Hydrolases, nucleic acids encoding them and methods for making and using them
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12Q-001/34
C12Q-001/00
C12N-009/14
C12N-001/20
C12N-015/00
C12P-007/00
C12P-021/04
C12P-021/06
C07H-021/04
출원번호
US-0527333
(2012-06-19)
등록번호
US-8465942
(2013-06-18)
발명자
/ 주소
Dayton, Christopher L. G.
Barton, Nelson
Bueno, Analia
Cuenca, Joslin G.
Hitchman, Tim
Kline, Katie A.
Lyon, Jonathan
Miller, Mark L.
Wall, Mark A.
출원인 / 주소
Bunge Oils, Inc.
대리인 / 주소
Jones Day
인용정보
피인용 횟수 :
0인용 특허 :
132
초록▼
Provided are hydrolases, including lipases, saturases, palmitases and/or stearatases, and polynucleotides encoding them, and methods of making and using these polynucleotides and polypeptides. Further provided are polypeptides, e.g., enzymes, having a hydrolase activity, e.g., lipases, saturases, pa
Provided are hydrolases, including lipases, saturases, palmitases and/or stearatases, and polynucleotides encoding them, and methods of making and using these polynucleotides and polypeptides. Further provided are polypeptides, e.g., enzymes, having a hydrolase activity, e.g., lipases, saturases, palmitases and/or stearatases and methods for preparing low saturate or low trans fat oils, such as low saturate or low trans fat animal or vegetable oils, e.g., soy or canola oils.
대표청구항▼
1. A method for biocatalytic synthesis of a structured lipid comprising the following steps: (a) providing a polypeptide having a palmitase activity, said polypeptide being selected from the group consisting of isolated, synthetic and recombinant polypeptides having a palmitase activity and said pol
1. A method for biocatalytic synthesis of a structured lipid comprising the following steps: (a) providing a polypeptide having a palmitase activity, said polypeptide being selected from the group consisting of isolated, synthetic and recombinant polypeptides having a palmitase activity and said polypeptide either i. being encoded by a nucleic acid comprising a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO:1 and having at least one base residue change recited in Table A, Table B, or Table C below, wherein the nucleic acid encodes at least one polypeptide having a palmitase activity, TABLE Aaminoacidoriginalnewresidueaminoaminooriginalnewpositionacidacidcodoncodon 7YLTACCTT 15ALGCCCTG 15AMGCCATG 16DWGATTGG 31MIATGATT 32GEGGCGAG 32GPGGCCCT 34LMCTGATG 43LICTGATT 46FFTTCTTT 48ACGCCTGT 48AMGCCATG 48ATGCCACT 49DNGACAAT 49DRGACCGT 49DSGACTCT 52AMGCCATG 68SFTCGTTT 68SYTCGTAT 85RACGGGCT 85RDCGGGAT 85RQCGGCAG 85RSCGGTCT 85RTCGGACG 85RYCGGTAT 95EKGAGAAG 92AVGCGGTT 92AEGCGGAG 95EDGAGGAT 95EAGAGGCT 96AKGCGAAG 96ARGCGAGG 97ASGCCTCG101KRAAGCGT104VLGTGTTG113YLTATCTT116EAGAGGCG116ECGAGTGT116EDGAGGAT116EFGAGTTT116EIGAGATT116EIGAGATT116ELGAGCTT116ENGAGAAT116EQGAGCAG116ESGAGAGT116ETGAGACT116EVGAGGTT116EWGAGTGG116EYGAGTAT117LMCTGATG120KRAAGAGG133SAAGTGCT136ASGCGTCG137GFGGCTTT139LMCTCATG140HRCACAGG142NWAACTGG144AIGCGATT144ALGCGTTG144AMGCGATG144AVGCGGTG149EHGAGCAT150AIGCGATT150AMGCGATG150AWGCGTGG153SNAGCAAT153SGAGCGGT158NDAACGAC162PGCCGGGT162PKCCGAAG162PSCCGTCG162PSCCGTCG162PSCCGTCG183VIGTGATT166QACAGGCG166QECAGGAG166QTCAGACG167IFATTTTT167IKATTAAG167ILATTCTG167IRATTCGT167IYATTTAT172RHCGCCAT172RKCGCAAG172RLCGCCTT172RYCGCTAT180LKCTCAAG180LRCTCAGG185ACGCGTGT185ANGCGAAT190EAGAAGCG190EKGAAAAG190EMGAAATG190EQGAACAG190ERGAAAGG200LICTAATT200LVCTAGTA200LVCTAGTT201EYGAGTAT203AHGCGCAT203APGCGCCG203ARGCGAGG207MLATGCTT214THACCCAT214TKACCAAG214TRACCAGG214TSACCTCG214TVACCGTT215GAGGGGCG222LICTGATT225ASGCGTCT163RYCGGTAT163RMCGGATG163RTCGGACG163RLCGGTTG163RCCGGTGT 