A highly selective synthesis of N-acetyllactosamine catalyzed by immobilised β-galactosidase from Bacillus circulans

https://doi.org/10.1016/S1381-1177(99)00112-5Get rights and content

Abstract

Immobilisation of the β-galactosidase of Bacillus circulans on Eupergit C gave a highly selective and stable biocatalyst. The immobilised enzyme catalysed the transfer of the β-galactosyl residue of p-nitrophenyl β-d-galactopyranoside to N-acetylglucosamine with high selectivity for transfer to the 4-position of the glycosyl acceptor.

Introduction

N-Acetyl-d-lactosamine (N-acetyllactosamine) is well known as a representative core structure in oligosaccharide components of glycoproteins and glycolipids [1], [2]. Organic chemical methods for obtaining it have been developed [3], [4], but they are characterized by various elaborate protection and deprotection procedures. On the other hand, enzymatic synthesis using glycosyl transferases has also been reported [5], [6]. In this type of enzymatic synthesis, glycosyl transferases are widely used to perform regiospecific galactosylation and sialylation on a preparative scale [7], but these enzymes belong to the Leloir pathway and are difficult to obtain and have limited stability. Moreover, they require expensive cofactors as glycosyl donors. Glycosyl hydrolases (glycosidases) can also be used to synthesise oligosaccharides in a kinetically controlled reaction, where a glycosyl donor is used to transfer its glycosyl residue to a sugar acceptor present in the reaction medium [8], [9], [10]. In spite of the increased amount of work carried out with glycosyl hydrolases, their main drawback is a lack of regioselectivity, which limits their use for synthetic purposes. In an important contribution to glycosidase-catalysed oligosaccharide synthesis, Usui et al. [11], [12] reported for the first time the use of a β-galactosidase from Bacillus circulans to synthesise some β-d-(1→4) galactosyl disaccharides bearing a GlcNAc or a GalNAc residue at the reducing end. Some β-(1–6) linkages were produced as well, but to the best of our knowledge, it was the first time that a preparative scale β-d-galactosyltransfer has been shown to occur preferentially at the O-4 position using a galactosyl hydrolase. There is considerable synthetic interest in the use of galactosidase to perform this type of galactosyl transfer. Recently, it has been shown that the use of the thioethyl glycoside of N-acetylglucosamine as acceptor and p-nitrophenyl β-d-galactoside as donor gave complete selectivity for 1→4 transfer (Scheme 1) [13].

Immobilisation of the enzyme can offer several advantages such as enhancing operational stability, continuous operation, repeated usage of the derivative and retention of the biocatalyst in the reaction vessel [14].

Because of its synthetic usefulness, we have investigated the immobilisation of β-galactosidase from B. circulans on Eupergit C. We found that it can be usefully applied as a recoverable and reusable enzyme for the synthesis of N-acetyllactosamine with high regioselectivity.

Section snippets

Materials

The commercially available β-galactosidase (E.C. 3.2.1.23, from B. circulans, Biolacta) was a gift from Daiwa Kasei, Osaka, Japan. Eupergit C was also a gift from Röhm Pharma, Weiterstadt. All other chemicals were obtained from commercial sources.

Analytical methods

NMR spectra were recorded on Bruker AC-400 or 250 MHz spectrometers. The structure of the enzymatically synthesised disaccharides were assigned by proton–proton shift correlation, carbon–proton shift correlation and DEPT-experiments; (1→4) and (1→6)

Influence of immobilisation of the enzyme on the selectivity of the process

The β-galactosidase from B. circulans has been proven to be a valuable biocatalyst for galactosyl transfer from suitable donors onto a variety of substrates. With N-acetylglucosamine or N-acetylglucosamine glycosides as acceptors, β-d-(1→4) transfer product predominates giving N-acetyllactosamine or N-acetyllactosamine glycosides, respectively. Some β-(1–6) galactosyl transfer was also observed [13], [16], [17].

Commercially available crude β-d-galactosidase, free and immobilised on Eupergit C,

Conclusions

All results presented in this work suggest that β-galactosidase from B. circulans immobilised on Eupergit C could be usefully applied as a recoverable and reusable enzyme for the synthesis of N-acetyllactosamine with high regioselectivity.

Acknowledgements

This work was supported by a Marie Curie Research Training grant from European Commission. Project ERB 4001GT.

References (19)

  • E.J. Toone et al.

    Tetrahedron

    (1989)
  • S.D. David et al.

    Adv. Carbohydr. Chem. Biochem.

    (1991)
  • K.G.I. Nilsson

    Carbohydr. Res.

    (1987)
  • T. Usui et al.

    Carbohydr. Res.

    (1993)
  • G. Vic et al.

    Tetrahedron: Asymmetry

    (1996)
  • E. Katchalski-Katzir

    Trends Biotechnol.

    (1993)
  • A. Vetere et al.

    Biochem. Biophys. Res. Commun.

    (1996)
  • S. Takayama et al.

    Bioorg. Med. Chem. Lett.

    (1996)
  • A. Varki

    Glycobiology

    (1993)
There are more references available in the full text version of this article.

Cited by (31)

  • Efficient synthesis of N-acetyllactosamine using immobilized β-galactosidase on a novel 3D polymer support

    2022, Enzyme and Microbial Technology
    Citation Excerpt :

    Three parallel reactions were carried out under the same operating conditions and all the data were obtained in average. The specific purification operation method of LacNAc was according to reference [32]. Reusability of the immobilized β-galactosidase was assayed according to the yield of LacNAc.

  • Enzymatic and chemoenzymatic synthesis of human milk oligosaccharides and derivatives

    2022, Carbohydrate Polymers
    Citation Excerpt :

    Notably, the regioselectivity of enzyme could be even changed by the effect of the immobilization. It was found that β-D-(1 → 4) galactosyl disaccharide could be selectively synthesized with no 1 → 6 isomer detected in the presence of immobilized enzyme while there is no regioselectivity via free enzyme catalyzation (Hernaiz & Crout, 2000). In conclusion, enzyme immobilization can greatly facilitate the synthesis of HMOs.

  • Two-step enzymatic strategy for the synthesis of a smart phenolic polymer and further immobilization of a β-galactosidase able to catalyze transglycosydation reaction

    2018, International Journal of Biological Macromolecules
    Citation Excerpt :

    However, the selectivity with respect to Gal–β(1–3)–GlucNAc formation remained after re-use. Similar stability data have been obtained using this enzyme immobilized on agarose gel [14] or the immobilization of other β-galactosidase isoforms from B. circulans in different supports such as porous polymers or Eupergit–C [36,38]. In conclusions, an efficient and sustainable enzymatic procedure to obtain an immobilized biocatalyst was successfully developed.

  • Bacillus circulans β-galactosidase catalyses the synthesis of N-acetyl-lactosamine in a hydro-organic medium via a steady-state ordered Bi Bi reaction mechanism

    2012, Journal of Molecular Catalysis B: Enzymatic
    Citation Excerpt :

    BcβGal is a retaining β-galactosidase that is assumed to operate through a double displacement mechanism, both steps involving a transition state with a substantial oxocarbenium ion character [15–18]. According to studies on the kinetics of the hydrolysis or transglycosylation activity of various glycosidases, a glycosidase-catalysed reaction involves either a ping pong Bi Bi [19–23] or an ordered (Uni Bi or Bi Bi) kinetic mechanism [10,24–26]. The ping pong and ordered Bi Bi mechanisms are both special multisubstrate reactions.

View all citing articles on Scopus
View full text