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