[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 01/2012; 13(8):9673-91. · 2.46 Impact Factor
[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 01/2012; 76:37-43. · 2.82 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 01/2012; 7(11):e49788. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Electrochemical DNA biosensor was successfully
developed by depositing the ionic liquid (e.g., 1-ethyl-3-
methylimidazolium trifluoromethanesulfonate ([EMIM]
[Otf])), ZnO nanoparticles, and chitosan (CHIT) nanocomposite
membrane on a modified gold electrode (AuE). The
electrochemical properties of the [EMIM][Otf]/ZnO/CHIT/
AuE for detection of DNA hybridization were studied. Under
optimal conditions using cyclic voltammetry, the target DNA
sequences could be detected in the concentration range of
1.0×10−18 to 1.82×10−4 mol L−1, and with the detection limit of 1.0×10−19 mol L−1. This DNA biosensor detection
approaches provide a quick, sensitive, and convenient method
to be used in the identification of Trichoderma harzianum
Journal of Solid State Electrochemistry 01/2012; 16(1-2.234):273-282. · 2.28 Impact Factor
[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 01/2012; 13(7):9207-17. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oleyl Esters (OEs) are newly synthesized esters from different fatty acids of different chain length and oleyl alcohol using Novozyme 435 as catalyst. The phase behavior of these esters were determined by constructing the pseudoternaryphase diagrams of OEs/Tween 80/water at 25.0 ± 0.5°C. Compositions from the isotropic and homogeneous region were selected for characterization.The shortest OEs chain represents the most stable nanoemusions system with the smallest droplet for both isotropic and homogeneous regions.The results from simulation study showed that the shape of emulsion droplet was in spherical shape with the values of eccentricity (e)0.11 to 0.17.
Journal of Dispersion Science and Technology - J DISPER SCI TECH. 01/2012;
[Show abstract][Hide abstract] ABSTRACT: A thermophilic Bacillus stearothermophilus F1 produces an extremely thermostable serine protease. The F1 protease sequence was used to predict its three-dimensional (3D) structure to provide better insights into the relationship between the protein structure and biological function and to identify opportunities for protein engineering. The final model was evaluated to ensure its accuracy using three independent methods: Procheck, Verify3D, and Errat. The predicted 3D structure of F1 protease was compared with the crystal structure of serine proteases from mesophilic bacteria and archaea, and led to the identification of features that were related to protein stabilization. Higher thermostability correlated with an increased number of residues that were involved in ion pairs or networks of ion pairs. Therefore, the mutants W200R and D58S were designed using site-directed mutagenesis to investigate F1 protease stability. The effects of addition and disruption of ion pair networks on the activity and various stabilities of mutant F1 proteases were compared with those of the wild-type F1 protease.
Journal of Microbiology and Biotechnology 01/2012; 22(1):34-45. · 1.40 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 01/2012; 13(9):11666-80. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ergosteryl oleate, a sterol ester, was synthesized using Lipozyme TLIM (immobilized Thermomyces lanuginose lipase) and Novozym 435 (immobilized Candida antarctica lipase B) as the biocatalysts for the reaction. Response surface methodology (RSM) with a three-factor-five-level central composite rotatable design (CCRD) was employed to study and optimize the reaction conditions. The effect of three main reaction parameters including time, temperature, and amount of enzyme on the synthesis of ester was analyzed. A modified two factorial model for Novozym 435, and a modified quadratic polynomial model for Lipozyme TLIM were fitted to the data with an R2 of 0.9861 and 0.9401, respectively. Temperature was the most significant parameter in the synthesis of ester by both enzymes. A high temperature was required for Lipozyme TLIM to obtain a high subsrate conversion whereas Novozym 435 worked best at lower temperatures. Using Lipozyme TLIM resulted in a higher ester yield (72.4%) compared to Novozym 435 (61.6%). In addition, required enzyme amount and time was lower in Lipozyme TLIM-catalyzed reaction. A good correspondence was observed between the actual yields and values predicted by the statistical models. The generated model can be applied to predict the ester yield within the given range of effective parameters.
Biocatalysis and Agricultural Biotechnology. 01/2012; 1(1):51–56.
[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 01/2012; 79:61-65. · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Palm oil esters (POEs), which are newly synthetic liquid wax esters with HLB value of 9.34, were proposed as a lipophilic phase for formation of nanoemulsion. Phase diagrams show domination of two-phase regions at all emulsification temperatures ranging from 30 to 80°C. Spontaneous and temperature-induced emulsification, high-shear and high pressure homogenization were utilized to form nanoemulsion. However, only high pressure homogenization successfully produced droplets sizes in the nano range. Thus, it was used to optimize the stability properties of POEs nanoemulsion. The manipulation of processing temperatures during the formation of emulsions could be used in lowering the droplets size of the emulsion.
Journal of Dispersion Science and Technology 01/2012; 33. · 0.60 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 01/2012; 13(8):9572-83. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A nano-emulsion system was developed for pesticide formulation. Pseudoternary phase diagrams were constructed consisting emulsion system of long-chain fatty acid methyl esters (LFAMEs)/mixed surfactant/water and a quarternary component, glyphosate isopropylamine (IPA) as a herbicide active. Isotropic (L) regions were formed in the phase diagrams using mixed surfactant long-chain alkylpolyglucosides (LAPG) and ethoxylated 3-(3-hydroxypropyl)-heptamethyltrisiloxane (organosilicone) at the ratios of 9:1, 8:2 and 7:3. Pre-formulation concentrates were chosen from the L regions with less than 20% (w/w) of inerts (LFAMEs + mixed surfactant) and were characterized with regard to particle size, particle aging rate and thermostability study. A pre-formulation concentrate with the lowest aging rate and stable at high temperature (54 °C) was selected for the mechanisms study of the pre-formulation concentrate in conjunction with the development of nano-emulsion formulation. The transmission electron microscopy (TEM) result showed that the pre-formulation concentrate appeared as a polymerized multi-connected network. Upon water dilution of the pre-formulation concentrate with gentle stirring (low-energy emulsification method), well-dispersed nanoparticles were formed with no needle structure being observed. The nano-emulsion particles were incorporated well with the glyphosate IPA thus inferring that this nano-emulsion system could ameliorate the bioactivity and bioavailability of the herbicide.
