Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase mutants with improved racemization activity

Article abstract of DOI:10.1016/j.molcatb.2015.02.012

Controlled racemization of enantiopure alcohols is a key step in dynamic kinetic resolution. We recently reported the racemization of enantiopure phenyl-ring-containing alcohols using W110A Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (W110A TeSADH), which relies on selectivity mistakes. Trp-110 lines the large pocket of the active site of TeSADH, which allows W110A TeSADH mutant to accommodate phenyl-ring-containing substrates in Prelog mode, albeit with selectivity mistakes. Here, we report the racemization of enantiopure phenyl-ring-containing alcohols using several Trp-110 mutants of TeSADH in the presence of the oxidized and reduced forms of nicotinamide-adenine dinucleotide. We observed a noticeable enhancement in racemization efficiency when W110G TeSADH was used compared with W110Q, W110M, W110L, W110I, and W110V. This observation was anticipated because the W110G mutation is expected to open the large pocket of the active site to a greater extent compared to other mutants of TeSADH at W110. Both enantiomers of 1-phenyl-2-propanol and 4-phenyl-2-butanol were fully racemized by W110G TeSADH. We also constructed a triple mutant of TeSADH, W110A/I86A/C295A, by site-directed mutagenesis with the aim of opening the two pockets of the active site of TeSADH. The W110A/I86A/C295A mutant was employed to racemize enantiopure phenyl-ring-containing alcohols. The current study demonstrates that W110G and W110A/I86A/C295A TeSADH are more efficient catalysts for the racemization of enantiopure secondary alcohols than the previously reported mutant W110A TeSADH [6].

Full text of DOI:10.1016/j.molcatb.2015.02.012

Accepted Manuscript
Title: Thermoanaerobacter ethanolicus secondary alcohol
dehydrogenase mutants with improved racemization activity
Author: Musa M. Musa Jay M. Patel Christopher M. Nealon
Chang Sup Kim Robert S. Phillips Ibrahim Karume
PII:
S1381-1177(15)00053-3
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.02.012
MOLCAB 3116
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
27-11-2014
10-2-2015
21-2-2015
Please cite this article as: M.M. Musa, J.M. Patel, C.M. Nealon, C.S. Kim, R.S. Phillips,
I. Karume, Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase mutants
with improved racemization activity, Journal of Molecular Catalysis B: Enzymatic
(2015), http://dx.doi.org/10.1016/j.molcatb.2015.02.012
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase mutants
with improved racemization activity
mutant TeSADH
NADP+, NADPH
mutant TeSADH
NADP+, NADPH
OH
OH
OH
OH
O
R1
R2
R1
R2
R1
R2
R1
R2
R1
R2
(S)-alcohol
(>99% ee)
(R)-alcohol
(>99% ee)
OH
R1
R2
rac-alcohol
1
Page 1 of 18

Thermoanaerobacter ethanolicus Secondary alcohol dehydrogenase mutants
with improved racemization activity
Highlights
 Racemization of enantiopure phenyl-ring-containing alcohols using different mutants of
TeSADH at Trp-110 site.
 Racemization of enantiopure phenyl-ring-containing alcohols using W110A/I86A/C295A
TeSADH.
 W110G TeSADH and W110A/I86A/C295A TeSADH are more efficient catalysts for
racemization than other W110 mutants.
2
Page 2 of 18

Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase mutants
with improved racemization activity
Musa M. Musa,*,a Jay M. Patel,b Christopher M. Nealon,b Chang Sup Kim,c Robert S. Phillipsb,d
and Ibrahim Karumea
a Department of Chemistry, King Fahd University of Petroleum and Minerals, Dhahran 31261,
KSA. E-mail: musam@kfupm.edu.sa
b Department of Chemistry, University of Georgia, Athens, GA 30602, USA
c
Department of Chemical and Biological Engineering, Hanbat National University, Daejeon
305-719, South Korea
d Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602,
USA
Corresponding author. Fax: +966-13-860-4277; Tel: +966-13-860-7343; E-mail address:
musam@kfupm.edu.sa (Musa M. Musa)
Keywords: alcohol dehydrogenase, racemization, redox reactions, secondary alcohols, site-
directed mutagenesis
3
Page 3 of 18

