Abstract
The presence of isoforms of β-glucosidase has been reported in some grasses such as sorghum, rice and maize. This work aims to extract and characterize isoform II in β-glucosidase from S. edule. A crude extract was prepared without buffer solution and adjusted to pH 4.6. Contaminating proteins were precipitated at 4 °C for 24 h. The supernatant was purified by chromatography on carboxymethyl cellulose (CMC) column, molecular exclusion on Sephacryl S-200HR, and exchange anionic on QFF column. Electrophoretic analyzes revealed a purified enzyme with aggregating molecular complex on SDS-PAGE, Native-PAGE, and AU-PAGE. Twelve peptides fragments were identified by nano liquid chromatography-tandem mass spectrometry (nano LC-ESI-MS/MS), which presented as 61% identical to Cucurbita moschata β-glucosidase and 55.74% identical to β-glucosidase from Cucumis sativus, another Cucurbitaceous member. The relative masses which contained 39% hydrophobic amino acids ranged from 982.49 to 2,781.26. The enzyme showed a specificity to β-d-glucose with a Km of 4.59 mM, a Vmax value of 104.3 μM∙min−1 and a kcat of 10,087 μM∙min−1 using p-nitrophenyl-β-D-glucopyranoside. The presence of molecular aggregates can be attributed to non-polar amino acids. This property is not mediated by a β-glucosidase aggregating factor (BGAF) as in grasses (maize and sorghum). The role of these aggregates is discussed.
Highlights
Introduction βGlucosidases (E.C. 3.2.1.21) are enzymes that hydrolyze glycosidic bonds to release residues, non-reducing sugar terminal glycosides, oligosaccharides, and aglycones
We report on the presence of non-polar amino acids (Table 2) which could participate in the formation of hydrophobic interactions, and point to β-glucosidase it as an isoform (II) as a pore-forming protein of molecular aggregates
We obtained a homogeneous purified preparation of the isoform II, which forms molecular complexes of high molecular weight, with aggregating properties. These complexes were not found in previous studies of isoform I [10]. We attribute this property to the content of non-polar amino acids that form hydrophobic, intra and intermolecular interactions
Summary
Glucosidases (E.C. 3.2.1.21) are enzymes that hydrolyze glycosidic bonds to release residues, non-reducing sugar terminal glycosides, oligosaccharides, and aglycones. The wide distribution of β-glucosidases in various organisms, both eukaryotes, and prokaryotes has been frequently mentioned Their existence and high molecular preservation indicates the importance that of these molecules in the biological processes of various organisms, in plants, in which a variety of functions have been reported. Among these processes participation in secondary metabolism, in the defense process [1], catabolism and lignification of the cell wall, symbiosis [2,3,4,5,6], biosynthesis and abscisic acid catabolism (ABA) [7,8], and antioxidant synthesis [9] are noted. Numerous cases of multiple enzyme isoforms have been demonstrated in plants such as sorghum, rice or maize. The function is attributed to several factors such as specificity towards one or a group of structurally related substrates, the site where it is synthesized in tissue or organelles, and the interaction with individual-specific substrates
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