Recombinant chimeric α-amylase (AmyP-Cr) was constructed by a catalytic core of α-amylase (AmyP) from a marine metagenomic library and a starch-binding domain (SBD) of α-amylase from Cryptococcus sp. S-2. The molecular fusion did not alter optimum pH, optimum temperature, hydrolysis products, and an ability of preferential and rapid degradation towards raw rice starch, but catalytic efficiency and thermostability were remarkably improved compared with those of the wild-type AmyP. AmyP-Cr achieved the final hydrolysis degree of 61.7 ± 1.2% for 10% raw rice starch and 47.3 ± 0.8% for 15% raw rice starch after 4 h at 40 °C with 1.0 U per mg of raw starch. The catalytic efficiency was very high, with 3.6-4.0 times higher than that of AmyP. The enhanced catalytic efficiency was attributed to the better thermostability and the higher adsorption and disruption to raw rice starch caused by SBD. The properties of AmyP-Cr open a new way in terms of a new design of raw rice starch processing.

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.