Protein Engineering of Bacillus α-Amylase to Improve Thermostability and Low Water Activity: A bioinformatics approach

Background: α-Amylase produced by Bacillus licheniformis (BLA) is widely used in starch industries.Water is so crucial for α-Amylase activity; due to the low amount of available water in the starch industry, itcan lead to denaturation of the enzyme. In this study, we attempt to reduce BLA aggregation and improvelow water activity by protein engineering without reducing its activity by computational methods.Material and Methods: As surface residues are responsible in aggregation and water activity, the surfaceamino acids were defined by PyMol software (PDB ID: ۱VJS). Aggrescan webserver was used to definehotspot aggregation residues. The surface amino acids of the α-Amylase which did not locate in the activesite of the enzyme and were hotspot aggregation residues, replaced with glutamic and aspartic acid asnegative amino acids by Chimera software. The stability and solubility of engineered α-Amylase comparedto native form were done by Protparam and Protein-sol, respectively. To analyze secondary protein Psipredwebserver was applied. Moreover, molecular dynamic simulation was done by Gromacs package to assessthe stability of the tertiary structure of mutant form compared to native BLA during the time.Result and Discussion: According to the active site analysis of BLA, surface Amino acid Q۳۴۰E, S۳۵۶D,G۴۷۴E, L۳۱۸E, and S۳۱۰E have neutral charges and are recognized as hot spots of aggregation that waschanged to negative charge amino acids. Physicochemical properties analysis, protein solubility, andaggregation potential discovered that these properties of engineered enzyme improved from engineeredmutant compared to native form. Also, secondary structure evaluation indicates that these mutations causedno change in protein structure which was proved by molecular dynamics simulation during time. So, thesemutations could reduce BLA aggregation and improve low water activity although experimental analysisneeds to prove it.

کلیدواژه ها:

α-Amylase ، protein engineering ، low water activity ، In Silico