Download Gene Cloning, Expression, and Substrate Specificity of an Imidase

CURRENT MICROBIOLOGY Vol. 55 (2007), pp. 61–64
DOI: 10.1007/s00284-005-0455-6
Current
Microbiology
An International Journal
ª Springer Science+Business Media, LLC 2007
Gene Cloning, Expression, and Substrate Specificity of an Imidase from
the Strain Pseudomonas putida YZ-26
Ya-wei Shi, Li-fang Cui, Jing-ming Yuan
Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of the National Ministry of Education,
Shanxi University, 92 Wucheng Road, Taiyuan, Shanxi 030006, PRC
Received: 22 November 2005 / Accepted: 30 June 2006
Abstract. A gene-encoding imidase was isolated from Pseudomonas putdia YZ-26 genomic DNA using
a combination of polymerase chain reaction and activity screening the recombinant. Analysis of the
nucleotide sequence revealed that an open reading frame (ORF) of 879 bp encoded a protein of 293
amino acids with a calculated molecular weight of 33712.6 kDa. The deduced amino-acid sequence
showed 78% identity with the imidase from Alcaligenes eutrophus 112R4 and 80% identity with Nterminal 20 amino-acid imidase from Blastobacter sp. A17p-4. Next, the ORF was subcloned into vector
pET32a to form recombinant plasmid pEI. The enzyme was overexpressed in Escherichia coli and
purified to homogeneity by Ni2+–NTA column, with 75% activity recovery. The subunit molecular mass
of the recombinant imidase as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis
was approximately 36 kDa, whereas its functional unit was approximately 141 kDa with four identical
subunits determined by size-exclusion chromatography. The purified enzyme showed the highest
activity and affinity toward succinimide, and some other substrates, such as dihydrouracil, hydantoin,
succinimide, and maleimde, were investigated.
Imidases from bacteria are attracting increasing attention as novel agents for the production of useful organic
acids as well as new tools for fine enzymatic synthesis
of chiral compounds. These include unnatural amino
acid [1], pyruvate [2], and 3-carbamoyl-alpha-picolinic
acid [3], all of which are critical building blocks for
semisynthetic antibiotics, pesticides, and food additives.
Imidase—which is also known as dihydropyrimidinase,
hydantoinase, dihydropyrimidinase, and amidohydrolase
because of its bound-substrate specificity [4, 5]—has
been shown to participate in pyrimidine metabolism in
vivo or in bioconversion of organic acids in vitro [6, 7].
Most of the imide-hydrolyzing enzymes share limited
sequence homology and have different substrate charts.
In general, they are dimer or tetramer with a 50- to 60kD subunit [4, 5], with the exception of two imidases
being reported so far as having a lower subunit molecular mass of 30 to 40 kDa. Contrary to known imidase,
Correspondence to: Y.-W. Shi; email: [email protected]
an imidase with a 35-kD subunit from Blastobacter sp.
A17p-4, which was first purified and characterized by
Ogawa et al., preferably hydrolyses cyclic imides and
does not accept 5¢-monosubstituted hydantoins as substrate [8, 9]. The imidase gene from the strain Alcaligenes eatrophus 112R4 was first isolated and expressed in
Escherichia coli [9]. In bacteria, imidase is involved in
ring-opening hydrolysis of cyclic imides to half-amides,
and the resulting half-amide is hydrolyzed by halfamidase to dicarboxylates, which then undergoes further
transformation through tricarboxylic acid (TCA) cyclic
reactions [8]. On the basis of our work on D-hydantoinase from the strain P. putdia YZ-26, a third imidase
with low subunit molecular weight is reported herein.
Materials and Methods
Bacterial strains, plasmids, and medium. The P. putdia strain YZ26 used was screened and identified by our laboratory. E. coli was used
as host in the cloning and expressing procedures. The thioredoxindeleted pET32a (Novagen) was used as expression vector. pUC118 and
62
polymerase chain reaction (PCR) primers were purchased from
TaKaRa (Dalian Branch, China). YCG medium (0.5% yeast, 0.5%
peptone, 0.5% glycerol, 0.2% K2HPO4, and 0.1% DL-hydantoin,
adjusted to pH 7.0) was used as screening medium for activity assay on
microtiter plate. The recombinant strain pEI/E. coli BL21 was
employed for expressing the enzyme in Lura-Bertani (LB) medium.
