Functional expression of human liver cytosolic b-glucosidase
in
Pichia pastoris
Insights into its role in the metabolism of dietary glucosides
Jean-Guy Berrin
1,2
, W. Russell McLauchlan
1
, Paul Needs
1
, Gary Williamson
1
, Antoine Puigserver
2
,
Paul A. Kroon
1
and Nathalie Juge
1,2
1
Nutrition, Health and Consumer Sciences Division, Institute of Food Research, Norwich, UK;
2
Institut Me
Â
diterrane
Â
en de Recherche
en Nutrition, Faculte
Â
des Sciences et Techniques de Saint-Je
Â

b-
D
-glucosides, b-
D
-galactosides, b-
L
-xylosides, b-
D
-arabino-
sides), similar to the native enzyme. For the ®rst time, we
show that the human enzyme has signi®cant activity towards
many common dietary xenobiotics including glycosides of
phytoestrogens, ¯avonoids, s imple phenolics and cyanogens
with higher apparent anities (K
m
) and speci®cities (k
cat
/K
m
)
for d ietary xenobiotics than f or other aryl-glycosides. These
data indicate that human CBG hydrolyses a broad range of
dietary glucosides a nd may play a critical role in xenobiotic
metabolism.
Keywords: heterologous expression; xenobiotic metabolism,
¯avonoids; iso¯avones; ®rst-pass metabolism.
b-Glucosidases (b-
D
-glucoside glucohydrolase; EC 3.2.1.21)
are members of glycosyl hydrolase families 1 and 3 [1,2].

ogy t o family 1 g lycosyl hydrolase and is also predicted to
occur in the cytosol of certain human cells [6,7] where it
might have a role in human aging [6].
Finally, a b-glucosidase, termed cytosolic b-glucosidase,
is present in the liver, kidney, intestine and spleen of
humans. This c ytosolic b-glucosidase (CBG) h as been
puri®ed from human liver and partially characterized
[8±10]. It is a 53-kDa monomeric protein with a pI of
 4.7, a broad and near-neutral pH optimum, and a broad
speci®city w ith respect to the g lycone moiety of substrates.
Human CBG hydrolyses synthetic aryl glycosides (including
4-nitrophenyl and 4-methylumbelliferyl monoglycosides)
[9], but no physiological substrate h as been found and the
function in vivo has yet to be determined. However, during
our research into the mechanisms underlying the absorption
and metabolism of dietary ¯avonoids and iso¯avones, we
demonstrated that crude protein e xtracts derived from
human liver and small intestine tissues ef®ciently hydrolysed
a range of foo d-borne phytochemical (¯avonoid and
iso¯avone) glucosides [11]. The effects of s peci®c enzyme
inhibitors appeared to indicate that the majority of
Correspondence to P. A. Kroon, Nutrition, Health & Consumer Sci-
ences Division, Institute of Food Research, Colney Lane, Norwich,
NR4 7UA, UK. Fax: + 44 1603 255038, Tel.: + 44 1603 255236,
E-mail:
Abbreviations: AOX1, a lcohol oxidase; BMGY, bu ered minimal
glycerol-complex medium; BMMY, buered minimal methanol-
complex medium; ESI, electrospray ionization; CBG, cytosolic
b-glucosidase; cbg-1, cDNA encoding CBG; reCBG, recombinant
CBG; LPH, lactase-phlorizin hydrolase; 4NP, 4-nitrophenol; YNB,

