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US 2010/0055085 A1Published application
Hydrolases, nucleic acids encoding them and methods for making and using them
AssigneeVerenium Corporation
InventorsTim HITCHMAN, Christopher L. G. Dayton, Katie A. Kline, Jonathan Lyon, Mark A. Wall, Nelson R. Barton
Life of this patent
Estimating the term…
Abstract
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.
Claims31
1:
- An isolated, synthetic or recombinant nucleic acid comprising
- (a) a nucleic acid (polynucleotide) encoding at least one polypeptide, wherein the nucleic acid comprises a sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to:
- (i) SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19, or,
- (ii) the nucleic acid of SEQ ID NO:1 having one or more nucleotide changes (or the equivalent thereof) encoding one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four or more or all the amino acid changes (or the equivalent thereof) as set forth in Table 3 or Table 4,
- wherein the nucleic acid of (i) or (ii) encodes at least one polypeptide having a hydrolase activity, or encodes a polypeptide or peptide capable of generating a hydrolase specific antibody (a polypeptide or peptide that acts as an epitope or immunogen),
- (b) the nucleic acid (polynucleotide) of (a), wherein the sequence identities are determined:
- (A) by analysis with a sequence comparison algorithm or by visual inspection, or (B) over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more residues, or the full length of a cDNA, transcript (mRNA) or gene,
- (c) the nucleic acid (polypeptide) or (a) or (b), wherein, the sequence comparison algorithm is a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall -p blastp -d “nr pataa” -F F, and all other options are set to default,
- (d) a nucleic acid (polynucleotide) encoding at least one polypeptide or peptide having a hydrolase activity, wherein the nucleic acid comprises a sequence that hybridizes under stringent conditions to the complement of the nucleic acid of (a), (b) or (c), wherein the stringent conditions comprise a wash step comprising a wash in 0.2×SSC at a temperature of about 65° C. for about 15 minutes,
- (e) a nucleic acid (polynucleotide) encoding at least one polypeptide having a hydrolase, e.g. a lipase, a saturase, a palmitase and/or a stearatase activity, wherein the polypeptide comprises the sequence of SEQ ID NO:2, or enzymatically active fragments thereof, having at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, or more or all the amino acid changes (or the equivalent thereof) as set forth in Table 3 or Table 4,
- (f) a nucleic acid (polynucleotide) encoding at least one polypeptide having a hydrolase activity, wherein the polypeptide comprises the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20 or enzymatically active fragments thereof,
- (g) (A) the nucleic acid (polynucleotide) of any of (a) to (f) and encoding a polypeptide having at least one conservative amino acid substitution and retaining its hydrolase activity, or, (B) the nucleic acid of (g)(A), wherein the at least one conservative amino acid substitution comprises substituting an amino acid with another amino acid of like characteristics;
- or, a conservative substitution comprises:
- replacement of an aliphatic amino acid with another aliphatic amino acid;
- replacement of a serine with a threonine or vice versa;
- replacement of an acidic residue with another acidic residue;
- replacement of a residue bearing an amide group with another residue bearing an amide group;
- exchange of a basic residue with another basic residue;
- or replacement of an aromatic residue with another aromatic residue,
- (h) the nucleic acid (polynucleotide) of any of (a) to (g) encoding a polypeptide having a hydrolase activity but lacking a signal sequence,
- (i) the nucleic acid (polynucleotide) of any of (a) to (h) encoding a polypeptide having a hydrolase activity further comprising a heterologous sequence,
- (j) the nucleic acid (polynucleotide) of (i), wherein the heterologous sequence comprises, or consists of a sequence encoding:
- (A) a heterologous signal sequence, (B) the sequence of (A), wherein the heterologous signal sequence is derived from a heterologous enzyme, or, (C) a tag, an epitope, a targeting peptide, a cleavable sequence, a detectable moiety or an enzyme, or,
- (k) a nucleic acid sequence (polynucleotide) fully (completely) complementary to the sequence of any of (a) to (j).
21 more elements
2:
- The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the hydrolase activity comprises a lipase activity, a saturase, a stearatase or a palmitase activity,
- wherein optionally, the saturase activity comprises selectively hydrolyzing at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the saturated fatty acid esters, wherein optionally, the hydrolyzed esters may be esters of saturated fatty acids and glycerol, umbelliferol or other alcohols,
- wherein optionally, the palmitase activity comprises selectively hydrolyzing fatty acids such that at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the fatty acids hydrolyzed are palmitic acid, or
- wherein optionally, the stearatase activity comprises selectively hydrolyzing fatty acids such that at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the fatty acids hydrolyzed are stearic acid.
