Novel Hydrolases from Thermophilic Filamentous Fungi for Enantiomerically and Enantiotopically Selective Biotransformations

Article abstract of DOI:10.1002/adsc.200303027

Fourteen thermophilic filamentous fungi were cultivated on two shake flask media and the supernatants were assayed for lipase/carboxylesterase activities using olive-oil, p-nitrophenyl palmitate and p-nitrophenyl butyrate as substrates. The crude enzyme p

Full text of DOI:10.1002/adsc.200303027

FULL PAPERS
Novel Hydrolases from Thermophilic Filamentous Fungi for
Enantiomerically and Enantiotopically Selective
Biotransformations
a, b
c
c
c
a
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Viktoria Bodai, Reka Peredi, Jozsef Balint, Gabriella Egri, Lajos Novak,
b,
a,
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Gyˆrgy Szakacs, * Laszlo Poppe *
a
Institute for Organic Chemistry and Research Group for Alkaloid Chemistry of the Hungarian Academy of Sciences,
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Budapest University of Technology and Economics, Gellert ter 4, 1111 Budapest, Hungary
Phone: ( ‡ 36)-1/463 3297, fax: ( ‡ 36)-1/463 3297; e-mail: poppe@mail.bme.hu
b
c
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Department of Agricultural Chemical Technology, Budapest University of Technology and Economics, Gellert ter 4,
1111 Budapest, Hungary
Fax: ( ‡ 36)-1/463 2598; e-mail: gszakacs@mail.bme.hu
Department of Organic Chemical Technology, Budapest University of Technology and Economics, 1521 Budapest,
PO Box 91, Hungary
Received: January 29, 2003; Accepted: March 25, 2003
Abstract: Fourteen thermophilic filamentous fungi testing the biocatalytic abilities of these preparations.
were cultivated on two shake flask media and the The tested biocatalysts proved to be comparable to
supernatants were assayed for lipase/carboxylesterase the commercially available enzymes with respect to
activities using olive-oil, p-nitrophenyl palmitate and the degree of enantiomer selectivity, whereas they
p-nitrophenyl butyrate as substrates. The crude en- exhibited a wider range of enantiotopic selectivity
zyme powders (acetone precipitated supernatants) than the most common commercial enzymes.
were tested as biocatalysts in organic solvents. Kinetic
resolution of racemic 1-phenylethanol (rac-1) and Keywords: asymmetric catalysis; enzyme catalysis;
desymmetrisation of 2-acyloxypropan-1,3-diols (3a,b ) hydrolases; kinetic resolution; thermophilic filamen-
by acetylation with vinyl acetate were chosen for tous fungi
Introduction
the organisms and, in general, are more heat stable than
those of the mesophilic fungi. Therefore, thermophilic
The development of novel biocatalytic methods is a fungi are potential sources of thermotolerant enzymes
continuously growing area of chemistry, microbiology of scientific and commercial interest. Some extracellular
and genetic engineering due to the fact that biocata- enzymes from thermophilic fungi are being produced
lysts are selective, easy-to-handle and environmentally commercially, and a few others have commercial
friendly.[1] Industrial applications are widespread as prospects.[5]
biocatalytic steps are already being used to manufacture
Lipases represent the most important group of
a wide range of products, including drugs, agricultural biocatalysts for biotechnological applications.[6] Al-
chemicals, organics, fine chemicals and plastics.[2] Be- though several lipases were isolated from thermophilic
cause the demand for the enantiopure form of the chiral fungi, only few, e.g., Thermomyces lanuginosus (for-
compounds has been increasing rapidly, novel micro- merly Humicola lanuginosa) or Rhizomucor miehei
organisms and/or their enzymes are the subjects of have been commercialised[7] and investigated as syn-
screening to produce such chemicals.[1,2]
thetic biocatalysts.[8,9] Genes encoding these enzymes
Enzyme preparations from fungi has been studied have also been cloned into recombinant hosts.[7] Immo-
since the early 1950×s.[3] Several fungal biocatalysts are bilisation,[10,11] genetic modifications by site directed
commercially available and they are valuable tools for mutagenesis,[12] gene shuffling[13] or directed evolu-
the synthetic chemists.[4] Thermophilic fungi are the only tion[14] of these recombinant enzymes have been used
representatives of eukaryotic organisms that can grow at to modify/enhance their practical applicability.
temperatures above 458C with a maximum temperature
Because some lipases from thermophilic fungi are
of growth extending up to 60 to 628C.[5] Their extra- highly valuable biocatalysts, less studied thermophilic
cellular enzymes display activity maxima that are close fungi for organic synthesis have been chosen to inves-
to or above the optimum temperature for the growth of tigate their extracellular lipase/carboxylesterase pro-
Adv. Synth. Catal. 2003, 345, 811 818
DOI: 10.1002/adsc.200303027
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Viktoria Bodai et al.
