Binding Phenol Oxidizing Enzyme-Peptide Complexes

Abstract

The present application relates to peptides which bind to a target stain, phenol oxidizing enzyme-binding peptide complexes wherein the binding peptide is attached to the C-terminus of the phenol oxidizing enzyme or is inserted or substituted into the phenol oxidizing enzyme. In a preferred embodiment the phenol oxidizing enzyme is a laccase specifically Stachybotrys oxidase B and variants thereof. The invention provides expression vectors comprising the phenol oxidizing enzyme-binding peptide complex as well as host cells comprising the vectors.

1 . A binding peptide having the amino acid sequence illustrated in any one of SEQ ID NOS: 2 through 433 wherein said peptide binds to a carotenoid compound. 
     
     
         2 . The binding peptide of  claim 1 , wherein said peptide is selected from the group consisting of SEQ ID NOS: 4, 16, 24, 77, 92, 94, 104, 105, 120, 198, 233, 237, 243, 247, 256, 279, 293, 300, 304, 317, 340, and 428. 
     
     
         3 . The binding peptide of  claim 2 , wherein said peptide is selected from the group consisting of SEQ ID NOS: 4, 16, 24, 92, 94, 104, 105, 120, 198, 233, 247, 256, 279, 293, 300, 304, and 317. 
     
     
         4 . The binding peptide of  claim 1 , further comprising a cysteine amino acid residue added to each end of the binding peptide. 
     
     
         5 . A binding peptide comprising a repeatable motif selected from the group consisting of SAPA, TAPP, APAL, PPP, PPPP, SSPH, SSP, SSK, SPT, LPAQ, PPPL, PTPL, SPTT, PLVP, PLP, YTKP, SLH, SLLNA, SPL, SNLA, SPLTQ, TTT, AARND, AARN, ARND, LSPG, NPNN, NLAT, NTS, PHSM, PPWM, PTSP, TGGA, YLPS, YTKP, PGSL, APS, TPV, TTTS and LNAT, wherein said binding peptide has 6 to 15 amino acid residues and binds to a carotenoid stain on a fabric. 
     
     
         6 . A polynucleotide sequence encoding the binding peptide of  claim 1 . 
     
     
         7 . A polynucleotide sequence encoding the binding peptide of  claim 5 . 
     
     
         8 . A phenol oxidizing enzyme-peptide complex comprising a phenol oxidizing enzyme and a peptide comprising the amino acid sequence illustrated in any one of SEQ ID NOS: 2 through 433. 
     
     
         9 . The phenol oxidizing enzyme-peptide complex of  claim 8 , wherein the binding peptide is selected from the group consisting of SEQ ID NOS: 4, 16, 24, 92, 94, 104, 105, 120, 198, 233, 247, 256, 279, 293, 300, 304, and 317. 
     
     
         10 . The phenol oxidizing enzyme-peptide complex of  claim 8 , wherein the peptide is attached to the phenol-oxidizing enzyme at the C-terminus. 
     
     
         11 . A laccase-peptide complex comprising a laccase obtainable from a  Stachybotrys  species and a peptide comprising an amino acid sequence illustrated in any one of SEQ ID NOS: 2 through 433. 
     
     
         12 . The laccase-peptide complex of  claim 11 , wherein the peptide has an amino acid sequence illustrated in any one of SEQ ID NOs: 4, 16, 24, 92, 94, 104, 105, 120, 198, 233, 247, 256, 279, 293, 300, 304, and 317. 
     
     
         13 . The laccase-peptide complex of  claim 11 , wherein the laccase has the amino acid sequence illustrated in SEQ ID NO: 1 or a variant thereof, said variant having at least 75% sequence identity to the amino acid sequence illustrated in SEQ ID NO: 1 and said variant is capable of modifying the color associated with colored compounds. 
     
     
         14 . The laccase-peptide complex of  claim 13  comprising a variant of SEQ ID NO: 1, wherein said variant differs from SEQ ID NO: 1 in at least one of the positions 48, 67, 70, 76, 83, 98, 115, 119, 134, 171, 175, 177, 179, 188, 236, 246, 253, 269, 272, 296, 302, 308, 318, 329, 331, 346, 348, 349, 365, 390, 391, 394, 404, 415, 423, 425, 428, 434, 465, 479, 481, 483, 499, 550, 562, 570, and 573 or sequence positions corresponding thereto and wherein said complex is capable of modifying the color associated with colored compounds. 
     
