Pseudomonas aeruginosa Recombinant L-asparaginase: PEGylation with Low Molecular Weight Polyethylene Glycol, Molecular Dynamics Simulation, In vitro and In vivo Serum half-life and Biochemical Characterization.

BACKGROUND: Microbial L-asparaginase (L-ASNase, EC 3.5.1.1) is a pivotal biopharmaceutical drug-protein that catalyzes the hydrolysis of the non-essential amino acid L-asparagine (L-Asn) into L-aspartic acid (L-Asp) and ammonia , resulting in deplenishing the cellular L-Asn pool, which leads to the ultimate death of the L-asparagine synthetase (L-ASNS) deficient cancerous cells. OBJECTIVE: This study aimed to investigate the impact of conjugating low molecular weight polyethylene glycol to recombinant P. aeruginosa L-ASNase by examining the pharmacokinetic properties, affinity towards the substrate, and enzyme stability prior to and following the reaction. METHODS: The recombinant P. aeruginosa L-ASNase was affinity purified and then PEGylated by attaching polyethylene glycol (MW= 330 Da) site-specifically to the protein's N-terminus end. After which, the PEGylated L-ASNase was examined by SDS-PAGE (15%), FTIR, and UV/Vis spectrophotometry and subsequently biochemically characterized. RESULTS: The Km and Vmax values of free P. aeruginosa rL-ASNase were determined to be 0.318 ±1.76 mM and 2915 µmol min-1and following the PEGylation, they were found to be 0.396 ±1.736 mM and 3193 µmol min-1, respectively. Polyethylene glycol (330 Da) has markedly enhanced LASNase thermostability at 37, 45, 50, and 55 °C, as opposed to the free enzyme, which retained 19.5% after 1 h of incubation at 37 °C. The PEGylated L-ASNase was found to be stable upon incubation with human serum for 28 h, in contrast to the sharp decline in the residual bioactivity of the free rL-ASNase after 4 h incubation. Accordingly, an in vivo study was used for validation, and it demonstrated that PEGylated rL-ASNase exhibited longer bioactivity for 24 h, while the free form's activity vanished entirely from the rats' blood sera after 8 h. Molecular dynamics simulation indicated that PEG (330 Da) has affected the hydrodynamic volume of L-ASNase and increased its structural stability. Docking analysis has explored the position of PEG with respect to binding sites and predicted a similar binding affinity to that of the free enzyme. CONCLUSION: For the first time, recombinant L-ASNase was modified by covalently attaching PEG (330 Da). The resultant novel proposed PEGylated rL-ASNase with remarkably increased stability and prolonged in vivo half-life duration, which could be considered an alternative to mitigate the high molecular weight of PEGylation's drawbacks.

SNP (A > G - rs13057211) but not GT(n) polymorphism in HMOX-1 promotor gene is associated with COVID-19 mortality.

INTRODUCTION: COVID-19 causes severe inflammatory respiratory distress syndrome. The global pandemic caused millions of cases of morbidity and mortality worldwide. Patients may present with variable symptoms including dyspnea, fever, and GIT manifestations. The HMOX-1 gene is located on the long (q) arm of chromosome 22 at position 12.3. HMOX-1 is expressed in all mammalian tissues at basal levels and is considered as a stress response enzyme. HMOX-1 has a specific polymorphic site with variable GT(n) repeats at the promotor region. Several authors evaluated the HMOX-1 GT(n) promoter polymorphism in different inflammatory conditions. We evaluated HMOX-1 promoter polymorphism in relation to serum Hemoxygenase level and inflammatory makers (CRP, Ferritin, PCT, IL-6 and D-dimer) in patients affected by SARS-COV-2 disease. SUBJECTS AND METHODS: Ninety patients confirmed to be infected with COVID-19 were followed up till the study end point (recovery and discharge or death). HMOX-1 promotor GT(n) polymorphism was evaluated using Sanger sequencing. HMOX-1 enzyme serum level was measured by ELISA and the level of different inflammatory markers was assessed by available commercial kits. RESULTS: A novel Single nucleotide polymorphism (SNP) (A > G) - rs13057211 in the GT(n) region of HMOX-1 promoter gene was found in 40 (61.5%) COVID-19 patients out of the studied 65 patients. This (A > G) SNP was associated with higher mortality rate in COVID-19 as it was detected in 27 patients (75% of the patients who succumbed to the disease) (p = 0.021, Odds ratio = 3.7; 95% CI:1.29-10.56). Serum IL-6 (Interleuken-6) was positively correlated the length of Hospital Stay (LOHS) and procalcitonin (PCT); (p = 0.014, r: 0.651 and p < 0.001, r:0.997) respectively while negatively correlated with levels of HMOX-1 enzyme serum level (p = 0.013, r: -0.61). CRP correlated positively with LOHS (p = 0.021, r = 0.4), PCT (p = 0.044, r = 0.425) and age (p < 0.001, r = 0.685). Higher levels of D-Dimer and PCT were observed in patients with the long repeat. There was no significant difference between patients who recovered and those who died from COVID-19 as regards HMOX-1 level and GT(n) polymorphism. CONCLUSION: We report a novel SNP (A > G, rs13057211) in the GT(n) region of HMOX-1 promoter gene that was associated with mortality in COVID-19 patients, however no significant difference was found in HMOX-1 serum level or HMOX-1 (GT)n repeats within the studied groups.

