Heterologous expression of a thermostable α-glucosidase from Geobacillus sp. Strain HTA-462 by Escherichia coli and its potential application for isomaltose–oligosaccharide synthesis

Article abstract of DOI:10.3390/molecules24071413

Isomaltose–oligosaccharides (IMOs), as food ingredients with prebiotic functionality, can be prepared via enzymatic synthesis using α-glucosidase. In the present study, the α-glucosidase (GSJ) from Geobacillus sp. strain HTA-462 was cloned and expressed in Escherichia coli BL21 (DE3). Recombinant GSJ was purified and biochemically characterized. The optimum temperature condition of the recombinant enzyme was 65 ^?C, and the half-life was 84 h at 60 ^?C, whereas the enzyme was active over the range of pH 6.0–10.0 with maximal activity at pH 7.0. The α-glucosidase activity in shake flasks reached 107.9 U/mL and using 4-Nitrophenyl β-D-glucopyranoside (pNPG) as substrate, the K_m and Vmax values were 2.321 mM and 306.3 U/mg, respectively. The divalent ions Mn²⁺ and Ca²⁺ could improve GSJ activity by 32.1% and 13.8%. Moreover, the hydrolysis ability of recombinant α-glucosidase was almost the same as that of the commercial α-glucosidase (Bacillus stearothermophilus). In terms of the transglycosylation reaction, with 30% maltose syrup under the condition of 60 ^?C and pH 7.0, IMOs were synthesized with a conversion rate of 37%. These studies lay the basis for the industrial application of recombinant α-glucosidase.

Full text of DOI:10.3390/molecules24071413

molecules
Article
Heterologous Expression of a Thermostable
α-Glucosidase from Geobacillus sp. Strain HTA-462
by Escherichia coli and Its Potential Application
for Isomaltose–Oligosaccharide Synthesis
Fan Zhang, Weiyang Wang, Fatoumata Binta Maci Bah, Chengcheng Song, Yifa Zhou, Li Ji *
and Ye Yuan *
Jilin Province Key Laboratory on Chemistry and Biology of Changbai Mountain Natural Drugs, School of Life
Sciences, Northeast Normal University, Changchun 130024, China; zhangf508@nenu.edu.cn (F.Z.);
wangwy576@nenu.edu.cn (W.W.); mariamatimbi@yahoo.com (F.B.M.B.); songcc225@nenu.edu.cn (C.S.);
zhouyf383@nenu.edu.cn (Y.Z.)
* Correspondence: jil132@nenu.edu.cn (L.J.); yuany268@nenu.edu.cn (Y.Y.); Tel.: +86-0431-85098212 (Y.Y.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 11 March 2019; Accepted: 7 April 2019; Published: 10 April 2019
Abstract: Isomaltose–oligosaccharides (IMOs), as food ingredients with prebiotic functionality, can be
prepared via enzymatic synthesis using
α-glucosidase. In the present study, the α-glucosidase
(GSJ) from Geobacillus sp. strain HTA-462 was cloned and expressed in Escherichia coli BL21 (DE3).
Recombinant GSJ was purified and biochemically characterized. The optimum temperature condition
of the recombinant enzyme was 65 ◦C, and the half-life was 84 h at 60 ◦C, whereas the enzyme was
active over the range of pH 6.0–10.0 with maximal activity at pH 7.0. The
α-glucosidase activity
in shake flasks reached 107.9 U/mL and using 4-Nitrophenyl -D-glucopyranoside (pNPG) as
β
substrate, the Km and Vmax values were 2.321 mM and 306.3 U/mg, respectively. The divalent
ions Mn2+ and Ca2+ could improve GSJ activity by 32.1% and 13.8%. Moreover, the hydrolysis
ability of recombinant α-glucosidase was almost the same as that of the commercial α-glucosidase
(Bacillus stearothermophilus). In terms of the transglycosylation reaction, with 30% maltose syrup
◦
under the condition of 60 C and pH 7.0, IMOs were synthesized with a conversion rate of 37%.
These studies lay the basis for the industrial application of recombinant α-glucosidase.
Keywords: α-glucosidase; isomaltose-oligosaccharides; heterologous expression; transglycosylation;
thermostability
1. Introduction
Isomalto–oligosaccharides (IMOs) are a group of functional oligosaccharides with a degree of
polymerization (DP) ranging 2–10, which consist of glucosyl saccharide units linked by
linkages [ ]. Isomaltose, panose, isomaltotriose and tetrasaccharides are defined as the main functional
components of IMOs [ ]. As functional oligosaccharides, which are non-digestible in nature, IMOs
have the function of regulating intestinal flora by promoting the proliferation of Bifidobacterium and
Lactobacillus in the human intestinal tract [ ], and also have the additional properties of: low caloric
value [ ], the promotion of gastrointestinal peristalsis, and the improvement of constipation and lipid
metabolism [ ]. Based on these functions, IMOs are widely utilized as prebiotics, food additives and
α-1, 6-glycosidic
1
2
3
4
5,6
feed ingredients [7–10].
IMOs are commonly produced from starch via enzymatic conversion using
α-amylase,
β-amylase, pullulanase and transglucosidase [11]. The typical process for IMO production usually
comprises three steps: (1) liquefaction, where starch is liquefied by a thermostable
α-amylase
Molecules 2019, 24, 1413; doi:10.3390/molecules24071413
www.mdpi.com/journal/molecules

