A newly isolated thermostable lipase from Bacillus sp

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• A thermophilic lipolytic bacterium, Bacillus sp. L2, was isolated from a Malaysian hot spring.
• Bacillus sp. L2 belongs to Group 5, a coherent group of thermophilic bacilli with high 16S rRNA sequence similarity (98.5-99.2%).

Purpose of the Study:

• To clone and characterize the L2 lipase gene from Bacillus sp. L2.
• To overexpress and purify the recombinant L2 lipase.
• To determine the biochemical and biophysical properties of the purified L2 lipase.

Main Methods:

• Polymerase chain reaction (PCR) was used for lipase gene cloning.
• Sequence analysis identified an open reading frame (ORF) of 1251 bp encoding 417 amino acids.
• Recombinant lipase was overexpressed, purified, and characterized using spectroscopy (CD) and activity assays across various temperatures and pH levels.

Main Results:

• The L2 lipase gene's ORF codes for a mature protein of 388 amino acids.
• Recombinant lipase exhibited a 178-fold increase in activity and was purified to homogeneity.
• The purified lipase demonstrated optimal activity at 70°C and pH 9, with a melting temperature (Tm) of 59.04°C. It showed significant inhibition by EDTA (100%) and other agents (PMSF, pepstatin-A, 2-mercaptoethanol, DTT >40%). Secondary structure analysis revealed 38.6% α-helices, 2.2% ß-strands, 23.6% turns, and 35.6% random conformations.

Conclusions:

• The L2 lipase is a robust thermophilic enzyme with potential for industrial applications.
• The characterization provides valuable insights into its structure-function relationship and stability.
• Further studies can explore its specific substrate specificities and applications in biocatalysis.