The Purification And Interaction Between Limit Dextrinase And Its Inhibitors From Beer Malt

As the key factor affecting the production of a high degree of fermentation andlight beer, limit dextrinase （LD） plays an important role in the brewing of beer. In thispaper, higher purity of LD and its inhibitors were purified from201106GairdnerAustralia barley malt, and the characteristics of LD and its inhibitors were studiedafter purification. In the buffer, the interaction mechanism between LD and itsinhibitors were explored preliminary, and the role of dithiothreitol （DTT） and releasemechanism from the bound LD to free one were discussed, too. The study will notonly provide theoretical basis and process methods for getting highly purified LD andits inhibitors, but will also offer materials to further study the characteristics of LD ofbeer malt at home. The most important thing is to provide a reliable theoretical basisfor the beer saccharification process to achieve enzyme regulation.In this paper, the separation and purification of malt LD were conducted bysaturated-precipitation method using25%-35%-80%ammonium sulfate, SephadexG-25gel chromatography, DEAE Sepharose FF ion exchange chromatography andSephadex G-100gel chromatography. The final protein solution was verified bySDS-PAGE and IEF with one band, which proved that the enzyme was purified tohomogeneity. Its molecular weight was about101.9kDa, and isoelectric point wasabout4.66. The protein concentration and LD activity were measured after each stepof purification and the specific activity, fold and yield of each step was calculated. Inthe end, a164.2-fold purification with a yield of16.5%was obtained. Proteinconcentration was measured in terms of Lowry method using casein as the standard;LD activity was measured by red Pullulan method. The basic properties of LD were studied. The maximal activity approximatelyoccurred at50and pH5.5. LD is a heat-sensitive enzyme, and its activity decreasedrapidly when the temperature exceeds50. It retained at least70%original activityfor1hr in the pH range from5.0-9.0. At pH5.5and50the K_mfor amylopectin was3.3426mg/mL and the V_maxwas0.8456mg/（min mL debranching enzyme）. Theenzyme was activated strongly by Ca²⁺, Mg²⁺and Mn²⁺（up to150.2%）, what’s more,Ca²⁺was shown to be an excellent protector of the enzyme at a temperature up to80.The separation and purification of LD inhibitors are conducted by crude extract,heated extract, Carboxymethyl cellulose CM-52ion-exchange chromatography andSephadexG-100gel chromatography. The two LD inhibitor mixtures were obtainedand their molecular weights were about15.8kDa, isoelectric point was about6.76and7.28, respectively. Finally, a39.9-fold purification with a yield of14.1%wasobtained.The study of the characteristic of LD inhibitor found that the LD activity wasinhibited by its inhibitor at a broad range （pH4.0-8.0） and the maximal inhibitory pHwas about5.5. Inactivation of the LD can be reversed by warming the LD/inhibitormixture at40in the presence of BSA and DTT. For example, approximately66.5%of the original enzyme activity was regained after8h.The regulatory mechanism of LD was studied in the condition in vitro. In thewarm environment（40）, BSA buffer system, the free LD can be released by DTTfrom bound LD. The free LD was not a sulfhydryl enzyme. In the system of this study,a hypothesis was proposed about the release of free LD from bound LD by DTT, thatwas to say, bound LD, which was activated or modified by BSA, was reducted to freeLD through DTT.