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MINIREVIEW
Cholesterol oxidase:biotechnological applications
Loredano Pollegioni,LucianoPiubelliandGianluca Molla
Dipartimento di Biotecnologie e Scienze Molecolari, Universita
`
degli studi dell’Insubria, Varese, Italy, and Centro di Ricerca Interuniversitario
in Biotecnologie Proteiche ‘‘The Protein Factory’’, Politecnico di Milano and Universita
`
degli studi dell’Insubria
Introduction
In eukaryotes, cholesterol is essential for maintaining
cell membrane structure and for synthesizing a number
of important compounds. Moreover, improper mainte-
nance of cholesterol concentrations can severely
affect the physiological function of an organism. Cho-
lesterol oxidase (ChOx, EC 1.1.3.6), or more precisely,
3b-hydroxysterol oxidase, is a bacterial enzyme that
has proven to be very useful in biotechnological appli-
cations related to the detection (and conversion) of
cholesterol and to the disruption of cholesterol-con-
taining membranes. As described in the two accompa-
nying reviews in this miniseries [1,2], ChOx catalyzes
the oxidation of the C3-OH group of cholesterol (and
other sterols) to give the corresponding D
5
-3-ketone
(cholest-5-en-3-one) and its isomerization to D
4
-3-ke-
tone (cholest-4-en-3-one, see Scheme 1 in the first mini-
review [1]). In bacteria, ChOx is the first enzyme in the
catalytic pathway that yields propionate and acetate as
final products. Importantly, there is no mammalian
homolog of ChOx.
Since 1973, ChOx has been used in clinical chemistry
to measure serum cholesterol. ChOx research has
grown largely in the past several years: a PubMed
search for the term ‘cholesterol oxidase’ yielded 124,
212 and > 220 papers in the periods 1980–1989, 1990–
1999 and 2000–2008, respectively. ChOx is produced
by two types of bacteria: (a) nonpathogenic bacteria,
which utilize cholesterol as a carbon source; and (b)
Keywords
biocatalysis; cholesterol; cholesterol
determination; diagnostic enzyme; enzyme
biotechnology; flavoproteins; virulence
Correspondence
Loredano Pollegioni, Dipartimento di
Biotecnologie e Scienze Molecolari,
Universita
`
degli studi dell’Insubria,
via J.H. Dunant 3, 21100 Varese, Italy
Fax: 0332 421500
Tel: 0332 421506
E-mail: loredano.pollegioni@uninsubria.it
(Received 23 July 2009, revised
2 September 2009, accepted 10
September 2009)
doi:10.1111/j.1742-4658.2009.07379.x
Cholesterol oxidase is a bacterial FAD-containing flavooxidase that catalyzes
the first reaction in cholesterol catabolism. Indeed, this enzyme catalyzes two
reactions: the oxidation of the C
3
-OH group of cholesterol (and other sterols)
to give cholest-5-en-3-one; and its isomerization to cholest-4-en-3-one. In the
past several years, the structural and functional characterization of choles-
terol oxidase has been developed together with its application as a biological
tool. Cholesterol oxidase has been used in biocatalysis for the production of
a number of steroids, as an insecticidal protein against boll weevil larvae and,
in particular, as a diagnostic enzyme for determining serum levels of choles-
terol. These applications prompted various laboratories worldwide to isolate
this flavooxidase from different sources and to improve its properties by pro-
tein engineering, further increasing our knowledge on its structure–function
relationships. These studies also discovered new physiological roles for cho-
lesterol oxidase (e.g. in virulence and as an antifungal sensor). We assume
that the investigations of cholesterol oxidase and its applications will con-
tinue to grow quickly in the near future, in particular to uncover unexpected,
new areas of application.
Abbreviations
BsChOx, Brevibacterium sterolicum cholesterol oxidase; ChOx, cholesterol oxidase (EC 1.1.3.6); ReChOx, Rhodococcus equi cholesterol
oxidase; ShChOx, Streptomyces hygroscopicus cholesterol oxidase.
FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS 6857
pathogenic bacteria, which require ChOx for infection
of the host macrophage because of its ability to alter
the physical structure of the lipid membrane by con-
verting cholesterol into cholest-4-en-3-one (see below).
Both pathogenic and nonpathogenic bacteria upregu-
late the expression of ChOx in the presence of choles-
terol.
This review summarizes our knowledge concerning
the production of ChOx and its main uses. By under-
standing the structure–function relationships of this
flavoenzyme and the mode of action by which it alters
the physical properties of lipid membranes, together
with its overexpression in recombinant systems, we
may discover new and ⁄ or optimized applications of
ChOx.
Sources of ChOx
A list description of ChOx-containing microorganisms
was published in 2000 [3]. This enzymatic activity is
produced by a variety of microorganisms that are found
in different environments: a detailed list is reported in
the Supplementary materials section. Microorganisms
such as Arthrobacter, Rhodococcus equi, Nocardia
erythropolis, Mycobacterium and Nocardia rhodochrous
produce an intracellular ⁄ membrane ChOx, while
Pseudomonas, Schizophyllum commune, Brevibacteri-
um sterolicum, Streptoverticillum cholesterolicum and
Streptomyces violascens produce an extracellular
enzyme. As a general rule, ChOx enzymes require the
prosthetic group FAD for their activity (both as a cova-
lently or noncovalently bound cofactor). An exception
to this could be the ChOx isolated from c-Proteobacteri-
um Y-134, which was reported to contain covalently
bound FMN as a cofactor [4]. This enzyme also differs
in substrate specificity compared with other known
ChOxs (e.g. it is not active on pregnenolone and
ergosterol, see Table 1).
