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

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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, Hg2+, Ag2+, and Cu2+. The activity lost by EDTA was completely regained by addition of Zn2+. 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 Zn2+ as a metal ion suggested that the enzyme belongs to class II α-Mannosidase.

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Abbreviations

PKM1 :

Periyakulam1

DE-52:

DiEthyl cellulose-52

Con A Sepharose:

Concanavalin A Sepharose

SW:

Swainsonine

DMNJ:

Deoxymannojirimycin

Glu2SI:

Glucosyl Salicilyl Imine

GSI:

Galactosyl Salicilyl Imine

GNI:

Galactosyl Naphthyl Imine

Glu2NI:

Glucosyl Naphthyl Imine

MNI:

Mannosyl Naphthyl Imine

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Acknowledgments

KKT thanks CSIR, India for Senior Research Fellowship. The authors are grateful to Prof. C.P.Rao and his lab members at IIT Mumbai for providing the synthetic glycoconjugates used in this study.

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Correspondence to Kiran Kumar Tejavath.

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Tejavath, K.K., Nadimpalli, S.K. Purification and characterization of a class II α-Mannosidase from Moringa oleifera seed kernels. Glycoconj J 31, 485–496 (2014). https://doi.org/10.1007/s10719-014-9540-z

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Keywords

  • Moringa oleifera
  • α-Mannosidase
  • Con A Sepharose 4B gel
  • DE-52 (DiEthyl cellulose)
  • PAS Staining