A carbonyl reductase from: Candida parapsilosis ATCC 7330: Substrate selectivity and enantiospecificity

Article abstract of DOI:10.1039/c7ob00340d

Candida parapsilosis ATCC 7330, a rich source of highly stereospecific oxidoreductases, catalyzes oxidation-reduction of a plethora of compounds yielding industrially important intermediates. An (S)-specific carbonyl reductase (SRED) purified and characterized from this yeast is reported here. (R)-Specific carbonyl reductase (CpCR) was reported by us earlier. SRED asymmetrically reduces ketones with excellent enantiospecificity (ee > 99%) and α-ketoesters with higher catalytic activity but moderate enantiospecificity (ee 70%) in the presence of NADPH. Minimal activity is shown towards the reduction of aldehydes. While the reduction of α-ketoesters with SRED can occur with either NADPH or NADH, for ketone reduction SRED requires NADPH specifically. SRED with a subunit molecular weight of 30 kDa shows optimal activity at pH 5.0 and 25 °C, and its activity is affected by Cu²⁺. Taken together, SRED and CpCR offer substrates which on asymmetric reduction give products of opposite absolute configurations.

Full text of DOI:10.1039/c7ob00340d

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DOI: 10.1039/C7OB00340D
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A carbonyl reductase from Candida parapsilosis ATCC 7330:
Substrate selectivity and enantiospecificity
a
a,b
Sneha Sudhakara and Anju Chadha*
Received 00th January 20xx,
Accepted 00th January 20xx
Candida parapsilosis ATCC 7330, a rich source of highly stereospecific oxidoreductases, catalyzes oxidation-reduction of a
plethora of compounds yielding industrially important intermediates. An (S)-specific carbonyl reductase (SRED) purified
and characterized from this yeast is reported here. (R)-specific carbonyl reductase (CpCR) was reported by us earlier. SRED
asymmetrically reduces ketones with excellent enantiospecificity (ee > 99%) and α-ketoesters with higher catalytic activity
but moderate enantiospecificity (ee 70%) in the presence of NADPH. Minimal activity is shown towards reduction of
aldehydes. While the reduction of α-ketoesters with SRED can occur with either NADPH or NADH, for ketone reduction
SRED requires NADPH specifically. SRED with subunit molecular weight of 30 kDa shows optimal activity at pH 5.0 and 25
DOI: 10.1039/x0xx00000x
www.rsc.org/
o
2+
C, and its activity is affected by Cu . Taken together, SRED and CpCR offer substrates which on asymmetric reduction
give products of opposite absolute configurations.
scope builds its way to a valuable biocatalyst in synthesizing
Introduction
important chiral molecules. For example, carbonyl reductase
S1 purified from Candida magnoliae AKU4643 was co-
expressed with glucose dehydrogenase (GDH) in E. coli HB101
Candida parapsilosis ATCC 7330 (Cp) is an efficient biocatalyst
to prepare industrially important chiral building blocks as
1
shown by us. The reactions involved are mainly oxidation and
-1
produced 208
g
L
of optically pure (S)-4-chloro-3-
precursor for HMG-CoA
(R)-carbonyl reductase (RCR) and (S)-
hydroxybutanoate (S-CHBE),
a
reduction which occur by deracemization, asymmetric
1
1,12
reductase inhibitors.
reduction and enantioselective oxidation. Deracemization of
2
carbonyl reductase (SCR) isolated and characterized from
Candida parapsilosis CCTCC M203011 were used in an efficient
one-step production of (S)-1-phenyl-1,2-ethanediol (S-PED)
racemic
ethyl
mandalate
and
ethyl-3-hydroxy-3-
3
phenylpropionate in whole cells follows a stereoinversion
mechanism with the aid of stereospecific oxidoreductases. We
7
,10,13-15
from (R)-1-phenyl-1,2-ethanediol (R-PED).
A bacterial β-
have also shown that asymmetric reduction can result in either
4
R)- or (S)-alcohols depending on a variety of factors. In order
ketoacyl-ACP reductase (FabG) from Bacillus sp. ECU0013 co-
(
-1
expressed with GDH in E. coli reduced 620 g L of ethyl-2-oxo-
-phenylbutanoate (EOPB) to (S)-ethyl-2-hydroxy-4-
phenylbutanoate (S-EHPB). Optically pure PED and EHPB
to understand the basis for their stereospecificities and their
substrate selectivity, the next logical progression was to purify
these carbonyl reductases and reconstitute deracemization
reactions in vitro. The present study reports the isolation and
purification of an (S)-specific carbonyl reductase (SRED) which
is quite different from its (R)-specific counterpart, CpCR which
was isolated from this yeast, crystallized (1.86 Å resolution)
4
1
6-18
are used in the synthesis of Propiconazole (fungicide) and
Benazepril (anti-hypertensive) respectively. Also an (R)-specific
carbonyl reductase (CprCR) from Candida parapsilosis co-
expressed with GDH in E. coli Rosetta (DE3) cells
-1
5
asymmetrically reduced 100 g L of N-Boc-3-piperidone to (S)-
N-Boc-3-hydroxypiperidine, chiral intermediate for the
synthesis of Ibrutinib used for the treatment of lymphoma.
and characterized. The (R)-specific, Zn-dependent enzyme
a
reduces aldehydes and α-ketoesters with high catalytic activity
6
but shows minimal activity towards ketones.
