Purification, characterization, and localization of linamarase in cassava

We have purified cassava (Manihot esculenta) linamarase to apparent homogeneity using a simplified extraction procedure using low pH phosphate buffer. Three isozymes of cassava linamarase were identified in leaves based on differences in isoelectric point. The enzyme is capable of hydrolyzing a number of β-glycosides in addition to linamarin. The enzyme is unusually stable and has a temperature optimum of 55°C. Immunogold labeling studies indicate that linamarase is localized in the cell walls of cassava leaf tissue. Since linamarin must cross the cell wall following synthesis in the leaf for transport to the root, it is likely that linamarin must cross the cell wall in a nonhydrolyzable form, possibly as the diglucoside, linustatin. In addition, we have quantified the levels of linamarin and linamarase activity in leaves of cassava varieties which differ in the linamarin content of their roots. We observed no substantial differences in the steady state linamarin content or linamarase activity of leaves from high or low (root) cyanogenic varieties. These results indicate that the steady state levels of linamarin and linamarase in leaves of high and low cyanogenic varieties are not correlated with the varietal differences in the steady state levels of linamarin in roots.     

New chemiluminescence assay for linamarin

A new chemiluminescence assay was developed for the quantitative determination of linamarin, a cyanogenic glucoside present in cassava. The assay involved hydrolysis of linamarin by a specific enzyme, linamarase, to release glucose, which was then quantitated by a chemiluminescence system consisting of glucose oxidase-peroxidase-luminol. The new assay was more sensitive than the conventional spectrophotometric method for quantitating linamarin in cassava extracts. However, the following agents were found to interfere with the new assay: Vanadate, Mg2+, and Cu2+, were inhibitory to the luminescence of the H2O2-peroxidase-luminol system used in the coupling reaction, whereas EDTA and EGTA activated the system. In addition, Hg2+, which inhibits glucose oxidase, and Tris ion, which inhibits linamarase, both interfered with the new assay.     

Pattern of enzyme changes in rabbits administered linamarin or potassium cyanide

Diseases like tropical ataxic neuropathy and endemic goitre have been reported to have definite correlation with a chronic ingestion of cassava (Manihot esculenta Crantz). The toxicity of cassava has been attributed to its two cyanogenic glycosides, linamarin and lotaustralin. In this study, an attempt has been made to understand the pattern of changes in certain clinically significant enzymes brought about by the chronic administration of sublethal doses of linamarin to rabbits. The profound elevation in rhodanese activity observed in the linamarin and cyanide treated rabbits indicated the attempt of the tissues to detoxify cyanide. That intact linamarin could be hydrolysed in vivo was a significant finding from the study. The mode of toxicity of linamarin was similar to that of cyanide by producing a gradual shift from aerobic to anaerobic metabolism.     

An enzyme-bound linamarin indicator paper strip for the semi-quantitative estimation of linamarin

Summary An enzyme-bound linamarin indicator paper strip was developed which was based on the hydrolysis of linamarin by cassava leaf linamarase and the detection of the cyanide released by alkaline picrate reagent. The linamarase could be stabilized with gelatin or gelatin in combination with polyvinylpyrrolidone-10 or trehalose. A positive reaction was observed within 15 minutes at 37°C and it could detect linamarin concentration as low as 0.5 to 1 mM. The indicator strip could be used to estimate linamarin content in cassava semiquantitatively.     

Overexpression of hydroxynitrile lyase in cassava roots elevates protein and free amino acids while reducing residual cyanogen levels

