Catalytic transformation of HODAs using an efficient meta-cleavage product hydrolase-spore surface display system

Article abstract of DOI:10.1016/j.molcatb.2014.02.014

The accumulation of 2-hydroxy-6-oxohexa-2,4-dienoic acids (HODAs) in the process of aromatics transformation will hinder the mineralization rate. In this study, a novel type of biocatalyst, meta-cleavage product (MCP) hydrolase (MfphA and BphD) displayed on the surface of Bacillus subtilis 168 spores, was developed for the transformation of HODAs. The successful display of CotG-MfphA and CotG-BphD fusion protein on the surface of spore were confirmed by western blot analysis and activity measurement. The optimal transformation conditions by spore surface-displayed MfphA and BphD were found to be 70 C and pH 7. The thermal and pH stability analysis exhibited that spore surface-displayed MfphA and BphD were stable and retained more than 80% of relative activities even at 80 C and pH 10. Meanwhile, recycling experiments showed that the conversion percentage of HODA by surface-displayed MfphA and BphD were not significantly decreased throughout the reutilization process, which still retained 45% and 70% at the tenth cycle, respectively. To the best of our knowledge, this is the first report concerning the B. subtilis 168 spore surface-displayed MCP hydrolases. The high activities and good recycle performance suggested that this novel biocatalyst system could serve as a suitable alternative for HODAs transformation.

Full text of DOI:10.1016/j.molcatb.2014.02.014

Accepted Manuscript
Title: Catalytic Transformation of HODAs Using an Efficient
meta-Cleavage Product Hydrolase-Spore Surface Display
System
Author: Yuanyuan Qu Jingwei Wang Zhaojing Zhang
Shengnan Shi Duanxing Li Wenli Shen E. Shen Jiti Zhou
PII:
S1381-1177(14)00058-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2014.02.014
MOLCAB 2896
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
4-12-2013
20-2-2014
21-2-2014
Please cite this article as: Y. Qu, J. Wang, Z. Zhang, S. Shi, D. Li, W. Shen, E. Shen, J.
Zhou, Catalytic Transformation of HODAs Using an Efficient meta-Cleavage Product
Hydrolase-Spore Surface Display System, Journal of Molecular Catalysis B: Enzymatic
(2014), http://dx.doi.org/10.1016/j.molcatb.2014.02.014
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Catalytic Transformation of HODAs Using an Efficient meta-Cleavage Product
Hydrolase-Spore Surface Display System
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Yuanyuan Qu 1 *, Jingwei Wang 1, Zhaojing Zhang 1, Shengnan Shi 2, Duanxing Li 1, Wenli Shen 1, E Shen 1, Jiti Zhou 1
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1 Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and
Technology, Dalian University of Technology, Dalian 116024, China
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2 State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China
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* Corresponding author
Mailing address: School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China
Tel: (86) 411-84706251
Fax: (86) 411-84706252
E-mail: qyy@dlut.edu.cn
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Abstract
The accumulation of 2-hydroxy-6-oxohexa-2,4-dienoic acids (HODAs) in the process of
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aromatics transformation will hinder the mineralization rate. In this study, a novel type of biocatalyst,
meta-cleavage product (MCP) hydrolase (MfphA and BphD) displayed on the surface of Bacillus
subtilis 168 spores, was developed for the transformation of HODAs. The successful display of
CotG-MfphA and CotG-BphD fusion protein on the surface of spore were confirmed by western blot
analysis and activity measurement. The optimal transformation conditions by spore surface-displayed
MfphA and BphD were found to be 70 oC and pH 7. The thermal and pH stability analysis exhibited
that spore surface-displayed MfphA and BphD were stable and retained more than 80% of relative
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activities even at 80 C and pH 10. Meanwhile, recycling experiments showed that the conversion
percentage of HODA by surface-displayed MfphA and BphD were not significantly decreased
throughout the reutilization process, which still retained 45% and 70% at the tenth cycle, respectively.
To the best of our knowledge, this is the first report concerning the B. subtilis 168 spore
surface-displayed MCP hydrolases. The high activities and good recycle performance suggested that
this novel biocatalyst system could serve as a suitable alternative for HODAs transformation.
