Screening of alkalophilic starch Degraders and characterization of Enzymes

Abstract

Screening for starch degraders from soil resulted in the isolation of 10 positive strains. Strain RCP6 was selected as the best Cyclodextrin Glycosyltransferase(CGTase) producer. The cultural,morphological and biochemical characteristics of the bacterial isolates were studied. The isolate RCP6 was characterized by 16S rRNA sequencing and was identified as Bacillus oshimensis. The CGTase production is dependent on the bacterial strain,medium composition and culture conditions; hence media optimization studies were carried out in shake flask conditions using conventional method. Production of CGTase from B.oshimensis was optimized using the Taguchi method. The design contained a total of 9 experiments,which employed the selected media components. The optimal values of these variables for maximum production of CGTase were comprised of containing 1% tapioca starch,0.5%peptone,0.5% yeast extract,0.2%ammonium dihydrogen phosphate, 0.14%MgSO4.7H2O and 1%Na2CO3. The CGTase activity of 4.5 U/ml was achieved after media optimization. The growth kinetics of CGTase producer B.oshimensis was studied using the optimized and basal media and it was found that the production of and#946;CD was increased using the optimized media. The strain improvement study was carried out using Nitrous acid. The mutation studies had a negative effect on the and#946;CD production in comparison to its production using optimized media. The synthesis of and#946;CD in presence of polar organic solvent was also studied that resulted in 45% conversion of starch to and#946;CD with 5% ethanol. The enzyme was purified by ammonium sulphate precipitation along with starch adsorption and DEAE cellulose. The purity was found to increase by 2.5fold with 11% yield. The characterization of the enzyme was carried out for pH and temperature and was found to be optimum at pH 8 and 37deg. Continuous production of and#946;CD was carried out by immobilizing the B.oshimensis cells on loofa sponge. Influence of immobilization time on and#946;CD production resulted in viability of B.oshimensis cells with 30days.