Effect Of High Pressure (HP) Treatment On Product Specificity Of α-CGTase

Recently, cyclodextrins have a broad application prospect in food, medicine, chemicalindustry, agriculture, etc, this makes cyclodextrin glycosyltransferase (CGTase), which is animportant enzyme for the production of cyclodextrin, become a research hotspot. HavingCGTase with higher efficiency, stonger specificity and better stability, is aways the goal ofcurrent industrial of cyclodextrins. In the present study, it has confirmed that high pressure(HP) processing can improve the stability and the reaction rate of certain enzyme at a certainpressure, but there is not so much research involving the CGTase systematically. Takingbacillus macerans α-CGTase as the research object, this paper studied the effect of HP on thecyclization activity, the specificity, the yield of the product, and the conformation of theα-CGTase, besides the effect of HP on the cyclization reaction system of α-CGTase was alsostudied.α-CGTase was purified by (NH4)2SO4fractional precipitation, desalination columnHiTrap Desalting and ion exchange chromatography HiPrep Q FF, and the purity of it wasidentified by SDS-PAGE. The results indicated that the specific activity of purified α-CGTasewas190.76U/mg after three-step purification, and was purified up to14.04, it presented asingle band on the protein electrophoresis. All these indicated that the enzyme was highlypurified after the three-step purification. The optimum temperature, optimum pH of α-CGTaseafter treated by HP were also studied. The results indicated that the optimum temperature andoptimum pH of α-CGTase after treated by HP didn’t change, were60°C,6.0separately.Firstly, the effect of HP on α-CGTase with different conditions was also studied. It showedthat: at200MPa, using different temperature treat α-CGTase, with the increase of temperature,cyclization activity and product specificity of α-CGTase would have an increase at first, thenreduced, and the highest point were both at40°C, at the same time the yield of cyclizationactivity decreased while the The holding temperature gradually increased; with the increase ofpressure, the cyclization activity of α-CGTase increased during0-200MPa, and then itreduced rapidly when pressure was over200MPa, the yield of product decreased whilepressure increasing gradually, meanwhile, the trend of the product specificity was just theopposite while the pressure increased gradually; at200MPa, adopting different dwell time,cyclization activity of α-CGTase first increased, with the extension of the holding time, thendecreased, when the dwell time was10min, the enzyme got the highest cyclization activity;however, the product specificity and product yield of α-CGTase decreased with the extensionof the dwell time.Secondly, the effect of HP on conformation of α-CGTase was studied by analyzingCircular Dichroism (CD) and fluorescence spectra of high pressure α-CGTase. the resultsshowed that the pressure would affect the protein secondary structure, changed proteinconformation near the substrate binding site, increased the exposure of hydrophobic aminoacids, the cavity near the catalytic sites in the substrate combination grooves was compressed,coupling and disproportionation reaction were suppressed, thus α-CGTase product specificitywas improved. Thirdly, the effect of HP on catalytic reaction system of α-CGTase was studied, theresults showed that cyclization activity would decrease and the product specificity wouldenhance with the increase of pressure. Under the conditions of200MPa,60°C, reaction time30min, the reaction got the highest yield.