Comparison of C-S lyase in Lentinus edodes and Allium sativum

The characteristics of C-S lyase in Lentinus edodes (shiitake) were compared with those in Allium sativum (garlic). C-S lyase mRNA from shiitake was hybridized with the garlic C-S lyase cDNA fragment, being almost the same length as that from garlic. The isoelectric point of the C-S lyase from shiitake was between pH 4 and 5, while that from garlic was over a wider range between pH 4 and 8. Different from the C-S lyase from garlic, that from shiitake was not a glycoprotein without being stained by PAS, and was not bound to the anti-garlic C-S lyase antibody. Similar to garlic C-S lyase, shiitake C-S lyase comprised a homodimer, and its molecular mass was 84 kDa. However, the N-terminal amino acid sequences of each subunit of shiitake C-S lyase were totally different from those of garlic C-S lyase.     

C-S lyase activities in leaves of crucifers and non-crucifers, and the characterization of three classes of C-S lyase activities from oilseed rape (Brassica napus L.)

C-S lyases in plants are involved in primary and secondary metabolism, and in glucosinolate-containing species may be involved in glucosinolate biosynthesis. Extracts from oilseed rape ( Brassica napus ) leaves were assayed for several C-S lyase activities. Four activities [using L -cystine, L -cystathionine, S -(2-benzothiazolyl)- L -Cys (SBC) and S -benzyl- L -Cys] were investigated in detail. All are developmentally regulated (highest in youngest leaves), and differentially inhibited by iodoacetamide, N -ethylmaleimide (NEM) and ethylenediaminetetraacetic acid (EDTA). Thermal stabilities and pH optima were also distinct. Competitive inhibition of the SBC lyase activity with a variety of sulphur-containing compounds indicated that cystine lyase contributes to SBC degradation, and this enzyme may cleave a wide range of compounds, both aliphatic and aromatic, but other 'SBC lyases' were also present. Putative aromatic glucosinolate intermediates were cleaved by the rape enzymes. Developmental and biochemical studies indicate that at least three classes of C-S lyase activity are present in rape leaves: cystathionine β -lyase, cystine lyase and a group of relatively non-specific lyases. C-S lyase preparations from other glucosinolate- and non-glucosinolate-containing species were capable of cleaving a number of aliphatic and aromatic conjugates. The highest activities were detected in glucosinolate-containing species and Allium cepa (onion). C-S lyase activities in non-glucosinolate-containing species (tobacco, Nicotiana tabacum, and barley, Hordeum vulgare ) were much lower.     

Localization of C-S lyase activity in the root cells ofAlbizzia lophanta

Summary The distribution of C-S lyase activity in root cells ofAlbizzia lophanta Benth. plantlets was investigated histochemically. H₂S formed upon cleavage of exogenously applied L-cysteine was precipitated by Pb⁺⁺ in a capture reaction at the site of its formation. Enzyme activity was found to be localized at the root tip and in a layer of cortex cells adjacent to the endodermis throughout the whole length of the root. Distinct areas within the exodermis, distributed in a regular pattern on the root surface, also exhibited the specific reaction. In vivo roots ofAlbizzia lophanta actively excrete the strongly smelling methylene dithiol, formed by enzymatic cleavage of djenkolic acid, the natural substrate of C-S lyase inAlbizzia. The physiological meaning of this compound, as well as the localization and intracellular distribution of C-S lyase activity are discussed.     

Dipeptide precursor of garlic odour in Marasmius species

γ-Glutamyl-marasmine, a new natural dipeptide containing an unusual cysteine sulphoxide moiety has been isolated from the Basidiomyceteous mushrooms Marasmius alliaceus, M. scorodonius and M. prasiosmus, which are known for their garlic like odour. It is shown that this compound is the common natural precursor and that its two step enzymatic cleavage leads to the odorous substances. In the first step γ-glutamyl -marasmine is cleaved by a γ-glutamyl transpeptidase. The formed marasmine is split in a second enzymatic reaction by a C-S lyase into pyruvic acid, ammonia and an unstable sulfur compound, which decomposes to form the odorous secondary products.     

