Formulations containing engkabang fat and engkabang fat esters, F10 and E15 respectively were prepared using a high-shear homogenizer, followed by a high-pressure homogenizer. Both formulations were stable at room temperature, at 45 degrees C, and after undergoing freeze-thaw cycles. The particle sizes of F10 and E15 after high pressure were 115.75 nm and 148.41 nm respectively. The zeta potentials of F10 and E15 were -36.4 mV and -48.8 mV respectively, while, the pH values of F10 and E15 were 5.59 and 5.81 respectively. The rheology of F10 and E15 showed thixotropy and pseudoplastic behavior respectively. There were no bacteria or fungal growths in the samples. The short-term moisturizing effect on 20 subjects analyzed by analysis of variance (ANOVA), gave p-values of 7.35 x 10(-12) and 2.77 x 10(-15) for F10 and E15 respectively. The hydration of the skins increased after application of F10 and E15 with p-value below 0.05.
Critical to the applications of proteins in non-aqueous enzymatic processes is their structural dynamics in relation to solvent polarity. A pool of mutants derived from Geobacillus zalihae T1 lipase was screened in organic solvents (methanol, ethanol, propanol, butanol and pentanol) resulting in the selection of six mutants at initial screening (A83D/K251E, R21C, G35D/S195 N, K84R/R103C/M121I/T272 M and R106H/G327S). Site-directed mutagenesis further yielded quadruple mutants A83D/M121I/K251E/G327S and A83D/M121I/S195 N/T272 M, both of which had improved activity after incubation in methanol. The km and kcat values of these mutants vary marginally with the wild-type enzyme in the methanol/substrate mixture. Thermally induced unfolding of mutants was accompanied with some loss of secondary structure content. The root mean square deviations (RMSD) and B-factors revealed that changes in the structural organization are intertwined with an interplay of the protein backbone with organic solvents. Spatially exposed charged residues showed correlations between the solvation dynamics of the methanol solvent and the hydrophobicity of the residues. The short distances of the radial distribution function provided the required distances for hydrogen bond formation and hydrophobic interactions. These dynamic changes demonstrate newly formed structural interactions could be targeted and incorporated experimentally on the basis of solvent mobility and mutant residues.
Lipolytic enzymes with cold adaptation are gaining increasing interest due to their biotechnological prospective. Previously, a cold adapted family I.3 lipase (AMS8 lipase) was isolated from an Antarctic Pseudomonas. AMS8 lipase was largely expressed in insoluble form. The refolded His-tagged recombinant AMS8 lipase was purified with 23.0% total recovery and purification factor of 9.7. The purified AMS8 lipase migrated as a single band with a molecular weight approximately 65kDa via electrophoresis. AMS8 lipase was highly active at 30°C at pH 10. The half-life of AMS8 lipase was reported at 4 and 2h under the incubation of 30 and 40°C, respectively. The lipase was stable over a broad range of pH. It showed enhancement effect in its relative activity under the presence of Li(+), Na(+), K(+), Rb(+) and Cs(+) after 30min treatment. Heavy metal ions such as Cu(2+), Fe(3+) and Zn(2+) inhibited AMS8 activity. This cold adapted alkalophilic AMS lipase was also active in various organic solvent of different polarity. These unique properties of this biological macromolecule will provide considerable potential for many biotechnological applications and organic synthesis at low temperature.
