Food Biotechnology

ISSN: 0890-5436 (Print) 1532-4249 (Online) Journal homepage: http://www.tandfonline.com/loi/lfbt20

Single-site mutation of C363G or N463T

strengthens thermostability improvement of
IG181–182 deleted acidic α-amylase from deep-sea
thermophile Geobacillus sp.

Yanyun Gao, Mengmeng Huang, Xiaoyue Sun, Xiaoxu Zhang, Yuanxing

Zhang, Xiangshan Zhou & Menghao Cai

To cite this article: Yanyun Gao, Mengmeng Huang, Xiaoyue Sun, Xiaoxu Zhang,
Yuanxing Zhang, Xiangshan Zhou & Menghao Cai (2017) Single-site mutation of C363G
or N463T strengthens thermostability improvement of IG181–182 deleted acidic α-amylase
from deep-sea thermophile Geobacillus sp., Food Biotechnology, 31:1, 57-71, DOI:
10.1080/08905436.2016.1276462

To link to this article: http://dx.doi.org/10.1080/08905436.2016.1276462

Published online: 19 Feb 2017.

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FOOD BIOTECHNOLOGY
2017, VOL. 31, NO. 1, 57–71
http://dx.doi.org/10.1080/08905436.2016.1276462

Single-site mutation of C363G or N463T strengthens

thermostability improvement of IG181–182 deleted acidic
α-amylase from deep-sea thermophile Geobacillus sp.
Yanyun Gaoa, Mengmeng Huanga, Xiaoyue Suna, Xiaoxu Zhanga,
Yuanxing Zhanga,b, Xiangshan Zhoua, and Menghao Caia
a
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology,
Shanghai, China; bShanghai Collaborative Innovation Center for Biomanufacturing, Shanghai, China

ABSTRACT KEYWORDS
The acidic α-amylase Gs4j-AmyA from deep-sea thermophile Acid α-amylase;
Geobacillus sp. is more acid-resistant than the commercial sources Geobacillus sp; single-site
as it displays more than 50% of the optimum at a range of pH 4.5– mutation; thermophile;
7. This may allow the removal of the step of pH adjustment in thermostability
starch processing and save costs. Unfortunately, this amylase is
not very stable at 90–95°C. Therefore, to develop a new commer-
cial acidic α-amylase targeted to the food industry, it is necessary
to further improve the thermostability of the α-amylase Gs4j-
AmyA. In this study, 11 different α-amylase mutants were
obtained by site-mutagenesis. Among them, the mutant IG181–
182* (IG181–182 deletion) showed significant improvement in
thermostability, whose half-life at 70°C was 63.4 times longer
than the wild type. Interestingly, single-site mutants C363G and
N463T showed no enhancement on thermostability, while the
half-lives of the combination mutants IG181–182*/C363G and
IG181–182*/N463T at 70°C were further extended by 16.8 and
38.7%, respectively. Unfortunately, the catalytic constants (kcat) of
the mutants C363G, N463T, IG181–182*/C363G and IG181–182*/
N463T declined by 59, 49, 37 and 16%, respectively. The optimum
temperature (65–70°C) and pH (5.5–5.6) of the mutants was
unchanged. The thermostability improvement by IG181–182
deletion could be strengthened by synchronous C363G or
N463T mutation.

Introduction
α-Amylase (endo-1,4-α-D-glucan glucanohydrolase; EC 3.2.1.1) indistinc-
tively catalyzes the hydrolysis of 1,4-α-D-glucosidic bonds in starch and
related carbohydrates, releasing α-anomeric products. The acidic α-amylase
is intensively used in the food industry including baking, brewing, starch
syrups and the production of cakes (Paula and Pérola, 2010). In food industry
practice, pH adjustment is often inevitable because commercial α-amylases

CONTACT Xiangshan Zhou and Menghao Cai xszhou@ecust.edu.cn; cmh022199@ecust.edu.cn State Key
Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road,
Shanghai 200237, China.
Color versions of one or more of the figures in this article can be found online at www.tandfonline.com/lfbt.
© 2017 Taylor & Francis
58 Y. GAO ET AL.

