Electrochemistry of complex formation of carbaryl with ds-DNA using [Ru(bpy)2dppz]2+ as probe

An electrochemical competition method was used to study the interaction of carbaryl with natural double-stranded DNA (ds-DNA). Layer-by-layer films of negatively charged natural ds-DNA and polycationic poly (diallyldimethylammonium chloride) were assembled on the surface of a glassy carbon electrode. The DNA intercalator [Ru(bpy)₂(dppz)]²⁺ (bpy?=?2, 2′-bipyridine, dppz?=?dipyrido [3, 2-a: 2′,3′-c] phenazine) was chosen as an electrochemical probe. Tripropylamine was used as an electron donor to chemically amplify the oxidation current of the probe. In order to examine the effects of substituting group on the binding interaction of carbaryl with DNA, the interaction of naphthalene or α-naphthol with DNA was also studied by square wave voltammetry (SWV). The values of binding constant K _b of the three compounds to DNA are determined, which fall in the range of (0.2?×?10⁵) to (1.3?×?10⁵)?M^?1. The correlation suggests that the functional groups may play an important role in the DNA/analyte competition binding interaction. We demonstrated that it is conducive to the combination of small molecules and DNA when the functional groups are hydrophobic and have the lone-pair electrons as the electron donor. Furthermore, UV-absorption and fluorescence intensity of Ru-dppz decreases in the presence of carbaryl. These characteristics strongly support the intercalation of carbaryl into double-stranded DNA.     

Human Saliva-Based Quantitative Monitoring of Clarithromycin by Flow Injection Chemiluminescence Analysis: A Pharmacokinetic Study

Human saliva quantitative monitoring of clarithromycin (CLA) by chemiluminescence (CL) with flow injection analysis was proposed for the first time, which was based on the quenching effect of CLA on luminol–bovine serum albumin (BSA) CL system with a linear range from 7.5?×?10^?4 to 2.0 ng/ml. This proposed approach, offering a maximum sample throughput of 100 h^?1, was successfully applied to the quantitative monitoring of CLA levels in human saliva during 24 h after a single oral dose of 250 mg intake, with recoveries of 95.2～109.0 % and relative standard deviations lower than 6.5 % (N?=?7). Results showed that CLA reached maximum concentration of 2.28?±?0.02 μg/ml at approximately 3 h, and the total elimination ratio was 99.6 % in 24 h. The pharmacokinetic parameters including absorption rate constant (0.058?±?0.006 h^?1), elimination rate constant (0.149?±?0.009 h^?1) and elimination half-life time (4.66?±?0.08 h) were obtained. A comparison of human saliva and urine monitoring was also given. The mechanism study of BSA–CLA interaction revealed the binding of CLA to BSA is an entropy driven and spontaneous process through hydrophobic interaction, with binding constant K _BSA–CLA of 4.78?×?10⁶ l/mol and the number of binding sites n of 0.82 by flow injection–chemiluminescence model. Molecular docking analysis further showed CLA might be in subdomain IIA of BSA, with K _BSA–CLA of 6.82?×?10⁵ l/mol and ΔG of ?33.28 kJ/mol.     

Synthesis,characterization, molecular modeling,and DNA interaction studies of a Cu(II) complex containing drug of chronic hepatitis B: adefovir dipivoxil

A new copper(II) complex [Cu(adefovir)₂Cl₂], where adefovir = adefovir dipivoxil drug, was synthesized and characterized by using different physicochemical methods. Binding interaction of this complex with calf thymus DNA (ct-DNA) has been investigated by multi-spectroscopic techniques and molecular modeling study. The complex displays significant binding properties of ct-DNA. The results of fluorescence and UV–vis absorption spectroscopy indicated that, this complex interacted with ct-DNA in a groove-binding mode, and the binding constant was 4.3(±0.2) × 10⁴ M^?1. The fluorimeteric studies showed that the reaction between the complex and ct-DNA is exothermic (ΔH = 73.91 kJ M^?1; ΔS = 357.83 J M^?1 K^?1). Furthermore, the complex induces detectable changes in the CD spectrum of ct-DNA and slightly increases its viscosity which verified the groove-binding mode. The molecular modeling results illustrated that the complex strongly binds to the groove of DNA by relative binding energy of the docked structure ?5.74 kcal M^?1. All experimental and molecular modeling results showed that the Cu(II) complex binds to DNA by a groove-binding mode.     

