Abstract
α-galactosidase AgaB of Bacillus stearothermophilus was subjected to directed evolution in an effort to modify its regioselectivity. The wild-type enzyme displays a major 1,6 and minor 1,3 regioselectivity. We used random mutagenesis and staggered extension process (StEP) to obtain mutant enzymes displaying modified regioselectivity. We developed a screening procedure allowing first the elimination of AgaB mutants bearing the 1,6 regioselectivity and secondly the selection of those retaining a 1,3 regioselectivity. Our results show that, among the evolved enzymes that have lost most of their activity towards the 1,6 linkage both in hydrolysis and in synthesis, one (E901) has retained its 1,3 activity. However the transglycosylation level reached by this mutant is quite low versus that of the native enzyme. This work constitutes the first example of modification of glycosylhydrolase regioselectivity by directed evolution.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Dwek RA, Glycobiology: Toward understanding the function of sugars, Chem Rev 96, 683–720 (1996).
Reddy G, Jain RK, Bhatti BS, Matta KL, Synthesis of α-Gal→β-Gal→GlcNAc sequence employing methyl-2,3,4,6-tetra-O-(4-methoxybenzyl)-1-thio-β-D-galactopyranoside as an efficient glycosyl donor, Carbohydr Res 263, 67–77 (1994).
Zhu T, Boons G-H, Two directional, convergent synthesis of a pentasaccharide that is involved in the hyperacute rejection response in xenotransplantation from man to pig, J Chem Soc, Perkin Trans I, 857–61 (1998).
Schmidt RR, New methods for the synthesis of glycosides and oligosaccharides. Are there alternatives to the Koenigs-Knorr methods, Angew Chem, Int Ed Engl 25, 212–36 (1986).
Ichikawa Y, Look GC, Wong C-H, Enzyme-catalyzed oligosaccharide synthesis, Anal Biochem 202, 215–38 (1992).
Heidlas JE, Lees WJ, Whitesides GM, Practical enzyme based synthesis of uridine-5′-diphosphogalactose and uridine-5′-diphospho-N-acetylgalactosamine on a gramscale, JOrg Chem 57, 152 (1992).
Hokke CH, Zervosen A, Elling L, Joziasse DH, Van den Eijnden DH, One pot enzymatic synthesis of Galα1-3galβ1-4GlcNAc sequence with in situ UDP-Gal regeneration, Glycoconjugate J 13, 687–92 (1996).
Yoon J-H, Ajisaka K, The synthesis of galactopyranosyl derivatives with b-galactosidases of different origins, Carbohydr Res 292, 153–63 (1996).
Vetere A, Paoletti S, Complete synthesis of 3′-sialyl-N-acetyllactosamine by regioselective transglycosylation, FEBS Lett 399, 203–6 (1996).
Vic G, Scigelova M, Hastings JJ, Howarth OW, Crout DHG, Glycosydase catalysed synthesis of oligosaccharides: trisaccharides with the α-D-Gal-(1->3)-D-Gal terminus responsible for the hyperacute rejection response in cross-species transplant rejection from pigs to man, Chem Commun 1473–4 (1996).
Vic G, Hastings JJ, Crout DHG, Glycosidase-catalysed synthesis of glycosides by an improved procedure for reverse hydrolysis: application to the chemoenzymatic synthesis of galactopyranosyl-(1->4)-O-alpha-galactopyranoside derivatives, Tetrahedron: Asymm 7, 1973–84 (1996).
Fang J, Xie W, Li J, Wang PG, Chemical and enzymatic synthesis of glycoconjugate 3: synthesis of lactosamine by thermophilic galactosidase catalysed galactosylation on a multigram scale, Tetrahedron Lett 39, 919–22 (1998).
Singh S, Scigelova M, Vic G, Crout DHG, Glycosidase-catalysed oligosaccharide synthesis of di-, tri-and tetra-saccharides using the n-acetylhexosaminidase from Aspergillus oryzae and the betagalactosidase from Bacillus circulans, J Chem Soc, Perkin Trans 1, 1921–26 (1996).
