Regio- And stereoselective subterminal hydroxylations of n-decane by fungi in a liquid-liquid interface bioreactor (L-L IBR)

Article abstract of DOI:10.1246/bcsj.82.105

This article may be the first report to describe the excellent regio- and stereoselective subterminal hydroxylations of n-alkane with microorganisms. Approximately 2000 fungal strains were screened for the regioselective hydroxylation of n-decane with a s

Full text of DOI:10.1246/bcsj.82.105

© 2009 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 82, No. 1, 105–109 (2009)
105
Regio- and Stereoselective Subterminal Hydroxylations of n-Decane
by Fungi in a LiquidÍLiquid Interface Bioreactor (LÍL IBR)
Shinobu Oda,*1 Kunio Isshiki,2 and Shinichi Ohashi1
1Genome Biotechnology Laboratory, Kanazawa Institute of Technology, 3-1 Yatsukaho, Hakusan 924-0838
2Bioresource Laboratories, Mercian Corporation, 1808 Nakaizumi, Iwata 438-0078
Received June 18, 2008; E-mail: odas@neptune.kanazawa-it.ac.jp
This article may be the first report to describe the excellent regio- and stereoselective subterminal hydroxylations of
n-alkane with microorganisms. Approximately 2000 fungal strains were screened for the regioselective hydroxylation of
n-decane with a solidÍliquid interface bioreactor, which is a microbial transformation device on an interface between an
agar plate and an organic phase (n-decane). Although Beauveria bassiana ATCC 7159, a typical fungus having a strong
and versatile hydroxylating ability, mainly produced a mixture of 2-, 3-, 4-, and 5-decanone regioisomers into a n-decane
layer, Monilliela sp. NAP 00702 and Rhizopus oryzae R-38-8 typically accumulated 4- and 5-decanols into the n-decane
layer, respectively. Although a small amount of 4- and 5-decanones were also produced, the regioselectivity at C-4 and
C-5 positions of n-decane reached 99 and 90%, respectively. Furthermore, the hydroxylation of n-decane with Monilliela
sp. NAP 00702 stereoselectively proceeded to afford almost 100% ee of (Õ)-4-decanol.
Fungi have various cytochrome P-450 systems, versatile
hydroxylating enzyme systems, to afford many useful hy-
droxylated products. For examples, plant pathogenic Cunning-
hamella, Curvularia, and Botrytis have been applied to various
hydroxylations of natural or artificial substances1Í3 besides
some harmless fungi such as Aspergillus, Absidia, Mucor, and
Rhizopus.4Í7 Concerning the hydroxylation of methylene
groups, microbial hydroxylation preferentially occurs at acti-
vated positions, such as benzyl and aryl sites in general.8,9
Hydroxylation of non-activated carbons is very difficult by
either chemical or enzymatic procedures.
However, it is well known that n-alkanes, consisting of non-
activated methyl and methylene groups, are degraded via
terminal and/or subterminal hydroxylation by various micro-
organisms.10 As for the hydroxylation of n-alkanes by bacteria,
alkane hydroxylase in Pseudomonas putida, which consists
of integral membrane monooxygenase, rubredoxin, and
rubredoxin reductase, catalyzes the terminal hydroxylation of
n-alkanes.11,12 While Brevibacterium erythrogenes,13 Micro-
coccus cerificans,14 Nocardia salmonicolor,15 and Corynebac-
terium sp.16,17 also hydroxylate a terminal methyl group of n-
alkanes, Arthrobacter sp. catalyzes the subterminal hydrox-
ylation of n-hexadecane to afford 2-, 3-, and 4-hexadecanols.18
Recently, it has been reported that an engineered cytochrome
P450BM-3 catalyzes both terminal and subterminal hydrox-
ylations of n-alkanes.19
group,25 Cunninghamella blakesleeana hydroxylates both
terminal methyl and subterminal methylene groups of n-
alkanes.26,27
As for the subterminal hydroxylation of n-alkanes, there is
no report of successful highly regio- and stereoselective
hydroxylations of n-alkanes. In this article, we demonstrate
the first report of regio- and stereoselective hydroxylation of
n-decane with fungi in a liquidÍliquid interface bioreactor (LÍL
IBR), which is a unique bioconversion system using fungal
cells growing on an interface between a liquid medium and a
hydrophobic organic solvent.28,29 In this system, fungal cells
are spontaneously immobilized on the liquidÍliquid interface
by the aid of ballooned microsphere particles to differentiate
naturally. The cells efficiently convert hydrophobic substrates
dissolved in the organic solvent to hydrophobic products. The
accumulation of the product in the organic solvent reaches a
higher level compared to emulsion and organicÍaqueous two-
liquid-phase systems, or a solidÍliquid interface bioreactor
(SÍL IBR).30Í34 Using the LÍL IBR system, Monilliela sp. NAP
00702 and Rhizopus oryzae R-38-8 catalyzed regioselective
hydroxylation of n-decane to afford 4- and 5-decanol in high
yields, respectively. Furthermore, the regioselective subtermi-
nal hydroxylation by Monilliela sp. NAP 00702 stereoselec-
tively proceeded to afford almost 100% ee of (Õ)-4-decanol.
