His-Tag Effect on Biochemical Properties of B. Subtilis US572 α-Amylase Produced in E. coli: Application of the Recombinant Enzyme in Breadmaking

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.

KEYWORDS:

• α-amylase
• heterologous expression
• C-terminal his-tag
• biochemical characterization
• molecular modeling
• breadmaking

Acknowledgments

We are grateful to the RANDA Society of Tunisia (www.Randa.tn) for flour samples. Thanks to Eng. Besma AYADI and Randa’s lab personnel for their help in the AmyKS application in breadmaking.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was supported by the Tunisian Ministry of Higher Education and Scientific Research under the contract program LMBEE-CBS/code: LR15CBS06-2015-2018 and the “National Agency for the Promotion of Scientific Research (ANPR)” for the MOBIDOC scholarships 2017-2019.