Sequence and domain arrangements influence mechanical properties of elastin‐like polymeric elastomers

Elastin is the polymeric, extracellular matrix protein that provides properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Elastin assembles by crosslinking through lysine residues of its monomeric precursor, tropoelastin. Tropoelastin, as well as polypeptides based on tropoelastin sequences, undergo a process of self‐assembly that aligns lysine residues for crosslinking. As a result, both the full‐length monomer as well as elastin‐like polypeptides (ELPs) can be made into biomaterials whose properties resemble those of native polymeric elastin. Using both full‐length human tropoelastin (hTE) as well as ELPs, we and others have previously reported on the influence of sequence and domain arrangements on self‐assembly properties. Here we investigate the role of domain sequence and organization on the tensile mechanical properties of crosslinked biomaterials fabricated from ELP variants. In general, substitutions in ELPs involving similiar domain types (hydrophobic or crosslinking) had little effect on mechanical properties. However, modifications altering either the structure or the characteristic sequence style of these domains had significant effects on such properties. In addition, using a series of deletion and replacement constructs for full‐length hTE, we provide new insights into the role of conserved domains of tropoelastin in determining mechanical properties. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 392–407, 2013.     

Overwintering strategy of endoparasitoids in Chilo suppressalis: a perspective from the cold hardiness of a host

Although parasitoids ultimately kill their host, koinobiont parasitoids must protect not only themselves but also their hosts against extreme environments. In this study, the parasitism rate of Chilo suppressalis Walker (Lepidoptera: Pyralidae) was investigated, and the average body weights, supercooling points, and concentrations of glycerol (acting as a cryoprotectant) in the hemolymph were compared between parasitized and non‐parasitized larvae. Five species of koinobiont endoparasitoids parasitized the overwintering C. suppressalis larvae and the total parasitism rate was 47.6% (n = 1 537). Average body weight of parasitized larvae was significantly lower than that of non‐parasitized larvae, and the parasitism rate of the lighter group (20–30 mg) was highest. The supercooling point of parasitized C. suppressalis larvae (?15.7 ± 0.3 °C) was significantly lower than that of the non‐parasitized larvae (?14.3 ± 0.2 °C). In addition, supercooling points were not correlated with body weights between parasitized and non‐parasitized larvae, indicating that cold hardiness of parasitized larvae was enhanced by endoparasitoids. Furthermore, the concentration of glycerol in the hemolymph was significantly higher in parasitized larvae (205.0 ± 7.1 μmol ml^?1) than in non‐parasitized larvae (169.8 ± 14.4 μmol ml^?1), which suggests that the mechanism that decreases the supercooling point of parasitized larvae was associated with glycerol. All these results indicated that the cold hardiness of parasitized C. suppressalis larvae was enhanced by their endoparasitoids, which benefitted overwintering endoparasitoids.     

A plant‐traits approach to assessing the success of alien weed species in irrigated Mediterranean orchards

In addition to their impact on natural habitats, invasive alien plants can have a significant negative effect on agricultural systems and cause economic losses. Flood‐irrigated orchards in the Mediterranean Basin are vulnerable to the invasion of alien weeds, primarily because of the traditional management practices used in the orchards, which are characterized by high soil moisture during the dry summer period, nutrient availability and high levels of disturbance. This study sought to determine whether their biological traits can explain the success of alien weed species. To answer this question, 408 floristic relevés were conducted in 136 flood‐irrigated orchards on the Plains of Lleida (Catalonia, NE of Spain). Richness and cover of native and alien weeds were compared. Furthermore, a set of biological traits were compared between successful and non‐successful weeds for the whole data and separately between native and alien weeds using logistic regression and classification trees. In flood‐irrigated orchards, alien species covered most of their area, even though the richness of alien species was lower than that of the native species. The most important species were C4 species with seeds dispersed by water, and on the other hand, rosulate and caespitose‐reptant hemicryptophytes with long flowering period. Most of these traits fitted with those of the invasive alien weeds, which were mostly C4 species with seeds dispersed by water. Perennial life form characterized successful native weeds. In this study, we discuss how the traditional management of flood irrigation in fruit‐tree orchards favours invasive alien weeds that have specific traits, acting as a reservoir for the spread of alien weeds into other crops and surrounding riparian habitats. We also propose changing management practices in order to avoid the selection of alien weeds and to promote native species.     

