Site-Specific, covalent immobilization of dehalogenase ST2570 catalyzed by formylglycine-generating enzymes and its application in batch and semi-continuous flow reactors

Article abstract of DOI:10.3390/molecules21070895

Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ) exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570.

Full text of DOI:10.3390/molecules21070895

molecules
Article
Site-Specific, Covalent Immobilization of
Dehalogenase ST2570 Catalyzed by
Formylglycine-Generating Enzymes and Its
Application in Batch and Semi-Continuous
Flow Reactors
Hui Jian, Yingwu Wang *, Yan Bai, Rong Li and Renjun Gao *
Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Science,
Jilin University, Changchun 130012, China; jianhui18686@163.com (H.J.); by0330@126.com (Y.B.);
ryong_li@163.com (R.L.)
*
Correspondence: wyw@jlu.edu.cn (Y.W.); gaorj@jlu.edu.cn (R.G.); Tel.: +86-138-4407-2416 (Y.W.);
86-431-8515-5212 (R.G.); Fax: +86-431-8515-5200 (R.G.)
+
Academic Editor: Roberto Fernandez-Lafuente
Received: 2 April 2016; Accepted: 5 July 2016; Published: 11 July 2016
Abstract: Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues
within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine
(FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was
introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of
the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from
Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ)
exhibited significant enzymological properties, such as new free aldehyde groups, a high level of
protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization
support for its large surface and excellent thermal and chemical stability. It was functionalized
with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was
immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided
the chemical denaturation that occurs in general covalent immobilization and resulted in better
fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold
higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free
ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of
batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption,
which loses 40% of its original activity when used for the second time. It is remarkable that the
site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse
in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale
production and application of site-specific, covalently immobilized ST2570.
Keywords: immobilization; dehalogenase; formylglycine-generating enzyme; Sulfolobus tokodaii
1
. Introduction
Enzymes are biocatalysts that catalyze reactions under mild conditions with high efficiency, and
stereospecificity or enantioselectivity, and they also reduce or avoid adverse conditions. Enzymes are
used in organic synthesis, brewing, food, medicine and other fields. However, enzymes exhibit limited
mechanical and storage stability for some reactions and cannot be reused. With the emergence of protein
modification and immobilization techniques, enzymes have become conducive to automatisation and
continuous use for industrial production.
Molecules 2016, 21, 895; doi:10.3390/molecules21070895
www.mdpi.com/journal/molecules

