Biotransformation of cyclohexene to value-added oxygenated derivative catalyzed by chloroperoxidase in the presence of small quantities of quaternary ammonium salts

Article abstract of DOI:10.1016/j.apcata.2011.05.014

Biotransformation of cyclohexene catalyzed by chloroperoxidase (CPO) from Caldariomyces fumago was employed to prepare value-added oxygenated derivative: trans-1,2-cyclohexanediol (CHD) using H₂O₂ as oxidants. The conversion of substrate was enhanced to 89.2% in the presence of small quantities of quaternary ammonium salts (QAS) compared to that of only 48.1% in aqueous phosphate buffer after 150 min. The enhancement was dependent on the concentration and alkyl group length of QAS. QAS were found playing multiple functions in reaction media: phase transfer catalysis and tuning the composition of product. UV-vis, fluorescence and circular dichroism (CD) spectral assay were employed to investigate the effect of QAS on micro-environment around active center and structure of protein. The strengthening of α-helix structure of CPO and more exposure heme for easily access of substrate was found responsible for the improvement of catalytic performance of CPO. Moreover, the determined kinetic parameters indicated the affinity and selectivity of CPO to substrate was improved according to the observation of a decrease of Michaelis constant K_m while an increase of the second order rate constants k_cat/K_m. The strategy reported in this work is environment-friendly, straightforward and applicable to large scale preparation.

Full text of DOI:10.1016/j.apcata.2011.05.014

Applied Catalysis A: General 401 (2011) 204–209
Contents lists available at ScienceDirect
Applied Catalysis A: General
journal homepage: www.elsevier.com/locate/apcata
Biotransformation of cyclohexene to value-added oxygenated derivative
catalyzed by chloroperoxidase in the presence of small quantities of quaternary
ammonium salts
Huizhen Zhanga, Yucheng Jianga,b,∗, Mancheng Hua,b, Shuni Lia,b, Quanguo Zhaia,b
a School of Chemistry and Materials Science, Shaanxi Normal University, Xi’an, 710062, PR China
b Key Laboratory of Macromolecular Science of Shaanxi Province, Shaanxi Normal University, Xi’an, 710062, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Biotransformation of cyclohexene catalyzed by chloroperoxidase (CPO) from Caldariomyces fumago was
employed to prepare value-added oxygenated derivative: trans-1,2-cyclohexanediol (CHD) using H2O2
as oxidants. The conversion of substrate was enhanced to 89.2% in the presence of small quantities of
quaternary ammonium salts (QAS) compared to that of only 48.1% in aqueous phosphate buffer after
150 min. The enhancement was dependent on the concentration and alkyl group length of QAS. QAS were
found playing multiple functions in reaction media: phase transfer catalysis and tuning the composition
of product. UV–vis, fluorescence and circular dichroism (CD) spectral assay were employed to investigate
the effect of QAS on micro-environment around active center and structure of protein. The strengthening
of ␣-helix structure of CPO and more exposure heme for easily access of substrate was found responsible
for the improvement of catalytic performance of CPO. Moreover, the determined kinetic parameters
indicated the affinity and selectivity of CPO to substrate was improved according to the observation of a
Received 25 January 2011
Received in revised form 24 April 2011
Accepted 14 May 2011
Available online 20 May 2011
Keywords:
Biotransformation
Chloroperoxidase
Cyclohexene
Quaternary ammonium salts
decrease of Michaelis constant Km while an increase of the second order rate constants kcat/Km
.
The strategy reported in this work is environment-friendly, straightforward and applicable to large
scale preparation.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
[18] etc., the preparation processes are troublesome and expensive,
and moreover, these catalytic systems are generally performed in
The catalytic transformations of olefin, which are the main
crude oil and natural gas constituents, into value-added oxy-
genated derivatives have been extensively studied over the last
few decades [1–3], for example, the study on oxidation of cyclohex-
ene arouses many interests due to that epoxycyclohexane (ECH) is
an essential organic intermediate in the production of fine chemi-
cals. However, the traditional chemical procedures for synthesis of
ECH produced a great deal of environmentally undesirable wastes.
