Abstract
Backgroundβ-amylase (EC 3.2.1.2) is an exo-enzyme that shows high specificity for cleaving the α-1,4-glucosidic linkage of starch from the non-reducing end, thereby liberating maltose. In this study, we heterologously expressed and characterized a novel β-amylase from Bacillus aryabhattai.ResultsThe amino acid-sequence alignment showed that the enzyme shared the highest sequence identity with β-amylase from Bacillus flexus (80.73%) followed by Bacillus cereus (71.38%). Structural comparison revealed the existence of an additional starch-binding domain (SBD) at the C-terminus of B. aryabhattai β-amylase, which is notably different from plant β-amylases. The recombinant enzyme purified 4.7-fold to homogeneity, with a molecular weight of ~ 57.6 kDa and maximal activity at pH 6.5 and 50 °C. Notably, the enzyme exhibited the highest specific activity (3798.9 U/mg) among reported mesothermal microbial β-amylases and the highest specificity for soluble starch, followed by corn starch. Kinetic analysis showed that the Km and kcat values were 9.9 mg/mL and 116961.1 s− 1, respectively. The optimal reaction conditions to produce maltose from starch resulted in a maximal yield of 87.0%. Moreover, molecular docking suggested that B. aryabhattai β-amylase could efficiently recognize and hydrolyze maltotetraose substrate.ConclusionsThese results suggested that B. aryabhattai β-amylase could be a potential candidate for use in the industrial production of maltose from starch.
Highlights
Starch is well known for its easy availability, renewability, and low cost [1, 2], as well as its versatility as a biomaterial used in foods, textiles, pharmaceuticals, and adhesives, and as starting material for alcohol-based fuels [3]
After searching for putative β-amylases against the B. aryabhattai genome, we identified a hypothetical protein annotated as β-amylase (WP_033580731.1), with sequence alignment showing that the protein shared highest similarity (80.73%) with that of a well-characterized β-amylase from Bacillus flexus
In the presence of a variety of dextrins, Bacillus aryabhattai β-amylase (AmyBa) exhibited higher relative activity for those with a high Dextrose equivalent (DE) (10–15: 65.2%) and lower for relative activity for those with a lower DE (8–10: 47.8%). These results indicated that recombinant AmyBa showed efficient hydrolysis ability toward soluble starch, cornstarch and dextrin (DE 10–15), which was similar with previous studies evaluating β-amylases from B. flexus, B. polymyxa, barley, wheat and soybean [35, 39]
Summary
Starch is well known for its easy availability, renewability, and low cost [1, 2], as well as its versatility as a biomaterial used in foods, textiles, pharmaceuticals, and adhesives, and as starting material for alcohol-based fuels [3]. Available starches are obtained from various sources, including wheat and corn (cereals), potato (tubers), and cassava (root) [4]. Β-amylase is strongly associated with fruit development, ripening, seed germination, and abiotic stress response. Plants, such as sweet potato, soybean, and barley, are frequently used as resources of β-amylase production in industrial fields [6, 12]. Plant-sourced β-amylases have drawbacks, including their requirement for large amounts of grains, complex preparation processes, low storage stability, and high production cost, which restrict their further application. It is urgent to search for new β-amylase resources
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