Screening the imidase. Genomic DNA from P. putdia YZ-26 was
isolated according to the method of Lewinton [10] and digested with
EcoRI for 6 hours at 37C. Approximately 2 to 9 kb DNA fragments
recovered from agarose gel were ligated into the corresponding site of
pUC118 and transformed into E. coli JM109. The transformants were
initially screened on LB agar plate containing X-gal, IPTG, and 100lg
/mL ampicillin. White transformants were picked out and inoculated
into a microtiter plate (96 wells) containing 50 ll liquid YCG medium
for each well and incubated at 37C overnight. Afterward, imidase
activity per well was detected by a colorimetric method using DLhydantoin as substrate [11]. Positive recombinants in wells developed
an unequivocal yellow colour, whereas negative recombinants
produced a reddish colour or were colourless.
DNA sequence analysis and subcloning. The imidase gene DNA
sequence of a positive recombinant (pUS804) was analyzed by
TaKaRa. The deduced amino-acid sequence of the open reading
frame (ORF) was aligned using the ProSite database at ExPASY. The
ORF of imidase from pUS804 was reamplified by PCR using forward
primer (5¢-CGGAATTCATGGCCAAGGAAATC-3¢) and reverse
primer (5¢-CCGAAGCTTTCACTTCTTGCGCGG-3¢). Additional
restriction sites for EcoRI and HindIII were introduced for
subsequent cloning of the gene. The amplification was performed
with 30 cycles at 94C for 1 minute, 50C for 1 minute, and 72C for 1
minute, and extension for 10 minutes by using a PTC-200 Peltier
Thermal Cycler. The amplified fragment, digested with EcoRI and
HindIII, was inserted into the corresponding sites of the modified
vector pET32a to form recombinant plasmid pEI.
Expression and purification of imidase. Recombinant plasmid was
transformed into E. coli BL21 (DE3) host cells for large-scale protein
production. The host cells containing the recombinant plasmid were
cultured in LB medium supplemented with 100 lg/mL ampicillin on a
shaker at a speed of 170 rpm at 37C. When the optical density
(A600nm) of cells reached approximately 0.6 to 0.8, 0.2 5mM IPTG was
added, and the cells were continuously incubated at 37C for 4 hours
under the same conditions to induce imidase production. Cells were
harvested by centrifugation at 4,000 rpm for 15 minutes, and the pellet
was suspended in 50 mM Tris-HCl (pH 8.0) containing 500 mM NaCl
and further disrupted by ultrasonication (SONICS Vibra Cell VC 455,
Germany). After centrifugation at 16,000 rpm (Sorvall SS-34 rotor) for
30 minutes at 4C, the supernatant was applied to an Ni2+-NTA affinity
column and washed thoroughly with binding buffer (50 mM Tris-HCl
[pH 8.0] 500 mM NaCl, and 50 mM imidazole). Then the target protein
was eluted out with elution buffer (50 mM Tris-HCl [pH 8.0]
containing 500 mM NaCl and 1 M imidazole). The enzyme was
collected and stored at 4C for further analysis.
Assay of imidase activity. Imidase activity was measured by a
spectrometric method using DL-hydantoin as substrate [11]. The
reaction rate was determined by monitoring the absorbance change at
430 nm and the concentration of the product N-carbamyl-glycine was
determined from a standard calibration plot. In the case of succinimide
or maleimide being used as substrate, high-performance liquid
chromatography (HPLC) was used to determine imidase activity. An
appropriate amount of imidase was mixed with 20 mM succinimide or
maleimide dissolved in 100 mM Tris-HCl (pH 8.0) or in 50 mM
CURRENT MICROBIOLOGY Vol. 55 (2007)
phosphate buffer pH 6.5, respectively, at a total volume of 200 ll. The
reaction mixture was incubated at 37C for 10 minutes and stopped by
boiling for 5 minutes. After centrifugation, the supernatant was
analyzed on an HPLC system (Waters) equipped with a Symmetry C18
column (4.6 mm x 150 mm, 5 lm). The mobile phase used in this
process was a mixture of methanol and 50 mM phosphate buffer (pH
6.5) (5:95 v/v) at a flow rate of 1.0 ml/min. The ultraviolet detector
was set at 210 nm for succinimide and at 255 nm for maleimide. One
unit of enzyme was defined as the amount of the enzyme that catalyzes
substrate to form the product at a rate of 1 lmol /min under the assay
conditions described previously.
