Production and purification of extracellular pullulanase by Klebsilla aerogenes NCIM 2239

The present study was carried out to optimize the production of pullulanase from Klebsilla aerogenes NCIM 2239, a new strain of the bacterium. The organism was screened for its ability to synthesize and secrete extracellular pullulanase, by analysis of pullulan degrading activity. Environmental (pH and temperature) and nutritional (carbon and nitrogen) factors influencing growth and product formation were optimized through fermentation trials in shake flasks. Maximum pullulanase production (78.62 U/ml) was observed at 48 h. Optimized pH and temperature were 7.0 and 37°C, respectively. Different carbon sources (pullulan, starch, sucrose, maltose and glucose) and nitrogen sources (peptone, beef extract, yeast extract and casein) were analysed for maximum production of pullulanase. This bacterium was found to exhibit maximum pullulanase production using starch and peptone as carbon and nitrogen sources, respectively. In addition, pullulanase production by solid substrate fermentation (SSF) was investigated using K. aerogenes NCIM 2239. The extracellular pullulanase was purified by ultrafiltration method of membrane separation. The pullulanase activity after ultrafiltration was 130.21 U/ml when compared with its crude (83.08 U/ml). Key words: Pullulanase, α-1,6 glucosidic linkages, Klebsilla aerogenes NCIM 2239, solid state fermentation, ultrafiltration.