Enhanced Production of Thermostable α-Amylase in Bacillus subtilis

Introduction: The increasing industrial demand for thermostable amylases necessitates the exploration of new sources, especially from understudied regions like Uzbekistan, which may offer novel, reliable, and stable enzyme characteristics. The objective of this study was to screen, identify, and characterize bacterial species producing thermostable α-amylase, clone its corresponding gene, and test B. subtilis ۱۶۸ htr۹ mutant hosts for recombinant enzyme overproduction.Materials and Methods: Bacterial isolates with amylase-producing capabilities were selected using a plate assay, and their taxonomy was confirmed through ۱۶S rRNA sequencing. The α-amylase activity was measured using the ۳,۵-dinitrosalicylic acid method. The B۶-۱-۵-amyL vector was specifically engineered to clone and express the α-amylase gene in expression hosts.Results: This study presents the first isolation and characterization of bacterial strains from the soils of Uzbekistan that produce thermostable α-amylase. Following a screening process, two isolates, ۱۰۴.K and amyR, displaying high amylolytic activity, were selected and identified as Bacillus licheniformis ۱۰۴.K and Bacillus velezensis amyR, respectively. The crude α-amylases from both strains exhibited maximum activity at ۹۰ °C and ۷۰ °C, respectively. The α-amylase gene from B. licheniformis ۱۰۴.K was cloned into a newly developed B۶-۱-۵ shuttle vector. Notably, overexpression of this amylase gene in Bacillus subtilis ۱۶۸ htr۹ ehp۲۴۱ led to a ۱.۱۴-fold and ۳۷۳-fold increase in amylase activity compared to the B. subtilis ۱۶۸ htr۹ and B. licheniformis ۱۰۴.K strain.Conclusions: The B. licheniformis ۱۰۴.K strain was selected for its thermostable α-amylase, and the gene was successfully cloned and expressed in Escherichia coli and B. subtilis ۱۶۸ strains. These findings hold promise for enhancing the catalytic efficiency of recombinant α-amylase using computational biology methods and promoting B. subtilis ۱۶۸ for large-scale production of recombinant enzymes.

کلیدواژه ها:

Thermostable α-amylase ، isolation ، Cloning ، Expression ، Bacillus licheniformis ، Bacillus subtilis