Optimizing of Cultural Conditions for the Production of Pectinolytic Enzyme from Marine Actinomycetes by Submerged Fermentation Along With Statistical Approach

Abstract

Actinomycetes species possess excellent enzymatic potential and are active degraders of pectic substances. Pectinases catalyze the hydrolysis of pectin (polygalacturonic acid) to galacturonic acid residues. The pectinase enzyme produced from Actinomycetes are highly stable and can withstand broader range of pH and moreover thermo-stable property makes an advantage over pectinase produced by other species. So, Actinomycetes are choosen to produce pectinase as it has numerous benefits and optimistic alternative than other species in future. This pectinase can be industrially used in extraction and clarification processes. In the present study, 52 Actinomycetes isolates from four locations along Visakhapatnam coast sample at a region (1 feet) of Bay of Bengal were recovered. A maximum of 9 isolates from the sample of Rushikonda Beach, 3 from Kursura, 27 from the sample of Thenneti and 13 from the R.K. Beach samples were obtained. All isolates were maintained on Starch casein Agar medium. Purified Actinomycetes isolates were subjected to pectinolytic activity by growing them on pectin agar plates and the pectinolytic activity was detected by visualizing a clear zone around the colony using iodine flooding method. Out of the 52 isolates, Thenneti sample isolates were found effective for hydrolysis of pectin. The isolate E5 has shown good zone of inhibition and was subjected to secondary screening under submerged fermentation. Physico-chemical parameters such as fermentation time, temperature, pH, inoculum age, inoculum volume were varied to analyze the optimum enzyme production. The highest yield of the pectinase was obtained at an initial medium pH of 6, when incubated at 35⁰C for 6 days with a 5 day old culture and 5ml of the volume i.e., 159.65U/ml. The effect of carbon and nitrogen supplements revealed that Ammonium sulfate (nitrogen source) enhanced the pectinase activity to 187.79U/ml. A 25 factorial Central Composite Design (CCD) using Response Surface Methodology (RSM) was employed to optimize 5 variables (fermentation time, temperature, pH, inoculum age and inoculum volume). According to the results of Response Surface Methodology, a maximum yield of 202.7955 U/ml of pectinase was produced.