Cloning and expression of L-asparaginase from Bacillus tequilensis PV9W and therapeutic efficacy of Solid Lipid Particle formulations against cancer

Abstract

L-asparaginase, a therapeutic involved in cancer therapy, from Bacillus tequilensis PV9W (ansA gene) was cloned and over expressed in Escherichia coli BL21 (DE3), achieved the aim of maximizing the yield of the recombinant enzyme (6.02 ± 1.77 IU/mL) within 12 h. The native L-asparaginase of B. tequilensis PV9W was encapsulated using solid lipid particles by hot lipid emulsion method, which is reported for first time in this study. Subsequently, the lipid encapsulated L-asparaginase (LPE) was characterized by SEM, UV-Vis spectroscopy, FT-IR, SDS-PAGE and its thermo stability was also analyzed by TGA. Further characterization of LPE revealed that enzyme was highly stable for 25 days when stored at 25 °C, showed high pH (9) tolerance and longer trypsin half-life (120 min). In addition, the cytotoxic ability of LPE on HeLa cells was highly enhanced compared to the native L-asparaginase from Bacillus tequilensis PV9W. Moreover, better kinetic velocity and lower Km values of LPE aided to detect L-asparagine in cell extracts by Differential Pulse Voltammetry (DPV) method. The LPE preparation also showed least immunogenic reaction when tested on normal macrophage cell lines. This LPE preparation might thus pave way for efficient drug delivery and enhancing the stability of L-asparaginase for its therapeutic applications.

Development and characterization of a novel l-asparaginase/MWCNT nanobioconjugate

“…The original MWCNTs (without enzyme) display almost no weight loss up to 700 C, being practically completely burned above 800 C. Aer this temperature a plateau is reached, corresponding to 3.8% of the initial mass, due to the existence of ashes resulting from the pyrolysis of impurities. The TG prole of ASNase has been previously reported by Shakambari et al 55 revealing an accentuated mass loss starting at 250 C, which is clearly observed in the TG prole of the ASNase-MWCNTs bioconjugate, corresponding to the thermal decomposition of the enzyme (Fig. 8b).…”

Superior operational stability of immobilized l-asparaginase over surface-modified carbon nanotubes

“…The first weight loss is detected at c.a. 250 °C, which is attributed to the thermal decomposition of the enzyme 44 , while the second weight loss at 600 °C is recognized as the simultaneous pyrolysis of ASNase and MWCNTs, attaining a plateau at a temperature near to 700 °C. This plateau corresponds to 33.8% of the initial mass of the enzyme-support complex.…”

Hydrophobically Modified Cellulose Nanofibers‐Enveloped Solid Lipid Microparticles for Improved Antioxidant Cargo Retention

“…[ 5 ] Meanwhile, recent studies show that the utilization of PEGylated macromolecular surfactants resulted in the production of thermodynamically stable SLPs. [ 6 ] Owing to the effective chain conformation of PEG blocks, they can not only prevent particle aggregation via the antifouling effect, but also prolong the blood circulation time. [ 7 ] Despite these advantages, most surfactants used for the production of SLPs have low molecular weights or relatively weak intermolecular attractions.…”