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to produce dextrin; (2) saccharification, where the dextrin is then saccharified by
β-amylase
and pullulanase; (3) transglycosylation, where
(saccharified products) into IMOs [12]. Since commercial
are well-studied and their industrial uses are relatively mature, studying the transglycosylation
reaction catalyzed by -glucosidase is the key step in IMO production. -glucosidase (EC 3.2.1.20,
GH13) catalyzes the liberation of glucose from non-reducing terminals of substrates [13]. In addition
to the hydrolysis activity, some -glucosidases, for instance, those from Aspergillus niger [14],
α
-glucosidase is used for converting maltose
α
-amylase, -amylase and pullulanase
β
α
α
α
Bacillus stearothermophilus [15] and Xantophyllomyces dendrorhous [16], possess transglycosylation
activity, which can transfer a glucosyl residue to the 6-OH group of the non-reducing glucose unit and
yield IMOs [17].
To date, a large number of
and characterized. Aspergillus niger
activity, and was hence successfully applied to produce IMOs [18
α
-glucosidases with transglycosylation activity have been identified
-glucosidase was reported to have high transglycosylation
19]. With the increasing commercial
α
,
interest and industrial application prospects,
α
-glucosidase production has drawn extensive attention.
◦
Generally,
α
-glucosidase from A. niger is stable below 50 C, restricting the current commercial
◦
transglycosylation reaction optimally operated at around 50 C. However, if the temperature of
the transglycosylation reaction can be increased, the faster reaction rate, higher conversion rate and
decreased viscosity of the substrate would be advantageously achieved. Previously, α-glucosidase from
Geobacillus sp. strain HTA-462 (an isolate from Mariana Trench sediment) was characterized, and the
results showed that the wild-type enzyme exhibited good thermostability and good transglycosylation
activity [20], indicating that GSJ has potential application in the IMO industry.
In the present study, for the first time, E. coli BL21 (DE3) was selected as the host strain for the
expression of the gene encoding
α-glucosidase (GSJ) from Geobacillus sp. strain HTA-462. The ability
of the purified recombinant -glucosidase to synthesize IMOs was investigated and our studies aimed
α
to provide a platform for the development of GSJ into an industrial enzyme.
2. Results
2.1. Expression and Purification of α-Glucosidase
The prokaryotic expression system (E. coli BL21 (DE3)/pET28a-gsj) was established for the
inducible expression of GSJ. The synthesized gene was sub-cloned into the EcoR I and Xho I sites
of pET-28a (+). The transformation of the designated pET28a/gsj into E. coli BL21 (DE3) yielded
the recombinant strain. Since we used the T7 promoter-based expression system in this study,
isopropyl
The expression efficiency of the engineered E. coli was explored in a shake flask. Under the optimized
culture conditions, the intracellular -glucosidase activity kept rising rapidly and reached a maximum
β-D-1-thiogalactopyranoside (IPTG) was used for the induction of protein overexpression.
α
value of 107.9 U/mL at 30 h (Figure 1).
The enzyme purification process is summarized in Table 1. The N-terminal his-tagged
recombinant α-glucosidase from the cell-free extract was purified through a combination of heat
treatment and chromatographic separation. GSJ was purified 6.6-fold, with a specific activity
of 283.3 U/mg and an overall recovery of 73.7%. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis showed that the purified recombinant enzyme was detected
as a single protein band with an apparent molecular mass of about 70 kDa, and the exact molecular
weight, determined using MALDI-TOF mass spectrometry, was 68.9 KDa, matching the theoretically
calculated value (Figure 2B).

