Purification and characterization of a class II α-Mannosidase from Moringa oleifera seed kernels

Abstract

α-Mannosidase (EC. 3.2.1.114) belonging to class II glycosyl hydrolase family 38 was purified from Moringa oleifera seeds to apparent homogeneity by conventional protein purification methods followed by affinity chromatography on Con A Sepharose and size exclusion chromatography. The purified enzyme is a glycoprotein with 9.3 % carbohydrate and exhibited a native molecular mass of 240 kDa, comprising two heterogeneous subunits with molecular masses of 66 kDa (α-larger subunit) and 55 kDa (β-smaller subunit). Among both the subunits only larger subunit stained for carbohydrate with periodic acid Schiff's staining. The optimum temperature and pH for purified enzyme was 50 °C and pH 5.0, respectively. The enzyme was stable within the pH range of 3.0-7.0. The enzyme was inhibited by EDTA, Hg(2+), Ag(2+), and Cu(2+). The activity lost by EDTA was completely regained by addition of Zn(2+). The purified enzyme was characterized in terms of the kinetic parameters K m (1.6 mM) and V max (2.2 U/mg) using para-nitrophenyl-α-D-mannopyranoside as substrate. The enzyme was very strongly inhibited by swainsonine (SW) at 1 μM concentration a class II α-Mannosidase inhibitor, but not by deoxymannojirimycin (DMNJ). Chemical modification studies revealed involvement of tryptophan at active site. The inhibition by SW and requirement of the Zn(2+) as a metal ion suggested that the enzyme belongs to class II α-Mannosidase.

Infrared radiation drying of Moringa oleifera grains for use in water treatment

“…Similar results were described by Tejavath & Nadimpalli (2014) and Jain et al (2019), who presented higher values of protein extraction and enzymatic activity, respectively, at temperature of 50 °C; below 50 °C, protein extraction and enzymatic activity increased with increasing temperature. Increases in temperature are responsible for protein folding and structural changes, which are processes that may have affected the protein concentration.…”

“…However, according to Ghebremichael et al (2005), protein is thermoresistant after treatment for 5 h at 95 °C. A recent study showed a drastic reduction in protein extraction at temperatures from 70 °C (Jain et al, 2019) and in enzymatic activity at temperatures from 80 °C (Tejavath & Nadimpalli, 2014). According to Jain et al (2019), this is due to protein denaturation.…”

Moringa oleifera (drumstick tree) seed coagulant protein (MoCP) binds cadmium - preparation and characterization of nanoparticles

“…The importance of M. oliefera plant and its seed material as an interesting source to study various aspects of phytochemistry, proteins and medicine has been growing over the past few years (45). Our laboratory also has isolated and purified from the seeds of this plant a coagulant protein MoCP and also raised an antibody for the same (38) additionally a α-mannosidase was also purified from the seeds and biochemically characterized (46).Our recent interest has also been to explore and identify what components in some seed materials are involved in removing metals from aqueous solutions. As a first step towards this, we recently characterized the proteins and polysaccharides from Strychnos potatorum seeds (31).…”

Mass spectrometry combined with affinity probes for the identification of CP4 EPSPS in genetically modified soybeans

“…Lectins are proteins that interact reversibly and selectively with specific residues of carbohydrates present in glycoconjugates . This specificity of lectins for a particular carbohydrate provides the basis for probing different types of glycoproteins from complex mixtures.…”

“…30,31 Lectins are proteins that interact reversibly and selectively with specific residues of carbohydrates present in glycoconjugates. 32 This specificity of lectins for a particular carbohydrate provides the basis for probing different types of glycoproteins from complex mixtures. Concanavalin A (Con A), a lectin from Canavalia ensiformis or Canavalia gladiate, has found broad use in the purification of several glycopreins.…”