Sequence optimization and heterologous expression of xanthine oxidase inhibitory peptides in Escherichia coli

Abstract

To solve the problem of low efficiency and purity in preparation of active peptides through enzymatic hydrolysis, the xanthine oxidase (XO) inhibitory peptides were optimized according to structure-activity relationship and a heterologous expression system for these peptides was constructed. The XO inhibitory peptide AEAWMWR (IC₅₀ = 1.76 mM), which exhibited enhanced activity, was obtained by optimizing AEAQMWR (IC₅₀ = 8.85 mM) in this research. The optimized peptide AEAWMWR exhibited approximately a 5-fold increase in activity compared to the template peptide AEAQMWR. The optimization results indicated that replacing the non-hydrophobic amino acids in the middle of the sequence with W or adding W to the C-terminal of the sequence effectively improved the activity of peptides. Additionally, to further achieve low-cost and rapid preparation of the peptides AEAQMWR and AEAWMWR, the recombinant plasmids containing fusion proteins of tandem repetitive peptides were designed and expressed in Escherichia coli. The recombinant peptides AEAQMWR (IC₅₀ = 8.19 mM) and AEAWMWR (IC₅₀ = 1.57 mM) exhibited significant activity. These results demonstrate that rational optimization and microbial synthesis of peptides can efficiently prepare bio-active peptides.