Structure, function and application of serine carboxypeptidase-like acyltransferases from plants / 生物工程学报

Plant serine carboxypeptidase-like acyltransferases (SCPL-AT) have similar structural characteristics and high homology compared to the serine carboxypeptidase. They can transfer the acyl from acyl glucose esters to many natural products, participate in the acylation modification of plant secondary metabolites, enrich the structural diversity of natural products, and improve the physicochemical properties such as water solubility and stability of compounds. This review summarizes the structural characteristics, catalytic mechanism, functional characterization, and biocatalytic applications of SCPL-AT from plants. This will help to promote the functional characterization of these acyltransferase genes and the biosynthesis of useful plant secondary metabolites by synthetic biotechnology.

Advances in plant heavy metal transporter P1B-ATPases / 生物工程学报

P1B-ATPases are a group of proteins that can transport heavy metal ions across membranes by hydrolyzing ATP and they are a subclass of the P-type ATPase family. It was found that P1B-ATPases are mainly responsible for the active transport of heavy metal ions in plants and play an important role in the regulation of heavy metal homeostasis in plants. In this paper, we dissusses the mechanism of P1B-ATPases from the structure and classification of P1B-ATPases, and review the current research progress in the function of P1B-ATPases, in order to provide reference for future research and application of P1B-ATPases in improving crop quality and ecological environment management.

Potential of antioxidant enzymes in depicting drought tolerance of wheat (Triticum aestivum L.).

In an effort to determine the biochemical markers for identifying genotypes before sowing for drought tolerance, changes in activities of antioxidant enzymes were determined in the seedlings of five drought-tolerant and five drought-sensitive wheat (Triticum aestivum L.) genotypes, each with different genetic background growing under normal and water deficit conditions induced by 6% mannitol. In comparison with non-stressed seedlings, the catalase (CAT) activity was upregulated by more than 50% in the roots of water-stressed seedlings in drought-tolerant genotypes. Water deficit stress also led to the upregulation of ascorbate peroxidase (APX) in the endosperms and glutathione reductase (GR), CAT and peroxidase (POD) in the shoots of stressed seedlings in drought-tolerant genotypes. Superoxide dismutase (SOD) activity was very low in roots and shoots and showed non-significant increase under water-stress in tolerant genotypes. Out of five specified enzyme activities (CAT in roots and shoots, APX in endosperms, GR and POD in shoots), if any three are upregulated in the specified tissues under water deficit conditions, the genotype is likely to be drought-tolerant. Wheat seedlings with low GR and APX activities and high POD activity in shoots with a low ratio of GR activity of shoot to root of non-stressed seedlings are likely to perform better under rainfed conditions. The observed data showed that status of antioxidant enzymes could provide a meaningful tool for depicting drought tolerance of a wheat genotype.

Conservación del ombusillo, planta amenazada de la provincia de Buenos Aires, Argentina / Conserving ombusillo, an endangered plant from the province of Buenos Aires, Argentina

Phytolacca tetramera Hauman "ombusillo", es una especie vegetal endémica del SE de la Provincia de Buenos Aires, Argentina, que se halla en peligro crítico de extinción. Su principal factor de amenaza es la reducción del hábitat por acción antrópica. Esta especie presenta principios activos fungicidas y, posiblemente, dada su afinidad con otras especies del mismo género, presente compuestos antivirales, antitumorales, bactericidas e insecticidas. Se realizaron ensayos de macropropagación con distintas concentraciones de reguladores de crecimiento de tipo auxínicos que muestran claramente un enraizamiento óptimo correspondiente a segmentos de ejes aéreos vegetales “estacas” sometidas a 300 ppm de ácido indol butírico y a segmentos de tallos subterráneos sin aplicación de hormonas. Así mismo, se realizaron ensayos de germinación, en condiciones de luz y de oscuridad, comprobándose que las semillas presentan fotoblastismo positivo con un porcentaje de germinación del 65%, el cual disminuye enormemente luego del año de cosecha.

Phytolacca tetramera Hauman "ombusillo" is an endemic plant species which is in critical danger of becoming extinct; it comes from the south-east of the province of Buenos Aires. The main factor threatening this species is the reduction of its natural environment by antropic action.This species has antifungal properties and, due to its relationship with other species from the same genus, it could also have antiviral, antitumour, antibacterial and insecticidal compounds. Macropropagation experiments were carried out using different concentrations of auxinic growth regulators. Segements of aerial axis “stakes” treated with 300 ppm of indol-butiric acid and segments of underground stems without hormonal treatment provided optimum rooting. Germination experiments in dark and light conditions were also carried out, finding that seeds showed positive photoblastisme with a 65% germination rate which declined considerably after the crop had been harvested.

