[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 three-electrode system amperometric biosensor that consisted of screen-printed carbon paste working electrode (SPE), a commercial platinum rod counter electrode vs Ag/AgCl reference electrode was developed for rapid determination of histamine in prawns. The biosensor was designed from entrapping diamine oxidase (DAO) enzyme in a poly(2-hydroxyethyl methacrylate) (photoHEMA) film prepared via a simple and one-step direct photocuring process on a carbon paste screen-printed electrode (SPE). The photoHEMA film exhibited water absorption of 34.14% after four hours exposure to water and no leaching of DAO was observed from the hydrogel film. The histamine biosensor showed response time of < 50 s with a linear response range from 0 to 60 ppm histamine (R2 of 0.9946). The sensitivity of the biosensor was of 5.56 nAppm-1, with a limit detection of 0.65 ppm histamine. The performance of the fabricated biosensor in the analysis of histamine in tiger prawn (Penaeus monodon) samples was comparable to high performance liquid chromatography (HPLC). The three-electrode system was then converted to an all-screen-printed histamine biosensor by printing onto a polyester substrate with carbon paste to form the working and counter electrodes and Ag/AgCl paste as the reference electrode. The performance of all-screen-printed histamine biosensor was evaluated using potassium hexacyanoferrate (III) as a mediator deposited electrochemically on the carbon-paste SPE. The presence of this mediator demonstrated improvement to the response of the allscreen- printed histamine biosensor.
International journal of electrochemical science 04/2012; 7(2.808):4702 - 4715. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enzyme immobilisation technology is an effective means to improve sugar ester production through the employment of biocatalysts. In the present study, immobilisation of Candida rugosa (CRL) lipase onto amino-activated mica is performed via covalent bonding (namely Amino-CRL) and the cross-linking of lipases into nano-reactors through physical adsorption (namely NER-CRL). Free and immobilised lipases were tested for their esterification activities. Specific activities for Amino-CRL and NER-CRL increased by 2.4 and 2.6-fold, respectively, upon immobilisation. Extending this work, immobilised lipases have novel capabilities in the synthesis of sugar esters. The optimised conditions for sugar fatty acid ester syntheses are 48 h at 2:1 of molar ratio of lactose sugar to capric acid at 55 °C. Furthermore, a high operational stability with half-lives of over 13 and 10 runs was achieved for NER-CRL and Amino-CRL, respectively, indicating the efficiency of the immobilisation process.
[Show abstract][Hide abstract] ABSTRACT: A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 °C and showed stability over a temperature range of 40-60 °C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8-9. Metal ions such as Ca(2+), Mn(2+), Na(+), and K(+) enhanced the lipase activity, but Mg(2+), Zn(2+), and Fe(2+) inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate inhibition was observed in the presence of PMSF at a similar concentration, indicating that serine residues are involved in its catalytic action. Further, the activity was not impaired by water-miscible solvents, including methanol, ethanol, and acetone.
The Protein Journal 02/2012; 31(3):229-37. · 1.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The T(m) for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 °C, 70.59 °C and 68.54 °C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher T(m) as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 Å using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 Å, b = 81.16 Å and c = 100.14 Å. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically.
International Journal of Molecular Sciences 01/2012; 13(1):943-60. · 2.46 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 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. · 3.53 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: 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