FULL PAPERS
duction. In order to obtain additional data for such have been evaluated as synthetic biocatalysts as yet.[8,9]
thermotolerant hydrolases, these fungal enzymes were Therefore, we thought it worthwhile to characterise the
also evaluated for enantiomer and enantiotopic selec- biocatalytic abilities of enzymes from thermophilic
tive biotransformations.
fungi by two typical enantioselective processes. Enan-
tiomer selectivity was evaluated by kinetic resolution of
racemic 1-phenylethanol (1), and the enantiotopic
selectivity by desymmetrisation of 2-acyloxypropane-
1,3-diols (3a,b ), both by enzymatic acetylation with
Results and Discussion
Among the thermophilic fungal strains selected for our vinyl acetate.
lipase/carboxylesterase study, Chaetomium thermophi-
The enantiomers of1-phenylethanol (1)are of interest
lum,[15,16] Humicola grisea,[17] Humicola insolens,[11] Pae- since they are used as chiral reagents for the determi-
cilomyces sp.,[13,18,19] Sporotrichum thermophile,[20] Talar- nation of enantiomeric purity and for resolution of
omyces emersonii,[21,22,23] Talaromyces thermophilus,[17,21] acids[26] or for asymmetric opening of cyclic anhydrides
Thermoascus aurantiacus,[15,16] Thermoascus thermophi- and of epoxides.[27] The enzymatic kinetic resolution of
lus[15,16,17,24] and Thermomyces lanuginosus[11],[16],[21] have racemic 1-phenylethanol (1) or its esters is well docu-
been reported for such enzyme production. However, mented.[1] Therefore, acetylation of rac-1 with vinyl
only a few of the above fungi have been used for the acetate in hexane was chosen for testing the enantiomer
industrial production of lipase or have had their selectivity of our enzyme preparations (Scheme 1,
enzymes tested thoroughly for organic synthesis. It is Table 2). The degree of enantiomer selectivity (E) was
noteworthy that extremely stable microenzymes (1.6 to precisely calculated[28] from the conversion-enantiomer-
4.1 kDa) with esterase activity were found in Talaro- ic composition data obtained by GC on a chiral sta-
myces emersonii and Emericella nidulans.[23] According tionary phase. For comparison and for unambiguous
to our best knowledge, lipase/carboxy ester hydrolase assignment of the absolute configurations of the pro-
activity of Myceliophthora thermophila and Thermo- duced acetate and alcohol enantiomers, acylation of rac-
mucor indicae-seudaticae has not yet been published.
1 with vinyl acetate in hexane by Lipozyme TL IM
In a previous screen of thermophilic fungi, lipase (Thermomyces lanuginosus lipase immobilised on silica
activity was detected in the culture filtrates of fungi by granulation) has also been performed on both small
within 48 h of incubation at 458C.[17] The presence of and preparative scales.
lipids in the growth medium enhanced the production of
Our data (Table 2; only the reactions exceeding 5%
extracellular lipases but the lack of lipids in the growth conversion after 168 h are listed) indicate that many of
medium did not prevent lipase synthesis.[17] In our study, the isolates were capable of performing enantiomer-
fourteen different thermophilic fungal strains were selective acetylation of the test substrate rac-1 with (R)-
cultured in two different media which contained olive- enantiomer preference exhibiting enantiomer selectiv-
oil to induce the lipase production (Table 1). The lipase/ ities from E ˆ 1.1 up to 231. Although the observed
carboxylesterase activities in the supernatants obtained enantiomer selectivity for Lipozyme TL IM under these
from the cultures after incubating at 458C for 72 h were non-optimised conditions was quite high (E ˆ 102),
determined by three different activity tests. The lipase several of our enzyme preparations exhibited even
activity was characterised by a titrimetric method using higher selectivities [e.g., Myceliophthora thermophila
olive oil as substrate and by a spectrophotometric test TUB-F-39 (LIP2): E ˆ 163; Humicola insolens CBS-
ˆ
using p-nitrophenyl palmitate as substrate. The general 147.64 (LIP2): E
171; Chaetomium thermophilum
carboxylesterase activity was determined by spectro- TUB-F-69 (LIP2): E ˆ 231]. In addition, not only the
photometry using p-nitrophenyl butyrate as substrate. selectivity but also the productivity (50% conversion
In line with previous observations made during lipase within 72 h) of the enzyme preparation from Chaeto-
activity screening,[25] in several instances practically no mium thermophilum TUB-F-69 (LIP2) proved to be
activity with olive oil was determined for preparations appropriate for synthetic purposes.
