Abstract
ABSTRACT The gene encoding Bacillus subtilis α-amylase was cloned into pET-21a (+). The expression level of the recombinant enzyme was 10.7-fold higher than the expression level of the native one (0.13 mg mL−1). The recombinant enzyme (His6-rAmyKS) was purified in one step using Ni-NTA column affinity with a specific activity of 664.28 U.mg−1. The biochemical properties of the His6-rAmyKS were determined and compared to those of the non-tagged enzyme. Interestingly, differences were found between the two enzymes mainly for the optimal temperature and pH. Experimental tests and molecular modeling confirmed that the extra residues (C-terminal His-tag fusion peptide and cleavage thrombin site) could be responsible for the slight increase in total activity and the improvement of biochemical properties of the His-tagged enzyme compared to the native one. The His6-rAmyKS was used as an additive in breadmaking. It showed a significant effect in improving the dough texture and the bread quality.
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