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Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD).Methods: In this study, the heavy and light chains of ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4°C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains’ interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for ranibizumab production.

We report cloning and expressing of recombinant human VEGF-A165, fused at the N-terminal with Hydrophobin II (HFBII) from Trichoderma reseei, in yeast Pichia pastoris. We validated the construct using SDS-PAGE and ELISA against VEGF-A165 and efficiently performed protein purification and enrichment based on HFBII counterpart and using an aqueous two-phase system (ATPS) with nonionic surfactant X-114. We studied the effects of various culture medium additives and interaction effects of positive factors to increase the recombinant HFBII-VEGF-A165 production. Supplementing the Pichia pastoris cell culture medium with Mg2+, Polysorbate 20 (PS 20), and 4-phenylbutyrate (PBA) improved the expression of the chimeric protein. Orthogonal experiments showed that the optimal condition to achieve maximal HFBII-VEGF-A165 production was with the addition of PBA, PS 20, and MgSO4. Under this condition, the production of the target protein was 4.5 times more than that in the medium without the additives. Overall, our approach to produce chimeric HFBII-VEGF-A165 and selectively capture it in ATPS is promising for large-scale protein production without laborious downstream processing.

Exact monitoring of the key process variables, such as specific growth rate (μ), is essential for effective process control, and maintenance of the optimal conditions for the production of recombinant proteins. In this study, a novel and confident on-line μ-stat approach for setting up methanol feeding strategy is described that is based on the consumption of ammonium hydroxide as feedback control. For this purpose a new and robust control with a time-based stable control structure was used, which had the ability to reconstruct the controller. Moreover, the MLP3 neural network was applied to adjust and optimize the performance of the robust control system. To evaluate the cited strategy, the overproduction of human recombinant alpha 1-antitrypsin (A1AT) under the control of alcohol oxidase 1 promoter (pAOX1) was investigated. On-line control of the μ at the optimal amount (0.03 1/h) led to 120 g/L dry cell weight and 324 mg/L of A1AT concentration. In comparison with traditional protocols of methanol feeding, the obtained product concentration demonstrated a significant improvement. It is suggested that this new presented method has a remarkable potential to be used for the overexpression of different recombinant proteins in the host Pichia pastoris as well as for process development in other hosts.

Abstract Biogenic amines (BAs) are low molecular weight organic bases formed by natural amino acids decarboxylation and trigger an array of toxicological effects in humans and animals. Bacterial amine oxidases enzymes are determined as practical tools to implement the rapid quantification of BAs in foods. Our study set out to obtain a new efficient, amine oxidase enzyme for developing new enzyme-based quantification of histamine. The soils from different sources were screened using histamine as sole carbon and nitrogen sources, and histamine oxidase producing bacteria were selected and identified using specific primers for histamine oxidase (HOD) gene. The HOD gene of six strains, out of 26 isolated histamine-utilizing bacteria, were amplified using our designed primers. The HOD enzyme from Glutamicibacter sp. N1A3101, isolated from nettle soil, was found to be thermostable and showed the highest substrate specificity toward the histamine and with no detected activity in the presence of putrescine, cadaverine, spermine, and spermidine. Its oxidation activity toward tyramine was lower than other HOD reported so far. The isolated enzyme was stable at 60 °C for 30 min and showed pH stability ranging from 6 to 9. Furthermore, we indicated the induction of identified HOD activity in the presence of betahistine as well, with nearly equal efficiency and without the consumption of the substrate.

Patients with β-thalassemia major (β-TM) show ineffective erythropoiesis and iron overload, which is the leading cause of mortality and organ injury. The present study aimed to investigate the relationships between two iron regulatory hormones, hepcidin and erythroferrone (ERFE) levels, and iron status parameters in Iraqi patients with β-TM. Iron status parameters and hormones were measured in 60 patients and compared with 30 healthy controls. The results indicated significant changes in different iron status parameters, while ferritin with the ∼11-fold increase showed the most change. Significant reduction in hepcidin and an increase in ERFE levels were detected in patients when compared to the control group, while no direct correlation was identified with the other measured iron status parameters. The receiver operating characteristic (ROC) analysis showed that the z-score of the composite of ERFE + ferritin has a full diagnostic ability for β-TM. In conclusion, our findings indicated the correlation between different iron status parameters and ferritin as the leading predictor of iron overload and two main iron regulatory hormones.