Production and properties of three pectinolytic activities produced byAspergillus niger in submerged and solid-state fermentation

Three extracellular pectinases were produced byAspergillus niger CH4 by submerged and solid-state fermentation, and their physicochemical and kinetic properties were studied. The highest productivities of endo- and exo-pectinase and pectin lyase were obtained with solid-state fermentation. The kinetic and physicochemical properties of these enzymes were influenced by the type of culture method used. All activities were very different in terms of pH and temperature optima, stability at different pH and temperature values and affinity for the substrate (K _m values). In solid-state fermentation, all pectinase activities were more stable at extreme pH and temperature values but theK _m values of endo-pectinase and pectin lyase were higher with respect to those activities obtained by the submerged-culture technique. The pectin lyase activity obtained by the submerged-culture technique showed substrate inhibition but the enzyme obtained by solid-state fermentation did not. Electrophoresis, using sodium dodecyl sulphate/polyacrylamide gel with enzymatic extracts obtained for both culture methods, showed the same number on protein bands but some differences were found in their electrophoretic position. The results obtained in this work suggest that the culture method (submerged or solid-state) may be responsible for inducing changes in some of the pectinolytic enzymes produced byA. niger.     

Highly alkaline pectate lyase Pel-4A from alkaliphilic Bacillus sp. strain P-4-N: its catalytic properties and deduced amino acid sequence

The gene for a highly alkaline pectate lyase, Pel-4A, from alkaliphilic Bacillus sp. strain P-4-N was cloned, sequenced, and overexpressed in Bacillus subtilis cells. The deduced amino acid sequence of the mature enzyme (318 amino acids, 34 805 Da) showed moderate homology to those of known pectate lyases in the polysaccharide lyase family 1. The purified recombinant enzyme had an isoelectric point of pH 9.7 and a molecular mass of 34 kDa, and exhibited a very high specific activity compared with known pectate lyases reported so far. The enzyme activity was stimulated 1.6 fold by addition of NaCl at an optimum of 100 mM. When Pel-4A was stored at 50°C for 60 h, striking stabilization by 100 mM NaCl was observed in a pH range from 5 to 11.5, whereas it was stable only around pH 11 in the absence of NaCl. Received: June 10, 2000 / Accepted: October 3, 2000     

Latency of Infection in Unripe Avocados by Colletotrichum gloeosporioides is Not Due to Inadequate Enzyme Potential

Colletotrichum gloeosporioides produced exo-pectin lyase and protease in a) liquid cultures with incorporated washed cell wall material from unripe or ripe avocado and b) autoclaved immature fruit. The activity of exo-pectin lyase and protease produced in liquid cultures incorporating washed cell walls from immature fruits was almost the same as when washed cell walls from ripe fruits were incorporated. Ripe fruit tissue rotted by the fungus contained exo-pectin lyase, endo-polygalacturonase (endo-PG) and protease. The endo-PG was found to be endogenous to avocado fruit, and had a pH optimum of 5.5. The pH optima of exo-pectin lyase and protease were 8.5 and 7.5 respectively in all three enzyme preparations. All these enzyme preparations completely macerated avocado fruit tissue discs in vitro in less than 3 h of incubation but not potato tuber discs. Neither immature nor ripe fruit contained substances, proteinaceous or otherwise, which could inhibit the exo-pectin lyase or protease activity of these preparations. The results indicated that C. gloeosporioides possesses sufficient enzyme potential to invade cell walls of unripe fruit and that the fruit tissue does not have a mechanism to inactivate such enzymes.     

Arabidopsis mutants in the C-S lyase of glucosinolate biosynthesis establish a critical role for indole-3-acetaldoxime in auxin homeostasis

We report characterization of SUPERROOT1 (SUR1) as the C-S lyase in glucosinolate biosynthesis. This is evidenced by selective metabolite profiling of sur1, which is completely devoid of aliphatic and indole glucosinolates. Furthermore, following in vivo feeding with radiolabeled p-hydroxyphenylacetaldoxime to the sur1 mutant, the corresponding C-S lyase substrate accumulated. C-S lyase activity of recombinant SUR1 heterologously expressed in Escherichia coli was demonstrated using the C-S lyase substrate djenkolic acid. The abolishment of glucosinolates in sur1 indicates that the SUR1 function is not redundant and thus SUR1 constitutes a single gene family. This suggests that the "high-auxin" phenotype of sur1 is caused by accumulation of endogenous C-S lyase substrates as well as aldoximes, including indole-3-acetaldoxime (IAOx) that is channeled into the main auxin indole-3-acetic acid (IAA). Thereby, the cause of the "high-auxin" phenotype of sur1 mutant resembles that of two other "high-auxin" mutants, superroot2 (sur2) and yucca1. Our findings provide important insight to the critical role IAOx plays in auxin homeostasis as a key branching point between primary and secondary metabolism, and define a framework for further dissection of auxin biosynthesis.     

