Ahmet Tülek

Thermophilic bacterial lipases must combine structural stability with the conformational flexibility required for interfacial activation, yet the local organization of their metal-binding sites remains incompletely understood. Here, we determined two X-ray crystal structures of the family I.5 lipase from Geobacillus kaustophilus ( Gk _Lip ). The 2.47-Å orthorhombic model contains a Ca²⁺ ion at the conserved metal-binding site, whereas the 2.30-Å anisotropic triclinic model contains four non-equivalent protein chains in which no Ca²⁺ ion was modeled. Gk _Lip retains the canonical α/β-hydrolase fold, the Ser114-Asp318-His359 catalytic triad, the Phe17/Gln115 oxyanion-hole region, an extended lid, and a conserved Zn²⁺-binding site. In the orthorhombic structure, Gly287, Glu361, Asp366, and Pro367 contribute to the Ca²⁺ coordination environment. Pro367 adopts a Cγ-endo pucker in the Ca²⁺-containing structure; three Ca²⁺-free chains also adopt the endo pucker, whereas one adopts a Cγ-exo pucker. The presence of both Pro367 pucker states in the Ca²⁺-free structure is consistent with calcium preferentially stabilizing a pre-existing, coordination-compatible conformation. This local organization is further supported by an Arg35-centered network of direct and water-mediated contacts. Comparison with the 94%-identical open G. thermocatenulatus lipase reveals substantial lid rearrangement despite strong conservation of the protein core. Together, these structures highlight localized conformational variability at the calcium-binding region within an otherwise highly conserved thermophilic lipase scaffold.

Thermophilic bacterial lipases must combine structural stability with the conformational flexibility required for interfacial activation, yet the local organization of their metal-binding sites remains incompletely understood. Here, we determined two X-ray crystal structures of the family I.5 lipase from Geobacillus kaustophilus (GkLip). The 2.47-Å orthorhombic model contains a Ca2+ ion at the conserved metal-binding site, whereas the 2.30-Å anisotropic triclinic model contains four non-equivalent protein chains in which no Ca2+ ion was modeled. GkLip retains the canonical α/β-hydrolase fold, the Ser114-Asp318-His359 catalytic triad, the Phe17/Gln115 oxyanion-hole region, an extended lid, and a conserved Zn2+-binding site. In the orthorhombic structure, Gly287, Glu361, Asp366, and Pro367 contribute to the Ca2+ coordination environment. Pro367 adopts a Cγ-endo pucker in the Ca2+-containing structure; three Ca2+-free chains also adopt the endo pucker, whereas one adopts a Cγ-exo pucker. The presence of both Pro367 pucker states in the Ca2+-free structure is consistent with calcium preferentially stabilizing a pre-existing, coordination-compatible conformation. This local organization is further supported by an Arg35-centered network of direct and water-mediated contacts. Comparison with the 94%-identical open G. thermocatenulatus lipase reveals substantial lid rearrangement despite strong conservation of the protein core. Together, these structures highlight localized conformational variability at the calcium-binding region within an otherwise highly conserved thermophilic lipase scaffold.

Efficient enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from natural sources remains a major challenge for sustainable ω-3 fatty acid production. This study reports the immobilization of Rhizomucor miehei lipase (RML) on multi-walled carbon nanotubes (MWCNTs) using three distinct coupling chemistries, including genipin (MWCNT/Gen@RML), glutaraldehyde (MWCNT/Glu@RML), and glyoxyl (MWCNT/Gly@RML). The resulting nanostructured biocatalysts were systematically evaluated for the selective enrichment of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) from commercial fish oil. The immobilized RML derivatives were characterized using FTIR, SEM, SEM-EDS, and TGA analysis. The maximum immobilized protein amounts were approximately 8.4, 8.1, and 8.6 mg g⁻ ¹ support for MWCNT/Gen@RML, MWCNT/Glu@RML, and MWCNT/Gly@RML, respectively, when 10 mg of protein was initially loaded per gram of support. The optimal pH was 7.5 for free RML and all immobilized RML derivatives, and the optimal temperatures were 45 °C for free RML, 55 °C for MWCNT/Glu@RML, and 60 °C for MWCNT/Gen@RML and MWCNT/Gly@RML. Thermal stability improved markedly for all immobilized derivatives, increasing by approximately 34.6, 25.5, and 44.4 fold for MWCNT/Gen@RML, MWCNT/Glu@RML, and MWCNT/Gly@RML, respectively at 60 °C. Kinetic analysis indicated that MWCNT/Glu@RML achieved the highest catalytic efficiency ( k _cat / K _m ) of 19.2 mM⁻ ¹ min⁻ ¹ , while MWCNT/Gen@RML exhibited superior reusability, retaining 75% of its initial activity after five cycles. During fish oil hydrolysis, MWCNT/Gen@RML provided the fastest midterm accumulation (92.85% total EPA + DHA at 36 h), whereas Glu-RML delivered the highest endpoint yield (82.33% at 48 h). The immobilized systems also enabled selective enhancement of either DHA or EPA, depending on coupling agent type and enzyme loading. Overall, the use of functionalized MWCNTs as supports, combined with mild natural and synthetic coupling agents produced stable high-performing lipase biocatalysts, and offered a sustainable route for the concentration of ω-3 fatty acids under mild, environmentally friendly conditions.

L-asparaginase (L-ASNase) is an enzyme that depletes asparagine, a key amino acid for cancer cell survival, producing aspartic acid and ammonia. Beyond its food industry applications, L-ASNase is a clinically important agent against acute lymphoblastic leukemia (ALL). In this study, L-ASNase was produced and purified from Candida utilis via solid-state fermentation. Optimization on wheat bran identified 2 mL inoculation volume, 60% moisture, and a 4-day fermentation period as the optimal conditions, yielding 172.5 U/mL activity. The purified enzyme was tested against glioblastoma (GBM) cell lines, showing IC₅₀ values of 0.4 U/mL for U87MG and 1.8 U/mL for T98G, with minimal toxicity toward normal HaCaT cells. Apoptotic effects were confirmed by DAPI/F-actin and Giemsa staining, while wound healing and clonogenic assays revealed inhibition of cell migration and colony formation. RT-qPCR analysis demonstrated downregulation of the Survivin gene, a key survival regulator. These findings highlight L-ASNase’s potent antiproliferative, anti-migratory, and pro-apoptotic effects, underscoring its potential as an adjuvant therapy for GBM.