US20050282248A1 - Chemically modified mutant serine hydrolases show improved catalytic activity and chiral selectivity - Google Patents

Chemically modified mutant serine hydrolases show improved catalytic activity and chiral selectivity Download PDF

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US20050282248A1
US20050282248A1 US10/791,093 US79109304A US2005282248A1 US 20050282248 A1 US20050282248 A1 US 20050282248A1 US 79109304 A US79109304 A US 79109304A US 2005282248 A1 US2005282248 A1 US 2005282248A1
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oxazolidinone
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phenyl
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John Jones
Michael Dickman
Richard Lloyd
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Priority to US13/561,257 priority patent/US8357524B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus

Definitions

  • This invention pertains to the field of serine hydrolases.
  • this invention pertains to serine hydrolases that have been mutated to introduce one or more cysteines which are then chemically derivatized. These chemically modified mutants demonstrate altered enzymatic activity.
  • Enzymes are now widely accepted as useful catalysts in organic synthesis.
  • U.S. Pat. No. 5,208,158 describes chemically modified detergent enzymes where one or more methionines have been mutated into cysteines.
  • the cysteines are subsequently modified in order to confer upon the enzyme improved stability towards oxidative agents.
  • improved stability is often a desirable property, it is also often desirable to alter other enzymatic properties (e.g. specificity, catalytic activity, stereoselectivity, etc.).
  • the mutants are serine hydrolases in which one or more amino acid residues (preferably residues in a subsite, e.g. S 1 , S 1 ′, or S 2 ) are replaced with a cysteine where the cysteine is chemically modified by replacing the thiol hydrogen in the cysteine with a substituent group providing a thiol side chain comprising a moiety selected from the group consisting of a polar aromatic substituent, an alkyl amino group with a positive charge, a chiral substituent, a heterocyclic substituent, and a glycoside.
  • Preferred serine hydrolases of this invention catalyze a transamidation or a transpeptidation or a transesterification reaction and in a most preferred embodiment is stereoselective in this catalysis.
  • Particularly preferred serine hydrolases include alpha/beta serine hydrolases, a subtilisin type serine proteases, and chymotrypsin serine proteases, with subtilisin being a particularly preferred serine protease.
  • Preferred amino acids selected for replacement with cysteine include asparagine, leucine, methionine, and serine.
  • Preferred sites for replacement e.g. in subtilisin type enzymes
  • amino acid 156 in the S1 subsite amino acid 166 in the S1 subsite, amino acid 217 in the S1′ subsite, amino acid 222 in S1′ subsite and amino acid 62 in the S2 subsite.
  • Preferred substituents include an oxazolidinone, a C 1 to C 15 alkyl amino group with a positive charge, and a glycoside (e.g., a monosaccaharide, a disaccharide, and an oligosaccharide comprising pentoses and hexoses) (see, e.g., FIG. 2 ).
  • a glycoside e.g., a monosaccaharide, a disaccharide, and an oligosaccharide comprising pentoses and hexoses
  • preferred substituents include (R)-2-methoxy-2-phenyl-ethyl-thiol, (S)-2-methoxy-2-phenyl-ethyl-thiol, (R)-2-hydroxy-2-phenyl-ethyl-thiol, (S)-2-hydroxy-2-phenyl-ethyl-thiol, N-(3′-thio-propyl)-2-oxazolidinone, N-(3′-thio-propyl)-(S)-4-phenyl-2-oxazolidinone, N-(3′-thio-propyl)-(R)-4-benzyl-2-oxazolidinone, N-(3′-thio-propyl)-(S)-4-benzyl-2-oxazolidinone, N-(2′-thio-ethyl)-(R)-4phenyul-2-oxazolidinone, N-(2′-thio-ethyl-(
  • this invention provides a chemically modified mutant subtilisin.
  • the modified subtilisin has one or more amino acid residues selected from the S1, S1′, or S2 subsites replaced with a cysteine, where the cysteine is modified by replacing the thiol hydrogen in the cysteine with a substituent group providing a thiol side chain comprising a moiety selected from the group consisting of a polar aromatic substituent, an alkyl amino group with a positive charge, an alkyl group bearing a negatively charged moiety, and a glycoside.
  • Particularly preferred cysteine substitution(s) are at amino acid 62, amino acid 156, amino acid 166, amino acid 217, and amino acid 222.
  • Preferred substituents are as described above and herein.
  • This invention also provides a method of forming a peptide bond.
  • the methods preferably involve contacting a compound comprising an ester substrate with a serine hydrolase and/or a chemically modified mutant subtilisin as described herein and a primary amine under conditions whereby the hydrolase or modified subtilisin the formation of a peptide bond.
  • a preferred ester substrate is an acyl donor and a primary amine is an acyl acceptor (e.g. an amino acid amide). Where the acyl acceptor is an amino acid amide the amino acid can be a D or an L amino acid and can optionally be present in a peptide.
  • the ester substrate can be a D or an L amino acid ester and can optionally be present in a peptide.
  • this invention provides methods of resolving racemic primary and secondary alcohols using a transesterification reaction. These methods involve contacting the racemic primary or secondary alcohol with a serine hydrolase and/or a modified mutant subtilisin as described herein and an acyl donor whereby said serine hydrolase catalyzes a transesterification reaction resolving the racemic primary or secondary alcohol.
  • Preferred primary or secondary alcohols include, but are not limited to, an aliphatic alcohol, an aromatic alcohol, and a heterocyclic alcohol.
  • Particularly preferred primary or secondary alcohols include, but are not limited to 2-phenyl-1-propanol, 2-methyl-1-pentanol, and 2 octanol.
  • Preferred acyl donors include, but are not limited to carboxylic acid esters (e.g., including but not limited to alkyl, aralkyl such as benzyl, esters) and activated esters (e.g., mono-, and/or di-, and/or tri-haloalkyl).
  • Particularly preferred modified mutant enzymes include, but are not limited to L217C—(CH 2 ) 2 —SO 3 ⁇ , N62C— (CH 2 ) 2 —SO 3 ⁇ , and N62C—S—CH 3 .
  • this invention provides methods of attaching a chiral moiety to a substrate via a transamidation, a transesterification, or a transpeptidation reaction. These methods involve contacting a substrate (e.g., a peptide, an amino acid, etc.) having a reactive site suitable for a transesterification or a tansamidation, and the moiety with a catalytic serine hydrolase as described herein whereby the chiral moiety is covalently coupled to the substrate.
  • a substrate e.g., a peptide, an amino acid, etc.
  • Preferred chiral moieties include, but are not limited to D amino acids, L-amino acids, acyclic aliphatics, a cyclic aliphatics, aralkyl R-carboxylic acids, aralkyl S-carboxylic acids, aromatic R-carboxylic acids, and aromatic S-carboxylic acids.
  • the reaction is preferential for a moiety of one chirality. Particularly where the reaction is a transesterification the transesterification preferably results in an enantiomerically biased product.
  • This invention also provides methods of incorporating an amino acid into a polypeptide. These methods involve contacting an amino acid ester with a catalytic serine protease as described herein and an amino acid primary amine under conditions whereby the serine hydrolase catalyzes the formation of a peptide bond between the amino acid of the amino acid ester and the amino acid of the amino acid amine.
