Apple-tree shoots and transformed carrot and apple roots used as biocatalysts in enantioselective acetate hydrolysis, alcohol oxidation and ketone reduction

Article abstract of DOI:10.1135/cccc19981655

(±)-1-Phenylethyl (1), (±)-1-(1-naphthyl)ethyl (2), (±)-1-(2-naphthyl)ethyl (3) and (±)-menthyl (4) acetates were hydrolyzed using apple-tree shoots and hairy roots of carrot and apple-tree to afford alcohols, which, subsequntly, in the same environment, were oxygenated to ketones. Pure (S)-1-(2-naphthyl)ethanol ((S)-(-)-7), (R)-1-phenylethyl acetate ((R)-(+)-1), (R)-1-(1-naphthyl)ethyl acetate ((R)-(+)-2). (S)-1-phenylethanol ((S)-(-)-5) and (-)-menthol (1R,25,5R)-(-)-8) have been produced.

Full text of DOI:10.1135/cccc19981655

Biotransformations
1655
APPLE-TREE SHOOTS AND TRANSFORMED CARROT AND APPLE
ROOTS USED AS BIOCATALYSTS IN ENANTIOSELECTIVE ACETATE
HYDROLYSIS, ALCOHOL OXIDATION AND KETONE REDUCTION*
a
b
Agnieszka MIRONOWICZ and Krystyna KROMER
a Department of Chemistry, Agricultural University of Wroclaw, ul. Norwida 25, 50-375 Wroclaw,
Poland; e-mail: amiron@ozi.ar.wroc.pl
b Botanical Garden of Wroclaw University, ul. Sienkiewicza 23, 50-335 Wroclaw, Poland
Received May 13, 1998
Accepted July 21, 1998
Dedicated to Dr Jan Fajkos on the occasion of his 75th birthday.
(±)-1-Phenylethyl (1), (±)-1-(1-naphthyl)ethyl (2), (±)-1-(2-naphthyl)ethyl (3) and (±)-menthyl (4)
acetates were hydrolyzed using apple-tree shoots and hairy roots of carrot and apple-tree to afford
alcohols, which, subsequntly, in the same environment, were oxygenated to ketones. Pure (S)-1-(2-
naphthyl)ethanol ((S)-(–)-7), (R)-1-phenylethyl acetate ((R)-(+)-1), (R)-1-(1-naphthyl)ethyl acetate
((R)-(+)-2), (S)-1-phenylethanol ((S)-(–)-5) and (–)-menthol (1R,2S,5R)-(–)-8) have been produced.
Key words: Biotransformations; Enantioselective hydrolysis and reduction; Enzymatic resolution;
Plant tissue cultures; Hairy roots; Shoots; Malus pumila; Daucus carota.
Esters of secondary alcohols are transformed both by microorganisms2,3 and plants4–6
.
In our earlier studies some parts of plants7–9, cell cultures of carrot10 and whole plants10
had been used for biotransformation of title substrates. The biotransformations resulted
in separation of enantiomers.
The aim of this study was to establish the ability of apple-tree roots and shoots and
carrot-roots in vitro culture to transform racemic esters.
The plant tissues transformed by Agrobacterium rhizogenes and hairy roots obtained
in this way seem to be a promising tool in modern biotechnology. Hairy roots are char-
acterized by a very efficient biomass increase and when the enzymes are released to the
medium, it is possible to use them efficiently after immobilization in bioreactors. The
comparison of the results of biotransformation carried out by underground and over-
* Part XLVI in the series Biotransformations; Part XLV see ref.1.
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Mironowicz, Kromer:
ground parts (root, bulb, shoot and fruit) of the same plant is likely to provide more
information on the potential role of its enzyme system in biocatalysis.
In the present investigation, the explants of the same (apple and carrot8,10) plants:
genetically modified (Agrobacterium rhizogenes) and non-transformed carrot (Daucus
carota) and apple-tree (Malus pumila) roots (underground parts of plant) as well as
apple shoots (overground parts) were used. Only a few papers on the comparison of the
abilities of various parts of a plant to transform exogenous substrates have been pub-
lished so far. In this study, racemic acetates of secondary alcohols and methyl acetate
were used as substrates. Such a selection of the substrates allowed us to examine the
enantioselectivity of the enzyme systems of apple and carrot tissues.