The synthetic analogue to biogenic apatite, hydroxyapatite (HA) has a number of physicochemical properties that make it an attractive candidate for diagnosis, treatment of disease and augmentation of biological tissues. Here we describe some of the recent studies on HA, which may provide bases for a number of new medical applications. The content of this review is divided to different medical application modes utilizing HA, including tissue engineering, medical implants, controlled drug delivery, gene therapies, cancer therapies and bioimaging. A number of advantages of HA over other biomaterials emerge from this discourse, including (i) biocompatibility, (ii) bioactivity, (iii) relatively simple synthesis protocols for the fabrication of nanoparticles with specific sizes and shapes, (iv) smart response to environmental stimuli, (v) facile functionalization and surface modification through noncovalent interactions, and (vi) the capacity for being simultaneously loaded with a wide range of therapeutic agents and switched to bioimaging modalities for uses in theranostics. A special section is dedicated to analysis of the safety of particulate HA as a component of parenterally administrable medications. It is concluded that despite the fact that many benefits come with the usage of HA, its deficiencies and potential side effects must be addressed before the translation to the clinical domain is pursued. Although HA has been known in the biomaterials world as the exemplar of safety, this safety proves to be the function of size, morphology, surface ligands and other structural and compositional parameters defining the particles. For this reason, each HA, especially when it comes in a novel structural form, must be treated anew from the safety research angle before being allowed to enter the clinical stage.

The Cardiovascular disease (CVD) is a main cause of worldwide morbidity and mortality overwhelms anyof the circulatory system disease. Therefore, The aims of presented study were to assess whether an Afamin,and Preptin levels are associated with Cardiovascular diseases; to find if there is an association betweenAfamin and preptin levels with insulin resistance in progressive of patients with cardiovascular diseasesbefore and after treatment by catheterization.A case-control study,comprised of 60 patients diagnosed as cardiovascular disease(30 male,30 female),their ages ranged between 35-65 years old, and were matched with patients age and number of male andfemale of 60 healthy control. The estimation of the levels of biochemical parameters in the patients andcontrol groups revealed a significant elevations (P

The authors thank Science et Surface company (Lyon-France) for SEM micrographs. Carole Chaix is thanked for fruitful discussions, Carole Farre for SEM observations, Gaetan Raimondi and Colin Minini for preparation and characterization of AuNPs.; International audience; During the last two decades, phenolic compounds have become the subject of intense research in the environmental preservation field. Phenols are included on the lists of priority pollutants in many countries and their determination is required. In this work, a fast, sensitive miniaturized whole cell conductometric biosensor was developed for the determination of phenol. The biosensor assembly was prepared by immobilizing Pseudomonas sp. (GSN23) bacteria, from oil refinery contaminated soils, on the surface of gold interdigitated microelectrodes. The results obtained with conductometric measurement allowed sensitive detection of phenol from 1 to 300 mg L-1 (10-3187 mu M), with a detection limit (LOD) of 0.2 mg L-1 (2 mu M). Furthermore, the bacterial biosensor was successfully applied to the determination of phenol in spiked river samples.Capsule abstract: A conductometric bacterial biosensor based on Pseudomonas sp. (GSN23) bacteria, from oil refinery contaminated soils has been developed. The detection limit for phenol is 2 mu M.