Nanoparticles fabricated from the bioactive tilapia scale collagen for wound healing: Experimental approach.

The creation of innovative wound-healing nanomaterials based on natural compounds emerges as a top research goal. This research aimed to create a gel containing collagen nanoparticles and evaluate its therapeutic potential for skin lesions. Collagen nanoparticles were produced from fish scales using desolvation techniques. Using SDS PAGE electrophoresis, Fourier transform infrared spectroscopy (FTIR) as well as the structure of the isolated collagen and its similarities to collagen type 1 were identified. The surface morphology of the isolated collagen and its reformulation into nanoparticles were examined using transmission and scanning electron microscopy. A Zeta sizer was used to examine the size, zeta potential, and distribution of the synthesized collagen nanoparticles. The cytotoxicity of the nanomaterials was investigated and an experimental model was used to evaluate the wound healing capability. The overall collagen output from Tilapia fish scales was 42%. Electrophoretic patterns revealed that the isolated collagen included a unique protein with chain bands of 126-132 kDa and an elevated beta band of 255 kDa. When compared to the isolated collagen, the collagen nanoparticles' FTIR results revealed a significant drop in the amide II (42% decrease) and amide III (32% decrease) band intensities. According to SEM analysis, the generated collagen nanoparticles ranged in size from 100 to 350 nm, with an average diameter of 182 nm determined by the zeta sizer. The produced collagen nanoparticles were polydispersed in nature and had an equivalent average zeta potential of -17.7 mV. Cytotoxicity study showed that, when treating fibroblast cells with collagen nanoparticle concentrations, very mild morphological alterations were detected after human skin fibroblasts were treated with collagen nanoparticles 32 µg/ml for 24 hours, as higher concentrations of collagen nanoparticles caused cell detachment. Macroscopical and histological investigations proved that the fabricated fish scale collagen nanoparticles promoted the healing process in comparison to the saline group.

Synergistic effect of Dactolisib/Lys05 combination on autophagy in A549 cells.

Effective therapeutic strategies are urgently required to enhance the prognosis of patients suffering from KRAS mutations. Owing to the undruggable nature of KRAS, targeting downstream signaling pathways, namely PI3K/AKT/mTOR, shows antiproliferative and apoptotic effects. Unfortunately, targeting this pathway upregulates autophagy, contributing to reduced drug efficacy. Therefore, it was reasonable to use a combination of kinase inhibitors and autophagy inhibitors to achieve a higher therapeutic benefit. The impact of Dactolisib, a dual PI3K/mTOR inhibitor, and Lys05, a dimeric chloroquine, was tested on the survival of breast cancer MCF-7 and lung cancer A549 cells. The dose selection for the optimal effect of the Dactolisib/Lys05 combination was determined using CompuSyn software. This combinatorial effect was evaluated using various methodologies, such as expression profile analysis for autophagic, proliferative, and apoptotic markers. These effects were corroborated by ELISA, Western blot, and flow cytometry using the Annexin V-FITC apoptosis detection kit. A549 cells treated in a 2:1 ratio of Lys05 and Dactolisib demonstrated a synergistic effect on cell death, proliferation, and apoptotic gene markers, in addition to its effect on autophagic gene and protein markers, showing an enhanced effect compared to monotherapy. Therefore, the PI3K/AKT kinase inhibitor/autophagy inhibitor combination establishes higher therapeutic benefits on A549 cells compared to kinase inhibitor monotherapy.