Determinação de polifenóis (taninos) em produtos alimentícios (chás) usando biossensor de Polifenol oxidase, obtida de extrato bruto da casca de banana nanica (Musa acuminata) e caracterização desse biossensor / Determination of polyphenols (tanines) in foods products (teas) using a biosensor of Polyphenol oxidase, obtained of crude extract of banana nanica peel (Musa acuminata) and characterization this biosensor

Introdução - Extrato bruto da casca de banana nanica (Musa acuminata); melhor fonte de enzima Polifenol oxidase (PFO) [EC.1.14.18.1] foi estudado como material biocatalítico para a oxidação aeróbica de substratos fenólicos. Materiais e Métodos - O extrato bruto de PFO foi obtido como em Perone et al.14 (2000). A atividade da enzima PFO e proteína total foram determinadas nesse extrato. Foi construído um biossensor desse extrato bruto da casca de banana nanica com 75 unidades de PFO, imobilizada com reagente glutaraldeído. Resultados - Esse biossensor, sensível a polifenóis, foi caracterizado e apresentou pH ótimo de imobilização da enzima igual a 6,5 e sensibilidade acentuada para o substrato catecol. Também foi utilizado no estudo da determinação da concentração de taninos em amostras de diversos tipos de chás. Conclusões - Foi verificado que a porcentagem de erro comparando com o método espectrofotométrico apresentou valores menores que 1,0% estando, portanto, de acordo com o procedimento padrão oficial. Comparando os resultados obtidos com esse biossensor e o de extrato bruto da polpa de banana nanica observamos, melhor tempo de armazenamento das membranas com a casca do que com a polpa, e uma diminuição significativa na quantidade de extrato imobilizado. Assim, conclui-se que o extrato de PFO da casca é melhor fonte de enzima do que a polpa e, portanto, será usado na construção dobiossensor. A vantagem do método amperométrico apresentado é possuir baixo custo, rapidez nas determinações e boa sensibilidade comparado com métodos cromatográficos.

Introduction - Crude extract of banana nanica (Musa acuminata); the best source of enzyme Poliphenol oxidase (PPO) [EC.1.14.18.1] was studied as biocatalytic material to the aerobic oxidation of phenolics substrates. Materials and Methods - The crude extract of I was done the same as at Perone et al.14 (2000). The activity from the enzyme PPO and total protein were determined in this extract. It has been built a biosensor of this crude extract from the peel of stunded banana with 75unities of PPO immobilized with glutaraldeyde reagent. Results - This biosensor, sensitive to poliphenol, was characterized and presented immobilizing optimium pH of the enzyme equal to 6.5and acute sensibility to its catechol substrate. It was also used at the study of the determination of tanines concentration in samples of many kinds of tea. Conclusions - It was verified that percentage of error comparing with the spectrophotometric method, has presented lower than 1,0% values according to the standard methods. Comparing the results obtained with this biosensor and the crude extract of the pulp of banana nanica, it was observed the better stock time of the membranes with the peel than with the pulp, and significative diminishing of the amount of immobilized extract. So, we conclude that the extract of PPO from the peel is better source of enzyme than the pulp and it will be used at the construction of the biosensor. The advantage of the amperometrics methods presented is to obtain low cost, fast determination and good sensibility compared to cromatographics methods.

Root-surface phosphatase activity in shrublands across a European gradient: effects of warming.

Root-surface phosphatase activities were measured in natural and semi-natural shrublands across an European climatic gradient of temperature and rainfall including Wales (WL), Denmark (DK), Netherlands (NL), Hungary (HU), Italy (IT) and Spain (SP). In each site a warming experiment was conducted since 1999 or 2001 by means of passive night-time warming using reflective curtains that covered the vegetation at night. The treatments increased yearly average soil temperatures around 0. 8 degrees C in most of sites. Root-surface phosphatase activity values ranged between 56 mg PNP g(-1) h(-1) in IT and 3.5 mg PNP g(-1) h(-1) in HU. Warming had no effect on root-surface phosphatase activity across the sites and only in Hungary a slight increase was detected. Plants at Mediterranean sites (IT, SP) showed a higher root-surface phosphatase activity than plants at temperate sites (WL, NL, DK). We suggest it might be an adaptation of plant species evolved under Mediterranean climate that allows them a) to compensate in wet period for the decrease in phosphatase activity, and thus P uptake, during drought periods, and/or b) to benefit from soluble organic P flushes following the frequent drying-rewetting episodes experienced by soils in Mediterranean ecosystems.

