Purification and characterization of a novel β-1,3-1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei with high specific activity and its gene sequence

Production, purification, and characterization of a novel β-1,3-1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei CAU432 were investigated. High-level extracellular β-1,3-1,4-glucanase production of 6230 U/mL was obtained when oat flour (3%, w/v) was used as a carbon source at 50 °C. The crude enzyme was purified to homogeneity with a specific activity of 28818 U/mg. The molecular weight of purified enzyme was estimated to be 35.4 kDa and 33.7 kDa by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.5 and 60 °C, respectively. The K(m) values of purified β-1,3-1,4-glucanase for barley β-glucan and lichenan were 2.0 mM and 1.4 mM, respectively. Furthermore, the gene (RmLic16A) encoding the β-1,3-1,4-glucanase was cloned and its deduced amino acid sequence showed the highest identity (50%) to characterized β-1,3-1,4-glucanase from Paecilomyces thermophila. The high-level production and biochemical properties of the enzyme enable its potential industrial applications.     

Origin and cytology of a novel cytotype of Allium tuberosum Rottl. ex Spreng. (2n = 48)

Naturally occurring/spontaneously produced polyploids with six/more genomes are rarely found in Alliums. A hexaploid form of Allium tuberosum with 2n = 48 chromosomes has been isolated for the first time amongst the open-pollinated seedlings of a hypotetraploid plant (2n = 4x = 31); latter being the seed-derived product of a normal tetraploid stock (2n = 4x = 32) growing in Jammu University Botanical Garden. Except for the guard cells and pollen grains that are of increased size, this form compared to its progenitor is dwarf, has smaller leaves and bears inflorescences with few flowers. This plant is also different from its progenitor in having nearly one-fourth (27.8 %) of its pollen mother cells (PMCs) with varying chromosome number viz. 27–64, with the remaining cells having somatic or double the somatic number of chromosomes. To assess the nature of hexaploid form, its chromosomes were studied for morphological details, putative grouping and pairing properties during reduction division. Morphological similarity in the chromosomes of the present cytotype and its progenitor, arrangement of 48 chromosomes in eight groups of six chromosomes each and presence of 21.88 % euploid cells with eight hexavalents pointed towards the autopolyploid nature of the present strain. Regarding the origin of this strain, observation made on the meiosis in the two sex mother cells of the progenitor provides some clues. In the later plant, presence of most of embryo-sac mother cells with 62 chromosomes that showed 31:31 segregations and existence of majority of the PMCs with 31 chromosomes exhibiting erratic segregations indicate that the hexaploid strain has probably originated as a result of the fusion of reduced male (n = 17) and unreduced female gamete (2n = 31).     

Molecular and cytogenetic characterization of a collection of bahiagrass (Paspalum notatum Flügge) native to Uruguay

Paspalum notatum is a subtropical grass present throughout America, and one of the main constituents of the natural grasslands in Uruguay. An apomictic autotetraploid (2n?=?4x?=?40) is the most frequent cytotype. The occurrence of sexual diploids (2n?=?2x?=?20) has also been reported as well as the occasional presence of apomictic triploids and pentaploids in Argentina. In this study, ISSR (inter simple sequence repeats) molecular markers were used to analyze the genetic variability of 210 P. notatum individuals from a collection from Uruguay. Cytometric analyses and chromosome counts were used to assess the ploidy level of the individuals. All plants from Uruguay analyzed were tetraploid. Intra- and inter-population variability was found both in genomic DNA content and at the genotypic level. Several multilocus genotypes were shared among individuals within populations and among populations over moderate geographical ranges, at the same time, very dissimilar genotypes were found within the same population. Part of the genetic variance among populations can be explained by a broader scale geographic structure which is partly coincident with the traditionally recognized grassland management regions. In spite of the apparently high degree of genetic admixture within populations, groups of related genotypes seem to follow a broader geographical structure in the area under study. These results suggest that an efficient collection strategy for this apomictically reproducing species should include carefully planned intra- and inter-population sampling. A broader scale regional sampling strategy should also be considered although further studies will be required to define genetic structure at this level.     

