Recombinant Expression and Characterization of a Novel Thermo-Alkaline Lipase with Increased Solvent Stability from the Antarctic Thermophilic Bacterium Geobacillus sp. ID17.

Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE

Original Publication: Basel, Switzerland : MDPI, [2000-

Geobacillus*/enzymology ; Geobacillus*/genetics ; Lipase*/genetics ; Lipase*/chemistry ; Lipase*/metabolism ; Lipase*/isolation & purification ; Enzyme Stability* ; Solvents*/chemistry ; Recombinant Proteins*/chemistry ; Recombinant Proteins*/metabolism ; Recombinant Proteins*/genetics; Antarctic Regions ; Hydrogen-Ion Concentration ; Bacterial Proteins/genetics ; Bacterial Proteins/chemistry ; Bacterial Proteins/metabolism ; Kinetics ; Substrate Specificity ; Temperature ; Escherichia coli/genetics ; Escherichia coli/metabolism

Lipases are enzymes that hydrolyze long-chain carboxylic esters, and in the presence of organic solvents, they catalyze organic synthesis reactions. However, the use of solvents in these processes often results in enzyme denaturation, leading to a reduction in enzymatic activity. Consequently, there is significant interest in identifying new lipases that are resistant to denaturing conditions, with extremozymes emerging as promising candidates for this purpose. Lip7, a lipase from Geobacillus sp. ID17, a thermophilic microorganism isolated from Deception Island, Antarctica, was recombinantly expressed in E. coli C41 (DE3) in functional soluble form. Its purification was achieved with 96% purity and 23% yield. Enzymatic characterization revealed Lip7 to be a thermo-alkaline enzyme, reaching a maximum rate of 3350 U mg ^-1 at 50 °C and pH 11.0, using p-nitrophenyl laurate substrate. Notably, its kinetics displayed a sigmoidal behavior, with a higher kinetic efficiency ( k cat / K m ) for substrates of 12-carbon atom chain. In terms of thermal stability, Lip7 demonstrates stability up to 60 °C at pH 8.0 and up to 50 °C at pH 11.0. Remarkably, it showed high stability in the presence of organic solvents, and under certain conditions even exhibited enzymatic activation, reaching up to 2.5-fold and 1.35-fold after incubation in 50% v / v ethanol and 70% v / v isopropanol, respectively. Lip7 represents one of the first lipases from the bacterial subfamily I.5 and genus Geobacillus with activity and stability at pH 11.0. Its compatibility with organic solvents makes it a compelling candidate for future research in biocatalysis and various biotechnological applications.

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ANID Proyecto de Exploración 2022 13220108 Agencia Nacional de Investigación y Desarrollo

Keywords: biocatalyst; biotechnology; esterase; extremophile; extremozyme; protein purification

EC 3.1.1.3 (Lipase)
0 (Solvents)
0 (Recombinant Proteins)
0 (Bacterial Proteins)

Date Created: 20240727 Date Completed: 20240727 Latest Revision: 20240730

20240731

PMC11277018

10.3390/ijms25147928

39063171