Stereoselective reduction of carbonyl compounds using the cell-free extract of the earthworm, Lumbricus rubellus, as a novel source of biocatalyst

Article abstract of DOI:10.1271/bbb.60408

We found the reducing activity toward carbonyl compounds in the cell-free extract of the earthworm, Lumbricus rubellus. The earthworm extract had a reducing activity for keto esters in the presence of NADH or NADPH as a coenzyme. The earthworm extract red

Full text of DOI:10.1271/bbb.60408

Biosci. Biotechnol. Biochem., 70 (12), 3077–3080, 2006
Note
Stereoselective Reduction of Carbonyl Compounds
Using the Cell-Free Extract of the Earthworm,
Lumbricus rubellus, as a Novel Source of Biocatalyst
y
1;
2
Kohji ISHIHARA and Nobuyoshi NAKAJIMA
1Department of Life Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan
2Industry, Government, and Academic Promotional Center, Regional Cooperative Research Organization,
Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197, Japan
Received July 18, 2006; Accepted August 4, 2006; Online Publication, December 7, 2006
[doi:10.1271/bbb.60408]
We found the reducing activity toward carbonyl
compounds in the cell-free extract of the earthworm,
Lumbricus rubellus. The earthworm extract had a
reducing activity for keto esters in the presence of
NADH or NADPH as a coenzyme. The earthworm
extract reduced ethyl 3-methyl-2-oxobutanoate to the
corresponding alcohol with a high enantiomeric excess
(91%, R-form) at 50 ꢀC in the presence of NADH. In
particular, ethyl 2-oxoheptanoate was exclusively re-
duced to the corresponding (R)-hydroxyl ester with a
high enantiomeric excess (> 99%).
also useful biocatalysts.15,16) Furthermore, they reported
that one of the earthworm serine proteases acts on
the hydrolysis of triacylglycerols.17) However, little
information is known about the possibility that the
earthworm is a source of biocatalyst in biotransforma-
tions.
Here we report the reduction of carbonyl compounds
using the cell-free extract of the earthworm, L. rubellus.
p-ABSF (4-(2-aminoethyl)benzenesulfonate fluo-
ride), a trypsin inhibitor (from soybean), and ethyl
pyruvate were purchased from Wako Pure Chemicals,
Tokyo, Japan. Ethyl 3-methyl-2-oxobutanoate was
obtained from Aldrich Chemicals, Boston, MA, USA.
Ethyl benzoylformate and ethyl 2-methyl-3-oxobuta-
noate were purchased from Tokyo Kasei Kogyo, Tokyo,
Japan. Other ꢀ-keto esters and ꢀ-hydroxy esters were
synthesized according to standard methods.4) The earth-
worms (L. rubellus, brand name ‘‘Rintarou’’) were
purchased from Nobeoka Asahi Senni (Miyazaki, Japan;
E-mail: nakamura.yr@om.asahi-kasei.co.jp). NADPH
and NADH were obtained from Kohjin, Tokyo, Japan.
All other chemicals used in this study were of analytical
grade and are commercially available.
One hundred earthworms (42 g of fresh L. rubellus)
were suspended in 95 ml of 50 mM potassium phosphate
buffer (KPB) (pH 7.0). This suspension was cooled
below 4 ꢀC and subsequently homogenated using a
household mixer for 60 s at room temperature. Cell
debris in the homogenate was removed by centrifugation
at 10;000 ꢁ g for 20 min at 4 ꢀC. p-ABSF (final con-
centration, 10 mM) and a soybean trypsin inhibitor (final
concentration, 0.1 mM) were added as protease inhib-
itors. The mixture solution was stirred gently for 30 min
in an ice-bath, and then the solution was centrifuged at
13;000 ꢁ g for 30 min at 4 ꢀC. The supernatant (98.5 ml)
served as the crude cell-free extract. The reducing
activity of the cell-free extract was spectrophotometri-
cally determined. The reaction mixture, in a total
Key words: earthworm; biocatalyst; keto ester; stereo-
selective reduction; cell-free extract
Biotransformations of exogenous substrates have
been widely studied in order to synthesize chiral
compounds.1–3) Microbial reduction of carbonyl com-
pounds is one of the convenient methods for obtaining
optically pure alcohols. For example, bakers’ yeast
(Saccharomyces cerevisiae) has often been used for the
reduction of keto esters to obtain optically active
hydroxyl esters.4,5) Furthermore, other microorganisms
(such as yeast,6) aerobic bacteria,7–9) and microalgae10)
)
or plant cultured cells3) that can catalyze the stereo-
selective reduction of keto esters are also used for the
preparation of chiral hydroxyl esters. As described
above, the biotransformation using microorganisms or
plant cells as biocatalysts has been widely investigated,
however, the bioconversion using other organisms, such
as invertebrates, has rarely been reported.
The earthworm belongs to the annelida phylum (in the
animalia kingdom) and is a decomposer in the global
ecosystem. It has long been used as a drug in Chinese
medicine under the name of ‘‘Jiryu.’’11,12) Nakajima et
al. reported that an earthworm, Lumbricus rubellus,
secreted alkaline serine proteases having a potent
fibrinolytic activity,13,14) and that these proteases were
y
To whom correspondence should be addressed. Fax: +81-86-256-9496; E-mail: ishihara@dls.ous.ac.jp