generally become inactivated below pH 5.0 and the pH of natural starch slurry
is 4.5 (Prasanna, 2005; Sivaramakrishnan et al., 2006). In order to reduce the
cost of labor and resources, it is necessary to develop novel types of α-amylase
with better acid-resistance and thermostability than those currently used in the
food industry (Sharma and Satyanarayana, 2013).
Based on the superior performances of BLA (Bacillus licheniformis α-amy-
lase) (Declerck et al., 2000; Suvd et al., 2001), the thermostability mechanism of
α-amylase has been extensively studied. The X-ray crystal structures of some
α-amylase molecules have been obtained, including BLA, BSA (B. subtilis
α-amylase), and BStA (B. stearothermophilus α-amylase) (Machius et al.,
1995; Fujimoto et al., 1998; Suvd et al., 2001). The structures of α-amylases
are generally similar, with the same three conserved structure domains despite
the fact that the properties of amylases vary a great deal. Among the three
conserved domains (Janeček et al., 2014), domain B is the key to thermo-
stability and keeps stable under high temperatures when binding with Ca2+
(Nielsen and Borchert, 2000; Priyadharshini and Gunasekaran, 2007). The
substitution of residues involved in the salt-bridges, Ca2+-binding, potential
deamination and oxidation process with similar amino acids tends to improve
the thermostability of α-amylases (Declerck et al., 2003).
A new acid-resistant thermostable α-amylase Gs4j-AmyA has been
recently discovered from deep-sea thermophile Geobacillus sp. (Jiang et al.,
2015). The new acidic α-amylase Gs4j-AmyA from Geobacisllus sp. has some
desirable properties including acid-resistance and high raw starch hydrolysis.
Gs4j-AmyA is more acid-resistant than the commercial sources as it displays
more than 50% of the optimum at a range of pH 4.5–7 (Jiang et al., 2015).
This may allow removal of the step of pH adjustment and save costs.
However, it is not stable at 90–95°C. This has reduced its practical utility
in applications requiring high-temperature hydrolytic efficiency. This
enzyme is a proper starting point from which to design a more thermostable
acidic α-amylase. The thermal stability can be improved by different pro-
cesses, such as immobilization, chemical modification, protein engineering
and by different additives. Protein engineering, including site-mutagenesis
and directed-evolution, is a relatively popular and routine practice for both
academic and industrial needs (Dey et al., 2016). Moreover, based on the
relatively clear molecular mechanism of thermostable amylases (Declerck
et al., 2002), the structure-guided site-mutagenesis is a preferential method
to improve the thermal stability of the α-amylase, rather than directed
evolution. Based on the studies on thermostability of BLA and BStA, this
research aimed to improve the thermostability of the α-amylase of
Geobacillus sp. with different site-mutagenesis strategies. Interestingly, it
was found that mutations C363G and N463T promoted the improvement
of thermostability by IG deletion. This research potentially provides a refer-
ence for other α-amylase-targeted evolution studies and some experimental
 FOOD BIOTECHNOLOGY 59

evidence for thermal inactivation mechanism (Tomazic and Klibanov, 1988)

of the thermostable α-amylase.

Materials and methods

Selection of target residues
Sequence alignment by the software GENE DOC was used to identify the
amino acid sequence similarity between different amylases. Some amino
acids related to thermostability in the amylase BLA could be obtained,
according to the description of mutagenesis in the database UniProt
(http://www.uniprot.org/uniprot/P06278) and some references (Suzuki
et al., 1989; Conrad et al., 1995; Declerck et al., 2000, 2003).

Site-directed mutagenesis
One-step PCR method was carried out using PrimeSTAR HS DNA polymer-
ase with recombinant plasmid pET-28a (+)-ompA-Gs4j-amyA plasmid (Jiang
et al., 2015) as template. The oligonucleotides used for site-directed muta-
genesis are listed in Table 1. The treated PCR products with DpnI restriction
enzyme (NEB) were transformed into Escherichia coli Top10 and confirmed
by sequencing (Sonny Sequencing Inc. or GENEWIZ Inc., China).

Table 1. Oligonucleotides used for site-directed mutagenesis.

