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PRBI-9196; No. of Pages 8 ARTICLE IN PRESS

Process Biochemistry xxx (2011) xxx–xxx

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Induction of NAD+ dependent alcohol dehydrogenases with activity towards long

chain aliphatic alcohols in mesophilic, thermophilic and extreme thermophilic
microorganisms
Lorena Álvarez ∗ , Fernando Acevedo, Andrés Illanes
School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Joint Doctoral Program on Biotechnology PUCV-UTFSM, Av. Brasil 2147, PO Box 4059, Valparaiso,
Chile

a r t i c l e i n f o a b s t r a c t

Article history: The production of alcohol dehydrogenases with the capacity of oxidizing long chain aliphatic alcohols
Received 2 December 2010 by mesophilic, thermophilic and hyperthermophilic strains induced by aliphatic alcohols and alkanes
Received in revised form 22 February 2011 with 2–24 carbon atoms has been studied. Seven strains were tested: the mesophilic yeast Candida trop-
Accepted 8 March 2011
icalis ATCC20336, the thermophilic bacterium Thermus AB1and five hyperthermophilic bacterial strains:
Thermus thermophilus HB27, HNI11, NR17, PRQ16 and PRQ25. Only C. tropicalis ATCC20336, Thermus AB1
Keywords:
and T. thermophilus PRQ25 produced alcohol dehydrogenases with the capacity of oxidizing aliphatic
Long chain fatty acids
alcohols with 22 and 24 carbon atoms, after induction with 1-hexacosanol, 1-docosanol and 1-eicosanol,
Polycosanols
Alcohol dehydrogenase
respectively. Higher initial reaction rates of oxidation of docosanol and eicosanol were obtained with the
Thermus enzyme from C. tropicalis ATCC20336, with values of 196.9 and 218.8 mmol NADH min−1 g of protein−1 ,
Thermus thermophilus respectively.
Behenic acid © 2011 Elsevier Ltd. All rights reserved.
Lignoceric acid

1. Introduction
The long chain fatty acids, behenic acid (C22 H44 O2 ) and ligno-
ceric acid (C24 H48 O2 ), are valuable products in pharmaceutical [1,2]
and health-care preparations [3,4] and as additives in fuels [5] and
foods [6]. Extraction from natural products is not an option since
these fatty acids are extremely scarce in nature: behenic acid is
found at levels of 1–5% in peanut skin and 0.5% in canola, sesame
and olive seeds, while lignoceric acid is found at even lower levels
in some vegetable oils [7]. Chemical oxidation of polycosanols has
been studied as one technological option for producing long chain
fatty acids. The chemical oxidation of octadecanol and eicosanol
to the corresponding stearic and arachidic acids has been studied
using quaternary ammonium peroxotungstophosphate as catalysts
in the first case [8], and an ionic liquid composed by Aliquat and
polyperoxometalate in the second [9].
As an alternative to chemical catalysis, enzymatic oxidation
stems as an interesting option. In fact, enzyme E.C.1.1.1.192 (long-
chain fatty acids alcohol dehydrogenase) is reported to be active
on the oxidation of hexadecanol to palmitic acid. This enzyme
is an oxidoreductase requiring the stoichiometric cofactor NAD+
which is reduced to NADH+ H+ , two moles of NAD+ being required
∗ Corresponding author. Tel.: +56 32 2273642; fax: +56 32 2273803.
E-mail address: lorena.alvarez.a@mail.pucv.cl (L. Álvarez).
per mole of acid produced [10]. At present, the alcohol dehy-
drogenases produced by Candida tropicalis and Candida lipolytica
are the only commercially available enzymes capable of oxi-
dizing aliphatic alcohols up to 16 carbon atoms. Horse liver
alcohol dehydrogenase has been reported to oxidize docosanol and
tetracosanol, although at lower rates than small chain aliphatic
alcohols [11].
Some induction studies have been conducted on enzyme pro-
duction related to v- and b-oxidation reactions with n-alkanes
as sole carbon source. Alkanes are first hydroxylated by the
cytochrome P450 system and NADPH-cytochrome P450 reductase
system, which is located in the endoplasmic reticulum (micro-
some), to the corresponding long chain aliphatic alcohols. These
alcohols are not only degraded via b-oxidation but also utilized
for the biosynthesis of lipids in mitochondria and endoplasmic
reticulum, after dehydrogenation by long-chain fatty acids alcohol
dehydrogenase, aldehyde dehydrogenase and possibly an alcohol
oxidase [12–14].
Some yeast strains belonging to the genus Candida produce v
and b diacids when cultured in n-alkanes or fatty acids as sole
carbon source. Alcohol dehydrogenases were induced capable of
oxidizing aliphatic alcohols from 8 to 16 carbon atoms, when a
mixture of n-alkanes from 10 to 13 carbon atoms was used as sole
carbon source in the cultivation of C. tropicalis ATCC 20336 and
C. lipolytica NRRL Y-6795. The highest activity was obtained using
tetradecanol as substrate of the enzymatic reaction. The enzymes

