Biochemical and Biophysical Research Communications
X-ray structure and characterization of a thermostable lipase from Geobacillus thermoleovorans
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.
References (40)
- et al.
Extremozymes—biocatalysts with unique properties from extremophilic microorganisms
Curr. Opin. Biotechnol.
(2014) - et al.
Cloning, expression and applicability of thermo-alkali-stable xylanase of Geobacillus thermoleovorans in generating xylooligosaccharides from agro-residues
Bioresour. Technol.
(2012) - et al.
Significant improvement of Geobacillus thermoleovorans CCR11 thermoalkalophilic lipase production using Response Surface Methodology
New Biotechnol.
(2011) - et al.
Identification and over-expression of a thermostable lipase from Geobacillus thermoleovorans Toshki in Escherichia coli
Microbiol. Res.
(2008) - et al.
Zinc in lipase L1 from Geobacillus stearothermophilus L1 and structural implications on thermal stability
FEBS Lett.
(2005) - et al.
Novel zinc-binding center and a temperature switch in the Bacillus stearothermophilus L1 lipase
J. Biol. Chem.
(2002) - et al.
Activation of bacterial thermoalkalophilic lipases is spurred by dramatic structural rearrangements
J. Biol. Chem.
(2009) - et al.
Selective concentration of EPA and DHA using Thermomyces lanuginosus lipase is due to fatty acid selectivity and not regioselectivity
Food Chem.
(2013) - et al.
[20] Processing of X-ray Diffraction Data Collected in Oscillation Mode, Methods in Enzymology
(1997) - et al.
Biocatalysis for industrial production of fine chemicals
Curr. Opin. Biotechnol.
(1999)
Crystal structure of a thermostable lipase from Bacillus stearothermophilus P1
J. Mol. Biol.
Extremozymes: a potential source for industrial applications
J. Microbiol. Biotechnol.
Enzymes from extreme environments and their industrial applications
Front. Bioeng. Biotechnol.
Draft genome sequence of entomopathogenic brevibacillus laterosporus strain lak 1210, an alkaliphilic chitin degrader
Genome Announc.
Thermoadaptation trait revealed by the genome sequence of thermophilic Geobacillus kaustophilus
Nucleic Acids Res.
Complete genome sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7
DNA Res.
Twenty years of bacterial genome sequencing
Nat. Rev. Microbiol.
Lipases and their industrial applications
Appl. Biochem. Biotechnol.
Bacterial lipases as potential industrial biocatalysts: an overview
Res. J. Microbiol.
Gene cloning, expression, immobilization and characterization of endo-xylanase from Geobacillus sp. TF16 and investigation of its industrial applications
J. Mol. Catal. B Enzym.
Cited by (18)
Structure-guided rational design of the Geobacillus thermoglucosidasius feruloyl esterase GthFAE to improve its thermostability
2022, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Thermophilic enzymes enable high working temperatures that adapt to the harsh conditions of industrial processes, reduce the risks of microbial contamination, improve the reusability and determine the economic feasibility [9]. Thermostable enzymes are mainly discovered by two ways: identified from thermophilic bacteria and enzyme engineering including random mutagenesis, directed evolution, rational design and semi-rational design [10–14]. Among these engineering strategies, computer-aided rational design guided by protein structure, would be most attractive in recent years, due to its efficiency and cost-saving, with more computational tools and structural information available.
Beneficial effects of dietary β-glucan on growth and health status of Pacific white shrimp Litopenaeus vannamei at low salinity
2019, Fish and Shellfish ImmunologyCitation Excerpt :Bacillus not only can promote growth, but also can enhance immunity and improve disease resistance [78,79]. Geobacillus has thermophilic, facultative anaerobic properties and contains a variety of thermostable enzymes used to degrade cellulose and starch [80–82]. Chitinase is a key enzyme to digest chitin in crustaceans and is closely related to the growth and development of crustaceans, molt and immune function [83].
Sequence identification and in silico characterization of novel thermophilic lipases from Geobacillus species
2024, Biotechnology and Applied BiochemistryAn Appraisal on Prominent Industrial and Biotechnological Applications of Bacterial Lipases
2023, Molecular BiotechnologyHotspots and Mechanisms of Action of the Thermostable Framework of a Microbial Thermolipase
2022, ACS Synthetic Biology