The crystal structure of the industrially important Aspergillus oryzae β-galactosidase has been determined at 2.60Å resolution. The Ao-β-gal is a large (985 residues) monomeric multi-domain enzyme that has a catalytic α/β-barrel domain. An electron density map revealed extensive N-glycosylation between the domain interfaces suggesting that the oligosaccharide-chains would have a stabilizing role for the structure of Ao-β-gal. Comparison of structure with other β-galactosidase structures of glycoside hydrolase family 35 revealed a number of hydrophobic residues, which may contribute favorably to the stabilization of the structure. The role of a high number of acidic residues in Ao-β-gal is also discussed.

The cDNA coding for a mature protein of 123 amino acids, containing all of the structural features of catalytically active group II sPLA2, has been amplified. The gene has been cloned into the bacterial expression vector pET-21a(+), which allows protein over-expression as inclusion bodies and enables about 3mg per litre of pure refolded fully active enzyme to be obtained. Recombinant expression of chPLA2-IIA in E-coli shows that the enzyme is Ca(2+) dependent, maximally active at pH 8-9, and hydrolyses phosphatidylglycerol versus phosphatidylcholine with a 15-fold preference. The ability to express reasonably large amounts of the sPLA2 Group IIA, compared to that obtained with the classical purification will provide a basis for future site directed mutagenesis studies of this important enzyme.

Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in Km values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1.

Conjugation of various arginine-rich peptide sequences to vectors based on 10kDa polyethylenimine (PEI) and its hydrophobic derivative (hexanoate-PEI) was investigated as a strategy for improving pDNA and siRNA transfection activities. Six different arginine-histidine (RH) sequences and two arginine-serine (RS) sequences with a range of R/H ratios were designed and coupled to PEI and hexanoate-PEI. All arginine-rich peptide derivatives of PEI significantly enhanced luciferase gene expression compared to PEI 10kDa alone. Hexanoate-PEI derivatives exhibited higher transfection activity than underivatized PEI vectors. Improved transfection activity may have resulted at least in part from use of higher vector/DNA ratios made possible by reduced cytotoxicity of vectors, and to use of vectors with higher molecular weights. Vectors that were the most efficient in pDNA delivery and transfection were also the most effective in siRNA delivery and protein expression knock down.

Propolis is a gum gathered by honey bees from various plants; the honey bees use propolis to seal holes in their honey combs, smooth out the internal wall and protect the entrance against intruders. It is composed of 50% resin (flavonoids and related phenolic acid), 30% wax, 10% essential oils, 5% pollen and 5% various organic components. As a natural mixture, propolis is widely used in medicine, cosmetics and food. So far no attempts have been yet made to make use of propolis in the realm of textile finishing. Current work presents the first systemic study targeted to build up a scientific basis for production of cotton textiles having antibacterial activity and other useful properties by making use of propolis as eco-friendly finish within the scope of green strategy. Propolis extract solution (70/30 ethanol/water) of 10% concentration was prepared as the stock. Different amounts of the latter were used along with a crosslinking agent and catalyst for treatment of cotton fabrics as per pad-dry-cure technique. Antibacterial activity of the so treated fabrics was obtained through monitoring the efficiency of the interaction of propolis with cotton cellulose. This interaction was expressed as inhibition zone diameter after the treated fabrics were exposed to (G+ve) and (G-ve) bacteria. Other properties include crease recovery, tensile strength and elongation at break. Factors affecting these properties such as type, nature and concentration of the crosslinking agent, concentration of propolis, and conditions of curing were investigated. In addition characterization of the propolis containing modified cotton fabrics including demonstration of the antibacterial activity, SEM, FTIR, durability to washing, UV protection and water repellency were performed. Based on results obtained, it is concluded that application of propolis along with glyoxal and Al2(SO4)3catalyst using pad-dry (3min/80°C), cure (5/140°C) bring about cotton textile with superior antibacterial activity, water repellent and ease of care characteristics as well as UV protection. Tentative mechanism of the reaction of propolis with cotton in the presence of glyoxal was also reported.