Keywords:
Surface
display
system;
meta-Cleavage
product
hydrolase;
2-Hydroxy-6-oxohexa-2,4-dienoic acid (HODA); Catalytic transformation
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1. Introduction
2-Hydroxy-6-oxohexa-2,4-dienoic acid (HODA) and substituted HODAs are the meta-cleavage
products in the microbial metabolic pathway of aromatic compounds such as catechols and
polychlorinated biphenyls [1,2]. Many studies reported that the HODAs accumulation occurred in
the transformation process of polychlorinated biphenyls, which could limit the aromatics
mineralization process [3,4]. Thus, it is desirable to remove the HODAs from the polluted
environments. Several meta-cleavage product (MCP) hydrolases (EC 3.7.1.8) that catalyzed
hydrolytic reaction of C5-C6 bond cleavage of HODAs have been reported, such as BphD from
Burkholderia cepacia LB400, BphD from Rhodococcus sp. RHA1, MhpC from Escherichia coli
W3110, MfphA and BphD from Dyella ginsengisoli LA-4 [2,5-9]. The research on the
transformation of HODAs was generally performed with purified MCP hydrolases or whole cells as
biocatalysts [2,7,10]. However, the purification of enzymes is time-consuming and costly.
Furthermore, purified enzymes are unstable for harsh environment and easily lose their activity [11].
In addition to the HODAs biodegradation process, MCP hydrolases also show potential application
in fine chemical industry field due to their versatile functions such as hydroxamic acid formation,
carbon-carbon bond formation, and hydrolysis of esters and thioesters [8,12]. Therefore, developing
a novel biocatalytic system for MCP hydrolases is highly recommended for both pollutants
degradation and fine chemicals production purposes.
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Recently, surface display has emerged as an important technology, which has a wide range of
biotechnological applications, including the development of vaccines, peptide and antibody libraries,
bioremediation, biocatalysis, and biosensing [13-17]. Among numerous systems that have been
employed in surface display, bacterial spore surface display systems have been widely used due to
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the advantages of high stability and their robust resistance to heat, radiation and chemicals in a harsh
environment [18,19]. Spores of Bacillus species, including B. subtilis, B. clausii, B. coagulans, B.
cereus, and B. natto, are generally recognized as safe bioresources and used as additives in food, oral
bacteriotherapy and bacterioprophylaxis [18]. Among all the Bacillus species, B. subtilis is widely
studied for surface display due to the detailed knowledge of its spore structure and availability, as
well as the mature genetic manipulation [20,21]. B. subtilis spores are surrounded by a coat, which is
a proteinaceous structure with two layers and composed of at least 20 polypeptides [22]. Of these,
CotB, CotC and CotG have successfully been used as anchoring motifs for display of either antigens
or bioactive molecules such as Neu5Ac aldolase, β-galactosidase, and NADPH-cytochrome P450
oxidoreductase [11,12,23,24]. However, the MCP hydrolases displayed on the surface of spores have
not been investigated as far as we know.
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In this work, a novel surface display system based on B. subtilis 168 for MCP hydrolases using
CotG as an anchoring motif was constructed, and the transformation characteristics of HODAs have
been investigated. Furthermore, the thermal stability, pH stability and reusability of the surface
display system were also studied, which displayed higher stability/reusability than purified and
SBA-15 immobilized enzymes. Our work highlights the potential application of B. subtilis surface
display system on the transformation of HODAs.
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2. Materials and methods
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2.1 Chemicals
HODA, 5-methyl-HODA, 5-fluoro-HODA, 5-chloro-HODA, and 5-bromo-HODA were
enzymatically synthesized using BphC as previously reported [25]. Catechol, 4-methylcatechol,
4-fluorocatechol,
4-chlorocatechol,
4-bromocatechol,
kanamycin
and
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isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from J&K Scientific Ltd. (China). Hind
III, Spe I and Xho I were purchased from TaKaRa (Dalian, China), which were used according to the
instructions of the manufacturer. The other chemicals were of analytical grade.
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2.2 Bacterial strain and growth condition
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B. subtilis 168 was cultivated in glucose-yeast extract-salt (GYS) medium as previous described
[11]. After cultivation in GYS medium at 37 °C for 24 h, the spores and sporangial cells of B. subtilis
168 with recombinant plasmids were harvested by centrifugation and resuspended with 0.1 M
phosphate sodium buffer (pH 7.0). The purification of spore was carried out as previously described
without phenylmethylsulfonyl fluoride (PMSF) [11].