A high salt tolerant neutral protease from Aspergillus Oryzae: Purification, characterization and kinetic properties

A neutral high salt tolerant protease from Aspergillus oryzae CICIM F0899 which could be used for soy sauce production and other relevant applications under high-salt conditions was purified to homogeneity through ammonium sulfate precipitation, ion-exchange chromatography and gel filtration chromatography with overall recovery of 2%. Its molecular weight was estimated to be 50 kDa by SDS-PAGE. The optimum pH and temperature for activity of the extracellular protease of A. oryzae CICIM F0899 were shown to be between 7.0–9.0, and 50°C, respectively. The protease behaved high salt tolerance in 18% NaCl and retained 72% of initial activity after 14 days, indicating the high stability. The enzyme activity was inhibited by metal ions such as Al³⁺ and Ag⁺, and slightly activated by Mn²⁺ and Cu²⁺. A kinetic model incorporating the Debye-Hückel limiting law was proposed for A. oryzae CICIM F0899 protease hydrolysis of casein at ionic strength NaCl from 0.10 to 3.18 M. It was found that, with the higher ionic strength, the Michaelis constant K _m of the protease monotonically increased while the turnover number k _cat decreased in accordance with first order kinetic model. The high-salt tolerant protease has been demonstrated to be promising for the soy sauce production process.     

An amylase from fresh fruiting bodies of the monkey head mushroom Hericium Erinaceum

An amylase with a molecular mass of 55 kDa and an N-terminal sequence exhibiting similarity to enzyme from Bacteroides thetaitaomicron was isolated from fruiting bodies of the monkey head mushroom Hericium erinaceum. The purification scheme included extraction with distilled water, ion exchange chromatography on DEAE-cellulose and SP-sepharose, and gel filtration by FPLC on Superdex 75. The amylase of H. erinaceum was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and eluted with 0.2 M NaCl in the same buffer. The enzyme was subsequently adsorbed on SP-Sepharose in 10 mM ammonium acetate buffer (pH 4.5) and eluted with 0.3 M NaCl in the same buffer. This fraction was subsequently subjected to gel filtration on Superdex 75. The first peak eluted had a molecular mass of 55 kDa in SDS-PAGE. The amylase of H. erinaceum exhibited a pH optimum of 4.6 and a temperature optimum of 40°C. The enzyme activity was enhanced by Mn²⁺ and Fe³⁺ ions, but inhibited by Hg²⁺ ions.     

Paenibacillus brassicae sp. nov., isolated from cabbage rhizosphere in Beijing, China

A novel Gram-positive, rod-shaped, motile, spore-forming, nitrogen-fixing bacterium, designated strain 112^T, was isolated from cabbage rhizosphere in Beijing, China. The strain was found to grow at 10–40 °C and pH 4–11, with an optimum of 30 °C and pH 7.0, respectively. Phylogenetic analysis based on a fragment of the full-length 16S rRNA gene sequence revealed that strain 112^T is a member of the genus Paenibacillus. High levels of 16S rRNA gene similarities were found between strain 112^T, Paenibacillus sabinae DSM 17841^T (97.82 %) and Paenibacillus forsythiae DSM 17842^T (97.22 %). However, the DNA–DNA hybridization values between strain 112^T and the type strains of these two species were 10.36 and 6.28 %, respectively. The predominant menaquinone was found to be menaquinone 7 (MK-7). The major fatty acids were determined to be anteiso-C_15:0 and C_16:0. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unknown aminophospholipids. The cell wall peptidoglycan was found to contain meso-diaminopimelic acid. The DNA G+C content was determined to be 55.4 mol%. On the basis of its phenotypic characteristics, 16S rRNA gene sequence analysis and the value of DNA–DNA hybridization, strain 112^T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus brassicae sp. nov. is proposed. The type strain is 112^T (= ACCC 01125^T = DSM 24983^T).     

Directed evolution and characterization of atrazine chlorohydrolase variants with enhanced activity

Atrazine chlorohydrolase (AtzA, EC 3.8.1.8) has attracted widespread interests as it catalyzes conversion of toxic atrazine to nontoxic hydroxyatrazine and can be used in the biodegradation of atrazine. To facilitate this application, a Haematococcus pluvialis-based method was applied to screen AtzA variants from a random mutagenesis library. Eight variants with enhanced enzyme activity were obtained. They showed 2.7- to 5.0-fold increase in specific activity compared with the wild type. Sequencing revealed that the two most active variants contained single substitution at Val12 and Leu395, respectively, while several improved variants contained substitutions at the four sites of Met315, His399, Asn429, and Val466 simultaneously, indicating that these residues contribute to the enzyme activity of AtzA. Kinetic analysis showed that five variants decreased the K _m value 0.6- to 0.9-fold, whereas all the variants increased the catalytic efficiency (k _cat/K _m value) 2.5- to 4.1-fold compared to the wild type. The modeled three-dimensional structure showed that AtzA is comprised of a typical (β/α)₈ domain of the amidohydrolase superfamily and a dual β-sheet domain. An iron ion and five ligand-binding residues are located in the β-barrel core of the (β/α)₈ domain. Some substituted residues are involved in hydrogen bond formation in the (β/α)₈-neighboring β-sheet.