S. Zuo et al. / Journal of Molecular Catalysis B: Enzymatic 109 (2014) 122–129
123
◦
1
428 [27], B. subtilis B11-06 [18], Thermococcus kodakaraensis KOD1
steps were performed at 4 C. Cells were harvested by centrifuga-
tion at 8000 g for 10 min. The centrifuged cell pellet was washed
twice with lysis buffer (50 mM Tris–HCl buffer, 100 mM NaCl, pH
7.5) and then was resuspended in the lysis buffer. For extraction
[
1], and Yersinia pseudotuberculosis [28]. Thermostability property
is an important issue for the application of industrial enzymes.
The screening and characterization of thermostable l-asparaginase
show potential for the application of the enzyme, especially in
food industry. Typically, the l-asparaginases from the hyperther-
mophilic archaeon strains, Py. furiosus [26] and Tco. kodakaraensis
KOD1 [1], have been determined having remarkable stability at
high temperatures.
In this study, an l-asparaginase with known NCBI accession
number (YP 002959808.1) was characterized from a novel hyper-
thermophilic archaeon, Thermococcus gammatolerans EJ3, which
◦
of crude enzyme, cells were disrupted by sonication at 4 C for
6 min (pulsations of 3 s, amplify 90) using a Vibra-Cell
TM
72405
Sonicator. The cell lysate was then centrifuged at 20,000× g for
◦
20 min at 4 C to remove insoluble cell debris. The resultant
supernatant was loaded onto Chelating Sepharose Fast Flow resin
column (1.0 cm × 10.0 cm), which was chelated with Ni
2+
and
pre-equilibrated with Binding Buffer (50 mM Tris–HCl buffer,
500 mM NaCl, pH 7.5). The unbounded proteins were washed with
the Binding Buffer, followed by a wash with a Washing Buffer
(50 mM Tris–HCl buffer, 500 mM NaCl, 50 mM imidazole, pH 7.5)
to remove weakly bound impurities. Then, the bound recombinant
l-asparaginase was eluted from the column with an Elution Buffer
(50 mM Tris–HCl buffer, 500 mM NaCl, 500 mM imidazole, pH
◦
thrives in temperatures between 55–95 C [29]. Herein, the Tco.
gammatolerans l-asparaginase was characterized having promising
thermostability and high catalytic activity toward l-asparagine. The
enzymatic properties and kinetic parameters were investigated in
detail, and were compared to other microbial l-asparaginases.
−
1
7
.5). The column was operated at a flow rate of 1 mL min . The
2
. Experimental
eluted fractions containing l-asparaginase activity were pooled
and buffer-exchanged in Sample Buffer (50 mM Tris–HCl buffer,
2.1. Reagents, plasmid and strains
pH 7.5) using a 10-kDa MW cut-off ultrafiltration membrane at
◦
4
C.
The resin (the Chelating Sepharose Fast Flow) for protein
purification, Superdex 200 10/300 GL colume, and Gel Filtration
Calibration Kit HMW (high molecular weight) were obtained from
GE (Uppsala, Sweden). Standard proteins (low range) for sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
were obtained from Sangon Biological Engineering Technology
and Services (Shanghai, China). Electrophoresis reagents were
purchased from Bio-Rad. Isopropyl -d-1-thiogalactopyranoside
2
.4. SDS-PAGE and gel filtration chromatography
To analyze the purity and integrity of Tco. gammatolerans
l-asparaginase, SDS-PAGE was performed in a 5% (w/v) polyacryl-
amide stacking gel and a 12% (w/v) separating gel to analyze the
purified enzyme, and the bands were stained with Coomassie Bril-
liant Blue G250 and were destained with an aqueous mixture of
(
IPTG) and all chemicals used for enzyme assays and characteriza-
1
0% (v/v) methanol/10% (v/v) acetic acid.