  • Preferred amino acid esters are acyl donors and preferred amino acid amines are acyl acceptor(s).
  • the amino acid amide can be a D or an L amino acid amide and may optionally be present in a peptide.
  • the amino acid ester may be a D or an L amino acid ester and may optionally be present in a peptide.
  • Particularly preferred hydrolases include, but are not limited to alpha/beta serine proteases, subtilisin type serine proteases, and chymotrypsin serine proteases with subtilisins being most preferred serine hydrolases.
  • the amino acid replaced with a cysteine preferably amino acid in the S1, S1′, or S2 subsite (e.g., subtilisin residues 156, 166, 217, 222, and 62) and/or preferably an asparagine, a leucine, a methionine, and a serine.
  • a cysteine preferably amino acid in the S1, S1′, or S2 subsite (e.g., subtilisin residues 156, 166, 217, 222, and 62) and/or preferably an asparagine, a leucine, a methionine, and a serine.
  • Particularly preferred substituents are as described herein.
  • the methods may further involve screening the modified serine hydrolase for an activity selected from the group consisting of a transesterification activity, a transamidation activity, and a transpeptidation activity.
  • the screens may optionally include a screen for stereoselectivity.
  • polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the term may also include variants on the traditional peptide linkage joining the amino acids making up the polypeptide.
  • residue refers to natural, synthetic, or modified amino acids.
  • enzyme includes proteins that are capable of catalyzing chemical changes in other substances without being permanently changed themselves.
  • Tthe enzymes can be wild-type enzymes or variant enzymes.
  • Enzymes within the scope of the present invention include, but are not limited to, pullulanases, proteases, cellulases, amylases, isomerases, lipases, oxidases, oxidoreductases, hydrolases, aldolases, ketolases, glycosidases, oxidoreductases, hydrolases, aldolases, ketolases, glycosidases, lyases, ligases, transferases, and ligases.
  • a “mutant enzyme” is an enzyme that has been changed by replacing an amino acid residue with a cysteine (or other) residue.
  • a “chemically modified” enzyme is an enzyme that has been derivatized to bear a substituent not normally found at that location in the enzyme.
  • a “chemically modified mutant enzyme” or “CMM” is an enzyme in which an amino acid residue has been replaced with another amino acid residue (preferably a cysteine) and the replacement residue is chemically derivatized to bear a substituent not normally found on that residue.
  • thiol side chain group “thiol containing group”, and thiol side chain” are terms that can be used interchangeably and include groups that are used to replace the thiol hydrogen of a cysteine.
  • the thiol side chain group includes a sulfur atom through which the thiol side chain group is attached to the thiol sulfur of the cysteine.
  • the “substitutent” typically refers to the group remains attached to the cysteine through a disulfide linkage formed by reacting the cysteine with a methanesulfonate reagent as described herein. While the term subsitutent preferably refers just to the group that remains attached (excluding its thiol group), the substituent can also refer to the entire thiol side chain group. The difference will be clear from the context.
  • the “binding site of an enzyme” consists of a series of subsites across the surface of the enzyme.
  • the substrate residues that correspond to the subsites are labeled P and the subsites are labeled S.
  • the subsites are labeled S 1 , S 2 , S 3 , S 4 , S 1 ′, and S 2 ′.
  • S 1 , S 2 , S 3 , S 4 , S 1 ′, and S 2 ′ A discussion of subsites can be found in Siezen et al. (1991) Protein Engineering, 4: 719-737, and Fersht (1985) Enzyme Structure and Mechanism, 2nd ed. Freeman, N.Y., 29-30.
  • the preferred subsites include S 1 , S 1 ′, and S 2 .
  • stereoselectivity when used in reference to an enzyme or to a reaction catalyzed by an enzyme refers to a bias in the amount or concentration of reaction products in favor of enantiomers of one chirality.
  • a stereoselective reaction or enzyme will produce reaction products that predominate in the “D” form over the “L” form (or “R” form over the “S” form) or conversely that predominate in the “L” form over the “D” form (or “S” form over the “R” form).
  • the predominance of one chirality is preferably a detectable predominance, more preferably a substantial predominance, and most preferably a statistically significant predominance (e.g. at a confidence level of at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98%).
  • amino acid ## or “amino acid ## in the XX subsite” is intended to include the amino acid at the referenced position (e.g. amino 156 of B. lentus subtilisin which is in the S 1 subsite) and the amino acids at the corresponding (homologous) position in related enzymes.
  • a “serine hydrolase” is a hydrolytic enzyme utilizing an active serine side chain to serve as a nucleophile in a hydrolytic reaction. This term includes native and synthetic serine hydrolases as well as enzymes engineered to perform the reverse reaction, e.g., for synthetic purposes.
  • alphalbeta serine hydrolases are a family of serine hydrolyases based on structural homology to enzymes including wheat germ serine carboxypeptidase II (see, e.g., Liao et al. (1992) Biochemistry 31: 9796-9812; Ollis et al. (1992) Protein Engineering, 5: 197-211).
  • subtilisin type serine proteases refer to a family of serine hydrolyases based on structural homology to enzymes in including subtilisin BPN′ (Bott et al. (1988) J. Biol. Chem. 263: 7895-7906; Siezen and Leunissen (1997) Protein Science 6: 501-523)
  • Subtilisins are bacterial or fungal proteases which generally act to cleave peptide bonds of proteins or peptides.
  • subtilisin means a naturally-occurring subtilisin or a recombinant subtilisin. A series of naturally-occurring subtilisins is known to be produced and often secreted by various microbial species.
  • subtilisins in this series exhibit the same or similar type of proteolytic activity.
  • This class of serine proteases shares a common amino acid sequence defining a catalytic triad which distinguishes them from the chymotrypsin related class of serine proteases.
  • the subtilisins and chymotrypsin related serine proteases have a catalytic triad comprising aspartate, histidine and serine.
  • the relative order of these amino acids, reading from the amino to carboxy terminus is aspartate-histidine-serine.
  • subtilisin In the chymotrypsin related proteases, the relative order, however, is histidine-aspartate-serine. Thus, subtilisin herein refers to a serine protease having the catalytic triad of subtilisin related proteases.
  • chymotrypsin serine protease family refers to a family of serine hydrolyases based on structural homology to enzymes including gamma chymotrypsin (Birktoft and Blow (1972) J. Molecular Biology 68: 187-240).
  • oxazolidinone refers to a compound including an oxazolidine ring and containing a keto group.
  • glycoside refers to a group of organic compounds that can be resolved by hydrolysis into sugars and other organic substances (e.g. aglycones).
  • Preferred glycosides are acetals that are derived from a combination of various hydroxy compounds with various sugars. They may be designaged individually as glucosides, mannosides, galactosides, etc.
  • Preferred glycosides include, but are not limited to monosacharrides and oligosaccharides, including pentose and hexose saccharides, including glucose and mannose containing saccharides.
  • Resolving a recemic mixture refers to racemic primary and secondary alcohols resolving racemic primary and secondary alcohols
  • FIG. 2 illustrates synthesis scheme 1; the modification of SBL mutants with chiral auxiliaries.
  • FIG. 3 illustrates synthesis scheme 2; the synthesis of mandelate-based ligands.
