Isoforms of Linamarase in Cassava (Manihot esculenta)

Linamarase (EC. 3.2.1.21) was purified from different tissues of cassava (leaf, rind and tuber) to compare the kinetic properties and characteristics of the enzyme in these tissues. Purified enzyme preparation appeared as single band of average molecular size 70 kD in SDS-PAGE gels. The kinetic properties of linamarase with respect to pH and temperature indicated that tuber linamarase possessed a broader pH optimum and higher temperature stability as compared to leaf and rind enzymes. Differences in Km values for linamarin were observed with leaf linamarase having the highest Km value (500 μM) followed by rind (400 μM) and then tuber (250 μM) linamarases. Rind enzyme appeared to be less susceptible to urea denaturation than the leaf enzyme. Comparison of elution profiles from DEAE-Cellulose indicated that the relative amounts of the various ionic forms of the enzyme differed in the case of each tissue. Elution patterns of the enzyme from Con A-Sepharose also differed, suggesting difference in glycosylation among leaf, rind and tuber enzymes. This was confirmed by carbohydrate analysis which showed that the tuber linamarase contained significantly higher amount of protein bound carbohydrate. These results suggest the possible occurrence of different forms of linamarase in cassava.     

肌苷酶电极生物传感器

为了构建肌苷酶电极生物传感器,以固定化核苷磷酸化酶(EC 2.4.2.1)、黄嘌呤氧化酶(EC 1.2.3.2)与过氧化氢电极组成电流型酶电极生物传感器,用于检测肌苷片中的肌苷,其输出电流可达500nA.结果发现,肌苷测定的线性范围为1-268 mg/L,精度:RSD小于0.14%,响应时间:60 s,使用寿命大于25 d,实际测定肌苷片中肌苷含量回收率:100.8%.由此表明:采用双酶电极法测定肌苷片中的肌苷含量,由于酶促反应专一性高、样品不需分离直接进样分析、处理条件温和、反应时间短暂因而结果较为可靠.     

Transglucosylation of tertiary alcohols using cassava beta-glucosidase

We have compared the ability of beta-glucosidases from cassava, Thai rosewood, and almond to synthesize alkyl glucosides by transglucosylating alkyl alcohols of chain length C(1)-C(8). Cassava linamarase shows greater ability to transfer glucose from p-nitrophenyl-beta-glucoside to secondary alcohol acceptors than other beta-glucosidases, and is unique in being able to synthesize C(4), C(5), and C(6) tertiary alkyl beta-glucosides with high yields of 94%, 82%, and 56%, respectively. Yields of alkyl glucosides could be optimized by selecting appropriate enzyme concentrations and incubation times. Cassava linamarase required pNP-glycosides as donors and could not use mono- or di-saccharides as sugar donors in alkyl glucoside synthesis.     

Detection of selection in populations of white clover (Trifolium repens L.)

Seed populations of white clover polymorphic for the presence/absence of both ovariogenic glucosides and the hydrolysing enzyme linamarase, were introduced into three natural populations. Over the first six months of life a significant increase in the frequency of linamarase containing individuals occurred. Estimated selection coefficients against plants lacking linamarase were in the region of 0.3. This result may have been due to selection at the enzyme locus alone, or to selection favouring cyanogenic individuals which possess both cyanogenic glucosides and enzyme.     

In-situ localization of cyanogenic β-glucosidase (linamarase) gene expression in leaves of cassava (Manihot esculenta Cranz) using non-isotopic riboprobes

The release of hydrogen cyanide (cyanogenesis) from damaged plant tissue depends upon the sequential action of a β-glucosidase and an α-hydroxynitrilase on cyanoglucosides. The non-isotopic digoxigenin labelling system was used to visualize the presence of cyanogenic β-glucosidase (linamarase) mRNA in cells of young leaves of Manihot esculenta Cranz (cassava). Strong hybridization to antisense riboprobes produced from the cDNA clone pCAS5, indicates localization of linamarase gene expression in laticifers (latex vessels). This is supported by the demonstration of linamarase mRNA in exuded latex. In contrast, in-situ localization of the control gene pGLF4, showed expression in all leaf mesophyll cells. High levels of linamarase activity were demonstrated in the latex of leaf petioles and this activity was shown to be dependent on the presence of attached leaflets. Assays of α-hydroxynitrilase activity in exuded latex and whole leaves shows that, unlike linamarase, this enzyme is present at very low levels in latex and must be located elsewhere in the leaf.     

