2.4.

Chitinase expression and purification

Expression
The wild-type and 128mt genes
were fused to a C-terminal His-tag

Expression ve
Inserted into the pFLAG-CTS
chiHind3 and c

E. coli
Top10

Cultured in LB
medium overnight
Transferring into 500 mL LB medium
Containing 100 μg/mL ampicillin.

Culture (37°C)
OD600 = 1,0
Isopropylthio-βgalactoside (IPTG) at
concentration of 1 mM

Culturing (28°C, 4h)

Crude
enzyme
2.4. Chitinase expression and purification

Purification
Crude
enzyme

Centrifugation ( 8000
rpm, 4°C for 20 min)

Resuspending in 1–
2 mL lysis buffer

Sonication ( 60 amplitude, pulses of 6 s,

Centrifugation ( 10 000
rpm, 4°C for 20 min)

Supernatant
Soluble chitinase

Purification Using Ni-NTA resins accordi

protocol.

Washed

Eluted

Concentrated using
Vivaspin

Enzyme (–20°C in sodium phosphate

solutions
2.5 Chitinase assay

0.4 µg pure enzyme,

0.008–0.7 mM substrate,
p-NP-(GlcNAc)2, 100
mM sodium phosphate
buffer (pH 6.0)

total volume of 100 μL

Incubation ( 37°C,
30 min)

Adding 10 μL 1 N
NaOH

Recording
absorbance at 405 nm

- Using a standard curve for pNP.

- Before determining the kinetic parameters, it was
established that product formation was linear over
time under the conditions of this assay.
- Reaction conditions were such that the amount of
substrate converted was below 20% of the total
amount of substrate added.
- The values of the kinetic constants were
calculated from the Michaelis-Menten plot using a
non-linear curve fitting method (GraphPad Prism
5)
TLTK: Tạp chí Công nghệ Sinh học 15(3): 571-57
 DNA template, dNTPs,
dGTP, dCTP, dTTP, MgCl2,
MnCl2, Tag DNA
Polymerase, Primers

Heat (95°C, 2m)

40 cycles of denat-
uration (95°C, 30s)

Annealing (55°C, 5s)

Extension (72°C,2m)
Tris- HCl PCR
MnCl2 DNaseI
pH 7,4 product

ice-cooled
Fermentation
0.5 M EDTA
(15oC, 7m)
pH 8.0.

Isolate by
polyacrylamide gel

Fragment reassembly
and PCR

Purification

Vecto
Cloning
pFLAG-CTS

Transform into E. coli

TOP10
(Electroporation)