[Show abstract][Hide abstract] ABSTRACT: Background
Rheumatoid arthritis (RA) is an autoimmune disease with unknown etiology. Anticitrullinated protein autoantibody has been documented as a highly specific autoantibody associated with RA. Protein arginine deiminase type 4 (PAD4) is the enzyme responsible for catalyzing the conversion of peptidylarginine into peptidylcitrulline. PAD4 is a new therapeutic target for RA treatment. In order to search for inhibitors of PAD4, structure-based virtual screening was performed using LIDAEUS (Ligand discovery at Edinburgh university). Potential inhibitors were screened experimentally by inhibition assays.
Twenty two of the top-ranked water-soluble compounds were selected for inhibitory screening against PAD4. Three compounds showed significant inhibition of PAD4 and their IC50 values were investigated. The structures of the three compounds show no resemblance with previously discovered PAD4 inhibitors, nor with existing drugs for RA treatment.
Three compounds were discovered as potential inhibitors of PAD4 by virtual screening. The compounds are commercially available and can be used as scaffolds to design more potent inhibitors against PAD4.
[Show abstract][Hide abstract] ABSTRACT: The crystallization of proteins makes it possible to determine their structure by X-ray crystallography, and is therefore important for the analysis of protein structure-function relationships. L2 lipase was crystallized by using the J-tube counter diffusion method. A crystallization consisting of 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl was found to be the best condition to produce crystals with good shape and size (0.5 × 0.1 × 0.2 mm). The protein concentration used for the crystallization was 3 mg/mL. L2 lipase crystal has two crystal forms, Shape 1 and Shape 2. Shape 2 L2 lipase crystal was diffracted at 1.5 Å and the crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 72.0, b = 81.8, c = 83.4 Å, α = β = γ = 90°. There is one molecule per asymmetric unit and the solvent content of the crystals is 56.9%, with a Matthew's coefficient of 2.85 Å Da(-1). The 3D structure of L2 lipase revealed topological organization of α/β-hydrolase fold consisting of 11 β-strands and 13 α-helices. Ser-113, His-358 and Asp-317 were assigned as catalytic triad residues. One Ca(2+) and one Zn(2+) were found in the L2 lipase molecule.
International Journal of Molecular Sciences 12/2012; 13(7):9207-17. DOI:10.3390/ijms13079207 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Palm oil-based esters (POEs) are unsaturated and non-ionic esters with a great potential to act as chemical penetration enhancers and drug carriers for transdermal drug nano-delivery. A ratio of palmitate ester and nonionic Tween80 with and without diclofenac acid was chosen from an experimentally determined phase diagram. Molecular dynamics simulations were performed for selected compositions over a period of 15 ns. Both micelles showed a prolate-like shape, while adding the drug produced a more compact micellar structure. Our results proposed that the drug could behave as a co-surfactant in our simulated model.
International Journal of Molecular Sciences 12/2012; 13(8):9572-83. DOI:10.3390/ijms13089572 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Here, we describe an improved enzyme-facilitated epoxidation of 1-nonene using a conventional water bath shaker at ambient temperature. Enzymes were used to produce peroxy acids instantly from hydrogen peroxide (H2O2) and various perhydrolysis substrates. The peroxy acid generated was then utilised directly for in-situ oxidation of 1-nonene to 1-nonene oxide. Various parameters affecting the reaction were studied such as the nature of the peroxy acids, organic solvents, enzyme sources and enzyme concentrations. The highest conversion rate was achieved using phenylacetic acid as an oxygen carrier. 1-Nonene was converted most efficiently with 95% of the maximum yield by Novozym 435, an immobilised Candida antarctica lipase B, using dichloromethane as the reaction media. A minimum amount (16 mg, 1.4% w/w) of Novozym 435 was needed to maintain catalytic activity (160.0 Ug−1). In addition, a simple and rapid Gas chromatography mass spectroscopy selective ion monitoring (GC-MS SIM) method was developed using a HP-5ms column for determining 1-nonene oxide. The method was found to be linear in the range of 29.9 to 298.8 mg/L with R2 = 0.9981.
