This study aimed to screen alpha-amylase producing microorganisms from honey as 
a low water activity medium, a suitable source for selecting stable and 
cost-beneficial bacterial enzyme production systems. Plackett-Burman method was 
used to select twelve effective factors including pH, inoculum size, 
temperature, time, corn starch, KH2PO4, peptone, MgSO4, CaCl2, NaCl, glycerin, 
and yeast extract concentrations on bacterial alpha-amylases production yield. 
The Box-Behnken method was utilized to optimize the level of selected 
significant factors. The stability of bacterial alpha-amylases was also 
determined in low pH and high-temperature conditions. In addition, in silico 
study was used to create the alpha-amylase structure and study the stability in 
high-temperature and low water available condition. Among all isolated and 
characterized microorganisms, Bacillus megaterium produced the highest amount of 
alpha-amylases. The in silico data showed the enzyme 3D structure similarity to 
alpha-amylase from Halothermothrix orenii and highly negative charge amino acids 
on its surface caused the enzyme activity and stability in low water conditions. 
Based on Box-Behnken results, the temperature 35 °C, pH 6 and starch 40 g/l were 
determined as the optimum level of significant factors to achieve the highest 
alpha-amylases unit (101.44 U/ml). This bacterial alpha-amylases enzyme showed 
stability at pH 5 and a range of temperatures from 40 to 60 °C that indicates 
this enzyme may possess the potential for using in industrial processes.