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https://www.abbs.info/ e-mail:[email protected] ISSN 0582-9879 |
Purification and Characterization of Alcaligenes
faecalis Penicillin G Acylase Expressed in Bacillus subtilis
ZHOU Zheng1, ZHOU Li-Ping1#,
CHEN Mei-Juan1$, ZHANG Yan-Liang1
LI Ren-Bao1, YANG Sheng2, YUAN Zhong-Yi1*
(
1Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences, the Chinese Academy of Sciences, Shanghai 200031, China;
2Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological
Sciences,
the Chinese Academy of Sciences, Shanghai 200031, China )
Abstract
The Alcaligenes faecalis PGA gene encoding heterodimeric penicillin
G acylase (PGA) was cloned and successfully expressed in Escherichia coli
andBacillus subtilis respectively. In contrast to E.coli hosts where
the enzymes were retained in the periplasm, B. subtilis cell secreted
the recombinant enzyme into the medium. Contrary to limited expression yield
of E. coli (pETAPGA), PGA extracellularly expressed by B. subtilis (pBAPGA)
and B. subtilis (pMAPGA) reached the highest yield of 653 u/L. This
yield increased 109-fold higher than the native expression of A. faecalis
CICC AS1.767. The enzyme was fractionated with (NH4)2SO4 and purified
by DEAE-Sepharose CL-6B with a yield of 81%. The purified enzyme had a specific
activity of 1.469 u/mg. Furthermore, some enzyme characteristics, such as
the pH and temperature optimum, the stability against organic solvent and
the ratio of cepholexin synthesis to hydrolysis were determined. By overexpressing
A. faecalis PGA in B. subtilis and purifying secreted enzyme
from culture medium one could readily obtain a large amount of an alternative
source of PGA.
Key words penicillin
G acylase; Alcaligenes faecalis; Bacillus subtilis
Since 1960s, penicillin
G acylase (PGA EC 3.5.1.11) has been used to carry out N-deacylations for
industrial production of 6-APA and 7-ADCA. More recently, developments showed
that the same enzyme could also be exploited successfully in synthesis of
a large variety of semi-synthetic β-lactam antibiotics based on its catalysis
of the reverse reaction. In view of drawbacks of the conventional chemical
processes, such as the high toxicity of some reagents and the requirement
for a lot of reaction steps, the chemical synthesis processes are being replaced
by enzyme-catalyzed processes. Nowadays, enhancements of the production capability
of commercially used PGA have become the focus of interest[1]. In recent years,
a variety of strategies have been designed to increase the production levels
of PGA by screening conventional mutant strains, or, by using DNA manipulation
to construct recombinant PGA overproducing strains. High-level extracellular
expression of Bacillus megaterium PGA (BmPGA) has been successfully performed
in our laboratory[2].
Besides having similar substrate preferences, enzymes of PGA diverse group
are evolutionarily related. In the recent past, the PGA from Alcaligenes
faecalis (AfPGA) is widely investigated. The enzyme possesses a clear
industrial advantage over other well-characterized penicillin G acylases.
(1) AfPGA has an attractive prospect to be used in enzymatic hydrolysis of
various side chains for its relative higher specificity constant[3]. (2) It
possesses higher ability of β-lactam antibiotic synthesis in aqueous organic
solvents[4]. (3) This enzyme is a better producer of chiral intermediates[4].
(4) With higher thermostability and probable higher stability against anhydrous
media, AfPGA is a more attractive candidate in synthetic conversions[5]. However,
sources of the enzyme are still limited nowadays. AfPGA gene has been expressed
in Escherichia coli system but successful expressions in other expression
systems have not been reported till now. Moreover, no data are given as to
the yield of PGA obtained in E.coli system[6,7]. So we intend to construct
an organism that can produce PGA constitutively and secrete it extracellularly
for large-scale production.
In this report, a high-level expression system was developed. The study reported
the cloning of an A. faecalis CICCAS1.767 PGA gene, the extracellular
PGA expression in B. subtilis and the procedure of purification. Furthermore,
conditions of PGA expression in different systems were investigated. Some
enzyme characteristics, such as the pH and temperature optimum, the stability
against organic solvent and the ratio of cepholexin synthesis to hydrolysis,
were also determined.
