Chemical modification of lipase for rational enhancement of enantioselectivity

Article abstract of DOI:10.1246/cl.150667

Chemical modifications of the I287C mutant of a Burkholderia cepacia lipase afforded various I287C-X conjugates, among which I287C-PAA bearing an N-phenylacetamide (PAA) moiety showed excellent enantioselectivity and catalytic activity for secondary alcohols. Site-directed chemical modifications are powerful tools to control enantioselective biocatalysis.

Full text of DOI:10.1246/cl.150667

CL-150667
Received: July 14, 2015 | Accepted: July 24, 2015 | Web Released: October 5, 2015
Chemical Modification of Lipase for Rational Enhancement
of Enantioselectivity
Tadashi Ema* and Hiroki Inoue
Division of Applied Chemistry, Graduate School of Natural Science and Technology,
Okayama University, Tsushima, Okayama 700-8530
(E-mail: ema@cc.okayama-u.ac.jp)
Chemical modifications of the I287C mutant of a Burkhol-
also allowed us to create variants that are superior to the wild-
type enzyme.7b,7c
deria cepacia lipase afforded various I287C-X conjugates,
among which I287C-PAA bearing an N-phenylacetamide (PAA)
moiety showed excellent enantioselectivity and catalytic activity
for secondary alcohols. Site-directed chemical modifications are
powerful tools to control enantioselective biocatalysis.
Random or site-directed mutagenesis is certainly the most
effective method for the alteration of enzyme structures, leading
to the improvement of enzymatic functions such as catalytic
activity and/or enantioselectivity.5,7 However, only twenty
natural amino acids can be introduced by mutagenesis, which
restricts diversity. With this limitation in mind, we decided to
investigate the potential of chemical modifications. Chemical
modifications can introduce various organic groups into en-
zymes to give a variety of hybrid biocatalysts, some of which
may fit a particular type of substrate. A number of methods for
chemical modifications have been developed.8,9 Among them,
we focused on cysteine-specific chemical modifications.9 The
thiol group of the cysteine residue is reactive for nucleophilic
aromatic substitution (SNAr),10 conjugate addition,11 the radical
reaction,12 disulfide bond formation,8a,9 and SN2 reaction.9,13
The high selectivity of the thiol group in these reactions should
be useful for the creation of hybrid biocatalysts with improved
enantioselectivity. It is a challenge to rationally improve or
control the enantioselectivity of enzymes by chemical modifi-
cations because successful examples of rational alterations
are limited.8a,14 Here we employed an I287C variant of a
Burkholderia cepacia lipase because position 287 is a hot spot
for the rational control of enantioselectivity for secondary
alcohols (Figure 1).6,7 The single I287C mutant was used to
prepare several chemically modified lipases (I287C-X), and
the effects of chemical modifications on catalytic activity and
enantioselectivity were examined. Among them, I287C-PAA
with an N-phenylacetamide (PAA) moiety showed excellent
enantioselectivity and catalytic activity for secondary alcohols.
We newly prepared the I287C variant of the lipase from
Burkholderia cepacia (NBRC 14595) via site-directed muta-
genesis, heterologous expression, in vitro refolding, and chro-
matographic purification.7 The catalytic activity of this I287C
variant was slightly lower than that of the wild-type enzyme but
was comparable to that of the I287A variant.7 This result
strongly suggests that an activator protein, which was produced
and mixed with the denatured lipase according to the previously
reported protocol,7 achieved the successful refolding of the
lipase including a disulfide bond formation. Therefore, among
the three cysteine residues in the I287C variant, Cys287 bears a
free thiol group while the other two cysteine residues (Cys190
and Cys270) are properly connected to each other via a disulfide
bond.
Lipases are useful biocatalysts that show high catalytic
activity and enantioselectivity especially for secondary alcohols
under mild reaction conditions (Scheme 1).1 Unlike other
enzymes, lipases can work in both aqueous and non-aqueous
media. Obviously, lipases are one of the most useful biocata-
lysts. However, they do not always give satisfactory results
toward unnatural substrates. In such cases, the solvent,2 temper-
ature,3 acylating agent,4 and enzyme structure5 are changed to
gain better outcomes. Figure 1 shows a transition-state model
proposed to rationalize the high enantioselectivity (R-preference)
of lipases for a wide range of secondary alcohols (Scheme 1).6
Based on this transition-state model, we have rationally altered
the structure of a lipase. For example, the I287F variant of a
Burkholderia cepacia lipase showed higher enantioselectivity
for 1-phenylethanol than the wild-type enzyme.7a Further studies
OH
OAc
OH
lipase
+
M
M
M
L
L
L
AcOCH=CH2
(R)-ester
(S)-alcohol
Scheme 1. Lipase-catalyzed kinetic resolution of secondary
alcohols.
