Appl Microbiol Biotechnol

DOI 10.1007/s00253-013-4842-9

MINI-REVIEW

An overview on alcohol oxidases and their potential

applications
Pranab Goswami & Soma Sekhar R. Chinnadayyala &
Mitun Chakraborty & Adepu Kiran Kumar &
Ankana Kakoti

Received: 7 December 2012 / Revised: 6 March 2013 / Accepted: 7 March 2013

# Springer-Verlag Berlin Heidelberg 2013

Abstract Alcohol oxidases (Alcohol: O2 Oxidoreductase;
EC 1.1.3.x) are flavoenzymes that catalyze the oxidation of
alcohols to the corresponding carbonyl compounds with a
concomitant release of hydrogen peroxide. Based on sub-
strate specificity, alcohol oxidases may be categorized
broadly into four different groups namely, (a) short chain
alcohol oxidase (SCAO), (b) long chain alcohol oxidase
(LCAO), (c) aromatic alcohol oxidase (AAO), and (d) sec-
ondary alcohol oxidase (SAO). The sources reported for
these enzymes are mostly limited to bacteria, yeast, fungi,
plant, insect, and mollusks. However, the quantum of re-
ports for each category of enzymes considerably varies
across these sources. The enzymes belonging to SCAO
and LCAO are intracellular in nature, whereas AAO and
SAO are mostly secreted to the medium. SCAO and LCAO
are invariably reported as multimeric proteins with very
high holoenzyme molecular masses, but the molecular char-
acteristics of these enzymes are yet to be clearly elucidated.
One of the striking features of the alcohol oxidases that
make them distinct from the widely known alcohol dehy-
drogenase is the avidly bound cofactor to the redox center of
these enzymes that obviate the need to supplement cofactor
during the catalytic reaction. These flavin-based redox en-
zymes have gained enormous importance in the develop-
ment of various industrial processes and products primarily
for developing biosensors and production of various
Authors Soma Sekhar R. Chinnadayyala and Mitun Chakraborty
contributed equally to this work.
P. Goswami (*) : S. S. R. Chinnadayyala : M. Chakraborty :
A. K. Kumar : A. Kakoti
Department of Biotechnology, Indian Institute of Technology
Guwahati, Guwahati 781 039 Assam, India
e-mail: pgoswami@iitg.ernet.in
Present Address:
A. K. Kumar
Codon Biosciences Pvt Ltd, Panjim, Goa 403001, India
industrially useful carbonyl compounds. The present review
provides an overview on alcohol oxidases from different
categories focusing research on these oxidases during the
last decade along with their potential industrial applications.
Keywords Alcohol oxidase . Short chain alcohol oxidase .
Long chain alcohol oxidase . Aromatic alcohol oxidase .
Secondary alcohol oxidase . Cholesterol oxidase . Polyvinyl
alcohol oxidase . Veratryl alcohol oxidase . Vanillyl alcohol
oxidase
Introduction
The direct conversion of alcohols to their corresponding
carbonyl compounds in nature is catalyzed primarily by
two different enzymes, namely alcohol dehydrogenase
(ADH) and alcohol oxidase (AOX). ADHs (Alcohol:
NAD+ oxidoreductases; EC 1.1.1.1) are widely distributed
in nature and are one of the most studied enzymes (Smidt et
al. 2008). AOX catalyzes the conversion of alcohols into
corresponding aldehydes or ketones, but not the reverse
r e ac t i o n s i m i l a r t o th a t c a t a l y z e d by t h e A D H
(Scheme 1a). There is a distinction in their cofactor require-
ment as well: AOX requires flavin-based cofactors, while
ADH requires NAD-based cofactors. The FAD in AOX is
avidly associated with the redox center of the enzyme and is
involved in transferring the hydride ion originated from
alcohol substrate to molecular oxygen leading to the forma-
tion of H 2 O 2 (Scheme 1b). The NAD + /NADH (or
NADP+/NADPH) involved in ADH catalysis is a strong
redox pair, the midpoint potential being −0.32 volts (Unden
and Bongaerts 1997), which makes NAD+ (or NADP+) a
strong oxidizing agent that accepts the hydride ion directly
from the substrate during the catalysis and generating the
corresponding reduced form, NADH/NADPH. In addition

Scheme 1 a Alcohol dehydrogenase (ADH) as catalyst and b alcohol
oxidase (AOX) as catalyst; R′=−H or any other alkyl/aryl group
to the aforementioned two biocatalytic routes for oxidation
of alcohols, peroxidatic oxidative of catalase and cyto-
chrome P450 catalyzed oxidation of alcohols to carbonyl
compounds has been also reported. A portion of the H2O2
formed when certain oxidases aerobically oxidize their sub-
strates is utilized by the catalase for the oxidation of ethanol
to acetaldehyde (Keilin and Hartree 1945). Cytochrome
P450 is involved in the oxidation of secondary alcohols to
corresponding ketones in mouse hepatic microsomes
(Matsunaga et al. 1998). The studies on the latter two routes
of biocatalytic oxidation of alcohols are still in its nascent
state.
Activity of AOX was initially reported by Farmer et al.
(1960) in a culture of Polystictus versicolor (a synonym of
Trametes versicolor). In a later report, Janssen et al. (1965)
described the isolation of an enzyme from a basidiomycete
that catalyzes the oxidation of methanol and other lower
primary alcohols to the corresponding aldehydes and H2O2.
Subsequently, they designated the enzyme as 'alcohol oxi-
dase, purified it to the crystalline state, and discovered FAD
as the prosthetic group of the enzyme (Janssen and Ruelius
1968). AOX activity is measured by monitoring (a) the
consumption of oxygen (Blasig et al. 1988) or (b) the
production of H2O2 in the reaction mixture. The concentra-
tion of H2O2 is usually measured by coupled enzymatic
methods that make use of horseradish peroxidase (HRP),
which oxidizes many chromogenic substrates during the
reduction of H2O2. Kemp et al. (1988) used the 2,2′-azino-
bis(3-ethylbenzothiazoline-6-sulphonic acid)/peroxidase-
linked assay for measuring AOX activity. HRP catalyzed
co-oxidation of phenol-4-sulfonic acid and 4-aminoantipyrine
is also reported to assay H2O2 (Vojinovic et al. 2004) for
measuring AOX activity (Azevedo et al. 2004). Based on
the substrate specificity, AOXs (EC 1.1.3.x) may be catego-
rized broadly into four groups: (a) short chain alcohol oxidase
(SCAO), (b) long chain alcohol oxidase (LCAO), (c) aromatic
alcohol oxidase (AAO), and (d) secondary alcohol oxidase
Appl Microbiol Biotechnol
(SAO). Rapid unveiling of the basic information on
these groups of enzymes has recently drawn a wide
attention for their possible applications in different
fields. The periodic review of the works on AOXs is
mainly limited to methanol oxidase, aryl alcohol oxidase, and
cholesterol oxidase. We report here a general overview on
different categories of AOXs including their potential appli-
cations in various fields by focussing the works mostly pub-
lished during the last decade and referring the readers to the
available review papers while discussing each of the
categories.
Short chain alcohol oxidase
Alcohol oxidase (EC 1.1.3.13), methanol oxidase, or etha-
nol oxidase that catalyzes the oxidation of lower chain
length alcohol substrates in the range of C1-C8 carbons
belongs to the category of SCAO (review: Tani et al.
1972; Sahm 1977; Woodward 1990; van der Klei et al.
1991; Ozimek et al. 2005). Yeasts and fungi are well-
known sources of SCAOs. There are also limited reports
on SCAOs from mollusk (Grewal et al. 2000). Among the
various sources, Pichia pastoris, Pichia putida, Pichia
methanolica, Hansenula polymorpha, Poria contigua, Can-
dida boidinii, Candida methanosorbosa, Cladosporium
fulvum, Aspergillus ochraceus, Aspergillus terreus,
Gloeophyllum trabeum, Achatina achatina, Achatina fulica,
Arion ater, Helix aspersa, Polyporus obtuses, Peniophora
gigantea, and Thermoascus aurantiacus are prominent, and
some of them are also reported in the last decade (Alvarado-
caudillo et al. 2002; Ko et al. 2005; Srisuk et al. 2006;
Kumar and Goswami 2006; Isobe et al. 2007; Daniel et al.
2007; Gvozdev et al. 2010).
SCAOs are intracellular enzymes located mostly in the
microbody/peroxisome of yeasts and in membranous loca-
tions of mollusks. The enzyme is an octameric protein with
the exceptions of A. ochraceus AIU 031 where tetrameric
nature has been demonstrated (Isobe and Kawakami 2007).
The subunit molecular masses of the SCAOs range from 65
to 80 kDa. The co-factor FAD is non-covalently bound to
the protein. In the methanol oxidases from methylotrophic
yeasts, the mFAD, a modified analog of FAD, is also addi-
tionally present. mFAD is assumed to be formed by the
spontaneous modification of FAD in the active center of
the enzyme and differs from FAD in the stereoposition of
the hydroxylic group in acyclic carbohydrate chain (Kellogg
et al. 1992). This modification of FAD slightly decreases the
Vmax however significantly reduces the Km of the enzyme
for the substrate methanol. These effects are of significant
physiological relevance in the cultures of low methanol
concentration. Depending on the source of yeast species,
oxygen stable flavin semiquinone moiety is present in
amounts varying from four to five molecules per AOX
Appl Microbiol Biotechnol
octamer; their moiety is however not involved in enzyme
catalysis (Mincey et al. 1980).
SCAO are usually active over a pH range of 6–9 with few
exceptions bearing activity up to pH 10. The optimum temper-
atures widely reported for the activity of SCAO are 25 to 30 and
37 °C with deviation to 50 °C and 50–55 °C for the enzyme
from C. methanosorbosa (Suye 1997) and A. ochraceus AIU
031 (Isobe et al. 2007), respectively. The widely reported in-
hibitors for SCAO are p-chloromercuribenzoic acid
(PCMB), H2O2, Cu2+, 1,10 phenanthroline, cyclopropanone,
5,5′-dithiobis-(2-nitrobenzoic acid), and 4-hydroxy-2-butynal.
Additionally, inhibitors moderately reported for the enzymes
are FeSO4, NiCl2, Ag+1, diethyldicarbonate, iodoacetate, so-
dium acetate, hydroquinone, KCN, phenylhydrazine, formal-
dehyde, hydroxylamine, acetamide, and NaN3.
The AOX protein and the regulation of the genes
encoding AOX have been studied for several reasons
(Holzmann et al. 2002; Gurkan et al. 2004). The promoter
that controls AOX expression is one of the strongest and
tightly controlled promoters in yeasts (Review: Hartner and
Glieder 2006). Several genes encoding AOX proteins that
have been cloned to date are mostly from the Pichia species
such as, Pichia angusta, P. pastoris, P. methanolica, and
Pichia pinus. Other sources reported in a later period are C.
boidinii (Sakai and Tani 1992), C. fulvum (Segers et al.
2001), Helminthosporium victoriae (Soldevila and Ghabrial
2001), Penicillium chrysogenum (Holzmann et al. 2002) and
Aspergillus nidulans (GenBank GI:40747510). Although
AOX proteins from filamentous fungi show a high degree
of homology (65–70 % identity) to those of methylotrophic
yeast species, their function is not related to methanol me-
tabolism as in yeasts. AOX activity in the cells of A. terreus
is induced by non-methanolic growth substrates (Kumar and
Goswami 2006). Expression of AOX gene is subject to
strong carbon catabolite repression. Under such conditions,
the AOX promoter sequence is organized into nucleosomal
structures (Costanzo et al. 1995). Prior to initiation of
AOX synthesis, the gene needs to be liberated from
nucleosomal packaging by a chromatin remodeler. The
ultimate level of AOX expression strongly depends on
the cultivation conditions, and differences exist in the
regulation of AOX expression in different yeast species.
Moreover, the components involved in the regulation of
AOX catabolite repression have been identified which in-
cludes a hexose transporter (Stasyk et al. 2004), a glucokinase,
and a hexokinase (Kramarenko et al. 2004; Laht et al. 2002;
Karp et al. 2003).
The AOX proteins, which belong to the group of
glucose-methanol-choline (GMC) family, have an FAD-
binding domain, a flavin attachment loop, and a substrate-
binding domain. In addition to these domains, there are two
minor domains, namely an FAD-covering loop and an ex-
tended FAD-binding domain. The FAD-binding domain is
the most conserved region and consists of an ADP-binding
motif (βαβ) and a characteristic nucleotide-binding site
GXGXXG (amino acids 13–18 of AOX) (Ozimek et al.
2005). Two inserts have also been identified in the
substrate-binding domain that are probably involved in
AOX-octamer formation. Boteva et al. (1999) created a
model of AOX based on the known structure of glucose
oxidase (GOX) from Aspergillus niger which shares maxi-
mum homology with Hansenula polymorpha AOX (25 %
identity). The conformation of AOX is irreversibly changed
following the dissociation of FAD, which is deeply buried in
the protein matrix (Zlateva et al. 2001).
Multiple AOX isoforms have been reported in several
strains of P. methanolica. In a study, a total of nine isoforms
were identified based on their electrophoretic mobility
which have identical subunit molecular weights but differ
in their pI (4.1–5.2). The reason for such molecular hetero-
geneity has been attributed to the occurrence of two AOX
genes (Nakagawa et al. 1996) differing in some amino acids
in the encoded proteins. Gruzman et al. (1996) confirmed
the occurrence of two types of subunits in the enzyme and
demonstrated that the octameric AOX isozymes are formed
as a result of different combinations of the subunits. In some
past studies, two highly homologous genes with different
promoters and transcription levels in P. pastoris were doc-
umented. These two genes, however, did not result in AOX
isozyme formation, instead are correspondingly expressed
into major and minor proteins with AOX activity. The minor
protein is the product of one of the genes transcribed on a
much lower level than the other gene (Koutz et al. 1989).
Notably, although H. polymorpha and C. boidinii are known
to have only one AOX gene, two AOX active proteins are
expressed in these strains (Lee and Komagata 1983). Effort
has been made to elucidate the crystal structure of AOX
from P. pastoris. The spatial structure of the enzyme could
not be further refined, and the possible reason for failure is
ascribed to the intermolecular microheterogeneity of the
enzyme (Gvozdev et al. 2010).
Long chain alcohol oxidase
Several terminologies, namely fatty alcohol oxidase (FAO),
long chain fatty acid oxidase, long chain fatty alcohol oxi-
dase, have been reported for LCAO (EC:1.1.3.20). These
enzymes catalyze alcohol substrates with carbon chain
length of usually above C6. The existence of LCAO was
first established in Torulopsis candida (Ilchenko 1984) and
Candida guilliermondii (Krauzova et al. 1984). The purifi-
cation and properties of a LCAO that oxidizes both the long
chains of saturated and unsaturated alcohol substrates from a
plant source, Tanacetum vulgare, were reported by
Banthorpe et al. (1976). The cotyledons of Simmondsia
chinensis containing FAO and fatty alcohol dehydrogenase

