(r)-hydroxynitrile lyase from brassicaceae

Abstract

The invention concerns a polypeptide which can be isolated from the Brassicaceae family and which has at least the activity of a hydroxynitrile lyase (HNL). The hydroxynitrile lyase of the invention is the first HNL from the Brassicaceae family. The plants ( Arabidopsis ) from which this enzyme or its gene is isolated is also described as non-cyanogenic. All HNL-containing plants described so far are cyanogenic plants and so it has until now been assumed that only cyanogenic plants contain hydroxynitrile lyases. Surprisingly, it transpires that a polypeptide (AtHNL) of the invention is (R)-selective. The amino acid sequence gives a theoretical molecular weight of 29.2 kDa for the AtHNL subunit. The calculated molecular mass of the protein of approximately 30 kDa can be confirmed by SDS gel electrophoresis.

1 . An isolated polypeptide, wherein said polypeptide is from a Brassicaceae family of plants, and has at least activity of a hydroxynitrile lyase. 
     
     
         2 . The isolated polypeptide according to  claim 1 , wherein the plants are of genus  Arabidopsis , in particular of species  Arabidopsis thaliana.    
     
     
         3 . The isolated polypeptide according to  claim 1 , wherein the polypeptide catalyzes at least a synthesis of chiral cyanohydrins from aldehydes or ketones and hydrocyanic acid. 
     
     
         4 . The isolated polypeptide according to  claim 1 , wherein the polypeptide catalyzes at least cleavage of a cyanohydrin into an aldehyde or ketone and hydrocyanic acid. 
     
     
         5 . The isolated polypeptide according to  claim 1 , wherein the polypeptide is (R)-selective. 
     
     
         6 . The isolated polypeptide according to  claim 1 , wherein the polypeptide is allocated to a α/β-hydrolase family. 
     
     
         7 . A polypeptide which comprises at least amino acid sequence according to SEQ ID NO: 1. 
     
     
         8 . A polypeptide which differs from the polypeptide according to  claim 7  by one or more amino acid replacement(s), wherein said replacement(s) are:
 a) asparagine in position 112 for threonine (N12T) or alanine (N12A);   b) tyrosine in position 14 for cysteine (Y14C) or alanine (Y14A); and/or   c) leucine in position 129 for tryptophan (L129W).   
     
     
         9 . A polypeptide which differs from the polypeptide according to  claim 7  by deletion, insertion and/or substitution of at least one and at most 100 amino acids, preferably 1 to 50 amino acids, particularly preferably 1 to 20 amino acids, and in particular 1 to 10 amino acids. 
     
     
         10 . A protein, in particular a fusion protein, which comprises at least one polypeptide according to  claim 1 . 
     
     
         11 . An isolated nucleic acid molecule which comprises at least one nucleotide sequence for synthesis of at least one polypeptide, wherein the nucleotide sequence is selected from the group consisting of:
 a) a nucleotide sequence which codes for a polypeptide according to  claim 1 ;   b) a nucleotide sequence which codes for a polypeptide which comprises at least amino acid sequence of SEQ ID NO: 1;   c) a nucleotide sequence which comprises SEQ ID NO: 2;   d) a nucleotide sequence which codes for fragments of the polypeptide coded by the nucleotide sequences of a), b) or c), wherein the fragments have the catalytic activity of the polypeptides coded by the nucleotide sequences of a), b) or c);   e) a nucleotide sequence which differs from the nucleotide sequences of a), b), c) or d) by replacement of at least one codon for a synonymous codon;   f) a nucleotide sequence, a complementary strand of which hybridizes with the nucleotide sequences of a), b), c) or d) and which codes for at least one polypeptide which has a catalytic activity of the polypeptide coded by the nucleotide sequences of a), b), c) or d);   g) a nucleotide sequence which has at least 85%, preferably 90%, in particular 95% identity with the nucleotide sequence of a), b), c) or d) and which codes for at least the polypeptide which has the catalytic activity of the polypeptide coded by the nucleotide sequences of a), b), c) or d); and   h) a nucleotide sequence which corresponds to a complementary strand of the nucleotide sequence of a) to g).   
     
     
         12 . The nucleic acid molecule according to  claim 11 , wherein the nucleic acid molecule is operatively coupled with at least one regulatory sequence. 
     
     
         13 . The nucleic acid molecule according to  claim 12 , wherein the regulatory sequence comprises a promoter sequence and/or a transcription termination sequence and/or a regulator gene. 
     
     
         14 . The nucleic acid molecule according to  claim 11 , wherein the nucleic acid molecule is coupled with at least one further nucleotide sequence which codes for a further polypeptide. 
     
     
         15 . A polypeptide, encoded by a gene which comprises the nucleic acid molecule according to  claim 11 . 
     
     
         16 . A vector for the synthesis of a polypeptide in a suitable cell, which comprises the nucleic acid molecule according to  claim 11  in expressible form. 
     
     
         17 . A cell which contains the nucleic acid molecule according to  claim 11  and/or a vector comprising said nucleic acid molecule in an expressible form. 
     
     
         18 . A cell culture which comprises cells according to  claim 17 . 
     
     
         19 . A method for the synthesis of chiral cyanohydrins from at least one carbonyl compound and hydrocyanic acid comprising:
 providing said at least one carbonyl compound and said hydrocyanic acid and carrying out a reaction in presence of the polypeptide of  claim 1  or a protein, in particular a fusion protein, comprising said polypeptide.   
     
     
         20 . A method for the synthesis of chiral cyanohydrins from at least one carbonyl compound and hydrocyanic acid comprising:
 providing said at least one carbonyl compound and said hydrocyanic acid and carrying out a reaction, in the presence of the cell according to  claim 17  or a cell culture comprising said cell.   
     
     
         21 . The method according to  claim 20 , wherein an aliphatic or aromatic aldehyde is used as the carbonyl compound. 
     
     
         22 . The method according to  claim 20 , wherein an aliphatic or aromatic ketone is used as the carbonyl compound. 
     
     
         23 . A method for cleavage of cyanohydrins into at least one carbonyl compound and hydrocyanic acid, wherein the cleavage is carried out in presence of a polypeptide according to  claim 1 , or a protein, in particular a fusion protein, comprising said polypeptide. 
     
     
         24 . A method for cleavage of cyanohydrins into at least one carbonyl compound and hydrocyanic acid, wherein said cleavage is carried out in presence of a cell according to  claim 17  or a cell culture comprising said cell.