  • FIG. 4 illustrates synthesis scheme 3; the synthesis of oxazolidinone-based ligands.
  • FIG. 5 illustrates synthesis scheme 4; the synthesis of indanol-based ligands.
  • FIG. 6A illustrates a comparison of N62C CMM specificity constants.
  • FIG. 6B illustrates a comparison of S166C CMM specificity constants.
  • FIG. 6C illustrates a comparison of 217C CMM specificity constants.
  • FIG. 7A illustrates the changes in esterase to amidase activity ratios in S166C CMMs.
  • FIG. 7B illustrates the changes in esterase to amidase activity ratios in L217C CMMs.
  • FIG. 8 illustrates a reaction scheme for the transesterification of N-acetyl-1-phenylalanine vinyl ester with an alcohol using a chemically modified mutant enzyme as a catalyst.
  • CCMs chemically modified mutant enzymes
  • Preferred modified enzymes of this invention maintain a high degree of stereoselectivity in the reaction.
  • the chemically modified mutant enzymes of this invention comprise a serine hydrolase in which one or more residues in one or more subsite(s) are mutated to a cysteine and the cysteine is derivatized (e.g. with a methanesulfonate reagent) to provide a substituent coupled in place of the thiol hydrogen on the cysteine.
  • the site(s) of mutation and the substituents are selected to produce an enzyme that maintains a higher degree of stereoselectivity than the wild type enzyme in a transesterification, transamidation, or transpeptidation reaction.
  • the mutant enzymes are usefully in a wide variety of contexts including, but not limited to peptide synthesis, transesterification, resolution of enantiomers via stereoselective catalysis of racemic esters or amides and related groups, detergents and other cleaning materials, textile treatments, feed additives, and the like. Because of their stereoselectivity, the mutant enzymes are particularly useful as reagents that catalyze steps in organic syntheses. If desired, the mutant enzymes produce an enantiomerically purer reaction product and, in certain preferred embodiments, can be used to catalyze reactions that are otherwise difficult.
  • the enzymes can be used to catalyze a transamidation reaction where a “D” amino acid is coupled to an “L” amino acid.
  • the modified enzyme has high esterase and low amidase activity.
  • Preferred enzymes for modification according to this invention include the serine hydrolases.
  • the serine hydrolases are a class of hydrolytic enzymes characterized by a hydrolytic enzymes that posses a catalytic triad composed of a serine, histidine and a carboxylate amino acid (either aspartic or glutamic acid), and which catalyze the hydrolysis, and microscopic reverse reactions thereof, of carboxylic acid derivatives including, but not restricted to, esters, peptides and amides.
  • Preferred serine hydrolases comprising this invention include the trypsin-chymotrypsin proteases, the subtilisin proteases, and the alpha/beta hydrolases.
  • the enzyme is protease, more preferably a subtilisin (e.g. a Bacillus lentis subtilisin).
  • the subtilisins are alkaline serine proteases that are finding increasing use in biocatalysis, particularly in chiral resolution, regioselective acylation of polyfunctional compounds, peptide coupling, and glycopeptide synthesis. The latter two applications are of particular interest because they provide an alternative to site-directed mutagenesis for introducing unnatural amino acids into proteins.
  • serine hydrolases for use in this invention include, but are not limited to Rick to provideall serine hydrolase comprising enzymes that belong to the subtilisin class (subtilases), ⁇ / ⁇ hydrolases or trypsin/chymotryspsin families of structurally serine hydrolase enzymes.
  • residues for modification in the serine hydrolase are rationally selected.
  • Particularly preferred amino acid residues selected for modification include residues expected to be important discriminatory sites within the subsites. Such resides are determined from mutagenesis experiments where the subsite residues are systematically mutagenized and the effect of such mutagenesis on binding specificity and/or enzymatic activity is determined.
  • important residues can be identified from inspection of crystal structures and/or from predicted protein folding or protein-protein interactions determined using protein modeling software (e.g., Quanta (Molecular Simulations Inc.) and Frodo (academic software).
  • residues are selected where introduction of a substituent, which can be, but is not restricted to being, small, bulky, hydrophobic or hydrophilic, or charged, is expected to change the conformation of the binding site.
  • a substituent which can be, but is not restricted to being, small, bulky, hydrophobic or hydrophilic, or charged, is expected to change the conformation of the binding site.
  • such residues typically lie in the S1, S1′, or S2 subsites although it will be appreciated that in certain cases, alteration of residues in other subsites can also produce dramatic effects.
  • preferred residues for mutation include, but are not limited to residues 156 and 166 in the S1 subsite, residues 217 and 222 in the S1′ subsite and residue 62 in the S2 subsite Leu96, Val 104, Ile107, Phe189 and Tyr209 or residues at homologous positions within the subsites of other subtilisin-type serine proteases.
  • serine hydrolase is a trypsin-chymotrypsin type serine hydrolase
  • preferred residues for mutation include Tyr94, Leu99, Gln175, Asp189, Ser190 and Gln192 of trypsin or residues at homologous positions within the sub sites of other trypsin-chymotrypsin-type serine proteases.
  • preferred residues for mutation include Trp104, Thr138, Leu144, Val154, Ile189, Ala 225, Leu278 and Ile185 of Candida antartica lipase (Protein Data Bank entry 1 tca) or residues at homologous positions within the subsites of other alpha/beta type serine hydrolases.
  • amino acids replaced in the enzyme 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 enzyme preferably the S1, S1′ or S2 subsites.
  • subtilisin the amino acids to be replaced are N62, L217, M222, S156, S166, site 104, site 107 (S4), site 96 (S2), site 189(S2′), and site 209 (S1′/S3′) or their homologues where the numbered position corresponds to naturally occurring subtilisin from Bacilus amyloliquefacients or to equivalent amino acid residues in other subtilisins such as Bacillus lentus subtilisin.
  • the mutants described herein are most efficiently prepared by site-directed mutagenesis of the DNA encoding the wild-type enzyme of interest (e.g. Bacillus lentis subtilisin). Techniques for performing site-directed mutagenesis or non-random mutagenesis are known in the art. Such methods include, but are not limited to alanine scanning mutagenesis (Cunningham and Wells (1989) Science, 244, 1081-1085), oligonucleotide-mediated mutagenesis (Adeliman et al. (1983) DNA, 2, 183), cassette mutagenesis (Wells et al. (1985) Gene, 344: 315) and binding mutagenesis (Ladner et al. WO 88/06630).
  • site-directed mutagenesis of the DNA encoding the wild-type enzyme of interest
  • Techniques for performing site-directed mutagenesis or non-random mutagenesis are known in the art. Such methods include, but are not limited to alanine scanning muta
  • the substitute amino acid residue (e.g. cysteine) is introduced to the selected target site by oligonucleotide-mediated mutagenesis using the polymerase chain reaction technique.
  • the gene encoding the desired native enzyme (e.g. subtilisin) is carried by a suitable plasmid. More preferably, the plasmid is an expression vector, e.g., a plasmid from the pBR, pUC, pUB, pET or pHY4 series.
  • the plasmid can be chosen by persons skilled in the art for convenience or as desired.
  • the fragment containing the selected mutation site is cleaved from the gene encoding the subject enzyme by restriction endonucleases is used as the template in a modified PCR technique (see, Higuchi et al. (1988) Nucleic Acid Res., 16, 7351-7367).
