[show abstract][hide abstract] ABSTRACT: The Cu 2+ dependent DNA cleavage DNAzyme is the unique example of known DNAzymes. The most established method for measuring the kinetics of deoxyribozyme reactions is based on the use of radiolabeled substrates. This method entails the cumbersome sampling, electrophoretic separation, and quantitation of the reaction products, as well as the continuous loss of substrate due to decay of the radioactive label. In this study we represent accurate, fast and inexpensive methods for kinetic study of the DNAzyme which is based on spectroscopic techniques. One of these methods is based on the SYBR gold extrinsic fluorescence. This probe has high affinity to double stranded DNA compared to single stranded DNA. Continuous Hyperchromicity assay (CHA) is used in second method which is a function of DNA hypercromicity that observed when double stranded DNA transformed to single stranded DNA. Using this methods can be traced DNAzyme substrate hybridization and product formation Simultaneously with reaction progress. Under conditions where the product release faster than enzymatic reaction, production release speed which is traced with these methods determined enzymatic reaction kinetic. These methods in addition to the kinetic study of the restriction DNAzyme can be used to study of other DNAzymes that has nucleic acid substrate. Comparison of the results show that the continuous hyperchromicity assay is more accurate than the extrinsic fluorescence method using SYBR gold because CHA method is DNA intrinsic physicochemical property result without interference of disturbing factors such as SYBR gold.
1st Tabriz International Life Science Conference & 12th Iran Biophysical Chemistry Conference; 05/2013
[show abstract][hide abstract] ABSTRACT: Structure-function relationships underlying laccases properties are very limited that makes these enzymes interesting for protein engineering approaches. Therefore in the current study, a thermostable laccase that was isolated from Bacillus sp.HR03 with the ability of bilirubin oxidation beside its laccase and tyrosinase activity is used. The extensive application of this enzyme is limited by its low expression level in Escherichia coli. Based on sequence alignments and structural studies, three single amino acid substitutions, D500G, D500E, D500S and a glycine insertion, are introduced using site-directed mutagenesis to evaluate the role of Asp(500) located in the C-terminal segment close to the T1 copper center. Substitution of aspartic acid with less sterically hindered, conserved residue such as glycine increase kcat (2.3 fold) and total activity (7.3 fold) which is accompanied by a significant increase in the expression level up to 3 fold. Biochemical characterization and structural studies using far-UV CD and fluorescence spectroscopy reveals the importance of C-terminal copper-binding loop in the laccase functional expression and catalytic efficiency. Kinetic characterization of the purified mutants toward 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ) and bilirubin, shows that substrate specificity is left unchanged.
International journal of biological macromolecules 05/2013; · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Laccases (benzenediol oxygen oxidoreductases, EC 220.127.116.11) are important multicopper enzymes that are used in many biotechnological processes. A recombinant form of laccase from Bacillus sp. HR03 was overexpressed in Escherichia coli BL-21(DE3). Inclusion body (IB) formation happens quite often during recombinant protein production. Hence, developing a protocol for efficient refolding of proteins from inclusion bodies to provide large amounts of active protein could be advantageous for structural and functional studies. Here, we have tried to find an efficient method of refolding for this bacterial enzyme. Solubilization of inclusion bodies was carried out in phosphate buffer pH 7, containing 8M urea and 4mM β-mercaptoethanol and refolding was performed using the dilution method. The effect of different additives was investigated on the refolding procedure of denaturated laccase. Mix buffer (phosphate buffer and citrate buffer, 100mM) containing 4mM ZnSO4 and 100mM sorbitol was selected as an optimized refolding buffer. Also Kinetic parameters of soluble and refolded laccase were analyzed.
Enzyme and microbial technology. 05/2013; 52(6-7):325-30.
[show abstract][hide abstract] ABSTRACT: The effect of proline on refolding and unfolding kinetics as well as activity of Lipase from Pseudomonas fluorescens was determined using stopped-flow fluorescence and UV/Vis absorbance spectroscopy. Enzyme assay at different concentrations of proline revealed that activity of enzyme reaches maximum at 0.6M concentration of proline. Kinetic measurements showed that refolding rate is considerably accelerated in the presence of 0.6M proline. Unfolding kinetic traces were fitted to double exponential function, and it was revealed that lipase molecules were unfolded via two different pathways. Fast unfolding rate constant decreased, while slow one did not change significantly upon addition of proline.