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doi:10.1016/j.procbio.2011.03.002

Please cite this article in press as: Álvarez L, et al. Induction of NAD+ dependent alcohol dehydrogenases with activity towards
long chain aliphatic alcohols in mesophilic, thermophilic and extreme thermophilic microorganisms. Process Biochem (2011),
doi:10.1016/j.procbio.2011.03.002
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Fig. 1. Effect of addition of inducer alcohols on cell growth. (a) Thermus thermophilus HB27, (b) Thermus thermophilus HN1.11, (c) Thermus thermophilus NR17, (d) Thermus
thermophilus PRQ16, (e) Thermus thermophilus PRQ25, (f) Thermus AB1 and (g) Candida tropicalis ATCC 20336. Induction was done by adding 0.025, 0.05, 0.5, 1 and 2% of
primary aliphatic alcohols ethanol (C2 ), 1-tetradecanol (C14 ), 1-hexadecanol (C16 ), 1-eicosanol (C20 ), 1-docosanol (C22 ) and 1-tetracosanol (C24 ) to the culture medium and
fermentations were conducted for 96 h. As controls, strains were grown at the same conditions without the addition inducer. Each bar represents an average of the two
experiments with its standard deviation.

from both strains were NAD+ dependent and localized in the micro-
some, mitochondria and peroxisome, together with other enzymes
like aldehyde dehydrogenase acting on long-chain aldehydes, acyl
CoA synthetase and those involved in fatty acid b-oxidation, indi-
cating that all of them are involved in fatty acid degradation [10,12].
Different NAD(P)+ dependent alcohol dehydrogenases have been
also produced using alcohols as sole carbon and energy source
[15–17].
Several studies on the characterization of alcohol dehydroge-
nases from thermophilic and hyperthermophilic strains have been
conducted in recent years. More than 20 thermophilic archae and
17 thermophilic bacteria have been identified as alcohol dehydro-

Please cite this article in press as: Álvarez L, et al. Induction of NAD+ dependent alcohol dehydrogenases with activity towards
long chain aliphatic alcohols in mesophilic, thermophilic and extreme thermophilic microorganisms. Process Biochem (2011),
doi:10.1016/j.procbio.2011.03.002
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Fig. 2. Effect of addition of inducer alkanes on cell growth. (a) Thermus thermophilus HB27, (b) Thermus thermophilus HN1.11, (c) Thermus thermophilus NR17, (d) Thermus
thermophilus PRQ16, (e) Thermus thermophilus PRQ25, (f) Thermus AB1 and (g) Candida tropicalis ATCC 20336. Induction was done by adding 0.025, 0.05, 0.5, 1 and 2% of
alkanes n-tetradecane (C14 ), n-heptadecane (C17 ), n-octadecane (C18 ), n-docosane (C22 ) and n-tetracosane (C24 ) to the culture medium and fermentations were conducted for
96 h. As controls, strains were grown at the same conditions without the addition inducer. Each bar represents an average of the two experiments with its standard deviation.

genase producers. Thermophilic organisms have been isolated from
natural habitats like hydrothermal vents, marine volcanic sedi-
ments, hot springs and also from petroleum reservoirs, digesters
and leach piles. Most thermophilic organisms produce various
types of dehydrogenases [18,19] whose production can be opti-
mized by using different strategies of microbial cultivation [20].
The objective of this work was to develop a strategy for
the production of microbial alcohol dehydrogenases by fer-
mentation with the capacity to oxidize long-chain aliphatic
alcohols by induction with primary aliphatic alcohols from
2 to 24 carbon atoms and alkanes from 14 to 24 carbon
atoms.

Please cite this article in press as: Álvarez L, et al. Induction of NAD+ dependent alcohol dehydrogenases with activity towards
long chain aliphatic alcohols in mesophilic, thermophilic and extreme thermophilic microorganisms. Process Biochem (2011),
doi:10.1016/j.procbio.2011.03.002
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Table 1
Effect of addition of alcohol inducers on the production of alcohol dehydrogenases with activity towards aliphatic alcohols. Induction was done by adding 0.05% of aliphatic
alcohols to each culture medium. Batch fermentations were conducted for 96 h. Enzyme activity (initial rate of oxidation) was determined at 30 ◦ C, 50 ◦ C and 70 ◦ C for the
alcohol dehydrogenases produced by Candida tropicalis, Thermus AB1 and Thermus thermophilus, respectively.