  • an oligonucleotide containing the desired mutation is used as a mismatch primer to initiate chain extension between 5′ and 0.3 PCR flanking primers.
  • the process includes two PCR reactions. In the first PCR, the mismatch primer and the 5′ primer are used to generate a DNA fragment containing the desired base substitution. The fragment is separated from the primers by electrophoresis. After purification, it is then used as the new 5′ primer in a second PCR with the 3′ primer to generate the complete fragment containing the desired base substitution. After confirmation of the mutation by sequencing, the mutant fragment is then inserted back to the position of the original fragment.
  • a cassette mutagenesis method may be used to facilitate the construction and identification of the cysteine mutants of the present invention.
  • the gene encoding the serine hydrolase is obtained and sequenced in whole or in part.
  • the point(s) at which it is desired to make a mutation of one or more amino acids in the expressed enzyme are identified.
  • the sequences flanking these points are evaluated for the presence of restriction sites for replacing a short segment of the gene with an oligonucleotide which when expressed will encode the desired mutants.
  • restriction sites are preferably unique sites within the serine hydrolase gene so as to facilitate the replacement of the gene segment.
  • any convenient restriction site which is not overly redundant in the hydrolase gene may be used, provided the gene fragments generated by restriction digestion can be reassembled in proper sequence. If restriction sites are not present at locations within a convenient distance from the selected point (e.g., from 10 to 15 nucleotides), such sites are generated by substituting nucleotides in the gene in such a fashion that neither the reading frame nor the amino acids encoded are changed in the final construction.
  • the task of locating suitable flanking regions and evaluating the needed changes to arrive at two convenient restriction site sequences is made routine by the redundancy of the genetic code, a restriction enzyme map of the gene and the large number of different restriction enzymes. Note that if a convenient flanking restriction site is available, the above method need be used only in connection with the flanking region which does not contain a site.
  • Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished e.g., M113 primer extension in accord with generally known methods.
  • the restriction sites flanking the sequence to be mutated are digested with the cognate restriction enzymes and the end termini-complementary oligonucleotide cassette(s) are ligated into the gene.
  • the mutagenesis is enormously simplified by this method because all of the oligonucleotides can be synthesized so as to have the same restriction sites, and no synthetic linkers are necessary to create the restriction sites.
  • a suitable DNA sequence computer search program simplifies the task of finding potential 5′ and 3′ convenient flanking sites.
  • any mutation introduced in creation of the restriction site(s) are silent to the final construction amino acid coding sequence.
  • a candidate restriction site 5′ to the target codon a sequence preferably exists in the gene that contains at least all the nucleotides but for one in the recognition sequence 5′ to the cut of the candidate enzyme.
  • the blunt cutting enzyme SmaI CCC/GGG
  • N if N needed to be altered to C this alteration preferably leaves the amino acid coding sequence intact.
  • subtilisin gene from Bacillus lentus (“SBL”).
  • the gene for SBL is cloned into a bacteriophage vector (e.g. M13mp19 vector) for mutagenesis (see, e.g. U.S. Pat. No. 5,185,258).
  • Oligonucleotide-directed mutagenesis is performed according to the method described by Zoller et al. (1983) Meth. Enzymol., 100: 468-500.
  • the mutated sequence is then cloned, excised, and reintroduced into an expression plasmid (e.g. plasmid GG274) in the B. subtilis host.
  • PEG 50%) is added as a stabilizer.
  • the crude protein concentrate thus obtained is purified by first passing through a SephadexTM G-25 desalting matrix with a pH 5.2 buffer (e.g. 20 mM sodium acetate, 5 mM CaCl 2 ) to remove small molecular weight contaminants. Pooled fractions from the desalting column are then applied to a strong cation exchange column (e.g. SP SepharoseTM FF) in the sodium acetate buffer described above and the SBL is eluted with a one step gradient of 0-200 mM NaCl acetate buffer, pH 5.2. Salt-free enzyme powder is obtained following dialysis of the eluent against Millipore purified water and subsequent lyophilization.
  • a pH 5.2 buffer e.g. 20 mM sodium acetate, 5 mM CaCl 2
  • the purity of the mutant and wild-type enzymes, which are denatured by incubation with a 0.1 M HCl at 0° C. for 30 minutes is ascertained by SDS-PAGE on homogeneous gels (e.g. using the PhastTM system from Pharmacia, Uppsala, Sweden).
  • the concentration of SBL is determined using the Bio-Rad (Hercules, Calif.) dye reagent kit which is based on the method of Bradford (1976) Anal. Biochem., 72: 248-254). Specific activity of the enzymes is determined as described below and in the examples.
  • kits for site-directed mutagenesis are commercially available (see, e.g. TransfomerTM Site-Directed Mutagenesis Kit available from Toyobo).
  • the mutated protein is expressed from a heterologous nucleic acid in a host cell.
  • the expressed protein is then isolated and, if necessary, purified.
  • the choice of host cell and expression vectors will to a large extent depend upon the enzyme of choice and its source.
  • a useful expression vector contains an element that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in a host cell independent of the genome of the host cell, and preferably one or more phenotypic markers that permit easy selection of transformed host cells.
  • the expression vector may also include control sequences encoding a promoter, ribosome binding site, translation initiation signal, and, optionally, a repressor gene, a selectable marker or various activator genes.
  • nucleotides encoding a signal sequence may be inserted prior to the coding sequence of the gene.
  • a gene or cDNA encoding a mutated enzyme to be used according to the invention is operably linked to the control sequences in the proper reading frame.
  • Suitable host cells include bacteria such as E. coli or Bacillus , yeast such as S. cerevisiae , mammalian cells such as mouse fibroblast cell, or insect cells.
  • bacteria such as E. coli or Bacillus
  • yeast such as S. cerevisiae
  • mammalian cells such as mouse fibroblast cell, or insect cells.
  • a bacterial expression system is used.
  • the host is Bacillus .
  • Protein expression is performed by processes well known in the art according to factors such as the selected host cell and the expression vector to culture the transformed host cell under conditions favorable for a high-level expression of the foreign plasmid.
  • one particularly preferred expression system is plasmid GG274 which is then expressed in a B. subtilis host.
  • substitutents can be used to modify the cysteine(s) introduced into the serine hydrolase.
  • preferred substituents are those that improve stereoselectivity of the enzyme in a transesterification and/or a transamidation and/or a transpeptidation reaction.
  • Preferred substituents are bulky (e.g. at least about 4-6 angstroms in one dimension and/or consisting of three of more atoms in a linear, cyclic or branched conformation), and/or hydrophobic, and/or charged.
  • the substituents include polar aromatic groups (e.g. derivatized benzenes such as fluorobenzene, chlorobenzene, derivatized 5 member rings, oxazolidadones, etc.).
  • Other preferred substituents include alkyl amino groups with a positive charge (e.g. C 1 to C 50 , more preferably C 1 to C 30 and most preferably C 1 to C 15 alkyl amino groups with a positive charge) and glycosides (e.g. mono or oligosaccharrides derived from pentoses and hexoses and derivatives therof). Where transesterification activity is desired, particularly preferred embodiments include alkyl groups (e.g.