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Figure 1. Recombinant
batch cultivations of recombinant E. coli in shake flasks:
concentration of recombinant GSJ. ( ) The intracellular enzyme activity of recombinant GSJ. (
α
-glucosidase (GSJ) production from E. coli BL21 (DE3). (
A
) Time profiles for
, the protein
) Sodium
•
, OD600 of the bacteria cells;
N
B
C
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of recombinant GSJ. Samples
were run on a 10% SDS-PAGE gel. Proteins were visualized using Coomassie Brilliant Blue G-250.
Lane M, molecular weight markers; Lane 1, E. coli BL21-pET28a; Lane 2-7, intracellular supernatant of
recombinant E. coli after isopropyl β-D-1-thiogalactopyranoside (IPTG) induction at 0 h, 6 h, 12 h, 21 h,
27 h and 33 h.
Figure 2.
markers; Lane 1, E. coli BL21-pET28a; Lane 2, crude enzyme; Lane 3, heat treatment; Lane 4, purified
GSJ. (
) MALDI-TOF analyses of recombinant GSJ. The major signals are labelled as [M + 2H]2+ and
[M + H]+, respectively.
(A) The purification of recombinant GSJ from E. coli BL21 (DE3). Lane M, molecular weight
B
Table 1. Purification scheme of recombinant GSJ.
Total Protein
Total Activity
(U)
Specific Activity
(U/mg)
Purification
Fold
Purification
Yield (%)
(mg)
Crude enzyme
Heat treatment
Ni-Sepharose
90.90
25.05
10.08
3876.1
3206.9
2856.0
42.64
128.0
283.3
1.0
3.0
6.6
100.0
82.7
73.7

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2.2. Characterization of the Recombinant Enzymes
2.2.1. Temperature Optimum and Thermostability
The optimum temperature curve of the purified enzyme indicated that the activity increased with
temperature until it peaked at 65 ◦C, and the enzyme retained above 50% activity between 55 ◦C and
◦
75 C (Figure 3A).◦The thermostability of the recombinant enzyme was determined at pH 7.0 and
◦
temperatures of 60 C and 65 C. As shown in Figure 3C, the enzyme still retained 90% of activity after
◦
◦
30 h at 60 C, and with a half-time of 84 h. Upon incubation at 65 C, the enzyme showed 57% residual
activity after 18 h. A GSJ with good thermostability and high optimum temperature is beneficial for
applications operating at a high temperature for a long time in industry.
2.2.2. pH Optimum and Stability
The enzyme exhibited high enzymatic activity (>75%) between pH 6.0 and 9.0, and the optimum
pH was 7.0 (Figure 3B). Furthermore, GSJ was stable at a wide range of pH values, of 6.0–10.0.
Additionally, the GSJ retained 93.9% and 84.1% after incubation for 48 h and 72 h at pH 7.0, respectively.
It also retained almost initial activity after incubation for 72 h at pH 10.0 (Figure 3D).
Figure 3. Effect of temperature and pH on the activity and stability of recombinant GSJ. (
A) Optimum
temperature. Activity was measured between 30 ◦C and 90 ◦C at pH 7.0 for 10 min. The activity at
optimum temperature was defined as 100%. (
B
) Thermostability. Assays were carried out in 50 mM
◦
◦
◦
phosphate buffer (pH 7.0) at 65 C for 10 min after the incubation of the enzyme at 60 C (
•
) or 65 C
) Optimum pH. Assays were carried
out at 65 ◦C for 10 min in buffers ranging in pH from 4.0 to 11.0. The activity at optimum pH was
(N). The activity without heat treatment was defined as 100%. (C
defined as 100%. (
ranging from pH 4.0–11.0 at 4 C for 24–72 h.
D
) pH stability. Assays were carried out after the incubation of the enzyme in buffers
◦
2.2.3. Kinetic Studies
The kinetic parameter of the enzyme was analyzed using 4-Nitrophenyl β-D-glucopyranoside
(pNPG) as the substrate at 65 ◦C and pH 7.0 (Figure 4). As shown in Table 2, the kinetic constant Km
was found to be 2.32 mM and the catalytic efficiency kcat/km was 151.6 mM−1s−1
.