Generally, the presence of cholesterol (or of a similar
compound) in the growth medium induces the produc-
tion of ChOx. Among the ChOx-producing bacteria,
R. equi (previously identified as B. sterolicum ATCC
21387) [5] exhibits high levels of ChOx activity, but com-
mercial use of this microorganism is limited because of
its highly pathogenic nature (see below). This enzyme is
the product of the gene choE and it is homologous to
other secreted ChOxs identified in Streptomyces spp. (see
Fig. S1). This protein also exhibits significant similarities
to putative ChOxs encoded by Mycobacterium tubercu-
losis and Mycobacterium leprae. Rhodococcus erythropo-
lis ChOx was produced (using cholesterol solubilized in
Tween-80 in the culture medium) both as an intracellular
enzyme and an extracellular enzyme: the maximal
production of both forms of ChOx was obtained after
70 h of growth, using a spray-dry method of preparation
of cholesterol (up to 365 UÆL
)1
and 1.7 UÆg
)1
of cells)
[6]. Rhodococcus (Nocardia) erythropolis IMET 7185 also
produces ChOx: the addition of 1 gÆL
)1
of cholesterol
results in a fivefold increase in ChOx production, up to
3.3 UÆg
)1
of cells [7]. The soil-isolated strain Rhodococ-
cus sp. GK1 produces ChOx both as membrane-bound
and extracellular forms [8]. The synthesis of ChOx
depends on the presence of either phytosterols or hex-
anoate as the sole carbon source; under these conditions,
the membrane-bound ChOx was produced up to
100 UÆg
)1
of cells and 400 UÆL
)1
[8]. By contrast, the
synthesis of ChOx from Burkholderia cepacia (wrongly
classified as Pseudomonas sp. strain ST-200 and corre-
sponding to the choS gene, Fig. S1) is not induced by
cholesterol and this bacterial strain produced only up to
13 UÆL
)1
[9]. The highest production levels have been
reported in the nonpathogenic Streptomyces sp., reach-
ing up to 2500 UÆL
)1
in fermentation broth in terms
of purified protein [10] (for the sake of comparison, the
activity values not measured at 25 °C have been cor-
rected for the effect of temperature).
Because of the moderate productivity of the various
microorganisms used and the requirement of adding
cholesterol to culture media to induce ⁄ enhance produc-
tion, the cost of ChOx remained relatively high [6,8].
The yield of ChOx production was enhanced by alter-
ing the composition of the growth medium and the
physical parameters of culture. In S. commune, ChOx
production depends on the amount of oleic acid in the
culture broth but, interestingly, this applies to the level
of insoluble oleic acid rather than to the soluble com-
ponent adsorbed onto the cell [3]. A detailed investiga-
tion of the growth conditions was also reported for
R. equi no. 23 grown in a batch fermentor: by adjust-
ing the pH (from 6.5 to 7.5), the temperature (from 39
to 37 °C) and the agitation speed (from 200 to
300 rpm) after 24 h of growth, maximal production of
ChOx (340 UÆL
)1
, corresponding to 11 UÆh
)1
ÆL
)1
) was
reached in 30 h of culture at an aeration rate of
5LÆmin
)1
[11].
To overcome the difficulties related to overproduc-
tion of this flavoenzyme in the original organism,
ChOx genes from several sources have been cloned
and expressed as recombinant proteins. However,
most of these recombinant microorganisms produce
ChOx at levels close to those of the original source.
An exception to this is the expression of the gene
encoding Rhodococcus equi cholesterol oxidase
(ReChOx) in Streptomyces lividans that resulted in a
yield of protein production 85-fold higher than
that from the natural organism [12]. Its expression in
Biotechnological applications of sterol oxidase L. Pollegioni et al.
6858 FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS
S. lividans (and in Escherichia coli) was accomplished
by using various deletions within the 5¢-flanking (non-
coding) region. When the ReChOx protein was fused
with the N-terminal part of the lacZ protein, large
amounts of inactive enzyme were produced as ‘inclu-
sion bodies’. In contrast, by using a gene with less
than 256 bp at the 5¢-flanking region, enzyme could be
efficiently produced in S. lividans, secreting
30000 UÆL
)1
into the culture medium. Recently,
ReChOx was also efficiently synthesized in E. coli
under the control of the pT7lac promoter in the
pET28 plasmid (up to 3.7 UÆmg
)1
of protein was
Table 1. Relative activity of ChOx from different sources. In all cases, activity on cholesterol (k
cat
value) was taken to be 100%.
Substrate ReChOx
a
BsChOx
b
ShChOx
b
c-Proteobacterium
Y-134
c
Chromobacterium
DS-1
d
CHO-U CHO-A
Cholesterol
HO
100 100 100 (100) 100 (100) 100 100 100
7a-or7b-OH-
Cholesterol
HO
HO
– 99, 82 – – – – –
7-Keto-
cholesterol
HO
O
–61 – – – – –
Ergosterol
HO
051 – – <3 0 12
b-Sitosterol
HO
19 65 – – 19 29 80
Pregnenolone
HO
O
22 90 70 (42) 70 (30) 0 0 95
Cholestanol
HO
13 – 80 (46) 119 (300) – – 95
a
Left column: 0.6% Triton X-100, pH 7.5 [70]; right column: 0.7% propan-2-ol, pH 7.5 [71].
b
100 mM potassium phosphate, pH 7.5, 0.4% Thesit (data in parenthesis were obtained in 500 mM potassium phosphate) [72,73].
c
100 mM potassium phosphate, pH 7.0, 0.05% Triton X-100, pH 7.5 [4,74].
d
50 mM sodium phosphate buffer, pH 7.0, 64 mM sodium cholate, 0.34% Triton X-100 [24].
