Studies On Screening Of Pullulanase-producing Strains And Properties Of The Pullulanase

In this research,62 pullulananse-producing strains were isolated from samples taken from Haikou, Sanya, Danzhou in Hainan province. After screening, one highly pullulananse-producing strain BCT-1 was obtained. The initial enzyme activity of this strain is 0.357U/ml.According to the characteristics of morphology and bacteria colony, physiology and biochemistry identification and 16S rDNA gene sequence analysis, the strain was preliminarily identified. It was observed that the cells of this strain were short rod shaped, Gram-, non-motile, no spores, no flagella. The isolated colonies in the medium plate is white or pale yellow, viscous, the edge of the cell is not sharp, the surface of the cell is ragged and no pigment secretion. The results of 25 physiological and biochemical identification indicated that this strain was basically similar with Acinetobacter.16S rDNA gene sequence analysis indicated that the similarity of the strain BCT-1 and Acinetobacter calcoaceticu reached 97.8%. Based on the above results, the strain BCT-1 was identified as Acinetobacter sp and named as Acinetobacter sp. BCT-1.One-factor experiment and Response Surface Analysis (RSA) were carried out to optimize the medium composition and fermentation conditions of BCT-1. The optimum medium and fermentation conditions were:corn starch 19.97g/L; yeast extract 10.45g/L; sulfate ammonium 5g/L; MgSO4·7H2O 0.5g/L; FeSO4·7H2O 0.01 g/L, initial pH 5.4; amount of inoculation is 8%,500 mL flask containing medium 150 mL, incubation temperature is 30℃and shaking at 200 r/min, fermentation time is 72 h. The optimum pullulanase activity was 2.656 U/ml, which was 7.45 times of the initial pullulanase activity,0.357 U/ml.The pullulanase enzyme was purified by sulphate precipitation, Sephadax G-75 gel filtration, ion-exchange chromatography on DEAE sepharose Fast Flow. The molecular weight of the pullulanase was estimated to be 58 KDa by SDS-PAGE. The specific activity is 9.97U/mg, comparing with the initial activity 1.57 U/mg, purified fold is 6.35, and recovery percent is 6.2%. The pullulanase of the optimum temperature is 50℃. When the enzyme was incubated at 55℃for 20min, there was 55% residual activity. The enzyme was active in the range of pH6.0-9.0. Ca2+, Mn2+, Mg2+ activated the pullulanase activity, while Ba2+, Fe2+, Cu2+, Co2+, Zn2+ and Hg2+ inhibited the enzyme activity. In the test of substrate specificity, the pullulan was the optimum substrate to this enzyme, followed by soluble starch, sticky rice starch, corn starch, amylopectin, potato starch.