Lactones are the compounds occurring in apple fruits. Apple (fruit) possesses the
enzymatic system capable of lactonizing exogenous hydroxyacids11. The enzymatic
system of the carrot is capable of a transformation of steroids5,12. Therefore, in our
investigations, ricinoleic (12-hydroxyoleic) acid and testosterone were also subjected to
the transformation with the use of a carrot and an apple shoot and root cultures.
EXPERIMENTAL
Chromatography
GC: Hewlett–Packard 5890, FID, carrier gas – H2 using Chrompack WCOT Fused Silica, Chirasil-Val
(0.25 mm × 25 m) capillary column for 1 (column temperature 125 °C, injection temperature 250 °C),
for 2 and 3 (column temperature 100 °C/5 min; 0.5 °C/1 min; 116 °C const., injection temperature
250 °C) and Chirasil-DEX CB (0.25 mm × 25 m) for 4 (column temperature 80 °C/1 min; 1 °C/1 min;
110 °C const., injection temperature 250 °C).
TLC: Silica gel 60 F254 pre-coated aluminium sheets (layer thickness 0.2 mm, Merck) with n-hexane-
EtOAc (8 : 1) for 1 and 2, n-hexane–Et2O (5 : 1) for 3 and benzene–EtOAc (10 : 3) for 4.
CC: silica gel with n-hexane–acetone (20 : 1 for 1, 30 : 1 for 2 and 35 : 1 for 3).
Biocatalysts
Cultures of M7 Apple Rootstock Shoots as Biocatalysts
Proliferating cultures of M7 apple shoots were obtained from isolated meristems. Meristems were
collected from field growing plants in the period of their intensive growth in June and July. After
being disinfected, isolated meristems were inoculated on Murashige Skoog (MS) medium13 contain-
ing 0.2 mg/dm3 of kinetin and 0.2 mg/dm3 of indole-3-acetic acid (IAA). For multiplication purposes,
developing shoots were transferred on the Quoirin and Lepoivre (QL) medium14 with half a macro-
nutrient concentration, enriched with benzyladenine (BA, 0.5 mg/dm3), indole-3-butyric acid (IBA,
0.1 mg/dm3) and gibberellic acid (GA3, 2.0 mg/dm3). Saccharose (20 g/dm3) and agar (10 g/dm3)
were added to the medium. Before sterilization, pH was adjusted to 5.8–6.0 with 0.1 M NaOH. Cul-
tures were maintained in a growth chamber at 25–27 °C and illuminated (2 500 lx) by using fluores-
cent Day Light type tubes in a 16 h photoperiod.
Apple shoots 2.0–2.5 cm long were taken from 10 week old culture. They were inoculated in 200 cm3
flasks containing 25 cm3 of a liquid medium. Ten shoots were transferred to each flask and then
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Biotransformations
1657
substrates were added. The medium composition was the same as that stimulating multiplication of
shoots, the only difference being the agar omission.
Transformation of Plant Tissues with A4 Agrobacterium rhizogenes Strain
Carrot – Daucus carota. Edible roots of carrot after being disinfected (20% Monochloramine, 20 min)
were cut into 1.5 cm slices. Explants of dimensions 1.0 × 1.5 cm, consisting of phloem, xylem and
cambium, were isolated. The apical ends of the explants were immersed into a 7-day culture of A4
Agrobacterium rhizogenes strain and inoculated on MS agar medium with inverted orientation. For
the growth of bacteria population Rhizogelose medium (in 1 dm3: yeast extract 5 g, casamino acids
0.5 g, mannitol 8 g, (NH4)2SO4 2 g, NaCl 5 g, Bactoagar 15 g, biotin 0.8 cm3/dm3, pH 6.6) was used.