Biocatalysis and Biotransformation 11/2012; 30(5-6). DOI:10.3109/10242422.2012.743121 · 0.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 11/2012; 7(11):e49788. DOI:10.1371/journal.pone.0049788 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A thermostable and organic solvent-tolerant lipase produced by Aneurinibacillus thermoaerophilus strain
HZ was purified and characterised. The lipase was purified to apparent homogeneity with two steps: anion
exchange chromatography on Q-Sepharose and gel filtration on Sephadex-G75. A final specific activity of
43.5 U/mg was obtained with an overall recovery of 19.7% and 15.6 purification fold. The molecular mass
of the HZ lipase was estimated to be 50 kDa. The optimum pH for the activity of the purified HZ lipase
was 7.0. The stability showed a broad range of pH values between pH 4.0 and 9.0 at 30 ◦C. The purified HZ
lipase exhibited an optimum temperature of 65 ◦C with a half-life of 3 h and 10 min at 65 ◦C. The activity of
the purified HZ lipase was stimulated in the presence of Ca2+. Organic solvents such as dimethyl sulfoxide
(DMSO), methanol, n-tetradecane and n-hexadecane enhanced the lipase activity. Studies on the effect
of oil showed that the lipase preferred natural oil, such as sunflower oil, over synthetic substrates.
Process Biochemistry 11/2012; 48(1):169–175. DOI:10.1016/j.procbio.2012.11.002 · 2.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: N-trans feruloyltyramine amide was successfully synthesized from 4-hydroxy-3-methoxycinnamic acid and tyramine hydrochloride in a one-step lipase catalysed reaction. The use of immobilized lipase, lipozyme TL IM as the catalyst in the reaction allowed simple isolation of the enzyme from the products and other components in the reaction mixture. N-feruloyltyramine amide was characterized using Fourier Transform Infrared (FTIR) Spectroscopy, Proton Nuclear Magnetic Resonance (1H NMR) and elemental analysis. Under optimized conditions 93.5% yield was obtained when the process was carried out for 48 h using a molar ratio of cinnamic acid:tyramine HCl, 6:1 at 40°C. In addition, a rapid simple and sensitive HPLC-UV method was developed for the determination of N-feruloyltyramine using an ®Rp-8 endcapped column. The optimum mobile phase used was acetonitrile:disodium hydrogen phosphate, 30:70(v/v.). N-feruloyltyramine amide was detected at a retention time of 12 min. The calibration curve was linear over the range of 5.27–12.30 × 10−4 M with correlation factor r = 0.9958. Consequently, the method was considered valid for quantitative analysis samples of N-trans-feruloyltyramine amide.
Biocatalysis and Biotransformation 09/2012; 30(4). DOI:10.3109/10242422.2012.701623 · 0.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role of Q114 in imparting enantioselectivity on T1 lipase. The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced. The enantioselectivity of the thermophilic lipases, T1, Q114L and Q114M correlated better with log p as compared to the dielectric constant and dipole moment of the solvents. Enzyme activity was good in solvents with log p < 3.5, with the exception of hexane which deviated substantially. Isooctane was found to be the best solvent for the esterification of (R,S)-ibuprofen with oleyl alcohol for lipases Q114M and Q114L, to afford E values of 53.7 and 12.2, respectively. Selectivity of T1 was highest in tetradecane with E value 49.2. Solvents with low log p reduced overall lipase activity and dimethyl sulfoxide (DMSO) completely inhibited the lipases. Ester conversions, however, were still low. Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M. The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.
International Journal of Molecular Sciences 09/2012; 13(9):11666-80. DOI:10.3390/ijms130911666 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PpCHS is a member of the type III polyketide synthase family and catalyses the synthesis of the flavonoid precursor naringenin chalcone from p-coumaroyl-CoA. Recent research reports the production of pyrone derivatives using either hexanoyl-CoA or butyryl-CoA as starter molecule. The Cys-His-Asn catalytic triad found in other plant chalcone synthase predicted polypeptides is conserved in PpCHS. Site directed mutagenesis involving these amino acids residing in the active-site cavity revealed that the cavity volume of the active-site plays a significant role in the selection of starter molecules as well as product formation. Substitutions of Cys 170 with Arg and Ser amino acids decreased the ability of the PpCHS to utilize hexanoyl-CoA as a starter molecule, which directly effected the production of pyrone derivatives (products). These substitutions are believed to have a restricted number of elongations of the growing polypeptide chain due to the smaller cavity volume of the mutant's active site.