Though many effective and recyclable catalysts, especially bio-
mimetic catalysts for oxidation of cyclohexene were prepared,
such as iron(III) porphyrin complex [4–6], Manganese(III) por-
phyrin [7–9], cobalt porphyrin [10], cobalt metal–organic complex
[11–13], zinc porphyrin complexes[14], transition-metal substi-
tuted ␣-titanium arsenate [15], molybdenum(VI) compounds [16],
Fe/CeO2 [17], and Ti(IV)-monosubstituted Keggin polyoxometalate
volatile and toxic organic solvents. An environment-friendly pro-
cess for the oxidative transformation of olefin is highly desirable.
Enzyme-catalyzed reactions are arousing more and more inter-
ests in the industrial synthesis due to its efficiency, selectivity, mild
conditions besides no potential pollution. Chloroperoxidase (CPO)
from the fungus Caldariomyces fumago is considered to be probably
the most versatile member of the heme protein family. In addition
to functioning as a halogenating enzyme and a classical peroxidase,
CPO carries out cytochrome P450 oxygen insertion reactions. From
the viewpoint of biocatalysis, the most important catalytic activity
of CPO is epoxidations, hydroxylations, and sulfoxidations [19–23].
However, CPO-catalyzed reactions are usually in aqueous solu-
tion. The low aqueous solubility of organic substrates often limits
CPO for broader applications. Several strategies were proposed to
deal with this problem, for example, imidazole ionic liquids were
used as co-solvent [24,25], CPO was interface-bound for interfa-
cial epoxidation of styrene [26], or directed-evolution of CPO was
designed for improving epoxidation catalysis [27]. In this paper,
oxidation of cyclohexene was carried out in the presence of small
amount quaternary ammonium salts (QAS) in order to enhance
the conversion of cyclohexene and improve the catalytic perfor-
∗
Corresponding author. Tel.: +86 29 85307765; fax: +86 29 85307774.
E-mail addresses: zhanghzer@stu.snnu.edu.cn (H. Zhang), jyc@snnu.edu.cn
(Y. Jiang), hmch@snnu.edu.cn (M. Hu), lishuni@snnu.edu.cn (S. Li),
zhaiqg@snnu.edu.cn (Q. Zhai).
0926-860X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.apcata.2011.05.014

H. Zhang et al. / Applied Catalysis A: General 401 (2011) 204–209
205
mance of CPO. QAS were found playing multiple functions in this
biotransformation, including phase transfer catalysis, tuning the
composition of products and inducing a catalytically favorable
structure of enzyme.
(300 MHz Bruker), or resolved by methanol for analysis by gas
chromatography–mass spectrometry with electron impact ioniza-
tion (GC–EI/MS).
The configuration of diols was confirmed by its melting point.
The synthetic strategy of biotransformation of cyclohexene by
CPO was environment-friendly, straightforward and applicable to
large scale preparation of value-added oxygenated derivatives form
hydrocarbons in crude oil and natural gas.
2.4. Calculation of substrate conversion and product yield
The product in organic phase and in remained aqueous phase
was combined for calculation of substrate conversion. The yield
and selectivity were calculated respectively as follows:
2. Experimental
ꢀ
ꢁ
cyclohexene converted (mol)
cyclohexene used (mol)
2.1. Materials
cyclohexene conversion (%) =
Chloroperoxidase was isolated from the growth medium of
C. fumago according to the method established by Morris and
Hager [28] with minor modifications, using acetone rather than
ethanol in the solvent fractionation step. The enzyme solution
was concentrated to 8.4 mg mL−1 CPO with Rz 1.18 (Rz = purity
standard = A398/A280 = 1.44 for pure enzyme) and an activity of
9912 U/mL based on the standard monochlorodimedon (MCD)
assay [29].
All the chemical regents, such as potassium hydrogen phos-
phate, potassium dihydrogen phosphate, hydrogen peroxide (30%
in aqueous solution) were obtained from Xi’an Chemical Co. Ltd.;
Quaternary ammonium salts: tetramethylammonium bromide
(TMABr), tetraethylammonium bromide (TEABr), tetrapropylam-
monium bromide (TPABr), tetrabutylammonium bromide (TBABr)
were from Sinopharm Chemical Reagent Co. Ltd. All these chemicals
are of analytical grade unless otherwise indicated.
× 100%
ꢀ
ꢁ
product formed (mol)
cyclohexene used (mol)
product yield (%) =
× 100%
ꢀ
ꢁ
product formed (mol)
cyclohexene converted (mol)
product selectivity (%) =
× 100%
2.5. Determination of kinetic parameters
The reaction catalyzed by CPO displayed Michaelis–Menten
kinetic characteristics. Michaelis constant Km and Vmax values in
pure buffer and in the presence of quaternary ammonium salts
were obtained by linear regression analysis of the double reciprocal
Lineweaver–Burk plots [31].
