Improved thermostability of Thermobifida fusca mannanase obtained by error-prone PCR mutagenesis

A thermostable β-Mannanase (ManB, PDB code 1BQC, gene code KJ806638) from Thermobifida fusca was expressed in Escherichia coli BL21. The error-prone PCR of ManB gene were adopted to obtain three special mutants with the sites of N15I (M1), D40G/T159I (M2), and H28Q/V57D (M3). It was found that the optimum temperature of wild type ManB was 72.5 °C, whereas of two mutants M1 and M2 were 77.5 °C and mutant M3 was 70 °C. The apparent melting temperatures (T_m) measured by differential scanning fluorimetry (DSF). The T_m values of ManB was 61.9 °C, as well the mutants M1, M2 and M3 were 63.2 °C, 63.4 °C and 48.7 °C, respectively. With guar gum as the substrate, the K_m of the original ManB and the mutants M1, M2 and M3 were 0.74, 0.79, 1.28, 0.70 mg/ml, as well the specific activity of them were 1293, 1344, 1728, 446 IU/mg, respectively. Stick models and molecular dynamics analysis show that structural stabilization, particularly in the active cleft region, improves the thermostability of the enzyme. Those specific mutated sites of N15I (M1), D40G/T159I (M2), and H28Q/V57D (M3) are novel and provide the additional structure understanding of mannanase 1BQC, as well the GROMACS module demonstrated the molecular structure modification of mannanase in the active cleft region effect the stability significantly.