International Journal of Molecular Sciences 08/2012; 13(8):9673-91. DOI:10.3390/ijms13089673 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enzymatic production of fatty acid esters from the esterification of oleyl alcohol with various fatty acids was investigated by using two new tetraethylammonium amino acid ionic liquids-coated Candida rugosa lipase (ILs-CRL) as biocatalysts in hexane. Both enzyme derivatives were prepared by mixing Candida rugosa lipase with tetraethylammonium l-histidinate (IL1) and tetraethylammonium l-asparaginate (IL2). The ILs-CRL system containing the equivalent protein concentration as in CRL showed higher esterification activity especially on medium chain fatty acids (C 12 –C 16) as compared to non-coated CRL. Hydrophilicity of ILs may play an important role in hydrogen bonding with enzyme surface and consequently stabilize the ILs-CRL.
Journal of Molecular Catalysis B Enzymatic 07/2012; 79:61-65. DOI:10.1016/j.molcatb.2012.03.003 · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dicarboxylic acid esters are one of the important classes of synthetic lubricants which exhibit good oxidation and thermal stability, low volatility, shear stability, high viscosity index and low toxicity. In this work, enzymatic synthesis of dioleyl adipate ester was performed in batch mode with a stirred tank reactor (STR). Immobilized Candida antarctica lipase B was used as the biocatalyst for the esterification reaction. Response surface methodology (RSM) based on a fourfactor-five-level central composite rotatable design (CCRD) was employed to model and analyze the reaction. The effects of all investigated parameters including amount of enzyme (1-10%w/w), temperature (35-75°C), time (30-480 min) and impeller speed (1-500 rpm) were significant (P<0.0001). All factors positively affected the yield with reaction time having the most significant effect. The highest obtained conversion yield was 96.2% under experimental conditions of 60.0°C, 323.7 min, amount of enzyme 6.0%w/w of the substrates and 500 rpm impeller speed. Verification experiments exhibited good correspondence between actual yields and the values predicted by the statistical model. The predictive model was successfully applied for optimization of upscale synthesis of adipate ester.
Chemical Product and Process Modeling 07/2012; 7(1). DOI:10.1515/1934-2659.1483
[Show abstract][Hide abstract] ABSTRACT: An electrochemical biosensor was developed based on formaldehyde dehydrogenase immobilized with Nafion membrane for determination of formaldehyde in fish. The enzyme was immobilized through the entrapment technique and measured based on the reduction of -nicotinamide adenine dinucleotide. The response time of the formaldehyde biosen-sor was <1 min, with an optimum pH of 8. The optimum enzyme loading and NAD + concentrations were found at 30 mg/mL and 0.5 mM, respectively. Using the formaldehyde biosensor, a linear response of formaldehyde showed a range of 0.1 to 10 ppm and a detection limit of 0.016 ppm. In application of Nash method, the samples were stored at 4 o C ± 1 for 10 days. With the two combined methods, a linear correlation coefficient with R 2 = 0.9982 (y = 0.956x -0.014) was found. The developed formaldehyde biosensor showed a good reproducibility, long storage stability (more than 6 months stored at 4 o C), and also effective monitoring of formaldehyde level in Indian mackerel (Rastrelliger kanagurta) fish.
