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Process Biochemistry xxx (2011) xxx–xxx

Contents lists available at ScienceDirect

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

1 Induction of NAD+ dependent alcohol dehydrogenases with activity towards long

2 chain aliphatic alcohols in mesophilic, thermophilic and extreme thermophilic
3 microorganisms
4 Lorena Álvarez ∗ , Fernando Acevedo, Andrés Illanes
5 School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Joint Doctoral Program on Biotechnology PUCV-UTFSM, Av. Brasil 2147, PO Box 4059, Valparaiso,
6 Chile
7

8 a r t i c l e i n f o a b s t r a c t
9
10 Article history: The production of alcohol dehydrogenases with the capacity of oxidizing long chain aliphatic alcohols
11 Received 2 December 2010 by mesophilic, thermophilic and hyperthermophilic strains induced by aliphatic alcohols and alkanes
12 Received in revised form 22 February 2011 with 2–24 carbon atoms has been studied. Seven strains were tested: the mesophilic yeast Candida trop-
13 Accepted 8 March 2011
icalis ATCC20336, the thermophilic bacterium Thermus AB1and five hyperthermophilic bacterial strains:
14 Thermus thermophilus HB27, HNI11, NR17, PRQ16 and PRQ25. Only C. tropicalis ATCC20336, Thermus AB1
15 Keywords:
and T. thermophilus PRQ25 produced alcohol dehydrogenases with the capacity of oxidizing aliphatic
16 Long chain fatty acids
alcohols with 22 and 24 carbon atoms, after induction with 1-hexacosanol, 1-docosanol and 1-eicosanol,
17 Polycosanols
18 Alcohol dehydrogenase
respectively. Higher initial reaction rates of oxidation of docosanol and eicosanol were obtained with the
19 Thermus enzyme from C. tropicalis ATCC20336, with values of 196.9 and 218.8 ␮mol NADH min−1 g of protein−1 ,
20 Thermus thermophilus respectively.
21 Behenic acid © 2011 Published by Elsevier Ltd.
22 Lignoceric acid

23 1. Introduction per mole of acid produced [10]. At present, the alcohol dehy- 44

drogenases produced by Candida tropicalis and Candida lipolytica 45

24 The long chain fatty acids, behenic acid (C22 H44 O2 ) and ligno- are the only commercially available enzymes capable of oxi- 46

25 ceric acid (C24 H48 O2 ), are valuable products in pharmaceutical [1,2] dizing aliphatic alcohols up to 16 carbon atoms. Horse liver 47

26 and health-care preparations [3,4] and as additives in fuels [5] and alcohol dehydrogenase has been reported to oxidize docosanol and 48

27 foods [6]. Extraction from natural products is not an option since tetracosanol, although at lower rates than small chain aliphatic 49

28 these fatty acids are extremely scarce in nature: behenic acid is alcohols [11]. 50

29 found at levels of 1–5% in peanut skin and 0.5% in canola, sesame Some induction studies have been conducted on enzyme pro- 51

30 and olive seeds, while lignoceric acid is found at even lower levels duction related to ␻- and ␤-oxidation reactions with n-alkanes 52

31 in some vegetable oils [7]. Chemical oxidation of polycosanols has as sole carbon source. Alkanes are first hydroxylated by the 53

32 been studied as one technological option for producing long chain cytochrome P450 system and NADPH-cytochrome P450 reductase 54

33 fatty acids. The chemical oxidation of octadecanol and eicosanol system, which is located in the endoplasmic reticulum (micro- 55

34 to the corresponding stearic and arachidic acids has been studied some), to the corresponding long chain aliphatic alcohols. These 56

35 using quaternary ammonium peroxotungstophosphate as catalysts alcohols are not only degraded via ␤-oxidation but also utilized 57

36 in the first case [8], and an ionic liquid composed by Aliquat and for the biosynthesis of lipids in mitochondria and endoplasmic 58

37 polyperoxometalate in the second [9]. reticulum, after dehydrogenation by long-chain fatty acids alcohol 59

38 As an alternative to chemical catalysis, enzymatic oxidation dehydrogenase, aldehyde dehydrogenase and possibly an alcohol 60

39 stems as an interesting option. In fact, enzyme E.C.1.1.1.192 (long- oxidase [12–14]. 61

40 chain fatty acids alcohol dehydrogenase) is reported to be active Some yeast strains belonging to the genus Candida produce ␻ 62

41 on the oxidation of hexadecanol to palmitic acid. This enzyme and ␤ diacids when cultured in n-alkanes or fatty acids as sole 63

