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C. Yao et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 105–110
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.2%, (NH ) SO 0.35%, Na HPO 0.2%, KH PO 0.2%, MgSO ·7H O
lipase activity were pooled, concentrated in a dialysis bag (7 kDa
molecular weight cut-off) by PEG20000, and dialyzed against the
same Tris–HCl buffer overnight. (NH ) SO was added to the dia-
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.05%) which contains 15% of toluene or 15% of DMSO. The cul-
tivation vessel was plugged with a chloroprene-rubber stopper
to prevent evaporation of the organic solvent. Cultivations were
conducted in 10 ml of medium in 50 ml test tubes for 2 days with
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lyzed sample to a concentration of 0.7 M, and then loaded onto
Phenyl Sepharose FF column which had been equilibrated with
the same Tris–HCl buffer containing 0.7 M (NH ) SO . After wash-
◦
shaking at 180 rpm at 30 C. After twice transferring to the fresh
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enrichment medium containing same organic solvent, the broths
were diluted and spread on LB-tributyrin agar plates (yeast extract
ing with 2 bed volumes of 50 mM Tris–HCl buffer and, separately,
with the same buffer containing 20% ethanol, the bound proteins
were eluted with the same Tris–HCl buffer containing 40% alco-
hol, dialyzed, and then lyophilized. Purified lipase was analyzed by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) according to
the method described by Laemmli [13].
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.0%, tryptone 0.5%, NaCl 1%, tributyrin 0.5%, and agar 1.8%), and
halo-forming colonies were isolated after incubation overnight. The
colonies were then inoculated to the optimized culture medium
(
Olive oil 1%, yeast extract 0.5%, tryptone 0.5%, urea 0.5%, KH PO4
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0
.2%, MgSO ·7H O 0.05%, Triton X-100 0.05%, pH 7.0) and incubated
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◦
at 30 C, 180 rpm. Lipase activity of the cultured supernatant was
measured spectrophotometrically. The solvent-stable lipases were
subsequently confirmed by determining the residual activity after
incubation of the crude enzyme with 25% (v/v) of various solvents.
The lipase with broad tolerance to various organic solvents was
selected for the following research.
2.7. Cloning of lipase YCJ01
The Coomassie-stained band of the purified lipase YCJ01 was
excised from the gel and submitted to the National Center of
Biomedical Analysis (NCBA, Beijing) for analysis of trypsin-digested
protein fragments by LC/MS/MS analysis, and the sequences of
the fragments were submitted to the Mascot program for possible
identity matching. Since some fragments of the lipase were iden-
tical to the lipase from B. cenocepacia AU1054, the forward primer
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.3. 16S rDNA gene of strain YCJ01 and identification of the strain
ꢀ ꢀ
LU1-5 -CATGCATGGCCAAATCGATGC-3 and the reverse primer
The nucleotide sequence of 16S rDNA gene of strain YCJ01 has
ꢀ
ꢀ
been assigned GenBank Accession No. JQ733582. The strain YCJ01
was identified by combining the analysis of the Biolog Automated
Microbiology Identification System (BIOLOG USA) and 16S rDNA
sequence.
LD1-5 -TCACACGCCCGCGAGTTTCAA-3 were designed based on
the nucleotide sequence of the B. cenocepacia AU1054 lipase (NCBI
Accession No. YP 624306). PCR amplification was carried out with
the primers using the genomic DNA of strain YCJ01 as template.
The PCR product was purified and ligated into a PMD18-T simple
vector and then sequenced.
2
.4. Culture conditions for producing lipase
The inoculums were prepared by transferring loopfuls of fresh B.
2.8. Lipase YCJ01 gene sequence
ambifaria YCJ01 cells cultured on LB agar plates into an LB medium,
◦
followed by incubation at 30 C with shaking at 180 rpm for 12 h.
The nucleotide sequence of lipase YCJ01 has been assigned Gen-
Bank Accession No. JQ733583.
The 40 ml of the optimized culture medium in a 250 ml Erlenmeyer
flask was seeded with 2 ml of the inoculum. The incubation was
carried out at 30 C with shaking at 180 rpm. After 48 h incubation,
the supernatant of the culture was obtained by centrifugation and
was directly used as crude lipase for purification.
◦
2.9. Effect of pH and temperature on the activity and stability of
lipase YCJ01
To study the effect of pH on lipase activity, the lipase was assayed
at various pH values (6.5–9.5). pH stability was tested by preser-
ving the enzyme in buffers of various pH values varying from pH
2
.5. Assay for lipase activity and substrate specificity
◦
Lipase activity was measured spectrophotometrically using a
5.4 to 10.6 at 30 C for 1 h, and the residual activity was measured
modified assay based on the hydrolysis of p-nitrophenyl palmi-
tate (pNPP) [12]. The reaction mixture was composed of 240 l of
pNPP solution and 10 l of lipase solution. The pNPP solution was
prepared as follows: 1 ml isopropanol (to improve the solubility
of substrates) containing 8 mol pNPP was added to 9 ml 0.05 M
sodium phosphate buffer (pH 7.5) supplemented with 0.1% gum
arabic and 0.6% Triton X-100. The enzyme reaction was incubated
according to the pNPP method.
To study the effect of temperature on lipase activity, the enzyme
activity assay was conducted at various temperatures from 30 to
◦
75 C at pH 7.5. The thermostability of the enzyme was checked by
◦
incubating the enzyme at different temperatures (30–80 C) at pH
7.0 for 3 h. The residual lipase activity was assayed under standard
conditions.
◦
at 50 C for 10 min. The p-nitrophenol (pNP) liberated was quanti-
fied spectrophotometrically at 410 nm. One unit of lipase activity
was defined as the amount of enzyme that produced 1 mol of
p-nitrophenol (pNP) under the conditions mentioned above.
Substrate specificity of the enzyme was determined using p-
nitrophenyl esters with different chain lengths (C2, C4, C8, C10,
C14, C16, C18) as described above. The substrates dissolved in iso-
propanol were all at the same mole number (8 mol/ml).
2.10. Effect of metal ions, inhibitors and detergents on enzyme
activity
The effect of metal ions and inhibitors on lipase activity was
studied by preincubating the lipase with 1 and 10 mM of the ions or
◦
inhibitors, or 0.1% and 0.5% surfactants for 1 h at 30 C. The residual
lipase activity was measured by the pNPP method.
2.6. Purification of lipase YCJ01
2.11. Organic solvent stability of the lipase
The crude lipase was obtained as described in Section 2.4, and
then loaded onto a DEAE Sepharose FF column equilibrated with
0 mM Tris–HCl buffer (pH 7.25). The column was washed with
two bed volumes of the same buffer and, separately, with the
buffer containing 0.2 M NaCl, and then the bound proteins were
eluted by 1.0 M NaCl in the same Tris–HCl buffer. Fractions with
The effects of organic solvents with different log P values at 25%
(v/v) and 40% (v/v) concentration on the activity and stability of
the purified lipase were investigated following Ogino et al. [14].
Aliquots (1 ml) of organic solvents were added to the purified lipase
solution (3 ml), or 1.6 ml of organic solvent to 2.4 ml of the purified
lipase with 0.05–0.1 mg/ml protein concentration in a sealed glass
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