Expression,Purification, and Characterization of NADP+-Dependent Malic Enzyme from the Oleaginous Fungus Mortierella Alpina

Malic enzymes are a class of oxidative decarboxylases that catalyze the oxidative decarboxylation of malate to pyruvate and carbon dioxide, with concomitant reduction of NAD(P)⁺ to NAD(P)H. The NADP⁺-dependent malic enzyme in oleaginous fungi plays a key role in fatty acid biosynthesis. In this study, the malic enzyme-encoding complementary DNA (cDNA) (malE1) from the oleaginous fungus Mortierella alpina was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant protein (MaME) was purified using Ni-NTA affinity chromatography. The purified enzyme used NADP⁺ as the cofactor. The K _m values for l-malate and NADP⁺ were 2.19?±?0.01 and 0.38?±?0.02 mM, respectively, while the V _max values were 147?±?2 and 302?±?14 U/mg, respectively, at the optimal condition of pH 7.5 and 33 °C. MaME is active in the presence of Mn²⁺, Mg²⁺, Co²⁺, Ni²⁺, and low concentrations of Zn²⁺ rather than Ca²⁺, Cu²⁺, or high concentrations of Zn²⁺. Oxaloacetic acid and glyoxylate inhibited the MaME activity by competing with malate, and their K _i values were 0.08 and 0.6 mM, respectively.     

Spectroscopy studies on DNA binding of two ruthenium complexes [Ru(MeIm)4(L)]2+ (L = iip and tip)

Two ruthenium(II) complexes [Ru(MeIm)₄(L)]²⁺ (L?=?2-(imidazo-4-group)-1H-imidazo-[4,5-f][1,10]phenanthroline, 2-(thiophene-2-group)-1H-imidazo[4,5-f][1,10]phenanthroline, MeIm?=?1-methylimidazole) have been synthesized according to literature and structurally characterized. The interaction of the complexes with calf thymus DNA has been explored using electronic absorption titration, competitive binding experiment, circular dichroism, thermal denaturation, and viscosity measurements. The results show that both complexes could bind DNA in a intercalation mode and the DNA-binding affinity of [Ru(MeIm)₄(tip)]²⁺ (K _b?=?(7.2?±?0.3)?×?10⁵?(mol?L^?1)^?1) is greater than that of [Ru(MeIm)₄(iip)]²⁺ (K _b?=?(6.1?±?0.2)?×?10⁵?(mol?L^?1)^?1).     

Binding interaction and conformational changes of human serum albumin with ranitidine studied by spectroscopic and time-resolved fluorescence methods

This study was designed to examine the interaction of histamine H₂-receptor antagonist drug ranitidine (RTN) with human serum albumin by multi-spectroscopic methods. The experimental results showed the involvement of dynamic quenching mechanism which was further confirmed by lifetime spectral studies. The binding constants (K _a) at three temperatures (288, 298, and 308 K) were 2.058 ± 0.020, 4.160 ± 0.010 and 6.801 ± 0.011 × 10⁴ dm³ mol^?1, respectively, and the number of binding sites (m) were 1.169, respectively; thermodynamic parameters ΔH ⁰ (44.152 ± 0.047 kJ mol^?1), ΔG ⁰ (?26.214 ± 0.040 kJ mol^?1), and ΔS ⁰ (236.130 ± 0.025 J K^?1 mol^?1) were calculated. The distance r between donor and acceptor was obtained (r = 3.40 nm) according to the Förster theory of non-radiative energy transfer. Synchronous fluorescence, CD, AFM and 3D fluorescence spectral results revealed the changes in secondary structure of the protein upon interaction with RTN. A molecular modeling study further confirmed the binding mode obtained by the experimental studies.     

