Rational mutagenesis of cyclodextrin glucanotransferase at the calcium binding regions for enhancement of thermostability

Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as "parent CGTase" herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.     

Alkaline weight reduction and dyeing properties of black dope-dyed poly(ethylene terephthalate) microfibre fabrics

Sea–island-type, black dope-dyed poly(ethylene terephthalate) microfibre fabrics were compared with normal sea–island-type PET microfibres in terms of their thermal properties, alkaline weight reduction, and dyeing properties. A comparison of the weight reduction behaviours of the two microfibre yarns revealed that the microfibre yarn containing carbon black exhibited a higher dissolution speed than that containing no carbon black. The incorporation of carbon black particles in the polyester fibre matrix therefore accelerates the degradation of the less crystalline sea component during alkali treatment. A cationic dye staining method was used to monitor the alkaline dissolution behaviour of the sea–island-type PET microfibres, and compared with the K/S values and the colour difference, the f_k value is the most-desirable indicator for determination of the termination point of the alkaline dissolution process. The colour strength and the colour fastness of the dope-dyed sea–island-type PET microfibre fabrics are superior to those of the normal type.     

The Oxidation of Hydrophobic Aromatic Substrates by Using a Variant of the P450 Monooxygenase CYP101B1

The cytochrome P450 monooxygenase CYP101B1, from a Novosphingobium bacterium is able to bind and oxidise aromatic substrates but at a lower activity and efficiency than norisoprenoids and monoterpenoid esters. Histidine 85 of CYP101B1 aligns with tyrosine 96 of CYP101A1, which, in the latter enzyme forms the only hydrophilic interaction with its substrate, camphor. The histidine residue of CYP101B1 was mutated to phenylalanine with the aim of improving the activity of the enzyme for hydrophobic substrates. The H85F mutant lowered the binding affinity and activity of the enzyme for β-ionone and altered the oxidation selectivity. This variant also showed enhanced affinity and activity towards alkylbenzenes, styrenes and methylnaphthalenes. For example the rate of product formation for acenaphthene oxidation was improved sixfold to 245 nmol per nmol CYP per min. Certain disubstituted naphthalenes and substrates, such as phenylcyclohexane and biphenyls, were oxidised with lower activity by the H85F variant. Variants at H85 (A and G) designed to introduce additional space into the active site so as to accommodate these larger substrates did not improve the oxidation activity. As the H85F mutant of CYP101B1 improved the oxidation of hydrophobic substrates, this residue is likely to be in the substrate binding pocket or the access channel of the enzyme. The side chain of the histidine might interact with the carbonyl groups of the favoured norisoprenoid substrates of CYP101B1.     

Synthetic Modular Antibody Construction by Using the SpyTag/SpyCatcher Protein-Ligase System

Efforts to engineer recombinant antibodies for specific diagnostic and therapy applications are time consuming and expensive, as each new recombinant antibody needs to be optimized for expression, stability, bio-distribution, and pharmacokinetics. We have developed a new way to construct recombinant antibody-like “devices” by using a bottom-up approach to build them from well-behaved discrete recombinant antibody domains or “parts”. Studies on antibody structure and function have identified antibody constant and variable domains with specific functions that can be expressed in isolation. We used the SpyTag/SpyCatcher protein ligase to join these parts together, thereby creating devices with desired properties based on summed properties of parts and in configurations that cannot be obtained by using genetic engineering. This strategy will create optimized recombinant antibody devices at reduced costs and with shortened development times.     

Influence of Different Additives on the Interaction of Quinolone Antibiotic Drug with Surfactant: Conductivity and Cloud Point Measurement Study

Conductometric and cloud point (CP) measurement studies have been performed to investigate the interaction of tetradecyltrimethylammonium bromide (TTAB) and Triton® X-100 (TX-100) with ciprofloxacin hydrochloride (CFH) in different solvents over the temperature range of 295.15–315.15 K. CFH is used for the treatment of various bacterial infections. The observed critical micelle concentration (CMC) values of TTAB were found to be reduced in the presence of electrolytes (Na₂SO₄/Na₃PO₄), and this reduction proceeds with the elevation of salt concentration. The order of the CMC of TTAB follows the trend: > >. The observed CMC values of TTAB were found to increase with increasing temperature and decrease with increasing concentration of CFH in aqueous medium. The values of Gibbs free energy of micellization () for the TTAB/TTAB + CFH mixture were found to be negative, implying spontaneous micellization. The estimated CP of TX-100 decreases with increasing concentration of TX-100 in aqueous medium. The CP values first decrease with increasing concentration of CFH and then increase at higher concentration of CFH almost in all cases investigated. The values of free energy of clouding were found to be positive in all cases studied implying that phase separation of TX-100 was nonspontaneous. The other thermodynamic parameters associated with the micellization of TTAB and the phase separation of TX-100 were estimated and explained.     

