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In this study, chitosan/curcumin (CS/Cur) and chitosan/papain (CS/Pa) nanoparticles were prepared and then characterized by Fourier transform infrared (FTIR), X-ray diffraction (XRD), and differential light scattering (DLS). Subsequently, release rate, porosity, swelling, degradability, anti-inflammatory, antioxidant, antibacterial, and cell viability tests were conducted to investigate and compare the healing potential of the nanoparticles for various types of wounds. The results of FTIR, XRD, and DLS indicated that the nanoparticles were manufactured correctly with a hydrodynamic diameter of 429 nm (CS/Cur) and 460 nm (CS/Pa), and zeta potential of 4.32 mV (CS/Cur) and 7.57 mV (CS/Pa). The release rate results indicated a higher release rate in a basic environment (pH 8.4) for curcumin, a higher release rate for papain in an acidic environment (pH 6.4), and the Korsmeyer-Peppas model for the release of curcumin and papain. The results indicated that CS/Cur with 41.6% antioxidant activity, high antibacterial effect, and cell growth up to 616% during 7 days, was more effective than CS/Pa. In comparison, CS/Pa (with a porosity of 70.5% and a swelling rate of 1392%) was more advantageous than CS/Cur in terms of porosity and swelling. In addition, CS/Cur was as effective as CS/Pa in terms of degradation and anti-inflammatory properties. In conclusion, the outcomes represented that the CS/Cur and CS/Pa nanoparticles improved wound healing, and each was suitable for specific wounds and wound healing stages., Competing Interests: Conflict of interestThe authors declare that there are no conflicts of interest., (© King Abdulaziz City for Science and Technology 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