On the other hand, while 2- and 3-decanol were produced by
Colletotrichum linicolum and Aspergillus tunetana with mod-
erate regioselectivities, Trichoderma sp. afforded 1-decanol
regioselectively (Figure 1).
On the other hand, some yeasts also catalyze monoterminal,
diterminal, and/or subterminal hydroxylations of n-alkanes.
For example, Candida intermedia,20 C. parasilosis,21 C. guil-
liermondii,22 and Lodderomyces elongisporus23 hydroxylate a
terminal methyl group of n-alkanes, while Candida lipolytica
hydroxylates both terminal and C-2 methylene groups of
n-alkanes.24 Concerning fungal hydroxylations of n-alkanes,
while Cladosporium resinae hydroxylates a terminal methyl
Experimental
Screening of n-Decane-Hydroxylating Fungi.
All fungi
tested were stocked in the Bioresource Laboratories of Mercian
Corporation, containing type culture strains from ATCC (American
Type Culture Collection), NRRL (Agricultural Research Service
Published on the web January 14, 2009; doi:10.1246/bcsj.82.105

106
Bull. Chem. Soc. Jpn. Vol. 82, No. 1 (2009)
Microbial Hydroxylation of n-Decane
Culture Collection), and NBRC (NITE Biological Resource
Center; former IFO). All strains were stocked on modified
Sabouraud agar plates consisting of 40.0 g of glucose, 10.0 g of
Bacto peptone, 5 mg of FeSO4¢7H2O, 20 mg of MnSO4¢5H2O,
10 mg of CaCl2, and 15.0 g of agar in 1.0 L of deionized water
(pH 6.0) at 4 °C. Approximately 2000 strains were screened with
the SÍL IBR (vessel, glass vial; volume, 50 mL; diameter, 3 cm)
consisting of the modified Sabouraud agar plate (volume, 10 mL;
surface area, 7.1 cm2) and n-decane (1.5 mL). Three pieces
(approximately 2 mm © 2 mm) of each fungal mat were inoculated
on the surface of the agar plate with a long toothpick, and
precultivation was done at 25 °C by allowing the plate to stand for
3 days. After the precultivation, 1.5 mL of n-decane was added
onto the surface of a fungal mat. The stationary incubation was
continued at 25 °C for 7 days. After the incubation, the n-decane
layer in the vessel was directly analyzed by gas chromatography.
The column (0.25 mm i.d. © 60 m) contained SUPELCOWAX-10
(Supelco Co., Ltd., Bellefonte, PA). The column temperature was
raised from 80 to 110 °C (2 °C minÕ1), held at 110 °C for 5 min,
raised from 110 to 120 °C (2 °C minÕ1), held at 120 °C for 5 min,
raised from 120 to 150 °C (6 °C minÕ1), and held at 150 °C for
10 min. The injector and detector temperatures were 155 and
160 °C, respectively. The carrier gas and split ratio were He
(20 s minÕ1) and 1:100, respectively. The retention times of 5-, 4-,
3-, and 2-decanones and 5-, 4-, 3-, 2-, and 1-decanols were 21.22,
21.36, 23.47, 25.66, 31.63, 31.79, 32.79, 34.37, and 42.54 min,
respectively. The products were identified by GC-MS (Turbo Mass
Gold, Perkin-Elmer., Inc., Waltham, MA) by comparison with
authentic samples and an attached database. The GC-MS measure-
ments were acquired under the following conditions; ionization
was by electron impact, ionization voltage was 0.6 eV, ion source
temperature was 50 °C. GC conditions were identical for both
qualitative and quantitative analysis.
Comparison of n-Decane-Hydroxylating Abilities of Mon-
illiela sp. NAP 00702 between Emulsion and LÍL IBR Systems.
