Improving effect of a natural α-amylase inhibitor on the baking quality of wheat flour containing malted barley flour

Abstract

Lipid transfer proteins (LTPs) are soluble plant peptides endowed with biological activities applicable in agronomy, industry, and medicine. We assessed in this work the inhibition of human and intestinal insect α-amylases by rVu-LTP. The native Vu-LTP coding sequence was inserted into pET-32 EK/LIC and produced in Escherichia coli fused with Trx and His tags (rVu-LTP-Trx-His). rVu-LTP-Trx-His was purified by Ni+-chromatography and cleaved with enterokinase to remove the tags. rVu-LTP was purified on a C18 reversed-phase column. The natural Vu-LTP was purified by chromatography steps from seeds extract. rVu-LTP translation and folding were analysed by Western blotting with antibodies against Vu-LTP and circular dichroism by comparing the spectra of Vu-LTP and rVu-LTP. Human salivary and beetles Callosobruchus maculatus and Tribolium castaneum gut α-amylases were tested for inhibition by rVu-LTP. rVu-LTP was correctly produced, translated, and folded in bacterial cytoplasm. rVu-LTP inhibited 78.1% of human salivary amylase activity at 20 μM, at 40 μM inhibited 65.7% of intestinal α-amylases activity from C. maculatus and was unable to inhibit the T. castaneum gut α-amylases. The results presented by this work suggest biotechnological application of rVu-LTP in medicine to treat obese patients and in agriculture to improve grain quality protecting plants from insect attack.