19
Such applications of biocatalysts in industries make the
process greener.
Y. Nie et al., reported purification of multiple (S)-specific
carbonyl reductases from Candida parapsilosis such as SCR,
SCR1, SCR2, SCR3, SCRII and an (R)-specific carbonyl reductase
With the intention of understanding the mechanism of the
enantiospecific preference of these enzymes, the (S)-specific
carbonyl reductase was purified and characterized. This
enzyme had opposite enantio-preference compared to CpCR
7
-10
(RCR).
Many carbonyl reductases from Candida sp. have
been purified and characterized. The detailed study of the
enzyme with its biochemical characterization and substrate
6
and reduced ketones unlike CpCR and both the enzymes show
activity towards α-ketoesters.
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Table 1 Specific activity of SRED determined for carbonyl substrates
Results and discussion
S
No
Substrate
α-Ketoesters
Ethyl-2-oxo-4-
Substrate
(mM)
NADPH
(mM)
Specific activity
-1
(Umg )
Substrate selectivity and enantiospecificity
1
1
0.05
1.17 ± 0.19
The activity of purified SRED, a member of short-chain
dehydrogenase/reductase (SDR) family (see protein
overexpression and purification section) as determined for α-
ketoesters, ethyl-2-oxo-4-phenylbutanoate (EOPB) and ethyl-
phenylbutanoate
2
-oxo-2-phenylacetate (EOPA), ketones such as 2-
hydroxyacetophenone (2HAP), 2-chloroacetophenone
2ClACP), acetophenone (ACP), p-bromoacetophenone, p-
2
Ethyl-2-oxo-2-
phenylacetate
1
0.05
2.31 ± 0.31
(
methylacetophenone and aldehydes, benzaldehyde and
anisaldehyde is presented in Table 1. Aldehydes are poor
substrates as compared to ketones as is the case with most of
7
,8
the reported SDRs. On the contrary, CpCR which is a
Ketones
2-Hydroxyacetophenone
medium-chain dehydrogenase/reductase (MDR) displayed
6
higher activity with aldehydes than ketones. For ketones,
3
4
5
6
4
4
4
4
0.20
0.20
0.20
0.20
3.68 ± 0.32
3.15 ± 0.10
0.59 ± 0.10
1.66 ± 0.26
SRED showed highest activity with 2HAP followed by 2ClACP.
There was 6-fold decrease in the activity with ACP as
compared to 2HAP. It is known that the substrate binding site
in alcohol dehydrogenase consists of two hydrophobic
substrate binding pockets which differ from one another in
2-Chloroacetophenone
2
0
volume and affinity towards the R groups of the substrate. In
substrates such as 2HAP and 2ClACP, the presence of an
electronegative atom like oxygen and chlorine respectively at
C2 position showed much better activity than ACP with methyl
group at C2 position. There could be a possibility for an
electrostatic interaction in the small group binding pocket that
facilitates better binding and hence better activity of 2HAP and
Acetophenone
2ClACP. SRED showed no activity with ortho- and minimal
activity with meta- substituents of ACP (results not shown).
This could be due to the steric effect of the substitution. But
para-bromoacetophenone showed ~ 3 fold increase in activity
and para-methylacetophenone showed 0.6 fold decrease in
activity with respect to ACP. The electron withdrawing and
electron donating nature of bromo- and methyl- respectively
at para position of ACP can explain this. For aldehyde and
ketone substrates, identical assay conditions were followed.
However, for α-ketoesters, EOPB and EOPA, SRED had high
catalytic activity and the assay conditions were modified i.e.
decrease in concentrations of substrate, NADPH and the
amount of enzyme to increase the reaction time in order to
monitor it experimentally. Between EOPB and EOPA, EOPA
showed greater activity implying less the number of carbons
between the phenyl and carbonyl group, better was the
p-bromoacetophenone
7
p-methylacetophenone
4
0.20
0.34 ± 0.05
Aldehydes
Benzaldehyde
8
9
4
4
0.20
0.20
0.57 ± 0.07
0.47 ± 0.13
6
activity which was in contrast to CpCR (MDR) and an aldo-keto
Anisaldehyde
2
1
reductase (AKR) from Neurospora crassa (NcCR). In summary,
for SRED, the relative activities are as follows: α-ketoesters >
ketones > aldehydes.
