Enhanced biosurfactant production through cloning of three genes and role of esterase in biosurfactant release

Abstract

Background: Biosurfactants have been reported to utilize a number of immiscible substrates and thereby facilitate the biodegradation of panoply of polyaromatic hydrocarbons. Olive oil is one such carbon source which has been explored by many researchers. However, studying the concomitant production of biosurfactant and esterase enzyme in the presence of olive oil in the Bacillus species and its recombinants is a relatively novel approach.

Results: Bacillus species isolated from endosulfan sprayed cashew plantation soil was cultivated on a number of hydrophobic substrates. Olive oil was found to be the best inducer of biosurfactant activity. The protein associated with the release of the biosurfactant was found to be an esterase. There was a twofold increase in the biosurfactant and esterase activities after the successful cloning of the biosurfactant genes from Bacillus subtilis SK320 into E.coli. Multiple sequence alignment showed regions of similarity and conserved sequences between biosurfactant and esterase genes, further confirming the symbiotic correlation between the two. Biosurfactants produced by Bacillus subtilis SK320 and recombinant strains BioS a, BioS b, BioS c were found to be effective emulsifiers, reducing the surface tension of water from 72 dynes/cm to as low as 30.7 dynes/cm.

Conclusion: The attributes of enhanced biosurfactant and esterase production by hyper-producing recombinant strains have many utilities from industrial viewpoint. This study for the first time has shown a possible association between biosurfactant production and esterase activity in any Bacillus species. Biosurfactant-esterase complex has been found to have powerful emulsification properties, which shows promising bioremediation, hydrocarbon biodegradation and pharmaceutical applications.

Figure 1

MultAlin (Multiple Sequence Alignment) of sfp, sfp0 and srfA gene sequences with esterase gene sequences from NCBI database. The consensus sequences are shown in the last line of the alignment table. Bacillus = Bacillus sp. NK13 esterase gene (Liu et al. 2005, PubMed Accession number: DQ196347). B. clausii = Bacillus clausii KSM-K16 esterase gene (Hakamada et al. 1994, PubMed Accession number: AP006627). sfp, sfp0 and srfA are gene sequences responsible for biosurfactant production in Bacillus subtilis SK320.

Figure 2

Effect of Proteinase K on purified biosurfactant. The purified biosurfactant was incubated at 37°C with various proteinase K concentrations for 10, 30, 60 and 120 min. The sample was analyzed for biosurfactant activity at 550 nm.

Figure 3

Effect of lipase on purified biosurfactant. The purified biosurfactant was incubated at 37°C with various lipase concentrations for 30, 60, 90 and 120 min. The sample was analyzed for biosurfactant activity at 550 nm.