L. Pollegioni et al. Biotechnologicalapplications of sterol oxidase
FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS 6859
produced in the crude extract and 2200 UÆL
)1
was
produced in the fermentation broth) [13,14], as well
as in Chromobacterium DS-1 ChOx (1870 UÆL
)1
was
produced in the fermentation broth and 2.3 UÆmg
)1
of
protein was produced in the crude extract) [15]. The
only data concerning the production of true B. steroli-
cum ChOx (i.e. that containing the FAD cofactor
covalently linked to the apoprotein moiety) were
obtained for E. coli using the pET24 plasmid (up to
4UÆmg
)1
of protein was produced in the crude extract
and 12000 UÆL
)1
was produced in the fermentation
broth) [F. Volonte
`
, L. Pollegioni, G. Molla, F. Mari-
nelli and L. Piubelli, personal communication].
The Streptomyces ChOx gene was also overexpressed
in S. lividans by subcloning the choP-choA operon into
a multicopy shuttle vector, which produced a 70-fold
greater amount of ChOx than the original organism
(i.e. up to 4400 UÆL
)1
in the fermentation broth) [16].
Interestingly, more than 90% of the protein produced
is extracellular. The same ChOx was then produced in
E. coli JM109 under the P8 promoter, reaching a
specific activity in the crude extract of 9.7 UÆmg
)1
of
protein after 3 days of growth [17]. A detailed review
of the methods for extracting and purifying ChOx has
been published previously [3].
ChOx as a biological tool
Effect of detergents and solvents on ChOx
activity and stability
ChOx is an enzyme that interacts with membranes and
micelles and acts on hydrophobic substrates whose sol-
ubility is increased by using detergents and ⁄ or solvents.
These compounds also affect the activity ⁄ stability of
the flavoprotein. One of the first investigations on the
effect of detergents on ReChOx activity showed a sig-
moidal behavior versus cholesterol concentration at a
Triton X-100 concentration of ‡ 0.3% (the corre-
sponding Hill plot gave an n-value of 4) with a con-
comitant increase in the K
m
value [18]. The transition
from a simple Michaelis–Menten behaviour to a sig-
moidal behavior was also recently observed for the
V121A mutant of Streptomyces hygroscopicus choles-
terol oxidase (ShChOx) at a fixed Triton X-100 con-
centration of 0.077% (the activity decreases with an
increase in Triton X-100 concentration) [19]. Val121 in
ShChOx is located in a hydrophobic loop near the
active site, which should play an important role in
extracting cholesterol from micelles: the V121A substi-
tution negatively affects this step only if cholesterol is
solubilized using nonionic detergents (no sigmoidal
behavior is observed when cholesterol is solubilized in
propan-2-ol). For example, the enzymatic activity of
Nocardia ChOx is induced upon the formation of
mixed micelles of cholesterol with molecules of the
detergent Surfal: the enzyme is half activated at a
detergent concentration corresponding to its critical
micelle concentration [20].
In 1988, Cees Veeger’s group published a very
important work regarding clarification of the effect of
the solvent ⁄ detergent on the reaction catalyzed by
ChOx [21]. The dependence of catalytic activity of No-
cardia ChOx on the composition of a solution com-
posed of n-hexane, propan-2-ol and water (a
detergentless microemulsion) revealed two maxima.
The maximal catalytic activity in ternary systems is
equal to that determined in aqueous solution, whereas
the K
m
value increases with solvent concentration
because of a preferential interaction with the surround-
ing organic solvent with respect to the hydrophobic
ChOx active site. By contrast, the stability profile
shows a single sharp maximum corresponding to the
microemulsion region of the phase diagram [21]; in
any case, the storage stability is higher in aqueous
solution. We have also systematically studied the effect
of the nonionic detergents Thesit and Triton X-100,
and of propan-2-ol (used as a substrate solubilizer), on
ShChOx and Brevibacterium sterolicum cholesterol oxi-
dase (BsChOx) activity [22]. At a low concentration of
Thesit, activity increases for both enzymes, whereas at
higher detergent concentrations (‡ 2–5%) the opposite
effect occurs. On the other hand, Triton X-100 inacti-
vates both enzymes at all concentrations. We thus
deduced that these surfactants exert their effect by
interacting with the enzymes and not by affecting
micellar phenomena. Analogously, the increase in con-
centrations of propan-2-ol (or other organic solvents)
up to 5–10% (v ⁄ v) induces an increase in the activ-
ity of both ShChOx and BsChOx and a decrease at
higher solvent concentration. A significant difference
between the two ChOx enzymes emerges when stability
is analyzed as a function of concentration of propan-2-
ol: BsChOx is rapidly inactivated, whereas for ShChOx
70% of the initial activity still remains after 5 h in the
presence of 30% propan-2-ol [22]. This observation is
in line with the stability to detergents reported in
Fig. 1 (see below).