(Moreau and Huang 1979) also oxidize fatty alcohols
formed from the hydrolysis of stored wax esters during
germination. The plant species Arabidopsis thaliana (Cheng
et al. 2004) and Lotus japonicus (Zhao et al. 2008) have also
been reported as sources of LCAO. However, FAOs are
mostly reported from yeast species, such as Candida
bombicola (Hommel and Ratledge 1990), Candida cloacae
(Vanhanen et al. 2000), Candida tropicalis (Kemp et al.
1988), Candida apicola (Hommel et al. 1994), and Yarrowia
lipolytica (Kemp et al. 1990). The LCAO from Candida
species catalyze the oxidation of long chain alcohols in
mono-terminal, di-terminal, and sub-terminal positions (Blasig
et al. 1988; Kemp et al. 1988) and plays an important role in
long chain fatty acid metabolism (Cheng et al. 2005). Many
filamentous fungi such as A. terreus (Kumar and Goswami
2006), Cladosporium species (Goswami and Cooney 1999),
Aspergillus species (Savitha and Ratledge 1991), and YR-1
strain (Robelo et al. 2004) Mucor circinelloides YR-1 (Silva-
Jiménez and Zazueta-Sandoval 2005) produce LCAO during
growth on hydrocarbon substrates.
The LCAO has been reported in different subcellular
locations such as mitochondrial, microsomal, and
glyoxysomal membrane fractions (Mauersberger et al.
1987; Krauzova et al. 1985; Kumar and Goswami 2006;
Goswami and Cooney 1999; Moreau and Huang 1979,
1981). The presence of FAO activities in either particulate
or soluble fractions from a cell-free extract was also reported
(Silva-Jiménez et al. 2009).
The molecular mass and the subunit number of LCAO
were found to largely differ from SCAO, being lower than
the latter. The LCAO from C. cloacae, (Vanhanen et al.
2000) C. tropicalis (Kemp et al. 1988; Dickinson and
Wadford 1992), and T. vulgare (Banthorpe et al. 1976) are
dimeric in nature with sub-unit molecular masses ranging
from 70 to 94 kDa. The enzymes are heterodimeric in nature
with the exception of C. tropicalis AOX, which is
homodimeric. The AOX isolated from the microsome of
n-hexadecane-grown A. terreus showed highest affinity for
n-heptanol (Km =0.498 mM, Kcat =2.7×102 s−1) and high
aggregating properties (Kumar and Goswami 2008). In this
flavoenzyme, flavin is non-covalently associated with the
AOX protein where two FAD-binding domains have been
identified (Kumar and Goswami 2009).
The activity of LCAO above 45 °C is generally not
known and has been described as highly sensitive to tem-
perature (Kumar and Goswami 2006). The enzymes are
usually active within the range of neutral pH value to around
pH 9 with a wider range of pH (4.8–10) for AOX from T.
vulgare (Banthorpe et al. 1976). LCAO activities from n-
alkane-grown C. tropicalis and Y. lipolytica rapidly de-
creased on exposure to light. The rate of inactivation of
the enzyme depended upon the intensity and wavelength
of the incident light, but was diminished under anaerobic
Appl Microbiol Biotechnol
conditions (Kemp et al. 1990). In addition to light, other
inhibitors have also been reported for the LCAO such as
PCMB for S. chinensis (Moreau and Huong 1981). Apart
from flavin-based co-factors, few reports on NAD(P)+, ri-
boflavin, 2,6-dichloro-4-((4-hydroxyphenyl)imino)-2,5-
cyclohexadien-1-one), potassium ferricyanide (Moreau and
Huang 1979, 1981) as cofactors are also available for
LCAOs.
Cloning and sequencing of two FAO genes from C.
cloacae (FERM O-736) and a single FAO gene from C.
tropicalis (NCYC 470) have been reported (Vanhanen et al.
2000). The open reading frames (ORFs) of FAO1 and FAO2
from C. cloacae were 2,094 and 2,091 bp, respectively,
while the ORF of FAO from C. tropicalis (NCYC 470)
was 2,112 bp. FAO of C. tropicalis shared 60.6 and 61.
7 % nucleotide identities and 74.8 and 76.2 % amino acid
sequence similarities with C. cloacae FAO1 and FAO2,
respectively. The FAO1 gene was successfully expressed
in Escherichia coli by Slabas et al. (1999). Full-length
cDNA of L. japonicus FAO (LjFAO1) protein has been also
expressed in E. coli (Zhao et al. 2008). The gene contains
three exons and two introns and was expressed in the whole
plant, with the highest expression level in the apex. The
LCFAO responded to cold stress. The active LjFAO1 pro-
tein showed substrate specificities toward 1-dodecanol, 1-
hexadecanol, and 1,16-hexadecanediol with Km values ∼59.
6, ∼40.9, and ∼19.4 μM, respectively. Cheng et al. (2004)
had purified the His-tagged recombinant protein from A.
thaliana expressed in E. coli. Several other LCAO from
plant T. vulgare and in yeast C. cloacae were purified to
homogeneity. Partial purification of LCAOs was done in
yeast C. tropicalis, plant S. chinensis, fungi Y. lipolytica
(Moreau and Huang 1979), and A. terreus (Kumar and
Goswami 2008).
Secondary alcohol oxidase
AOXs, such as polyvinyl alcohol oxidase (PAO) and cho-
lesterol oxidase (ChOx), which catalyze the oxidation of
secondary alcohols to the corresponding ketones, fall under
this category. PAO (polyvinyl-alcohol: oxygen oxidoreduc-
tase: EC 1.1.3.30) activity was initially reported in the
culture supernatant of polyvinyl alcohol (PVA)-degrading
Pseudomonas strains by Suzuki (1976) and Watanabe et al.
(1976). Since then, many PAO have been reported from
Pseudomonas species (Morita et al. 1979; Shimao et al.
1985; Kawagoshi and Fujita 1997). The other sources for
the enzyme are limited, such as Penicillium sp. (Qian et al.
2004) and Sphingopyxis sp. (Yamatsu et al. 2006). The
enzyme oxidizes PVA to form diketone structures in PVA
(Sakai et al. 1986; Kawai and Hu 2009). Two different
oxidative pathways involved in PAO catalyzed degradation
of PVA were proposed (Suzuki 1976; Morita and Watanabe
Appl Microbiol Biotechnol
1977). Both PVA inducible and constitutive PAOs are
reported from the Pseudomonas strains. PVA-degrading en-
zymes are monomeric in nature and are reported as extra-
cellular, periplasmic, or membrane bound (Shimao et
al.1983, 1985; Zhang et al. 2006). A PAO with Mw of
∼40 kDa and pI of 4.5 was purified from a bacterial symbi-
otic mixed culture (Sakai et al. 1985). One atom of non-
heme iron was detected per molecule of the enzyme, and the
enzyme catalyzes the oxidation of various low molecular
weight secondary alcohols as well. A SAO activity detected
in the microsome of A. terreus was active against various
secondary alcohol substrates such as isopropanol, isoamyl
alcohol, 2-octanol, cyclooctanol, 3-octanol, and 2-
dodecanol (Kumar and Goswami 2006, 2008). Until now,
limited literatures are available on the basic biochemical
characteristics and properties of PAO. The enzymes are
active within the pH range of 5–10. EDTA and Hg2+ are
common inhibitors of PAO from different Pseudomonas
species, while BaCl2, MnCl2, NiCl2, O-phenanthroline,
Pb2+, Zn2+, hydroxyl amine, salicylaldoxine Cu2+, Fe2+,
Sn2+, and iodoacetamide are also reported as inhibitors for
some of the PAO (Morita et al. 1979; Kawagoshi and Fujita
1997; Suzuki 1978; Shimao et al. 1983).
ChOx (cholesterol: oxygen oxidoreductases: EC 1.1.3.6)
catalyzes the oxidation of 3β-hydroxysteroids and the isom-
erization of the intermediate, Δ5-6-ene- 3β-ketosteroid
(cholest-5-en-3-one), to produce Δ3-4-ene- 3β-ketosteroid
(cholest-4-en-3-one) (CEO) (Smith and Brooks 1975).
However, Molnar and group first reported a bacterial en-
zyme that oxidizes cholesterol to 6β-hydroperoxycholest-4-
en-3-ne (HCEO) but not the CEO produced by most ChOxs.
This HCEO is formed by the oxygenation of the sixth
position on the cholesterol nucleus by the ChOx (Molnar
et al. 1993). Since then, two distinctive types of bacterial
ChOxs have been found—one forms CEO and the other
forms HCEO as the major product (review: MacLachlan et
al. 2000; Doukyu 2009; Kreit and Sampson 2009). ChOx
was first isolated from Proactinomyces (Rhodococcus)
erythropolis by Turfitt (1944a, b). Later, the enzyme has
been isolated from various microbial strains such as
Arthrobacter, Brevibacterium, Corynebacterium, Mycobac-
terium, Nocardia, Rhodococcus, Streptomyces, and
Streptoverticillium, gram-negative bacteria such as
Burkholderia/Pseudomonas, Chromobacterium, and eu-
karyotic microorganism such as basidiomycetes,
Schizophyllum, etc. Although no mammalian homolog of
ChOx has been reported, there are mammalian enzymes that
catalyze the same chemistry as part of steroid biosynthesis.
The molecular weights of ChOxs are mostly in the range
of 40–63 kDa. Generally, microorganisms secrete ChOx to
the growth medium. However, microorganisms such as
Rhodococcus strains produce both membrane/cell-bound
and extracellular ChOxs (Sojo et al. 1997; Ahmad and
Goswami 2013). Microbial ChOxs generally have neutral
pH optima and possess stability over a wide pH range. The
temperature optima of the enzymes generally fall in the
range of 40–60 °C with exception of the enzyme from
Streptomyces fradiae whose optimum temperature is 70 °C
(Tabatabaei Yazdi et al. 2001). Highest thermostability of
the enzyme that retained 80 % of its original activity even at
85 °C after 30 min is also reported from a Chromobacterium
sp. (Doukyu et al. 2008). The thermal stability of these
enzymes is probably influenced by the structural stability
of the β-sheet at high temperatures (Doukyu et al. 2009).
The thermostability of ChOx is also substantially enhanced
by random mutagenesis (Nishiya et al. 1998). Some of the
prominent inhibitors of ChOxs are Hg2+, Ag+, FeCl3, and
FeSO4 for Streptomyces violascens (Tomioka et al. 1976)
and CuSO4 for Streptoverticillium cholesterolieum (Inouye
et al. 1982). PCMB partially reduces the activity of ChOx
from Arthrobacter simplex (Liu et al. 1988) and
Brevibacterium sterolicum (Uwajima et al. 1974). The en-
zymes from a Pseudomonas sp. and γ-Proteobacterium
were partially activated by Mn2+ (Lee et al. 1989; Isobe et
al. 2003). A detergent-tolerant ChOx was reported from γ-
Proteobacterium Y-134 (Isobe et al. 2003) where the en-
zyme retained more than 80 % of its original activity in 0.
5 % Triton X-405 and sodium cholate after incubation for
1 h at 60 °C. The activity of ChOx is generally influenced
by organic solvents. The activity of the B. sterolicum en-
zyme is rapidly inactivated, whereas the Streptomyces
hygroscopicus enzyme retained 70 % of the initial activity
after 5 h in the presence of 30 % propan-2-ol at 25 °C
(Pollegioni et al. 1999). Commercially available ChOx,
including Streptomyces sp., Cellulomonas sp., Nocardia
sp., Nocardia erythropolis, and Pseudomonas fluorescens,
were inactivated by 50 % volume of dimethylsulfoxide,
methanol, ethanol, acetone, isopropanol, ethyl acetate, or
butanol after incubation for 24 h at 37 °C. By contrast,
DS-1 and Burkholderia cepacia ST-200 enzymes were sta-
ble in the presence of all solvents except for acetone.
Doukyu et al. (2009) observed that the Km values of ChOx
from Nocardia species were relatively lower than the corre-
sponding enzymes from R. erythropolis, Chromobacterium
sp., B. cepacia, P. fluorescens, and Streptomyces sp. The
Vmax and Vmax/Km values of the DS-1 enzyme were the
highest among the enzymes tested.
ChOxs are of two types, class I and class II that corre-
spondingly contains non-covalently and covalently linked
FAD cofactor to the protein (Croteau and Vrielink 1996;
Sampson and Vrielink 2003). Some of the organisms such
as B. sterolicum possess both the classes of enzymes. The
class I enzyme belongs to the GMC oxidoreductase family,
while the class II enzyme belongs to the VAO family. Class I
enzymes have been identified mostly in actinomycetes such
as Streptomyces sp., B. sterolicum, Rhodococcus sp., and

