Overexpression of a recombinant wild-type and His-tagged
Bacillus
subtilis
glycine oxidase in
Escherichia coli
Viviana Job, Gianluca Molla, Mirella S. Pilone and Loredano Pollegioni
Department of Structural and Functional Biology, University of Insubria, Varese, Italy
We have cloned the gene coding for t he Bacillus subtilis
glycine oxidase (GO), a new flavoprotein that oxidizes gly-
cine and sarcosine to the corresponding a-keto a cid,
ammonia a nd hydrogen peroxide. By inserting the DNA
encoding for GO into the multiple cloning site of the
expression vector pT7.7 we produced a recombinant plasmid
(pT7-GO). The pT7-GO encodes a fully active fusion protein
with six additional residues at the N-terminus of GO
(MARIRA). In BL21( DE3)pLysS Escherichia coli cells, and
under optimal isopropyl thio-b-
D
-galactoside induction
conditions, soluble and active chimeric GO was e xpressed up
to 1.14 U g
)1
of cell (and a fermentation yield of 3.82 UÆL
)1
of fermentation broth). A n N-terminal His-tagged protein
(HisGO) w as also successfully expressed in E. coli as a
soluble protein and a fully active holoenzyme. HisGO
represents  3.9% of the total soluble protein content o f t he
cell. The His-tagged GO was purified in a single step by
nickel-chelate chromatography to a specific activity of
1.06 UÆmg

primary and secondary amines to give a-keto acids,
ammonia and hydrogen peroxide (L. Pollegioni, unpub-
lished r esults). SOX catalyses the o xidative demethylati on of
sarcosine (N-methylglycine) to form glycine and formalde-
hyde. Similarly, DAAO catalyses the oxidative d eamination
of neutral and (with a lower efficiency) basic
D
-amino acids
to give the corresponding a-keto acids and ammonia. In
both cases, the reduced coenzyme is re-oxidized by
molecular oxygen to yield H
2
O
2
. Although DAAO and
SOX s how a wide s ubstrate t olerance, they do not efficiently
oxidize glycine.
The present aim of this project is to elucidate the
structure–function relationships in GO, with the ultimate
goal of clarifying the modulation of the substrate specificity
in enzymes (GO, DAAO and SOX) that catalyse reactions
on similar compounds. Although extensive information on
the functional and structural characteristics of DAAO and
SOX is available, there is little knowledge of GO p roperties
and reactions. There is also a biotechnological aspect to this
project, as the stereoselective reaction catalysed by DAAO
is of considerable importance in biotechnology and indus-
try, e.g. the bioconversion of cephalosporin C to glutaryl-
7-amino cephalosporanic acid [3,4]. Industrial interest i n t he
discovery of amino-acid oxidase activities with new, wider

-aspartate oxidase; DMGDH, dimethylglycine
dehydrogenase; SDH, sarcosine dehydrogenase; PIPOX, pipecolate
oxidase; INT, iodonitrotetrazolium chloride.
(Received 23 O ctober 2001, revised 7 January 2002, accepted 16
January 2002)
Eur. J. Biochem. 269, 1456–1463 (2002) Ó FEBS 2002
Pharmacia Biotech and hydroxyapatite was from Bio-Gel-
HTP. All purification steps were p erformed using a n
A
¨
KTA-FPLC system (Amersham Pharmacia Biotech).
The plasmid DNA was extracte d and purified from E. coli
cells using the FlexiPrep Kit and the DNA was extracted
from the gel us ing the Sephaglass BandPrep K it (Amersham
Pharmacia Biotech). The pT7.7A plasmid was from USB;
BL21(DE3)pLysS E. coli cells were from Novagen Inc.
Assay for GO activity
GO activity was assayed using a Hansatech oxygen
electrode equipped with a thermostat to measure the oxygen
consumption at p H 8.5 and 25 °Cwith10m
M
sarcosine as
substrate. One unit of GO is defined as the amount of
enzyme that converts 1 lmol of substrate (sarcosine or
oxygen) per minute at 25 °C. The pH effect on GO activity
and s tability was dete rmined using a multicomponent
buffer: 15 m
M
Tris, 15 m
M