ABSTRACT
Controlled racemization of enantiopure alcohols is a key step in dynamic kinetic resolution. We
recently reported the racemization of enantiopure phenyl-ring-containing alcohols using W110A
Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (W110A TeSADH), which
relies on selectivity mistakes. Trp-110 lines the large pocket of the active site of TeSADH, which
allows W110A TeSADH mutant to accommodate phenyl-ring-containing substrates in Prelog
mode, albeit with selectivity mistakes. Here, we report the racemization of enantiopure phenyl-
ring-containing alcohols using several Trp-110 mutants of TeSADH in the presence of the
oxidized and reduced forms of nicotinamide-adenine dinucleotide. We observed a noticeable
enhancement in racemization efficiency when W110G TeSADH was used compared with
W110Q, W110M, W110L, W110I, and W110V. This observation was anticipated because the
W110G mutation is expected to open the large pocket of the active site to a greater extent
compared to other mutants of TeSADH at W110. Both enantiomers of 1-phenyl-2-propanol and
4-phenyl-2-butanol were fully racemized by W110G TeSADH. We also constructed a triple
mutant of TeSADH, W110A/I86A/C295A, by site-directed mutagenesis with the aim of opening
the two pockets of the active site of TeSADH. The W110A/I86A/C295A mutant was employed
to racemize enantiopure phenyl-ring-containing alcohols. The current study demonstrates that
W110G and W110A/I86A/C295A TeSADH are more efficient catalysts for the racemization of
enantiopure secondary alcohols than the previously reported mutant W110A TeSADH [6].
4
Page 4 of 18

1. Introduction
Deracemization of enantiomers is an ideal alternative to the well-known industrial kinetic
resolution (KR) approach, which is limited to 50% chemical yield with high enantiomeric purity
and therefore requires separation of starting materials from products. By contrast, dynamic
kinetic resolution (DKR) is a deracemization method that involves KR and in situ racemization
of the slowly reacting enantiomer, thus enabling complete conversion of a racemic starting
material to a single enantiopure product. Developing mild controlled racemization approaches is
crucial to enabling the development of new DKR methods. Metal catalysts have been used for
controlled racemization in DKR [1,2]. Most established racemization methods involve harsh
conditions that use acids or bases and are therefore unsuitable for use in DKR. Modern advances
in biotechnology allow enzymes to be altered to improve their effectiveness in organic synthesis
[3]. Because nature has a limited requirement for racemization, there is no known enzyme with
physiological racemase activity for secondary alcohols that is not accompanied by another
functionality [4]. Alcohol dehydrogenases (ADHs) have been employed to racemize enantiopure
alcohols [5,6,7,8]. Enzyme-catalyzed racemization represents a greener alternative to metal-
catalyzed racemization. For effective racemization, an ADH with low stereoselectivity is
required. However, only a few nonselective ADHs are known because the primary goal was to
search for highly selective enzymes [5].
Secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (TeSADH; EC
1.1.1.2), a nicotinamide-adenine dinucleotide phosphate (NADP+)-dependent ADH, has high
thermal stability and tolerance to elevated concentrations of organic solvents, making it a
favorable biocatalyst for organic synthesis [9,10,11,12]. Several mutants of TeSADH have been
designed with expanded substrate specificity compared to the wild type enzyme [13,14].
5
Page 5 of 18

Temperature [15,16], pressure [17], mutation [18,19] and reaction medium [20] influence the
stereospecificity of TeSADH. We recently reported a racemization approach for enantiopure
phenyl-ring-containing alcohols using W110A TeSADH in the presence of reduced and oxidized
forms of cofactor (i.e., NADPH and NADP+), shown in Scheme 1 [6]. TeSADH follows Prelog’s
rule; the stereoselectivity toward S-configured alcohols is higher if the bulkier alkyl group of the
alcohol has higher Cahn-Ingold-Prelog priority. Although ADHs are known for their high
enantioselectivities, selectivity mistakes can occur, leading to lower enantioselectivities. The
accumulation of selectivity mistakes leads to the formation of racemic mixtures under
appropriate conditions and time. Racemization is a thermodynamically downhill process (ΔG ≈ -
RT ln 2) because of the entropy increase associated with the formation of two enantiomers from
one [21].
OH
OH
O
W110A TeSADH
W110A TeSADH
R1
R2
R1
R2
R
R2
(S)-alcohol
(R)-alcohol
NADP+
NADPH
NADP+
NADPH
Scheme 1. W110A TeSADH-catalyzed racemization of enantiopure phenyl-ring-containing
secondary alcohols.
We recently created six mutants of TeSADH at the Trp-110 site to enhance the
enantioselectivity of the reduction of phenyl-ring-containing ketones [22]. In this study, we
evaluated the effect of these small alterations in the active site of TeSADH on the efficiency of
the racemization reactions of enantiopure phenyl-ring-containing secondary alcohols. We also
6
Page 6 of 18