Chemical Modification of Ethyl Cellulose-Based Highly Porous Membrane for the Purification of Immunoglobulin G

The chemical modification of developed ethyl cellulose-based membrane was carried out to make it suitable for bioseparation. The different reagents were used for the modification of membrane to couple protein A (PA) to study the purification of immunoglobulin G (IgG) from blood. The chemical modification was carried out using relatively simple and mild reaction conditions. The attenuated total reflectance Fourier transform infrared analysis of chemically modified membrane showed new peak at 1,596.06 and 1,716.49 cm^?1. The scanning electron microscopy of PA-coupled membrane, which was used for IgG purification showed open pores and 950?±?21.5 LMH (L?m^?2?h^?1) operational flux at 0.5-bar out pressure. The flux of unmodified membrane was 1,746?±?18.5 LMH at 0.5-bar out pressure. The equilibrium adsorption concentration (318.5?±?5.9 μg?cm^?2) was obtained at 3 h. The adsorption character of PA-coupled membrane was consistent with the Langmuir adsorption model and the non-specific binding was 67.08?±?1.3 μg?cm^?2. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed similar purification pattern for purified IgG from human serum and commercial preparation of IgG. All the results have suggested a high potential of PA-coupled ethyl cellulose-based membrane for large-scale purification of IgG.     

Direct electrochemistry and electrochemical biosensing of glucose oxidase based on CdSe@CdS quantum dots and MWNT-modified electrode

The direct electron transfer of glucose oxidase (GOx) was achieved based on the immobilization of CdSe@CdS quantum dots on glassy carbon electrode by multi-wall carbon nanotubes (MWNTs)-chitosan (Chit) film. The immobilized GOx displayed a pair of well-defined and reversible redox peaks with a formal potential (E ^θ’) of ?0.459 V (versus Ag/AgCl) in 0.1 M pH 7.0 phosphate buffer solution. The apparent heterogeneous electron transfer rate constants (k _s) of GOx confined in MWNTs-Chit/CdSe@CdS membrane were evaluated as 1.56 s^?1 according to Laviron's equation. The surface concentration (Γ*) of the electroactive GOx in the MWNTs-Chit film was estimated to be (6.52?±?0.01)?×?10^?11?mol?cm^?2. Meanwhile, the catalytic ability of GOx toward the oxidation of glucose was studied. Its apparent Michaelis–Menten constant for glucose was 0.46?±?0.01 mM, showing a good affinity. The linear range for glucose determination was from 1.6?×?10^?4 to 5.6?×?10^?3?M with a relatively high sensitivity of 31.13?±?0.02 μA?mM^?1?cm^?2 and a detection limit of 2.5?×?10^?5?M (S/N=3).     

Antibacterial,nuclease, and SOD-mimic behaviors of copper(II) complexes of norfloxacin and phenanthrolines

Square-pyramidal complexes [Cu(NFL)(A ⁿ )Cl]?·?5H₂O (A ^{n ?}=?phenanthroline derivatives and NFL?=?deprotonated norfloxacin) have been synthesized and characterized. Interactions with Herring Sperm DNA and pUC19 DNA have been investigated. Mode and extent of interaction was measured by the perturbation in absorbance of complexes in the absence and presence of DNA. Hydrodynamic volume change and gel electrophoretic results were also kept under consideration. Synthesized complexes bind to DNA via intercalation with binding constant 0.875–1.446?×?10⁴?(mol?L^?1)^?1 based on bathochromism and hypochromism observed. Intercalative binding of complexes with DNA was further supported by relative viscosity, where 5 intercalates more strongly with most increase in relative viscosity, and K _b value of 1.446?×?10⁴?(mol?L^?1)^?1. Evaluation of electrophoretic separation of plasmid on agarose gel reveals that 5 cleaves more efficiently. Square-pyramidal geometry at the metal center supports superoxide-dismutase (SOD)-mimic behavior in addition to an electron-withdrawing group on the ancillary ligand stabilizing Cu–O bonding.     

A systematic capillary electrophoresis study on the effect of the buffer composition on the reactivity of the anticancer drug cisplatin to the DNA model 2′-deoxyguanosine 5′-monophosphate (dGMP)