  • C 1 to C 50 more preferably C 1 to C 30 and most preferably C 1 to C 15 alkyl groups
  • a negative charge e.g. SO 3 ⁇ , and other sulfur acids, CO 2 ⁇ , and other acidic species including phopsphorus acid moieties, etc.
  • Typical oxazolidinones for use in this invention include, but are not limited to, (R)-2-methoxy-2-phenyl-ethyl-thiol, (S)-2-methoxy-2-phenyl-ethyl-thiol, (R)-2-hydroxy-2-phenyl-ethyl-thiol, (S)-2-hydroxy-2-phenyl-ethyl-thiol, N-(3′-thio-propyl)-2-oxazolidinone, N-(3′-thio-propyl)-(S)-4-phenyl-2-oxazolidinone, N-(3′-thio-propyl)-(R)-4-benzyl-2-oxazolidinone, N-(3′-thio-propyl)-(R)-4-benzyl-2-oxazolidinone, N-(3′-thioxazolidinone, N-(3′-thio-propyl)-
  • Particular preferred embodiments include, but are not limited to, the substituents illustrated in FIG. 2 and Other particularly preferred embodiments include, but are not limited to, the substituents illustrated in FIG. 2 and any of the commonly available chiral auxiliaries and ligands applied in asymmetric synthesis.
  • the R group on cysteines provides a convenient relatively reactive thiol group (—SH) that can be exploited for coupling a desired substituent to the cysteine.
  • the substitutent of interest is provided, derivatized as a methanethiosulfonate reagent which, when reacted with the cysteine, results in the substituent of interest covalently coupled to the cysteine by a disulfide linkage (—S—S—).
  • chemical modification with the methanethiosulfonate reagent(s) is carried out as described by Berglund et al. (1997) J. Am. Chem. Soc., 119: 5265-5255 and DeSantis et al. (1998) Biochemistry, 37: 5968-5973. Briefly, 200 ⁇ L of a 1 M solution of the methanethiosulfonate (MTS) reagent is added to a solution (5-10 mg/mL, 3.5 mL) of the cysteine mutant in 70 mM CHES, 5 nM MES, 2 mM CaCl 2 , pH 9.5. The MTS reagent is added in two portions over 30 minutes.
  • MTS methanethiosulfonate
  • Reaction mixtures are kept at 20° C. with continuous end-over-end mixing. Reactions are monitored by following the specific activity (e.g. with suc-AAPF-pNA) and by tests for residual free thiol (e.g. with Ellman's reagent).
  • the reaction mixture is loaded on a SephadexTM PD-10 G25 column with 5 mM MES and 2 mM CaCl 2 , pH 6.5. The protein fraction is then dialyzed against 1 mM CaCl 2 and the dialysate is lyophilized.
  • the reactive groups may be derivatized with appropriate blocking/protecting groups to prevent undesired reactions during the coupling.
  • the thiol group(s) on these cysteines may be derivatized with appropriate protecting groups (e.g. (e.g. benzyl, trityl, tert-butyl, MOM, acetyl, thiocarbonate, thiocarbamate, and others).
  • the chemically modified mutants are typically screened for the activity or activities of interest.
  • activities include amidase activity, esterase activity, the ratio of amidase to esterase activity, stereoselectivity, transesterification, transamidation, transpeptidation, and the like.
  • Assays for such activities are well known to those of skill in the art.
  • k cat /K M is obtained in a microtiter plate format, from the rate of product formation (v) using the limiting case of the Michaelis-Menten equation at low substrate concentration as an approximation (Equation 1 where [S] and [E] are the substrate and enzyme concentrations, respectively): V ⁇ (K cat /K M )[S][E] for [S] ⁇ K M .
  • Enzyme stock solutions are prepared in 5 mM 4-morpholineethanesulfonic acid (MES) with 2 mM CaCl 2 , pH 6.5 at about 5 ⁇ 10 ⁇ 7 M for amidase and about 5 ⁇ 10 ⁇ 8 M for esterase assays substrate solutions are prepared in dimethyl sulfoxide (DMSO).
  • MES 4-morpholineethanesulfonic acid
  • DMSO dimethyl sulfoxide
  • the amidase substrate sucAAPF-pNa stock is 1.6 mM which give s 0.8 mM in the well.
  • the esterase substrate isosuccinyl-alanine-alanine-proline-phynylalanine-thiobenzyl ester (sucAAPF-SBn) stock solution is 1.0 mM, which gives 0.05 mM in the well.
  • Tris buffer for the esterase assay contains 0.375 nM DTNB. This buffer should be used immediately as the DTNB decomposes within a few hours due to the high pH of the buffer.
  • a sample of each enzyme solution ( ⁇ 150 ⁇ L) is placed in a well in the 1st, 5th, or 9th column of an enzyme loading plate.
  • Rows A to g contain enzymes, and row H contains MES buffer.
  • Rows A to g contain enzymes, and row H contains MES buffer.
  • Rows A to g contain enzymes, and row H contains MES buffer.
  • Rows A to g contain enzymes, and row H contains MES buffer.
  • 10 ⁇ L of substrate solution and 180 ⁇ L of buffer are dispensed into wells along columns to be used in a run.
  • Columns 14 on the assay plate contain four replicates of the enzymes in column 1 of the loading plate; columns 5-8 contain four replicates of the enzymes in column 5 of the loading plate.
  • Reactions are initiated by transferring 10 ⁇ L of enzyme solution from the loading plate to the assay plate with an 8-channel pipette. For amidase assays, four columns are initiated for one run. For esterase assays, two columns are initiated for a run.
  • the time delay between addition of enzyme to the first column and onset of reading is about 22-30 seconds (amidase) and 10-15 seconds (esterase).
  • the pate is placed on a Titertech Multiscan MCC340 reader (programmed in the kinetic mode, filter 414 mm, lag time 0.0 minutes, interval 5 seconds with automatic background subtraction of blank row H) (Labsystems, Finland) and is read for 1.0 minute (amidase) or 30 seconds (esterase).
  • Prolonged reading, past the nearly linear part of the progress curve) up to ⁇ 50% conversion provides an underestimate of the rate.
  • the output from the reader represents the average rate of change in absorbance at 4114 nm min ⁇ 1 , measured at 5 second intervals, of the total time programmed.
  • other catalytic activities are assayed (e.g. transamidation, transpeptidation, transesterification).
  • substrate specificity and/or stereoselectivity is also determined.
  • stereoselectivity can be determined according to a number of methods known to those of skill in the art. In one embodiment, stereoselectivity is determined by using stereoselective liquid or gas chromatographic procedures (e.g., using Chiralcel columns, Daicel Chemical Industries, Ltd.) as described in the examples.
  • subtilisins can catalyze peptide bond formation starting from an ester substrate, by first forming an acyl enzyme intermediate which then reacts with a primary amine to form the peptide product.
  • preferred enzymes have high esterase activity to promote acyl enzyme formation and then low amidase activity to minimize hydrolysis of the peptide bond of the desired product.
  • subtilisins do not meet this requirement and in one embodiment the improvement of the esterase to amidase selectivities of subtilisins is one feature of the present invention.
  • Another particularly preferred feature of this invention is the improved stereoselectivity obtained with the modified mutant enzymes.
  • the modified mutant enzymes can be utilized to resolve racemic alcohols and to stereoselectively acylate prochiral and meso diols.