report a triple mutant of TeSADH and demonstrate its effectiveness in the racemization of
enantiopure alcohols.
2. Experimental section
2.1. General
Capillary gas chromatographic measurements were performed on a gas chromatograph (GC)
equipped with a flame ionization detector and an HP chiral-20B column (30 m, 0.32 mm [i.d.],
0.25 μm film thickness) using Helium as the carrier gas. Nuclear Magnetic Resonance spectra
were recorded on a 500 MHz spectrometer at 500 MHz (1H) and at 125 MHz (13C) at room
temperature using the solvent peak as an internal standard. Details of GC and GC-MS analysis
have been provided in supplementary information. Commercial grade solvents were used without
further purification. Candida antarctica lipase B (CALB, Novozyme 435), NADP+, NADPH,
isopropenyl acetate, (R)-1-phenyl-2-propanol [(R)-1a] (>99% ee), (S)-1-phenyl-2-propanol [(S)-
1a] (>99% ee), (rac)-1-phenyl-2-propanol [(rac)-1a], (rac)-4-phenyl-2-butanol [(rac)-2a], (R)-4-
phenyl-2-butanol [(R)-2a] (>99% ee), (S)-4-phenyl-2-butanol [(S)-2a] (>99% ee), 4-(4ˈ-
methoxyphenyl)-2-butanone, and 1-phenyl-2-butanone were used as purchased from commercial
sources. (R)-2a (>99% ee), (S)-2a (72.7% ee), and (R)-1-phenyl-2-butanol [(R)-4a] (98% ee)
were prepared by lipase-catalyzed kinetic resolution of their racemates as reported [22]. (S)-4-
(4ˈ-methoxyphenyl)-2-butanol [(S)-3a] (91% ee) was prepared by the W110A TeSADH-
catalyzed reduction of the corresponding ketone as reported [23].
2.2. Methods
7
Page 7 of 18

2.2.1. Preparation, gene expression and purification of mutants
All point mutations were introduced by PCR-amplified oligonucleotide-directed mutagenesis
using a modified QuikChange site-directed mutagenesis protocol (Agilent) and confirmed by
DNA sequencing. The wild-type T. ethanolicus adhB gene for TeSADH in the pADHB1M1-kan
plasmid (a pBluescriptII KS(+)-kanamycin derivative, Burdette et al.) was used as the template
[11,24]. W110G, W110V, W110I, W110Q, W110L, W110M TeSADH (W: tryptophan, G:
glycine, V: valine, I: isoleucine, Q: glutamine, L: leucine, M: methionine) were prepared,
expressed and purified as reported [22]. To prepare the double mutant, I86A/C295A TeSADH, a
C295A mutation (C: cysteine) was introduced in the single mutant I86A [14] using the forward
mutagenic primer 5′- ATAAAAGGCGGGCTAGCCCCCGGTGGACGTCT and its reverse
complement as the reverse mutagenic primer (the mutated nucleotides are bold and underlined).
To prepare the triple mutant, W110A/I86A/C295A TeSADH, an additional mutation (W110A)
was introduced in the double mutant using the forward mutagenic primer 5′-
TGGAATGCTGGCAGGCGCGAAATTTTCGAATGTA and its reverse complement as the
reverse mutagenic primer. W110A/I86A/C295A TeSADH was then expressed and purified as
reported [22], with the exception that the heat treatment was performed at 65°C instead of 70°C.
Briefly, the TeSADH gene was expressed in E. coli DH5α under the control of the lacZ promoter
on the pBluescriptII KS(+) plasmid. Enzyme concentrations were determined by the Bradford
protein assay [25].
2.2.2. General procedure for mutant TeSADH-catalyzed racemization
Enantiopure alcohol (0.015 mmol), NADP+ (0.5 mg), NADPH (1.0 mg), mutant TeSADH (0.2
mg in Tris-HCl buffer solution, 50 mM, pH 8.0), Tris-HCl buffer solution (950 μL, 50 mM, pH
8
Page 8 of 18