The stability of ChOxs from different sources, in the
presence of several detergents, was also compared after
1 h of incubation at 30, 45 and 60 °C [4,23,24]. The sta-
bility at 30 °C was similar among the ChOxs analyzed
(the only exception was observed in the presence of
‡ 0.1% SDS that fully inactivated all enzymes except
for those from Burkholderia and Chromobacterium
DS-1), with the most significant changes being observed
Biotechnological applications of sterol oxidase L. Pollegioni et al.
6860 FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS
after 1 h of incubation at 60 °C in the presence of
nonionic detergents (see Fig. 1, which reports additional
data from our experiments on covalent BsChOx and its
noncovalent variant H69A mutant). At 0.5% Triton
X-100, all ChOxs assayed showed a high stability, while
in the presence of sodium cholate, only the ChOxs from
Proteobacterium Y-134, Chromobacterium DS-1 and
Pseudomonas were stable. Interestingly, the ChOxs from
Proteobacterium Y-134 and Chromobacterium DS-1
were also more stable to temperature than all other
ChOxs: they maintained more than 90% of the original
activity after 1 h of incubation at 60 °C. The results
obtained using BsChOx were significantly different from
those observed with other ChOxs. Both wild-type and
H69A BsChOxs showed very low stability under the
conditions tested: the residual activity after 60 min of
incubation at 60 °C was £ 3% in all cases, despite the
presence of the covalent FAD linkage in the wild-type
BsChOx. Analogously, a systematic comparison was
carried out to determine the stability of ChOxs, from
different sources, after 12–24 h of exposure to solvents
at 37 °C [23,24]. Interestingly, only the enzyme from
Burkholderia is not inactivated by 33% (v ⁄ v) acetone
and propan-2-ol.
Uses in biocatalysis
Many Actinobacteria are highly efficient in oxidizing
the 3-OH group of D
5
-hydroxysteroids coupled to
D
5
fi D
4
isomerization, thus providing valuable inter-
mediates for industrial steroid drug production. For
example, R. equi DSM 89-133 was used to convert cho-
lesterol and other sterols to androst-4-ene-3,17-dione
and androsta-1,4-diene-3,17-dione [25]: up to 82% of
cholesterol was converted when the growth medium was
supplemented with acetate. The synthesis of 7b-hy-
droxytestosterone was achieved by incubating a C
8
-ester
at the C-17 position of 5-androstene-3b,7b,17b-triol-17-
caprylate with ChOx and subsequently eliminating the
intermediate ester with porcine lipase [26]. Analogously,
in the bioconversion of cholesterol into bile acids,
ReChOx was used to convert 3b,7a -cholest-5-ene-3,7-
diol to 7a-hydroxycholest-4-en-3-one, obtaining a yield
higher than 90% [27]. As another alternative, ShChOx
was used to prepare unsaturated C21 triols as reference
standards to study adrenal steroid production in
Smith-Lemli-Opitz syndrome, with the ultimate aim of
developing a prenatal or a postnatal diagnostic method
[28]. R. erythropolis ChOx was employed for the
preparative oxidation of cyclic allylic, bicyclic (e.g.
10b-methyl-D
1(9)
-2b-octalol) and tricyclic alcohols, as
well as of a synthon to synthesize several ergot alkaloids
[29]. This work also demonstrated a lack of enantiospec-
ificity for the steroids. Recently, ChOx from Chromo-
bacterium DS-1 was reported to catalyze the oxidation
of cholesterol to hydroperoxy-cholest-4-en-3-one with
the consumption of 2 mol of O
2
[24].
A
B
C
Fig. 1. Comparison of the stability of ChOxs, from different
sources, in the absence (A) and in the presence of 0.1% (B) and
0.5% (C) Triton X-100 (black bars) or sodium cholate (white bars).
The enzyme (0.1 UÆmL
)1
) was incubated at 60 °C for 60 min in
100 m
M potassium phosphate, pH 7.0, before the enzymatic activ-
ity assay was performed: the residual activity was calculated as a
percentage of the enzyme activity without heating and incubation.
Data for Pseudomonas (Pse), Nocardia (Noc), Rhodococcus (Rho),
Streptomyces (Str) and c-Proteobacterium Y-134 (Y-134) are from
Isobe et al. [4] and for Chromobacterium sp. DS-1 are from Doukyu
et al. [24]. Bre, BsChOx. n.d., not determined.
L. Pollegioni et al. Biotechnologicalapplications of sterol oxidase
FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS 6861
With the ultimate goal of modifying the relative
ratios of specific plant sterols to stanols [i.e. the hydro-
genated forms having the C-5 double bond reduced
(and which attracted attention because of their benefi-
cial effect in reducing serum and low-density lipopro-
tein cholesterol levels)], it is noteworthy that ShChOx
was used for engineering oil seeds from rapeseed and
soybean [30].
Clinical uses
ChOx is a useful analytic tool for determining choles-
terol in various samples: (a) total and esterified serum;
(b) from low-density lipoproteins to high-density lipo-
proteins; (c) on the cell membrane of erythrocytes (and
of other cells and cellular compartments); and (d) in
gall stones and in human bile.
Normal human blood serum contains less than
5.2 mm (200 mgÆdL
)1
) of cholesterol; in plasma,
lipoproteins contain cholesteroland about 70% is
esterified by fatty acids. Determining the concentration
of serum cholesterol is fundamental in the assessment
of a variety of diseases (e.g. in atherosclerosis and
other lipid disorders) and for estimating the risk of
thrombosis, myocardial infarction, etc. The risk for
Alzheimer disease is also related to hypercholesterol-
emia via mechanisms involving oxidative stress: this
disease is characterized by the accumulation of
amyloid b-peptide (a 39–43 amino acid peptide) in the
neocortex, which is connected to peroxidative damage.