2.2. Procedure for enzymatic epoxidation of cyclohexene
ꢂ
ꢃ
Km
ꢀ−1
=
S
[ ]
−1 + Vmax
−1
Vmax
Cyclohexene (7.50 mmol) was put into 10.0 mL 0.1 M aqueous
phosphate buffer (pH 5.0) in a tinfoil-wrapped 25 mL round bot-
tom flask and magnetically stirred at 35 ◦C till the solution was
clear (for about 20 min). CPO (0.25 ␮mol) was added in the solu-
tion and continued stirring for 10 min. Then, H2O2 (14.8 mmol in
total) was added at every 10 min interval, each time about 835 ␮L
H2O2 (2.96 mol L−1). The procedure was the same in the case of QAS
presence.
Kinetic assay was carried out over the cyclohexene concen-
tration range of 0.005–0.05 mol L−1 in the presence of QAS at its
optimum concentration. The turn over number, kcat was calculated
according to the following equation:
Vmax = kcat[E0]
E0 was the initial concentration of CPO.
The reaction was quenched after 150 min stirring by a satu-
rated sodium sulfite solution and extracted 3 times by ethyl acetate.
The combined organic extract was purified by rotatory evaporation
(0.09 MPa, 45 ◦C), and further dried by anhydrous MgSO4. Mean-
while, the remained aqueous solution was extracted 5 times by
dioxane to separate the product in aqueous phase because CPO
and QAS were not soluble in solvent with low dielectric con-
stant (ε, εdioxane = 2.01). The combined extracts were processed
by rotary evaporation to remove dioxane. The obtained solid was
dried firstly; then, a further purification was performed through
recrystallization by ethyl acetate. Characterization, determination
of products both in organic and remained phase was carried out,
and calculation of substrate conversion and product yield was made
accordingly.
2.6. Spectrogram collection
1.2 ␮M CPO was prepared in pure 0.1 mol L−1 phosphate buffer
or in the presence of QAS (pH 5.0). The samples were excited at
280 nm, and fluorescence spectra were registered from 300 nm to
420 nm.
0.8 ␮M CPO was prepared in pure 0.1 mol L−1 phosphate buffer
or in the presence of QAS (pH 5.0) for UV–vis and circular dichroism
(CD) spectral assay.
3. Results and discussion
The Br− in QAS would not compete with cyclohexene in the con-
dition of pH 5.0 in this work, because halogenation catalyzed by CPO
occurred under condition at pH < 3. The acidic media was benefi-
cial for proton shuttle, which was vital for the transformation of
intermediate to halogenation product [30].
3.1. Products CPO-catalyzed oxygenation
The results of 1H NMR and GC–EI/MS measurements were:
the product in organic phase was epoxycyclohexane: 1H NMR
(300 MHz, CDCl3) ı 3.13 (s, 1H), 2.02–1.87 (m, 1H), 1.81 (d,
J = 14.2 Hz, 1H), 1.41 (d, J = 6.2 Hz, 1H), 1.25 (d, J = 6.3 Hz, 1H); MS
(+ev) m/z: 97, 83, 69, 54, 41.
In the presence of QAS while CPO absence, no expected products
were detectable, however, the pH of solution decreased.
The product in remain aqueous phase was diols: 1H NMR
(300 MHz, CDCl3) ı 3.35 (s, 1H), 2.65 (s, 1H), 1.96 (s, 1H), 1.70 (s,
1H), 1.26 (d, J = 3.1 Hz, 2H); MS (+ev) m/z: 116, 98, 84, 70, 55, 41.
The melting point of diols was 104 ◦C. It was trans-1,2-
cyclohexanediol (CHD) (reported data was 104–105 ◦C [32]).
2.3. Characterization of products
The solid in organic phase or in remained aqueous phase
was resolved by chloroform-d (CDCl3) for 1H-NMR analysis

206
H. Zhang et al. / Applied Catalysis A: General 401 (2011) 204–209
Fig. 1. ECH yield dependence on reaction time in buffer. Reaction conditions:
0.25 ␮mol CPO, 7.50 mmol cyclohexene, 14.8 mmol H2O2 in total, 0.1 mol L−1 phos-
phate buffer, pH 5.0 at 35 ◦C.