Name Sequence Tm (°C)
RGI179–181Q 5′-GAGCCGCATTTACAAATTCCAAGGCAAAGCGTGGGATTGG-3′ 69
5′-CCAATCCCACGCTTTGCCTTGGAATTTGTAAATGCGGCTC-3′
K272A 5′-GGAGCTATGACATCAACGCCTTGCACAATTACATTACG-3′ 66
5′-CGTAATGTAATTGTGCAAGGCGTTGATGTCATAGCTCC-3′
IG181–182* 5′-CATTTACAAATTCCGCGGCAAAGCGTGGGATTGGGAAGTAG-3′ 69
5′-CTACTTCCCAATCCCACGCTTTGCCGCGGAATTTGTAAATG-3′
N193F 5′-GGGAAGTAGACACGGAATTCGGAAACTATGACTACTTAATG-3′ 66
5′-CATTAAGTAGTCATAGTTTCCGAATTCCGTGTCTACTTCCC-3′
T191R 5′-GTGGGATTGGGAAGTAGACAGGGAAAACGGAAACTATGAC-3′ 68
5′-GTCATAGTTTCCGTTTTCCCTGTCTACTTCCCAATCCCAC-3′
R157Y 5′-CTACTCCAGCTTTAAGTGGTACTGGTACCATTTTGACGGCG-3′ 69
5′-CGCCGTCAAAATGGTACCAGTACCACTTAAAGCTGGAGTAG-3′
A184T 5′-CCGCGGCATCGGCAAAACGTGGGATTGGGAAGTAGACAC-3′ 72
5′-GTGTCTACTTCCCAATCCCACGTTTTGCCGATGCCGCGG-3′
Q336E 5′-CATGACACCGAACCCGGCGAAGCGCTGCAGTCATGGGTC-3′ 74
5′-GACCCATGACTGCAGCGCTTCGCCGGGTTCGGTGTCATG-3′
Q128R 5′-CAATCCGTCCGACCGCAACCGAGAAATCTCGGGCACCTATC-3′ 73
5′-GATAGGTGCCCGAGATTTCTCGGTTGCGGTCGGACGGATTG-3′
C363G 5′-CAGGAAGGATACCCGGGCGTCTTTTATGGTGAC-3′ 67
5′-GTCACCATAAAAGACGCCCGGGTATCCTTCCTG-3′
N463T 5′-GGAGTGACACCGTCACCATCACCAGTGATGGATGGGGGGAATTC-3′ 73
5′-GAATTCCCCCCATCCATCACTGGTGATGGTGACGGTGTCACTCC-3′
Nucleotides printed in bold and underline were the positions chosen for mutation. Tm: Temperature of
melting, that is, a temperature at which a double-helix of the DNA sequence is denatured.
60 Y. GAO ET AL.

Subsequently, the purified mutant plasmids from E. coli Top10 were trans-
formed into E. coli BL21 (DE3) pLysS for expression of Gs4j-AmyA mutants.
Combination mutants were constructed through the same method with
single mutagenesis using the mutant plasmids as template.

Analysis of thermostability of mutant α-amylases

The recombinant E. coli BL21(DE3)pLysS was cultured (exposed to the air) at
37°C in Luria-Bertani (LB) medium containing 50 μg/ml of kanamycin.
When the OD600 reached 0.6–0.8, 1 mM isopropyl-β-d-thiogalactopyranoside
(IPTG) was added to induce α-amylase expression at 37°C. After 48 h, 1 ml
of culture broth containing the secreted enzymes of interest was centrifuged
at 12,000 g for 1 min to remove cells.
The thermostability of α-amylases in the supernatant was determined
initially according to the residual enzyme activity after incubation in acetate
buffer (25 mM sodium acetate buffer, pH 5.5) for 10 min at 100°C (in a
boiling water bath). Then, the enzyme activity was measured at pH 5.5 and
65°C. Relative enzyme activities were calculated and the activity of the
enzyme without incubation was defined as 100%.