The Role of Pyrazolo[3,4-d]pyrimidine-Based Kinase Inhibitors in The Attenuation of CCl4-Induced Liver Fibrosis in Rats.

Liver Fibrosis can be life-threatening if left untreated as it may lead to serious, incurable complications. The common therapeutic approach is to reverse the fibrosis while the intervention is still applicable. Celecoxib was shown to exhibit some antifibrotic properties in the induced fibrotic liver in rats. The present study aimed to investigate the possible antifibrotic properties in CCl4-induced liver fibrosis in male Sprague-Dawley rats compared to celecoxib of three novel methoxylated pyrazolo[3,4-d]pyrimidines. The three newly synthesized compounds were proved to be safe candidates. They showed a therapeutic effect against severe CCl4-induced fibrosis but at different degrees. The three compounds were able to partially reverse hepatic architectural distortion and reduce the fibrotic severity by showing antioxidant properties reducing MDA with increasing GSH and SOD levels, remodeling the extracellular matrix proteins and liver enzymes balance, and reducing the level of proinflammatory (TNF-α and IL-6) and profibrogenic (TGF-ß) cytokines. The results revealed that the dimethoxy-analog exhibited the greatest activity in all the previously mentioned parameters compared to celecoxib and the other two analogs which could be attributed to the different methoxylation patterns of the derivatives. Collectively, the dimethoxy-derivative could be considered a safe promising antifibrotic candidate.

Structure-Guided Engineering of a Family IV Cold-Adapted Esterase Expands Its Substrate Range.

Cold active esterases have gained great interest in several industries. The recently determined structure of a family IV cold active esterase (EstN7) from Bacillus cohnii strain N1 was used to expand its substrate range and to probe its commercially valuable substrates. Database mining suggested that triacetin was a potential commercially valuable substrate for EstN7, which was subsequently proved experimentally with the final product being a single isomeric product, 1,2-glyceryl diacetate. Enzyme kinetics revealed that EstN7's activity is restricted to C2 and C4 substrates due to a plug at the end of the acyl binding pocket that blocks access to a buried water-filled cavity. Residues M187, N211 and W206 were identified as key plug forming residues. N211A stabilised EstN7 allowing incorporation of the destabilising M187A mutation. The M187A-N211A double mutant had the broadest substrate range, capable of hydrolysing a C8 substrate. W206A did not appear to have any significant effect on substrate range either alone or when combined with the double mutant. Thus, the enzyme kinetics and engineering together with a recently determined structure of EstN7 provide new insights into substrate specificity and the role of acyl binding pocket plug residues in determining family IV esterase stability and substrate range.

Theophylline-encapsulated Nile Tilapia fish scale-based collagen nanoparticles effectively target the lungs of male Sprague-Dawley rats.

Nile Tilapia fish scale collagen has high biodegradability, excellent biocompatibility, and low antigenicity. We assessed both the encapsulation efficiency of theophylline into Nile Tilapia fish scale-based collagen nanoparticles and their stability as a pulmonary drug delivery system in male Sprague-Dawley rats. The present study has demonstrated the successful encapsulation of theophylline into the synthesised nanoparticles as shown by spectrophotometric analysis, light microscope, scanning electron microscope, transmission electron microscope, and dynamic light scattering. The antibacterial activity of the nanoparticles improves with increasing their concentrations. Intratracheal treatment of rats using theophylline-encapsulated nanoparticles reduced the levels of creatinine, alanine transaminase, and aspartate transaminase, compared to the control group. Nevertheless, nanoparticles combined with theophylline exhibited no effects on cholesterol and triglycerides levels. Histopathological examination revealed typical uniform and diffuse thickening of the alveolar walls with capillary oedema in treated rats. We concluded that the synthesised collagen nanoparticles appropriately target the lungs of male Sprague-Dawley rats when delivered via a nebuliser, showing good tolerability to lung cells. However, dose ratio of collagen nanoparticles to theophylline needs further evaluation. The nanoprecipitation method may be optimised to involve poorly water-soluble inhaled drugs, and avoid the drawbacks of traditional drug delivery.