42 is an oxidoreductase requiring the stoichiometric cofactor NAD+ carbon source. Alcohol dehydrogenases were induced capable of 64

43 which is reduced to NADH+ H+ , two moles of NAD+ being required oxidizing aliphatic alcohols from 8 to 16 carbon atoms, when a 65

mixture of n-alkanes from 10 to 13 carbon atoms was used as sole 66

carbon source in the cultivation of C. tropicalis ATCC 20336 and 67

∗ Corresponding author. Tel.: +56 32 2273642; fax: +56 32 2273803. C. lipolytica NRRL Y-6795. The highest activity was obtained using 68

E-mail address: lorena.alvarez.a@mail.pucv.cl (L. Álvarez). tetradecanol as substrate of the enzymatic reaction. The enzymes 69

1359-5113/$ – see front matter © 2011 Published by Elsevier Ltd.

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
 G Model
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2 L. Álvarez et al. / Process Biochemistry xxx (2011) xxx–xxx

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.

70 from both strains were NAD+ dependent and localized in the micro- also produced using alcohols as sole carbon and energy source 76

71 some, mitochondria and peroxisome, together with other enzymes [15–17]. 77

72 like aldehyde dehydrogenase acting on long-chain aldehydes, acyl Several studies on the characterization of alcohol dehydroge- 78

73 CoA synthetase and those involved in fatty acid ␤-oxidation, indi- nases from thermophilic and hyperthermophilic strains have been 79

74 cating that all of them are involved in fatty acid degradation [10,12]. conducted in recent years. More than 20 thermophilic archae and 80

75 Different NAD(P)+ dependent alcohol dehydrogenases have been 17 thermophilic bacteria have been identified as alcohol dehydro- 81

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
 G Model
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L. Álvarez et al. / Process Biochemistry xxx (2011) xxx–xxx 3

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.

82 genase producers. Thermophilic organisms have been isolated from The objective of this work was to develop a strategy for 88

83 natural habitats like hydrothermal vents, marine volcanic sedi- the production of microbial alcohol dehydrogenases by fer- 89

84 ments, hot springs and also from petroleum reservoirs, digesters mentation with the capacity to oxidize long-chain aliphatic 90

85 and leach piles. Most thermophilic organisms produce various alcohols by induction with primary aliphatic alcohols from 91

86 types of dehydrogenases [18,19] whose production can be opti- 2 to 24 carbon atoms and alkanes from 14 to 24 carbon 92

87 mized by using different strategies of microbial cultivation [20]. atoms. 93

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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4 L. Álvarez et al. / Process Biochemistry xxx (2011) xxx–xxx

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 (␮mol 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

94 2. Materials and methods Madrid, Madrid, Spain). Mesophilic yeast C. tropicalis (ATCC20336) was provided by 111
ATCC. 112
95 2.1. Materials

96 2.1.1. Chemicals
97 Nicotinamide adenine dinucleotide (NAD+ ) was purchased from Jülich Fine 2.2. Methods 113
98 Chemicals. Diethylene glycol, bis(2-methoxyethyl) ether (diglyme), benzenecar-
99 boximidamide (benzamidine), bovine serum albumin (BSA), Coomassie blue 2.2.1. Culture media and conditions of cultivation 114
100 G-250, 1-octanol, 1-tetradecanol, 1-hexadecanol, n-tetradecane, n-heptadecane, n- Inocula were prepared for all microorganisms in the same cultivation media 115
101 octadecane, n-dodecane and n-tetradecane were purchased from Sigma–Aldrich described below, but in the case of C. tropicalis no Tween was added. Cells were 116
102 Chem. Co. Tryptone, yeast extract, ethanol, 1-dodecanol and 1-eicosanol were pur- harvested in mid-exponential phase and each cultivation flask was inoculated with 117
103 chased from Merck, docosanol was purchased from Sasol Germany GmbH and 20 ml of cell suspensions containing 0.3 mg cell dry weigh/ml. 118
104 tetracosanol was purchased from ABCR GmbH & CoKG. All other reagents were of All microorganisms were cultivated in 500 ml shake flasks containing 200 ml 119
105 analytical grade. of medium on a rotary shaker at 200 rpm. T. thermophilus HB27, HN1.11, NR17, 120
PRQ16, PRQ25, and Thermus AB1 strain were cultivated on Thermus culture 121
106 2.1.2. Microorganisms medium consisting of: 8 g/l tryptone, 4 g/l yeast extract, 3 g/l NaCl, 0.59 g/l 122
107 Thermus thermophilus HB27 (ATCC BA-163), HN1.11, NR17, PRQ16, PRQ25, and 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 123
108 Thermus AB1 strain were kindly provided by Drs. José Berenguer and Daniel Vega FeSO4 ·7H2 O, 0.019 g/l MnCl2 ·4H2 O, 0.032 g/l ZnSO4 ·7H2 O, 0.005 g/l CoCl2 ·6H2 O, 124
109 (Universidad Autónoma de Madrid). These strains belong to the culture collec- 0.0035 g/l CuSO4 ·5H2 O in distilled water at pH 7.5. Temperature was 70 ◦ C for HB27, 125
110 tion of the Centro de Biología Molecular Severo Ochoa (Universidad Autónoma de HN1.11, NR17, PRQ16 and PRQ25, and 50 ◦ C for AB1. 126

