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Protease is a kind of enzyme with different applications in medicine and industry. The limitation of the enzymes in industry is lack of tolerance for harsh conditions (high temperature, pH, and low water condition). This study intends to evaluate in silico study and screen the alkaline serine protease producing bacteria from honey and optimize the production of low water active alkaline serine protease. Plackett–Burman method was applied to improve effective factors on protease production, including pH, inoculum size, temperature, time, soybean protein, KH2PO4, MgSO4, CaCl2, NaCl, Glycerin and glucose concentration. Further, the Box–Behnken method was used to optimize the significant level of selected factors. Besides, in silico study was utilized to create the enzyme structure and investigate the stability in harsh conditions. The results showed that the highest protease production belongs to Bacillus sp. Khoz1 closed Bacillus safensis, Also temperature, glucose and soybean protein concentration were three significant factors for protease production and the optimized level for them were 35 °C, 0.5 g/l, and 38.32 g/l, respectively. The microorganism was able to produce protease until 98.36 U/ml and it was 98.68% similar to B. safensis. The stability of microbial alkaline serine protease was also determined in high pH and temperature conditions. The best stable condition for isolated protease was pH 9 and temperature at 50 °C. The in silico data showed that this protease has similar 3D structure to Bacillus subtilis Subtilisin E and highly charge amino acids on its surface were that caused this enzyme still activate and stable in low water condition. [ABSTRACT FROM AUTHOR]