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Abstract Dopamine is one of the significant neurotransmitters and its monitoring in biological fluids is a critical issue in healthcare and modern biomedical technology. Here, we have developed a dopamine biosensor based on surface plasmon resonance (SPR). For this purpose, the carboxymethyl dextran SPR chip was used as a surface to immobilize laccase as a bioaffinity recognition element. Data analysis exhibited that the acidic pH value is the optimal condition for dopamine interaction. Calculated kinetic affinity (KD) (48,545 nM), obtained from a molecular docking study, showed strong association of dopamine with the active site of laccase. The biosensor exhibited a linearity from 0.01 to 189 μg/ml and a lower detection limit of 0.1 ng/ml (signal-to-noise ratio (S/N) = 3) that is significantly higher than the most direct dopamine detecting sensors reported so far. Experiments for specificity in the presence of compounds that can co-exist with dopamine detection such as ascorbic acid, urea and l-dopa showed no significant interference. The current dopamine biosensor with high sensitivity and specificity, represent a novel detection tool that offers a label-free, simple procedure and cost effective monitoring system.

Abstract Spinal cord injury healing has been shown to be aided by chondroitinase ABC I (cABCI) treatment. The transport of cABCI to target tissues is complicated by the enzyme's thermal instability; however, cABCI may be immobilized on nanosheets to boost stability and improve delivery efficiency. This investigation's goal was to assess the immobilization of cABC I on graphene oxide (GO). for this purpose, GO was produced from graphene using a modified version of Hummer’s process. the immobilization of cABC I on GO was examined using SEM, XRD, and FTIR. The enzymatic activity of cABC I was evaluated in relation to substrate concentration. The enzyme was then surface-adsorption immobilized on GO, and its thermal stability was examined. As compared to the free enzyme, the results showed that the immobilized enzyme had a greater Km and a lower Vmax value. The stability of the enzyme was greatly improved by immobilization at 20, 4, 25, and 37 °C. For example, at 37 °C, the free enzyme retained 5% of its activity after 100 min, while the immobilized one retained 30% of its initial activity. The results showed, As a suitable surface for immobilizing cABC I, GO nano sheets boost the enzyme's stability, improving its capability to support axonal regeneration after CNC damage and guard against fast degradation.

The main shortcoming of the Surface Plasmon Resonance (SPR) method is its inability to detect low molecular weight (

Abstract Multiple sclerosis (MS) is a chronic inflammatory disease, which leads to focal demyelination in the brain and spinal cord. Studies showed that iron released during the course of myelin breakdown exacerbates tissue damage, which is in agreement with the features of iron-dependent cell death, ferroptosis. Here, we aimed to investigate the possible contribution of ferroptosis in the demyelinated optic nerve, and to explore the effectiveness of ferroptosis inhibitor, deferiprone (DFP), on the extent of demyelination, inflammation and axonal damage. For this purpose, focal demyelination was induced by injection of lysolecithin (LPC), into the optic nerve of male C57BL/6J mice. Afterward, optic nerves were harvested at different time points from as early as 6 h up to 7 days post-LPC injection. Next, to evaluate the effectiveness of DFP two groups of animals received daily intraperitoneal injection of DFP for 3 or 7 continuous days. Vehicle groups received saline. Iron deposition was observed at different time points post-LPC injection from 6 h to 7 days post injection. Examining ferroptosis markers showed a significant reduction in glutathione content along with increased level of malondialdehyde and upregulated ferroptosis marker genes at early time points after injection. Besides, DFP treatment during the inflammatory phase of the model resulted in decreased microgliosis and inflammation. Reduced demyelination, microgliosis and astrogliosis was shown in mice that received DFP for 7 days. Moreover, DFP protected against axonal damage and retinal ganglion cells loss. Our results suggest the possible contribution of ferroptosis pathway in the process of demyelination. The therapeutic strategies targeting iron deposition, e.g. DFP treatment might thus represent a promising therapeutic target for patients with MS.

