Biotechnological
Communication
Biosci. Biotech. Res. Comm. 9(3): 357-365 (2016)
Site-directed mutagenesis and thermal stability
analysis of phytase from
Escherichia coli
Jie Zhang*
1
, Yue Liu
1
, Shuping Gao
1
, Li Zhu
1
, Wei Li
1
, Xuewen Tian
2
and
Yuanyuan Liu
3
1
Key Laboratory of Biomedical Engineering & Technology of Shandong High School, Shandong Wanjie
Medical College, Zibo, 255213, China
2
Sports Science Research Center of Shandong Province, Jinan, 250102, China
3
School of Nursing, Weifang Medical University, Weifang, 261042, China
ABSTRACT
This study aims to obtain a phytase with thermal stability and acid resistance for potential industrial production.
The phytase gene appA was optimized according to Pichia pastoris and modi ed by site-directed mutagenesis,
which replaces speci c residues with another amino acid. Escherichia coli DH5 cells (cultured at 37 °C in Luria
broth media) were the host strain for recombinant DNA manipulation. P. pastoris GS115 and the plasmid pPIC9 were
the host strain and expression vector, respectively, for heterologous phytase gene expression. Through site-directed
mutagenesis, we obtained six mutants, namely, M1, C2, K24E, K43E, M2, and M4 of which the mutants M2 and M4
maintained higher activity in a wider reaction temperature range than other mutants. The mutants M1 and K24E
showed strong thermostability and retained more than 60% activity after heat treatment for 20 min (even at 90 °C).
We screened mutants that expressed phytase, which can withstand the high-temperature feed pelleting process and
retain a high level of phytase activity at the low pH of the monogastric gut environment.
KEY WORDS: PHYTASE; DESIGNATED MUTATIONS; THERMAL STABILITY;
ESCHERICHIA COLI
357
ARTICLE INFORMATION:
*Corresponding Author: wqlzq@gmail.com
Received 2
nd
Sep, 2016
Accepted after revision 25
th
Sep, 2016
BBRC Print ISSN: 0974-6455
Online ISSN: 2321-4007
Thomson Reuters ISI ESC and Crossref Indexed Journal
NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006
© A Society of Science and Nature Publication, 2016. All rights
reserved.
Online Contents Available at: http//www.bbrc.in/
IN TRODUCTION
Phytases (myo -inositol-hexaphosphate phosphohy-
drolase) catalyze the hydrolysis of phytate, the major
form of phosphorus storage in cereals and legumes
(Pandee, et al., 2011), into inositol and phosphoric acid
in a stepwise manner (Greiner, et al., 2001, Kim, et al.,
2008), thus increasing the available phosphorus con-
tent and decreasing the af nity of phytate to minerals
and proteins (Pandee, et al., 2011, Zhang, et al., 2007).
358 MUTATION AND PHYTASE THERMAL STABILITY FROM
ESCHERICHIA COLI
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Jie Zhang et al.
The addition of phytase in animal feed could not only
improve the utilization rate of phytate phosphorus in
feed efficiently but also signi cantly reduce the envi-
ronmental pollution from phytate phosphorus excreted
by swine and poultry, (Haefner, et al., 2005, Leytem, et
al., 2008and Kim, et al., 2010).Research on phytases has
recently focused on its application to human food and
the synthesis of lower inositol phosphates (Guerrero-
Olazaran, et al., 2010). Commercially produced phytases
are currently used worldwide. However, one of the prob-
lems that phytase still faces is its poor thermal stabil-
ity. Heat resistance of the phytase enzymes are desirable
during the pelleting process of phytase additives, which
can allow it to withstand high temperatures of 60 °C to
95 °C lasting at least 30 s, (Pandee, et al., 2011).
However, this characteristic is rarely found among
naturally occurring sources (Zhang, et al., 2007). There-
fore, site-directed mutagenesis (Kim, et al., 2008, Tran,
et al., 2011), error-prone PCR (Liao, et al., 2012), DNA
shuf ing, exon shuf ing, stagger extension processes
(Zhu, et al., 2010), and substituting residues (Zhang, et
al., 2007) have been used to enhance the thermal stabil-
ity of phytase.Phytases are diffusely distributed among
plants, animal tissues, and microorganisms (Guerrero-
Olazaran, et al., 2010, Liao, et al., 2012).
