Production and Characterization of Pullulanase from Bacillus licheniformis using various Organic Substrates

Abstract

The present study was conducted to find out cost effective organic substrate and optimum conditions for the production of industrially important pullulanase enzyme from previously isolated Bacillus licheniformis-C1. Optimization of different parameters like temperature, pH, nitrogen sources and starch supplementation were carried out. Acetone precipitation was used to partially purify the enzyme from culture supernatant and then further purified by gel filtration chromatography technique. Finally, characterization of crude, partial and purified enzyme was done on different parameters like temperature, pH and effect of different metal ions on enzyme activity was measured. According to the results obtained it was observed that Bacillus licheniformis-C1 is an efficient producer of pullulanase enzyme when initially screened on pullulan containing media plate with 3 cm clear zone around the colony. It is also observed that Bacillus licheniformis-C1 can efficiently grow on corn bran and produces pullulanase enzyme 16 U/mg at 55oC and at pH 6.0.The peptone (5g/l) and starch supplementation (5g/l) were also increases the pullulanase production. The specific activity of pullulanase enzyme was increased upto 30 U/mg after partial purification with 80% acetone. Further purification with gel filtration chromatography increases specific activity of enzyme upto 190 U/mg by using Sephadex G-100. Finally the crude filtrate, partially purified and purified enzyme were characterized and have maximum specific activity at temperature 50oC with optimum pH 5 for crude filtrate and 60°C with optimum pH 5.0 for partial and purified enzyme. According to our observations Fe2+, Hg+ and anionic detergent like SDS completely inhibit the enzyme activity, while Zn2+, Mg2+, and sodium iodide (NaI) showed slight inhibition on pullulanase activity. In addition, Ca2+ and ethylenediaminetetraacetic acid (EDTA) have no noticeable effect on enzyme activity. So, pullulanase from Bacillus licheniformis-C1 is Ca+ independent enzyme. Therefore, it was concluded that pullulanase from thermophilic Bacillus licheniformis-C1 has various applications in industries especially in starch processing industry.