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
 G Model
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L. Álvarez et al. / Process Biochemistry xxx (2011) xxx–xxx 5

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 (␮mol 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

127 C. tropicalis ATCC20336 was cultivated on a minimum medium which con- time zero may fully exploit the effect of the inducer but an early addition might 153
128 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 produce inhibition on cell growth, so we chose also the addition at mid exponen- 154
129 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, tial phase were the culture is growing vigorously. Samples were taken throughout 155
130 0.03 g/l ZnSO4 ·7H2 O, 0.03 g/l CoCl2 , 0.003 g/l CuSO4 ·5H2 O, 0.04 ␮g/l folic acid, the fermentation and enzyme produced was determined evaluating its activity over 156
131 0.4 ␮g/l pantothenic acid, 0.01 ␮g/l biotin, 1 ␮g/l niacin, 0.1 ␮g/l tiamin B1 and 0.5% docosanol and tetracosanol. 157
132 (v/v) de Tween 80 in distilled water at pH 6.0. Temperature was 30 ◦ C and Tween 80
133 was added to increase substrate solubility. Inducers were added to each cultivation 2.2.4. Preparation of crude enzyme extracts 158
134 medium as described in Section 2.2.2. Cell growth was monitored by measuring the turbidity at 650 nm. Cells were 159
harvested during the stationary phase of growth and after freezing at −18 ◦ C, they 160
135 2.2.2. Conditions of induction were resuspended in a 1/1 volume of 10 mM sodium phosphate buffer pH 7.5 with 161
136 Induction was done by adding 0.025, 0.05, 0.5, 1 and 2% of primary 10 mM NaCl and 3 mM benzamidine, and disrupted by mechanical abrasion with 162
137 aliphatic alcohols or alkanes to each culture medium. Ethanol, 1-tetradecanol alumina powder (0.5 g/ml). The soluble fraction obtained after removal of cell debris 163
138 (C14 H30 O), 1-hexadecanol (C16 H34 O), 1-eicosanol (C20 H42 O), 1-docosanol (C22 H46 O), by centrifugation (15,000 × g, 15 min, 4 ◦ C) was used as source of the alcohol dehy- 164
139 1-tetracosanol (C24 H50 O) and alkanes n-tetradecane (C14 H30 ), n-heptadecane drogenases. 165
140 (C17 H35 ), n-octadecane (C18 H38 ), n-docosane (C22 H46 ) and n-tetracosane (C24 H50 )
141 were used as inducers. As control experiments, strains were grown at the same 2.2.5. Protein determination 166
142 conditions and in the same media without the addition of inducer. Protein was determined by the method of Bradford [21] using Coomassie blue 167
G-250 dye as reagent and bovine serum albumin (BSA) as standard, by measuring the 168
143 2.2.3. Time of addition of inducers on the production of alcohol dehydrogenases absorbance at 595 nm at 25 ◦ C in a Jenway Spectrophotometer (model 6715 UV/VIS). 169
144 The effect of the time of addition of inducer and harvest time was studied in all
145 strains, for determining the conditions at which they produced higher enzyme activ- 2.2.6. Determination enzyme activity with aliphatic alcohols 170
146 ities over tetracosanol and docosanol. Induced cells (adapted inoculum) were used as Alcohol dehydrogenase activity of the different crude extracts was ana- 171
147 inoculums in these studies. Batch fermentations were conducted for 180 h in 500 ml lyzed by the increase in absorbance at 340 nm corresponding to the formation 172
148 shake flasks with 200 ml culture medium, at 70 ◦ C for T. thermophilus HB27, HN1.11, of NADH concomitant to the oxidation of the aliphatic alcohols. A sample of 173
149 NR17, PRQ16 and PRQ25 and 50 ◦ C for Thermus AB1, on a rotary shaker at 200 rpm, enzyme preparation (50–200 ␮l) was added to a spectrophotometer cell with 174
150 and at 30 ◦ C for C. tropicalis ATCC20336 on a rotary shaker at 180 rpm. Inducers at 2 ml of 0.2 mM substrates (ethanol, octanol, dodecanol, tetradecanol, hexadecanol, 175
151 0.05% (w/v) concentration were added at the beginning of the fermentation and docosanol and tetracosanol) in 100 mM phosphate buffer pH 7.0 with 0.6% diglyme 176
152 during mid-exponential growth phase (after about 12 h of cultivation). Addition of and 100 ␮l of 100 mM NAD+ . Initial rates of oxidation of aliphatic alcohols were 177

