Skip to main content
SpringerLink
Log in

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

  • Published:
Glycoconjugate Journal Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

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

References

  1. Snaith, S.M.: Characterization of jack-bean alpha-D-mannosidase as a zinc metalloenzyme. Biochem. J. 147(1), 83–90 (1975)

    PubMed  CAS  PubMed Central  Google Scholar 

  2. Hossain, M.A., Nakamura, K., Kimura, Y.: alpha-mannosidase involved in turnover of plant complex type N-glycans in tomato (Lycopersicum esculentum) fruits. Biosci. Biotechnol. Biochem. 73(1), 140–146 (2009)

    Article  PubMed  CAS  Google Scholar 

  3. Gnanesh Kumar, B.S., Pohlentz, G., Mormann, M., Siva Kumar, N.: Characterization of alpha-mannosidase from Dolichos lablab seeds: partial amino acid sequencing and N-glycan analysis. Protein Expr. Purif. 89(1), 7–15.

  4. Li, Y.T.: Studies on the glycosidases in jack bean meal. I. Isolation and properties of alpha-mannosidase. J. Biol. Chem. 242(23), 5474–5480 (1967)

    PubMed  CAS  Google Scholar 

  5. Paus, E.: alpha-Mannosidase from Phaseolus vulgaris. Composition and structural properties. Eur. J. Biochem. 73(1), 155–161 (1977)

    Article  PubMed  CAS  Google Scholar 

  6. Curdel, A., Petek, F.: Purification and properties of alpha-D-mannosidase from the germinated seeds of Medicago sativa (alfalfa). Biochem. J. 185(2), 455–462 (1980)

    PubMed  CAS  PubMed Central  Google Scholar 

  7. Tulasi, R.B., Nadimpalli, S.K.: Purification of alpha-mannosidase activity from Indian lablab beans. Biochem. Mol. Biol. Int. 41(5), 925–931 (1997)

    PubMed  CAS  Google Scholar 

  8. Ohtani, K., Misaki, A.: Purification and characterization of α-D-galactosidase and α-D-mannosidase from papaya (Carica papaya) seeds. Agric. Biol. Chem. 47, 2441–2451 (1983)

    Article  CAS  Google Scholar 

  9. Kishimoto, T., Hori, H., Takano, D., Nakano, Y., Watanabe, M., Mitsui, T.: Rice alpha-mannosidase digesting the high mannose glycopeptide of glutelin. Physiol. Plant. 112(1), 15–24 (2001)

    Article  PubMed  CAS  Google Scholar 

  10. Subha Mahadevi, A., Vegiraju Suryanarayana, R., Siva Kumar, N.: Alpha-mannosidase from the seeds of Triticale. J. Biochem. Mol. Biol. Biophys. 6(6), 397–400 (2002)

    Article  PubMed  CAS  Google Scholar 

  11. Kestwal, R.M., Bhide, S.V.: Purification and partial characterization of alpha-D-mannosidase from Erythrina indica seeds. Indian J. Biochem. Biophys. 42(3), 156–160 (2005)

    PubMed  CAS  Google Scholar 

  12. Ahi, A.P., Gonnety, J.T., Faulet, B.M., Kouamé, L.P., Niamké, S.: Biochemical characterization of two α -mannosidases from breadfruit (Artocarpus communis) seeds. Afr. J. Biochem. Res. 1(6), 106–116 (2007)

    Google Scholar 

  13. Einhoff, W., Rudiger, H.: Isolation of the Canavalia ensiformis seed alpha-mannosidase by chromatography on concanavalin A, the lectin from the same plant, without involving its sugar binding site. Biol. Chem. Hoppe Seyler 367(4), 313–320 (1986)

    Article  PubMed  CAS  Google Scholar 

  14. Kimura, Y., Hess, D., Sturm, A.: The N-glycans of jack bean alpha-mannosidase. Structure, topology and function. Eur. J. Biochem. 264(1), 168–175 (1999)

    Article  PubMed  CAS  Google Scholar 

  15. Blom, H., Reyes, F., Carlsson, J.: Purification and characterization of an alpha-mannosidase from the tropical fruit babaco (Vasconcellea x heilbornii Cv. babaco). J. Agric. Food Chem. 56(22), 10872–10878 (2008)

    Article  PubMed  CAS  Google Scholar 

  16. Burrows, H.M., Rastall, R.A.: Essential carboxyl and tryptophan residues in jack bean α-d-Mannosidase. Phytochemistry 48(2), 257–260 (1998)

    Article  CAS  Google Scholar 

  17. Kestwal, R.M., Konozy, E.H., Hsiao, C.D., Roque-Barreira, M.C., Bhide, S.V.: Characterization of alpha-mannosidase from Erythrina indica seeds and influence of endogenous lectin on its activity. Biochim. Biophys. Acta 1770(1), 24–28 (2007)