Protein purification
The α-amylases in the cell free supernatant were roughly concentrated by the
ratio of 15:1 (The volume changed approximately from 500–33 ml) by ultra-
filtration (MWCO = 10 kDa) with the Lab-Scale TFF System (Millipore,
Germany). During ultrafiltration, the solution of α-amylases was changed
from LB medium to the buffer (10 mM NaH2PO4, 10 mM Na2HPO4, 0.5 M
NaCl, 20 mM imidazole at pH 7.4). Gs4j-AmyA and its mutants were purified
with a Bio-Rad Biologic LP System by using a Ni2+-charged 1 ml of Bestarose
6FF Crude column (Bestchrom Biotech) at a flow rate of 1 ml/min. The
recombinant α-amylases were eluted according to Jiang et al. (2015) with
buffer (10 mM NaH2PO4, 10 mM Na2HPO4, 0.5 M NaCl, 250 mM imidazole
at pH 7.4). Active fractions were transferred into ultrafiltration devices, added
with appropriate amount of the phosphate buffer solution (PBS) (137 mM
NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4), and centrifuged at
5000 g to remove the imidazole in the elution buffer. The sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was per-
formed on a 12% running gel (1.6 ml water, 2 ml 30% acrylamide solution,
1.3 ml 1.5 mol/l Tris [pH 8.8], 50 μl 10% SDS, 50 μl 10% ammonium persulfate,
2 μl TEMED) and protein bands were visualized by staining with Coomassie
brilliant blue R-250. The protein concentration was determined by Bradford
method of a kit (Tiangen Biotech, Beijing, China).
 FOOD BIOTECHNOLOGY 61

Measurement of enzymatic activity of purified α-amylases

The enzyme activity was determined by measuring the amount of reducing
sugar released during enzymatic hydrolysis of 1% soluble starch in acetate
buffer (pH 5.5) at 65°C for 10 min. A control without enzyme addition was
used. The reducing sugar was determined by 3,5-dinitrosalicylic acid (DNS)
method (Ghose, 1987). One unit of α-amylase was defined as the amount of
enzyme that liberates 1 μmol of reducing sugars per minute under the assay
condition.
The thermostability of the enzyme solution was assessed by incubating in
acetate buffer (pH 5.5) for appropriate time at 70, 85 and 95°C. Then the
remaining enzyme activity was determined at 65°C. Enzyme inactivation
kinetics equation was expressed as the following Eq. (1) (Ghose, 1987):
ln ðEt =E0 Þ ¼ kd t (1)

where kd is enzyme inactivation constant, t is time of heat treatment, E0 and

Et are the relative activities at initial time and that time, respectively. And kd
can be calculated as the slope of ln (Et/E0) versus time t. Thus, half-life of the
enzyme was obtained by the following Eq. (2):
t1=2 ¼ ðln 2Þ=kd (2)

The optimal temperature of purified enzyme was determined by enzyme

assay in acetate buffer (pH 5.5) at various temperatures within the range of
30–100°C. For each α-amylase, the highest activity at any temperatures (WT,
70°C; IG181–182*, 65–70°C; C363G, 70°C; N463T, 70°C; IG181–182*/
C363G, 70°C; IG181–182*/N463T, 70°C) was designated 100%. In the case
of IG181–182*, the average value of enzyme activities at 65 and 70°C was
designated 100%. Different buffers (25 mM acetate buffer, pH 4.0–5.8; 25
mM phosphate buffer, pH 5.8–8.0) were used to determine the optimal pH at
the optimum temperature of each α-amylase.
The kinetic parameters (Km, Vmax, kcat, and kcat/Km) of the wild type and
mutants were determined in the acetate buffer (pH 5.5, 25 mM sodium
acetate buffer) at 65°C. The enzyme was maintained at a proper concentra-
tion and soluble starch was added to 1–20 mg/ml. The Km and Vmax were
acquired from a Michaelis Menten fitting plot by the software Origin 8.6.

Statistical analysis
All experiments were independently conducted at least three times, and the
results were expressed as means ± standard deviations (SD). Statistical
analyses were performed with the Student’s t-test; a p value less than 0.05
was considered statistically significant.
62 Y. GAO ET AL.