Strain Alcohol inducers Substrate of enzymatic reaction

Enzyme activity (mmol min−1 g of protein−1 )

Ethanol Octanol Dodecanol Tetradecanol Hexadecanol Docosanol Tetracosanol

Thermus thermophilus HB27 None 24.8 0 0 0 0 0 0

C2 1.1 0 0 0 0 1.6 7.7
C14 0 0 0 0 0 0 0
C16 0 0 5.8 0 0 0 0
C20 0 0 0 0 0 5.9 16.8
C22 0 7.5 0 0 0 8.8 0
C24 0 13.3 0 0 4.7 6.2 7.9
Thermus thermophilus HN1.11 None 27.4 0 0 0 0 0 0
C2 18.9 12.6 0 0 0 0 0
C14 0 0 0 0 0 0 0
C16 0 0 0 0 0 0 0
C20 0 0 0 0 0 0 0
C22 0 0 0 0 0 13.4 15.1
C24 0 0 0 0 17.9 17.8 19.3
Thermus thermophilus NR17 None 5.8 1.4 0 0 0 0 0
C2 5.4 0 0 0 0 0 0
C14 0 0 0 0 0 0 0
C16 0 0 0 0 0 0 0
C20 0 0 0 0 1.3 10.5 10.8
C22 0 0 0 0 5.8 7.8 7.7
C24 0 0 0 0 7.2 8.8 0
Thermus thermophilus PRQ16 None 9.3 9.2 10.1 0 11.8 0 0
C2 176.6 60.2 87.4 19.0 46.8 0 0
C14 0 0 0 0 0 0 0
C16 62.6 8.5 12.9 33.5 70.1 0 0
C20 6.5 4.8 1.6 1.1 16.1 16.4 10.4
C22 8.2 0 0 2.6 1.1 9.4 4.5
C24 7.0 0 0 0 7.6 8.8 9.7
Thermus thermophilus PRQ25 None 10.6 0 0 0 0 0 0
C2 5.0 0 0 0 0 0 0
C14 0 0 0 0 0 0 0
C16 0 0 64.9 0 0 0 0
C20 9.2 0 0 15.0 29.2 27.4 36.4
C22 0 0 0 11.6 0 24.1 15.7
C24 0 0 0 8.9 31.0 20.9 21.6
Thermus AB1 None 6.9 5.8 0 0 0 0 0
C2 14.7 13.2 2.3 0 0 4.4 7.7
C14 0 0 0 0 0 0 0
C16 0 0 0 0 0 0 0
C20 4.5 4.0 4.8 0 0 3.8 6.9
C22 0 1.0 1.9 1.9 1.0 20.4 42.9
C24 0 6.4 21.2 15.7 17.2 15.5 25.8
Candida tropicalis ATCC 20336 None 7.6 0 0 0 0 0 0
C2 14.4 12.9 0 0 0 0 0
C14 5.2 4.4 3.6 0 0 0 0
C16 54.0 69.8 65.1 86.7 145.9 196.9 218.8
C20 0 0 0 0 0 0 0
C22 0 0 0 5.3 6.1 5.4 6.4
C24 0,5 0 0 0 0 4,2 5,0

2. Materials and methods Madrid, Madrid, Spain). Mesophilic yeast C. tropicalis (ATCC20336) was provided by
ATCC.
2.1. Materials

