Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Der f 21 (rDer f 21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDer f 21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19 M Tris-HCl pH 8.0, 32% PEG 400 at 293 K. X-ray diffraction data were collected to 1.49 Å resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25 Å, β = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blo t 5 (45%) and Blo t 21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDer f 21 is likely to be a dimeric protein.
A new subfamily of glycosyl hydrolase family GH13 was recently proposed for α-amylases from Anoxybacillus species (ASKA and ADTA), Geobacillus thermoleovorans (GTA, Pizzo, and GtamyII), Bacillus aquimaris (BaqA), and 95 other putative protein homologues. To understand this new GH13 subfamily, we report crystal structures of truncated ASKA (TASKA). ASKA is a thermostable enzyme capable of producing high levels of maltose. Unlike GTA, biochemical analysis showed that Ca(2+) ion supplementation enhances the catalytic activities of ASKA and TASKA. The crystal structures reveal the presence of four Ca(2+) ion binding sites, with three of these binding sites are highly conserved among Anoxybacillus α-amylases. This work provides structural insights into this new GH13 subfamily both in the apo form and in complex with maltose. Furthermore, structural comparison of TASKA and GTA provides an overview of the conformational changes accompanying maltose binding at each subsite.
C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.
TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced in Escherichia coli and purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%(v/v) ethylene glycol and 5%(v/v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space group P3121, with unit-cell parameters a = b = 126.62, c = 95.63 Å.
Melioidosis is an infectious disease caused by the pathogenic bacterium Burkholderia pseudomallei. Whole-genome sequencing revealed that the B. pseudomallei genome includes 5855 coding DNA sequences (CDSs), of which ∼25% encode hypothetical proteins. A pathogen-associated hypothetical protein, BPSL1038, was overexpressed in Escherichia coli, purified and crystallized using vapour-diffusion methods. A BPSL1038 protein crystal that grew using sodium formate as precipitant diffracted to 1.55 Å resolution. It belonged to space group C2221, with unit-cell parameters a = 85.36, b = 115.63, c = 46.73 Å. The calculated Matthews coefficient (VM) suggests that there are two molecules per asymmetric unit, with a solvent content of 48.8%.
The advancements in material science and technology have made polyurethane (PU) one of the most important renewable polymers. Enhancing the physio-chemical and mechanical properties of PU has become the theme of this and many other studies. One of these enhancements was carried out by adding starch to PU to form new renewable materials called polyurethane-starch composites (PUS). In this study, PUS composites are prepared by adding starch at 0.5, 1.0, 1.5, and 2.0 wt.% to a PU matrix. The mechanical, thermal, and morphological properties of PU and PUS composites were investigated. Scanning electron microscope (SEM) images of PU and PUS fractured surfaces show cracks and agglomeration in PUS at 1.5 wt.% starch. The thermo-mechanical properties of the PUS composites were improved as starch content increased to 1.5 wt.% and declined by more starch loading. Despite this reduction, the mechanical properties were still better than that of neat PU. The mechanical strength increased as starch content increased to 1.5 wt.%. The tensile, flexural, and impact strengths of the PUS composites were found to be 9.62 MPa, 126.04 MPa, and 12.87 × 10(-3) J/mm², respectively, at 1.5 wt.% starch. Thermal studies showed that the thermal stability and crystallization temperature of the PUS composites increased compared to that of PU. The loss modulus curves showed that neat PU crystallizes at 124 °C and at 127 °C for PUS-0.5 wt.% and rises with increasing loading from 0.5 to 2 wt.%.
Changes in molecular structure configuration during strain induced crystallisation of an amorphous Poly(Lactic Acid) (PLA 4032D) polymer was monitored in-situ by simultaneously recording the wide angle x-ray scattering (WAXS) and small angle x-ray scattering (SAXS) patterns together with polymer deformation images and force data. The amorphous chain orientation from the beginning of deformation until the onset of crystallisation was studied from the WAXS patterns. The true mechanical behaviour described by the true stress-true strain curve related to an amorphous chain orientation exhibited a linear behaviour. Approaching critical amorphous orientation, the true stress-true strain curve deviated from linear into non-linear behaviour. After the onset of crystallization, when the deformed polymer became a semicrystalline state, the true mechanical behaviour exhibited true strain hardening which greatly affected by the formation of the morphology. The gradual true strain hardening was associated with the formation of micro-fibrillar structure containing thin crystallite morphology whilst sharp increased in true strain hardening was associated with the formation of stacked lamellar morphology in the form of macro-lattice structure. The study was accomplished by the application of high brilliance synchrotron radiation at beamline ID2 of ESRF, Grenoble in France and the usage of the high contrast resolution of WAXS and SAXS charge-couple device (CCD) camera as well as 40 milliseconds temporal resolution of data acquisition system.
