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, 116 (4), 1219-25

Cyanogenesis in Cassava. The Role of Hydroxynitrile Lyase in Root Cyanide Production

Cyanogenesis in Cassava. The Role of Hydroxynitrile Lyase in Root Cyanide Production

WLB White et al. Plant Physiol.

Abstract

In the cyanogenic crop cassava (Manihot esculenta, Crantz), the final step in cyanide production is the conversion of acetone cyanohydrin, the deglycosylation product of linamarin, to cyanide plus acetone. This process occurs spontaneously at pH greater than 5. 0 or enzymatically and is catalyzed by hydroxynitrile lyase (HNL). Recently, it has been demonstrated that acetone cyanohydrin is present in poorly processed cassava root food products. Since it has generally been assumed that HNL is present in all cassava tissues, we reinvestigated the enzymatic properties and tissue-specific distribution of HNL in cassava. We report the development of a rapid two-step purification protocol for cassava HNL, which yields an enzyme that is catalytically more efficient than previously reported (Hughes, J., Carvalho, F., and Hughes, M. [1994] Arch Biochem Biophys 311: 496-502). Analyses of the distribution of HNL activity and protein indicate that the accumulation of acetone cyanohydrin in roots is due to the absence of HNL, not to inhibition of the enzyme. Furthermore, the absence of HNL in roots and stems is associated with very low steady-state HNL transcript levels. It is proposed that the lack of HNL in cassava roots accounts for the high acetone cyanohydrin levels in poorly processed cassava food products.

Figures

Figure 1
Figure 1
A, Coomassie blue-stained SDS-PAGE profile of isolated cassava leaf HNL obtained from the ammonium sulfate fraction (5 μg of total protein). The predicted molecular mass is 28.5 kD. The polypeptide profile of the crude, low-pH buffer extract yields essentially two proteins, linamarase and HNL (Mkpong et al., 1990). B, Native molecular weight (MW) of cassava HNL as determined by gel-permeation chromatography. Protein molecular mass standards were: RNase A, 13.7 kD; chymotrypsinogen A, 25 kD; ovalbumin, 43 kD; and BSA, 67 kD. HNL and linamarase eluted at 50.1 and 138 kD, respectively. ⋄, HNL; ▪, standards; and ▵, linamarase.
Figure 2
Figure 2
Substrate-dependent enzyme kinetics for the isolated cassava leaf HNL. Open circles indicate substrate concentration-dependent rate kinetics for the purified enzyme. Inset shows a double reciprocal plot of the data.
Figure 3
Figure 3
Western-blot analysis of the distribution and abundance of HNL in different cassava tissues. Each tissue lane contained 30 μg of total soluble protein. The primary antibody was generated against purified cassava leaf HNL. Lane L, Leaf; lane S, stem; lane RR, root rind; and lane RP, root parenchyma. Lanes 1 to 6, Purified HNL equivalent to 0.084, 0.168, 0.336, 0.672, 1.34, and 2.68 μg of HNL, respectively.
Figure 4
Figure 4
Immunofluorescent localization of HNL in cassava leaf tissue. The primary antibody was generated against cassava leaf HNL and the secondary antibody was labeled with fluorescein. Preimmune sera did not cross-react with the cassava tissue (data not shown). The yellow fluorescence associated with the central vascular bundle and the epidermal cells is the result of autofluorescence. In some cells a yellow-orange or red punctate fluorescence is due to the combined fluorescence emission of chloroplasts (red) and fluorescein (green).
Figure 5
Figure 5
Northern-blot analysis of HNL transcript abundance in different cassava tissues. Lane R, Root; lane S, stem; and lane L, leaf tissue. Equal loadings (based on A280 readings and ethidium bromide staining intensity of rRNAs) of total RNA are present in each lane.

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