Purification, Characterization, and Specificity Determination of a New Serine Protease Secreted by Penicillium waksmanii

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 °C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl₂, KCl, and BaCl, and partially inhibited by CuCl₂, CoCl₂ and totally inhibited by AlCl₃ and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k _cat/K _m of 10,666 mM^?1?s^?1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k _cat/K _m of 7,500 mM^?1?s^?1. Basic and acidic side chain-containing amino acids performed best at subsite S₁. Subsites S₂, S₃, S^′ ₂, and S^′ ₁, S^′ ₃ showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k _cat/K _m were observed for the subsites S₂, S₃, and S^′ _2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level.     

Surface modification of cotton nanocrystals with a silane agent

The research herewith aims at obtaining cellulose nanocrystals with a reduced hydrophilic surface character using a silane with isocyanate groups (isocyanatepropyltriethoxysilane), which are very reactive to hydroxyl groups and thus, are readily able to react with the low quantity of free hydroxyl groups present in the cellulose nanocrystal surfaces, therefore, promoting surface modification. Cellulose nanocrystals were obtained by hydrochloric acid hydrolysis of cotton fiber and were characterized by X-ray diffraction, Fourier transform infrared spectroscopy (FTIR) and solid state ²⁹Si nuclear magnetic resonance (NMR) and their morphologies were investigated by scanning and transmission electron microscopy techniques. The nanocrystals presented a needle-like geometry with a 10 nm approximate diameter and a 166 nm average length. FTIR, ²⁹Si NMR and silicon mapping images showed that nanocrystal surface chemical modification was successfully achieved. Also, the results confirm that the chemical modification occurred mainly at the nanocrystal surface, keeping the morphological integrity of the nanocrystals. The applied methodology for surface modification of the cellulose nanocrystals provided nanofillers with more appropriate surface characteristics that allow the dispersion in polymeric matrices and the adhesion at filler-matrix interface to be obtained. This may result in a better performance of these nanocrystals as reinforcing agents of hydrophobic polymer matrices.     

Influence of the layered silicate type on the structure, morphology and properties of cellulose acetate nanocomposites

This paper deals with the effect of different montmorillonite source clays, including pristine and organophilic montmorillonites, on the structure, morphology and properties of cellulose acetate (CA)/clay nanocomposites. In this study, the nanocomposites were prepared by melt extrusion in the presence of the environmentally friendly triethyl citrate plasticizer. The structure and morphology of the materials were analysed by X-ray diffraction and scattering (SAXS), X-ray microtomography and energy filtered transmission electron microscopy (EFTEM). SAXS and EFTEM results indicated that the nanocomposite morphologies were made up of tactoids together with exfoliated clay platelets in different proportions depending on the clay type. It can be concluded that well distributed clay tactoids and platelets can be achieved in CA nanocomposites prepared by melt extrusion and consequently property improvements can be found by using pristine or organophilic clays. In this case, the addition of a plasticizer, able to intercalate in the clay gallery, seems to be sufficient to promote the clay delamination mechanism under shearing inside the cellulose acetate matrix.     

New insight into the mechanism of action of wasp mastoparan peptides: lytic activity and clustering observed with giant vesicles

