Chinese mitten crab Eriocheir sinensis is an important economic species, but it largely remains unknown for the genome-wide identification of digestive enzyme genes, gene expression pattern and their activities during the molting cycle of E. sinensis. Therefore, this study was conducted to investigate the diversity of major digestive enzyme genes, their dynamic expression patterns as well as the digestive enzyme activities during the molting cycle; the binding sites and affinities of major digestive enzyme-substrate were also predicted using molecular docking analysis. The results revealed that there were 4 α-amylase genes, 14 trypsin genes, and 7 lipase genes identified in E. sinensis. Among them, Es-Amylase4, Es-Trypsin11-I7, and Es-Lipase1 exhibited the highest transcriptional expression levels respectively, with Es-Amylase4 and Es-Lipase1 peaking in stage C and Es-Trypsin11-I7 in stage D. The α-amylase and lipase activities peak in stage C, while trypsin activity was highest in stage A/B. During molting process, the transcriptional expression levels of 4 α-amylase isoforms and 3 lipase isoforms positively correlated with enzyme activity changes, and the expression of Es-lipase1-mRNA was significantly higher than the other lipase transcripts (P < 0.05). As lipase is one of most important digestive enzymes during food digestion and there are two transcription isoforms with significantly higher expression levels than the others. There were 1 and 3 hydrogen bonds for Es-lipase1- triglyceride and Es-lipase7- triglyceride complexes, respectively, while the binding affinities of Es-lipase1- triglyceride and Es-lipase7- triglyceride were − 8.0 kcal/mol and − 7.1 kcal/mol, respectively. In conclusion, crustaceans have numerous digestive enzymes with a high degree of polymorphism, but there are 3, 2, and 1 highly expressed gene in E. sinensis contributed to the major enzyme activities of α-amylase, trypsin, and lipase, respectively. For α-amylase and lipase, both gene expression and their enzyme activities peaked at stage C, which was highly related to food intake and specific molting stage.
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