Cassava is the major source of calories for more than 250 million Sub-Saharan Africans, however, it has the lowest protein-to-energy ratio of any major staple food crop in the world. A cassava-based diet provides less than 30% of the minimum daily requirement for protein. Moreover, both leaves and roots contain potentially toxic levels of cyanogenic glucosides. The major cyanogen in cassava is linamarin which is stored in the vacuole. Upon tissue disruption linamarin is deglycosylated by the apolplastic enzyme, linamarase, producing acetone cyanohydrin. Acetone cyanohydrin can spontaneously decompose at pHs >5.0 or temperatures >35°C, or is enzymatically broken down by hydroxynitrile lyase (HNL) to produce acetone and free cyanide which is then volatilized. Unlike leaves, cassava roots have little HNL activity. The lack of HNL activity in roots is associated with the accumulation of potentially toxic levels of acetone cyanohydrin in poorly processed roots. We hypothesized that the over-expression of HNL in cassava roots under the control of a root-specific, patatin promoter would not only accelerate cyanogenesis during food processing, resulting in a safer food product, but lead to increased root protein levels since HNL is sequestered in the cell wall. Transgenic lines expressing a patatin-driven HNL gene construct exhibited a 2-20 fold increase in relative HNL mRNA levels in roots when compared with wild type resulting in a threefold increase in total root protein in 7 month old plants. After food processing, HNL overexpressing lines had substantially reduced acetone cyanohydrin and cyanide levels in roots relative to wild-type roots. Furthermore, steady state linamarin levels in intact tissues were reduced by 80% in transgenic cassava roots. These results suggest that enhanced linamarin metabolism contributed to the elevated root protein levels.     

The linamarase of Mucor circinelloides LU M40 and its detoxifying activity on cassava

Mucor circinelloides LU M40 produced 12·2 mU ml⁻¹ of linamarase activity when grown in a 3 l fermenter in the following optimized medium (g l⁻¹ deionized water): pectin, 10·0; (NH₄)₂SO₄,
1·0; KH₂PO₄, 2·0; Na₂HPO₄, 0·7; MgSO₄.7H₂O, 0·5; yeast extract, 1·0; Tween-80,
1·0, added after 48 h of fermentation. The purified linamarase was a dimeric protein with a molecular mass of 210 kDa; the enzyme showed optimum catalytic activity at pH 5·5 and 40 °C and had a wide range (3·0–7·0) of pH stability. The enzyme substrate specificity on plant cyanogenic glycosides was wide; the K_m value for linamarin was 2·93 mmol l⁻¹. The addition, before processing, of the fungal crude enzyme to cassava roots facilitated and shortened detoxification; after 24 h of fermentation, all cyanogenic glycosides were hydrolysed.     

Cyanogenic potential in cassava and its influence on a generalist insect herbivore Cyrtomenus bergi (Hemiptera: Cydnidae)

The hypothesis that cyanogenic potential in cassava is a defense mechanism against arthropod pests is one of the crucial questions relevant to current efforts to reduce or eliminate cyanogenic potential (CNP) in cassava. The generalist arthropod Cyrtomenus bergi, which attacks cassava roots, was used in a bioassay relating oviposition and survival to CNP, concentration of nonglycosidic cyanogens, and linamarase (beta-glycosidase) activity in twelve selfed cassava siblings and their parental clone, which has segregated for different levels of cyanogenesis. Electron microscopic evaluation revealed an intracellular pathway of the stylet of C. bergi in the cassava root tissue to rupture cell walls. This feeding behavior causes cyanogenesis and increased linamarin content in the hemolymph of C. bergi while feeding on a cyanogenic diet. This diet resulted in a significant reduction in oviposition, especially at levels of CNP above 150 ppm (expressed as hydrogen cyanide) on fresh weight basis (or 400 ppm on dry weight basis) in cassava roots. An exponential decline in oviposition was observed with increasing levels of CNP, beginning 12 d after exposure to the cyanogenic diet. Cyanogenic potential and dry matter content showed a positive effect on survival. No relationship was found between concentrations of nonglycosidic cyanogens or linamarase activity in the cassava root and either oviposition or survival. According to our results, there is a significant difference between potentially noncyanogen and high cyanogen clones, but there may not be a significant difference between potentially noncyanogen and low cyanogen clones. Consequently, more frequent outbreaks or higher levels of damage might not be anticipated in potentially noncyanogen cassava clones than that anticipated in low cyanogenic clones. The negative effect of cyanogenesis on oviposition concurrent with a positive effect on survival of this pest is most likely the result of a physiological trade-off between survival and oviposition. The question of whether ovipositional rates could be recovered after a long-term exposure to cyanide remains unanswered.     

