Purification, Characterization, and Cloning of α-Hydroxynitrile Lyase from Cassava (Manihot esculenta Crantz)
ABSTRACT alpha-Hydroxynitrile lyase (HNL, acetone cyanohydrin lyase, EC 188.8.131.52) was purified to homogeneity from young leaves of the cyanogenic tropical crop plant cassava (Manihot esculenta Crantz). The purified protein is a homo-trimer with a subunit relative molecular mass of 28,500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The active protein is not glycosylated and does not contain a flavin group. HNL exhibits complex kinetics which vary according to substrate concentration and may be related to aggregation of the enzyme. HNL activity against two natural substrates, acetone cyanohydrin and 2-butanone cyanohydrin, and one nonphysiological substrate, 2-pentanone cyanohydrin, was demonstrated. N-terminal and internal peptide sequences, obtained from HNL digested with the endoproteinase Glu-C, were used to design degenerate oligonucleotide primers for polymerase chain reaction with single-strand cDNA, using purified mRNA from cotyledons as template. The resulting DNA fragment was used to probe a cassava cotyledon cDNA library. Four cDNA clones were isolated, sequenced, and shown to contain derived amino acid sequences identical to those obtained from the purified protein.
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- "The resulting acetone cyanohydrin is unstable and degrades spontaneously at pH levels higher than 5.0 or temperatures greater than 35°C, or enzymatically by hydroxynitrile lyase in leaves, releasing hydrogen cyanide and acetone (Cutler and Conn, 1981; Hughes et al., 1994; White et al., 1994, 1998; White and Sayre, 1995). However, cyanide release generally does not happen in intact cells because linamarin is localized in the vacuole, while the deglycosylase, linamarase, is localized in the cell wall and in laticifers (Mkpong et al., 1990; Hughes et al., 1994; McMahon et al., 1995). "
ABSTRACT: One of the major constraints facing the large-scale production of cassava (Manihot esculenta) roots is the rapid postharvest physiological deterioration (PPD) that occurs within 72 h following harvest. One of the earliest recognized biochemical events during the initiation of PPD is a rapid burst of reactive oxygen species (ROS) accumulation. We have investigated the source of this oxidative burst to identify possible strategies to limit its extent and to extend cassava root shelf life. We provide evidence for a causal link between cyanogenesis and the onset of the oxidative burst that triggers PPD. By measuring ROS accumulation in transgenic low-cyanogen plants with and without cyanide complementation, we show that PPD is cyanide dependent, presumably resulting from a cyanide-dependent inhibition of respiration. To reduce cyanide-dependent ROS production in cassava root mitochondria, we generated transgenic plants expressing a codon-optimized Arabidopsis (Arabidopsis thaliana) mitochondrial alternative oxidase gene (AOX1A). Unlike cytochrome c oxidase, AOX is cyanide insensitive. Transgenic plants overexpressing AOX exhibited over a 10-fold reduction in ROS accumulation compared with wild-type plants. The reduction in ROS accumulation was associated with a delayed onset of PPD by 14 to 21 d after harvest of greenhouse-grown plants. The delay in PPD in transgenic plants was also observed under field conditions, but with a root biomass yield loss in the highest AOX-expressing lines. These data reveal a mechanism for PPD in cassava based on cyanide-induced oxidative stress as well as PPD control strategies involving inhibition of ROS production or its sequestration.Plant physiology 06/2012; 159(4):1396-407. DOI:10.1104/pp.112.200345 · 7.39 Impact Factor
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- "When the plant tissue is broken down, cyanogenic glucosides, composing of linamarin and lotaustralin, are hydrolyzed to glucose cyanohydrin compounds by linamarase, a specific enzyme found in the plant cell wall (Hughes et al., 1992; McMahon , White, & Sayre, 1995). The cyanohydrin is decomposed by enzyme or alkaline condition (pH > 5) to hydrogen cyanide (Hughes, Carvahlo, & Hughes, 1994; White, McMahon, & Sayre, 1994). "
ABSTRACT: This research investigated the kinetics of cyanide oxidation by ozone in cassava starch production process. An acrylic column, equipped with a gas sparger located at the bottom of the column, were used as a reactor. Cassava starch solutions with the cyanide concentrations of 10, 20, 30, 40mg/l were prepared from fresh cassava roots. The experiments were performed at the ozone generation rates of 7.4, 15.0, 22.6, and 30.0gO3/h. The results showed that the cyanide concentration sharply decreased during the first 30s of the reaction time, and after that the concentration slightly decreased. Moreover, cyanohydrin, which could not be oxidized by ozone, was not completely converted to hydrogen cyanide. The kinetics of cyanide oxidation was first order with respect to cyanide and zero order with respect to ozone. The rate constant obtained from the first order equation of cyanide oxidation with ozone was 2.76min−1.Journal of Food Engineering 02/2008; 84(4):563-568. DOI:10.1016/j.jfoodeng.2007.06.015 · 2.58 Impact Factor
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- "Linamarin Acetone cyanohydrin None Rice protein (unknown function)  Sorghum bicolour—SbHNL (Gramineae)   Dhurrin (S)-4-hydroxymandelonitrile None Wheat Serine carboxypeptidases (60%)  Sorghum vulgare—SvHNL (Gramineae)  Dhurrin (S)-4-hydroxymandelonitrile None Serine carboxy-peptidases  Manihot esculenta—MeHNL (Euphorbiaceae)   Linamarin "
ABSTRACT: Hydroxynitrile lyases are the versatile group of enzymes, which play a significant defensive role in plant system against microbial attack. In chemical industries, hydroxynitrile lyase is used as an important industrial biocatalyst for the synthesis of chiral cyanohydrins by exploiting the reversible enzymatic reaction. Cyanohydrins are biologically active compounds used in synthesis of β-amino alcohols, α-hydroxy ketones and α-hydroxy acids, which have importance as fine chemicals, pharmaceuticals and agrochemicals. NMR and inhibition studies reveled the involvement of different amino acids at the active site and proved that the hydroxynitrile lyases generally utilize acid/base catalysis mechanism. Protein engineering and site directed mutagenesis have been used to change the active site and alter the substrate specificity of various hydroxynitrile lyases. Many recombinant hydroxynitrile lyases have been expressed in Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris.Enzyme and Microbial Technology 08/2005; 37(3-37):279-294. DOI:10.1016/j.enzmictec.2005.04.013 · 2.97 Impact Factor