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Caldicellulosiruptor bescii, the most thermophilic cellulolytic bacterium, is rich in hydrolytic and accessory enzymes that can degrade untreated biomass, but the precise role of many these enzymes is unknown. One of such enzymes is a predicted GDSL lipase or esterase encoded by the locus Athe_0553. In this study, this probable esterase named as Cbes-AcXE2 was overexpressed in Escherichia coli. The Ni-NTA affinity purified enzyme exhibited an optimum pH of 7.5 at an optimum temperature of 70 °C. Cbes-AcXE2 hydrolyzed p-nitrophenyl (pNP) acetate, pNP-butyrate, and phenyl acetate with approximately equal efficiency. The specific activity and K for the most preferred substrate, phenyl acetate, were 142 U/mg and 0.85 mM, respectively. Cbes-AcXE2 removed the acetyl group of xylobiose hexaacetate and glucose pentaacetate like an acetyl xylan esterase (AcXE). Bioinformatics analyses suggested that Cbes-AcXE2, which carries an SGNH hydrolase-type esterase domain, is a member of an unclassified carbohydrate esterase (CE) family. Moreover, Cbes-AcXE2 is evolutionarily and biochemically similar to an unclassified AcXE, Axe2, of Geobacillus stearothermophilus. Thus, we proposed a novel family of carbohydrate esterase for both Cbes-AcXE2 and Axe2.

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.

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