Studies On Meiothermus Rosaceus WQBGR2-3 Producing Thermostable Pullulanase From Hot Spring In Yunnan And The Enzyme Properties

Pullulanase is one of debranching enzymes, which can hydrolyze the smallest substrates and make use of starch maximally cooperating withα-amylase andβ-amylase, making the use of starch ultimately. So it is widely used in food, detergent, textile, biofuel etc. industries that translate starch into sugar solution. Because of the saccharification condition, pullulanase should be thermostable and acid-resistant. So it is important to explore such enzyme. Till now, so many microorganisms have been found to produce pullulanase, but only the pullulanase from B. acidopullulyticus is widely and commercially used explored by NOVO. The study on pullulanase in China is only the beginning. As their activity are not high or stable, none of the pullulanase have be used from China.Twenty strains without endospore isolated from Yunnan hot spring were screened which could produce pullulanase. In this paper, Iodine vapor method for screening the microorganism producing extracellular amylolytic enzyme was improved and simplified. Staining with 0.04g iodine for 1 minute is suitable to visualize halos in the plates whose diameter are 6 centimeters and less harm to the strains in several minutes. Different strains showed different resistant ability to iodine but all can be alive vapored for a few minutes. We isolated out 5 strains by this way and chose the strain WQBGR2-3 for further study by DNS method. The enzyme activity was increased from 1.409U/mL to 4.452U/mL by substrate inducing.After the steps of centrifugal separation, (NH4)₂SO₄ treatment, gel chromotograph , ion exchange, gel recycled by Native-PAGE, the fermentation broth was purified for 10.88 fold. The specific activity is from 2.65 U/mg to 28.82 U/mg. The molecular weight detected by SDS-PAGE is about 81.7KDa. Purified enzyme can be got by gel recycle.The properties of Pul-WQBGR2-3 were studied. The optimum temperature is 75℃, and the enzyme can be thermostable in 50℃for a long time. The half life of the enzyme is 30 minutes in 70℃. The optimum pH is 5.5 and can conserve some activity in pH4.0-7.0. So it is a thermostable and acid-resisitant enzyme. Mn²⁺ is the activator , Cu²⁺, Co²⁺ can inhibite the activity obviously. The enzyme can hydrolysis theα-1,6-glycosidic linkages of pullulan and amylopectin as well asα-1,4-glycosidic linkages. So it may be the class II. Pul-WQBGR2-3 can be used with glucoamylase for its high specific activity and acid-resisitant property.The bacteria WQBGR2-3 is Meiothermus rosaceus according to the morphological, physiological, biochemical characteristics and 16S rDNA etc. identification, and its evolution distance is farther than the typical stain RH9901. The secondary structure of helix 12 is 4 {3} 4(4), as well as Me. rosaceus, Me. ruber, Me. chliarophilus, and helix 17 is 5[2]2(7), like Me. cerbereus, Me. chliarophilus, Me. ruber and Me. taiwanensis, but different from Me. rosaceus' 3[1]2[1]2(10). So it is one pullulanase- producing strain further in evolution distance than the typical strain RH9901 of Me. rosaceus. This is the first report of pullulanase in Meiothermus.