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Figure 4. Lineweaver–Burk plot of recombinant GSJ.
Table 2. Kinetic parameter of recombinant GSJ.
Vmax (U
−1
−1
)
Substrate
Km (mM)
kcat (s
)
kcat/Km (mM−1s
−1
mg
)
pNP-α
-D-glucopyranoside
2.321
306.3
352
151.6
2.2.4. Metal Requirements
Previously, Mn2+ was reported to stimulate the activity of
α
-glucosidase from
Thermotoga maritima [21], Ca2+ was required for the catalytic efficiency of the enzyme from
E. coli [22], and Mucor racemosus α-glucosidase was able to be stimulated by Al3+, Ca2+, Mg2+ and
Na+ [23]. In this study, the effect of metal ions on the activity of GSJ was analyzed by preincubating the
enzyme with 1 mM metal ions at 65 ◦C for 1 h. As shown in Figure 5, Mg2+ and K+ did not affect the
enzyme activity of GSJ, Cu2+, Zn2+ and Ni2+ strongly inhibited the enzyme activity by 12.3%–38.6%,
while Fe2+ almost completely inhibited the enzyme activity. However, its activity was significantly
improved by 113.8% and 132.1% in the presence of Ca2+ and Mn2+
.
Figure 5. Effect of metal ions on recombinant GSJ. The activity was assayed after the incubation of the
◦
enzyme (30 ng) in 50 mM phosphate buffer (pH 7.0) containing 1 mM metal ion at 65 C for 1 h.
2.2.5. Substrate Specificity
Nine pNP derivatives were chosen as substrates to determine the substrate specificity of GSJ.
As the results show in Table 3, GSJ exhibited high activity and specificity toward pNP
enzyme showed no activity against the other eight pNP derivatives (pNP Glu, pNP
pNP Gal, pNP Ara, pNP Rha, pNP Man, pNP Xyl). The enzyme exclusively hydrolyzed
4-linked terminal glucose residue.
α
Glu, whereas the
Arap, pNP Gal,
-1,
β
α
α
β
α
α
β
β
α

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Table 3. Substrate specificity of recombinant GSJ on pNP derivatives.
Substrate
Relative Activity (%)
pNP-α-D-glucopyranoside
pNP-β-D-glucopyranoside
pNP-α-L-arabinofuranoside
pNP-α-L-arabinopyranoside
pNP-α-D-galactopyranoside
pNP-β-D-galactopyranoside
pNP-α-L-rhamnopyranoside
pNP-β-D-mannopyranoside
pNP-β-D-xylopyranoside
100.0
4.1
6.6
0.6
2.7
0.0
6.8
0.0
3.9
2.3. Hydrolysis Activity
α
-glucosidase is a typical exo-type glycosidase, which could cleave the terminal
α
-1, 4-glucosidic
linkage from the non-reducing ends of oligosaccharides to form glucose. To determine the
hydrolysis activity of the recombinant GSJ, commercial -amylase was utilized to degrade soluble
starch to oligosaccharides, which was subsequently hydrolyzed to glucose by the recombinant
GSJ/commercial -glucosidase (Bacillus. Stearothermophilus, E-TSAGS, Megazyme, Ireland), and the
degradation products were analyzed by high-performance anion-exchange chromatography (HPAEC).
When using -amylase alone, the degradation products were mainly oligosaccharides and a small
amount of glucose. However, when -amylase and GSJ/commercial -glucosidase were combined,
α
α
α
α
α
the degradation products were almost entirely glucose (Figure 6), suggesting that the recombinant
enzyme has high hydrolysis activity, the same as the commercial enzyme.
Figure 6. Identification of the hydrolysis products by HPAEC-PAD-200. The hydrolysis products
of soluble starch by commercial
α
-amylase and recombinant GSJ (
A
), commercial
α-amylase and
commercial
of glucose.
α
-glucosidase (
B
), commercial -amylase ( ). ( ) Soluble starch. (E
α
C
D
). Standard samples

