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W. Tao et al. / Process Biochemistry 50 (2015) 2121–2127
In this study, a novel cephalosporin-deacetylating AXE from
It was considered as the AXE crude enzyme. The protein concen-
tration of the supernatant was determined by the Bradford method
[17].
Bacillus subtilis CICC 20034 with high activity and stability toward
cephalosporin C and 7-aminocephalosporanic acid was reported
[15], and the ability of B. subtilis CICC 20034 AXE to catalyze the
perhydrolysis of acetyl substrates to produce PAA was investigated.
The ability of the bacteria to increase the commercial production of
PAA in an aqueous environment with optimized reaction conditions
was also studied.
2.5. Immobilization of AXE
Before immobilization, acrylate amino resin MI-BS1 (Novocata,
China) was activated by the following procedure: The resins were
washed with 20 mM Tris–HCl buffer (pH 8.0) and were suspended
in the same buffer containing glutaraldehyde (1% v/v) with stirring
at 250 rpm and 25 ◦C for 1 h. Then, the remaining glutaraldehyde
solution was removed thoroughly, and the resins were washed with
distilled water before use and stored at 4 ◦C. For the immobilization
of AXE, crude AXE solution (0.1 g of protein) and 10 g of activated
resins were mixed with 50 mL of 100 mM Tris–HCl buffer (pH 7.4)
and incubated at 35 ◦C for 6 h. After reaction, the immobilized AXE
was recovered by filtration and stored in 100 mM Tris–HCl buffer
(pH 7.4) at 4 ◦C until use.
2. Materials and methods
2.1. Cloning AXE gene (Cah) into the expression vector pET28a
The Cah gene from B. subtilis encoding AXE [15] was ampli-
fied with two primers designed to generate NcoI and XhoI sites
(forward, 5ꢀ-CATGCCATGGGCATGCAATTATACGACT-3ꢀ and reverse,
5ꢀ-CCCTCGAGGCCTTTC AGATGCGCTT-3ꢀ). The amplified gene was
digested with NcoI and XhoI and inserted into the expression vec-
tor pET28a (Novagen) to generate a pET28a-Cah plasmid. Then, the
recombinant plasmid was transformed into Escherichia coli BL21
(DE3).
2.6. Evaluation of commercial lipases and recombinant AXE for
perhydrolysis reaction
The reaction was performed in a 5-mL test tube with a reaction
volume of 1 mL containing glycerol triacetate (250 mM), hydrogen
peroxide (1 M), and 0.3 mg of commercial enzymes or recombinant
AXE in 100 mM Tris–HCl buffer (pH 7.4) at 25 ◦C for 10 min.
2.2. Expression and production of recombinant AXE
In order to reduce the cost of enzyme production, autoinduction
medium was used to replace the expensive conventional IPTG (iso-
propyl -d-1-thiogalactopyranoside) induction. Considering the
fact that the amount of glycerin, glucose, and lactose present in
the medium could affect the cell growth and enzyme production
[16], the culture medium was optimized by the orthogonal test in
three factors and three levels: 15 g/L peptone, 25 g/L yeast extract,
and 10 g/L NaCl; 0.1–0.3% (v/v) glycerin, 0.1–0.3% (w/v) glucose, and
0.2–0.4% (w/v) lactose. Shake flasks were used for optimizing the
medium.
In practice, AXE was produced in a 5-L jar fermentor as follows:
The seed medium for the recombinant E. coli was Luria–Bertani
(LB) broth; the recombinant cells were incubated at 37 ◦C for
12 h in a 250-mL rotary shaker (200 rpm) containing 50 g/mL of
kanamycin. The seed culture (2%, v/v) was then added to 3 L of
the optimized fermentation medium. The agitation rate was main-
tained at 200 rpm, air was sparged at a rate of 1.5 v/v/m, and the
cultures were incubated at 30 ◦C for 16 h. Bacterial growth was then
Comparison of kinetic parameters between free enzymes and
immobilized AXE was performed using the monochlorodimedone
assay [18] to measure the initial rates of enzymes with substrates
(glycerol triacetate, hydrogen peroxide, and ethyl acetate) at 25 ◦C
and pH 6.5. The apparent KM values were obtained using the
Michaelis–Menten equation.
2.8. Perhydrolysis
Perhydrolysis of acetate esters catalyzed by the recombinant
enzyme AXE was performed in a 5-mL test tube containing a reac-
tion volume of 1 mL via different reactions. Conditions such as
different acetate esters, concentration of the substrate, amount of
enzyme, temperature, buffer pH, and reaction time were optimized.
The reusability of the immobilized AXE was investigated at the end
of each reaction, which was removed from the reaction medium
by centrifugation and reused after being thoroughly rinsed with
100 mM Tris–HCl buffer (pH 7.4).
monitored by measuring the A600
.
2.3. Enzyme assays
The cells collected from the culture broth were suspended in
100 mM Tris–HCl buffer (pH 7.4) and were disrupted by sonica-
tion; supernatants were collected by centrifugation (10,000 × g for
30 min). AXE activity was assayed as described earlier [15]. One
unit of enzyme activity (U) was defined as the quantity of enzyme
required to release 1 mol of p-nitrophenol per minute at 40 ◦C for
10 min.
2.9. Determination of PAA by high-performance liquid
chromatography
In general, PAA is analyzed with its redox properties by oxi-
dizing methyl p-tolyl sulfide (MTS) to the corresponding sulfoxide
(MTSO). Thus, PAA was quantitatively measured by estimating
the MTSO content with high-performance liquid chromatography
(HPLC) analysis, as described by Shaw and Hager [19]. However,
after complete consumption of PAA, the excess H2O2 present con-
tinued to oxidize MTS. In order to avoid a steady increase in the
MTSO peak due to MTS oxidation by excess H2O2, triphenylphos-
phine (TPP) was used to initiate the second derivatization step,
which was then oxidized to triphenylphosphine oxide (TPPO) by
the excess H2O2. Thus, PAA could be determined as follows: 0.1 mL
of the sample solution was mixed with 0.1 mL of 20 mM MTS in
acetonitrile and 0.3 mL of deionized water in the dark, at 25 ◦C for
10 min. Then, 0.4 mL of acetonitrile and 0.1 mL of a 10 mM solution
2.4. Preparation of AXE crude enzyme
A concentrated cell suspension in a 3-L culture of E. coli
BL21-pET28a-Cah was obtained by continuous centrifugation at
8000 rpm (centrifuge model Avanti J-26XP, Beckman Coulter, Inc.,
USA). Then, the cells were washed with 100 mM Tris–HCl buffer
(pH 7.4) and were concentrated 10 times with the same buffer.
Subsequently, the cells were disrupted through a high-pressure
homogenizer (model AH100B, ATS Engineering Inc., Germany) at
800 bar and centrifuged for 20 min at 8000 rpm to remove cell
debris. The supernatant was collected and stored at 4 ◦C until use.