1 Materials and Methods
1.1 Plasmids, strains and culture conditions
Alcaligenes faecalis CICC AS1.767 was obtained from Institute of Industrial
Microbiology, Shanghai, China. E. coli XL1-Blue, JM109(DE3) and B.subtilis
WB600 were used as the hosts. pUC18 was from ATCC. pWB980 and pMA5 were both
derivative plasmids of pUB110 with both p43 and HpaII as promoters respectively.
Escherichia coli XL1-Blue, JM109(DE3) and B.subtilis WB600 were cultivated
with aeration at 37 °C in Luria broth (LB). Ampicillin (100 g/L) and kanamycin
(50 g/L) were added to the medium when E. coli XL1-Blue, JM109(DE3) and B.subtilis
WB600 harboring the recombinant plasmid were selected, respectively. The E.
coli engineering strain for expression grew in fermentation medium A consisted
with 1.5 g/L yeast extract, 0.5 g/L NaCl and 5 g/L glycerol. The B.subtilis
strain expressed PGA in fermentation medium B consisted with 5 g/L peptone,
5 g/L yeast extract, 1 g/L NaCl and 15 g/L starch.
1.2 Reagent
The restriction enzymes, Pfu polymerase, large fragment of DNA polymerase
I, calf Intestinal Alkaline Phosphatase, and T4 DNA ligase were purchased
from TaKaRa (Dalian, China) and were used according to the instruction of
the manufacturer. Isopropylthio-beta-D-galactoside (IPTG) was from Sangon
(Shanghai, China). 6-nitro-3-phenylacetaminobenzoic acid (NIPAB) was from
Dongfeng Reagent Factory (Shanghai, China). Other reagents were of AR grade.
The DNA sequencing reactions were conducted by using PRISMTM Dye Terminator
Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems). The data were
collected and analyzed with a ABI PRISMTM 310 DNA Sequencer.
1.3 Enzyme assay
PGA activity was assayed by a modification of the colorimetric method[8].
One unit of enzyme was defined as the amount of enzyme catalyzing the hydrolysis
of 1 micromole of NIPAB per minute at 37 °C. Protein determination was carried
out using the Folin-phenol reagents.
1.4 Gene isolation and plasmid constructions
A. faecalis CICC AS1.767 was grown for 24 h in LB medium for isolation
of chromosomal DNA. The AfPGA gene was obtained by PCR amplification. Following
oligos (restriction sites for Nde I and BamHI are underlined) were designed
according to sequence of GenBank AFU93881: 5′-GGCCATATGCAGAAAGGGCTTGTTC-3′and
5′-CCGGATCCCTAAGGCTGAGGCTGAATC-3′，The 5′- primer contained an initial
signal (ATG) whereas the 3′- primer consisted of a translational stop signal
(TAA). The PCR reactions was performed with 3 min at 94 °C, 30 s at 94 °C,
30 s at 55 °C, 5 min at 72 °C for 30 cycles. The PCR products were blunt-end
ligated into a pBluscript (SK) vector linearized with SmaIto construct pBAPGA
and then sequenced (GenBank accession No.AF455356). To construct expressing
plasmid pMAPGA and pETAPGA, a 2.5 kb NdeI-BamHI fragment of plasmid pBAPGA
was inserted respectively into plasmid pMA5 and pET24a(+) which were digested
in the same way. The AfPGA gene was then modified of shortening the signal
sequence by PCR reaction using a newly designed 5′- primer (restriction site
for HindIII is underlined): 5′-AAGCTTTTGCCCAAG-TGCAGTCGGTAGAG-3′. The
PCR products were cloned into plasmid pUC980, a plasmid derived from pWB980
by inserting the linearized pUC18 into multiple cloning site of EcoRI.
