Identification Of A Nitrilase From B. Japonicum By Rational Genome Mining And Methods Of Use
OwnerDunming Zhu, Ling Hua +2inventors
InventorsDunming Zhu, Ling Hua, Edward Biehl, Chandrani Mukherjee
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Abstract
The present disclosure relates to methods of rational genome mining. A method may include narrowing the number of clones that would otherwise need to be screened and/or identifying a gene with a desired catalytic activity. The disclosure also relates to a nitrile hydrolase from Bradyrhizobium japonicum USDA110 first identified by rational genome mining. In addition, the disclosure relates to nitrilase bll6402 and catalytically active variants capable of converting an α-hydroxy nitriles, a β-hydroxy nitrile and/or an α,ω-dinitrile to a carboxylic acid.
Claims44
A purified nitrilase, said nitrilase comprising a polypeptide having the amino acid sequence of SEQ ID NO:
- 2.
A purified nitrilase, wherein said nitrilase:
- comprises a polypeptide having an amino acid sequence that is more than about 95% identical to SEQ ID NO:
- 2, and
- has a catalytic activity sufficient to convert, in a solvent at a pH between about 5 and about 9 and a temperature of less than about 50° C., at least about 50% of a nitrile to a carboxylic acid.
A purified nucleic acid, wherein said purified nucleic acid:
- comprises a sequence that is more than about 75% identical to SEQ ID NO:
- 1, and
- encodes a nitrilase, wherein said nitrilase has a catalytic activity sufficient to convert, in a solvent at a pH between about 5 and about 9 and a temperature of less than about 50° C., at least about 50% of a nitrile to a carboxylic acid.
A method of converting a nitrile to a carboxylic acid, said method comprising:
- contacting a nitrile with a nitrilase under conditions that permit hydrolysis,
- wherein said nitrilase comprises a polypeptide having an amino acid sequence that is more than about 95% identical to SEQ ID NO:
- 2 and having a catalytic activity sufficient to convert, in a solvent at a pH between about 5 and about 9 and a temperature of less than about 50° C., at least about 50% of a nitrile to a carboxylic acid.
A method of converting a β-hydroxy nitrile to a corresponding β-hydroxycarboxylic acid, said method comprising:
- contacting a β-hydroxy nitrile with a nitrilase under conditions that permit conversion of the β-hydroxy nitrile to the corresponding β-hydroxycarboxylic acid,
- wherein said nitrilase comprises a polypeptide having an amino acid sequence that is more than about 95% identical to SEQ ID NO:
- 2 and having a catalytic activity sufficient to convert, in a solvent at a pH between about 5 and about 9 and a temperature of less than about 50° C., at least about 50% of a nitrile to a carboxylic acid.
A method of converting an α,ω-dinitrile to a corresponding ω-cyanocarboxylic acid, said method comprising:
- contacting an α,ω-dinitrile with a nitrilase under conditions that permit conversion of the β-hydroxy nitrile to the corresponding ω-cyanocarboxylic acid,
- wherein said nitrilase comprises a polypeptide having an amino acid sequence that is more than about 95% identical to SEQ ID NO:
- 2 and having a catalytic activity sufficient to convert, in a solvent at a pH between about 5 and about 9 and a temperature of less than about 50° C., at least about 50% of a nitrile to a carboxylic acid.
A method of producing a nitrilase operable to convert, in a solvent at a pH between about 5 and about 9 and a temperature of less than about 50° C., at least about 50% of a nitrile to a carboxylic acid, said method comprising:
- contacting a microorganism with a nucleic acid comprising an expression control sequence operably linked to a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:
- 2 under conditions that permit expression of the nucleotide sequence and production of the nitrilase.
A method of identifying a nucleic acid comprising a sequence encoding an enzyme having a desired substrate and/or product specificity, said method comprising:
- selecting a genome mining database query selected from the group consisting of a text-based query, a sequence-based query, and combinations thereof;
- running the genome mining database query against a database comprising a nucleotide sequence or series of nucleotide sequences that, in the aggregate, correspond to at least 75% of the genome of an organism, to produce at least one candidate nucleotide sequence;
- examining at least one nucleotide sequence flanking each at least one candidate nucleotide sequence and determining whether the at least one flanking nucleotide sequence comprises an open reading frame or a predicted open reading frame;
- and
- determining whether the polypeptide encoded by each actual or predicted open reading frame is operable to produce or predicted to be operable to the desired substrate or a derivative thereof and/or consume the desired product or a derivative thereof,
- wherein a candidate nucleotide sequence is identified as encoding an enzyme having the desired substrate and/or product specificity where at least one flanking nucleotide sequence comprises an actual or predicted open reading frame encoding a polypeptide that is operable to produce or predicted to be operable to the desired substrate or a derivative thereof and/or consume the desired product or a derivative thereof.
A method of predicting whether a nucleic acid comprises a sequence encoding an enzyme having a desired substrate and/or product specificity, said method comprising:
- selecting a genome mining database query selected from the group consisting of a text-based query, a sequence-based query, and combinations thereof;
- running the genome mining database query against a database comprising a nucleotide sequence or series of nucleotide sequences that, in the aggregate, correspond to at least 75% of the genome of an organism, to produce at least one candidate nucleotide sequence;
- examining at least one nucleotide sequence flanking each at least one candidate nucleotide sequence and determining whether the at least one flanking nucleotide sequence comprises an open reading frame or a predicted open reading frame;
- and
- determining whether the polypeptide encoded by each actual or predicted open reading frame is operable to produce or predicted to be operable to the desired substrate or a derivative thereof and/or consume the desired product or a derivative thereof,
- wherein a candidate nucleotide sequence is predicted to encode an enzyme having the desired substrate and/or product specificity where at least one flanking nucleotide sequence comprises an actual or predicted open reading frame encoding a polypeptide that is operable or predicted to be operable to produce the desired substrate or a derivative thereof and/or consume the desired product or a derivative thereof.
Dependencies are inferred from the claim text.