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

Abstract

Background Lipolytic enzymes of hyperthermophilic archaea generally prefer small carbon chain fatty acid esters (C 2 –C 12 ) 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 Archaeglobus fulgidus . 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 Thermococcus onnurineus (strain NA1) 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— Thermomyces lanuginosus (TLIP). Results TON-LPL and rc-TGL were cloned and overexpressed in E. coli, 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 opt ). 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. Conclusions 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. Electronic supplementary material The online version of this article (10.1186/s13068-019-1452-5) contains supplementary material, which is available to authorized users.

Structural dynamics and mechanistic action guided engineering of lipolytic enzymes

“…The new thermoalkaliphilic lipolytic enzyme can be designed in the following ways: (i) inserting the C‐terminal extended region of MAS1 into LipA at the same place and substituting the residue Gly153/Cys153 for proper disulfide bond formation, (ii) inserting N‐terminal extended region of MAS1 into LipA at the same place and substituting the residue Ser32/Cys32 for proper disulfide bond formation, and (iii) creating the metal ion center at the C terminus mimics to MAS1 by substituting the residue. In recent years, many studies have been reported on lipolytic enzymes using the MD approach to alter the enzyme properties 13,47 . Singh et al explored the thermostability properties of Bacillus subtilis lipase using the MD simulation approach 48 .…”

“…In recent years, many studies have been reported on lipolytic enzymes using the MD approach to alter the enzyme properties. 13,47 Singh et al explored the thermostability properties of Bacillus subtilis lipase using the MD simulation approach. 48 Recently, Dong et al improved the thermostability of LipA.…”

Rebuilding the lid region from conformational and dynamic features to engineering applications of lipase in foods: Current status and future prospects

“…Swapping lids with non‐lipase lids such as esterases and carbohydrases may have great advantages in designing a hyperfunction lipase for industrial applications. For example, the monoacylglycerol lipase from Thermococcus onnurineus was constructed into a triacylglycerol lipase by replacing lid swapping with the lid domain of TLL (Soni et al., 2019). In addition, the constructed lipase was shown with high activity toward castor oil and has a potential application in castor oil biorefinery to obtain value‐added chemicals.…”

Trends in lipase engineering for enhanced biocatalysis

“…For most lipases, entry to the active site comprising of a catalytic triad is protected by a lid structure that consists of α‐helices, joined by a loop to the body of a lipase. Many lipase variants have been generated by methods (Table I), like insertion of a disulfide bridge in the hinge region of the lid, lid swapping [14], and so on, altering its substrate specificity, activity profile, and thermostability (Fig. 2).…”