  • the stereoselective modified enzymes of this invention can also be used to catalyze the formation of peptide linkages with particular chiral moieties.
  • the coupling of D amino acids in peptide synthesis protocols has proven problematic.
  • the modified enzymes of this invention provide a convenient and efficient mechanism to preferentially couple a D- or an L-amino acid to an individual amino acid or to an amino acid present in a polypeptide.
  • Enzymatic peptide coupling is an attractive method for preparation of a variety of peptides because this method requires minimal protection of the substrate, proceeds under mild conditions, and does not cause racemization (Wong et al. (1994) pages 41-130 In: Enzymes in Synthetic Organic Chemistry , Pergamon Press, Oxford).
  • the chemically modified mutant enzymes of this invention can incorporate D-amino acid esters as acyl donors in peptide synthesis or an ⁇ -branched amino acid amide as acyl acceptor in peptide synthesis to give a variety of dipeptides. These reaction are not possible with the wild-type enzymes.
  • modified enzymes of the present invention can be used in organic synthesis to, for example, catalyze a desired reaction and/or to favor a certain stereoselectivity.
  • modified enzymes of this invention can also be utilized in more “traditional” applications.
  • the modified enzymes of this invention e.g. in particular the proteases and/or lipases
  • These detergent cleaning compositions or additives can also include other enzymes such as known proteases, amylases, cellulases, lipases or endoglycosidases as well as builders and stabilizers.
  • the modified subtilisins are used in formulating various detergent compositions.
  • a number of known compounds are suitable surfactants useful in such detergent compositions. /These include nonionic, anionic, cationic, anionic, or zwitterionic detergents (see, e.g., U.S. Pat. Nos. 4,404,128, and 4,261,868).
  • a suitable detergent formulation is that described in example 7 of U.S. Pat. No. 5,204,015.
  • the modified enzymes of this invention may provide improved was performance in a detergent composition (as compared to previously known additives). Improves wash performance typically refers to increased cleaning of certain modified enzyme-sensitive stains such as grass or blood, as determined by a standard evaluation procedure (e.g. light reflectance) after a standard wash cycle.
  • modified enzymes of the present invention may be used for any purpose that the native or wild-type enzymes are used.
  • these modified enzymes can be used, for example, in bar or liquid soap applications, dish care formulations, contact lens cleaning solutions or products, peptide synthesis, feed applications such as feed additives or preparation of feed additives, waste treatment, textile application such as the treatment of fabrics, and as fusion-cleavage enzymes in protein production.
  • the modified enzymes of this invention are used in a method of treating a textile.
  • the methods involve contacting a chemically modified mutant enzyme of this invention with a textile under conditions effective to produce a textile resistant to certain enzyme-sensitive stains (e.g. grass or blood stains).
  • the method can be used to treat, for example, silk or wool.
  • Enzyme treatments of such fabrics are know to those of skill in the art and are described for example in Research Disclosure 216,034, European Patent application No: 134,267, U.S. Pat. No. 4,533,359, and European Patent application 3244,259.
  • the modified enzymes of this invention are used in the preparation of an animal feed, for example, a cereal-based feed.
  • the enzyme can be incorporated into essentially any cereal feed, e.g. a cereal comprising one or more of wheat, barley, maize, sorghum, rye, oats, triticale, and rice.
  • a cereal component of a cereal-based feed constitutes a source of protein, it is usually necessary to include species of supplementary protein in the feed such as those derived form fish meal, meat, or vegetables.
  • Sources of vegetable proteins include, but are not limited to soybeans, rape seeds, canola, soybean meal, rapeseed meal, and canola meal.
  • the inclusion of a modified enzyme in an animal feed can enable the crude protein value and/or the digestibility and/or the amino acid content of the feed to be increased. This permits a reduction in the amounts of alternative protein sources and/or amino acid supplements that are added to the feed.
  • kits for synthesizing and/or screening modified mutants of this invention preferably include one or more mutant serine hydrolases having one or more amino acid residues substituted with a cysteine as described herein.
  • the kits may additionally include one or more methane sulfonate reagents as described herein that can be used to derivatized the mutant serine hydrolase.
  • Such kits may additionally include one or more reagents and/or apparatus for performing such derivitizations.
  • kits can include appropriate substrates and/or reactants for screening the chemically modified mutant enzyme for one or more desired activities as described herein.
  • kits for the use of the modified mutant enzymes described herein preferably contain one or more containers containing one or more of the chemically modified mutant serine hydrolases as described herein.
  • kits can also include appropriate reagents and/or substrates to use the modified enzymes in one or more of the reactions described herein.
  • kits may include instructional materials containing directions (i.e., protocols) for the practice of the syntheses, uses or assay methods described herein.
  • the instructional materials provide protocols derivatizing the mutant enzyme with one or more of the methane sulfonate reagents described herein.
  • the instructional materials may provide protocols describing the use of the modified enzyme in catalyzing formation of a peptide bond.
  • the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
  • Methanethiosulfonate reagents may be used to introduce virtually unlimited structural modifications in enzymes via reaction with the thiol group of cysteine.
  • the covalent coupling of enantiomerically pure (R) and (S) chiral auxiliary methanethiosulfonate ligands to cysteine mutants of subtilisin Bacillus lentus induces spectacular changes in catalytic activity between diastereomeric enzymes.
  • Amidase and esterase kinetic assays using a low substrate approximation were used to establish k cat /K M values for the chemically modified mutants, and up to 3 fold differences in activity were found between diastereomeric enzymes.
  • Enantiomerically pure MTS ligands, 1a-i, ( FIG. 2 ) were synthesized and used to chemically modify the N62C, S156C, S166C and L217C mutants of SBL. These residues were targeted on the basis of SBL's x-ray crystal structure (X-ray structure solved by Rick Bott at Genencor International Inc. Brookhaven data base entry IJEA of SBL). N62C is in the S 2 pocket near His-64 (nomenclature according to Schechter and Berger (1967) Biochem. Biophys. Res. Commun. 27: 157-162). S156C and S166C are at the bottom of the S 1 pocket. However, S156C is surface exposed and S166C is buried pointing into the pocket. L217C is found in S 1 ′ which is where the leaving group is bound. A kinetic assay of amidase and esterase activity was conducted on these new diastereomeric CMMs in order to investigate their properties and to probe any changes in selectivity.
  • the mesylate was converted to bromide, (R)-10 (73%), by the action of LiBr in refluxing acetone, and methanethiosulfonate, (R)-1a, was formed in 84% yield from bromide, (R)-10, using NaSSO 2 CH 3 in DMF.
  • the methanethiosulfonate (S)-1a was made in an analogous fashion from (S)-mandelic acid (see Scheme 2, FIG. 3 ).
  • Oxazolidinones have been widely used as chiral auxiliaries in asymmetric synthesis, and the degree of asymmetric induction can be excellent in chemical transformations ranging from alkylations to aldol reactions to Diels-Alder additions (Gage and Evans (1990) Org. Synth., 68: 77-91; Ager et al. (1997) Aldrichimica Acta, 30: 3-12).
