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This study describes the hyaluronic acid (HA) production by S. zooepidemicus ATCC 43079, and the effect of the hyaluronidase enzyme on HA levels. The hyaluronidase production, glucose consumption, and lactate formation were recorded during fermentation. The HA production, and productivity at different amounts of glucose, yeast extract and pH were evaluated by response surface statistical approach in presence of 6-O-palmytoil-l-ascorbic acid as a chemical inhibitor for biocatalyst hyaluronidase. Under optimum conditions, HA production was increased two-fold from 190 ± 17 mg L-1 in basal medium to 384.6 ± 7.5 mg L-1 in the optimized medium containing enzyme inhibitor. Furthermore, the results indicated that the chemical inhibitor could suppress the biocatalyst activity and prevent the HA loss at the end of the exponential phase of S. zooepidemicus ATCC 43079.

Superoxide dismutases (SODs) (EC 1.15.1.1) are well known antioxidant enzymes that play critical roles in cellular defenses of living organisms against harmful superoxide radicals during oxidative stress. This study details on cloning, biochemical and functional characterization of an iron containing type superoxide dismutase (SOD) from a novel thermophilic bacteria Cohnella sp. A01 (CaSOD). The secondary and three dimensional structure of the protein were predicted. CaSOD gene was subsequently cloned into pET-26b(+) expression vector and expression of the recombinant protein (rCaSOD) was optimized in E. coli BL21 (DE3) and the purified recombinant SOD showed a single band with an apparent molecular weight of 26 kDa by SDS-PAGE. The half-life and thermodynamic parameters including ΔH⁎, ΔS⁎, and ΔG⁎ were 187 min at 60 °C, 7.3 kJ.mol−1, −76.8 kJ.mol−1.°K−1, and 84.1 kJ.mol−1, respectively. The rCaSOD exhibited catalytic activity in a very broad range of pH (6.0–10.0) and temperatures (35–75 °C), as well as stability in a broad pH range, from 3.0 to 11.0, and wide range of temperature, different concentrations of detergent agents, metal ions, organic solvents and other chemicals. The results suggest that this novel enzyme could be used for various industrial applications in cosmetic, food, and pharmaceutical industries.

The biodetoxification of cyanide-rich wastewater has been suggested as an appropriate technique due to its environmental friendliness and cost effectiveness. In this research, Enterobacter zs that was newly isolated from cyanide-polluted wastewater was selected to catalyze cyanide via an enzymatic mechanism. Enzyme was purified and its activity was also determined by ammonia assay. Subsequently, the operational procedure was optimized to enhance cyanide biodegradation at variable pH values, temperatures and cyanide concentrations using response surface methodology (RSM). The results revealed that the interactions between pH and temperature, as well as those between pH and cyanide concentration, were significant, and the concentration of cyanide in a 650 mg.L-1 solution was decreased by 73%. According to this study, it can be proposed that due to its higher activity level compared with those of similar enzymes, this enzyme can prove useful in enzymatic biodegradation of cyanide which is a promising approach in the treatment of industrial effluent.

A novel xylanase gene of 870bp was isolated and cloned from the thermophilic bacteria Cohnella sp.A01 that encodes an enzyme comprising 290 amino acid residues and belonging to the GH10 family of xylanases. The enzyme was efficiently expressed in Escherichia coli BL21, then purified using one-step chromatography. The recombinant XynB-A01 not only exhibited significant thermostability but also showed to be stable in a broad range of pH (3.0–10.0), the presence of some detergents, and organic solvents. Although XynB-A01 demonstrated significant activity toward both beechwood and oat spelt xylans, it showed a stronger affinity and higher activity toward the former. HPLC analysis of beechwood xylan hydrolysis revealed that xylotetraose, xylobiose, and xylose are the major hydrolysis products. The investigation of XynB-A01 in pulp biobleaching showed effectiveness in the release of reducing sugars and chromophores. Simple purification, stability over a broad range of pH and temperature, cellulase-free character, and the capacity to produce xylooligosaccharides are some of the advantages of the xylanase of Cohnella sp.A01, suggesting that it may be a promising candidate for biobleaching of paper pulps, producing xylooligosaccharides, and applying in other industries.