In case of SDRs such as NADPH-dependent carbonyl reductases
8
from Candida parapsilosis CCTCC M203011 (SCRs), Candida
1
1,22
benzaldehyde and ACP. The trend of higher activity for
ketoesters followed by 2ClACP and aryl substituted ACP was
also followed by carbonyl reductase from Candida krusei SW
magnoliae AKU 4643 (S1 & S4)
and NADH-dependent
2
carbonyl reductase from Kluyveromyces aestuarii (KaCR1),
3
higher activity was shown towards ketoester substrates and
very minimal activity was shown towards aryl substituted
2
4
25
2
026
and
Sporobolomyces
salmonicolor
(SSCR).
Interestingly, SSCR showed higher activity with o- and m- than
p- substituted ACP.
2
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Comparing the two carbonyl reductases SRED and CpCR from Cofactor selectivity
the same yeast Cp, the substrate specificity trend was very
different. SRED preferred EOPA, EOPB (ketoesters), then 2HAP, SRED reduced 2HAP only in presence of NADPH. Carbonyl
ClACP, p-bromoactophenone, ACP, p-methylacetophenone reductases SCR and SCR1-3 from Candida parapsilosis also
ketones) and benzaldehyde, anisaldehyde (aldehydes). But preferred NADPH for the asymmetric reduction of prochiral
2
(
highest activity of CpCR was observed with aldehydes followed carbonyl compounds including 2HAP and EOPB. The authors
6
by α-ketoesters. Minimal activity of CpCR was found with related the presence of the positively charged residue His
ketones unlike SRED. These reductases belong to different (His70 in SCR1, His68 in SCR2, SCR3, SCR) to play a role in the
families. Taken together, these have a reasonable substrate affinity of the enzyme for NADPH, rather than NADH, by
bank which on asymmetric reduction gives products of forming a salt bridge with the phosphate moiety at the 2’-
7
,8
opposite absolute configurations.
position of AMP. His 68 is present in SRED but showed
The whole cells of Cp are established excellent catalysts for activity in presence of both NADPH and NADH with respect to
1
carrying out wide range of chemical transformations. The 14 the substrate, EOPB. This difference in selectivity of cofactor
h culture of Cp asymmetrically reduced phenyl glyoxal (PG) to depending on the type of substrate may indicate that not only
o
S)-PED completely in 3 h at 25 C with 99% ee and 2HAP is the the presence of specific amino acid in the enzyme that
(
intermediate. The whole cells reduced the aldehyde function interacts with the cofactor is important but the nature of
first to the intermediate in 10 min and then the keto group substrate and its interaction with the cofactor also influences
2
6
was reduced in 170 min. When the keto intermediate, 2HAP cofactor selectivity. Similar observation was made with CpCR
was given as a substrate to the isolated SRED, incomplete from Cp which preferred NADPH for the reduction of
conversion was observed in 3 h with > 99% ee of the formed aldehydes but showed dual cofactor specificity for reduction of
6
(S)-alcohol. Thus to ensure complete conversion the reaction α-ketoesters.
time was extended. In addition to this, the reaction conditions
were varied such as pH (5, 7.5) and co-solvents (DMSO, IPA). Protein overexpression and purification
Complete conversion of 2HAP to (S)-PED was observed in 10 h
with > 99% ee in 0.1 M HEPES buffer, pH 7.5 and co-solvent SRED was overexpressed in E. coli BL21 (DE3) cells using
IPA. This is because at pH 7.5, cofactor regeneration occurs by genomic DNA of Cp as the template. The translated protein
oxidation of IPA (co-substrate) to acetone which is not possible sequence was identical to that of the carbonyl reductase (SCR)
at pH 5 and with DMSO as co-solvent. The extended reaction from Candida parapsilosis CCTCC M203011 which belongs to
time could be due to the shortage of the cofactor unlike the the SDR. This implied SRED to be an SDR with identical
7
case with the whole cells. Thus, the purified SRED reinforced conserved motifs.
SRED was purified by affinity
the mechanism of asymmetric reduction of PG to (S)-PED in chromatography with N-terminal GST tag (cleaved using
the host Cp. Formation of (S)-PED has also been reported by PreScission protease). The purified fraction (25-fold purity)
enzymes SCR and SCRII (> 99% ee), SCR 1 – 3 (ee not contained SRED in ~20% yield (2 – 4 mg from 1 L culture). The
7
-9
mentioned). The same reaction conditions were followed subunit molecular weight as observed by 12% SDS PAGE was ~
and 2ClACP was reduced by SRED to its (S)-alcohol with ee > 30 kDa (Fig. 1A), in agreement with its theoretical value (279
9
9%.
amino acid residues) similar to the carbonyl reductases (SDRs)
7
EOPB and EOPA (α-ketoesters) were asymmetrically reduced from Candida parapsilosis CCTCC M203011 (SCR), Candida
1
1,22
1
00% and 40% by SRED using the optimized reaction magnoliae AKU 4643 (S1 & S4),
Kluyveromyces aestuarii
30,31
SRED is smaller
2
3
conditions respectively in 6h with 70% ee for both. The (KaCR1), Pichia stipitis (PsCR I & PsCR II).