  • Subtilisin mutants produced as described above, were modified with the homochiral MTS reagents. Characterization of the new CMMs was done by PMSF titration (Hsia et al. (1996) Anal. Biochem., 242: 221-227) of their active sites, Ellman's titration (Ellman et al. (1961) Biochem. Pharmacol., 7: 88-95) of residual thiol ( ⁇ 2% in all cases), ES-MS after FPLC purification (mol. wt. ⁇ 6 mass units in all cases), and by nondenaturing gradients gels which all showed one band.
  • Assay errors are the mean standard errors from sets of six replicates.
  • b k cat /K M obtained by full kinetic run of 8 substrate concentrations and calculation of independent k cat and K M values. Errors were obtained from the curve-fitting errors in k cat and K M .
  • Chiral auxiliaries are employed in asymmetric organic synthesis to block one diastereotopic face of a molecule thus forcing the reaction to the other face which results in the formation of solely one diastereomer.
  • the covalent coupling of enantiomerically pure (R) and (S) chiral auxiliary MTS ligands to SBL cysteine mutants has caused remarkable changes in enzyme activity. We can attribute these changes uniquely to the difference in spatial orientation at the ligand stereocenter when comparing diastereomeric enzymes. The extraordinary differences in catalytic activity between diastereomeric enzymes can be compared in FIGS. 6A, 6B , and 6 C.
  • N62C—(R)-e is both an excellent amidase (2.2 fold better than WT) and an excellent esterase (3.9 fold better than WT).
  • the (S)-diastereomer is a good amidase (308 mM ⁇ 1 s ⁇ 1 ) and esterase (6564 mM ⁇ 1 s ⁇ 1 ), but not as good as the (R)-diastereomer.
  • N62C—(R)-e and N62C-c show significantly higher k cat and lower K M values than WT giving overall 5.4 fold and 3.7 fold respectively better esterase activity than WT.
  • the N62C—(S)-e CMM does not display these characteristics.
  • Modifications at S166C produced many sets of diastereomeric CMMs with large differences in activity.
  • the 1a, 1b, 1f, 1g, 1h and 1i modifications produced CMMs with greater then 2 fold variances between diastereomeric CMMs.
  • the largest difference of any set of CMMs was achieved with S166C-b which has a [k cat /K M (R)]/[k cat /K M (S)] ratio of 3.2.
  • the modifications with the phenyl and benzyl oxazolidinones at S166C reverse which diastereomeric CMM has greater catalytic activity in a way similar to the same modifications at N62C.
  • S166C—(S)-g and S166C—(S)-i are good esterases (4069 mM ⁇ 1 s ⁇ 1 and 4556 mM ⁇ 1 s ⁇ 1 respectively) and have high esterase/amidase ratios of 110 and 97 making them good candidates as peptide ligation catalysts ( FIG. 7A ).
  • S166C—(S)-a and S166C—(S)-b have relatively high esterase/amidase ratios (48 and 62) compared to S166C (4) and WT, but these two CMMs are very poor esterases.
  • the (S)-ligand consistently gives a CMM with a higher esterase to amidase ratio than the (R)-ligand, except in the case of the if where the two diastereomeric enzymes have similar ratios.
  • L217C—(S)-d has a very high esterase k cat /K M (9296 mM ⁇ 1 ⁇ l) and a low amidase value (104 mM ⁇ 1 s ⁇ 1 ) giving it a relatively high esterase/amidase ratio of 89.
  • L217C—(R)-f has a similar ratio of 88 and a good esterase k cat /K M (6435 mM ⁇ 1 s ⁇ 1 ).
  • N62C—(R)-e was particularly remarkable. It's amidase k cat /K M was 1.56 fold better than it's diastereomer, N62C—(S)-e, and 3 fold better than WT. Also, the esterase k cat /K M of N62C—(R)-e was 2.6 fold better than it's diastereomer and 5.4 fold better than WT.
  • N62C, L217C, S166C, and S156C mutants of subtilisin Bacillus lentus were prepared and purified by the general method (Stabile et al. (1996) Bioorg. Med. Chem. Lett. 6: 2501-2506). Spectrophotometric measurements were made on a Perkin-Elmer Lamda 2 spectrophotometer.
  • HRMS data were acquired using a Micromass 70-250S (double focussing) mass spectrometer for EI spectra and a Micromass ZAB-SE for FAB spectra.
  • Enantiomeric excesses of methanethiosulfonates ((R)-1a, (S)-1a, (R)-1b and (S)-1b) were determined by HPLC on a Chiralcel OJ column using a hexane:isopropanol eluent system.
  • Enantiomeric excesses (ee) of bromides ((R)-18, (S)-18, (R)-19, (S)-19, (R)-20, (S)-20, (R)-21, (S)-21, (R)-22, (S)-22, (R)-25 and (S)-25) were determined by HPLC on a Chiralcel OD column using the same eluent system.
  • (S)-3 was prepared in the same manner as the (R)-3. From (S)-mandelic acid (4.00 g, 26.29 mmol) was obtained (S)-1 (1.301 g, 30%).
  • (R)-2-methyloxymethoxy-2-phenyl-1-ethylmethanethiosulfonate, (R)-12 was prepared in the same manner as (R)-1a.
  • (R)-10 1.58 g, 5.948 mmol was converted to (R)-12 (1.005 g, 61%).
  • (s)-4 was prepared in the same manner as (R)-4. From (S)-mandelic acid (3.176 g, 20.87 mmol) was obtained crude (s)-4 (3.45 g, quantitative) which was used directly in the next step.
  • (R)-21 N-(3′-bromoethyl)-(R)-4-phenyl-2-oxazolidinone, (R)-21, was prepared in the same manner as 17, except 10 eq of 1,2-dibromoethane and 3 eq of KOH were used. From (R)-4-phenyl-2-oxazolidinone (0.261 g, 1.599 mmol) was obtained (R)-21 (0.387 g, 90%, ee ⁇ 98%), as a colorless oil.
  • N-(3′-bromoethyl)-(R)-4-benzyl-2-oxazolidinone, (R)-22 was prepared in the same manner as 17, except 10 eq of 1,2-dibromoethane and 3 eq of KOH were used. From (R)-4-benzyl-2-oxazolidinone (0.386 g, 2.178 mmol) was obtained (R)-22 (0.372 g, 60%, ee ⁇ 98%), as a colorless oil.
  • reaction solution was purified on a disposable desalting column (Pharmacia Biotech PD-10, Sephadex G-25 M) pre-equilibrated with MES buffer (5 mM MES, 2 mM CaCl 2 , pH 6.5).
  • MES buffer 5 mM MES, 2 mM CaCl 2 , pH 6.5.
  • the CMM was eluted with MES-buffer (5.0 mL), dialyzed (MWCO 12 14,000) against MES buffer (10 mM MES, 1 mM CaCl 2 , pH 5.8) then flash frozen and stored at ⁇ 20° C.
  • Modified enzymes were analyzed by nondenaturing gradient (8-25%) gels at pH 4.2, run towards the cathode on the Pharmacia Phast-Systemä, (Pharmacia Application File No. 300) and appeared as one single band. Each of the CMMs was analyzed in parallel with its parent cysteine mutant and the WT enzyme.