Molecular mass of the enzyme. The apparent molecular mass of the
subunit was estimated on sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE) [12]. The native molecular mass of the
imidase was determined by size-exclusion chromatography with a
prepacked Superose12 (10/30) column pre-equilibrated and eluted with
50 mM Tirs-HCl and 100 mM NaCl (pH 8.0). The column was
calibrated by standard proteins, including trypsinogen (24 kDa),
alcohol dehydrogenase (41 kDa), egg albumin (45 kDa), bovine serum
albumin dimer (136 kDa), and transferrin dimer (160 kDa). The native
molecular mass of the enzyme was calculated from Kav plot against
the logarithm of standard protein.
Results and Discussion
Cloning imidase gene from the strain P. putdia
YZ26. Genomic DNA extracted from P. putdia YZ26 was randomly cut by EcoRI, and fragments with a
length of approximately 2 to 9 kb recovered from
agarose gel were inserted into vector pUC118 and
then transformed into E. coli JM109. By hydantoinase
activity assay of approximately 2000 colonies on a 96well microtiter plate, 1 positive colony with higher
hydantoinase activity was selected. The plasmid
extracted from this colony was designated pUS804,
which contained a 2.69-kb inserted fragment
(GenBank accession no. DQ093858). DNA sequence
analysis showed that an ORF of 879 bp was involved
in this fragment, which corresponded to 293 amino
acids with a calculated molecular mass of 33712.6
kDa (Fig. 1). The deduced amino-acid sequence
showed 78% identity with the imidase (291 amino
acids) from A. eatrophus 112R4 [9] and 80% identity
with the 20 N-terminal amino acids of imidase from
Blastobacter sp. A17p-4 [8]. However, no similarity
was found compared with any known 50- to 60-kDa
imide-hydrolyzing enzymes on the protein sequence
database available from the Internet using the BLAST
search engine [13]. To express the gene, the ORF was
reamplified by PCR with pUS804 as template, and the
resultant PCR product was inserted into the modified
pET32a to form recombinant plasmid pEI. After being
introduced into E. coli BL21, the strain pEI/E. coli
BL21 expressed the active imidase in LB medium,
resulting in a dense band of 36 kDa on SDS-PAGE
(see next section).
Y.-W. Shi et al.: Imidase Gene Cloning, Expression, and Substrate Specificity
63
Fig. 2. SDS-PAGE analysis of expression product and imidase purification. Lane 1 = Culture of uninduced cells. Lane 2 = Culture of
induced cells. Lane 3 = Supernatant after sonication and centrifugation. Lane 4 = Sample eluted from Ni2+-NTA column.
Fig. 1. DNA sequence analysis of 2.69 kb fragment from P. putida
YZ-26 genomic DNA. The ORF was shown to be 1282 bp to 2163 bp
and the corresponding amino acids were deduced. This sequence was
deposited to GenBank under accession no. DQ093858.
Expression and purification of imidase. Recombinant
E. coli BL21(pEI) could express imidase in soluble
form, and the activity reached 5.19 U/ml cultured cell as
described in Materials and Methods. (His)6-tagged
imidase was subsequently purified using an Ni2+-NTA
agarose, which occurred as a single band with an
apparent molecular mass of 36 kDa as determined by
SDS-PAGE (Fig. 2). Overall recovery of the enzymatic
activity by one-step affinity purification was 75%
(Table 1).