After 2–3 weeks formed hairy roots were cut out and transferred to a liquid QL medium with half a
macronutrient concentration, containing IAA in the amount of 1.0 mg/dm3. In order to eliminate the
Agrobacterium, two antibiotics were added to the medium: Carbeniciline and Cefotaxim (Polfa, Tar-
chomin, Poland), 500 mg/dm3 of each. After 8 days (two changes of medium), the transformed roots
were subcultured to a medium without antibiotics and the sterility of cultures was subsequently vis-
ually checked.
Non-transformed roots were obtained from the same explants as the transformed ones. They were
cultured on the agar MS medium with a half concentration of macronutrients and the addition of IBA
(0.5 mg/dm3). The roots regenerated from explants on that medium and those obtained via the so-
matic embryogenesis were used for the comparative studies.
According to Pierik15 adventitious roots are mainly formed on the basal side of explants, which
results in more efficient formation of the roots when the apolar inoculation is used (upside down),
due to better oxygen availability.
M 7 Apple rootstocks – Malus pumila. Transformed apple roots were obtained by the infection of
the shoot sections followed by their inoculation in inverted orientation on a medium lacking phyto-
hormons, in which the concentration of salts was half of that of the QL medium. After 6 weeks, the
arising hairy roots were transferred to the medium containing antibiotics. Non-transformed com-
parative roots were induced on the QL medium with a half concentration of macronutrients and the
addition of IBA (1.0 mg/dm3).
The cultures of hairy roots and those genetically unchanged of both species under investigation
serving for biotransformation, were grown on a liquid QL medium with the reduced amount of mac-
ronutrients (1/2) and some addition of IAA (1 mg/dm3).
Transformations of root tissues was confirmed by opine test16. For biotransformation experiments
to each flask 5–7 fragments of the hairy roots 3–6 cm long were used.
Biotransformation
The 200 cm3 flasks containing plant material (approximately 0.01–0.28 g DW) were placed on
a shaker (WU 4 type, approximately 80 rpm, 8 h a day) at 25–27 °C and illuminated by the light of
intensity of 1 500 lx. Ten days after the placement of the plant material in a fresh medium (25 cm3)
5 mg of the substrate (0.003 mol of 1, 0.0023 mol of 2 and 3, 0.0025 mol of 4) dissolved in 0.5 cm3
of acetone was added. Dissolving of the substrates one day before application prevented the culture
from possible contamination that could be introduced with the chemicals used. The ten days’ period
of the delay was necessary to adapt the cultures to a fresh medium and, as well, to eliminate probable
contamination. After 14 days of incubation with substrates, the medium and plant material were ex-
tracted separately with CHCl3. As it was established that the transformation takes place both in plant
tissues and in the medium due to the presence of exzoenzymes, in the next biotransformations they
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Mironowicz, Kromer:
were both extracted together. The composition of the reaction mixture was determined by TLC and
GC.
The following amounts of plant material per one flask (dry mass, 105 °C, 12 h) after the experi-
ment were determined: apple shoots – 0.28 g, transformed and non-transformed carrot and apple
roots – 0.01–0.014 g. Each reaction for every substrate was carried out repeatedly in 3–7 flasks to
confirm the results and/or to obtain sufficient amounts of products for further study. Stability of the
substrates in the media was checked.
The amount of the substrate used for biotransformation (5 mg/25 cm3 of medium) was established
on the basis of the previous experiments10 with the use of the whole, intact ferns as biocatalysts
transforming substrates 1–4. At the substrate concentration of 0.12% (30 mg/25 cm3 of medium) the
survival of plants during the biotransformation was 5 to 54 days, depending on the substrate used and
the fern species (Nephrolepis exaltata, N. cordifolia and Cyrtomium falcatum). For a 14-day transfor-
mation, with the use of the material under examination, the concentration of the substrate (5 mg/25 cm3)
was chosen, at which 2/3 of the plant biomass survived (few signs of necrosis of leaves were ob-
served).
Under the same conditions, using the same biocatalysts, an attempt to transform a ricinoleic acid
and testosterone was made (5 mg/0.5 cm3 of acetone, 25 cm3 of a medium in 200 cm3 flasks).
