Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of ␣-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form of Bacillus deramificans pullulanase was recently constructed to enhance the thermostability and catalytic efficiency of the enzyme (X. Duan, J. Chen, and J. Wu, Appl Environ Microbiol 79:4072-4077, 2013, http://dx.doi.org/10.1128/AEM.00457-13). In the present study, three N-terminally truncated variants of this DM that lack the CBM41 domain (DM-T1), the CBM41 and X25 domains (DM-T2), or the CBM41, X25, and X45 domains (DM-T3) were constructed. Upon expression, DM-T3 existed as inclusion bodies, while 72.8 and 74.8% of the total pullulanase activities of DM-T1 and DM-T2, respectively, were secreted into the medium. These activities are 2.8-and 2.9-fold that of the DM enzyme, respectively. The specific activities of DM-T1 and DM-T2 were 380.0 ؋ 10 8 and 449.3 ؋ 10 8 U · mol
؊1, respectively, which are 0.94-and 1.11-fold that of the DM enzyme. DM-T1 and DM-T2 retained 50% of their activity after incubation at 60°C for 203 and 160 h, respectively, which are 1.7-and 1.3-fold that of the DM enzyme. Kinetic studies showed that the K m values of DM-T1 and DM-T2 were 1.5-and 2.7-fold higher and the K cat /K m values were 11 and 50% lower, respectively, than those of the DM enzyme. Furthermore, DM-T1 and DM-T2 produced D-glucose contents of 95.0 and 94.1%, respectively, in a starch saccharification reaction, which are essentially identical to that produced by the DM enzyme (95%). The enhanced secretion and improved thermostability of the truncation mutant enzymes make them more suitable than the DM enzyme for industrial processes. P ullulanase (EC 3.2.1.41), which catalyzes the hydrolysis of ␣-1,6-glucosidic linkages in pullulan, amylopectin, and the dextrins of amylopectin, is a well-known starch-debranching enzyme (1). It belongs to the ␣-amylase family, which is identified as glycoside hydrolase family 13 in the CAZy database. When combined with ␣-amylase, -amylase, glucoamylase, or cyclodextrin glucosyltransferase, pullulanase can be used in the production of glucose, fructose, maltose, cyclodextrins, and amylose (2, 3). The addition of pullulanase allows the reaction time to be reduced, the substrate concentration to be increased, and the conversion rate to be improved (4, 5). Recently, pullulanase has been used as a biocatalyst for the production of fuel ethanol, low-calorie beers, resistant starch, maltotriose, and other important products (6, 7). With this plethora of potential industrial applications, pullulanase production has attracted a great deal of attention in recent years (8,9).A large number of microorganisms, including mesophiles, thermophiles, and hyperthermophiles, have been shown to produce pullulanase (10, 11). Although numerous pullulanases have been identified and heterologously expressed (12)(13)(14), reports have shown that the secretion ratio of recombinant pullulanase is low. Low extracellular prod...