Thermal behaviour and crystallization analysis of ethylene-propylene (EP) copolymer and EP-styrene terpolymer

Polyolefin copolymers have been an important commercial product since their invention. Hence, it is crucial to study their co- and terpolymers due to their extensive use. In this paper, in situ synthesis of ethylene-propylene (EP) copolymer, its terpolymer with styrene, and composites with nickel-chromium (NiCr) layered double hydroxide (LDH) has been reported along with their thermal properties. Styrene had a significant impact on the activity, increasing the yield by 195% and 235% with an addition of 0.5 and 1.0 mL styrene, respectively, compared to neat EP. The crystallinity, melting temperature, and thermal stability decreased due to styrene; nevertheless, it performed better compared to a similar work of terpolymer where α-olefin was the third monomer. The incorporation of NiCr LDH as a drop-in filler during in situ polymerization affected adversely the thermal stability of the terpolymer. However, the ultrasonication treatment improved the thermal stability of the final product.     

Effect of operating factors on liquid–liquid mass transfer and dispersion pattern of sedimentary liquid in a mechanically stirred vessel

Liquid–liquid dispersion and mass transfer were investigated in mechanically stirred vessels without baffles by changing operation factors such as an impeller rotation speed, off-bottom clearance, volumetric liquid ratio, etc. The dispersion regime was categorized into five groups: the sedimentary liquid was kept at the vessel bottom (I), partially elevated without any collision (II), partially dispersed by colliding with the impeller bottom (III), both liquids were partially dispersed by collisions with impeller blades (III’), and the sedimentary liquid was completely dispersed (IV). The dispersion switched to I → II → III → IV with the increasing rotation speed and decreasing off-bottom clearance. The liquid–liquid mass transfer rate was significantly enhanced with the collision of the sedimentary liquid with the impeller bottom, and subsequently increased with the increasing rotation speed, volumetric liquid ratio, and vessel diameter and with the decreasing off-bottom clearance. A multiple regression analysis method was applied to determine the mass transfer rates of III and III’.     

Enrichment,in vitro,and quantification study of antidiabetic compounds from neglected weed Mimosa pudica using supercritical CO2 and CO2-Soxhlet

Supercritical fluid extraction (SFE) using carbon dioxide (CO₂) and liquid CO₂ using Soxhlet (CO₂-Soxhlet) extraction were employed to extract three (3) antidiabetic compounds viz. stigmasterol, quercetin, and avicularin from Mimosa pudica. Various extraction parameters were studied. Extracts were analyzed pharmacologically, qualitatively and quantitatively to ascertain enrichment levels. All three antidiabetic compounds were effectively enriched under optimized conditions of temperature 60°C, pressure 40 MPa, co-solvent ratio 30%, and CO₂ flow rate of 5 ml min^?1. SFE was found to be the better method for enrichment of the antidiabetic compounds than the CO₂-Soxhlet method. Extraction conditions were seen to affect the enrichment of desired compounds.     

An investigation of sound absorption coefficient on sisal fiber poly lactic acid bio‐composites

In this research, the mechanical, acoustical, thermal, morphological, and infrared spectral properties of untreated, heat and alkaline‐treated sisal fiber‐reinforced poly‐lactic‐acid bio‐composites were analyzed. The bio‐composite samples were fabricated using a hot press molding machine. The properties mentioned above were evaluated and compared with heat‐treated and alkaline‐treated sisal fibers. Composites with heat‐treated sisal fibers were found to exhibit the best mechanical properties. Thermo‐gravimetric analysis (TGA) was conducted to study the thermal degradation of the bio‐composite samples. It was discovered that the PLA‐sisal composites with optimal heat‐treated at 160°C and alkaline‐treated fibers possess good thermal stability as compared with untreated fiber. The results indicated that the composites prepared with 30wt % of sisal had the highest sound absorption as compared with other composites. Evidence of the successful reaction of sodium hydroxide and heat treatment of the sisal fibers was provided by the infrared spectrum and implied by decreased bands at certain wavenumbers. Observations based on scanning electron microscopy of the fracture surface of the composites showed the effect of alkaline and heat treatment on the fiber surface and improved fiber‐matrix adhesion. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42470.     

Synthesis and Biological Evaluation of Ferrocenylquinoline as a Potential Antileishmanial Agent

The emergence of resistance against antileishmanial drugs in current use necessitates the search for new classes of antileishmanial compounds. Herein we report the design, synthesis, and evaluation of a novel ferrocenylquinoline for activity against Leishmania donovani. 7‐Chloro‐N‐[2‐(1H‐5‐ferrocenyl‐1,2,3‐triazol‐1‐yl)ethyl]quinolin‐4‐amine ( 1 ) was generated by coupling an iron(II) ethynylferrocene species with 4‐(2‐ethylazido)amino‐7‐chloroquinoline using click chemistry. The synthesized compound 1 was tested for its antileishmanial activity using both promastigote and amastigote stages of L. donovani. Compound 1 showed promising anti‐promastigote activity, with an IC₅₀ value of 15.26 μM and no cytotoxicity toward host splenocytes. From the battery of tests conducted in this study, it appears that this compound induces parasite death by promoting oxidative stress and depolarizing the mitochondrial membrane potential, thereby triggering apoptosis. These results suggest that ferrocenylquinoline 1 is a suitable lead for the development of new antileishmanial drugs.