1.5 Expression and purification
The B. subtilis clone was grown in 3 ml LB for 18 h at 37 °C. 0.5 ml
of the culture was added to the 50 ml fermentation medium B in 250 ml shake
flask. Fermentation was performed on a rotary shaker at 37 °C for 86 h. The
culture was centrifuged. The supernatant was precipitated by 20%－55% saturation
of ammonium sulfate. This fraction was collected, dissolved in 0.05 mol/L
sodium phosphate buffer (pH 7.0) and dialyzed. Subsequently, dialyzate was
loaded on DEAE-Sepharose CL-6B column and eluted with a linear gradient of
0 to 500 mmol/L NaCl buffer. Protein purified was monitored by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The E. coli clone grown in 3 ml LB over night was withdrawn and added into
50 ml fermentation medium A and incubated on a shaker at 37 °C. While cell
density reached A600=0.6, IPTG was added to final concentration of 0.5 mmol/L
and the fermentation continued for 3 h. The cells were collected by centrifugation
at 4000 g 1.6Evaluation of optimal pH, optimal temperature and stability in
dimethyl formamide (DMF)
The effect of pH on enzyme was determined by varying the pH of the buffer
system between 5.0 and 10.0, with an increment of 0.5－1.0. Effect of temperature
on enzyme was studied between 25 °C and 67 °C with gradual increments. Stability
in organic solvent expressed as residual enzyme activity, was measured by
incubating the enzyme in 50%DMF / 10 mmol/L sodium phosphate buffer (pH 6.4),
55 °C, in different ratios for 20, 40, 50 and 60 min.
Fig.1 Structures of
expression vectors pMAPGA, pBAPGA and pETAPGA for A.faecalis PGA gene were
constructed
For vectors employed to express other PGA on the basis of pMA5 and pET24a(+),
AfPGA gene was replaced by homological ones.1.7Assay of synthesis/hydrolysis
ratio (S/H)
Enzymatic synthesis of cephalexin with substrates of 0.2 mol/L 7-aminodeacetoxyce-phalosporanic
acid (7-ADCA) and 0.4 mol/L D-phenylglycine were carried out in 50 mmol/L
phosphate buffer at pH 7.0 at 4 °C. Substrates and products of enzymatic synthesis
were analyzed by reversed-phase HPLC using a C18 column with Shimadzu delivery
system LC-10AS and a Shimadzu UV SPD-10AV detector set at 215 nm as a modified
method described by Hernández-Jústiz et al[KG-*3〗.[9]. All compounds were
eluted with 50 mmol/L sodium phosphate buffer (pH 7.0) (mobile A) and 80%
acetonitrile (mobile B). The following gradient was used: 0－4 min elution
with 100% mobile A, 4－8 min gradual decrease to 80% mobile A, 8－16 min elution
with 80% mobile A, 16－20 min elution with 100% mobile A. Concentrations of
substrates and products were calculated from standard curves performed with
standard solutions. The S/H ratio was caculated when 10% of the 7-ADCA had
been consumed.
2 Results
2.1 Cloning and DNA sequencing of the AfPGA gene
AfPGA is a novel enzyme with many attractive characteristics whose gene is
recently revealed[5]. The PGA gene of A. faecalis CICC AS1.767 was
cloned using PCR. The sequence was submitted to GenBank and the accession
number was AF455356. Amino acid sequence alignment with the PGA sequence of
Genbank AFU93881, which was derived from A. faecalis ATCC 19018, demonstrated
existence of significant homology as well as evident divergency. These enzymes
just shared the 94.6% protein sequences identity (89.8% DNA sequences identity).
Further alignment revealed that AfPGA amino acids were highly conserved to
the PGA from gram-negative bacteria of Escherichia coli, Kluyvera citrophila
and Providencia rettgeri, but largely different to the PGA from gram-positive
bacteria of Bacillus megaterium and Arthrobacter viscosus (Pairwise identity
of the α subunit sequences ranged from 32% to 47% and β subunit sequences,
from 33% to 41%). Although totally 44 differences of amino acid residues existed
between two sequences, no more residue difference except for Aα34T and Iβ77M
occurred in conserved clusters.
2.2 Expression and purification of AfPGA
E. coli-B.subtilis shuttle plasmids of pUC980 and pMA5 were selected to express
AfPGA in this study. By constructing plasmid of pMAPGA, complete AfPGA gene
with signal peptide sequence was placed between HpaII expression-secretion
cassettes. In addition, the signal peptide sequence of PGA was removed using
PCR. The 2.5 kb insert was ligated into the multiple cloning site region downstream
of the B.subtilis p43 promoter and the SecB signal sequence of pUC980 at the
HindIII/BamHI restriction sites (Fig.1). Both of the derived plasmids, pMAPGA
and pBAPGA were transformed to B.subtilis host of WB600[10] and transformants
with high-level extracellular expressions of PGA were designated SIBSB300
and SIBSB301. Fermentation of strains SIBSB 300 and 301 was performed for
86 h. Under optimized conditions, productivity of PGA reached levels of 172
u/L and 653 u/L respectively, which increased by 29-fold and 109-fold compared
to the native expression in A. faecalis (6 u/L) (Fig.2).