attributes that renders it an attractive host for the expression
and production of CBG: it can b e grown conveniently to
high den sity levels in a simple and inexpensive medium; it is
able to carry out certain post-translational modi®cation
events such as proteolytic maturation, glycosylation and
disul®de bond formation; under the co ntrol of t he ef®cient
and highly regulated promoter of the alcohol oxidase gene,
AOX1, it c an secrete p roteins to very h igh levels [18±20].
In this report, we show that puri®ed recombinant CBG
possesses similar physical and enzymatic properties to CBG
isolated from human liver. Furthermore, we investigated the
speci®city of the human CBG with r espect to the glycone
and aglycone moieties, and in particular characterized the
ef®ciency of the enzyme in hydrolysing a broad r ange of
dietary xenobiotic glycosides. The potential role for human
CBG in xenobiotic metabolism and uptake is also discussed.
MATERIALS AND METHODS
Materials and strains
The Zero Blunt
TM
TOPO
TM
PCR cloning vector and the
pHIL-S1 shuttle vector [32] were purchased from Invitrogen
(San Diego, CA, USA). Restriction endonucleases and
DNA modifying enzymes were purchased from Promega
(Madison WI, USA) and used according to the manufac-
turer's recommendation. Escherichia c oli DH5 (supE44,
hsdR17, recA1, endA 1, gyrA96, th i-1, relA1) and TOP10
(F

Abingdon, Oxford, UK). Mandelonitrile-b-
D
-glucopyrano-
side (prunasin), mandelonitrile-b-
D
-gentiobioside (amyg-
dalin), 1,4-benzenediol-b-
D
-glucopyranoside (arbutin),
guiacol-b-
D
-glucopyranoside (salicin), 2,4-dinitrophenyl-2-
¯uoro-2-deoxy-b-
D
-glucopyranoside, and the nitrophenyl
glycosyl derivatives were obtained from Sigma Aldrich
(Poole, Dorset, U K).
Synthesis of quercetin-7-
O
-b-
D
-glucopyranoside (Q7Glc)
3¢,4¢,4,5-Tetrabenzoylquercetin [21] (100 mg, 139 lmol),
2,3,4,6-tetra-O-acetyl-a-
D
-glucopyranosyl b romide (170 mg,
3eq.),Ag
2
CO
3

(50 mL), and H
2
O ( 50 mL), and t hen dried (MgSO
4
). The
evaporated residue was stirred into 1
M
NaOH ( 50 mL)
under Ar (0°, 9 0 m in), warmed to room temperture, heated
at re¯ux (20 min), and cooled. Dowex 50 W resin (H
+
form, 70 mL) was added. Filtrate and washings (50%
aqueous MeOH, 100 mL) were evaporated, dissolved in
10% aqueous MeOH (300 mL), and washed CH
2
Cl
2
(3 ´ 80 mL). The aqueous phase was evaporated, taken
up in MeOH (2.5 m L) and puri®ed by HPLC. Yield 7 m g,
12%.
1
H-NMR (CD
3
OD): d 7.74 (d, 1 H, J
2¢,6¢
2.0 Hz, H-2¢),
7.65 (dd, 1 H, J
6¢,5¢
7.6 Hz, H-6¢), 6.88 (d, 1 H, H-5¢), 6.74 (d,
1H,J

mixedwithanequivalentvolumeofethyleneglycoland
stored at )20 °C.
Isolation, sequencing and analysis of
cbg-1
from a human cDNA library
The full length cDNA encoding for human CBG w as
isolated from a human liver kTriplEx
TM
cDNA library
(Clontech, Palo Alto, CA, USA) by h ybridization screening
using a 900-bp o ligonucleotide probe ampli®ed from the
cDNA library by PCR using d egenerate primers designed
against conserved regions in domains II I and IV of human
LPH [22] and guinea pig CBG [23]. The sequence of forward
primer HCG/F2 was 5¢-TAYCGNTTYTCNATHTCN
TGG-3¢. The sequence o f the reverse p rimer HCG/R3 was
5¢-NCCNTTYTCNGTRATRTA-3¢. PCR was p erformed
using 1 lL of library lysate, 20 p mol of primers HCG/F2
and HCG/R3, 0.2 m
M
dNTPs, 2.5 U of Taq polymerase
(Amersham Pharmacia B iotech) 1 0 m
M
Tris/HCl, pH 9 .0,
50 m
M
KCl, 3.5 m
M
MgCl
2