95EK163RF183VI TABLE BoldnewAAoldnewAAcodoncodon#codoncodon#GCGGCT 35AGCAGT108GCGGCT 35GTGGTT102GCGGCT 35ACCACG188GCGGCT 35GTGGTT183GCGGCT 35GTGGTT102GCGGCT 35GTGGTT102GCGGCT 35GCGGCT 35GTGGTT183GCGGCT 35GTCGTG128GCGGCT 35AGTTCT133GCGGCT 35AGCAGT108GCGGCT 35GGCGGA 45GCGGCT 35GGCGGA 45GCGGCT 35GGCGGA 45GGCGGA 45GTGGTT102GGCGGA 45GGCGGA 45GTGGTT102GCGGCT 35GGCGGA 45GCGGCT 35GGCGGA 45GCGGCT 35GGCGGA 45GCGGCT 35GGCGGA 45GCGGCT 35GGCGGA 45GGCGGA 45GCGGCT 35GGCGGA 45GCGGCT 35GGCGGA 45GCGGCT 35GGCGGA 45 GCGGCT 35GGCGGA 45GCGGCT 35GGCGGA 45GCGGCT 35GGCGGA 45GCGGCT 35GGCGGA 45GCGGCT 35GGCGGA 45GCGGCT 35CTGCTT117GCGGCT 35CTGCTT117GCGGCT 35AGCAGT108GCGGCT 35GTGGTT102GTGGTT102AGCAGT108GCGGCT 35GTGGTT102GCGGCT 35GTGGTT102GCGGCT 35AGCAGT108GCGGCT 35CTGCTT117CTGCTT117CGGAGG126AGCAGT108CTGCTT117AGCAGT108CTGCTT117GCGGCT 35GCGGCT 35CTGCTT117GCGGCT 35GTGGTT102oldnewAAoldnewAAcodoncodon#codoncodon#GTGGTT183ACCACG188AGCAGT108CTGTTG124ACCACG188AGCAGT108GTCGTG128AGCAGT108CTGTTG124GTGGTT102CTGTTG124AGTTCT133GTGGTT183ACCACG188CTGTTG124AGTTCT133GTGGTT102AGCAGT108CTGCTT117CTGTTG124AGCAGT108CTGCTT117CTGCTT117GTCGTG128GTGGTT102AGCAGT108CTGCTT117AGCAGT108CTGCTT117CTGCTT117CTGTTG124AGCAGT108CTGCTT117AGCAGT108CTGCTT117GTGGTT102CTGCTT117AGCAGT108CTGCTT117CTGCTT117ACCACG188AGCAGT108CGGAGG126AGCAGT108AGTTCT133CTGTTG 90GTGGTT183AGCAGT108GTCGTG128GTGGTT102GTCGTG128GTGGTT102AGCAGT108ACCACG188GTGGTT183ACCACG188CTGCTT117CTGTTG124AGCAGT108CTGCTT117CTGCTT117CGGAGG126CTGCTT117GTGGTT183CTGCTT117CTGTTG124CTGCTT117GTCGTG128CTGTTG124AGTTCT133AGTTCT133CGCCAC172GTGGTT183ACCACG188CTGTTG124CGGAGG126CTGCTT117ACCACG 188GTGGTT183CTGCTT117AGTTCT133oldnewAAoldnewAAcodoncodon#codoncodon#GTCGTG128AGTTCT133GTCGTG128AGTTCT133GTCGTG128GTGGTT183ACCACG188CTGCTT117CGGAGG126GTGGTT183CGGAGG126ACCACG188AGTTCT133CTGCTT117CGGAGG126 ACCACG188GTCGTG128AGTTCT133CTGTTG124AGTTCT133AGTTCT133ACCACG188CTGTTG124CGGAGG126ACCACG188GTCGTG128AGTTCT133AGTTCT133GTGGTT183ACCACG188CGGAGG126GTCGTG128GTCGTG128AGCAGT153CGGAGG126AGTTCT133ACCACG188GTGGTT183ACCACG188AGTTCT133GTGGTT183ACCACG188 GTCGTG128GTGGTT183GTGGTT183ACCACG188oldnewAAoldnewAAcodoncodon#codoncodon#GTGGTT183GTGGTT183AGTTCT133ACCACG188ACCACG188GTCGTG128AGTTCT133GTGGTT183CCACG188ACCACG188ACCACG188ACCACG188oldnewAAcodoncodon#ACCACG188 TABLE Cold codonnew codonold AAnew AAAA #CCGTCGPS162ACGATGTM 22AGCGGCSG153GAAAAAEK190CGCCACRH172ATGATAMI 31GTGATGVM 83CTAATALI200GCAGTAAV211ACGATGTM 22orii. having at least 85% sequence identity to SEQ ID NO:2, and having at least one amino acid residue change recited in Table A, or Table C above, oriii. comprising an amino acid sequence of SEQ ID NO:2 but also comprising at least one of amino acid residue modification D61A; D61E; R72E; R72K; E116A; E116Q; E116R; E116T; E116V; S133A; I151G; I151A; V163R; D164R, or a combination thereof, oriv. comprising an amino acid sequence of SEQ ID NO:2 but also comprising at least one of amino acid residue modification 120L; V62S; G77P; V83C; D88H; Y113G; E116T; E116G; H140K; K146S; I167S; L180E; E194M; A211Q; S212Y; G215C; G215V; G215W; A218H; A218S; V223A; A225M; A225Q, or