## 3 Results
### Binding Ability of the Digested Peptides to Angiotensin-Converting Enzyme  
The released oligopeptides (2 \(\leq\) peptide length \(\leq\) 5) by in silico digestion of 16 proteins were docked to angiotensin-converting enzyme. Table 1 presents the high-affinity oligopeptides with a binding score of \(<\)\(-\)110. The results indicated that mussel-derived NADH dehydrogenase possessed the largest F value (0.0721), followed by razor-clam-derived cytochrome b, NADH dehydrogenase, and ATP synthase, as well as mussel-derived AMP-activated protein kinase, which also had the largest F values (>0.06). There were 18 top high-affinity oligopeptides with a binding score of \(<\)\(-\)180 (Table 2). Subsequently, seven peptides (IQPEW, PICQF, VQPEW, SCCGY, SSGAW, VICGH, and ISGTF) were synthesized, and their ACE inhibition activities are presented in Figure 2. The data indicated that SCCGY, PICQF, and SSGAW had strong inhibition activities, with inhibition percentages of 96.04%, 89.755, and 82.72%, respectively, at the concentration of 2 mM. The IC50 values of SCCGY, SSGAW, IQPEW, PICQF, and VQPEW were determined as 0.09883, 0.2529, 0.6098, 0.4131, and 1.189 mM, respectively. Figure 1: In silico hydrolysis of marine shellfish proteins. Sixteen proteins from four species of marine shellfish (razor claims (**A**), claims (**B**), scallops (**C**), and mussels (**D**)) were selected, their amino acids sequences were retrieved from the NCBI database ([[https://www.ncbi.nlm.nih.gov/protein/](https://www.ncbi.nlm.nih.gov/protein/)]([https://www.ncbi.nlm.nih.gov/protein/](https://www.ncbi.nlm.nih.gov/protein/)), accessed on 5 March 2022), and their in silico hydrolysis by gastrointestinal enzymes (pepsin, trypsin, and chymotrypsin) (no order) was performed using the BIOPEF-UWM database ([[https://biochemia.uwm.edu.pl/biopep-uwm](https://biochemia.uwm.edu.pl/biopep-uwm)]([https://biochemia.uwm.edu.pl/biopep-uwm](https://biochemia.uwm.edu.pl/biopep-uwm)), accessed on 20 March 2022). The interactions of SCCGY and PICQF with the target protein ACE are shown in Figures 3 and 4. For SCCGY and ACE (Figure 3A), seven pairs of hydrogen bonds were formed: Cys 2 (peptide) vs. Cys 3 (peptide), Gly 4 (peptide) vs. Ala354 (ACE), Gly 4 (peptide) vs. His353 (ACE), Gly 4 (peptide) vs. His513 (ACE), Gly 4 (peptide) vs. Tyr5 (peptide), Gly 4 (peptide) vs. Gln281 (ACE), and Gly 4 (peptide) vs. Asp415 (ACE). There were eight residues involved in hydrophobic contacts: His387, Phe512, Ser355, Ala356, His410, Val518, Phe457, and Phe 527 (Figure 3B). For PICQF and ACE (Figure 4A), three pairs of hydrogen bonds existed: Cys 3 (peptide) vs. Ala 356 (enzyme), Gln 4 (peptide) vs. \begin{table}
\begin{tabular}{l l c c} \hline \hline
**Peptides** & **Sources** & **Protein** & **Docking Score** \\ \hline IQPEW & Razor clam, Clam & Cytochrome b & \(-\)201.7 \\ PICQF & Razor clam & Cytochrome b & \(-\)206 \\ SPVIF & Razor clam & Cytochrome b & \(-\)190.5 \\ VPVDF & Razor clam & NADH dehydrogenase & \(-\)189.4 \\ SCCGY & Razor clam & ATP synthase & \(-\)187.7 \\ SIQGF & Razor clam & ATP synthase & \(-\)186 \\ SSGAW & Clam & Cytochrome b & \(-\)184.5 \\ DIAIW & Scallop & Cytochrome b & \(-\)180.6 \\ CQCVF & Scallop & Cytochrome b & \(-\)187.5 \\ SVGVY & Scallop & NADH dehydrogenase & \(-\)180.2 \\ VICGH & Scallop & ATP synthase & \(-\)181.9 \\ VIDVW & Scallop & Superoxide dismutase & \(-\)192.7 \\ PTVF & Scallop & AMP-activated protein kinase & \(-\)180 \\ ISGTF & Scallop & AMP-activated protein kinase & \(-\)182.3 \\ SVGPW & Mussel & Cytochrome b & \(-\)192.2 \\ ICTY & Mussel & Cytochrome b & \(-\)180.2 \\ VQPEW & Mussel & Cytochrome b & \(-\)199.6 \\ IVPTR & Mussel & NADH dehydrogenase & \(-\)181.7 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Top high-affinity oligopeptides. Figure 2: Inhibitory activities of the synthesized peptides on angiotensin-converting enzyme (**A**) and IC50 values (mM) (**B**).