exhibiting activity on other substrates. The results listed
2-Substituted-propane-1,3-diols are important chiral
in Table 1 strongly support that most of the fungal building blocks of numerous biologically active mole-
strains produced more than one extracellular enzyme
exhibiting carboxy ester hydrolase activity. It is clearly
seen, e.g., for Talaromyces emersonii NRRL-3221 or
Talaromyces thermophilus NRRL-2155, that the esterase
vs. lipase activity pattern was dependent on the culturing
conditions. In many cases growth in LIP1 medium
favoured the esterase production, whereas culturing in
LIP2 medium increased the lipase-like activity.
Although hydrolases (lipases/esterases) were detect-
ed in numerous thermophilic fungi strains, only a few (rac-1).
Scheme 1. Enzymatic acetylation of racemic 1-phenylethanol
812
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Novel Hydrolases from Thermophilic Filamentous Fungi
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Table 1. Lipase and carboxylesterase activities of thermophilic filamentous fungi.
Fungal strain (Medium)
Precipitate [mg/mL][a]
Hydrolase activity[b] [mU/mL]
Olive oil
p-NPP
p-NPB
Chaetomium thermophilum TUB-F-69 (LIP1)
Chaetomium thermophilum TUB-F-69 (LIP2)
Humicola grisea var. thermoidea CBS-183.64 (LIP1)
Humicola grisea var thermoidea CBS-183.64 (LIP2)
Humicola insolens CBS-147.64 (LIP1)
4.5
1.0
2.8
5.0
1.4
3.4
7.8
3.9
16.0
13.5
1.3
2.3
0.3
0.2
3.3
2.4
1.6
13.5
1.3
1.0
2.3
1.9
1.1
4.6
2.4
1.1
1.6
2.6
0
0
0
0
0
0
0
0
0
0
0
9.3
0.2
0
0
2.7
0
0
0
97.3
15.3
429.0
188.0
55.2
22.4
84.5
100.2
0.35
9.5
30.0
94.7
80.1
76.4
5.1
4.9
7.3
28.8
5.9
7.8
34.5
3.9
104.0
1.7
4.6
26.0
0
109.6
73.3
210.0
526.0
96.3
309.0
191.3
320.4
1.4
Humicola insolens CBS-147.64 (LIP2)
Myceliophthora thermophila TUB-F-39 (LIP1)
Myceliophthora thermophila TUB-F-39 (LIP2)
Paecilomyces sp. TUB-F-70 (LIP1)
Paecilomyces sp. TUB-F-70 (LIP2)
6.4
Sporotrichum thermophile ATCC-36.347 (LIP1)
Sporotrichum thermophile ATCC-36.347 (LIP2)
Sporotrichum thermophile WFPL-264 A (LIP1)
Sporotrichum thermophile WFPL-264 A (LIP2)
Talaromyces emersonii NRRL-3221 (LIP1)
Talaromyces emersonii NRRL-3221 (LIP2)
Talaromyces thermophilus NRRL-2155 (LIP1)
Talaromyces thermophilus NRRL-2155 (LIP2)
Thermoascus aurantiacus TUB-F-43 (LIP1)
Thermoascus aurantiacus TUB-F-43 (LIP2)
Thermoascus thermophilus NRRL-5208 (LIP1)
Thermoascus thermophilus NRRL-5208 (LIP2)
Thermomucor indicae-seudaticae NRRL-6429 (LIP1)
Thermomucor indicae-seudaticae NRRL-6429 (LIP2)
Thermomyces lanuginosus ATCC-38.905 (LIP1)
Thermomyces lanuginosus ATCC-38.905 (LIP2)
Thermomyces lanuginosus CBS-224.63 (LIP1)
Thermomyces lanuginosus CBS-224.63 (LIP2)
13.8
63.5
68.4
66.8
15.3
5.8
6.7
6.0
23.5
18.0
30.0
0
42.5
0.6
20.7
3.1
16.9
16.9
0
1.8
3.1
0
0
20.3
17.9
0
12.8
31.5
[a]
Mass of dried precipitate from 1 mL of supernatant of the corresponding culture.
For activity tests, see Experimental Section. Activities below 0.1 mU/mL are reported as 0.