Abstract Background Colorectal cancer is one of the most common cancer and the third leading cause of death worldwide. Increased generation of reactive oxygen species (ROS) is observed in many types of cancer cells. Several studies have reported that an increase in ROS production could affect the expression of proteins involved in ROS-scavenging, detoxification and drug resistance. Nuclear factor erythroid 2 related factor 2 (Nrf2) is a known transcription factor for cellular response to oxidative stress. Several researches exhibited that Nrf2 could exert multiple functions and expected to be a promising therapeutic target in many cancers. Here, Nrf2 was knocked down in colorectal cancer cell line HT29 and changes that occurred in signaling pathways and survival mechanisms were evaluated. Methods The influence of chemotherapy drugs (doxorubicin and cisplatin), metastasis and cell viability were investigated. To explore the association between specific pathways and viability in HT29-Nrf2 −, proteomic analysis, realtime PCR and western blotting were performed. Results In the absence of Nrf2 (Nrf2 −), ROS scavenging and detoxification potential were dramatically faded and the HT29-Nrf2 − cells became more susceptible to drugs. However, a severe decrease in viability was not observed. Bioinformatic analysis of proteomic data revealed that in Nrf2 − cells, proteins involved in detoxification processes, respiratory electron transport chain and mitochondrial-related compartment were down regulated. Furthermore, proteins related to MAPKs, JNK and FOXO pathways were up regulated that possibly helped to overcome the detrimental effect of excessive ROS production. Conclusions Our results revealed MAPKs, JNK and FOXO pathways connections in reducing the deleterious effect of Nrf2 deficiency, which can be considered in cancer therapy.

Background and objectives: Urease, that catalyzes the hydrolysis of urea, has received substantial attention for its impact on living organisms’ health and human life quality. Urease inhibitors play important role in management of different diseases including gastritis and other gastrointestinal disorders. In the present study, a new surface plasmon resonance-based biosensor was designed to discover new urease inhibitors. Methods: The biosensor surface was prepared by the covalent immobilization of urease on carboxymethyldextran hydrogel (CMD 500D) via its primary amine groups. Results: The biosensor combined with an orthogonal enzyme inhibition assay was utilized for screening of 40 traditional medicinal plant extracts against Jack-bean urease. Among them, Laurus nobilis leaf extract displayed a high affinity with the immobilized urease; therefore, its active compound (quercetin) was isolated and identified as a urease inhibitor. The equilibrium constant (KD) and Gibbs free energy (ΔGbinding) values for the interaction of quercetin with urease were obtained to be 55 nM and -41.62 kJ/mol, respectively. The results of molecular docking analysis also confirmed our findings. Conclusion: This SPR-based biosensor represents a new, fast, reliable, and an accurate technique for the identification of new urease inhibitors or novel 'lead' compounds from natural resources.

Abstract Herein proteomic profiling of the rat hippocampus from the kindling and pilocarpine models of epilepsy was performed to achieve new potential targets for treating epileptic seizures. A total of 144 differently expressed proteins in both left and right hippocampi by two-dimensional electrophoresis coupled to matrix-assisted laser desorption-mass spectrometry were identified across the rat models of epilepsy. Based on network analysis, the majority of differentially expressed proteins were associated with Ca2+ homeostasis. Changes in ADP-ribosyl cyclase (ADPRC), lysophosphatidic acid receptor 3 (LPAR3), calreticulin, ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), synaptosomal nerve-associated protein 25 (SNAP 25) and transgelin 3 proteins were probed by Western blot analysis and validated using immunohistochemistry. Inhibition of calcium influx by 8-Bromo-cADP-Ribose (8-Br-cADPR) and 2-Aminoethyl diphenylborinate (2-APB) which act via the ADPRC and LPAR3, respectively, attenuated epileptic seizures. Considering a wide range of molecular events and effective role of calcium homeostasis in epilepsy, polypharmacy with multiple realistic targets should be further explored to reach the most effective treatments.

