High-level Expression and Characterization of Thermostable

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High-level Expression and Characterization of Thermostable
Proceedings of the 55th International Convention of Society of Wood Science and Technology
August 27-31, 2012 - Beijing, CHINA
High-level Expression and Characterization of
Thermostable Esterase from Thermoanaerobacter
Tengcongensis in Escherichia coli
Ruobing Deng, Xun Li∗, Hong Gao, Fei Wang
China College of Chemical Engineering, Nanjing Forestry University,
Nanjing 210037, China
Jiangsu Key Lab of Biomass-Based Green Fuels and Chemicals,
Nanjing 210037, China
Abstr act
A novel thermostable esterase from Thermoanaerobacter tengcongensis MB4T was
successfully over-expressed in Escherichia coli and characterized. Two plasmids pET28a (T7
strong promoter) and pTrc99A (trc strong promoter) were used as the expression vectors. The
results indicated that plasmid pTrc99A was more suitable for this esterase expression and the
efficiency of the trc promoter for our analysis is much higher than T7 promoter. The
recombinant esterase, having a molecular mass of 43 kDa determined with SDS-PAGE, was
purified to homogeneity through heat treatment and DEAE-SepharoseCL-6B. The
pTrc99A-est esterase showed maximum activities towards short-chain p-NP esters (C4). The
esterase was optimally active at 700C (over 15 min) and at pH 7.5. It is highly thermostable,
with a residual activity greater than 80% after incubation at 700C for more than 2 h.
Key words:
Esterase, over-expression, characterization, Thermoanaerobacter tengcongensis, E.coli
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Introduction
Lipolytic enzymes (EC 3.1.1.x), represent a hydrolases group, which specifically works over
carboxylic ester, and hence has a potentially broad spectrum of biotechnological uses.
Lipolytic enzymes can be classified into three main groups: esterase/carboxylesterase (EC
3.1.1.1, carboxyl ester hydrolases), which prefer water-soluble substrates and catalyze the
hydrolysis of glycerol esters with short acyl chain (≤10 carbon atoms) to partial gly cerides
and fatty acids; true lipases (EC 3.1.1.3, triacylglycerol hydrolases), which display maximal
activity towards water- insoluble long-chain triglycerides(≥10 carbon atoms); and various
types of phospholipase (Arpigny et al. 1999, Pleiss, J. et al. 1998). The three-dimensional (3D)
structures of both enzymes show the characteristic -hydrolase fold (David L. et al. 1992).
Recently, esterase has been identified containing a Gly-x-x-Leu motif (Wei Y. et al. 1995) as
well as enzymes showing high homology to class α/β-lactamases (Petersen et al. 2001).
Moreover, Esterase plays a major role in degradation of synthetic materials and the synthesis
of optically pure compounds, perfumes, and antioxidants (Panda, T. et al. 2005). As
applications for esterase are found in various fields, there are growing interest in this enzyme.
Furthermore, the esterase in this report is not a true lipase but a novel thermostable esterase
(Zhang, J. et al. 2003), which have been isolated from thermophilic organism,
Thermoanaerobacter tengcongensis (Xue, Y. et al. 2001), and this kind of enzymes have been
found a number of commercial applications because of their overall inherent stability
( Demirijan, D. et al. 2001), such as the field of the petroleum, chemical and pulp and paper
industries, for example, thermostable enzymes have been used for the elimination of sulphur
containing pollutants through the biodegradation of compounds like dibenzothiophene
( Bahrami, A. et al. 2001). Besides, the gene of this enzyme has been cloned and expressed in
E.coli by using the plasmid pET23b as the vector (Zhang, J. et al. 2003), E. coli remains the
most widely used host for recombinant protein expression. It is easy to transform, grows
quickly in simple media, and requires inexpensive equipment for growth and storage.
Different strategies like promoter strength (Boer, H. et al. 1983), Combination of plasmids
(Khushoo, A. et al. 2005) were used to overcome the limitations.
In this work, we compared the expression of the thermostable esterase from
Thermoanaerobacter tengcongensis cloned in two different plasmids pET28a (T7 strong
promoter) and pTrc99A (trc strong promoter) in order to find a suitable plasmid for high-level
expression of this esterase. As a result we found that pTrc99A (trc strong promoter) was more
suitable for this esterase expression. Then we purified and characterized the esterase cloned in
plasmid pTrc99A. This knowledge may be applied in the strategy for the selection of suitable
plasmid for this esterase expression.
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Results
Cloning and Overexpression of Est from T. tengcongensis in E. coli
Genomic DNA obtained from the T. tengcongensis was used as a PCR template. A DNA
fragment of about 1100 bp length was obtained with PCR amplification with primers
described above, and was confirmed by the sequence analysis. The expression plasmid
pTrc99A-est and pET28a-est were successfully constructed and were transformed into E. coli
TOP10 and E. coli BL21DE3 respectively for protein expression. For strain
Top10/pTrc-99A-est, the expression of the esterase was confirmed by SDS-PAGE analysis
(Fig.1, lane 1). And the molecular weight of this protein (43 kDa) on SDS-PAGE (Fig.1, lane
2) was found consistent with the expected molecular weight size. As a negative control, no T.
tengcongensis esterase protein or activity was detected in the vector-transformed strain (Fig.1,
lane 1). However, the result of the enzyme expression in strain BL21DE3/pET-28a-est (Fig.2),
shows that the esterase was not successfully over-expressed with pET-28a in E. coli.
Figure 1. Overexpression and purification of T. tengcongensis lipase (est) in E. coli. Lane M1,
Lane M2, protein molecular weight markers (in kDa); lane 1, total proteins from strain
Top10/pTrc-99A; lane 2 total proteins from strain Top10/pTrc-99A::est; lane 3, supernatant
of concentrated proteins fromTop10/ pTrc-99A::est. lane 4, purified lipase.