     
         15 . The laccase-peptide complex of  claim 18 , wherein the laccase variant comprises a sequence that differs from that of SEQ ID NO: 1 in at least one of the positions 188, 254, 272, 346, 348, 394 and 425 or sequence positions corresponding thereto. 
     
     
         16 . An expression vector comprising a polynucleotide encoding the phenol oxidizing enzyme-peptide complex of  claim 8 . 
     
     
         17 . A host cell comprising the vector of  claim 16 . 
     
     
         18 . A laccase-peptide complex comprising a peptide having the amino acid sequence illustrated in any one of SEQ ID NOs:2-433 and a laccase, wherein said laccase comprises the amino acid sequence illustrated in SEQ ID NO: 1 or a variant thereof, wherein said variant differs in at least one of the positions 188, 254, 272, 346, 348, 394 and 425 of SEQ ID NO: 1. 
     
     
         19 . A method of enhancing the binding of a laccase enzyme to a target stain comprising;
 a) obtaining a peptide according to  claim 1 ,   b) combining said peptide with a laccase to form a laccase-peptide complex, and   c) exposing a target stain to the laccase-peptide complex under suitable conditions to allow the complex to bind with the target stain.   
     
     
         20 . The method according to  claim 19 , wherein the target stain is a carotenoid compound. 
     
     
         21 . The method according to  claim 19 , wherein the peptide is selected from the group consisting of SEQ ID NOS: 4, 16, 24, 92, 94, 104, 105, 120, 198, 233, 247, 279, 293, 300, 304, and 317. 
     
     
         22 . The method according to  claim 19 , wherein the laccase is an enzymatically active laccase having the amino acid sequence illustrated in SEQ ID NO: 1 or a variant thereof, said variant having at least 75% sequence identity to the amino acid sequence illustrated in SEQ ID NO: 1 and which differs in at least one of the positions 48, 67, 70, 76, 83, 98, 115, 119, 134, 171, 175, 177, 179, 188, 236, 246, 253, 269, 272, 296, 302, 308, 318, 329, 331, 346, 348, 349, 365, 390, 391, 394, 404, 415, 423, 425, 428, 434, 465, 479, 481, 483, 499, 550, 562, 570, and 573 or sequence positions corresponding thereto and wherein said variant is capable of modifying the color associated with a targeted stain. 
     
     
         23 . A detergent composition comprising
 a) one or more surfactants and   b) the phenol oxidizing enzyme-peptide complex of  claim 8 , wherein said complex selectively binds to a target stain during a wash cycle that includes agitation.   
     
     
         24 . An enzymatic composition comprising
 a) one or more surfactants and   b) the phenol oxidizing enzyme-peptide complex of  claim 8 .   
     
     
         25 . A method for bleaching a carotenoid stain on a fabric or surface comprising contacting the stain with the enzyme composition of  claim 24 . 
     
     
         26 . A method for producing a host cell comprising a polynucleotide encoding a laccase-peptide complex, comprising the steps of:
 (a) obtaining a polynucleotide encoding a laccase having at least 68% identity to the amino acid sequence disclosed in SEQ ID NO: 1;   (b) obtaining a polynucleotide encoding a binding peptide having an amino acid sequence as illustrated in any one SEQ ID NOS: 2-433;   (c) conjugating the polynucleotide of step (a) with (b);   (d) introducing said conjugated polynucleotide into the host cell; and   (e) growing said host cell under conditions suitable for the production of said laccase-peptide complex.   
     
     
         27 . A method of using a binding peptide to target a stain on a textile comprising
 a) obtaining a binding peptide as illustrated in any one of SEQ ID NOS: 2-433;   b) exposing said binding peptide to a target stain, wherein said binding peptide binds to said stain and not to said textile.   
     
     
         28 . The method according to  claim 27 , wherein the binding peptide is, selected from the group consisting of SEQ ID NOS: 4, 16, 24, 92, 94, 104, 105, 120, 198, 233, 247, 256, 279, 293, 300, 304, and 317. 
     
     
         29 . A method of enhancing the selectivity of a phenol oxidizing enzyme to a target stain which comprises,
 a) derivatizing a laccase with a binding peptide as illustrated in any one of SEQ ID NOS: 2-433 to form a laccase-peptide complex; and   b) exposing the laccase-peptide complex to a target stain, wherein selectivity of the laccase-peptide complex to the target stain is greater than the selectivity of a nonderivatized laccase having the same amino acid sequence as the laccase of the laccase-peptide complex.   
     
     
         30 . The method according to  claim 29 , wherein the stain is a carotenoid compound stain.