Microbial Cell Factories最新文献

Background: Cyanobacteria and their bioactive components represent a promising potential for the green synthesis of nanoparticles. This work investigates the physicochemical characteristics and biological activities of silver nanoparticles (PC-AgNPs) synthesized using phycocyanin, a photosynthetic pigment extracted from the cyanobacterium Arthrospira platensis Pc-005.

Results: The phycocyanin-derived AgNPs were characterized using UV-Vis spectroscopy, transmission electron microscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction (XRD), Energy Dispersive X-Ray Spectroscopy (EDX), and Dynamic light scattering (DLS). Physicochemical analysis confirmed the formation of PC-AgNPs, and revealed their nanoscale size, crystalline nature and purity. PC-AgNPs demonstrated a UV-Vis absorption peak at 410 nm. TEM analysis revealed that phycocyanin-derived AgNPs were spherical in shape with an average diameter of 20.0 ± 4.2 nm. XRD and EDX analyses confirmed the crystallinity and purity of PC-AgNPs. FTIR analysis revealed the involvement of phycocyanin functional groups in AgNPs formation. The results demonstrated that PC-AgNPs exhibited strong antimicrobial activity against opportunistic pathogenic bacteria Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, as well as fungi Aspergillus flavus, Penicillium aurantioviolaceum, and Trichoderma viride. The findings indicated that significantly higher concentration of PC-AgNPs was needed to achieve antifungal activity compared to antibacterial activity. The PC-AgNPs also affected the activity of crucial antioxidant enzymes, such as superoxide dismutase and catalase, which play key role in the bacterial defense against oxidative stress induced by AgNPs. Moreover, PC-AgNPs demonstrated significant concentration-dependent anticancer activity against A549 adenocarcinoma cells. They also inhibited key angiogenic factors, including VEGF-A and MMP-2, as well as inflammation-related markers such as TNF-α and COX-2.

Conclusion: Thus, this study provides a comprehensive physicochemical and biological characterization of silver nanoparticles derived from the phycocyanin of A. platensis. The PC-AgNPs exhibit a broad spectrum of biological activities, underscoring their promising potential for innovative applications in biomedical and biotechnological fields.

Background: The development of antimicrobial treatments as alternatives to antibiotics to combat antimicrobial-resistant (AMR) bacteria is a global priority. Antimicrobial peptides and proteins such as Host Defense Peptides (HDPs) and endolysins are one of the alternatives that are being explored. HDPs are small, cationic, and amphiphilic antimicrobial peptides derived from the innate immune system exhibiting a broad-spectrum antimicrobial activity. On the other hand, endolysins are enzymes produced by bacteriophages to hydrolyze the bacterial peptidoglycan layer, offering more specific antimicrobial activity than HDPs. While short peptides can be chemically synthesized, this approach presents several limitations, and recombinant production is also being used. Escherichia coli is the most used bacterial expression system for protein production. Alternative systems based on Generally Recognized as Safe (GRAS) microorganisms such as Lactic Acid Bacteria (LAB) have also been employed. However, so far, no comparative studies have evaluated the production and activity of antimicrobial proteins expressed in E. coli versus LAB and this study aims to address that gap.

Results: To evaluate potential differences in the production of antimicrobial proteins using E. coli and two LAB (Lactococcus lactis and Lactiplantibacillus plantarum) hosts, various proteins were evaluated. These included two HDPs fused to a GFP, two multidomain HDP-based proteins and one endolysin. The results revealed a clear influence of the expression system on the quality of HDP-based protein, including both GFP fusions and multidomain constructs. Protein yield was higher in E. coli and all HDP-based proteins exhibited higher antimicrobial activity when expressed in E. coli compared to L. lactis and L. plantarum. In contrast, endolysin activity was comparable when produced in E. coli and L. lactis.

Conclusions: These results demonstrate that the choice of bacterial expression host significantly affects not only the yield but, more importantly, the antimicrobial activity of HDP-based proteins. For these proteins, the antimicrobial activity was consistently higher when produced in E. coli. In contrast, endolysins exhibited similar characteristics regardless of whether they were expressed in E. coli or in L. lactis.

Background: The production of recombinant proteins in Escherichia coli (E. coli) is often hampered by the formation of inclusion bodies. While fusion tags can enhance solubility, existing systems are hampered by a lack of standardization, with tags scattered across disparate plasmid backbones and inconsistent cloning sites, complicating parallel screening.

Results: To address this, we constructed a standardized series of expression vectors, termed pNX, by incorporating nine small fusion tags (SUMO, LD, ACP, BCCP, GB1, Fh8, SmbP, TolA, and TrxA) into a uniform pET-28b backbone. Each pNX vector features an identical configuration: a T7 promoter, an N-terminal fusion tag, a synthetic linker, a TEV protease cleavage site, and a multiple cloning site (MCS) flanked by dual 6×His tags. We evaluated this system using four model proteins (EcFabG, eGFP, XccXanA2, and XccXanL). Our results showed that specific tags significantly improved both the expression level and solubility of the target proteins without compromising their biological activity. Notably, the lipoyl domain (LD) was identified, to our knowledge for the first time, as an effective solubility enhancer. The standardized MCS enabled rapid, parallel cloning, facilitating the efficient screening of optimal fusion partners.

Conclusions: The pNX vector series provides a versatile and efficient platform for enhancing the soluble expression of challenging recombinant proteins in E. coli, streamlining the empirical identification of ideal fusion tags.

L-asparaginase is a crucial enzyme used in chemotherapy regimens for the treatment of acute lymphoblastic leukemia (ALL), its incorporation in the pediatric treatment protocols helped in achieving a high cure rate. However, immunogenic side-effects restrict its application and frequently result in stopping treatment. There is a current need for the identification of novel L-asparaginase with improved properties and lower adverse effects compared to those available in the market. L-asparaginase gene from Stenotrophomonas maltophilia (S. maltophilia), an isolated organism that was mentioned as a novel and excellent source for L- asparaginase, was cloned and expressed using E. coli DH5α and E. coli BL21(DE3). Investigations of different conditions of expression of recombinant L-asparaginase in E. coli BL21(DE3) using Box-Behnken design predicted maximum expression at 37 °C temperature, 250 rpm agitation, 0.83 mM isopropylthio-β-D-galactoside (IPTG) concentration after incubation for 17 h. The optimized expression conditions were validated using L-asparaginase activity assay. The obtained recombinant protein was purified using Ni-NTA spin column. SDS-PAGE demonstrated a single band of 17 KDa apparent molecular weight. The kinetic parameters were determined, and they exhibited a low Km value of 2.94 × 10^- 2 M and Vmax of 14.73 IU/ml. Cytotoxicity on various cell lines was tested in relation to marketed E. coli L-asparaginase and exhibited low IC50 of 1.92 IU/ml and 2.03 IU/ml for HEP-G2 and K-562 cell lines, respectively. Additionally, mice treated with recombinant L-asparaginase displayed a significantly lower immunological response (IgG) compared to mice treated with marketed E. coli L-asparaginase (p-value < 0.0001). These findings demonstrate the potentiality of recombinant L-asparaginase for its development as a chemotherapeutic drug.