This study aimed to fabricate gum Arabic (GA)-coated Fe 3 O 4 nanoparticles bearing numerous active aldehyde groups on their surface, followed by an assessment of their capability as a magnetic support for the covalent immobilization of the trypsin enzyme for the first time. FT-IR, XRD, TGA, and SEM results demonstrated the successful synthesis of GA-coated Fe 3 O 4 nanoparticles, along with the covalent immobilization of the enzyme onto the support. Immobilization enhanced the relative enzymatic activity across a range of aqueous solution pH levels (ranging from 4 to 11) and temperatures (ranging from 20 to 80 °C) without altering the optimum pH and temperature for trypsin activity. Kinetic studies using Michaelis-Menten plots revealed changes in kinetic parameters, including a lower V max and higher K m for immobilized trypsin compared to the free enzyme. The immobilization onto magnetic gum Arabic nanoparticles resulted in an improved stability of trypsin in the presence of various solvents, maintaining a stability order comparable to that of the free enzyme due to the stabilizing effect of the support. The reusability results showed that the immobilized enzyme can retain over 93% of its activity for up to 15 cycles., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Herein, 5-fluorouracil and shikonin (extracted from Fusarium tricinctum) were loaded in chitosan/pectin nanoparticle (CS/PEC-NPs), prepared by blending (B-CS/PEC-NPs) and coating (C-CS/PEC-NPs) methods. The nanoparticles characterized by Fourier Transform Infrared (FTIR), X-ray diffraction (XRD), Energy-dispersive X-ray (EDX), Scanning Electron Microscope (SEM) and Differential Light Scattering (DLS). Then, some properties of the nanoparticles such as drug release rate and the nanoparticles cytotoxicity were studied. The FTIR, XRD, EDX, SEM and DLS results showed that the nanoparticles synthesized properly with an almost spherical morphology, an average size of 82-93 nm for B-CS/PEC-NPs, an average diameter of below 100 nm (mostly 66-89 nm) for C-CS/PEC-NPs, and hydrodynamic diameter of 310-817 nm. The drug release results indicated the lower release rate of drugs for B-CS/PEC-NPs relative to C-CS/PEC-NPs at different pHs, high release rate of drugs for the nanoparticles in the simulated large intestinal fluids containing pectinase, and Korsmeyer-Peppas model for release of the drugs. The results showed more cytotoxicity of B-CS/PEC-NPs containing drugs, especially B-CS/PEC-NPs containing both drugs (B-CS/PEC/5-FU/SHK-NPs) after treating with pectinase (IC 50 of 18.6 μg/mL). In conclusion, despite the limitation of C-CS/PEC-NPs for simultaneous loading of hydrophilic and hydrophobic drugs, B-CS/PEC-NPs showed suitable potency for loading and targeted delivery of the drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this study, the process of manufacturing nanohydrogels containing papain and how to release it was investigated. Chitosan nanohydrogels and chitosan-polyethylene glycol hybrid nanohydrogels were used to entrapment of papain as a protein model. In order to evaluate and confirm different properties of nanohydrogels such as size, shape, the rate of swelling and flexibility, different methods was used. The maximum amount of papain entrapment was observed in 0.75% concentration of chitosan and 1% concentration of sodium Tripolyphosphate (TPP) as linker. The results of scanning electron microscope (SEM) and X-ray diffraction (XRD) patterns showed that nanohydrogels containing papain on a nano scale are very porous and swollen. Differential scanning calorimetry (DSC) thermograms analysis showed that nanohydrogels have relatively good water absorption capacity. Also, by adding polyethylene glycol to chitosan, the melting temperature of hybrid nanohydrogels decreased and this can be a reason for the formation of flexible structures in these nanohydrogels. In chitosan nanohydrogels, the highest release rate of papain was observed at pH lower than 7 and high temperatures, but by adding polyethylene glycol to the chitosan, in addition to increasing papain release, a proper and continuous release of papain was observed at temperature and pH close to physiological conditions, especially at low ratios of polyethylene glycol. According to the present results, hybrid nanohydrogels can have a good potential in protein delivery systems in terms of structure and release., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Endophytic fungi are microorganisms that are considered as a potential source of natural compounds, and can be applied in various industries. The aims of this research were molecular identification of endophytic fungi isolated from the Gundelia tournefortii stems, and investigation their biological activities as well as phenolic and fatty acid profile. Surface sterilized stems of G. tournefortii were placed on potato dextrose agar (PDA) to isolate the fungal endophytes. Genomic DNA was extracted by CTAB method, and PCR amplification was performed by ITS 1 and ITS 4 as primers. The enzyme production of endophytic fungi was determined based on the formation of a clear zone that appeared around the colonies of fungus. The anti-oxidant activity was evaluated by measuring the amount of free radicals DPPH. Also, the total phenol and flavonoid contents were measured obtained by Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively. Moreover, the separation and identification of phenolic acids and fatty acids were done by HPLC and GC, respectively. Phylogenetic analysis was done based on the Internal Transcribed Spacer (ITS) region, and five isolates were identified as following: Aspergillus niger, Penicillium glabrum, Alternaria alternata, A. tenuissima, and Mucor circinelloides. Evaluation of the enzymatic properties showed that P. gabrum (31 ± 1.9 mm), and A. niger (23 ± 1.7) had more ability for producing pectinase and cellulase. The anti-oxidant activity of isolates showed that A. alternata extract (IC 50  = 471 ± 29 µg/mL) had the highest anti-oxidant properties, followed by A. tenuissima extract (IC 50  = 512 ± 19 µg/mL). Also, the extract of A. alternata had the greatest amount of total phenols and flavonoids contents (8.2 ± 0.4 mg GAL/g and 2.3 ± 0.3 mg QE/g, respectively). The quantification analysis of phenolic acid showed that rosmarinic acid, para-coumaric acid, and meta-coumaric acid (42.02 ± 1.31, 7.53 ± 0.19, 5.41 ± 0.21 mg/g, respectively) were the main phenolic acids in the studied fungi. The analysis of fatty acids confirmed that, in all fungi, the main fatty acids were stearic acid (27.9-35.2%), oleic acid (11.3-17.3%), palmitic acid (16.9-23.2%), linoleic acid (5.8-11.6%), and caprylic acid (6.3-10.9%). Our finding showed that endophytic fungi are a source of bioactive compounds, which could be used in various industries. This is the first report of endophytic fungi associated with G. tournefortii, which provides knowledge on their future use on biotechnological processes., (© 2024. The Author(s).)