lowing oligonucleotides: YJBr-up (5¢-GCCAT
GAATTC
GCGCTATGAAAAGGCATTATGAAGCAGTGG-3¢)
derived from the 5¢ end and YJBr-down (5¢-CCGAT
GAATTCCATCATATCTGAACCGCCTCCTTGCG-3¢)
derived from the 3¢ end of nucleotide sequ ence of yjbR gene
(the sequence recognized by EcoRI restriction enzyme is
underlined). This amplification yielded a product of
1139 bp representing the entire GO gene. The PCR p roduct
was digested with t he restriction enzyme EcoRI, i solated by
agarose gel electrophoresis and inserted i n the unique EcoRI
site of the multiple cloning site of the expression vector
pT7.7 downstream of t he T7 RNA polymeras e promoter t o
produce the recombinant p lasmid pT7-GO. The correct
orientation of the insert was checked by restriction digestion
with the e nzyme HindIII. Both strands of the resulting
plasmid were automatically sequenced: the nucleotide
sequence was identical to t he known sequence of the yjbR
gene [1].
Expression and purification of GO in
E. coli
The pT7-GO expression plasmid was amplified in t he E. co li
strain JM109 and then transferred, for p rotein production,
to the host BL21(DE3)pLysS E. coli strain. Cells carrying
the recombinant plasmid were grown at 37 °CinLuria–
Bertani, 2 · Luria–Bertani, 2 · YT [5] or terrific broth
media containing ampicillin (100 lgÆmL
)1
final concentra-
tion) and c hloramphenicol (34 lgÆmL

lysis buffer per gram of E. coli cells). The i nsoluble fraction
of the l ysate w as removed by centrifugation a t 39 000 g for
40 min at 4 °C.
The cell homogenate, obtained by French P ress lysis of
 34 g of E. coli cells under the conditions reported a bove,
was precipitated with ammonium sulfate at 30% of
saturation (164 gÆL
)1
). The supernantant was then brought
to 45% of saturation (86 gÆL
)1
). After centrifugation, the
protein pellet was re-suspended a nd dialysed against 5 0 m
M
potassium phosphate buffer, pH 7.0, 2 m
M
EDTA, 1 0%
glycerol a nd 5 m
M
2-mercaptoethanol (buffer A). After
dialysis and c entrifugation, the enzyme solution w as ap plied
to a DEAE-Sepharose Fast-Flow c olumn (1.6 · 21 cm)
and eluted with a 10–20% linear gradient using buffer A to
which 1
M
NaCl was added. Fractions containing GO
activity were pooled and concentrated using a n Amicon cell
concentrator equipped with a YM30 membrane. The
sample was then loaded onto a hydroxyapatite column
(1.6 · 12 cm) and GO eluted, using a 50 m

stored at )20 °C.
HisGO preparation, expression and purification
The G O DNA obtained by EcoRI digestion of the original
pT7-GO plasmid (see above) was inserted into the EcoRI
site of the pT7-DBam/Hind plasmid [6]. The resulting
recombinant plasmid, defined as pT7-HisGO, encodes for
an additional N -terminal sequence c ontaining one methi-
onine and six histidine residues (Fig. 1). The correct
insertion of the GO gene was checked by digestion with
the restriction enzymes NdeIandBamHI/ScaI and by a
PCR reaction, using the XbaI [6] and YJBr-down oligonu-
cleotidesasaprimer.
The pT7-HisGO expression plasmid was transferred
to the host BL21(DE3)pLysS E. coli strain for protein
production. Recombinan t cells were grown at 3 7 °Cin
2 · Luria–Bertani medium containing ampicillin and
Ó FEBS 2002 A His-tagged chimeric glycine oxidase (Eur. J. Biochem. 269) 1457
chloramphenicol (100 lgÆmL
)1
and 34 lgÆmL
)1
final con-
centration, respectively) and induced at D
600
¼ 0.8 by
adding isopropyl thio-b-
D
-galactoside at a final concentra-
tion of 1 m
M

phosphate b uffer, pH 8.5, containing 2 m
M
EDTA, 5 m
M
2-mercaptoethanol and 10% glycerol.
Substrate specificity
Two d ifferent methods were u sed to i nvestigate the substr ate
specificity of GO.
Activity stain using iodonitrotetrazolium chloride (INT)
on protein samples separated by native PAGE. After the
electrophoretic separation, each single lane was incubated
for2hinthedarkat37°C in a solutio n containing 75 m
M
sodium pyrophosphate, pH 8.5, 100 m
M
substrate, 5 l
M
FAD a nd 0.1 mgÆmL
)1
INT (dissolved in pure ethanol).
The activity was revealed as a pink band on the gel in the
position corresponding to the G O.
Spectrophotometric determination of GO activity meas-
uring the hydrogen peroxide produced in the presence of
different substrates using a 96-well ELISA plate. Each
assay well has 200 lL of a solution containing 10 m
M
or
90 m
M

