Lactobacillus casei a1-6 fucosidase E274A

Abbreviation 

AlfC-E274A

Source

Lactobacillus casei

Activity

Transfer Fuc-a-F to GlcNAc of GlcNAc-peptide

Expression

&

Purification

Expression system:    pCPD with C-terminal CPD and His6-tag , E. coli BL21(DE3)

Induction condition:  0. 2 mM IPTG, 16 °C, 20 h, 200 rpm, Luria LB broth

Purification: 20 mg/L culture

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 6 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

40-50 °C

7.0 - 9.0

100 mM Tris-HCl (pH 6.5), 20 mM donor, 10 mM acceptor, incubate at 42 °C for 1 h.

Temperature

pH

Metal ion:

Example condition:   

GDP-Fuc

GlcNAc-Asn

GlcNAc-peptide

N-glycan-peptide

Donor:

Acceptor:

Kinetics:

Substrate Specificity

  • Li C., Zhu S., Ma C. and Wang L. Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N‑Glycopeptides and N‑Glycoproteins. J AM Chem Soc, 2017, 139, 15074. 

References