Production and application of α-amylase, native AmyJ33-ABC, from Bacillus siamensis JJC33M in gelatinized potato starch and its industrial potential.

Abstract

α-Amylases (EC 3.2.1.1) are endoenzymes that hydrolyze α-1,4-glycosidic bonds in starch to produce maltooligosaccharides with broad industrial applications (food, textile, fermentation, biofuels). Most α-amylases act only on gelatinized starch, but Bacillus siamensis JJC33M secretes a native enzyme (AmyJ33-ABC) active on both gelatinized and raw starch. The growth of B. siamensis JJC33M was evaluated, showing µ = 0.55 h⁻¹, Y_p/s = 0.13 g/g, Y_x/s = 0.24 g/g, Y_p/x = 0.55 g/g, and Q_p = 0.063 g/Lh, values comparable with other native production systems. AmyJ33-ABC was partially purified and characterized. The enzyme displayed optimal activity at pH 5.0 and 80 °C, with K_m = 1.47 mg/mL, V_max = 39.37 U/mg, and catalytic efficiency K_cat/K_m = 22.31 s⁻¹ mg⁻¹ mL, comparable with another native systems. At optimal conditions, it hydrolyzed 57.5% of gelatinized potato starch, generating glucose, maltose, maltotriose, maltotetraose, and minor maltooligosaccharides up to DP7. Structural modeling confirmed the canonical GH13 fold (A/B and C domains) and revealed three aromatic-rich surface-binding sites (SBS) located near the catalytic triad. These SBS may explain the enzyme activity on raw starch despite lacking a carbohydrate-binding module (CBM). AmyJ33-ABC combines dual activity on gelatinized and raw starch, acidic pH preference, and high-temperature optimum. These distinctive features highlight its potential for starch bioconversion in bakery, syrup, and related industries.