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Enzyme immobilization may alter enzyme property and performance in many different ways,
which depends on the methods used, a proper immobilization may improve stability and, in certain
cases, activity, while a bad selection of the immobilization strategy may drive to the enzyme
inactivation and even decrease enzyme stability [1]. Physical methods including physical adsorption
and entrapment etc., which are simple, however the immobilized enzymes with those methods maybe
leaked out from the matrix during operation [
chemical covalent binding sometimes may result in a certain loss of observed activity, however, the
chemical stability will be significantly enhanced and the enzyme leakage could be minimized [ ].
2]. The immobilization of enzymes via multi-point
3
In addition, immobilization may provide other benefits such as creating compatible environment
for enzyme, and avoiding multi-layer packing of enzymes etc., such that improved specific activity
may be observed after immobilization in certain cases [4]. Enzyme orientation during immobilization
may play a critical role in the recovered activity, for example, when the substrate is large and the
active center is oriented towards the surface of the support this may raise some steric hindrances,
or when the interaction of a specific area of the enzyme with the enzyme may drive to their
inactivation [
inactivation caused by chemical reactions. Various techniques have been proposed for the chemical
modification of proteins [ ]. Introduction of short peptides to the target protein is a commonly
5]. Modifying enzymes by adding a tag can maintain enzyme activity and avoid enzyme
6
useful method to achieve selective and site-specific modification of a proteins. With this method,
target proteins can be recognized selectively and modified by a co-expressed enzyme in vivo or
in vitro [7–13]. The reaction between the protein and the supports is therefore more controlled.
Table 1 lists the enzyme-mediated systems used for the modification of target proteins. Prokaryotic
formylglycine-generating enzyme (FGE) is used for protein labeling and was first identified in 2003 [14].
Several researchers reported that FGE can recognize a six-residue sulfatase submotif (L
oxidize the cysteine residue selectively within the motif to form an aldehyde-bearing formylglycine
FGly) residue [15 16]. The target protein can be fused with a LCTPSR submotif for modification by a
CTPSR) and
(
,
co-expressed FGE. Thus, the free aldehyde group on the surface of the target protein can be used for
site-specific covalent binding to amino-functionalised fluoresceins or immobilization supports through
a Schiff base reaction [10].
Haloacid dehalogenases exhibit a potential for pharmaceutical and chemical industrial use and
for the remediation of contaminated land [17]. In previous work, haloacid dehalogenase from the
thermophile archaeon Sulfolobus tokodaii str.7 (ST2570) was cloned and characterized [18]. This enzyme
belongs to the L-2-halogen acid (L-2-HAD) superfamily and is vital for industrial applications
because of its efficient catalytic activity, stereoselectivity, thermal stability and tolerance to a wide pH
range. As a monomer globin, ST2570 was a suitable model enzyme for testing site-specific covalent
immobilization [19].
Many scientists have successfully fused a sulfatase sub-motif (also called an “aldehyde tag”) to
target proteins by PCR reaction using a specific primer design before the construction of an expression
plasmid. In this study, the pET28a plasmid was reconstructed, as shown in Scheme 1A. The gene
encoding the peptide LCTPSR was introduced at different positions in pET28a, and a new set of vectors
were obtained: pET28aDH, pET28aCQ, pET28aNQ and pET28aDQ. Thus, the sulfatase submotif
can be easily fused to any target protein at different positions without designing specific primers.
Two expression plasmids containing the ST2570 gene and FGE gene would be transformed into bacteria
for co-expression. The host that retains both plasmids will co-express ST2570 and FGE in the presence
of L-pectinose and isopropyl β-D-1-thiogalactopyranoside (IPTG). In this study, four proteins that are
either wildtype enzymes or modified enzymes with aldehyde groups in different positions will be
obtained (Scheme 1B).

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Table 1. Enzymes-mediated systems used in protein modification.
Enzymes
Recognition Motif
Mechanism of Enzyme-Mediated System
Ref(s)
A cysteine residue in sortase catalyzes the cleavage of the peptide bond between
the threonine and glycine residues within the motif. Sortase will accept the
N-terminus of an oligoglycine nucleophile.
sortase A
LPXTG
[7]
TG2 mediates the incorporation of functionalized amines into a high-affinity
Gln-substrate peptide tag.
microbial transglutaminase (mTG)
farnesyltransferase (PFTase)
WALQRPH
CAAX
[8]
[9]
PFTase catalyzes the transfer of a farnesyl isoprenoid group from farnesyl
pyrophosphate to a sulfur atom present in the cysteine residue of the CAAX box.
FGE converts cysteine to formylglycine (FGly) within the LCTPSR motif.
Proteins bearing a unique aldehyde group were chemically modified by
selective reaction with hydrazide- or aminooxy-functionalized reagents.
formylglycine generating enzyme (FGE)
biotin ligase (BirA)
LCXPXR
[10]
[11]
BirA catalyzes the sequence-specific ligation of the biotin or the ketone isostere
of biotin to the lysine side-chain of a 15-amino-acid acceptor peptide (AP).
GLNDIFEAQKIEWHE
LplA catalyzes the specific attachment of octanoic acid, 6-thio-octanoic acid,
selenolipoic acid and alkyl azide to an engineered LplA acceptor peptide (LAP).
The alkyl azide was selectively derivatized with cyclooctyne conjugates to
various probes.
microbial lipoic acid ligase (LplA)
N-myristoyl transferase (NMT)
DEVLVEIETD
K
AVLEVPGGEEE
[12]
[13]
NMT specifically attaches myristate or modified myristate to peptides with
amino-terminal Gly residues. Peptides with Asn, Gln, Ser, Val or Leu
penultimate to the amino terminal Gly are substrates.
GXXXS