Over-expression of hydroxynitrile lyase in transgenic cassava roots accelerates cyanogenesis and food detoxification

Cassava (Manihot esculenta, Crantz) roots are the primary source of calories for more than 500 million people, the majority of whom live in the developing countries of Africa. Cassava leaves and roots contain potentially toxic levels of cyanogenic glycosides. Consumption of residual cyanogens (linamarin or acetone cyanohydrin) in incompletely processed cassava roots can cause cyanide poisoning. Hydroxynitrile lyase (HNL), which catalyses the conversion of acetone cyanohydrin to cyanide, is expressed predominantly in the cell walls and laticifers of leaves. In contrast, roots have very low levels of HNL expression. We have over-expressed HNL in transgenic cassava plants under the control of a double 35S CaMV promoter. We show that HNL activity increased more than twofold in leaves and 13-fold in roots of transgenic plants relative to wild-type plants. Elevated HNL levels were correlated with substantially reduced acetone cyanohydrin levels and increased cyanide volatilization in processed or homogenized roots. Unlike acyanogenic cassava, transgenic plants over-expressing HNL in roots retain the herbivore deterrence of cyanogens while providing a safer food product.     

Compartmentation of cyanogenic glucosides and their degrading enzymes

Whereas high activities of β-glucosidase occur in homogenates of leaves of Hevea brasiliensis Muell.-Arg., this enzyme, which is capable of splitting the cyanogenic monoglucoside linamarin (linamarase), is not present in intact protoplasts prepared from the corresponding leaves. Thus, in leaves of H. brasiliensis the entire linamarase is located in the apoplasmic space. By analyzing the vacuoles obtained from leaf protoplasts isolated from mesophyll and epidermal layers of H. brasiliensis leaves, it was shown that the cyanogenic glucoside linamarin is localized exclusively in the central vacuole. Analyses of apoplasmic fluids from leaves of six other cyanogenic species showed that significant linamarase activity is present in the apoplasm of all plants tested. In contrast, no activity of any diglucosidase capable of hydrolyzing the cyanogenic diglucoside linustatin (linustatinase) could be detected in these apoplasmic fluids. As described earlier, any translocation of cyanogenic glucosides involves the interaction of monoglucosidic and diglucosidic cyanogens with the corresponding glycosidases (Selmar, 1993a, Planta 191, 191–199). Based on this, the data on the compartmentation of cyanogenic glucosides and their degrading enzymes in Hevea are discussed with respect to the complex metabolism and the transport of cyanogenic glucosides.     

Co-occurrence of Valine/Isoleucine-derived and cyclopentenoid cyanogens in a Passiflora species

The valine/isoleucine-derived cyanogenic glycosides linamarin and lotaustralin have been isolated together with the cyclopentenoid cyanogen passibiflorin from Passiflora lutea. This is only the second report of the production of cyanogenic glycosides from more than one biosynthetic pathway in individuals of a single species.     

Pattern of enzymes involved in cyanogenesis and HCN metabolism in cell cultures of Phaseolus lunatus L. varieties

Callus and cell suspension cultures were established from root and shoot tips of aseptically-grown seedlings of highly cyanogenic Phaseolus lunatus L. varieties. The content of cyanogenic glucosides in the explanted seedling sections decreased during storage, the derived callus cells were free of cyanogenic glycosides. In spite of the non-existence of cyanogenic glucosides, the cyanogen degrading linamarase, the cyanide detoxifying enzyme -cyanoalanine synthase and also hydroxynitrile lyase were still present in suspension cultures. The linamarase activity equalled the total -glucosidase activity, of which up to 80% was found in the culture medium. In contrast the -cyanoalanine synthase and the hydroxy nitrile lyase were entirely localized in the cell biomass.~Botanical Institute, Technical University Braunschweig     

An enzyme-immobilized microplate for determination of linamarin for large number of samples

Summary An enzyme-immobilized microplate for determination of linamarin was prepared by covalently linking cassava leaf linamarase to the microplate. For linamarin determination, cassava roots were homogenised in 0.1 Mo-phosphoric acid and the filtrate adjusted to pH 6 with NaOH prior to adding into the wells. The cyanide released was then determined spectrophotometrically. One nmol linamarin can be detected. The microplate method is suitable for analysis of large number of samples and is useful for screening purposes.     

