Study On Screening And Characteristic Of Strains Of Producing Pullulanase

Pullulanase is a debranching enzyme which has important uses and good prospects in the industries of food, textile, pharmaceutical, detergent and feed. This paper has studied screening of pullulanase producing strains, mutation, production of fermentation research and enzymatic properties of pullulanase, in order to provide theoretical basis for the industrial production and application of pullulanase. The results were as follows:1. Screening strains which produced pullulanase from different samples by transparent circle method and submerged fermentation method. Study on the enzymatic properties of the strains, and combined morphological identification and molecular identification. In the final, the marine bacteria Pseudoalteromonas sp. M25, which was non-pathogenic, producing alkaline pullulanase and fewer reported, was chose for further research.2. The Pseudoalteromonas sp. M25 was mutagenized by UV, UV-LiCl and DES. The results showed that the the optimum UV mutagenesis processing time is 25 s, the best concentration of lithium chloride is 0.4% and the optimum DES processing time is 15 min. Finally, the mutant ULD-4 which had good genetic stability was obtained. The fermentation of strain ULD-4 was determined that it still remained 94.18% of enzyme producing capacity after 5 generations. The pullulanase activity of the mutant was increased to 1.78 U/mL,0.96 times higher than that of the original strain which was 0.91 U/mL.3. The culture medium and the culture condition of the mutant ULD-4 were optimized. The optimal culture medium was as follows (g/L):potato starch 15.0, yeast extract 10.0, KH2PO41.0, FeSO4-7H2O 0.01, NaCl 26.5, MgCl2·7H2O 2.1, KCl0.9, CaCl2-H2O 1.2, MgSO4·7H2O 4.2, pH 7.0. The fermentation condition were as follows:fermentation temperature 30℃, inoculation quantity 1.0%, liquid volume 50 mL/250 mL, shaking speed 200 r/min and the fermentation time 18 h. The pullulanase activity of the strain after optimization was 2.85 U/mL, increased 60.11% than before optimization which was 1.78 U/mL, and 2.13 times higher than that of the original strain.4. Researched on the enzymatic properties of pullulanase, we learned that the optimum temperature of pullulanase produced by strain ULD-4 was 50℃. The pullulanase had good stability of temperature, maintaining more than 90% of enzyme activity after heating 2 h at 30℃-50℃. The optimal reaction pH of the pullulanase was 8.0. The pH stability was stable in the range of pH 6.0 to 9.0, which can keep 75% enzyme activity after deal with 4 h. In the test of substrate specificity, the pullulan was the best substrate to this enzyme. Determined from Lineweaver-Burk plots, the kinetic parameters Km and Vmax for the enzyme activity on pullulan were 3.70 mg/mL and 3.96 U/(min·mL). The pullulanase was stimulated by Ca2+, Mn2+ and Mg2+, while Zn2+, Gu2+, Co2+ and Ni2+ inhibited the enzyme activity.