5
moderate enantiospecificity could be due to the presence of than CpCR which is 40 kDa and a dimer. Most purified SDRs
8
,11,22
an ethyl ester group (hydrophobic) in EOPB against oxygen of are reported in either dimeric or tetrameric forms.
SRED
hydroxyl (polar) in 2HAP as the small group. In EOPB, a had an apparent molecular weight of ~ 58 kDa when eluted
competition is involved between phenyl and ester moiety, through gel filtration column indicating that SRED could be a
both being hydrophobic, in binding in the substrate binding homodimer. However, SRED on native PAGE showed a major
pockets leading to lower enantiospecificity, as explained by T. band comparable to that of a BSA dimer (132 kDa) indicating
2
7
Ema et al. However, 40 h culture of Cp asymmetrically that it could be a tetramer (Fig. 1B). In 2007, Y. Nie et al.,
28
reported that purified CPADH (renamed as SCR later) existed
o
reduced EOPB to (S)-EHPB in 4 h at 25 C with 99% ee.
7
Deracemization of (RS)-EHPB to (S)-EHPB and (RS)-EHPA to (S)- as a monomer. But in 2008, R. Zhang et al., described SCR as a
o
EHPA was carried out by 24 h culture of Cp in 1 h at 25 C.
29
homotetramer in its crystal structure (PDB 3CTM) and in
Evidently, in the whole cells, different reductases are induced solution. However the functional unit was shown to be a dimer
at different time intervals that catalyze these transformations. (A-C type). The need for dimer-dimer interface, formed due to
On the other hand, purified CpCR reduced EOPB to (R)-EHPB overlapping of N-terminal peptide, the helix αG and the strand
1
4
with > 99% ee.
βG with no hydrogen bond formation, is unclear. Thus, the
In essence, SRED showed higher catalytic activity and tetramer formation could be concentration dependent with no
moderate specificity with α-ketoesters as compared to obvious role in protein function. That SRED is a dimer as per
moderate catalytic activity and excellent specificity with gel filtration could be due to the dilution of protein in the
ketones.
column which is not the case with native PAGE. (S)-1-
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Phenylethanol dehydrogenase from denitrifying bacterium
strain EbN1 showed a similar discrepancy in the apparent
molecular weight which was 59 kDa by gel filtration and 93
kDa by native PAGE. This behavior was attributed to the
interaction of the enzyme with the column material which led
3
2
to its retardation.
Fig. 2 Effect of pH and temperature on SRED. (A) Activity-pH profile.
(
B) Activity-temperature profile. Relative activity was expressed as a
percentage of the maximum activity under experimental conditions.
Effect of various metal ions on the enzyme activity
It is well known that zinc forms an integral part of structure
and function of alcohol dehydrogenases. Zinc coordinates with
the oxygen of the substrate, acts as a Lewis acid facilitating the
3
7
proton relay via side chain of Ser and His (acts as a base). We
Fig. 1 Analysis by electrophoresis. (A) SDS-PAGE analysis. Lane 1, cell- have noticed decrease in activity of Zn-dependent CpCR by
6
free extract; Lane 2, flow through; Lane 3, eluate containing SRED with addition of chelators which was reversed by addition of zinc.
GST tag (56 kDa); Lane 4, SRED and GST after cleavage; Lane 5, GST (26 Consequently the effect of metal ions on SRED activity for
kDa); Lane 6, purified SRED (30 kDa); Lane 7, protein ladder. (B) Native reduction of 2HAP was investigated. Divalent cations such as
2
PAGE analysis. Lane 1, 10 μg of SRED; Lane 2, 5 μg of SRED; Lane 3, 20 Ca , Co , Mg , Mn , Ni , Zn did not have any effect on the
+
2+
2+
2+
2+
2+
2
+
3+
μg of BSA.
SRED activity. However, Cu and Fe reduced the activity of
SRED by around 25% and 40% respectively (Table 2). Also, the
metal chelator, EDTA (1 mM) reduced the SRED activity by 15%
pH and temperature for optimal enzyme activity
(result not shown).