Cytochromes P-450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin. Cloning, functional expression in Pichia pastoris, and substrate specificity of the isolated recombinant enzymes

The first committed steps in the biosynthesis of the two cyanogenic glucosides linamarin and lotaustralin in cassava are the conversion of L-valine and L-isoleucine, respectively, to the corresponding oximes. Two full-length cDNA clones that encode cytochromes P-450 catalyzing these reactions have been isolated. The two cassava cytochromes P-450 are 85% identical, share 54% sequence identity to CYP79A1 from sorghum, and have been assigned CYP79D1 and CYP79D2. Functional expression has been achieved using the methylotrophic yeast, Pichia pastoris. The amount of CYP79D1 isolated from 1 liter of P. pastoris culture exceeds the amounts that putatively could be isolated from 22,000 grown-up cassava plants. Each cytochrome P-450 metabolizes L-valine as well as L-isoleucine consistent with the co-occurrence of linamarin and lotaustralin in cassava. CYP79D1 was isolated from P. pastoris. Reconstitution in lipid micelles showed that CYP79D1 has a higher k(c) value with L-valine as substrate than with L-isoleucine, which is consistent with linamarin being the major cyanogenic glucoside in cassava. Both CYP79D1 and CYP79D2 are present in the genome of cassava cultivar MCol22 in agreement with cassava being allotetraploid. CYP79D1 and CYP79D2 are actively transcribed, and production of acyanogenic cassava plants would therefore require down-regulation of both genes.     

Light affects in vitro organogenesis of Linum usitatissimum L. and its cyanogenic potential

The relationships between organogenesis of oil flax (Linum usitatissimum L., cv. ‘Szafir’) in vitro, cyanogenic potential (HCN-p) of these tissues and light were investigated. Shoot multiplication obtained on Murashige and Skoog medium containing 0.05 mg L^?1 2,4-dichloro-phenoxyacetic acid and 1 mg L^?1 6-benzyladenine (BA), was about twice higher in light-grown cultures than those in darkness. Light-grown explants showed also higher rate of roots regeneration (in medium containing 1 mg L^?1 α-naphtaleneacetic acid and 0.05 mg L^-1 BA) than dark-grown ones. The cyanogenic potential (expressed both as linamarin and lotaustralin content and linamarase activity) of flax cultured in vitro was tissue-specific and generally was higher under light conditions than in darkness. The highest concentration of linamarin and lotaustralin was detected in light-regenerated shoots, and its amount was twice as high as in roots, and about threefold higher than in callus tissue. The activities of linamarase and β-cyanoalanine synthase in light-regenerated organs were also higher than those in darkness. Thus, higher frequency of regeneration of light-grown cultures than dark-grown ones seems to be correlated with higher HCN-p of these tissues. We suggest that free HCN, released from cyanoglucosides potentially at higher level under light conditions, may be involved in some organogenetic processes which improve regeneration efficiency.     

The Linamarin beta-Glucosidase in Costa Rican Wild Lima Beans (Phaseolus lunatus L.) Is Apoplastic

Analysis of mesophyll protoplasts and cell wall extracts of leaf discs of Costa Rican wild lima bean (Phaseolus lunatus L.) shows that the linamarase activity is confined to the apoplast. Its substrate linamarin, together with the related enzyme hydroxynitrile lyase, is found inside the cells. This compartmentation prevents cyanogenesis from occurring in intact tissue, and suggests that linamarin has to be protected during any translocation across the linamarase rich apoplast.     

Cyanogenic glycosides: a case study for evolution and application of cytochromes P450