Background: Human alpha 1-antitrypsin (AAT) is a monomeric glycosylated protein; it is the potent inhibitor of a whole range of serine proteases and protects tissues against their destructive effects. The human plasma-derived AAT, which is currently used to augment the AAT level in patients, is limited due to high cost and source limitation. Recombinant production of AAT can be considered as a potential alternative. Objectives: This study aims to develop and optimize a new chemically defi ned medium based on an elemental analysis of the yeast Pichia pastoris for an effi cient culture of the recombinant yeast-producing secretory AAT. Materials and Methods: An elemental analysis of Carbon (C), Hydrogen (H), Nitrogen (N), Sulfur (S); CHNS in its abbreviated form, and metallic elements was performed to determine the exact molecular constituent of the P. pastoris. The medium components were selected according to the obtained formula; they were optimized by the response surface methodology (RSM). The grown yeast cell was measured at the end of 18 h glycerol batch culture. The amounts of AAT production and elastase inhibitory capacity (EIC) were measured at the end of three days' methanol feeding. Results: The optimized medium compositions consist of glycerol (40 g.L-1), KH2PO4 (24.78 g.L-1), NaCl, (0.88 g.L-1), MgSO4 .7H2 O (1.95 g.L-1), (NH4 )2 SO4 (22.76 g.L-1), and trace elements (20 mL.L-1). The presented quadratic models show that KH2 PO4 and (NH4)2 SO4, are the most abundant ones in the P. pastoris biomass and have the greatest effect on the cell growth, EIC, and AAT protein production responses. Conclusions: According to the results of this study, it can be concluded that the characterizing cell composition formula could be considered as an appropriate method to design culture media in order to improve cell growth and productivity. Compared to the common P. pastoris chemically defi ned media, FM22 and BSM, production of AAT protein increased by 1.5 and 1.4 times, respectively, in this new medium.

Dunaliella salina is a halotolerant microalga with high protein content. Until now, there was no report on biological properties of peptides prepared by enzymatic digestion of D. salina proteins. T...

Cheese production is relied upon the action of Rennet on the casein micelles of milk. Chymosin assay methods are usually time consuming and offline. Herein, we report a new electrochemical technique for studying the proteolysis of K-casein. The interaction of rennet and its substrate was studied by fast Fourier transform continuous cyclic voltammetry (FFTCCV) based on a determination of k-casein in aqueous solution. FFTCCV technique is a very useful method for studying the enzymatic procedures. Fast response, no need of modified electrodes or complex equipment is some of FFTCCV advantages. Various concentrations of enzyme and substrate were selected and the increase in the appearance of charged species in solution as a result of the addition of rennet was studied. Data obtained using FFTCCV technique were also confirmed by turbidity analysis. The results show that rennet proteolysis activity occurs in much shorter time scales compare with its aggregation. Hence, following the appearance of charged segments as a result of proteolysis could be under consideration as a rapid and online method.

Although the use of silver nanoparticles (AgNPs) has substantial benefits, their entrance into the environment, food chain, and human body and their toxicity have come under serious scrutiny. Multiple noncovalent attractive forces between AgNPs and bio-macromolecules are responsible for immediate corona formation upon exposure to biological tissue. Here, the influence of AgNPs with neuro-enzyme Acetylcholinesterase (AChE) was investigated. AgNPs to enzyme ratio had an effect on the enzyme and features of the treated samples. It was also observed that time increments had a positive effect on the size of AgNPs and caused an increase in their initial size. In other words, smaller AgNPs resulted in size increments after interaction with enzymes, while the larger ones showed size decrements. The nano-crystalline AgNPs were identified in x-ray powder diffraction analyses before and after treatment with AChE. The (220) crystalline plane is related to the internal crystallinity of cubic Ag. The results show that the interaction between AChE and AgNPs could lead not only to a decrease in AChE activity, but also to a reduction in the crystallinity and stability of AgNPs. The circular dichroism demonstrates that the secondary structure of AChE also declined after 30 min of incubation with AgNPs at 37 °C.

Here, we report the development of a new polyacrylamide (PAAm)/starch nanofibers’ blend system and highlight its potential as substrate for efficient enzyme immobilization. PAAm was synthesized and blended with starch. The final blend was then electrospun into nanofibers. The response surface methodology was used to analyze the parameters that control nanofiber’s diameter. Electrospun mat was then modified either by cross-linking or phytase immobilization using silane coupling agent and glutaraldehyde chemistry. Physico-chemical properties of blends were investigated using spectroscopic and thermal studies. The evaluation of immobilized enzyme kinetics on both pure and the starch blended PAAm nanofibers was performed using Michaelis–Menten kinetic curves. Fourier transform infrared spectroscopy results along with differential scanning and X-ray diffraction confirmed that blending was successfully accomplished. TGA analysis also demonstrated that the presence of starch enhances the thermal degradability of PAAm nanofibers. Finally, it was shown that addition of starch to PAAm increases the efficacies of enzyme loading and, therefore, significantly enhances the activity as well as kinetics of the immobilized enzyme on electrospun blend mats.

