Co-production of Pectinase and Biosurfactant by the Newly Isolated Strain Bacillus subtilis BKDS1

Abstract

Aim: To isolate pectinase producing bacterial strains from various samples and to investigate the co-production of pectinase and biosurfactant by this isolated strain. Study Design: Isolation, screening, selection and identification of pectinolytic bacteria. Production of pectinase and analysis of exo-pectinase types. Partial purification by ammonium sulphate precipitation and dialysis. The study also analyses the capability of the selected strain for biosurfactant production. Place and Duration of Study: Department of Life Sciences, University of Calicut, Kerala 673635, India, between July 2013 and November 2014. Methodology: The pectinolytic bacteria was isolated and screened by iodine plate assay method. The enzyme production was confirmed by 3,5-dinitrosalicylic acid (DNS) method. The potent enzyme producing strain was biochemically characterised and identified by 16S rRNA gene sequence analysis. Thiobarbituric acid (TBA) and DNS assay were performed to test the types of exo-pectinase produced. Partial purification of the enzyme was done by ammonium sulphate precipitation. Biosurfactant production by the strain was tested by methods like foam formation, Original Research Article Kavuthodi et al.; BMRJ, 10(2): 1-12, 2015; Article no.BMRJ.19627 2 drop collapse test, oil displacement test, microplate assay, hemolytic assay, penetration assay, emulsification activity and bacterial adhesion to hydrocarbon (BATH) assay. Results: Thirty six pectinolytic bacterial strains were screened from the collected samples. Four isolates were selected on the basis of zone size (20 mm to 26 mm) in well plate screening method. In which, the isolate showed maximum enzyme production in DNS assay (0.707 U/mL) is the same which exhibited larger pectin depolymerization area (26 mm) in well diffusion assay. This strain was identified as Bacillus subtilis BKDS1 using 16S rDNA based molecular technique. The optimum incubation time was found to be 72 h for the maximum enzyme production (1.288 U/mL). Assay of exo-pectinase revealed that, the organism was able to produce polygalacturonase (PG), pectin lyase (PNL) and pectate lyase (PEL). In ammonium sulphate precipitation, the enzyme activity was found in 40 -100% salt saturation fractions. On further analysis, it was observed that the organism also produced biosurfactant along with pectinase in the same culture medium. Conclusion: The present study indicates the possibility of an integrated process for obtaining pectinase enzymes and biosurfactants in the same culture media. So this could greatly increase the economic viability of the isolated strain.