The amyloid b-peptide forms complexes with Cu
2+
ions, which oxidize cholesterol into cholest-4-en-3-one,
thus mimicking the activity of ChOx. In fact, brain
tissues from Alzheimer disease patients had a cholest-
4-en-3-one content 2-fold higher than brain tissues
from controls [31].
The chemical methods for determining serum choles-
terol (e.g. Liebermann–Burchard reactions) were ousted
in 1973 by Richmond [32]: he had demonstrated that
ChOx from Nocardia can be used to measure serum
cholesterol by assaying the amount of hydrogen perox-
ide produced. Hydrogen peroxide is reacted with quadri-
valent titanium and xylenol orange, yielding a colored
product measured at 550 nm. This method relied on the
nonenzymatic hydrolysis (alkaline saponification) of the
cholesterol esters. The same enzyme was then used for
the serum cholesterol assay by directly measuring the
absorbance of the product cholest-4-en-3-one at
240 nm, following a lengthy 2-hour incubation period
and subsequent product extraction [33]. Total choles-
terol could also be determined in serum (including the
esters) by employing a totally enzymatic method in
which cholesterol hydrolase (EC 3.1.1.13) was used to
hydrolyze cholesterol esters [34]. Free cholesterol is then
oxidized by ChOx to produce hydrogen peroxide, which
is finally assessed enzymatically with horseradish
peroxidase by the oxidative coupling of 4-aminoantipy-
rine and phenol: this results in a quinoneimine dye (with
an absorption maximum at 500 nm). This method is
characterized by the advantages of simplicity and little
interference, and is highly reproducible: it is still
routinely used in clinical laboratories. The colorimetric
method can also accurately determine serum cholesterol
levels but, because of interference from pigments, it is
difficult to assess bile cholesteroland thus an electro-
chemical method based on oxygen consumption by
ChOx reaction is better [35]. The different methods of
determining serum cholesterol are reported in Table 2.
Recently, the performance of the end point versus the
kinetic method for enzymatic assay of cholesterol was
compared [36]. The end point method is more accurate
and precise; however, the kinetic method shows a lower
sensitivity to interfering substances and analysis times
are shorter.
The method of Allain et al. [34] for assaying high-
density lipoprotein cholesterol was improved by
employing polyethylene glycol-modified ChOx and
cholesterol esterase [37]. The serum, to which a-cyclo-
dextran sulfate and a small amount of dextran sulfate
was added, was directly analyzed without precipitating
lipoprotein micelles, and the results compared favour-
ably with the previous method. Subsequently, the same
group reported on an automated method for measur-
ing serum low-density lipoprotein cholesterol without
ultracentrifugation separation [38]. This result was
achieved by using a nonionic surfactant, polyoxyethyl-
ene-polyoxypropylene block copolyether, magnesium
ions and a sodium salt of sulfated cyclic maltohexaose,
a-cyclodextrin sulfate. Polyethylene glycol-modified
ChOx andcholesterol esterase were then used for the
amperometric determination of high-density lipopro-
tein cholesterol in 1–2 lL of serum [39].
For many years, ChOx was used in a great variety
of biosensors based on different detection systems. For
example, ChOx immobilized on a nylon membrane
was used to build a fiberoptic biosensor based on the
change in fluorescence of an oxygen-sensitive dye [40];
the analytical range is 0.2–3 mm, and the steady-state
signal is achieved in 7–12 min. Subsequently, a choles-
terol sensor based on the electrochemical reduction of
oxygen was developed using a bilayer-film coating: this
sensor is less sensitive to organic interferences [41].
ChOx was immobilized on nylon nets placed over the
membrane of an oxygen probe or over the cellulose
acetate membrane of a hydrogen peroxide detector
and then covered with a polycarbonate membrane [42].
Biotechnological applications of sterol oxidase L. Pollegioni et al.
6862 FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS
When incorporated in a flow injection system, the
hydrogen peroxide-based device was fast, reproducible
and linear over a 0.5–5 mm cholesterol concentration
range. An amperometric biosensor was also obtained
by reconstituting the apoprotein of Pseudomonas fluo-
rescens ChOx with a FAD monolayer [43]: the sensor
shows high sensitivity and selectivity towards electroac-
tive interference. Most recently, self-assembled mono-
layers have been used to improve response and binding
in biosensors based on immobilized ChOx. The prop-
erties of biosensors for determining cholesterol using
ChOx immobilized by silane-based self-assembled
monolayers onto an indium-tin oxide surface have
been compared [44]. An amperometric-rotating biosen-
sor using immobilized ChOx, cholesterol esterase and
peroxidase was also produced; this biosensor contains
a micropacked column with immobilized ascorbate
oxidase to eliminate l-ascorbic acid interference [45].
By use of this device, cholesterol can also be deter-
mined in the range of 1.2 lm–1 mm with a lifetime of
25 days of use. Furthermore, ChOx was also used to
develop a sensor for determining total cholesterol in
different food samples [46].