Antimicrobial peptides of the mastoparans family exert their bactericidal activity by binding to lipid membranes, inducing pores or defects and leaking the internal contents of vesicles and cells. However, this does not seem to be the only mechanism at play, and they might be important in the search for improved peptides with lower undesirable side effects. This work deals with three mastoparans peptides, Polybia-MP-1(MP-1), N2-Polybia-MP-1 (N-MP-1), and Mastoparan X (MPX), which exhibit high sequence homology. They all have three lysine residues and amidated C termini, but because of the presence of two, one, and no aspartic acid residues, respectively, they have +2, +3, and +4 net charges at physiological pH. Here we focus on the effects of these mastoparans peptides on anionic model membranes made of palmitoleyoilphosphatidylcholine (POPC) and palmitoleyoilphosphatidylglycerol (POPG) at 1:1 and 3:1 molar ratios in the presence and in the absence of saline buffer. Zeta potential experiments were carried out to measure the extent of the peptides' binding and accumulation at the vesicle surface, and CD spectra were acquired to quantify the helical structuring of the peptides upon binding. Giant unilamellar vesicles were observed under phase contrast and fluorescence microscopy. We found that the three peptides induced the leakage of GUVs at a gradual rate with many characteristics of the graded mode. This process was faster in the absence of saline buffer. Additionally, we observed that the peptides induced the formation of dense regions of phospholipids and peptides on the GUV surface. This phenomenon was easily observable for the more charged peptides (MPX > N-MP-1 > MP-1) and in the absence of added salt. Our data suggest that these mastoparans accumulate on the bilayer surface and induce a transient interruption to its barrier properties, leaking the vesicle contents. Next, the bilayer recovers its continuity, but this happens in an inhomogeneous way, forming a kind of ply with peptides sandwiched between two juxtaposed membranes. Eventually, a peptide-lipid aggregate forming a lump is formed at high peptide-to-lipid ratios.     

Probing the steric limits of rhodium catalyzed hydrophosphinylation. P-H addition vs. dimerization/oligomerization/polymerization

The reactivity of secondary phosphine oxides containing bulky organic fragments in hydrophosphinylation reactions has been investigated using several rhodium based catalysts. Upon heating in a focused microwave reactor, HP(O)(2-C₆H₄Me)₂ adds to prototypical terminal alkynes affording a complex mixture containing 1,2 and 1,1-addition products. Addition of a second ortho-substituent (HP(O)Mes₂) completely suppresses the hydrophosphinylation reaction for alkyl and aryl substituted alkynes. Variations in the temperature, catalyst loading, solvent, and microwave power were unable to induce an addition reaction in the case of HP(O)Mes₂. While this secondary phosphine oxide did not participate in the hydrophosphinylation reaction, it promoted the polymerization of phenylacetylene. HP(O)R₂ substrates are not commonly thought of as innocent ligands for rhodium complexes in reactions involving alkynes due to facile hydrophosphinylation. While this is certainly true for diphenylphosphine oxide, the chemistry presented herein suggests that HP(O)Mes₂ and related bulky secondary phosphine oxides have great potential as valuable ligands for rhodium catalyzed transformations involving alkynes due to their lack of reactivity towards the addition reaction.     

Core-softened potentials,multiple liquid–liquid critical points,and density anomaly regions: An exact solution

The pressure versus temperature phase diagram of a system of particles interacting through a multiscale shoulder-like potential is exactly computed in one dimension. The N-shoulder potential exhibits N density anomaly regions in the phase diagram if the length scales can be connected by a convex curve. The result is analyzed in terms of the convexity of the Gibbs free energy.     

Mercapto-modified copolymers in polymer blends, 1. Functionalization of EPDM with mercapto groups and its use in NBR/EPDM blends

Ethylene-propylene-diene rubber (EPDM) was reacted with thioacetic acid in the presence of 2,2′-azoisobutyronitrile (AIBN), giving rise to thioacetate-modified EPDM (EPDMTA). The extent of the incorporation was studied as a function of both thioacetic acid and AIBN concentration and reaction time. The products were analyzed by FTIR spectroscopy, sulfur content, and ¹H NMR spectroscopy. The hydrolysis of EPDMTA in a NaOH methanolic solution resulted in mercapto-modified EPDM (EPDMSH). The addition of 5 wt.-% of this functionalized copolymer to a sulfur-vulcanized nitrile rubber (NBR)/EPDM (70 : 30 wt.-%) blend accelerated the vulcanization process as indicated by oscillating disk rheometer analysis. In addition, an improvement of mechanical and ageing properties was also observed.