Homotetrameric β-xylosidase from Selenomonas ruminantium (SXA) is one of the most efficient enzymes known for the hydrolysis of cell wall hemicellulose. SXA shows a rapid rate of activity loss at temperatures above 50°C. In this study, we have introduced two inter-subunit disulfide bridges with one point mutation. Lys237 was chosen to be replaced with cysteine since it interacts with the same residue in the opposite subunit. While pH optimum, temperature profile and catalytic efficiency of the mutated variant were similar to the native enzyme, the mutated enzyme showed about 40% increase in thermal stability at 55°C. Our results showed that introduction of a single residue mutation in structure of SXA results in appearance of two disulfide bonds at dimer-dimer interface of the enzyme. Coarse-grained molecular dynamics (CG-MD) simulations also proved lower amounts of root mean square fluctuation (RMSF) for position 237 and potential energy for mutated SXA. Based these results, we suggest that choosing a correct residue for mutation in multi subunit proteins results in multiple site conversions which equals to several simultaneous mutations. Furthermore, CG-MD simulation in agreement with experimental methods showed higher thermostability of mutated SXA which proved applicability of this simulation for thermostability analysis.

Beta thalassemia major (β-TM) disorder characterized by the lack, or severe reduction in the production of hemoglobin β-globin chains. The standard protocol for the management of β-TM is blood transfusion and iron chelation therapy to reduce the iron overload state. The present study aimed to investigate the relationships between two iron regulatory hormones, hepcidin (HEPC) and erythroferrone (ERFE) levels and iron status parameters (ISPs) in Iraqi patients with β-TM. ISPs and hormones were measured in sixty patients and compared with thirty healthy controls. The results indicated significant changes in different iron status parameters, while ferritin (FRT) with the ~11 fold increase showed the most change. Significant reduction in HEPC and increase in ERFE levels were detected in patients as compared to the control group, while no direct correlation was identified with the other measured ISPs. Receiver operating characteristic (ROC) analysis showed that the z-score of the composite of ERFE+FRT has a full diagnostic ability for β-TM. In conclusion, our finding indicated the correlation between different ISPs, FRT as the leading predictor of iron overload and tow main iron regulatory hormones.

Physical adsorption of lipase from Thermomyces lanuginosus onto single-layer sheets of graphene oxide (GO) was studied using the response surface methodology to evaluate the physicochemical factors – temperature, pH, ionic strength, and concentration – affecting the enzymatic activity and the immobilization efficiency. The immobilization efficiency and the activity of the enzyme were inversely proportional to each other. Specifically, higher pH values increased the immobilization efficacy, but produced changes in the aggregation state and secondary structure of the enzyme, thus decreasing its activity. Lower pH values, in turn, reduced the immobilization efficacy, but increased the activity of the adsorbed lipase. The adsorbed and the free lipase were followed during 600 ns and 3.5 μs, respectively, in molecular dynamics (MD) simulations. MD trajectories showed that irreversible adsorption freezes the enzyme in a state with a correctly opened catalytic cavity, while the active site remains without a direct interaction with the GO adsorbent. In contrast to the interfacial activation of lipases in a hydrophobic environment, where the catalytic pocket attaches to the hydrophobic surface, the adsorption onto GO made the active site of the lipase accessible by altering the tertiary structure of the enzyme, leading to a higher catalytic efficiency. Experimental investigations confirmed that the physical adsorption onto GO induces tertiary structure changes in the lipase and protects it from H2O2 by accepting the oxidative damage upon itself. In summary, the physical adsorption of the lipase onto GO is mainly affected by pH and could possibly provide a spreadable and robust catalytic interface for biotechnological applications.