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2.4. Transglycosylation Activity of Recombinant α-Glucosidase
To determine the transglycosylation activity of GSJ, the recombinant
α
-glucosidase was incubated
with 30% (w/v) maltose solution at 60 ◦C. The progress of the reaction was monitored by HPAEC,
and IMO standards were used to identify the retention time of the components. As shown in Figure 7,
after 2 h of the reaction, more than 80% of the maltose was consumed, and glucose and a series of
transglycosylation products (isomaltose, panose and isomaltotriose) were formed and clearly assigned
by HPAEC. Isomaltose, panose and isomaltotriose were measured for total IMOs, and IMOs were
synthesized with approximately 37% conversion in 2 h by the recombinant enzyme.
Figure 7. Identification of the transglycosylation products by HPAEC-PAD-200. (
A
) Standard samples
of glucose (1), isomaltose (2), isomaltotriose (3), maltose (4) and panose (5). (B) maltose substrate
(control group). (C) Transglycosylation products by recombinant GSJ.
3. Discussion
Due to the transglycosylation activity,
-amylase and pullulanase hydrolyses starch to maltose, which subsequently transglycosylates to
IMOs by -glucosidase. Currently, A. niger -glucosidase has been applied to produce IMOs in
α
-glucosidase is utilized in IMO production [24]. α-amylase,
β
α
α
industry, and the relatively mature technology is used by Amano Enzymes Inc., Japan [19]. IMOs,
one of the emerging prebiotics, have proved their beneficial effects on bifidogenic flora, bowel function
and metabolism, as well as on the immune system [17]. In response to consumers paying more
attention to personal health and well-being and increasing demand, the production of IMOs has been
promoted in recent years. In order to enhance the IMO yield, many technologies have been developed,

Molecules 2019, 24, 1413
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such as heterologous expression and the immobilization of
α
-glucosidase, the combination of different
enzymes, and enzymes being bound to living cells. Recombinant enzyme technology was reported to
significantly improve IMO production [12].
Thermostable enzymes, which have the advantage of high reactivity, better yield, high stability,
low viscosity and little contamination, are important and highly attractive candidates for industrial
applications [25]. Previously, α-glucosidase from the Geobacillus sp. strain HTA-462 was identified and
characterized, and the encoding gene was sequenced and expressed in Bacillus subtilis ISW1214 [20].
E. coli is the preferred host for recombinant protein expression due to its high-level production of
heterologous proteins, fast growth and simple culture conditions. Therefore, in order to improve
the enzyme activity and study of the production of IMOs by GSJ, in the present study the gene
encoding α-glucosidase from Geobacillus sp. strain HTA-462 was cloned and expressed in E. coli BL21
(DE3). The enzyme activity of the recombinant GSJ in shake flasks reached 107.9 U/mL. However,
the biochemical properties of the GSJ expressed in E. coli revealed dissimilarity from the wild-type
◦
enzyme. The optimum temperature of the wild-type GSJ was 60 C, while, in contrast, the recombinant
GSJ showed a shift in optimum temperature to 65 ◦C. The optimal pH of the wild-type GSJ was 9.0 in
the glycine-NaOH buffer, while the optimum pH of the recombinant GSJ was 7.0 in phosphate buffer,
and it maintained more than 70% activity ranging from pH 6.0 to 10.0. Significantly, recombinant GSJ
exhibited higher thermostability. At 60 ◦C, the half-life of the recombinant GSJ was 84 h, which was
6.3-fold greater than that of the wild-type. The big difference in thermostability was probably caused
by the detection at different pHs and in different buffer systems. Notably, in comparison to the
recombinant α-glucosidase from A. niger, the recombinant GSJ exhibited greater thermostability with a
longer half-life, which was 28-fold greater than that of the A. niger
60 C) [18]. The good thermal stability suggests that GSJ has potential for application in IMO industries.
Both the hydrolysis and transglycosylation activities of the recombinant GSJ were determined.
α
-glucosidase (half-life of 3.0 h at
◦
α
-amylase and the recombinant
to convert soluble starch. The results showed that both the
-glucosidase could almost completely hydrolyze the soluble starch to glucose in only 1 h, indicating
that the recombinant GSJ had good hydrolysis activity towards the terminal -1, 4-glycosidic bond.
With regard to the transglycosylation activity, the IMOs were synthesized by GSJ with a conversion rate
of 37% when using maltose as a substrate. In industry, the yield of IMOs produced by -glucosidase is
α
-glucosidase/commercial
α
-glucosidase were simultaneously used
α
-glucosidase and the commercial
α
α
α
generally reported to be about 60%. Although the IMO yield in our study was not ideal, optimizing the
conditions of the transglycosylation reaction may raise the yields further. Additionally, because of its
good thermal stability, the recombinant GSJ could be immobilized to recycle and reuse it, which could
reduce the production costs.
4. Materials and Methods
4.1. Cloning and Expression of GSJ in E. coli BL21 (DE3)
The nucleotide sequence of a 1668-bp fragment encoding GSJ gene (AB154818) was synthesized
by Synbio Technologies Inc. (Jiangsu, China). The synthesized GSJ was digested using EcoR I and Xho I,
and then cloned into a pET-28a (+) vector with a strong T7 promoter, and the recombinant plasmid
was transformed into the E. coli BL21 (DE3) for overexpression. A single colony of E. coli BL21 (DE3)
harboring the recombinant plasmid was inoculated into a ◦10 mL Luria–Bertani (LB) culture-grown
medium containing 0.1 mg/mL kanamycin and grown at 37 C. The overnight culture was diluted into
50 mL terrific broth (TB), when the cell density was OD600 0.5–0.6, the expression of the recombinant
α-glucosidase was induced by 0.4 mM IPTG for continuous 36 h cultivation at 25 ◦C.
4.2. Assay of α-Glucosidase Activity
The hydrolysis activity of
the amount of p-nitrophenol (pNP) released from p-nitrophenyl-
α
-glucosidase was determined spectrophotometrically by measuring
-glucopyranoside (pNPG, Sigma).
α