The F1 liquid medium consisted of 20.0 g of potato starch, 10.0 g
of glucose, 20.0 g of soy protein (SoyproÓ, Inui Co., Ltd., Osaka),
1.0 g of KH2PO4, 0.5 g of MgSO4¢7H2O in 1.0 L of deionized
water (pH 6.0) was used for seed cultivation. As for the emulsion
system (Figure 2), 4 mL of a 3-day seed broth, which was prepared
by shaken cultivation (25 °C, 220 rpm), was inoculated to 50 mL of
the modified Sabouraud liquid medium. After precultivation at
25 °C with agitation (300 rpm) for 3 days, 20 mL of n-decane was
added to the broth and incubation was continued at 25 °C
with agitation (300 rpm) for 9 days. As for the LÍL IBR system
(Figure 2), 4 mL of a 3-day broth was inoculated to the modified
Sabouraud liquid medium containing 1.0 g of ballooned poly-
acrylonitrile microsphere (MFL-80GCA, CaCO3-coated type;
mean diameter, 20 µm; density, 0.2; Matsumoto Yushi-Seiyaku,
Co., Ltd., Osaka). After 3-day stationary precultivation at 25 °C,
20 mL of n-decane was added onto the surface of a fungus-
microsphere mat and incubation was continued at 25 °C by
allowing the reactor to stand for 9 days. Products in the n-decane
layer were directly determined by gas-chromatography in the
above-mentioned manner. The enantiomeric excess of 4-decanol
produced was also directly determined by gas-chromatography.
The column (0.25 mm i.d. © 30 m) contained ¢-DEXÓ 325
(Supelco Co., Ltd.). The column, injector, and detector temper-
atures were 160, 165, and 170 °C, respectively. The carrier gas
and split ratio were He (20 s minÕ1) and 1:100, respectively.
The retention times of (Õ)- and (+)-4-decanol were 17.61 and
17.89 min, respectively.
Comparison of n-Decane-Hydroxylating Abilities of Rhizo-
pus oryzae R-38-8 among Emulsion, SÍL IBR, and LÍL IBR
Systems. Schematic diagrams of emulsion, SÍL IBR, and LÍL
IBR systems are shown in Figure 2. As for the emulsion system,
4 mL of a homogenate of a fungal mat formed on the agar plate for
7 days was added to 60 mL of the modified Sabouraud liquid
medium. After 3-day precultivation at 25 °C with agitation
(300 rpm), 20 mL of n-decane was added to the broth and
cultivation was continued for 5 days. Concerning the SÍL IBR
system, 2 mL of the homogenate was inoculated onto the surface of
the modified Sabouraud agar plate (volume, 60 mL; surface area,
38.5 cm2). After 3-day stationary precultivation at 25 °C, 20 mL of
n-decane was added onto the surface of a fungal mat and
cultivation was continued for 5 days. As for the LÍL IBR system,
4 mL of the homogenate was added to 60 mL of the liquid medium
containing 1.2 g of ballooned polyacrylonitrile microsphere (MFL-
80GTA, talc-coated type; mean diameter, 20 µm; density, 0.2;
Matsumoto Yushi-Seiyaku, Co., Ltd., Osaka). After 3-day sta-
Liquid-Liquid Interface Bioreactor
(L-L IBR)
Oxygen
Colletotrichum
Monilliela
Air
Decanols
Decanones
n-Decane
layer
n-Decane
Fungus-MS mat
Rhizopus
Aspergillus
Trichoderma
Liquid
medium
Water Nutrients
MS, ballooned microsphere
Figure 1. Principle of liquidÍliquid interface bioreactor
(LÍL IBR) and its application to regioselective hydrox-
ylations of n-decane with fungi. A fungus-microsphere mat
located on an interface between a liquid medium and
n-decane regioselectively hydroxylates n-decane.
Emulsion
S-L IBR
L-L IBR
A
A
F
A
B
D
G
D
E
B
C
A, Deep Petri dish (height, 5 cm; diameter, 7 cm); B, liquid medium; C, magnet;
D, n-decane layer; E, fungal mat; F, agar plate; G, fungus-microsphere mat
Figure 2. Schematic diagrams of emulsion, SÍL IBR, and LÍL IBR systems. All systems are shown as vertical sections.