The role of oxidative stress is one of the most important factors in the development of cancer. In this study, the catalase activity and total antioxidant capacity in NHL patients were measures and compared with those in healthy subjects. The population of this study consisted of 25 patients with non-Hodgkin's lymphoma, diagnosed based on pathological findings. Total antioxidant capacity (TAC) and catalase catalytic activity (CAT) were determined by spectrophotometry. Based on the results, the CAT activity was significantly lower and TAC was lower (0.29±0.04 vs. 2.69±0.15; P-value=0.01) in NHL patients than in healthy subjects. The findings showed that these markers could be used as a prognosis in non-Hodgkin's lymphoma that oxidative stress may also be related to the NHL or increase the risk of disease.

Objectives Extensive applications of peroxidase (POX) have raised the global market demand at a considerable rate during the forecast period of 2020 - 2025. Nonetheless, the large-scale POX preparation still relies on the extraction from agricultural products, while there is an accumulative driving force toward employing biotechnological processes with agricultural hassle free identity. In pursuit of this trend; Results, A novel heme peroxidase was purified to homogeneity (MW of 40 kD) from the callus culture of Ocimum basilicum L. in darkness on Murashige-Skoog medium supplemented by 2,4-dichlorophenoxyacetic acid (10-6 M) and kinetin (10-5 M). The highest activity of the purified peroxidase (ObPOX) was observed in Tris-base buffer at pH 7.5 and 80 °C. ObPOX showed high stability over pH(s) 5 to 7.5 and temperatures of 15 to 60 °C. ObPOX specific activity was 1245.142 AU mg-1 in the presence of phenol, 4 times higher than that of HRP. ObPOX showed moderate affinity for guaiacol (Km = 21.5 mM), but obtained an exceptionally high specificity constant (kcat/Km = 66743.7 s-1M-1) for GASA (4-[(4-Hydroxy-3-methoxyphenyl) azo]-benzenesulfonic acid), the introduced substrate for determination of blood sugar. Applying ObPOX instead of HRP in glucose measurements of the real samples improved the regression constant of the correlation diagram between the tests and the lab results from 0.958 to 0.981. Conclusion Physicochemical properties of ObPOX as well as the growth rate of basil callus (5.04 g L-1 per day) and the yield of ObPOX production (35 mg per 100 g dry biomass per subculture) designates O. basilicum cell culture for large-scale production of a robust peroxidase.

Thermostability and substrate specificity of proteases are major factors in their industrial applications. rEla is a novel recombinant cysteine protease obtained from a thermophilic bacterium, Cohnella sp.A01 (PTCC No: 1921). Herein, we were interested in recombinant production and characterization of the enzyme and finding the novel features in comparison with other well-studied cysteine proteases. The bioinformatics analysis showed that rEla is allosteric cysteine protease from DJ-1/ThiJ/PfpI superfamily. The enzyme was heterologously expressed and characterized and the recombinant enzyme molecular mass was 19.38 kD which seems to be smaller than most of the cysteine proteases. rEla exhibited acceptable activity in broad pH and temperature ranges. The optimum activity was observed at 50℃ and pH 8 and the enzyme showed remarkable stability by keeping 50% of residual activity after 100 days storage at room temperature. The enzyme Km and Vmax values were 21.93 mM, 8 U/ml, respectively. To the best of our knowledge, in comparison with the other characterized cysteine proteases, rEla is the only reported cysteine protease with collagen specificity. The enzymes activity increases up to 1.4 times in the presence of calcium ion (2 mM) suggesting it as the enzyme’s co-factor. When exposed to surfactants including Tween20, Tween80, Triton X-100 and SDS (1% and 4% v/v) the enzyme activity surprisingly increased up to 5 times.

Glutamate carboxypeptidase is a bacterial enzyme of metallopeptidase superfamily. This enzyme is an exo-peptidase that catalyzes the hydrolysis of glutamate residues at the C-terminus of folic acid. The rCP302 is a novel zinc ion-dependent recombinant glutamate carboxypeptidase derived from a thermophilic bacterium, Cohnella sp. A01 (PTCC No: 1921). By simulating the structure of rCP302, analyzing its activity in various environmental settings, and contrasting it with that of related enzymes, we wanted to evaluate the heterologous production, purification, and characterization of this enzyme. The bioinformatics study showed that rCP302 had maximum similarity to M20 family of metallopeptidases. The purified rCP302 molecular weight was about 41.6 kDa. The optimum temperature and pH for the catalytic activity of rCP302 were 50 °C and 7.2, respectively. Fluorescence spectroscopy data elucidated the secondary structure of rCP302 and determined conformational changes caused by alterations in ambient conditions. Using folate as a substrate, K