GO was determined on a purified soluble protein sample
using an automated protein sequencer (Procise Model
492, Applied Biosystems). The
BLAST
program (http://
www.ncbi.nlm.nih.gov/blast/Blast.cgi) [9] was used to
search for proteins showing sequence similarity. Multiple
sequence alignments were performed with the
CLUSTALW
program ( />[10].
RESULTS
Cloning of
B. subtilis
GO DNA and protein sequence
comparison
To clone the gene encoding the B. subtilis GO, the genomic
DNA was amplified by PCR using the oligonucleotides
derived from the sequence of the yjbR gene of GO from
B. subtilis [1]. Currently, t here are three proteins in the
nonredundant Protein Data B ank that have been classified
asGO.InadditiontotheenzymefromB. subtilis,the
proteins from Bacillus halodurans and Thermoplas ma
volcanium (accession nos BAB05153 and NP_111169,
respectively) have been also included in the database. The
overall sequence identity w ith B. subtilis GO is modest
(27% an d 22%, respectively), although a higher conserva-
tion is evident at their N-termini (containing the Rossman
fold fingerprint m otif GXGXXG, w hich is in volved in
binding of the ADP moiety of FAD) [11] and for the 70
residues at their C-termini (for th is latter region a 31–40%

D
-amino acid that modulates NMDA neurotransmission. A
partial sequence comparison between GO and SOX or
DAAO proteins [12,13] of t he regions containing the h ighly
conserved residues that play a key role in catalysis is
reportedinFig.2A,B.
Table 1.
BLAST
results o f s earch for ide ntity an d homology with B. subtilis GO. The p ercentages refer t o t he 369 amino acids of GO. Th e h omology
score is considered the sum of identical and s trongly similar a mino acids. Th e proteins i dentifi ed by the code ÔNP_Õ have been recognized following
the complete sequence of the genome of the correspon ding organism; therefore, their activity was never proven. TSOX, b subunit of hetero-
tetrameric sarcosine oxidase.
Entry code Organism Protein Identity (%) Homology (%)
BAB05153 Bacillus halodurans GO 27 51.8
NP_111169 Thermoplasma volcanium GO 21.7 48.0
NP_107418 Mesorhizobium loti TSOX 24.4 45.5
NP_126006 Pyrococcus abyssi TSOX 26.8 49.9
P40875 Corynebacterium sp. P-1 TSOX 23.8 48.8
P23342 Bacillus sp. NS-129 MSOX 21 43
P40859 Bacillus sp. B-0618 MSOX 20.6 43.4
P24552 Fusarium solani DAAO 18.4 39.3
P80324 Rhodotorula gracilis DAAO 18.4 38
P31228 Bos taurus DASPO 18.4 39
Q63342 Mus musculus DMGDH 24.7 49
NP_106044 Mesorhizobium loti SDH 27.6 49.6
NP_057602 Homo sapiens PIPOX 21.4 46.9
Fig. 2. Details of the multiple sequence alignment f or GO from B. subtilis with sequences of S OX (A) and DAAO (B) and comparison of the 3D
structure o f t he active site with those o f other enzymes (C). Residues marked with (*) i ndicate identity (
:) indicate strongly similar, and ( .)indicate
weakly si milar. The b oxes identify the amino acids present in the active s ite of M SOX [12] and of DAAO [15,16]. (C) C omparison of t he active site