2.1.1. Chemicals
Nicotinamide adenine dinucleotide (NAD+ ) was purchased from Jülich Fine
Chemicals. Diethylene glycol, bis(2-methoxyethyl) ether (diglyme), benzenecar-
boximidamide (benzamidine), bovine serum albumin (BSA), Coomassie blue
G-250, 1-octanol, 1-tetradecanol, 1-hexadecanol, n-tetradecane, n-heptadecane, n-
octadecane, n-dodecane and n-tetradecane were purchased from Sigma–Aldrich
Chem. Co. Tryptone, yeast extract, ethanol, 1-dodecanol and 1-eicosanol were pur-
chased from Merck, docosanol was purchased from Sasol Germany GmbH and
tetracosanol was purchased from ABCR GmbH & CoKG. All other reagents were of
analytical grade.
2.1.2. Microorganisms
Thermus thermophilus HB27 (ATCC BA-163), HN1.11, NR17, PRQ16, PRQ25, and
Thermus AB1 strain were kindly provided by Drs. José Berenguer and Daniel Vega
(Universidad Autónoma de Madrid). These strains belong to the culture collec-
tion of the Centro de Biología Molecular Severo Ochoa (Universidad Autónoma de
2.2. Methods
2.2.1. Culture media and conditions of cultivation
Inocula were prepared for all microorganisms in the same cultivation media
described below, but in the case of C. tropicalis no Tween was added. Cells were
harvested in mid-exponential phase and each cultivation flask was inoculated with
20 ml of cell suspensions containing 0.3 mg cell dry weigh/ml.
All microorganisms were cultivated in 500 ml shake flasks containing 200 ml
of medium on a rotary shaker at 200 rpm. T. thermophilus HB27, HN1.11, NR17,
PRQ16, PRQ25, and Thermus AB1 strain were cultivated on Thermus culture
medium consisting of: 8 g/l tryptone, 4 g/l yeast extract, 3 g/l NaCl, 0.59 g/l
KH2 PO4 , 2.55 g/l (NH4 )2 SO4 , 0.23 g/l MgSO4 ·7H2 O, 0.18 g/l CaCl2 ·2H2 O, 0.046 g/l
FeSO4 ·7H2 O, 0.019 g/l MnCl2 ·4H2 O, 0.032 g/l ZnSO4 ·7H2 O, 0.005 g/l CoCl2 ·6H2 O,
0.0035 g/l CuSO4 ·5H2 O in distilled water at pH 7.5. Temperature was 70 ◦ C for HB27,
HN1.11, NR17, PRQ16 and PRQ25, and 50 ◦ C for AB1.

Please cite this article in press as: Álvarez L, et al. Induction of NAD+ dependent alcohol dehydrogenases with activity towards
long chain aliphatic alcohols in mesophilic, thermophilic and extreme thermophilic microorganisms. Process Biochem (2011),
doi:10.1016/j.procbio.2011.03.002
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Table 2
Effect of the addition of alkane inducers on the production of alcohol dehydrogenases with activity towards aliphatic alcohols. Induction was done by adding 0.05% of
n-alkanes to each culture medium. Batch fermentations were conducted for 96 h. Enzyme activity (initial rate of oxidation) was determined at 30 ◦ C, 50 ◦ C and 70 ◦ C for the
alcohol dehydrogenases produced by Candida tropicalis, Thermus AB1 and Thermus thermophilus respectively.

Strain Alkane inducers Substrate of enzymatic reaction

Enzyme activity (mmol min−1 g of protein−1 )
Ethanol Octanol Dodecanol Tetradecanol Hexadecanol Docosanol Tetracosanol

Thermus None 24.8 0 0 0 0 0 0

thermophilus HB27 C14 3.4 0 0 0 0 0 0
C17 0 0 0 0 0 9.9 14.0
C18 4.6 0 0 0 0 3.2 12.8
C22 3.8 0 0 0 0 8.9 14.5
C24 3.3 0 0 0 7.6 7.9 18.1
Thermus thermophilus HN1.11 None 27.4 0 0 0 0 0 0
C14 0 0 0 0 0 0 0
C17 0 0 0 0 0 0 0
C18 22.2 0 0 0 15.8 18.8 16.3
C22 0 0 0 0 0 21.4 20.1
C24 0 0 0 0 0 19.7 21.0
Thermus thermophilus NR17 None 5.8 1.4 0 0 0 0 0
C14 10.8 9.5 0 0 0 1.5 2.3
C17 8.1 3.9 0 0 0 4.1 5.4
C18 6.5 1.7 1.4 0 0 5.2 5.0
C22 2.0 0 0 0 0 7.0 5.5
C24 0 0 0 0 2.4 5.9 7.1
Thermus thermophilus PRQ16 None 9.3 9.2 10.1 0 11.8 0 0
C14 0 4.4 0 0 0 9.0 9.1
C17 0 0 0 0 0 9.8 9.8
C18 0 8.4 0 0 6.1 10.5 11.1
C22 0 0 0 0 0 10.8 15.6
C24 6.9 9.7 0 0 0 9.7 15.5
Thermus thermophilus PRQ25 None 10.6 0 0 0 0 0 0
C14 12.7 0 0 0 0 25.2 8.1
C17 2.0 0 0 0 0 26.8 31.3
C18 1.5 0 0 0 0 18.3 22.2
C22 1.6 0 0 0 0 15.7 32.4
C24 2.7 0 0 0 0 27.2 30.1
Thermus AB1 None 6.9 5.8 0 0 0 0
C14 0 16.6 2.3 11.1 19.2 0 0
C17 0 0 0 0 0 2.2 12.5
C18 5.0 0 0 0 0 8.5 11.3
C22 3.0 0 0 0 0 29.2 16.8
C24 0 2.9 2.0 0 14.9 16.2 18.7
Candida tropicalis ATCC 20336 None 7.6 0 0 0 0 0 0
C14 0.9 1.3 0 0 0 0 0
C17 5.8 0 0 0 0 0 0
C18 0 0 0 0 0 0 0
C22 0 0 0 0 0 0 0
C24 0 0 0 0 0 0 0