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2.3 Plasmids and strain construction
To display mfphA and bphD on the surface of B. subtilis 168 spores, we constructed two genetic
fusions: cotG-mfphA and cotG-bphD. The DNA fragment with the cotG promoter and structure gene
from the genome of B. subtilis 168 was amplified by primers P1 and P2, digested with Hind III and
Spe I, and ligated into pEasy-Blunt cloning vector to generate the plasmid pEasy-Blunt-cotG. The
mfphA gene was amplified using primers P3 and P4 using plasmid pEBLX-mfphA as the template,
digested with Spe I and Xho I, and cloned into the same restriction endonuclease sites of plasmid
pEasy-Blunt-cotG to yield pEasy-Blunt-cotG-mfphA (a flexible linker [Gly-Gly-Gly-Gly-Ser] was
inserted at the C terminus of the cotG structural gene product). The bphD gene was amplified using
primers P5 and P6 with plasmid pET28a-bphD as the template, and digested as described with
pEasy-Blunt-cotG-mfphA to yield pEasy-Blunt-cotG-bphD.
The fusion genes cotG-mfphA and cotG-bphD were digested with Hind III and Xho I, and inserted
into the shuttle vector pEB03 using the same enzymes to yield recombinant plasmids
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pEB03-cotG-mfphA and pEB03-cotG-bphD. The recombinant plasmids pEB03-cotG-mfphA and
pEB03-cotG-bphD were then separately transformed into B. subtilis 168 by electroporation
transformation [26]. Spectinomycin (50 μg/mL) was added for selection of recombinant B. subtilis
168 harboring shuttle plasmid pEB03-cotG-mfphA or pEB03-cotG-bphD.
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2.4 Western blot analysis
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Spore coat protein was extracted from 100 μL purified spore suspension (1×1010 spores/mL) by
sodium dodecyl sulfate (SDS)-dithiothreitol (DTT) treatment at 65°C [27]. The western blot analysis
was performed using standard protocols [24]. The penta-His antibody (TaKaRa, Dalian, China) and
HRP-rabbit anti-mouse IgG (TaKaRa, Dalian, China) were used for immunodetection of the fusion
protein. The color reaction of horseradish peroxidase was done according to the manufacturer’s
instructions.
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2.5 Activity assays of spore surface-displayed MfphA and BphD
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The standard activity assays were performed at 30 C in a total volume of 2.0 mL, consisting of
phosphate sodium buffer (0.1 M, pH 7.0), HODAs (40 μM), and the purified spore suspension
containing MfphA/BphD. The reaction was initiated by adding the purified spore suspension
containing MfphA/BphD. After 10 min incubation, the reaction mixture was centrifuged (8,739×g,
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15 min, 20 C). The residual concentration of HODAs was measured by UV-vis spectrophotometer
(JASCO V-560, Japan) at specific wavelengths (HODA at 375 nm, 5-methyl-HODA at 381 nm,
5-fluoro-HODA at 385 nm, 5-chloro-HODA at 380 nm, and 5-bromo-HODA at 380 nm). Extinction
coefficients of the yellow-colored HODAs were determined as described by Zhou et al. [9]. One unit
of enzyme activity was defined as the amount of enzyme required to consume 1 μmol of the HODA
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per minute. The number of spores was calculated by direct counting with a Burker chamber under an
optical microscope.
2.6 Biotransformation of HODA by spore surface-displayed MfphA and BphD
To optimize the biotransformation conditions, 10 mL of reaction mixture in a 50 mL Erlenmeyer
flask was used with variations as follows. Both the concentrations of spore surface-displayed MfphA
and BphD were 2 U/mL. Temperatures ranged from 30 to 80 oC. The buffer solutions with different
pH used in this study were 0.1 mM PBS (pH 7) and 0.2 mM Tris-HCl (pH 8-10). Conversion rate (%)
was calculated the following equation:
A0 − A1
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Conversion rate (%) =
×100%
A0
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where A0 and A1 represent the initial and residual concentration of HODAs, respectively. All of
the experiments were performed in duplicates and the average values were used in calculations.