The most prevalent phytases are from bacteria, yeast,
and fungi (Liao, et al., 2012). Among many phytases,
Escherichia coli phytase AppA as a bifunctional enzyme
(Greiner, et al., 1993) has a great potential for indus-
trial applications due to its wide range of active acid
pH, high speci c activity for phytate, and resistance to
pepsin digestion (Greiner, et al., 1993, Luo, et al., 2007).
Thus, the product of the appA gene was chosen as a can-
didate to enhance the thermal stability of phytase and to
promote the residual activity of phytase during the pel-
leting process, which can reach temperatures as high as
70 °C to 90 °C (Zhang, et al., 2007). The phytase gene has
1233 bp and the protein it encodes has 410 amino acids.
The deduced amino acid sequence of appA contains an
RHGXRXP motif in the N-terminal and HD motif in the
C-terminal, which are characteristics in common with
histidine acid phosphatases, (Kim, et al., 2006, Pandee,
et al., 2011, Yao, et al., 2012, Fan, et al., 2013 and Roy,
et al., 2016).
Pich ia pastoris is a euka ryotic expression system that
was proposed in the 1980s and has since been widely
used for the production of various recombinant heter-
ogeneous proteins (Chang, 2008). By 2005, P. pastoris
had been used to successfully express more than 500
exogenous proteins (Cereghino, et al., 2000), many of
which had reached 10 mg·m L
−1
(Cregg, et al., 2000). As
one of the most ideal exogenous protein expression sys-
tems, P. pastoris has the advantage of being simple to
operate, being highly stable, having high expression lev-
els, and having large quantities of secretion (Li, et al.,
2001).
Based on the previous reports of enhancing the ther-
mal stability of phytase, (Kim, et al., 2008, Zhang, et al.,
2007, Zhu, et al., 2010, Liao, et al., 2012, Tran, et al.,
2011, Noorbatcha, et al., 2013, Rocky-Salimi, et al., 2016
and Tan et al., 2016), we chose six amino acid residues
sites which maybe change thermal stability by chang-
ing residual charge. In this study, phytase gene appA
was optimized according to P. pastoris and was modi ed
by site-directed mutagenesis. Site-directed mutagenesis
of the cloned appA gene was used to replace speci c
residues with another amino acid. The mutant genes
were expressed in P. pastoris GS115, and the recombi-
nant phytases were subjected to detailed assays of their
thermostability, pH dependence of enzyme activity, and
their Michaelis constant. The biochemical characteristics
of these recombinant phytases were compared to obtain
a phytase with excellent quality, which may have poten-
tial for industrial production.
MATERIAL AND METHODS
E. coli DH5 c ells (TaKaRa, Japan) were the host strain
for recombinant DNA manipulation and were cultured
at 37 °C in Luria broth media. P. pastoris GS115 and the
plasmid pPIC9 (Invitrogen, USA) was the host strain and
expression vector, respectively, for heterologous phytase
gene expression. Plasmid vector pGEM-T Easy (Promega,
USA) containing a fusion gene of the syntheti c phytase
gene appA and the synthetic signal peptide (designated
MF4I; maintained in our laboratory) was used as a PCR
template for site-directed mutagenesis. Oligonucleotide
primers for gene cloning and site-directed mutagenesis
were designed and synthesized by TaKaRa (Japan).
The phytate substrate (sodium salt, P0109) was pur-
chased from Sigma (USA). EcoRI, NotI, BamHI, BglII, Taq
DNA polymerase, T
4
ligase, a nd dNTP were purchased
from Promega (USA). Yeast extract–peptone–dextrose
medium, minimal dextrose (MD) medium, buffered glyc-
erol complex (BMGY) medium, and buffered methanol
complex (BMMY) medium were prepared according to
the P. pastoris Expression Kit manual (Invitrogen 2002).
All other chemicals were of analytical grade and were
commercially available.