Electrospinning, a simple and versatile method to fabricate nanofibrous supports, has attracted attention in the field of enzyme immobilization. Biocomposite nanofibers were fabricated from mixed PVA/BSA solution and the effects of glutaraldehyde treatment, initial BSA concentration and PVA concentration on protein loading were investigated. Glutaraldehyde cross-linking significantly decreased protein release from nanofibers and BSA loading reached as high as 27.3% (w/w). In comparison with the HRP immobilized into the nascent nanofibrous membrane, a significant increase was observed in the activity retention of the enzyme immobilized into the PVA/BSA biocomposite nanofibers. The immobilized HRP was able to tolerate much higher concentrations of hydrogen peroxide than the free enzyme and thus the immobilized enzyme did not demonstrate substrate inhibition. The immobilized HRP retained∼50% of the free enzyme activity at 6.4mM hydrogen peroxide and no significant variation was observed in the KM value of the enzyme for hydrogen peroxide after immobilization. In addition, reusability tests showed that the residual activity of the immobilized HRP were 73% after 11 reuse cycles. Together, these results demonstrate efficient immobilization of HRP into electrospun PVA/BSA biocomposite nanofibers and provide a promising immobilization strategy for biotechnological applications.

Polymeric nanofiber prepares a suitable situation for enzyme immobilization for variety of applications. In this research, we have fabricated polyvinyl alcohol (PVA)/malonic acid nanofibers using electrospinning. After fabrication of nanofibers, the effect of air, nitrogen, CO 2 , and argon DBD (dielectric barrier discharge) plasmas on PVA/malonic acid nanofibers were analysed. Among them, air plasma had the most significant effect on glucose oxidase (GOx) immobilization. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrum analysis and X-ray photoelectron spectroscopy (XPS) results revealed that in case of air plasma modified nanofibers, the carboxyl groups on the surface are increased. The scanning electron microscopy (SEM) images showed that, after GOx immobilization, the modified nanofibers with plasma has retained its nanofiber structure. Finally, we analysed reusability and storage stability of GOx immobilized on plasma modified and unmodified nanofibers. The results were more satisfactory for modified nanofibers with respect to unmodified ones.

The cellulase component ratio affects the bioethanol yield in the bioethanol process. A method to increase the enzymatic degradation efficiency of cellulose is by preparing a cocktail of involved enzymes. The aim of this study is cocktail preparation and optimization which will reduce the cost of bioethanol process hydrolysis. Moreover, best enzyme composition and ratio depends on the substrate and type of the performed pretreatment. In the present study, response surface methodology (RSM) was used to optimize the ratio of three cellulase involved enzymes, including Exo-1,4-β-D-glucanases II (CBHII), Endo-1,4-β-D-glucanases II (EGII) and 1,4-β-D-glucosidases I (BGI), for degradation of pretreated bagasse. A cellulase activity based on the glycan residue formation was monitored using high performance Liquid Chromatography-Mass Spectrometry (LC-ESI/MS). In addition to cocktail production, the influential parameters affecting the process were studied and the best rate of enzymatic cocktail was adjust...

β-xylosidase and several other glycoside hydrolase family members, including xylanase, cooperate together to degrade hemicelluloses, a commonly found xylan polymer of plant-cell wall. β-d-xylosidase/α-l-arabinofuranosidase from the ruminal anaerobic bacterium Selenomonas ruminantium (SXA) has potential utility in industrial processes such as production of fuel ethanol and other bioproducts. The optimized synthetic SXA gene was overexpressed in methylotrophic Pichia pastoris under the control of alcohol oxidase I (AOX1) promoter and secreted into the medium. Recombinant protein showed an optimum pH 4.8 and optimum temperature 50 °C. Furthermore, optimization of growth and induction conditions in shake flask was carried out. Using the optimum expression condition (pH 6, temperature 20 °C and 1% methanol induction), protein production was increased by about three times in comparison to the control. The recombinant SXA we have expressed here showed higher turnover frequency using ρ-nitrophenyl β-xylopyranoside (PNPX) substrate, in contrast to most xylosidase experiments reported previously. This is the first report on the cloning and expression of a β-xylosidase gene from glycoside hydrolase (GH) family 43 in Pichia pastoris. Our results confirm that P. pastoris is an appropriate host for high level expression and production of SXA for industrial applications.