a combination thereof;(b) providing a composition comprising a triacylglyceride (TAG);(c) contacting the polypeptide of step (a) with the composition of step (b) under conditions wherein the polypeptide hydrolyzes a palmitic acid residue at the Sn1 or Sn3 position of the triacylglyceride (TAG), thereby producing a 1,2 diacyglycerol (1,2-DAG) or 2,3-diacylglyceride (2,3-DAG);(d) promoting acyl migration in the 1,2-DAG or 2,3-DAG of step (c) under kinetically controlled conditions, thereby producing a 1,3-DAG,(e) providing an R1 ester;(f) providing an R1-specific hydrolase, and(g) contacting the 1,3-DAG of step (d) with the R1 ester of step (d) and the R1-specific hydrolase of step (e) under conditions wherein the R1-specific hydrolase catalyzes esterification of the Sn2 position, thereby producing the structured lipid. 2. The method of claim 1, wherein the R1-specific hydrolase is an Sn2-specific lipase. 3. The method of claim 1, wherein the structured lipid is selected from the group consisiting of a cocoa butter alternative, a synthetic cocoa butter, a natural cocoa butter, 1,3-dipalmitoyl-2-oleoylglycerol, 1,3-distearoyl-2-oleoylglycerol, 1-palmitoyl-2-oleoyl-3-stearoylglycerol and 1-oleoyl-2,3-dimyristoylglycerol. 4. The method of claim 1, wherein said R1 ester comprises one or more of an omega-3 fatty acid, an omega-6 fatty acid, an omega-9 fatty acid, or stearic acid. 5. The method of claim 1, wherein said R1 ester comprises a moiety selected from the group consisting of alpha-linolenic acid, stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, oleic acid, palmitoleic acid, and stearic acid. 6. The method of claim 1, wherein said synthesized structured lipid has a lower saturated fat content than said triacylglyceride. 7. The method of claim 1, wherein said synthesized structured lipid has a lower trans fat content than said triacylglyceride. 8. The method of claim 1, wherein the polypeptide is encoded by a nucleic acid comprising a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO:1 and having at least one base residue change recited in Table A, Table B or Table C, wherein the nucleic acid encodes at least one polypeptide having a palmitase activity. 9. The method of claim 1, wherein the polypeptide has at least 85% sequence identity to SEQ ID NO:2, and has at least one amino acid residue change recited in Table A or Table C. 10. The method of claim 1, wherein the polypeptide comprises an amino acid sequence of SEQ ID NO:2 but also comprising at least one of amino acid residue modification D61A; D61E; R72E; R72K; E116A; E116Q; E116R; E116T; E116V; S133A; I151G; I151A; V163R; D164R, or a combination thereof. 11. The method of claim 1, wherein the polypeptide comprises an amino acid sequence of SEQ ID NO:2 but also comprising at least one of amino acid residue modification I20L; V62S; G77P; V83C; D88H; Y113G; E116T; E116G; H140K; K146S; I167S; L180E; E194M; A211Q; S212Y; G215C; G215V; G215W; A218H; A218S; V223A; A225M; A225Q, or a combination thereof. 12. The method of claim 1, wherein the nucleic acid sequence is the sequence of SEQ ID NO:23. 13. The method of claim 1, wherein the nucleic acid sequence is codon optimized for expression in Pseudomonas fluorescens.
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