Cycloamylose Production From Debranched Starch By Thermus Aquaticus4-α-glucanotransferase

Native starches from different sources were used as substrates to produce cycloamylosein this study. First, these starches were debranched by isoamylase and then they were treatedby4-α-glucanotransferase (4αGT). The purity of4αGT was identified by SDS-PAGE and thereducing powers of these starches during debranching process were measured to confirm theoptimum debranching conditions. Meanwhile, the optimum conditions for cycloamyloseproduction were investigated and the differences of conversion yields of cycloamylose inwater reaction system and DMSO reaction system were compared. In addition, the differencesof conversion yields of cycloamylose from different starches were also compared. Finally,amylose and amylopectin from amylomaize were separated to study the effect of lineardextrins from debranched amylopectin on cycloamylose production. And the minimumdegree of polymerization (DP) of cycloamylose from Thermus aquaticus4αGT treatment wasconfirmed.4αGT was purified by Ni-NTA column and the purity of it was identified by SDS-PAGE.The results indicated that the enzyme presented a single band on the protein electrophoresis.This indicated that the enzyme was highly purified after the three-step purification. Theenzyme property of4αGT was also studied. The results indicated that the optimumtemperature and optimum pH of4αGT were75℃and7.5, separately. It was stable between60℃and80℃. And it was also stable when pH was between7.0and8.0. The minimumglycosyl donor of4αGT was maltose when it catalyzed transglycosylation reaction.The reducing powers of amylomaize under different enzyme dosages and differentreaction times were measured. The results indicated that amylomaize was completelydebranched when the enzyme dosage was7.5U/g and the reaction time was12h.The conversion yields of cycloamylose under different enzyme dosages and differentreaction times were measured to confirm the optimum conditions for cycloamyloseproduction. The results indicated that the maximum conversion yield was achieved when theenzyme dosage was10U/g and the reaction time was12h. As the high solubility and theauxo-action of DMSO on enzyme activity of4αGT, DMSO reaction system was widely usedin practical production of cycloamylose. In this study, the conversion yields of cycloamylosefrom amylomaize in these two reaction systems were compared because of the potentialtoxicity of DMSO. The results indicated that the maximum conversion yield of cycloamylosein DMSO reaction system was only1.67%higher than that in water reaction system. Thisindicated that DMSO reaction system can be replaced by water reaction system.The changes of conversion yields of cycloamylose after starch debranching were studiedand the results indicated: the maximum conversion yield of cycloamylose from amylomaizewas24.55%, while after debranching of amylomaize, it increased to45.58%; the maximum conversion yield of cycloamylose from corn starch was20.30%, while after debranching ofcorn starch, it increased to33.60%; the maximum conversion yield of cycloamylose fromcassava starch was16.82%, while after debranching of cassava starch, it increased to32.40%.All these indicated that the debranching of starch was an effective method to improve theconversion yield of cycloamylose.The changes of conversion yields of cycloamylose from amylose and amylopectin(amylomaize) in different reaction times were studied. The results indicated that the lineardextrins from debranched amylopectin had a synergistic effect on cycloamylose productionduring the reaction period from6h to12h.In addition, by comparing the DP distributions of cycloamylose in different reactionconditions, we can confirm that the minimum DP of cycloamylose from Thermus aquaticus4αGT treatment was5regardless of reaction conditions. This was different from previousresearch.