Fig. 2. Effect of additive concentration on CHD yield. Reaction conditions: 0.25 ␮mol
CPO, 7.50 mmol cyclohexene, 14.8 mmol H2O2, 0.1 mol L−1 phosphate buffer,
pH 5.0 at 35 ◦C. Additive concentration: TMABr: 0.0325–0.162 mol L−1
, TEABr:
3.2. CPO-catalyzed epoxidation of cyclohexene in aqueous buffer
0.0238–0.162 mol L−1, TPABr: 0.0188–0.162 mol L−1, TBABr: 0.0155–0.162 mol L−1
.
The influence of oxidant concentration, solution pH and CPO
consumption was investigated in order to optimize the reac-
tion process in aqueous solution. The results showed that the
reaction condition should be controlled around pH 5.0. High con-
centration of H2O2 was not favorable for this enzymic reaction.
This was due to the CPO inactivation by concentrated H2O2.
The inactivation, generally happened in heme proteins such as
cytochrome P450, horseradish peroxidase and CPO, was mainly
due to internal oxidative destruction of the porphyrin moiety
[33–35]. So, the total H2O2 was added intermittently to keep
the H2O2 concentration low in reaction solution to suppress this
inactivation.
An increasing–decreasing pattern versus reaction time was
found for ECH accumulation. The yield reached maximum within
60 min with the selectivity higher than 85%, and then started
dropping due to spontaneous hydrolytic opening of epoxide
ring.
The cyclohexene conversion and ECH yield, however, reached
to only 43.7% and 38.5% at optimum conditions after 60 min
(Fig. 1). Then, the substrate conversion increased only a lit-
tle when the reaction time continued. It reached 48.1% after
150 min. The low conversion of cyclohexene was mainly due to
the poor solubility of hydrophobic substrate in aqueous reaction
solution.
3.4. The promotion effect of QAS on CPO-catalyzed oxygenation of
cyclohexene
One role of QAS was phase transfer catalyst. Quaternary
ammonium salts are hydrophilic, but they have a hydropho-
bic alkyl chain. They would take hydrophobic cyclohexene
by their hydrophobic chain from organic phase into aque-
ous phase, where CPO and H2O2 were, to complete reaction
efficiently (Scheme 1).
Moreover, it was speculated these additives could make it easier
for substrate binding to CPO simply by producing a more hydropho-
bic microenvironment around the active site. In heme peroxidases,
the heme edge is usually available for substrate interactions. But the
opposite is true in CPO, in which the heme edge is not accessible
directly. It was deeply inside the protein molecule, and there was a
channel connecting the surface to heme [36]. It was difficult for the
additives with longer hydrophobic chain to access the active site
due to the spatial resistance. However, if the alkyl was too short,
they could not carry substrate transferring phase successfully. So,
TPABr, with the most suitable hydrophobic chain, was the most
effective additives (Fig. 3).
The second role of QAS played here was tuning the composi-
tion of product. The main product was ECH and CHD respectively
in the absence and presence of QAS after same reaction time
3.3. CPO-catalyzed oxygenation of cyclohexene in the presence of
QAS
Quaternary ammonium salts, having both the hydrophilic
cationic head group and a hydrophobic alkyl chain, was introduced
into the reaction media as additives in order to improve the sol-
ubility of cyclohexene in aqueous reaction media. As expected,
cyclohexene conversion was enhanced to 89.2% after 150 min, but
the main product was CHD instead of ECH with the yield of 79.3%
and selectivity of 88.9%.
The CHD yield depended greatly on QAS concentration: an effect
of promotion was observed at its low concentration while inhi-
bition occurred at its high concentration (Fig. 2). The optimum
concentration was small: 0.130 mol L−1 TMABr, 0.0952 mol L−1
TEABr, 0.0751 mol L−1 TPABr and 0.0620 mol L−1 TBABr respec-
tively, which was used in all the following experiments without
further indication.
Scheme 1. Phase transfer catalysis effect played by quaternary ammonium salts
(QAS: quatemary ammonium salts).

H. Zhang et al. / Applied Catalysis A: General 401 (2011) 204–209
207
Table 1
Reaction kinetics in the absence of quaternary ammonium salts.
