SDS-PAGE analysis of Recombinant <i>Tm</i>Cel12B.

<p>Lane M, Protein marker; Lane 1, Supernatant after ultrosonication; Lane 2, suspensions after ammonium sulfate precipitation; Lane 3, treated by heat at 75°C for 15 min; Lane 4, purified enzyme after purification kit.</p

Site-Directed Mutagenesis of a Hyperthermophilic Endoglucanase Cel12B from <i>Thermotoga maritima</i> Based on Rational Design

<div><p>To meet the demand for the application of high activity and thermostable cellulases in the production of new-generation bioethanol from nongrain-cellulose sources, a hyperthermostable β-1,4-endoglucase Cel12B from <i>Thermotoga maritima</i> was selected for further modification by gene site-directed mutagenesis method in the present study, based on homology modeling and rational design. As a result, two recombinant enzymes showed significant improvement in enzyme activity by 77% and 87%, respectively, higher than the parental enzyme <i>Tm</i>Cel12B. Furthermore, the two mutants could retain 80% and 90.5% of their initial activity after incubation at 80°C for 8 h, while only 45% for 5 h to <i>Tm</i>Cel12B. The <i>K</i>_m and <i>V</i>_max of the two recombinant enzymes were 1.97±0.05 mM, 4.23±0.15 μmol·mg^-1·min^-1 of <i>Tm</i>Cel12B-E225H-K207G-D37V, and 2.97±0.12 mM, 3.15±0.21 μmol·mg^-1·min^-1 of <i>Tm</i>Cel12B-E225H-K207G, respectively, when using CMC-Na as the substrate. The roles of the mutation sites were also analyzed and evaluated in terms of electron density, hydrophobicity of the modeled protein structures. The recombinant enzymes may be used in the hydrolysis of cellulose at higher temperature in the future. It was concluded that the gene mutagenesis approach of a certain active residues may effectively improve the performance of cellulases for the industrial applications and contribute to the study the thermostable mechanism of thermophilic enzymes.</p></div

Structures of site-directed amino acid residues with other vicinal residues by H-bond.

<p>A, B: Glutamic acid 225 was mutated into Histidine. C, D: Aspartic acid 37 was mutated into Valine. E, F: Lysine 207 was mutated into Glutamic acid.</p

Homology model of <i>Tm</i>Cel12B.

<p>A: The secondary structural elements and loops are spectrum-colored according to their positions in the amino acid sequence. The 14 β-strands are pack against each other into two anti-parallel sheets. B: Active site Glu131 and Glu229 was colored green.</p

Conserved amino acid residues of thermostable endoglucanase.

<p>The conserved amino acid residues with green color are mostly located in the active cleft. A: front view. B: side view.</p

Structure-based sequence of <i>Tm</i>Cel12B and other amino acid sequences with known protein crystal structures.

<p>The secondary structure elements of the <i>Tm</i>Cel12B proteins homology modelled are drawn at the top of the alignment. The aligned protein sequences with their PDB access codes indicated in parentheses.</p