High thermostability of the enzymes is one of the distinguishing characteristics that increase their industrial utility. In the current research work, rigidifying the flexible amino acid residues of a Show more
High thermostability of the enzymes is one of the distinguishing characteristics that increase their industrial utility. In the current research work, rigidifying the flexible amino acid residues of a lysophospholipase (Pa-LPL) from Pyrococcus abyssi was used as a protein engineering approach to improve its thermostability. A truncated variant of Pa-LPL (t-LPL∆12) was constructed by trimming its 12 amino acid residues (50-61) through overlap extension PCR. The truncated enzyme worked optimally at 65°C and pH 6.5 with remarkable thermostability at 65°C-85°C. In comparison to wild-type Pa-LPL, 5.8 and 1.2-fold increase in half-life (t Show less
Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve Show more
Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35-95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35-95 °C with first order rate constant (k Show less