TM
Terminator Cycle Sequencing kit and an ABI 373 DNA
sequencer. Sequence analysis was carried out using the
Wisconsin
GCG V
10.1 software package (Genetics Computer
Group, Madison, Wisconsin, USA) and sequence align-
ments using
BLAST
v2.0 [24].
Construction of the pHIL-S1/
cbg-1
expression plasmid
The pHIL-S1-derived expression plasmid w ith the cDNA
insert encoding human CBG is shown in Fig. 1. The DNA
manipulations were carried out using standard procedures
[25]. The cDNA fragment (1407 bp) containing the cbg-1
coding region was ampli®ed by PCR from the TriplEx clone
by using Pfu DNA polymerase (Stratagene) and the
upstream primer (5¢-TTTTTT
CTCGAGAAGCTTTCC
CTGCAGGAT-3¢) and downstream primer (5¢-TTTTT
T
GGATCCCTACAGATGTGCTTCAAGGCC-3¢), thus
introducing XhoIandBamHI sites, respectively (underlined)
at each end of the gene. The 5¢ terminus of this construct was
designed to introduce t he Pichia phosphatase sign al
sequence cleavage site (Ala-Arg) in frame with the cbg-1
coding sequence (Fig. 1). As the native PHO1 signal
sequence cleavage site contains a g lutamate re sidue imme-

et al. [25]. Transformants were grown in liquid bacterial
cultures, recombinant plasmids isolated using Q iagen col-
umns (Mini-Prep kit), and identity c hecked by restriction
mapping to yield pHIL-S1/cbg-1.
Transformation of
Pichia pastoris
and selection
of a recombinant clone
Transformation of the P. pa storis strain (his4)/GS115 [26]
and screening were achieved using the spheroplast proce-
dure [27], modi®ed as described previously [28]. Brie¯y,
pHIL-S1/cbg-1 ( 1 lg) as well as the pHIL-S1 vector, as
negative control, were digested with BgIII prior to trans-
formation by the spheroplast method. After screening f or
methanol sensitive clones, Mut
s
colonies were used to
inoculate 10 mL BMGY pH 6 . After 2 d ays with shaking at
250 r.p.m., 30 °C, the cells were pelleted a nd resuspended in
2 mL BMMY. Following another 5 days at 30 °C, the
culture w as centrifuged and the amount of reCBG in the
supernatant w as estim ated b y activity measurement ass ays
using 4NPGlc a s substrate.
Expression of
cbg-1
in
P. pastoris
and isolation
of reCBG
Large-scale expression was achieved using 250 mL cultures

-glucopyranoside (4NPGlc; 10 m
M
)in
50 m
M
sodium-phosphate buffer (pH 6.5) at 37 °Cis
determined at 400 n m using the molar extinction coef®cient
for 4NP of 18 300
M
)1
ácm
)1
. The p H optimum for r eCBG
was determined b y m easuring the b-glucosidase activity in
50 m
M
sodium phosphate (pH range 2.8±7.6). The thermal
stability of CBG was assessed by measuring the residual
b- glucosidase activity (4NPGlc as substrate) follow ing incu-
bation (30 m in) of CBG samples at various temperatures
(23±70 °C). The activity of puri®ed CBG towards various
nitrophenyl glycosides (a-
D
-glucopyranoside, a-
D
-glucopyr-
anoside, a-
D
-galactopyranoside, a-
L