3078
K. ISHIHARA and N. NAKAJIMA
Table 1. Relative Activity of the Cell-Free Extract of the Earth-
worma,b
volume of 1.0 ml, contained 5.0 mM of the substrate,
0.15 mM of the coenzyme in 0.1 M KPB (pH 7.0), and a
limited amount of the enzyme solution. Consumption of
the reduced coenzyme was followed using a Beckman
DU-640 spectrophotometer at 340 nm at 37 ꢀC. One unit
(U) of enzyme activity was defined as the amount of
enzyme catalyzing the oxidation of 1 mmol NAD(P)H
per min under the specified conditions.
Relative rate (%)c
NADH NADPH
Relative rate (%)c
NADH NADPH
Substrate
Substrate
O
O
O
100
30
46
33
11
13
26
5
2
0
0
0
3
1
0
0
0
CO2Et
O
O
O
O
O
O
O
O
OAc
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
In a polypropylene tube were placed the cell-free
extract (0.1 units), NAD(P)H (11 mmol), the substrate
(10 mmol), and 0.1 M KPB (pH 7.0, total volume, 0.45
ml). The mixture was gently shaken at 37 ꢀC or 50 ꢀC.
After 6 h, the mixture was filtered using an Extrelutꢀ NT
(Merck, Darmstadt, Germany) short column, extracted
with ether, and then concentrated under reduced pres-
sure. The conversions of the products and chemical
yields were determined by GLC equipped with a cap-
illary column (DB-WAX) using an internal standard.
The enantiomeric excesses (e.e.) of the products were
determined using a GLC equipped with an optically
active capillary column (Chirasil-DEX CB, 0:25 mm ꢁ
25 m and Gamma DEXꢁ 225, 0:25 mm ꢁ 25 m). The
absolute configuration of the isomer was determined by
comparing its retention time with those of authentic
samples prepared according to standard methods.4)
The cell-free extract of the earthworm was tested for
its reducing ability toward various carbonyl compounds.
The results of the reducing activities in the presence of
NAD(P)H are summarized in Table 1.
10
OMe
Cl
O
O
30
Ph
Ph
38
O
O
19
13
0
0
Ph
O
10
6
5
4
0
0
0
0
Ph CO2Et
Ph
O
O
O
Cl
OEt
OEt
O
O
O
4
3
2
1
0
0
0
0
O
O
O
OEt
aThe reducing activity (relative activity) was measured in 0.1 M KPB
(pH 7.0) at 37 ꢀC.
bThe substrate concentration was 5 mM.
cRelative rates were determined by setting the activity of ethyl pyruvate in
the presence of NADH to 100.
It was found that the extract of the earthworm had
a reducing activity toward various ꢀ- and ꢁ-keto esters
in the presence of both NADH and NADPH as the
coenzyme. One hundred earthworms (42 g) gave a
supernatant (98.5 ml), and the activity toward ethyl
pyruvate in the presence of NADPH was 1.12 units/ml
of the extract (total activity, 1:10 ꢁ 102 units). The
specific activity was 1:53 ꢁ 10ꢂ1 units/mg of protein.
In the reducing activity toward ethyl pyruvate, the
specific activity of the earthworm extract (1:53 ꢁ
10ꢂ1 units/mg) had a higher value than that of the
yeast cell-free extract (4:86 ꢁ 10ꢂ2 units/mg).18) This
high specific activity of the earthworm extract is an
advantage for the reduction of ꢀ-keto esters. As the alkyl
chain length of the substrate molecule (ꢀ-keto esters)
become longer, the reducing activity decreased. No
catalytic activity toward alkyl phenyl ketones, methyl
ketones, or other ketones (such as 1-methoxy-2-prop-
anone, phenacyl chloride and menthone) was detected.
No coenzyme regeneration (reduction of NAD(P)þ) was
observed in the presence of D-glucose, glycerol, meth-
anol, ethanol, 1-propanol, 2-propanol, cyclopentanol,
cyclohexanol, or lactate as a substrate (data not shown).
The stereoselectivity of the reduction toward ꢀ-keto
esters was investigated by a GLC analysis, as shown in
Table 2.
low. The selectivity in the reduction of ethyl 3-methyl-2-
oxobutanoate was improved by raising the reaction
temperature from 37 to 50 ꢀC. Ethyl 2-oxoheptanoate
was converted by the earthworm extract to the corre-
sponding alcohol with a high conversion ratio and high
e.e., but the yield of the alcohol was less than 60% (data
not shown). To clarify the high selectivity during the
reduction biotransformation of the hydroxyl ester as a
substrate was then carried out (see Table 3).
The e.e. of the residual hydroxyl ester shifted to the R-
form with the reaction time, in particular, the shift of
ethyl 2-hydroxyheptanoate was remarkable. These re-
sults indicate that the enantioselective reduction of the
substrate (ethyl 2-oxoheptanoate) and setereoselective
hydrolysis of the produced alcohol (ethyl 2-hydroxy-
heptanoate) take place during the biotransformation of
ethyl 2-oxoheptanoate using the earthworm extract. The
stereoselective hydrolysis is probably involved in the
activity of a protease, isozyme C, in the earthworm
having an esterase-like activity.16) In fact, Ushio et al.
reported that bakers’ yeast catalyzed the enantioselec-
tive hydrolysis of ꢀ-hydroxy ester and that the hydrol-
ysis activity was inhibited by butylsulfonyl fluoride as
an esterase inhibitor.19)
Aliphatic ꢀ-keto esters were reduced by the earth-
worm extract in high conversion ratios, however, the
enantioselectivities of the produced hydroxyl esters were
Thus, we have demonstrated the reduction of carbonyl
compounds, such as keto esters, using the cell-free

