The reverse synthesis action of pullulanase was verified at high substrate concentration. In addition to increasing the sugar yield, pullulanase reduces reaction time, allows for high substrate concentrations, and facilitates maltose production with 50 % of the normal glucoamylase concentration. In the grain industry, pullulanase is often used with glucoamylase or β-amylase to produce high glucose and maltose syrups. Pullulanase is a debranching enzyme that can degrade the α-1,6-linkages of pullulan, amylopectin, and other branched polysaccharides. In a starch saccharification reaction, the dextrose equivalents of M1, M3, and M5 proteins were 94.7, 94.5, and 93.1 %, respectively, which were also essentially identical to that of WT (93.6 %). The optimal temperature and pH for purified preparations of M1, M3, and M5 were similar to those of the WT enzyme. After fermentation, about 56.6 and 93.4 % of the total activity of PelB-M1 and PelB-M5 were transferred to the periplasm, respectively, followed by cell lysis and leakage of the partial enzyme into the extracellular medium. PelB-M1 and PelB-M5 were transported to the periplasmic space, where PelB is part of the PelB-pET28a(+) construct, and PelB-M3 (ΔX25) and PelB-WT variants were largely retained in the cytoplasm. The enhanced expression of soluble protein is the main reason for these improved activities. The activities of M1 (ΔCBM41) and M5 (ΔCBM41ΔX25) variants were 2.9- and 2.4-fold that of wild-type (WT) enzyme, respectively. BaPul possesses a complex modular architecture that consists of CBM41-X45a-X25-X45b-CBM48-GH13.
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