Development of a Triticale‐Based Amylolytic Biocatalyst for Starch Hydrolysis With Applications in Brewing Wort Sugar Enhancement

ABSTRACT This study aimed to develop a biocatalyst derived from triticale (X Triticosecale Wittmack) malt germinated for 5–8 days, by extracting α‐amylase, β‐amylase, and amyloglucosidase through aqueous methods, purifying them using aqueous two‐phase systems (ATPSs), and co‐immobilizing the enzymes on a gelatin support cross‐linked with CaCl 2 . After 7 days of germination, the enzyme extracts showed maximum activities of 549.6 CU/g for α‐amylase, 54.8 BU/g for β‐amylase, and 0.11 U/g for amyloglucosidase. The type 3 ATPS (30% ethanol/18% citrate) enabled recovery yields of 97% for α‐amylase and 68.6% for β‐amylase, with purification factors of 2.8 and 1.1, respectively. The purified enzymes exhibited optimal catalytic activity at 70°C and pH 5–6 (α‐amylase) and at 60°C and pH 6–7 (β‐amylase). Kinetic parameters indicated high substrate affinity ( K m < 200 mg/mL), with improved values after immobilization (α‐amylase: K m 118.2 mg/mL, V max 15.6 mg/min; β‐amylase: K m 101.9 mg/mL, V max 23.1 mg/min). The cross‐linked gelatin support demonstrated a water absorption capacity of 469% and a solubility of 61% after 24 h, with immobilization efficiencies exceeding 100%. In barley wort mashing trials, the immobilized enzyme consortium increased the release of reducing sugars by 11.8 g/L in the first cycle and maintained an additional 5.8 g/L after five reuse cycles. These findings demonstrate the potential of a robust, stable, and reusable biocatalyst for industrial applications in brewing processes and starch hydrolysis.