2
Y. Gong et al. / Journal of Molecular Catalysis B: Enzymatic 109 (2014) 1–8
the method of choice to engineer this particular property
[13–15].
(1–3 amino acids substitutions per protein) for the 1.5 kb parent
gene. The mutagenic PCR products were gel purified, digested with
SalI and NotI, and ligated into the vector pET28a which was digested
with the same two restriction enzymes. E. coli BL21 (DE3) cells were
transformed with the resultant mutant plasmids and plated onto
LB agar medium supplemented with 50 g/mL of Kanamycin.
Directed evolution does not require detailed information of
structures or accurate prediction on amino acid substitutions at
proper sites [16]. It involves generating a vastly mutated genes
library of interest by random mutagenesis such as error-prone
polymerase chain reaction (epPCR) or DNA shuffling, followed
by screening mutants with specified criteria for desired proper-
ties. This approach has been particularly successful in improving
matically enhanced by 110-fold through combining mutations
from epPCR and in vivo shuffling with several mutants constructed
by site-directed mutagenesis. A thermostable fungal cellobiohy-
drolase (Cel6A) [18] was engineered by random mutagenesis and
recombination of beneficial mutations, which could reduce the
reaction time for 10-fold in 75 ◦C than that of the wild type
enzyme in 60 ◦C for avicel hydrolysis. Crystal structures showed
the enhanced hydrophobic interactions and confined loop confor-
mations contribute to the thermostability. These successful studies
prompted us to generate thermostable BSE variants via directed
evolution.
In the present work, we performed one round of directed evo-
lution via random mutagenesis, in which three variants with better
the three variants were combined together by site-directed muta-
genesis, affording BSEV4. It is reported that the protein stability is
related to the rigidity, while the activity is related to the flexibility
[19]. We hypothesize that, increasing the rigidity might lead to a
higher stability. When the rigidity increases, the flexibility could
decline, which might lead to the loss of activity. Thus, our goal is to
find a variant with higher thermostability, while avoiding signifi-
cant loss of the activity. In our study, BSEV4 showed 4.7 ◦C increase
in T5105 than that of the wild type BSE, due to the additional introduc-
tion of ionic bonds, hydrogen bonds and hydrophobic interactions,
making BSEV4 a more desirable biocatalyst in practical application.
2.3. Screening for thermostable mutants
The screening for BSE mutants with improved thermostabi-
lity was performed in 96-well microplates. The colonies harboring
mutant esterase genes were transferred into 96-well plates, with
several colonies carrying parent gene of BSE as a control. The
colonies were grown overnight at 37 ◦C and 220 rpm. These plates
served as master plates and they were suspended with glycerol
(final concentration, 8%) and stored at −80 ◦C. An appropriate
amount of suspension was transferred into 600 L fresh medium,
cultivated at 37 ◦C and 220 rpm for 2 h. Then the enzyme production
was induced by addition of IPTG (final concentration, 0.5 mM). After
cultivation for another 10 h at 30 ◦C and 220 rpm, the microplates
were centrifuged at 1660 × g for 10 min at 4 ◦C, and the supernatant
was discarded. The cell pellets in microplates were frozen at −80 ◦C
for 2 h and thawed at room temperature for 20 min. To each well,
200 L lysis buffer was added, containing 5 mM Tris–HCl (pH 8.5),
0.075% lysozyme and 1 U mL−1 DNase I. Then the plates were incu-
bated for 1 h at 37 ◦C, and the cell suspensions were diluted by 1:5
with 5 mM Tris–HCl (pH 8.5), and centrifuged again at 1660 × g for
20 min. The supernatant was treated at 55 ◦C for 15 min in a thermal
cycler and then the residual activity was measured.
The reaction mixture for screening consisted of 220 L solu-
tion in each well [20], including 20 L double indicators containing
0.5 mg/mL bromothymol blue and phenol red, respectively (dis-
solved in 5 mM Tris–HCl, pH 8.0), 80 L 100 mM calcium chloride
(dissolvedin 5 mM Tris–HCl, pH8.0), 50 Ldl-menthylacetatesolu-
tion (200 mM dissolved in DMF), 50 L Tris–HCl buffer (5 mM, pH
8.0) and 20 L enzyme solution. The color change was observed at
630 nm for 15 min at 30 ◦C.
2. Experimental
2.4. Site-directed mutagenesis
2.1. Materials and chemicals
Site-directed mutagenesis was used to introduce amino acids
for the generation of BSEV4. The site-directed mutagenesis was
performed by using the KOD-Plus-Neo polymerase. The reaction
mixture contained 1× buffer with 1.5 mM MgSO4, 0.2 M of each
dNTP, 1 M fw- and rev-primer, 1.0 U KOD-Plus-Neo DNA poly-
merase and 50 ng template DNA (BSE gene in plasmid pET28a [7]).
The extension reaction was initiated by pre-heating the reaction
mixture to 94 ◦C for 2 min. After adding the DNA polymerase, the
reactions were carried out 20 cycles of heating at 98 ◦C for 10 s,
annealing at 55–65 ◦C for 30 s according to the melting tempera-
ture of the primer pair, followed by elongation at 68 ◦C for 3.5 min.
The template DNA was digested with 10 U DpnI for 1 h at 37 ◦C. Two
microliters of the plasmids containing the mutated BSE gene was
transformed into 50 L competent cells of E. coli BL21 (DE3).
Tryptone and yeast extract were obtained from Oxoid (Shanghai,
China). All restriction endonucleases and T4 DNA ligase were
purchased from TaKaRa (Dalian, China). The recombinant plas-
mids of wild type BSE (GenBank accession number: KM203868)
in pET28a were the products of our previous study [8]. E. coli strain
BL21 (DE3) from Tiangen (Shanghai, China) was used as the host
strain for gene cloning and expression. The strain was routinely
grown in Luria–Bertani medium at 37 ◦C unless stated otherwise.
Kanamycin (50 g/mL) was used for the selection of recombinant
strains of E. coli. The pET28a expression vector was purchased from
Novagen (Shanghai, China). rTaq DNA polymerase and the restric-
tion enzymes (DpnI, SalI and NotI) were obtained from TaKaRa.
High fidelity DNA polymerase, KOD-plus-Neo, was purchased from
Toyobo (Shanghai, China).
2.5. Expression and purification of parental BSE and its variants
2.2. Construction of random mutagenesis library
The entire procedure was performed at 4 ◦C. Cell pellets were
collected, suspended in 0.1 M potassium phosphate buffer (pH 7.4,
plus 0.5 M sodium chloride) and lysed by 99 rounds of sonication,
each working for 6 s and stop for 4 s, with an ultrasonic oscillator
(JY92-II, Scientz Biotech. Co., Ltd.). After centrifugation at 10,000 × g
for 20 min, the supernatant was used for enzyme purification. The
purification was carried out as described previously [21].
Random mutagenesis library was constructed by epPCR. Plas-
mid pET28a-BSE containing the wild type esterase gene was
used as the template for the first generation of random muta-
genesis. Primers 5ꢀ-ACGCGTCGACATGACTCATCAAATAGTAACG-3ꢀ
and 5ꢀ-AAGGAAAAAAGAGCGGCCGCTTATTCTCCTTTTGAAGGGAA-
3ꢀ were used as forward and reverse primers, respectively. MnCl2
(0.15 mM) was used to obtain the desired level of mutagenesis rate