2HAP was used as a standard substrate for measuring the Furthermore, the effect of metal ions on the secondary
activity of SRED unless mentioned otherwise. For the optimal structure of SRED was studied by circular dichroism (CD) to
activity of the purified SRED, the effect of pH and temperature correlate with its activity. Examining the molar ellipticities (θ)
on its activity for the reduction of 2HAP was monitored in the at 217 nm and 222 nm of SRED in presence of these metal salts
o
range of pH 4.5 - 10 and 20 – 50 C respectively. 0.1 M of (Fig. 3) revealed information about the effect of metal ions on
different buffers were used with overlapping buffer range for the secondary structural components such as α-helix (at 222
determining the activity. Maximal activity was observed at pH nm) and β-sheet (at 217 nm). After addition of the cofactor
5.5 (Fig. 2A). The activity decreased by 50% with pH 7 – 7.5 and NADPH, the negative value of θ increased at both 217 and 222
no activity was observed beyond pH 9. Given the reported nm indicating that there was increase in orderliness in β-sheet
1
1
mechanism of these carbonyl reductases, it is reasonable to and α-helix respectively. This was as expected. Similarly,
expect the activity in the acidic range. In acidic pH, Tyr is most of the metal salts showed increase in the negative value
protonated allowing the proton abstraction by oxygen of the of θ with respect to the control (only SRED) except in case of
1
1
7
carbonyl group from the substrate. Other reported SDRs S1,
CuSO as reported for SCR enzyme.
4
2
2
23
S4, KaCR1 have their optimal pH between 5.5 – 6.5. The Reduction of 2HAP was also studied in presence of 1 mM of
o
activity of SRED was optimum between 25 – 30 C (Fig. 2B). Cu , Fe , Fe , Zn and EDTA with the optimized reaction
2+
2+
3+
2+
2
+
3+
2+
However, reductases S1, S4, KaCR1 have reported their conditions. Fe , Fe , Zn and EDTA had no effect on the
optimal temperature in the higher range between 45 – 55 reduction of 2HAP (> 99% conversion) when compared with
o
11,22,23
C.
o
Beyond 50 C, SRED was found to be inactive but was the control (without metal salt). However, in presence of Cu
2+
2
only 27% conversion was observed. Cu is one of the thiol
+
stable at the ambient temperatures.
+
+
24
specific metal ion, Ag and Hg being the others. It is
interesting to know that OR2T11, a human olfactory thiol
receptor had a strong copper effect for the compound t-butyl
mercaptan (TBM) and copper was found to coordinate with
sulfur atoms of TBM, Met, Cys and nitrogen atom of His
3
8
residues. Since SRED has 2 Met and 5 Cys residues in its
primary sequence, there is a possibility of copper coordinating
with the sulfur atoms and altering the structure (Fig. 3)
4
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thereby reducing the activity of this enzyme. Similar inhibitory amplified product was digested with BamHI and XhoI, and was
effect of copper was found in carbonyl reductases from ligated in to the expression vector, pGEX-6-P-1 by directional
7
Candida parapsilosis CCTCC M203011, Kluyveromyces cloning. The clone was verified by DNA sequencing from
2
3
24
aestuarii, Candida krusei SW 2026, Kluyveromyces lactis Shrimpex (India) and was transformed in E. coli BL21 (DE3).
3
NRIC 1329, Candida viswanathii MTCC 5158.
9
40
The successfully transformed BL21 cells containing SRED gene
Presence of EDTA made no difference to SRED activity were grown in Luria Bertani media until the OD600 reached 0.6
o
indicating the enzyme could be metal independent similar to – 0.8 at 37 C. Upon standardization of protein over-expression
7
reductases from Candida parapsilosis CCTCC M203011 and using different induction parameters such as IPTG (isopropyl β-
4
0
Candida viswanathii MTCC 5158.
D-1-thiogalactopyranoside) concentration,
incubation
temperature and time, the soluble fraction of protein gave
o
better yield at 0.3 mM IPTG at 23 C incubated for 18 h.
Table 2 Effect of metal salts on SRED activity
Protein purification was performed using a fast protein liquid
chromatography (FPLC) system (ÄKTA purifier, GE Biosciences).
Entry
Metal salt
Control
Relative specific activity(%)
100.0 ± 3.7
o
All the purification steps were carried out at 4 C using
1
2
3
4
5
6
7
8
CaCl
CoCl
CuSO
2
89.8 ± 1.2
phosphate buffer saline with 10% glycerol. The cleared lysate
after ultra-sonication and centrifugation was loaded onto a
GSTrap FF column. The recombinant protein was eluted with
50 mM Tris (pH 8.0) containing 25 mM glutathione. SRED with
GST tag at N-terminal was digested with PreScission protease
2
91.4 ± 8.0
4
74.8 ± 3.1
FeCl
3
61.1 ± 7.6
a
ND
FeSO
4
o
MgSO
4
4
98.6 ± 5.3
96.1 ± 2.7
90.0 ± 1.9
99.2 ± 5.5
for 12–16 h at 4 C. The PreScission protease digested samples
MnSO
were loaded again onto GSTrap FF column and the flow-
through was collected that contained 30 kDa SRED. This
protein solution was concentrated and quantified (Bradford
1
1
0
1
NiCl
2
ZnSO
4
a
41
ND: not detectable
method). The purified protein samples were analyzed by
SDS–PAGE followed by Coomassie Brilliant Blue R250 staining.