ChOx activity has also been employed to analyze
cholesterol in cultured cells by means of an HPLC
method. At first, the cholesteryl esters were quantita-
tively hydrolyzed using cholesterol esterase, and then
total cholesterol was converted by Nocardia ChOx to
cholest-4-en-3-one before detection at 240 nm. In 3 min,
free and total cholesterol can be measured in as few as
5000 cells of human monocyte-derived macrophages
[47]. Subsequently, the same assay was developed using
a fluorimetric method: cholesteroland related sterols
have been assayed in sickle-cell and healthy erythrocytes
[48]. Other examples of cholesterol analysis in cells have
been briefly described [3]. Interestingly, the surface area
of a cell can be easily estimated by using ChOx to deter-
mine cell-surface cholesterol. For example, an area of
17500 lm
2
was calculated for human fibroblasts from
the cholesterol concentration in the plasma membrane
(equivalent to 44 fmolÆcell
)1
) and a known choles-
terol ⁄ phospholipid molar ratio of 0.8 [49].
Table 2. Alternative ChOx-based methods for determining cholesterol concentration.
Molecule assayed Method Detection limit Notes Reference
Cholest-4-en-3-one HPLC with detection under UV
light (240 nm)
0.15 nmol Determination of unsaponifiable
lipids and thoracic duct lymph
chylomicrons
[75]
Hydrogen peroxide Amperometric assay using
immobilized ChOx and cholesterol
esterase
1 lmolÆL
)1
Determination of both free and
total cholesterol in blood serum
[76]
Hydrogen peroxide Chemiluminescent detection of
the reaction using lucigenin
2.6 lmolÆL
)1
Requires accurate adjustment of
the pH to 11.75–11.9
[77]
Hydrogen peroxide Temperature-enhanced
chemiluminescent flow system
using bis-(2,4,6-trichlorophenyl)
oxalate-perylene in the presence
of Triton X-100 (detection
at 500 nm)
19 lmolÆL
)1
Significant increase in the
signal-to-noise ratio
[78]
Hydrogen peroxide Fluorimetric determination (excitation
320 nm; emission 400 nm) using
4-hydroxyphenylacetic acid
0.5 nmolÆL
)1
Measurements can be performed
on 1 lL of serum
[79]
Hydrogen peroxide Spectrophotometric determination
of the reaction with
4-aminophenazone plus phenol
(at 520 nm) using immobilized
ChOx, cholesterol esterase and
horseradish peroxidase on
arylamine glass beads
54 lmolÆL
)1
The co-immobilized enzymes did
not show loss of activity after 300
uses
[80]
Electron transfer from ChOx Electrochemical determination
using electron mediators (i.e.
1-methoxy-5-methylphenazinium,
thionine, etc.)
0.25–2.5 mmolÆL
)1
The measurement is altered by the
dissolved oxygen and requires
‡ 10 min to obtain a steady-state
response. No effect of classical
interference was observed in the
colorimetric assays.
[81]
L. Pollegioni et al. Biotechnologicalapplications of sterol oxidase
FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS 6863
ChOx in agriculture
Genetically modified plants that produce insecticidal
proteins (e.g. the Bacillus thuringiensis toxin) are now
available to control insect pests of several major crops.
In 1993, from a random screening of > 10000 filtrates
from microbial fermentations, Monsanto Co. (St Louis,
MO, USA) discovered a highly efficient protein in cul-
ture filtrates that killed boll weevil (Anthonomus grandis
Boheman) larvae [50]. Histological and biochemical
studies identified the protein as ChOx: purified ChOx is
active against boll weevil larvae at a 50% lethal con-
centration (LC
50
) of 20.9 lgÆmL
)1
, which is comparable
to the bioactivity of B. thuringiensis proteins against
other insect pests. The ChOx gene (Monsanto named it
choM, see Fig. S1), from the Streptomyces sp. strain
A19249, exhibits 85% DNA sequence identity and
89% amino acid sequence similarity with known Strep-
tomyces sp. SA-COO (choA gene). In addition to boll
weevil, several lepidopterans were negatively affected
by the presence of ChOx at a dietary concentration of
0.001%. Adding the products of cholesterol oxidation
by ChOx (i.e. cholest-en-3-one and hydrogen peroxide)
to the diet, and pretreating the diet with the enzyme,
excluded insecticidal effects caused by the ingestion of
toxic compounds. However, the boll weevil larvae are
acutely sensitive to ingested ChOx because it induces
lysis at the midgut epithelium. Boll weevil adults are
insensitive to ingested ChOx, although the fecundity of
adult females was greatly reduced if 50 lgÆmL
)1
of the
enzyme was present in the diet [51]. ChOx reduced sub-
sequent oviposition (up to 83% in eggs laid) and larval
survival (97% reduction as compared to controls)
because of poorly developed ovaries and few develop-
ing oocytes. ChOx was expressed in transformed
tobacco plants, and the synthesis levels in leaf tissues
routinely ranged from approximately 5–50 lgof
enzyme per g fresh weight. In the absence of a chloro-
plast-targeting sequence, ChOx production resulted in
severe abnormalities in plant development and fertility.
When produced as a fusion with a chloroplast-targeting
peptide, synthesis of the mature and the full-length
enzyme did not cause the deleterious phenotypic effect
observed with untargeted ChOx [52]. Transgenic leaf
tissues expressing ChOx exerted insecticidal activity
against boll weevil larvae. When produced in the cyto-
sol, or when targeted to chloroplasts, ChOx metabo-
lizes phytosterols in vivo. Transgenic plants expressing
ChOx in cytosol accumulated low levels of saturated
sterols (stanols), while the transgenic plants expressing
chloroplast-targeted ChOx maintained a greater accu-
mulation of stanols and appeared phenotypically and
developmentally normal. It was proposed that ChOx
could modify sterol ratios, thus influencing cell
division, or could affect brassinosteroid biosynthesis in
steroid hormones.