L-glutaminase importance to use in the food industry and medicine has attracted much attention. Enzymes stability has always been a challenge while working with them. We heterologously expressed and characterized a novel stable L-glutaminase from an extremophile bacterium (Cohnella sp. A01, PTCC No: 1921). Km, Vmax, catalytic efficiency and specific activity of rSAM were respectively 1.8 mM, 49 µmol/min, 1851 1/(S.mM) and 9.2 IU/mg. Activation energy for substrate to product conversion and irreversible thermo-inactivation were respectively 4 kJ/mol and 105 kJ/mol from the linear Arrhenius plot. rSAM had the highest activity at temperature 50 °C, pH 8 and was resistant to a wide range of temperature and pH. In compare to the other characterized glutaminases, rSAM was the most resistant to NaCl. Mg2+, glycerol, DTT, and BME enhanced the enzyme activity and iodoacetate and iodoacetamide inhibited it. rSAM had only been partially digested by some proteases. According to the Fluorimetry and Circular dichroism analysis, rSAM in pH range from 4 to 11 and temperatures up to 60 °C had structural stability. A cysteine residue in the enzyme active site and a thiol bond were predicted upon the modeled tertiary structure of rSAM. Present structural studies also confirmed the presence of a thiol bond in its structure.

This study describes the hyaluronic acid (HA) production by

The biodetoxification of cyanide-rich wastewater has been suggested as an appropriate technique due to its environmental friendliness and cost effectiveness. In this research

Cellulases are hydrolytic enzymes with wide scientific and industrial applications. We described a novel cellulase, CelC307, from the thermophilic indigenous Cohnella sp. A01. The 3-D structure of the CelC307 was predicted by comparative modeling. Docking of CelC307 with specific inhibitors and molecular dynamic (MD) simulation revealed that these ligands bound in a non-competitive manner. The CelC307 protein was purified and characterized after recombinant expression in Escherichia coli (E. coli) BL21. Using CMC 1% as the substrate, the thermodynamic values were determined as Km 0.46 mM, kcat 104.30 × 10–3 (S−1), and kcat/Km 226.73 (M−1 S−1). The CelC307 was optimally active at 40 °C and pH 7.0. The culture condition was optimized for improved CelC307 expression using Plackett–Burman and Box–Behnken design as follows: temperature 20 °C, pH 7.5, and inoculation concentration with an OD600 = 1. The endoglucanase activity was positively modulated in the presence of Na+, Li+, Ca2+, 2-mercaptoethanol (2-ME), and glycerol. The thermodynamic parameters calculated for CelC307 confirmed its inherent thermostability. The characterized CelC307 may be a suitable candidate for various biotechnological applications.

Hyaluronic acid (HA) is a natural polymer with various molecular weights that specify multiple biological roles. Traditionally, HA is obtained from animal waste and conventional pathogenic streptococci. However, there are challenges in these processes such as the presence of exotoxins, hyaluronidase, and viral contamination. In order to reduce these problems, this study was conducted to produce HA using recombinant bacterium that is generally recognized as safe (GRAS), and thereafter increase production through experimental design. At first, some lactic acid bacteria were screened and evaluated for HA production. Accordingly, among the selected bacteria, Lactobacillus acidophilus PTCC1643 produced about 0.25 g HA/L in the 48th hour of cultivation, and was thus selected as an alternative host for heterologous HA production. An expression vector containing HA synthase genes was transformed into L. acidophilus by electroporation. Consequently, HA production increased to 0.4 g/L. Eventually, response surface method (RSM) was used, which increased HA production to 1.7 g/L. This is approximately 7-fold higher than that produced at first. The resulting HA was characterized by FTIR spectroscopy and its molecular weight was estimated using agarose gel electrophoresis. In conclusion, L. acidophilus could be a safe, effective, and novel HA producer with industrial potential and commercial prospects.

Cyanide compounds are hazardous compounds which are extremely toxic to living organisms, especially free cyanide in the form of hydrogen cyanide gas (HCN) and cyanide ion (CN−). These cyanide compounds are metabolic inhibitors since they can tightly bind to the metals of metalloenzymes. Anthropogenic sources contribute significantly to CN− contamination in the environment, more specifically to surface and underground waters. The treatment processes, such as chemical and physical treatment processes, have been implemented. However, these processes have drawbacks since they generate additional contaminants which further exacerbates the environmental pollution. The biological treatment techniques are mostly overlooked as an alternative to the conventional physical and chemical methods. However, the recent research has focused substantially on this method, with different reactor configurations that were proposed. However, minimal attention was given to the emerging technologies that sought to accelerate the treatment with a subsequent resource recovery from the process. Hence, this review focuses on the recent emerging tools that can be used to accelerate cyanide biodegradation. These tools include, amongst others, electro-bioremediation, anaerobic biodegradation and the use of microbial fuel cell technology. These processes were demonstrated to have the possibility of producing value-added products, such as biogas, co-factors of neurotransmitters and electricity from the treatment process.