Overexpression of alginate lyase of <Emphasis Type="Italic">Pseudoalteromonas elyakovii</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>, purification,and characterization of the recombinant alginate lyase

The alyPEEC gene encoding alginate lyase from marine bacterium Pseudoalteromonas elyakovii IAM 14594 was subcloned into pBAD24 with arabinose promoter and sequenced, and overexpressed in TOP10 strain of E. coli after arabinose induction. Expression levels of alyPEEC gene in E. coli cells were over 39.6-fold higher than those in P. elyakovii IAM 14594 cells. The molecular mass of purified alginate lyase from the engineered E. coli cells was estimated to be 32.0 kDa. Optimum pH and temperature of the alginate lyase activity were 7.0 and 30 °C, respectively. The enzyme was unstable on heating and in acidic and alkaline solution. The enzyme activity was stimulated by the MgCl₂, NaCl, KCl, CaCl₂, BaCl₂ and MnCl₂, but was inhibited by the addition of 1.0 mM of EGTA, EDTA, SDS, ZnSO₄, AgNO₃, and CoCl₂. All the alginate, polyM and polyG could be converted into oligosaccharides with more than tetrasaccharides by the purified recombinant alginate lyase, suggesting that the recombinant alginate lyase produced by the engineered E. coli has highly potential application in seaweed genetics, food and pharmaceutical industries.     

Endo-polygalacturonate lyase of Cytophaga johnsonii

Summary An extracellular endopolygalacturonate lyase of Cytophaga johnsonii was purified from the culture filtrate. It appeared to be homogeneous as judged by polyacrylamide gel electrophoresis at pH 8.6 as well as pH 4.3. The purified enzyme had a pH optimum around 9.0 and required Ca⁺⁺ ions for its maximum activity. The apparent K _mfor polygalacturonic acid was found to be 0.22%. Both paper and column chromatography indicated formation and accumulation of an unsaturated monomer along with unsaturated di-, tri-, tetra- and pentamers from polygalacturonic acid by the enzyme action, indicating that the enzyme cleaved the substrate randomly in a non-hydrolytic manner. The glycosidic linkage next to the non-reducing end of polygalacturonic acid was not resistant to attack by this enzyme unlike in other known polygalacturonate lyases.Abbreviations PG lyase Polygalacturonate lyase - Tris Tris (hydroxymethyl) aminomethane     

Partial purification and properties of pectin lyase from Penicillium expansum

A pectin lyase, poly(methoxygalacturonide) lyase, EC 4.2.2.10, from a culture filtrate of Penicillium expansum was partially purified 33-fold with 7.3% yield. The enzyme was monomeric with a molecular mass of 36.5 kDa. The enzyme did not contain pectate lyase activity and degraded citrus and apple pectin best at pH 7.0 and 40 to 45°C. The K _m for citrus pectin was 9 mg ml^-1.     

Erratum to: Extracellular protein production and morphogenesis of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in submerged culture

Along with a brief review of Lentinula edodes (shiitake mushroom) submerged cultivation history within the framework of important extracellular proteins biosynthesis, this study contains the authors’ own results. The possibility of regulating the lectin activity of shiitake using the synthetic components is shown. The time course of lectin production in culture liquid of L. edodes in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. A relationship between the chemical composition of nutrient medium, the activity of extracellular lectins of L. edodes, and the formation of pigmented mycelial film in liquid culture has been found. The formulation of medium, on which the brown mycelial film appears in several days of submerged cultivation, is proposed. The results obtained make a contribution to the present notion of biochemical processes that give rise to the occurrence of the aforesaid morphological structure of shiitake. Finally, two extracellular lectins from the submerged culture of L. edodes have been isolated and purified to homogeneity. Their physicochemical properties and composition have been studied.     

Production of lyases byPhoma exigua associated with seed-rot ofVigna radiata

Phoma exigua associated with seed-rot ofVigna radiata produced lyases which varied with the media tested. The production of lyases was higher in pectin-supplemented media.Vigna seed meal medium was not suitable for induction of lyase production. The pectin lyase and pectate lyase was maximum after 11 d of incubation by which time the pH was shifted to alkaline side. Temperature of 25 °C and pH 9 was found to be optimum for the activity of pectin lyase and pectate lyase. Fungicides (antracol and panoctine), phenols (pyrocatechol and gallic acid) and growth substances (gibberellic acid and yeast extract) adversely affected the enzyme secretion.     