Cytological, anatomical and biochemical studies on the side-effect of some pesticides

The invisible side-effects of triazophos and spinosad [insecticides], thiophanate-methyl [fungicide] and 2,4-D [herbicide] on plant were carried out using the bioassay test system of Vicia faba root tips to detect their cytotoxicity. Also, effects on the anatomical and biochemical characteristics were investigated. The obtained data showed that a linear decrease in mitotic index [MI] with increasing pesticide concentration. In addition, a linear correlation between the concentration and percentage of chromosomal abnormalities was recorded in the presently treated pesticides. The EC50 values caused undivided to 50% of total root tip cells were 3.61x10[-6], 1.31x10[-3], 4.85x10[-3] and 35.5 ppm for spinosad, thiophanate-methyl, 2,4-D and triazophos, respectively. The results also revealed that all tested pesticides found to decrease the growth of seedlings by 50 to 60% compared to the control. The normal tissues of stem and root similar to control were obtained in the case of insecticide spinosad and tested fungicide thiophanate-methyl while, malformation of the parenchyma cells and dialysis of the cell walls were observed in the case of herbicide 2,4-D. All tested pesticides significantly decreased the protein content in shoot and root except the herbicide 2,4-D in the case of shoot tissue. The insecticide spinosad increased the DNA content while, insecticide triazophos, fungicide thiophanate-methyl and herbicide 2,4-D reduced the DNA content of shoot and root tissues. It is clear that the effect of tested pesticides depends on pesticide type and plant tissue. In addition, the peroxidase enzyme was high sensitive to the tested pesticides. In similar, all of the tested pesticides shown to be highly esterase inhibitors in the case of shoot tissues. It was clear that there were 5 bands of protein pattern in control sample. The protein bands identical with 95.9 and 56.2 KDa were recorded in all treatment samples. In contrast, the protein band of 40.4 KDa was completely disappeared from the homogenates of all tested treatments, Peroxidase isozyme [58.9 KDa] was appeared in tested fungicide and herbicide treatments. While, the band [30.8 KDa] was disappeared in all treatments. Two major esterase isozymes corresponded to 68.3 and 27.8 KDa were presented. These isozymes were appeared in all pesticide treatments except in the case of spinosad. In general, according to the results of present study it can be stated that; the evaluation of pesticides hazardous effects depends on their side visible effects on the non-target organism did not enough. The invisible side-effect is very important in this response

Phylogenetic tree construction of butyrylcholinesterase sequences in life forms.

Butyrylcholinesterase is an enzyme with few known physiological functions. It is related to acetylcholine that was shown to be expressed in a variety of life forms. We performed a search using the human butyrylcholinesterase gene (HGNC:983;MIM:177400), and found the sequence in a broad spectrum including plants, bacteria and animals. Therefore butyrylcholinesterase appears to have evolved early in evolution, and to have been conserved.

A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis

Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100°C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure.

Physico-chemical and functional characterization of a high molecular weight carboxymethylcellulase from Cuscuta reflexa.

Carboxymethylcellulase (CMCase) was extracted and purified from an angiosperm parasite Cuscuta reflexa free from beta-glucosidase and other enzyme activities. The molecular mass and Stokes' radius of the purified enzyme are 144 kDa and 44 A, respectively. The diffusion coefficient and frictional ratio of the enzyme were 5.15 x 10(-7) cm2/sec and 1.27. The SDS-PAGE revealed homotetrameric nature of the enzyme with a subunit molecular mass of 35 +/- 1 kDa. Titration against DTNB and NBS revealed 19 sulfhydryl groups and 8 tryptophan groups, respectively, per mole of the enzyme. A sharp pH optimum at 5.0 was obtained. Cuscuta CMCase activity is unique amongst plant endoglucanases in being stimulated by Mg2+ and Mn2+ ions and by various thiols. Reaction product analysis, mode of enzyme action and substrate specificity test suggest the endo- nature of the purified CMCase. The enzyme showed K(m) value of 26 +/- 1 mg/ml for carboxymethylcellulose (sodium salt).