As a safe substitute for hydroquinone, β-arbutin, a natural plant substance, and its synthetic counterpart, α-arbutin, are used in depigmentation formulations. However, there are debatable points regarding the impact of arbutin on tyrosinase and the pigmentation process. To shed light on this issue, the effects of Pyrus biossieriana leaves extract (PbLE) and β-arbutin, extracted from PbLE, on mushroom tyrosinase (MT) were comprehensively examined. The study was focused on cresolase activity as the characteristic reaction of a tyrosinase. Kinetics studies disclosed that β-arbutin can modulate MT monophenolase activity from inhibition to activation or vice versa. β-Arbutin inhibited L-tyrosine (LTy) oxidation at concentrations 0.3 mM but it increased (more than 400%) the enzymatic oxidation of L-tyrosine at the concentrations 0.3 mM. An opposite pattern (activation then inhibition) was observed when a synthetic substrate was used instead of LTy. Computational studies, focused on the heavy chain of MT, indicated that β-arbutin effect could be overruled by the enzyme's ability to provide the ligand with a non-specific binding site (MTPc). A plausible mechanism was presented to show the influence of MTPc on the substrate pose in the active site. The possible determinant correlation between the findings of this research and the current studies on human tyrosinase role in the pigmentation process has been presented.

BackgroundSHuffle is a suitableEscherichia coli(E. coli) strain for high yield cytoplasmic soluble expression of disulfide-bonded proteins such as Insulin due to its oxidative cytoplasmic condition and the ability to correct the arrangement of disulfide bonds. Lispro is an Insulin analog that is conventionally produced inE. colias inclusion bodies (IBs) with prolonged production time and low recovery. Here in this study, we aimed to optimize cultivation media composition for high cell density fermentation of SHuffle T7E. coliexpressing soluble Lispro proinsulin fused to SUMO tag (SU-INS construct) to obtain high cell density fermentation.ResultsFactors including carbon and nitrogen sources, salts, metal ions, and pH were screened via Plackett–Burman design for their effectiveness on cell dry weight (CDW) as a measure of cell growth. The most significant variables of the screening experiment were Yeast extract and MgCl2concentration, as well as pH. Succeedingly, The Central Composite Design was utilized to further evaluate and optimize the level of significant variables. The Optimized media (OM-I) enhanced biomass by 2.3 fold in the shake flask (2.5 g/L CDW) that reached 6.45 g/L (2.6 fold increase) when applied in batch culture fermentation. The efficacy of OM-I media for soluble expression was confirmed in both shake flask and fermentor.ConclusionThe proposed media was suitable for high cell density fermentation ofE. coliSHuffle T7 and was applicable for high yield soluble expression of Lispro proinsulin.

Background: The activities of programmed cell death 1 (PD-1) and programmed death ligand-1 (PD-L1) have already been identified in various cancers. However, in non-Hodgkinʼs lymphoma (NHL), the prognostic value of PD-1/PD-L1 gene polymorphisms and expression levels remains unclear. Objectives: The present study aimed to investigate the relationship between the genetic polymorphisms of PD-1/PD-L1 genes and NHL in the Iranian population. Methods: Four single-nucleotide polymorphisms (SNPs) of PD-1/PD-L1 genes were examined in 134 NHL patients and 134 healthy controls using polymerase chain reaction-restriction fragment length polymorphism. The expression levels of PD-1/PD-L1 genes were analyzed using real-time polymerase chain reaction. Results: The obtained results of the current study demonstrated that PD-L1 rs2890685 (A>C) SNP (P

Background, Programed cell death 1 (PD-1) and its ligand (PD-L1) activity have already detected in various cancers. In non-Hodgkin lymphoma (NHL), however, the prognostic value of PD-1/PD-L1 genes polymorphisms and expression levels remains unclear. In the present study we aimed to investigate the relationship between genetic polymorphisms of PD-1/PD-L1 genes and non-Hodgkin lymphoma in Iranian papulation. Methods, four single nucleotide polymorphisms of the PD-1/PD-L1 genes, including 134 NHL patients and 125 healthy controls were examined using PCR-RFLP method. The expression level of PD-1/PD-L1 genes were analyzed using real time PCR method.Results, our data demonstrated that the PD-L1 rs2890685 (A>C) SNP (pConclusion, Collectively, our finding demonstrated that the functional PD-L1 rs2890685 polymorphism was associated with NHL risk, suggesting that genetic variant of PD-L1 might be a possible prognosis marker for prediction of NHL risk and its development.