International journal of biological macromolecules 01/2013; · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Like many other bacterial lipases Pseudomonas aeruginosa lipase has a very strong structure-function relationship. Herein, the effect of structural compactness of lipase has been investigated on its activity. Pseudomonas aeruginosa HR59 was isolated from burn infection as a new and inexpensive source. Bacterial lipase was extracted from this strain and purified by ion-exchange chromatography. Two divalent metal ions (calcium and magnesium) and three polar organic solvents (methanol, ethanol and iso- propanol) were used to study the structure-function relationship. Results of this study revealed that these additives activated enzyme by reducing the helix content of enzyme. Moreover, thermo stability of the enzyme decreased in the presence of calcium and magnesium ions; whereas it increased upon interaction with polar organic solvents under heat-induced denaturation. Results of this investigation encourage utilization of these activity enhancers for various medical and industrial applications.
International journal of biological macromolecules 12/2012; · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: A sequence of 10 amino acids at the C-terminus region of methylglyoxal synthase from Escherichia coli (EMGS) provides an arginine, which plays a crucial role in forming a salt bridge with a proximal aspartate residue in the neighboring subunit, consequently transferring the allosteric signal between subunits. In order to verify the role of arginine, the gene encoding MGS from a thermophile species, Thermus sp. GH5 (TMGS) lacking this arginine was cloned with an additional 30 bp sequence at the 3´-end and then expressed in form of a fusion TMGS with a 10 residual segment at the C-terminus (TMGS(+)). The resulting recombinant enzyme showed a significant increase in cooperativity towards phosphate, reflected by a change in the Hill coefficient (nH) from 1.5 to 1.99. Experiments including site directed mutagenesis for Asp-10 in TMGS and TMGS(+), two dimentional structural survey, fluorescence and irreversible thermoinactivation were carried out to confirm this pathway. [BMB Reports 2012; 45(12): 748-753].
[show abstract][hide abstract] ABSTRACT: Candida antarctica lipase B (CALB) belongs to psychrophilic lipases which hydrolyze carboxyl ester bonds at low temperatures. There have been some features reported about cold-activity of the enzyme through experimental methods, whereas there is no detailed information on its mechanism of action at molecular level. Herein, a comparative molecular dynamics simulation and essential dynamics analysis have been carried out at three temperatures (5, 35 and 50uC) to trace the dominant factors in the psychrophilic properties of CALB under cold condition. The results clearly describe the effect of temperature on CALB with meaningful differences in the flexibility of the lid region (a5 helix), covering residues 141–147. Open-closed conformations have been obtained from different sets of long-term simulations (60 ns) at 5uC gave two reproducible distinct forms of CALB. The starting open conformation became closed immediately at 35 and 50uC during 60 ns of simulation, while a sequential open-closed form was observed at 5uC. These structural alterations were resulted from a5 helical movements, where the closed conformation of active site cleft was formed by displacement of both helix and its side chains. Analysis of normal mode showed concerted motions that are involved in the movement of both a5 and a10 helices. It is suggested that the functional motions needed for lypolytic activity of CALB is constructed from short-range movement of a5, accompanied by long-range movement of the domains connected to the lid region.
PLoS ONE 07/2012; 7(7):e40327. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Beyond the preservation of genetic information in the double helix structure in cells, DNA could also have catalytic role in the single stranded form taking different 3-D structures. This artificial catalytic biomolecule is known as DNAzyme or deoxyribozyme. The Cu 2+ dependent DNA cleavage DNAzyme is the unique example of known DNAzymes. It is introduced as a restriction DNAzyme due to the site specific cleavage of single strand DNA molecule. It is also used as Cu 2+ nanobiosensor in aqueous solutions by modification of DNAzyme molecule with florescence dye. Herein, we studied the structure and catalytic function of DNAzyme using Uv-visible and extrinsic fluorescence spectroscopy. Hyperchromic and hypochromic effects of DNA have been traced by Uv-visible spectroscopy to investigate structure, hybridization phenomenon and catalytic function. Absorbance intensity at 260 nm decreases upon hybridization of DNAzyme with substrate (hypochromic effect), which increased upon addition of cofactor and starting catalytic activity (hyperchromic effect). This result confirmed the efficiency of this spectroscopic technique for kinetic and function study of the DNAzyme compared with conventional methods. The optimum pH and thermal conditions for hybridization of restriction DNAzyme and its catalytic activity was also determined. The results are well in accordance with pervious findings reported by R. R Breaker. The extrinsic fluorescence study of the DNAzyme hybridization and its catalytic function by SYBR GOLD show consistency with Uv-visible experiment results, however this technique offers higher sensitivity compared to that of Uv-visible spectroscopy. Fluorescence intensity of DNAzyme-substrate increased upon hybridization, and decreased when the catalytic activity started. Our findings suggest that spectroscopic techniques, particularly extrinsic fluorescence spectroscopy using SYBR GOLD could be a good alternative to conventional methods for studying kinetic and catalytic activity of DNAzyme, being affordable and time saving.