The development of DNA-targeted next-generation platinum-based anticancer chemotherapeutics is often accompanied by studies on the reactivity to DNA models. However, the incubation conditions used in literature vary widely, and some of the buffer/salts used are known to form complexes with Pt. Such coordination can influence the binding process and also the adducts formed. In a systematic approach, studies on the binding of cisplatin (1 mM) to dGMP (2 mM) in a series of different incubation solutions of relevance to biological systems are reported, employing capillary zone electrophoresis (CZE) with UV/vis and electrospray ionization–mass spectrometric (ESI-MS) detectors. Kinetic experiments performed with CZE–UV showed a high reactivity of dGMP to cisplatin in pure water (τ _1/2?=?4.1?±?0.7 h) but a significantly slowed down in a solution containing a carbonate/phosphate buffer supplemented with NaCl, resulting in a half-life of dGMP of 25?±?3 h. Especially carbonate had a major impact on the binding, though no coordination to the metal center was detectable with the methods used. The only adducts containing buffer components were (phosphate)Pt and tris(ammine)Pt species, as identified by means of CZE–ESI-MS, in addition to the main adduct [Pt(NH₃)₂(dGMP)₂???4H⁺]^2? and other less abundant Pt-containing species.     

Plant Regeneration Through Callus Organogenesis and True-to-Type Conformity of Plants by RAPD Analysis in Desmodium gangeticum (Linn.) DC.

An efficient plant regeneration protocol was established for an endangered ethnomedicinal plant Desmodium gangeticum (Linn.) DC. Morphogenic calli were produced from 96 % of the cultures comprising the immature leaf explants on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (4.0 mg?l^?1) in combination with 6-benzylaminopurine (BA; 0.8 mg?l^?1). For callus regeneration, various concentrations of BA (1.0–5.0 mg?l^?1) or thidiazuron (TDZ; 1.0–5.0 mg?l^?1) alone or in combination with indole-3-acetic acid (IAA; 0.2–1.0 mg?l^?1) were used. Highest response of shoot regeneration was observed on MS medium fortified with TDZ (4.0 mg?l^?1) and IAA (0.5 mg?l^?1) combination. Here, 100 % cultures responded with an average number of 22.3 shoots per gram calli. Inclusion of indole-3-butyric acid in half MS medium favored rooting of recovered shoots. Out of 45 rooted plants transferred to soil, 40 survived. Total DNA was extracted from the leaves of the acclimatized plants of D. gangeticum. Analysis of random amplified polymorphic DNA using 13 arbitrary decanucleotide primers showed the genetic homogeneity in all the ten plants regenerated from callus with parental plant, suggesting that shoot regeneration from callus could be used for the true-to-type multiplication of this plant.     

Cell-Free Supernatants Obtained from Fermentation of Cheese Whey Hydrolyzates and Phenylpyruvic Acid by Lactobacillus plantarum as a Source of Antimicrobial Compounds, Bacteriocins, and Natural Aromas

Cheese whey hydrolyzates supplemented with phenylpyruvic acid (PPA) and commercial nutrients can be efficiently metabolized by Lactobacillus plantarum CECT-221 to biosynthesize some compounds with attractive applications in the food market. The main metabolites of cell-free extracts were antimicrobial compounds such as phenyllactic acid (PLA) and lactic acid (LA). The production of PLA by L. plantarum CECT-221 was evaluated in the Man–Rogosa–Sharpe broth supplemented with two biosynthetic precursors: phenylalanine or PPA. Using 30.5 mM PPA, the microorganism increased sevenfold the concentration of PLA producing 16.4 mM PLA in 46 h. A concentration of 40 mM PPA was a threshold to avoid substrate inhibition. The biosynthesis of whey hydrolyzates as a carbon source was enhanced by fed-batch fermentation of PPA; the average productivity of PLA increased up to 45.4?±?3.02 mM after 120 h with a product yield of 0.244 mM mM^?1; meanwhile, LA reached 26.1?±?1.3 g L^?1 with a product yield of 0.72 g g^?1. Cell-free fed-batch extracts charged in wells showed bacteriocin activity with halos of 7.49?±?1.44 mm in plates inoculated with Carnobacterium piscicola and antimicrobial activity against Staphylococcus aureus (11.54?±?1.14 mm), Pseudomonas aeruginosa (10.17?±?2.46 mm), Listeria monocytogenes (7.75?±?1.31 mm), and Salmonella enterica (3.60?±?1.52 mm). Additionally, the analysis of the volatile composition of the headspace of this cell-free extract revealed that L. plantarum is a potential producer for natural aromas, such as acetophenone, with high price in the market. This is the first report of PLA production from cheese whey and PPA. The extracts showed bacteriocin activity and potential to be applied as an antimicrobial in the elaboration of safer foods.     