(50 m
M
NaCl/P
i
, pH 6.5; ®nal c oncentration d imethylsulf-
oxide < 2%, v/v), equilibrated at 37 °C , and reactions
started with the addition of enzyme (0.1±1 lgin10lL) in a
®nal volume o f 100 lL. Reactions were terminated by the
addition of acetonitrile/1% aqueous tri¯uoroacetic acid
(50 : 50 , v/v; 100 lL), ®ltered and analysed by reversed-
phase HPLC with online diode-array detection using a
LUNA C-18 co lumn (4.6 ´ 25 mm, 5 lm; Phenomonex,
Maccles®eld, UK) with an injection volume of 20 lL.
Solvents A (water/tetrahydrofuran/tri¯uoroacetic acid,
98 : 2 : 0.1 v/v), B (acetonitrile), C (water/tri¯uoroacetic
acid, 99.9 : 0.1), and D (methanol/tri¯uoroacetic acid,
99.9 : 0.1) were run at a ¯ow rate of 1 mLámin
)1
.The
following gradients were used: incubations containing
arbutin or salicin as substrate; 100% C initial, i ncreasing
D t o 2 0% (10 min), 50% ( 15 min), 100% (5 min), held at
100% (5 min); cyanodin glycosides; 5% B/95% A initial
(5 min), increasing B to 20% (10 m in), 90% (10 min), held
at 90% (5 m in); iso¯avonoid, mandelonitrile and dihydr-
ochalcone glycosides, 17% A/83% B initial (1 min),
increasing B to 90% (10 min), held at 90% (4 min). The
column was re-equilibrated (5 m in) in the appropriate
starting solvent conditions following gradient development.
Standard curves were constructed using HPLC grade

incubation periods (see Fig. 3) was determined by adding
252 J G. Berrin et al. (Eur. J. Biochem. 269) Ó FEBS 2002
20 lL of the enzyme/inhibitor mixture to 180 lLof
substrate (4NPGlc), incubating for 30 min at 37 °C, and
measuring the rel ease of 4NP.
Protein assays and protein sequencing
Total protein in crude and semipuri®ed samples was
estimated using the Pierce Protein Assay Reagent with
BSA as standard. For puri®ed reCBG, total protein was
calculated using an e xtinction coef®cient at 2 80 nm
(122 120
M
)1
ácm
)1
) derived fro m the a mino-acid composi-
tion for the primary s tructure for reCBG. Protein sequenc-
ing was performed at the Protein Sequencing & Peptide
Synthesis Facility (John Innes C entre, Norwich, UK) using
an ABI 4 91 Procise sequencer.
Gel electrophoresis
SDS/PAGE was routinely p erformed using 12% homoge-
neous Tris/glycine gels (Novex, Frankfurt, Germany)
according to the manufacturer's instructions, a nd stained
with Coomassie Blue. Molecular m asses were estimated
from plots of log(M
r
) vs. migration for a series of known
standard proteins (LMW Marker Kit; Amersham Pharma-
cia Biotech). Isoelectric focusing w as performed using 5%

lian liver CBGs [9,12,14,23,30]. We were unable to obtain
an N-terminal sequence for the puri®ed enzyme probably
because, as with guinea-p ig CBG [23], the N-terminus was
blocked.
Human liver
cbg-1
cDNA cloning and sequence analysis
A human liver cDNA library was screened b y a c onven-
tional a pproach using a 900-bp
32
P-labelled DNA fragment
from human CBG. This DNA probe was ampli®ed by PCR
from the c DNA library using two degenerate oligonucleo-
tide primers d esigned against consensus sequences from the
coding regions of domains III and IV of human lactase
phlorizin hydrolase (LPH) [22] and guinea pig cytosolic
b-glucosidase [23]. Five cDNA clones were isolated and
sequenced. The largest clone was found t o contain an ORF
of 1407 nucleotides encoding a p rotein of 496 a mino acids
with a calculated molecular mass of 53.7 kDa. A single
putative glycosylation site was located at N47 of the
deduced amino-acid sequence within the motif KNQT. No
signal sequence was apparent which indicates, as expected,
CBG is located in the cytosol. The nucleotide a nd amin o-
acid sequence has been submitted to the GenBank seq uence
data bank and is available under a ccession number
AF317840.
The primary sequence for human CBG s hared extensive
sequence homology with other mammalian b-glucosidases.
CBG shared 79% nucleotide similarity and 83.6 % ami no-

