A Study on the Transglycosylation Activity of beta-Galactosidases Employing Different Donors

R. Peinsipp*[1], K. Dax[1], T. Lischnig[2] and W. Steiner[2]

[1]Institut fuer Organische Chemie der Technischen Universitaet Graz, Stremayrgasse 16, A-8010 Graz, Austria [2]Institut fuer Biotechnologie der Technischen Universitaet Graz, Petersgasse 12, A-8010 Graz, Austria


Glycosidase-catalysed transglycosylation is a promising alternative to classical chemical glycosylation methods. Enzymes show - depending on their source and the kind of glycosyl donor used - different transglycosylation activities. Looking for synthetically useful enzyme preparations, including those from new sources, we started a programme towards the elucidation of their transglycosylation capacities.

Galactosyl donor Product Aspergillus orycae1 Penicillium simplicissimum2 Almond Meal
lactose b-D-Gal(1-6)a-D-Glc-OMe 4 20 % 27 % 17 %
4-nitrophenyl- b-D-galactopyranoside b-D-Gal(1-6)-a-D-Glc-OMe4
25 %
54 %
25 %
56 %
12 %
12 %
2,4-dinitrophenyl- b-D-galactopyranoside b-D-Gal(1-6)-a-D-Glc-OMe4
32 %
62 %
28 %
57 %
20 %
21 %

Almond meal almost exclusively catalysed the synthesis of b-D-Gal(1-6)a-D-Glc-OMe, whereas both fungal galactosidases gave a reasonable proportion of disaccharides involving secondary hydroxyl groups (all possible regioisomeres could be isolated, Fig. 1).

Scheme 1

Employing lactose as donor we could isolate b-D-Gal(1-6)-b-D-Gal(1-4)-D-Glc as a side product (lactose serves as donor as well as acceptor, Fig. 2).

Scheme 2

The donor 2,4-dinitrophenyl b-D-galactopyranoside 5 gave the best yields in all investigated cases. 2,4-Dinitrophenyl 2-deoxy-2-fluoro-b-D-glucopyranoside has been found to be a mechanism-based inhibitor 6, and - to the best of our knowledge - 2,4-dinitrophenyl b-D-glycosides have never been used as donors in glycosidase-catalysed transglycosylation reactions so far.

As kinetically controlled reactions allow the use of crude enzyme preparations or culture filtrates as catalysts, we employed almond meal on the one hand and a crude culture filtrate of Penicillium simplicissimum on the other hand. In the latter case a novel b-galactosidase with high transglycosylation activity was detected.


Breeding of Penicillium simplicissimum:

Breeding conditions:
2,5% corn cobs, 1,2% carob seeds, 3% Solulyst A St, 0,5% NH4NO3 and 0,25% KH2PO4. The b-galactosidase activity of the culture filtrate used was about 4 u/ml. Attempts to increase the b-galactosidase activity were not successful so far.

Enzymatic reactions:

donor (0,2 mol/l), methyl a-D-glucopyranoside (1,5 mol/l), phosphate buffer (50 mmol/l), pH=5, 20 C

The course of reactions was followed by means of HPLC and stopped at maximum yield by heating to 90 C for 10 minutes.

2,4-dinitrophenyl b-D-galactopyranoside and 4-nitrophenyl b-D-galactopyranoside:
RP-18 column (25cm x 4mm)
flow: 1ml/min
solvent: acetonitrile:water=10:90 (v/v)
All other substrates and products:
aminopropylated silica column, Nucleosil 100-5NH2 (25cm x 4mm)
flow: 1ml/min
solvent: acetonitrile:water=80:20 (v/v)

Separation of the transglycosylation products:

The isolation of all products was possible by using a charcoal-Celite column, which was prepared as follows: equal parts by weight of dry charcoal and Celite were slurried in water and packed into a glass column (50cm x 3,5cm). After denaturation of the enzyme the reaction mixture was applied to the column and eluted with a linear gradient from 0 to 30%(v/v) EtOH in water. The fractions containing transglycosylation products were collected, concentrated and rechromatographed on the same column (elution with a gradient from 6,5 to 10%(v/v) EtOH in water).

References and Footnotes:
  1. obtained by Sigma, grade XI
  2. obtained by Sigma
  3. yields determined by means of HPLC
  4. total yields include all transglycosylation products formed; determined as consumption of the donor minus liberated galactose
  5. synthesized according to F. Ballardie et. al., J.C.S. Perkin I (1973) 2418-2419
  6. S. G. Withers et al., Biochemistry 31 (1992) 9970-9978

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