In this work, a novel membrane crystallization system was used to crystallize micro-sized seeds of piroxicam monohydrate by reverse antisolvent addition. Membrane crystallization seeds were compared with seeds produced by conventional antisolvent addition and polymorphic transformation of a fine powdered sample of piroxicam form I in water. The membrane crystallization process allowed for a consistent production of pure monohydrate crystals with narrow size distribution and without significant agglomeration. The seeds were grown in 350 g of 20:80w/wacetone-water mixture. Different seeding loads were tested and temperature cycling was applied in order to avoid agglomeration of the growing crystals during the process. Focused beam reflectance measurement (FBRM); and particle vision and measurement (PVM) were used to monitor crystal growth; nucleation and agglomeration during the seeded experiments. Furthermore; Raman spectroscopy was used to monitor solute concentration and estimate the overall yield of the process. Membrane crystallization was proved to be the most convenient and consistent method to produce seeds of highly agglomerating compounds; which can be grown via cooling crystallization and temperature cycling.
Poly(lactic acid) (PLA)-based nanocomposites filled with graphene nanoplatelets (xGnP) that contains epoxidized palm oil (EPO) as plasticizer were prepared by melt blending method. PLA was first plasticized by EPO to improve its flexibility and thereby overcome its problem of brittleness. Then, xGnP was incoporated into plasticized PLA to enhance its mechanical properties. Plasticized and nanofilled PLA nanocomposites (PLA/EPO/xGnP) showed improvement in the elongation at break by 3322% and 61% compared to pristine PLA and PLA/EPO, respectively. The use of EPO and xGnP increases the mobility of the polymeric chains, thereby improving the flexibility and plastic deformation of PLA. The nanocomposites also resulted in an increase of up to 26.5% in the tensile strength compared with PLA/EPO blend. XRD pattern showed the presence of peak around 26.5° in PLA/EPO/xGnP nanocomposites which corresponds to characteristic peak of graphene nanoplatelets. Plasticized PLA reinforced with xGnP showed that increasing the xGnP content triggers a substantial increase in thermal stability. Crystallinity of the nanocomposites as well as cold crystallization and melting temperature did not show any significant changes upon addition of xGnP. However, there was a significant decrease of glass transition temperature up to 0.3wt% of xGnP incorporation. The TEM micrograph of PLA/EPO/xGnP shows that the xGnP was uniformly dispersed in the PLA matrix and no obvious aggregation was observed.
Star-shaped polymers have vast potential in bioapplication due to their architecture. In this study, the suitability of ring opening polymerization (ROP) technique to synthesis star-shaped poly(caprolactone) and the thermal properties of the synthesized star-shaped polymers were demonstrated. The 4 -arm star- shaped of poly(caprolactone) (4s PCL) with -OH terminal and average molecular weight (Mn) of 5000, 10000, and 15000 g/mol were synthesized via ROP of ԑ-caprolactone (ԑ-CL) using a symmetric pentaerythritol (PET) as the core. Different molecular weights were obtained by using different ratios of ԑ-CL and PET in the presence of catalyst, stannous octoate (Sn(Oct)2). The FTIR spectra showed the presence of bands of methylene group of polymer repeating chain which confirm ROP of the ԑ- caprolactone. The average molecular weight (Mn) determined from proton nuclear magnetic resonance (1H NMR) analysis showed that all 4s PCL have approximately the same molecular weight as the theoretical values. All polymers obtained had high yield with >85%. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) analysis showed that there were no significance different in the thermal properties of the synthesized polymers. A single step degradation for all 4s PCL was observed and the crystallization melting point of the polymers was within the range of melting point of PCL.
Sulfur has been highly sought by many industries everywhere around the world for various applications. This exceptionally useful element has been largely manufactured especially in powder form each year reflecting its increase in demand in line with technological advancement and uses for various product applications. The manufacturing processes include mining as well as chemical reactions in Claus Process. Rubber industries normally use abundance of sulfur in their latex compound to introduce vulcanization. The geneal concern of sulfur for rubber vulcanization is dispersibility in the rubber matrix due to improper optimization of its preparation process prior to latex compounding. Another crucial issue is crystallization of soluble sulfur from its insoluble origin, either during or post-rubber vulcanization that constitutes to formation of sulfur bloom that grows on the surface of the rubber articles. It is known that both issues are related to the process conditions and compounding recipe that could not be fully solved. Various studies have been conducted to minimize such occurences – from process optimization to sulfur chemistry itself – and of continuous improvement and innovation to solve various threats in sulfur applications. This paper reviews on detailed description on elemental sulfur, of respective industrial applications, and most importantly highlights on sulfur trends and issues normally encountered.