SITE-DIR ECTED MUTAGENESIS
Double-stranded methylat ed plasmid pGEM-T E asy con-
taining the appA gene was utilized to encode the mutant
phytases. Six pairs of complementary primers (Table 1)
containing the desired point mutation were employed
to genera te the necessary mutants (K24E, K43E, W46E,
Q62W, A73P, and K75C). The many mutants of Citrobac-
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS MUTATION AND PHYTASE THERMAL STABILITY FROM
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359
Jie Zhang et al.
ter braakii phytase in Patent PCT/EP2011/054639 were
analyzed, and the part of the same site also generated
in phytase AppA to know their effect on Escherichia
coli phytase. The mutagenesis primers were extended
by PrimeSTAR®HS DNA polymerase in a thermocycling
process (94 °C for 4 min, 20 cycles at 94 °C for 10 s,
55 °C for 15 s, and 72 °C for 4 min). The product was
treated with DpnI at 37 °C for 1 h to remove methylated
and hemimethylated DNA template strands. The nicked
plasmid DNA containing the desired mutations was then
conveyed into E. coli DH5 cells, where the nick was
repaired by the cell.
CONSTRUCTION OF EXPRESSION VECTOR
Expression vectors pPIC9-appAm(X) were constituted by
digesting the recombinant plasmids pGEM-T Easy con-
taining mutant genes with NotI-EcoRI and EcoRI-BamHI,
respectively, obtaining appA
m
(X) and the synthetic sig-
nal peptide, and then inserting it into pPIC9 (Invitrogen)
digested with NotI-BamHI. The recombinant plasmids
pPIC9-appA
m
(X) were linearized with BglII and transformed
into P. pastoris GS115 (In vitrogen) using a LiCl method.
SC REENING AND EXPRESSION
The transformants were screened using SDS-PAGE and
the phytase activity assay. After being grown on the MD
plate and cultured in BMGY/BMMY medium at 30 °C,
six positive tr ansformants containing different muta-
tions were successfully obtained (M1, K24E, K43E, C2,
M2, and M4). To generate the enzyme, P. pastoris GS115
was c ultivated at 30 ° C, 200 rp m, and pH 6.0 in 50 mL
BMGY for pr oliferation in an Erlenmeyer  ask for 48
h; P. pastoris GS115 was then cultivated at 30 °C, 200
rpm, and pH 6.0 in 50 mL BMMY for the expression of
phytases in an Erlenmeyer  ask for 48 h.
PU RIFICATION O F RECOMBINANT PHYTASES
Purifying the re combinant proteins involves the precipi-
tation of ammonium sulfate followed by two chroma-
tographic steps. Ammonium sulfate precipitation of the
recombinant proteins was achieved by  rst saturating
the crude culture  ltrate to 80%; th e p recipitate is sub-
sequently collected by centrifugation and concentrated
at a speed of 10,000 rpm for 10 min. The precipitate was
subsequently dissolved in ac etate buffer (pH 4.5). The
traces of ammonium sulfate in the resuspended phytase
solution were removed by dialysis (2 h, three acetate
buffer washings).
A 1 mL Resource S column was equilibrated in
the a cetate buffer, and the dialyzed pr otein samples
were applied at a  ow rate of 0.6 mg·mL
−1
. A linear
sodium chloride gradient (0.2–1.0 M) was developed
in 10 min by using the same buffer. The activity was
eluted as a single component. In the  nal chromato-
graphic step, samples from the previous step were passed
through the Superdex 75 column at a  ow rate of 0.6
mg·mL
−1
. Activity was eluted as a single peak (at peak),
and the active fractions were pooled in the following
test.
ENZYME ACTIVITY ASSAY
Phytase activity was assayed using the molybdenum
blue method. The enzyme reaction was performed in 1
mL of 0.1 M sodium acetate buffer using sodium phytate
(6.25 mM) as a substrate at 37 °C. The reaction was ter-
minated by adding 1 mL of 10% (w/v) trichloroacetic
acid. One unit of phytase activity was de ned as the
amount of enzyme required to liberate 1 μmol of phos-
phate per minute under assay conditions. Protein was
quanti ed using the BCA assay (Fermentas). All deter-
minations were performed three times.