Nanocomposites containing different effective materials have various effects, such as antioxidant, and anti-inflammatory activity, which are desirable for wound dressing. Herein, nanocomposites based on chitosan/reduced graphene oxide (CS/rGO) containing curcumin (CS/rGO/Cur), curcumin and papain (CS/rGO/Cur/Pa), curcumin, papain, and collagen peptide (CS/rGO/CP/Cur/Pa), prepared using ionic gelation method and characterized by Fourier Transform Infrared (FTIR), Differential Light Scattering (DLS), X-ray diffraction (XRD), and Scanning Electron Microscope (SEM). Subsequently, the nanocomposite's potential for wound healing was studied through parameters such as porosity, swelling, degradability, anti-inflammatory, antioxidant, antibacterial, cell viability, and in-vivo. The results of FTIR, XRD, SEM, and DLS showed that the nanocomposites synthesized properly with an almost spherical morphology, an average diameter of below 100 nm (mostly 40-85 nm), and a hydrodynamic diameter of 455-616 nm. The various tests demonstrated the nanocomposite's effectiveness in wound healing. The results showed that CS/rGO/CP/Cur/Pa increased the anti-inflammatory and cell viability up to 99.7 % and 395 %, respectively, which is higher than others. Animal tests on rats showed that CS/rGO/CP/Cur/Pa accelerated the wound healing rate up to 70 %. In conclusion, the results showed that the nanocomposites based on CS/rGO significantly improve wound healing, and the presence of collagen peptides boost their wound healing potency., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The immobilized enzymes' properties can be affected by cross-linkers on the surface of supports. To study how cross-linkers alter enzymes function, chitosan-coated magnetic nanoparticles (CMNPs) with immobilized papain were prepared using glutaraldehyde and or genipin, and then, the properties of the nanoparticles and the immobilized enzymes were assessed. The Scanning Electron Microscope (SEM), Fourier Transform Infrared (FTIR), and X-Ray Diffraction (XRD) results showed that the CMNPs were prepared and papain molecules were immobilized on CMNPs by glutaraldehyde (CMNP-Glu-Papain) or by genipin (CMNP-Gen-Papain). Also, the results associated with enzymes activity indicated that the immobilization by glutaraldehyde and genipin increased the pH optimum of papain from 7 to 7.5 and 9, respectively. The kinetic results indicated that the immobilization by genipin slightly affects the enzyme affinity to the substrate. The stability results showed that CMNP-Gen-Papain has more thermal stability than CMNP-Glu-Papain and papain immobilization on CMNPs by genipin leads to stabilization of the enzyme in the presence of polar solvents, probably due to the more hydroxyl groups on CMNPs activated by genipin. In conclusion, this study suggests that there is a relationship between the types of cross-linker on the surface of supports, and the mechanism of action, kinetic parameters, and the stability of immobilized papain., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Pseudomonas aeruginosa is the leading cause of nosocomial infections, especially in people with a compromised immune system. Targeting virulence factors by neutralizing antibodies is a novel paradigm for the treatment of antibiotic-resistant pseudomonas infections. In this respect, exotoxin A is one of the most potent virulence factors in P. aeruginosa. The present study was carried out to identify a novel human scFv antibody against the P. aeruginosa exotoxin A domain I (ExoA-DI) from a human scFv phage library., Methods: The recombinant ExoA-DI of P. aeruginosa was expressed in E. coli, purified by Ni-NTA column, and used for screening of human antibody phage library. A novel screening procedure was conducted to prevent the elimination of rare specific clones. The phage clone with high reactivity was evaluated by ELISA and western blot., Results: Based on the results of polyclonal phage ELISA, the fifth round of biopanning leads to the isolation of several ExoA-DI reactive clones. One positive clone with high affinity was selected by monoclonal phage ELISA and used for antibody expression. The purified scFv showed high reactivity with the recombinant domain I and full-length native exotoxin A., Conclusions: The purified anti-exotoxin A scFv displayed high specificity against exotoxin A. The human scFv identified in this study could be the groundwork for developing a novel therapeutic agent to control P. aeruginosa infections.

