, followed by lyophilization overnight which gave the modified mutant (14.3 mg). The specific activity was 64.3 U / mg when compared to the Cys mutant (47.1 U / mg).

Measurement of the Activity of Modified Proteases The activity, including the kcat, KM and kcat / KM kinetic parameters were measured for the hydrolysis of succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide from the synthetic peptide substrate using the method described in Bonneau, P. et al. (1991) J. Am. Chem. Soc., 113 (3): 1030. Briefly, a small aliquot of solution was added in the presence of a variant of subtilisin to a 1 cm test tube containing the substrate dissolved in a buffer solution of sodium phosphate 0.1 M, pH 7.5, containing 0.5M NaCl and 1 % of DMSO, and treated with thermostat at 25 ° C or similarly to a pH of 8.6, a buffer solution of 0.1 M tris containing 0.05% Tween® 80 and 1% DMSO. The progress of the reaction is monitored spectrophotometrically verifying the absorbance of p-nitroaniline from the reaction product at 410 nm using a Perkin Elmer? 2 spectrophotometer (? e4io 8800 M "1 cm" 1). The kinetic parameters were obtained by measuring the initial velocities at substrate concentrations of 0.25 mM-4.0 mM (eight concentrations) and these data are adjusted to the Michaelis-Menten equation. Table 1 shows the abbreviations for certain thiosulfonates. Table 2 shows the kinetic parameters of the subtilisins of jB. modified lentus (SBL) and the precursor subtilisin (SBL-WT) at pH 7.5. The modified enzymes were prepared as described above after site-directed mutagenesis to replace the amino acid of interest with a cysteine. The kinetic parameters were determined at pH 7.5 as described above. The precursor protease was a subtilisin of Bacillus lentus (SBL-WT).

Table 1 Abbreviation Structure -SBn -SCH3C6H5 -Siso-butyl -SCH2CH (CH3) 2 -Sneo-pentyl -SCH2C (CH3) 3 -SCH2cyclohexyl -SCH2-C-C6H ?? -Slither -S-n-C? OH2? Table 2 Example 4 Alteration of the Specificity of Subtilisin of B. lentus Changes in substrate specificity, particularly the specificity of the Si subsite, can be shown using various boronic acids as competitive inhibitors. Four of the modified S156C mutants and three of the modified S166C mutants described above were evaluated using boronic acid inhibitors. The modified mutants were S156C-SMe, Sl56C-SBn, S156C-SCH2CH2S03", S156C-SCH2CH2NH3 +, S166C-SCH2CH2S03", S166C-SCH2CH2NH3", and S166C-SBn. Boronic acids were prepared, and their inhibition constants were measured at pH 8.6 (Waley (1982) Biochem J. 205: 631-33), as previously described in Seufer-Wasserthal et al (1994) Bioorganic and Medicinal Chemistry 2: 35-48) The results are shown in the Figures 1 and 2.

Example 5 Washing Performance Test The washing performance of several of the modified enzymes described in the previous examples was evaluated by measuring the removal of stains from fabric samples of EMPA 116 (blood / milk / carbon black on cotton) (Testfabrics, Inc., Middlesex, NJ 07030) which have been pre-whitened as follows: in a 4-liter beaker, 1.9 grams of perborate tetrahydrate, 1.4 grams of perborate monohydrate and 1 gram of TAED (tetraacetylenediamine) are dissolved in 3 liters of deionized water at 60 ° C for 1 minute with shaking. 36 samples of EMPA 116 fabric are added and stirred for 3 minutes. The fabric samples were rinsed immediately with cold deionized water for 10 minutes. The fabric samples were laid flat on absorbent paper towels to dry overnight. Five pre-blanched EMPA 116 fabric samples were placed in each pot of a Model 7243S Tergotometer (United States Testing Co., Inc., Hoboken, NJ) containing 100 ml of water, 3 gpg of hardness (Ca ++; Mg ++; 3: 1; p: p), 0.67 g of detergent with bleach and the appropriate enzyme. The detergent base was WFKl detergent from wfk-Testgewebe GmbH, Adlerstrasse 42, Postfach 13 07 62, D-47759 Krefled, Germany.