Mycobacterium sp. Additionally, the class I enzymes from
some sources (Navas et al. 2001) showed a consensus
sequence for FAD binding, GlyXGlyXXGly, in the N-
terminal region. The analysis of class I from Streptomyces
sp. revealed that His447 and Glu361 residues are involved
in the activity for oxidation and isomerization (Yue et al.
1999), and these residues are conserved in many of them.
The phylogenetic tree analysis showed that the enzymes can
be further divided into two groups: class I-1 and I-2. Unlike
the class I-1 enzymes, the class I-2 enzymes lack a signal
sequence, suggesting a cytoplasmic localization (Navas
et al. 2001). Class II enzymes have been reported from
B. sterolicum, R. erythropolis, Burkholderia sp.,
Chromobacterium sp., and Pseudomonas aeruginosa. X-
ray crystallographic (Coulombe et al. 2001) structure of B.
sterolicum enzyme (class II) suggested covalent attachment
of FAD to an active-site histidine via the C8 α group of the
flavin isoalloxazine ring. This covalent interaction is corre-
lated to redox potential and stability of the enzyme
(Caldinelli et al. 2005). In addition, Glu475 and Arg477,
located at the active-site cavity, were suggested to constitute
gate functioning in the control of oxygen access (Piubelli et
al. 2008). These amino acid residues are conserved in the
sequence of the class II enzymes.
Aromatic alcohol oxidase
Aromatic alcohol oxidase or aryl alcohol oxidase (AAO;
aryl-alcohol:oxygen oxidoreductase: EC 1.1.3.7) catalyzes
the oxidation of aromatic primary alcohol to aromatic alde-
hyde (review: Hernández-Ortega et al. 2012a). Veratryl al-
cohol oxidase and vanillyl alcohol oxidase (VAO; EC:1.1.3.
38) also belong to this oxidoreductase family (reviews: van
den Heuvel et al. 2001b, 2004). Notably, the VAO is active
over a wide range of phenolic compounds and catalyzes
oxidation, deamination, demethylation, dehydrogenation,
and hydroxylation reactions. For detail about the catalytic
activity of VAO for other functional groups, we refer readers
the above review papers by van den Heuvel et al.
AAO is mostly isolated and characterized from several
fungal strains such as Bjerkandera adusta, Pleurotus
eryngii, Pleurotus ostreatus, Pleurotus sajor-caju, Pleurotus
pulmonarius, Penicillium simplicissimum, Geotrichum
candidum, Phanerochaete chrysosporium, Brachypsectra
fulva, T. versicolor, Fusarium solani, Rigidoporus
microporus, and Botrytis cinerea. The other well-known
sources of this enzyme are insects such as Chrysomela
populi, Phratora vitellinae, and a land slug such as Arion
ater. AAOs from different fungi are involved in the
ligninolytic system together with peroxidase or laccase by
providing H2O2 produced from the oxidation of benzyl
alcohols (Ruiz-Duenas and Martinez 2009). AAO and
laccase/peroxidase cooperate in the production of hydroxyl
Appl Microbiol Biotechnol
radical, which is thought to be involved in the initial attack
on lignocellulose. Further, it is suggested that AAO prevents
repolymerization of laccase oxidation products and sustain
the lignin degradation process. Many of the lignin-
degrading fungi produce soluble AAOs. The important char-
acteristic of AAO is its extracellular production except for
few cases, such as from A. ater, where the enzyme is present
in the membrane. Veratryl alcohol, anisyl alcohol, and
4-(methoxymethyl) phenol are reported as inducer of many
VAOs. Under anaerobic conditions, certain Pseudomonas
species produce a VAO analog with a tetrameric α2β2
structure. These flavocytochromes share similar substrate
specificity with VAO and are involved in the biodegradation
of different phenolic compounds. These enzymes use their
heme-containing subunit instead of natural oxygen as the
terminal electron acceptor.
The optimum temperature for AAO activity from
many microorganisms falls in the range of 25 to 45 °C
(Asada et al. 1995; Ruiz-Duenas et al. 2006; Kumar
and Rapheal 2011; Tamboli et al. 2011) with few ex-
ceptions for fungal AAO which showed the optimum at
55 °C (Guillen et al. 1990). Majority of the AAO are
active within a neutral to slightly acidic pH range (5.5–
7.5), whereas few are active in a more alkaline range.
Phenol, Ag+, NaN3, Pb2+, ρ-methoxylbenzyl alcohol, anisic
acid, methoxybenzylamine, 4-Allyl-2,6-dimethoxyphenol,
HgCl2, and PCMB are the inhibitors reported for some
AAOs. Sodium azide, EDTA, L-cysteine, and dithiothreitol
are also reported recently as potent inhibitors of AAO
(Tamboli et al. 2011).
AAOs from most of the fungal sources are monomeric
glycoprotein with molecular weights in the range of 65.3 to
78 kDa. The enzyme from P. simplicissimum, however, is an
inducible intracellular homooctamer, with holoenzyme mo-
lecular mass of 510 kDa (Fraaije et al. 1995), whereas the
enzyme from C. populi and P. vitellinae are homotetramers
with subunit molecular mass ∼79 kDa (Bruckmann et al.
2002). Initially, the AAO protein sequences were analyzed
from P. eryngii, P. pulmonarius (Varela et al. 2000b; 2001)
and B. adusta (Romero et al. 2010). Cloning of AAO from
A. terreus has been recently reported from the author’s lab
(GenBank GI: JX139751:2012). The number of sequences
however has been rapidly increased with the increasing
basidomycetes genome in databases including around 50
sequences annotated as putative AAO. Hernández-Ortega
et al. (2012a) compared 112 GMC oxidoreductase se-
quences from 31 basidomycetes species including 40 AAO
and 37 methanol oxidase sequences and demonstrated phy-
logenetic distance of AAO from other GMC enzymes types
including methanol oxidases. Recombinant AAO from P.
eryngii has been expressed and purified from both bacterial
and fungal expression systems (Varela et al. 2001; Ruiz-
Duenas et al. 2006). Native or recombinant AAO has also
Appl Microbiol Biotechnol
been purified or partially purified from A. terreus (Kumar and
Goswami 2009), Chrysomela tremulae, C. populi (Michalski
et al. 2008) and P. sajor-caju (Bourbonnais and Paice 1988).
The cDNA sequence of P. pulmonarius AAO consisted of 593
amino acids including a signal peptide. Comparison with
other AOXs showed highly conserved amino acid sequences
in the N-terminal and C-terminal regions, in spite of differ-
ences in substrate specificities.
Two prominent crystal structures of aryl alcohol oxidase
from P. eryngii (Varela et al. 2000a) and P. simplicissimum
(Mattevi et al. 1997) are available in the Protein Data Bank
(PDB). The structure of P. eryngii AAO (PDB id 3FIM)
revealed significant similarities with the structures of ChOx
(PDB id 3COX) from B. sterolicum (Li et al. 1993) and GOX
(PDB id 1GAL) from A. niger (Hecht et al. 1993). In all these
three proteins, which belong to GMC oxidoreductase family,
the type of FAD bonding is non-covalent, and FAD-binding
sequence code is GXGXXG (X represents variable amino
acid residue). There is, however, a difference in the FAD-
binding fold among them, which is β-α-β fold for the AAO
(PDB id 3FIM), whereas ρ-hydroxy-benzoate hydroxylase
fold for the later two proteins. Interestingly, there are consid-
erable dissimilarities between 1VAO and 3FIM. Unlike 3FIM,
1VAO belongs to ρ-cresol methylhydrolase (PCMH) family
and the FAD-bonding type in it is covalent (with His422).
Moreover, in 1VAO the FAD-binding fold comprises two α+
β domains, and the FAD-binding sequence code is
P(X)6GXN. The secondary structure view of AAO (PDB id:
3FIM), VAO (PDB id: 1VAO), ChOx (PDB id: 3COX), and
GOX (PDB id: 1GAL) is shown in Fig. 1. In AAO (Fig. 1a)
Fig 1 Secondary structure
view of a AAO (PDB id: 3FIM)
from P. eryngii. b VAO chain A
and B (PDB id: 1VAO) from P.
simplicissimum. c ChOx (PDB
id: 3COX) from B. sterolicum,
d GOX (PDB id: 1GAL) from
A. niger retrieved from PDB. α-
helices, β-sheets, and loops are
depicted in red coil ribbon, blue
sheets ribbon, and ash-grey
color, respectively. The FAD
and substrate-binding pockets a b
are highlighted in green grids.
FAD is highlighted in yellow,
and the isoalloxazine ring faces
the substrate-binding pocket in
all the structures. Structures are
created using Molegro Virtual
Docker (Thomsen and
Christensen 2006), Molegro-A
CLC bio company, Denmark
c d
and GOX (Fig. 1d), the substrate-binding pocket is separated
from the FAD-binding site, whereas in VAO (Fig. 1b) and
ChOx (Fig. 1c), the active site shares the same pocket with the
FAD. The FAD molecule in AAO interacts with the protein
through a network of H-bonds predominantly involving the
main-chain NH and CO groups of residues located at the N-
terminus and several water molecules. The FAD is stabilized
by some important residues located in the βαβ fold adapted
by the N-terminal region in the protein. The putative active-
site cavity is located in the vicinity of the FAD isoalloxazine
ring and near two histidines that could contribute as catalytic
bases to activate alcohol. Moreover, the active site of the
native enzyme is solvent inaccessible and controls substrate
specificity by providing a “size exclusion mechanism.” The
additional two structural elements existing in the surroundings
of its active site that modulate the access of substrates are
absent in the structure of GOX (Ferreira et al. 2009). From the
molecular docking studies on AAO crystal structure, the rea-
son for its inability to bind secondary alcohols efficiently was
identified (Hernández-Ortega et al. 2011a). The small space
available at the bottom part of the active site cavity restricts
large substituents at the Cα position. As discussed above, the
cofactor, flavin or FAD, in AAO is either covalently or non-
covalently bound to the protein. The covalent flavinylation is
reported to enhance the oxidative power of VAO (Fraaije et al.
2003).
Analysis of crystal structures of AAO, GOX, ChOx, and
choline oxidase showed a highly conserved catalytic site,
suggesting a similar oxidation mechanism. The mechanism
of AAO catalyzed alcohol oxidation was revealed from
Chain B Chain A
Active
site
FAD
and
active
site
FAD
binding
site
FAD
and
active
Active
site
site
FAD
binding
site

