1996. Characterization, purification, and molecular cloning of hydroxynitrile lyase from cassava

1996. Characterization, purification, and molecular cloning of hydroxynitrile lyase from cassava


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ABSTRACT

Cassava (Manihot esculenta Crantz) is a staple food crop in Africa. The cyanogenic glycoside, linamarin, is stored in cassava cells and probably functions as an herbivore deterrent. When cassava roots are prepared for human consumption, linamarin and its degradation products, acetone cyanohydrin and hydrogen cyanide, can remain in the food products. Exposure to these cyanogens has been linked to epidemics of neurological disorders in Africa. Acetone cyanohydrin is the main contributor to cyanide exposure from cassava consumption.

We purified hydroxynitrile lyase, which converts acetone cyanohydrin to acetone and hydrogen cyanide, from cassava leaf apoplast extracts and whole leaves. The enzyme was a homodimer of 29 kD subunits. The pH and temperature optima were 5.0 and 30°C, respectively. The enzyme displayed typical Michaelis-Menten kinetics. Activity was saturated by 20 mM acetone cyanohydrin and the Km was 0.925 mM. Hydroxynitrile lyase maintained over 50 percent of its optimal activity at pH 4.0-5.5 and temperatures 4-45°C. Also, the enzyme was not permanently inactivated by 24 hours of exposure to low pHs (4.0-6.0).

Anti-hydroxynitrile lyase antibodies were raised and used to localize the enzyme. Using FITC-labeled secondary antibodies, hydroxynitrile lyase was found in the cell walls of leaf epidermal, parenchyma, and mesophyll cells. Western blots demonstrated the presence of hydroxynitrile lyase in leaves and not stems or roots.

The gene encoding hydroxynitrile lyase was isolated from a cassava leaf cDNA library. The 1.1 kb cDNA clone encoded a 258 amino acid protein. The derived amino acid sequence had 78 percent identity to rubber tree (Hevea brasiliensis) hydroxynitrile lyase and 30 percent identity and 56 percent similarity to two pathogen induced response proteins from rice (Oryza sativa). Southern blot analysis supported the presence of one copy of the hydroxynitrile lyase gene in the cassava genome. Little expression of the hydroxynitrile lyase gene (1.3 kb mRNA) was found in cassava stems and roots  .