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Scheme 1. Schematic of the E. coli co-expression system used for the aldehyde tagging of proteins
in vivo. ( ) the reconstructed plasmids; ( ) the expression of four kinds ST2570 that are either wildtype
A
B
enzymes or modified enzymes with aldehyde groups in different positions; (C) the one-step specific
immobilization of ST2570; (D) the immobilized ST2570.
It is well-known that both the method and the support used might impact the efficiency of
enzyme immobilization. Inorganic supports can form manageable particles with large surface areas,
are reactive towards derivatizing/functionalizing agents and present excellent thermal and microbial
resistance, reasonable mechanical strength and chemical resistance under operational conditions.
2
On the other hand, mesoporous supports not only exhibit a very high specific surface (up to 500 m /g),
but can also host the enzyme molecules within pores and protect against physical and mechanical
damages [20]. Fortunately, SBA-15 has all the advantages above and was selected as the support.
Amino functionalized SBA-15 is also low-cost and convenient for the one-step specific immobilization
of ST2570 (Scheme 1C).
2
. Results and Discussion
2
.1. Expression and Purification
The aldehyde tag (LCTPSR) was ligated into the C or N-terminal or both terminals of ST2570
using the reconstructed plasmid pET28a. However, results showed that the aldehyde tag variably
affected the protein expression level and enzyme activity (Figure 1). Unlike wildtype proteins, all of
the aldehyde-tagged ST2570 reduced the level of expression, which can evidently affect the enzyme
activity. The C-terminal tag improved the activity of enzyme, despite a reduced level of expression,
but the enzyme with double-sided aldehyde tags lost almost all activity. These results indicated that
the fused polypeptide tag affects protein folding and the tertiary structure.
2
.2. Labeling of Recombinant Proteins with Fluorescence Probe
Although many protein modification methods were developed, these methods usually resulted in
the selective labeling of a given functional group rather than the site-specific labeling of one residue
in a given protein [ ]. Thus, the fused aldehydes were used for site-specific protein modification
8
and identification with an aminooxy functionalised probe. A panel of aldehyde-tagged proteins was
incubated with Alexa Fluor 647 C5-aminooxyacetamide and the reaction was analyzed by SDS-PAGE

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and Odyssey Near Infrared Laser Imaging system (Figure 2). In contrast, the wildtype protein
without an aldehyde tag demonstrated no signals, the C-terminal modified proteins showed strong
signals. Additionally, the N-terminal and double-terminal modified proteins exhibited weaker signals.
Some LCTPSR peptide tags in these proteins may be covered during the process of protein folding,
and could not be modified by the co-expressed FGE. We selected ST2570CQ for immobilization because
of its excellent activity and the availability of free aldehyde groups.
Figure 1. Effect of the fused aldehyde tag on the expression level and activity of the recombinant
protein ST2570.
Figure 2. Selective labelling of aldehyde tagged proteins with Alexa Fluor 647 C5-aminooxyacetamide.
2
.3. Modification of SBA15
Silica-based mesoporous materials have been used as enzyme supports, biomarkers and
biosensors in medicine. However, its direct use as a support for enzyme immobilization is
limited because of its inert nature. Thus, chemically modifying the surface of these materials is
necessary to introduce an active group, and the most common modifier is aminopropyltriethoxysilane
(
APTES, C H NO Si) [21–23]. As shown in Table 2, SBA-15 exhibited improved loading capacity,
9 23 3
and the enzyme showed better immobilization efficiency (IE) but worse retention of activity (R)
when immobilization was performed using support with either deficient or excessive modification.
This outcome could be due to the random combination and aggregation of enzymes through physical
absorption, which can enhance the steric hindrance effect and prevent the formation of the monolayer.
Moreover, over-modification with APTES will result in increased steric hindrance because of the high
protein density. Therefore, SBA-15 modified with 2% APTES (v/v) was selected for the next experiment.
Table 2. Modification of SBA-15 with different ratios of APTES and ethanol.
Ratio of APTES and
Ethanol % (v/v)
Loading Capacity
mg/gcarrier
Specific
Activity U/mg
Specific Activity
U/gcarrier
Immobilization
Efficiency %
Retention of
Activity %
0
2
6
4.31
3.17
4.37
4.23
103
443.93
448.97
474.93
478.78
90.79
66.31
91.42
88.49
57.17
78.61
60.32
60.81
141.63
108.68
109.56
1
0