Abstract Background Since vascular endothelial growth factor (VEGF) is a significant regulator of cancer angiogenesis, it is essential to develop a technology for its sensitive detection. Herein, we sensitized a chemiluminescence (CL) immunoassay through the combination of H2O2-sensitive TGA-CdTe quantum dot (QD) as signal transduction, dextran as a cross-linker to prepare enzyme-labeled antigen and the ultrahigh bioactivity of catalase (CAT) as reporter enzyme. Results Under the optimized experimental conditions, the chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) method can detect VEGF in the excellent linear range of 2–35,000 pg mL−1, with a detection limit (S/N = 3) of 0.5 pg mL−1 which was approximately ten times lower than the commercial colorimetric immunoassay. This proposed method has been successfully applied to the clinical determination of VEGF in the human serum samples, and the results illustrated an excellent correlation with the conventional ELISA method (R2 = 0.997). The suitable recovery rate of the method in the serum ranged from 97 to 107%, with a relative standard deviation of 1.2% to 13.4%. Conclusions The novel immunoassay proposes a highly sensitive, specific, and stable method for very low levels detection of VEGF that can be used in the primary diagnosis of tumors. With the well-designed sensing platform, this approach has a broad potential to be applied for quantitative analysis of numerous disease-related protein biomarkers for which antibodies are available.

Chondroitinase ABCI (cABCI) is a drug enzyme that can be used to treat spinal cord injuries. Due to low thermal stability of cABCI, this enzyme was immobilized on Fe3O4 nanoparticle to increase its thermal stability. The size and morphology, structure and magnetic property of the Fe3O4 nanoparticles were characterized by the analyses of SEM, XRD and VSM, respectively, and FTIR spectroscopy was employed to confirm the immobilization of cABCI on the surface of Fe3O4 nanoparticles. The results indicated that the optimum conditions for pH, temperature, cABCI-to-Fe3O4 mass ratio and incubation time in immobilization process were 6.5, 15 °C, 0.75 and 4.5 h, respectively, and about 0.037 mg cABCI was bound to 1 mg of Fe3O4 nanoparticles at these conditions. The value of Vmax was the same for free and immobilized cABCI, but Km value for immobilized cABCI was 1.6 times higher than that for free one. The storage stability of immobilized cABCI was significantly enhanced at low temperatures, e.g. free cABCI retained 19% of its activity after six days at –20 °C, while the immobilized one retained 96% of its activity. In vitro release of cABCI from Fe3O4 particles showed that about 94% of the enzyme was released after 6 h.

Background and objective: Biosurfactants are increasingly used by food industries due to their low toxicities and unique structures. In this study, biosurfactants were produced and characterized for the first time using acidic bacteria isolated from acidic hot springs in Bushehr Province, Iran. Material and methods: Screening and identification of the most efficient species for biosurfactant production were carried out on 12 bacterial species using several experiments such as hemolysis, surface tension, emulsification index and diameter of clear zone. In addition to biosurfactant production, kinetics, stability and structural and thermal analysis were carried out for the bacterial strains using thin layer chromatography, Fourier Transform Infrared, nuclear magnetic resonance and differential scanning calorimetry. Results and conclusion: The biosurfactant from the selected bacteria (0.1 g l-1) was thermally stable at 120°C for 30 min. Stability at temperatures up to 140°C was confirmed using differential scanning calorimetry. The most significant novelty included the fact that the surface property was preserved until an osmolarity of 4% w v-1. Decreased surface tension and the emulsification potential were only reported at concentrations higher the highlighted concentration. Biological assay showed that Staphylococcus aureus was susceptible to produced biosurfactants, while no susceptibility was seen in Escherichia coli. Degeneration of SW480 cell line exposed to 0.601 µg µl-1 of the biosurfactant was detected after 24 h. The structural analysis showed that the biosurfactant was similar to surfactin as a food bioemulsifier. Conflict of interest: The authors declare that they have no conflict of interest.

Precisely selective interactions of peptides with their unique binding partners represent an out-standing starting point for designing novel therapeutics. It is well established that peptides with a variety of critical physiological functions and specific mechanisms of action offer distinct ad-vantages, including excellent safety and higher efficiency over traditional small molecule thera-peutics. Certain intrinsic weaknesses of naturally occurring peptides such as negligible plasma half-life, low bioavailability, and potential immunogenicity have limited their administration as medicines. Nanotechnology has expanded several promising strategies to address the limitations associated with therapeutic peptides. This review aims to perform a state-of-the-art summary of the strategies that are actively used to develop efficient formulations of nanosystem based pep-tide medicines. We first focus on the recent advances and updates on peptide-based nanomedi-cines. Then we indicate how nanosystems improved the functionality of therapeutic peptides and what the future opportunities and challenges of developments in the field of therapeutic pep-tides are. Potential noninvasive delivery platforms for peptide incorporated nanoparticles through alternative administration routes are also discussed.