  • CMMs Prior to ES-MS analysis, CMMs were purified by FPLC (BioRad, Biologic System) on a Source 15 RPC matrix (17-0727-20 from Pharmacia) with 5% acetonitrile, 0.01% TFA as the running buffer and eluted with 80% acetonitrile, 0.01% TFA in a one step gradient. Electrospray mass spectra were recorded on a PE SCIEX API III Biomolecular Mass Analyzer.
  • the active enzyme concentration was determined as previously described (Hsia et al. (1996) Anal. Biochem. 242: 221-227) by monitoring fluoride release upon enzyme reaction with a-toluenesulfonyl fluoride (Aldrich Chemical Co. Inc.) as measured by a fluoride ion sensitive electrode (Orion Research 96-09).
  • the active enzyme concentration determined in this way was used to calculate kinetic parameters for each CMM.
  • a general run consisted of equilibrating six plastic cuvettes containing 980 ⁇ L of 0.1 M Tris, 0.005% Tween 80 at pH 8.6 to 25° C.
  • the substrate (10 ⁇ L) in DMSO was added and the cuvette was shaken twice before returning it to the machine for zeroing.
  • the enzyme (10 ⁇ L) in 20 mM MES, 1 mM CaCl 2 at pH 5.8 was added and the cuvette was returned to the machine with a eight sec delay.
  • the initial rate data was recorded and used to calculate k cat /K M . Esterase data was adjusted to account for background hydrolysis of the substrate.
  • Michaelis-Menten constants were measured at 25° C. by curve fitting (GraFit® 3.03) of the initial rate data determined at eight concentrations (0.05 mM-3.0 mM) of the N-Suc-AAPF-pNA substrate for amidase activity and eight concentrations (0.015 mM-2.0 mM) of the N-Suc-AAPF-SBn substrate for esterase activity.
  • a combined site-directed mutagenesis and chemical modification strategy was used to create superior enzyme catalysts for the resolution of racemic primary and secondary alcohols using a transesterification reaction.
  • the chemically modified mutant N62C—S—CH 3 of subtilisin Bacillus lentus catalyze the transesterification of N-acetyl-L-phenylalanine vinyl ester with ⁇ -branched primary alcohols faster than wild type.
  • the cysteine mutant, M222C of subtilisin Bacillus lentus gave higher yields (90% and 92% yields with 1-phenylethanol and 2-octanol respectively versus 19% and 10% for wild-type) and better enantioselectivity than wild-type when secondary alcohols were used.
  • Hydrolase-catalyzed transesterifications are widely employed to resolve racemic alcohols and to stereoselectively acylate prochrial and meso diols (Faber (1996) Biotransformations in Organic Chemistry, 3rd Ed., Springer-Verlag, Heidelberg).
  • serine proteases have found limited application in comparison to lipases and esterases (Id.).
  • One reason for this is the high substrate specificity of many serine proteases compared to other hydrolases (Faber supra., Sears and Wong (1996) Biotechnol. Prog., 12: 423433).
  • Cysteine mutants of SBL and chemically modified mutants were prepared and characterized as described above and in Berglund et al. (196) Bioorg. Med. Chem. Lett., 6: 2507-2512) and the best esterases among them were selected for comparative evaluation (Plettner et al. (198) Bioorg. Med. Chem. Lett., 8: 2291-2296).
  • N62C—S—(CH 2 ) 2 —SO 3 ⁇ gave a higher yield of product than WT when 2-phenyl-1-propanol was the nucleophile.
  • N52-C—S—(CH 2 ) 2 —SO 3 ⁇ as catalyst gave a significant improvement in the des of the product ester (41%) over WT (26% de).
  • Only one CMM catalyst, N62C—S—CH 3 gave marked increases in product yield for the two primary alcohols (97% for 2-phenyl-1-propanol and 79% for 2-methyl-1-pentanol). No changes in stereochemical preferences from WT were observed for any of the CMMs. TABLE 4 Yields and d.e.
  • Both enzymes catalyzed the transesterification of primary and secondary alcohols faster than WT and with de's that were comparable to WT. Remarkably, they gave much higher yield of product ester than WT when the sterically hindered secondary alcohols were used as nucleophiles.
  • M222C gave almost quantitative yield product ester with 1-phenylethanol and an excellent yield (92%) of ester with 2-octanol. M222C improved the de of product ester to above 90% for both secondary alcohols and N62C—S—CH 3 gave product ester in 97% de for 2-octanol.
  • N62C—S—CH 3 and M222C were seen to be better transesterification catalysts than WT. The reasons for this appear to be different.
  • N62C—S—CH 3 catalyzed the transesterification of primary alcohols with 2 in higher yield and in shorter time than M222C, but the reverse was true for secondary alcohols where M222C efficiently coupled 1-phenylethanol and 2-octanol with 2 in 98% and 92% yields respectively.
  • WT gives lower yields with secondary alcohols because branching at the ⁇ -carbon of the alcohol is poorly tolerated by the S 1 ′ pocket (nomenclature according to Schechter and Berger (1967) Biochem. Biophys. Res.
  • Residue 222 of SBL is at the boundary between the S 1 - and S 1 ′-pockets, a region in close proximity to a location where the nucleophile would approach the acyl-enzyme intermediate in order to deacylate the enzyme and complete the catalytic cycle. Therefore, it is reasonable to expect that if methionine is replaced by the smaller cysteine at position 222, a larger space in this critical region would permit more sterically hindered nucleophiles to react with the acyl-enzyme intermediate. This is exactly what was observed for M222C catalyzed reactions of secondary alcohols.
  • N62C—S—CH 3 gave considerably higher yields than WT with secondary alcohols. Further more, this CMM catalyzed the transesterification of primary alcohols much faster than either WT or M222C. It is probable that N62C—S—CH 3 catalyzed transesterification faster than M222C or WT because of a higher turnover rate (Plettner et al. (198) Bioorg. Med. Chem. Lett., 8: 2291-2296), but that in the case of secondary alcohols, the improved catalytic efficiency could not entirely overcome the negative steric hindrance factors.
  • N62C—S—CH 3 and M222C are superior transesterification catalysts to WT, with N62C—S—CH 3 giving higher yields in a shorter reaction time in transesterification reactions that WT when primary alcohols are used with 2 as acyl donor.
  • M222C itself has been found to be an excellent catalyst for the transesterification of secondary alcohols.

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US13/405,083 US20120156721A1 (en) 1998-11-10 2012-02-24 Chemically modified mutant serine hydrolases show improved catalytic activity and chiral selectivity
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ATE433485T1 (de) 1998-11-10 2009-06-15 Univ Toronto Chemisch modifizierte mutante serinhydrolasen
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WO2000028007A3 (en) 2000-07-27
DK1129180T3 (da) 2009-08-24
AU3099000A (en) 2000-05-29
US20090075329A1 (en) 2009-03-19
ATE433485T1 (de) 2009-06-15
AU772427B2 (en) 2004-04-29
US8148128B2 (en) 2012-04-03
EP1129180A2 (de) 2001-09-05
US20120295329A1 (en) 2012-11-22
JP4932990B2 (ja) 2012-05-16
DE69940978D1 (de) 2009-07-23
US8357524B2 (en) 2013-01-22
EP1129180B1 (de) 2009-06-10
US20120156721A1 (en) 2012-06-21
CA2348014A1 (en) 2000-05-18
WO2000028007A2 (en) 2000-05-18

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