Control biotransformation with the use of enzymes secreted to the medium. Before adding the sub-
strate to the plant culture, the biocatalysts were removed and, after 14 days, the culture was extracted
with CHCl3.
RESULTS AND DISCUSSION
The following racemic acetates were used for biotransformations: 1-phenylethyl (1),
1-(1-naphthyl)ethyl (2), 1-(2-naphthyl)ethyl (3) and menthyl (4). Each acetate was hy-
drolyzed to an appropriate alcohol which was subsequently oxygenated to a ketone
(Scheme 1).
OH
OH
C
O
C
OAc
Ar
C
CH
3
H C
3
Ar
Ar
CH
3
Ar
CH CH
+
+
3
H
H
racemic
( _ ) 1: S-(-) and R-(+)
+
S-(-)-5
S-(-)-6
S-(-)-7
R-(+)-5
R-(+)-6
R-(+)-7
9
( _ ) 2: S-(-) and R-(+)
10
11
+
( _ ) 3: S-(-) and R-(+)
+
1, 5, 9:
Ar = phenyl
2, 6, 10:
3, 7, 11:
Ar = 1-naphthyl
Ar = 2-naphthyl
+
+
+
OAc
OAc
OH
OH
O
1S, 2R, 5S-(+)-8
12
1R, 2S, 5R-(-)-4
1S, 2R, 5S-(+)-4
1R, 2S, 5R-(-)-8
SCHEME 1
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Biotransformations by Apple-Tree Shoots
Biotransformations of acetates (±)-1, (±)-2, (±)-3 and (±)-4 by apple-tree shoots result
in hydrolysis yielding 98% of 5, 15% of 6, 9% of 7 and 11% of 8, respectively. The
alcohol obtained in this way was oxidized to ketone only in the case of acetate (±)-1:
the transformation products are enantiomeric alcohols 5 and acetophenone (9) yielding
2%; other alcohols 6, 7, 8 are not oxidize to ketones.
Both the alcohols (±)-5, (±)-6, (±)-7 and (±)-8 and ketones 9, 10, 11 and 12 were also
subjected to transformations under the same conditions. The results obtained in the
experiments confirmed that only 1-phenylethanol ((±)-5) and acetophenone (9) were
transformed, whereas the other alcohols and ketones remained untransformed. During
a 14-day period of transformation of alcohol (±)-5 2% of acetophenone (9) was ob-
tained. (S)-(–)-Alcohol was oxidized a slightly faster than its (R)-enantiomer, predomi-
nating in the reaction mixture (e.e. 16%). Acetophenone (9), transformed under the
same conditions, was reduced to alcohol 5 (2%). The formation of (S)-(–)-alcohol was
faster (e.e. 60%).
The 14-day transformation of racemic 1-phenylethyl acetate ((±)-1) by apple-tree
shoots resulted in total transformation of the substrate and for this reason, the products
of transformations were analysed after 1, 2 and 5 days of incubation (Fig. 1).
Initially, the amount of acetophenone (9) produced in hydrolysis of 1-phenylethyl
acetate ((±)-1) increases, but then it decreases due to the reduction to alcohol 5, in
which (S)-enantiomer predominates (e.e. 11%).
Separation of enantiomers occurs during the hydrolysis of 1-phenylethyl acetate ((±)-1)
(Fig.1): (S)-(–) acetate is totally transformed after 50 h, while 20% of pure (R)-(+)
enantiomer (e.e. 100%) remains unreacted and can easily be separated using column
chromatography8.
100
80
60
40
20
0
0
2
4
6
8
10
12
14
16
Time, days
FIG. 1
Biotransformation of 1-phenylethyl acetate 1 in the presence of apple shoots; ∆ S-(–)-1, * 9, × R-(+)-1,
◆ R-(+)-5, ■ S-(–)-5
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Mironowicz, Kromer:
The hydrolysis of racemic menthyl acetate ((±)-4) also results in separation of
enantiomers: after 14-day transformation, the only enantiomer produced (11%) is
(1R,2S,5R)-(–)-8 (e.e. 100%).