Determination of enzyme activity
The reductase activity of the enzyme was determined by
monitoring the decrease in the absorbance of NADPH at 340
o
6
nm spectrophotometrically at 25 C. The standard assay
mixture consisted of 0.1 M citrate buffer (pH 5), appropriate
amount of the protein, 4 mM of the aldehyde/ketone
substrate and 0.2 mM of NADPH. For, α-ketoester substrates,
the assay mixture constituted 0.1 M citrate buffer (pH 5),
Fig. 3 The plot of molar ellipticities at 217 nm and 222 nm against appropriate amount of the protein, 1 mM of the substrate and
SRED with different metal salts.
Experimental
Materials
0.05 mM of NADPH. One unit of the enzyme activity was
defined as the amount of enzyme that oxidizes 1 µmol of
NADPH per minute at the specified assay condition.
Effect of pH and temperature on the enzyme activity
All the carbonyl substrate molecules were procured and some The enzyme activity was calculated by performing the
alcohol products were synthesized by the reported standard assay (as mentioned in the previous section) with
methodology (as given in the supplementary data file). 2HAP as the substrate. To determine the pH for the optimum
Coenzymes (NADH, NADPH) were purchased from SRL (India). activity the assay was performed in 0.1 M buffer, pH ranging
GSTrap FF column was purchased from GE Healthcare Life from 4.5 to 10, using citrate for pH 4.5 – 6, phosphate for pH 6
Sciences (India). BLUltra pre-stained protein ladder for SDS-gel – 8, HEPES for pH 7 – 8, Tris-Cl for pH 7 – 9 and Glycine - NaOH
electrophoresis was purchased from GeneDireX, Inc. (India). All for pH 9 – 10. The optimal enzyme activity was also
o
determined by varying temperature from 20 – 50 C.
other reagents were of analytical grade.
Protein expression and purification
Effect of various metal ions on the enzyme activity
The genomic DNA of Candida parapsilosis ATCC 7330 was used The enzyme activity in presence of 1 mM of various metal salts
as a template for amplifying SRED gene using the primers F- 5’- was estimated by the standard assay (as mentioned in
ATCGGATCCGATGGGCGAAATCGAATCTTATTG-3’ (BamHI) and determination of enzyme activity section) with 2HAP as the
7
R-5’-TGACTCTCGAGTGGACACGTGTATCCACCGTC-3’(XhoI). The substrate and 0.18 mM of SRED. The metal salts that were
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Organic & Biomolecular Chemistry
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DOI: 10.1039/C7OB00340D
ARTICLE
Journal Name
used for the assay were CaCl
2
, CoCl
2
, CuSO
4
, FeCl
3
, FeSO
4
,
with the corresponding standard racemic alcohols (refer
4 4 2 4
MgSO , MnSO , NiCl , ZnSO . The enzyme activity was also supplementary data file).
determined in presence of chelating agent EDTA (1 mM). The
effect of metal salts and EDTA on the secondary structure of Optimization of pH, co solvent and reaction time for the
SRED, indirectly relating to its activity, was examined by Jasco reduction of 2-hydroxyacetophenone (2HAP) and ethyl-2-
J-815 CD spectrometer. The experiment was performed with oxo-4-phenylbutanoate (EOPB)
2.6 mM of SRED in 20 mM of citrate buffer (pH 5) with 0.5 mM
of NADPH. Different metal salts of final concentration 15 mM 4 mM of a model ketone substrate, 2-hydroxyacetophenone
were added separately to the solution and the measurement and an α-ketoester, ethyl-2-oxo-4-phenylbuanoate were
was taken from 200 – 250 nm.
reduced separately by 0.6 U of SRED, 2 mM NADPH in 0.1 M
buffer. The conversion of the substrate was maximized by
varying the buffer pH, co-solvent and reaction time. 0.1 M
citrate buffer, pH 5 and 0.1 M HEPES buffer, pH 7.5 with co-
Stereospecificity and cofactor selectivity
The enantiospecificity and cofactor selectivity of the enzyme solvent IPA and DMSO were used by examining conversion up
was determined by the reduction of the carbonyl substrates in to 10 h. After the reaction, the mixture was extracted and
presence of NAD(P)H. The reaction mixture (1 ml) consisted of analyzed for conversion and enantiomeric excess as explained
0.1 M HEPES buffer (pH 7.5), 5 mM of the carbonyl substrate in in the previous section.
isopropanol (10% v/v), 0.3 U of the purified SRED and 2 mM of
o
NAD(P)H. The reaction mixture was incubated at 25 C for 10 h Conclusion
with shaking followed by extraction with ethyl acetate (2
times) and analysis.