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.     

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.     

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.     

Biochemical characterisation of the Li locus,which controls the activity of the cyanogenic β-glucosidase in Trifolium repens L.

The cyanogenic -glucosidase (linamarase) was purified from white clover leaf tissue. The enzyme is a homodimer with a molecular weight of 105 300–103 400 daltons estimated from molecular exclusion chromatography. The effect of buffer ions on the pH optimum and charge properties of the enzyme are presented. A combination of molecular exclusion chromatography and CM cellulose ion exchange chromatography purified linamarase 16 fold to a single 62 000 dalton polypeptide on SDS polyacrylamide gel electrophoresis. This polypeptide represented about 5% of the total soluble leaf protein and can be seen as a prominent band in SDS polyacrylamide gel electrophoresis of crude leaf extracts from Li Li plants. Screening backcross progeny showed that extracts from li li plants, which have no linamarase activity, lack this 62 000 dalton polypeptide. Linamarase is the major glycoprotein in white clover leaf extracts which binds to Concanavalin A-Sepharose.     

A molecular and biochemical analysis of the structure of the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Cranz).

The cyanogenic beta-glucosidase (linamarase) of cassava is responsible for the first step in the sequential break-down of two related cyanoglucosides. Hydrolysis of these cyanoglucosides occurs following tissue damage and leads to the production of hydrocyanic acid. This mechanism is widely regarded as a defense mechanism against predation. A linamarase cDNA clone (pCAS5) was isolated from a cotyledon cDNA library using a white clover beta-glucosidase heterologous probe. The nucleotide and derived amino acid sequence is reported and five putative N-asparagine glycosylation sites are identified. Concanavalin A affinity chromatography and endoglycosidase H digestion demonstrate that linamarase from cassava is glycosylated, having high-mannose-type N-asparagine-linked oligosaccharides. Consistent with this structure and the extracellular location of the active enzyme is the identification of an N-terminal signal peptide on the deduced amino acid sequence of pCAS5.     

Determination of urate in undiluted whole blood by enzyme electrode

An amperometric enzyme electrode is described for the assay of urate in undiluted, unstirred whole blood. The electrode used Aspergillus flavus uricase (EC.1.7.3.3) cross-linked to bovine serum albumin by means of glutaraldehyde, sandwiched between a dimethyldichlorosilane-treated microporous polycarbonate membrane and an inner cellulosic H2O2-selective membrane. The resulting device had a low pH dependence, was capable of repeated use in blood, and gave an acceptable correlation with a standard spectrophotometric method. Electrode steady state and dynamic response were found to be dependent upon the amount of enzyme loading, and could be further optimised by the incorporation of catalase in the enzyme layer.     

Linamarase and other β-glucosidases are present in the cell walls of Trifolium repens L. leaves

Linamarase (EC 3.2.1.21) is a specialized -glucosidase that hydrolyses the cyanogenic glucoside linamarin. Two clones of Trifolium repens L. derived from natural populations, of which one clone exhibited linamarase activity, were used in a comparative study to try to establish the localization of linamarase and other -glucosidases. Two methods were used: the first one was vacuum infiltration of intact leaf cells, followed by centrifugation. A significant amount of linamarase and -glucosidase activity could be extracted from intact tissue by a 0.25 M NaCl solution, indicating that these activities are localized in the apoplast. The second method, immuno-cytofluorescense of microtome sections, confirmed this. It was found that linamarase and other -glucosidases are present in the cell walls, especially those of the epidermal cells, and in the cuticle. However their presence in the cell walls of other tissues i.e. walls of the vessels, could not be excluded. No difference in distribution could be detected between linamarase and other -glucosidases.     

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.     

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.     

