Mapping the substrate selectivity of novel lipase from pseudozyma hubeiensis SY62

Full text of DOI:10.1002/bkcs.10918

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DOI: 10.1002/bkcs.10918
BULLETIN OF THE
S. Park
KOREAN CHEMICAL SOCIETY
Mapping the Substrate Selectivity of Novel Lipase from Pseudozyma
hubeiensis SY62
*
Seongsoon Park
Department of Chemistry, Center for NanoBio Applied Technology, Institute of Basic Sciences, Sungshin
Women’s University, Seoul 01133, Republic of Korea, *Eail: spark@sungshin.ac.kr
Received July 20, 2016, Accepted August 5, 2016
Keywords: Biocatalyst, Chiral alcohol, Kinetic resolution, Lipase, Selectivity
Biocatalysts are one of the strongest tools in syntheses of
chiral building blocks.1 The preparation of chiral building
blocks is an important step in drug syntheses because the
stereochemistry of drug serves a critical role in many drug
actions.2 Numerous biocatalysts have been isolated from
microorganism, plants, and animals.3 A traditional method
to obtain biocatalysts is their direct isolation and purifica-
tion from microorganisms, or tissues of animals and plants.
In current days, more popular approaches are based on
molecular biology technique. Researchers have used direct
cloning the corresponding genes of the biocatalysts from
various organisms, and expressed them in microorganisms
such as yeast and E. coli. More recently, a new approach to
obtain novel biocatalysts has been employed based on
searching the genetic information in the banks of genome
or protein sequences according to increase of genomic
information.3,4 Instead of the direct cloning genes from
organisms, researches chemically synthesize the target
genes, which are identified from the genome or protein
sequence pool. The synthetic genes can be used to express
biocatalysts in microorganisms. Our research group has
also used such approach to find novel biocatalysts, which
are homologous lipases of Candida antarctica lipase B
(CAL-B, also called as Pseudozyma antarctica lipase B).5
CAL-B possesses remarkably high enantioselectivity
towards chiral sec-alcohols, and thus has been widely used
in the syntheses of enantiopure sec-alcohols or correspond-
ing esters.6 Six sequences possessing above 60% identity
with that of CAL-B were identified through protein
sequence comparison in the protein sequence bank, and
four lipases were functionally expressed in E. coli. Lipase
from Pseudozyma hubeiensis SY62 (PHL) exhibited the
highest hydrolysis activity among four enzymes. In the cur-
rent investigation, the detailed study of the substrate selec-
tivity and enantioselectivity of the novel lipase (PHL) was
performed with the reactions for a series of substrates to
serve useful information as a biocatalyst.
The two moieties enter into two different binding pockets
of lipase: namely, the acyl and alcohol binding pockets.
Hence, each lipase often shows the different selectivities
according to the chain length of the acyl or alcohol moi-
eties. For identification of the chain length selectivity of
PHL, a series of ethyl esters and acetates with varied car-
bon chain lengths were chosen (Scheme 1).
The substrate library was examined for the hydrolytic
activity of PHL by a colorimetric assay method, which is
based on a pH indicator (4-nitrophenol).7 Hydrolysis of an
ester compound by PHL produces protons because of acid
generation, and the produced protons cause a decrease in
the absorbance of 4-nitrophenol at 404 nm. The absorbance
change was monitored by a microplate reader. Assay with
acetates (1a–g in Scheme 1) exhibited that PHL accepted
most substrates, and higher activity was observed as the
length of the carbon chain of the alcohol moiety increases
(Figure 1). This implies that the active site of PHL can well
accommodate a long and linear carbon chain in the alcohol
binding pocket. On the other hand, screening towards an
ethyl ester library (2a–h in Scheme 1) exhibited a different
trend. The maximum activity of PHL was observed with
the reaction by the substrate 2d (C6) (Figure 2). This repre-
sents that the acyl binding pocket possesses a limited space
and can accept a linear acyl group up to 2d without severe
steric clash. However, a longer acyl group than C6 may
undergo distortion and bending to get into the acyl binding
pocket. Hence, the distortion is presumably responsible for
the retardation of the reaction.
A homology model supports the hypothesis. The amino
acid sequence of PHL possesses 74% sequence identity
with the CAL-B sequence. It implies that a homology
model of PHL can be generated with high accuracy because
homology modeling with a template possessing above 50%
sequence identity can provide a high quality model. A
homology model was generated by the SWISS-MODEL
(https://swissmodel.expasy.org) (Figure 3(a)). Obviously,
the homology model of PHL is resembled with the crystal
structure of CAL-B (RMSD = 0.418 Å for Cα atoms,
Figure S1, Supporting information). Based on the model
structure, a tetrahedral intermediate of PHL was created
The role of lipases in nature is hydrolysis of ester com-
pounds such as lipids. The ester compounds possess two
moieties (i.e., the acyl and alcohol moieties), which are
present on each side of the scissile bond (i.e., ester bond).
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Scheme 1. Substrates used to survey the selectivity of PHL.
Figure 1 Specific activity of PHL for hydrolyses of alkyl acetates.