Table 1: Pr imers used for site-directed mutagenesis
Primer Name Primer Sequence (5’3’)
AppA (W46E)-Reverse CCTCTAGGTGTCAACTCACCCAGCTTGACTGGC
AppA (W46E)-Forward GCCAGTCAAGCTGGGTGAGTTGACACCTAGAGG
AppA (Q62W)-Reverse GCAACAAGACGCTGTCTCCAGTAGTGAC
AppA (Q62W)-Forward GTCACTACTGGAGACAGCGTCTTGTTGC
AppA (A73P)-Reverse GTGGACAACCCTTCTTGGGCAACAATCC
AppA (A73P)-Forward GGATTGTTGCCCAAGAAGGGTTGTCCAC
AppA (K75C)-Reverse GATTGTGGACAACCACACTTGGGCAACAATCC
AppA (K75C)-Forward GGATTGTTGCCCAAGTGTGGTTGTCCACAATC
AppA (K24E)-Reverse GTTGGGTGGCCTCGGTTGGTGCTCTAAC
AppA (K24E)-Forward GTTAGAGCACCAACC GAGGCCACCCAAC
AppA (K43E)-Reverse CAACCCAGCTCGACTGGCCAGGTTG
AppA (K43E)-Forward CAACCTGGCCAGTCGAGCTGGGTTG
360 MUTATION AND PHYTASE THERMAL STABILITY FROM
ESCHERICHIA COLI
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Jie Zhang et al.
OPTIMUM PH AND PH STABILITY
The optimum pH was determined by incubating the
puri ed phytase with sodium phytate in the following
buffers: pH 2.0–3.5 (0.05 M glycine-HCl), pH 4.0–6.0
(0.05 M sodium a cetate-acetic acid), and pH 6.5–8.0
(0.05 M Tris-HCl). These assays were performed at 37
°C for 5 min. For assays o f pH stability, the enzy me was
incubated at 37 °C in the same buffers over the range
of pH 2.0–8.0 for 2 h, and residual enzyme activity was
measured under standard conditions (optimum pH, 37
°C, 30 min).
OPTIMUM TEMPERATURE AND THERMAL
STABILITY
Enzyme activity was measured at nine differen t tem-
peratures ranging from 37–80°C to determine optimal
temperature. The temperatures chosen were 37, 40, 45,
50, 55, 60, 65, 70, and 80 °C. The tests were performed
using standard buffers. Thermal stability was measured
by assessing residual enzyme activity under standard
conditions (optimum pH, 37 °C, 30 min) followin g incu-
bation of the enzyme at six different temperatures. The
enzyme s amples were incubate d at 37, 50, 60, 70, 80, or
90 °C for 20 min, respectively. The enzyme samples were
then chilled on melting ice for 1 h before the test.
KINETIC MEASUREMENTS
V
max
and K
m
values for each phytase were determined at
37 °C in 0.05 M sodium acetate (pH 4.0 to 4.5, according
to the optimum pH of each enzyme) with 0.125–5.0 mM
sodium phytate as substrate. The initial reaction rates
were assayed for a 7 min period. The V
max
and K
m
were
estimated by Lineweaver–Burk plots.
STRUCTUR AL ANALYSIS OF MUTAN T PHYTASE
Basing on the crystal structur e of phytase AppA (Golo-
van, et al., 2000), structure analysis was applied to the
mutant that showed the most pot ential as a phytase-
producing strain. Three-dimensional structure predic-
tion was done using the program Swiss-PdbViewer 3.7.
RESULTS AND DISCUSSION
SITE-DIR ECTED MUTAGENESIS AND
CONSTRUCTION OF EXPRESSION VECTOR
Using a site-directed mutagenesis method, a total of six
mutants were obtained, including four single mutants
(K24E, K43E, W46E, and Q62W), one double mutant
(W46E and Q72W), and one quadruple mutant (W46E,
Q62W, A73P, and K75C; Table 2). Mutants were gener-
ated by introducing residual substitutions (K24E, K43E,
W46E, Q62W, A73P, and K75C) into the previously syn-
thetic phytase gene appA. The names of the mutants,
including the mutant sites, are presented in Table 2. The
designated mutations in each variant were veri ed by
DNA sequencing.
SCREENING AND EXPRESSION
The genes coding the protein without a native signal
sequence were inserted into vector pPIC9. Similar to
homologous recombination, the mutant genes with the
signal sequence of yeast -mating factor were inte-
grated into the genome of P. pastoris so that proteins
could be expressed s tably. After transforming the result-
ant recombinant plasmids, six p ositive transformants
that expressed phytase were screened by phytase activ-
ity assay and SDS-PAGE. The positive transformant
for each mutant, which was grown on the MD plate,
was transferred to BMGY and proliferated in an Erlen-
meyer  ask for 48 h. After continuous cultivation in
BMMY for 48 h, the supernatant protein had reached
1.0–1.3 mg·mL
−1
. The crude enzyme solution was pro-
duced after centrifuging supernatant protein in the cul-
ture medium (10,000 g for 10 min). The crude enzyme
solution of recombinant proteins from M1, C2, K24E,
K43E, M2, and M4 were  nally obtained using the same
method.