first international BiochemicalPhyisics congress; 06/2012
[show abstract][hide abstract] ABSTRACT: Bacillus licheniformis alpha-amylase (BLA), a thermophilic counterpart of Bacillus amyloliquefaciens alpha-amylase (BAA), is an appropriate model for the design of stabilizing mutations in BAA. BLA has 10 more histidines than BAA. Considering this prominent difference, in the present study, three out of these positions (I34, Q67, and P407; located in the thermostability determinant 1 region and Ca-III binding site of BAA) were replaced with histidine in BAA, using the site-directed mutagenesis technique. The results showed that the thermostability of P407H and Q67H mutants had increased, but no significant changes were observed in their kinetic parameters compared to that of the wild type. I34H replacement resulted in complete loss of enzyme activity. Moreover, fluorescence and circular dichroism data indicated a more rigid structure for the P407H variant compared with that of the wild-type BAA. However, the flexibility of Q67H and I34H mutants increased in comparison with that of wild-type enzyme.
Journal of Microbiology and Biotechnology 05/2012; 22(5):592-9. · 1.40 Impact Factor
[show abstract][hide abstract] ABSTRACT: The unique morphology of anisotropic rod-shaped gold nanostructures has offered new prospects for biomedical and biosensing applications. This study investigates the interaction of two types of rod-shaped nanostructures, gold nanorods and gold nanorices with lysozyme as a model protein, comparing the probable structural, activity and kinetic stability alterations. Circular dichroism spectropolarimeter revealed that lysozyme retains high fraction of its native conformation in the presence of both nanostructures, with a slight increase in the helical and beta content. Upon the protein adsorption on both types of nanorods, kinetic studies showed maintenance of enzymatic activity, together with increase in the enzymatic affinity and kinetic stability at high temperature. Comparatively, gold nanorice induced better effect on the activity and stability of enzyme than that of gold nanorod. This study might open new insight into potential applications of gold nanorods as nanocarriers for genes and drugs; provided that the toxicological aspect of cationic surfactant-coated nanostructure is taken into consideration.
International journal of biological macromolecules 04/2012; 51(1-2):91-6. · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ionic liquids (ILs) have gained increasing attention as solvents in chemical and biotechnological applications.
Since alcohol dehydrogenases are of prime interest in industrial field, the present study has been conducted to
study the influence of imidazolium based ionic liquids on kinetics, structure and stability of the thermophilic
Thermoanaerobacter brockii (TBADH) alcohol dehydrogenase. Our results exhibited that the ionic liquids could
affect kinetic parameters and stability, but not the tertiary structure. Through the determination of inhibition
profile, which revealed mixed inhibition, Ki (affinity of IL for the enzyme) and KI (affinity of IL for
enzyme–substrate complex) values were calculated. Structural analysis using crystallographic data from
protein data bank elucidated the structural details responsible for different responses of alcohol dehydrogenases
toward ionic liquids. Finally, enhanced stability in [MIm][Cl] was discussed.