In this work supercapacitive performance of electrochemically synthesized Poly pyrrole/Manganese dioxide nano wire (Ppy/NwMnO2) was studied by classical and modern electrochemical techniques. NwMnO2 synthesized hydrothermally and distributed in pyrrole solution for electrochemical synthesis. Scanning electron microscopy (SEM) graphs show the uniform distribution of nanowires on the structure of polymer matrix. The specific capacitance of Ppy and Ppy/NwMnO2 were calculated 109 and 203 F/g respectively. Galvano static charge discharge and electrochemical impedance spectroscopy (EIS) are another electrochemical techniques that used for study the supercapacitive behavior of composite electrode. Enhancement the capacitance, conductivity and stability of composite electrode through continues CVs are the features of using nano materials in the structure of polymer film. Fast fourier transform continuous cyclic voltammetry (FFTCCV) studies presented in this work for highlight the performances of composite electrode. Study the nature of composite electrode continuously is one of the advantages that achieved by FFTCCV technique.

AOX1 promoter (pAOX1) is a robust inducible promoter highly preferred for the production of recombinant proteins in Pichia pastoris (P. pastoris). However, repression by other carbon sources and induction by methanol, which is a fire hazard chemical and undesirable for industrial production, are remarkable drawbacks in large-scale use of this promoter. Hence, novel strong regulatory promoters are highly desired. In the present study, the promoter region of methanol oxidase gene (pMOX), from Hansenula polymorpha, was explored for the heterologous expression of foreign proteins in protease deficient and wild type P. pastoris strains. The promoter region of MOX was isolated and replaced with the pAOX1 in the pPINK-HC plasmid. The activity of pMOX and pAOX1 was compared using endoglucanase 3 (CMC3) and endoglucanase II (EgII) enzymes as the reporter proteins. Evaluation of carbon sources on pMOX activity showed complete inactivation in the presence of xylose and sorbitol and high activity by glycerol, glucose and methanol feeding. Furthermore, the results indicated that increasing the gene dosage and using protease deficient-trait significantly increased CMC3 and EgII expression under the control of pMOX. In conclusion, in this study, a new small powerful and methanol-free promoter is introduced for recombinant protein production in yeast P. pastoris.

Atmospheric pressure cold plasma (ACP) is introduced as a useful tool in a variety of biological applications. Proteins are the most abundant macromolecules in living systems with a central role in all biological processes. These organic molecules are modified by ACP exposure that is responsible for many of ACP’s biological effects. This study evaluated the effect of ACP on the production of recombinant phytase in yeast Pichia pastoris (P. pastoris) as well as the structure and function of the phytase enzyme. The results indicated that yeast cells treated with ACP, directly or indirectly, produced higher amounts of recombinant phytase, which was associated with the time of ACP treatment. The exposure of commercial phytase solution with ACP caused a significant increase in the enzyme activity (125%) after 4 hours. Evaluation of the phytase solution by far- and near-UV circular dichroism (CD) and fluorescence analysis indicated that this protein maintained its secondary structure when exposed to ACP while the tertiary structure was slightly unfolded. The effects of heat and H2O2 on the phytase structure and function were compared with the effect of ACP treatment. The modification of Cys, Tyr and Trp amino acids upon reactive oxygen/nitrogen spices was simulated using a molecular dynamics approach. RMSF and RMSD analysis suggested that this structural alteration occurs owing to changes made by reactive species in accessible amino acids.

β-xylosidase is an essential enzyme for breakdown of xylan to d-xylose. It has a significant potential application value for medicine, food, paper and pulp, and biofuel industries. Due to the negative consequences caused by buried free cysteine residues, mutational substitution of such residues is often accompanied by a notable increase in thermal stability. To characterize the role of cysteine residues in the structure, function and stability of Selenomonas ruminantium β-d-Xylosidase (SXA), we prepared and evaluated wild-type and four cysteines- deficient SXA proteins. Buried cysteine residues were replaced with. In comparison with the wild-type, the Km values of the mutants remained relatively constant while their kcat values decreased. The C101V and C286V displayed higher thermal stability than the wild-type at 55 and 60 °C. Conformational changes of the secondary and tertiary structure as derived from circular dichroism and fluorescence spectroscopy revealed that changing a buried cysteine to a hydrophobic residue could lead to an increase in thermal stability with minimal perturbation of the wild-type protein structure. In addition to experimental methods, the stability of WT SXA and C101V and C286V mutants at 333 K was also studied by MD simulation. Our theoretical data had a good agreement with the experimental results.