s
transformants were
grown under noninduced conditions (MGY or BMGY) and
then transferred to medium containing methanol (MMY or
BMMY). Routine activity assays against pNP-b-
D
-gluco-
pyranoside were u sed for the selection of clones with h igh
b-glucosidase productivity. b-Glucosidase activ ity was
found only when rich m edium (BMGY/BMMY) was used
for induction of CBG expression. However, as P. p ast oris
secretes endogenous b-glucosidase activity into the medium,
although at very low level, it was important to discriminate
between the recombinant and endogenous activities. This
was achieved using the ¯avonol glucoside Q4¢Glc, which is a
substrate for human liver CBG (Table 2) but not for
P. pastoris endogenous b-glucosidase, as demonstrated
using media from P. pastoris transformedwithpHIL-S1
lacking the CBG cDNA insertion (data not shown). Hence,
Ó FEBS 2002 Xenobiotic metabolism by a human b-glucosidase (Eur. J. Biochem. 269) 253
although both the Pichia endogenous b-glucosidase and the
human reCBG hydrolysed 4NPGlc, the use of Q4¢Glc
con®rmed that the increased level of b-glucosidase activ ity
was due to the secretion of the human recombinant enzyme.
A representative His
+
Mut
s
transformant was selected for
production of recombinant CBG (reCBG) in shake-¯ask

pI 4.7 and 4.8 (Fig. 2B), in good agreement with that
obtained for CBG isolated from human liver. Conventional
Edman sequencing of reCBG indicated a single N-terminal
sequence (REAFP) demonstrating that there had been
correct processing of the PHO1 signal sequence (Fig. 1).
The b-glucosidase inhibitor, 2,4-dinitroph enyl-2-¯uoro-
2-deoxy-b-
D
-glucopyranoside, was a potent inhibitor of
reCBG (Fig. 3). Incubation of reCBG (0.35 l
M
®nal
concentration) in the presence of 1 and 5 l
M
inhibitor
reduced the b-glucosidase activity in a time-dependent
manner; 36 and 70% of the b-glucosidase activity remain ed
following 30 and 50 min incubation with 1 and 5 l
M
inhibitor, respectively. b-Glucosidase activity was not
recovered following extensive dialysis of the inhibited
enzyme, indicating that inhibition was essentially irrevers-
ible. The highest rates for hydrolysis of 4NPGlc over 10 min
were obtained at 50 °C, 2.3-fold faster than at 37 °Cand
4-fold faster than at 58 °C (re¯ecting thermal inactivation).
The enzyme was relatively stable at 37 °C as more than 80%
activity remained after 24 h at this temperature. The pH
optimum for b oth r eCBG and human liver CBG w as 6.5,
with ³ 70% o f optimum activity maintained over the pH
range 5.0±7.5, but < 4% at pH 4.0.

-fucopyranoside > a-
L
-arabinopyrano-
side > b-
D
-glucopyranoside > b-
D
-galactopyranoside >
b-
D
-xylopyranoside > b-
L
-arabinopyranoside. These data
are in general agreement with those obtained by Daniels
et al. [9] and con®rm that CBG has a broad speci®city that
can accommodate several glycones in the active site,
including b-
D
-linked pentose and hexose s ugars and a-
L
-
or b-
L
-linked arabinopyranosides, although several other
a-linked sugar derivatives (pentose and hexose) are not
hydrolysed by reCBG. Although we detected no me asure-
able release of 4NP from 4NP-b-
D
-mannopyranoside, we
were able to con®rm [16] that this compound was an