[b]
(3b) with vinyl acetate (Scheme 2, Tables 3 and 4) have
been chosen for testing the enantiotopic selectivities of
the novel biocatalysts from the thermophilic fungi as
well.
Because the configuration of (R)-4a was previously
determined by correlation with compounds of ambig-
uous low optical rotation data,[32a] the absolute config-
uration of (R)-4a was unambiguously re-determined by
chemical correlation starting from (R)-(5)[33] via ben-
zoylation and subsequent catalytic hydrogenation
(Scheme 3) before performing the enzymatic reactions
with 3a. Afterwards, asymmetric acylation reactions of
the prochiral diol (3a) were performed (Scheme 2,
Table 3) to test the enantiotopic selectivity of the
Scheme 2. Enzymatic acetylation of 2-acyloxypropane-1,3-
diols (3a,b ).
cules, such as phospholipids,[29] PAF (platelet-activating thermophilic fungal enzymes. The results of these
factor)[30] or b-blockers.[31] Our group has recently reactions proved also to be quite promising. The
published results on enantiotopic selective enzymatic prochiral diol (3a) was accepted as substrate by many
acetylation of prochiral 2-acyloxypropan-1,3-diols[32] preparations. The asymmetric acylation of 3a with
which proved to be useful for the preparation of such Talaromyces emersonii NRRL-3221 (LIP2) proceeded
chiral building blocks in optically active form. There- with the same enantiotopic preference and almost as
fore, acylation reactions of 2-benzoyloxypropane-1,3- selectively as the reaction with the best commercial
diol (3a) and 2-(p-toluenesulfonyl)oxypropane-1,3-diol enzyme PPL[32a] (94% ee vs. 96% ee of (R)-4a, respec-
Adv. Synth. Catal. 2003, 345, 811 818
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Table 2. Acetylation of racemic 1-phenylethanol (rac-1) with thermophilic fungal enzymes.
Enzyme
t [h]
c [%][a]
ee [%][a]
E[b]
Fungal strain (Medium)
Chaetomium thermophilum TUB-F-69 (LIP2)
Humicola insolens CBS-147.64 (LIP2)
Myceliophthora thermophila TUB-F-39 (LIP2)
Chaetomium thermophilum TUB-F-69 (LIP1)
Lipozyme TL IM
Humicola grisea var. thermoidea CBS-183.64 (LIP2)
Thermomyces lanuginosus CBS-224.63 (LIP1)
Thermomyces lanuginosus CBS-224.63 (LIP2)
Thermomyces lanuginosus ATCC-38.905 (LIP1)
Talaromyces emersonii NRRL-3221 (LIP2)
Sporotrichum thermophile ATCC-36.347 (LIP2)
Sporotrichum thermophile WFPL-264 A (LIP1)
Thermoascus aurantiacus TUB-F-43 (LIP2)
Sporotrichum thermophile WFPL-264 A (LIP2)
72
24
96
72
20
50
11
5
50
47
14
6
10
5
14
24
26
5
96.4
98.7
98.7
94.3
95.0
97.7
97.1
95.6
91.5
82.1
76.1
41.7
10.3
0.9
231
171
163
123
102
101
72
50
25
12
7.5
96
168
168
168
168
72
72
168
72
2.8
1.3
1.0
31
[a]
Conversion and enantiomeric composition of the produced acetate [(R)-2] were determined by GC on chiral stationery
phase, for details see Experimental Section.
[b]
ˆ
The degree of enantiomer selectivity (E) was calculated by using the equation E ln[1 c(1 ‡ ee(P))]/ln[1 c(1 ee(P))]
as defined for the irreversible enantiomer selective transformations.[28]
Table 3. Acetylation of 2-benzoyloxypropane-1,3-diol (3a) with thermophilic fungal enzymes.
Enzyme
t [h]
4a
Fungal strain (Medium)
m [mg]
Yield [%]
Config.