S. Oda et al.
Bull. Chem. Soc. Jpn. Vol. 82, No. 1 (2009)
107
Table 1. Screening of Fungi Catalyzing Regioselective Hydroxylation of n-Decane
Product concentration/mg mLÕ1
Strain
5K
4K
3K
2K
5A
4A
3A
2A
1A
Beauveria bassiana ATCC 7159
Trichoderma sp. f 16062
Colletotrichum linicolum G-4
Aspergillus tunetana var. pallidas A-124
Monilliela sp. NAP 00702
2.60
ndb)
nd
tr
nd
1.02
nd
nd
0.09
0.22
tr
1.98
nd
0.03
0.26
tr
3.39
nd
0.08
0.06
tr
0.30
nd
0.03
0.12
0.05
7.65
0.20
tra)
0.11
0.41
1.44
0.83
0.33
tr
1.16
1.60
0.04
0.11
0.32
tr
2.52
0.11
nd
nd
1.78
nd
0.17
nd
Rhizopus oryzae R-38-8
2.88
nd
nd
0.07
0.18
a) tr, Trace amount. b) nd, not detected. 5K, 5-decanone; 4K, 4-decanone; 3K, 3-decanone; 2K, 2-decanone; 5A, 5-decanol;
4A, 4-decanol; 3A, 3-decanol; 2A, 2-decanol; and 1A, 1-decanol. After 3-days precultivation, 1.5 mL of n-decane was added
onto a fungal mat (surface area, 7.1 cm2) and incubation was statically done at 35 °C for 7 days.
tionary precultivation at 25 °C, 20 mL of n-decane was added onto
the surface of a fungus-microsphere mat. Incubation was continued
at 25 °C by allowing the reactor to stand for 5 days. Products in the
n-decane layer were directly analyzed by gas-chromatography in
the above-mentioned manner.
C-2 and C-3 positions of n-decane at moderate regioselectivity.
Trichoderma sp. f16062 regioselectively catalyzed terminal
hydroxylation of n-decane to afford 1-decanol. As for the
production of 5-decanol, many Rhizopus strains also exhibited
superior regioselectivity for the subterminal hydroxylation of
n-decane as shown in Table 2. Although oxidation of alkanols
to alkanones proceeded in part, the accumulation of all alkanols
reached over 1 g LÕ1 in the SÍL IBR because of its effective
toxicity alleviation effect. Especially, R. oryzae R-38-8 accu-
mulated 7.65 mg of 5-decanol in 1 mL of n-decane layer in
spite of the high biotoxicity of the alkanol.41
Results and Discussion
Since the early nineteen-sixties, numerous reports of
assimilation and oxidation of n-alkanes by microorganisms
have been published.10,35 Terminal and subterminal hydroxyla-
tions are initial steps of degradation of n-alkanes with the aid of
alkane hydroxylase or cytochrome P-450 systems. While the
terminal hydroxylation by microorganisms is highly regiose-
lective in general, the regioselectivity of microbial subterminal
hydroxylation is very low. Moreover, microbial subterminal
hydroxylation of fatty acids, linear-chain compounds similar
to n-alkanes, also proceeds non-regioselectively.36Í38 Thus,
recognition of the difference of hydroxylated positions in
enzymatic and chemical reactions is very difficult. Toward the
above-mentioned traditional information, we tried to discover
regio- and stereoselective subterminal hydroxylation of n-
alkanes with fungi.
Comparison of n-Decane-Hydroxylating Abilities of
Monilliela sp. NAP 00702 between Emulsion and LÍL
IBR Systems. It was reported that the LÍL IBR enabled the
higher accumulation of hydrophobic products such as 2-ethyl-
1-hexanol28 and (S)-benzoin29 compared with emulsion, or-
ganicÍaqueous two-liquid-phase, and SÍL IBR systems. Thus,
the regioselective subterminal hydroxylation of n-decane with
Monilliela sp. NAP 00702 was applied to the emulsion and the
LÍL IBR systems. As shown in Figure 3A, although initial
hydroxylation rate in the emulsion system was higher than
that in the LÍL IBR system, 4-decanol and 4-decanone were
degraded after the 4th day in the emulsion system. It was
assumed that 4-decanol was degraded via BaeyerÍVilliger
oxidation of 4-decanone in the emulsion system likewise in
some other microorganisms.42,43 Thus, the n-decane layer in the
emulsion system with vigorous agitation might not sufficiently
function as a reservoir of 4-decanol in the same way as a
dispersed organicÍaqueous two-liquid-phase system.44,45
On the other hand, the accumulation of 4-decanol continued
after the 4th day in the LÍL IBR system. It is easily assumed
that a stationary n-decane layer acts as a reservoir of 4-decanol
and 4-decanone to alleviate the degradation of these products.