-mannopyranoside (10 m
M
)].
Hydrolysis of xenobiotic glycosides by reCBG
The a bility of CBG to hydrolyse a variety of glucosides was
assessed using a wide variety of aglycone structures that
were linked to sugars through various positions on the
aglycone (Tables 2 and 3, Fig. 4 ). The analysis was
performed in order to (a) assess the capacity of CBG to
hydrolyse a variety of plant-derived glycosides which are
commonly ingested by humans, and (b) determine some
relationships between aglycone structure a nd CBG speci-
®city. CBG hydrolysed e f®ciently many of t he compounds
tested, demonstrating lower apparent af®nities (K
m
)and
higher speci®city constants (k
cat
/K
m
) than those obtained
using various nitrophenyl glycosides (compare with data in
Table 1 ). b-
D
-Glucosides of ¯avones, iso¯avones and
¯avonols were hydrolysed p articularly ef®ciently. For
example, the estimates of apparent af®nity and speci®city
constant obtained using the ¯avone glucoside luteolin-4¢-
Glcassubstrate(10l
M

(m
M
)
k
cat
/ K
m
(m
M
)1
ás
)1
)
4NP-b-
D
-fucopyranoside 10.7  0.0 0.37  0.01 28.9
4NP-a-
L
-arabinopyranoside 5.97  0.45 0.57  0.08 10.4
4NP-b-
D
-glucopyranoside 12.1  0.3 1.76  0.15 6.9
4NP-b-
D
-galactopyranoside 17.6  0.3 3.14  0.15 5.6
4NP-b-
D
-xylopyranoside 0.75  0.02 1.58  0.14 0.48
4NP-b-
L

k
cat
/K
m
(m
M
)1
ás
)1
)
Simple Phenolics
Salicyl alcohol-Glc (salicin) 0.171 ND ND ND
Hydroquinone-Glc (arbutin) 0.015 ND ND ND
Iso¯avones (phytoestrogens)
Genistein-7-Glc (genistin) 1.73 1.53  0.04 35  2.9 44
Daidzein-7-Glc (daidzin) 2.75 3.55  0.16 118  11 30
Daidzein-7-MalGlc 0.038 0.24  0.01 3230  130 0.075
Flavonols
Quercetin-4¢-Glc (spiraeoside) 1.19 1.08  0.02 31.8  2.9 34
Quercetin-7-Glc 0.77 0.69  0.02 42.2  3.2 16
Quercetin-3,4¢-diGlc 0.21
b
0.30  0.01 274  21 1.1
Flavones
Apigenin-7-Glc (apigetrin) 1.30 1.53  0.05 21.5  1.6 71
Luteolin-4¢-Glc 1.30 1.17  0.01 10  0.06 117
Luteolin-7-Glc 2.85 3.05  0.07 50  3.2 61
Luteolin-3¢,7-diGlc 1.46
c
ND ND ND

aglycone structure ( Table 3).
CBG demonstrated remarkable speci®city with respect to
the position of glycosylation. For example, although gluco-
sides formed in the 4¢- and 7-position of quercetin were
ef®ciently hydrolysed, the 3-glucoside was not a substrate f or
the enzyme. Indeed, no activity could b e detected on any of
the glucosides c onjugated at the 3-position in the C-ring of
¯avonoids (Table 3). CBG was most active on substrates
conjugated at the 4¢-compared to the 7-position as evidenced
by a lower K
m
and a higher k
cat
/K
m
. It was possible t o
determine the relative effects of aglycone structure o n the
apparent af®nity and speci®city constant using (iso)¯avo-
noids conjugated in the 7 -position. Values for K
m
varied
20-fold, k
cat
5-fold and k
cat
/K
m
14-fold. Some of the
differences could be ascribed to s ingle substitution differ-
ences between otherwise s imilar aglycones, for example t he