One of the advantages of surface plasmon resonance is its sensitivity and real-time analyses performed by this method. These characteristics allow us to further investigate the interactions of challenging proteins like Rap1-interacting factor 1 (Rif1). Rif1 is a crucial protein responsible for regulating different cellular processes including DNA replication, repair, and transcription. Mammalian Rif1 is yet to be fully characterized, partly because it is predicted to be intrinsically disordered for a large portion of its polypeptide. This protein has recently been the target of research as a potential biomarker in many cancers. Therefore, finding its most potent interacting partner is of utmost importance. Previous studies showed Rif1’s affinity towards structured DNAs and amongst them, T6G24 was superior. Recent studies have shown mouse Rif1 (muRif1) C-terminal domain’s (CTD) role in binding to G-quadruplexes (G4). There were many concerns in investigating the Rif1 and G4 interaction, which can be minimized using SPR. Therefore, for the first time, we have assessed its binding with G4 at nano-molar concentrations with SPR which seems to be crucial for its binding analyses. Our results indicate that muRif1-CTD has a high affinity for this G4 sequence as it shows a very low KD (6 ± 1 nM).

Background: Somatic ACE is a two-domain protein, C and N which are resulted from gene duplication. Presence of two active sites with particular properties, demonstrates functional significance of each domain. Increased levels of circulating N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), could be the result of ACE N-domain selective inhibition. Moreover, ACE C-domain specific inhibitors are able to inactivate bradykinin and inhibit the conversion of angiotensin I to angiotensin II in order to regulate blood pressure as well as reduced side effect profiles. Methods: The present study was designed to determine ACE domain specificity of the novel ACE inhibitor, onopordia which was recently isolated from Onopordon acanthium L. The ACE inhibition activity was determined using Abz-SDK (Dnp)P-OH and Abz-LFK(Dnp)-OH as ACE domain selective substrates. IC50 values of onopordia determined and compared with those of captopril as the standard. Results: IC50 values of onopordia for ACE N and C- domains were 180 µM and 244 µM respectively which demonstrated approximately similar affinity of the mentioned compound to ACE C and N-domains. A pharmacophore model was further generated based on onopordia interactions with the relevant ACE domain active sites. Conclusion: According to onopordia interactions in the ACE C and N-domain active sits, it is a potential to generate more potent and also specific inhibitor based on this new scaffold by doing accurate adjustments. Therefore, this study provides the molecular basis for further designing ACE inhibitors, which are new therapeutics in combating tissue fibrosis diseases.

In this work, CKCr-6A strain was found to be highly resistant to some toxic heavy metals such as Cr+6, Cr+3, Cu+2, Co+2, Cd+2, Pb+2, Hg+2, U+6, tellurium, and selenite. Herein, high chromate tolerance of an isolated strain is reported with a high minimum inhibitory concentration value of 80,000 mg/L and the effective parameters (pH, temperature, shaking, and glucose concentration) were selected for Cr(VI) removal by this isolated strain. Cr(VI) elimination by the target strain increased with glucose addition to the culture medium. We optimized the possible parameters and their interactions using design experimental software. After optimization, this strain showed high efficiency in detoxifying chromate; this could reduce up to 100 mg/L of Cr(VI) to Cr(III) over 3 h. The CKCr-6A strain exhibited ability to in vitro reduction after 3 h and repeated removing of Cr(VI) without any amendment of nutrients, suggesting its possible application in continuous bioremediation.

Nanoparticles (NPs) functionalized with antibodies (Abs) on their surface are used in a wide range of bioapplications. Whereas the attachment of antibodies to single NPs to trigger the internalization in cells via receptor-mediated endocytosis has been widely studied, the conjugation of antibodies to larger NP assemblies has been much less explored. Taking into account that NP assemblies may be advantageous for some specific applications, the possibility of incorporating targeting ligands is quite important. Herein, we performed the effective conjugation of antibodies onto a fluorescent NP assembly, which consisted of fluorinated Quantum Dots (QD) self-assembled through fluorine–fluorine hydrophobic interactions. Cellular uptake studies by confocal microscopy and flow cytometry revealed that the NP assembly underwent the same uptake procedure as individual NPs; that is, the antibodies retained their targeting ability once attached to the nanoassembly, and the NP assembly preserved its intrinsic properties (i.e., fluorescence in the case of QD nanoassembly).

