Improvement of Thermal Stability via Outer-Loop Ion Pair Interaction of Mutated T1 Lipase from Geobacillus zalihae Strain T1

Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The Tm for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 °C, 70.59 °C and 68.54 °C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher Tm as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 Å using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 Å, b = 81.16 Å and c = 100.14 Å. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically

Impact of Dermatophagoides pteronyssinus mite body raw material on house dust mite allergy diagnosis in a Serbian population

House dust mite (HDM) allergy has different clinical and immunological patterns in different geographic regions. The impact of raw material of commercial Dermatophadoides pteronyssinus (Acari: Pyroglyphidae) mite bodies on the quality of allergen extracts for allergy diagnosis in the Serbian population has not been previously evaluated. House dust mite bodies obtained from manufacturers in Europe, South America and Australia were used in the preparation of allergen extracts for in vivo diagnosis and serological analysis in a group of 14 HDM-allergic adults. In the group of mite-allergic patients, there was no statistically significant difference in skin test reactivity (Wilcoxon matched pairs test) among the three HDM body extract preparations. In a CAP inhibition assay, two extracts (A and C) achieved maximum inhibition of gt 90%, whereas extract B demonstrated a different inhibition slope and lower inhibition potential (80%). However, a remarkable difference in immunoglobulin E reactivity using Western blot analysis with individual patients' sera was observed in one of the preparations (extract B). These findings emphasize the need for the careful selection of starting material for the preparation of HDM diagnostic reagents intended for use in patients from geographically distinct regions as these preparations can have implications on the selection criteria for patient-tailored immunotherapy of HDM allergy