In this study we prepare a nanocomposite substrate from poly lactic acid (PLA) and graphene oxide (GO) using electrospinning. Determining the possible placement of graphene oxide nano-sheets in the electrospun fiber mats is the main goal of this work. The investigative methods employed include scanning electron microscopy (SEM), atomic force microscopy (AFM) micrographs, tensile modulus and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR). It is shown that the localization of nanoparticle by different methods controls different final properties. We performed the study of the mechanical properties, surface chemical structure and topology of obtaining nanofiber mats. Studies showed that the location of GO sheets depends on the lateral size of them and based on this claim we estimated three possible locations for them: (1) small GO sheets (less than 200 nm) fully or partially inside the electrospun fibers, (2) some larger ones rolling around and on the fibers surface under the high applied voltage, and (3) parts of large sheets bridging between the fibers.

Protein composition and molecular weight play an important role in the digestibility of microalgae proteins. In this study for the first time, proteinous materials of Dunaliella salina and Spirulina platensis were extracted and purified by fast protein liquid chromatography. Then, they are affected by trypsin and chymotrypsin as indicator intestinal enzymes. The results showed that the extracted protein from S. plantesis (ProS) was more rapidly hydrolysed than proteins from D. salina (ProD) because of their lower molecular weight and likely their greater flexibility and open structure. Also, the extent of hydrolysis by trypsin and chymotrypsin of ProS were higher and faster than ProD due to the more number of hydrolytic sites in ProS for both enzymes. The catalytic efficiency and kcat displayed that ProS were more suitable substrate than ProD for intestinal enzymes. The results exhibited that chymotrypsin can act better and faster than trypsin on peptide bonds of proteins.

EndoglucanaseII (Cel5A) of Trichoderma reesei is widely used industrially with the high catalytic efficiency, but it is not stable high temperatures. Structural comparison with the closest thermophilic endoglucanase homolog, Cel5A from Thermoascus aurantiacus, demonstrates disulfide bond differences. Replacement of Cysteine99 with Valine and Cysteine323 with Histidine by site directed mutagenesis caused elimination of two disulfide bonds. Recombinant expression in Pichia pastoris showed the catalytic efficiency (kcat/Km) increment toward CMC for single mutant enzymes, C99V and C323H, about 1.87 and 1.3 folded respectively. This indicates that the elimination of disulfide bond in substrate binding cleft around the catalytic domain of mutant EndoglucanaseII may be increased the flexibility of protein, to form a suitable E–S complex. In direct contrast with previous studies suggesting the existence of disulfide bonds increase the protein stability, the results showed mutant endoglucanase enzymes with disulfide bond reduction have higher thermal stability. The thermal stability of C99V and C323H in 80 °C were increased 2.4 and 2.34 folded, respectively. In this project, theoretical data had a good agreement with the experimental results. Because of high enzyme activity and thermal stability, both of C99V and C323H mutant have high potential suitable for different industrial applications.

There are different methods to modify polymer surfaces for biological applications. In this work we have introduced air-dielectric barrier discharge (DBD) plasma at atmospheric pressure as an economical and safe method for modifying the surface of electrospun chitosan/PEO (90/10) nanofibers for acetylcholinesterase (AChE) immobilization. According to the contact angle measurement results, the nanofibers become highly hydrophilic when they are exposed to the DBD plasma for 6 min in compared to unmodified membrane. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) results reveal hydroxyl, C O and NH 3 + polar groups increment after 6 min plasma treatment. Contact angle measurements and ATR-FTIR results are confirmed by X-ray photoelectron spectroscopy (XPS). AChE at pH 7.4 carries a negative charge and after immobilization on the surface of plasma-treated nanofibrous membrane attracts the NH 3 + group and more enzyme activity is detected on the plasma-modified nanofibers for 6 min in compared to unmodified nanofibers. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used for the surface topography and morphology characterization. The results have proved that air-DBD plasma is a suitable method for chitosan/PEO nanofibrous membrane modification as a biodegradable and functionalized substrate for enzyme immobilization.