Premature Ovarian Insufficiency (POI) is viewed as a type of infertility in which the menopausal status occurs before the physiological age. Several therapeutic strategies have been introduced in clinic for POI treatment, although the outputs are not fully convincing. Platelet-rich plasma (PRP) is a unique blood product widely applied in regenerative medicine, which is based on the releasing of the growth factors present in platelets α-granules. In the current investigation, we examined the effectiveness of PRP as a therapeutic alternative for POI animals. POI in Wistar albino rats was induced by daily intraperitoneal (IP) administration of gonadotoxic chemical agent, 4-vinylcyclohexene dioxide (VCD) (160 mg/ kg) for 15 consecutive days. After POI induction, the PRP solution was directly injected intra-ovarian in two concentrations via a surgical intervention. Every two weeks post-injection, pathological changes were monitored in the ovaries using Hematoxylin-Eosin staining method, until eight weeks. Follicle Stimulating Hormone (FSH) content in serum was measured, together with the expression of the angiogenic-related transcripts ANGPT2 and KDR by real-time qPCR. Furthermore the fertility status of the treated rats was evaluated by mating trials. Histopathological examination revealed successful POI induction via the depletion of morphologically normal follicles in rats following VCD treatment compared to the control rats. The injection of PRP at two concentrations reduced the number and extent of the follicular atresia and inflammatory responses (p < 0.05). The expression of both ANGPT2 and KDR transcripts were significantly increased in POI rats due to enhanced inflammation, while these values were modulated after PRP administration (p < 0.05) compared to POI rats. FSH showed a decreased trend in concentration eight weeks after PRP treatment, but not statistically significant (p > 0.05). Nevertheless, a clear improvement in litter counts was found in POI rats receiving PRP compared to the non-treated POI group, being able to consider PRP as a facile, quick, accessible, safe and relatively cheap alternative therapeutic strategy to revert POI-related pathologies.

Pectinolytic enzymes that catalyze the breakdown of substrates containing pectin are widespread. Pectinases have potential applications in various industries, including food, animal feed, textile, paper, and fuel. In this study, one hundred bacterial isolates were collected from Marand city farmlands (Azarbaijan-E-Sharqi, Iran) and screened by MP medium on the base of pectinase activity considering the significance of pectinases. The results depicted that three isolates showed the most pectinase activity (more massive halo). The biochemical and molecular test results showed that the three screened bacteria were Enterobacter and named Enterobacter sp. MF41, Enterobacter sp. MF84, and Enterobacter sp. MF90. Enterobacter sp. MF84 had the largest halo, so this strain was selected for the study of its produced pectinase. The results exhibited that the produced enzyme has optimum temperature and pH for activity at 30 °C and in 9, respectively. Finally, the enzyme production by Enterobacter sp. MF84 is optimized using response surface methodology (RSM) considering four factors (NH 4 Cl, K 2 HPO 4 , pectin, and incubation time) as variables. The results showed that the optimization procedure increased the enzyme production up to 12 times (from 1.16 to 14.16 U/mg). The Pareto analysis revealed that ammonium chloride has a significant role in decreasing the enzyme production, probably by inducing the nitrification pathway enzymes in the presence of organic nitrogen in Enterobacter sp. MF84.

Immobilization of enzyme onto nanoparticles such as chitosan can have biotechnological importance. In this study, chitosan nanoparticles (ChNPs) were prepared by Ionic gelation method and Endoglucanase Cel9A from Alicyclobacillus acidocaldariius (AaCel9A) immobilized on the nanoparticles. The FTIR results showed that the enzymes were immobilized on the ChNPs. The dynamic light scattering and scanning electron microscope (SEM) results illustrated that the AaCel9A-ChNPs approximately had 40 nm diameters. For optimizing enzyme immobilization, response surface methodology was employed using different variables (pH, enzyme immobilization time, and enzyme to ChNPs ratio [E/Cs]). The results showed that the high immobilization efficiency was achieved in pH 7, E/Cs of 0.4 in 2.63 hr. The enzyme activity results showed that, immobilization increased optimum pH for activity (from 6.5 to 7.5) and the enzyme K m (from 3.703 to 12.195 [mg/ml]), which make it suitable to use in some industries such as detergents., (© 2020 American Institute of Chemical Engineers.)