Studies of O,O-dimethyl α-(2,4-dichlorophenoxyacetoxy)ethylphosphonate (HW02) as a new herbicide. 1. Synthesis and herbicidal activity of HW02 and analogues as novel inhibitors of pyruvate dehydrogenase complex

On the basis of the previous work for optimization of O,O-diethyl α-(substituted phenoxyacetoxy)alkylphosphonates, further extensive synthetic modifications were made to the substituents in alkylphosphonate and phenoxy moieties of the title compounds. New O,O-dimethyl α-(substituted phenoxyacetoxy)alkylphosphonates were synthesized as potential inhibitors of pyruvate dehydorogenase complex (PDHc). Their herbicidal activity and efficacy in vitro against PDHc were examined. Some of these compounds exhibited significant herbicidal activity and were demonstrated to be effective inhibitors of PDHc from three different plants. The structure-activity relationships of these compounds including previously reported analogous compounds were studied by examining their herbicidal activities. Both inhibitory potency against PDHc and herbicidal activity of title compounds could be increased greatly by optimizing substituent groups of the title compounds. O,O-Dimethyl α-(2,4-dichlorophenoxyacetoxy)ethylphosphonate (I-5), which acted as a competitive inhibitor of PDHc with much higher inhibitory potency against PDHc from Pisum sativum and Phaseolus radiatus than from Oryza sativa , was found to be the most effective compound against broadleaf weeds and showed potential utility as herbicide.     

Isotopic Model (NISOTOP) used to Investigate 15N-Urea Transformations in the Presence of Phenanthrene,Chrysene and Benzo(a)pyrene in a Soil–Plant System

An isotopic model (NISOTOP) has been developed to investigate the effect of the addition to soil of xenobiotics on urea hydrolysis, N mineralization and immobilization, nitrification and plant uptake of nitrogen in a soil-plant system, after addition of ¹⁵N enriched compounds. Therationale of the model follows from the errors in %¹⁵N abundance (^15N _D) and N concentration (C_N) determinations which cause high variability coefficients in the calculation of the amount of nitrogen present in the different compounds derived from the added ¹⁵N enriched urea. The extent of these errors, besides depending on C_N and ^15N _D errors, will also depend on natural ¹⁵N and ¹⁵N of the added compound, and therefore on the experimental conditions. The model is described by 18 first-order differential equations and is numerically solved by Euler's method with a time increment of 0.01 day. As an illustration, the model is applied to the effect of phenanthrene, chrysene and benzo(a)pyrene to a soil-plant system, following the addition of ¹⁵N-urea. These compounds have been chosen as examples of molecules having 3, 4 and 5 fused aromatic rings and are hereafter collectively referred to as PAHs. PAHs at the rate of 2 mg kg^-1 soiland ¹⁵N-urea at the rate of 166.7 mg N kg^-1 soil were added to wheat pots. At harvesting (after 14 days from plantation) the dry matter yield, the total N content and the N concentration of the wheat seedlings were not statistically affected by addition of the PAHs (P = 0.05). The efficiency of N uptake, that is the percentage of fertilizer taken up by the plants at harvesting in the absence of PAHs was 47.3%, while it was 11.7, 15.2 and 14.8% in the presence of phenanthrene, chrysene and benzo(a)pyrene,respectively. The computation of the first-order rate constants of the N transformations showed that N mineralization, nitrification and N-uptake were affected by the addition of phenanthrene, chrysene and benzo(a)pyrene, whilst benzo(a)pyrene inhibited urea hydrolysis more than phenanthrene and chrysene.     

Photochemical oxidation of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) in soils — a tool to assess their degradability?

To determine the degradability of PAHs and PCBs for soil remediation or ecotoxicological risk assessment, a simple method is needed. We tested the suitability of photocatalytic oxidation for this purpose. We determined the concentrations of 20 PAHs and 12 PCBs in four mineral topsoil horizons, six organic horizons, and four particle‐size fractions of each of three soils before and after UV irradiation with TiO₂ as a catalyst in suspension. Preliminary experiments showed that in dry soil no photooxidation occurred, but after 48 h of irradiation in suspension the PCB concentrations decreased by up to 40—50 %, while the PAH concentrations did not change significantly. In contrast to this, 95—100 % of PAH and PCB standards spiked on quartz sand were degraded within 8 h, indicating that sorption to organic matter limited degradation of PAHs and PCBs in soil suspensions. There was no difference in the degradation among different individual PAHs and PCBs, respectively, indicating that the degradation did not occur in dissolved state, but in association with soil organic matter. In all samples except one, the degradation of PCBs (10—80 % loss of initial concentrations) was higher than those of the PAHs (0—40 % loss). This suggests that the accessibility of PCBs for OH· radicals generated during irradiation was higher, or the oxidation of PAHs was limited by the properties of the sorbing organic matter. Thus, the tested method was not suitable to predict biodegradability, because it did not reflect the differences in degradability of individual compounds.     