kinetic studies, which was defined as the hydride transfer
from substrate to flavin N5 concerted with proton abstrac-
tion from ∞ hydroxyl by a catalytic base (Ferreira et al.
2009). Specific His residues have been identified as the
most likely catalytic bases in AAO and choline oxidase
(Hernández-Ortega et al. 2011b, 2012b; Ghanem and Gadda
2005). The substrate specificity studies on P. eryngii AAO
demonstrated that the extension of the aromatic system in
the substrate benzyl alcohol favors the enzyme action.
Moreover, the oxidation rates are strongly affected by the
nature, position, and number of the aromatic-ring substitu-
ents. In this line, β-naphthylmethanol was identified as the
most readily oxidized substrate (Ferreira et al. 2005). Gener-
ally, electron donor substituents promote the alcohol oxida-
tion, whereas electron-withdrawing substituents produce the
reverse effect. An interesting functional activity of AAO is its
minor activity towards aromatic aldehydes (Guillen et al.
1992). It was noted that electron-withdrawing substituents
promoted aldehyde oxidation by AAO, while electron donors
caused the opposite effect, in contrast to that observed for
alcohols. The enzyme activity on these aldehydes is correlated
with their hydration degree in water, which is identified as the
activating molecule in the oxidation (Ferreira et al. 2010).
Aldehyde is also accepted as substrate by choline oxidase,
which also belongs to GMC oxidoreductase family in which
case betaine aldehyde formed as the oxidation product of its
primary substrate choline is the substrate (Fan et al. 2006).
A comparative analysis among the four categories of
AOX enzymes (Table 1) shows that source organisms, lo-
cations, nature of the protein molecules, primary substrates,
and type of inhibitors significantly vary across these redox
enzymes, even though their common function is the catalyt-
ic oxidation of non-aromatic hydroxyl to the corresponding
carbonyl functional groups.
Potential applications of AOX
The alcohol oxidase attains high industrial interest because
of its various favorable properties such as (1) its catalytic
power to oxidize various alcohols irreversibly and selective-
ly, (2) its easy availability since these enzymes are widely
produced by different aerobic microorganisms, making the
production of these enzymes possible in large scale, and (3)
it does not require any external co-factors for the catalysis.
The potential areas of application of AOXs as revealed from
literature are biosensors, biocatalytic synthesis of flavor and
fragnance compounds, organic synthesis of optically pure
compounds, and to a limited extent the bioremediation of
hydrocarbon contamination. Prompt detection of ethanol,
methanol, cholesterol, ethylene glycol, and iso-propanol in
plasma and other body fluids is a demanding job in clinical,
toxicological, and forensic laboratory works along with food
and fermentation technology. Many amperometry-based
Appl Microbiol Biotechnol
alcohol biosensors have been reported, and most of them
rely on the detection of co-substrate O2 or co-product H2O2
involved in the AOX catalyzed oxidation of alcohols (re-
view: Azevedo et al. 2005). AOX-based alcohol biosensor
relying on the detection of O2 has been reported widely
including few in the last decade (Akyilmaz and Dinckaya
2005). The oxygen-based sensor, however, has some prac-
tical inconveniences and limitations such as low accuracy
and reproducibility and high minimum detectable concen-
tration. Many first-generation amperometric AOX biosen-
sors for alcohol using H2O2 as electroactive species have
been reported in the last decade (Carelli et al. 2006; Alpeeva
et al. 2005; Shkotova et al. 2006; Smutok et al. 2006).
However, the requirement of high overpotential for the
oxidation of H2O2 causes electrochemical interference in
the detection. A way to decrease the necessary applied
potential is to use electron transfer mediator that shuttles
the electrons between the flavin redox center of AOX and
the electrode and thereby debarring the oxygen to act as an
electron acceptor, a principle widely known as second-
generation biosensors. In this line, a ferrocene mediator-
based biosensor with low working potential has been
reported that improves the electrochemical selectivity and
the detection limits of the biosensor (de Prada et al. 2003).
The susceptibity of the mediator to dissolute in the sample
solution, however, poses hurdle for sensitive detection of
target analytes. Significantly low flavin midpoint redox
potentials in AOX enzymes, such as for ChOx (Motteran
et al. 2001) and AAO (Munteanu et al. 2008), are likely to
contribute to the criteria suitable for developing third-
generation alcohol biosensors. Notably, third-generation
biosensors offer highly selective and sensitive detection of
target analytes in the samples.
The application of AOX into commercialy successful
instruments has been hampered mainly by their poor stabil-
ity. One of the major focuses of the AOX-based biosensor is
to prolong the activity of this complex enzyme on the sensor
platform as many AOX-based biosensors exhibited varying
operational stability (Shkotova et al. 2006; Carelli et al.
2006; Barsan et al. 2008). The stability of the enzymes for
application in bio-analytical systems may be enhanced by
using suitable biocompatible matrices and immobilization
techniques. It is worth mentioning that nanomaterials such
as gold nanoparticles, carbon nanotubes, etc. have recently
attracted attention for developing efficient and stable
bioelectrode for biosensor applications. A third-generation
P. pastoris AOX-based biosensor using AOX-MWCNT-
based composite was used for detecting alcohol in real
samples (Das and Goswami 2013). The bioelectrode
retained ∼90 % of the original response after 4 weeks when
stored in 4 °C. Gouveia-Caridade et al. (2008) developed an
alcohol biosensor in which AOX was immobilized with
BSA on solid MWCNT–carbon film electrode. The authors
Table 1 A comparative account on some general characteristics among different categories of AOXs
Characteristics SCAO
Source organism Yeasta filamentous fungib
Localization Intracellular: peroxisomesa
Nature of the protein Homooctomer (subunit Mw
molecule ~65–80 kDa)a
Primary substrates Methanol, ethanola
Optimum pH range ~6.0–8.5a
Optimum 25–30 °Ca, 37 °Cb
temperature range
Inhibitors H2O2a, 1,10-phenanthrolineb,
Cu2+b, 1,4-Butynediolc ,
4-Hydroxy-2-butynalc,
Cyclopropanolc,
5,5'-dithiobis(2-
nitrobenzoate)d
Widely reported or intensively studied data are only included. General enzyme inhibitors are excluded from the list
The superscript alphabets denote the citations in the reference column against each characteristic in the table
LCAO SAO
Yeastc, filamentous fungid, Bacteriaf
plante
Intracellular: microsomeb, Extracellulard
glyoxisomec
Dimeric (subunit Mw Monomeric (Mw~40–85 kDa)c.
~70–94 kDa)b For ChOx: ~45–63kDad
Long chain alcohols Polyvinyl alcoholc, 2o alcohols:
(C8-C16)b chain length C4-C12d. For ChOxe:
β-Cholesterol, β-sitosterol,
β-stigmasterol
~6–9.5b ~6.5–10c For ChOx: 6–8d
20–30 °Cc 40–50°Cd For ChOx: 40–60e
Light (λ450 nm)e, λ405 nm +O2f BaCl2g,MnCl2g, NiCl2g,
o-phenanthrolineh, Zn2+ h,
hydroxylaminei, Co2+ i,
salicylaldoxinei, Sn2+ j, Fe2+ j.
For ChOxk: AgNO3, FeCl3, FeSO4,
1-Fluoro-2,4-dinitrobenzen
AAO
Fungig, insecth, molluski
Extracellulare
Momomeric (Mw ~66–80 kDa)e
Benzyl-, 3- and 4- methoxy
benzyl-,2,4-dimethoxybenzyl
alcohol, veratryl alcohol,
cinnamyl alcohol, naphthyl
methanol f.
5–8.5e
25–45 °Cf
2-Hydroxybenzyl alcoholl, 3-
Phenyl-1-propanolm, 4-anisic
acidm, 4-methoxy
benzylaminem, phenolm,
toluenem, benzylmethyl
etherm, Ag+ n, Pb2+ n
References
a
Couderc and Baratti 1980, bKumar and
Goswami 2006, cKemp et al. 1990, dSavitha
Appl Microbiol Biotechnol
and Ratledge 1991,eBanthorpe et al. 1976,
f
Suziki 1976, gOkamoto and Yanase 2002,
h
Michalski et al. 2008, iMann et al. 1989
a
van der Klei et al. 1991, bKumar and
Goswami 2008, cMoreau and Hang 1979,
d
Shiamo et al. 1985, eVarela et al. 2000
a
Couderc and Baratti 1980, bBanthorpe et al.
1976, cSakai et al. 1985, cKawagoshi and
Fujita 1997, dLi et al. 2010, dGhasemian et
al. 2009, dFukuyama and Miyake 1979.
e
Guillen et al. 1992,eMichalski et al. 2008
a
Janssen and Ruelius 1968, b Kemp et al.
1988, cSakai et al. 1985, dKumar and
Goswami 2006, eDoukyu 2009,
f
Hernández-Ortega et al. 2012a
a
Suye 1997, bBanthorpe at al. 1976, cMorita
et al. 1979, cShimao et al. 1983,
d
MacLachlan et al. 2000, eBourbonnais and
Paice 1988 eGuillen et al. 1990, eFerreira et
al. 2005
a
Isobe et al. 2009,bCouderc and Baratti 1980,
c
Banthorpe et al. 1976, dSakai et al. 1985,
e
Doukyu 2009 eLi et al. 2010, fRuiz-Dueñas
et al. 2006, fSaparrata and Guillen 2005,
f
Kumar and Rapheal 2011, fAsada et al.
1995
a
Couderc and Baratti 1980, b Yamada et al.
1979, cvan der Klei et al. 1990, dGvozdev et
al. 2010, eDickinson and Wadforth 1992,
f
Kemp et al. 1990,gShimao et al. 1983,
h
Morita et al. 1979, iSuzuki 1978,
j
Kawagoshi and Fujita 1997, kTomioka et
al. 1976, kDoukyu 2009, lMichalski et al.
2008, mFerreira et al. 2005; Guillen et al.
1990n