Background: Infertility is a major problem worldwide. Various strategies are being used to develop better treatments for infertility and The most trending strategy is the stem cell therapy. In this study, the literature on stem cell therapy for ovarian disorders is summarized with analysis of current developments., Objective: Different published studies on stem cell-based therapy for the treatment of various types of ovarian insufficiency and disorders such as Premature Ovarian Insufficiency (POI) in the affected female population in animal or human clinical studies are systematically reviewed., Methods: We monitored five databases, including PubMed, Cochrane, Embase, Scopus, and ProQuest. A comprehensive online search was done using the criteria targeting the application of stem cells in animal models for menopause. Two independent reviewers carefully evaluated titles and abstracts of studies. The stem cell type, source, dosage, route of administration were highlighted in various POI animals models. Non-relevant and review articles were excluded., Outcomes: 648 published studies were identified during the initial comprehensive search process from which 41 were selected according to designed criteria. Based on our analysis, stem cells could accelerate ovarian tissues rejuvenation, regulate systemic sex-related hormones levels and eventually increase fertility rate., Conclusion: The evidence suggests that stem cell-based therapies could be considered as an alternative modality to deal with women undergoing POI., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Objective: We explored detrimental effects of VCD on non-ovarian tissues such as kidneys and liver 14 days post-drug administration. Twelve rats were randomly assigned into two groups. In VCD group, rats received 160 mg/kgbw VCD intraperitoneally for 15 consequent days. Control rats were injected with VCD-free normal saline. At the respective time point, rats were euthanized, blood and tissue samples were collected. H&E staining was performed to evaluate pathological changes. Serum level of ALT, AST, creatinine and urea were also measured., Results: Histological analysis revealed hyperemia and follicular atresia in the ovaries, indicating successful POF induction in rats. In renal tissue, extensive tubular necrosis, focal hemorrhage, hyaline casts, and interstitial nephritis were observed. Analysis of hepatic tissue showed numerous hemorrhagic foci, chronic cholangitis, and hepatocyte necrosis, indicating apparent VCD toxicity of both hepatic and renal tissues. The biochemical evaluation revealed a tendency of increase in ALT, AST, creatinine, and Urea in VCD-treated rats; however, the values did not reach significant level. In conclusion, the induction of POF in rats by VCD correlates with renal and hepatic damages. Commensurate with data from this study, any conclusions from experiments based on VCD-induced premature ovarian failure rats should be reported with caution.

The immobilization of enzymes improves their stability in non-conventional media such as organic solvents. In this work, the effects of solvents (DMSO, methanol, ethanol, and n -propanol) on the endoglucanase Cel9A activity and stability were studied. Then, the enzymes were stabilized by its immobilization on chitosan nanoparticles and also using polyols (sorbitol and glycerol) against organic solvents. The SEM results illustrated that the chitosan nanoparticles had about 40 nm diameter. The results indicated that the organic solvents, especially n -propanol, decreased the activity of the free and immobilized enzymes. The reduced activity of the immobilized enzyme was less than that of the free enzyme. Our studies about the enzymes' stability showed that the free and immobilized enzymes in hydrophobic solvents (with high log P ) had the lowest stability compared to other solvents as we observed the half-life of the free enzyme in n -propanol solvent was 2.84 min, and the half-life of the immobilized enzyme was 4.98 min in n -propanol and ethanol solvents 4.50 min. Analysis of the combinatory effects of polyols (sorbitol and glycerol) and the solvents on the stability revealed that sorbitol and glycerol had the most stabilizing effect on the free enzyme in hydrophilic (DMSO) and hydrophobic ( n -propanol) solvents, respectively. However, the stabilizing effects of polyols in the immobilized enzyme were independent of the solvents' hydrophobicity (or log P ) due to the hydrophilic properties of chitosan nanoparticles. Therefore, one can conclude that the physiochemical properties of nanoparticles (such as hydrophilicity) influence the stabilizing effects of polyols on immobilized enzyme., Competing Interests: Conflict of interestThe authors declare that there are no conflicts of interest.