L-asparaginase from Dickeya chrysanthemi: expression, purification and cytotoxicity assessment.

Microbial L-asparaginases are aminohydrolases that hydrolyze L-asparagine to L-aspartate. They are used to treat acute lymphoblastic leukemia and Hodgkin's lymphomas and in food industries. Increasing demand for L-ASNases is therefore needed. In the current study, the recombinant L-ASNase from Dickeya chrysanthemi (DcL-ASNase) was cloned into pET28a (+) expression vector and expressed in Escherichia coli as a 6His-tagged fusion protein and purified using Ni2+ chelated Sepharose chromatography resin, yielding a highly purified enzyme. Kinetics analysis allowed the determination of its substrate specificity and the physicochemical parameters that affect enzyme activity. The enzyme showed operational stability at 37 °C and 45 °C. The immunogenicity of the purified DcL-ASNase was evaluated by measuring the IgG and IgM levels in rats after injection. The cytotoxicity DcL-ASNase in selected cancer cell lines and peripheral blood monocytes was determined. The results showed that the enzyme induces pleiotropic effects, including significant morphological changes and the formation of apoptotic bodies. No cytotoxic effects were observed in peripheral blood monocytes at the same concentrations. In addition, gene expression analysis by RT-PCR of apoptotic biomarkers (Bax, survivin, and Ki-67) allowed the study of the apoptotic mechanism induced by DcL-ASNase on THP-1 cells.

Structure and in silico simulations of a cold-active esterase reveals its prime cold-adaptation mechanism.

Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/ß hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7's closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7's cold adaptation rather than changes in dynamics.

Pseudomonas aeruginosa recombinant L-asparaginase: Large scale production, purification, and cytotoxicity on THP-1, MDA-MB-231, A549, Caco2 and HCT-116 cell lines.

In previous studies Pseudomonas aeruginosal-ASNase complete coding sequence gene, 984 bp (GenBank accession number KU161101.2) was isolated by PCR, cloned into pET28a(+) vector, expressed in E. coli DE3(BL21) pLysS, purified to apparent homogeneity and biochemically characterized. In the present work we highlight large scale production, affinity purification of the recombinant enzyme, effect of osmolytes on the stability of the l-ASNase and cytotoxicity on different cancer cell lines. Successful overexpression was achieved in E. coli as a 6-His-Tag fusion protein after 18 h of induction with lactose at a concentration of 2 g/L in fermentation medium and at 37 °C. The recombinant enzyme was purified to homogeneity using Ni2+ chelated Fast Flow Sepharose resin with 19758.8 specific activity and 10.28 purification fold. With respect to the effect of osmolytes on the stability of the purified enzyme, the majority of the tested osmolytes namely 5% maltose, 5% mannitol, 30% glycerol and 5% BSA were found to increase the stability of the recombinant l-ASNase as compared to the free enzyme. Triple negative breast cancer cell line, MDA-MB-231 treated with recombinant l-ASNase showed significant morphological changes and the IC50 of the purified enzyme was found to be 3.1 IU. Human leukemia cell line, THP-1 treated with l-ASNase showed apoptotic bodies and morphological changes with IC50 of the purified enzyme 1.75 IU. Moreover, the purified recombinant l-ASNase was found to induced cytotoxic effects on colorectal adenocarcinoma cell line, Caco-2 with IC50 of 68.28 IU. Results of apoptosis assay on THP-1 cells revealed that the purified l-ASNase induced early and late apoptosis at 14.16% and 7.56 respectively as compared to the control untreated cells.

Heat shock protein 90α inhibitor, PU-H71 in combination with DHEA promoting apoptosis in triple-negative breast cancer cell line MDA-MB-231.

