D. Hu, et al.
ProcessBiochemistryxxx(xxxx)xxx–xxx
for the regeneration of NAD(P)H in the organic/aqueous biphasic
system.
The recombinantly expressed SyGDH and SyS1 harboring a 6×His
tag at its N-terminus were purified by affinity chromatography using a
nickel-nitrilotriacetic acid (Ni-NTA) column (Tiandz, Beijing, China).
The purity and the concentration of SyGDH and SyS1 were analyzed by
SDS-PAGE and the BCA-200 protein assay kit (TaKaRa, Dalian, China),
respectively.
The whole genome of T. acidophilum DSM 1728 has been sequenced,
in which three putative SDR family GDH genes (GenBank accession nos.
AL445065: Ta0747 and Ta0754; AL445063: Ta0191) and one putative
medium-chain dehydrogenase/reductase (MDR) family GDH gene
(AL445065: Ta0897) have been identified [18]. Both Ta0754 and
Ta0191 genes were expressed in E. coli, respectively, while other two
genes have not been heterogeneously expressed. The expressed Ta0754
and Ta0191 GDHs exhibited NAD+ and NADP+ dependences, respec-
tively, but both low specific activities towards D-glucose (3.5 and 3.1
U/mg) [19,20]. To date, several studies have been performed on the
characterization of purified T. acidophilum GDH (TaGDH) [21], the
expression of TaGDH gene (GenBank: X59788, encoding 353 residues)
in E. coli [22], the determination of TaGDH crystal structure [23], and
the application of TaGDH in coenzyme regeneration [24]. However, the
amino acid sequence of TaGDH in the C-terminus is significantly dif-
ferent from that deduced from the putative MDR family GDH gene
(Ta0897, encoding 361 residues).
In our previous studies, a SyGDH-encoding gene Sygdh was syn-
thesized based on the putative GDH gene (Ta0897) in T. acidophilum
genome. Meanwhile, the SyS1-encoding gene, Sys1 was synthesized
with optimized codons based on the carbonyl reductase gene from
Candida magnoliae (GenBank: AB036927) [25]. In this work, both Sygdh
and Sys1 were separately expressed and coexpressed in E. coli
BL21(DE3). The pH and temperature properties, substrate and coen-
zyme specificity, and organic solvent tolerance of purified SyGDH were
characterized. Additionally, the whole cells of E. coli/Sygdh-Sys1 coex-
pressing SyGDH and SyS1 were applied to the asymmetric reduction of
m-chlorophenacyl chloride (m-CPC) and α-bromoacetophenone (α-
BAP) coupled with NADPH regeneration in situ in an organic solvent/
phosphate buffer system. Our studies not only completed the catalytic
properties of the robust SyGDH, but also provided a reference for the
application of GDHs in the coenzyme regeneration.
2.3. Activity assays of SyGDH and SyS1
The SyGDH activity was assayed at 40 °C in a 96-well plate, in which
each well contained 50 mM glucose and 2 mM NADP+ in 50 mM
phosphate buffer (pH 7.5). Then, the reaction, in a final volume of
220 μL, was initiated by the addition of a certain amount of purified
SyGDH, and continuously monitored for an increase in OD340 using a
Synergy™ H4 multi-mode microplate reader (BioTek, Winooski, VT).
Similarly, for the SyS1 activity assay, each well contained 20 mM α-BAP
and 2 mM NADPH in the same buffer and a certain amount of purified
SyS1 in a final volume of 220 μL, and the decrease in OD340 was mea-
sured [25]. One activity unit (U) was defined as the amount of enzyme
catalyzing the reduction of 1 μmol NADP+ per minute (for SyGDH) or
the oxidation of 1 μmol NADPH per minute (for SyS1) under the given
assay conditions.
2.4. pH and temperature properties of the purified SyGDH
The pH optimum of purified SyGDH was determined under the
standard assay conditions, except for 50 mM different buffers
(Na2HPO4–citric acid: pH 4.0–7.0 and Tris−HCl: pH 7.5–9.0) were
used. To evaluate the pH stability, aliquots of SyGDH solution were
incubated in the absence of substrate, in a pH range of 4.0–9.0 and at
40 °C for 1 h. Additionally, aliquots of purified SyGDH were incubated
at pH values of 4.5, 5.5, 6.5 and 7.5 at 25 °C, respectively, for 1, 2, 4, 6,
8, 10, and 12 h. Then, the residual activity was measured under the
standard assay conditions.
