Abstract: Raw starch degrading enzymes are α-amylases with the ability to hydrolyze raw starch. It can degrade raw starch granules below the starch pasting temperature and suitable for cold hydrolysis of starch. The raw starch hydrolase AmyZ1 is derived from a marine microorganism and is not thermally stable. The mock structure of AmyZ1 was obtained by homology modeling. AmyZ1∶ΔTG is a mutant enzyme with deletion of the flexible region was constructed by sentinel mutation. The catalytic property, Ca2+ dependence, and ability to hydrolyze high concentration raw maize starch of the mutant were evaluated. The optimum temperature of AmyZ1∶ΔTG was 45 ℃, and the enzyme activity was maintained above 60% in the range of 35 ℃-55 ℃. The thermal stability of AmyZ1∶ΔTG was improved and the half-lives of 30 ℃ and 35 ℃ reached 18 h and 15 h, which were 10 and 15 times of AmyZ1, respectively. The decreased flexibility of the protein resulted in a decrease in the catalytic efficiency of AmyZ1∶ΔTG. Exogenous Ca2+ has less effect on the catalytic performance of AmyZ1∶ΔTG, but it can improve the thermal stability of AmyZ1∶ΔTG, compared to that of AmyZ1. Using 300 g/L corn raw starch as the substrate, the hydrolysis rate of AmyZ1∶ΔTG was 40%, which was similar to AmyZ1.

Key words: raw starch α-amylase, thermal stability, Ca2+-dependence