2.7 Stability of spore surface-displayed MfphA and BphD
The thermal stabilities of spore surface-displayed MfphA and BphD were determined by
incubating the reaction mixtures at 30, 40, 50, 60, 70 and 80 oC for 10 min at pH 7, and then residual
activities against HODA were determined. The variable pH stability studies were performed by
incubating the surface-displayed MfphA and BphD at different buffers (pH from 7 to 10) for 60 min
at 70 oC. Reusability experiments were conducted by reusing the spore surface-displayed MfphA and
BphD for ten cycles. After each biotransformation batch, the reaction mixtures were centrifuged and
the supernatants were removed.
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3. Results
3.1 Construction of surface display system
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To construct the surface display system, the outer coat protein CotG (23.9 kDa) expressed at the
coat of B. subtilis 168 was used as an anchoring motif for the display of MfphA and BphD. A flexible
linker composed of 5 amino acids (Gly-Gly-Gly-Gly-Ser) was inserted between the C terminus of
cotG gene product and the N terminus of mfphA and bphD gene product to obtain the cotG-mfphA
and cotG-bphD fusion gene, respectively. For the detection of surface displayed protein, the genes
mfphA and bphD fused to a DNA fragment encoding six histidines (His6) at N terminus were
amplified, respectively. The His6 was served as an epitope for the rabbit anti-His probe antibody. A
cotG-specific promoter was employed in the surface display system as previously reported [10].
Western blot analysis of spore coat proteins purified from recombinant strains revealed the presence
of a 58 kDa band (lane 3) which reacted with rabbit anti-His probe antibody (Figure 1). The
molecular weight of CotG-MfphA and CotG-BphD was about 58 kDa. Thus, the 58 kDa bands in
lane 3 could be the fusion protein of CotG-MfphA and CotG-BphD with a His6 at N terminus.
Meanwhile, no signal was detected in the spores of B. subtilis 168 (lane 2), indicating that there was
no His6 in the proteins of B. subtilis 168. Thus, it was confirmed that MfphA and BphD were
successfully displayed on the spore surface of B. subtilis 168.
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In order to further investigate if surface display system was successfully constructed, the
activities of spore surface-displayed MfphA and BphD against HODAs were also examined. The
reaction scheme of HODAs by spore surface-displayed MfphA and BphD was shown in Figure 2.
Since not all proteins were extractable from the B. subtilis spore coat, the units of MfphA and BphD
were normalized with respect to the numbers of spores. The spores not harboring the recombinant
plasmid had no activities (data not shown). As seen in Table 2, both spore surface-displayed MfphA
and BphD showed high activities against HODAs. BphD exhibited the highest activity (1.32 10-8 U
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spore-1) against HODA in 10 min which was 1.02, 1.11, 1.08, and 1.05-fold higher than that of
5-methyl-HODA, 5-fluoro-HODA, 5-chloro-HODA, and 5-bromo-HODA, respectively. A similar
trend was also observed for MfphA, which was consistent with the free MfphA expressing in E. coli
BL21 (DE3) [9]. Moreover, the activity of BphD against HODA was 1.29-fold higher than that of
MfphA. These results indicated that both MfphA and BphD were successfully displayed in an active
form.
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3.2 The effects of temperature and pH on HODAs transformation
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To investigate the HODA transformation characterizations by surface-displayed MfphA and
BphD, the effects of temperature and pH on HODAs transformation were tested. As shown in Figure
3 and 4, using HODA (40 μM) as substrate, the highest conversion efficiency of HODA (40 μM) was
detected at 70 oC and pH 7 by both MfphA and BphD, corresponding to 70% and 92%, respectively.
In the temperature ranging from 30 to 80 oC, the conversion efficiencies by MfphA and BphD were
more than 60% and 50%, respectively. Within the pH range of 7 to 10, the conversion efficiencies of
HODA by MfphA and BphD were 60% and 80%, respectively. These results indicated that the spore
surface-displayed MfphA and BphD exhibited high biotransformation capability towards HODA
over wide ranges of temperature and pH.