1 more element
3:
- The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the hydrolase activity comprises
- (a) hydrolyzing a triacylglycerol to a diacylglycerol and a free fatty acid, or, hydrolyzing a triacylglycerol to a monoacylglycerol and free fatty acids, or, hydrolyzing a diacylglycerol to a monoacylglycerol and free fatty acids, or, hydrolyzing a monoacylglycerol to a free fatty acid and a glycerol, or, comprises hydrolyzing a triacylglycerol (TAG), a diacylglycerol (DAG) or a monoacylglycerol (MAG),
- (b) synthesizing a triacylglycerol from a diacylglycerol or a monoacylglycerol and free fatty acids,
- (c) synthesizing 1,3-dipalmitoyl-2-oleoylglycerol (POP), 1,3-distearoyl-2-oleoylglycerol (SOS), 1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS) or 1-oleoyl-2,3-dimyristoylglycerol (OMM), long chain polyunsaturated fatty acids, arachidonic acid, docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA),
- (d) hydrolase activity that is triacylglycerol (TAG), diacylglycerol (DAG) or monoacylglycerol (MAG) position-specific, wherein optionally the hydrolase activity is Sn2-specific, Sn1- or Sn3-specific,
- (e) hydrolase activity that is fatty acid specific, wherein optionally the fatty acid is a palmitic, stearic or other saturated fatty acid,
- (f) modifying oils by hydrolysis, alcoholysis, esterification, transesterification or interesterification,
- (g) hydrolase activity that is regio-specific or chemoselective, wherein optionally the hydrolase activity comprises synthesis of enantiomerically pure chiral products, or,
- (h) synthesis of umbelliferyl fatty acid (FA) esters.
6 more elements
4-5. (canceled)
6:
- An expression cassette, a vector or a cloning vehicle comprising a nucleic acid comprising a sequence as set forth in claim 1, wherein optionally the cloning vehicle comprises a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificial chromosome, wherein optionally the viral vector comprises an adenovirus vector, a retroviral vector or an adeno-associated viral vector, and optionally the cloning vehicle comprises a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).
7:
- A transformed cell comprising a nucleic acid comprising a sequence as set forth in claim 1, or an expression cassette, vector or cloning vehicle as set forth in claim 6, wherein optionally the cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.
8:
- A transgenic non-human animal comprising a sequence as set forth in claim 1, wherein optionally the animal is a mouse.
9:
- A transgenic plant comprising a sequence as set forth in claim 1,
- (i) wherein optionally the plant is a corn plant, a sorghum plant, a potato plant, a tomato plant, a wheat plant, an oilseed plant, a rapeseed plant, a soybean plant, a rice plant, a barley plant, a grass, or a tobacco plant,
- (ii) wherein optionally the seed is rice, a corn seed, a wheat kernel, an oilseed, a rapeseed, a soybean seed, a palm kernel, a sunflower seed, a sesame seed, a rice, a barley, a peanut or a tobacco plant seed.