Reduction of Carbonyl Compounds Using Earthworm Extract
3079
Table 2. Redcution of ꢀ-Keto Esters Using the Cell-Free Extract of Lumbricus rubellusa
37 ꢀC
50 ꢀC
Substrate
NADH
NADPH
NADH
NADPH
Conv. (%) e.e. (%)b (R/S)b Conv. (%) e.e. (%)b (R/S)b Conv. (%) e.e. (%)b (R/S)b Conv. (%) e.e. (%)b (R/S)b
O
>99
>99
>99
>99
>99
45
7
(S)
(S)
(S)
(S)
(R)
>99
>99
>99
>99
>99
69
29
(S)
(S)
(S)
(R)
(R)
>99
>99
>99
>99
>99
30
11
7
(S)
(R)
(R)
(S)
(R)
>99
>99
>99
>99
>99
64
5
(S)
(S)
(S)
(R)
(R)
CO2Et
O
O
O
O
O
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
10
10
10
21
59
15
1
6
>99
>99
50
>99
11
61
(R)
(R)
>99
20
40
(R)
(R)
>99
91
41
(R)
(R)
>99
86
10
(R)
(R)
O
46
24
8
7
Ph CO2Et
aThe cell-free extract (0.1 units), substrate (10 mmol), coenzyme (NADH or NADPH) (11 mmol), and 0.2 M potassium phosphate buffer (pH 7.0, total volume 0.45 ml)
were incubated for 6 h.
bEnantiomeric excesses and absolute configuration were measured by GLC analysis using optically active capillary columns.
Table 3. Biotransformation of ꢀ-Hydroxy Esters in the Earthworm
Extracta
Acknowledgments
This work was supported by financially by the Grant
After 1 h
After 2 h
After 6 h
Substrate
from Japan Bioindustry Association (2005). This work
was partially supported financially by The Asahi Glass
Foundation (2003–2005), Japan.
e.e. (%)b (R/S)b e.e. (%)b (R/S)b e.e. (%)b (R/S)b
OH
1
8
(R)
(S)
(R)
(R)
2
7
(R)
(S)
(R)
(R)
6
3
(R)
(S)
(R)
(R)
CO2Et
OH
CO2Et
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OH
CO2Et
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