Identification and thermoadaptation engineering of thermostability conferring residue of deep sea bacterial α-amylase AMY121

• Lys209 was identified as thermostability conferring residue for the first time.
• Systematic mutagenesis on Lys209 revealed its structural role.
• Thermostability of AMY121 improved 3.4 folds by semi-rational protein design.

Protein engineering on hotspot residues is acknowledged as an effective way to improve the stability and activity of enzymes. Searching for the key spot of a protein is a critically important but time-consuming process. In our previous study, an InDel site neighbouring residue Lys209 of the deep sea bacterial amylase AMY121 was proposed as one putative determinant for thermostability by homology sequence comparison and biochemical analysis [1]. Here, the structural stabilizing role of residue Lys209 was verified, and semi-rational protein design was applied to further improve capacity of higher temperature adaptation. Systematic mutagenesis study on Lys209 of amylase AMY121 revealed that all of the nineteen substitution mutants on the 209th site led to stability loss compared with wild type enzyme, indicating the crucial role of residue Lys209 in protein structural stabilizing. Four Lys209 neighboring sites were then selected and site directed saturation mutagenesis libraries were constructed for high through put thermostability screening. Two thermostability enhanced mutants Y187E and K205L were obtained from 1600 mutant colonies. Temperature properties comparison of wild type AMY121 and its variants showed Y187E and K205L possessed better kinetic thermal stability. The optimum temperature Y187E and K205L both increased by 5 °C. T5015 values of Y187E and K205L also increased to 0.63 and 8.52 °C, respectively. Structural basis for the difference in thermostability of wild type enzyme and its variants was further analyzed by computer modeling, and increasing in number of salt bridges and hydrophobic interactions around Lys209 was proposed as the main driving force for better compact protein structure of the enzyme.

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