| Popis: |
A novel lipase gene, Lip-1420, was isolated from a metagenomics library constructed from soil samples from reed marshes and from Mt. Jumbong in Korea consisting of 112,500 recombinant plasmids. A selected recombinant plasmid, Lip-1420, was further analyzed which exhibited the maximum lipolytic activity among 18 recombinant fosmids that showed lipolytic activity. Among them, DNA sequence analysis of a 5,513 bp subclone in pUC119 Lip-1420-sub revealed that 11 ORFs were included in the gene according to the blast search from GenBank. The transformant of Lip-1420-ORF3 exhibited lipolytic activity, and plasmid pET21a(+)-Lipase-6H was constructed and transferred to the expression hostE. coliBL21(DE3). Finally, the lipase Lip1420 was purified from a FPLC system, and the recombinant enzyme was kept in a −70 ºC freezer at a concentration of 1 mg/mL in a buffer solution (50 mM Tris-HCl and 0.15 M NaCl, pH 7.4). The Lip-1420 gene was registered in GenBank (MH628529), and the purified enzyme had an optimal activity at 40 °C and pH 8.0. Kinetic analysis of the Lip-1420 lipase with the substratep-nitrophenyl palmitate was performed at 40°C and pH 8.0, and theKmandVmaxvalues of the protein were determined to be 0.268 mM and 1.821 units, respectively. The purified Lip-1420 exhibited the maximum enzymatic activity towardsp-nitrophenyl palmitate, indicating that it is an esterase.IMPORTANCEThis study provides the knowledge to develop a new lipase from the metagenomics library of rhizosphere in Korea using an activity-based method. In addition, the knowledge gained from this study has allowed us to understand that the natural ecosystems are still an unknown genetic material storage report in relation to industrially useful biocatalysts, which are still rich in biodiversity. Moreover, alkaline lipase has great potential for applications in the detergent industry. Especially in the major part of the global industrial enzyme market with high growth potential. It is used, for example, in detergent additives, biopolymer synthesis, and biodiesel production, synthesis of optically pure compounds and food additives as well as in the paper industry, perfume and fragrance blends, biological purification and waste treatment. |