noted a significant increase in sensitivity but poor stability
of the biosensor. Various optical biosensors for alcohol have
also been reported in the past using O2 sensitive com-
pounds, mostly different ruthenium organic complex
(Mitsubayashi et al. 2003) to construct the biosensors. Hack
et al. (2007) reported that a dipstick designed to detect
ethanol in saliva was able to cross-react and show a positive
color change with very low concentrations of methanol but
not ethylene glycol.
The substrate specificity of ChOx has prompted its use in
clinical determination of serum cholesterol levels as part of
human disease monitoring and treatment (Review:
Pollegioni et al. 2009; Arya et al. 2008). A great deal of
work on optical, first- and second-generation amperometry-
based cholesterol biosensors has been done. The majority of
the cholesterol biosensors involve electrochemical
transducer-based detection of H2O2. The third-generation
biosensors are however difficult to achieve due to the deep
embedment of the redox center of ChOx in the protein core.
The amperometric biosensor research is currently focusing
upon developing new materials that offer promise to solve
the problem of low enzyme activity and stability (Sarma et
al. 2009). The applications of various nano-materials to
establish the direct electrochemistry of ChOx for developing
cholesterol biosensors have been reported in the recent past
(Saxena et al. 2011a, b; Dhand et al. 2008; Gopalan et al.
2009; Roy et al. 2006; Tan et al. 2005; Yang et al. 2006a).
Optical cholesterol biosensors which relied on fluorescence
properties of ruthenium complexes have been developed
(Wu and Choi 2003; Wang et al. 2004). The enzymatic
reaction of ChOx may be coupled with the conversion of
an indicator dye into a chromogen, and the concept has been
used for cholesterol sensing (Arya et al. 2007; Law et al.
1997; Malik and Pundir 2002). Kouassi et al. (2005) have
reported the determination of cholesterol via detection of
increase in the absorption intensity for 4-cholesten-3-one
produced during enzymatic reaction. Besides this, the SPR
technique has also been used for developing cholesterol
biosensors (Arya et al. 2006, 2007d; Solanki et al. 2007).
ChOx has also been used as a probe to detect the interaction
of cholesterol with phospholipids (Lange et al. 2005) and
the eukaryotic membrane structure (Rouquette-Jazdanian et
al. 2006). The investigation of the membrane structure such
as lipid raft is useful to study membrane function such as
signal transduction, protein and lipid sorting, cellular entry
by toxins and viruses, and viral budding.
Conversion of alcohols to aldehydes and ketones is
of significant industrial importance. The chemical
methods commonly practiced for this conversion (Zhan
and Thompson 2004) are not regarded as ideal methods
owing to either or all of the drawbacks such as need of
expensive oxidizing agents, generation of large amounts
of metal waste, and non-eco-friendly reagents and
Appl Microbiol Biotechnol
reaction parameters. The selectivity and room tempera-
ture reaction in aqueous environment make AOX a
potential catalyst for organic synthesis. AOX from the
P. pastoris oxidizes wide spectrums of unbranched pri-
mary alcohols, including propargyl alcohol, 2-
chloroethanol, 2-cyanoethanol, to aldehydes leading to
important synthetic intermediates (Dienys et al. 2003).
The high yield conversion of a wide range of alcohol
substrates to aldehydes using AOX from A. terreus has
been reported (Kakoti et al. 2012), which also described
the production of n-heptanal—an important starting ma-
terial for several aroma and flavor chemicals. The poly-
urethane foam immobilized enzyme retained ∼60 % of
the initial activity till the fifth reaction cycle, thus
providing high cumulative yield of the product. The
re-specificity of AOXs from different leaf beetles larvae
including Phaedon armoraciae and P. vitellinae has also
been reported (Veith et al. 1996, 1997). Also, the
stereoselectivity of AOX present in C. boidinii (Kraus
and Simon 1975) and pheromone glands of several
moths such as Manduca sexta (Hoskovec et al. 2002)
is well described. AOX present in the pheromone glands
of several moths catalyzes the production of fatty alde-
hydes inluding fatty aldehydes of various degree of
unsaturation from different fatty alcohols (Fang et al.
1995). The oxidation of betaine aldehyde by choline
oxidase is of considerable research interest for the de-
velopment of therapeutic agents for pathogenic bacteria
and for the engineering of drought and temperature
resistance in economically relevant crops (Fan et al.
2006).
The importance of lignin-degrading fungi for their AAO-
based biocatalytic production of various flavors and aromas
has also drawn attention in the field. Many of these fungi are
able to produce important aroma compounds, namely, van-
illin, benzaldehyde p-anisaldehyde, and its halogenated de-
rivatives, by the action of AAO and mycelium-associated
other redox enzymes. The production of vanillin through
biotransformation route from benzenoid and other precur-
sors (Priefert et al. 2001) could be improved either by
genetic modification of the microbial strains (Plaggenborg
et al. 2006; Di Gioia et al. 2011) or by using natural or
evolved variants of VAO (van den Heuvel et al. 2001a,
2004). Many industrially important non-phenolic aromatic
aldehydes are also the potential biocatalytic product of AAO
from B. adusta (Romero et al. 2009). AAO is also reported
as a potential biocatalyst for stereoselective biotransforma-
tion for the synthesis of various secondary alcohols
(Hernández-Ortega et al. 2012a).
ChOx present in isolate or in whole cell has been used for
bioconversion of many 3β-hydroxysteroids, which in turn
can be used for the synthesis of steroid hormones and other
pharmaceutical steroids (Lee and Biellmann 1988; Mahato
Appl Microbiol Biotechnol
and Garai 1997; Guo et al. 2003). The enzyme also exhibits
insecticidal activity against lepidopteran cotton insect pests,
including tobacco budworm, corn earworm, and pink boll-
worm. ChOx from Streptomyces natalensis has been de-
scribed as a key enzyme in the biosynthesis of the polyene
macrolide pimaricin (Mendes et al. 2007; Aparicio and
Martín 2008), which is a macrolide antifungal agent widely
used in the food industry. ChOx is also thought to contribute
to the pathogenecity of many pathogenic organisms (Navas
et al. 2001; Brzostek et al. 2007); hence, the enzyme may
also be a potential target for new antibiotics.
The oxidation of alcohols is one of the critical steps
occurring during the microbial degradation of aliphatic hy-
drocarbons. The cytochrome P450/alkane monooxygenase
catalyzed alcohol products of alkanes are further oxidized
by AOX in case of filamentous fungi. Broad substrate-
specific cytochrome P450/alkane monooxygenase and
AOX enzymes in the hydrocarbon-degrading fungi A.
terreus were isolated, and their biocatalytic products formed
during the oxidation of aliphatic hydrocarbons were charac-
terized (Vatsyayan et al. 2008; Kumar and Goswami 2006).
The application potentials of these AOX redox systems for
bioremediation technology are however yet to be systemat-
ically explored.
Immobilization of enzyme on a suitable carrier facilitates
the stability of ChOx and is widely considered as an impor-
tant criterion for process economy. Dienys et al. (2003)
immobilized AOX by glutaraldehyde-activated macroporous
cellulose and retained the activity even up to eight
cycles of the reaction for the oxidation of n-butanol
and 2-methoxyethanol. A giant multilammellar vesicle
as encapsulating and stabilizing matrix for the multimeric
AOX protein from A. terreus was recently demonstrated.
Further, the constructed vesicles were used as a container
for the AOX-HRP coupled reaction to probe the pres-
ence of alcohol in the samples using optical detection
method (Virk et al. 2012). DEAE dextran/lactitol has
been identified as a promising immobilization system for
retaining the AOX activity (Gibson et al. 1993). Azevedo et al.
(2004) had devised an in situ stabilization strategy for AOX
through its immobilization onto controlled pore glass in mini
packed-bed bioreactors. This strategy led to high operational
stabilities and was successfully applied in a FIA system for
ethanol analysis.
Conclusion and future direction of research
Among the four categories of AOXs, SCAO and LCAO are
more complex, and their molecular characteristics are not
adequately known. As a whole, the AOX enzymes are less
explored on their structure, function, and applications as
compared to their complementary alcohol dehydrogenase
enzymes. To bring these groups of redox enzymes into the
working protocol, further investigation is needed to eluci-
date the molecular characteristics, structure–function rela-
tionship, and their native role in the respective host
organisms with the help of the advanced techniques and
protocols available currently in the field of molecular biol-
ogy, protein sciences, and bioinformatics. The application
potential of this group of redox enzymes is enormous, as
validated through the volume of publications in the area of
biosensors and biocatalytic production of various carbonyl
compounds useful in flavor, pharmaceutical and clinical
industries. Significantly low midpoint potentials of the re-
dox center of these flavin-based enzymes may offer the
possibility of developing third-generation label-free, sensi-
tive, and interference-free biosensors for various alcohols of
clinical and industrial importance. However, to use the
isolated enzymes in the relevant applications, operational
and storage stability of these labile enzymes need to be
improved. Parallel research on advance biocompatible ma-
terial and chemical sciences in addition to the application of
conventional molecular biology techniques may offer solu-
tions to improve the above mentioned traits to qualify them
as efficient biocatalyst for numerous applications.
Acknowledgments We acknowledge the financial assistance from
Department of Biotechnology, India and editorial help of Madhuri
and Santhosh to carry out the work.
References
Ahmad S, Goswami P (2013) Enhanced production of cell-bound
cholesterol oxidase from Rhodococcus sp. NCIM 2891 by the
statistical method. Ann Microbiol 63:199–205
Akyilmaz E, Dinckaya E (2005) An amperometric microbial biosensor
development based on Candida tropicalis yeast cells for sensitive
determination of ethanol. Biosens Bioelectron 20:1263–1269
Alpeeva IS, Vilkanauskyte A, Ngounou B, Csoregi E, Sakharov IY,
Gonchar M, Schuhmann W (2005) Bi-enzyme alcohol biosensors
based on genetically engineered alcohol oxidase and different
peroxidases. Microchim Acta 152:21–27
Alvarado-Caudillo Y, Bravo Torres JC, Novoa VZ, Silva JH, Torres-
Guzman JC, Gutierrez-Corona JF, Zazueta-Sandoval R (2002)
Presence and physiologic regulation of alcohol oxidase activity
in an indigenous fungus isolated from petroleum-contaminated
soils. Appl Biochem Biotech 98:243–256
Aparicio JF, Martín JF (2008) Microbial cholesterol oxidases: biocon-
version enzymes or signal proteins? Mol Biosyst 4:804–809
Arya SK, Datta M, Malhotra BD (2008) Recent advances in choles-
terol biosensor. Biosens Bioelectron 23:1083–1100
Arya SK, Solanki PR, Singh RP, Pandey MK, Datta M, Malhotra BD
(2006) Application of octadecanethiol self-assembled monolayer
to cholesterol biosensor based on surface plasmon resonance
technique. Talanta 69:918–926
Arya SK, Solanki PR, Singh SP, Kaneto K, Pandey MK, Datta M,
Malhotra BD (2007) Poly-(3-hexylthiophene) self-assembled
monolayer based cholesterol biosensor using surface plasmon
resonance technique. Biosens Bioelectron 22:2516–2524

