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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.

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.

A new strain of Influenza A Virus (IAV), so-called "H7N9 Avian Influenza", is the first strain of this virus in which a human is infected by transmitting the N9 of influenza virus. Although continuous human-to-human transmission has not been reported, the occurrence of various H7N9-associated epidemics and the lack of production of strong antibodies against H7N9 in humans warn of the potential for H7N9 to become a new pandemic. Therefore, the need for effective vaccination against H7N9 as a life-threatening viral pathogen has become a major concern. The current study reports the design of a multi-epitope vaccine against Hemagglutinin (HA) and Neuraminidase (NA) proteins of H7N9 Influenza A virus by prediction of Cytotoxic T lymphocyte (CTL), Helper T lymphocyte (HTL), IFN-γ and B-cell epitopes. Human β-defensin-3 (HβD-3) and pan HLA DR-binding epitope (PADRE) sequence were considered as adjuvant. EAAAK, AAY, GPGPG, HEYGAEALERAG, KK and RVRR linkers were used as a connector for epitopes. The final construct contained 777 amino acids that are expected to be a recombinant protein of about ~ 86.38 kDa with antigenic and non-allergenic properties after expression. Modeled protein analysis based on the tertiary structure validation, docking studies, and molecular dynamics simulations results like Root-mean-square deviation (RMSD), Gyration, Root-mean-square fluctuation (RMSF) and Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) showed that this protein has a stable construct and capable of being in interaction with Toll-like receptor 7 (TLR7), TLR8 and m826 antibody. Analysis of the obtained data the demonstrates that suggested vaccine has the potential to induce the immune response by stimulating T and Bcells, and may be utilizable for prevention purposes against Avian Influenza A (H7N9).

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.

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.

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.

Lipases from Bacillus thermocatenulatus are a member of superfamily of α/β hydrolase, but there are structural differences between them. In this work, we focused on the α5 helix of B. thermocatenulatus lipase (BTL2) which is absent in canonical α/β hydrolase fold. In silico study showed that the α5 helix is a region that causes disorder in BTL2 protein. So, the α5 helix (residues 131 to 150) has been deleted from the B. thermocatenulatus lipase gene (BTL2) and the remain (Δα5-BTL2) has been expressed in Pichia pastoris yeast. The α5 deletion results in increase of enzyme-specific activity in the presence of tributyrin, tricaproin, tricaprylin, tricaprin, trilaurin, and olive oil (C18) substrates by 1.4-, 1.7-, 2.0-, 1.2-, 1.75-, and 1.95-fold, respectively. Also, deletion leads to increase in enzyme activity in different temperatures and pHs, whereas it did not significantly affect on enzyme activity in the presence of organic solvents, metal ions, and detergents.

Bacillus thermocatenulatus lipase 2 (BTL2), a thermoalkalophilic lipase, is the best studied enzyme for its particular properties, which make it useful in different industries. Displacement of conserved phenylalanine 17 (Phe-17) residue in the active site of BTL2 has a critical role in oxyanion hole formation, which is important for enzyme activity. In this study, to facilitate oxyanion hole formation, Phe-17 was substituted with Alanine residue (F17A). The best structures of the opened form of the native and mutated lipases were garnered based on the crystal structures of 2W22. To evaluate catalytic activity, both lipases were docked to a set of ligands using Hex 6.3 software. Following in silico study, both native and mutant btl2 genes were cloned and expressed in Pichia pastoris. Based on the results obtained, the mutation increased lipase lipolytic activity against most of the applied substrates, especially for tributyrin and tricaprylin, by 1.9 and 2.15 fold, respectively. However, optimum temperature and pH were the same for both lipases (60 °C and pH 8.0). As previously reported, it is believed that F17A mutation simplifies oxyanion hole formation and declines steric hindrance in the enzyme active site, which might ultimately lead to more efficient accessibility of substrates.

Pepsin-solubilized collagen (PSC) was extracted from the skin of sea cucumber Holothuria parva and was fractionally characterized. The PSC from H. parva skin consisted of three α1 chains (α1)3, in contrast to calf skin collagen type I with two α1 and one α2 chains (α1)2α2 with approximately 130 kDa each. The maximum transition (Tm) and denaturation temperature (Td) of PSC were determined to be 46.94 and 32.5 °C, respectively. The amino acid composition analysis revealed that glycine, proline, alanine, and hydroxyproline were the abundant amino acids available in extracted PSC. The results showed that the isolated collagen from H. parva has some similar characteristics to previously reported collagens used in food and pharmaceutical industries.

Lipases are one of the highest value commercial enzymes as they have broad applications in detergent, food, pharmaceutical, and dairy industries. To provide chimeric Bacillus thermocatenulatus lipase (BTL2), the completely 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. For this purpose, three mutations including A112G, H113E, and Q115A were inserted in the conserved pentapeptide sequence of btl2 gene. Based on the crystal structures of 2W22, the best structure of opened form of the chimeric lipases were garnered using the MODELLER v9.10 software. The native and chimeric lipases were docked to a set of ligands, and a trial version of Molegro Virtual Docker (MVD) software was used to obtain the energy values. Docking results confirmed chimeric lipase to be better than the native lipase. Following the in silico study, cloning experiments were conducted and expression of native and chimeric btl2 gene in Pichia pastoris was performed. The native and chimeric lipases were purified, and the effect of these mutations on characteristics of chimeric lipase studied and then compared with those of native lipase. Chimeric lipase exhibited 1.6-fold higher activity than the native lipase at 55 °C. The highest percentage of both lipases activity was observed at 60 °C and pH of 8.0. The ion Ca2+ slightly inhibited the activity of both lipases, whereas the organic solvent enhanced the lipase stability of chimeric lipase as compared with the native lipase. According to the results, the presence of two glycine residues at the conserved pentapeptide region of this chimeric lipase (112Gly-Glu-Ser-Ala-Gly116) may increase the flexibility of the nucleophilic elbow region and affect the enzyme activity level.

For the first time, a polygalacturonase from the culture broth of Tetracoccosporium sp. was isolated and incubated at 30 degrees C in an orbital shaker at 160 rpm for 48 h. The enzyme was purified by ammonium sulfate precipitation and two-step ion-exchange chromatography and had an apparent molecular mass of 36 kDa, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Its optimum activity was at pH 4.3 and 40 degrees C, and the Km and Vmax values of this enzyme (for polygalacturonic acid) were 3.23 mg/mL and 0.15 micromol/min, respectively. Ag+, Co2+, EDTA, Tween-20, Tween-80, and Triton X-100 stimulated polygalacturonase activity whereas Al3+, Ba2+, Ca2+, Fe2+, Fe3+, Ni2+, Mg2+, Mn2+, and SDS inhibited it. In addition, iodoacetamide and iodoacetic acid did not inhibit enzyme activity at a concentration of 1 mM, indicating that cysteine residues are not part of the catalytic site of polygalacturonase. We studied the kinetic properties and thermal inactivation of polygalacturonase. This enzyme exhibited a t1/2 of 63 min at 60 degrees C and its specific activity, turnover number, and catalytic efficiency were 6.17 U/mg, 113.64 min-1, and 35.18 mL/(min.mg), respectively. The activation energy (DeltaE#) for heat inactivation was 5.341 kJ/mol, and the thermodynamic activation parameters DeltaG#, DeltaH#, and DeltaS# were also calculated, revealing a potential application for the industry.