PURIFICATION OF RECOMBINANT PHYTASES
The phytases obtained from the supernatants were pur i-
ed to homogeneity by ammonium sulfate precipitation,
anion exchange chromatography, and gel  ltration on
Superdex 75 column ( Fig. 1). Only one peak with high
phytase activity was obtained when samples were chro-
matographed on gel  ltration. After three puri cation
steps, the recombinant proteins were puri ed 28.6-fold
from the crude extract with a  nal yield of 17%. The
puri cation factor and yield are related to elution pro-
cedure (Zhang, et al., 2010). The puri cation scheme of
the phytase is summarized in Table 3.
Table 2: Name of the mutant
including the mutant site
Name Mutant site
M1 W46E
C2 Q62W
K24E K24E
K43E K43E
M2 W46E, Q62W
M4 W46E, Q62W, A73P, K75C
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS MUTATION AND PHYTASE THERMAL STABILITY FROM
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Jie Zhang et al.
OPTIMUM PH AND PH STABILITY
As shown in Fig. 2a, the optimal reaction pH for the
mutant K24E was 4.0, whereas that of WT and other
mutant phytas es was 4.5. All phytase activities showed
one sharp peak at the optimal pH, and the relative activ-
ities of the phytases decreased signi cantly when the
pH value exceeded the optimal pH. After incubating in
the same buffer s ystem at different pH levels for 2 h,
the enzymes were puri ed and t he phytase activity was
tested under standard conditions (o ptimum pH, 37 °C,
30 min). The effects of pH on the stability of phytase
are shown in Fig. 2b. P hytase AppA and its mutants had
excellent pH stability (except for mutants K43E and C2),
and a fter incubation at 37 °C in different buffers ranging
from pH 2.0 to 8.0 for 2 h, the r esidual enzyme activity
was kept above 80%. A buffer of pH 2.0 or pH 8.0 might
not affect the enzyme activity signi cantly, but for the
mutants C2 and K43E, enzyme activity residue was less
than 30% after incubating with buffer of pH 8.0.
A: Optimal reaction pH of Mutant K24E is a round
4.0; Optimal reaction pH of AppA and other mutants
(M1, C2, M2, M4 and K43E) is a round 4.5. The every
assay value was averaged from three different experi-
ments. B: Except for mutant K43E and C2, Phytase appA
and mutants (M1, M2, M4 and K43E) all had a pH stabil-
ity and maintained more than 80% enzymatic activity
after 2 hours when they were place in pH 2.0-8.0 buffer
solution except for K43E and C2. The every assay value
was averaged from three different experiments.
OPTIMUM TEMPERATURE AND THER MAL
STABILITY
The opti mal reac tion temperature was 60 °C for the
mutants M1, C2, K24E, and K43E as well as for the
phytase AppA; however, the optimal temperature was
65 °C for the mutants M2 and M4. For the muta nts
M1, C2, K24E, and K43E, and the phytase AppA, the
enzyme activity decreased signi cantly when the reac-
tion temperature was lower than 55 °C or higher than
60 °C. For the mutants M2 and M4, the enzyme activ-
ity decreased distinctly when the reaction temperature
was lower or higher than 65 °C. As shown in Fig. 3,
the mutants M2 and M4 maintained higher activity
at a wider reaction temperature range than any other
mutants (M1, C2, K24E, or K43E) including the wild-
type phytase AppA. The phytase activity of wild -type
Table 3: Puri cation scheme of phytase
Step Total
activities
(/U·mL−1)
Total protein
(m/mg)
Speci c activity
(/U·mg−1)
Puri cation
(fold)
Recovery
(%)
Crude enzymes 93,866 19.56 4800 1 100
(NH
4
)
2
SO
4
(80%) 91,096 11.16 8157 1.7 97
Resource S 4561 1.53 27,818 5.8 46
Superdex-75 17,486 0.13 134,508 28.6 17
The every assay value was averaged from three different experiments.
