Assessment of structural behaviour of a new L-asparaginase and SAXS data-based evidence for catalytic activity in its monomeric form.

The present study reports the structural and functional characterization of a new glutaminase-free recombinant L-asparaginase (PrASNase) from Pseudomonas resinovorans IGS-131. PrASNase showed substrate specificity to L-asparagine, and its kinetic parameters, Km, Vmax, and kcat were 9.49 × 10-3 M, 25.13 IUmL-1 min-1, and 3.01 × 103 s-1, respectively. The CD spectra showed that PrASNase consisted of 18.5 % helix, 21.5 % antiparallel sheets, 4.2 % parallel sheets, 14 % turns, and rest other structures. FTIR was used for the functional characterization, and molecular docking predicted that the substrate interacts with serine, alanine, and glutamine in the binding pocket of PrASNase. Differing from known asparaginases, structural characterization by small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC) unambiguously revealed PrASNase to exist as a monomer in solution at low temperatures and oligomerized to a higher state with temperature rise. Through SAXS studies and enzyme assay, PrASNase was found to be mostly monomer and catalytically active at 37 °C. Furthermore, this glutaminase-free PrASNase showed killing effects against WIL2-S and TF-1.28 cells with IC50 of 7.4 µg.mL-1 and 5.6 µg.mL-1, respectively. This is probably the first report with significant findings of fully active L-asparaginase in monomeric form using SAXS and AUC and demonstrated the potential of PrASNase in inhibiting cancerous cells, making it a potential therapeutic candidate.

Description of lipase producing novel yeast species Debaryomyces apis f.a., sp. nov. and a modified pH indicator dye-based method for the screening of lipase producing microorganisms.

Four yeast strains were isolated from the gut of stingless bee, collected in Churdhar, Himachal Pradesh, India. Physiological characterization, morphological examination, and sequence analysis of small subunit ribosomal RNA (18S rRNA) genes, internal transcribed spacer (ITS) region, and D1/D2 domain of the large subunit rRNA gene revealed that the four strains isolated from the gut of stingless bee belonged to the Debaryomyces clade. Strain CIG-23HT showed sequence divergence of 7.5% from Debaryomyces nepalensis JCM 2095T, 7.8% from Debaryomyces udenii JCM 7855T, and Debaryomyces coudertii JCM 2387T in the D1/D2 domain. In the ITS region sequences, strain CIG-23HT showed a 15% sequence divergence from Debaryomyces nepalensis JCM 2095T and Debaryomyces coudertii JCM 2387T. In 18S rRNA gene sequence, the strain CIG-23HT showed 1.14% sequence divergence from Debaryomyces nepalensis JCM 2095 and and Debaryomyces coudertii JCM 2387, and 0.83% sequence divergence from Debaryomyces hansenii NRRL Y-7426. Strain CIG-23HT can utilize more carbon sources than closely related species. The findings suggest that strain CIG-23HT is a novel species of the genus Debaryomyces, and we propose to name it as Debaryomyces apis f.a., sp. nov. The holotype is CBS 16297T, and the isotypes are MTCC 12914T and KCTC 37024T. The MycoBank number of Debaryomyces apis f.a., sp. nov. is MB836065. Additionally, a method using cresol red and Bromothymol blue pH indicator dyes was developed to screen for lipase producers, which is more sensitive and efficient than the currently used phenol red and rhodamine B dye-based screening methods, and avoids the problem of less differentiable zone of hydrolysis.

Specific ion effects on adsorption of lysozyme on functionalized SBA-15 mesoporous silica.

Ordered mesoporous materials (OMMs) have a pore size suitable to host proteins. Previous works have shown how to tune the amount of adsorbed protein by changing pH or ionic strength of the adsorbing solution. Here we investigated the adsorption of lysozyme on a functionalized SBA-15 (SBA-15-NH(2)) as a function of added salts. For the first time, it was ascertained that the amount of adsorbed protein follows a reversed Hofmeister series for anions (sodium salts), SCN(-) > ClO(4)(-) > Br(-) > NO(3)(-) > Cl(-) > SO(4)(2-), whereas for cations (chloride salts) the sequence was Na(+) > Li(+) > K(+) > Cs(+). These findings not only demonstrate a specific effect of the Na(+) SCN(-) ion pair in favoring the adsorption at a solid surface but confirm also the role of the biologically important sodium ions. In addition, the process was found to be more effective at 0.2 M than at 0.8 M, thus indicating that adsorption also depends on the added salt concentration.