Asada Y, Watanabe A, Ohtsu Y, Kuwahara M (1995) Purification and
characterization of an aryl-alcohol oxidase from the lignin-
degrading basidiomycete Phanerochaete chrysosporium. Biosci
Biotechnol Biochem 59:1339–1341
Azevedo AM, Prazeres F, Cabral JMS, Fonseca LP (2005) Ethanol
biosensors based on alcohol oxidase. Biosens Bioelectron
28:235–247
Azevedo AM, Vojinovic V, Cabral JMS, Gibson TD, Fonseca LP (2004)
Operational stability of immobilized horseradishperoxidase
in mini-packed bed bioreactors. J Mol Catal B: Enzymatic
28:12112–8
Banthorpe DV, Cardemil E, Del C, Contraras M (1976) Purification
and properties of alcohol oxidase from Tanacetum vulgare.
Phytochem 15:391–394
Barsan MM, Brett CMA (2008) An alcohol oxidase biosensor using
PNR redox mediator at carbon film electrodes. Talanta 74:1505–
1510
Blasig R, Mauersberger S, Reige P, Schunck WH, Jockisch W, Franke
P, Muller HG (1988) Degradation of long chain n-alkanes by the
yeast Candida maltosa. Oxidation of n-alkanes and intermediates
using microsomal membrane fractions. Appl Microbiol
Biotechnol 28:589–597
Boteva R, Visser A, Filippi B, Vriend G, Veenhuis M, van der Klei IJ
(1999) Conformational transitions accompanying oligomerization
of yeast alcohol oxidase, a peroxisomal flavo-enzyme. Biochem
38:5034–5044
Bourbonnais R, Paice MG (1988) Veratryl alcohol oxidases from the
lignin degrading basidiomycete Pleurotus sajorcaju. Biochem J
255:445–450
Bruckmann M, Termonia A, Pasteels JM, Hartmann T (2002) Charac-
terization of an extracellular salicyl alcohol oxidase from larval
defensive secretions of Chrysomela populi and Phratora
vitellinae (Chrysomelina). Insect Biochem Molec 32:1517–1523
Brzostek A, Dziadek B, Rumijowska-Galewicz A, Pawelczyk J, Dziadek
J (2007) Cholesterol oxidase is required for virulence of Mycobac-
terium tuberculosis. FEMS Microbiol Lett 275:106–112
Caldinelli L, Iametti S, Barbiroli A, Bonomi F, Fessas D, Molla G,
Pilone MS, Pollegioni L (2005) Dissecting the structural determi-
nants of the stability of cholesterol oxidase containing covalently
bound flavin. J Biol Chem 280:22572–22581
Carelli D, Centonze D, De Giglio A, Quinto M, Zambonin PG (2006)
An interference free first generation alcohol biosensor based on a
gold electrode modified by an over oxidised non-conducting
polypyrrole film. Anal Chim Acta 565:27–35
Cheng Q, Liu HT, Bombelli P, Smith A, Slabas AR (2004)
Functional identification of AtFao3, a membrane bound long
chain alcohol oxidase in Arabidopsis thaliana. FEBS Lett
574:62–68
Cheng Q, Sanglard D, Vanhanen S, Liu HT, Bombelli P, Smith A,
Slabas AR (2005) Candida yeast long chain fatty alcohol
oxidase is a c-type haemoprotein and plays an important role
in long chain fatty acid metabolism. Biochim Biophys Acta
1735:192–203
Costanzo G, Di Mauro E, Negri R, Pereira G, Hollenberg C (1995)
Multiple overlapping positions of nucleosomes with single in vivo
rotational setting in the Hansenula polymorpha RNA polymerase
II MOX promoter. J Biol Chem 270:11091–11097
Couderc R, Baratti J (1980) Oxidation of methanol by the yeast, Pichia
pastoris: purification and properties of alcohol oxidase. Agric
Biol Chem 44:2279–2289
Coulombe R, Yue KQ, Ghisla S, Vrielink A (2001) Oxygen access to
the active site of cholesterol oxidase through a narrow channel is
gated by an Arg-Glu pair. J Biol Chem 276:30435–30441
Croteau N, Vrielink A (1996) Crystallization and preliminary X-ray
analysis of cholesterol oxidase from Brevibacterium sterolicum
containing covalently bound FAD. J Struct Biol 116:317–319
Appl Microbiol Biotechnol
Daniel G, Volc J, Filonova L, Plihal O, Kubátová E, Halada P (2007)
Characteristics of Gloeophyllum trabeum alcohol oxidase, an
extracellular source of H2O2 in brown rot decay of wood. Appl
Environ Microbiol 73:6241–6253
Das M, Goswami P (2013) Direct electrochemistry of alcohol oxidase
using multiwalled carbon nanotube as electroactive matrix for
biosensor application. Bioelectrochem 89:19–25
de Prada AGV, Pena N, Mena ML, Reviejo AJ, Pingarron JM (2003)
Graphite/Teflon composite bienzyme amperometric biosensors
for monitoring of alcohols. Biosens Bioelectron 18:1279–1288
Dhand C, Arya SK, Datta M, Malhotra BD (2008) Polyaniline-carbon
nanotube composite film for cholesterol biosensor. Anal Biochem
383:194–199
Dickinson FM, Wadforth C (1992) Purification and some properties of
alcohol oxidase from alkane-grown Candida tropicalis. Biochem
J 282:325–331
Dienys G, Jarmalavicius S, Budriene S, Citavicius D, Serekaite J
(2003) Alcohol oxidase from yeast Pichia pastoris a potential
catalyst for organic synthesis. J Mol Catal B: Enzym 21:47–49
Di Gioia D, Luziatelli F, Negroni A, Ficca AG, Fava F, Ruzzi M (2011)
Metabolic engineering of Pseudomonas fluorescens for the pro-
duction of vanillin from ferulic acid. J Biotechnol 156:309–316
Doukyu N, Shibata K, Ogino H, Sagermann M (2008) Purification and
characterization of Chromobacterium sp. DS-1 cholesterol oxi-
dase with thermal, organic solvent, and detergent tolerance. Appl
Microbiol Biotechnol 80:59–70
Doukyu N (2009) Characteristics and biotechnological applications of
microbial cholesterol oxidases. Appl Microbiol Biotechnol
83:825–837
Doukyu N, Shibata K, Ogino H, Sagermann M (2009) Cloning, sequence
analysis, and expression of a gene encoding Chromobacterium sp.
DS-1 cholesterol oxidase. Appl Microbiol Biotechnol 82:479–
490
Fan F, Germann MW, Gadda G (2006) Mechanistic studies of choline
oxidase with betaine aldehyde and its isosteric analogue 3,3-
dimethylbutyraldehyde. Biochemistry 45:1979–1986
Fang N, Teal PEA, Doolittle RE, Tumlinson JH (1995) Biosynthesis of
conjugated olefinic systems in the sex pheromone gland of female
tobacco hornworm moths, Manduca sexta (L.). Insect Biochem
Mol Biol 25:39–48
Farmer VC, Henderson ME, Russell JD (1960) Aromatic alcohol
oxidase activity in the growth medium of Polystictus versicolor.
Biochem J 74:257–262
Ferreira P, Medina M, Guillén F, Martínez MJ, van Berkel WJH,
Martínez AT (2005) Spectral and catalytic properties of aryl-
alcohol oxidase, a fungal flavoenzyme acting on polyunsaturated
alcohols. Biochem J 389:731–738
Ferreira P, Hernández-Ortega A, Herguedas B, Martínez AT, Medina M
(2009) Aryl-alcohol oxidase involved in lignin degradation: a
mechanistic study based on steady and pre-steady state kinetics
and primary and solvent isotope effects with two different alcohol
substrates. J Biol Chem 284:24840–4847
Ferreira P, Hernández-Ortega A, Herguedas B, Rencoret J, Gutiér-
rez A, Martínez MJ, Jiménez-Barbero J, Medina M, Martínez
AT (2010) Kinetic and chemical characterization of aldehyde
oxidation by fungal aryl-alcohol oxidase. Biochem J
425:585–593
Fraaije MW, van den Heuvel RHH, Mattevi A, van Berkel WJH (2003)
Covalent flavinylation enhances the oxidative power of vanillyl-
alcohol oxidase. J Mol Catal B: Enzym 21:43–46
Fraaije MW, Veeger C, van Berkel WJH (1995) Substrate specificity of
flavin-dependent vanillyl alcohol oxidase from Penicillium
simplicissimum. Eur J Biochem 234:271–277
Fukuyama M, Miyake Y (1979) Purification and some properties of
cholesterol oxidase from Schizophyllum commune with covalently
bound flavin. J Biochem 85:1183–1193
Appl Microbiol Biotechnol
Gibson TD, Hulbert JN, Woodward JR (1993) Preservation of shelf life
of enzyme based analytical systems using a combination of
sugars, sugar alcohols and cationic polymers or zinc ions. Anal
Chim Acta 279:185–192
Ghanem M, Gadda G (2005) On the catalytic role of the conserved
active site residue His466 of choline oxidase. Biochem 44:893–
904
Ghasemian A, Yazdi M, Sepehrizadeh Z, Yazdi Z, Zarrini G (2009)
Overexpression, one-step purification, and characterization of a
type II cholesterol oxidase from a local isolate Rhodococcus sp.
PTCC 1633. World J Microbiol Biotechnol 25:773–779
Gopalan AI, Lee KP, Ragupathy D (2009) Development of a stable
cholesterol biosensor based on multi-walled carbon nanotubes-
gold nanoparticles composite covered with a layer of chitosan-
room-temperature ionic liquid network. Biosens Bioelectron
24:2211–2217
Goswami P, Cooney JJ (1999) Sub-cellular localization of enzymes
involved in oxidation of n-alkane by Cladosporium resinae. Appl
Microbiol Biotechnol 51:860–864
Gouveia-Caridade C, Pauliukaite R, Brett CMA (2008) Development
of electrochemical oxidase biosensors based on carbon nanotube-
modified carbon film electrodes for glucose and ethanol.
Electrochim Acta 53:6732–6739
Grewal N, Parveen Z, Large A, Perry C, Connock M (2000) Gastropod
mollusc aliphatic alcohol oxidase: subcellular localisation and
properties. Comp Biochem Physiol Part B 125:543–554
Gruzman MB, Titorenko VI, Ashin VV, Lusta KA, Trotsenko YA
(1996) Multiple molecular forms of alcohol oxidase from the
methylotrophic yeast Pichia methanolica. Biochem (Moscow)
61:1537–1544
Guillen F, Martinez AT, Martinez MJ (1990) Production of hydrogen
peroxide by aryl-alcohol oxidase from the ligninolytic fungus
Pleurotus eryngii. Appl Microbiol Biotechnol 32:465–469
Guillen F, Martinez AT, Martinez MJ (1992) Substrate specificity and
properties of the aryl-alcohol oxidase from the ligninolytic fungus
Pleurotus eryngii. Eur J Biochem 209:603–611
Guo LW, Wilson WK, Pang J, Shackleton CH (2003) Chemical syn-
thesis of 7- and 8-dehydro derivatives of pregnane-3, 17alpha, 20-
triols, potential steroid metabolites in Smith-Lemli-Opitz syn-
drome. Steroids 68:31–42
Gurkan C, Symeonides SN, Ellar DJ (2004) High-level production in
Pichia pastoris of an anti-p185HER-2 scFv using an alternative
secretion expression vector. Biotechnol Appl Biochem 39:115–
122
Gvozdev AR, Tukhvatullin IA, Gvozdev RI (2010) Purification and
properties of alcohol oxidase from Pichia putida. Biochem
(Moscow) 75:242–248
Hack JB, Early J, Brewer KL (2007) An alcohol oxidase dipstick
rapidly detects methanol in the serum of mice. Academ Emerg
Med 14:1130–1134
Hartner FS, Glieder A (2006) Regulation of methanol utilisation path-
way genes in yeasts. Microb Cell Fact 5:39. doi:10.1186/1475-
2859-5-39
Hecht HJ, Kalisz HM, Hendle J, Schmid RD, Schomburg D (1993)
Crystal structure of glucose oxidase from Aspergillus niger re-
fined at 2.3 A resolution. J Mol Biol 229:153–172
Hernández-Ortega A, Borrelli K, Ferreira P, Medina M, Martínez AT,
Guallar V (2011a) Substrate diffusion and oxidation in GMC
oxidoreductases: an experimental and computational study on
fungal aryl-alcohol oxidase. Biochem J 436:341–350
Hernández-Ortega A, Lucas F, Ferreira P, Medina M, Guallar V,
Martínez AT (2011b) Modulating O2 reactivity in a fungal
flavoenzyme: involvement of aryl-alcohol oxidase Phe-501 con-
tiguous to catalytic histidine. J Biol Chem 286:41105–41114
Hernández-Ortega A, Ferreira P, Martínez AT (2012a) Fungal
aryl-alcohol oxidase: a peroxide-producing flavoenzyme
involved in lignin degradation. Appl Microbiol Biotechnol
93:1395–1410
Hernández-Ortega A, Ferreira P, Merino P, Medina M, Guallar V,
Martínez AT (2012b) Stereoselective hydride transfer by aryl-
alcohol oxidase, a member of the GMC superfamily. Chem Bio
Chem 13:427–435
Holzmann K, Schreiner E, Schwab H (2002) A Penicillium
chrysogenum gene (AOX) identified by specific induction upon
shifting pH encodes for a protein which shows high homology to
fungal alcohol oxidases. Curr Genet 40:339–344
Hommel RK, Lassner D, Weiss J, Kleber HP (1994) The inducible
microsomal fatty alcohol oxidase of Candida (Torulopsis)
apicola. Appl Microbiol Biotechnol 40:729–734
Hommel R, Ratledge C (1990) Evidence for two fatty alcohol oxidases
in the biosurfactant-producing yeast Candida (Torulopsis)
bombicola. FEMS Microbiol Lett 70:183–186
Hoskovec M, Luxova A, Svatos A, Boland W (2002) Biosynthesis of
sex pheromones in moths: stereochemistry of fatty alcohol oxida-
tion in Manduca sexta. Tetrahedron 58:9193–9201
Ilchenko AP (1984) Oxidase of higher alcohols in the yeast Torulopsis
candida grown on hexadecane. Mikrobiol 53:903–906
Inouye Y, Tagnchi K, Fujii A, Ishimaru K, Nakamura S, Nomi R
(1982) Purification and characterization of extracellular 3β-
hydroxysteroid oxidase produced by Streptoverticillium
cholesterolieum. Chem Pharm Bull 30:951–958
Isobe K, Shoji K, Nakanishi Y, Yokoe M, Wakao N (2003) Purification
and some properties of cholesterol oxidase stable in detergents from
gamma-proteobacterium Y-134. J Biosci Bioeng 95:257–263
Isobe K, Kato A, Ogawa J, Kataoka M, Iwasaki A, Hasegawa J,
Shimizu S (2007) Characterization of alcohol oxidase from As-
pergillus ochraceus AIU 031. J Gen Appl Microbiol 53:177–183
Isobe K, Kawakami Y (2007) Preparation of convection interaction
media isobutyl disc monolithic column and its application to
purification of secondary alcohol dehydrogenase and alcohol
oxidase. J Chromatography:A 1144:85–89
Isobe K, Takahashi T, Ogawa J, Kataoka M, Shimizu S (2009) Pro-
duction and characterization of alcohol oxidase from Penicillium
purpurescens AIU 063. J Biosci Bioeng 107:108–112
Janssen FW, Kerwin RM, Ruelius HW (1965) Alcohol oxidase, a
novel enzyme from a basidiomycete. Biochem Biophys Res
Commun 20:630–634
Janssen FW, Ruelius HW (1968) Alcohol oxidase, a flavoprotein from
several basidiomycetes species: crystallization by fractional pre-
cipitation with polyethylene glycol. Biochim Biophy Acta
151:330–342
Kakoti A, Kumar AK, Goswami P (2012) Microsome bound alcohol
oxidase catalyzed production of carbonyl compounds from alco-
hol substrates. J Mol Catal B: Enz 78:98–104
Karp H, Jarviste A, Kriegel TM, Alamäe T (2003) Cloning and biochem-
ical characterization of hexokinase from the methylotrophic yeast
Hansenula polymorpha. Curr Gent 44:268–276
Kawagoshi Y, Fujita M (1997) Purification and properties of polyvinyl
alcohol oxidase with broad substrate range obtained from Pseu-
domonas vesicularis var. povalolyticus PH. World J Microbiol
Biotechnol 13:273–277
Kawai F, Hu X (2009) Biochemistry of microbial polyvinyl alcohol
degradation. Appl Microbiol Biotechnol 84:227–237
Keilin D, Hartree EF (1945) Properties of catalase catalysis of coupled
oxidation of alcohols. Biochem J 39:293–300
Kellogg RM, Kruizinga W, Bystrykh LV, Dijkhuizen L, Harder W
(1992) Structural analysis of a stereochemical modification of flavin
adenine dinucleotide in alcohol oxidase from methylotrophic yeast.
Tetrahedron 48:4147–4162
Kemp GD, Dickinson FM, Ratledge C (1988) Inducible long chain
alcohol oxidase from alkane-grown Candida tropicalis. Appl
Microbiol Biotechnol 29:370–374