Due to the lack of markers (ER, PR, and HER-2/Neu) for the molecular-targeted therapies triple-negative breast cancer (TNBC) is more challenging than other subtypes of breast cancer. Moreover, the conventional chemotherapeutic agents are still the mainstay of most therapeutic protocols and eventually turn into a refractory drug-resistance , hence, more efficient therapeutic regimens are urgently required. The present study aimed to elucidate the effects of PU-H71 combined with DHEA on triple-negative breast cancer cell line MDA-MB-231 and to assess the synergy using the Chou-Talalay method. The combined therapy controlled the expression of an array of antioxidants and metabolizing enzymes, leading to the induction of oxidative stress which in turn induced apoptotic cell death. Our results indicated that the combined treatment with PU-H71 and DHEA exerts a synergistic anti-tumor effect on MDA-MB-231 triple-negative breast cancer cell line.

Bacillus sonorensis L. Asparaginase: Cloning, Expression in E. coli and Characterization.

L-asparaginases (L-ASNases; EC 3.5.1.1) are aminohydrolases that catalyze the hydrolysis of L-asparagine (L-Asn) to L-aspartic acid and ammonia, resulting in the death of acute lymphoblastic leukemic cells and other blood cancer cells. In this study, Bacillus sonorensis (accession number MK523484) uncharacterized L-ASNase gene (accession number MN562875) was isolated by polymerase chain reaction (PCR), cloned into pET28a (+) vector, and expressed in Escherichia coli as a cytosolic protein. The recombinant enzyme was purified by affinity chromatography at 23.79-fold and 49.37% recovery. Denaturing polyacrylamide gel (10%) analysis of the purified enzyme resulted in a single protein band at 36 kDa that immunoreacted strongly with 6His-tag monoclonal antibody. The purified enzyme exhibited optimal activity at 45 °C and pH 7.0 and retained 92% and 85% of its initial activity after incubation for 60 min at 37 °C and 45 °C, respectively. The purified enzyme exhibited substrate specificity toward L-asparagine and low glutaminase activity (15.72%) toward L-glutamine at a concentration of 10 mM. The Km and Vmax values were 2.004 mM and 3723 µmol min1-, respectively.

Prodigiosin/PU-H71 as a novel potential combined therapy for triple negative breast cancer (TNBC): preclinical insights.

Prodigiosin, a secondary metabolite red pigment produced by Serratia marcescens, has an interesting apoptotic efficacy against cancer cell lines with low or no toxicity on normal cells. HSP90α is known as a crucial and multimodal target in the treatment of TNBC. Our research attempts to assess the therapeutic potential of prodigiosin/PU-H71 combination on MDA-MB-231 cell line. The transcription and protein expression levels of different signalling pathways were assessed. Treatment of TNBC cells with both drugs resulted in a decrease of the number of adherent cells with apoptotic effects. Prodigiosin/PU-H71 combination increased the levels of caspases 3,8 and 9 and decreased the levels of mTOR expression. Additionally, there was a remarkable decrease of HSP90α transcription and expression levels upon treatment with combined therapy. Also, EGFR and VEGF expression levels decreased. This is the first study to show that prodigiosin/PU-H71 combination had potent cytotoxicity on MDA-MB-231 cells; proving to play a paramount role in interfering with key signalling pathways in TNBC. Interestingly, prodigiosin might be a potential anticancer agent to increase the sensitivity of TNBC cells to apoptosis. This study provides a new basis for upcoming studies to overcome drug resistance in TNBC cells.

Highly efficient Pyrococcus furiosus recombinant L-asparaginase with no glutaminase activity: Expression, purification, functional characterization, and cytotoxicity on THP-1, A549 and Caco-2 cell lines.

L-Asparaginase (L-ASNase EC 3.5.1.1) is considered as an important biopharmaceutical drug enzyme in the treatment of childhood acute lymphoblastic leukemia (ALL). In the present study, Pyrococcus furiosus L-ASNase gene was cloned into pET26b (+), expressed in E. coli BL21(DE3) pLysS, and purified to homogeneity using Ni2+ chelated Fast Flow Sepharose resin with 5.7 purification fold and 23.9% recovery. The purified enzyme exhibited a molecular weight of ~33,660 Da on SDS-PAGE and showed maximal activity at 50 °C and pH 8.0. It retained 98.3% and 60.7% initial activity after 60 min at 37 °C and 50 °C, respectively. The recombinant enzyme showed highest substrate specificity towards L-ASNase substrate, while no detectable specificity was observed for l-glutamine, urea, and acrylamide at 10 mM concentration. The Km and Vmax of the purified recombinant enzyme as calculated using Lineweaver-Burk plot were determined to be 1.623 mM and 105 µmol min-1 mg-1, respectively. Human leukemia cell line THP-1 treated with recombinant L-ASNase showed significant morphological changes, and the IC50 of the purified enzyme was found to be 0.8 IU. Moreover, the purified recombinant L-ASNase induced cytotoxic effects on lung adenocarcinoma A549 and colorectal adenocarcinoma Caco-2 cell lines with IC50 of 1.78 IU and 30 IU, respectively.