To this detergent base, the following additions were made: Sodium perborate monohydrate and sodium perborate tetrahydrate are obtained from Degussa Corporation, Ridgefield Park, NJ 07660. TAED (tetraacetylenediamine) was obtained from Warwick International, Limited, Mostyn, Hollywell, Clwyd CH8 9HE, England. The pre-bleached EMPA 116 fabric samples are washed in detergent with 0.1 ppm of enzyme for 20 minutes at 20 ° C and subsequently rinsed twice for 5 minutes in 1000 ml of water. The fabric samples were dried and pressed, and the reflectance of the fabric samples was measured using the L value of the laboratory scale of a Minol Chroma Meter, Model CR-200 (Minolta Corporation, Ramsey, NJ 07446). The operation is reported as the percentage of removal of spots and the percentage of removal of spots in relation to the B protease. Len your natural. The percentage of removal of the spots is calculated using the equation: (L value of samples (value of washed fabric samples) of unwashed cloth) X 100 (L value of samples of (L-value of samples without EMPA 221 unstained) - unwashed) Table 3 Example 7 Altering the pH Profile of a Precursor Subtilisin To examine the effects of chemical modification on the SBL pH profile, seven modified N62C mutants were made as described above. The 0.02M ethylene diamine buffer solutions 0.05M ionic strength (adjusted with KCl) were used with 1.25 x 10 ~ 4 M succinyl-AAPF-pNA substrate and Kcat / KM measurements were performed as described above. The Kcat / KM value reflects the pKa of His64, part of the catalytic triad for SBL, in the free enzyme and is not affected by non-productive binding modes, Fersht, A.E. (1985) (Enzime Structure and Mechanism 2 / a. Ed., Freeman (New York). The pKa was calculated using Graphit (McGeary &; Associates, Middletown CT). The change in the pKa is reflected in a change in the pH profile of the SBL. The representative pH profiles for SBL N62C-Scyclohexyl (N62C-Scy) and SBL-WT are shown in Figure 3 ([E] = 1x10-7 to 5xlO-8M at 25 ° C). The points were made in duplicate. Table 4 shows the pKa of His64, the change in pKa from the wild type B. lentus (WT) and the Kcat / KM for seven modified S62 N62C mutants.

Table 4 As shown in Table 4, a very dramatic 0.5 unit decrease is observed in the pKa of His64 for the SBL modified with N62C-Sciclohexyl when compared to that of the wild type. As such, it is possible to design altered pH profiles without altering the surface charge. Although the invention has been described in relation to specific embodiments thereof, it will be understood that it is capable of further modifications and this application is proposed to cover any variations or adaptations of the invention that follow, in general, the principles of the invention. and including such deviations from the present disclosure that come within the known or customary practice within the art to which the invention pertains and when they can be applied to the essential features described hereinafter, and as follows in the scope of the attached claims. All publications and patents or applications referred to in the above specification are incorporated herein for reference.

It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following

Claims (42)

1. A modified enzyme wherein one or more amino acid residues are replaced by cysteine residues, characterized in that the cysteine residues are modified by replacing the hydrogen in the thiol with a substituent group that provides a thiol side chain selected from the group consisting of: ) -SR1R2, wherein R1 is an alkyl and R2 is a charged or polar portion; b) -SR3, wherein R3 is a substituted or unsubstituted phenyl; c) -SR4, wherein R4 is substituted or unsubstituted cyclohexyl; and d) -SR5, wherein R5 is alkyl with 10 to 15 carbon atoms.

2. The modified enzyme according to claim 1, characterized in that R1 is alkyl with 1 to 10 carbon atoms.

3. The modified enzyme according to claim 1, characterized in that R2 is positively charged.

4. The modified enzyme according to claim 3, characterized in that R2 is NH3 +.

5. The modified enzyme according to claim 1, characterized in that R2 is negatively charged.

6. The modified enzyme according to claim 5, characterized in that R2 is S03".

7. The modified enzyme according to claim 1, characterized in that the enzyme is a protease.

8. The method according to claim 7, characterized in that the protease is a subtilisin of Bacill used in your.

9. The modified protease according to claim 7, characterized in that the amino acid replaced with a cysteine is an amino acid selected from the group consisting of asparagine, leucine, methionine and serine.

10. The modified protease according to claim 9, characterized in that the asparagine is in a subsite of the protease.

11. The modified protease according to claim 10, characterized in that the subsite is S2.

12. The modified protease according to claim 11, characterized in that asparagine is in position 62.

13. The modified enzyme according to claim 1, characterized in that it further comprises an alkyl group, R, before R3 or R4 to form -SRR3 or -SRR4.

14. The modified enzyme according to claim 13, characterized in that R is alkyl with 1 to 10 carbon atoms.

15. A modified enzyme in which one or more amino acid residues are replaced by cysteine residues, characterized in that the cysteine residues are modified by replacing the thiol hydrogen of the cysteine residue with a substituent group that provides a thiol side chain -SR6, in where R6 is an alkyl with 1 to 6 carbon atoms, and wherein the amino acid residue is selected from the group consisting of asparagine, leucine and serine.