Synthesis,structure, spectroscopic,and DNA binding properties of Co(III) and Ni(II) complexes with ((E)-2-(amino((pyridin-2-ylmethylene)amino)methylene)maleonitrile)

Two new complexes, [Co(L)₂]Cl·(MeOH)₂ (1) and [Ni(L)₂]₄·EtOH (2) (L?=?(E)-2-(amino((pyridin-2-ylmethylene)amino)methylene)maleonitrile), were synthesized and characterized by X-ray crystallography, IR, UV, and fluorescence spectroscopy. According to X-ray crystallographic studies, each metal was six-coordinate with six nitrogens from two ligands. Both complexes form two-dimensional supramolecular networks via hydrogen bonding and π–π interactions. Ultraviolet and visible spectra showed that absorptions arise from π–π ^?, MLCT, and d–d electron transitions. Fluorescence spectroscopy revealed moderate intercalative binding of these two complexes with EB–DNA, with apparent binding constant (K _app) values of 9.14?×?10⁵ and 3.20?×?10^5?M^?1 for Co(III) and Ni(II) complexes, respectively. UV–visible absorption spectra showed that the absorption of DNA at 260?nm was quenched for 2 but quenched then improved for 1 with addition of complexes, tentatively attributed to the effect of the combined intercalative binding and electrostatic interaction for 1.     

Complexation of p-Sulphonato-calix[6]arene by Glycine, Glycyl-glycine, and Glycyl-glycyl-glycine in Aqueous Solution

The complexing ability of p-sulphonato-calix[6]arene towards glycine, glycyl-glycine, and glycyl-glycyl-glycine has been evaluated at pH?=?1.8 and 7.9 using UV?CVis spectrophotometry. At these pHs the guest molecules are in their cationic and zwitterionic forms, respectively. The results showed that the host is capable of complexing with the guests in 1:1 guest-to-host ratios. Formation constants of the systems have been determined at different temperatures (20?±?0.1 to 40?±?0.1?°C). Considering the formation constant values, the binding selectivity of the host towards the guests is in the order glycyl-glycyl-glycine?>?glycyl-glycine?>?glycine. The thermodynamic parameters have been evaluated and are interpreted in terms of the importance of the various interactions responsible for the complexation. A roughly linear relationship between ??H ^o and T??S ^o has been observed for the studied systems and is discussed.     

Cytochrome c embraced in sodium dodecyl sulfate nano-micelle as a homogeneous nanostructured peroxidase

A homogeneous nanostructured enzyme (artificial peroxidase, AP) with suitable catalytic efficiency was generated using bovine heart cytochrome c (Cyt c) and sodium dodecyl sulfate nano-micelles in 50?mM phosphate buffer pH 10.5 at 25?°C. The Michaelis?CMenten (K _m) and catalytic rate (k _cat) of the AP were determined to be 21.6?±?1.2???M and 0.474?±?0.013?s^?1, respectively. The catalytic efficiency of the AP was 0.0219?±?0.002???M^?1s^?1, which was 30?±?1.5?% as efficient as the native horseradish peroxidase (HRP). The mean diameter of AP was measured to be 6.4?nm using dynamic light scattering technique. The UV?CVis spectrometry, circular dichroism, surface tension, isothermal titration calorimetric and electrochemistry methods were utilized for additional characterization of the AP. Together our results suggest that the AP generated here can be used in place of HRP in industrial and commercial fields under some extreme conditions.     

Adsorption and thermodynamics of biosurfactant,surfactin, monolayers at the air-buffered liquid interface

Adsorption of surfactin, a powerful lipopeptide biosurfactant, at the air-liquid interface has been investigated in this article. The adsorption took place from buffered solutions containing relatively high concentrations of surfactin co- and counterions. Dynamic surface tension measurements were used to follow the self-assembly of surfactin at the interface until equilibrium surface pressure Π ^e is reached at a given surfactin concentration (C _s). Gibbs adsorption equation in conjunction with the Langmuir adsorption isotherm was used to predict surfactin surface excess as a function of the biosurfactant concentration up to the critical micelle concentration (CMC). The predicted surface excess at saturation (Γ _∞ ) is 1.05?±?0.05 μmol m^?2, corresponding to an area per molecule (A _∞ ) of 159?±?8 Å². The adsorption equilibrium constant (K?=?(1.5?±?0.6)?×?10⁶ M^‐?1) was also estimated from the nonlinear regression of Π ^e???C _s data in region B of the Π ^e???ln?C _s plot. The value of K suggests that surfactin has strong affinity for the interface, which is in line with its known high surface activity. Gibbs elasticity (E _G) of the interfacial surfactin monolayers, which is an important thermodynamic property, was also predicted at different surfactin concentrations. The limiting value (at the CMC) of E _G was found to be 183 mN m^?1, which is comparable to those reported in the literature for similar systems. The findings reported in this work reveal that the surface tension measurements coupled with appropriate theoretical analysis could provide useful information comparable to those obtained using highly sophisticated techniques.     