against pure B. subtilis GO did not recognize DAAO a nd
MSOX (a faint band is only observed when a large amount
of TSOX is used, data not shown). Analogously, anti-
DAAO IgG did not recognize GO, MSOX or TSOX.
Expression of
B. subtilis
GO gene in
E. coli
and
GO purification
The DNA encoding for G O was inserted in the EcoRI site
of the multiple cloning site of the expression vector pT7.7
downstream of the T7 RNA polymerase promoter to
produce the recombinant plasmid pT7-GO. Because of the
presence of an ATG c odon upstream of the cloning site, t his
procedure produces a fusion protein; six additional residues
are a dded a t t he N-terminus of the protein before the
original starting methionin e. The new ATG starting codon
is positioned 8 bp downstream from the ribosomal binding
site. The recombinant plasmid pT7-GO was used to
transform BL2 1(DE3)pLysS E. coli cells. A significant
increase in GO synthesis was observed immediately after
the addition of 1 m
M
isopropyl thio-b-
D
-galactoside in cell
cultures transformed with the pT7-GO expression vector
and grown on Luria–Bertani m edium, as indicated by native
PAGE and GO activity staining. The highest level of GO

)1
protein). GO accumulated to 1% of total
E. coli soluble proteins in the crude extract.
Recombinant GO was purified from E. coli cells by
precipitation with ammonium sulfate twice and then a
three-step chromatography procedure (see Materials and
methods). Ab out 13 mg of enzyme (90% homogenous)
were obtained starting from 3 4 g of cell paste (Table 2).
The total yield of the purification was about 20%. The
N-terminal sequence of the first eight amino acid s of the
recombinant GO was determined. This sequence is unique
and was identical to that inferred from the nucleotide
sequence [1]; protein sequencing o f 0.2 nmol of the purified
enzyme resulte d in the A RIRAMKR s equence, confirming
the presence of five of the six additional residues at the
N-terminus of recombinant chimeric GO (the starting
methionine is not present).
The recombinant GO produced in E. coli was purified
as a holoenzyme with spectral p roperties typical of the
flavin-containing oxidases (absorbance maxima at 457 nm
and 376 nm and a A
274
/A
456
¼ 9). Na tive GO is a
180-kDa homotetramer, as determined by SDS/PAGE
(46.6 ± 1.3 kDa) and gel permeation chromatography on
a Superdex 200 column (187.6 ± 2.3 kDa). The recombin-
ant enzyme catalysed the oxidation of sarcosine and glycine
(specific activity of 0.57 and 0 .43 UÆmg

HisGO
Crude extract 86.6 2188 0.039 1.0 100
HiTrap Chelating 86.3 80 1.06 27.2 98
1460 V. Job et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Addition of exogenous FAD or FMN to the assay mixture
did not increase enzyme activity, indicating a tight binding
of the coenzyme to t he protein moiety.
Preparation, overexpression and purification
of the chimeric His-tagged GO
The scheme of the strategy devised for obtaining a plasmid
coding for the chimeric His-tagged GO is reported in the
Materials and methods section; the map of the resulting
expression vector pT7-HisGO is given in Fig. 1. In these
experiments the conditions for the expression of GO in
E. coli BL21(DE3)pLysS cells were optimized. Cells were
grown at 37 °C and induced with 1 m
M
isopropyl thio-b-
D
-
galactoside at an D
600
¼ 0.8; the growth temperature was
then decreased to 30 °C a nd the cells were harvested 24 h
after induction. The protein was overexpressed under the
reported co nditions and was totally soluble and thus
recovered in the crude extract. In addition, the chimeric
HisGO was entirely present as an active holoenzyme.
Therefore, the addition o f exogenous FAD o r FMN to the
assay m ixture did n ot increase the activity a t all. The enzyme

from the E. coli cell paste). Rema rkably, a nd due to the fast
procedure used, we obtained a f ully active holoenzyme with
a specific activity that was twofold higher than that of the
wild-type recombinant enzyme (1.06 vs. 0.57 UÆmg
)1
pro-
tein). In addition, the total recovered enzyme a ctivity
obtained starting from a similar amount of E. coli cell paste
are higher for the HisGO than for the wild-type enzyme.
Properties of the His-tagged chimeric GO
The molecular mass of HisGO, as determined by SDS/
PAGE, is slightly higher than the value calculated from
the amino-acid sequence (49.4 ± 1.1 k Da vs. 42.66 kDa,
respectively). U nder native conditions, the molecular mass
of the His-tagged GO is 166.4 ± 11.1 kDa, as determined
by gel permeation chromatography. The elution volume
(R
t
¼ 13.4 ± 0.2 mL) is not dependent on the protein
concentration in the range 0.01–12 mgÆmL
)1
. T he result is
consistent with the presence in solution of a stable
homotetramer. The theoretical isoelectric points of GO
and HisGO are 6.14 and 6.34, respectively. However, we
were not able to determine this under any of the present
experimental conditions due to the instability of the protein
at a pH < 7 (see below). The purified holoenzyme shows
the typical absorbance spectrum of F AD-containing
proteins, with two well-resolved peaks in the visible region