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3.3 Stability of spore surface-displayed MfphA and BphD
The stability of surface display system is one of the most important parameters for application
[28]. The stability of spore surface-displayed MfphA and BphD was examined. Figure 5 showed the
residual relative activities of the MfphA and BphD after 60 min incubation at different temperatures.
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The surface-displayed MfphA and BphD were stable at 30 oC, 40 C, 50 C and 60 oC. Both of the
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surface-displayed enzymes maintained more than 90% of corresponding initial activities at 30 C in
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60 min. Surprisingly, even the temperature was increased to 70 C and 80 C, the spore
surface-displayed MfphA and BphD did not show much loss of activity (less than 20%). As shown in
Figure 6, within the pH range of 7 to 10, both the surface-displayed MfphA and BphD were stable
and retained a relative activity of more than 85% in 60 min.
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3.4 Reusability of spore surface-displayed MfphA and BphD
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The reusability of the surface-displayed protein was also an important index during the process of
application. To investigate the reusability of spore surface-displayed MfphA and BphD, the reactions
were conducted under the optimal transformation conditions (Figure 7). After each reaction cycle,
the spore surface-displayed MfphA and BphD were isolated by centrifugation and washed with 0.1
M phosphate sodium buffer (pH 7.0). The spore surface-displayed MfphA and BphD maintained
high relative activities towards HODA within 3 reaction cycles. From the fourth to the tenth cycle,
the catalytic efficiencies of surface-displayed MfphA and BphD were gradually decreased to 45%
and 70% by MfphA and BphD, respectively, at the end of tenth cycle.
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4. Discussion
The meta-cleavage product (MCP) hydrolases (EC 3.7.1.9), which are identified in the
degradation pathway of aromatic compounds, can catalyze the rare carbon-carbon bond cleavage of
the 2-hydroxy-6-oxohexa-2,4-dienoate (HODA) [2,5-9]. Moreover, they could also catalysis C-C
bond formation in organic media [12]. In previous studies, free MCP hydrolases, immobilized MCP
hydrolases, and whole cells containing MCP hydrolases were used for HODAs degradation [2,7,10].
In recent years, biological surface display has been used in various areas. Many enzymes such as
levansucrase, organophosphorous hydrolase, and lipase have been displayed on the cell surface in
Gram-negative bacteria [13,29,30]. In contrast to Gram-negative bacteria, the cell surface display in
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Gram-positive bacteria enjoyed some advantages, which made the bacteria suitable for variety of
biotechnological applications [31]. However, only a few existing studies of the cell display of
contaminate-specific proteins onto Gram-negative bacteria were available [11,32]. Besides, the cell
display in Gram-negative bacteria mainly focused on biosynthesis and heavy metal ions removal
[11,32,33]. Little information on the biodegradation by displaying contaminate-specific proteins in
Gram-negative bacteria has been reported. In this study, we introduced a novel surface display
system using B. subtilis 168 for displaying MCP hydrolases and investigated the catalytic capability
of this system using HODA and substituted HODAs as substrates. To our best knowledge, this was
the first report about displaying MCP hydrolases on the surface of bacterial spores to degrade
HODAs.
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CotG, a 23.9-kDa outer coat protein, was a convincing example of a coat protein that resulted in
the display of functional enzymes [11,12]. In this study, we used CotG as the anchoring motif for the
display of MCP hydrolases on the surface of B. subtilis 168 spores. In the previous study, the
strategies of genetic construction to display the heterogenous protein on spores were utilizing
low-copy-number expression vector, thus low activity of the displayed enzymes were generally
obtained [11]. For example, Xu et al., compared the expression vectors pHP13 (low-copy-number)
and pEB03 (high-copy-number) for the displaying Neu5Ac aldolase, indicating that the activity of
Neu5Ac aldolase obtained using pEB03 as the expression vector was 6-fold higher than that obtained
using pHP13 as the expression vector [11]. Thus, the vector pEB03 with high-copy-number has been
used as the expression vector for displaying the fusion genes cotG-mfphA and cotG-bphD to ensure
effective expression of the enzymes during the sporulation process. This surface display system was
similar with that reported for displaying Neu5Ac aldolase on the surface of B. subtilis WB 600
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spores [11]. A 58 kDa fusion protein obtained by western blot analysis confirmed that MfphA and
BphD were successfully displayed on the spore surface of B. subtilis 168, due to the His6 of this
fusion protein reacted with rabbit anti-His probe antibody.