The substrate selectivity and enantiospecificty of the purified
SRED from Candida parapsilosis ATCC 7330 revealed that SRED
preferred α-ketoesters > ketones > aldehydes. This reductase
asymmetrically reduced ketones with excellent ee > 99% and
Biotransformation and substrate selectivity
Bioreduction of different carbonyl substrates were carried out α-ketoesters with moderate ee 70% in the presence of NADPH
in 1 ml reaction mixture with 0.1 M HEPES buffer (pH 7.5), 5 to their (S)-alcohols. SRED, together with CpCR, from the same
mM of the carbonyl substrate in 10% v/v isopropanol (IPA), 0.3 source, which shows opposite enantiospecificity, offers the
o
U of the purified SRED and 2 mM of NADPH incubated at 25 C possibility of synthesis of numerous optically pure industrially
with shaking. After the reaction time, the unreacted substrate important chiral building blocks.
and the formed product were extracted using ethyl acetate (2
times). The extract was dried over anhydrous sodium sulphate, Acknowledgements
concentrated using rotavapor and analyzed.
Conversion was determined by GC-MS (Shimadzu GC-2010 One of the authors (Sneha Sudhakara) expresses her gratitude
TM
Plus) using Supelco Omegawax 320 capillary column (30 m X to the IIT Madras, India, for the fellowship. Also we thank DST-
0
.32 mm X 0.25 μm film thickness) with injector / ion source FIST facility at Department of Biotechnology, IIT Madras for CD
o
o
temperature of 250 C / 250 C and temperature profile of 50 spectrometer. We thank Dr. Nidhi Aggarwal, Post-Doctoral
o
o
o
-1
C for 2 min, increased to 210 C at the rate of 4 C min and Fellow, Weizmann Institute of Science, Israel for her help in
then held for 6 min. The retention time of ketones 2HAP, cloning and Mr. Rahul Choudhury, Junior Research Fellow,
ClACP and the corresponding alcohols were 30.9 min, 30.4 Department of Biotechnology, IIT Madras for his help in
2
min and 40.2 min, 33.0 min respectively. α-ketoesters EOPB, synthesis.
EOPA and the corresponding α-hydroxyesters had retention
times of 35.0 min, 30.0 min and 36.8 min, 32.6 min References
respectively. All the carbonyl substrates and the alcohol
products were characterized by MS (refer supplementary data
file).
1
2
3
4
5
A. Chadha, S. Venkataraman, R. Preetha and S. K. Padhi,
Bioorg. Chem., 2016, 68, 187–213.
The enantiomeric excess of the alcohols PED (S- 9.5 min; R- 7.4
min) and 2-chloro-1-phenylethanol (S- 9.7 min; R- 7.5 min)
were determined by HPLC (Jasco PU-1580 with PDA detector)
using Chiralcel OB-H column (Daicel, 0.46 cm X 25 cm), mobile
B. Baskar, N. G. Pandian, K. Priya and A. Chadha,
Tetrahedron, 2005, 61, 12296–12306.
S. K. Padhi, N. G. Pandian and A. Chadha, J. Mol. Catal. B-
Enzym., 2004, 29, 25–29.
-1
phase of hexane:isopropanol = 90:10 at 1 ml min . For α-
hydroxyesters ethyl-2-hydroxy-4-phenylbutanoate (S- 11.2
min; R- 17.7 min) and ethyl-2-hydroxy-2-phenylacetate (S- 10.7
min; R- 19.3 min), enantiomeric excess was calculated using
Chiralcel OD-H column (Daicel, 0.46 cm X 25 cm), mobile phase
T. Kaliaperumal, S. N. Gummadi and A. Chadha,
Tetrahedron: Asymmetry, 2011, 22, 1548–1552.
N. Aggarwal, P. K. Mandal, N. Gautham and A. Chadha,
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 2013,
69, 313–315.
-1
of hexane:isopropanol = 98:02 at 1 ml min . The chiral
products were characterized by matching their retention time
6
7
N. Aggarwal, PhD Thesis, Indian Institute of Technology
Madras, 2013.
Y. Nie, Y. Xu, X. Q. Mu, H. Y. Wang, M. Yang and R. Xiao,
6
| J. Name., 2012, 00, 1-3
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Orga Pn l iec a s&e Bd oi o nmo to al ed cj uu sl ta mr Ca rhg ei nms istry
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DOI: 10.1039/C7OB00340D
Journal Name
ARTICLE
Appl. Environ. Microbiol., 2007, 73, 3759–3764.
Y. Nie, R. Xiao, Y. Xu and G. T. Montelione, Org. Biomol.
45, 82–93.
8
9
1
1
33
34
35
36
J. Peters, T. Minuth and M. Kula, Enzyme Microb. Technol.,
1993, 15, 950-958.
Chem., 2011, 9, 4070–8.
R. Zhang, Y. Geng, Y. Xu, W. Zhang, S. Wang and R. Xiao,
Bioresour. Technol., 2011, 102, 483–489.
Y. Nie, Y. Xu, M. Yang and X. Q. Mu, Lett. Appl. Microbiol.,
Y. H. Choi, H. J. Choi, D. Kim, K. N. Uhm and H. K. Kim, Appl.
Microbiol. Biotechnol., 2010, 87, 185–193.
