Cloning and expression of (R)-hydroxynitrile lyase from Linum usitatissimum (flax)

The gene encoding for (R)-hydroxynitrile lyase ((R)-HNL) from Linum usitatissimum has been cloned by polymerase chain reaction using 3',5'-RACE (rapid amplification of cDNA ends). The resulting clone contained an open reading frame of 1266 bp corresponding to a protein of 422 amino acids (45.8 kDa), which shows significant homologies to zinc-dependent formaldehyde dehydrogenases and alcohol dehydrogenases from various organisms. The dimeric active enzyme was expressed in Escherichia coli as N-terminal hexa-histidine fusion protein allowing the purification of homogeneous protein in one step. The formation of inclusion bodies could be reduced using a thioreductase deficient E. coli strain as a host and performing expression of (R)-HNL at 28 degrees C. Under these conditions recombinant (R)-HNL was obtained with a specific activity of 76 U/mg. (C) 1999 Elsevier Science B.V. All rights reserved.