(Table 2). CBG was tested for
activity against a series of ¯avonol glycosides that differed
only in the glycone moiety [Q3Glu, Q3Gal, Q3Xyl, Q3Ara,
Q3GlA, Q3Rha and Q3GlcMal; K3Glc, K3GlA and
k3(pCA)Glc]. However, we were not able to assess the
effects of the glycone moiety in this way as none of these
compounds were substrates. These data indicate that
¯avonoid-3-glycosides are not substrates for C BG.
DISCUSSION
The m echanism by w hich xenobiotics are metabolized and
absorbed in humans has re ceived much attention due to the
high levels of plant-derived compounds that are ingested
orally and bioactive, or which g enerate potentially t oxic or
bene®cial metabolites [38±41]. The vast majority of t hese
compounds are in the form of b-glycosides (most commonly
b-
D
-glucosides) and hydrolysis to release the relatively more
hydrophobic aglycone is, almost without exception, a
prerequisite to metabolism, conjugation and excretion. It
has been commonly thought that hydrolysis of ingested
glycosides occurs only in the colon, facilitated by microbial
b-glucosidases. However, there is clear evidence to show
that uptake via the c olon is not the only route for dietary
xenobiotics to enter the general circulation. Firstly, phenyl-
glycosides can be actively transported i nto small intestinal
enterocytes by hexose transporters such as the sodium-
dependent glucose transporter (SGLT1 [42±45]). Secondly,
pharmacokinetic data indicate that absorption of many
xenobiotic glycosides occurs very rapidly f ollowing inges-

Cyanidin-3,5-diGlc
Fig. 4. Structure s of the xenobiotic aglycones, potential substrates for
cytosolic b-glucosidase. (A) quercetin (R1, OH; R2, OH), ap igenin (R1,
H, R2, H), luteolin (R1, H, R2, OH); (B) naringenin (R, H ), eriodictyol
(R, OH); (C) daidzein (R, H), genistein (R, OH); (D) h ydroquinone;
(E) salicyl alcohol; (F) mandelonitrile.
256 J G. Berrin et al. (Eur. J. Biochem. 269) Ó FEBS 2002
cDNA was c loned f rom a human liver c DNA library and
expressed heterologously.
The recombinant protein, produced in P. pastoris,was
very similar to CBG isolated from human liver according to
various criteria (electrophoretic mobility, isoelectric point,
speci®c activity towards 4NPGlc). Furthermore, reCBG
hydrolysed various aryl-glycosides ef®ciently and was
inhibited in a time-dependent manner by 2,4-dinitrophe-
nyl-2-¯uoro-2-deoxy-b-
D
-glucopyranoside ( a known mech-
anism-based b-glucosidase inhibitor). This is the ®rst re port
describing expression of a b-glucosidase gene in the
methylotrophic yeast P. pastoris ,anorganismthathas
shown great potential for heterologous protein expression
[19,20,50]. Expression facilitated the puri®cation of CBG
and allowed characterization in s ome detail, especially with
respect to its glycone/aglycone speci®city a nd ability to
catalyse the hydrolysis of dietary xenobiotic glycosides. This
is also the ®rst report describing heterologous expression of
a mammalian CBG, and will facilitate identi®cation of
putative endogenous substrate(s).
CBG f ul®ls many of the criteria required f or an enzyme

glycosides at appreciable rates and with micromolar af®nity
constants, and have suggested a role for this enzyme in
xenobiotic metabolism.
ACKNOWLEDGEMENTS
The a uthors thank Dr N. Lambe rt for assistance with the puri®cation
of CBG, Dr M. J . Naldrett (Jo hn I nnes C entre, Norwic h, UK) for
protein sequ encing, J . Eagle s for mass spectro scopy, S . D upont and
K. O'Leary for kind gifts of ¯avonoid glycosides and quercetin
glucuronides, respectively, Dr A.J. Day for useful discussions, and the
Anatomic Gift Fo undation (Maryland, USA) for the sample of
human liver. This work was funded b y a Biotechnology and
Biological Sciences Research Council Competitive Strategic Grant
and a Europ ean Union F rame work V G rant ( POLYBIND; QLKI-
1999±00505) to J .G.B.
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