10:
- (canceled)
11:
- An isolated, synthetic or recombinant polypeptide comprising a sequence
- (a) having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or has 100% (complete) sequence identity to:
- (i) the amino acid sequence of SEQ ID NO:2, or enzymatically active fragments thereof, and having at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four or more or all of the amino acid residue changes (or the equivalent thereof) as set forth in in Table 3 or Table 4, or
- (ii) the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20, or enzymatically active fragments thereof,
- wherein the polypeptide or peptide of (i) or (ii) has a hydrolase activity, or the polypeptide or peptide is capable of generating a hydrolase specific antibody (a polypeptide or peptide that acts as an epitope or immunogen),
- (b) the polypeptide or peptide of (a), wherein the sequence identities are determined:
- (A) by analysis with a sequence comparison algorithm or by a visual inspection, or (B) over a region of at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 100, 150, 200, 250, 300 or more amino acid residues, or over the full length of the polypeptide or peptide or enzyme, and/or enzymatically active subsequences (fragments) thereof,
- (c) the polypeptide or peptide of (b), wherein the sequence comparison algorithm is a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall -p blastp -d “nr pataa” -F F, and all other options are set to default;
- (d) an amino acid sequence encoded by the nucleic acid of claim 1, wherein the polypeptide has (i) a hydrolase activity, or, (ii) has immunogenic activity in that it is capable of generating an antibody that specifically binds to a polypeptide having a sequence of (a), and/or enzymatically active subsequences (fragments) thereof;
- (e) the amino acid sequence of any of (a) to (d), and comprising at least one conservative amino acid residue substitution, and the polypeptide or peptide retains a hydrolase activity;
- (f) the amino acid sequence of (e), wherein the conservative substitution comprises replacement of an aliphatic amino acid with another aliphatic amino acid;
- replacement of a serine with a threonine or vice versa;
- replacement of an acidic residue with another acidic residue;
- replacement of a residue bearing an amide group with another residue bearing an amide group;
- exchange of a basic residue with another basic residue;
- or, replacement of an aromatic residue with another aromatic residue, or a combination thereof,
- (g) the amino acid sequence of (f), wherein the aliphatic residue comprises alanine, valine, leucine, isoleucine or a synthetic equivalent thereof;
- the acidic residue comprises aspartic acid, glutamic acid or a synthetic equivalent thereof;
- the residue comprising an amide group comprises asparagine, glutamine or a synthetic equivalent thereof;
- the basic residue comprises lysine, arginine, histidine or a synthetic equivalent thereof;
- or, the aromatic residue comprises phenylalanine, tyrosine, tryptophan or a synthetic equivalent thereof;
- (h) the polypeptide of any of (a) to (f) having a hydrolase activity but lacking a signal sequence,
- (i) the polypeptide of any of (a) to (h) having a hydrolase activity and further comprising a heterologous sequence;
- (j) the polypeptide of (i), wherein the heterologous sequence comprises, or consists of (A) a heterologous signal sequence, (B) the sequence of (A), wherein the heterologous signal sequence is derived from a heterologous enzyme, and/or, (C) a tag, an epitope, a targeting peptide, a cleavable sequence, a detectable moiety or an enzyme;
- or,
- (k) comprising an amino acid sequence encoded by the nucleic acid of claim 1.
23 more elements
12:
- The isolated, synthetic or recombinant polypeptide of claim 11, wherein the hydrolase activity comprises a lipase activity, a saturase, a stearatase or a palmitase activity,
- wherein optionally, the saturase activity comprises selectively hydrolyzing at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the saturated fatty acid esters, wherein optionally, the hydrolyzed esters may be esters of saturated fatty acids and glycerol, umbelliferol or other alcohols,
- wherein optionally, the palmitase activity comprises selectively hydrolyzing fatty acids such that at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the fatty acids hydrolyzed are palmitic acid, or
- wherein optionally, the stearatase activity comprises selectively hydrolyzing fatty acids such that at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the fatty acids hydrolyzed are stearic acid.
1 more element
13:
- The isolated, synthetic, or recombinant polypeptide of claim 11, wherein the hydrolase activity comprises
- (a) hydrolyzing a triacylglycerol to a diacylglycerol and a free fatty acid, or, hydrolyzing a triacylglycerol to a monoacylglycerol and free fatty acids, or, hydrolyzing a diacylglycerol to a monoacylglycerol and free fatty acids, or, hydrolyzing a monoacylglycerol to a free fatty acid and a glycerol, or, comprises hydrolyzing a triacylglycerol (TAG), a diacylglycerol (DAG) or a monoacylglycerol (MAG),
- (b) synthesizing a triacylglycerol from a diacylglycerol or a monoacylglycerol and free fatty acids,
- (c) synthesizing 1,3-dipalmitoyl-2-oleoylglycerol (POP), 1,3-distearoyl-2-oleoylglycerol (SOS), 1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS) or 1-oleoyl-2,3-dimyristoylglycerol (OMM), long chain polyunsaturated fatty acids, arachidonic acid, docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA),
- (d) hydrolase activity that is triacylglycerol (TAG), diacylglycerol (DAG) or monoacylglycerol (MAG) position-specific, wherein optionally the hydrolase activity is Sn2-specific, Sn1- or Sn3-specific,
- (e) hydrolase activity that is fatty acid specific, wherein optionally the fatty acid is a palmitic, stearic or other saturated fatty acid,
- (f) modifying oils by hydrolysis, alcoholysis, esterification, transesterification or interesterification,
- (g) hydrolase activity that is regio-specific or chemoselective, wherein optionally the hydrolase activity comprises synthesis of enantiomerically pure chiral products, or,
- (h) synthesis of umbelliferyl fatty acid (FA) esters.