O
NH
–
O
(solvent)
H
N
H
O
N
+
N
O
I287X
His286
O
H
X
N
H
Ser87
R1
His86
HN
O
N
R2
HN
O
H
H
O
–
H
O
N
N
O
HN
bulkiness
The I287C variant was subjected to chemical modifications
with six agents (IPAA, NEM, MSBT, BnBr, MSPOD, and
MVK) (Scheme 2). Except for MVK, a solution of the chemical
modification agent in DMSO was added to a solution of the
I287C mutant in 10 mM phosphate buffer (pH 7.0), while MVK,
R1 > R2 : faster-reacting enantiomer
R1 < R2 : slower-reacting enantiomer
Figure 1. Transition-state model for lipases toward secondary
alcohols, where residue 287 is added to the original version.
1374 | Chem. Lett. 2015, 44, 1374–1376 | doi:10.1246/cl.150667
© 2015 The Chemical Society of Japan

Table 2. Kinetic resolution of 1a with altered lipasesa
OH
OAc
OH
I287C-X
chemical
modification
+
SH
S
X
AcOCH=CH2
i-Pr2O, 30 °C, 8 h
1a
(R)-2a
(S)-1a
Entry
I287C-X
c/%b
E
1
2
3
4
5
6
7
I287C
20
41
24
36
47
34
34
5
I287C
I287C-X
I287C-PAA
I287C-NEM
I287C-BT
I287C-Bn
I287C-POD
I287C-MVK
29
20
16
11
6
H
N
N
I
O
N
O
MeO2S
O
Et
S
IPAA
NEM
MSBT
38
O
Br
O
MeO2S
Ph
aConditions: lipase I287C-X (200 mg, 0.5% (w/w) enzyme/Toyonite-
200M), 1a (0.50 mmol), vinyl acetate (1.0 mmol), molecular sieves 3A
N
N
BnBr
MVK
MSPOD
b
(3 pieces), dry i-Pr2O (5 mL), 30 °C, 8 h. Conversion.
Scheme 2. Preparation of I287C-X with chemical modification
agents.
used for comparison. The enantioselectivity was evaluated by
the E value.15 The results are shown in Table 2. The I287C
mutant gave a 20% conversion and an E value of 5 (Entry 1).
This low E value is reasonable because the steric hindrance that
is important for enantioselectivity (Figure 1) is decreased by the
replacement of Ile287 with less bulky cysteine. In contrast, to
our delight, I287C-PAA gave an E value of 29, which is almost
six times higher than that of the I287C mutant (Entry 2). This
Table 1. ESI-MS of altered lipases
Entry I287C-X
Expected valuea
Observed valueb
1
2
3
4
5
6
7
I287C
33277
33410
33402
33410
33367
33421
33347
33277
33410
33404
33410
33277 + 90n (n = 1-6)
33277 + 144n (n = 1-5)
33277 + 70n (n = 19)
I287C-PAA
I287C-NEM
I287C-BT
I287C-Bn
I287C-POD
I287C-MVK
change of the E value from 5 to 29 amounts to an energetic
¹1 7c
difference of 1.0 kcal mol
,
clearly indicating the effective-
ness of the chemical modification. Furthermore, I287C-PAA
showed a twofold conversion (41%) as compared with the I287C
mutant (20%), which indicates the enhanced activity of I287C-
PAA for 1a. The bulky organic group at position 287 may
suppress the fluctuation of the catalytic residue His286 to
enhance the catalytic activity (Figure 1). I287C-NEM and
I287C-BT also showed three to four times greater E values than
the I287C mutant (Entries 3 and 4). I287C-Bn showed twofold E
value (Entry 5), whereas I287C-POD showed no enhancement
in the E value (Entry 6). I287C-MVK with nineteen MVK
moieties exhibited seven times higher E value than the I287C
mutant, which is an unexpected but interesting result although
the effect of each MVK moiety attached to the protein is unclear
(Entry 7). All the results strongly suggest that the organic groups
introduced into Cys287 acted as stereochemical controllers.
In view of the above results, we selected I287C-PAA as the
best hybrid biocatalyst and examined the substrate scope of
I287C-PAA. The results are shown in Table 3. Surprisingly, in
all cases for 1a-1g, I287C-PAA gave higher E values than the
I287C mutant (Entries 1-7). In particular, I287C-PAA exhibited
excellent enantioselectivity (E > 200) for 1b, 1d, 1f, and 1g.