Industrial Crops and Products 11/2011; 36:607-613. · 3.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This article describes the development of environmentally friendly nano-emulsion system for water-soluble herbicide application. Pseudoternary phase diagrams were established in the emulsion system of fatty acid methyl esters (FAMEs)/alkylpolyglucosides (APG) and/or 3-(3-hydroxypropyl)-heptamethyltrisiloxane (organosilicone)/water encompassed with 41% (w/w) glyphosate isopropylamine (IPA) as herbicide active. Pre-formulations were selected from isotropic (L) region in the phase diagrams and their emulsion system characteristics were determined. The microemulsion systems were chosen and then dispersed into water using low-energy stirring method (200rpm for 5min). Oil-in-water (O/W) nano-emulsions were formed with particle sizes of diameter less than 200nm. The nano-emulsion systems showed significantly lower surface tension than a commercial formulation (Roundup®). In the biological application study, treatments of nano-emulsion formulations and Roundup® were applied on narrow-leaved weed Eleusine indica. Multiple doses of glyphosate IPA of the treatments were applied for the construction of dose–response curves for determination of effective dose (ED50). The nano-emulsion formulation showed lower ED50 was 0.40kg a.e./ha in controlling the weed than Roundup® was 0.48kg a.e./ha. This finding suggested that the possibility of using nano-emulsion system to increase penetration and uptake of glyphosate IPA.
Pesticide Biochemistry and Physiology 10/2011; 102:19-29. · 2.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The utilization of natural mica as a biocatalyst support in kinetic investigations is first described in this study. The formation of lactose caprate from lactose sugar and capric acid, using free lipase (free-CRL) and lipase immobilized on nanoporous mica (NER-CRL) as a biocatalyst, was evaluated through a kinetic study. The apparent kinetic parameters, K(m) and V(max), were determined by means of the Michaelis-Menten kinetic model. The Ping-Pong Bi-Bi mechanism with single substrate inhibition was adopted as it best explains the experimental findings. The kinetic results show lower K(m) values with NER-CRL than with free-CRL, indicating the higher affinity of NER-CRL towards both substrates at the maximum reaction velocity (V(max,app)>V(max)). The kinetic parameters deduced from this model were used to simulate reaction rate data which were in close agreement with the experimental values.
Bioscience Biotechnology and Biochemistry 08/2011; 75(8):1446-50. · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incubation with 1.0mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092 U mg(-1)), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65°C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50°C for more than 150 min.
[Show abstract][Hide abstract] ABSTRACT: Electrochemical DNA biosensor was fabricated using the ZnO nanoparticles/chitosan (CHIT) nanocomposite
membrane on modified gold electrode (AuE) as the working electrode. The ZnO/CHIT was used as a modified-AuE for
the immobilization of the single-stranded DNA probe. This particular DNA biosensor provided some advantages such as
the biocompatibility of the ZnO nanoparticles, good film forming ability of CHIT, and the high conductivity of AuE.
Methylene blue was used as the electrochemical indicator for monitoring the hybridization reaction following the
hybridization of the target DNA sequence. Differential pulse voltammetry was used for recording the electrochemical
response of MB. The specific target DNA sequence could be detected in the concentration range of 1.0 x 10-14 to 1.82 x
10-4 mol L-1, with the detection limit at 1.0 x 10-15 mol L-1. This novel approach of constructing an electrochemical
biosensor allowed the hybridization of synthetic target DNA. In addition, it also facilitated hybridization with template—
DNA taken from real samples. The results proved that the ZnO/CHIT/AuE electrode has the potential for the sensitive
detection of specific sequence related to a Trichoderma harzianum gene.
Current Analytical Chemistry 07/2011; 7(2.134):296- 305. · 1.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aneurinibacillus thermoaerophilus strain AFNA as a novel isolated extracellular thermostable organic solvent tolerant lipase producing bacterium was employed in the present study. The lipase production of strain AFNA and its correlation with bacterial growth was studied via a modeling assessment by response surface methodology (RSM) and artificial neural network (ANN) techniques. The best achieved models were multilayer full feed forward incremental back propagation network and modified cubic response surface model (mRSM) using backward elimination. The highest lipase specific activity (13.1 Umg-1) and bacterial growth (OD600 = 3.0) were obtained at technically similar: growth temperature (53 and 53ºC), inoculum size (2.6 and 3.0%), agitation rate (118 and 115 rpm) and initial pH (7.0 and 7.2) but different medium volume (139 and 87 ml) and incubation period (48 and 38 hrs), respectively. In addition, the importance of effective parameters on the bacterial growth and lipase production was studied where pH and inoculum size were the most and the least effective factors, respectively. Significant correlation between lipase production and bacterial growth was observed when Bivariate correlation was employed to analyse the data. As a conclusion, lipase production was the result of a synergistic combination of effective parameters interactions and these parameters were in equilibrium.
Electronic Journal of Biotechnology 06/2011; 14(4). · 0.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant, and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at OD(600) induction time of 0.5 was found to be the best condition for chalcone synthase secretion.
Applied biochemistry and biotechnology 06/2011; 165(2):737-47. · 1.94 Impact Factor