Homology modeling of tertiary structure and key amino acid analysis

The online software Swiss-Model (http://swissmodel.expasy.org/) was used to
predict the structure of Gs4j-AmyA (Benkert et al., 2011). The theoretical
structure of Gs4j-AmyA was obtained through homology modeling with the
Swiss-Model server by using the crystal structure of the α-amylase (PDB ID:
4UZU) from G. stearothermophilus as template (Offen et al., 2015). There
was 99.58% sequence identity between model and template. Mutated key
amino acids analysis and annotation were carried out with software Pymol
and Swiss-Pdb-Viewer.

Results
Key amino acids for thermostability
As a result of the extraordinary thermostability of BLA, corresponding amino
acid residues in Gs4j-AmyA were selected with reference to the amino acids
related to thermostability in BLA (Declerck et al., 2000, 2003). According to
the results of sequence alignments (Fig. 1a), the identity between BLA and
Gs4j-AmyA was 58%. The key amino acids for thermostability in BLA were
predicted to benefit Gs4j-AmyA because of the conservation of the three-
dimensional structure of α-amylases. RGI179-181Q and K272A in Gs4j-
AmyA were selected according to the research on thermostability of chimeric
enzymes of BLA and BAA (B. amyloliquefaciens α-amylase) (Suzuki et al.,
1989; Conrad et al., 1995). The deletion of the two amino acids IG181–182
had been confirmed to improve the thermostability of α-amylases in Bacillus
KSM-1378 (Igarashi et al., 1998) and B. stearothermophilus US100 (Ali et al.,
2006; Khemakhem et al., 2009). Besides, the mutated amino acids, beneficial
to thermostability in BLA, were predicted to be applied to the α-amylase
Gs4j-AmyA. Consequently, IG181–182* (Ali et al., 2006), R157Y, A184T
(Frantzen et al., 1999), N193F, T191P, Q336E and Q128R (Declerck et al.,
2000, 2002, 2003; Machius et al., 2003) in Gs4j-AmyA were identified as
potential mutating sites to improve thermostability.
Sequence alignments (Fig. 1b) of Gs4j-AmyA and BStA showed that there
were only three different amino acids including one in signal peptide. The α-
amylase from B. stearthermophilus US100 appeared more thermostable than
Gs4j-AmyA. According to the research results of Jiang et al. (2015) and Ali
et al. (2006), there was hardly any residual activity of Gs4j-AmyA after
incubation at 90°C for 10 min, while the BStA remained at about 60%
relative activity after incubation at 100°C. Notably, only two amino acid
sites (C363G and N463T) in the sequences without signal peptides were
different (Fig. 1b). In addition, the amino acids C and N contributed to the
irreversible inactivation of B. stearthermophilus α-amylase (Tomazic and
Klibanov, 1988). Therefore, the two different amino acids C363G and
 FOOD BIOTECHNOLOGY 63

Figure 1. Sequence alignments of Gs4j-AmyA and two other α-amylases. (a) Alignment of amino
acid sequences of Gs4j-AmyA and BLA, an α-amylase from Bacillus licheniformis (P06278).
(b) Alignment of amino acid sequences of Gs4j-AmyA and BstA, an α-amylase from B. stear-
othermophilus US100 strain (NCBI GenBank accession No. CAB93517). Amino acid sequences were
aligned by the software GENE DOC.

N463T were predicted to be key amino acids related to thermostability and

selected as key amino acids for thermostability.

Preliminary selection of beneficial mutants

After the mutant supernatant was obtained, preliminary selection was carried
out by determining the remaining enzyme activity after incubating the
supernatant at 100°C for 10 min. As shown in Fig. 3a, two mutants
RGI179–181Q and IG181–182* remained about 75% relative enzyme activity
in fermentation supernatant, indicating a significant enhancement on ther-
mostability (p < 0.001) compared to 17% remaining activity of the wild type.
In fact, the mutated amino acid site of the two mutants RGI179–181Q and
IG181–182* was the same, site 179. The amino acid at 179 in RGI179–181Q
was Q, while the corresponding amino acid at 179 in the mutant IG181–182*
was R. Besides, the mutations of N193F, C363G and N463T also showed
obvious improvement in thermostability compared with the wild type
(Fig. 3a). The remaining enzyme activity increased from 17% in the wild
64 Y. GAO ET AL.

type to 26% (p = 0.035) in N193F, 31% (p = 0.003) in C363G and 33% (p =

0.002) in N463T. Finally, the top three thermostable mutants IG181–182*,
C363G and N463T were selected for further research. The combination
mutants IG181–182*/C363G and IG181–182*/N463T were then obtained to
investigate their synergetic function.

