Figure 1.
Congo red plate assay for evaluating protein stability at elevated temperature.
E. coli BL21 transformants (1×105 CFU), harboring pTrc99A, pTCel5A, pTCel5A_1R4, pTCel5A_2R1, and pTCel5A_2R2, were spotted onto LB-ampicillin agar plates, cultivated at 37°C for 6 h, and then induced with 0.5 mM IPTG for 2 h. The plates were overlaid with 0.5% CMC, and incubated at 65°C for 30 min. Finally, plates were stained with Congo red and destained with 1 M sodium chloride.
Figure 2.
In vitro thermotolerance assay for wild-type Cel5A and its mutants.
Enzyme was incubated at 65°C for 20 min and residual activity was determined using CMC as a substrate at optimum reaction conditions. Residual enzyme activity (%) = (Enzyme activity (U/mL) at t = 20 min/Enzyme activity (U/mL) at t = 0 min)×100. The error bars represent the standard deviation of triplicate measurements.
Table 1.
Mutations obtained after random mutagenesis.
Figure 3.
SDS-PAGE profile for expression and purification of Cel5A_2R2-CBM6 fusion protein.
Lanes are as follows: W, whole cell proteins; S, soluble proteins; P, cell pellet proteins; HP, His-tag purified protein; and M, molecular weight marker.
Figure 4.
Optimum temperature and pH for Cel5A_2R2 and Cel5A_2R2-CBM6 fusion protein.
Symbols are as follows: Cel5A_2R2 (Open circles) and Cel5A_2R2-CBM6 (Closed circles). The error bars represent the standard deviation of triplicate measurements.
Table 2.
Specific enzyme activity of Cel5A_2R2 and Cel5A_2R2-CBM6 on various soluble and insoluble cellulosic substrates.
Figure 5.
TLC analysis of hydrolysis products of cellotriose, cellotetraose, cellopentaose, cellohexaose, CMC, PASC, filter paper, Avicel, and p-NPC.
A: Hydrolysis (1 h) products of cellotriose and cellotetraose, B: Hydrolysis (1 h) products of cellopentaose and cellohexaose, C: Hydrolysis (5 h) products of CMC and PASC, D: Hydrolysis (16 h) products of filter paper and Avicel, and E: Hydrolysis (1 h) product of p-NPC. M: Standard marker, where G1 to G6 represent glucose, cellobiose, cellotriose, cellotetraose, cellopentasoe, and cellohexaose. Cello-oligosaccharides, CMC, PASC, and p-NPC were treated with 0.1 nmol of Cel5A_2R2-CBM6 at 55°C. The same reaction was performed using Avicel and filter paper with 1.0 nmol of Cel5A_2R2-CBM6. Reactions were performed in the absence (−) and presence (+) of the enzyme.
Figure 6.
Cel5A mutations covered in this work mapped onto the model of the Cel5A catalytic domain.
Cellobiose at the reaction cavity is displayed as ball-and-sticks (carbon in yellow and oxygen in red). Mutated residues in each mutant are shown as sticks in different colors: D45G in orange from 1R1, V108G, and L240Q in green from 1R2, D275G in cyan from 1R3, N252D in hot pink from 1R4, D40E in purple-blue from 1R5, T195A in magenta from 2R1, F90L in blue from 2R2, the common mutation V256A in red.
Figure 7.
Synergistic interaction of cellobiohydrolase (CbhA) from C. thermocellum with Cel5A_2R2 parent protein and Cel5A_2R2-CBM6 fusion protein.
The error bars represent the standard deviation of triplicate measurements.