Fig.2 Growth and A.
faecalis PAG production curve of WB600 (pWAPGA) and WB600 (pMAPGA) fermentation
Symbol of circle represent WB600 (pWAPGA); symbol of square represent WB600
(pMAPGA). Cell density (○, □); enzyme activity (●, ■).Based on similar expression
manipulation, microorganisms containing PGA genes derived from different origin
were obtained (Table 1). In this study, although BmPGA yields produced by
SIBSB305 reached optimum of 8 u/ml, it was only 1.3 folds higher than the
expression level (6 u/ml) provided by the parental B.megaterium strain, which
extracellularly expressed PGA. Therefore, PGA yield enhancement of SIBSB300
is much higher. Furthermore, We have attempted to express other PGA in B.subtilis
but unfortunately failed. Among all the PGA from the gram-negtive bacteria,
AfPGA was proved the unique example that could be expressed in B. subtilis.
All PGA investigated in this study were successfully expressed in E. coli
under the control of T7 promoter. However, such expression needed induction
of expensive IPTG and, expressed enzymes were located in periplasm[6]. AfPGA
expression in E. coli at 37 °C just achieved limited productivity of 150 u/L,
which were 4 folds lower than yield of SIBSB300.
Purification of AfPGA from the SIBSB300 culture broth by centrifugation, ammonium
sulfate precipitation and ion exchange chromatography achieved approximately
86-fold purification and overall yield of 81% (Table 2). The 23 kD and 65
kD bands corresponded with the α subunit and β subunits are clear (Fig.3).
2.3 Characterization of AfPGA and BmPGA
The isolated recombinant AfPGA was characterized toward the pH and temperature
Table 1 Strains, plasmids and PGA genes used in this study
| Organism | Plasmid | PGA Gene | Strain/original vector | Induction | PGA yield (u/L) |
Ya | Rb |
| SIBSB300 | pMAPGA | A. faecalis | WB600/ pMA5 | No | 653 | 150.7 | 108.8 |
| SIBSB301 | pWAPGA | A. faecalis | WB600/ pWB980 | No | 172 | 33.4 | 28.7 |
| SIBSB302 | pMEPGA | E. coli | WB600/ pMA5 | No | 0 | 0 | 0 |
| SIBSB303 | pMPPGA | P.rettgeri | WB600/ pMA5 | No | 0 | 0 | 0 |
| SIBSB304 | pMKPGA | K.citrophila | WB600/ pMA5 | No | 0 | 0 | 0 |
| SIBSB305 | pMMPGA | B.megaterium | WB600/ pMA5 | No | 8000 | 1650.1 | 1.3 |
| SIBSE301 | pETAPGA | A. faecalis | JM109(DE3)/pET24a(+) | IPTG | 150 | ND | 25.0 |
| SIBSE302 | pETEPGA | E. coli | JM109(DE3)/pET24a(+) | IPTG | 320 | ND | ND |
| SIBSE303 | pETPPGA | P.rettgeri | JM109(DE3)/pET24a(+) | IPTG | 993[11] | ND | 66.2 |
| SIBSE304 | pETKPGA | K.citrophila | JM109(DE3)/pET24a(+) | IPTG | 840 | ND | ND |
aRelative yield of PGA (Unit per gram dry cell). LbRatio of PGA production
of engineering strain and wild type strain. ND: not determined
Table 2 Purification
of AfPGA from the SIBSB300 culture broth
| Purification step | Volume (ml) |
Total protein(mg) | Total activity(u) |
Specific activity(u/mg) | Purification factor |
Recovery(%) |
| Crude broth supernatant | 150 | 2102.5 | 35.7 | 0.017 | 1.0 | 100 |
| (NH4)2SO4 fractionation | 15 | 128.7 | 31.8 | 0.247 | 14.5 | 89 |
| DEAE-Sepharose CL-6B | 4 | 19.6 | 28.8 | 1.469 | 86.4 | 81 |
Fig.3 SDS-PAGE of A.faecalis
penicillin G acylases
1, purified A. faecalis PGA. 2, fraction of (NH4)2SO4 precipitate;
M, marker. Subunits of PGA were marked by arrows.
optimum, the stability
against organic solvent and the S/H ratio. The recombinant BmPGA was selected
as a control example because the well studied enzyme was a representative
member in gram-positive bacteria PGA family and could also be expressed in
B. subtilis[12,13].
Fig.4 Relative activities
of PGA at different pH (A) and different temperature (B)
■, A. faecalis PGA; □, B.megaterium PGA.
