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Background: Multi-drug resistant bacteria hazards to the health of humans could be an agent in the destruction of human generation. Natural products of Bacillus species are the main source to access progressive antibiotics that can be a good candidate for the discovery of novel antibiotics. Wild honey as a valuable food has been used in medicine with antimicrobial effects., Objective: Bacillus strains isolated from wild honey were evaluated for the potential antimicrobial activity against human and plant bacterial and fungal pathogens., Methods: Three bacterial isolates were identified as strain Khuz-1 (98.27% similarity with Bacillus safensis subsp. Safensis strain FO-36b T ), strain Khuz-2 (99.18% similarity with Bacillus rugosus strain SPB7 T ), and strain Khuz-3 (99.78% similarity with Bacillus velezensis strain CR-502  T ) by 16S rRNA gene sequences. The strains were characterized by their ability to inhibit the growth of human and phytopathogenic fungi., Results: The results indicated that B. rugosus strain Khuz-2 inhibited the growth of phytopathogenic and human fungal more effective than other ones. It seems that the strain Khuz-2 has a suitable antimicrobial and antifungal potential as a good candidate for further pharmaceutical research., Conclusion: Based on the results of GC-MS, Pyrrolo [1,2-a] pyrazine-1,4-dion, hexahydro-3-(2-methylpropyle) (PPDHM) was the major compound for all strains which have a various pharmacological effect. Isolation and identification of beneficial bacteria from natural sources can play an important role in future pharmaceutical and industrial applications., (© 2022. The Author(s).)

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, KH 2 PO 4 , peptone, MgSO 4 , CaCl 2 , 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.