Enzymatic characterization of thermostable mutants

According to SDS-PAGE analysis (Fig. 2), the purity of α-amylases almost
reached the electrophoretic pure level with few impure protein bands. The
thermostability of the wild type and mutants was indicated by their half-lives
at three different temperatures of 70, 85 and 95°C.
The half-lives of the mutants at 70°C were compared with the wild type
(Fig. 3b). Among single-point mutations, the mutant IG181–182* showed
significant improvement in thermostability, and its half-life at 70°C was 63.4
times (p < 0.001) longer than the wild type. However, the thermostability of
C363G and N463T mutants showed small insignificant enhancement. The
half-life of the mutant C363G at 70°C increased by 20.9% (p = 0.001), and the
increase of the half-life of the mutant N463T was not statistically significant
(p = 0.216). Interestingly, compared to the mutant IG181–182*, the half-lives
of the combination mutants IG181–182*/C363G and IG181–182*/N463T at
70°C increased by 16.8% (p < 0.001) and 38.7% (p < 0.001), respectively.
The half-lives of the mutants at 85°C (Fig. 3c) and 95°C (Fig. 3d) showed
similar changes to that at 70°C. The mutation C363G did not change the
half-lives of Gs4j-AmyA at 85 and 95°C. Unfortunately, the mutation N463T
had poor stability at higher temperatures, and its half-lives at 85 and 95°C

Figure 2. SDS-PAGE analysis of the purified wild-type Gs4j-AmyA and mutants. Lane M: mole-
cular protein marker; Lane 1: wild-type Gs4j-AmyA; Lane 2–6: mutants of Gs4j-AmyA including
IG181–182*, C363G, N463T, IG181–182*/C363G, IG181–182*/N463T.
 FOOD BIOTECHNOLOGY 65

Figure 3. Preliminary selection of thermostable mutants (a) and characterization of the wild
type and mutants (b–f). (a) Residual activity of 11 mutants after treatment at 100°C for
10 min. WT indicated the wild type Gs4j-AmyA from Geobacillus sp. For each α-amylase, the
enzyme activity in fermentation supernatant before incubation was defined as 100%. (b), (c),
(d): Half-lives (t1/2) of the wild type and mutants at different temperatures of 70, 85 and
95°C. (e) Effect of temperature on the activities of thermostable mutants. For each amylase,
the highest activity at any temperature was designated 100%. (f) Effect of pH on the
activities of thermostable mutants. Different buffers (25 mM acetate buffer, pH 4.0–5.8;
25 mM phosphate buffer, pH 5.8–8.0) were used to determine the optimum pH at the
optimum temperature of each amylase. The highest enzyme activity at any pH values in the
buffer (pH 4.0–5.8) was designated 100%.
66 Y. GAO ET AL.