C. tropicalis ATCC20336 was cultivated on a minimum medium which con-
tained: 6.4 g/l glycerol, 1.6 g/l (NH4 )2 SO4 , 13.9 g/l KH2 PO4 , 2 g/l Na2 HPO4 , 1.25 g/l
MgSO4 ·7H2 O, 0.12 g/l CaCl2 ·2H2 O, 0.09 g/l FeSO4 ·7H2 O/l, 0.02 g/l MnCl2 ·4H2 O,
0.03 g/l ZnSO4 ·7H2 O, 0.03 g/l CoCl2 , 0.003 g/l CuSO4 ·5H2 O, 0.04 mg/l folic acid,
0.4 mg/l pantothenic acid, 0.01 mg/l biotin, 1 mg/l niacin, 0.1 mg/l tiamin B1 and 0.5%
(v/v) de Tween 80 in distilled water at pH 6.0. Temperature was 30 ◦ C and Tween 80
was added to increase substrate solubility. Inducers were added to each cultivation
medium as described in Section 2.2.2.
2.2.2. Conditions of induction
Induction was done by adding 0.025, 0.05, 0.5, 1 and 2% of primary
aliphatic alcohols or alkanes to each culture medium. Ethanol, 1-tetradecanol
(C14 H30 O), 1-hexadecanol (C16 H34 O), 1-eicosanol (C20 H42 O), 1-docosanol (C22 H46 O),
1-tetracosanol (C24 H50 O) and alkanes n-tetradecane (C14 H30 ), n-heptadecane
(C17 H35 ), n-octadecane (C18 H38 ), n-docosane (C22 H46 ) and n-tetracosane (C24 H50 )
were used as inducers. As control experiments, strains were grown at the same
conditions and in the same media without the addition of inducer.
2.2.3. Time of addition of inducers on the production of alcohol dehydrogenases
The effect of the time of addition of inducer and harvest time was studied in all
strains, for determining the conditions at which they produced higher enzyme activ-
ities over tetracosanol and docosanol. Induced cells (adapted inoculum) were used as
inoculums in these studies. Batch fermentations were conducted for 180 h in 500 ml
shake flasks with 200 ml culture medium, at 70 ◦ C for T. thermophilus HB27, HN1.11,
NR17, PRQ16 and PRQ25 and 50 ◦ C for Thermus AB1, on a rotary shaker at 200 rpm,
and at 30 ◦ C for C. tropicalis ATCC20336 on a rotary shaker at 180 rpm. Inducers at
0.05% (w/v) concentration were added at the beginning of the fermentation and
during mid-exponential growth phase (after about 12 h of cultivation). Addition of
time zero may fully exploit the effect of the inducer but an early addition might
produce inhibition on cell growth, so we chose also the addition at mid exponen-
tial phase were the culture is growing vigorously. Samples were taken throughout
the fermentation and enzyme produced was determined evaluating its activity over
docosanol and tetracosanol.
2.2.4. Preparation of crude enzyme extracts
Cell growth was monitored by measuring the turbidity at 650 nm. Cells were
harvested during the stationary phase of growth and after freezing at −18 ◦ C, they
were resuspended in a 1/1 volume of 10 mM sodium phosphate buffer pH 7.5 with
10 mM NaCl and 3 mM benzamidine, and disrupted by mechanical abrasion with
alumina powder (0.5 g/ml). The soluble fraction obtained after removal of cell debris
by centrifugation (15,000 × g, 15 min, 4 ◦ C) was used as source of the alcohol dehy-
drogenases.
2.2.5. Protein determination
Protein was determined by the method of Bradford [21] using Coomassie blue
G-250 dye as reagent and bovine serum albumin (BSA) as standard, by measuring the
absorbance at 595 nm at 25 ◦ C in a Jenway Spectrophotometer (model 6715 UV/VIS).
2.2.6. Determination enzyme activity with aliphatic alcohols
Alcohol dehydrogenase activity of the different crude extracts was ana-
lyzed by the increase in absorbance at 340 nm corresponding to the formation
of NADH concomitant to the oxidation of the aliphatic alcohols. A sample of
enzyme preparation (50–200 ml) was added to a spectrophotometer cell with
2 ml of 0.2 mM substrates (ethanol, octanol, dodecanol, tetradecanol, hexadecanol,
docosanol and tetracosanol) in 100 mM phosphate buffer pH 7.0 with 0.6% diglyme
and 100 ml of 100 mM NAD+ . Initial rates of oxidation of aliphatic alcohols were