ChOx in virulence
ChOx is an interesting pharmaceutical target for treat-
ing bacterial infections. R. equi is a Gram-positive coc-
cobacillus that resides within macrophages of the host.
It is a common soil organism that frequently infects
young horses; the most common manifestation is a
chronic suppurative bronchopneumonia with abscess
formation and cavitary pneumonia (for a review, see
[53]). Since 1967, this organism has also been reported
to infect humans, being frequently diagnosed as an
opportunistic pathogen in immunocompromised
patients, 70% of whom are infected with HIV [54].
The clinical manifestations of R. equi infections are
different: the most frequent form is severe pyrogranul-
omatous pneumonia. Among the candidate virulence
factors of this pathogenic actinomycete, in vitro data
suggested that during R. equi infection of the host cell,
membrane lysis is facilitated by the induction of extra-
cellular ChOx [55]. Mutational analysis indicated that
ChOx is the membrane-damaging factor responsible
for the synergistic hemolytic reaction elicited by
R. equi in the presence of sphingomyelinase C-produc-
ing bacteria, such as Listeria ivanovii, Bacillus cereus
and Staphylococcus aureus [55]. The membrane-damag-
ing activity of R. equi requires the presence of bacterial
sphingomyelinase C, thus indicating that the ChOx
substrate is not directly accessible to the enzyme in
intact membranes. A detailed description of the role of
ChOx in the virulence of R. equi, as well as recently
reported criticisms [56], has been reported previously
[2].
ChOx as antifungal sensor
Streptomyces natalensis ChOx (encoded by the pimE
gene; Fig. S1) plays a main role in the biosynthesis of
the polyene macrolide pimaricin [57] (the details of
ChOx involvement in this biosynthetic process have
been reported previously [2]). This 26-member tetraene
macrolide antifungal antibiotic is widely used in the
food industry (to prevent contamination of cheese and
other nonsterile food with mold) and in the treatment of
fungal keratitis because it interacts with membrane ster-
ols (ergosterol is the major sterol found in fungal mem-
branes), altering the membrane structure and causing
cell leakage. Interestingly, putative ChOx-encoding
genes are present in other known biosynthetic gene clus-
ters of antifungal polyketides, such as filipin (pteG) and
Biotechnological applications of sterol oxidase L. Pollegioni et al.
6864 FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS
rimodicin ⁄ CE-108 (rimD). All these polyketides are
elicited by soil bacteria against their fungal competitors,
whose membranes contain ergosterol, representing a
selective advantage for the producing organism.
Protein engineering studies
Improvement in thermal stability
The temperature sensitivity of ChOxs from a number
of sources has been compared recently [23]. The ther-
mal stability (as well as the stability at alkaline pH) of
ShChOx is not optimal for use as a diagnostic enzyme
(discussed above). In order to improve its properties, a
random mutagenesis approach by error-prone PCR of
the choA gene was used [58]. Following screening of the
mutant libraries at 50 °C, four single point mutants
(S103T, V121A, R135H and V145E ShChOx, see the
sequence numbering reported in Fig. S1) were isolated
whose kinetic properties resemble those of the wild-type
enzyme but whose thermal stability is increased. In par-
ticular, the V145E ShChOx shows a marked increase in
thermal stability and an enlarged range of optimal pH
(from acid to alkali).The single mutations identified by
random mutagenesis were combined. All the mutations,
except for R135H, had an additive effect: the double
mutant S103T ⁄ V145E was the most improved ShChOx
because it shows unaltered activity after 4 weeks at
40 °C. Concerning the rationale of the observed
changes, the C c atom of the threonine residue in S103T
ShChOx seems to be accommodated in a relatively
large cavity, thus strengthening the atomic packing
(Fig. 2A). The V145E substitution introduces an addi-
tional hydrogen bond and a salt bridge with the side
chain of R417, a residue close to the FAD cofactor.
These new interactions could help to stabilize the native
conformation, thus increasing the thermal resistance
and yielding a marked change in the optimal pH.
BsChOx is a monomeric flavoenzyme containing
one molecule of FAD covalently linked to H69
(mature protein numbering, corresponding to H121 on
the full-length polypeptide; Fig. S1). The covalent link
is eliminated by the H69A substitution: wild-type and
H69A BsChOx do not show significant structural
A
B
Fig. 2. (A) Details of the substitutions intro-
duced in ShChOx (Protein Data Bank, PDB
code: 1mxt) by random mutagenesis and
resulting in a higher stability to temperature
and to pH [58]. Mutated residues are col-
ored according to the reporting authors as in
Fig. S1. FAD (yellow) and 3-beta-hydroxy-5-
androsten-17-one (AND, gray) are shown in
CPK representation. Left: details of the
regions surrounding two selected mutations,
as reported by Nishiya et al. [58]. The
molecular surface is shown in light brown.
(B) Comparison of the active site of ReChOx
(PDB code: 1coy) and the active site of
ShChOx (PDB code: 1mxt), highlighting the
position important for substrate specificity.
Residues of the ReChOx active site are
numbered as reported by Li et al. [68];
residues of the ShChOx active site are
numbered as reported in Lario et al. [69]
(representation licorice) to which were
added residues mutated as described in
Xiang & Sampson [64] (representation ball-
and-stick). Proposed hydrogen bonds are
represented in pink. The ligand AND was
placed in the ShChOx active site according
to the position occupied in the active site of
ReChOx.