These results clearly demonstrate that I287C-PAA is an excellent
hybrid biocatalyst although it was created by a simple chemical
modification. Although I287C-PAA exhibited a higher E value
for 1c than the I287C mutant, I287C-PAA showed a lower
conversion than the I287C mutant. I287C-PAA also showed
good enantioselectivity for 1e. The fact that I287C-PAA showed
higher enantioselectivity for 1b-1g than for 1a can be explained
by an increased steric hindrance between the PAA moiety of
lipase and the more bulky substituent of the (S)-enantiomer of
1b-1g (Figure 1). The reaction time for 1b was shorter than
that for 1a probably because the nucleophilicity of the hydroxy
aThe values expected for the protein without the N-terminal Met. bThe
values obtained by the deconvoluted mass spectra.
which is water soluble, was directly added to the lipase solution.
The mixture was stirred at 4 °C or room temperature for several
hours. After a small amount of sample was taken for the ESI-MS
analysis, the chemically modified lipase was immobilized on
Toyonite-200M, a porous ceramic support with an organic group
at the surface, according to the previously reported procedure.7
The analytical sample was desalted, concentrated, and then
analyzed by the ESI-MS measurement. The results are summa-
rized in Table 1. The I287C mutant showed an expected peak
at 33277 (Entry 1), while I287C-PAA had an expected peak at
33410 (Entry 2), which clearly indicates the successful forma-
tion of the I287C-PAA conjugate. I287C-NEM and I287C-BT
exhibited an expected peak indicating the formation of the
corresponding conjugate (Entries 3 and 4) although a peak of the
unreacted I287C mutant was also observed at 33277. On the
other hand, I287C-Bn showed peaks suggesting that several
(one to six) benzyl groups were attached (Entry 5), while
I287C-POD also exhibited peaks suggesting that several (one to
five) 5-phenyl-1,3,4-oxadiazole (POD) moieties were attached
(Entry 6). Interestingly, I287C-MVK had a peak that was greater
than expected by 1260, which suggests that nineteen MVK
moieties were attached (Entry 7). It is most likely that BnBr,
MSPOD, and MVK were not cysteine-selective but reacted
nonselectively with several amino acid residues.
We next investigated the abilities of the chemically modified
lipases (I287C-X) as biocatalysts. The lipase-catalyzed kinetic
resolution of 1-phenylethanol (1a) was conducted with vinyl
acetate in dry i-Pr2O at 30 °C for 8 h. The I287C variant was
Chem. Lett. 2015, 44, 1374–1376 | doi:10.1246/cl.150667
© 2015 The Chemical Society of Japan | 1375

Table 3. Comparison between I287C-PAA and I287C in the
kinetic resolution of 1a
Liese, K. Seelbach, C. Wandrey, Wiley-VCH, Weinheim, 2006.
doi:10.1002/3527608184. d) Biocatalysis in the Pharmaceutical and
Biotechnology Industries, ed. by R. N. Patel, CRC Press, Florida,
2007. e) Practical Methods for Biocatalysis and Biotransformations,
ed. by J. Whittall, P. Sutton, John Wiley & Sons, West Sussex,
2010. doi:10.1002/9780470748589. f) K. Faber, Biotransformations in
Organic Chemistry: A Textbook, 6th ed., Springer-Verlag, Berlin, 2011.
doi:10.1007/978-3-642-17393-6.
a) A. M. Klibanov, Nature 2001, 409, 241. b) T. Itoh, E. Akasaki, K.
Kudo, S. Shirakami, Chem. Lett. 2001, 262. c) T. Itoh, S. Han, Y.
Matsushita, S. Hayase, Green Chem. 2004, 6, 437. d) T. Matsuda, T.
Harada, K. Nakamura, Green Chem. 2004, 6, 440. e) T. Matsuda,
J. Biosci. Bioeng. 2013, 115, 233. f) H. N. Hoang, T. Matsuda,
Tetrahedron Lett. 2015, 56, 639. g) Y. Kitamoto, Y. Kuruma, K. Suzuki,
T. Hattori, J. Org. Chem. 2015, 80, 521.
a) T. Sakai, I. Kawabata, T. Kishimoto, T. Ema, M. Utaka, J. Org. Chem.