The preparation of biocatalysts based on immobilized trypsin is of great importance for proteomic research, industrial applications and organic synthesis. Here in, we have developed a facile method to immobilize trypsin on magnetic nanoparticles. Fe 3 O 4 nanoparticles were synthesized by co-precipitating Fe 2+ and Fe 3+ in an ammonia solution and then coated by silicon dioxides were developed by sol-gel method. The silica-coated Fe 3 O 4 nanoparticles were further modified with 3-aminopropyltriethoxysilane, resulting in attaching of primary amine groups on the surface of the particles. Trypsin from porcine pancrease was then immobilized on the magnetic core-shell particles by using glutaraldehyde as a cross-linker. The synthesis steps and characterizations of immobilized trypsin were examined by FT-IR, XRD, TGA, EDX and SEM. The results showed that the enzyme immobilization increased the enzyme activity in different pHs and temperatures, without any changes in the optimum pH and temperature for enzyme activity. The Kinetic results showed that the enzyme immobilization decreased and increased V max and K m values, respectively. The stability results showed that the enzyme immobilization improved trypsin thermostability in the absence and presence of 10% (v/v) of the used solvents (DMF, THF, DMSO, ACN and 1, 4-Dioxane). The reusability results indicated that the immobilized enzyme maintained 85% of its activity after 6 periods of activity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) has an Ig-like domain and the enzyme stability is dependent to calcium. In this study the effect of calcium on the structure and stability of the wild-type enzyme and the truncated form (the wild-type enzyme without Ig-like domain, AaCel9AΔN) was investigated. Fluorescence quenching results indicated that calcium increased and decreased the rigidity of the wild-type and truncated enzymes, respectively. RMSF results indicated that AaCel9A has two flexible regions (regions A and B) and deleting the Ig-like domain increased the truncated enzyme stability by decreasing the flexibility of region B probably through increasing the hydrogen bonds. Calcium contact map analysis showed that deleting the Ig-like domain decreased the calcium contacting residues and their calcium binding affinities, especially, in region B which has a role in calcium binding site in AaCel9A. Metal depletion and activity recovering as well as stability results showed that the structure and stability of the wild-type and truncated enzymes are completely dependent on and independent of calcium, respectively. Finally, one can conclude that the deletion of Ig-like domain makes AaCel9AΔN independent of calcium via decreasing the flexibility of region B through increasing the hydrogen bonds. This suggests a new role for the Ig-like domain which makes AaCel9A structure dependent on calcium.

Background: In this study, we have experimentally investigated the effects of different osmolytes including sucrose, sorbitol, urea, and guanidinium chloride (GdmCl) on the stability and structure of the Mycobacterium tuberculosis pyrazinamidase (PZase). PZase converts pyrazinamide to its active form., Methods: In addition, in order to gain molecular insight into the interactions between osmolytes and PZase, we have conducted 1000-ns molecular dynamics simulations., Results: The results indicated that sucrose and sorbitol increase the stability and compactness of the enzyme, whereas in the presence of urea and GdmCl, PZase loses its stability and compactness. Furthermore, the activity of PZase in the presence of sucrose was more than the other solutions. The energetic analyses imply that the electrostatic and van der Waals interactions are the major factors in the osmolyte-PZase interactions. Sorbitol and sucrose, as protective osmolytes, protect the protein structure by utilizing the van der Waals interaction from denaturation. In addition, urea molecules affect the structure of the protein using the hydrogen bonds and van der Waals interactions., Conclusion: The results show that the most important factor in the denaturing effect of GdmCl is the strong interactions of positively charged guanidinium ions with the aspartate and glutamate residues., Competing Interests: There are no conflicts of interest

