Catalytic efficiency and thermal stability promotion of the cassava linamarase with multiple mutations for better cyanogenic glycoside degradation.

In our previous study, we found that cassava cyanogenic glycosides had an acute health risk. Therefore, to solve this problem, the improvement of specific degradation of cyanogenic glycosides of cassava linamarase during processing is the key. In this study, the catalytic activity and thermal stability of enzymes were screened before investigating the degradation efficiency of cyanogenic glycosides with a cassava linamarase mutant K263P-T53F-S366R-V335C-F339C (CASmut) -controlled technique. The CASmut was obtained with the optimum temperature of 45 °C, which was improved by 10 °C. The specific activity of CASmut was 85.1 ± 4.6 U/mg, which was 2.02 times higher than that of the wild type. Molecular dynamics simulation analysis and flexible docking showed there were more hydrogen bonding interactions at the pocket, and the aliphatic glycoside of the linamarin was partially surrounded by hydrophobic residues. The optimum conditions of degradation reactions was screened with CASmut addition of 47 mg/L at 45 °C, pH 6.0. The CASmut combined with ultrasonication improved the degradation from 478.2 ± 10.4 mg/kg to 86.7 ± 7.4 mg/kg. Those results indicating the great potential of CASmut in applying in the cassava food or cyanogenic food. However, challenges in terms of the catalytic mechanism research is worthy of being noticed in further studies.