Introduction: L-Asparaginase is an anti-neoplastic drug used in lymphoblastic leukemia chemotherapy. Nowadays, this enzyme derived from bacterial sources, mostly L-asparaginase II from Escherichia coli and in lesser amount L-asparaginase of Erwinia sp. has medical utilization. The long-term usage of these agents leads to allergic reactions; therefore new L- asparaginase with new immunological characteristics is required. Halophilic bacteria might contain L-asparaginase with novel immunological properties that can be used in hypersensitive patients. Materials and methods: In this study, the production of L-asparaginase by 130 halophilic and halotolerant bacteria isolated from saline environments in Iran was screened. Modified M-9 agar medium was used for qualitative analysis. In the selected strain, growth curve plotting, asparaginase activity assay and analysis for the effect of some factors on the production and enzyme activity were done. Results: 40 strains produced L-asparaginase. Most of L-asparaginase producers were member of genus Halomonas and Marinobacter (21.4%). GBP­x3 is potent bacteria in L-asparaginase production which belonged to Vibrio sp. based on 16S rRNA analysis. Optimized factors for L-asparaginase production were 350C (0.65 IU/ml), pH 8 (0.926 IU/ml) and 2.5% NaCl (0.903 IU/ml). The highest activity of L-asparaginase was in 350C (0.781 IU/ml), pH 8 (0.82 IU/ml) and without NaCl (0.806 IU/ml). Discussion and conclusion: The aim of this study was to screen the production of L-asparaginase through 130 halophilic and halotolerant bacteria which were isolated from saline environments in Iran. The results of this work indicate that the bacterium, Vibrio sp. GBP­x3 displays to be potent for asparaginase production.

Proteins from their native conformation convert into highly ordered fibrillar aggregation under particular conditions; that are described as amyloid fibrils. α-Synuclein (α-Syn) is a small natively unfolded protein that its fibrillation is the causative factor of Parkinson's disease. One important approach in the development of therapeutic agents is the use of small molecules (such as flavonoids) that could specifically and efficiently inhibit the aggregation process. In this study the effect of few herbal extract (Berberis, Quercus robur, Zizyphus vulgaris, Salix aegyptica) containing flavonoids were investigated on fibril formation of α-syn by using conventional methods such as ThT fluorescence, circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). The interaction of extracts were also analysed by surface plasmon resonance (SPR). Among extracts, Salix aegyptica revealed the highest inhibitory effect on fibril formation. As expected, Salix aegyptica extract also exhibited the highest affinity toward α-syn. Cell viability using MTT assay revealed that fibrils alone were more toxic than those containing the extract. Overall, we demonstrated that the affinity of compounds used in this study corresponds to their ability to arrest fibrillation and reduce cellular toxicity of α-syn fibrils.

Introduction: Excessive use of tellurite nowadays, has suffered the environment from the toxic effects of the oxyanion. Hence, biological treatment of polluted areas is considered as an environmentally friendly and inexpensive method. Although the toxic effects of tellurite for most microorganisms have been reported, but several species of the bacteria including the halophilic bacteria used in this project can overcome the toxicity of the oxyanion by its reduction to the elemental form. The aim of this study was to identify the mechanism (s) involved in tellurite detoxification. Materials and methods: In order to enhance and maintain enzymatic activity during purification, the test conditions and enzyme production by the strain were optimized. The optimization was done by One Factor at A Time (OFAT) method. Several factors, including: time, various percentages of inoculum, range of pH, concentration of tellurite and various salts effects were optimized. For assurance tellurite removal was examined during the experiment. The enzyme was purified by various methods, including ammonium sulphate precipitation and hydrophobic interaction column chromatography in which the Concentration of 1.4 M saturated ammonium sulphate was applied. The purity of the enzyme was assessed by SDS-PAGE during each phase. Results: The optimum conditions obtained showed that at 30 hours, 3% inoculum, pH 7.5, without tellurite and with 5% NaCl the highest enzyme activity and tellurite removal are observed. Purification of the enzyme greatly reduced the concentration of unrelated proteins and caused a concentrated band which could be one subunit of the enzyme targeted. The partially purified enzyme’s fraction was shown to have nitrate and selenite reductase activity other than tellurite reductase activity. Discussion and conclusion: This study is an approach to the identification of the halophilic microorganisms Physiology and enzymes involved in restoring tellurite. The production of the enzyme responsible for this phenomenon has been optimized and partially purified.

