Microalgae starch is receiving increasing attention as a renewable feedstock for biofuel production. Raw microalgae starch from Tetraselmis subcordiformis was proven to be very efficiently hydrolyzed by an α-amylase (AmyP) of glycoside hydrolase subfamily GH13_37 below the temperature of gelatinization (40 °C). The hydrolysis degree reached 74.4 ± 2.2% for 4% raw microalgae starch and 53.2 ± 1.7% for 8% raw microalgae starch after only 2 h. The hydrolysis efficiency was significantly stimulated by calcium ions. The enzyme catalysis of AmyP and its mutants (Q306A and E347A) suggested that calcium ions contributed to the hydrolysis of cyclic structures in raw microalgae starch by a distinctive calcium-binding site Ca2 of AmyP. The study explored raw microalgae starch as a new resource for cold enzymatic hydrolysis and extended our knowledge on the function of calcium in amylolytic enzyme.

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