Figure 3 (a) A homolog model of PHL. (b) A tetrahedral interme-
diate model. The catalytic triad (Asp188-His224-Ser105), the oxy-
anion hole (Gln106 and Thr40), Thr138, Gln157, and the
substrate (pentyl hexanoate) were represented by a stick model.
The terminal carbon atom of the acyl group of the substrate pos-
sesses hydrophobic interactions with the side chains of the Thr138
and Gln157 residues (yellow color).
chosen and transesterification with vinyl butyrate was car-
ried out (Table 1). The medium substituent at the chiral
center of the sec-alcohols was fixed to a methyl group and
the large substituent was varied. The reactions were con-
ducted with an immobilized PHL on Celite9 at 25ꢀC in t-
butyl methyl ether (MTBE). The enantioselectivity was
determined by enantiomeric ratio (E), which was calculated
using the method of Chen et al. from the enantiomeric
excesses of both starting alcohol and ester product.8 PHL,
similar to most lipases including CAL-B, exhibited R-
stereoselectivity towards all substrates.10 PHL exhibited
moderate or low enantioselectivity (E = 19 and 5) when the
large substituent is an ethyl or acetylene group (3a and 3b,
respectively). However, when one more carbon was
attached to the alcohol chain (3c), PHL exclusively reacted
with (R)-enantiomer to produce enantiopure (R)-4c (ee =
99.9%), and the reaction conversion also increased. PHL
exhibited high enantioselectivity towards rac-3d bearing an
Figure 2 Specific activity of PHL for hydrolyses of ethyl esters.
with pentyl hexanoate (Figure 3(b)). The docking structure
exhibited that the acyl moiety (C6) of pentyl hexanoate
binds well without severe steric clash, and the substrate
possesses a hydrophobic interaction with the side chains of
Thr138 and Gln157 (Figure 3(b)). The model structure sug-
gests that if one more carbon attaches to the acyl moiety,
the acyl moiety should be distorted to avoid steric clash.
The distortion presumably requires additional steric energy
at the transition state, and thus the reaction becomes
slower.
One of the valuable properties of lipase is high enantios-
electivity towards chiral sec-alcohols. To evaluate the enan-
tioselectivity of PHL, eight sec-alcohols (3a–h) were
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Table 1. Enantioselectivity of PHL towards sec-alcohols
should not bear a bulky group at the β-position from the
hydroxyl group.
O
H
O
O
H
O
PHL (20 mg)
+
+
O
Experimental
R
Pr
tBuOMe (1 mL)
O
R
R
25 °C
rac-3a-h
(0.1 mmol)
(R)-4a-h
(S)-3
Rates of Ester Hydrolysis. Rates of ester hydrolysis were
measured calorimetrically as reported in the literature.7 The
reaction mixtures contained 1.25–12.5 mM of substrates.
(0.3 mmol)
H
O
H
O
H
O
OH
H
O
Enantioselectivity of PHL for Transesterification of Sec-
Alcohols. The reaction mixture was prepared by mixing a
rac-alcohol (3a–h, 0.1 mmol), vinyl butyrate (39 μL,
0.3 mmol), and immobilized PHL (20 mg) in t-butyl
methyl ether (MTBE, 1 mL). The reaction mixture was
shaken at 200 rpm. A little amount of the reaction mixture
(200 μL) was retrieved and dissolved in MTBE (1 mL).
After centrifugation of the diluted reaction mixture to
remove the immobilized PHL, the clear solution was ana-
lyzed by a GC (Agilent 6890N; Agilent Technologies,
Seoul, Korea) equipped with a chiral capillary column
(Cyclosil-B 30 m × 0.25 mm). The GC analysis condition
for the reaction with 3e: initial column temperature 80ꢀC
for 5 min, ramp up to 120ꢀC at a rate of 2.5ꢀC/min, and
then held at 120ꢀC for 15 min.
3a
3b
3c
3d
3e
H
O
H
O
H
O
l
C
3f
3g
3h
Substrate
Time (h)
% ee of (R)-4a
Conv. (%)b
E
3a
3b
3c
3d
3e
3f
3g
3h
a
16
16
16
44
16
16
44
16
82.4
58.9
41
38
49
10
40
29
2
20
5
>200
>200
>200
>200
>200
>200
>99.9
>99.9
>99.9
>99.9
>99.9
>99.9
Acknowledgment. This research was supported by the
Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Minis-
50
The ee values were determined by GC.
try
of
Science,
ICT
and
Future
Planning
b
Conversions were calculated from enantiomeric excesses of both the
(2014R1A2A2A01004836).
starting materials and products.8
Supporting Information. Additional supporting informa-
tion is available in the online version of this article.
iso-propyl group, although it shows low conversion (10%
for 44 h). Besides, PHL well discriminated the enantiomers
when a methyl group for the medium substituent and a
phenyl group or phenyl derivatives for the large substituent
were present (3d–h). However, 3g shows low conversion
(2% for 44 h).
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useful guidance regarding synthetic applications. Similar to
other lipases, PHL preferred ester substrates carrying longer
carbon chain alcohol moieties. As the carbon chain length
of alcohols increases, PHL exhibited not only higher reac-
tion rate in hydrolysis of esters but also higher conversion
in transesterification of sec-alcohol. In contrast, PHL
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