Purification And Characterization Of A Novel Debranching Enzyme

Bacillus sp. was isolated from a highway soil sample in Qing dao by researcher Zhao Xianchun of Centre of Biomass Resource of School of Biotechnology, Jiangnan University. Due to its relatively high Starch Debranching enzyme(DBE) activity, this strain can be applied in factories as a new source to synthesis DBE. For the purpose of improving the activity and stability of DBE produced by the strain above, we purified DBE from Bacillus sp. and studied its basic properties.Purified strain was inoculated into induction medium and cultured under 37℃on 180rpm shaking table for 36h. The strain Bacillus sp.CBB272 with highest DBE activity was chosen as start strain after secondary screening. Rejuvenate the strain 272 by a serial passages till the activity reached a stable level. After 11 passages, the DBE activity increased by 1.28 times.Preparing crude enzyme in 15L computer controlled fermentor. Purified strain of two-step culturing, lasting 24h and 12h respectively, was added into 9L medium with an inoculation amount of 10%. After 36h fermentation at 37℃, the 9L product was concentrated to 1.6L crude enzyme with a protein amount of 1.3mg/mL by centrifuge and ultra filtration.The target protein was precipitation between 30%-80% ammonium sulfate saturation and the sediment was redissolved in 20mM Tris-HCl buffer(pH 8.2). The protein solution was run through Sephacryl S-100 gel filtration, DEAE FF ion exchange colume and Mono Q ion exchange column. The final protein solution was verified on SDS-PAGE with one band, which proved that the enzyme was purified to homogeneity. The protein concentration and DBE activity were measured after each step of purification and the specific activity, fold and yield of each step was calculated. In the end, a 369.21-fold purification with a yield of 19.2% was obtained. Protein concentration was measured in terms of Bradford method using BSA as the standard; DBE activity was measured by Iodine method.The basic properties of DBE was studied. The maximal activity occurred at 70℃and pH 6.0. It was stable at a temperature between 30℃and 70℃, and retained at least 70% original activity for 60min in the pH range from 4.5-9.0. At pH 6.0 and 50℃the Km for支链淀粉was 4.0324mg/mL and the Vmax was 0.1841mg/(min·mL debranching enzyme). The enzyme was activated strongly by Ca2+, Mg2+ and Mn2+ (up to 141.31%), what is more, Ca2+,was shown to be an excellent protector of the enzyme at a temperature up to 80℃.The homogenous Starch Debranching Enzyme and its basic properties were obtained through this study, which laid a foundation for the future study of DBE amino acid sequence and the relationship of DBE structure & function. Accordingly, we can increase the output and quality of DBE by modifying the enzyme to enhance its activity and stability.