Inside the cells, proteins are surrounded by mixtures of different osmolytes. However, our current understanding of the combinatorial effects of such mixtures on the stability of proteins remains elusive. In the present study, the stability and structure of recombinant pyrazinamidase (PZase) from Mycobacterium tuberculosis were analyzed in the presence of stabilizing osmolytes (sorbitol, sucrose and glycerol) and alcohols (methanol, ethanol, isopropanol and n-propanol). The far-UV and near-UV circular dichroism (CD), intrinsic fluorescence and thermostability results indicated that methanol, unexpectedly, has stronger effect on destabilization of the enzyme compared to ethanol which has larger log P. Interestingly, the relative half-life of PZase was longer in mixtures methanol with the osmolytes, sorbitol or sucrose (expectedly), or glycerol (unexpectedly), compared to other alcohols. Molecular dynamics simulation results showed that methanol increases the flexibility of region 5-40 and loop 51-71 in the PZase, which are potentially crucial for the stability and activity of the enzyme, respectively. Our results indicated that methanol can interact with PZase via hydrophobic interactions and hydrogen bonds, and therefore resulting in destabilization of the structure of the enzyme. In addition, glycerol probably increases the stability of the enzyme in methanol by disrupting the unfavorable hydrophobic interactions and hydrogen bonds., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective/background: Mycobacterium tuberculosis pyrazinamidase (PZase) is known an enzyme that is involved in degradation of pyrazinamide to ammonia and pyrazinoic acid. Pyrazinamide is an important first-line drug used in the short-course treatment of tuberculosis. Previous investigations have indicated that the pyrazinamide (PZA)-resistant M. tuberculosis strains are caused by point mutations in the PZase enzyme which is the activator of the prodrug PZA. Although the general fold of PZase was determined, the structural and functional properties of the enzyme in solution were not understood very well. In this study, the PZase enzyme was overexpressed and purified. In addition, two polyols, namely sorbitol and glycerol, were chosen to study their effects on the structure, dynamics, and stability of the enzyme. To gain a deeper insight, molecular dynamics simulation and spectroscopic methods, such as fluorescence spectroscopy and circular dichroism (CD), were used., Methods: The genes were cloned in Escherichia coli BL21 (DE3), harboring the recombinant pET-28a (+) plasmid, overexpressed and purified by Ni-NTA Sepharose. The far UV-visible CD spectra were measured by a Jasco-810 spectropolarimeter. The intrinsic fluorescence spectra were measured on a Cary Varian Eclipse spectrofluorometer. For molecular dynamics (MD) simulations, we have applied GROMACS4.6.5., Results: The results showed that glycerol and sorbitol increased the enzyme activity up to 130% and 110%, respectively, at 37°C. The stability of PZase was decreased and the half-life was 20 min. Glycerol and sorbitol increased the PZase half-life to 99 min and 23 min, respectively. The far UV CD measurements of PZase indicated that the CD spectra in glycerol and sorbitol give rise to an increase in the content of α-helix and β-sheets elements. The average enzyme root mean square deviation (RMSD) in sorbitol solution was about 0.416nm, a value that is higher than the enzyme RMSD in the pure water (0.316). In dictionary of protein secondary structure (DSSP) results, we observed that the secondary structures of the protein are partially increased as compared to the native state in water. The experimental and simulation data clearly indicated that the polyols increased the PZase stabilization in the order: glycerol>sorbitol., Conclusion: It can be concluded that the native conformation of the enzyme was stabilized in the sorbitol and glycerol and tend to exclude from the PZase surface, forcing the enzyme to keep it in the compactly folded conformation. The glycerol molecules stabilized PZase by decreasing the loops flexibility and then compacting the enzyme structure. It appears that more stability of PZase in glycerol solution correlates with its amphiphilic orientation, which decreases the unfavorable interactions of hydrophobic regions., (Copyright © 2016.)

The effect of glycerol and sorbitol on the stability of porcine pancreas trypsin was investigated in this work. Molecular dynamics simulation and thermostability results showed that trypsin has two flexible regions, and polyols (sorbitol and glycerol) stabilize the enzyme by decreasing the flexibility of these regions. Radial distribution function results exhibited that sorbitol and glycerol were excluded from the first water layer of the enzyme, therefore decrease the flexibility of the regions by preferential exclusion. Also, results showed that the stabilization effect of sorbitol is more than glycerol. This observation could be because of the larger decrease in the fluctuations of trypsin in the presence of sorbitol. We also examined the role of solvent's hydrophobicity in enzyme stabilization by sorbitol and glycerol. To do so, the thermostability of trypsin was evaluated in the presence of solvents with different hydrophobicity (methanol, ethanol, isopropanol and n-propanol) in addition to the polyols. Our results depicted that glycerol is a better stabilizer than sorbitol in the presence of hydrophobic solvents (n-propanol), whereas sorbitol is a better stabilizer than glycerol in the presence of hydrophilic solvents (methanol)., (© 2015 International Union of Biochemistry and Molecular Biology, Inc.)

Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) is a monomeric enzyme with 537 residues. This enzyme has an Ig-like domain in the N-terminus of the catalytic domain. In this study, the role of the Ig-like domain on the activity, stability, and structural rigidity of AaCel9A and the effect of calcium on enzyme activity and stability were examined by comparing a truncated enzyme with deletion of the Ig-like domain (AaCel9AΔN) to the wild-type enzyme. Our results showed that the deletion of the Ig-like domain increased the catalytic efficiency of the truncated enzyme up to threefold without any significant changes in the K m of the enzyme. Furthermore, pH and temperature optimum for activity were shifted from 6.5 to 7.5 and from 65 to 60 °C, respectively, by deletion of the Ig-like domain. The thermal stability and fluorescence quenching results indicated that the stability and rigidity of the truncated enzyme have been more than that of the wild-type enzyme. Calcium similarly increased the catalytic efficiency of the enzymes (up to 40 %) and remarkably raised the stability of the AaCel9A compared to the AaCel9AΔN. This shows that Ig-like domain has a role in the increase of the enzyme stability by calcium in the wild-type enzyme.