Intercropping garlic at different planting times and densities for insect pest or crop yield and value management in tobacco fields

Although the densities of tobacco pests have been decreased in garlic‐tobacco fields, further studies are needed to judge the effects of garlic transplanting densities or times on tobacco pests in tobacco fields. Therefore, field experiments were conducted in Liancheng County in Longyan City, Fujian Province, in China in 2014 and 2015. Myzus persicae (Sulzer) abundance, the species or abundance ratios of enemies and pests, the intercropping effects and the tobacco yield and crop value showed that the effects of transplanting tobacco 10 days after garlic transplantation at a density of 5.85 individual plants per square meter on pests were stronger than those of other treatments. Aphid abundance was significantly lower in transplanting tobacco 10 days after garlic transplantation at a density of 5.85 individual plants per square meter than in the other treatments. The ratio between enemies and pests in transplanting tobacco 10 days after garlic transplantation at a density of 5.85 individual plants per square meter was higher than those in the other treatments. The intercropping effects of transplanting tobacco 10 days after garlic transplantation at a density of 5.85 individual plants per square meter on Myzus persicae, Spodoptera litura (Fabricius), Heliothis assulta Guenee and Nezara viridula Linnaeus were significantly stronger than those of the other treatments, whereas the effects of transplanting tobacco 15 days after garlic transplantation at a garlic density of 5.85 individual plants per square meter on Agrotis ypsilon (Rottemberg) were significantly stronger. Additionally, the yield and crop value of transplanting tobacco 10 days after garlic was transplanted at a density of 5.85 individual plants m^?2 were higher than those of the other treatments. Therefore, our study demonstrated that the model of transplanting tobacco 10 days after garlic was transplanted at a density of 5.85 individual plants m^?2 is an optimal management strategy to control flue‐cured tobacco pests and to acquire higher crop yield in garlic‐tobacco fields.     

Optimization of culture media for solid-state culture of<Emphasis Type="Italic">Pleurotus ferulae</Emphasis>

In order to elucidate the possibility of artificial production ofP. ferulae by solid-state culture, the optimization of culture conditions was carried out. When NH₄H₂PO₄ and CaCO₃ were used in the cultures using test tube with 30 g ofPopulus sawdust at 25°C±1 in the dark, the favored mycelial growth was observed with 1% of NH₄H₂PO₄ and the production of polysaccharide was 7.85 mg/100 mg of mycelium with 1% of CaCO₃. The mixtures of 80% ofPopulus sawdust and 20% of rice bran at 60% of water content were determined to be optimal for the production of fruiting bodies in the sawdust culture. When three treatments containing various ratios of garlic powder were conducted, yields of fruiting bodies were drastically higher than those of synthetic mixture without garlic powder. The highest yield (143 g/bag) was obtained with 7% garlic powder. The yield of synthetic mixture containing 7% of garlic powder was 83% higher than that of sawdust culture. The reason why garlic powder did support growth was not clear but it is possible that garlic powder might contain effective components for the formation of fruiting body. The optimal synthetic mixture composition consisted of cotton seed 77%, lime 6.4%, K₂HPO₄ 0.2%, KH₂PO₄ 0.2%, CaHPO₄, 0.2%, corn flour 4%, wheat flour 5%, and garlic powder 7%.     

Characterization of alginate lyase gene using a metagenomic library constructed from the gut microflora of abalone

A metagenomic fosmid library was constructed using a genomic DNA mixture extracted from the gut microflora of abalone. The library gave an alginate lyase positive clone (AlyDW) harboring a 31.7-kbp insert. The AlyDW insert consisted of 22 open reading frames (ORFs). The deduced amino acid sequences of ORFs 11–13 were similar to those of known alginate lyase genes, which are found adjacent in the genome of Klebsiella pneumoniae subsp. aerogenes, Vibrio splendidus, and Vibrio sp. belonging to the phylum Gammaproteobacteria. Among the three recombinant proteins expressed from the three ORFs, alginate lyase activity was only observed in the recombinant protein (AlyDW11) coded by ORF 11. The expressed protein (AlyDW11) had the highest alginate lyase activity at pH 7.0 and 45°C in the presence of 1 mM AgNO₃. The alginate lyase activity of ORF 11 was confirmed to be endolytic by thin-layer chromatography. AlyDW11 preferred poly(β-d-mannuronate) as a substrate over poly(α-l-guluronate). AlyDW11 contained three highly conserved regions, RSEL, QIH, and YFKAGVYNQ, which may act to stabilize the three-dimensional conformation and function of the alginate lyase.     