Glutatión S-transferasas en no-vertebrados y en mamíferos: su importancia en la detoxificación de insecticidas / The Glutathione S-transferases (GST) of non-vertebrate organisms and mammals

Las Glutatión S-transferasas (GST) de organismos no-vertebrados no han sido estudiadas con la misma intensidad que las de mamíferos. El interés en las GST en no-vertebrados radica en su importancia como protección bioquímica de los organismos expuestos a compuestos químicos ambientales. En efecto, se ha observado que niveles elevados de GST podrían estar asociados con la tolerancia a pesticidas. La intención de esta actualización es revisar el nivel de conocimiento actual sobre estas enzimas en no-vertebrados, comparándolas con las de mamíferos. Evaluar la contribución de estos estudios al conocimiento del rol de las glutinatión transferasas en general, e intentar discernir la dirección de las futuras investigaciones en este campo

Carbonic anhydrase: a multigene-multifunctional enzyme

Carbonic anhydrase is a zinc metalloenzyme that catalyzes the simple interconversion between carbon dioxide (CO2) and bicarbonate (HCO3). Seven genes encode the CA isozymes in vertebrates. They are single chain peptides termed CAI-VII. One CA isozyme is present in teleost fish. Three isozymes clearly appear together in birds. All seven types appear in mammals. Despite the great similarity among these isozymes, they present strong differences with respect to their kinetic properties. Many physiological and biochemical processes are related to the activity of CA isozymes.

Distribution of urease in the seeds of some Egyptian species of malvaceae and tiliaceae

In this study, urease content was screened in 20 species of three tribes of Tiliaceae and four tribes of Malvaceae. Data showed some correlations at the tribal level in both Tiliaceae and Malvaceae. In Tiliaceae, the highest urease level was recorded in Triumfetta rhomboidea [Tribe Triumfetteae], while the lowest values were recorded in Grewia species [Tribe Grewieae] and Corchorus species [Tribe Enteleae] gave intermediate urease levels. In Malvaceae, Pavonia triloba [Tribe Ureneae] contained the highest urease level [243 units] among the wild species of the family, while Abutilon pannosum [Tribe Abutileae] gave the lowest [1.07 units] activity. In this family, the highest specific activity recorded in cultivated specific were 8.7 and 7.1 units mg-1 protein for Gossypium barbadense cv. Giza 51 [Tribe Hibisceae] and Hibiscus sabdariffa [Tribe Hibisceae], respectively, while it was 5.3 units mg-1 protein in the wild species [Pavonia triloba]. Difference in urease level in the seeds of the studied plants proved its value for a better understanding of the affinities among the taxa of Tiliaceae and Malvaceae and could be applied for chemical taxonomy of the flowering plants

Pectolytic and cellulolytic activity of enzymes secreted by erwinia dieffenbachia, causing stem soft rot on dieffenbachia plants

Fourteen isolates of Erwinia dieffenbachia were isolated from dieffenbachia stem rot, four of them were rated as highly, moderately and weakly virulent and were active in secreting pectin methylesterase, polygalacturonase and cellulase enzymes in vivo and in vitro. They varied in their enzyme production. The highly virulent isolate produced in the culture filtrates 3-5 times higher PME, PG and cellulase activities than the low virulent one. Dieffenbachia stems inoculated with any of the tested isolates showed higher activity of such enzymes than healthy stems. A correlation between the PME, PG and cellulase activities and the pathogenicity of the tested pathogen isolates was detected

A biochemical comparison between latex from Carica Candamarcensis and C. papaya

1. A group of plant proteinases is present mainly in the unripe fruit of the papaya tree (Carica papaya), which is a member of the genus Carica. C. candamarcensis is another species that belongs to this group. Its latex contains several proteinases displaying high proteolytic activity. 2. We used several electrophoretic techniques to compare the protein composition of the latex from the two species. Acid electrophoresis followed by staining or Western blot revelated a total of 17 proteins in C. candamarcensis and 7 proteins in C. papaya. Some of the proteins observed in C. papaya have been previously reported in the literature. 3. Electrophoresis on denaturing gels, followed by staining or Western blot revealed the presence of 14 proteins in C. candamarcensis and 6 proteins in C. papaya. Non-equilibrium isoelectrofocusing of the latex from both species showed a larger array of proteins in C. candamarcensis. The analysis of esterase and proteolytic activities on gel fractions after electrophoresis revealed the presence of distinct areas presenting enzyme activity. Some proteins detected in C. candamarcensis have different mobilities when compared with from C. papaya. 4. These results support the view that latex from C. candamarcensis contains a wider diversity of proteins compared to C. papaya, and that some of the proteins not in C. papaya present esterase and proteolytic activity

Inactivation of glyceraldehyde-3-phosphate dehydrogenase with SH-reagents and its relationship to the protein quaternary structure.