Time (min)
Conversion (%)
Selectivity (%)
Yield (%)
OH
OH
OH
O
O
OH
10
20
30
60
90
25.8
31.3
36.6
43.7
46.6
48.1
92.6
90.5
90.2
88.1
62.5
19.5
–
–
–
5.23
31.2
66.9
23.9
28.3
33.0
38.5
29.1
9.38
–
–
–
2.29
14.5
32.2
150
Reaction conditions: 0.25 ␮mol CPO, 7.50 mmol cyclohexene, 14.8 mmol H2O2 in total, 0.1 mol L−1 phosphate buffer, pH 5.0 at 35 ◦C.
Table 2
Reaction kinetics in the presence of quaternary ammonium salts.
Time (min)
Conversion (%)
Selectivity (%)
Yield (%)
OH
OH
OH
OH
O
O
10
20
30
60
90
50.4
58.1
65.6
72.2
84.7
89.2
53.7
52.3
42.2
25.7
11.9
4.02
39.2
43.4
47.1
71.5
82.5
88.9
27.1
30.4
27.7
18.6
10.1
3.59
19.8
25.2
30.9
51.6
69.9
79.3
150
Reaction conditions: 0.25 ␮mol CPO, 7.50 mmol cyclohexene, 14.8 mmol H2O2, 0.1 mol L−1 phosphate buffer, Additive concentration: TPABr 0.0751 mol L−1
.
(Tables 1 and 2). ECH would hydrolyze to CHD with the assist of
H3O+ in aqueous solution. In the later stage of reaction, the pH was
found decreased about 1.0 unit, which was the main reason for pro-
motion of epoxide hydrolysis. The increase of H+ was postulated
coming from the following two ways: one was the hydrolysis of N
atom with positive charge in QAS (N+ would seize OH− in H2O and
release H+); the other was the produced adipinic acid [37], which
was testified by a control experiment: a little part of cyclohexene
could be oxidized opening to adipinic acid by H2O2 in the presence
of QAS without CPO.
3.5. The structure changes and catalytic performance of CPO in
the presence of QAS
UV–vis spectrum assay was employed for the investigation
of heme micro-environment of CPO in the presence of QAS. The
UV–vis spectrum of CPO is caused mainly by porphyrin. There are
two nearly degenerate ꢁ → ꢁ* (a1u, a2u) transitions give rise to
a B (or Soret) band at 380–420 nm and Q (or visible) band(s) in
500–600 nm region. The maximum is Soret band at ꢂmax = 398 nm
[38]. The results revealed that the absorption at 398 nm was
increased, while no shift of peak position and no new absorption
peak appeared, which indicated no heme derivate was formed. The
increase of absorption should be attributed to the change of micro-
environment around heme. The crystal structures of CPO showed
the heme edge was not accessible directly, and there was a small
opening above the heme which could allow access of substrate to
active center. But the size and structure of substrates would be
restricted by this channel. The increased absorption at 398 nm sug-
gested that the heme ring was more exposure, so it was easier
for CPO to bind with substrates in such environment. Moreover,
TPABr corresponded to the maximum increase (Fig. 4a). Fig. 4b
showed if the additive concentration was higher than the optimum,
a decrease of 398 nm absorption would occur, probably due to a
destruction of the heme.
Like most protein molecule, the intrinsic fluorescence depends
mainly upon tryptophan (Trp), tyrosine (Tyr) and phenylalaline
(Phe) residue [39]. Fluorescence efficiency of them was 100:9:0.5.
So the dominant fluorophore in CPO was Trp. The maximum emis-
sion of CPO was around 337 nm. Fig. 5a showed an obvious increase
of emission intensity in the presence of QAS (especially in TPABr
media), while no red-shift of this emission peak was accompa-
nied. This indicated there was no obvious unfolding of enzyme
molecule. The increase of fluorescence yield was speculate to be
attributed to the energy transfer from Tyr to Trp, resulting in a flu-
orescence quenching of Tyr while an increase of Trp fluorescence
[40]. This implied the ␣-helix structure of CPO was strength-
ened in additives media, resulting in the distance between Tyr
Fig. 3. CHD yield in the presence of different quaternary ammonium salts or in pure
buffer. Reaction conditions: 0.25 ␮mol CPO, 7.50 mmol cyclohexene, 14.8 mmol
H2O2, 0.1 mol L−1 phosphate buffer, pH 5.0 at 35 ◦C. Additive concentration: TMABr:
0.130 mol L−1, TEABr: 0.0952 mol L−1, TPABr: 0.0751 mol L−1, TBABr: 0.0620 mol L−1
.