The prepared protein and protein-ligand complexes (protease-SDS and protease-tween complexes) were used in MD simulation, performed using GROMACS v 4.6.5 with CHARMM36 all-atom force field. The CGenFF server (https://cgenff.umaryland.edu/) provides topologies and parameters of ligands compatible with the CHARMM36 all-atoms force field. Protein and protein-ligand complexes were soaked in a cubic box of water molecules, and the charges on the protein were neutralized by the addition of Na+ and Cl- ions. The energy of the system was minimised using the steepest descent algorithm to eliminate bad contact and clashes. The NVT and NPT ensembles were used during the equilibration to achieve the desired temperature (373.15 K for protease and 300 K for protease-ligand complex) and pressure (1 bar) for 100 ps and restraint forces of 1000 kJ/mol. Finally, 40 ns MD run were performed in triplicate after releasing all restraints. All bonds were constrained by the LINCS algorithm.

After the cultivation period, cells were collected by centrifugation at 3500 × g for 30 min at 4 °C and resuspended in 4 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole and 0.05% Tween 20 at a pH of 7). The mixture was sonicated with 4 × 40 sec pulses followed by 20 sec rest between cycles at 4 °C. The crude extracts were then centrifuged at 9000 × g for 30 min at 4 °C, and the resulting supernatant was heat-treated in a hot water bath at 90 °C for 15 min. Insoluble material was separated by centrifugation at 13000 × g for 10 min, and the supernatant, containing the enzyme, was analyzed on a 12% SDS–polyacrylamide. The purified elution fractions were dialysed overnight in a 50 mM potassium-phosphate buffer (pH=7) at 4 °C.

The nucleotide sequence of Coh01147, previously obtained from Cohnella sp. A01 (Genbank Accession No: QEV88423) was translated to the amino acids using Expasy translate tool (http://web.expasy.org/translate). The resulting protein sequencewas used for further in silico analysis of the Cohnella sp. A01 derived protease 1147 protein. The biochemical properties of the protein, including molecular weight, isoelectric point, instability index, aliphatic coefficient, and grand average of hydropathy (GRAVY), were calculated using Expasy ProtParam tool (http://web.expasy.org/protparam). The presence of putative signal peptide and the location of its cleavage site was predicted using SignalP 5.0 server (http://www.cbs.dtu.dk/services/SignalP). pFam (http://pfam.xfam.org/search) and MEROPS (http://MEROPS.sanger.ac.uk) databases were used for sequence alignment and identifying the peptidase family. The phylogenetic relationships of the protein sequence were analysed using ClustalW in MEGA 7 programs. Multiple alignments of the resulting protein family of protease 1147 (from MEROPS) was carried out using COBALT (https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi), and the conserved areas were determined using DNAMAN software (www.lynnon.com). BLOSUM62 substitution matrix and a gap penalty = 12 were utilised in the search.

Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/β sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, Km, and kcat, kcat/Km values of 13.72 mM, 3.143 × 10-3 (s-1), and 0.381 (M-1 S-1) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60°C with the inactivation rate constant (kin) of 2.10 × 10-3 (m-1), and half-life (t1/2) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100°C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications.

Chitinases with high thermostability are important for many industrial and biotechnological applications. This study was conducted to enhance the stability of Serratia marcescens B4A chitinase by site directed mutagenesis of G191 V. Further characterization showed that the thermal stability of the mutant showed marked increase of about 5 and 15 fold at 50 and 60 °C respectively, while the optimum temperature and pH was retained. Kinetic analysis showed decreased Km and Vmax of the mutant in comparison with the wild type chitinase of about 1.3 and 3 fold, respectively. Based on structural prediction, it was speculated that this replacement shortened an important loop concomitant with the extension of adjacent β sheets. Accordingly, a higher thermostability of G191 V up to 90 °C supporting the decreased flexibility of unfolded state was also indicated. Finally, a practical proof of kinetic and thermal stabilization of chitinase was provided through decreased flexibility and entropic stabilization of its surface loops.