Current Analytical Chemistry 07/2012; 8(4-2.134). DOI:10.2174/157341112803216843 · 1.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose – The purpose of this paper is to describe a capacitive biosensor device consisting of an enzyme electrode and a simple detector which has been developed for histamine measurement. Design/methodology/approach – In this analysis, degradation of histamine through enzymatic reaction produces signal that is monitored using a simple detector equipped with “astable” multivibrator operation circuit (in capacitor-resistor circuit). Findings – Different frequency (f) readings have been obtained for glucose, alcohol and histamine in different concentration levels, showing the ability of this simple device system to measure their dielectric constant (k) as formulated by the equation f=(1.44d)/ [kA (R1+2R2)]. The analysis using smaller electrode gap (d) produces higher value of f, indicating that d, is directly proportional to f. For histamine, by using immobilized enzyme electrode, the results show that the change of dielectric properties during the 300-second reaction period could also be monitored. A linear relationship is obtained between concentration and frequency from 50 to 200?ppm. Practical implications – Based on this result, an enzyme electrode and “astable” operation circuits have the potential to be used in the development of a simple capacitive biosensor device. Originality/value – The paper is an outcome of experimental work carried out to observe capacitive sensing behavior using an immobilized enzyme, to measure biological samples, especially histamine.
[Show abstract][Hide abstract] ABSTRACT: The activation of lipases has been postulated to proceed by interfacial activation, temperature switch activation, or aqueous activation. Recently, based on molecular dynamics (MD) simulation experiments, the T1 lipase activation mechanism was proposed to involve aqueous activation in addition to a double-flap mechanism. Because the open conformation structure is still unavailable, it is difficult to validate the proposed theory unambiguously to understand the behavior of the enzyme. In this study, we try to validate the previous reports and uncover the mystery behind the activation process using structural analysis and MD simulations. To investigate the effects of temperature and environmental conditions on the activation process, MD simulations in different solvent environments (water and water-octane interface) and temperatures (20, 50, 70, 80, and 100°C) were performed. Based on the structural analysis of the lipases in the same family of T1 lipase (I.5 lipase family), we proposed that the lid domain comprises α6 and α7 helices connected by a loop, thus forming a helix-loop-helix motif involved in interfacial activation. Throughout the MD simulations experiments, lid displacements were only observed in the water-octane interface, not in the aqueous environment with respect to the temperature effect, suggesting that the activation process is governed by interfacial activation coupled with temperature switch activation. Examining the activation process in detail revealed that the large structural rearrangement of the lid domain was caused by the interaction between the hydrophobic residues of the lid with octane, a nonpolar solvent, and this conformation was found to be thermodynamically favorable.
Protein Science 06/2012; 21(8):1210-21. DOI:10.1002/pro.2108 · 2.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Pesticides are developed with carriers to improve their physicochemical properties and, accordingly, the bioefficacy of the applied formulation. For foliar-applied herbicide, generally less than 0.1% of the active ingredient reaching the target site could reduce pesticide performance. Recently, a carrier of nanoemulsion consisting of oil, surfactant and water, with a particle size of less than 200 nm, has been shown to enhance drug permeability for skin penetration in pharmaceutical delivery systems. In the present work, the aim was to formulate a water-soluble herbicide, glyphosate isopropylamine (IPA), using a green nanoemulsion system for a biological activity study against the weeds creeping foxglove, slender button weed and buffalo grass.
RESULTS: The nanoemulsion formulations displayed a significantly lower spray deposition on creeping foxglove (2.9–3.5 ng cm−2), slender button weed (2.6–2.9 ng cm−2) and buffalo grass (1.8–2.4 ng cm−2) than Roundup® (3.7–5.1 ng cm−2). The visible injury rates of weeds treated with the nanoemulsion formulations were statistically equivalent to those relating to Roundup® at 14 days after treatment, with a control range of 86.67–96.67%.
CONCLUSION: It was hypothesised that the significant difference in spray deposition with equal injury rates can be attributed to enhanced bioactivity of the nanoemulsion formulations. This initial discovery could be the platform for developing better penetration of agrochemical formulations in the future. Copyright
Pest Management Science 05/2012; 69(1)(1). DOI:10.1002/ps.3371 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues, Val, Met, Leu, Ile, Trp, and Phe into Gln 114 of T1 lipase. The in silico investigations accurately predicted the enzymatic characteristics of the mutants in the experimental studies and provided rationalization for some of the experimental observations. Substitution with Leu successfully improved the conformational stability and enzymatic characteristics of T1 lipase. However, replacement of Gln114 with Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability, diminished lipase activity and inferior enzymatic characteristics. These results suggested that the substitution of a larger residue in a densely packed area of the protein core can have considerable effects on the structure and function of an enzyme. This is especially true when the residue is next to the catalytic serine as demonstrated with the Phe and Trp mutation.
