Pepsin assay kit

Activity of the recombinant API protein in inhibiting protease (pepsin) was tested as described previously [25 (link)]. Fifty micrograms of recombinant API and the same weight of pepsin (Ameresco, Kaysville, USA) were dissolved in 10 mM acetate (pH 2.0) and incubated for 30 min at 37 °C, the residual enzyme activity of pepsin was determined by Pepsin assay kit (Nanjing Jiancheng Bio, Nanjing, China) according to the manufacturer’s instructions. Breifly, 150 μg of hemoglobin (Nanjing Jiancheng Bio, Nanjing, China) dissolved in the same buffer, was added to the above reaction and incubated for another 30 min at 37 °C and the resulting products were analyzed by 12% SDS-PAGE. The inhibition efficiencies for API in different reactive pH (2–10) and temperature (37–100 °C) were also checked. Trypsin (Ameresco, Kaysville, USA) was chosen as a control in this study. Each experiment was run in triplicate.

In this study, the enzymatic activities of pepsin, lipase, and α-amylase in the intestine and stomach were detected using a Pepsin Assay Kit, a Lipase Assay Kit, and an α-Amylase Assay Kit (Nanjing Jiancheng, Bioengineering Institute, China). The enzymatic activities of ACP, AKP, LDH, SOD, CAT, GPT, and GOT in the gill, brain, intestine, stomach, kidney, liver, and plasma were detected using an Acid Phosphatase Assay Kit, an Alkaline Phosphatase Assay Kit, a Lactate Dehydrogenase Assay Kit, a Superoxide Dismutase Assay Kit, a Catalase (CAT) Assay Kit, an Alanine Aminotransferase Assay Kit, and an Aspartate Aminotransferase Assay Kit (Nanjing Jiancheng Bioengineering Institute, China). Total protein was determined with a Total Protein Assay Kit (Nanjing Jiancheng Bioengineering Institute, China). The operation steps were carried out according to the operation guide of the reagent kit, and the operation guide of the corresponding reagent kit can be searched for on the website (http://www.njjcbio.com/, accessed on 8 June 2023). In addition, the calculation formulas for these enzyme activities are detailed in the Supplemental Material.

In this study, the enzymatic activities of pepsin, α-amylase and lipase (LPS) in liver and intestine were assessed using the Pepsin Assay Kit, α-Amylase Assay Kit and Lipase Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Total protein (TP) was determined with the Total Protein Assay kit (Nanjing Jiancheng Bioengineering Institute, China). The enzymatic activities of Acid phosphatase (ACP), alkaline phosphatase (AKP), Peroxidase (POD), Superoxide Dismutase (SOD), Lysozyme (LZM), Glutathione peroxidase (GSH-Px), Glutathione reductase (GR) and Malondialdehyde (MDA) in the skin, gill, muscle, liver, and intestine were assessed using the Acid Phosphatase Assay Kit, Alkaline Phosphatase Assay Kit, Peroxidase Assay Kit, Superoxide Dismutase Assay Kit, Lysozyme Assay Kit, Glutathione Peroxidase Assay Kit, Glutathione reductase Assay Kit and Malondialdehyde Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The assays all were carried out according to the manufacturer’s protocols. In addition, the calculation formulas of these enzyme activities are detailed in the Supplementary Materials.