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2
.4. Immobilization of ST2570CQ
One-step immobilization and purification of ST2570CQ were simultaneously performed through
a Schiff base reaction between the amino groups in the support and the free aldehyde groups in the
enzyme. This approach avoids further purification of proteins before the immobilization. The amount
of enzymes bound to the carrier and the activity of the immobilized enzyme depend on conditions
during immobilization, such as the availability of linking groups, pH, temperature, coupling time and
initial enzyme amount. To achieve the optimal immobilization efficiency (IE), retention of activity (R)
and immobilized enzyme activity, the conditions for immobilization were optimized.
The ionization state of enzyme molecules was affected by the pH of the buffer used in the
immobilization process. Figure 3A shows the effect of pH on the activity of the enzymes during
immobilization. The relative activity of immobilized ST2570CQ on SBA15-NH did not vary from
2
pH 6.0 to 10.0. The highest activity for immobilized enzymes, was observed at pH 6.5. The relative
activity of the immobilized enzyme significantly increased when the initial crude ST2570CQ amounts
used in the immobilization were changed from 0 to 3.0 mg per 10 mg of the carrier. However, the
enzyme reached steady-state when its amounts was greater than 2.0 mg (Figure 3B). The operating
˝
temperature of immobilization was 10–37 C, and the immobilization time ranged from 1 h to 10 h
(
˝
Figure 3C,D). The optimal temperature for immobilization was 10 C for more than 3 h. The optimal
conditions for immobilization are 2 mg of crude enzyme per 10 mg of support in 50 mM PB buffer
˝
pH 6.5) and incubation for 3 h at 10 C. Under the optimal conditions of immobilization, the enzyme
(
loading capacity can be as much as 108 mg/g support, and the immobilized enzyme activity can be as
much as 450 U/g immobilized enzyme.
Figure 3. Conditions for the covalent immobilization of ST2570CQ on SBA-15. (
pH for the immobilization system using either, 50 mM PB buffer (pH 6.0–9.0) or 50 mM Gly-NaOH
buffer (pH 8.6–10.0); ( ) The optimization of the initial amount of enzyme per 10 mg support for
immobilization system; ( ) The optimization of temperature for immobilization system; ( ) The
A) The optimization of
B
C
D
optimization of time for the immobilization. The immobilization system consisted of 2 mL 50 mM PB
buffer (pH 6.0) containing 10 mg support in a 20 mL reactor. We set the best enzyme activity as 100%.
Three trials were performed for each group.