Please cite this article in press as: Álvarez L, et al. Induction of NAD+ dependent alcohol dehydrogenases with activity towards
long chain aliphatic alcohols in mesophilic, thermophilic and extreme thermophilic microorganisms. Process Biochem (2011),
doi:10.1016/j.procbio.2011.03.002
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PRBI-9196; No. of Pages 8 ARTICLE IN PRESS
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expressed in mmol NADH min−1 g−1 of protein and mmol NADH min−1 g−1 of cell dry
weight.
2.2.7. Data analysis
Experiments for the selection of inducers and microorganisms producing alcohol
dehydrogenase with the highest activity of oxidation of docosanol and tetradecanol
were done in duplicate and samples assayed in duplicate. Initial rate of alcohol oxi-
dation, expressed in mmol NADH min−1 g−1 of cell dry weight, was used as selection
parameter. An analysis of variance of means (ANOVA) was conducted, the F-test
in ANOVA indicating whether there are significant differences amongst the means.
The software STATGRAPHICS PLUS 5.1 was used for analysis of significant (P) level
through F-test. Only P < 0.05 were accepted as significant factors, for a 95% confi-
dence level. Subsequently, to determine which means are significantly different,
Multiple Range Test was performed using the Fisher’s least significant difference
method (LSD).
3. Results and discussion
T. thermophilus HB27, HN1.11, NR17, PRQ16, PRQ25, Ther-
mus AB1 and C. tropicalis ATCC20336 were selected for having
previously determined as producers of alcohol dehydrogenases
capable of oxidizing aliphatic alcohols of more than 6 carbon
atoms. Thermophilic and hyperthermophilic microorganisms are
quite appealing since their enzymes withstand higher tempera-
tures allowing the use of higher substrate concentrations, because
of the dramatic increase in solubility with temperature. This is
quite important, as becomes apparent by simple consideration of
Michaelis–Menten kinetics, but also because the use of organic sol-
vents to dissolve the very hydrophobic substrates can be avoided.
Moreover, enzymes produced by such organisms are more stable
than mesophilic enzymes, so that the biocatalyst is more efficiently
used and also the risk of microbial contamination is reduced. All
those are significant aspects from a process perspective.
3.1. Effect of inducers on cell growth
Induction of alcohol dehydrogenases was done by adding alco-
hols or alkanes of different chain length to the culture medium.
Therefore, the effect of such supplements on cell growth was deter-
mined. Firstly each inducer was tested as the sole carbon source
in the fermentation medium and in all cases no cell growth was
observed.
The effect of alcohols and alkenes supplementation to the cul-
ture media is presented in Figs. 1 and 2, respectively. Strains
responded differently to the addition of the different inducers.
Using alcohols as inducers, cell growth was reduced with respect
to the respective controls (no inducer added) in all strains with the
exception of T. thermophilus PRQ 25, Thermus AB1 and C. tropicalis
ATCC 20336.
On the other hand, growth of T. thermophilus HB27, HN1.11 and
PRQ25 increased with all alkanes used as inducers (see Fig. 2). Cell
growth of HB27 was 37% higher than the control when supple-
mented with n-docosane, while cell growth of PRQ25 increased by
24, 23 and 32% when supplemented with n-octadecane, n-docosane
and n-tetracosane respectively. Similar results have been reported
when the effect of alkane chain length was studied on the cellular
growth of Geobacillus thermoleovorans B23; in this case, an increase
in the production of proteins related to b oxidation was observed,
whose magnitude correlated with the chain length of the alkane
[22].
With respect to the effect of the concentration of inducer,
statistical analysis was performed as described in Section 2.2.7.
According to this analysis, best results in terms of cell growth were
obtained in most cases at inducer concentrations from 0.05 to 1%,
with a significant reduction at concentrations over 1% (Figs. 1 and
2). However, tetradecanol inhibited the growth of the strains even
at concentrations of 0.025%, except in the case of T. thermophilus
HB27 and Thermus AB1. Similar results were obtained with 0.025%
Please cite this article in press as: Álvarez L, et al. Induction of NAD+ dependent alcohol dehydrogenases with activity towards
long chain aliphatic alcohols in mesophilic, thermophilic and extreme thermophilic microorganisms. Process Biochem (2011),
doi:10.1016/j.procbio.2011.03.002
