Thermostability improvement of a streptomyces xylanase by introducing proline and glutamic acid residues.
Protein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability of Streptomyces sp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/D185P/S186E), and the mutant and wild-type enzymes were expressed in Pichia pastoris. Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner β-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability.
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
Kun
K
Wang
Huiying
H
Luo
Jian
J
Tian
Ossi
O
Turunen
Huoqing
H
Huang
Pengjun
P
Shi
Huifang
H
Hua
Caihong
C
Wang
Shuanghe
S
Wang
Bin
B
Yao
D019943:Amino Acid Substitution; D002151:Calorimetry; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D018698:Glutamic Acid / Q000235:genetics*; D006358:Hot Temperature; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011392:Proline / Q000235:genetics*; D011487:Protein Conformation; D015202:Protein Engineering; D055550:Protein Stability; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D018698:Glutamic Acid; D011392:Proline; D014995:Xylosidases