8.0), and acetonitrile (50 μL) were mixed in a 1.5-mL plastic tube. The reaction mixture was
shaken at 50°C and 200 rpm for 48 h, followed by extraction with ethyl acetate (2×500 μL). The
combined organic layer was dried with sodium sulfate and concentrated to dryness. The
remaining residue was treated with pyridine (two drops) and acetic anhydride (one drop) for 1 h
to convert the alcohols to their corresponding acetates. The acetate products were diluted with
CHCl3 prior to analysis on a GC equipped with a chiral column to determine ee.
2.2.3. Determination of the absolute configuration of alcohols
The racemization products were converted to the corresponding acetate derivatives, then
injected in a GC equipped with a chiral stationary phase. The resulting retention time was then
compared with the R- or S-acetate derivatives of standard samples of alcohols. The acetate
derivatives of the racemization products of enantiopure 1a and 2a were co-injected with the
acetate derivatives of standard samples of enantiopure alcohols. The derivatized racemization
products of (S)-3a were co-injected with a sample of (S)-3a prepared by W110A TeSADH-
catalyzed reduction of the corresponding ketone, which is know to produce (S)-3a [23]. The
acetate derivatives of racemization products of (R)-4a were co-injected with a sample prepared
by Lipase-catalyzed kinetic resolution of (rac)-4a. Details of chiral GC analysis, GC-MS
analysis have been provided in supplementary information.
3. Results and discussion
3.1. Racemization of (R)-1-phenyl-2-propanol and (S)-1-phenyl-2-propanol using different
mutants of TeSADH at the W110 site
9
Page 9 of 18

We examined racemization of enantiopure phenyl-ring-containing alcohols using W110X
TeSADH, where X= V, L, I, Q, M, or G. We utilized these mutants to racemize (R)-1-phenyl-2-
propanol [(R)-1a] and (S)-1-phenyl-2-propanol [(S)-1a], as shown in Table 1, to study the effect
of different mutations at W110 on the efficiency of racemization. Because the enzyme and its
W110 mutants obey Prelog's rule, the active site of TeSADH can be modeled as two pockets,
large and small [13,14,18,26]. These pockets play an essential role in aligning substrates to
achieve stereospecific reactions. W110 lies within the large pocket of TeSADH, and thus
substrates such as (S)-1a are expected to fit with the benzyl moiety in the large pocket and the
methyl group in the small pocket [13]. We selected the enantiopure forms of alcohol 1a as
models in this study because we previously determined that the enantioselective reduction of
phenylacetone using W110A TeSADH produces (S)-1a in only 37% ee [23]. This poor
enantioselectivity could be explained by the ability of phenylacetone to fit within the large
pocket in alternating modes, thus increasing “selectivity mistakes”.
Table 1. W110X TeSADH-catalyzed racemization of enantiopure 1-phenyl-2-propanol (X= V,
L, I, Q, M, or G).a
OH
OH
O
W110X TeSADH
W110X TeSADH
Ph
Ph
Ph
NADPH, NADP+
50oC
NADPH, NADP+
50oC
(R)-1a, >99% ee
(S)-1a, >99% ee
1b
Mutant
TeSADH
ee (%)b
E valuec
Entry
Substrate
1
2
3
4
5
(S)-1a
(R)-1a
(S)-1a
(R)-1a
(S)-1a
77.5 (S)
82.6 (R)
98.4 (S)
>99 (R)
87.4 (S)
W110V
134.5 ± 27.7
W110L
W110I
104.4 ± 42.1
80.3 ± 16.2
10
Page 10 of 18