1-hexadecanol that inhibited the growth of T. thermophilus HN1.11,
NR17, PRQ16 and Thermus AB1.
At concentrations higher than 0.5%, precipitation of the inducer
was observed in the culture medium at temperatures below 60 ◦ C,
which is the case for C. tropicalis. Ueda and Tanaka [10] reported
the use of 0.7% of a mixture of alkanes with 10–13 carbon atoms;
however, nutrients were hard to solubilize at 30 ◦ C, even with the
addition of Tween 80. On the hand, Kato et al. [22] supplemented
the growth medium with 0.1% (v/v) of pure alkanes, or mixtures
with 10–34 carbon atoms 70 ◦ C, cultivating the yeast for a period of
ten days. Inducer concentration of 0.05% (w/v) was selected as the
most appropriate for subsequent studies of production of alcohol
dehydrogenase.
3.2. Effect of inducers on the production of alcohol
dehydrogenases
Results of the effect of addition of alcohols as inducers on the
production of alcohol dehydrogenases with capacity of oxidizing
aliphatic alcohols are presented in Table 1 in terms of initial rates
of oxidation. Crude enzyme extracts were obtained after 96 h of fer-
mentation using 0.05% (w/v) of inducer (aliphatic alcohol or alkane)
in the culture medium. Enzyme extracts were obtained by cell dis-
ruption according to the methodology in Section 2.2.4.
Results of the activities of alcohol dehydrogenases, expressed
as initial rates of oxidation of ethanol, octanol, dodecanol, tetrade-
canol, docosanol and tetracosanol, induced with aliphatic alcohols
and alkanes are presented in Tables 1 and 2, respectively. As
seen in Table 1, higher activities were obtained over the aliphatic
alcohols 1-tetradecanol, 1-docosanol and 1-tetracosanol with the
alcohol dehydrogenases from C. tropicalis ATCC20336 induced with
hexadecanol, T. thermophilus PRQ25 induced with eicosanol and
Thermus AB1 induced with docosanol. On the other hand, as seen
in Table 2, higher activities over docosanol and tetracosanol were
obtained with alcohol dehydrogenases from T. thermophilus PRQ25
induced with n-heptadecane and n-tetradecane and from Thermus
AB1 induced with n-docosane.
These results show that, even though aliphatic alcohols have
some inhibitory effect on cell growth in non-adapted strains, they
are very good inducers of alcohol dehydrogenases with activity over
long-chain aliphatic alcohols.
Oxidation rate over tetracosanol obtained with the alcohol
dehydrogenase from C. tropicalis ATCC 20336 induced with 1-
hexadecanol (86.7 mmol NADH min−1 g−1 protein) was higher
than a value previously reported by Ueda and Tanaka (70 mmol
NADH min−1 g−1 protein) [10], where a mixture of alkanes with
10–13 carbon atoms was used as the sole carbon and energy source
[10]. Without induction, a basal level of activity was observed only
on ethanol, with the exception of Thermus AB1 where a basal activ-
ity over octanol was also observed and T. thermophilus PRQ16 who
departed from that pattern, expressing activity on ethanol, octanol,
dodecanol and hexadecanol.
There is some relationship between the length of the chain of
the inducer and the level of activity expressed on the substrate of
the corresponding chain length of the reaction in several strains
of Thermus; however, in the case of Candida this pattern is not
followed.
A statistic analysis was done, as described in Section 2.2.7,
to determine the best microorganisms and inducers in terms
of production of alcohol dehydrogenases with activity towards
long-chain aliphatic alcohols (docosanol and tetracosanol). Higher
oxidation rates were observed towards long chain aliphatic alco-
hols (docosanol and tetracosanol) with the alcohol dehydrogenase
from C. tropicalis ATCC20336 induced by 1-hexadecanol (C16 ), T.
thermophilus PRQ25 induced with 1-eicosanol (C20 ) and Thermus
AB1 induced with 1-docosanol (C22 ).
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Fig. 3. Effect of time of addition of inducer (docosanol) in the production of the
enzyme in Thermus AB1. Batch fermentations were conducted for 180 h in 500 ml
shake flasks with 200 ml culture medium at 50 ◦ C. Docosanol at 0.05% (w/v) was
added at (a) the beginning of fermentation and (b) during mid-exponential growth
phase (after about 12 h of cultivation). Cell samples were collected to measure the
enzyme activity on tetracosanol and docosanol as substrates. Biomass (N), docosanol
(d), tetracosanol (
), black arrow indicates the moment of addition of the inducer, obtained in all cases at 0.05% inducer concentration.
staple arrows point to the corresponding Y-axis. Each bar represents an average of
the two experiments with its standard deviation.
