Entrapment of Papain in Chitosan-Polyethylene Glycol Hybrid Nanohydrogels: Presenting a Model for Protein Delivery Systems.

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.

Enhancing Enzyme Stability and Functionality: Covalent Immobilization of Trypsin on Magnetic Gum Arabic Modified Fe3O4 Nanoparticles.

This study aimed to fabricate gum Arabic (GA)-coated Fe3O4 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 Fe3O4 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 Vmax and higher Km 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.

The Relationship Between the Cross-Linker on Chitosan-Coated Magnetic Nanoparticles and the Properties of Immobilized Papain.

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.

Isolation and characterizations of a novel recombinant scFv antibody against exotoxin A of Pseudomonas aeruginosa.

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.

Screening of pectinase-producing bacteria from farmlands and optimization of enzyme production from selected strain by RSM.

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 (NH4Cl, K2HPO4, 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.

Optimized immobilization of endoglucanase Cel9A onto glutaraldehyde activated chitosan nanoparticles by response surface methodology: The study of kinetic behaviors.

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 Km (from 3.703 to 12.195 [mg/ml]), which make it suitable to use in some industries such as detergents.

Immobilization of endoglucanase Cel9A on chitosan nanoparticles leads to its stabilization against organic solvents: the use of polyols to improve the stability.

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.

Ig-like Domain in Endoglucanase Cel9A from Alicyclobacillus acidocaldarius Makes Dependent the Enzyme Stability on Calcium.

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.

Deleting the Ig-Like Domain of Alicyclobacillus acidocaldarius Endoglucanase Cel9A Causes a Simultaneous Increase in the Activity and Stability.

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.

Alpha 1-antitrypsin activity is markedly decreased in Wegener's granulomatosis.

Alpha 1-antitrypsin (A1AT) is the most abundant proteinase inhibitor in plasma and the main inhibitor of Proteinase 3, the target antigen of antineutrophil cytoplasmic antibodies (ANCAs) that predominant in Wegeners' granulomatosis. Α1AT deficiency correlated with ANCA-associated vasculitis. This study explores the trypsin inhibitory capacity (TIC), specific activity, and phenotypic deficiency of Α1AT in Wegener's granulomatosis. Twenty-seven WG patients were studied. ANCA was tested by IIF and ELISA. Serum a1-anti-trypsin levels were quantified in WG patients and healthy controls by immunoturbidimetric assay. Serum TIC was assessed by the enzymatic colorimetric assay. Phenotypes of A1AT were detected by Isoelectric Focusing. A1AT concentration was equivalent in patients and controls; however, serum TIC (P = 0.001) and specific activity of A1AT (P = 0.001) were dramatically lower in WG patients. Five patients had deficient phenotypes of A1AT: MZ (n = 3), MS (n = 1) and SS (n = 1). This was correlated with an increase in the prevalence of deficient phenotypes of A1AT in WG (P = 0.01). Trypsin inhibitory capacity and specific activity of A1AT were decreased in WG patients and may be involve in disease pathogenesis and can worsen the clinical manifestations. This A1AT deficiency probably resulted from oxidative inactivation and/or enzymatic degradation of A1AT. This could result in localized deficiency of A1AT in vessel wall interfaces and lead to severe disease.

HIF-1α Overexpression Induces Angiogenesis in Mesenchymal Stem Cells.

Stem cell therapy continues to be an innovative and promising strategy for heart failure. Stem cell injection alone, however, is hampered by poor cell survival and differentiation. This study was aimed to explore the possibility of improving stem cell therapy through genetic modification of stem cells, in order for them to promote angiogenesis in an auto- and paracrine manner under hypoxic conditions. Hypoxia inducible factor-1α was overexpressed in bone marrow-derived mesenchymal stem cells (MSCs) by stable transduction using a lentiviral vector. Under hypoxic and normoxic conditions, the vascular endothelial growth factor (VEGF) concentration in the cells' supernatant was measured by an enzyme-linked immunosorbent assay. Migration was assayed by wound healing and c-Met expression by flow cytometry. Tube formation was evaluated on a Matrigel basement membrane. The concentration of VEGF was significantly increased in the supernatant of HIF-1α-overexpressing MSCs; this medium was significantly more effective in inducing endothelial cell migration compared to untransduced MSCs. Transduced cells showed increased levels of c-Met expression and were more efficient at tube formation. However, no indication of differentiation toward an endothelial phenotype was observed. This study indicated that genetic modification of MSCs by HIF-1α overexpression has the potential to improve components of the angiogenesis process under a hypoxic condition by paracrine and autocrine mechanisms.