16. A method of producing a modified enzyme, characterized in that it comprises: (a) providing an enzyme wherein one or more amino acids have been replaced with cysteine residues; and (b) replacing the thiol hydrogen with a substituent group that provides a thiol side chain selected from the group consisting of: (i) -SR ^ -R2, wherein R1 is an alkyl and R2 is a charged or polar moiety; (ii) -SR3, wherein R3 is a substituted or unsubstituted phenyl; (iii) -SR4, wherein R4 is substituted or unsubstituted cyclohexyl; and (iv) -SR5, wherein R5 is alkyl with 10 to 15 carbon atoms.

17. The method according to claim 16, characterized in that R1 is alkyl with 1 to 10 carbon atoms.

18. The method according to claim 16, characterized in that R2 is positively charged.

19. The method according to claim 18, characterized in that R2 is NH3 +.

20. The method according to claim 16, characterized in that R2 is negatively charged.

21. The method according to claim 20, characterized in that R2 is S03".

22. The method according to claim 16, characterized in that the enzyme is a protease.

23. The method according to claim 22, characterized in that the protease is a subtilisin of Bacillus lentus.

24. The method according to claim 16, characterized in that the amino acid replaced with a cysteine is an amino acid selected from the group consisting of asparagine, leucine, methionine, and serine.

25. The method according to claim 23, characterized in that asparagine is in a subsite of the protease.

26. The method according to claim 25, characterized in that the subsite is S2.

27. The method according to claim 26, characterized in that asparagine is in position 62.

28. The method according to claim 16, characterized in that it further comprises an alkyl group, R, before R3 or R4 to form -SRR3 or -SRR4.

29. The method according to claim 28, characterized in that R is alkyl with 1 to 10 carbon atoms.

30. A method of producing a modified enzyme, characterized in that it comprises: (a) providing an enzyme wherein one or more amino acids have been replaced with cysteine residues and wherein the amino acids are selected from the group consisting of asparagine, leucine and serine; and (b) replacing the thiol hydrogen of the cysteine residue with a substituent group that provides a thiol side chain -SR6, wherein R6 is an alkyl having 1 to 6 carbon atoms.

31. An additive for detergent, characterized in that it comprises the modified enzyme of claims 1 or 15.

32. An additive for food, characterized in that it comprises the modified enzyme of claims 1 or 15.

33. A composition for the treatment of a textile material, characterized in that it comprises the modified enzyme of claims 1 or 15.

34. The modified enzyme according to claim 1, characterized in that the enzyme is a subtilisin of Bacill us len, the amino acid is N62 and the side chain of thiol is -SRXR2 wherein R1 is CH2CH2 and R2 is S03".

35. The modified enzyme according to claim 1, characterized in that the enzyme is a subtilisin of Bacill used in thy, the amino acid is N62 and the side chain of thiol is -SRR3 wherein R is CH2 and R3 is C6 6H «5.

36. The modified enzyme according to claim 1, characterized in that the enzyme is a subtilisin of Bacill us i in thy, the amino acid is N62 and the side chain of thiol is -SR1R4 where R is CH2 and R4 is c-CeHn.

37. The modified enzyme according to claim 1, characterized in that the enzyme is a subtilisin of Bacillus lentus, the amino acid is N62 and the side chain of thiol is -SR5 wherein R5 is n-C? Or H2 ?.

38. The modified enzyme according to claim 1, characterized in that the enzyme is a subtilisin of Bacillus lentus, the amino acid is L217 and the side chain of thiol is -SR5 wherein R5 is n-C? Or H2 ?.

39. The modified enzyme according to claim 15, characterized in that the enzyme is a subtilisin of Bacillus lentus, the amino acid is N62 and the side chain of thiol is -SR6 wherein R6 is CH2C (CH3) 3.

40. The modified enzyme according to claim 15, characterized in that the enzyme is a subtilisin of Bacillus in it, the amino acid is N62 and the side chain of thiol is -SR6 wherein R6 is CsHu.

41. The modified enzyme according to claim 15, characterized in that the enzyme is a subtilisin of Bacill used in thy, the amino acid is L217 and the side chain of thiol is -SR6 wherein R6 is CH2C (CH3) 3.

42. The modified enzyme according to claim 15, characterized in that the enzyme is a subtilisin of Bacillus lentus, the amino acid is L217 and the side chain of thiol is -SR6 wherein R6 is C5Hn.