Furthermore, it was observed that the subterminal hydrox-
ylation of n-decane by Monilliela sp. NAP 00702 stereo-
selectively proceeded to afford (Õ)-isomer. The enantiomeric
excess of (Õ)-4-decanol was almost 100% as shown in
Figures 3B and 3C. As for the stereoselectivity of subterminal
hydroxylation of n-alkanes, it was reported that an engineered
cytochrome P450BM-3 (9-10A-A328V) afforded 55% ee of
(R)-2-decanol.19 It was also reported that the stereoselectivities
of subterminal hydroxylations of some carboxylic acids and n-
alkylbenzenes with other P-450 systems were high, however,
the regioselectivities of the hydroxylations were very low.46Í48
Thus, it is concluded that the hydroxylation of n-decane with
Screening of Microorganisms Catalyzing Regioselective
Subterminal Hydroxylation of n-Decane.
First, approx-
imately 2000 fungal strains were applied to the SÍL IBR
consisting of an agar plate and n-decane layer. The bioreactor is
an efficient and conventional device for the microbial trans-
formation of water-insoluble substrates because of the excellent
substrate- and/or product-toxicity alleviation effect and the
solubilization of the substrates.30Í34 The organic phase serves
as a reservoir and a solvent of toxic substrates and products. In
the case of this study, n-decane was used as the organic phase
and the substrate. As shown in Table 1, Beauveria bassiana
ATCC 7159 having a strong and versatile hydroxylating
activity39,40 mainly afforded a mixture of 2-, 3-, 4-, and 5-
decanones. It was confirmed that the fragmentation patterns of
each product was identical with corresponding authentic
sample and an attached database by the aid of GC-MS.
However, Monilliela sp. NAP 00702 and Rhizopus oryzae
R-38-8 regioselectively hydroxylated n-decane to afford mainly
4- and 5-decanol, respectively. The regioselectivities (alcohol
plus ketone per sum of all products) of Monilliela sp. NAP
00702 and R. oryzae R-38-3 reached to 99 and 90%,
respectively. On the other hand, Colletotrichum linicolum
G-4 and Aspergillus tunetana var. pallidas A-124 hydroxylated

108
Bull. Chem. Soc. Jpn. Vol. 82, No. 1 (2009)
Microbial Hydroxylation of n-Decane
Table 2. Product Profiles of Hydroxylation of n-Decane with Rhizopus spp.
Product concentration/mg mLÕ1
5K + 5A
/%
Strain
5K
4K
3K
2K
5A
4A
3A
2A
1A
R. oryzae R-38-8
R. oryzae R-25-3
Rhizopus sp. IFO 4789
R. oryzae R-35-5
Rhizopus sp. IFO 4723
R. oryzae R-28-5
2.88
2.17
1.78
1.83
1.52
1.32
1.54
3.20
1.16
2.54
3.49
2.17
1.85
1.68
0.90
0.78
0.69
0.38
tra)
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
ndb)
nd
nd
nd
nd
nd
nd
nd
nd
nd
tr
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
tr
nd
nd
nd
nd
nd
nd
nd
7.65
7.25
6.64
6.41
5.78
5.65
5.63
5.27
5.11
4.38
3.98
3.73
2.96
2.82
2.65
2.49
2.49
2.00
0.83
0.73
0.79
0.80
0.71
0.71
0.71
0.64
0.62
0.57
0.60
0.44
0.44
0.44
0.34
0.35
0.33
0.28
0.11
0.14
0.10
0.09
0.08
0.08
0.08
0.08
0.07
0.06
0.12
0.05
0.07
0.08
0.03
0.03
tr
0.07
0.11
0.06
0.06
0.05
0.05
0.05
0.06
0.05
0.03
0.06
0.03
0.04
0.04
tr
0.18
0.08
0.08
0.08
0.07
0.06
0.07
0.29
0.05
nd
0.30
0.19
0.12
0.10
0.05
0.04
0.03
nd
89.9
89.9
89.1
88.9
88.9
88.6
88.7
88.8
87.6
91.3
87.4
89.3
87.8
87.2
89.4
88.6
89.8
85.3
R. oryzae R-32-5
R. oryzae R-27-8
R. oryzae IFO 4728
R. oryzae R-12-10
R. oryzae IAM 6002
R. delemar IFO 4786
R. delemar IFO 4806
R. shanghaiensis IFO 4888
R. oryzae R-43-1
R. oryzae R-41-8
R. oryzae IFO 4716
R. oryzae IAM 6065
tr
nd
0.06
0.07
a) tr, Trace amount. b) nd, not detected. 5K, 5-decanone; 4K, 4-decanone; 3K, 3-decanone; 2K, 2-decanone; 5A, 5-decanol;
4A, 4-decanol; 3A, 3-decanol; 2A, 2-decanol; and 1A, 1-decanol. Reaction conditions were the same as in the footnote to
Table 1.