Background: Typically, non-cellulytic glucanase, including fungi and yeast cell wall hydrolyzing enzymes, are released by some symbiotic fungi and plants during the mycoparasitic fungi attack on plants. These enzymes are known as the defense mechanisms of plants. This study intends to investigate the biochemical properties of β-1,6-glucanase (bg16M) from native thermophilic bacteria, Cohnella A01. Methods: bg16M gene was cloned and expressed in E. coli BL21 (DE3). The enzyme was purified utilizing Ni-NTA nikcle sepharose column. Pustulan and laminarin were selected as substrates in enzyme assay. The purified bg16M enzyme was treated with different pH, temperature, metal ions, and detergents. Results The expressed protein, including 639 amino acids, showed a high similarity with the hydrolytic glycosylated family 30. The molecular weight of enzyme was 64 kDa, and purification yield was 46%. The bg16M demonstrated activity as 4.83 U/ml on laminarin and 2.88 U/ml on pustulan. The optimum pH and temperature of the enzyme were 8 and 50 °C, respectively. The enzyme had an appropriate stability at high temperatures and in the pH range of 7 to 9, showing acceptable stability, while it did not lose enzymatic activity completely at acidic or basic pH. None of the studied metal ions and chemical compounds was the activator of bg16M, and urea, SDS, and copper acted as enzyme inhibitors. Conclusion Biochemical characterization of this enzyme revealed that bg16M can be applied in beverage industries and medical sectors because of its high activity, as well as thermal and alkaline stability.

Cyanide is used in many industries despite its toxicity. Cyanide biodegradation is affordable and eco-friendly. Sampling from cyanide-contaminated areas from the Muteh gold mine and isolation of 24 bacteria were performed successfully. The selected bacteria—‘Bacillus sp. M01’—showed maximum tolerance (15 mM) to cyanide and deposited in Persian Type Culture Collection by PTCC No.: 1908. In the primary experiments, effective factors were identified through the Plackett–Burman design. In order to attain the maximum degradation by Bacillus sp. M01 PTCC 1908, culture conditions were optimized by using response surface methodology. By optimizing the effective factor values and considering the interaction between them, the culture conditions were optimized. The degradation percentage was calculated using one-way ANOVA vs t test, and was found to have increased 2.35 times compared to pre-optimization. In all of the experiments, R2 was as high as 91%. The results of this study are strongly significant for cyanide biodegradation. This method enables the bacteria to degrade 86% of 10 mM cyanide in 48 h. This process has been patented in Iranian Intellectual Property Centre under Licence No: 90533.

Increasing demand for efficient and environmentally benign oxidation technologies has resulted in a focus on the use of oxidoreductases. Laccases and tyrosinases, which utilize molecular oxygen and...

Laccase (EC 1.10.3.2; benzenediol; oxygen oxidoreductases) is a multi-copper oxidase that catalyzes the oxidation of phenols, polyphenols, aromatic amines, and different non-phenolic substrates with concomitant reduction of O2 to H2O. Enzymatic oxidation techniques have the potential of implementation in different areas of industrial fields. In this study, the Cohnella sp. A01 laccase gene was cloned into pET-26 (b+) vector and was transformed to E. coli BL21. Then it was purified using His tag affinity (Ni sepharose resin) chromatography. The estimated molecular weight was approximately 60 kDa using SDS-PAGE. The highest enzyme activity and best pH for 2,6-dimethoxyphenol (DMP) oxidation were recorded as 8 at 90 °C respectively. The calculated half-life and kinetic values including Km, Vmax, turn over number (kcat), and catalytic efficiency (kcat/Km) of the enzyme were 106 min at 90 °C and 686 μM, 10.69 U/ml, 20.3 S−, and 0.029 s−1 μM−1, respectively. The DMP was available as the substrate in all the calculations. Enzyme activity enhanced in the presence of Cu2+, NaCl, SDS, n-hexane, Triton X-100, tween 20, and tween 80, significantly. The binding residues were predicted and mapped upon the modeled tertiary structure of identified laccase. The remaining activity and structural properties of Cohnella sp. A01 laccase in extreme conditions such as high temperatures and presence of metals, detergents, and organic solvents suggest the potential of this enzyme in biotechnological and industrial applications. This process has been patented in Iranian Intellectual Property Centre under License No: 91325.

