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

Publication date: April 2016 Source:Journal of Molecular Catalysis B: Enzymatic, Volume 126 Author(s): Jian Yang, Lizhen Li, Yunzhu Xiao, Jie Li, Lijuan Long, Fazuo Wang, Si Zhang 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 te...
Source: Journal of Molecular Catalysis B: Enzymatic - Category: Biochemistry Source Type: research