Cyanogenic glycosides are ancient biomolecules found in more than 2,650 higher plant species as well as in a few arthropod species. Cyanogenic glycosides are amino acid-derived β-glycosides of α-hydroxynitriles. In analogy to cyanogenic plants, cyanogenic arthropods may use cyanogenic glycosides as defence compounds. Many of these arthropod species have been shown to de novo synthesize cyanogenic glycosides by biochemical pathways that involve identical intermediates to those known from plants, while the ability to sequester cyanogenic glycosides appears to be restricted to Lepidopteran species. In plants, two atypical multifunctional cytochromes P450 and a soluble family 1 glycosyltransferase form a metabolon to facilitate channelling of the otherwise toxic and reactive intermediates to the end product in the pathway, the cyanogenic glycoside. The glucosinolate pathway present in Brassicales and the pathway for cyanoalk(en)yl glucoside synthesis such as rhodiocyanosides A and D in Lotus japonicus exemplify how cytochromes P450 in the course of evolution may be recruited for novel pathways. The use of metabolic engineering using cytochromes P450 involved in biosynthesis of cyanogenic glycosides allows for the generation of acyanogenic cassava plants or cyanogenic Arabidopsis thaliana plants as well as L. japonicus and A. thaliana plants with altered cyanogenic, cyanoalkenyl or glucosinolate profiles.     

Characterization and expression profile of two UDP-glucosyltransferases, UGT85K4 and UGT85K5, catalyzing the last step in cyanogenic glucoside biosynthesis in cassava

Manihot esculenta (cassava) contains two cyanogenic glucosides, linamarin and lotaustralin, biosynthesized from l ‐valine and l ‐isoleucine, respectively. In this study, cDNAs encoding two uridine diphosphate glycosyltransferase (UGT) paralogs, assigned the names UGT85K4 and UGT85K5, have been isolated from cassava. The paralogs display 96% amino acid identity, and belong to a family containing cyanogenic glucoside‐specific UGTs from Sorghum bicolor and Prunus dulcis. Recombinant UGT85K4 and UGT85K5 produced in Escherichia coli were able to glucosylate acetone cyanohydrin and 2‐hydroxy‐2‐methylbutyronitrile, forming linamarin and lotaustralin. UGT85K4 and UGT85K5 show broad in vitro substrate specificity, as documented by their ability to glucosylate other hydroxynitriles, some flavonoids and simple alcohols. Immunolocalization studies indicated that UGT85K4 and UGT85K5 co‐occur with CYP79D1/D2 and CYP71E7 paralogs, which catalyze earlier steps in cyanogenic glucoside synthesis in cassava. These enzymes are all found in mesophyll and xylem parenchyma cells in the first unfolded cassava leaf. In situ PCR showed that UGT85K4 and UGT85K5 are co‐expressed with CYP79D1 and both CYP71E7 paralogs in the cortex, xylem and phloem parenchyma, and in specific cells in the endodermis of the petiole of the first unfolded leaf. Based on the data obtained, UGT85K4 and UGT85K5 are concluded to be the UGTs catalyzing in planta synthesis of cyanogenic glucosides. The localization of the biosynthetic enzymes suggests that cyanogenic glucosides may play a role in both defense reactions and in fine‐tuning nitrogen assimilation in cassava.     

Mechanisms of increased linamarin degradation during solid-substrate fermentation of cassava

Several fungi and bacteria, isolated from Ugandan domestic fermented cassava, released HCN from linamarin in defined growth media. In 72 h, a Bacillus sp. decreased the linamarin to 1% of initial concentrations, Mucor racemosus to 7%, Rhizopus oryzae and R. stolonifer to 30%, but Neurospora sitophila and Geotrichum candidum hardly degraded the linamarin. Adding pectolytic and cellulolytic enzymes, but not linamarase, to root pieces under aseptic conditions, led to root softening and significantly lower linamarin contents. Neurospora sitophila showed no linamarase activity, in contrast to M. racemosus and Bacillus sp., both of which were less effective in root softening and decreasing the root linamarin content. The most important contribution of microorganisms to linamarin decrease in the solid-substrate fermentation of cassava is their cell-wall-degrading activity, which enhances the contact between endogenous linamarase and linamarin.A.J.A. Essers and M.H.J. Bennik were and M.J.R. Nout is with the Department of Food Science, Wageningen Agricultural University, Bomenweg 2, 6703HD Wageningen, The Netherlands. A.J.A. Essers is now with the Department of Toxicology, Wageningen Agricultural University, PO Box 8000, 6700EA Wageningen, The Netherlands; M.H.J. Bennik is now with the Agrotechnological Research Institute, PO Box 17, 6700AA Wageningen, The Netherlands.