6
7
(R)-1a
(S)-1a
(R)-1a
(S)-1a
(R)-1a
(S)-1a
(R)-1a
(S)-1a
(R)-1a
92.6 (R)
83.6 (S)
82.3 (R)
61.4 (S)
72.8 (R)
6.3 (S) d
10.3 (R) d
0.2 (R)
W110Q
W110M
W110A
W110G
80.0 ± 17.5
16.3 ± 3.5
17.4 ± 4.7
9.0 ± 2.6
8
9
10
11
12
13
14
1.5 (R)
a Conditions: Reactions were performed at 50°C for 48 h using enantiopure 1a (2.0 mg, 0.015 mmol), NADP+ (0.5
mg), NADPH (1.0 mg), W110X TeSADH (0.2 mg), Tris-HCl buffer solution (950 μL, 50 mM, pH 8.0), and
acetonitrile (50 μL). b ee values were determined by chiral stationary phase GC of the corresponding acetate
derivatives. c E values were obtained from (J. M. Patel et al., Org. Biomol. Chem., 2014, 12, 5905-5910). d Results
for W110A TeSADH-catalyzed racemization were obtained from (M. M. Musa et al., Org. Biomol. Chem., 2013,
11, 2911-2915).
The racemization reactions of (S)-1a and (R)-1a were performed in Tris-HCl buffer
solution (50 mM, pH 8.0) containing acetonitrile (5%, v/v) to enhance the solubility of the
alcohol substrates. W110V TeSADH decreased the ee of (S)-1a and (R)-1a from >99% to 77.5%
and 82.6%, respectively. In a previous report [22], kinetic assays revealed that this mutant has a
kcat/Km value of 45,300 M-1s-1 for (S)-1a and an E value of 134.5 [E = (kcat/Km)S/(kcat/Km)R], an
indication that even a highly stereospecific mutant for (S)-1a, such as W110V TeSADH, can lead
to racemization if sufficient time is allowed. Under the same conditions and using W110L
TeSADH, a negligible decrease in ee was observed for (S)-1a and no change for (R)-1a after 48
h. Thus, the E value is likely not the only factor influencing racemization, and the stability of the
mutant may also be important. Racemization using W110I, W110Q, and W110M TeSADHs
yielded results similar to those obtained with W110V TeSADH. Surprisingly, even though
W110M has a significantly lower E value than W110V, W110I and W110Q, they all yielded
similar racemization results (Table 1). Among the six W110 mutants tested, W110G TeSADH
achieved the best racemization results with a racemic mixture obtained from the racemization of
11
Page 11 of 18

(S)-1a and (R)-1a after 48 h. This performance is explained by the bigger size of the larger
pocket of the active site in this mutant when compared to others, which enables it to recognize
both enantiomers of 1a (leading to more “selectivity mistakes”). The improved efficiency of
W110G TeSADH in racemization is consistent with the kinetic parameters shown in Table 1
because it exhibits the lowest E value among the six W110 mutants evaluated in the current
study. W110G TeSADH-catalyzed racemization reactions of (S)-1a and (R)-1a are also more
effective than those reported using W110A TeSADH [6], as shown in Table 1.
3.2. Racemization of enantiopure phenyl-ring-containing alcohols using W110G TeSADH and
W110V TeSADH
Encouraged by the results obtained with W110G TeSADH in the racemization of (R)-1a
and (S)-1a, we next used this mutant to racemize other phenyl-ring-containing enantiopure
alcohols, as shown in Table 2. Under the same conditions used for the racemization of
enantiopure 1a in Table 1, (S)-4-phenyl-2-butanol [(S)-2a] and (R)-4-phenyl-2-butanol [(R)-2a]
were fully racemized using W110G TeSADH. In a previous study, W110A TeSADH-catalyzed
racemization of (S)-2a and (R)-2a resulted in reductions of ee from 72.7% and 99% to 51.8% and
82.4%, respectively [6]. The ee of (S)-4-(4′-methoxyphenyl)-2-butanol [(S)-3a] was reduced
from 91% to 1.4% using W110G TeSADH compared to 44% when W110A TeSADH was used.
The W110G TeSADH-catalyzed racemization of (R)-1-phenyl-2-butanol [(R)-4a] reduced the ee
from 98% to 74%.
Table 2. Racemization of enantiopure phenyl-ring-containing alcohols using W110G TeSADH
and W110V TeSADH.a
12
Page 12 of 18