Molecular mass and form of imidase. The subunit
molecular mass of the (His)6-tagged imidase was
approximately 36 kDa (Fig. 2). It has been reported
that the functional unit of imidase from divergent
sources usually occurs as dimer or tetramer [4, 5]. As
such, size-exclusion chromatography was performed to
evaluate the molecular form of the native imidase on an
AKTA purifier. The result indicated that the retention
volume of the native enzyme (11.75 ml) is close to the
that of the bovine serum albumin (BSA) dimer (12.02
ml). Using the standard curve–deduced molecular mass
curve of standard proteins (Fig. 3), the molecular mass
of the native imidase was estimated to be 141 kDa. By
contrast to the trimer imidase from Blastobacter sp.
strain A17p-4 [8], the imidase isolated in the current
study corresponds to a tetramer of identical subunits.
Substrate specificity of imidase. Substrate specificity
of the imidase was tested with various compounds, such
as hydantoin, dihydrourarcil, succinimide, and
maleimide. Imidase demonstrated the highest activity
toward succinimide, which is in accordance to the
kinetic parameters listed in Table 2. Imidase
demonstrated 100- to 500-fold higher velocity in the
hydrolysis of simple cyclic imide (e.g., succinimide and
maleimide) than in the hydrolysis of cyclic ureides (e.g.,
hydantoin or dihydrourarcil). However, the optimal
substrate of most known imidases with 50- to 60-kDa
subunits is 5¢-substitute hydantoin or dihydrouracil, not
succimide. Similar results have also been observed in
imidase from Blastobacter sp. strain A17p-4 [8] and
from A. eatrophus 112R4 [9]. This implies that the
conformation of the active site of the imidase may prefer
the simple cyclic imide.
Conclusion
Just as D-hydantoinase with cyclic ureide as substrate
was reported in our previous work [14, 15], another
imidase with succinimide as optimal substrate was
achieved in the same strain, P. putida YZ-26. With respect to substrate specificity, kinetic parameters, subunit
molecular mass, and amino-acid sequence, this enzyme
is different from approximately 50- to 60-kDa imidases,
such as D-hydantoinase, which is the third imidase
found so far in bacteria. Our data demonstrate that two
different kinds of imidases can be present in Pseudomonas as reported in Blastobacter spp. [7].
64
CURRENT MICROBIOLOGY Vol. 55 (2007)
Table 1. Purification summary of the recombinant imidase (1 L culture)
Steps
Total protein
(mg)
Total activity
(lmol/min)
Specific activity
(lmol/min/mg)
Yield %
Fold of
purification
Extract
Ni2+—NTA
331.1
24.7
988.6
744.3
2.99
30.1
100
75,3
1.0
10.1
Literature Cited
Fig. 3. Determination of the molecular mass of the imidase by sizeexclusion chromatography. Imidase and standard proteins were applied
to a prepacked Sepharose 12 column, pre-equilibrated in 50 mM TrisHCl (pH 8.0) and 100 mM NaCl. Kav is defined as (Ve-Vo/Vi-Vo)
with Ve, Vi, and Vo as the elution volumes, the column volume (23.56
ml), and the void volume (7.86 ml), respectively. Protein standards
(with Mr and corresponding Kav in bracket) are (a) trypsinogen (24
kDa, 0.467), (b) alcohol dehydrogenase (41 kDa, 0.369), (c) chicken
egg albumin (45 kDa, 0.336) (d) bovine serum albumin (dimer 136
kDa, 0.265, and (e) transferrin (dimer 160 kDa, 0.226). The arrow
points to the value corresponding to the imidase (140 kDa, 0.248).
Table 2. Substrate specificity of the recombinant imidase
Substrates
km
(mM)
kcat
(min)1)
kcat/km
(mM)1min)1)
Hydantoin (100 mM)
Dihydrouracil (100 mM)
DL-5-Phenylhydantion (10 mM)
DL-p-Hydroxyphenylhydantion
(10 mM)
Succinimide (20 mM)
Maleimide (20 mM)
53.5
74.3
—
—
126.4
548.6
—
—
2.4
7.3
—
—
29.2
39.1
89641.0
28750.4
3061.4
735.5
ACKNOWLEDGMENTS
This research was supported by the grant from National Science
Foundation of Shanxi Province (NSFSX, 031042), PRC.
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