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In previous studies, MfphA and BphD have been purified and immobilized on SBA-15 and
carbon nanotubes [9,34]. Compared with these previous studies, spore surface-display based
immobilization represented a promising alternative for HODAs degradation, because it excluded the
purification and immobilization process. Meanwhile, the natural robustness of spores in the presence
of heat, solvent, pH, oxidizing agents and salts has been also reported [35]. In our previous studies,
the pH stability of the free and SBA-15 immobilized MfphA for HODA hydrolysis indicated that
both of them retained less than 20% of relative activity at pH 8 [9]. However, both the spore
surface-displayed MfphA and BphD were stable and retained more than 85% within the pH ranges
(from 7 to 10). In the case of thermostabilities, the immobilized MfphA on SBA-15 retained 33% of
its original activity at 50 oC [9]. Meanwhile, carbon nanotube immobilized BphD retained 40% of its
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original activity after incubation at 60 C for 25 min [34]. In this work, the spore surface-displayed
MfphA and BphD exhibited higher thermal resistance, which were very stable at mesophilic
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temperatures (from 30 to 50 C) and retained above 90% of their initial activity. These results
indicated that MfphA and BphD displayed on the surface of spores could enhance both thermal and
pH stabilities. Generally speaking, thermostability of an enzyme is often accompanied with good
performance under harsh conditions, as well as long-term survival under mild conditions [36]. As
shown in Figure 3, although the optimal reaction temperature was around 70 oC, the
surface-displayed MfphA and BphD also exhibited relatively high conversion percentage to HODA
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at 30 oC. In addition, they also displayed the best thermostabilities at 30 C. Therefore, it suggested
that this novel biocatalyst system could serve as a suitable alternative for HODAs transformation.
The reusability of a biocatalyst is one of the most important factors to be considered in its
industrial application. Spores have the characteristic of easy purification by centrifugation or
filtration. Therefore, the spore surface-displayed MfphA and BphD could be easily separated from
the catalytic system to be used for circular catalysis process [11]. The transformation rates of HODA
by MfphA and BphD were not significantly affected throughout the reutilization process, which still
retained 45% and 70% transformation activities of HODA by MfphA and BphD at the tenth cycle,
respectively. Previous studies indicated that the immobilized MfphA on SBA-15 retained 30%
activity at tenth cycle because the MfphA was only adsorbed by relatively weak electrostatic
interactions with the silica walls [9]. Surface-displayed Neu5Ac aldolase in the spores of B. subtilis
WB 600 could be used for up to 3 reaction cycles without a significant decrease in the conversion
rate [11]. Therefore, the spore-displayed enzymes could help to meet the ever-increasing industrial
demand for preparation and stabilization of biocatalysts and may be generally applicable to
numerous biocatalytic reactions.
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5. Conclusion
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Two novel systems using spore surface-displayed MfphA and BphD were constructed for the
transformation of HODA. Both MfphA and BphD were successfully displayed in an active form on
the spore surface of B. subtilis 168 according to the western blot analysis and activity measurement.
The conditions of HODA biotransformation by spore surface-displayed MfphA and BphD were
characterized. The thermal and pH stabilities of spore surface-displayed MfphA and BphD were both
improved. Meanwhile, the spore surface-displayed MfphA and BphD could be reused up to 10 times
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with 45% and 70% residual activities, respectively. This work will provide the potential application
of spore surface display system in the fields of pharmaceuticals, fine chemicals, agrochemicals, and
other demanding industries.
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Acknowledgements
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The authors gratefully acknowledge the financial supports from the National Natural Science
Foundation of China (No. 21176040; No. 51078054), and supports from the New Century Excellent
Talents in University (No. NCET-13-0077).