OH
OH
O
W110X TeSADH
W110X TeSADH
R1
R2
R1
R2
R1
R2
NADPH, NADP+
50 oC
NADPH, NADP+
50 oC
(S)-na
nb
(R)-na
ee (%)b
ee (%)b
Entry
R1
R2
Substrate
before
after
X = G
0.1 (R)
0.2 (R)
1.4 (S)
74 (R)
X = V
1
2
3
4
PhCH2CH2
PhCH2CH2
p-MeO-C6H4(CH2)2
PhCH2
CH3
CH3
CH3
(S)-2a
(R)-2a
(S)-3a
(R)-4a
72.7
>99
91
64.3 (S)
92 (R)
73.3 (S)
94.8 (R)
CH3CH2
98
a Conditions: Reactions were performed at 50°C for 48 h using enantiopure na (0.015 mmol), NADP+ (0.5 mg),
NADPH (1.0 mg), W110X TeSADH (0.2 mg), Tris-HCl buffer solution (950 μL, 50 mM, pH 8.0), and acetonitrile
(50 μL). b ee values were determined by chiral stationary phase GC of the corresponding acetate derivatives.
To validate the efficiency of W110G TeSADH for racemization, we conducted W110V
TeSADH-catalyzed racemization for the same enantiopure alcohol substrates under the same
reaction conditions used for W110G TeSADH. W110V TeSADH reduced the ee of (S)-2a from
72.7% to 64.3% and that of (R)-2a from >99% to 92%, as shown in Table 2. The ee of (S)-3a
was reduced from 91% to 73.3% in the W110V TeSADH-catalyzed racemization, whereas the ee
of (R)-4a was reduced from 98% to 94.8%. The results shown in Table 2 demonstrate that
W110G TeSADH is a remarkable catalyst for the racemization of phenyl-ring-containing
enantiopure alcohols. These results also indicate that a highly stereospecific mutant for (S)-
configured alcohols, such as W110V TeSADH, can still racemize enantiopure alcohols, albeit
less efficiently than W110G TeSADH.
3.3. Racemization of enantiopure phenyl-ring-containing alcohols using W110A/I86A/C295A
TeSADH
13
Page 13 of 18

To further lower the stereospecificity of W110A TeSADH toward phenyl-ring-containing
alcohols and thus improve its performance in racemization reactions, we constructed a triple
mutant, W110A/I86A/C295A TeSADH. This mutant was designed to expand both pockets of
TeSADH and consequently was predicted to increase the occurrence of selectivity mistakes. As
expected, using this mutant, the ee of (R)-1a and (S)-1a were reduced from >99% to
approximately 3% (Table 3) under the same conditions described for the other mutants in Tables
1 and 2. The W110A/I86A/C295A TeSADH-catalyzed racemization resulted in a decrease of ee
from 72.7% to 21.7% for (S)-2a and >99% to 8.2% for (R)-2a. The improved racemization of
(R)-2a in comparison with (S)-2a was previously observed when W110A TeSADH was used
with (S)-2a and (R)-2a but not (R)-1a and (S)-1a [6]. This is explained by the Prelog
stereopreference for this enzyme and the significantly higher E value of 2a compared to 1a [6].
The enzymatic oxidation of (R)-2a to the corresponding ketone (a difficult process) followed by
its enantioselective reduction to (S)-2a is expected to result in accumulation of the latter (i.e.,
more effective racemization). The W110A/I86A/C295A TeSADH-catalyzed racemization of (S)-
3a lowered its ee from 91% to 25.3%. The lower performance of W110A/I86A/C295A TeSADH
in the racemization of enantiopure 1a, 2a, and 3a compared to W110G TeSADH could be
explained by differences in the stability of the two mutants. The triple mutant is expected to be
less stable because the active site may be unable to bind Zn, which is essential for enzymatic
activity. However, the triple mutant TeSADH reduced the ee of (R)-4a from 98% to 47.7%
(Table 3, entry 6), superior to the racemization result obtained using W110G TeSADH (entry 4
of Table 2). W110A/I86A/C295A TeSADH is a more efficient catalyst for the racemization of
(S)-1a, (R)-1a, (S)-2a, (R)-2a, and (S)-3a than the previously reported mutant W110A TeSADH
[6].
14
Page 14 of 18