The arginine repressor (ArgR) of Escherichia coli binds to six L-arginine molecules that act as its co-repressor in order to bind to DNA. The binding of L-arginine molecules as well as its structural analogues is compared by means of computational docking. A grid-based energy evaluation method combined with a Monte Carlo simulated annealing process was used in the automated docking. For all ligands, the docking procedure proposed more than one binding site in the C-terminal domain of ArgR (ArgRc). Interaction patterns of ArgRc with L-arginine were also observed for L-canavanine and L-citrulline. L-lysine and L-homoarginine, on the other hand, were shown to bind poorly at the binding site. Figure A general overview of the sites found from docking the various ligands into ArgRc ( grey ribbons). Red coloured sticks: residues in binding site H that was selected for docking
The experiment aims to investigate the effect of high energy milling to the crystallite size of α-alumina. The starting material used is α-alumina powder with starting crystal size of 86nm. This powder was milled at different time ranges from 0 to 60 minutes and milling speed ranges from 400 rpm to 1100 rpm using a wet milling technique in corundum abrasive materials. The wet milling technique involved the use of water with the alumina to water ratio of 1:6.1. Samples prepared were then examined using the X-Ray Diffraction (XRD) to calculate the crystallite size and scanning electron microscope (SEM) was also used to determine changes in the morphology. Results from these analysis showed that the crystallite size will get smaller when milling speed and time of more than 600rpm and 30 minutes respectively were used. Optimum conditions to achieve the smallest crystal size of 79.7nm are 1000 rpm and 60 minutes.
The incorporation of magnesium ions into the calcium phosphate structure is of great interest for the development of artificial bone implants. This paper investigates the preparation of magnesium-doped biphasic calcium phosphate (Mg-BCP) via sol gel method at various concentrations of added Mg. The effect of calcinations temperature (ranging from 500 degrees C to 900 degrees C) and concentrations of Mg incorporated into BCP has been studied by the aid of XRD, TGA and infrared spectroscopy (IR) in transmittance mode analysis. The study indicated that the powder was pure BCP and Mg-BCP with 100% purity and high crystallinity. The results also indicated that beta-tricalcium phosphate (beta-TCP) phase can be observed when the powder was calcined at 800 degrees C and above.
Two types of palm oil and sal fat based cocoa butter equivalents, namely fCBE (produced by using co-fractionation method) and mCBE (produced by using conventional method) were prepared. Results showed that the fCBE had triglyceride composition and solidification characteristics closer to the Malaysian cocoa butter than the mCBE produced at the same yield percentage. Increasing acetone washing time had little effect on the fCBE if compared to the effect of increasing palm olein to sal fat blend ratio. Co-fractionation technique increase the compatibility between CBE component triglycerides. Thus, more palm oil can be incorporated in the preparation and the process can be carried out at not low temperature as compared to the conventional method.
Geobacillus zalihae sp. nov., which produces a putative thermostable lipase, represents a novel species, with type strain T1. The characterisation of this intrinsically thermostable T1 lipase either physicochemically or structurally is an important task. The crystallisation of T1lipase in space was carried out using a High-Density Protein Crystal Growth (HDPCG) apparatus with the vapour diffusion method, and X-ray diffraction data were collected. The microgravity environment has improved the size and quality of the crystals as compared to earth grown crystal. The effect of microgravity on the crystallisation of T1 lipase was clearly evidenced by the finer atomic details at 1.35 A resolution. Better electron densities were observed overall compared with the Earth-grown crystals, and comparison shows the subtle but distinct conformations around Na(+) ion binding site stabilized via cation-π interactions. This approach could be useful for solving structure and function of lipases towards exploiting its potentials to various industrial applications.