6 more elements
14-20. (canceled)
21:
- A heterodimer or a homodimer comprising a polypeptide as set forth in claim 11.
22:
- An immobilized polypeptide, wherein the polypeptide comprises a sequence as set forth in claim 11, or a subsequence thereof, wherein optionally the polypeptide is immobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an array or a capillary tube.
23:
- (canceled)
24:
- An isolated, synthetic or recombinant antibody that specifically binds to a polypeptide as set forth in claim 11, wherein optionally the antibody is a monoclonal or a polyclonal antibody.
25-27. (canceled)
28:
- A method of producing a recombinant polypeptide comprising the steps of:
- (a) providing a nucleic acid operably linked to a promoter, wherein the nucleic acid comprises a sequence as set forth in claim 1;
- and (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide,
- wherein optionally the method further comprises transforming a host cell with the nucleic acid of step (a) followed by expressing the nucleic acid of step (a), thereby producing a recombinant polypeptide in a transformed cell.
1 more element
29:
- (canceled)
30:
- A method of increasing thermotolerance or thermostability of a hydrolase polypeptide, the method comprising glycosylating a hydrolase polypeptide, wherein the polypeptide comprises at least thirty contiguous amino acids of a polypeptide as set forth in claim 11, thereby increasing the thermotolerance or thermostability of the hydrolase polypeptide,
- wherein optionally the hydrolase specific activity is thermostable or thermotolerant at a temperature in the range from greater than about −100° C. to about 115° C.
31:
- A detergent composition comprising a polypeptide as set forth in claim 11, wherein the polypeptide comprises a hydrolase activity, wherein optionally the hydrolase is a nonsurface-active hydrolase or a surface-active hydrolase, or optionally the hydrolase is formulated in a non-aqueous liquid composition, a cast solid, a granular form, a particulate form, a compressed tablet, a gel form, a powder, a gel, a hydrogel, a liposome, an aerosol, a paste or a slurry form.
32:
- A method for washing an object comprising the following steps:
- (a) providing a composition comprising a polypeptide having a hydrolase activity, wherein the polypeptide comprises a polypeptide as set forth in claim 11;
- (b) providing an object;
- and
- (c) contacting the polypeptide of step (a) and the object of step (b) under conditions wherein the composition can wash the object.
2 more elements
33:
- A method for stereoselectively hydrolyzing racemic mixtures of esters of 2-substituted acids comprising the following steps:
- (a) providing a hydrolase as set forth in claim 11, wherein the hydrolase is stereo selective;
- (b) providing a composition comprising a racemic mixture of esters of 2-substituted acids;
- and
- (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions wherein the polypeptide of step (b) can selectively hydrolyze the esters.
- wherein optionally the hydrolase is immobilized,
- and optionally the 2-substituted acid comprises a 2-aryloxy substituted acid, an R-2-(4-hydroxyphenoxy)propionic acid or a 2-arylpropionic acid, wherein optionally the 2-substituted acid comprises a ketoprofen.
4 more elements
34:
- A composition comprising a hydrolase as set forth in claim 11, the variant hydrolase of claim 35, or the enzyme generated by the method of claim 37,
- wherein optionally the composition is a detergent,
- wherein optionally the composition is a cosmetic or a cream,
- wherein optionally the composition is a pharmaceutical, a liposome, a tablet, a capsule, a formulation or a drug delivery agent,
- wherein optionally the composition is a food, feed, food supplement, feed supplement, dietary aid, dietary composition, or enzyme delivery matrix, and wherein optionally the food, feed, food supplement, feed supplement, dietary aid, dietary composition, or enzyme delivery matrix further comprises a nutritional base comprising a fat or a fat and no or little hydrolase, or optionally the hydrolase is activated by a bile salt, or optionally the food, feed, food supplement, feed supplement, dietary aid, dietary composition, or enzyme delivery matrix further comprises a cow's milk-based infant formula, or optionally the hydrolase can hydrolyze long chain fatty acids.