Table 3. W110A/I86A/C295 TeSADH-catalyzed racemization of enantiopure phenyl-ring-
containing alcohols.a
W110A/I86A/C295A
TeSADH
OH
W110A/I86A/C295A
TeSADH
OH
O
R1
R2
R1
R2
R1
R2
NADPH, NADP+
50 oC
NADPH, NADP+
50 oC
(S)-na
nb
(R)-na
ee (%)b
before
>99
ee (%)b
after
3.7 (S)
Entry
R1
R2
Substrate
1
2
3
4
5
6
PhCH2
CH3
CH3
CH3
CH3
CH3
(S)-1a
(R)-1a
(S)-2a
(R)-2a
(S)-3a
(R)-4a
PhCH2
>99
72.7
>99
91
3.4 (R)
21.7 (S)
8.2 (R)
PhCH2CH2
PhCH2CH2
p-MeOC6H4(CH2)2
PhCH2
25.3 (S)
CH3CH2
98
47.7 (R)
a Conditions: Reactions were performed at 50°C for 48 h using enantiopure na (0.015 mmol), NADP+ (0.5 mg),
NADPH (1.0 mg), W110A/I86A/C295A TeSADH (0.2 mg), Tris-HCl buffer solution (950 μL, 50 mM, pH 8.0), and
b
acetonitrile (50 μL). ee values were determined by chiral stationary phase GC of the corresponding acetate
derivatives.
W110G TeSADH and W110A/I86A/C295A TeSADH are more efficient catalysts for
racemization of enantiopure phenyl-ring-containing alcohols than the previously reported
W110A TeSADH [6]. They are also more efficient racemization catalysts for these substrates
than ADH-“A”, Lactobacillus kefir ADH, and KRED-118 in racemization of (R)-2-octanol using
the single enzymatic approach reported by Gruber et al. [5].
In all racemization reactions described in this report, the percentage of ketone formed due
to equilibrium with alcohols did not exceed 3%. Generating more mutants of TeSADH will be
crucial to alter its stereospecificity and improve its racemization performance. TeSADH, with its
15
Page 15 of 18

remarkable thermal stability and organic solvent tolerance [7-10,18], can thus be used in situ
with a KR method to achieve successful DKR.
4. Conclusion
In this study, the racemization of phenyl-ring-containing enantiopure alcohols was studied
using seven mutants of TeSADH. Among the tested mutants, six were at the W110 site (W110V,
W110L, W110I, W110Q, W110M, and W110G). W110G TeSADH exhibited a remarkable
improvement in the racemization of phenyl-ring-containing enantiopure alcohols compared to
the other five W110 mutants tested in this study, and racemic mixtures were obtained from
enantiopure (S)-1-phenyl-2-propanol, (R)-1-phenyl-2-propanol, (S)-4-phenyl-2-butanol, (R)-4-
phenyl-2-butanol, and (S)-4-(4′-methoxyphenyl)-2-butanol. W110G TeSADH is also more
efficient than the previously reported mutant W110A TeSADH in the racemization of
enantiopure phenyl-ring-containing alcohols. The current results indicate that the W110 site
plays a very important role in controlling the stereospecificity of TeSADH-catalyzed redox
reactions of phenyl-ring-containing alcohols and their corresponding ketones. We also evaluated
the racemization ability of W110A/I86A/C295A TeSADH using the same enantiopure alcohols
and obtained results similar to those obtained with W110G TeSADH. Two of the mutants
reported in the current study, W110G TeSADH and W110A/I86A/C295A TeSADH, are more
efficient catalysts in racemization of enantiopure alcohols than the previously reported W110A
TeSADH. This enhancement of the efficiency of the environmentally benign TeSADH-catalyzed
racemization may enable its use in situ with a KR method to achieve DKR.
16
Page 16 of 18

