Rice Bifunctional α-Amylase/Subtilisin Inhibitor: Cloning and Characterization of the Recombinant Inhibitor Expressed inEscherichia coli

Abstract

The complete nucleotide sequences of the cDNA and its gene that encode a bifunctional alpha-amylase/subtilisin inhibitor of rice (Oryza sativa L.) (RASI) were analyzed. RASI cDNA (939 bp) encoded a 200-residue polypeptide with a molecular mass of 21,417 Da, including a signal peptide of 22 amino acids. Sequence comparison and phylogenetic analysis showed that RASI is closely related to alpha-amylase/subtilisin inhibitors from barley and wheat. RASI was found to be expressed only in seeds, suggesting that it has a seed-specific function. A coding region of RASI cDNA without the signal peptide was introduced into Escherichia coli and was expressed as a His-tagged protein. Recombinant RASI was purified to homogeneity in a single step by Ni-chelating affinity column chromatography and characterized to elucidate the target enzyme. The recombinant inhibitor had strong inhibitory activity toward subtilisin, with an equimolar relationship, comparable with that of native RASI, and weak inhibitory activity toward some microbial alpha-amylases, but not toward animal or insect alpha-amylases. These results suggest that RASI might function in the defense of the seed against microorganisms.

Molecular cloning and characterization of a novel bi-functional α-amylase/subtilisin inhibitor from Hevea brasiliensis

“…Svendsen et al (1986) have cloned and expressed a 19.8 kDa barley protein (BASI) that inhibits α-amylase and the protease subtilisin. Yamasaki et al (2006) have cloned a 21.4 kDa of bi-functional α-amylase/subtilisin inhibitor from rice (RASI). The recombinant inhibitor has strong inhibitory activity towards subtilisin and weak inhibitory activity towards α-amylase.…”

Biochemical changes associated with gibberellic acid-like activity of smoke-water, karrikinolide and vermicompost leachate during seedling development ofPhaseolus vulgarisL.

“…Activity of alpha-amylase was determined according to Yamasaki et al (2006) with some modifications. The material was homogenized in K-Pi buffer (as for protein extraction) and the supernatant was used as a crude enzyme.…”

Abiotic stress responses in plant roots: a proteomics perspective

“…Moreover, disease/defense-related proteins such as glycosylated polypeptides, α-amylase/subtilisin inhibitors and chitinases were enhanced in abundance in wheat roots during flood condition (Kong et al, 2010). Among these, α-amylase/subtilisin inhibitors function in defense against micro-organisms (Yamasaki et al, 2006) and chitinase is involved in a defense mechanism against pathogens as well as abiotic stresses (Shibuya and Minami, 2001). Higher abundance of these proteins in the root thus indicated that molecular processes such as protein folding and degradation are involved in plant adaptive responses toward unfavorable environmental condition.…”