2 more elements
35:
- A method of generating a variant of a nucleic acid encoding a polypeptide with a hydrolase activity comprising the steps of:
- (a) providing a template nucleic acid as set forth in claim 1;
- and
- (b) modifying, deleting or adding one or more nucleotides in the template sequence, or a combination thereof, to generate a variant of the template nucleic acid,
- wherein optionally the method further comprises expressing the variant nucleic acid to generate a variant hydrolase polypeptide,
- and optionally the modifications, additions or deletions are introduced by a method comprising error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, Gene Site Saturation MutagenesisSM (GSSMSM), synthetic ligation reassembly (SLR or GeneReassembly) and a combination thereof, or, the modifications, additions or deletions are introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof, wherein optionally the method is iteratively repeated until
- (i) a hydrolase having an altered or different activity or an altered or different stability from that of a polypeptide encoded by the template nucleic acid is produced
- wherein optionally the variant hydrolase polypeptide is thermotolerant, and retains some activity after being exposed to an elevated temperature, or, optionally the variant hydrolase polypeptide has increased glycosylation as compared to the hydrolase encoded by a template nucleic acid, or optionally the variant hydrolase polypeptide has a hydrolase activity under a high temperature, wherein the hydrolase encoded by the template nucleic acid is not active under the high temperature,
- (ii) a hydrolase coding sequence having an altered codon usage from that of the template nucleic acid is produced,
- wherein optionally the method is iteratively repeated until a hydrolase gene having higher or lower level of message expression or stability from that of the template nucleic acid is produced, or,
- (iii) a hydrolase having altered substrate specificity or substrate preference is produced, wherein optionally the substrate specificity or substrate preference of the variant hydrolase comprises preferential or increased hydrolysis of palmitic acid from an oil, or, the substrate specificity or substrate preference of the variant hydrolase comprises preferential or increased hydrolysis of stearic acid from an oil, or the substrate specificity or substrate preference of the variant hydrolase comprises preferential or increased hydrolysis of palmitic and stearic acid from an oil, or,
- wherein optionally, the hydrolase of (i), (ii) or (iii), has at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four or more or all of the amino acid residue changes (or the equivalent thereof) as set forth in in Table 3 or Table 4.
9 more elements
36:
- (canceled)
37:
- A method for making an enzyme having an altered substrate specificity or substrate preference comprising:
- (a) providing a parental hydrolase as set forth in claim 11;
- and
- (b) making at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or more amino acid residue modifications to the parental hydrolase enzyme, wherein optionally the amino acid residue modifications correspond to the amino acid sequence mutations to SEQ ID NO:2 as shown in Table 3 or Table 4 (or the equivalent thereof), thereby generating an enzyme having an altered substrate specificity or substrate preference,
- wherein optionally the altered substrate specificity or substrate preference comprises preferential or increased hydrolysis of palmitic acid,
- wherein optionally the altered substrate specificity or substrate preference.comprises preferential or increased hydrolysis of stearic acid,
- wherein optionally the altered substrate specificity or substrate preference comprises preferential hydrolysis of a particular fatty acid, wherein optionally the fatty acid is a saturated fatty acid, wherein optionally the fatty acid is a linolenic acid, linoleic acid, oleic acid, palmitic acid or stearic acid.
4 more elements
38:
- A lipase comprising an amino acid sequence as set forth in SEQ ID NO:2 but also comprising at least amino acid residue modification D61A;
- D61E;
- R72E;
- R72K;
- E116A;
- E116Q;
- E116R;
- E116T;
- E116V;
- S133A;
- I15G;
- I151A;
- V163R;
- D164R, or the equivalent thereof, or a combination thereof,
- wherein optionally, the substrate specificity or substrate preference of the modified lipase comprises preferential or increased hydrolysis of a fatty acid as compared to the “parental” SEQ ID NO:2, wherein optionally the fatty acid is linolenic acid, linoleic acid, oleic acid, palmitic acid or stearic acid.
12 more elements
39:
- A lipase comprising an amino acid sequence as set forth in SEQ ID NO:2 but also comprising at least 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 the equivalent thereof, or a combination thereof,
- wherein optionally, the substrate specificity or substrate preference of the modified lipase comprises preferential or increased hydrolysis of a fatty acid as compared to the “parental” SEQ ID NO:2, wherein optionally the fatty acid is linolenic acid, linoleic acid, oleic acid, palmitic acid or stearic acid.
21 more elements
40-42. (canceled)