Expression, Characterisation and Homology Modelling of a Novel Hormone-Sensitive Lipase (HSL)-Like Esterase from Glaciozyma antarctica

Abstract

Microorganisms, especially those that survive in extremely cold places such as Antarctica, have gained research attention since they produce a unique feature of the protein, such as being able to withstand at extreme temperature, salinity, and pressure, that make them desired for biotechnological application. Here, we report the first hormone-sensitive lipase (HSL)-like esterase from a Glaciozyma species, a psychrophilic yeast designated as GlaEst12-like esterase. In this study, the putative lipolytic enzyme was cloned, expressed in E. coli, purified, and characterised for its biochemical properties. Protein sequences analysis showed that GlaEst12 shared about 30% sequence identity with chain A of the bacterial hormone-sensitive lipase of E40. It belongs to the H group since it has the conserved motifs of Histidine-Glycine-Glycine-Glycine (HGGG)and Glycine-Aspartate-Serine-Alanine-Glycine (GDSAG) at the amino acid sequences. The recombinant GlaEst12 was successfully purified via one-step Ni-Sepharose affinity chromatography. Interestingly, GlaEst12 showed unusual properties with other enzymes from psychrophilic origin since it showed an optimal temperature ranged between 50–60 °C and was stable at alkaline pH conditions. Unlike other HSL-like esterase, this esterase showed higher activity towards medium-chain ester substrates rather than shorter chain ester. The 3D structure of GlaEst12, predicted by homology modelling using Robetta software, showed a secondary structure composed of mainly α/β hydrolase fold, with the catalytic residues being found at Ser232, Glu341, and His371.

Cold Adaptation Strategies and the Potential of Psychrophilic Enzymes from the Antarctic Yeast, Glaciozyma antarctica PI12

“…These findings are linked to the increment of structure flexibility under cold conditions [40]. The increment of structural flexibility in cold adaptation is also supported by other findings such as G. antarctica esterase [41], exo-b-1,3-glucanase [42], laminarinase [43], β-mannanase [44], α-amylase [45] and lipase (HSL)-like esterase [46]. Structural elucidation of G. antarctica fuculose aldolase using X-ray crystallography data shows that the enzyme retains a highly conserved catalytic histidine triad, an increase in its non-polar residues on the protein surface and a lower number of arginine residues, features that serve for adaptation in the cold [47].…”

“…Interestingly, we found several intriguing and unusual findings where some G. antarctica enzymes deviate from common psychrophilic properties. As an example, the G. antarctica lipase-like esterase was found to work optimally at 50–60 °C [ 46 ] and cold-active type II 3-dehydroquinate dehydratase displayed maximal activity at 40 °C [ 84 ]. These show that at low temperatures, G. antarctica adopts its adaptive machinery by producing cold-active proteins such as chaperones and enzymes as part of its strategy of adaptation to low temperatures.…”

The Unusual Dominance of the Yeast Genus Glaciozyma in the Deeper Layer in an Antarctic Permafrost Core (Adélie Cove, Northern Victoria Land) Is Driven by Elemental Composition

“…The activity of GaDlh was inhibited in the presence of Zn and Cu2, while the presence of Mg and Ca determined only partial inhibition, respectively [72]. On the other hand, Na, K, Ca, and Mn enhanced the esterase activity of GlaEst12, while Mg, Ni, and Cu acted in a reverse manner by inhibiting the GlaEst12 activity [73].…”

“…Moreover, a study on a 3D model of a cold-active α-amylase (AmyPI12) produced by G. antarctica revealed the presence of binding sites for Ca and Na [71]. The effects of various elements on the activity of some cold-active hydrolases released by G. antarctica PI12, i.e., an esterase-like protein (GaDlh) and a hormone-sensitive lipase (HSL)-like esterase (GlaEst12) were recently tested [72,73]. The activity of GaDlh was inhibited in the presence of Zn and Cu2, while the presence of Mg and Ca determined only partial inhibition, respectively [72].…”

Editorial Catalysts: Special Issue on Novel Enzyme and Whole-Cell Biocatalysts

“…These two enzymes proved to be highly specific to laminarin and tolerant to high temperature, and are good candidates for application in biomass conversion. In addition, Tahir et al [6] clone and overexpress a novel hormone-sensitive lipase-like esterase from Glaciozyma antartica. Unlike other known enzymes, this protein demonstrates higher activity towards medium-chain ester substrates, rather than shorter chain esters, and increased stability at 60 • C, as well as alkaline pH conditions.…”