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

In this study,20 strains of pululanase-producing were isolated from the sample taken from the area near the starch factory and starch processing plant in Shandong.A strain with strong enzyme-producing capacity was obtained because of pullulan.Single colony,observation strain growth morphology,colony characteristics,the strain of the system classification is judged:this strain is Gram-positive bacilli,mycelium oval or short rod like,can produce spores,and have sportivity.The form of single colony is gray or white,sometimes slightly yellow,thicker,irregular and conical,and the edge is raised,the surface is smooth and no other pigment is secreted.The results of physiological and biochemical identification experiments show that the indexes are similar to Bacillus subtilis,and the strain is determined by 16S rDNA sequence analysis.The similarity between the sequence and Bacillus subtilis reached the highest affinity.The optimum medium and fermentation conditions of the fermentation medium were:soluble starch 15.0g/L,peptone 10.0g/L,ammonium sulfate 5.0g/L,sodium dihydrogen phosphate 1.0g/L,pH value of 5.The temperature is 37 degrees,200rpm,the inoculum volume is 1%?when the fermentation triangle bottle is 50mL,the liquid volume is 20mL?,the optimized enzyme production level is 4.3U/mL.After the supernatant was concentrated by freeze-drying,ammonium sulfate was precipitated and precipitated,then a series of separation and purification steps,such as dialysis,strong anion column chromatography,and so on,the activity of the purified enzyme was 9.3 U/mL and two times higher than that before purification.The enzymic molecular weight of the purified enzyme was determined to be about70kD by SDS-PAGE method.Then a series of exploration was made on the properties of the enzyme.The results showed that the enzyme activity reached its peak at 55 C,its heat resistance was poor,the enzyme solution was kept at 55 degree and 60 minutes after heat preservation,and the relative enzyme activity was only 65%.The optimum pH was about6.2,and the enzyme remained stable in the range of 5.5-6.2.The activity remained above63%after 90 minutes at pH5.4.K⁺,Ba²⁺,Zn²⁺and Na⁺significantly promoted the activity of enzymes.Cu²⁺,Ga²⁺,Mg²⁺,Sn²⁺,Fe²⁺and Cd²⁺ had different degrees of inhibition.