L. Pollegioni et al. Biotechnologicalapplications of sterol oxidase
FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS 6865
differences [59]. The mutant enzyme retains the oxida-
tion and the isomerization catalytic activities. How-
ever, the H69A BsChOx shows a 35-fold decrease in
maximal oxidation activity and a flavin midpoint
reduction potential of 100 mV lower ()204 mV ver-
sus )101 mV for wild-type ChOx) [60]. We demon-
strated that the covalent bond of the flavin in ChOx
represents a structural device for stabilizing the protein
tertiary structure [61]. In fact, the urea-induced unfold-
ing of H69A BsChOx occurred at significantly lower
urea concentrations ( 2 m lower), as did the tempera-
ture-induced unfolding ( 10–15 °C lower), than for
the wild-type enzyme.
Alteration of the substrate specificity
Table 1 describes the substrate preference of some
ChOxs (expressed as relative activity with respect to
cholesterol). In vivo random mutagenesis of the choA
gene decreased the K
m
value for cholesterol by 10-fold
without changing maximal activity: the evolved
ShChOx mutant contained the V145E ⁄ G405S substitu-
tions [62]. Based on sequence comparison between
Streptomyces and Rhodococcus ChOxs, six positions
were mutated in ShChOx [63]. Among the mutants
generated, the S379T variant showed a twofold higher
activity towards pregnenolone than cholesterol as a
substrate (interestingly, a threonine is present in the
same position in ReChOx, i.e., T387; see Fig. 2B). The
catalytic efficiency (k
cat
⁄ K
m
) of this mutant enzyme for
cholesterol and pregnenolone was two- and sixfold
higher, respectively, than that of the wild-type enzyme
[63]. Noteworthy is that the cavity around the side
chain of S379 in ShChOx is larger than that surround-
ing T387 in ReChOx (Fig. 2B). Structure-based
rational design of ShChOx was also used to identify
the residues involved in substrate binding [64]: M58,
L82, V85, M365, and F433 were identified as being in
direct contact with the C17 sterol tail (these residues
correspond to M95, L119, V122, M402 and F470 in
the full-length sequence reported in Fig. S1). Each
position was degenerated using NYS (N = A, C, G,
T; Y = C, T; S = C, G) codons to limit amino acids
to predominantly hydrophobic residues. The
L82A ⁄ V85T ⁄ F433L ShChOx mutant showed a twofold
increase in the k
cat
⁄ K
m
ratio with b-sitosterol and stig-
masterol, which differ from cholesterol for the
extended C17 tail.
By deleting the five residues belonging to the
active-site loop in ShChOx (mutant D79-83 corre-
sponding to sequence 115-119 in Fig. S1), the kinetic
efficiency (k
cat
⁄ K
m
ratio) was increased 5.6-fold for
dehydroepiandrosterone compared with the wild-type
enzyme, while the same ratio decreased 170-fold with
micellar cholesteroland up to 2800-fold with choles-
terol in a vesicle [2]. These results also suggest that
the tip of the active-site loop is necessary for the
packing with the C17-tail, being mainly responsible
for the substrate specificity at this position of ChOx
[65].
The oxidation and isomerization reactions catalyzed
by ShChOx could be separated by site-directed muta-
genesis. The N480A ⁄ Q mutants (corresponding to
position N522 in the full-length sequence reported in
Fig. S1) showed no oxidative activity but retained the
ability to isomerize cholest-5-en-3-one into cholest-4-
en-3-one [66]. However, the E361D ShChOx mutant
(corresponding to the E356D mutant in [66] and to
position 398 in the full-length sequence; Fig. S1)
retained the oxidative activity, but not the isomeriza-
tion activity [67]. Furthermore, the k
cat
⁄ K
m
ratio for
the oxidation reaction was increased sixfold in the
E356D ShChOx mutant and the k
cat
⁄ K
m
ratio for the
isomerization reaction was increased threefold in the
N480A mutant.
Conclusions
Certainly, future molecular biology, biochemical and
structural investigations will enable us to clarify the
role that ChOx activity plays in different microorgan-
isms and also to identify new functional roles for this
flavooxidase. Furthermore, in order to comprehend
why this enzyme exists as a flavoprotein containing
noncovalently or covalently linked FAD, we will also
need to understand its physiological role. These inves-
tigations will be carried out whilst optimizing known
applications of the ChOx reaction and developing new,
and more sophisticated, biotechnological uses of this
flavoenzyme. The latter hinges both on the discovery
of novel ChOx activities (see the recent identification
of ChOx from thermophilic bacteria) and on the engi-
neering of enzyme variants. We assume that the num-
ber of investigations of ChOx will continue to grow
quickly in the near future, in particular to identify
unexpected areas of application.
Acknowledgements
This work was supported by grants from Fondo di
Ateneo per la Ricerca. We are grateful for the support
from Consorzio Interuniversitario per le Biotecnologie
(CIB) and from the Research Center of Biotecnologie
per la Salute Umana. We would like to apologize to
many colleagues whose work we could not discuss in
detail because of space limitations.
Biotechnological applications of sterol oxidase L. Pollegioni et al.
6866 FEBS Journal 276 (2009) 6857–6870 ª 2009 The Authors Journal compilation ª 2009 FEBS
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