ee [%]
PPL: lipase from porcine pancreas[32a]
300
48
101
73
1
48
63
87
86
14
12
27
12
14
12
12
47
30
51
47
R
R
R
R
R
-
S
S
S
S
S
S
S
S
96
94
67
23
15
0
Talaromyces emersonii NRRL-3221 (LIP2)
Talaromyces emersonii NRRL-3221 (LIP1)
Sporotrichum thermophile ATCC-36.347 (LIP1)
Myceliophthora thermophila TUB-F-39 (LIP2)
Sporotrichum thermophile ATCC-36.347 (LIP2)
Chaetomium thermophilum TUB-F-69 (LIP2)
Humicola grisea var. thermoidea CBS-183.64 (LIP2)
Thermomucor indicae-seudaticae NRRL-6429 (LIP2)
Thermomyces lanuginosus CBS-224.63 (LIP1)
Paecilomyces sp. TUB-F-70 (LIP2)
120
168
168
168
168
168
168
168
72
42
126
101
202
78
5
12
16
29
53
53
59
71
65
103
101
96
Thermoascus thermophilus NRRL-5208 (LIP2)
Thermomyces lanuginosus ATCC-38.905 (LIP2)
Talaromyces thermophilus NRRL-2155 (LIP1)
168
15
21
70
tively). In addition, enzymes preferring the formation of for a highly selective optimised biotransformation
the opposite enantiomer (S)-4a up to 71% ee with either a fully optimised enzyme production process or
Talaromyces thermophilus NRRL-2155 (LIP1) were production of the enzyme in question by recombinant
also found. The results indicate that these thermophilic organisms overexpressing its gene might be necessary. It
fungi produce various hydrolases with different selec- is also shown that enzymes from closely related strains
tivity patterns not only the degree but also the sense of Talaromyces emersonii NRRL-3221 vs. Talaromyces
selectivity can differ which may enrich the selection of thermophilus NRRL-2155 can behave significantly
the synthetically useful biocatalysts. In some instances, differently.
data observed with the two different isolates from the
Because a wide range of enantiotopic selectivity was
same strain [e.g., Talaromyces emersonii NRRL-3221: observed in the asymmetric acetylation of 3a, it seemed
(LIP2), 94% ee vs. (LIP1), 67% ee of (R)-4a or worthwhile to investigate the acylation reaction of 3b as
Thermoascus thermophilus NRRL-5208: (LIP2), 53% well. The prochiral 3b has also been tested with a
ee vs. (LIP1), 71% ee of (S)-4a] clearly indicate that selection of commercially available enzymes for asym-
thermophilic fungi produce different extracellular hy- metric acylation,[32b] but only moderate enantiotopic
drolases with remarkably different selectivities. Thus, selectivities towards the formation of (S)-4b were found.
814
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Novel Hydrolases from Thermophilic Filamentous Fungi
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Table 4. Acetylation of 2-(4'-toluenesulfonyl)oxypropane-1,3-diol (3b) with thermophilic fungal enzymes.
Enzyme
t [h]
4b
Strain, Collection No (Medium)
m [mg]
Yield [%]
Config.
ee [%]
Talaromyces emersonii NRRL-3221 (LIP2)
Sporotrichum thermophile ATCC-36.347 (LIP1)
Thermomyces lanuginosus CBS-224.63 (LIP2)
Sporotrichum thermophile ATCC-36.347 (LIP2)
Talaromyces emersonii NRRL-3221 (LIP1)
Myceliophthora thermophila TUB-F-39 (LIP1)
Thermomyces lanuginosus CBS-224.63 (LIP1)
Talaromyces thermophilus NRRL-2155 (LIP2)
Humicola grisea var. thermoidea CBS-183.64 (LIP2)
Sporotrichum thermophile ATCC-36.347 (LIP2)
103
248
86
245
100
232
69
168
168
178
168
72
168
168
168
168
4
0.5
168
10
168
21
10
44
61
76
29
52
12
8
45
16
23
82
5
R
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
4
2
8
13
16
17
21
23
32
41
42
45
50
51
52
53
61
196
157
50
[32b]
Lipozyme IM
Thermoascus thermophilus NRRL-5208 (LIP1)
Thermomyces lanuginosus ATCC-38.905 (LIP2)
Thermoascus thermophilus NRRL-5208 (LIP2)
Paecilomyces sp. TUB-F-70 (LIP2)
52
100
174
78
24
31
28
45
Talaromyces thermophilus NRRL-2155 (LIP1)
74
vinyl acetate catalysed by a series of acetone-dried
extracellular enzyme preparations indicated that these
enzymes might be superior in synthetic biotransforma-
tions over the commercialised thermophilic fungal
lipases with respect to the degree of enantiomer
selectivity or direction/degree of enantiotopic selectiv-
ity.
Experimental Section
Scheme 3. Determination of the absolute configuration of
(R)-4a by chemical correlation with (R)-5.
