title: Construction of a novel lipolytic fusion biocatalyst GDEst-lip for industrial application.
abstract: The gene encoding esterase (GDEst-95) from Geobacillus sp. 95 was cloned and sequenced. The resulting open reading frame of 1497 nucleotides encoded a protein with calculated molecular weight of 54.7 kDa, which was classified as a carboxylesterase with an identity of 93-97% to carboxylesterases from Geobacillus bacteria. This esterase can be grouped into family VII of bacterial lipolytic enzymes, was active at broad pH (7-12) and temperature (5-85 °C) range and displayed maximum activity toward short acyl chain p-nitrophenyl (p-NP) esters. Together with GD-95 lipase from Geobacillus sp. strain 95, GDEst-95 esterase was used for construction of fused chimeric biocatalyst GDEst-lip. GDEst-lip esterase/lipase possessed high lipolytic activity (600 U/mg), a broad pH range of 6-12, thermoactivity (5-85 °C), thermostability and resistance to various organic solvents or detergents. For these features GDEst-lip biocatalyst has high potential for applications in various industrial areas. In this work the effect of additional homodomains on monomeric GDEst-95 esterase and GD-95 lipase activity, thermostability, substrate specificity and catalytic properties was also investigated. Altogether, this article shows that domain fusing strategies can modulate the activity and physicochemical characteristics of target enzymes for industrial applications.
authors.affiliation: Institute of Biosciences, Vilnius University, Sauletekio Avenue 7, 10257, Vilnius, Lithuania. renata.gudiukaite@gmail.com.
authors.forename: Renata
authors.initials: R
authors.lastname: Gudiukaite
authors.affiliation: Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Vilnius University, Sauletekio Avenue 7, 10257, Vilnius, Lithuania.
authors.forename: Mikas
authors.initials: M
authors.lastname: Sadauskas
authors.affiliation: Institute of Biosciences, Vilnius University, Sauletekio Avenue 7, 10257, Vilnius, Lithuania.
authors.forename: Audrius
authors.initials: A
authors.lastname: Gegeckas
authors.affiliation: Institute of Biosciences, Vilnius University, Sauletekio Avenue 7, 10257, Vilnius, Lithuania.
authors.forename: Vilius
authors.initials: V
authors.lastname: Malunavicius
authors.affiliation: Institute of Biosciences, Vilnius University, Sauletekio Avenue 7, 10257, Vilnius, Lithuania.
authors.forename: Donaldas
authors.initials: D
authors.lastname: Citavicius
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D056506:Geobacillus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008066:Lipolysis; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D002265:Carboxylic Ester Hydrolases; D008049:Lipase
keywords: Biotechnology of thermophiles; Fusion enzymes; Geobacillus esterases; Geobacillus lipases; Protein engineering