    Article  PubMed  CAS  Google Scholar 

  18. Misaki, R., Fujiyama, K., Yokoyama, H., Ido, Y., Miyauchi, K., Yoshida, T., Seki, T.: Characterization of almond alpha-mannosidase and its application for structure analysis of sugar chain. J. Biosci. Bioeng. 96(2), 187–192 (2003)

    Article  PubMed  CAS  Google Scholar 

  19. Kumar, A., Singhal, N.K., Ramanujam, B., Mitra, A., Rameshwaram, N.R., Nadimpalli, S.K., Rao, C.P.: C(1)-/C(2)-aromatic-imino-glyco-conjugates: experimental and computational studies of binding, inhibition and docking aspects towards glycosidases isolated from soybean and jack bean. Glycoconj. J. 26(4), 495–510 (2009)

    Article  PubMed  CAS  Google Scholar 

  20. Gnanesh Kumar, B.S., Pohlentz, G., Schulte, M., Mormann, M., Siva Kumar, N.: Jack bean alpha-mannosidase: Amino acid sequencing and N-glycosylation analysis of a valuable glycomics tool. Glycobiology 24(3), 252–261. doi:cwt106

  21. Tejavath, K.K., Nadimpalli, S.K.: Purification and biochemical Characterization of an acidic α-galactosidase from the Moringa oleifera seeds. Trends Carbohydr. Res. 5(2), 27–34 (2013)

    CAS  Google Scholar 

  22. Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976)

    Article  PubMed  CAS  Google Scholar 

  23. Dubois, M., Gilles, K., Hamilton, J.K., Rebers, P.A., Smith, F.: A colorimetric method for the determination of sugars. Nature 168(4265), 167 (1951)

    Article  PubMed  CAS  Google Scholar 

  24. Andrews, P.: Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem. J. 91(2), 222–233 (1964)

    PubMed  CAS  PubMed Central  Google Scholar 

  25. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259), 680–685 (1970)

    Article  PubMed  CAS  Google Scholar 

  26. Zacharius, R.M., Zell, T.E., Morrison, J.H., Woodlock, J.J.: Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 30(1), 148–152 (1969)

    Article  PubMed  CAS  Google Scholar 

  27. Gassenschmidt, U., Jany, K.D., Tauscher, B., Niebergall, H.: Isolation and characterization of a flocculating protein from Moringa oleifera Lam. Biochim. Biophys. Acta 1243(3), 477–481 (1995)

    Article  PubMed  Google Scholar 

  28. Woo, K.K., Miyazaki, M., Hara, S., Kimura, M., Kimura, Y.: Purification and characterization of a co(II)-sensitive alpha-mannosidase from Ginkgo biloba seeds. Biosci. Biotechnol. Biochem. 68(12), 2547–2556 (2004)

    Article  PubMed  CAS  Google Scholar 

  29. Waln, K.T., Poulton, J.E.: Partial purification and characterization of an α-d-mannosidase from mature seeds of Prunus serotina Ehrh. Plant Sci. 53(1), 1–10 (1987)

    Article  CAS  Google Scholar 

  30. Howard, S., He, S., Withers, S.G.: Identification of the active site nucleophile in jack bean alpha-mannosidase using 5-fluoro-beta-L-gulosyl fluoride. J. Biol. Chem. 273(4), 2067–2072 (1998)

    Article  PubMed  CAS  Google Scholar 

  31. Priya Sethu, K.M., Prabha, T.N.: Alpha-D-Mannosidase from Capsicum Annuum. Phytochemistry 44(5), 383–387 (1997)

    Article  CAS  Google Scholar 

  32. Nakajima, M., Imamura, H., Shoun, H., Wakagi, T.: Unique metal dependency of cytosolic alpha-mannosidase from Thermotoga maritima, a hyperthermophilic bacterium. Arch. Biochem. Biophys. 415(1), 87–93 (2003)

    Article  PubMed  CAS  Google Scholar 

  33. Kawar, Z., Karaveg, K., Moremen, K.W., Jarvis, D.L.: Insect cells encode a class II alpha-mannosidase with unique properties. J. Biol. Chem. 276(19), 16335–16340 (2001)

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  34. Howard, S., Braun, C., McCarter, J., Moremen, K.W., Liao, Y.F., Withers, S.G.: Human lysosomal and jack bean alpha-mannosidases are retaining glycosidases. Biochem. Biophys. Res. Commun. 238(3), 896–898 (1997)

    Article  PubMed  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Central University of Rajasthan, Kishangarh, Ajmer, 305817, Rajasthan, India

    Kiran Kumar Tejavath

  2. Protein Biochemistry and Glycobiology Laboratory, Department of Biochemistry, University of Hyderabad, Hyderabad, 500046, India

    Siva Kumar Nadimpalli

Authors
  1. Kiran Kumar Tejavath
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Siva Kumar Nadimpalli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kiran Kumar Tejavath.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10719-014-9540-z

Keywords

Search

Navigation