Journal of Molecular Liquids 03/2012; 170:66-71. · 1.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: It is the common feature of α-amylases that calcium ion is required for their structural integrity and thermal stability. All amylases have at least one Ca(2+) per molecule; therefore amino acids involved in calcium binding are specific and conserved. In this study, sequence analysis revealed the presence of EF-hand-like motif in calcium-binding loop of Bacillus megaterium WHO (BMW)-amylase that was previously isolated from BMW. The EF-hand motif and its variants (EF-hand-like motif) are the most common calcium-binding motifs found in a large number of protein families. To investigate the effect of calcium ion on the thermal stability and activity of BMW-amylase, we used site-directed mutagenesis to replace histidine 58 with Asp (D), Ile (I), Tyr (Y), Phe (F), and Arg (R) at the seventh position of EF-hand-like motif. Upon the addition of an extra DX unit to the calcium-binding loop in H58D variant, thermal stability, catalytic activity, and chelating power of the enzyme improved due to higher affinity toward calcium. H58D variant demonstrated calcium independency compared to the wild type and other created mutants. Conformational changes in the presence and absence of Ca(2+) were monitored using fluorescence technique.
[show abstract][hide abstract] ABSTRACT: It has been lately proposed that the interaction between like-charged residues stabilizes the native state of proteins. To explore this, we created a histidine-histidine pair in the Ca-III binding site of the Bacillus amyloliquefaciens α-amylase (BAA) and then examined the impact of this pairing on the BAA. For this purpose, we used site-directed mutagenesis (SDM) to substitute Pro407 with His, Ala, Gln, Arg, and Glu in the BAA. Subsequently, thermostability, kinetic parameters and structural properties of these variants were measured. Moreover, His-His pairing effect on the BAA thermostability was examined by simultaneous mutation of two residues (P407H/H406A and P407H/H406N). The data exhibited a significant improve in thermostability and structural features of enzyme by His replacement instead of Pro407. Other substitutions in this site did not have a significant effect on the enzyme properties, except for P407R, which yielded a partial improvement. The results also showed that the thermostabilities of double mutants significantly decreased compared with that of the P407H mutant. Moreover, the thermostability of P407H remarkably increased compared with that of other variants even in the absence of Ca(2+). Our data clearly demonstrated that His406-His407 pairing was the major cause for improved thermal stability.
International journal of biological macromolecules 01/2012; 50(4):1040-7. · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Artemin is an abundant thermostable protein in Artemia encysted embryos under stress. It is considered as a stress protein, as its highly regulated expression is associated with stress resistance in this crustacea. In the present study, artemin has been shown to be a potent molecular chaperone with high efficacy. Artemin is capable of inhibiting the chemical aggregation of proteins such as carbonic anhydrase (CA) and horseradish peroxidase (HRP) at unique molar ratios of chaperone to substrates (1:40 and 1:26 for CA and HRP, respectively). Furthermore, it can also enhance refolding yield of these substrates by nearly 50%. The refolding promotion of CA is checked and verified through a sensitive fluorimetric technique. Based on these experiments, artemin showed higher chaperone activity than other chaperones. The evaluation of artemin surface using ANS showed it to be highly hydrophobic, probably resulting in its high efficacy. These results suggest that artemin can be considered a novel low molecular weight chaperone.
The Protein Journal 12/2011; 30(8):549-57. · 1.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Laccases (benzenediol oxygen oxidoreductases, EC 18.104.22.168) are used in many biotechnological processes, including removal of polyphenols in beverages, decolorizing and detoxifying effluents, drug analysis and bioremediation. In the present work, we have tried to increase thermal stability of laccase from Bacillus HR03 using site directed point mutations. Glu188 was substituted with 2 positive (Lys and Arg) and one hydrophobic (Ala) residues. All mutations showed improved thermal stability. Thermal activation of laccase was also increased after introducing the mutations. Remarkably, the Glu188Lys variant showed 3-fold higher thermal activation and higher T(50) (5 °C) with respect to the native enzyme. Furthermore steady-state k(cat) and K(m) values were influenced despite the distance between the mutated position and the catalytic site. In Glu188Arg mutation, the k(cat) was improved 3-fold and K(m) reduced by 25%. Interestingly, all three variants showed higher stability against urea as a chemical denaturant. Structural analyses of the native and mutated variants were carried out using fluorescence and far-UV circular dichroism.
Enzyme and microbial technology. 10/2011; 49(5):446-52.
