Production and characterization of a plant α‐hydroxynitrile lyase in Escherichia coli

Abstract

Abstract:The coding sequence of the cyanogenic ␣-hydroxynitrile lyase gene of Manihot esculenta Crantz (cassava) was cloned in the plasmid vector pMal-c2 and expressed in Escherichia coli strain JM105. DNA sequencing showed that the recombinant plasmid contained the same sequence as the cDNA clone pHNL10. Peptide sequencing of the recombinant protein showed that the N-terminus was heterogeneous, with either four or six additional amino acid residues compared with the native protein. Circular dichroism spectra indicated similar secondary structure contents for both proteins. Enzyme assays showed that specific activity of native and recombinant proteins were 0.24 and 0.26 mmol CN − /mg/min, respectively; that both proteins had optimal activity at 40°C and pH 5.5; and that both proteins were inhibited by the serine protease inhibitor phenyl-methane sulfonyl flouride (PMSF). Isoelectric focusing of native and recombinant protein revealed multiple isoforms for both proteins; the recombinant protein had a more basic mean isoelectric point (pI) (5.1) than the native protein (4.5).

Expression of Hydroxynitrile Lyase fromManihot esculentain Yeast and Its Application in (S)-Mandelonitrile Production Using an Immobilized Enzyme Reactor

“…Although the gene has been cloned and expressed in Escherichia coli, research into (S)-cyanohydrin production using native MeHNL is still in progress. [5][6][7][8][9] HNL from a tropical rubber tree, Hevea brasiliensis, a highly homologous primary sequence to MeHNL, has also been expressed in microbial hosts. 10) In order to achieve a practical production system with MeHNL, here we describe high expression of the enzyme with recombinant yeast, and its application to commercial-scale (S)-mandelonitrile production were investigated.…”

Uneven twins: Comparison of two enantiocomplementary hydroxynitrile lyases with α/β-hydrolase fold

“…In citrate phosphate buffer, AtHNL exhibits a broad optimum between pH 5.75 and 6.5 and is almost inactive below pH 5, whereas MeHNL has a clear optimum at pH 5.75 and shows still 40% of its maximal activity at pH 4, which agrees well with the literature (Wajant et al, 1995;Hughes et al, 1997) (Fig. 2A).…”

Hydroxynitrile lyase catalyzed cyanohydrin synthesis at high pH-values

“…4 the pH-optima determined for the cleavage and synthesis reaction differ significantly. Both the cleavage of mandelonitrile and acetone cyanohydrin [28] showed a pH-optimum around pH 5.5, while a pH-optimum of pH [ 8 was found for the synthesis of 3-phenoxy-benzaldehyde cyanohydrin. Beyond pH 8 the non-enzymatic side-reaction overlaps again significantly the enzymatic reaction, in accordance with the cleavage assays.…”