The yield of hydrolysis of (±)-2, (±)-3, (±)-4 by apple shoots (the overground parts
of plant) is 6–7 times lower than that obtained in the presence of transformed apple
roots (Table I). It is also 4–8 times lower than that of the hydrolysis carried out by the
enzymes present in the fruit of the same plant8 (apples).
Biotransformation of all the acetates under investigation takes place inside the plant
tissues, which means that both the substrates and products are transported through the
cell membranes. It was also confirmed by separate extraction of the homogenized plant
material and of the medium. Quantitative analysis of the two extracts after the transfor-
mation of 1-phenylethyl acetate ((±)-1) shows that concentration of the products (in
particular acetophenone (9)) is higher in the plant material than in the medium.
Additionally, it was shown that enzymes released to the medium by a biocatalyst
(apple shoots) cause biotransformation of acetates 1–4. Substrates (1–4) were added to
the flasks containing the medium after the removal of the apple shoots having been
cultured there for 14 days and then shaken for next 14 days. The yield of acetate hydro-
lysis was then comparable to the yield obtained after the transformation in the presence
of apple shoots, which points to the uptake of the compounds added to the plant cul-
tures (the presence of substrates in apple shoots), and to enzyme secretion to the me-
dium as well.
In addition to the above, it was found that apple shoots are not able to transform
neither ricinoleic acid to lactone nor testosterone.
Biotransformations by Apple-Tree and Carrot Roots
Table I shows the results of biotransformations carried out by genetically modified
(hairy roots) and unmodified apple (Malus pumila) and carrot (Daucus carota) roots.
The results obtained in the study show that the enzyme system of apple roots is more
active than that of carrots. The suspended cell cultures of Daucus carota10, having been
studied earlier, were capable of hydrolysing the investigated acetates to a larger extent
(3–12 times) than the root cultures of the same plant.
Higher yields of hydrolysis of the acetates by transformed apple roots than those by
non-transformed can result from genetic modifications, e.g. T-DNA insertion from Ri
plasmid. Considering the results, one may assume that for the increase in the yields of
hydrolysis genes related with biosynthesis of auxin or opine in apple hairy roots are
responsible. Each hairy root, however, may represent high variability, which have not
been examined in our investigations so far. In the case of both types of carrot roots such
differences were not observed. It seems quite likely that this is associated with the
specificity of the plant species, which can be connected, for example, with limitation in
tryptophane availability.
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Mironowicz, Kromer:
The transformations carried out by apple root cultures result in the separation of
enantiomers due to the difference in the rate of hydrolysis. There are two possible
reaction paths: either pure enantiomer of 1 or 2 remains in the unreacted substrate, or
pure (or characterized by high optical purity) enantiomer of alcohol 7 or 8 is produced.
The transformations of alcohols (±)-5, (±)-6, (±)-7, (±)-8 and ketones 9, 10, 11 and
12 were studied separately, using each of the four types of root cultures, e.g. trans-
formed and non-transformed apple and carrot roots. Menthol ((±)-8) and menthone (12)
were not transformed at all, while (±)-5, (±)-6 and (±)-7 were transformed in negligible
amounts (1–6%; the oxidation of (S)-enantiomers was faster), or in trace amounts (10
and 11). The 1-phenylethanol ((±)-5) was oxidized (5%, without substrate enantioselec-
tivity) only by transformed carrot root culture.
Reduction of acetophenone (9) in the culture of transformed apple-tree roots ac-
counted for 50% of 5, with 8% excess of (R)-1-phenylethanol ((R)-(+)-5). Non-trans-
formed apple roots reduced acetophenone in 79% yield with 9% excess of the same
enantiomer, ((R)-(+)-5). The effect of carrot roots on acetophenone (9) was different:
transformed roots caused a reduction in trace amounts, while non-transformed roots
produced 60% of pure enantiomer (S)-(–)-5 (e.e. 100%).
Neither the hairy roots nor the non-transformed apple and carrot roots were able to
affect ricinoleic acid and testosterone under the conditions described above.
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