Effects of pure AcOH and mixed AcOH–CS (CS?=?CH3CN and THF) solvents on the rate of cleavage of phthalic anhydride (PAn)

The values of pseudo-first-order rate constants (k _obs) for the acetolysis of phthalic anhydride (PAn) increase from 6.60?×?10^?7 to 31.5?×?10^?7?s^?1 with the increase in temperature from 30 to 50?°C. These values of k _obs give activation parameters ?H* and ?S* as 14.4?±?0.4?kcal?mol^?1 and ?39.1?±?1.3?cal?K^?1?mol^?1, respectively. The values of k _obs remain essentially unchanged with the increase in the content of CS (CS?=?CH₃CN or THF) from 0 to 40?% v/v in mixed AcOH?CCS solvents. These observations have been explained qualitatively.     

Control of the Harmful Alga Microcystis aeruginosa and Absorption of Nitrogen and Phosphorus by Candida utilis

This study is aimed at controlling eutrophication through converting the nutrients such as nitrogen and phosphorus into microbial protein and simultaneously inhibiting the growth of Microcystis aeruginosa by Candida utilis. C. utilis and M. aeruginosa (initial cell density was 2.25?×?10⁷ and 4.15?×?10⁷ cells·mL^?1) were cultured together in the absence or presence of a carbon source (glucose) during a 10-day experiment. In the absence of carbon source, the measured removal efficiencies of NH₄ ⁺–N and PO₄ ^3?–P were 41.39?±?2.19 % and 82.93?±?3.95 %, respectively, at the second day, with the removal efficiency of 67.82?±?2.29 % for M. aeruginosa at the fourth day. In contrast, the removal efficiencies of NH₄ ⁺–N and PO₄ ^3?–P were increased to 87.45?±?4.25 % and 83.73?±?3.55 %, respectively, while the removal efficiency of M. aeruginosa decreased to 37.89?±?8.41 % in the presence of the carbon source (C/N?=?2:1). These results showed that the growth of M. aeruginosa was inhibited by C. utilis. Our finding sheds light on a novel potential approach for yeast to consume nutrients and control harmful algal during bloom events.     

Kinetics and Thermodynamics of Ethanol Production by Saccharomyces cerevisiae MLD10 Using Molasses

In this study, we have used ultraviolet (UV) and γ-ray induction to get a catabolite repression resistant and thermotolerant mutant with enhanced ethanol production along with optimization of sugar concentration and temperature of fermentation. Classical mutagenesis in two consecutive cycles of UV- and γ-ray-induced mutations evolved one best catabolite-resistant and thermotolerant mutant Saccharomyces cerevisiae MLD10 which showed improved ethanol yield (0.48?±?0.02 g g^?1), theoretical yield (93?±?3 %), and extracellular invertase productivity (1,430?±?50 IU l^?1 h^?1), respectively, when fermenting 180 g sugars l^?1 in molasses medium at 43 °C in 300 m³ working volume fermenter. Ethanol production was highly dependent on invertase production. Enthalpy (ΔH*) (32.27 kJ M^?1) and entropy (ΔS*) (?202.88 J M^?1 K^?1) values at 43 °C by the mutant MLD10 were significantly lower than those of β-glucosidase production by a thermophilic mutant derivative of Thermomyces lanuginosus. These results confirmed the enhanced production of ethanol and invertase by this mutant derivative. These studies proved that mutant was significantly improved for ethanol production and was thermostable in nature. Lower fermentation time for ethanol production and maintenance of ethanol production rates (3.1 g l^?1 h^?1) at higher temperature (43 °C) by this mutant could decrease the overall cost of fermentation process and increase the quality of ethanol production.