Polyvinylferrocenium modified Pt electrode for anaerobic glucose monitoring

An amperometric enzyme electrode for the determination of glucose under anaerobic solution conditions was developed by immobilizing glucose oxidase and then by adsorbing ferrocene in polyvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of ferrocene that the reduced flavin adenine dinucleotide centers of glucose oxidase was measured at a constant potential. The response characteristics of the enzyme electrode were investigated. The effects of the thickness of the polymeric film, the amount of the enzyme immobilized, the amount of the mediator, the glucose concentration, the applied potential, operating pH and temperature on the response of the enzyme electrode were studied. The response time and the optimum pH were found to be 30-40 s and pH 7.4 at 25 degrees C, respectively. The linear response was observed up to 5.0 mM glucose concentration that the produced detectable current was 0.0075 mM glucose concentration. The activation energy (E(a)) of immobilized enzyme reaction was calculated to be 41.3 kJ mol(-1) from the Arrhenius plot. The apparent Michaelis-Menten constant (K(Mapp)) was found to be 6.05 mM glucose according to the Lineweaver-Burk graph of the Michaelis-Menten equation under the optimum conditions. The interference signal due to the most common electrochemical interfering species was also evaluated.     

A new amperometric enzyme electrode for alcohol determination

A new enzyme electrode for the determination of alcohols was developed by immobilizing alcohol oxidase in polvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of enzymatically generated H(2)O(2) was measured at a constant potential of +0.70 V versus SCE. The effects of substrate, buffer and enzyme concentrations, pH and temperature on the response of the electrode were investigated. The optimum pH was found to be pH 8.0 at 30 degrees C. The steady-state current of this enzyme electrode was reproducible within +/-5.0% of the relative error. The sensitivity of the enzyme electrode decreased in the following order: methanol>ethanol>n-butanol>benzyl alcohol. The linear response was observed up to 3.7 mM for methanol, 3.0 mM for ethanol, 6.2 mM for n-butanol, and 5.2 mM for benzyl alcohol. The apparent Michaelis-Menten constant (K(Mapp)) value and the activation energy, E(a), of this immobilized enzyme system were found to be 5.78 mM and 38.07 kJ/mol for methanol, respectively.     

Polyvinylferrocenium immobilized enzyme electrode for choline analysis

The response characteristics of a new enzyme electrode for determining choline are reported. The enzyme electrode consists of a polyvinylferrocenium perchlorate coated Pt surface onto which the enzyme, choline oxidase, is attached. Choline oxidase catalyzes the oxidation of choline to betaine, producing H₂O₂. Current due to H₂O₂ oxidation catalyzed by polyvinylferrocenium centers was measured. The effects of choline concentration, the amount of enzyme immobilized and the operating pH and temperature on the response of the enzyme electrode were studied. The effects of interferents were also investigated. The response time was found to be 60–70 s and the upper limit of the linear working portion was found to be 1.2 mM choline concentration. The minimum substrate concentration that produced detectable current was 4.0×10⁻⁶ M choline concentration. The steady-state current of this enzyme electrode was reproducible within ±4.6% of relative error. The apparent Michaelis–Menten constant (K_Mapp) and the activation energy, E_a, of this immobilized enzyme system were found to be 2.32 mM and 38.91 kJ/mol, respectively.     

The role of polypyrrole as charge transfer mediator and immobilization matrix for d-fructose dehydrogenase in a fructose sensor

A fructose dehydrogenase (FDH) modified electrode is produced by the electroadsorption of a layer of FDH on a platinum electrode followed by the electropolymerization of a polypyrrole (PPy) film around and over the enzyme. This immobilizes and stabilizes the enzyme as well as providing an electron transfer pathway to the electrode. The amperometric response to fructose and the enzymatic activity are measured as a function of PPy film thickness. The electrode is shown to have a maximum response at a PPy thickness of approximately the thickness of the enzyme layer. A measure of the electrode efficiency is also obtained, this is the amperometric response to fructose as a percentage of that expected on the basis of the enzyme activity. The functioning of the electrode is also dependent on the counter-ion used for PPy polymerization. This is shown to be mainly related to the nucleation and growth of the PPy film in the interfacial region.     

Study of xanthine oxidase immobilized electrode based on modified graphite

Xanthine oxidase (E. C. 1.2.3.2) was immobilized by adsorption on electrochemically modified graphite plate to obtain an enzyme electrode. The current of the enzyme electrode in substrate (xanthine) solutions was found to be a result of the electrooxidation of H2O2 generated in the enzyme layer. The linearity of the amperometric signal was up to a substrate concentration of 65 microM at 0.6 V (vs. Ag/AgCl). The response time was 2 minutes. The enzyme electrode preserves 80% of its initial activity after a three-week storage in air at room temperature.