3.3. Effect of time of addition of inducers on the production of
alcohol dehydrogenases
The effectivity of induction may vary according to the moment
of addition of the inducer during fermentation; the inducers were
added at two different culture time, at the beginning of fermen-
tation and during mid-exponential growth phase (after about
12 h of cultivation). Thermus AB1was induced with docosanol,
T. thermophilus PRQ25 induced with eicosanol, and C. tropicalis
ATCC20336 with hexadecanol.
Results obtained with Thermus AB1 are presented in Fig. 3 show-
ing that the addition of the inducer at the beginning of fermentation
produced higher enzyme activities over tetracosanol and docosanol
than adding it during exponential growth. Higher values of initial
reaction rates over docosanol and tetracosanol were 1.00 ± 0.03 and
1.41 ± 0.02 mmol NADH min−1 g−1 cell, respectively, both obtained
after 120 h of fermentation. Similar results were obtained with
T. thermophilus PRQ25 and C. tropicalis ATCC20336, best results
being obtained with induction at the beginning of fermenta-
tion. Higher values of initial reaction rates over docosanol and
tetracosanol were obtained after 140 and 100 h of fermentation
for T. thermophilus PRQ25 and C. tropicalis ATCC20336, respec-
tively, being 1.32 ± 0.04 and 1.64 ± 0.03 mmol NADH min−1 g−1 cell
for T. thermophilus PRQ25 and 2.83 ± 0.05 and 3.20 ± 0.02 mmol
NADH min−1 g−1 cell for C. tropicalis ATCC20336, respectively (time
course of fermentations are not shown). Time of cell harvesting
is important; two different alcohol dehydrogenases from Ther-
moanaerobacter ethanolicus ATCC 31550 have been reported: one
with affinity for primary alcohols, like heptanol, which is produced
Please cite this article in press as: Álvarez L, et al. Induction of NAD+ dependent alcohol dehydrogenases with activity towards
long chain aliphatic alcohols in mesophilic, thermophilic and extreme thermophilic microorganisms. Process Biochem (2011),
doi:10.1016/j.procbio.2011.03.002
7
after exponential growth phase and another one with higher affin-
ity for secondary alcohols and long chain aliphatic alcohols which
is produced during exponential growth phase [23,24].
Cell growth was of Thermus AB1was lower when the inducer
was added at mid-exponential phase than at the beginning of fer-
mentation. This result suggests that inducers are being used also
as carbon and energy source for biomass production. On the other
hand, cell growth increased when cells adapted to the inducer were
used as inoculums. As shown in Figure 3, maximum cell growth of
Thermus AB1 induced with docosanol using an adapted inoculum
was 3.96 ± 0.08 g/l, while it was only 2.20 ± 0.06 g/l when using a
non-adapted inoculum (see Fig. 1f).
4. Conclusions
- It was possible to induce the production of alcohol dehydroge-
nases with activity over long-chain aliphatic alcohols (docosanol
and tetracosanol) by adding long chain aliphatic alcohols and
alkanes to the fermentation medium.
- The type of inducer, its concentration and time of addition are
most important parameters for the production of alcohol dehy-
drogenases with oxidation activity towards long-chain aliphatic
alcohols – a relationship was observed between the inducer
added and the substrate preference, with respect to the chain
length: long-chain aliphatic alcohols and alkanes induced higher
alcohol dehydrogenase activity towards long-chain alcohols and
short-chain alcohols and alkanes induced higher activity towards
short-chain alcohols, with the exception of the alcohol dehydro-
genases from C. tropicalis and T. thermophilus NR17 where such
pattern was not observed.
- A detrimental effect on cell growth was observed at inducer con-
centrations of 0.5% or higher, being the higher cell concentrations
- Best results in terms of alcohol dehydrogenase activity of oxida-
tion of aliphatic alcohols of 22 and 24 carbon atoms were obtained
with C. tropicalis ATCC 20336 induced with hexadecanol, Thermus
AB1 induced with docosanol and T. thermophilus PRQ25 induced
with eicosanol.
- Higher enzyme levels were obtained when the inducer is added at
the beginning of fermentation and cells are harvested at the onset
of stationary phase (between 100 and 140 h of fermentation).
- Results suggest that it is possible to modulate the kind of alcohol
dehydrogenase synthesized in terms of its activity with respect
to chain length of aliphatic alcohols by the use of inducers of a
corresponding chain length.
Acknowledgements
This work was funded by FONDEF Grant D04I1007 and CSIC
(Spain)-CONICYT (Chile) Grant 177-2007.
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