FIGURE 1: Puri cation and SDS-PAGE of phytase A: Puri cation of phytase
with the Sephdex G-75 gel chromatogram; B: Lane 1: M1; Lane 2: K24E;
Lane 3: K43E Lane 4:C2; Lane 5: M2; Lane 6: M4 Lane 7: phytase AppA.
Jie Zhang et al.
362 MUTATION AND PHYTASE THERMAL STABILITY FROM
ESCHERICHIA COLI
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
AppA decr eased sharply when inc ubated at 50 °C for
20 min, and when inc ubated at 70, 80, or 90 °C for 20
min, the phytase activity practically did not exist. Fur-
thermore, the mutant phytases exhibited improved ther-
mostability compared with AppA. The phytase acti vity
remained above 30% after heat treatment at 70, 80, to
90 °C for 20 min, among which the mutants M1 and
K24E showed strong thermostability and retained more
than 60% activity after heat treatment for 20 min even
at 90 °C.
A: Optimal reaction temperature of phytase AppA
mutants (M2 and M4) is a round 65 °C; AppA and other
mutant is a round 60 °C. The every assay value was
averaged from three different experiments. B: The activ-
ity of phytase AppA decreased by 50% under 60 °C for
20 minutes and was almost entirely lost under 70 °C,
80 °C and 90 °C for 20 minutes; mutant K43E and C2
decreased by 70% under 90 °C for 20 minutes; mutant
M1, M2, M4 and K24E decreased by 40% under 80 °C for
20 minutes. The every assay value was averaged from
three different experiments.
KINETIC MEASUREMENTS
Under standard conditions (optimum pH, 37 °C, 7 min),
the K
m
and V
max
values of WT enzymes and its mutants
were calculated using sodium phytate as the substrate
and based on the Lineweaver–Burk method (see Table
4). As indicated in Table 4, the K
m
and V
max
of AppA
were 0.45 mM·L
−1
and 3.85 mmol·m
−1
·mg
−1
, r espec-
tively, whereas the K
m
of mutant K24E was 0.32 mM·L
−1
,
which was the lowest, and the V
max
of K24E was 4.10
mmol·(m·mg)
−1
, which was the highest. This  nding
indicated that K2 4E has the most af nity and the most
catalytic ef ciency to sodium phytate.
STRUCTURAL ANALYSIS OF MUTANT PHYTASE
An analysis of all the previous experimental results and
data shows that the mutant K24E outstrips all the other
mutants because of its low optimal reaction pH and high
optimal reaction temperature, preferable pH stability,
and outstanding thermostability. Therefore, the mutant
K24E was chosen to demonstrate structural analysis by
using 3D conformation. Using SWISS-MODEL protein
data analysis (template: 1dkq.1.A), the three-dimen-
sional conformations of mutant K24E and AppA were
found to have the same structure, and their structural
domains and active sites were not different. As shown
in Fig. 4, the crystal structure of AppA consisted of an
-helix, a -sheet, and an irregular coil. In addition, a
deep concave pocket harboring the active enzyme site
FIGURE 2: Optimal reaction pH and pH stability of phytase AppA and its mutants
FIGURE 3: Optimal reaction temperature and thermal stability of phytase AppA
and its mutants.
Jie Zhang et al.
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS MUTATION AND PHYTASE THERMAL STABILITY FROM
ESCHERICHIA COLI
363
within the surface of the domains was found. Ly s24 sub-
stituted by Glu was proximal to the concave pocket and
was located at the surface region of the pocket struc-
ture’s edge and was close to the active center site.
Some research con rmed that Escherichia coli AppA
phytase’s C-terminal plays an important role in its ther-
mostability by the thermostable mutants Q307D, Y311K,
and I427L (Fei, et al., 2012). Subsequently, the team
also found that the salt bridge subtraction mutant E31Q
showed 13.96% thermostability decreasement, and the
salt bridge addition mutant Q307D showed 9.15% ther-
mostability enhancement than the wild-type both with-
out the pH and temperature optimum changed (Fei, et
al., 2013). Wu et al. found that Escherichia c oli phytase
mutants V89T, one from eleven mutants, exhibited
17.5% increase in catalytic activity (Wu, et al., 2014).
In this study, we sought methods that reformed the
phy tase AppA to enable it to withstand the high-tem-
perature feed pelleting process and retain a high level
of phytase activity at the low pH of the monogastric gut
environment (Yao, et al., 2013).