There is an increasing need for accurate and inexpensive glucometers as the world moves toward personalized medicine. Among the existing technologies, photometric based devices are more desired due to the cost-effectiveness, ease-of-use and the potential to be adopted in the smart-phone technology for remote sensing and self-monitoring purposes. However, the accuracy, precision, and reproducibility of the results of these devices are heavily dependent on the details of the chosen glucose measuring method. Considering the delicate problems with the current spectrophotometric methods, a new method was developed for more precise, accurate, and fast measurement of blood glucose via the coupled reactions of glucose oxidase and peroxidase using 4-[(4-Hydroxy-3-methoxyphenyl) azo]-benzenesulfonic acid (GASA) as the substrate. Stability of GASA and its oxidized products along with its direct and fast consumption by peroxidase, made it possible to determine blood glucose concentration in20 s with high reproducibility. The low detection limit of GASA method (0.36 mg dL

The use of herbal remedies is significantly considered in the atherosclerosis treatment, reduction of fatty elements, and prevention of activity of oxidative stress factors. The present study was conducted on 48 rats in 6 groups. The experimental and sham groups were fed with 2% cholesterol for 40 days; and experimental groups were separately treated by atorvastatin, quercetin, and hydroalcoholic extract for 4 weeks. After treatment procedure, some serum factors such as low‐density lipoprotein (LDL), total cholesterol (TC), malondialdehyde (MDA), and reactive oxygen species (ROS) were evaluated. Serum levels of LDL, TC, MDA, and ROS were significantly lower in experimental groups than sham group (p

Twelve bacterial strains isolated from shrimp farming ponds were screened for their growth activity on chitin as the sole carbon source. The highly chitinolytic bacterial strain was detected by qualitative cup plate assay and tentatively identified to be Cohnella sp. A01 based on 16S rDNA sequencing and by matching the key morphological, physiological, and biochemical characteristics. The cultivation of Cohnella sp. A01 in the suitable liquid medium resulted in the production of high levels of enzyme. The colloidal chitin, peptone, and K2HPO4 represented the best carbon, nitrogen, and phosphorus sources, respectively. Enzyme production by Cohnella sp. A01 was optimized by the Taguchi method. Our results demonstrated that inoculation amount and temperature of incubation were the most significant factors influencing chitinase production. From the tested values, the best pH/temperature was obtained at pH 5 and 70 °C, with Km and V max values of chitinase to be 5.6 mg/mL and 0.87 µmol/min, respectively. Ag+, Co2+, iodoacetamide, and iodoacetic acid inhibited the enzyme activity, whereas Mn2+, Cu2+, Tweens (20 and 80), Triton X-100, and EDTA increased the same. In addition, the study of the morphological alteration of chitin treated by enzyme by SEM revealed cracks and pores on the chitin surface, indicating a potential application of this enzyme in several industries.

Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are one of the highest value commercial enzymes as they have potential applications in biotechnology for detergents, food, pharmaceuticals, leather, textiles, cosmetics, and paper industries; and are currently receiving considerable attention because of their potential applications in biotechnology. Bacillus thermocatenulatus Lipase 2 (BTL2) is one of the most important research targets, because of its potential industrial applications. In this study, the effect of substitution Phe17 with Ser in mutated BTL2 lipase, which conserved pentapeptide (112Ala-His-Ser-Gln-Gly116) was replaced with similar sequences (207Gly-Glu-Ser-Ala-Gly211) of Candida rugosa lipase (CLR) at the nucleophilic elbow region. Docking results confirmed the mutated lipase to be better than the chimeric lipase. So, cloning was conducted, and the mutated and chimeric btl2 genes were expressed in Escherichia coli, and then the enzymes were purified by anion exchange chromatography. The mutation increased lipase lipolytic activity against most of the applied substrates, with the exception of tributyrin when compared with chimeric lipase. Further, the mutated lipase exhibited higher activity than the chimeric lipase at all temperatures. Optimum pH of the mutated lipase was obtained at pH 9.5, which was more than the chimeric one. Enzyme activity of the mutated lipase in the presence of organic solvents, detergents, and metal ions was also improved than the chimeric lipase.