As shown by Fig.4(A),
the pH optimums for both enzymes are pH 8.0. However, the temperature optimum
for AfPGA is 60 °C, and 45 °C for BmPGA [Fig.4(B)]. It has been proved that
the AfPGA exhibits higher thermostabiliy than E. coli PGA[5]. Considering
long-term catalysis, it is more important to evaluate the enzyme stability
against aqueous organic solvents. The enzymes were added into 50% aqueous
DMF and the residual enzyme activity were determined (Fig.5). In the presence
of 50% DMF, half activity of BmPGA was lost in 45 min and almost 100% of activity
was lost in 60 min. However, the half-life of the A. faecalis enzyme
was almost 60 min. Thus, the stability of the AfPGA is significantly higher
than that of the BmPGA.
Fig.5 Stabilities of
PGA in 50% aqueous DMF
■, A. faecalis PGA; □, B.megaterium PGA.
Fig.6 Relative S/H
ratio of penicillin G acylase from A. faecalis PGA (Af) and B. megaterium
PGA (Bm)
Pr, Kc, Ec are homologous enzymes from P. rettgeri, K. citrophilar and E.
coli.
The S/H ratio is a crucial
catalytic characteristic of PGA. The recombinant PGA was used to catalyze
synthesis of cephalexin. The initial ratio of cephalexin synthesis to hydrolysis
of AfPGA and BmPGA was almost the same but significantly lower than PGA from
other species (Fig.6).
3 Discussion
From a biotechnological point of view, the complex post-translational maturation,
i.e., the conversion of the PGA precursor into the mature enzyme is the limiting
factor for a recombinant PGA overproduction in E. coli[14]. Expression/secrection
systems such as Saccharomyces cerevisiae were able to overcome the present
limitations of the recombinant E. coli system[15]. E. coli expression system
has been found to succeed in producing the AfPGA. However, no other system
was used to express AfPGA ever before. In this study, we expressed the enzyme
in E. coli and B. subtilis. Contrary to what occured in E. coli, recombinant
AfPGA expressed in B. subtilis was secreted into the growth medium.
Moreover, B. subtilis could not express PGA from other gram-negative
bacteria. This result implies that the enzyme expression in B. subtilis
is very different from expressions of other evolutionarily related enzymes.
In this study, we also observed that the enzyme just had a poor synthesis
capability of β- lactam antibiotic in water. The result was opponent to the
opinion that the AfPGA possessed high S/H ratio in aqueous organic solvents
from van Langen et al.[4].
It had been reported that p43 expression-secretion cassette was preferred
to overproduce BmPGA[16] because this vegetative promoter was much stronger
for PGA expression than HpaII promoter. In our study, however, HpaII promoter
expressed AfPGA better than p43 promoter. Manipulation of substituting SecB
signal peptide for AfPGA signal peptide probably accelerates PGA maturation
processes such as signal peptide recognize and removal, or, cytoplasmic precursor
transportation and location.
Sequence analysis of the enzyme indicates that cysteines of Cβ493 and Cβ526,
which form a disulfide bridge. The disulfide bridge resulted in enzyme’s high
thermostability[5]. It was demonstrated in our study that the disulfide bridge
also contributes to the enzyme higher stability against organic solvent. However,
some proteins containing disulfide bonds are secreted very poorly by B.
subtilis because of the formation of incorrect disulfide bonds or the
lack of formation of such bonds[17]. That means disulfide bridges may has
negative effect on PGA expression.
This work provided an efficient production and recovery protocol of AfPGA.
Based on the successful high-level extracellular expression in B. subtilis,
efficiency of different promoters and different signal peptides were investigated
and a rapid isolation procedure for the secreted AfPGA was developed. One
could readily obtain a large amount of an alternative source of PGA using
this method.
Acknowledgements Dr. WONG Sui-Lam (University of Calgary, Canada) and
Dr. DARTOIS Veronique (MicroGenomics Inc., CA, USA) should be gratefully acknowledged
for their kindly providing plasmids of pWB980 and pMA5.
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