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2
.5. Characterization of Immobilized ST2570CQ
2
.5.1. pH and Thermo-Stability of Immobilized ST2570CQ
The pH and thermo-stability of an enzyme are important parameters for exploring potential
˝
industrial applications. ST2570 is a thermophilic protein that is stable at 70 C. In this part of the study,
˝
the thermal stability was investigated by incubating either the free or immobilized enzyme at 90 C for
a period of time. The remaining activity was monitored after each incubation time. All the immobilized
enzymes used were prepared with the optimal conditions described at the end of Section 2.4.
As shown in Figure 4A, when immobilized ST2570CQ was incubated for 2 h, it still retained 60%
of its activity, which was still three times higher than that of free enzyme. The stability of various
immobilized enzymes can be frequently improved by inorganic supports because inorganic supports
have greater dimensional stabilities [24]. Inorganic supports always are capable of forming manageable
particles with a high surface area, are reactive towards derivatizing/functionalizing agents and present
excellent thermal and microbial resistance, reasonable mechanical strength and chemical resistance
under the operational conditions [20]. The thermal stability of enzymes was speculated to drastically
increase if they are attached to a complementary surface of a relatively rigid support [25,26]. The
pH stability of free and immobilized enzymes is illustrated in Figure 4B. When the enzymes were
incubated in buffers with different pH values for 24 h, it was found that both the free enzyme and
the immobilized enzyme were stable when the pH value is higher than 7, indicating that no severe
conformational change, which affects the pH stability of ST2570, occurred during immobilization [26].
As for the free enzyme that already had the best pH stability, combining it with the support did not
improve the pH stability appreciably anymore.
Figure 4. The thermal and pH stability of free and immobilized ST2570CQ: (
free and site-specific immobilized ST2570CQ; ( ) The pH stability of free and immobilized ST2570CQ.
) free enzyme ( ) immobilized enzyme; 50 mM PB buffer (pH 6.0–9.0) and 50 mM Gly-NaOH buffer
pH 8.6–10.5). All the immobilized enzymes used were prepared with the optimal conditions described
A) The thermal stability of
B
(
(
ꢀ
at the end of Section 2.4. The conditions for the activity assay were constant stirring at 110 rpm for
˝
5
min in 70 C water-bathing constant temperature vibrator. Three trials were performed for each test.
2
.5.2. Operational Stabilities of Immobilized Enzyme in Batch and Semi-Continuous Reactors
Operational stability is the most important factor used to assess the feasibility of immobilized
enzymes for industrial productions. A number of investigations were previously carried out in this
area [21 27 28]. The operational stability of immobilized enzyme was determined in the batch and
,
,
semi-continuous flow reactors. The results were compared in terms of activity and cycling times of the
immobilized enzyme. To eliminate the influence of storage time on enzyme activity, all measurements
were carried out in the same day. In the batch reaction operation (Figure 5A), the enzyme activity

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remained above 95% of the original activity after five cycles in the reactors loaded with 10 mg of
immobilized enzyme. The result in this work shows a significant improvement in the operation stability
of immobilized ST2570CQ on SBA-15 in the batch reaction compared with the immobilized enzymes
in other reports [26–31]. The stability of immobilized ST2570CQ on SBA-15 by physical adsorption
was also examined in the batch wise reaction (Figure 5B); which turned out the physically immobilized
enzyme lost 40% of its original activity when used for the second time; and only retained less than
2
0% of its original activity in the fifth use; which may be ascribed to significant deprival of the enzyme
from the support during the reaction agitation and recovery process. On the contrary, the activity loss
of the chemical immobilized ST2570CQ on SBA-15 may be due to the deactivation of enzyme by strong
shaking during washing operation. The results demonstrate that the multi-point chemical binding of
ST2570CQ to SBA-15 provide a much stronger linkage and confinement for site-specific immobilized
ST2570CQ than the immobilized ST2570CQ by physical adsorption [32].
Figure 5. The effect of reuse on immobilized enzymes in reactors. The activity of site-specific
covalently-immobilized enzymes in a batch reactor (
A); the activity of enzyme immobilized by physical
adsorption in a batch reactors ( ); and the activity of site-specific covalently-immobilized enzymes in a
B
semi-continuous flow reactor (C). The tests were carried out at room temperature.
In a semi-continuous flow reactor, the immobilized ST2570CQ retained almost 100% of its
original activity after being used 10 times in the column (Figure 5C). The operational stability in
the semi-continuous flow reactor is better than the operational stability in the batch type reactor.
The difference in outcome was most likely because of the gradual loss of immobilized enzymes when
the enzymes were separated by centrifugation during each round in the batch reactor. This probably did
not occur in the semi-continuous flow reactor, in which the immobilized enzymes were not separated.
Operational stability improved when a semi-continuous flow reactor was utilized, in contrast with
the result of Bachas–Daunert [8]. Thus, the high operational stabilities obtained with immobilized
ST2570CQ preparations indicate that these immobilized enzyme preparations can successfully be used
for continuous production [28].