Native molecular mass was determined by ÄKTA Purifier Sys-
tion were at least of analytical grade obtained from Sigma (St. Louis,
MO, USA) and Sinopharm Chemical Reagent (Shanghai, China).
The pUC57 plasmid and pET-22b(+) expression vector were
obtained from Novagen (Darmstadt, Germany). The host strain
tem (GE Healthcare, Sweden) using Superdex 200 10/300 GL gel
filtration column (GE Healthcare, Sweden). The protein was eluted
with 150 mM NaCl in 50 mM Tris–HCl buffer (pH 7.5) at a flow rate
−
−
−
E. coli BL21(DE3) (F ompT hsdSB (rB mB ) gal dcm (DE3)) was from
−1
of 0.5 mL min
.
Merck (Darmstadt, Germany).
2.5. Enzyme assay
2.2. Gene cloning and heterologous expression
All studies were performed with the 6 × histidine-tagged
The whole genome of Tco. gammatolerans EJ3 has been
enzyme without cleavage of the tag, and the enzyme concentra-
tion was calculated on the basis of monomeric form of the protein
(
sequenced by Zivanovic et al. [29] and was released in Gen-
Bank (NCBI accession number: NC 012804). The gene (locus tag:
TGAM 1442) encoding the hypothetical protein (protein ID:
YP 002959808.1) was synthesized and incorporated with NdeI and
36.5 kDa). The l-asparaginase activity was measured by estimating
the amount of ammonia produced during the l-asparagine hydrol-
ysis reaction. Enzyme assay mixture of 400 L contained 0.02 M
l-asparagine, 0.05 M Tris–HCl (pH 7.5), and 10 L enzyme solu-
ꢀ
ꢀ
XhoI sites in the 5 - and 3 -terminal of the gene, respectively, and
then was cloned into pUC57 vector by Shanghai Generay Biotech
Co., Ltd (Shanghai, China). And then, the gene was sub-cloned
into E. coli expression plasmid vector, pET-22b(+), using NdeI and
XhoI sites, and an in-frame fusion 6 × histidine sequence at the C-
terminus of the DNA sequence was provided in the reconstructed
plasmid, named by pET-Thga-l-ASN. And the reconstructed plas-
mid was introduced into E. coli for l-asparaginase production.
Enzyme expression was performed using E. coli BL21 (DE3) as
host strain. Tco. gammatolerans l-asparaginase was expressed in LB
◦
tion. The reaction was performed at 85 C and pH 8.5 for 2.5 min
and was terminated by adding 100 L of 1.5 M trichloroacetic acid
(
TCA). Reaction mixture was centrifuged at 20,000× g and 150 L
of clear supernatant was added to 1.4 mL of deionized water and
00 L of Nessler’s reagent to measure the released ammonia
after l-asparagine hydrolysis. All the measurements were done
spectrophotometrically at 450 nm. One unit (U) of l-asparaginase
activity was defined as the amount of enzyme required to release
1 mol of ammonia per minute under the conditions mentioned
above.
2
−1
medium supplemented with 100 g mL ampicilin. After inocula-
◦
tion cells were grown to an optical density of 0.6 at 600 nm at 37 C,
IPTG was added to a final concentration of 1 mM, and the culture
◦
was grown further for l-asparaginase overexpression at 28 C for
2.6. Effect of pH and temperature
6
h.
The optimum pH was determined by assessing its activity at dif-
◦
2.3. Enzyme purification
ferent pHs at 85 C. Four buffer systems, including sodium citrate
buffer (50 mM, pH 4.0–6.0), sodium phosphate buffer (50 mM, pH
6.0–7.0), Tris–HCl buffer (50 mM, pH 7.0–9.0), and glycine–NaOH
buffer, (50 mM, pH 9.0–10.0), were used for measuring the opti-
mum pH of enzyme activity.
Tco. gammatolerans l-asparaginase was expressed as
× histidine-tagged fusion protein and the recombinant enzyme
6
2
+
was purified using Ni -affinity chormotography. All purification