decreased by 6.2% (p = 0.010) and 34% (p < 0.001), respectively. However,

two mutations C363G and N463T played an important role in improving
thermostability of Gs4j-AmyA when combining with the deletion of two
amino acids IG181–182. It showed that there was a synergistic function
between them. Similar to the results at 70°C, with IG181–182* as control,
the half-lives of the combined mutant amylases IG181–182*/C363G and
IG181–182*/N463T at 85°C increased by 7.8% (p = 0.031) and 15.5% (p =
0.001), and the half-lives of IG181–182*/C363G and IG181–182*/N463T at
95°C increased by 18.5% (p = 0.013) and 20.3% (p = 0.002), respectively.
Notably, single N463T mutation had no effect on the improvement of Gs4j-
AmyA stability at 95°C, while the combination mutation IG181–182*/N463T
manifested a synergistic function and obviously improved the amylase ther-
mostability at high temperature.
The changes of optimum temperature and pH with amylase mutation
had also been investigated. No obvious change in the optimum tempera-
ture of all mutations was found and mutants were stable at 65–70°C
(Fig. 3e). Likewise, the optimum pH value of the wild type and mutants
were stable at 5.5–5.6 (Fig. 3f), and the wild type and all mutants main-
tained more than 50% relative enzyme activity at a pH range of 4.2–7.4.
In summary, single-site and combination mutations in this study could
exert negligible influence on the optimum temperature and pH response
of amylase Gs4j-AmyA.
As illustrated in Table 2, the Km values of the mutants N463T and IG181–
182*/N463T increased by 48% (p < 0.001) and 37% (p < 0.001), respectively,
which indicated that the mutation N463T decreased the affinity of the
α-amylase for the substrate. The Km values of the other mutants IG181–
182*, C363G and IG181–182*/C363G were almost the same as that of the
wild type. Unfortunately, the catalytic constants (kcat) of the mutants C363G,
N463T, IG181–182*/C363G and IG181–182*/N463T declined by 59, 49, 37
and 16%, respectively. As a result, the kcat/Km values of the mutants C363G,
N463T, IG181–182*/C363G and IG181–182*/N463T decreased to different
extents. In other words, mutation C363G or N463T led to a decrease in the
catalytic efficiency in comparison to the wild type.

Table 2. Kinetic parameters of the wild type and mutants.

Enzyme Km (g/l) kcat (×105 min−1) kcat/Km (×105 l/(g▪min)
Wild type 2.7 ± 0.3 39.4 ± 1.2 14.6 ± 0.3
IG181–182* 2.8 ± 0.3 39.2 ± 1.3 14.2 ± 0.3
C363G 2.5 ± 0.5 16.2 ± 0.5 6.8 ± 0.1
N463T 4.0 ± 0.5 19.9 ± 0.8 4.9 ± 0.5
IG181–182*/C363G 2.2 ± 0.3 24.8 ± 0.7 11.4 ± 0.3
IG181–182*/N463T 3.7 ± 0.5 33.0 ± 0.9 9.1 ± 0.5
 FOOD BIOTECHNOLOGY 67

Figure 4. Model structure of Gs4j-AmyA showing mutant sites (a) and changes (H-bonds)
brought by substitution at N463 (b). (a) Structure of the wild-type amylase and sites of mutated
amino acids. Domain A, B, and C are shown in green, magenta, and cyan, respectively. Mutated
amino acids are shown in red, labelled by the name of amino acid residue, and numbered by the
sequence Gs4j-AmyA without the signal peptide. (b) H-bonds before and after substitution of N
by T. The cyan sticks show amino acid N in up figure or amino acid T in down figure, and the
green dash line and gray sticks indicate H-bonds and amino acids coordinating with N/T,
respectively.

Tertiary structure modeling analysis

The tertiary structure of the α-amylase Gs4j-AmyA was simulated through
homologue modeling by the online software Swiss-Model (http://www.swiss
model.expasy.org/). The file as PDB (Protein Data Bank) is shown by the
software Pymol in Fig. 4a. The mutant amino acids in this research were
labelled in the three-dimensional structure of the α-amylase Gs4j-AmyA.
Most key amino acids identified to be related to thermostability were
found or located in the domain B. Only two mutant amino acids Q336 and
C363 were in domain A. Besides, the amino acid C363 was near to the three
amino acids of the catalytic center (Fig. 5). The mutant site N463 was in
domain C, and the number of hydrogen bonds around N463 site decreased
from five to three because of the substitution with the amino acid T at the
site 463 (Fig. 4b). This might result in no thermostable improvement with its
single mutation.

Discussion
The thermostability of amylase Gs4j-AmyA was significantly improved by
the deletion of IG181–182. This result was in accordance with previous
68 Y. GAO ET AL.