The C-S lyases of higher plants: preparation and properties of homogeneous alliin lyase from garlic (Allium sativum)

Alliin lyase from garlic (Allium sativum) has been purified to homogeneity. The purification procedure involves the use of affinity chromatography on concanavalin A-Sepharose 4B. Addition of polyvinylpolypyrrolidone to the homogenizing medium greatly improves the specific activity of the extract. The enzyme is a glycoprotein as seen by its ability to bind to concanavalin A-Sepharose 4B and by its positive periodic acid-Schiff base stain. It has a carbohydrate content of 5.5%. Km values for this enzyme were estimated to be 5.7 mM for S-ethyl-L-cysteine sulfoxide and 3.3 mM for S-allyl-L-cysteine sulfoxide. The molecular weight of this garlic enzyme, as determined by gel filtration, was found to be 85,000; the molecule consists of two equal subunits of Mr 42,000. The amino acid content was found to be similar to that reported previously for onion alliin lyase, although there is twice as much tryptophan in the garlic alliin lyase as in the onion enzyme. By both chemical and spectral methods the enzyme was found to have two molecules of pyridoxal 5-phosphate per enzyme molecule, suggesting one per subunit. There are significant differences in the nature of these findings from those previously reported from this laboratory for the onion enzyme. Studies are in progress to compare further the alliin lyases from garlic and onion.     

Sugar Synthesis in Leptospira

The presence and some properties of the key enzymes of the glyoxylate cycle, isocitrate lyase (threo-D_s-isocitrate glyoxylate-lyase, EC 4.1.3.1) and malate synthase (L-malate glyoxylate-lyase (CoA-acetylating) EC 4.1.3.2), were investigated in Leptospira biflexa. Isocitrate lyase activity was found for the first time in the organism. The enzyme was induced by ethanol but not by acetate. The optimum pH was 6.8. The activity was inhibited by phosphoenolpyruvate, a specific inhibitor of isocitrate lyase. The optimum pH of malate synthase of L. biflexa was about 8.5. The K_m value for glyoxylate was 3.0 × 10^?3 M and the activity was inhibited by glycolate, the inhibitor. The results strongly suggested the presence of a glyoxylate cycle in Leptospira. The possibility that the glyoxylate cycle plays an essential role in the synthesis of sugars, amino acids and other cellular components as an anaplerotic pathway of the tricarboxylic acid cycle in Leptospira was discussed.     

The C-S Lyases of Higher Plants : Direct Comparison of the Physical Properties of Homogeneous Alliin Lyase of Garlic (Allium sativum) and Onion (Allium cepa)

Garlic and onion alliin lyases, although from closely related species, have many differences. The two enzymes differ in their K_m values, pH optima, and isoelectric points. There is a major difference in their molecular weight and subunit structure. The garlic holoenzyme has a molecular weight of 85,000 and consists of two subunits of molecular weight 42,000. The onion enzyme has a holoenzyme molecular weight of 200,000 composed of four subunits of molecular weight 50,000. The onion enzyme is much more difficult to dissociate into its subunits which suggests differences in subunit interaction between the two enzymes. The dimeric stucture of the garlic and the tetrameric structure of the onion enzyme is consistent with a coenzyme content (pyridoxal-5′-phosphate) equivalent to one mole per subunit. The two enzymes vary vastly in their spectra, the onion enzyme having a lower pyridoxal-5′-phosphate absorbance at 430 nanomoles and an inability to react with l-cysteine. Both enzymes are glycoproteins and bind to concanavalin A-Sepharose columns. The onion alliin lyase binds more tightly than the garlic enzyme. The amino acid content of both enzymes is similar as is the carbohydrate content. However, upon hydrolysis the onion lyase does yield more mannose units than the garlic enzyme which is consistent with the former's stronger affinity for concanavalin A.     

Physical properties of pectic polysaccharidases ofPseudomonas solanacearum from Nigeria

Pseudomonas solanacearum (obtained from Nigeria) produced certain pectic polysaccharidases when grown in aerobic batch cultures containing pectic substances. The pH optima of the enzymes were different. The optimum for polygalacturonase EC 3.2.2.15 was 5.5, and for pectate lyase EC 4.2.99.3 it was 8.5. The -1,4-glycosidic bonds between galacturonide units were cleaved at random, indicating the endo character of the enzymes. The pectic polysaccharidases were purified by (NH₄)₂SO₄ fractionation and by electrofocusing. Highest polygalacturonase activity and pectate lyase activity were obtained in the fractions at 41%–60% and 61%–80% (NH₄)₂SO₄ saturation, respectively. Polygalacturonase was resolved into two components with isoelectric points of 5.0 and 7.5; the isoelectric point of pectate lyase was 8.1.