Chiral enantiomers [Cu(phen)(L-threo)(H2O)]NO3 1 and [Cu(phen)(D-threo)(H2O)]NO3 2 (threo = threoninate) underwent aldol-type condensation with formaldehyde, with retention of chirality, to yield their respective enantiomeric ternary copper(II) complexes, viz. L- and D-[Cu(phen)(5MeOCA)(H2O)]NO3·xH2O (3 and 4; phen = 1,10-phenanthroline; 5MeOCA = 5-methyloxazolidine-4-carboxylate; x = 0-3) respectively. These chiral complexes were characterized by FTIR, elemental analysis, circular dichroism, UV-Visible spectroscopy, fluorescence spectroscopy (FL), molar conductivity measurement, ESI-MS and X-ray crystallography. Analysis of restriction enzyme inhibition by these four complexes revealed modulation of DNA binding selectivity by the type of ligand, ligand modification and chirality. Their interaction with bovine serum albumin was investigated by FL and electronic spectroscopy. With the aid of the crystal structure of BSA, spectroscopic evidence suggested their binding at the cavity containing Trp134 with numerous Tyr residues in subdomain IA. The products were more antiproliferative than cisplatin against cancer cell lines HK-1, MCF-7, HCT116, HSC-2 and C666-1 except HL-60, and were selective towards nasopharyngeal cancer HK-1 cells over normal NP69 cells of the same organ type.
A series of ternary copper(II)-1,10-phenanthroline complexes with glycine and methylated glycine derivatives, [Cu(phen)(aa)(H(2)O)]NO(3)·xH(2)O 1-4 (amino acid (aa): glycine (gly), 1; DL: -alanine (DL: -ala), 2; 2,2-dimethylglycine (C-dmg), 3; sarcosine (sar), 4), were synthesized and characterized by FTIR, elemental analysis, electrospray ionization-mass spectra (ESI-MS), UV-visible spectroscopy and molar conductivity measurement. The determined X-ray crystallographic structures of 2 and 3 show each to consist of distorted square pyramidal [Cu(phen)(aa)(H(2)O)](+) cation, a nitrate counter anion, and with or without lattice water, similar to previously reported structure of [Cu(phen)(gly)(H(2)O)]NO(3)·1½H(2)O. It is found that 1-4 exist as 1:1 electrolytes in aqueous solution, and the cationic copper(II) complexes are at least stable up to 24 h. Positive-ion ESI-MS spectra show existence of only undissociated [Cu(phen)(aa)](+) species. Electron paramagnetic resonance, gel electrophoresis, fluorescence quenching, and restriction enzyme inhibition assay were used to study the binding interaction, binding affinity and selectivity of these complexes for various types of B-form DNA duplexes and G-quadruplex. All complexes can bind selectively to DNA by intercalation and electrostatic forces, and inhibit topoisomerase I. The effect of the methyl substituents of the coordinated amino acid in the above complexes on these biological properties are presented and discussed. The IC(50) values (24 h) of 1-4 for nasopharyngeal cancer cell line HK1 are in the range 2.2-5.2 μM while the corresponding values for normal cell line NP69 are greater than 13.0 μM. All complexes, at 5 μM, induced 41-60 % apoptotic cell death in HK1 cells but no significant cell death in NP69 cells.
Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.
The binding selectivity of the M(phen)(edda) (M=Cu, Co, Ni, Zn; phen=1,10-phenanthroline, edda=ethylenediaminediacetic acid) complexes towards ds(CG)(6), ds(AT)(6) and ds(CGCGAATTCGCG) B-form oligonucleotide duplexes were studied by CD spectroscopy and molecular modeling. The binding mode is intercalation and there is selectivity towards AT-sequence and stacking preference for A/A parallel or diagonal adjacent base steps in their intercalation. The nucleolytic properties of these complexes were investigated and the factors affecting the extent of cleavage were determined to be: concentration of complex, the nature of metal(II) ion, type of buffer, pH of buffer, incubation time, incubation temperature, and the presence of hydrogen peroxide or ascorbic acid as exogenous reagents. The fluorescence property of these complexes and its origin were also investigated. The crystal structure of the Zn(phen)(edda) complex is reported in which the zinc atom displays a distorted trans-N(4)O(2) octahedral geometry; the crystal packing features double layers of complex molecules held together by extensive hydrogen bonding that inter-digitate with adjacent double layers via pi...pi interactions between 1,10-phenanthroline residues. The structure is compared with that of the recently described copper(II) analogue and, with the latter, included in molecular modeling.