Y. Ni, C.-X. Li, L.-J. Wang, J. Zhang and J.-H. Xu, Org. Biomol.
Chem., 2011, 9, 5463–5468.
0
1
2007, 44, 555–562.
M. Wada, M. Kataoka, H. Kawabata, Y. Yasohara, N. Kizaki,
J. Benach, S. Atrian, R. Gonzàlez-Duarte and R. Ladenstein,
J. Mol. Biol., 1999, 289, 335–355.
J. Hasegawa and S. Shimizu, Biosci. Biotechnol. Biochem.,
1
998, 62, 280–5.
37
38
B. V Plapp, Arch. Biochem. Biophys., 2010, 493, 3–12.
S. Li, L. Ahmed, R. Zhang, Y. Pan, H. Matsunami, J. L.
Burger, E. Block, V. S. Batista and H. Zhuang, J. Am. Chem.
Soc., 2016, 138, 13281–13288.
12
N. Kizaki, Y. Yasohara, J. Hasegawa, M. Wada, M. Kataoka
and S. Shimizu, Appl. Microbiol. Biotechnol., 2001, 55, 590–
5
95.
M. Yang, Y. Xu, X. Mu and R. Xiao, Front. Chem. Eng. China,
007, 1, 404–410.
1
3
4
39
H. Yamamoto, N. Kimoto, A. Matsuyama and Y. Kobayashi,
Biosci Biotechnol Biochem, 2002, 66, 1775–1778.
P. Soni, H. Kansal and U. C. Banerjee, Process Biochem.,
2007, 42, 1632–1640.
2
1
R. Zhang, G. Zhu, W. Zhang, S. Cao, X. Ou, X. Li, M. Bartlam, 40
Y. Xu, X. C. Zhang and Z. Rao, Protein Sci., 2008, 17, 1412–
1423.
41
M. M. Bradford, Anal. Biochem., 1976, 72, 248–254.
15
16
17
18
19
20
21
22
R. Zhang, Y. Xu, R. Xiao, B. Zhang and L. Wang, Microb. Cell
Fact., 2012, 11, 167.
Y. Xie, J. H. Xu and Y. Xu, Bioresour. Technol., 2010, 101,
1054–1059.
Y. Ni, C. X. Li, H. M. Ma, J. Zhang and J. H. Xu, Appl.
Microbiol. Biotechnol., 2011, 89, 1111–1118.
Y. Ni, C. X. Li, J. Zhang, N. D. Shen, U. T. Bornscheuer and J.
H. Xu, Adv. Synth. Catal., 2011, 353, 1213–1217.
J. Chen, M. Yan and L. Xu, World J. Microbiol. Biotechnol.,
2017, 33, 61-72.
J. Peters, T. Minuth and M. R. Kula, Biocatal.
Biotransformation, 1993, 8, 31–46.
N. Richter and W. Hummel, Enzyme Microb. Technol.,
2011, 48, 472–479.
M. Wada, H. Kawabata, A. Yoshizumi, M. Kataoka, S.
Nakamori, Y. Yasohara, N. Kizaki, J. Hasegawa and S.
Shimizu, J. Biosci. Bioeng., 1999, 87, 144–148.
23
H. Yamamoto, K. Mitsuhashi, N. Kimoto, A. Matsuyama, N.
Esaki and Y. Kobayashi, Biosci. Biotechnol. Biochem., 2004,
6
8, 638–649.
N. Li, Y. Ni and Z. Sun, J. Mol. Catal. B Enzym., 2010, 66,
90–197.
24
25
26
27
28
29
30
31
1
D. Zhu, Y. Yang, J. D. Buynak and L. Hua, Org. Biomol.
Chem., 2006, 4, 2690–2695.
P. Mahajabeen and A. Chadha, Tetrahedron Asymmetry,
2011, 22, 2156–2160.
T. Ema, H. Moriya, T. Kofukuda, T. Ishida and K. Maehara, J.
Org. Chem., 2001, 66, 8682–8684.
B. Baskar, N. G. Pandian, K. Priya and A. Chadha,
Tetrahedron Asymmetry, 2004, 15, 3961–3966.
A. Chadha and B. Baskar, Tetrahedron Asymmetry, 2002,
13, 1461–1464.
Q. Ye, M. Yan, L. Xu, H. Cao, Z. Li, Y. Chen, S. Li and H. Ying,
Biotechnol. Lett., 2009, 31, 537–542.
H. Cao, L. Mi, Q. Ye, G. Zang, M. Yan, Y. Wang, Y. Zhang, X.
Li, L. Xu, J. Xiong, P. Ouyang and H. Ying, Bioresour.
Technol., 2011, 102, 1733–1739.
32
H. W. Höffken, M. Duong, T. Friedrich, M. Breuer, B. Hauer,
R. Reinhardt, R. Rabus and J. Heider, Biochemistry, 2006,
This journal is © The Royal Society of Chemistry 20xx
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