"Recombinant cold -adapted halotolerant, organic solvent-stable esterase (estHIJ) from Bacillus halodurans.

Esterases and lipases enduring harsh conditions, including low temperature and extreme tolerance to organic solvents, have attracted great attention in recent times. In the current study, a full open reading frame of 747 bp that encodes a novel, cold-adapted esterase (estHIJ) of 248 amino acids from Bacillus halodurans strain NAH-Egypt was heterologously cloned and expressed in E. coli BL21 (DE3) Rosetta. Amino acid sequence analysis revealed that estHIJ belongs to family XIII of lipolytic enzymes, with a characteristic pentapeptide motif (G-L-S-L-G). The recombinant estHIJ was purified using Ni-affinity chromatography to homogeneity with purification fold, yield, specific activity, and molecular weight (MW) of 3.5, 47.5%, 19.8 U/mg and 29 kDa, respectively. The enzyme showed preferential substrate specificity towards pNP-acetate (C2), with catalytic efficiency of 46,825 min-1 mM-1 estHIJ displayed optimal activity at 30 °C and pH (7.0-8.0). estHIJ demonstrated robust stability in the presence of 50% (v/v) non-polar solvents and 4 M NaCl after 15 h and 6 h of incubation, respectively. The promising features of the recombinant estHIJ underpin its potential in several fields, e.g., the synthesis of pharmaceutical compounds and the food industry.

The Arabian camel, Camelus dromedarius interferon epsilon: Functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines.

The current study highlights, for the first time, cloning, overexpression and purification of the novel interferon epsilon (IFNƐ), from the Arabian camel Camelus dromedaries. The study then assesses the cytotoxicity of IFNε against two human breast cancer cell lines MDA-MB-231 and MCF-7. Full-length cDNA encoding interferon epsilon (IFNε) was isolated and cloned from the liver of the Arabian camel, C. dromedarius using reverse transcription-polymerase chain reaction. The sequence analysis of the camel IFNε cDNA showed a 582-bp open reading frame encoding a protein of 193 amino acids with an estimated molecular weight of 21.230 kDa. A BLAST search analysis revealed that the C. dromedarius IFNε shared high sequence identity with the IFN genes of other species, such as Camelus ferus, Vicugna pacos, and Homo sapiens. Expression of the camel IFNε cDNA in Escherichia coli gave a fusion protein band of 24.97 kDa after induction with either isopropyl ß-D-1-thiogalactopyranoside or lactose for 5 h. Recombinant IFNε protein was overexpressed in the form of inclusion bodies that were easily solubilized and refolded using SDS and KCl. The solubilized inclusion bodies were purified to apparent homogeneity using nickel affinity chromatography. We examined the effect of IFNε on two breast cancer cell lines MDA-MB-231 and MCF-7. In both cell lines, IFNε inhibited cell survival in a dose dependent manner as observed by MTT assay, morphological changes and apoptosis assay. Caspase-3 expression level was found to be increased in MDA-MB-231 treated cells as compared to untreated cells.

Expression and Functional Characterization of Pseudomonas aeruginosa Recombinant L.Asparaginase.

Recombinant l.asparaginase, L.ASNase, from Pseudomonas aeruginosa was purified using nickel affinity chromatography. The affinity purified L.ASNase exhibited a protein band with a molecular weight of 72.4 kDa on a native polyacrylamide gel and 36.276 kDa using SDS-PAGE. The activity of the purified L.ASNase was enhanced by Mg2+ and inhibited by Zn2+ at a concentration of 5 mM. The specificity of the recombinant L.ASNase towards different substrates was examined, and it was found that the enzyme showed the highest activity towards l.asparagine. Moreover, the enzyme showed lower activity towards other substrates such as L.glutamine, urea and acrylamide. The in vitro hemolysis assay revealed that the purified L.ASNase did not show hemolysis effect on blood erythrocytes. Serum and trypsin half-life of L.ASNase suggested that the recombinant L.ASNase retained 50% of its initial activity after 90 and 60 min incubation period in serum and trypsin separately.