Kemp GD, Dickinson FM, Ratledge C (1990) Light sensitivity of
the n-alkane-induced fatty alcohol oxidase from Candida
tropicalis and Yarrowia lipolytica. Appl Microbiol Biotechnol
32:461–464
Ko HS, Fujiwara H, Yokoyama Y, Ohno N, Amachi S, Shinoyama H,
Fujii T (2005) Inducible production of alcohol oxidase and cata-
lase in a pectin medium by Thermoascus aurantiacus IFO 31693.
J Biosci Bioeng 99:290–292
Koutz P, Davis GR, Stillman C, Barringer K, Cregg J, Thill G (1989)
Structural comparison of the Pichia pastoris alcohol oxidase
genes. Yeast 5:167–177
Kouassi GK, Irudayaraj J, McCarty G (2005) Examination of choles-
terol oxidase attachment to magnetic nanoparticles. J Nanobiotechnol
3:1–9
Kramarenko T, Karp H, Jarviste A, Alamäe T (2004) Sugar repression
in the methylotrophic yeast Hansenula polymorpha studied by
using hexokinase-negative, glucokinase-negative and double
kinase-negative mutants. Folia Microbiol 5:521–529
Kraus A, Simon H (1975) Stereochemistry and mechanism of the
methanol oxidase from Candida boidinii. Hoppe Seylers Z Phys-
iol Chem 356:1477–1484
Krauzova VI, Il'chenko AP, Sharyshev AA, Lozinov AB (1985) Pos-
sible pathways of the oxidation of higher alcohols by membrane
fractions of yeast culture on hexadecane and hexadecanol.
Biochim 50:726–732
Krauzova VI, Komarova GN, Il'chenko AP, Gulevskaia SA (1984)
Alcohol oxidation by Candida guilliermondii yeasts grown on
hexadecanol. Mikrobiol 53:621–627
Kreit J, Sampson NS (2009) Cholesterol oxidase: physiological func-
tions. FEBS J 276:6844–6856
Kumar AK, Goswami P (2006) Functional characterization of alcohol
oxidases from Aspergillus terreus MTCC 6324. Appl Microbiol
Biotechnol 72:906–911
Kumar AK, Goswami P (2008) Purification and properties of a novel
broad substrate specific alcohol oxidase from Aspergillus terreus
MTCC 6324. BBA-Protein Proteom 1784:1552–1559
Kumar AK, Goswami P (2009) Dissociation and reconstitution studies
of a broad substrate specific multimeric alcohol oxidase protein
produced by A. terreus. J Biochem 145:259–265
Kumar VV, Rapheal VS (2011) Induction and purification by three-
phase partitioning of aryl alcohol oxidase (AAO) from Pleurotus
ostreatus. Appl Biochem Biotechnol 163:423–432
Laht S, Karp H, Kotka P, Jarviste A, Alamäe T (2002) Cloning and
characterization of glucokinase from a methylotrophic yeast
Hansenula polymorpha: different effects on glucose repression in
H. polymorpha and Saccharomyces cerevisiae. Gene 296:195–203
Lange Y, Ye J, Steck TL (2005) Activation of membrane choles-
terol by displacement from phospholipids. J Biol Chem
280:36126–36131
Law WT, Doshi S, McGeehan J, McGeehan S, Gibboni D,
Nikolioukine Y, Keane R, Zheng J, Rao J, Ertingshausen G
(1997) Whole-blood test for total cholesterol by a self-metering,
self-timing disposable device with built-in quality control. Clin
Chem 43:384–389
Lee K, Biellmann JF (1988) Cholesterol conversion to δ4 -
cholestenone by cholesterol oxidase in polyphasic systems: ex-
tension to the selective oxidation of 7β-hydroxycholesterol. Tet-
rahedron 44:1135–1139
Lee JD, Komagata K (1983) Further taxonomic study of methanol-
assimilating yeasts with special references to electrophoretic com-
parison of enzymes. J Gen Appl Microbiol 29:395–416
Lee S, Rhee H, Tae W, Shin J, Park B (1989) Purification and character-
ization of cholesterol oxidase from Pseudomonas sp. and taxonomic
study of the strain. Appl Microbiol Biotechnol 31:542–546
Li B, Wang W, Wang F, Wei D (2010) Cholesterol oxidase ChoL is a
critical enzyme that catalyzes the conversion of diosgenin to 4-
Appl Microbiol Biotechnol
ene-3-keto steroids in Streptomyces virginiae IBL-14. Appl
Microbiol Biotechnol 85:1831–1838
Li J, Vrielink A, Brick P, Blow DM (1993) Crystal structure of
cholesterol oxidase complexed with a steroid substrate: implica-
tions for flavin adenine dinucleotide dependent alcohol oxidases.
Biochemistry 32:11507–11515
Liu WH, Meng MH, Chen KS (1988) Purification and some properties
of cholesterol oxidases produced by an inducible and a constitu-
tive mutant of Arthrobacter simplex. Agric Biol Chem 52:413–
418
MacLachlan J, Wotherspoon ATL, Ansell RO, Brooks CJW (2000)
Cholesterol oxidase: sources, physical properties and analytical
applications. J Steroid Biochem Mol Biol 72:169–195
Mahato SB, Garai S (1997) Advances in microbial steroid biotransfor-
mation. Steroids 62:332–345
Malik V, Pundir CS (2002) Determination of total cholesterol in serum
by cholesterol esterase and cholesterol oxidase immobilized and
co-immobilized on to arylamine glass. Biotechnol Appl Biochem
35:191–197
Mann V, Large A, Khan S, Malik Z, Connock MJ (1989) Aromatic
alcohol oxidase: a new membrane-bound H2O2-generating en-
zyme in alimentary tissues of the slug Arion ater. J Exp Zool
251:265–274
Matsunaga T, Kishi N, Tanaka H, Watanabe K, Yoshimura H,
Yamamoto I (1998) Major cytochrome P450 enzyme responsible
for oxidation of secondary alcohols to the corresponding ketones
in mouse hepatic microsomes: oxidation of 7-hydroxy-D8-tetra-
hydrocannabinol to 7-oxo-D8-tetrahydrocannabinol. Drug Metab
Dispos 26:1045–1047
Mattevi A, Fraaije MW, Coda A, van Berkel WJH (1997) Crystalliza-
tion and preliminary X-ray analysis of the flavoenzyme vanillyl
alcohol oxidase from Penicillium simplicissimum. Proteins
27:601–603
Mauersberger S, Kargel E, Matyashova RN, Muller HG (1987) Sub-
cellular organization of alkane oxidation in the yeast Candida
maltosa. J Basic Microbiol 27:565–582
Mendes MV, Recio E, Antón N, Guerra SM, Santos-Aberturas J,
Martín JF, Aparicio JF (2007) Cholesterol oxidases act as signal-
ing proteins for the biosynthesis of the polyene macrolide
pimaricin. Chem Biol 14:279–290
Michalski C, Mohagheghi H, Nimtz M, Pasteels J, Ober D (2008)
Salicyl alcohol oxidase of the chemical defense secretion of two
chrysomelid leaf beetles. Molecular and functional characteriza-
tion of two new members of the glucose-methanol-choline oxi-
doreductase gene family. J Biol Chem 283:19219–19228
Mincey T, Taylern G, Maldvan AS, Aleles RH (1980) Presence of a
flavin semiquinone in methanol oxidase. Biochem 77:7099–7101
Mitsubayashi K, Kon T, Hashimoto Y (2003) Optical bio-sniffer for
ethanol vapor using an oxygen-sensitive optical fiber. Biosens
Bioelectron 19:193–198
Molnar I, Hayashi N, Choi K, Yamamoto H, Yamashita M, Murooka Y
(1993) Bacterial cholesterol oxidases are able to act as
flavoprotein-linked ketosteroid monooxygenases that catalyse
the hydroxylation of cholesterol to 4-cholesten-6-ol-3-one. Mol
Microbiol 7:419–428
Moreau RA, Huang AHC (1981) Enzymes of wax ester catabolism in
jojoba. Methods Enzymol 71:804–813
Moreau RA, Huang AHC (1979) Oxidation of fatty alcohol in the
cotyledons of jojoba seedlings. Arch Biochem Biophys
194:422–430
Morita M, Hamada N, Sakai K, Watanabe Y (1979) Purification and
properties of secondary alcohol oxidase from a strain of Pseudo-
monas. Agric Biol Chem 43:1225–1235
Morita M, Watanabe Y (1977) A secondary alcohol oxidase: a com-
ponent of a polyvinyl alcohol degrading enzyme preparation.
Agric Biol Chem 41:1535–1537
Appl Microbiol Biotechnol
Motteran L, Pilone MS, Molla G, Ghisla S, Pollegioni L (2001)
Cholesterol oxidase from Brevibacterium sterolicum. The rela-
tionship between covalent flavinyla-tion and redox properties. J
Biol Chem 276:18024–18030
Munteanu FD, Ferreira P, Ruiz-Duenas FJ, Martinez AT, Cavaco-
Paulo A (2008) Bioelectrochemical investigations of aryl-
alcohol oxidase from Pleurotus eryngii. J Electroanal Chem
618:83–86
Nakagawa T, Uchimura T, Komagata K (1996) Isozymes of methanol
oxidase in a methanol utilizing yeast, Pichia methanolica IAM
12901. J Ferment Bioeng 81:498–503
Navas J, Gonzalez-Zorn B, Ladron N, Garrido P, Vazquez-Boland JA
(2001) Identification and mutagenesis by allelic exchange of
choE, encoding a cholesterol oxidase from the intracellular path-
ogen Rhodococcus equi. J Bacteriol 183:4796–4805
Nishiya Y, Harada N, Teshima SI, Yamashita M, Fujii I, Hirayama N,
Murooka Y (1998) Improvement of thermal stability of Strepto-
myces cholesterol oxidase by random mutagenesis and a structural
interpretation. Protein Eng 10:231–235
Okamoto K, Yanase H (2002) Aryl alcohol oxidases from the white-rot
basidiomycete Pleurotus ostreatus. Mycosci 43:391–395
Ozimek P, Veenhuis M, Van der Klei IJ (2005) Alcohol oxidase: a
complex peroxisomal, oligomeric flavoprotein. FEMS Yeast Res
5:975–983
Piubelli L, Pedotti M, Molla G, Feindler-Boeckh S, Ghisla S,
Pilone MS, Pollegioni L (2008) On the oxygen reactivity of
flavoprotein oxidases: an oxygen access tunnel and gate in
Brevibacterium sterolicum cholesterol oxidase. J Biol Chem
283:24738–24747
Plaggenborg R, Overhage J, Loos A, Archer JAC, Lessard P, Sinskey
AJ, Steinbuchel A, Priefert H (2006) Potential of Rhodococcus
strains for biotechnological vanillin production from ferulic acid
and eugenol. Appl Microbiol Biotechnol 72:745–755
Pollegioni L, Gadda G, Ambrosius D, Ghisla S, Pilone MS (1999)
Cholesterol oxidase from Streptomyces hygroscopicus and
Brevibacterium sterolicum: effect of surfactants and organic sol-
vents on activity. Biotechnol Appl Biochem 30:27–33
Pollegioni L, Piubelli L, Molla G (2009) Cholesterol oxidase: biotech-
nological applications. FEBS J 276:6857–6870
Priefert H, Rabenhorst J, Steinbüchel A (2001) Biotechnological pro-
duction of vanillin. Appl Microbiol Biotechnol 56:296–214
Qian D, Du G, Chen J (2004) Isolation and culture characterization of a
new poly vinyl alcohol degrading strain: Penicillium sp. WSH02–
21. World J Microbiol Biotechnol 20:587–591
Robelo CR, Novoa VZ, Sandoval RZ (2004) Effects of carbon source on
expression of alcohol oxidase activity and on morphologic pattern of
YR-1 strain, a filamentous fungus isolated from petroleum-
contaminated soils. Appl Biochem Biotechnol 113:116–171
Romero E, Ferreira P, Martínez AT, Martínez MJ (2009) New oxidase
from Bjerkandera arthroconidial anamorph that oxidizes both
phenolic and nonphenolic benzyl alcohols. Biochim Biophys
Acta 1794:689–697
Romero E, Martínez AT, Martínez MJ (2010) Molecular characteriza-
tion of a new flavooxidase from a Bjerkandera adusta anamorph.
In: Feijoo G, Moreira MT (eds) Proc OESIB, ISBN-13: 978-84-
614-2824-3 pp. 86–91
Rouquette-Jazdanian AK, Pelassy C, Breittmayer JP, Aussel C (2006)
Revaluation of the role of cholesterol in stabilizing rafts implicat-
ed in T cell receptor signaling. Cell Signal 18:105–122
Roy S, Vedala H, Choi W (2006) Vertically aligned carbon
nanotube probes for monitoring blood cholesterol. Nanotechnol
17:14–18
Ruiz-Dueñas FJ, Ferreira P, Martínez MJ, Martínez AT (2006) In vitro
activation, purification, and characterization of Escherichia coli
expressed aryl alcohol oxidase, a unique H2O2 producing enzyme.
Protein Expr Purif 45:191–199
Ruiz-Dueñas FJ, Martínez AT (2009) Microbial degradation of lignin:
how a bulky recalcitrant polymer is efficiently recycled in nature
and how we can take advantage of this. Microbial Biotechnol
2:164–177
Sahm H (1977) Metabolism of methanol by yeasts. Adv Biochem Eng
6:77–103
Sakai K, Hamada N, Watanabe Y (1985) Purification and properties of
secondary alcohol oxidase with an acidic isoelectric point. Agric
Biol Chem 49:817–825
Sakai Y, Tani Y (1992) Cloning and sequencing of the alcohol oxidase-
encoding gene (AOD1) from the formaldehyde-producing
asporogeneous methylotrophic yeast, Candida boidinii S2. Gene
114:67–63
Sakai K, Hamada N, Watanabe Y (1986) Degradation mechanism of
poly(vinyl alcohol) by successive reactions of secondary alcohol
oxidase and β-diketone hydrolase from Pseudomonas sp. Agric
Biol Chem 50:989–996
Sampson NS, Vrielink A (2003) Cholesterol oxidases: a study of
nature’s approach to protein design. Acc Chem Res 36:713–
722
Saparrata MC, Guillen F (2005) Ligninolytic ability and potential
biotechnology applications of the South American fungus
Pleurotus laciniatocrenatus. Folia Microbiol (Praha) 50:155–160
Sarma AK, Vatsyayan P, Goswami P, Minteer SD (2009) Recent
advances in material sciences for developing enzyme electrodes.
Biosens Bioelectron 24:2313–22
Savitha J, Ratledge C (1991) Alcohol oxidase of Aspergillus flavipes
grown on hexadecanol. FEMS Microbiol Lett 80:221–224
Saxena U, Chakraborty M, Goswami P (2011a) Covalent immobiliza-
tion of cholesterol oxidase on self-assembled gold nanoparticles
for highly sensitive amperometric detection of cholesterol in real
samples. Biosens Bioelectron 26:3037–3043
Saxena U, Das M, Ahmed S, Barbora L, Borthakur M, Verma A, Bora
U, Goswami P (2011b) Multiwalled carbon nanotube-based en-
zyme electrode for total cholesterol estimation in human serum. J
Exper Nanosci 6:84–95
Segers G, Bradshaw N, Archer D, Blissett K, Oliver RP (2001) Alco-
hol oxidase is a novel pathogenic factor for Cladosporium fulvum,
but aldehyde dehyrogenase is dispensible. Mol Plant Microbe In
13:367–77
Shimao M, Nishimura Y, Kato N, Sakazawa C (1985) Location of
polyvinyl alcohol oxidase produced by a bacterial symbiont,
Pseudomonas sp. strain VM15C. Appl Environ Microbiol
49:8–10
Shimao M, Tsuda T, Takahashi M, Kato N, Sakazawa C (1983)
Purification of membrane-bound polyvinyl alcohol oxidase in
Pseudomonas sp VM15C. FEMS Microbiol Lett 20:429–433
Shkotova LV, Soldatkin AP, Gonchar MV, Schuhmann W, Dzyadevych
SV (2006) Amperometric biosensor for ethanol detection based
on alcohol oxidase immobilized within electrochemically depos-
ited resydrol film. Mat Sci Eng C–Biomim Supramol Sys 26:411–
414
Silva-Jiménez H, Zazueta-Novoa V, Durón-Castellanos A, Rodríguez-
Robelo C, Leal-Morales CA, Zazueta-Sandoval R (2009) Intra-
cellular distribution of fatty alcohol oxidase activity in Mucor
circinelloides YR-1 isolated from petroleum contaminated soils.
Antonie Van Leeuwenhoek 96:527–535
Silva-Jiménez H, Zazueta-Sandoval R (2005) Intracellular fate of hy-
drocarbons: possible existence of specific compartments for their
biodegradation. Appl Microbiol Biotechnol 121–124:205–217
Slabas AR, Elborough K, Vanhanen S, West M, Cheng Q, Lindner N,
Casey J, Sanglard D (1999) International patent application WO
99/47685
Smidt OD, Preez JCD, Albertyn J (2008) The alcohol dehydrogenases
of Saccharomyces cerevisiae: a comprehensive review. FEMS
Yeast Res 8:967–978

