## 4 Discussion  
The main objective of this study is to introduce modifications in the 8S\(\alpha\) globulin of mung bean, leading to the expression and release of the hypocholesterolem peptide, IIAEK. Using protein engineering, specifically site-directed mutagenesis, base mutations were successfully introduced to the wild-type plasmid, producing a mutant plasmid capable of expressing the IIAEK bioactive peptide. In silico 3D modeling and prediction of the mutant 8S\(\alpha\) globulin indicated high homology (97.23%) and similarity (98.49%) with the wild-type template. Protein determination confirmed the expression of the mutant 8S\(\alpha\) globulin. Purification and digestion of protein further validated the expression and stability of the mutant 8S\(\alpha\) globulin. The identification of released IIAEK peptide was successfully done using LC-MS. Both Wt and Mut2 hydrolyses exhibited bile acid binding activity using the UPLC method. The binding capacity exhibited by the Wt digests still needs further proof to identify what factor contributes to this effect. [PERSON] et al. (2014) [40] reported that mung bean proteins exhibited hypocholesterolem activity, as evidenced by decreased plasma cholesterol and triacylglycerol in the experimental groups with mung bean supplementation. A recent study by [PERSON] et al. (2018) [41] found reduced plasma concentrations of total cholesterol and non-(high density lipoprotein)HDL \begin{table}
\begin{tabular}{l c c} \hline \hline
**Database/** & **Epitope Match** & **Amino Acid Sequence** & **Type** \\ \hline
**ADFS**[26] & & \\ _Bos taurus_ & \begin{tabular}{c} Bos d\_5.0101\(|\)EP00143\(|\)R00286.0 \\ associated with the reactive \\ patients’ group \\ \end{tabular} & \begin{tabular}{c} LQKWENDECAQKK \\ _IIAEKTK_ \\ \end{tabular} & Linear \\ (bovine milk) & \
\begin{tabular}{c} Bos d\_5.0101\(|\)EP00150\(|\)R00286.0 \\ detected as 3 contiguous spots in \\ peptide microarray \\ \end{tabular} &
\begin{tabular}{c} LCAQKK\(IIAEKTKIPAVFKD \\ ALNEN \\ AQKK\(IIAEKTKI\) \\ \end{tabular} & Linear \\
**Allerbase**[28] & & & \\ _Bos taurus_ & \
\begin{tabular}{c} Bos d 5.0101\(|\)AP00074\(|\)R00286.0 \\ (bovine milk) \\ \end{tabular} &
\begin{tabular}{c} AQKK\(IIAEKTKI\) \\ DQKWENDECAQKK \\ _IIAEKTK_ \\ \end{tabular} & Linear \\ \hline \hline \end{tabular}
\end{table} 
Table 8: Potential allergenicity analysis of the IIAEK peptide with existing IgE-binding epitopes. cholesterol, increased fecal cholesterol excretion, and reduced levels of asparagine aminotransferase and alanine aminotransferase enzymes in the liver, which suggested that mung beans exhibited significant hypocholesterolem activity. The identified binding capacity of the Mut2 hydrolysates was comparable to the \(\beta\)-lactoglobulin tryptic hydrolysate and casein tryptic hydrolysate which showed 47.4% and 35.1% binding capacity, respectively, as reported by [PERSON] et al., 2001 [PERSON] et al. (2001). Their study suggested that the inhibition of micellar solubility of cholesterol, which causes the suppression of cholesterol absorption by a direct interaction between cholesterol mixed micelles, and the \(\beta\)-lactoglobulin tryptic hydrolysate in the jejun epithelia is part of the underlying mechanism of the hypocholesterolem action of IIAEK. They speculated that IIAEK (lactostatin) may have influenced the reduction in cholesterol activity by increasing the amount of vesicular cholesterol rather than that of micellar cholesterol. Nonetheless, in a recent review by the same author [PERSON] et al. (2001), it was shown that the cholesterol-lowering peptide (lactostatin: IIAEK) operates via a new regulatory pathway in the calcium-channel-related mitogen-activated protein kinase (MAPK)-signaling pathway of cholesterol degradation. This, along with various in vitro and in vivo assays, points to the mechanism of IIAEK which involved the transactivation of the CYP7A1 gene (7\(\alpha\)-hydroxylase), which is the rate-limiting step of cholesterol degradation or bile acid synthesis.