Isomerases, nucleic acids encoding them and methods for making and using them
This invention relates generally to enzymes, polynucleotides encoding the enzymes having isomerase activity, e.g., racemase activity, e.g., amino acid racemase activity, alanine racemase activity, and/or epimerase activity, and/or catalyze the re-arrangement of atoms within a molecule, catalyze the conversion of one isomer into another, catalyze the conversion of an optically active substrate into a raceme, which is optically inactive, catalyze the interconversion of substrate enantiomers, catalyze the stereochemical inversion around the asymmetric carbon atom in a substrate having only one center of asymmetry, catalyze the stereochemical inversion of the configuration around an asymmetric carbon atom in a substrate having more than one asymmetric center, and/or catalyze the racemization of amino acids.
1. An expression cassette, a vector, or a cloning vehicle comprising an isolated, synthetic or recombinant polynucleotide that encodes a polypeptide having racemase activity, wherein the polynucleotide is selected from the group consisting of:
(a) a nucleic acid-sequence having at least 95%, 96%, 97%, 98%, or 99%, sequence identity to the full length nucleic acid sequence of SEQ ID NO: 411,
(b) a nucleic acid sequence that hybridizes under stringent conditions to full length nucleic acid sequence of SEQ ID NO:411 and the stringent conditions comprise a wash step comprising a wash in 0.2X SSC at a temperature of about 65° C. for about 15 minutes;
(c) the nucleic acid sequence of SEQ ID NO:411; and
(d) a nucleic acid sequence fully complementary to the sequence of any of (a) to (c).
2. The expression cassette, a vector, or a cloning vehicle of claim 1 , wherein the cloning vehicle comprises a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificial chromosome.
3. The cloning vehicle of claim 2 , wherein the viral vector comprises an adenovirus vector, a retroviral vector or an adeno-associated viral vector, or, the artificial chromosome comprises a bacterial artificial chromosome (BAC), a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).
4. An isolated host cell comprising the polynucleotide of claim 1 , wherein the host cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.
5. A method of producing a recombinant polypeptide comprising the steps of:
(a) providing the polynucleotide of claim 1 , operably linked to a promoter; and
(b) expressing the polynucleotide of step (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide,
and optionally the method further comprises transforming an isolated host cell with the polynucleotide of step (a) followed by expressing the nucleic acid of step (a), thereby producing a recombinant polypeptide in a transformed cell.
6. A method of generating a variant of the polynucleotide of claim 1 comprising the steps of: providing a template nucleic acid comprising the sequence of claim 1 ; and 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.
7. The polynucleotide of claim 1 , and encoding a variant polypeptide having at least one conservative amino acid substitution and the variant retains racemase activity.
8. The nucleic acid of claim 7 , wherein the at least one conservative amino acid substitution is selected from:
(a) substituting an amino acid with another amino acid of like characteristics;
(b) replacement of an aliphatic amino acid with another aliphatic amino acid;
(c) replacement of a Serine with a Threonine or vice versa;
(d) replacement of an acidic residue with another acidic residue;
(e) replacement of a residue bearing an amide group with another residue bearing an amide group;
(f) exchange of a basic residue with another basic residue; and
(g) replacement of an aromatic residue with another aromatic residue.
9. The polynucleotide of claim 1 , encoding a polypeptide having racemase activity but lacking a signal sequence, or a prepro domain.
10. The polynucleotide of claim 1 , encoding a polypeptide having racemase activity, and further comprising a heterologous sequence.
11. The polynucleotide of claim 10 , wherein the heterologous sequence is a heterologous domain, a heterologous catalytic domain (CD), or a combination thereof.
12. The polynucleotide of claim 11 , wherein the heterologous signal sequence, domain or catalytic domain (CD) is derived from a heterologous enzyme.
13. The polynucleotide of claim 10 , wherein the heterologous sequence is a tag, an epitope, a targeting peptide, a cleavable sequence, a detectable moiety or an enzyme.