Smith AG, Brooks CJ (1975) Studies of the substrate specificity of
cholesterol oxidase from Nocardia erythropolis in the oxidation
of 3-hydroxy steroids. Biochem Soc Trans 3:675–677
Smutok O, Ngounou B, Pavlishko H, Gayada G, Gonchar M,
Schuhmann W (2006) A reagentless bienzyme amperometric
biosensor on alcohol oxidase/peroxidase and an Os-complex
modifies electrodeposition paint. Sens Actuators B 113:590–598
Sojo M, Bru R, Lopez-Molina D, Garcia-Carmona F, Argüelles J
(1997) Cell-linked and extracellular cholesterol oxidase activities
from Rhodococcus erythropolis. Isolation and physiological char-
acterization. Appl Microbiol Biotechnol 47:583–589
Solanki PR, Arya SK, Nishimura Y, Iwamoto M, Malhotra BD (2007)
Cholesterol biosensor based on amino-undecanethiol self-
assembled monolayer using surface plasmon resonance technique.
Langmuir 23:7398–7403
Soldevila AI, Ghabrial SA (2001) A novel alcohol oxidase/RNA-
binding protein with affinity for mycovirus double-stranded
RNA from the filamentous fungus Helminthosporium
(Cochliobolus) victoriae: molecular and functional characteriza-
tion. J Biol Chem 276:4652–4661
Srisuk N, Limtong S, Yurimoto H, Sakai Y, Kato N (2006) Physiolog-
ical study and alcohol oxidase gene(s) of thermotolerant
methylotrophic yeasts isolated in Thailand. Kasetsart J (Nat Sci)
40:121–135
Stasyk OV, Stasyk OG, Komduur J, Veenhuis M, Cregg JM, Sibirny
AA (2004) A hexose transporter homologue controls glucose
repression in the methylotrophic yeast Hansenula polymorpha. J
Biol Chem 279:8116–8125
Suye S (1997) Purification and properties of alcohol oxidase from
Candida methanosorbosa M-2003. Curr Microbiol 34:374–377
Suzuki T (1978) Oxidation of secondary alcohol by polyvinyl alcohol-
degrading enzyme produced by Pseudomonas. Agric Biol Chem
42:1187–1194
Suzuki T (1976) Purification and some properties of polyvinyl alcohol
degrading enzyme produced by Pseudomonas. Agric Biol Chem
40:497–504
Tabatabaei Yazdi M, Zahraei M, Aghaepour K, Kamranpour N (2001)
Purification and partial characterization of a cholesterol oxidase
from Streptomyces fradiae. Enz Microb Technol 8:410–414
Tamboli DP, Telke AA, Dawkar VV, Jadhav SB, Govindwar SP (2011)
Purification and characterization of bacterial aryl alcohol oxidase
from Sphingobacterium sp. ATM and its uses in textile dye
decolorization. Biotechnol Biopro Engineer 16:661–668
Tan X, Li M, Cai P, Luo L, Zou X (2005) An amperometric cholesterol
biosensor based on multiwalled carbon nanotubes and organically
modified sol–gel/chitosan hybrid composite film. Anal Biochem
337:111–120
Tani Y, Miya T, Ogata K (1972) The microbial metabolism of metha-
nol. Agric Biol Chem 36:68–75
Thomsen R, Christensen MH (2006) MolDock: a new technique for
high-accuracy molecular docking. J Med Chem 49:3315–3321
Tomioka H, Kagawa M, Nakamura S (1976) Some enzymatic proper-
ties of 3β-hydroxysteroid oxidase produced by Streptomyces
violascens. J Biochem 79:903–915
Turfitt GE (1944a) Microbiological agencies in the degradation of
steroids: 1. The cholesterol-decomposing organisms of soils. J
Bacteriol 47:487–493
Turfitt GE (1944b) The microbiological degradation of steroids: 2.
Oxidation of cholesterol by Proactinomyces sps. Biochem J
38:492–496
Unden G, Bongaerts J (1997) Alternative respiratory pathways of
Escherichia coli: energetics and transcriptional regulation in re-
sponse to electron acceptors. Biochim Biophys Acta 1320:217–234
Uwajima T, Yagi H, Nakamura S, Terada O (1974) Properties of
crystalline 3β-hydroxysteroid oxidase of Brevibacterium
sterolicum. Agric Biol Chem 38:1149–1156
Appl Microbiol Biotechnol
van den Heuvel RHH, Fraaije MW, Laane C, van Berkel WJ (2001a)
Enzymatic synthesis of vanillin. J Agric Food Chem 49:2954–
2958
van den Heuvel RHH, Fraaije MW, Mattevi A, Laane C, van Berkel
WJH (2001b) Vanillyl-alcohol oxidase, a tasteful biocatalyst. J
Mol Catal B: Enzym 11:185–188
van den Heuvel RHH, van den Berg WAM, Rovida S, van Berkel WJH
(2004) Laboratory-evolved vanillyl-alcohol oxidase produces nat-
ural vanillin. J Biol Chem 279:33492–33500
van der Klei IJ, Bystryck LV, Harder W (1990) Alcohol oxidase from
Hansenula polymorpha CBS 4732. Methods Enzymol 188:420–
427
van der Klei IJ, Harder W, Veenhuis M (1991) Biosynthesis and
assembly of alcohol oxidase, a peroxisomal matrix protein in
methylotrophic yeasts: a review. Yeast 7:195–209
Vanhanen S, West M, Kroon JTM, Lindner N, Casey J, Cheng Q,
Elborough KM, Slabas AR (2000) A consensus sequence for
long-chain fatty-acid alcohol oxidases from Candida iden-
tifies a family of genes involved in lipid omega-oxidation
in yeast with homologues in plants and bacteria. J Biol Chem
275:4445–4452
Vatsyayan P, Kumar AK, Goswami P, Goswami P (2008) Broad
substrate Cytochrome-P450-monooxygenase activity in the cells
of Aspergillus terreus MTCC 6324. Biores technol 99:68–75
Varela E, Bockle B, Romero A, Martinez AT, Martinez MJ (2000a)
Biochemical characterization, cDNA cloning and protein crystal-
lization of aryl-alcohol oxidase from Pleurotus pulmonarius.
Biochim Biophys Acta 1476:129–138
Varela E, Jesús Martínez M, Martínez AT (2000b) Aryl-alcohol oxi-
dase protein sequence: a comparison with glucose oxidase and
other FAD oxidoreductases. Biochim Biophys Acta 1481:202–
208
Varela E, Guillen A, Martinez AT, Martinez MJ (2001) Expression of
Pleurotus eryngii aryl alcohol oxidase in Aspergillus nidulans:
purification and characterization of the recombinant enzyme.
Biochim Biophys Acta 1546:107–113
Veith M, Dettner K, Boland W (1996) Stereochemistry of an alcohol
oxidase from the defensive secretion of larvae of the leaf beetle
Phaedon armoraciae (Coleoptera: Chrysomelidae). Tetrahedron
52:6601–6612
Veith M, Oldham NJ, Dettner K, Pasteels JM, Boland W (1997)
Biosynthesis of defensive allomones in leaf beetle larvae: stereo-
chemistry of salicylalcohol oxidation in Phratora vitellinae and
comparison of enzyme substrate and stereospecificity with alco-
hol oxidases from several iridoid producing leaf beetles. J Chem
Ecol 23:429–443
Virk S, Baruah V, Goswami P (2012) Giant vesicles as encapsulating
matrix for stabilizing alcoholoxidase and as container for coupled
enzymatic reactions. Artif Cells Blood Substit Biotechnol.
doi:10.3109/10731199.2012.731413
Vojinovic V, Azevedo AM, Martins VCB, Cabral JMS, Gibson TD,
Fonseca LP (2004) Assay of H2O2 by HRP catalysed co-oxidation
of phenol-4-sulphonic acid and 4-aminoantipyrine: characterisa-
tion and optimisation. J Mol Catal B: Enzym 28:129–35
Wang S, Li S, Yu Y (2004) Immobilization of cholesterol oxidase on
cellulose acetate membrane for free cholesterol biosensor devel-
opment. Artif Cells Blood Substit Immobil Biotechnol 32:413–
425
Watanabe Y, Hamada N, Morita M, Tsujisaka Y (1976) Purification
and properties of a polyvinyl alcohol-degrading enzyme produced
by a strain of Pseudomonas. Arch Biochem Biophys 174:575–
581
Woodward JR (1990) Biochemistry and applications of alcohol oxi-
dase from methylotrophic yeasts. In: Codd GA, Dijkhuizen L,
Tabita FR, Hoff DMN (eds) Advances in autotrophic microbiol-
ogy and one-carbon metabolism. Springer, Leeds, pp 205–238
Appl Microbiol Biotechnol
Wu XJ, Choi MM (2003) Hydrogel network entrapping cholesterol
oxidase and octadecylsilica for optical biosensing in hydrophobic
organic or aqueous micelle solvents. Anal Chem 75:4019–4027
Yamada H, Shin KC, Kato N, Shimizu S, Tani Y (1979) Purification
and characterization of alcohol oxidase from Candida 25-A. Agric
Biol Chem 43:877–878
Yamatsu A, Matsumi R, Atomi H, Imanaka T (2006) Isolation and
characterization of a novel poly(vinyl alcohol)-degrading bacterium,
Sphingopyxis sp. PVA3. Appl Microbiol Biotechnol 72:804–811
Yang M, Yang Y, Liu Y, Shen G, Yu R (2006) Platinum nanoparticles-
doped sol–gel/carbon nanotubes composite electrochemical sen-
sors and biosensors. Biosens Bioelectron 21:1125–1131
Yue K, Kass IJ, Sampson N, Vrielink A (1999) Crystal structure
determination of cholesterol oxidase from Streptomyces and
structural characterization of key active site mutants. Biochemis-
try 38:4277–4286
Zhan BZ, Thompson A (2004) Recent developments in the aerobic
oxidation of alcohols. Tetrahedron 60:2917–2975
Zhang Y, Li Y, Shen W, Liu D, Chen J (2006) A new strain,
Streptomyces venezuelae GY1, producing a poly(vinyl alco-
hol)-degrading enzyme. World J Microbiol Biotechnol 22:625–
628
Zhao S, Lin Z, Ma W, Luo D, Cheng Q (2008) Cloning and charac-
terization of long-chain fatty alcohol oxidase LjFAO1 in Lotus
japonicas. Biotechnol Prog 24:773–779
Zlateva T, Boteva R, Filippi B, Veenhuis M, van der Klei IJ (2001)
Deflavination of flavo-oxidases by nucleophilic reagents.
Biochim Biophys Acta 1548:213–219