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The reaction mixture (200
µ
L) containing 10 mM pNPG, 15 mM phosphate buffer (pH 7.0) and
appropriately diluted enzyme, was incubated at 65 ◦C for 10 min. The reaction was terminated by
adding 50
µL of 1 M Na2CO3 and the liberated pNP was measured at 405 nm. One unit of
α
-glucosidase
activity was defined as the amount of enzyme required to release 1
conditions described above.
µmol pNP per minute under the
4.3. Enzyme Preparation and Purification
Cells were harvested and disrupted by sonication, and the intracellular soluble fraction was
centrifuged at 12,000 g for 10 min to remove insoluble cell debris and proteins. The crude extracts
×
of the enzymes were purified according to the following procedures. The intracellular soluble
◦
fraction was heated at 60 C for 20 min and centrifuged at 12,000 for 10 min. The sample was
then filtered with 0.22 µm filters (Jiangsu Green Union Science Instrument Co., Ltd.) followed
by loading onto a Ni-NTA column. The f◦ractions containing the target enzyme were pooled
and dialyzed against phosphate buffer at 4 C for 24 h. Proteins were monitored using sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Bradford method was used to
determine the protein concentrations [26].
4.4. Effects of Temperature and pH on the Enzyme Activity and Stability
Temperature optimum and thermostability. The optimal temperature of
α-glucosidase was
determined by measuring the enzyme activity between 30 ◦C and 80 ◦C at pH 7.0, using pNPG as the
substrate. The thermostability of α-glucosidase was determined by incubating the enzyme in 15 mM
sodium phosphate buffer (pH 7.0) at 60 ◦C and 65 ◦C, and samples were assayed for residual activity
at different intervals.
pH optimum and stability. pH optimum of α
-glucosidase was measured at 65 ◦C over the range
between 4.0–11.0 with increments of 1.0 pH unit, using 15 mM acetate buffer (pH 4.0–6.0), sodium
phosphate buffer (pH 6.0–9.0) and glycine-NaOH buffer (pH 9.◦0–11.0). To determine the pH stability,
the enzyme was incubated in the above various buffers at 4 C for 24–48 h, followed by residual
activity determination at pH 7.0 and 65 ◦C.
4.5. Metal Requirement
To determine the effect of metal ions on the recombinant α-glucosidase activity, the enzyme (30 ng)
was preincubated with 1 mM of each metal ion (Ca2+, Mg2+, Fe2+, Zn2+, Mn2+, Ni2+, Cu2+ and K+) in
15 mM sodium phosphate buffer (pH 7.0) for 1 h at 65 ◦C. The residual enzyme activity was assayed
at 65 ◦C.
4.6. Kinetic Parameters
Enzymatic assays for determining kinetic parameters were performed in the 15 mM sodium
◦
phosphate buffer (pH 7.0) at 65 C with 0.1–4.0 mM pNPG as substrate. The apparent kinetic constants
were calculated using the GraphPad software.
4.7. Substrate Specificity
The substrate specificity of purified GSJ was determined with various pNP derivatives (pNP
αGlu,
pNP
β
Glu, pNP Arap, pNP Gal, pNP Gal, pNP Ara, pNP Rha, pNP Man, pNP Xyl) as substrates.
α
α
β
α
α
β
β
The reaction contained 1 mM pNP-glycoside, 15 mM sodium phosphate buffer (pH 7.0) and 2
recombinant enzymes were carried out at 65 C for 10 min. All of the pNP derivatives were purchased
µ
g
◦
from Sigma (St. Louis, MO, USA).

