Detection of Asp371, Phe375, and Tyr376 Influence on GD-95-10 Lipase Using Alanine Scanning Mutagenesis.
GD-95-10 and GD-95-20 lipases are modified GD-95 lipase variants, which lack 10 and 20 C-terminal amino acids, respectively. Previous analysis showed that GD-95-10 lipase has higher activity than GD-95 lipase, while GD-95-20 lipase almost completely loses its activity. Analysis in silico suggested three conservative amino acids at region between 369 and 378 amino acids which can be relevant to the activity of GD-95-10 lipase. These amino acids have direct contacts with residues involved in substrate binding, stabilization of the serine loop or form oxyanion hole. In this work, the role of Asp371, Phe375, and Tyr376 on activity, functionality, and structure of GD-95-10 lipase was analyzed by Ala scanning mutagenesis. We showed that even a single mutation can impact the main structure and activity of Geobacillus lipases. Our experiments provide new knowledge about lipases from thermophilic Geobacillus bacteria and are important for protein engineering and synthetic biology. These enzymes and their engineering can be basis for future biocatalysts applied in production of biofuel or other industrial esters.
Department of Microbiology and Biotechnology, Faculty of Natural sciences, Vilnius University, Čiurlionio 21/27, 03101, Vilnius, Lithuania. renata.gudiukaite@gmail.com.
Renata
R
Gudiukaitė
Department of Microbiology and Biotechnology, Faculty of Natural sciences, Vilnius University, Čiurlionio 21/27, 03101, Vilnius, Lithuania.
Audrius
A
Gegeckas
Department of Microbiology and Biotechnology, Faculty of Natural sciences, Vilnius University, Čiurlionio 21/27, 03101, Vilnius, Lithuania.
Mikas
M
Sadauskas
Department of Microbiology and Biotechnology, Faculty of Natural sciences, Vilnius University, Čiurlionio 21/27, 03101, Vilnius, Lithuania.
Donaldas
D
Citavicius
D000409:Alanine; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics*; D016296:Mutagenesis; D013696:Temperature
D000596:Amino Acids; D008049:Lipase; D000409:Alanine
Alanine scanning mutagenesis; Biotechnology of thermophiles; C-terminal amino acids; Gene cloning and expression; Geobacillus lipases; Protein engineering