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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178 expressed in ␮mol NADH min−1 g−1 of protein and ␮mol NADH min−1 g−1 of cell dry 1-hexadecanol that inhibited the growth of T. thermophilus HN1.11, 241
179 weight. NR17, PRQ16 and Thermus AB1. 242

At concentrations higher than 0.5%, precipitation of the inducer 243

180 2.2.7. Data analysis
was observed in the culture medium at temperatures below 60 ◦ C, 244
181 Experiments for the selection of inducers and microorganisms producing alcohol
182 dehydrogenase with the highest activity of oxidation of docosanol and tetradecanol which is the case for C. tropicalis. Ueda and Tanaka [10] reported 245

183 were done in duplicate and samples assayed in duplicate. Initial rate of alcohol oxi- the use of 0.7% of a mixture of alkanes with 10–13 carbon atoms; 246
184 dation, expressed in ␮mol NADH min−1 g−1 of cell dry weight, was used as selection however, nutrients were hard to solubilize at 30 ◦ C, even with the 247
185 parameter. An analysis of variance of means (ANOVA) was conducted, the F-test addition of Tween 80. On the hand, Kato et al. [22] supplemented 248
186 in ANOVA indicating whether there are significant differences amongst the means.
187 The software STATGRAPHICS PLUS 5.1 was used for analysis of significant (P) level
the growth medium with 0.1% (v/v) of pure alkanes, or mixtures 249

188 through F-test. Only P < 0.05 were accepted as significant factors, for a 95% confi- with 10–34 carbon atoms 70 ◦ C, cultivating the yeast for a period of 250

189 dence level. Subsequently, to determine which means are significantly different, ten days. Inducer concentration of 0.05% (w/v) was selected as the 251
190 Multiple Range Test was performed using the Fisher’s least significant difference most appropriate for subsequent studies of production of alcohol 252
191 method (LSD).
dehydrogenase. 253

192 3. Results and discussion

3.2. Effect of inducers on the production of alcohol 254
193 T. thermophilus HB27, HN1.11, NR17, PRQ16, PRQ25, Ther- dehydrogenases 255
194 mus AB1 and C. tropicalis ATCC20336 were selected for having
195 previously determined as producers of alcohol dehydrogenases Results of the effect of addition of alcohols as inducers on the 256
196 capable of oxidizing aliphatic alcohols of more than 6 carbon production of alcohol dehydrogenases with capacity of oxidizing 257
197 atoms. Thermophilic and hyperthermophilic microorganisms are aliphatic alcohols are presented in Table 1 in terms of initial rates 258
198 quite appealing since their enzymes withstand higher tempera- of oxidation. Crude enzyme extracts were obtained after 96 h of fer- 259
199 tures allowing the use of higher substrate concentrations, because mentation using 0.05% (w/v) of inducer (aliphatic alcohol or alkane) 260
200 of the dramatic increase in solubility with temperature. This is in the culture medium. Enzyme extracts were obtained by cell dis- 261
201 quite important, as becomes apparent by simple consideration of ruption according to the methodology in Section 2.2.4. 262
202 Michaelis–Menten kinetics, but also because the use of organic sol- Results of the activities of alcohol dehydrogenases, expressed 263
203 vents to dissolve the very hydrophobic substrates can be avoided. as initial rates of oxidation of ethanol, octanol, dodecanol, tetrade- 264
204 Moreover, enzymes produced by such organisms are more stable canol, docosanol and tetracosanol, induced with aliphatic alcohols 265
205 than mesophilic enzymes, so that the biocatalyst is more efficiently and alkanes are presented in Tables 1 and 2, respectively. As 266
206 used and also the risk of microbial contamination is reduced. All seen in Table 1, higher activities were obtained over the aliphatic 267
207 those are significant aspects from a process perspective. alcohols 1-tetradecanol, 1-docosanol and 1-tetracosanol with the 268

alcohol dehydrogenases from C. tropicalis ATCC20336 induced with 269

208 3.1. Effect of inducers on cell growth hexadecanol, T. thermophilus PRQ25 induced with eicosanol and 270