1997, 62, 4906. b) T. Sakai, T. Kishimoto, Y. Tanaka, T. Ema, M. Utaka,
Tetrahedron Lett. 1998, 39, 7881.
a) T. Ema, S. Maeno, Y. Takaya, T. Sakai, M. Utaka, J. Org. Chem.
1996, 61, 8610. b) Y. Kita, Y. Takebe, K. Murata, T. Naka, S. Akai,
J. Org. Chem. 2000, 65, 83. c) S. Akai, T. Naka, T. Fujita, Y. Takebe, T.
Tsujino, Y. Kita, J. Org. Chem. 2002, 67, 411.
I287C-PAA
or I287C
OH
OAc
OH
+
R
R
R
AcOCH=CH2
i-Pr2O, 30 °C
1
(R)-2
I287C-PAA
c/%b
(S)-1
I287C
Time
/h
Entry
1
R
c/%b
E
2
E
1
2
3
4
5
6
7
1a
1b
1c
1d
1e
1f
Ph
8
41
44
18
50
43
35
44
29
>200
102
>200
111
>200
>200
20
34
50
47
36
30
34
5
34
8
88
7
79
126
4-Me-C6H4
3-Me-C6H4
4-Br-C6H4
3-Br-C6H4
2-naphthyl
(CH2)2Ph
5
19
10
9
13
5
3
4
1g
aConditions: I287C-PAA or I287C (200 mg, 0.5% (w/w) enzyme/
Toyonite-200M), 1 (0.50 mmol), vinyl acetate (1.0mmol), molecular
b
sieves 3A (3 pieces), dry i-Pr2O (5 mL), 30 °C. Conversion.
group of 1b was increased by the electron-donating methyl
group. It was also found that 1c and 1e with the substituent at
the meta position were less enantioselective than 1b and 1d with
the substituent at the para position. In addition, I287C-PAA took
a longer reaction time for 1c than for 1a. Probably, the PAA
moiety in I287C-PAA has a bad contact with the substituent
at the meta position of the (R)-enantiomers. 1f with the more
bulky substituent (naphthyl group) took a longer reaction time
probably because of steric repulsion with the lipase. On the
whole, I287C-PAA showed higher enantioselectivity and cata-
lytic activity than the I287C variant, and the enantioselectivity
was well controlled by the chemical modification of the hot spot
residue at position 287. Although I287C-PAA was less enantio-
selective for 1a (E = 29) than the wild-type enzyme (E = 68),7c
the enzymatic function of I287C-PAA for 1b-1g reached a
practical level (E > 100).
In summary, we have chemically modified a Burkholderia
cepacia lipase to create various hybrid biocatalysts. Among
them, the I287C-PAA conjugate, which was prepared by the
treatment of the I287C variant with 2-iodo-N-phenylacetamide,
showed excellent enantioselectivity for secondary alcohols. Site-
directed chemical modifications are powerful tools to control
enantioselective biocatalysis. This method will be useful even
when it is combined with directed evolution using random
mutagenesis.
5
a) M. Bocola, N. Otte, K.-E. Jaeger, M. T. Reetz, W. Thiel,
ChemBioChem 2004, 5, 214. b) Z. Qian, S. Lutz, J. Am. Chem. Soc.
2005, 127, 13466. c) J. D. Carballeira, P. Krumlinde, M. Bocola, A.
Vogel, M. T. Reetz, J.-E. Bäckvall, Chem. Commun. 2007, 1913.
d) M. T. Reetz, S. Prasad, J. D. Carballeira, Y. Gumulya, M. Bocola,
J. Am. Chem. Soc. 2010, 132, 9144. e) H. B. Brundiek, A. S. Evitt, R.
Kourist, U. T. Bornscheuer, Angew. Chem., Int. Ed. 2012, 51, 412. f) Q.
Wu, P. Soni, M. T. Reetz, J. Am. Chem. Soc. 2013, 135, 1872.
a) T. Ema, J. Kobayashi, S. Maeno, T. Sakai, M. Utaka, Bull. Chem. Soc.
Jpn. 1998, 71, 443. b) T. Ema, M. Jittani, K. Furuie, M. Utaka, T. Sakai,
J. Org. Chem. 2002, 67, 2144. c) T. Ema, K. Yamaguchi, Y. Wakasa, A.
Yabe, R. Okada, M. Fukumoto, F. Yano, T. Korenaga, M. Utaka, T.
Sakai, J. Mol. Catal. B: Enzym. 2003, 22, 181.
a) T. Ema, T. Fujii, M. Ozaki, T. Korenaga, T. Sakai, Chem. Commun.