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4.8. Hydrolysis Reaction
Commercial
starch and this mixture was incubated at 90 ◦C for 30 min. Then the mixture was incubated at 100 ◦C
for 20 min to inactivate the enzyme, and centrifuged at 12,000 g for 15 min. After adding 5 ug
α-amylase (Bacillus licheniformis, A3403, Sigma) was added into the 1% (w/v) soluble
×
recombinant GSJ or commercial α-glucosidase (Bacillus. Stearothermophilus, E-TSAGS, Megazyme,
Ireland), the supernatant was incubated at 60◦C. Following incubation for 1 h, the mixtures were
◦
incubated at 100 C for 5 min to inactivate the enzyme, and centrifuged at 12,000
The hydrolysis products were determined by HPAEC.
×
g for 15 min.
4.9. Transglycosylation Reaction
The reaction mixture, which consisted of 30% maltose and 5
buffer, was incubated at 60 C for 2 h. Reaction mixtures were incubated at 100 C for 5 min to
µ
g enzyme in sodium phosphate
◦
◦
inactivate the enzyme, and the samples were then centrifuged at 12,000
by HPAEC.
×
g for 10 min and analyzed
4.10. HPAEC Analysis
Both the hydrolysis products and transglycosylation products were analyzed using HPAEC.
The HPAEC-PAD analysis was performed on a Dionex ICS 5000 system with a CarboPac PA-200
column and pulsed electrochemical detector. The samples were eluted at 0.5 mL/min with a linear
gradient from 0 to 50 mM NaAc in 50 mM NaOH for 20 min, and then eluted by 200 mM NaOH and
400 mM NaAc for 10 min. The standards of glucose, maltose, isomaltose, panose and isomaltotriose
were obtained from Shanghai Hui Cheng Biological Technology Co. Ltd. (Shanghai, China).
4.11. MALDI-TOF Mass Spectrometry
The purified GSJ were dissolved into 0.1% trifluoroacetic acid (TFA). The reagent 2.5-dihydroxy
benzoic acid was dissolved in 30/70 (w/w) acetonitrile/0.1% TFA as the matrix. Equal volumes of
sample and matrix were mixed. The mixture (1 µL) was placed on an AnchorChip Standard (800 µm)
plate and examined using a BRUKER Autoflex Speed mass spectrometer. Data were recorded using
the FlexControl software and analyzed using FlexAnalysis software. Protein adduct ions [M + 2H]2+
or [M + H]+ were assigned.
5. Conclusions
In conclusion,
in E. coli, and the recombinant enzyme was characterized in detail. The
shake flasks was 107.9 U/mL. A detailed biochemical characterization showed that the recombinant
-glucosidase had high activity and stability at high temperatures and in a broad pH range.
Additionally, the GSJ showed high -1, 4-glucosidic hydrolysis and high transglycosylation activity,
and converted maltose to IMOs with a yield of 37%. These studies indicated that the recombinant
-glucosidase has a great potential for industrial-scale applications. To the best of our knowledge, this is
α
-glucosidase of the Geobacillus sp. strain HTA-462 was cloned and expressed
α
-glucosidase activity in
α
α
α
the first report to express the GSJ gene in E. coli and study IMO synthesis by the recombinant enzyme.
Author Contributions: F.Z., W.W. and C.S. performed the experiments; F.Z. analyzed the data and wrote the
manuscript; F.B.M.B. and Y.Z. revised the manuscript; F.Z., L.J. and Y.Y. designed the experiments.
Funding: This research was funded by the National Natural Science Foundation of China (No: 31770852), Natural
Science Foundation of Changchun City of China (17DY026) and“Fundamental Research Funds for the Central
Universities (2412018QD010)”.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2019, 24, 1413
11 of 12
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