Gene Expression and Characterization of Iturin A Lipopeptide Biosurfactant from <i>Bacillus aryabhattai</i> for Enhanced Oil Recovery

Abstract

Biosurfactants are eco-friendly surface-active molecules recommended for enhanced oil recovery techniques. In the present study, a potential lipopeptide (biosurfactant) encoding the iturin A gene was synthesized from Bacillus aryabhattai. To improvise the yield of the lipopeptide for specific applications, current research tends toward engineering and expressing recombinant peptides. An iturin A gene sequence was codon-optimized, amplified with gene-specific primers, and ligated into the pET-32A expression vector to achieve high-level protein expression. The plasmid construct was transformed into an E. coli BL21 DE3 host to evaluate the expression. The highly expressed recombinant iturin A lipopeptide was purified on a nickel nitrilotriacetic acid (Ni-NTA) agarose column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the purity and molecular mass of iturin A was 41 kDa. The yield of recombinant iturin A was found to be 60 g/L with a 6.7-fold increase in comparison with our previously published study on the wild strain. The approach of cloning a functional fragment of partial iturin A resulted in the increased production of the lipopeptide. When motor oil was used, recombinant protein iturin A revealed a biosurfactant property with a 74 ± 1.9% emulsification index (E24). Purified recombinant protein iturin A was characterized by mass spectrometry. MALDI-TOF spectra of trypsin digestion (protein/trypsin of 50:1 and 25:1) showed desired digested mass peaks for the protein, further confirming the identity of iturin A. The iturin A structure was elucidated based on distinctive spectral bands in Raman spectra, which revealed the presence of a peptide backbone and lipid. Recombinant iturin A was employed for enhanced oil recovery through a sand-packed column that yielded 61.18 ± 0.85% additional oil. Hence, the novel approach of the high-level expression of iturin A (lipopeptide) as a promising biosurfactant employed for oil recovery from Bacillus aryabhattai is not much reported. Thus, recombinant iturin A demonstrated its promising ability for efficient oil recovery, finding specific applications in petroleum industries.

Keywords

• iturin A
• lipopeptide
• biosurfactant
• expression
• MALDI-TOF
• Raman spectroscopy