Objective: Immunotoxins (ITs) have been developed for the treatment of cancer, and comprise of antibodies linked to toxins. Also vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis, and the blockade of VEGF receptor-2 (VEGFR2) inhibits angiogenesis and tumor growth. The aim of this study was to produce anti-VEGFR2/rPE (Pseudomonas exotoxin) 38 IT to test its cytotoxic activity and mechanism of action. Materials and Methods: In this basic research and experimental study, at first, DNA that encodes recombinant PE38 protein was inductively expressed in Escherichia coli (E.coli) and purified by nickel-sepharose chromatography and further analyzed by western blot. Then, for production of IT, rPE38 was chemically conjugated to anti-VEGFR2. The cytotoxicity response of IT treatment was evaluated by 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) test in Human Umbilical Vein Endothelial Cell (HUVEC) and Michigan Cancer Foundation-7 (MCF-7) (VEGFR2+) cell lines. The mechanism of IT cytotoxicity was observed by Annexin V staining and flow cytometry. Continuous variables were compared with the analysis of variance (ANOVA; for all groups). P values less than 0.05 were considered statistically significant. Results: SDS-PAGE showed 98% purity of rPE38 and IT. In vitro dose-dependent cytotoxicity assay demonstrated that anti-VEGFR2/PE38 is toxic to VEGFR2-positive cells. IT treatment significantly inhibited proliferation of HUVEC and MCF-7 in a VEGFR2-specific manner as compared with the control groups (p

Introduction: Laccases (benzene diol oxygen oxidoreductase: EC 1.10.3.2 are one of the multicopper oxidase family members that catalyze the oxidation of various phenolic compounds by using molecular oxygen Materials and methods Laccase gene was from strain GAZ23 amplified by cloning primers. PCR product cloned in the expression vector (pET21a) and transferred to BL21 strain of E. coli and sequence analysis were carried out. Biochemical properties were investigated using common laccase substrates, 2,2ˊ-azino-bis(3-ethylbenzothioazolin-6-sulphonicacid )ABTS). Results: 16S rRNA gene of strain GAZ23 isolated from Iran soils, showed high similarity to Bacillus pumilus (100%). The gene of the GAZ23 has an open reading frame composed of 1533 bases, which encode 510 amino acid residues. Discussion and conclusion: The laccase gene from GAZ23 shows 67% similarity with CotA from B. subtilis. The expression was performed under microaerobic condition and decreased temperature in order to obtain high amounts of soluble protein. This protein contains four histidine rich copper-binding domains.

The biodesulfurization (BDS) of sulfur compounds in fossil fuels is a process to reduce sulfur dioxide emissions that cause environmental pollution. Gordonia alkanivorans RIPI90A is able to convert dibenzothiophene, an organic sulfur compound in petroleum, to 2-hydroxybiphenyl (2-HBP) in 4S pathway. In this study, (DBT), DszA and DszB, DBT sulfone monooxygenase, and desulfinase were respectively isolated from G. alkanivorans RIPI90A. PCR amplified fragments were obtained by using primers designed based on known sequences from G. alkanivorans RIPI90A. They are identified as dszA and dszB and have shown high similarity compared to Rhodococcus erythropolis IGTS8 (88% for dszA and 88% for dszB). Subsequently, dszA and dszB genes were expressed under the control of T7 promoter in Escherichia coli. The recombinant proteins were purified to achieve homogeneity using Ni-agarose column chromatography. The molecular mass of the purified DszA and DszB were determined to be 51.9 and 39.2 kDa respectively by using SDS-polyacrylamide gel electrophoresis. DszA showed a Km of 0.14±0.005 mM and a maximal velocity of 0.004±0.0004 mM/min. DszB showed a wide substrate range in a way that all aromatic sulfonates compounds acted as its substrate; as it seemed the active site was suitable for the sulfonated aromatic rings. The Km and Vmax values of DszB were calculated to be 1.81±0.02 mM and 6.55 ± 0.005 µM/min respectively using 4-Amino-3-hydroxy-naphthalene-sulfonic acid as a substrate