Authentic 4-decanol
Emulsion
S-L IBR
L-L IBR
3
2
1
B
A
(-)-4A [17.61 min]
(+)-4A [17.89 min]
2
1
Monilliela sp. NAP 00702
C
(-)-4A [17.58 min]
3A [18.37 min]
2
4
6
8
5K
5A
Incubation period / day
Figure 4. Comparison of 5-decanol and 5-decanone pro-
duction with R. oryzae R-38-8 among emulsion, SÍL IBR,
and LÍL IBR systems. 5K, 5-decanone; 5A, 5-decanol.
While the emulsion system was conducted at 25 °C with
agitation (300 rpm) for 5 days, the SÍL and LÍL IBR
systems were statically conducted at 25 °C.
Figure 3. Time course of hydroxylation of n-decane with
Moniliella sp. NAP 00702 and chiral gas chromatogram of
4-decanol (4A) produced. (A) Time course of hydroxyla-
tion. Open circles, 4A produced in emulsion system;
closed circles, 4A produced in LÍL IBR system; open
triangle, 4-decanone (4K) produced in emulsion system;
closed triangle 4K produced in LÍL IBR system. (B)
Chiral gas chromatogram of authentic 4-decanol. (C)
Chiral gas chromatogram of 4-decanol produced by
Monilliela sp. NAP 00702. A small amount of 3-decanol
(3A) was also produced.
LÍL IBR system was significantly higher than those in the
emulsion and the SÍL IBR systems. In the emulsion system, the
accumulations of 5-decanol and 5-decanone were very low
level because R. oryzae R-38-8 formed a large aggregate in the
broth. It is well known that the control of fungal morphology is
a very important factor for fermentation and microbial trans-
formation.49,50 In conclusion, the easy morphology control of
R. oryzae R-38-8, the higher contents of the substrate and
oxygen in the organic phase, and the excellent toxicity
alleviation effect in the LÍL IBR system enables the higher
production of 5-decanol.
Monilliela sp. NAP 00702 may be the first report for the regio-
and stereoselective subterminal hydroxylation of n-alkanes
with microorganisms.
Comparison of n-Decane-Hydroxylating Abilities of
Rhizopus oryzae R-38-8 among Emulsion, SÍL IBR, and
LÍL IBR Systems. Efficient production of hydroxylation
product in the LÍL IBR system was also estimated for the
production of 5-decanol with R. oryzae R-38-8. As shown in
Figure 4, the accumulation of 5-decanol and 5-decanone in the
In order to gain a series of optically active decanol isomers
in higher yields, both the deletion of secondary alcohol
dehydrogenase and the selection of other fungi having excellent

S. Oda et al.
Bull. Chem. Soc. Jpn. Vol. 82, No. 1 (2009)
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22 H.-G. Müller, W.-H. Schunck, P. Riege, H. Honeck, Acta
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109
regio- and stereoselective C-2 and C-3 hydroxylation activities
must be achieved. Moreover, it is expected that a large scale
LÍL IBR system will be constructed. We have continued
various trials for resolving the above-mentioned tasks.
23 S. Mauersberger, W.-H. Schunck, H.-G. Müller, Appl.
Microbiol. Biotechnol. 1984, 19, 29.
24 M. J. Klug, A. J. Markovetz, J. Bacteriol. 1967, 93, 1847.
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28 S. Oda, K. Isshiki, Process Biochem. 2007, 42, 1553.
29 S. Oda, K. Isshiki, Biosci. Biotechnol. Biochem. 2008, 72,
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This study was mainly performed in the Bioresource
Laboratories, Mercian Corporation. The authors wish to thank
Mercian Corporation for permission of this work. We also
express our sincere thanks to Director Nobuo Ichimaru and Mr.
Tadashi Hiraide of Matsumoto Yushi-Seiyaku Co., Ltd., for the
kind gift of microspheres, and Miss Aki Kobayashi of Mercian
Corporation for helpful technical assistance.
30 S. Oda, H. Ohta, Biosci. Biotechnol. Biochem. 1992, 56,
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