[Show abstract][Hide abstract] ABSTRACT: Yeasts are a convenient platform for many applications. They have been widely used as the expression hosts. There is a need to have a new yeast expression system to contribute the molecular cloning demands. Eight yeast isolates were screened from various environment sources and identified through ribosomal DNA (rDNA) Internal Transcribed Spacer (ITS). Full sequence of the rDNA ITS region for each isolate was BLASTed and phylogenetic study was constructed by using MEGA4. Among the isolates, isolate WB from 'ragi' (used to ferment carbohydrates) could be identified as a new species in order Saccharomycetales according to rDNA ITS region, morphology and biochemical tests. Isolate SO (from spoiled orange), RT (rotten tomato) and RG (different type of 'ragi') were identified as Pichia sp. Isolates R1 and R2, S4 and S5 (from the surrounding of a guava tree) were identified as Issatchenkia sp. and Hanseniaspora sp., respectively. Geneticin, 50 µg/mL, was determined to be the antibiotic marker for all isolates excepted for isolates RT and SO which used 500 µg/mL and 100 µg/mL Zeocin, respectively. Intra-extracellular proteins were screened for lipolytic activity at 30°C and 70°C. Thermostable lipase activity was detected in isolates RT and R1 with 0.6 U/mg and 0.1 U/mg, respectively. In conclusion, a new yeast-vector system for isolate WB can be developed by using phleomycin or geneticin as the drugs resistance marker. Moreover, strains RT and R1 can be investigated as a novel source of a thermostable lipase.
[Show abstract][Hide abstract] ABSTRACT: The present invention discloses biologically pure cultures of Geobacillussp. strain ARM and Aneurinibacillus thermoaerophilus strain AFNA isolated from soil contaminated with oil; whereby the Geobacillus spstrain ARM is deposited under the accession number DSM 21496 at DeutscheSammlung von Mikroorganismen und Zellkulturen (DSMZ) and NCIMB 41583 at The National Collection of Industrial, Marine and Food Bacteria (NCIMB), UK, and Aneurinibacillus thermoaerophilusstrain AFNA is deposited under the accession number DSM 21497 at DeutscheSammlung von Mikroorganismen und Zellkulturen (DSMZ) Germany and NCIMB 41584 at The National Collection of Industrial, Marine and Food Bacteria (NCIMB), UK. Moreover, the Geobacillus sp. strain ARM and Aneurinibacillus thermoaerophilus strain AFNA is capable to produce a novel thermostable enzyme isolated and characterized from Geobacillus sp. Strain ARM and Aneurinibacillus thermoaerophilus strain AFNA.
In addition, the present invention provides the use of Geobacillus sp. strain ARM and Aneurinibacillus thermoaerophilus strain AFNA producing thermostable lipase, for industrial applications such as in food industry, oil processing, and production of surfactants, oil processing, detergents, pesticides, environmental management and leather industry. In particular the use of ARM lipase for industrial applications such as in food industry, oil processing, production of surfactants, oil processing, detergents, pesticides, environmental management and leather industry.
[Show abstract][Hide abstract] ABSTRACT: A fast and improved lipase-catalyzed synthesis of galactose oleate ester was performed in 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF 4 ]) ionic liquid with the addition of dimethylsulfoxide (DMSO) as a solubilizing agent and co-solvent; and Lipozyme RM IM (lipase from Rhizomucor miehei immobilized on macroporous anion exchange resin) as the biocatalyst. Different reaction parameters (type of solvent, type of enzyme, amount of enzyme, reaction time, temperature, stirring rate and sub-strate molar ratio) were studied. A high conversion (87%) was obtained after only 2 h at optimal synthesis conditions (1:20 DMSO:[Bmim][BF 4 ] ratio with 2% (w/w) Lipozyme RM IM, temperature 60 • C, stirring rate of 300 rpm and a molar ratio of galactose to oleic acid of 1:3).
Journal of Molecular Catalysis B Enzymatic 04/2012; 76:37-43. DOI:10.1016/j.molcatb.2011.12.004 · 2.13 Impact Factor