Acknowledgments
The authors acknowledge the support provided by King Abdulaziz City for Science and
Technology (KACST) through the Science and Technology Unit at King Fahd University of
Petroleum and Minerals (KFUPM), for funding this work through project No. 11-BIO1666-04,
as part of the National Science, Technology and Innovation Plan. This research was also partially
supported by the Basic Science Research Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education (NRF-2012R1A1A4A01012231).
References
[1] M.C. Warner and J.-E. Bäckvall, Acc. Chem. Res. 46 (2013) 2545-2555.
[2] S. Manzini, C.A. Urbina-Blanco, A. Poater, A.M.Z. Slawin, L. Cavallo, S.P. Nolan,
Angew. Chem. Int. Ed. 51 (2012) 1042-1045.
[3] U.T. Bornsheuer, G.W. Huisman, R.J. Kazlauskas, S. Luts, J.C. Moore, K. Robins, Nature
485 (2012) 185-194.
[4] B. Schnell, K. Faber, W. Kroutil, Adv. Synth. Catal. 234 (2003) 653-666.
[5] C.C. Gruber, B.M. Nestl, J. Gross, P. Hildebrandt, U.T. Borncheuer, K. Faber, W. Kroutil,
Chem. Eur. J. 13 (2007) 8271-8276.
[6] M.M. Musa, R.S. Phillips, M. Laivenieks, C. Vieille, M. Takahashi, S.M. Hamdan, Org.
Biomol. Chem. 11 (2013) 2911-2915.
[7] H. Yang, A. Jonsson, E. Wehtje, P. Adlerccreutz, B. Mattiasson, Biochim. Biophys. Acta
1336 (1997) 51-58.
[8] S. Kara, D. Spichermann, A. Weckbecker, C. Leggewie, I.W.C.E. Arends, F. Hollmann
ChemCatChem 6 (2014) 973-976.
17
Page 17 of 18

[9] C. Heiss, M. Laivenieks, J.G. Zeikus, R.S. Phillips, Bioorg. Med. Chem. 9 (2001) 1659-
1666.
[10] D. Burdette, J.G. Zeikus, Biochem. J. 302 (1994) 163-170.
[11] D. Burdette, C. Vieille, J.G. Zeikus, Biochem. J. 316 (1996) 115-122.
[12] M.M. Musa, K.I. Ziegelmann-Fjeld, C. Vieille, J.G. Zeikus, R.S. Phillips, Angew. Chem.
Int. Ed. 46 (2007) 3091-3094.
[13] K.I. Ziegelmann-Fjeld, M.M. Musa, R.S. Phillips, J.G. Zeikus, C. Vieille, Protein Eng.
Des. Sel. 20 (2007) 47-55.
[14] M.M. Musa, N. Lott, M. Laivenieks, L. Watanabe, C. Vieille, R.S. Phillips, ChemCatChem
1 (2009) 89-93.
[15] V.T. Pham, R.S. Phillips, L.G. Ljungdhal J. Am. Chem. Soc. 111 (1989) 1935-1936.
[16] C. Heiss, M. Laivenieks, G. Zeikus, R.S. Phillips, J. Am. Chem. Soc. 123 (2001) 345-346.
[17] J.M. Patel, R.S. Phillips, ACS catalysis 4 (2014) 692-694.
[18] C. Heiss, M. Laivenieks, J. Zeikus, R.S. Phillips, Bioorg. Med. Chem. 9 (2001) 1659-1666.
[19] A.E. Tripp, D.S. Burdette, J.G. Zeikus, R.S. Phillips, J. Am. Chem. Soc. 120 (1998) 5137-
5141.
[20] M.M. Musa, K.I. Ziegelmann-Fjeld, C. Vieille, R.S. Phillips, Org. Biomol. Chem. 6 (2008)
887-892
[21] E. Eliel, S.H. Wilen, M.P. Doley, Basic Organic Stereochemistry, Wiley, New York, 2001,
pp. 102-106.
[22] J.M. Patel, M.M. Musa, L. Rodriguez, D.A. Sutton, V. Popik, R.S. Phillips, Org. Biomol.
Chem. 12 (2014) 5905-5910.
[23] M.M. Musa, K.I. Ziegelmann-Fjeld, C. Vieille, J.G. Zeikus, R.S. Phillips, J. Org. Chem. 72
(2007) 30-34.
[24] D.S. Burdette, F. Secundo, R.S. Phillips, J. Dong, R.A. Scott, J.G. Zeikus, Biochem. J. 326
(1997) 717–724.
[25] M.M. Bradford, Anal. Biochem. 72 (1976) 248-254.
[26] E. Keinan, E.K. Hafeli, K.K. Seth, R. Lamed, J. Am. Chem. Soc. 108 (1986) 162-169.
18
Page 18 of 18