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2
.6. Catalytic Ability of Immobilized ST2570CQ
ST2570 was first reported in 2009, and it can selectively catalyze L-2-halogenated acid into
D-2-hydroxy alkyl acid (Scheme 2) [18]. The immobilized and free ST2570 exhibited different catalytic
abilities when L-2-haloalkanoic was used as a substrate. The highest conversion rate of L-2-haloalkanoic
catalyzed by the immobilized and free ST2570 were 49.74% and 40.94%, respectively. The improvement
of catalytic ability is attributed to the better thermal and operational stability of immobilized ST2570,
which are the most important factors during reactions performed with high temperatures and long
reaction times.
Scheme 2. Hydrolysis reaction catalyzed by L-2-dehalogenase.
3
. Experimental Section
3
.1. Materials
The L-2-haloacid dehalogenase (ST2570) from thermophile archaeon Sulfolobus tokodaiistr.7
(JCM 10545), plasmids pET28a, myc-his A Rv0712 (FGE) plasmid (add gene plasmid 16132),
Escherichia coli BL21(DE3) pLysS (Novagen, Madison, WI, USA), mesoporous Silica Support
SBA15 (550–600 m2
¨
g
´1
, 7–9 nm, 0.65–0.67 cm3
¨
g
´1
, Nanjing XFNANO Materials Tech Co., Ltd.,
Nanjing, China), (3-Aminopropyl)triethoxysilane (APTES, 98%, Aladdin, New York, NY, USA),
L-2-chloropropionic acid (Alfa Aesar, Ward Hill, MA, USA), acetonitrile (HPLC Grade, Fisher Scientific,
Waltham, MA, USA), ninhydrin reagent (2% (w/w) ethanol solution) and all other reagents were of
analytical grade.
3
.2. Methods
3
.2.1. Expression and Purification
To obtain sufficient amounts of target protein for the analysis, enzymes were produced by
E. coli BL21 (DE3) pLysS. The method was based on a two-plasmid system in BL21 (DE3) pLysS,
the first plasmid was the reconstructed pET28a with the gene encoding for the S. tokodaii L-2-haloacid
dehalogenase (ST2570), and the other plasmid co-expressed the formylglycine-generating enzyme
(FGE), which converts cysteine in the polypeptide fusion tag of LCTPSR to formylglycine (FGly) to
obtain ST2570 with or without the aldehyde groups in the right place.
The aldehyde tag gene sequence was introduced to pET28a by site-directed mutation to replace the
His-tag sequence. The plasmid pET28aDH was constructed by deleting the His-tag at the N-terminal of
the multiple cloning site in pET28a. The plasmid pET28aCQ was constructed by replacing the His-tag
at the C-terminal of the Multiple Cloning Site in pET28aDH with the gene encoding for LCTPSR.
The plasmid pET28aNQ was constructed by replacing the His-tag at the N-terminal of the Multiple
Cloning Site in pET28a with the sequence encoding LCTPSR. Lastly, the plasmid pET28aDQ was
constructed by replacing the His-tag at the C-terminal of the Multiple Cloning Site in pET28aNQ with
the sequence encoding LCTPSR.BL21(DE3) pLysS cells that carried plasmids pET28a (DH, CQ, NQ
˝
or DQ)-st2570 and myc-his A Rv0712 [33] were cultured in LB medium at 37 C until an OD600 of
0
0
.8 was obtained. The cells were induced by the addition of L-pectinose to a final concentration of
.2% for 30 min, induced further for 16 h by 0.5 mM IPTG at 18 C then harvested. The cell pellet was
˝
resuspended in 50 mM PB at pH 6.0, disrupted by ultrasonication in an ice bath and centrifuged at