Thermus AB1 induced with docosanol. On the other hand, as seen 271

209 Induction of alcohol dehydrogenases was done by adding alco- in Table 2, higher activities over docosanol and tetracosanol were 272

210 hols or alkanes of different chain length to the culture medium. obtained with alcohol dehydrogenases from T. thermophilus PRQ25 273

211 Therefore, the effect of such supplements on cell growth was deter- induced with n-heptadecane and n-tetradecane and from Thermus 274

212 mined. Firstly each inducer was tested as the sole carbon source AB1 induced with n-docosane. 275

213 in the fermentation medium and in all cases no cell growth was These results show that, even though aliphatic alcohols have 276

214 observed. some inhibitory effect on cell growth in non-adapted strains, they 277

215 The effect of alcohols and alkenes supplementation to the cul- are very good inducers of alcohol dehydrogenases with activity over 278

216 ture media is presented in Figs. 1 and 2, respectively. Strains long-chain aliphatic alcohols. 279

217 responded differently to the addition of the different inducers. Oxidation rate over tetracosanol obtained with the alcohol 280

218 Using alcohols as inducers, cell growth was reduced with respect dehydrogenase from C. tropicalis ATCC 20336 induced with 1- 281

219 to the respective controls (no inducer added) in all strains with the hexadecanol (86.7 ␮mol NADH min−1 g−1 protein) was higher 282

220 exception of T. thermophilus PRQ 25, Thermus AB1 and C. tropicalis than a value previously reported by Ueda and Tanaka (70 ␮mol 283

221 ATCC 20336. NADH min−1 g−1 protein) [10], where a mixture of alkanes with 284

222 On the other hand, growth of T. thermophilus HB27, HN1.11 and 10–13 carbon atoms was used as the sole carbon and energy source 285

223 PRQ25 increased with all alkanes used as inducers (see Fig. 2). Cell [10]. Without induction, a basal level of activity was observed only 286

224 growth of HB27 was 37% higher than the control when supple- on ethanol, with the exception of Thermus AB1 where a basal activ- 287

225 mented with n-docosane, while cell growth of PRQ25 increased by ity over octanol was also observed and T. thermophilus PRQ16 who 288

226 24, 23 and 32% when supplemented with n-octadecane, n-docosane departed from that pattern, expressing activity on ethanol, octanol, 289

227 and n-tetracosane respectively. Similar results have been reported dodecanol and hexadecanol. 290

228 when the effect of alkane chain length was studied on the cellular There is some relationship between the length of the chain of 291

229 growth of Geobacillus thermoleovorans B23; in this case, an increase the inducer and the level of activity expressed on the substrate of 292

230 in the production of proteins related to ␤ oxidation was observed, the corresponding chain length of the reaction in several strains 293

231 whose magnitude correlated with the chain length of the alkane of Thermus; however, in the case of Candida this pattern is not 294

232 [22]. followed. 295

233 With respect to the effect of the concentration of inducer, A statistic analysis was done, as described in Section 2.2.7, 296

234 statistical analysis was performed as described in Section 2.2.7. to determine the best microorganisms and inducers in terms 297

235 According to this analysis, best results in terms of cell growth were of production of alcohol dehydrogenases with activity towards 298

236 obtained in most cases at inducer concentrations from 0.05 to 1%, long-chain aliphatic alcohols (docosanol and tetracosanol). Higher 299

237 with a significant reduction at concentrations over 1% (Figs. 1 and oxidation rates were observed towards long chain aliphatic alco- 300

238 2). However, tetradecanol inhibited the growth of the strains even hols (docosanol and tetracosanol) with the alcohol dehydrogenase 301

239 at concentrations of 0.025%, except in the case of T. thermophilus from C. tropicalis ATCC20336 induced by 1-hexadecanol (C16 ), T. 302

240 HB27 and Thermus AB1. Similar results were obtained with 0.025% thermophilus PRQ25 induced with 1-eicosanol (C20 ) and Thermus 303

