Response surface methodology based optimized production, purification, and characterization of L-asparaginase from Fusarium foetens.

L-asparaginase is used as one of the prime chemotherapeutic agents to treat acute lymphoblastic leukemia. L-asparaginase obtained from bacteria exhibits hypersensitive reactions including various side effects. The present work aimed to optimize growth parameters for maximum production of L-asparaginase by Fusarium foetens through response surface methodology, its purification, and characterization. The optimization of L-asparaginase production by Fusarium foetens was initially done through a one-factor-at-a-time method. L-asparaginase production was further optimized using a central composite design based response surface methodology. The maximum L-asparaginase activity of 12.83 IU/ml was obtained under the following growth conditions; temperature-27.5 °C, pH-8, inoculum concentration-1.5 × 106 spores/ml, and incubation period-7 days. In comparison with the unoptimized growth conditions (4.58 IU/ml), the optimization led to a 2.65-fold increase in the L-asparaginase activity. The L-asparaginase from Fusarium foetens was purified 15.60-fold, with a yield of 39.89% using DEAE-cellulose column chromatography. After purification, the L-asparaginase activity was determined to be 127.26 IU/ml and the specific activity was found to be 231.38 IU/mg. The molecular mass was estimated to be approximately 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme showed optimum activity at pH 5, and a temperature of 40 °C. The enzyme showed 100% specificity towards L-asparagine and no activity towards L-glutamine. Its activity was enhanced by Mn2+, Fe2+, and Mg2, while it was inhibited by ß-mercaptoethanol and EDTA. The Km and Vmax of the purified L-asparaginase were found to be 23.82 mM and 210.3 IU/ml respectively. The results suggest that Fusarium foetens could be a potent candidate for the bioprocessing of L-asparaginase at a large scale.

Inhibition of sPLA2 enzyme activity by cell-permeable antioxidant EUK-8 and downregulation of p38, Akt, and p65 signals induced by sPLA2 in inflammatory mouse paw edema model.

The arachidonic acid (AA) metabolic pathway, plays a vital role in the production of eicosanoids by the action of pro-inflammatory secretory phospholipase A2 (PLA2 ). Release of eicosanoids is known to be involved in many inflammatory diseases. Identification of the inhibitory molecules of this AA pathway enzyme along with the regulation of intracellular signaling cascades may be a finer choice to develop as a powerful anti-inflammatory drug. In this regard, we have screened few cell-permeable antioxidant molecules Tempo, Mito-TEMPO, N,N'-Bis(salicylideneamino)ethane-manganese(II) (EUK)-134, and EUK-8 against pro-inflammatory sPLA2 s. Among these, we found EUK-8 is a potent inhibitor with its IC50 value ranges 0.7-2.0 µM for sPLA2 s isolated from different sources. Furthermore, docking studies confirm the strong binding of EUK-8 towards sPLA2 . In vivo effect of EUK-8 was studied in HSF-sPLA2 -induced edema in mouse paw model. In addition to neutralizing the edema, EUK-8 significantly reduces the phosphorylation level of inflammatory proteins such as p38 member of MAPK pathway, Akt, and p65 along with the suppression of pro-inflammatory cytokine (interleukin-6) and chemokine (CXCL1) in edematous tissue. This shows that EUK-8 not only inhibits the sPLA2 activity, it also plays an important role in the regulation of sPLA2 -induced cell signaling cascades. Apart from the sPLA2 inhibition, we also examine the regulatory actions of EUK-8 with other downstream enzymes of AA pathway such as 5-LOX assay in human polymorphonuclear leukocytes (PMNs) and COX-2 expression in carrageenan-λ induced paw edema. Here EUK-8 significantly inhibits 5-LOX enzyme activity and downregulates COX-2 expression. These data indicate that EUK-8 found to be a promising multitargeted inhibitory molecule toward inflammatory pathway. In conclusion, mitochondrial targeted antioxidant EUK-8 is not only the powerful antioxidant, also a potent anti-inflammatory molecule and may be a choice of molecule for pharmacological applications.