This work reports the modification of freeze/thaw poly(vinyl alcohol) hydrogel using citric acid as the bioactive molecule for hydroxyapatite formation in simulated body fluid. Inclusion of 1.3 mM citric acid into the poly(vinyl alcohol) hydrogel showed that the mechanical strength, crystalline phase, functional groups and swelling ability were still intact. Adding citric acid at higher concentrations (1.8 and 2.3 mM), however, resulted in physically poor hydrogels. Presence of 1.3 mM of citric acid showed the growth of porous hydroxyapatite crystals on the poly(vinyl alcohol) surface just after one day of immersion in simulated body fluid. Meanwhile, a fully covered apatite layer on the poly(vinyl alcohol) surface plus the evidence of apatite forming within the hydrogel were observed after soaking for seven days. Gel strength of the soaked poly(vinyl alcohol)/citric acid-1.3 mM hydrogel revealed that the load resistance was enhanced compared to that of the neat poly(vinyl alcohol) hydrogel. This facile method of inducing rapid growth of hydroxyapatite on the hydrogel surface as well as within the hydrogel network can be useful for guided bone regenerative materials.
The in situ polymorphic forms and thermal transitions of refined, bleached and deodorized palm oil (RBDPO), palm stearin (RBDPS) and palm kernel oil (RBDPKO) were investigated using coupled X-ray diffraction (XRD) and differential scanning calorimetry (DSC). Results indicated that the DSC onset crystallisation temperature of RBDPO was at 22.6°C, with a single reflection at 4.2Å started to appear from 23.4 to 17.1°C, and were followed by two prominent exothermic peaks at 20.1°C and 8.5°C respectively. Further cooling to -40°C leads to the further formation of a β'polymorph. Upon heating, a of β'→βtransformation was observed between 32.1 to 40.8°C, before the sample was completely melted at 43.0°C. The crystallization onset temperature of RBDPS was 44.1°C, with the appearance of the α polymorph at the same temperature as the appearance of the first sharp DSC exothermic peak. This quickly changed from α→β´ in the range 25 to 21.7°C, along with the formation of a small β peak at -40°C. Upon heating, a small XRD peak for the β polymorph was observed between 32.2 to 36.0°C, becoming a mixture of (β´+ β) between 44.0 to 52.5°C. Only the β polymorph survived further heating to 59.8°C. For RBDPKO, the crystallization onset temperature was 11.6°C, with the formation of a single sharp exothermic peak at 6.5°C corresponding to the β' polymorphic form until the temperature reached -40°C. No transformation of the polymorphic form was observed during the melting process of RBDPKO, before being completely melted at 33.2°C. This work has demonstrated the detailed dynamics of polymorphic transformations of PKO and PS, two commercially important hardstocks used widely by industry and will contribute to a greater understanding of their crystallization and melting dynamics.
Copolyesters of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) were produced by Cupriavidus sp. (USMAA2-4) (DSM 19379) from carbon sources of 1,4-butanediol and gamma-butyrolactone. The composition of copolyesters produced varied from 0 to 45 mol% 4HB, depending on the combination of carbon sources supplied. The P(3HB-co-4HB) films containing Mitragyna speciosa crude extract were prepared with the ratio varying from 10 to 40% (w/w). The in vitro crude extract release of the films was studied in 0.1M phosphate buffer (pH 7.4) at 37 degrees C. Although the release rate was slow, it was maintained at a constant rate. This suggests that the crude extract release was due to the polymer degradation because the amount of crude extract released was consistent. The amount of degradation was based on the films' dry weight loss, decrease in molecular weight and surface morphology changes. The degradation rate increased with the 4HB content. This showed that the polymer degradation is dependant on the molecular weight, crystallinity, thermal properties and water permeability. The different drug loading ratio which led to surface morphology changes also gave an effect on polymer degradation.
Different fractions of fully hydrogenated soybean oil (FHSBO) in soybean oil (10-30% w/w) and the addition of 1% salt (sodium chloride) were used to investigate the effect of high-pressure treatments (HP) on the crystallization behaviors and physical properties of the binary mixtures. Sample microstructure, solid fat content (SFC), thermal and rheological properties were analyzed and compared against a control sample (crystallized under atmospheric condition). The crystallization temperature (Ts) of all model fats under isobaric conditions increased quadratically with pressure until reaching a pressure threshold. As a result of this change, the sample induction time of crystallization (tc) shifted from a range of 2.74-0.82 min to 0.72-0.43 min when sample crystallized above the pressure threshold under adiabatic conditions. At the high solid mass fraction, the addition of salt reduced the pressure threshold to induce crystallization during adiabatic compression. An increase in pressure significantly reduced mean cluster diameter in relation to the reduction of tc regardless of the solid mass fraction. In contrast, the sample macrostructural properties (SFC, storage modulus) were influenced more significantly by solid mass fractions rather than pressure levels. The creation of lipid gel was observed in the HP samples at 10% FHSBO. The changes in crystallization behaviors indicated that high-pressure treatments were more likely to influence crystallization mechanisms at low solid mass fraction.