In this paper, a frequency-reactive power channel (FRCh) is proposed for improving the performance of conventional frequency control system in an islanded MicroGrid (MG). Using this channel, it will be possible to take advantage of reactive power management for frequency control, in addition to the conventional methods of using active power. In order to create the mentioned channel, a frequency-reactive power controller (FRC) is proposed for controlling the inverter-interfaced energy storage devices. The proposed controller reduces the MG frequency decline during contingencies. Moreover, it reduces the power of battery employed for the frequency control, while maintaining the customer voltage within permissible limits. Furthermore, this paper defines a new frequency-reactive power factor (FRF) capable of determining the best place for installation of FRC. This factor indicates the dependence of microgrid frequency on reactive power variations in each bus and identifies the buses which are more effective on the MG frequency. To demonstrate the effectiveness of proposed method and the accuracy of new factor, it is implemented on a test network in MATLAB/Simulink environment.

Secondary structure content of proteins in molten globule state is relatively constant while the quantity of tertiary structures clearly declines due to alterations in side-chain packing. In the present study, we analyze the MG state of lipase-3646 for the first time. We introduce lipase-3646 as an appropriate model for investigating the properties and behavior of a protein in MG state as well as folding pathway. Applying fluorescence spectroscopy we measured both intrinsic and extrinsic fluorescence of lipase-3646 in a pH range from 1.0 to 12.0. It was found that at pH 3.0 the protein acquires a MG state. Applying far-UV circular dichroism (CD), our analysis on the secondary structure of lipase-3646 revealed a slight change in the MG state intermediate (pH 3.0) compared to the native state (pH 8.5), which this amount of change is common for MG. Measurements in near-UV CD also showed a significant change in the enzyme conformation at pH 3.0 in comparison with the pH 8.5 wherein the protein acquires its native structure. Quenching the fluorescence by applying acrylamide, the amount 23 and 35 M(-1) were measured at pHs 8.5 and 3.0 respectively for stern-volmer constant (KSV). An increase in the enzyme molecular volume in the MG state was confirmed by gel filtration chromatography.

Lipases form one of the most important groups of biocatalysts used in biotechnology. We studied the lipase from the bacterium Cohnella sp. A01 due to the versatility of thermophilic lipases in industry. In this study lipase 3646 gene from the thermophilic bacterium Cohnella sp. A01 was expressed in Escherichia coli and the enzyme was purified by a two-steps anion exchange chromatography. The purified lipase appeared to have a molecular weight of approximately 29.5 kDa on SDS–PAGE. The values of K m and V max , calculated by the Michaelis–Menten equation, were 1077 μM and 61.94 U/mg, respectively. The kinetic characterization of the purified enzyme exhibited maximum activity at 70 °C and pH 8.5. Activities at 50, 55 and 60 °C for 120 min were measured 58%, 47% and 41%, respectively. The enzyme was also highly stable at the pH range of 8.5–10.0 for 180 min. The effect of EDTA indicated that the enzyme is not a metalloenzyme. The stability of lipase 3646 in the presence of organic solvents, detergents, metal ions and inhibitors suggested that this lipase could be exploited in certain industries such as detergent and leather. Lipase 3646 was determined structurally to be 37.5% α-helix, 12.8% β-sheet, 22.7% β-turn and 27% random coil.

Background: Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein’s structure and function. Objectives: We introduced a nested-SOE-PCR (N –SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR. Materials and Methods: Genomic DNA from Bacillus thermocatenulatus was extracted. Nested PCR was used to amplify B. thermocatenulatus lipase gene variants, namely wild type and mutant, using gene specific and mutagenic specific primers, followed by cloning in a suitable vector. Briefly in N-SOE-PCR method, instead of two pairs of primers, three pairs of primers are used to amplify a mutagenic fragment. Moreover, the first and second PCR products are slightly longer than PCR products in a conventional SOE. PCR products obtained from the first round of PCR are used for the second PCR by applying the nested and mutated primers. Following to the purification of the amplified fragments, they will be subject of the further purification and will be used as template to perform the third round of PCR using gene specific primers. In the end, the products will be cloned into a suitable vector for subsequent application. Results: In comparison to the conventional SOE-PCR, the improved method (i.e. N-SOE-PCR) increases the yield and specificity of the products. In addition, the proposed method shows a large reduction in the non-specific products. Conclusions: By applying two more primers in the conventional SOE, the specificity of the method will be improved. This would be in part due to annealing of the primers further inside the amplicon that increases both the efficiency and a better attachment of the primers. Positioning of the primer far from both ends of an amplicon leads to an enhanced binding as well as increased affinity in the third round of amplification in SOE.