Chitosan hydrogels may be formed by various mechanisms. In this study, we aimed to form hybrid polymer networks of chitosan with alginate using a crosslinker which enabled the covalent binding of the two macromolecules. The structural and thermal characterization of these hydrogels was performed by using Fourier transform infrared (FTIR) and differential scanning calorimeter (DSC). The morphological analysis of the crosslinked material was investigated by scanning electron microscopy (SEM) and a scanning probe microscope with atomic force microscope (AFM) attachment. The swelling properties of these gels were analyzed in water and in phosphate buffered saline (PBS) solution. The presence of alginate in a chitosan/alginate hydrogel was shown to support the hydrogel stability. Compared to chitosan/alginate (1/2) hydrogel prepared with 1wt% DCC, the swelling of chitosan/alginate (1/2) hydrogels prepared with 3wt% DCC was limited. To measure the degree of cell proliferation, the hydrogels were seeded with L929 mouse fibroblasts and cell numbers measured by neutral red uptake assay. The cell attachment was also followed by (SEM) photography. It was observed that chitosan/alginate (1/2) hydrogels with 1wt% (DCC) provides a better environment for cell attachment and proliferation. This study presents functional hydrogel formation by crosslinked chitosan and alginate, a novel biomaterial which also supports cell growth.

From the aerial parts of sage (Salvia officinalis L.) an arabino-(4-O-methyl-glucurono)-xylan (AGX) was isolated by alkaline extraction followed by precipitation with barium hydroxide solution. Polymer was isolated from sage as a light brown polysaccharide material of molecular mass (Mp) 84,000. Compositional analyses of sage AGX revealed xylose (81%), arabinose (10%), glucuronic acid (8%) and small amounts of hexoses (1%). Linkage sugar analyses showed the (1→4)-linked xylopyranosyl backbone with low degree of substitution (9-10%) at O-2 and O-3. Arabinofuranose residues were found as the terminal, 1,3-, 1,5- and 1,3,5-linked. NMR structural analyses of acidic oligomers, generated by partial acidic hydrolysis of AGX, confirmed a substitution of xylose residues by glucuronic acid and its 4-O-methyl derivate at O-2 at an average on every fourteenth xylose residue. NMR and FT-IR measurements, as well as a high negative optical rotation confirmed the β configuration of glycosidic linkages in AGX backbone.

The major objective of this work is to investigate the enhancing effect of β-cyclodextrin on the intestinal absorption of berberine hydrochloride, a P-glycoprotein (Pgp) substrate. The inclusion complexation behavior of BBH with β-CD was investigated by phase-solubility diagram, Fourier transform infrared spectroscopy, differential scanning calorimetry, X-ray powder diffractometry, NMR spectroscopy, and molecular modeling studies. Results indicated that the 1,3-benzodioxole of BBH was included into the cavity of β-CD to form an inclusion complex which exhibited higher dissolution rate than BBH in vitro. The intestinal absorption of the inclusion complex in rats was significantly higher than the free drug due to its solubilizing effect and Pgp modulatory activity. The mechanisms of β-CD on Pgp modulation were demonstrated by modifying the Pgp ATPase activity, the Pgp mRNA level and the Pgp expression.

The present work describes the characterization and thermal properties of hydrophobic starch obtained by the esterification of cassava starch with acyl imidazoles, acid chlorides and methyl ester derivatives of fatty acids with n-alkyl chains with 12-22 carbon atoms, in order to compare the dependence of their properties as a function of the length of the side chain and the methodology used for their synthesis. The n-acyl starches presented degrees of substitution (DS) between 0.06 and 1.2. Most of the derivatives obtained with acyl imidazoles were found to be stable at temperatures up to 300°C, whereas those synthesized with acid chlorides or methyl ester decomposed below. Finally, when the n-acyl starches were substituted with n-alkyl side chains of 16 or more carbon atoms, they were capable to crystallize in separate paraffinic phases independent of the starch backbone.

N-furoyl chitosan (NF-CS) and N-furoyl chitosan oligosaccharide (NF-COS) were prepared via acylation of chitosan and chitosan oligosaccharide. Their chemical structures and physical properties were characterized by Fourier transform infrared spectroscopy, (1)H NMR, X-ray diffraction, and elemental analysis. Results showed that the degrees of substitution of the derivatives were 0.68 and 0.72, respectively. Their antioxidant activities in vitro were further studied. The inhibition concentration of NF-CS at 50% (IC50) and the scavenging effect of NF-COS on DPPH were 1.3 and 0.76mgmL(-1), respectively. The derivatives also exhibited higher antioxidant activities than chitosan based on the reducing power and hydroxyl radical scavenging activities.