Pyrazinamide (PZA) is one the first line anti-tuberculosis drugs that require activation by the pyrazinamidase (PZase). Most PZA-resistant Mycobacterium tuberculosis strains have mutations in the pncA gene which encoding PZase that result in the reduction or loss of the enzyme activity. Herein, we have examined how various mutations, which have been found from the PZA-resistant M. tuberculosis strains in Iran, modify the PZase activity. To elucidate the possible role of these mutations, namely A143T (MUT1), L151S (MUT2), A143T/T168A/E173K (MUT3), in the bioactivity of the enzyme, the PZase and mutant genes were cloned, functionally expressed and biochemically and computationally characterized. In comparison to the PZase enzyme, the enzymatic efficiency of mutant enzymes was decreased, with MUT2 indicating the largest enzymatic efficiency reduction. Homology models of mutants were constructed based on the PZase X-ray crystal structure. Molecular modeling and substrate docking revealed that the wild-type has much stronger binding affinity to PZA than the mutants whereas MUT2 has the weakest binding affinity. In addition, the molecular dynamics simulations and the essential dynamics results illustrated that the positions of the 51st to 71st residues were more dynamics in MUT2 as compared to the other atoms in PZase, MUT1 and MUT3 which could decrease the K(m) and k(cat) values of the enzymes.

A sequence of 10 amino acids at the C-terminus region of methylglyoxal synthase from Escherichia coli (EMGS) provides an arginine, which plays a crucial role in forming a salt bridge with 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 MGS from a thermophile species, Thermus sp. GH5 (TMGS) lacking this arginine was cloned with an additional 30 bp sequence at the 3´-end and then expressed in form of a fusion TMGS with a 10 residual segment at the C-terminus (TMGS(+)). The resulting recombinant enzyme showed a significant increase in cooperativity towards phosphate, reflected by a change in the Hill coefficient (nH) from 1.5 to 1.99. Experiments including site directed mutagenesis for Asp-10 in TMGS and TMGS(+), two dimentional structural survey, fluorescence and irreversible thermoinactivation were carried out to confirm this pathway.

In order to clarify the impact of Ca-binding sites (Ca1 and 2) on the conformational stability of neutral proteases (NPs), we have analyzed the thermal, pH and organic solvent stability of a NP variant, V189P/A195E/G203D/A268E (Q-mutant), from Salinovibrio proteolyticus. This mutant has shown to bind calcium more tightly than the wild-type (WT) at Ca1 and to possess Ca2. Q-mutant was resisted against autolysis, thermoinactivation and pH denaturation in a Ca-dependent manner and exhibited better activity in organic solvents compared to the WT enzyme. These results imply that Ca1 and Ca2 are important for the conformational stability of NPs.

A gene encoding methylglyoxal synthase from Thermus sp. GH5 (TMGS) was cloned, sequenced, overexpressed, and purified by Q-Sepharose. The TMGS gene was composed of 399 bp which encoded a polypeptide of 132 amino acids with a molecular mass of 14.3 kDa. The K (m) and k (cat) values of TMGS were 0.56 mM and 325 (s(-1)), respectively. The enzyme exhibited its optimum activity at pH 6 and 75 degrees C. Comparing the amino acid sequences and Hill coefficients of Escherichia coli MGS and TMGS revealed that the loss of Arg 150 in TMGS has caused a decrease in the cooperativity between the enzyme subunits in the presence of phosphate as an allosteric inhibitor. Gel filtration experiments showed that TMGS is a hexameric enzyme, and its quaternary structure did not change in the presence of phosphate.

The effect of organic solvents (n-propanol, isopropanol, dimethylformamide and dimethylsulfoxide) on the structure, activity and stability of thermolysin was the focus of this investigation. Results show the ability of the solvents to cause mixed inhibition of thermolysin, which was indicated by kinetic and structural studies (near-UV CD spectra and intrinsic fluorescence). Inhibitory effect of the solvents increased with increments in solvents logP. Thermoinactivation of thermolysin was studied at 80 degrees C in 50% of solvents and showed that with the increase in solvent hydrophobicity, thermal stability of the enzyme decreased. For the stabilization of thermolysin at high temperature, additives such as glycerol, sorbitol and trehalose were employed. In the presence of DMF with a relatively low logP, trehalose was shown to be a good stabilizer, whereas glycerol had a marked stabilization effect in the presence of n-propanol and isopropanol with a relatively high logP. Consequently, it was concluded that the stabilizing effect of additives can be correlated with the logP of solvents.