[show abstract][hide abstract] ABSTRACT: Cauliflower-like DNAs are stem-loop DNAs that are fabricated periodically in inverted repetitions from deoxyribonucleic acid phosphates (dNTPs) by loop-mediated isothermal amplification (LAMP). Cauliflower-like DNAs have ladder-shape behaviors on gel electrophoresis, and increasing the time of LAMP leads to multiplying the repetitions, stem-loops, and electrophoretic bands. Cauliflower-like DNAs were fabricated via LAMP using two loop primers, two bumper primers, dNTPs, a λ-phage DNA template, and a Bst DNA polymerase in 75- and 90-min periods. These times led to manufacturing two types of cauliflower-like DNAs with different contents of inverted repetitions and stem-loops, which were clearly indicated by two comparable electrophoresis patterns in agarose gel. LAMP-fabricated DNAs and natural dsB-DNA (salmon genomic DNA) were dialyzed in Gomori phosphate buffer (10 mM, pH 7.4) to be isolated from salts, nucleotides, and primers. Dialyzed DNAs were studied using UV spectroscopy, circular dichroism spectropolarimetry, and fluorescence spectrophotometry. Structural analyses indicated reduction of the molecular ellipticity and extinction coefficients in comparison with B-DNA. Also, cauliflower-like DNAs demonstrated less intrinsic and more extrinsic fluorescence in comparison with natural DNA. The overwinding and lengthening of the cauliflower-like configurations of LAMP DNAs led to changes in physical parameters of this type of DNA in comparison with natural DNA. The results obtained introduced new biomolecular characteristics of DNA macromolecules fabricated within a LAMP process and show the effects of more inverted repeats and stem-loops, which are manufactured by lengthening the process.
Journal of biomolecular techniques: JBT 07/2011; 22(2):60-6.
[show abstract][hide abstract] ABSTRACT: The stability of enzymes with no reduction in their catalytic activity still remains a critical issue in industrial applications. Naturally occurring osmolytes are commonly used as protein stabilizers. In this study we have investigated the effects of sorbitol and trehalose on the structural stability and activity of Pseudomonas cepacia lipase (PCL), using UV-visible, circular dichroism (CD) and fluorescence spectroscopy. Surface plasmon resonance (SPR) technique was used to trace changes in the refractive index and dielectric constant of the environment. The results revealed that catalytic activity and intrinsic fluorescence intensity of PCL increased in the presence of both osmolytes. Far-UV CD spectra indicated that the protein has undergone some conformational changes upon interacting with these osmolytes. Increasing the concentration of sorbitol led to changes in the refractive index and consequently the dielectric constant of environment; whereas in the case of trehalose, such changes were not significant. Unfavorable interactions of trehalose with protein surface induced higher preferential exclusion from the enzyme-water interface than that of sorbitol. Results of this report could give further insights about the stabilization mechanism of osmolytes.
International journal of biological macromolecules 07/2011; 49(4):652-6. · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gold nanorods, with their unique morphology and optical properties have offered new prospects for biomedical and biosensing applications. Herein, the interaction between gold nanorods and a model protein has been monitored using spectroscopic techniques. The enzyme retains high fraction of its native structure with a slight increase in the helical content at the expense of β-turns. Kinetic investigations revealed notable increase of enzyme affinity for substrate without significant decrease in the V(max). Emission spectra of tryptophan residues in the presence of chaotropic agent highlighted the maintenance of internal quenching due to the induced compactness. Comparison of the gold nanorod treated lysozyme with free enzyme revealed higher thermodynamic stability under denaturing condition. Results from this study encourage the possibility of utilizing gold nanorods as promising nanocarrier candidates for a new generation of drug delivery applications.
International journal of biological macromolecules 06/2011; 49(4):629-36. · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: DNA nanotechnology is a novel approach for synthesis of DNA-based nanostructures. Stem-loops, nanojunctions, sticky-ends and periodic lengths of DNA are the most essential nanostructures in DNA nanofabrications. Loop-mediated isothermal amplification (LAMP) is a powerful technology for repetitive synthesis of double-stranded and cauliflower-like DNAs. The process leads to long and repetitive sequences of DNAs, which are fabricated via loop primers. The authors demonstrate here scanning tunneling micrographs of LAMP-synthesised DNAs deposited on highly ordered pyrolytic graphite. The scans are compared with natural DNAs. Scanning tunneling microscopy (STM) images indicated the creation of periodic long DNAs, stem-looped DNAs and three-way DNA nanojunctions. It is also suggested that such nanomaterials could be promising candidates for use in DNA-based nanodevices.
IET Nanobiotechnology 03/2011; 5(1):8. · 1.00 Impact Factor