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˝ ˝
2,000 rpm for 20 min at 4 C. The crude extract was incubated at 80 C for 30 min and centrifuged
1
˝
20 C. Protein concentrations were determined
again. The freeze-dried protein powder was stored at
using the Bradford method.
´
3
.2.2. Enzyme Assay
The 3 mL reaction system consisting of 50 mM Gly–NaOH buffer (pH 9.5) and 1 mM
˝
L-2-chloropropionic acid as a model substrate was incubated in an oscillating water bath at 70 C for
5
˝
min. The free or immobilized enzyme was added for further incubation in a water bath at 70 C and
shaken with a speed of 110 rpm for 5 min. The reaction was terminated by addition of 50 L of 1 M
µ
nitric acid and 2 mL of 6% ammonium ferric sulfate solution. The system was treated with 1 mL of 0.4%
mercury thiocyanate solution to produce a color reaction after removal of the immobilized enzyme.
Each sample was tested three times (p < 0.05). The OD460 value for each sample was tested using a
UV-1700 spectrophotometer (Shimazu, Kyoto, Japan), and the lactic acid yield combined with the
chlorine ion standard curve was calculated. One unit of activity is defined as the production of 1 µmol
˝
hydrochloric acid per min at 70 C and pH 9.5. The activity of free L-2-haloacid dehalogenase was given
as U/mgenzyme, and immobilized enzyme activities were expressed as U/mgenzyme or U/gsupport.
3
.2.3. Labelling of Recombinant Proteins with the Fluorescence Probe
The fluorescence-labeling reaction was performed by combining recombinant proteins and
C5-aminooxyacetamide [21] and the reactions were analyzed by SDS-PAGE and Odyssey Near Infrared
Laser Imaging system.
3
.2.4. Modification of SBA-15
SBA-15 was washed with distilled water and dried overnight at 60 C before salinisation. Different
˝
concentrations of APTES (0%, 2%, 6%, and 10%) was added to the dried 1 g SBA-15 in 50 mL of
˝
anhydrous ethanol (v/v) and then incubated in the oscillation incubator at 80 C and 200 rpm for 12 h.
SBA-15 was washed thoroughly with anhydrous ethanol and distilled water on a filtered funnel until
˝
no APTES was detected in the filtrate, and the supports were dried overnight at 100 C. The amount of
APTES in different solutions was determined using a ninhydrin-based monitoring system described
earlier by Özlem Alptekin [34]. The conditions used to initially optimize the activation of support was
˝
mg of ST2570CQ per 10 mg of SBA-15-NH incubated in 50 mM PB buffer (pH 6.5) at 10 C for more
2
2
than 6 h.
3
.2.5. Immobilization of ST2570CQ
The amino-functionalized SBA-15 (SBA15-NH ) (10 mg) was incubated with 2 mL of phosphate
2
˝
buffer (50 mM, pH 6.0) containing 2 mg of the heat-treated ST2570 at 10 C for 6 h of shaking at 150 rpm
and reduced with 1% (w/w) NaCNBH for 1 h. Aside from the target protein, other proteins without
3
aldehyde groups cannot establish a covalent link with SBA15-NH . Thus, the immobilized enzyme
2
was easily separated by centrifugation at 12,000 rpm for 10 min and washed twice with 1 M NaCl
solution, saturated ammonium sulfate solution, and distilled water to remove the other physically
adsorbed proteins. Immobilization efficiency (IE, %) and retention of activity (R, %) were calculated
as follows:
The activity of added enzyme ´ The remaining activity in the supernatat
IE “
ˆ 100%
(1)
(2)
The activity of added enzyme
The observed activity of immobilized enzyme
The theoretical activity of immobilized enzyme
R “
ˆ 100%

