Enhancing the secretion efficiency and thermostability of a Bacillus deramificans pullulanase mutant (D437H/D503Y) by N-terminal domain truncation

Abstract

Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of α-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 Km values of DM-T1 and DM-T2 were 1.5- and 2.7-fold higher and the Kcat/Km 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.

FIG 1

Schematic representations of pullulanase and truncated enzyme variants. Bars: white, CBM48 domain; light gray, X45 domain (including X45a and X45b); black, X25 domain; dark gray, CBM48 domain; diagonally lined, GH13 superfamily catalytic domain. DM-T1 is the mutant protein lacking the CBM41 domain. DM-T2 is the mutant protein lacking both the CBM41 and X25 domains. DM-T3 is the mutant protein lacking the CBM41, X25, and X45 domains.

FIG 2

SDS-PAGE analysis of the extracellular crude fractions (A) and purified enzymes (B) from the expression in E. coli of the DM enzyme and its truncated variants. Lanes: M, protein molecular mass markers; 1, crude DM enzyme; 2, crude DM-T1 enzyme; 3, crude DM-T2 enzyme; 4, purified DM enzyme; 5, purified DM-T2 enzyme; 6, purified DM-T1 enzyme. The values between the panels are molecular sizes in kilodaltons.

FIG 3

Effect of pH on the activities of pullulanase variants. Assays were carried at 60°C for 10 min in buffers with various pHs.

FIG 4

Effect of temperature on the activities and stabilities of pullulanase variants. (A) Optimal temperatures for pullulanase activity. Activity was measured in 50 mM sodium acetate buffer (pH 4.5) at 40 to 70°C for 10 min. (B) Thermostabilities of pullulanases. The activity of each recombinant enzyme prior to incubation at 60°C and pH 4.5 was defined as 100%. The error bars show the standard deviations of three replicates.

FIG 5

Glucose production by a combination of glucoamylase and pullulanase variants. Starch (30%) was incubated with 100 U of glucoamylase and 0.5 U of pullulanase at 60°C and pH 4.5. The amount of glucose generated (DX value) was measured by HPLC. The time course of saccharification was monitored at 60°C for 64 h. The error bars show the standard deviations of three replicates.