Purified GO shows a similar specific activity on sarcosine
and glycine as substrates (1.06 and 1.00 UÆmg
)1
protein,
respectively). Because of the sequence similarity of GO with
Fig. 3. Absorbance spectrum of the purified HisGO ( 12 l
M
)inthe
oxidized form (1) and in the reduced form (2), as obtained under
anaerobic conditions by t he addition of 20 m
M
glycine in 5 0 m
M
potas-
sium phosphate buffer pH 7.0, 5 m
M
2-mercaptoethanol, 2 m
M
EDTA
and 10% glycerol.
Ó FEBS 2002 A His-tagged chimeric glycine oxidase (Eur. J. Biochem. 269) 1461
a number of flavoenzymes (see Table 1), the substrate
specificity of the reaction catalysed by GO has been
investigated using several compounds that are known to
be substrates of DAAO, DASPO, SOX, DMGDH and
PIPOX. Rapid s creening was performed by measuring the
increase in absorbance at 440 nm following the horseradish
peroxidase assay coupled with o-dianisidine using a 96-well
ELISA plate. The results, reported as a percentage with
respect to t he absorbance change observed with sarcosine as

D
-isomer, as further demonstrated by the INT staining
assayreportedinFig.5,inset.
A comparison of t he activity measured u sing a similar
amount (5 mU) of GO, DAAO and MSOX on various
substrates showed that GO and DAAO oxidize
D
-proline,
D
-alanine and
D
-2-aminobutyrate with similar relative
efficiencies. Analogously, GO and MSOX show a fairly
similar activity o n s arcosine and N-ethylglycine. In contrast,
an appreciable activity on glycine, glycine-ester a nd
D
-pipecolic acid was only observed using GO. Interestingly,
D
-proline i s t he only amino acid in which the a-amino group
is involved in a covalent bond with the side chain and it is
the only compound that was oxidized by all three enzymes
used. I t i s also the only
D
-amino ac id that is oxidized
efficiently by both D AAO and
D
-aspartate oxidase [18].
CONCLUSIONS
The present data demonstrate the successful overexpression
of a chimeric B. subtilis GO in E. coli (upto3.9%oftotal

-valine; (10)
L
-valine, as substrate.
Fig. 4. Temperature–activity and temperature stability profiles (A) and
pH–activity and pH stability profiles of HisGO (B). Enzyme activity (d)
was assayed in the tem perature range 15 –60 °C. For thermostability
(s) e nzym e samples were incubated at th e indicated t empe rature for
30 minin50 m
M
potassium phosphate, pH 7.0, 10% glycerol a nd then
assayed using the O
2
consumption a ssay at 25 °C . Data are expressed
as per cent o f enzyme activity in t he standard assay; the l ines through
the data points have been obtained by smooth fitting. SEM is the
average of three determinations. (B) pH–activity and pH stability
profiles of HisGO. Enzyme a ctivity (d) was assayed in the pH range
4.8–12. For thermostability ( s) enzyme samples we re incubated a t the
indicated pH for 30 min in the multicomponent buffer (see Materials
and methods) and the n assayed usin g the stand ard O
2
consumption
assay at 25 °C. Data are exp ressed as per cent of enzyme activity in the
standard assay; th e lines through t he data points have be en obtained
by smooth fitting. The SEM is the average of three determinations.
1462 V. Job et al. (Eur. J. Biochem. 269) Ó FEBS 2002
conservation of active s ite residues suggest that GO would
exhibit significant structural similarity with enzymes acting
on sarcosine or sarcosine analogues (monomeric and tetra-
meric sarcosine oxidases and s arcosine and dimethylglycine

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Ó FEBS 2002 A His-tagged chimeric glycine oxidase (Eur. J. Biochem. 269) 1463