Figure 5. Location of the amino acid site C363 in the structure. The red arrow points to Cys363
shown in red stripe. Yellow sticks indicate the three catalytic amino acid sites Glu264, Asp234
and Asp331. Domain A, B, and C are shown in green, magenta and cyan, respectively.

studies involving α-amylases derived from B. amyloliquefaciens, B. licheni-

formis and B. stearothermophilus (Igarashi et al., 1998; Ali et al., 2006;
Khemakhem et al., 2009). It was reported that deletion of Ile-Gly (IG) loop
enhanced the stability of domain B, which improved the thermostability of
the mutant (Igarashi et al., 1998; Ali et al., 2006; Khemakhem et al., 2009).
It was undesired that the catalytic efficiency of the mutant C363G, N463T,
IG181–182*/C363G and IG181–182*/N463T decreased. The amino acid C363
was near the three catalytic amino acid sites in domain A (Fig. 5). The amino
acid N463 was in domain C which is related to substrate affinity (Raymond
et al., 2002). Substitutions of N463 and C363 seemed to affect the substrate
affinity with the enzyme molecule and catalytic progress, respectively. The vast
improvement on thermostability brought by deletion of IG181–182 seemed to
compensate for the decrease of catalytic efficiency to some degree.
Mutations C363G and N463T were very interesting. Single mutants C363G
and N463T showed little improvement in thermostability. However, when
synergistically functioning with IG181–182*, they further increased thermo-
stability of the amylase Gs4j-AmyA at different temperatures (Fig. 3b–d).
The thermo-inactivation mechanism of α-amylase was illustrated via insights
and experiences on BAA and BStA (Tomazic and Klibanov, 1988). Oxidation
of amino acid C with sulfydryl and deamination of amino acid N/Q were
both the cause of thermo-inactivation of thermostable α-amylases (Declerck
et al., 2000, 2002; Tomazic and Klibanov, 1988). From the perspective of
molecular structure of an amino acid, two mutants C363G and N463T
removed the sulfydryl and acyl amino groups from α-amylase molecule,
respectively. Theoretically, the substitution C363G or N463T could improve
thermostability of Gs4j-AmyA by eliminating the possibilities of cysteine
oxidation and asparagine deamination. Probably, the key domain, which is
domain B of α-amylase changed and before that the substitute G worked at
 FOOD BIOTECHNOLOGY 69

high temperatures, which could protect the residue at 363 from oxidation.
However, the substitution of amino acid C with G could further raise the
stability of the α-amylase molecule on the basis of stabilizing domain B by
deletion of IG181–182.
Substitution of N463 with T decreased the thermostability of Gs4j-AmyA
at 85 and 95°C (Fig. 3c, 3d), and this should probably be attributed to the
decreased number of H-bonds (from 5–3; Fig. 4b). However, the deletion of
IG181–182 enhanced the stability of domain B of the α-amylase molecule
structure. As a result, the potential deamination was eliminated and the
thermo-inactivation resisted, while the network of hydrogen bonds was no
longer important. Consequently, the thermostability of the wild type
decreased by single mutation of N463T, while the thermostability of
IG181–182* increased by synchronous mutation of N463T.
In fact, there were some successful examples that independent replacement
of such amino acids that were susceptible to oxidation and deamination
improved thermostability of amylases (Declerck et al., 2000; Rahimzadeh
et al., 2012). However, the domain B of the amylases like BLA (Declerck
et al., 2000) was originally stable. This tenet was bolstered by the synergetic
function of improving thermostability in this research.

Conclusion
The thermostability of amylase Gs4j-AmyA could be improved through
single-site mutations of IG181–182, C363G and N463T although the
improvement in N463T was not satisfactory at high temperature. However,
the synergetic function of the mutation C363G or N463T with the IG
deletion could always improve the thermostability, even more than the sum
of their separate action. The replacement of potential amino acids susceptible
to oxidation and deamination might be a way to improve the thermostability
of α-amylases. The research provides insights and a reference to improve
thermostability of other industrial α-amylases.

Acknowledgments
We thank Prof. Runying Zeng, Third Institute of Oceanography of China, for supply of the
strain and other help.

Funding
This work was financially supported by the Chinese National High Technology Research,
Development Program (No. 2014AA093501; 2012AA092103) and Grants of Young and
Middle-aged Leading Science and Technology Innovation Talents from Ministry of Science
and Technology of China.
70 Y. GAO ET AL.

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