Using site-directed mutagenesis, we successfully
screened six mutants (M1, C2, K24E, K43E, M2, and
M4). Phy tase AppA was puri ed to homogeneity by
ammonium sulfate precipitation, anion exchange chro-
matography, and gel  ltration on a Superdex 75 col-
umn. Using the same strategy, we also obtained puri-
ed protein from the six mutants. Subsequently, we
carried out experiments on optimal reaction pH and pH
stability, optimal reaction temperature, thermostability,
and the Michaelis constant. The six mutant enzymes
showed varying degrees of improvement in thermo-
stability compared with the wild-type enzyme, among
which the mutant K24E was the most stable even after
being heated to 80 °C or 90 °C for 20 min. As shown by
the results of the Michaelis constant, the K
m
of mutant
K24E was lower and the V
max
was higher than wild-type
AppA. Th e optimal reaction pH of K24E was 4.0 and
the residual activity of K24E remained above 90% after
tre atment in the pH range of 3.0–8.0 for 2 h. The optimal
reaction temperature of K24E was 60 °C, and the residual
activity of K24E remained above 60% after treatment in
70, 80, or 90 °C for 20 min. Using the Lineweaver–Burk
method, the K
m
and V
max
values of mutant K24E were
calculated to be 0.32 mM·L
−1
and 4.10 mmol·(m·mg)
−1
,
whereas K
m
and V
max
of AppA were 0.45 mM·L
−1
and 3.85
mmol·(m·mg )
−1
, respectively. The above results showed
the thermal resistance of mutant phytase AppA at 70-90
is higher than previously reported, (Liao, et al., 2013, Xu
et al., 2015 and Tan et al., 2016).
This result indicated that K24E has more af nity and
more catalytic ef ciency toward sodium phytate com-
pared with AppA. We applied a 3D structure analysis on
mutant K24E. SWISS-MODEL was employed for model
building of mutant K24E by using the X-ray structure of
phytase AppA as a template.
The validated mutant structure was aligned with the
wild-type structure by using SWISS-MODEL protein data
analysis. As shown in F ig. 4, the 3D conformations of
mutant K24E and AppA were found to have the same
structure, and a deep concave pocket harboring the
enzyme active site in the inner surface of the domains
was found. Mutant K24E was located at a supporting
point on the surface of the pocket edge structure and
close to the active center site. The replacement of Lys with
Glu, which has a smaller side chain, might reduce struc-
tural hindrances when combined with the substrate. This
phenomenon might be the reason behind the increased
af nity of enzyme and substrate and the decreased K
m
of
enzymatic reactions. The electrostatic interaction between
enzyme and substrate increased as the positively charged
Lys mutated into the negatively charged Glu. This inter-
FIGURE 4: Model of mutant K24E A: Model of mutant K24E; B: Model of AppA
Table 4: K
m
and V
max
of phytase AppA and its mutants
Sample AppA M1 C2 K24E K43E M2 M4
Km ( mM/L) 0.45 0.38 0.43 0.32 0.37 0.35 0.47
Vmax (mmol·m
−1
·mg
−1
) 3.85 2.86 2.16 4.10 4.04 3.18 2.04
The K
m
and V
max
value was averaged from three different experiments.
Jie Zhang et al.
364 MUTATION AND PHYTASE THERMAL STABILITY FROM
ESCHERICHIA COLI
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
action might be another reason for the increase of af nity
between phytase and its substrate. The isoelectric point of
Lys and Glu are pH 9.74 and 3.22, respectively. Substitu-
tion of Lys for Glu presumably contributes to the decrease
of the optimum reaction pH. Our results have showed that
phytase AppA mutant had excellent pH stability (Keeping
80% residual enzyme activity from pH 2.0 to 8.0 for 2 h
at 37° C), which is more favorable to apply to animal feed
than wild-type AppA and other phytase (Rocky-Salimi,
et al., 2016).
ACKNOWLEDGMENTS
This work was supported by grants from the Natural
Scienti c Foundation of Shandong Province, China
(ZR2014CM046, ZR2010CQ031 and ZR2015CL019) and
Collaborative Innovation Center of Chinese medicine
antivirus in Shandong University of Tranditional Chi-
nese Medicine (XTCX2014B01-07).
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