The temperature optimum of purified SyGDH was measured at the
optimum pH over temperatures ranging from 10 to 70 °C. To estimate
the thermostability, aliquots of purified SyGDH were incubated at
10–70 °C for 1 h, respectively. Additionally, the aliquots of purified
SyGDH were incubated at 40 °C, 50 °C and 60 °C at a pH of 7.5, re-
spectively, for 1, 2, 4, 6, 8, 10, and 12 h. Then, the residual activity was
measured under the standard assay conditions. The pH stability and
thermostability were defined as the temperature and pH range, re-
spectively, in which the residual activity of SyGDH was over 80% of its
original activity. The half-life was defined as the time, at which the
residual activity of SyGDH was 50% of its original activity.
2. Materials and methods
2.1. Strains, plasmids and chemicals
E. coli BL21(DE3) and single and double promoter plasmids (pET-
28a(+) and pETDuet-1) (Novagen, Madison, WI) were used for gene
expression. E. coli transformants, E. coli/Sygdh and E. coli/Sys1 (sepa-
rately expressing SyGDH and SyS1) and E. coli/Sygdh-Sys1 (coexpres-
sing two enzymes), were constructed and preserved in our lab [25]. E.
coli BL21(DE3) transformed with pET-28a(+) and pETDuet-1, desig-
nated as E. coli/pET-28a and E. coli/pETDuet, were used as the negative
control. Reduced and oxidized nicotinamide adenine dinucleotide
(phosphate), coenzymes NAD(P)H and NAD(P)+, were purchased from
YuanYe Biotechnology (Shanghai, China). Both m-CPC and α-BAP as
well as the corresponding racemic 2-chloro-1-(3-chlorophenyl)ethanol
(rac−CCE) and rac-BPE were purchased from Sun Chemical Tech-
nology (Shanghai, China).
2.5. Effects of metal ions and EDTA on the activity of SyGDH
To estimate the effects of metal ions and EDTA on the activity of
SyGDH, aliquots of purified SyGDH without preprocessing by EDTA
were incubated ZnCl2, FeCl2, CoCl2, MgSO4, LiCl, SnCl2, FeCl3, NaCl,
AlCl3, BaCl2, CaCl2, CuSO4, or EDTA solution (50 mM, dissolved in
water) at a final concentration of 2 mM, respectively, in 20 mM phos-
phate buffer (pH 7.5) at 40 °C for 1 h. Then, the residual enzyme ac-
tivity was measured under the standard assay conditions. Additionnly,
the activity of SyGDH incubated in the 2 mM EDTA was measured by
addtion of extra 5 mM ZnCl2. Enzyme without adding any additive was
used as the control.
2.2. Expression and purification
A single colony of E. coli transformant was inoculated into 2 mL LB
medium supplemented with 100 μg/mL kanamycin for E. coli/Sygdh or
with 100 μg/mL ampicillin for E. coli/Sys1 and /Sygdh-Sys1, and cul-
tured at 37 °C overnight as the seed culture. Then, 30 mL fresh LB
medium was inoculated with 2% (v/v) seed culture, and cultured until
the optical density at 600 nm (OD600) reached 0.6–0.8. The expression
of SyGDH and SyS1 and coexpression of both SyGDH and SyS1 were
induced, respectively, by addition of 0.6 mM IPTG at 25 °C for 10 h. The
induced E. coli cells were harvested by centrifugation, and resuspended
in 50 mM K2HPO4–KH2PO4 buffer (pH 7.0) to 100 mg wet cells/mL
unless stated otherwise.
2.6. Substrate and coenzyme specificities of SyGDH
The substrate specificity of SyGDH was investigated by measuring
its specific activities (U/mg protein) towards 50 mM different mono-
and di-saccharides (such as D-glucose, D-galactose, D-xylose, D-man-
nose, D-maltose, glucose 6-phosphate and sucrose) under the standard
assay conditions. The oxidation rate of D-glucose (μmol/min/mg
2