2005, 4650. b) T. Ema, S. Kamata, M. Takeda, Y. Nakano, T. Sakai,
Chem. Commun. 2010, 46, 5440. c) T. Ema, Y. Nakano, D. Yoshida, S.
Kamata, T. Sakai, Org. Biomol. Chem. 2012, 10, 6299.
a) B. G. Davis, Curr. Opin. Biotechnol. 2003, 14, 379. b) E. Baslé, N.
Joubert, M. Pucheault, Chem. Biol. 2010, 17, 213. c) Y. Takaoka, A.
Ojida, I. Hamachi, Angew. Chem., Int. Ed. 2013, 52, 4088.
6
7
8
9
J. M. Chalker, G. J. L. Bernardes, Y. A. Lin, B. G. Davis, Chem.®Asian
J. 2009, 4, 630.
10 a) N. Toda, S. Asano, C. F. Barbas, III, Angew. Chem., Int. Ed. 2013, 52,
12592. b) A. M. Spokoyny, Y. Zou, J. J. Ling, H. Yu, Y.-S. Lin, B. L.
Pentelute, J. Am. Chem. Soc. 2013, 135, 5946.
11 a) H.-Y. Shiu, T.-C. Chan, C.-M. Ho, Y. Liu, M.-K. Wong, C.-M. Che,
Chem.®Eur. J. 2009, 15, 3839. b) A. Abbas, B. Xing, T.-P. Loh, Angew.
Chem., Int. Ed. 2014, 53, 7491. c) G. Badescu, P. Bryant, J. Swierkosz,
F. Khayrzad, E. Pawlisz, M. Farys, Y. Cong, M. Muroni, N. Rumpf, S.
Brocchini, A. Godwin, Bioconjugate Chem. 2014, 25, 460.
This work was partly supported by JSPS KAKENHI Grant
Number 25288078. We are grateful to the SC-NMR Laboratory
of Okayama University for the measurement of NMR spectra.
We are also grateful to Ms. Tsugumi Shiokawa and Dr. Hiroko
Tada at Division of Instrumental Analysis for the ESI-MS
measurements.
12 a) M. L. Conte, S. Staderini, A. Marra, M. Sanchez-Navarro, B. G.
Davis, A. Dondoni, Chem. Commun. 2011, 47, 11086. b) L. Markey, S.
Giordani, E. M. Scanlan, J. Org. Chem. 2013, 78, 4270.
13 a) H. Jo, N. Meinhardt, Y. Wu, S. Kulkarni, X. Hu, K. E. Low, P. L.
Davies, W. F. DeGrado, D. C. Greenbaum, J. Am. Chem. Soc. 2012, 134,
17704. b) M. Zabet-Moghaddam, A. L. Shaikh, S. Niwayama, J. Mass
Spectrom. 2012, 47, 1546. c) R. I. Nathani, P. Moody, V. Chudasama,
M. E. B. Smith, R. J. Fitzmaurice, S. Caddick, Chem. Sci. 2013, 4, 3455.
14 a) M. Dickman, J. B. Jones, Bioorg. Med. Chem. 2000, 8, 1957. b) Y.
Ivarsson, M. A. Norrgård, U. Hellman, B. Mannervik, Biochim.
Biophys. Acta, Gen. Subj. 2007, 1770, 1374. c) F. López-Gallego, O.
Abian, J. M. Guisán, Biochemistry 2012, 51, 7028. d) A. Bautista-
Barrufet, F. López-Gallego, V. Rojas-Cervellera, C. Rovira, M. A.
Pericàs, J. M. Guisán, P. Gorostiza, ACS Catal. 2014, 4, 1004.
15 C.-S. Chen, Y. Fujimoto, G. Girdaukas, C. J. Sih, J. Am. Chem. Soc.
1982, 104, 7294.
Supporting Information is available electronically on J-STAGE.
References
1
a) Enzyme Catalysis in Organic Synthesis: A Comprehensive Handbook,
2nd ed., ed. by K. Drauz, H. Waldmann, Wiley-VCH, Weinheim, 2002.
doi:10.1002/9783527618262. b) U. T. Bornscheuer, R. J. Kazlauskas,
Hydrolases in Organic Synthesis: Regio- and Stereoselective Biotrans-
formations, 2nd ed., Wiley-VCH, Weinheim, 2006. doi:10.1002/
3527607544. c) Industrial Biotransformations, 2nd ed., ed. by A.
1376 | Chem. Lett. 2015, 44, 1374–1376 | doi:10.1246/cl.150667
© 2015 The Chemical Society of Japan