Yunnan Hot Spring The Thermus Thermal Stability Pullulanase Producing Strain Selection Of A Preliminary Study

Pullulanases (pullulan 6-glucanohydrolase; EC 3.2.1.41) are debranching enzymes that are able to hydrolyze the α-l,6 bonds of the linear α-glucan pullulan, producing maltotriose as the final product. Pullulanases are divided into two classes based on substrate specificity: (i) type I pullulanases or debranching enzymes, which specifically hydrolyze the a-1, 6 linkages in branched oligosaccharides, such as starch, amylopectin,and glycogen, forming linear α-1, 4-linked oligomers; and (ii) type II pullulanases or amylopullulanases, which cleave a-1, 6 glycosidic linkages in pullulan and branched substrates in addition to the α-1, 4 glycosidic linkages in polysaccharides other than pullulan. A number of pullulanases have been purified from different bacterial and archaeal sources and characterized. Most enzymes from thermophilic and hyperthermophilic microorganisms are type II pullulanases and have been isolated mainly from thermophilic archaea Thermostable type I pullulanases have been characterized from the aerobic moderately thermophilic bacteria Bacillus acidopullulolyticus and Bacillus flavocaldarius KP 1228, the thermophilic bacteria Thermus aguaticusYT-1, Thermus caldophilus GK-24, and Bacillus thermoleovorans, and the extreme anaerobic thermophilic bacteria Caldocellulosiruptor saccharolyticus, Fervidobacterium pennivorans, and Thermo toga maritima.It is said that Thurmus has little ability of hydrolyze Starch, so there must be relationship between Starch and the Enzyme of Thurmus. 100 strains of Thurmus had been isolated from different hot well of Tengchong and Eryuan, Yunnan. These bacterium had been done some experiments to testify they could produce amylase. Through the experiments different bacterium which could produce different amylase such as α - amylase, β- amylase, γ- amylase and pullulanase had been isolated.Through these experiments the relationship beteen bacterium and amylase is clear and the bacterium of WQBGR2-3 has more ability of producing pullulanase, which could hydrolyze Starch and Pullulan. The bacterium is a Gram-negative bacterium, and it is a bacillus. Starch and yeast have strong infection at the pullulanase yieldThrough the abduction the pullulanase yield increased. There arc three factor which has more effect on producing pullulanase, such as starch, peptone, yeast. The experiment of surface response has been designed. Through the experiment the prescription has been established: Starch 3. 73684g/L, yeast 0.918875g/L, and peptone 0.674l87g/L The bacterium incubate at 65℃ continuing about 40h, then the activity of this enzyme come to a head. The optimum temperature and pH of the enzyme is about 85℃ and pH 4.8.The enzyme could be isolated by Gelatin chromatography...