Cloning, expression and characterization of cold active esterase (EstN7) from Bacillus cohnii strain N1: A novel member of family IV.

Esterases and lipases from extremophiles have attracted great attention due to their unique characteristics and wide applications. In the present study, an open reading frame (ORF) encoding a novel cold active esterase (EstN7) from Bacillus cohnii strain N1 was cloned and expressed in Escherichia coli. The full-length esterase gene encoding a protein of 320 amino acids with estimated molecular weight of 37.0 kDa. Amino acid sequence analysis revealed that the EstN7 belongs to family IV lipases with a characteristic penta-peptide motif (GXSXG), the catalytic triad Ser, Asp, His and the conserved HGGG motif of the family IV. The recombinant enzyme was purified to apparent homogeneity using nickel-affinity chromatography with a purification fold of 5 and recovery 94.5%. The specific activity of the purified enzyme was 336.89 U/mg. The recombinant EstN7 showed optimal activity at 5 °C moreover, EstN7 displayed full robust stability in the presence of wide range of organic solvents. The purified enzyme had Km and Vmax of 45 ±â€¯0.019 µM and 1113 µmol min-1 mg-1, respectively on p-NP-acetate. These promising characteristics of the recombinant EstN7 would underpin its possible usage with high potential in the synthesis of fragile compounds in pharmaceutical industries.

Molecular cloning, structural modeling and production of recombinant Aspergillus terreusl. asparaginase in Escherichia coli.

l-Asparaginase (EC 3.5.1.1) is an important medical enzyme that catalysis the hydrolysis of l-asparagine to aspartic acid and ammonium. For over four decades l. asparaginase utic agent for the treatment of a variety of lymphoproliferative disorders and lymphoma such as acute lymphoblastic leukemia. In the present study A. terreus full length l. asparaginase gene, 1179bp was optimized for expression in Escherichia coli BL21 (DE3) pLysS. The full length A. terreusl. asparaginase gene encoding a protein of 376 amino acids with estimated molecular weight of 42.0kDa and a theoretical isoelectric point (pI) of 5.0. BLAST and phylogeny analysis revealed that the A. terreusl. asparaginase shared high similarity with other known fungal l. asparaginase (75% homology with A. nomius and 71% with A. nidulans). The recombinant protein was overexpressed in the form of amorphous submicron proteinaceous inclusion bodies upon induction with 1mM IPTG at 37°C for 18h.

Overexpression, purification and enzymatic characterization of a recombinant Arabian camel Camelus dromedarius glucose-6-phosphate dehydrogenase.

In a previous study the full-length open reading frame of the Arabian camel, Camelus dromedarius liver cytosolic glucose-6-phosphate dehydrogenase (G6PD) cDNA was determined using reverse transcription polymerase chain reaction. The C. dromedarius cDNA was found to be 1545 nucleotides (accession number JN098421) that encodes a protein of 515 amino acids residues. In the present study, C. dromedarius recombinant G6PD was heterologously overexpressed in Escherichia coli BL21 (DE3) pLysS and purified by immobilized metal affinity fast protein liquid chromatography (FPLC) in a single step. The purity and molecular weight of the enzyme were analyzed on SDS-PAGE and the purified enzyme showed a single band on the gel with a molecular weight of 63.0 KDa. The specific activity was determined to be 2000 EU/mg protein. The optimum temperature and pH were found to be 60 °C and 7.4, respectively. The isoelectric point (pI) for the purified G6PD was determined to be 6.4. The apparent Km values for the two substrates NADP+ and G6P were found to be 23.2 µM and 66.7 µM, respectively. The far-UV circular dichroism (CD) spectra of G6PD showed that it has two minima at 208 and 222 nm as well as maxima at 193 nm which is characteristic of high content of α-helix. Moreover, the far-UV CD spectra of the G6PD in the presence or absence of NADP+ were nearly identical.