Materials and Methods
The NMR spectra were recorded in CDCl3 on a Bruker DRX-
500 spectrometer (at 500 MHz for H and 125 MHz for 13C
1
spectra); chemical shifts are expressed in ppm (d scale). IR
spectra (film) were taken on a Specord 2000 Series spectro-
photometer; bands are listed in cmÀ1 (n). GC analyses were
carried out on HP 5890 or Agilent 4890D instruments
equipped with FID detector and HP Chiral column (30 m Â
0.32 mm, 0.25 mm film of 20% permethylated b-cyclodextrin;
HP Part No.: 190916-B213) using H2 carrier gas (oven: 1008C,
injector: 2508C, detector: 2508C, head pressure: 10 psi, 50:1
split ratio). Optical rotations were determined on a Perkin
Elmer 241 polarimeter. TLC was carried out on Kieselgel 60
F254 (Merck) sheets. Spots were visualised by treatment with
The best result with commercially available enzymes
was achieved with Lipozyme IM (immobilised Rhizo-
mucor miehei lipase),[32b] thus this reaction providing
(S)-4b with 41% ee is also listed in Table 4 for
comparison. Although a wider selectivity range than
with the previously tested commercial lipases[32b] was
observed [from (R)-4b of 4% ee with Talaromyces
emersonii NRRL-3221 (LIP2) to (S)-4b of 53% ee with
Talaromyces thermophilus NRRL-2155 (LIP1)], these
selectivities are still unsatisfactory for practical purpos- 5% ethanolic phosphomolybdic acid solution and heating of
the dried plates. All solvents were freshly distilled prior their
use.
es.
Racemic 1-phenylethanol (rac-1) and vinyl acetate used in
enantiomer selective acylations were products of Aldrich. 2-
Benzoyloxypropane-1,3-diol (3a) and 2-(4'-toluenesulfonyl)-
oxypropane-1,3-diol (3b) as test substrates for the enantiotopic
selective biotransformations were prepared by our established
methods.[32] Lipozyme TL IM was a product of Novozymes,
Denmark. The inorganic salts and materials for biomass
production were the corresponding products of Sigma, Aldrich
Conclusions
Our present study indicates that thermophilic filamen-
tous fungi are a promising and not yet fully exploited
source of hydrolases with valuable biocatalytic proper-
ties. The results from three stereoselective test reactions
acylation of rac-1, 3a and 3b in organic media with or Fluka.
Adv. Synth. Catal. 2003, 345, 811 818
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Viktoria Bodai et al.
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Microorganisms
Preparation of the Biocatalysts
Thermophilic fungi have been purchased from the following
culture collections: ATCC (American Type Culture Collec-
tion, Manassas, Virginia); CBS(Centraalbureau voor Schim-
melcultures, Utrecht, The Netherlands; NRRL (Northern
Regional Research Center, USDA, Peoria, Illinois); TUB
(Technical University of Budapest, Hungary); WFPL (Western
Forest Products Laboratory, Vancouver, Canada). The TUB
strains have the following accession numbers at the ATCC
The main portion of the supernatants was treated with acetone
(2 3 v/v). The forming precipitate was filtered and treated
with acetone (2 3 v/v). The residual solvent from the
precipitate was evaporated in vacuum and the resulting dry
powder was stored at 48C in sealed tubes. Many of the
preparations maintained almost unaltered biocatalytic activity
over a year.
ˆ
collection: Chaetomium thermophilum TUB F-69 ( ATCC
ˆ
58136), Myceliophthora thermophila TUB F-39 ( ATCC
Enantiomer-Selective Acetylations of Racemic 1-
Phenylethanol (rac-1)
ˆ
58152), Paecilomyces sp. TUB F-70 ( ATCC 58155) and
ˆ
Thermoascus aurantiacus TUB F-43 ( ATCC 58156).
Suspensions of the enzyme preparations (20 mg) with a
solution of rac-1 (20 mg) in hexane (2 mL) and vinyl acetate
(0.5 mL) were shaken at 1000 rpm in sealed glass vials at room
temperature for the time indicated in Table 2. The conversions
were checked by TLC (hexane-acetone, 10:4, v/v). At the
reaction time indicated in Tables 2, the enzyme was filtered off
and the enantiomeric compositions and the ratios of the
product and residual substrate fractions were analysed by GC
on a chiral stationary phase (Table 2). GC retention times:
7.64 min, (S)-2; 8.04 min, (R)-1; 8.23 min, (R)-2; 8.65 min, (S)-
1; GC molar response factor of acetate/alcohol (rac-2/rac-1):
1.18 [used for calculations of c and E throughout in Table 2].
Table 2 lists only those reactions which exhibited more than
5% conversion after 168 h.
