X-ray structure and characterization of a thermostable lipase from Geobacillus thermoleovorans

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Abstract

Thermo-alkalophilic bacterium, Geobacillus thermoleovorans secrets many enzymes including a 43 kDa extracellular lipase. Significant thermostability, organic solvent stability and wide substrate preferences for hydrolysis drew our attention to solve its structure by crystallography. The structure was solved by molecular replacement method and refined up to 2.14 Å resolution. Structure of the lipase showed an alpha-beta fold with 19 α-helices and 10 β-sheets. The active site remains covered by a lid. One calcium and one zinc atom was found in the crystal. The structure showed a major difference (rmsd 5.6 Å) from its closest homolog in the amino acid region 191 to 203. Thermal unfolding of the lipase showed that the lipase is highly stable with Tm of 76 °C. 13C NMR spectra of products upon triglyceride hydrolysate revealed that the lipase hydrolyses at both sn-1 and sn-2 positions with equal efficiency.

Introduction

Application of enzymes in industrial processes requires robust and thermostable enzymes that can perform in non-natural conditions [1]. Enzymes sourced from organisms that grow in extremes of conditions, i.e. Extremophiles, were very valuable in several industrial processes [2,3]. . Genomic approaches on extremophiles have further accelerated discovery of many novel enzymes [[4], [5], [6], [7]]. Among the enzymes, lipases are extensively used in many industrial processes including fabric cleaning, food formulations, chemical synthesis, leather making etc. To make these processes economically viable it is important to discover lipases that are stable and could be produced in large quantities [8,9]. Several thermo-stable enzymes, including lipases have been isolated from genus Geobacillus [[10], [11], [12]]. The particular strain of Geobacillus thermoleovorans, used in this study was isolated from a hot water spring in New Zealand [13].

Geobacillus thermoleovorans has been a source for several thermostable enzymes including some lipases [[14], [15], [16]]. The bacterium produces a mature lipase (will be referred as GTL) with 388 amino acids (43 kDa) along with a signal sequence consisting of 28 amino acids which facilitate its secretion (UniProt accession number Q8L1V2_GEOTH). GTL belongs to lipase family I.5. The catalytic serine (113S) in GTL is situated in Ala-Xaa-Ser-Xaa-Gly motif which is common to lipases isolated from gram positive bacteria [17]. Active triad (113S, 358H, 317D) observed in other homologous lipases is also conserved in this lipase sequence. GTL was expressed in E. coli cells and some preliminary characterizations were reported [18]. However, neither 3D structure nor the detail characterization of GTL is available to understand the basis for its thermostability.

Lipases homologous to GTL from other species of Geobacillus have been studied. These homologous lipases showed optimum activity at elevated temperature (60 °C–70 °C) and at alkaline pH (9–10) and are highly thermostable [19,20]. They are also known to hydrolyze lipids rich in saturated fatty acid such as beef tallow and bind divalent cations [21,22]. Structures of some of these homologous lipases have been reported both in lid closed and lid open conformation which provides valuable knowledge about the mechanism of thermo-stability as well as mechanism of interfacial activation [23,24]. GTL has an ability to discriminate against poly unsaturated fatty acids (PUFA) and could be a promising catalyst in enriching PUFAs in natural oils like fish oil, algal oil etc.

In this work we have cloned GTL gene and purified by hydrophobic interaction chromatography (HIC). The structure of GTL was solved by X-ray diffraction to a resolution of 2.14. Biochemical and biophysical properties of GTL were investigated and reported.

Section snippets

Materials

Geobacillus thermoleovorans was obtained from MTCC, Chandigarh (Acc. No. 4219). TritonX-100 and para-nitrophenyl butyrate (pNPB) were purchased from Sigma Aldrich and iso-propyl thiogalactosidase (IPTG) was from Siri organics, Hyderabad, India. Ethylene glycol and glycerol were supplied by Merck India. Enzymes used in cloning were purchased from NEB, USA. All other chemicals used are of analytical grade or higher.

Cloning GTL gene into pET vector

Gene encoding GTL along with the signal sequence was amplified from genomic DNA of

Secondary structure of GTL

CD spectra provide useful information on secondary structure of proteins. CD spectra of GTL show that the protein is well folded. The spectra clearly show negative peaks near 209 nm and 222 nm (Fig. 1a), which suggests that α-helix is the predominant secondary structure in GTL. Based on the CD spectra, K2D program calculated the secondary structure content of GTL to be 44% α-helix, 20% β-sheet and 36% coil which matched closely with that of the crystal structure [35].

Thermostability of GTL

Thermal denaturation of GTL

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

Authors acknowledge the indispensable effort of Dr R. Sankaranarayanan, Dr Anant B Patel Dr Lakshmi Prasanna G. and P Sambhavi towards the completion of this work and Indo-Australian Biotechnology Fund (GAP373) for the financial support. T. R. Moharana acknowledges research fellowship received from CSIR.

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