Response surface methodology to understand the anaerobic biodegradation of organochlorine pesticides (OCPs) in contaminated soil—significance of nitrate concentration and bioaccessibility

Purpose

Problems associated with Organochlorine pesticide (OCP)-contaminated soils have received wide attention. To understand the anaerobic biodegradation process constraints, innovative mathematical analysis methods are effective.

Materials and methods

Response surface methodology (RSM) and Tenax TA extraction method combined with the first-three-compartment model were employed to systematically investigate the role of nitrate concentration and bioaccessibility enhancer (methyl-β-cyclodextrin, MCD) in the anaerobic biodegradation of OCPs in contaminated soil.

Results and discussion

The sole addition of either KNO₃ or MCD could facilitate the anaerobic biodegradation of OCPs. The highest biodegradation for total OCPs, hexachlorocyclohexanes, endosulfans, and chlordanes were 71.6, 82.1, 68.3, and 55.6 %, respectively, when 20 mM KNO₃ and 3.0 % (w/w) MCD were applied simultaneously. As predicted by RSM, the theoretical maximum biodegradation for total OCPs ranged from 60 to 80 % when 20 to 25 mM KNO₃ and >2.5 % (w/w) MCD were applied simultaneously. Tenax TA extraction method demonstrated the enhancement of OCP bioaccessibility caused by MCD addition. Changes in the soil microbial activities also suggested the positive effects of adding suitable amounts of KNO₃ as a cosubstrate to facilitate the anaerobic biodegradation of OCPs.

Conclusions

The amount of KNO₃ and MCD are crucial in influencing OCP biodegradation. RSM was demonstrated to be a powerful tool to estimate and predicting the optimal OCP biodegradation under KNO₃ and MCD application simultaneously.     

Lestes dryas kirby—a declining species of dragonfly (odonata) in need of conservation: Notes on its status and habitat in England and Ireland

A study of Lestes dryas was made in 1978. None was found in ten localities in south east England, which had held populations in the period 1940–1947. Major changes had occurred in eight of the localities, and these could readily account for the disappearance of the insect from them. A search for L. dryas was made in apparently suitable habitat in the Cambridgeshire Fens but none was found. L. dryas was found on three out of eighty waterbodies visited in the west and south of Ireland. Neutral or slightly acid waters with extensive emergent vegetation, notably Equisetum fluviatile, Scirpus lacustris and Typha sp., and supporting the dragonflies Sympertrum sanguineum and/or Coenagrion pulchellum, appear to indicate a suitable habitat for the species. The decline of L. dryas appears to have been caused by a combination of factors: loss of habitat, periods of drought and small population numbers.     

A new method to measure allyl isothiocyanate (AITC) concentrations in mustard—Comparison of AITC and commercial mustard solutions as earthworm extractants

Earthworms are target organisms both for scientists studying the biological component of soils and for farmers concerned with monitoring the quality of their soils. Different expellants are used to extract earthworms from the soil but differences in chemical properties and efficiency between commercial mustard and allyl isothiocyanate (AITC) solutions remain unknown. The objectives of this study were to compare (i) the concentration of irritating product (allyl isothiocyanate AITC) in two expellant solutions (diluted mustard or AITC solution) and (ii) their efficiency in extracting earthworms from the soil.AITC concentration was analyzed according to a new method, based on AITC solvent extraction and HPLC quantification, in one commercial mustard brand to assess its variability within and between batches of jars. According to mustard spiking with AITC standard solution, extraction recovery was estimated as 98 ± 2%. Earthworm field data were collected in spring 2012 in 22 cultivated fields located in east Île-de-France, comparing pure AITC to commercial mustard solutions. Species diversity, abundance and biomass of earthworms per plot were measured.We showed that AITC concentration in commercial mustard varied according to the use by date but not according to the batch. We thus recommend using the freshest mustard available from the same batch. Moreover, AITC solution was found to be about four times more concentrated in AITC than the commercial mustard solution. Despite this result, no significant differences were found in the efficiency of commercial mustard or AITC solutions to bring earthworms to the soil surface in terms of abundance, biomass or diversity. We thus discuss the advantage and drawbacks of using both expellants in the field.