The fungi were grown and maintained on potato dextrose
agar (PDA) slants at 458C until appropriate sporulation then
stored at room temperature. Fully sporulated Petri plate PDA
cultures were harvested by washing with 0.1% Tween-80
containing water and the dense spore suspension was used for
shake flask inoculation. Viable spore content was determined
by serial dilution and colony forming unit (CFU) counting on
PDA at 458C.
Shake Flask Fermentation
A preparative scale conversion of rac-1 (200 mg) catalysed
by Lipozyme TL IM (200 mg) was also performed in hexane
(20 mL) and vinyl acetate (5 mL). After shaking the reaction
mixture at 1000 rpm, room temperature for 5 h, the enzyme
was removed by filtration. The solvent was distilled off from
the filtrate by rotary evaporation and the residue was
separated by vacuum chromatography (silica gel, hexane-
acetone, 10:1, v/v) to give alcohol (S)-1 (yield: 156 mg 78%;
23% ee by GC); [a]D22: À 11.1 (c 1.0, CHCl3) {lit.: [a]D: À 45.3
(CHCl3);[35] [a]D: À 53.5 (c 1.13, CHCl3);[36] [a]D25: À 55.1 (c 1.63,
CHCl3)[37]}; IR: n ˆ 3352, 3008, 2976, 2952, 1466, 1430, 1368,
1204, 1080, 1008, 900, 760, 690 cmÀ1; 1H NMR: d ˆ 1.513 (d, J ˆ
For shake flask fermentation, 150 mL medium was distributed
into cotton-plugged 500-mL Erlenmeyer flasks, and sterilised
at 1218C for 30 min. The flasks were inoculated to a final
concentration of 1 Â 106 spores of the respective fungus/mL,
and incubated on a rotary shaker at 458C and 300 rev/min for 3
days. Fourteen thermophilic filamentous fungal strains were
screened, each in two different shake flask media (LIP1 and
LIP2). The compositions were as follows (in g/L):
LIP1: olive oil, 10; sucrose, 10; defatted soybean meal, 2.5;
KH2PO4, 2; CaCO3, 1.5; (NH4)2HPO4, 1; Tween-80, 0.5;
MgSO4 ¥ 7 H2O, 0.5; NaCl, 0.5; NaNO3, 0.5; trace element
solution, 0.5.
LIP2: olive oil, 10; corn meal, 15; KH2PO4, 2; CaCO3, 2;
Tween-80, 2; (NH4)2HPO4, 1.5; corn steep liquor, 1; MgSO4 ¥
7 H2O, 0.5; NaCl, 0.5; KNO3, 0.5; trace element solution, 0.5.
Trace element solution (g/L): MnSO4, 1.6; ZnSO4 ¥ 7 H2O,
3.4; CoCl2 ¥ 6 H2O, 2; FeSO4 ¥ 7 H2O, 5.
ˆ
6.9 Hz, 3H, 2-CH3), 3.38 (br s, 1H, OH), 4.861 (q, J 6.3 Hz, 1H,
1-CH), 7.40 (mc, 5H, Ar H); 13C NMR: d ˆ 25.034, 69.843,
125.071, 126.862, 127.970, 145.525 and acetate (R)-2 (yield:
43 mg, 16%; 97% ee by GC); [a]D22: 99.0 (c 1.0, CHCl3) {lit.:
[a]2D0: 103.5 (c 1.0, CHCl3);[38] [a]D: 105.1 (c 1.3, CHCl3)[39]}; IR:
1
n ˆ 1740, 1456, 1376, 1244, 1208, 1064, 944, 760, 700 cmÀ1; H
NMR: d ˆ 1.574 (d, J ˆ 7.4 Hz, 2-CH3), 21.103 (s, 3H, Ac CH3),
ˆ
5.926 (q, J 6.9 Hz, 1H, 1-CH), 7.30 7.40 (m, 5H, Ar H);
13C NMR: d ˆ 21.416, 22.283, 72.241, 125.860, 127.632, 128.258,
141.422, 169.946; both as colourless oils. IR and NMR data
agreed with the reported spectra of (S)-1[40] and (R)-2.[41]
Lipase/Carboxylesterase Activity Tests from
Supernatants
The whole fermentation broths of the resulting 28 cultures
were centrifuged (3000 rpm, 10 min at 208C) and aliquots from
the supernatants were used for enzyme activity measurements.
