Recombinant thermotolerant alkaline lipase fromfor detergent and hard (Ras) cheese applications: cloning, expression, molecular docking, and characterization
Source: PubMed Central Open Access, NCBI / U.S. National Library of Medicine
Background Thermostable and alkaline lipases are of significant interest for industrial applications, particularly in detergents and food processing. This study aimed to isolate, clone, and express lipase-encoding genes from a potent bacterial source to produce a thermo-tolerant alkaline lipase with enhanced catalytic efficiency and practical applicability. Results Among several bacterial isolates, the most potent lipase producer was identified as, and its 16 S rRNA sequence was deposited in GenBank (). Three lipase-encoding genes (,, and) were successfully isolated, cloned, and heterologously expressed inBL21 (DE3). Their sequences were submitted to GenBank under accession numbers,, and, respectively. The recombinant lipase encoded by(rLipase) exhibited the highest activity (150 U/mL) compared with the native enzyme (56.2 U/mL). Molecular docking analysis demonstrated strong binding affinity of rLipase toward major fatty acid derivatives in olive oil, with the highest affinity for linoleic acid (− 8.0 kcal/mol), followed by oleic acid (− 7.8 kcal/mol) and palmitic acid (− 7.3 kcal/mol). These interactions were stabilized by hydrophobic interactions and hydrogen bonding, with key contributions from critical amino acid residues, particularly VAL250. The partially purified recombinant lipase (rLipase) exhibited a maximum activity of 320 U/mL at 80 °C and pH 9, demonstrating remarkable thermostability and alkaline tolerance. Functional evaluation showed that rLipase improved the
Abstract
Background Thermostable and alkaline lipases are of significant interest for industrial applications, particularly in detergents and food processing. This study aimed to isolate, clone, and express lipase-encoding genes from a potent bacterial source to produce a thermo-tolerant alkaline lipase with enhanced catalytic efficiency and practical applicability. Results Among several bacterial isolates, the most potent lipase producer was identified as, and its 16 S rRNA sequence was deposited in GenBank (). Three lipase-encoding genes (,, and) were successfully isolated, cloned, and heterologously expressed inBL21 (DE3). Their sequences were submitted to GenBank under accession numbers,, and, respectively. The recombinant lipase encoded by(rLipase) exhibited the highest activity (150 U/mL) compared with the native enzyme (56.2 U/mL). Molecular docking analysis demonstrated strong binding affinity of rLipase toward major fatty acid derivatives in olive oil, with the highest affinity for linoleic acid (− 8.0 kcal/mol), followed by oleic acid (− 7.8 kcal/mol) and palmitic acid (− 7.3 kcal/mol). These interactions were stabilized by hydrophobic interactions and hydrogen bonding, with key contributions from critical amino acid residues, particularly VAL250. The partially purified recombinant lipase (rLipase) exhibited a maximum activity of 320 U/mL at 80 °C and pH 9, demonstrating remarkable thermostability and alkaline tolerance. Functional evaluation showed that rLipase improved the detergent efficiency for oil stain-removal from cotton fabrics. In addition, supplementation with 0.4% rLipase accelerated Ras cheese ripening by shortening the maturation period from 120 to 90 days with maintaining the desired ripening process. Conclusions The recombinant lipase fromdemonstrated high thermal stability, alkaline tolerance, and strong catalytic efficiency. Its effectiveness in detergent formulations and cheese ripening highlights its potential as a versatile industrial biocatalyst for lipid bioconversion and related applications. Supplementary Information The online version contains supplementary material available at. Abs1