208
H. Zhang et al. / Applied Catalysis A: General 401 (2011) 204–209
Fig. 4. UV–vis spectrum of CPO (0.1 mol L−1 phosphate buffer, pH 5.0) in different quaternary ammonium salts at optimum concentration (a) or in the presence of TPABr
from 0 to 0.0939 mol L−1 (b).
Fig. 5. Fluorescent spectrum of CPO (0.1 mol L−1 phosphate buffer, pH 5.0) in different quaternary ammonium salts at optimum concentration (a) or in the presence of TPABr
from 0 to 0.0939 mol L−1 (b).
and Trp decreased. This conclusion was also confirmed by CD
assay.
yield. The improvement of CPO catalytic performance was char-
acterized by the decrease of Michaelis constant Km,app (apparent,
it was measured under the particular conditions of a defined con-
centration of the invariant substrate in the case of a two-substrate
enzyme), which indicated the affinity of CPO to substrate was
improved. The second order rate constants kcat/Km, a measure for
the catalytic efficiency and for the substrates specificity of the
enzyme, were also found to increase in the presence of additives
(especially in TPABr solution) [42–44].
Similarly, Fig. 5b showed a decrease in fluorescent intensity if
the additive concentration was higher than the optimum.
CD assay revealed the structure change of enzyme in this
process. In “far-UV” spectral region (190–250 nm), there was a
large negative absorption peak around 208 nm, which demon-
strated CPO was a typical ␣-helix protein. The most obvious
change appeared when QAS was introduced was that this nega-
tive absorption increased in the presence of additives (TPABr media
corresponded to maximum increase), implying the ␣-helix struc-
ture of CPO was strengthened (Fig. 6a)[41]. A same phenomenon
as above was observed in Fig. 6b that the negative absorption
began to decrease when additive concentration was higher than the
optimum.
Table 3
Kinetic parameters of cyclohexene epoxidation catalyzed by CPO at a given additive
concentration (optimum) at 35 ◦C.
Additives
Km,app (10−2 mol L−1
)
kcat (103 S−1
)
kcat/Km (mol−1 S−1 L)
The effect of QAS on CPO catalytic performance from the above
three-spectrum assay was consistent with each other, that is, TPABr
was the most efficient additive, and the concentration should be
controlled below optimum to ensure CPO holding the favorable
structure.
Furthermore, kinetic assay was carried out by measuring the
enzymic kinetic parameters (Table 3). The catalytic turnover fre-
quency (kcat) was increased, responding to the increase of product
Pure buffer
TMABr
TEABr
TPABr
TBABr
1.33 0.14
1.14 0.12
0.698 0.10
0.438 0.09
1.06 0.06
0.891 ( 0.16)
1.21 ( 0.11)
2.69 ( 0.12)
6.83 ( 0.07)
1.84 ( 0.07)
6.70 × 104
1.06 × 105
3.86 × 105
1.56 × 106
1.73 × 105
Reaction conditions: 14.8 mmol H2O2, 0.05 ␮mol CPO and the concentration of sub-
strate (cyclohexene) varied over the range of 0.005–0.05 mol L−1 at 35 ◦C. Additive
concentration: TMABr: 0.130 mol L−1, TEABr: 0.0952 mol L−1, TPABr: 0.0751 mol L−1
TBABr: 0.0620 mol L−1.
,

H. Zhang et al. / Applied Catalysis A: General 401 (2011) 204–209
209
Fig. 6. CD spectrum of CPO (0.1 mol L−1 phosphate buffer, pH 5.0) in different quaternary ammonium salts at optimum concentration (a) or in the presence of TPABr from 0
to 0.0939 mol L−1 (b).
4. Conclusion
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a value-added oxygenated
derivative, trans-1,2-cyclohexanediol, by CPO-catalyzed biotrans-
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media played two important roles: phase transfer catalysis and
tuning the composition of product.
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the enhancement of catalytic turnover frequency (kcat). Both the
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presence of quaternary ammonium salts.
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This work is supported by the National Natural Science Founda-
tion of China (20876094) and the Fundamental Research Funds for
the Central Universities (GK201001005).
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