Figure 2. Overexpression and purification of T. tengcongensis lipase (est) in E. coli. Lane M,
protein molecular weight markers; lane 1, total proteins from strain BL21DE3 (condon
plus)/pET-28a; lane 2 total proteins from strain BL21DE3 (condon plus)/pET-28a::est
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Enzyme pur ification
Purfication was achieved by heat treatment and DEAE Sepharose Fast Flow. Table 1 depicts
specific activity and the purification fold after these steps. The purity of the esterase after the
purification increased approximately 12-fold over the crude extract. The purified lipase
showed a single band in 12% SDS–PAGE gel (Fig.1, lane 4).
Total
Total
Specific
yield
Purification
protein acctivity activity (U/
(%)
(fold)
(mg)
(U)
mg)
Crude extract
329
2782
8
100
1
Heat treatment
44
1984
45
71
5
DEAE-SepharoseCL-6B
6
570
99
20
12
Table 1 Activity and yield of the recombinant esterase in purification procedure
Substr ates specificity
The esterase expressed with pTrc99a showed maximum activities towards
short-chain p-NP esters (C2 and C4), middle activities towards middle-chain p-NP
esters (C6 to C10) and little activity towards long-chain p-NP ester (C16) (Fig. 3).
However the esterase expressed with pET-28a showed little activity towards all of
these substrates.
Figure 3. Determination of the substrate specificity of the esterase (Est). The p-NP-acetate
(C2), p-NP-butyrate (C4), p-NP-caproate (C6), p-NP-caprate (C10), and p-NP-palmitate
(C16) were selected as the substrates. The reactions were conducted using the standard assay
conditions (see Materials and methods section), and the highest esterase activity towards
p-NP-butyrate (C4) was defined as the 100% level (about 99 U mg−1).
Effects of pH and temperature on the esterase activity
The pH profiles showed that the esterase was active over a broad pH range (pH 7.0–8.0) and
the optimum pH was 7.5 (Fig. 4B). The temperature activity curve for the recombinant lipase
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was shown in Fig. 4A. The temperature for the optimum of the esterase was 700C. The
thermal stability of the esterase was studied by measuring the residual activity after
incubation for different periods at temperatures ranging between 65 and 800C. The half life of
the thermostability at 800C was more than 60 min. (Fig. 4C).
Figure 4. Effects of temperature and pH on esterase activity and stability. (A) Determination
of esterase activity at various temperatures at pH 7.5 for 15 min, using p-NP- butyrate as a
substrate. The highest activity at 700C was defined as 100% level (5.014 U mL−1). (B)
Determination of esterase activity at various pHs at 650C for 15 min, using p-NP- butyrate as
a substrate. The highest activity at pH 7.5 was defined as 100% level (3.51 U mL−1). (C)
Determination of the effects of temperature on esterase stability by incubating the purified
esterase at various temperatures for up to 2 h and measuring the remaining activity. The
original enzyme activity before treatment was defined as 100% (4.01 U mL−1). (D)
Determination of the effects of pH on esterase stability by incubating the purified esterase at
various pHs for up to 2 h and measuring the remaining activity. The original enzyme activity
before treatment was defined as 100% (3.008 U mL−1).
Discussion
Our results showed that the recombinant esterase of T. tengcongensis MB4T was successfully
over-expressed in E. coli. The expression level of the esterase in pTrc99A was about 100
times higher than that in pET-28a. A number of central elements are essential in the design of
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recombinant expression systems (Baneyx, F. 1999, Jonasson, P. et al. 2002). Promoter
strength affects the amount of transcripts produced and thereby increasing the protein
expression. So the plasmids we selected in this study (pTrc99A and pET-28a) were both
controlled by strong promoter (trc and T7).
However the result shows that the efficiency of the trc promoter for our analysis is much
higher than T7 promoter. Briefly many different types of promoters can affect the level of gene
expression in E. coli and suitability of promoters is important for hith-level expression (Hannig,
G. and Makrides, S.C. 1998). Furthermore, the copy number of the expression vector is an
important factor for high-level expression (Kobayashi, M. et al. 1991). Plasmid pTrc99A is a
high-copy-number of about 100 (Amann, E. et al. 1988), while plasmid pET-28a is a
medium-copy-number of 40-50 copies/cell (Kleefeld, A. et al. 2009). It is consistent with the
result of our study. In addition, a highly repressible promoter is particularly important for case
in which the protein of interest is toxic or detrimental to the growth of the host cell (Hannig, G.
and Makrides, S.C. 1998, Amann, E. et al. 1988, Yike, I. et al. ). So the reason why pET-28a
can not be suitable for this esterase expression maybe the target protein expressed with
pET28a is toxic or detrimental to the growth of to the host cell E. coli BL21DE3.
The recombinant enzyme was purified by the heat treatment and DEAE Sepharose Fast Flow
presented as a single protein band on SDS-PAGE with a molecular weight of 43 kDa. In
addition, the result shows that this esterase is indeed highly thermostable. The studies on
substrate specificity demonstrated that the recombinant esterase had the strong catalytic
ability to the substrates with short carbon chain. These properties would be important for the
lipase to be applied in industry. However, modern methods of enzyme engineering, especially
directed evolution will certainly provide suitable esterase variants with increased use in
organic synthesis and other areas of application in the near future.
Acknowledgments
This work was supported by grants from the programs of State Forestry Administration,
China (No. 201004001, 2009-4-63), and a project funded by the priority academic program
development of Jiangsu Higher Education Institutions (PAPD).
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