In this work, activity of immobilized phytase on blended polyacrylamide (PAAm)/starch nanofibers produced via electrospinning was optimized by computer-aided statistical method of experimental design, response surface methodology (RSM). Thereafter, the role of polymer concentration and percentage of starch in period of enzyme activity were optimized using RSM. We employed these two polymers in blend format as two types of bio-compatible and biodegradable materials including sufficient chemical groups to be appropriate candidates for biological applications. Cross-linking treatments were performed using 3-aminopropyltriethoxysilan (APTES) and glutaraldehyde (GA), and finally phytase enzyme was successfully immobilized on the surface of membranes. First, we used scanning electron microscopy for morphological observation of blended nanofibers. According to the results obtained from response surface method and mean effect assessment, PAAm concentration and percentage of starch as process variables in the experimental design were successfully able to influence the biological properties of immobilized enzyme. Besides, it is also supposed that the presence of starch as a biodegradable and biocompatible natural polymer has a great effect on increasing the unit of enzyme’s activity on the membrane surface.

In this research, nanocomposite nanofibrous webs of poly(acrylic acid) (PAA)/multi-walled carbon nanotubes (MWNTs) were obtained via electrospinning. The effect of MWNTs concentration on the morphology and mechanical properties of PAA/MWNTs nanofibers was investigated by changing the MWNTs content from 0 to 5 wt%. The results showed that average diameter of nanofibers increased with increasing the MWNTs concentration and presence of MWNTs led to the enhancement of mechanical properties. Also, the results revealed that the strength, modulus, and elongation at break of samples increased 3.22, 2.70, and 4.27 fold, respectively, after adding 3 wt% of MWNTs. In addition, the effect of rotating speed of collector on the orientation of PAA nanofibers and its effect on mechanical properties was investigated. Scanning electron microscopy (SEM) studies demonstrated that the degree of nanofibers orientation increased with the augmentation of drum speed to 25 rps. Moreover, the average nanofibers diameter decreased with the increase of drum speed. Improvement of nanofiber orientation resulted in superior mechanical properties that is, higher strength and modulus of aligned nanofiber layers were obtained in comparison to nonaligned layers (12.6 and 26.6 fold, respectively). POLYM. COMPOS., 37:3149–3159, 2016. © 2015 Society of Plastics Engineers

The supercapacitive behavior of polypyrrole/reduced graphene oxide/Au nano particles (Ppy/rGO/AuNPs) as a ternary composite electrode was studied by cyclic voltammetry (CV), galvanostatic charge/discharge (CD), impedance spectroscopy (EIS) and finally fast Fourier transform continuous cyclic voltammetry (FFTCCV) techniques. A composite electrode was synthesized electrochemically in a 1 M KCl solution containing the pyrrole monomer (0.1 M), rGO/AuNPs (0.2% wt) and 5 mM sodium dodecyl sulfate (SDS). Based on the CV study, the specific capacitances of the Ppy and Ppy/rGO/AuNPs electrodes were calculated to be 190 and 310 F g−1, respectively. CD studies showed that using rGO/AuNPs in the structure of the composite electrode decreases the equivalent series resistance of composite film. FFTCCV is a novel and modern electrochemical technique that presents useful data for studying the performance of active materials. The results showed that after 1200 cycles the Ppy/rGO/AuNPs electrode can retain its capacitance compared with the Ppy electrode.

In this work, supercapacitive performance of polypyrrole copper chromate nano particles (Ppy/CuCr2O4 NPs) was studied. CuCr2O4 NPs with the average size of 20nm were synthesized simply by hydrothermal method and the composite electrodes were then electropolymerized on the surface of glassy carbon electrode. Common surface analysis techniques such as scanning electron microscopy (SEM), transmission electron microscopy(TEM) and Fourier transform infrared (FTIR) were used to study the morphology and structure of the composite. Furthermore, for electrochemical evaluation of composite electrodes, techniques including cyclic voltammetry (CV), galvanostatic charge discharge (CD) and impedance spectroscopy (EIS) were used. Using cyclic voltammetry, the specific capacitance values of Ppy and Ppy/CuCr2O4 NPs were calculated to be 109 and 508 F g-1, respectively. Results show that using CuCr2O4 NPs in the structure of polymeric films led to increased specific capacitance of composite electrodes more than four times that of poly pyrrole. Increasing the conductivity and stability of composite electrodes through continuous cycles are the other advantages of using CuCr2O4 NPs as active materials in a polymeric structure.