Inactivation of mung bean glyceraldehyde-3-phosphate dehydrogenase (GPDH) with excess iodoacetate or N-ethylmaleimide exhibits pseudo-first order kinetics at pH 7.3 and 8.6 in the absence and presence of NAD+, suggesting that all the reactive SH groups (four per tetrameric GPDH molecule) have equivalent reactivity towards these reagents. This is similar to the D2-symmetry conformation proposed on the basis of thermal inactivation data [Malhotra and Srinivasan, Arch. Biochem. Biophys. 236, 775-781 (1985)]. With p-chloromercury benzoate (p-CMB), the inactivation of GPDH is very fast and its kinetics can be monitored at low reagent concentration only. Keeping a high molar p-CMB: enzyme ratio (= 47), the kinetics were found to be biphasic, with half of the activity being lost in a fast and the remaining in a slow phase, characteristic of C2-symmetry conformation and half site reactivity. The p-CMB inactivation could be largely reversed on the addition of excess cysteine. A comparison of these data with literature reports on this and other GPDHs reveals that all reagents having large non-polar moieties exhibit half site reactivity with this enzyme.

Lysine- and threonine-sensitive aspartokinase isoenzymes from spinach leaves share common antigenic determinants.

The lysine- and threonine-sensitive isoenzymes of aspartate kinase were purified to homogeneity from spinach leaves and polyclonal antibodies were raised in rabbits. The antibodies were characterized by various immunological tests like Ouchterlonys-double-diffusion, titrations of the inhibition of enzyme activity and ELISA. The antibodies against the lysine-sensitive isoenzyme could recognise as little as 50 ng of the pure antigen protein and that against the threonine-sensitive form could recognise 200 ng of the protein in the ELISA tests. The immunological tests have also shown that the lysine and threonine sensitive isoenzymes of aspartate kinase share some common antigenic determinants and differ in others.

Studies on cinnamic acid-4-hydroxylase from wounded potato tissue: isolation and characterization of cinnamic acid-4-hydroxylase activation factor.

Cinnamic acid-4-hydroxylase activation factor has been found to be located in the supernatant fraction of wounded potato tissue homogenate in phosphate buffer. The factor has been purified to homogeneity as judged by SDS polyacrylamide gel electrophoresis, by heat treatment on boiling water-bath for 7.5 min followed by dialysis with cut off limit of 8 kDa and final separation by gel filtration on Sephadex G-50 column. Gel filtration resolved this into three active fractions of molecular mass 12500, 10000 and 8500 Da conjugated to a fluorescent compound and subsequently identified as a folate derivative. The amino acid analysis of polypeptide chains of these fractions revealed that the polypeptides were rich in glutamic and aspartic acids. The fluorescent moiety of the complex released from polypeptide chain of molecular weight 10000 by mild acid hydrolysis was able to support the growth of Lactobacillus casei ATCC 7469 which requires folic acid for its growth. On storage, this compound degraded into a number of fluorescent products identified as p-amino benzoic acid, p-amino benzoyl glutamic acid, pteroic acid and 6-methyl pterin indicating that the activation factor is a folic acid derivative conjugated with the polypeptide chain.

The phenyl propanoid pathway enzymes in Solanum tuberosum exist as a multienzyme complex.

The elution profile of the core sequence enzymes of the phenyl propanoid pathway, namely phenyl alanine ammonia lyase, t-cinnamic acid 4-hydroxylase and p-coumaryl CoA ligase, on AcA 34 column suggested the existence of a high molecular form (P1) and a low molecular form (P2) for all the three enzymes. All the P1 forms eluted together in same fractions, while the P2 forms eluted out according to their respective molecular mass. Rechromatography of P1 form under identical conditions showed a similar elution profile (Q1 and Q2 forms). Further, the Q1 form did not show any significant increase in specific activity when compared to the P1 form. These results suggested the possibility of these enzymes existing as a protein cluster. Further confirmation was obtained on repeated column chromatography of the Q1 form in presence of 0.1 M KCl which did not result in complete dissociation of the complex to its individual enzyme components. The identification of the subunit polypeptide of the individual enzyme components in the multi enzyme complex and the in vitro demonstration of the phenyl propanoid core pathway reaction sequence using phenylalanine alone as a substrate supplementing the required cofactors for appropriate reactions substantiated that at least the core enzymes of the phenyl propanoid sequence existed as a multi enzyme complex.