N-terminal domain replacement changes an archaeal monoacylglycerol lipase into a triacylglycerol lipase

• Published in: Biotechnology for biofuels 2019-05, Vol.12 (1), p.110-110, Article 110
• Main Authors: Soni, Surabhi, Sathe, Sneha S, Sheth, Rutuja R, Tiwari, Prince, Vadgama, Rajesh-Kumar N, Odaneth, Annamma Anil, Lali, Arvind M, Chandrayan, Sanjeev K
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Alternative energy sources; Amino acids; Archaea; Biochemistry; Biodiesel fuels; Biorefineries; Castor oil; Chains; Chemical properties; Chemical reactions; Chromatography; Domains; E coli; Engineering; Engineers; Enzymatic activity; Enzymes; Esterase; Esterases; Esters; Ethanol; Fatty acids; Gel chromatography; Gel filtration; Glycerol; Hydrolase; Hyperthermophilic archaea; Industrial applications; Lipase; Lipases; Monoglycerides; Mud-water interfaces; N-Terminus; Oil; Organic chemistry; Protein engineering; Protein structure; Proteins; Quaternary structure; rc-TGL; Residues; Substrate specificity; Substrates; Switches; Temperature; Thermococcus onnurineus (strain NA1); Thermomyces lanuginosus (TLIP); TON-LPL; Triacylglycerol lipase; Triglycerides; Trimers
• ISSN: 1754-6834; 1754-6834
• DOI: 10.1186/s13068-019-1452-5

Lipolytic enzymes of hyperthermophilic archaea generally prefer small carbon chain fatty acid esters (C -C ) and are categorized as esterases. However, a few have shown activity with long-chain fatty acid esters, but none of them have been classified as a true lipase except a lipolytic enzyme AFL from . Thus, our main objective is to engineer an archaeal esterase into a true thermostable lipase for industrial applications. Lipases which hydrolyze long-chain fatty acid esters display an interfacial activation mediated by the lid domain which lies over active site and switches to open conformation at the oil-water interface. Lid domains modulate enzyme activities, substrate specificities, and stabilities which have been shown by protein engineering and mutational analyses. Here, we report engineering of an uncharacterized monoacylglycerol lipase (TON-LPL) from an archaeon into a triacylglycerol lipase (rc-TGL) by replacing its 61 N-terminus amino acid residues with 118 residues carrying lid domain of a thermophilic fungal lipase- (TLIP). TON-LPL and rc-TGL were cloned and overexpressed in and the proteins were purified by Ni-NTA affinity chromatography for biochemical studies. Both enzymes were capable of hydrolyzing various monoglycerides and shared the same optimum pH of 7.0. However, rc-TGL showed a significant decrease of 10 °C in its optimum temperature (T ). The far UV-CD spectrums were consistent with a well-folded α/β-hydrolase fold for both proteins, but gel filtration chromatography revealed a change in quaternary structure from trimer (TON-LPL) to monomer (rc-TGL). Seemingly, the difference in the oligomeric state of rc-TGL may be linked to a decrease in temperature optimum. Nonetheless, rc-TGL hydrolyzed triglycerides and castor oil, while TON-LPL was not active with these substrates. Here, we have confirmed the predicted esterase activity of TON-LPL and also performed the lid engineering on TON-LPL which effectively expanded its substrate specificity from monoglycerides to triglycerides. This approach provides a way to engineer other hyperthermophilic esterases into industrially suitable lipases by employing N-terminal domain replacement. The immobilized preparation of rc-TGL has shown significant activity with castor oil and has a potential application in castor oil biorefinery to obtain value-added chemicals.