Molecular characterization of pectinase producing innovative bacterium, Aeromonas guangheii, isolated from the gut of fish, systomus sarana, RSM optimization for its enzyme production, the cloning and expression analysis of exo-pelA-gene and the recombinant protein purification

Abstract

During the present research, 11 gut bacteria were isolated from the fresh water fish, Systomus sarana (General name: olive barb) and upon screening, the strains produced extracellular pectinase enzyme. Among them, the SS6 strain was found to produce a high quantity of 208.731 U/mg pectinase. Through molecular characterization the SS6 strain was identified as Aeromonas guangheii. In this study, a group of controlled experimental factors were investigated to optimize through the response surface methodology with a Box-Behnken design. The optimal conditions were found to be; 2.11% of maltose, 2.20% of yeast extract, 6.5 of pH, and a temperature of 27.3 ºC at 32-h incubation. Under the above conditions, the activity of pectinase production was enhanced to 371 U/mg. The purified pectinase’s molecular weight was determined to be ~50 kDa (by 10% 2-D PAGE). The FT-IR analysis of the degraded pectin-products revealed the presence of six functional groups. Totally, nine peptides were identified from the purified pectinase enzyme through the MALDI-TOF-MS analysis and MASCOT tool was used to get the mass spectrum of the peak at 2211 of peptide that indicated the reference pectinase protein. The referenced gene primer (pectate lyases) was PCR amplified and their nucleotide sequence was analyzed. The exo-pelA gene was cloned in pREST vector, that was over expressed in Escherichia coli BL21. The ORF encoded for a protein of 425 amino acids with a predicted molecular weight of ~50 kDa. The present findings underline the potential of the fish-gut microbes as a source of biotechnologically important enzymes.