Weignerova L, Sedmara P, Hunkova Z, Halada P, Kren V, Casali M, Riva S, Enzymatic synthesis of iso-globotriose from partially protected lactose, Tetrahedron Lett 40, 9297–9 (1999).
Murata T, Usui T, Preparation of oligosaccharide units library and its utilization, Biosci Biotech Biochem 61, 1059–66 (1997).
Chiffoleau-Giraud V, Spangenberg P, Dion M, Rabiller C, Transferase activity of a b-Glycosidase from Thermus thermophilus. Specificities and limits. Application to the synthesis of α-(1,3)-disaccharides, Eur J Org Chem 757–63 (1999).
Usui T, Kubota S, Ohi H, A convenient synthesis of b-Dgalactosyl disaccharide derivatives using the α-D-galactosidase from Bacillus circulans, Carbohydr Res 244, 315–23 (1993).
Nilsson KGI, A simple strategy for changing the regioselectivity of glycosidase-catalysed formation of disaccharides, Carbohydr Res 167, 95–103 (1987).
Leung DW, Chen E, Goeddel DV, A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction, Technique 1, 11–15 (1989).
Stemmer WPC, Rapid evolution of a protein in vitro by DNA shuffling, Nature 370, 389–90 (1994).
Zhao H, Giver L, Shao Z, Affholter JA, Arnold FH, Molecular evolution by staggered extension process (StEP) in vitro recombination, Nat Biotechnol 16, 258–61 (1998).
Petrounia IP, Arnold FH, Design evolution of enzymatic properties, Curr Opin Biotech 11, 325–30 (2000).
Zhang J-H, Dawes G, Stemmer WPC, Directed evolution of a fucosidase from a-galactosidase by DNA shuffling and screening, Proc Natl Acad Sci USA 94, 4504–9 (1997).
May O, Nguyen PT, Arnold FH, Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine, Nat Biotechnol 18, 317–20 (2000).
Joo H, Lin Z, Arnold FH, Laboratory evolution of peroxidemediated cytochrome P450 hydroxylation, Nature 399, 670–3 (1999).
Spangenberg P, André C, Dion M, Rabiller C, Mattes R, Potential of new sources of a-galactosidases for the synthesis of disaccharides, Carbohydr Res 329, 65–73 (2000).
Janz J, Ganter C, Strezowski J, Mattes R. In Biochemical Engineering, Reuss M, Chmiel H, Gilles ED, Knackmuss HJ (eds), Stuttgart, G. Fischer, 1991, pp. 170–3.
Ganter C, Boćk A, Buckel P, Mattes R, Production of a thermostable, recombinant a-galactosidase suitable for raffinose elimination from sugar beet syrup, J Biotechnol 8, 301–10 (1988).
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JD, Smith JA, Struhl K. Current protocols in molecular biology, New York, Wiley, 1994.
Bradford MM, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem 72, 248–54 (1976).
Spangenberg P, Chiffoleau-Giraud V, André C, Dion M, Rabiller C, Probing the transferase activity of glycosidases by means of in situ NMR spectroscopy, Tetrahedron: Asymmetry 10, 2905–12 (1999).
Burstein C, Kepes A, The α-galactosidase from Escherichia coli K12, Biochim Biophys Acta 230, 52–63 (1971).
Fridjonsson O, Watzlawick H, Gehweiler A, Rohrhirsch T, Mattes R, Cloning of the gene encoding a novel thermostable agalactosidase from Thermus brockianus ITI360, Appl Environ Microbiol 65, 3955–63 (1999). Received 11 January 2001; revised 16 May 2001; accepted 30 May 2001 Modulation of the regioselectivity of a Bacillus a-galactosidase 223
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Dion, M., Nisole, A., Spangenberg, P. et al. Modulation of the regioselectivity of a Bacillus α-galactosidase by directed evolution. Glycoconj J 18, 215–223 (2001). https://doi.org/10.1023/A:1012448522187
Issue Date:
DOI: https://doi.org/10.1023/A:1012448522187