Abstract
Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca2+-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter Plac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65°C and retained ≥ 97% activity after incubation at 50°C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.
Highlights
Lipases are glycerol ester hydrolases that catalyze the hydrolysis or synthesis of a broad range of water insoluble esters
By using analysis of comparative sequence, genomic data, phylogenetic analysis, homology modeling method and characterization of the recombinant enzyme, we have reported the discovery of a novel lipase from a thermophilic bacterium belonging to an uncharacterized subfamily of family I
Arpigny and Jaeger [7] had classified bacterial lipolytic enzymes based on their amino acid sequences and biological properties
Summary
Lipases are glycerol ester hydrolases that catalyze the hydrolysis or synthesis of a broad range of water insoluble esters. They constitute the most important group of biocatalysts for biotechnological application. Lipolytic enzymes have been classified hitherto according to different criteria such as their known substrate specificity, interfacial activation and possessing a movable lid. The increasing knowledge at the atomic level of the 3-D structure of bacterial lipolytic enzymes using either X-ray crystallography or NMR spectroscopy has promoted an attempt to classify these enzymes according to their fold. The growing sequence information of cloned lipases and knowledge of protein 3-D structures has been used to identify possible sequence specific motifs. Lipases share two conserved features, including a serine in a highly conserved GXSXG pentapeptide motif and an aspartate or glutamate residue that is hydrogen bonded to a histidine to form a catalytic triad, and the oxyanion hole [7,8,9]
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