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Table 1 Strains, plasmids and primers used in this work
Strain, plasmid, primer
Description
Type strain
Source
Strain
Bacillus subtilis 168
Bacillus
Genetic
Stock Center (BGSC)
Plasmid
pEBLX-mfphA
pEBLX with gene mfphA
[7]
pET28a-bphD
pEasy-Blunt
pET28a with gene bphD
[7]
Cloning vector
Transgene
This study
This study
[10]
pEasy-Blunt-cotG-mfphA
pEasy-Blunt-cotG-bphD
pEB03
pEasy-Blunt with fusion gene cotG-mfphA
pEasy-Blunt with fusion gene cotG-bphD
High-copy-number E. coli-B. subtilis shuttle vector
pEB03 with fusion gene cotG-mfphA
pEB03 with fusion gene cotG-bphD
pEB03-cotG-mfphA
pEB03-cotG-bphD
Primers
This study
This study
P1 a
5´-GCCTTTAAGCTTAGTGTCCCTAGCTCCGAG-3´
5´-CTATTGACTAGTTGAACCCCCACCTCCTTTGTA
TTTCTTTTTGACTA-3´
This study
This study
P2 a,b
P3 a,c
5´-CTCCACTAGTCATCATCATCATCATCACATG
ACGGCACTTACCGA-3´
This study
P4 a
5´-CGATCTCGAGTCATTCATGCTTGAGAAAAT-3´
5´-GCCCGACTAGTCATCATCATCATCATCACAT
GTCCAAGCCAGATA-3´
This study
This study
P5 a,c
P6 a
5´-AATTCTCGAGTCAGGGCAGCGGCAGCGGTT-3´
This study
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a The italicized letters indicate the introduction of restriction sites.
b The underlined letters indicate the introduction of a flexible linker at the C terminus of the cotG structural gene product.
c The bold letters indicate the introduction of His-tag at the C terminus of the mfphA or bphD structural gene product.
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Table 2 The activity of MfphA and BphD displayed on the spore surface
Substrate
HODA
MfphA (10-8 U spore-1)
1.02 ± 0.08
BphD (10-8 U spore-1)
1.32 ± 0.24
5-methyl-HODA
5-fluro-HODA
5-chloro-HODA
5-bromo-HODA
1.00 ± 0.07
0.83 ± 0.04
0.89 ± 0.05
0.96 ± 0.06
1.29 ± 0.15
1.19 ± 0.11
1.22 ± 0.12
1.26 ± 0.14
2
3
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Figure Captions
Figure 1 The western blot analysis of recombinant spores expressing cotG-mfphA and cotG-bphD
fusion proteins. (A) line 1, molecular mass standards; line 2, spores of B. subtilis 168; line
3, spores expressing cotG-mfphA fusion protein; line 4, Perfect ProteinTM Marker. (B) line
1, molecular mass standards; line 2, spores of B. subtilis 168; line 3, spores expressing
cotG-bphD fusion protein; line 4, Perfect ProteinTM Marker.
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Figure 2 The reaction scheme of HODAs by spore surface-displayed MfphA and BphD.
Figure 3 Effect of temperature on the biotransformation of HODA by spore surface-displayed
MfphA (A) and BphD (B).
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Figure 4 Effect of pH on the biotransformation of HODA by spore surface-displayed MfphA (A) and
BphD (B).
o
Figure 5 Thermal stability of spore surface-displayed MfphA (A) and BphD (B). 30 oC (■); 40 C
(●); 50 oC (▲); 60 oC (▼); 70 oC (◆); 80 oC (◄).
Figure 6 The pH stability of spore surface-displayed MfphA (A) and BphD (B). pH 7 (■); pH 8 (▲);
pH 9 (●); pH 10 (▼).
Figure 7 Reuse of spore surface-displayed MfphA (A) and BphD (B) for the biotransformation of
HODA.
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1
2
(A)
3
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(B)
Figure 1
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-O
O
-O
O
O
O
H2O
R2
R1
OH
+
OH
MCP hydrolase R1
OH
R2
H
2
3
Figure 2
4
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(A)
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4
(B)
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Figure 3
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(A)
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(B)
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Figure 4
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(A)
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(B)
Figure 5
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(A)
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(B)
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Figure 6
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(A)
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(B)
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Figure 7
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Highlight
λ
λ
λ
An effective meta-cleavage product hydrolase-spore surface display system was constructed.
Thermostability, pH stability and reusability of BphD and MfphA were greatly improved by immobilization.
Surface displayed BphD and MfphA possessed high activities on various substrates.
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*Graphical Abstract (for review)
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