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),
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moanaerobacter ethanolicus ATCC 31550 have been reported: one 331

with affinity for primary alcohols, like heptanol, which is produced 332

after exponential growth phase and another one with higher affin- 333

ity for secondary alcohols and long chain aliphatic alcohols which 334

is produced during exponential growth phase [23,24]. 335

Cell growth was of Thermus AB1was lower when the inducer 336

was added at mid-exponential phase than at the beginning of fer- 337

mentation. This result suggests that inducers are being used also 338

as carbon and energy source for biomass production. On the other 339

hand, cell growth increased when cells adapted to the inducer were 340

used as inoculums. As shown in Figure 3, maximum cell growth of 341

Thermus AB1 induced with docosanol using an adapted inoculum 342

was 3.96 ± 0.08 g/l, while it was only 2.20 ± 0.06 g/l when using a 343

non-adapted inoculum (see Fig. 1f). 344

4. Conclusions 345

- It was possible to induce the production of alcohol dehydroge- 346

nases with activity over long-chain aliphatic alcohols (docosanol 347

and tetracosanol) by adding long chain aliphatic alcohols and 348

alkanes to the fermentation medium. 349

- The type of inducer, its concentration and time of addition are 350

most important parameters for the production of alcohol dehy- 351

drogenases with oxidation activity towards long-chain aliphatic 352

alcohols – a relationship was observed between the inducer 353

added and the substrate preference, with respect to the chain 354

length: long-chain aliphatic alcohols and alkanes induced higher 355

Fig. 3. Effect of time of addition of inducer (docosanol) in the production of the alcohol dehydrogenase activity towards long-chain alcohols and 356
enzyme in Thermus AB1. Batch fermentations were conducted for 180 h in 500 ml short-chain alcohols and alkanes induced higher activity towards 357
shake flasks with 200 ml culture medium at 50 ◦ C. Docosanol at 0.05% (w/v) was short-chain alcohols, with the exception of the alcohol dehydro- 358
added at (a) the beginning of fermentation and (b) during mid-exponential growth
genases from C. tropicalis and T. thermophilus NR17 where such 359
phase (after about 12 h of cultivation). Cell samples were collected to measure the
enzyme activity on tetracosanol and docosanol as substrates. Biomass (), docosanol pattern was not observed. 360

(䊉), tetracosanol (), black arrow indicates the moment of addition of the inducer, - A detrimental effect on cell growth was observed at inducer con- 361
staple arrows point to the corresponding Y-axis. Each bar represents an average of centrations of 0.5% or higher, being the higher cell concentrations 362
the two experiments with its standard deviation.
obtained in all cases at 0.05% inducer concentration. 363

- Best results in terms of alcohol dehydrogenase activity of oxida- 364

304 AB1 induced with 1-docosanol (C22 ). tion of aliphatic alcohols of 22 and 24 carbon atoms were obtained 365

with C. tropicalis ATCC 20336 induced with hexadecanol, Thermus 366

AB1 induced with docosanol and T. thermophilus PRQ25 induced 367

305 3.3. Effect of time of addition of inducers on the production of with eicosanol. 368
306 alcohol dehydrogenases - Higher enzyme levels were obtained when the inducer is added at 369

the beginning of fermentation and cells are harvested at the onset 370
307 The effectivity of induction may vary according to the moment of stationary phase (between 100 and 140 h of fermentation). 371
308 of addition of the inducer during fermentation; the inducers were - Results suggest that it is possible to modulate the kind of alcohol 372
309 added at two different culture time, at the beginning of fermen- dehydrogenase synthesized in terms of its activity with respect 373
310 tation and during mid-exponential growth phase (after about to chain length of aliphatic alcohols by the use of inducers of a 374
311 12 h of cultivation). Thermus AB1was induced with docosanol, corresponding chain length. 375
312 T. thermophilus PRQ25 induced with eicosanol, and C. tropicalis
313 ATCC20336 with hexadecanol.
314 Results obtained with Thermus AB1 are presented in Fig. 3 show- Acknowledgements 376

315 ing that the addition of the inducer at the beginning of fermentation
316 produced higher enzyme activities over tetracosanol and docosanol This work was funded by FONDEF Grant D04I1007 and CSIC 377

317 than adding it during exponential growth. Higher values of initial (Spain)-CONICYT (Chile) Grant 177-2007. 378

318 reaction rates over docosanol and tetracosanol were 1.00 ± 0.03 and
319 1.41 ± 0.02 ␮mol NADH min−1 g−1 cell, respectively, both obtained References 379
320 after 120 h of fermentation. Similar results were obtained with
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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