| Amylopectin is the major composition in both mass and granule structure of starch,which has complex semicrystalline architecture. Debranching enzyme is able to cleave-1,6-linkages in amylopectin, it is essential for starch saccharification. However, differencesin structure and catalytic function between debranching enzymes from different sourcesresults in various action modes and product composition. In this thesis, some characterizationof recombinant Thermobifida fusca isoamylase were mentioned after purification. At the sametime, instrumental analysis methods were used to elucidate the properties of isoamylase onfive starches.The recombinant strain was disrupted and centrifuged to get the crude enzyme solution.After purified by His Trap affinity chromatography and desalted by ultrafiltration,recombinant isoamylase showed one band on SDS-PAGE with molecular mass of around79kDa, which proved that the enzyme was purified to homogeneity. The specific activityreached to3440U·mg-1and a116.7-fold purification with a yield of12.5%was obtained.Some properties of the pure recombinant isoamylase were characterized:1) the optimumtemperature was60℃, after1h of incubation at60℃would make it lose merely7%of itsmaximal activity, and there was a good stability between30-60℃;2) the optimum pH was5.5and the enzyme displayed good stability between pH5.0and7.0;3) Li+, Ca2+, Mg2+, Zn2+,Mn2+and citrate activated the enzyme activity, Sn2+and Fe3+reduced the activity significantlyby more than55%of control levels. Furthermore,30mmol·L-1SDS and2mol·L-1urea led toevident loss of its activity. It was insensitive to K+, Na+, EDTA, Cu2+and Co2+.-Cyclodextrin and maltose up to a concentration of20mmol·L-1and20%, respectively, didnot influence the catalytic activity. A7%inhibition of the activity was observed in thepresence of50mmol·L-1-cyclodextrin;4) its Kmand Vmaxfor amylopectin was1.39mg·mL-1and3.87mg·min-1·(mg protein)-1, respectively;5) the results of substrate specificityshowed that the enzyme had high enzyme activity on amylopectin and with no hydrolysisaction on pullulan, demonstrated that it was an isoamylase-type debranching enzyme;6) highperformance liquid chromatography analyzed the hydrolyzates from various substrates, themain products of amylopectin was maltotriose. However, there was a proportion of glucoseliberated from soluble starch.Instrumental analysis methods resolved the hydrolysis properties of recombinantisoamylase on amylopectin, soluble starch, potato starch, maize starch and glutinous rice. Theresults showed that isoamylase was capable to hydrolyze these substrates effectively. Highperformance size exclusion chromatography spectrum illustrated that isoamylase cut off bothinner and outer branching linkages and the polymer structure was almost completelydestroyed. The population of starch fragments liberated was characterized by highperformance anion exchange chromatography with pulsed amperometric detector, the degreeof polymerization of final hydrolysates (Mw<10.5kDa) distributed mainly in15-35.Nevertheless, their hydrolysates varied in the length distribution and ratio of side chains.Released glucose, maltose and maltotriose demonstrated that the smallest units hydrolyzed by isoamylase were side chains with1-3glucose residues.1H nuclear magnetic resonancespectrum showed that isoamylase specifically and almost completely cleaved-1,6glycosidiclinkages and produced amylose chains or maltooligosaccharides with-or β-anomericreducing ends, but the proportion of-1,4glycosidic linkages almost remained the same afterhydrolyzed. The highest amount of end-products in potato starch, maize starch and glutinousrice was glucose, whereas it was maltose and maltotriose in soluble starch and amylopectin,respectively. This might caused by different composition and structure in various starches. |
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