Purification and Some Properties of α-Amylase Inhibitor from Avena Nuda
|Course||Biochemistry and Molecular Biology|
|Keywords||α- amylase inhibitor Purification Trypsin inhibitor Antifungal activity Chitinase|
a-amylase inhibitor (a-amylase inhibitor, a-AI) are glycoside hydrolase inhibitors, mainly in the endosperm of seeds.The content ofα-amylase inhibitor is positive correlation with the content of starch.a-AI are mainly from plants, microorganisms and artificial synthesis.α-amylase inhibitors from natural plant resources become a focus of research, with its advantages of wide material sources, minimal side-effect and environmental friendly.In this paper, a-amylase inhibitor was isolated by acetone derosination,30%-60% ammonium sulfate precipitation, Q-Sepharose fast flow column chromatography, Heparin-Sepharose fast flow column chromatography and Sephacryl-200 column chromatography from cold-resistant crops Avena nuda.With above processes, the overall recovery of enyzmatic activity was 1.23% and the specific activity achieved 26.43 IU/mg with more than 95% purity as estimated by SDS-PAGE analysis.The a-amylase inhibitor is a monomer with a molecular mass 23.76 kDa as determined by both Superosel2 size-exclusion chromatography and SDS-PAGE. The sulfhydryl groups of a-amylase inhibitor were determined by Ellman method, revealing that the a-amylase inhibitor contains 15 sulfhydryl groups, which formed about four pairs of disulfide bonds.Theα-amylase inhibitor not only inhibits the activity of a-amylase, but also inhibits the activity of trypsin, additionaly, the inhibitors also have anti-fungal activity (chitinase activity). Theα-amylase inhibitor was stable at pH 6.0-10.0 with its optimum pH at 8.0.The optimum temperature of the a-amylase inhibitor is at 40℃, It has a good thermal stability,when incubated in 100℃for half an hour, it still retain 50% of the inhibitory activity.Trypsin inhibitor activity of the a-amylase inhibitor was evaluated.For 95% puritya-amylase inhibitor, it has 16.13IU/mg for trypsin inhibitor activity.The trypsin inhibitor activity was less affected by temperature from 25℃to 55℃,while incubated in 65℃for half an hour, it retain 50% relative activity,The optimum temperature and pH for trypsin inhibitor activity is 45℃,pH8.0. The anti-fungal activity of a-amylase inhibitor was tested with Trametessp.SQ01, Chaetomiaceae, and Monascuspurpureus.The result was indicated that the most obvious inhibition phenomena is for Trametessp.SQ01,with the minimum inhibitory concentration 30μg/mL. Chitinase activity of the a-amylase inhibitor was also evaluated.The optimum temperature and pH of chitinase activity is at 45℃and 5.0. and the chitinase activity was stable at pH2.0-8.0 and below 55℃.1mM Mn2+ can significantly increase chitinase activity.