The hydrolase activities were determined by three different,
well established and properly adopted activity tests:
Method A: titrimetric method using olive oil as substrate;[34]
Method B: spectrophotometric method using p-nitrophenyl
palmitate as substrate;[25]
Enantiomeric Excess Determination of 4a and 4b
The racemic or optically active 3-acetoxy-2-acyloxypropan-1-
ols (4a or 4b, 9 12 mg), pyridine (25 mL) and 4-(N,N-
dimethylamino)pyridine (2 mg) were added to a solution of
5% (R)-MTPA-Cl in CCl4 (350 mL) and the mixture was heated
in a sealed ampoule at 508C for 3 h. The resulting mixture was
successively washed with 5% HCl solution (1 mL), saturated
Na2CO3 solution (1 mL) and brine (1 mL). The organic phase
was dried over Na2SO4 and the solvent was evaporated. The
Method C: spectrophotometric method using p-nitrophenyl
butyrate as substrate.[25]
816
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
Adv. Synth. Catal. 2003, 345, 811 818

Novel Hydrolases from Thermophilic Filamentous Fungi
FULL PAPERS
diastereomeric excess (de) values of the forming MTPA esters
(s, 3H, CH3), 3.73 (m, 2H, 3-CH2), 4.37 4.47 (dd, 2H, 1-CH2),
4.57 (dd, 2H, CH2-Ph), 5.48 (m, 1H, 2-CH), 7.26 8.06 (m, 10H,
2 Â Ar).
(MTPA-4a and MTPA-4b) were determined from their
1
1H NMR spectra (500 MHz, CDCl3). The following H NMR
signals were used for de determinations: MTPA-4a: 3.52 (s) and
3.54 (s); MTPA-4b: 1.93 (s); 1.96 (s).
(R)-3-Acetoxy-2-benzoyloxypropan-1-ol (R)-4a
To a solution of (R)-6 (1.64 g, 5.0 mmol) in i-PrOH (16 mL)
10% Pd-C catalyst (100 mg) was added and the resulting
mixture was hydrogenated at room temperature under atmos-
pheric pressure for 1 h. After the catalyst was removed by
filtration, the solvent was evaporated off from the filtrate in
vacuum and the residue was purified by vacuum chromatog-
raphy (silica gel, hexane-acetone, 10:1, v/v) to give (R)-4a as a
colourless oil, yield: 1.09 g (92%); [a]2D0: À 27.2 (c 1.0, MeOH);
the IR and 1H NMR spectra were indistinguishable from those
of product (R)-4a prepared with Talaromyces emersonii,
NRRL-3221 (LIP2).
Asymmetric Acetylation of the Prochiral Diols 3a and
3b
To the solution of prochiral diol 3a or 3b (200 mg) and vinyl
acetate (0.8 mL) in THF (2 mL) and hexane (2 mL) fungal
enzyme preparation was added (for quantities, see Tables 3and
4) and the mixture was stirred at room temperature (for
reaction times, see Tables 3 and 4). The conversion was
checked by TLC (hexane-acetone, 10:4, v/v). At the reaction
time indicated in Tables 3 and 4, the enzyme was filtered off,
the solvent was removed from the filtrate in vacuum and the
residue was purified by vacuum chromatography (silica gel,
hexane-acetone, 10:1, v/v) to give 4a or 4b (yield, configuration
and enantiomeric excess data are listed in Tables 3 and 4; data
are shown only for those reactions which exhibited more than
10% conversion after 168 h).
Yield, configuration and enantiomeric excess data for 4a are
listed in Table 3. As an example, data for (R)-4a prepared by
Talaromyces emersonii NRRL-3221 (LIP2) enzyme are given
here. Yield: 211 mg (87%); 94% ee by 1H NMR of MTPA-4a;
[a]D22: À 26.9 (c 1.0, EtOH), {lit.: [a]D: À 27.4 (c 1, EtOH), 96%
ee[32a]}; IR: n ˆ 3390, 2956, 2908, 2848, 1732, 1720, 1586, 1476,
1438, 1352, 1274, 1108, 1016, 948, 708 cmÀ1; 1H NMR: d ˆ 2.09
(s, 3H, CH3), 3.30 (br s, 1H, OH), 3.89 (m, 2H, 1-CH2), 4.43
Acknowledgements
The Hungarian OTKA Foundation (T-033112) and the Na-
tional R&D Project, Ministry of Education (NKFP3-35-2002)
are gratefully acknowledged for financial support. J. B. and
G. E. thank the OTKA for the postdoctoral fellowships (D-
32705 and D-29445). Thanks are due to Unilever Hungary Rt
for donation of an HP 5890 gas chromatograph.
ˆ
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ˆ
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ˆ
¬
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1602, 1584, 1453, 1372, 1265, 1124, 1034, 725 cmÀ1; H NMR:
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