Molecules 2016, 21, 895
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The conditions for the immobilization of ST2570CQ on SBA-15-NH , including pH, temperature,
2
time and initial amount of protein per 10 mg of support, were optimized. The activity of the
immobilized enzyme was determined for all the samples.
3
.2.6. Characterization of Immobilized ST2570CQ
In this part, the pH and thermal stabilities of the immobilized ST2570CQ were detected.
The thermal stabilities of free and immobilized enzymes were investigated by incubating both the
˝
free and immobilized enzymes at 90 C for different lengths of time. The remaining enzyme activity
was monitored after each incubation. After incubation in buffers with different pH values (pH 6.0–9.0
for 50 mM PB buffer; pH 8.6–10.5 for 50 mM Gly–NaOH buffer) for 24 h, the remaining activities of
free and immobilized enzymes were detected according to Section 3.2.2.
The operational stabilities in batch and semi-continuous reactors were also investigated. For batch
˝
reactors, all reactions were carried out in a vibrating water-bathing at a constant temperature of 70 C
and shaking at 110 rpm for 5 min. The reaction mixture consists of 3 mL of 50 mM Gly–NaOH solution
at pH 9.5 containing 1 mM L-2-chloropropionic and 10 mg of immobilized ST2570CQ. The enzyme
was recovered by centrifugation and washed with distilled water to prepare for the next cycle.
The product was then detected according to Section 3.2.2. For the semi-continuous flow reactor,
2
1
(
0 mg of immobilized ST2570CQ was loaded into a column (1 cm
ˆ
10 cm). The reaction mixture
mobile phase) consisting of 3 mL of 50 mM Gly-NaOH buffers (pH 9.5) and 1 mM L-2-halogenated acid
˝
was applied to a column and incubated for 10 min at 70 C. The reaction mixture was then allowed to
flow out of the column, then the product was then detected according to Section 3.2.2. The column was
washed with 5 mL distilled water five times to prepare for next cycle. All the immobilized enzymes
used for characterization were prepared with the optimal conditions described in Section 2.4.
3
.2.7. Catalytic Ability of Immobilized ST2570CQ
Rates of lactic acid production catalyzed by free and immobilized ST2570CQ were also measured
using high-performance liquid chromatography (HPLC). A mixture consisting of 1 mL of 50 mM
Gly-NaOH pH 9.5 containing 20 mM L-2-CPA was assigned as the control group, while 0.05 mg (9 U)
and 20 mg (9 U) free and immobilized ST2570CQ were included separately in experimental groups.
The conversion rate was calculated according to the following formula:
substrate pthe control groupq´the residue of substrate pthe experiment groupq
conversion rate p%q “
(3)
substrate pthe control groupq
The HPLC conditions are as follows: 4.6 mm
mobile phase: a mixture of 0.01 mol/L phosphate buffer (pH 2.0) and acetonitrile (8:2, v/v); flow rate:
.6 mL/min; UV detector wavelength: 210 nm; column temperature 18 C; volume of sample injection:
ˆ
250 mm Waters Nova-pak C18 HPLC column;
˝
0
1
0 µL.
4
. Conclusions
In this experiment, we prepared a panel of aldehyde tagged proteins using a pET28a plasmids
system that was designed in our previous work. The C-terminal aldehyde-tagged protein (ST2570CQ)
exhibited significant enzymatic properties, such as the generation of free aldehyde groups, a higher
level of protein expression and improved enzyme activity. ST2570CQ was covalently immobilized to
SBA-15 modified with 2% APTES ethanol solution (v/v) in the following optimal conditions: 2 mg of
˝
crude ST2570CQ per 10 mg of SBA15-NH in 50 mM PB buffer pH 6.5 and incubated at 10 C for 3 h.
2
The immobilized ST2570CQ exhibited a 3-fold increase in thermal stability and improved operational
stability and catalytic ability than free ST2570. These improved properties increase the potential of
site-specific, covalently immobilized ST2570 for industrial-scale production and application.
Acknowledgments: The authors are grateful to the National Natural Science Foundation of China (No. 20772046)
and the Jilin Province Natural Sciences Foundation (Grant 20120943) for financial support.

Molecules 2016, 21, 895
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Author Contributions: Hui Jian and Renjun Gao conceived and designed the experiments; Hui Jian, Yan Bai and
Rong Li performed the experiments; Renjun Gao and Yingwu Wang contributed reagents/materials/analysis
tools; Hui Jian wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).