Introduction: Halophiles, especially haloarchaea are one of the most important groups of extremophiles. Halophilic hydrolases have been studied worldwide and have been considered for biotechnology and industrial technologies. This study is the first report in amylopullulanase production in halophilic microorganisms.Materials and methods: A halophilic archaeon, Halorubrum sp. strain Ha25, produced extracellular halophilic organic solvent-tolerant amylopullulanase. The enzyme was purified using ethanol precipitation and anion exchange chromatography method. Molecular mass of purified enzyme was determined by SDS–PAGE method. After purification, the enzyme was characterized. To study the effects of organic solvents in the stability of the enzyme, the enzyme solution was incubated in the presence of various organic compounds and then, residual enzyme activity was measured. Mode of action of the enzyme was determined by thin-layer chromatography.Results: Molecular weight of the purified enzyme was estimated to be 140 kDa by SDS–PAGE method. Optimum temperature for amylolitic and pullulytic activities was 50 °C. Optimum pH for amylolitic activity was 7.0 and for pullulytic activity was 7.5. This enzyme was active over a wide range of concentrations (0-4.5 M) of NaCl. The effect of organic solvents on the amylolitic and pullulytic activities showed that this enzyme was more stable in the presence of non-polar organic solvents than polar solvents. The enzyme solely hydrolyzed pullulan and soluble starch to glucose.Discussion and conclusion: Halorubrum sp. strain Ha25 produces thermophilic and extremely halophilic amylopullulanase. The catalytic function under multi extreme condition of high temperature, high salinity, and low water activity might possess biotechnological and commercial values such as treatment waste solutions with starch residues, high salt content and solvents.

A sequence of 10 amino acids at the C-terminus region ofmethylglyoxal synthase from Escherichia coli (EMGS) providesan arginine, which plays a crucial role in forming a salt bridgewith a proximal aspartate residue in the neighboring subunit,consequently transferring the allosteric signal between subunits.In order to verify the role of arginine, the gene encoding MGSfrom a thermophile species, Thermus sp. GH5 (TMGS) lackingthis arginine was cloned with an additional 30 bp sequence atthe 3´-end and then expressed in form of a fusion TMGS with a10 residual segment at the C-terminus (TMGS+). The resultingrecombinant enzyme showed a significant increase in cooperativitytowards phosphate, reflected by a change in the Hillcoefficient (nH) from 1.5 to 1.99. Experiments including sitedirected mutagenesis for Asp-10 in TMGS and TMGS+, twodimentional structural survey, fluorescence and irreversiblethermoinactivation were carried out to confirm this pathway.

Thirty-five fungal strains which isolated from vegetable wastes, were screened for the use of polygalacturonic acid as the sole carbon source. Twenty-five isolates were positive for polygalacturonase activity in cup-plate assay, as evidenced by clear hydrolysation zones. The most productive strain was determined by measuring clear zones formed around colonies stained with ruthenium red. The highly pectinolytic fungal strain was tentatively identified as Tetraoccosporium sp. according to morphological characterization. The cultivation of the selected strain (Tetracoccosporiumsp.) in liquid media resulted in high quantities of polygalacturonase enzyme. Maximum polygalacturonase activity was reached in 48 h of growth in the pectate medium. The collected polygalacturonase had optimum activity at pH 5.0 and maximal activity of the enzyme was determined at 35 °C. Mn2+, Ag3+and surface active detergents such as tween 20 and triton X-100 increased the polygalacturonase activity by 37 % and EDTA enhanced the activity up to 125 %.

Sea hares have attracted the interest of many workers investigating chemical defense substances. Most of these substances are low molecular weight compounds derived from algal diets. Anticancer effects of a novel protein isolated from purple fluid of A. dactylomela are reported. The purification procedure consisted basically of ammonium sulphate fractionation, ion exchange and ultrafiltration techniques. For cytotoxicity effects, L929, K562, HL60 and NB4 cell lines and MTT assay were used. A protein of 60000 Da of the purple fluid of A. dactylomela had antiproliferative effects on the cell lines it was maximally active at 0.5-1.5 microgram/ml on NB4 cell line. Therefore, the purple fluid of A. dactylomela has a novel antiproliferative agent.