Enhanced Production of Thermostable α-Amylase in Bacillus subtilis

Kholikov, Askar; Nazirov, Muhammad Latif; Vokhidov, Khushnut; Kachan, Alexandr; Evtushenkov, Anatoli N.; Makhsumkhanov, Akhmadzhan

doi:10.30491/jabr.2025.487018.1803

Enhanced Production of Thermostable α-Amylase in Bacillus subtilis

Document Type : Original Article

Authors

Askar Kholikov ¹

Muhammad Latif Nazirov ¹

Khushnut Vokhidov ¹

Alexandr Kachan ²

Anatoli N. Evtushenkov ²

Akhmadzhan Makhsumkhanov ¹

¹ Institute of Microbiology, Uzbekistan Academy of Sciences, Tashkent, Uzbekistan

² Department of Molecular Biology, Belarusian State University, Minsk, Belarus

10.30491/jabr.2025.487018.1803

Abstract

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 168 htr9 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 16S rRNA sequencing. The α-amylase activity was measured using the 3,5-dinitrosalicylic acid method. The B6-1-5-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, 104.K and amyR, displaying high amylolytic activity, were selected and identified as Bacillus licheniformis 104.K and Bacillus velezensis amyR, respectively. The crude α-amylases from both strains exhibited maximum activity at 90 °C and 70 °C, respectively. The α-amylase gene from B. licheniformis 104.K was cloned into a newly developed B6-1-5 shuttle vector. Notably, overexpression of this amylase gene in Bacillus subtilis 168 htr9 ehp241 led to a 1.14-fold and 373-fold increase in amylase activity compared to the B. subtilis 168 htr9 and B. licheniformis 104.K strain.
Conclusions: The B. licheniformis 104.K strain was selected for its thermostable α-amylase, and the gene was successfully cloned and expressed in Escherichia coli and B. subtilis 168 strains. These findings hold promise for enhancing the catalytic efficiency of recombinant α-amylase using computational biology methods and promoting B. subtilis 168 for large-scale production of recombinant enzymes.

Keywords

Thermostable α-amylase

Isolation

Cloning

Expression

Bacillus licheniformis

Bacillus subtilis