Bifidobacterium infantis galactosyl-b1–3-N-acetyl-hexosamine phosphorylase

Abbreviation 

BiGalHexNAcP

Source

Bifidobacterium longum subsp. infantis (ATCC 15697)

Activity

Transfer Gal from Gal-1-P to GlcNAc./GalNAc via a b1,3-linkage

Expression

&

Purification

Expression system:    pET22b with C-terminal His6-Tag, E. coli Origami (DE3)

Induction condition:  0.2 mM IPTG, 16 °C, 20 h, 200 rpm, Luria LB broth

Purification

1) Suspend cells collected from 1 L medium in ice-cold 40 mL Buffer-A

2) Disrupt cells by a CF1 cell disruptor (Constant Systems), 20 kPsi, 4 °C

3) Centrifuge at 12000 x g, 4 °C, 30 min

4) Load supernatant to a 10 mL gravity column filled with 2 mL Ni-NTA resin (pre-equilibrated)

5) Wash with 50 mL Buffer-A, then 50 mM Buffer-B, finally elute with 10 mL Buffer-C.

6) Concentrate into 2.5 mL and change buffer to Buffer D using a PD-10 column. 

7) Store at 4 °C for up to 4 months, store at -80 °C for up to 1 year (avoid repeated freeze)

Buffers:   A: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole;

                B: A + 15 mM imidazole; C: A + 250 mM imidazole. 

                D: 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 20 % Glycerol

Optimal Condition

37 °C

5.0 - 6.5

MnCl2, MgCl2

50 mM MES-KOH (pH 6.5), 22 mM donor, 20 mM acceptor, 20 mM MnCl2, incubate at 37 °C for 1 h to overnight.

Temperature

pH

Metal ion:

Example condition:   

UDP-Gal

HexNAc monosaccharide

Donor:

Acceptor:

Substrate Specificity

  • Yu H., Thon V., et al. Highly Efficient Chemoenzymatic Synthesis of β1–3-Linked Galactosides. ChemComm, 2010, 46, 7507.

References