| Summary: | 碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 94 === Vigna radiata defensin 1 (VrD1) is a small, basic and cysteine-rich peptide of 46 amino acids. In former study, VrD1 was reported to exhibit insecticidal activity, and three dimensional structure of VrD1 have been determined by nuclear magnetic resonance (NMR) spectroscopy. However, the insecticidal mechanism of VrD1 is still indistinct. Our preliminary data showed that VrD1, which was purified from mung bean, inhibited Tenebrio molitor α-amylase. To elucidate the α-amylase inhibition mechanism of VrD1, recombinant VrD1 was constructed, expressed and purified from Escherichia coli. According to amino acid sequence analysis and protein structure comparison, specific residues involved in α-amylase inhibition were identified by site-directed mutagenesis. Eleven mutants were totally obtained and analyzed by circular dichroism (CD) for secondary structure and α-amylase activity assay for the inhibition function. The CD spectra showed that all recombinant VrD1 proteins have similar secondary structures. The results of α-amylase inhibition assay show that three mutants, K6A, R26E and R38A, significantly decrease in a-amylase inhibition. These three residues may play important roles in inhibitory function in VrD1.
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