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Screening procedures: Colonies were picked and transferred into
deep-well plates containing lysogeny broth (LB; 300 mL) with kana-
mycin (50 mgmLÀ1) and were cultured overnight at 378C with shak-
ing. An aliquot (120 mL) was transferred to glycerol stock plate and
was stored at À808C. Then, TB medium (800 mL) with 0.2 mm iso-
propyl b-d-thiogalactopyranoside (IPTG) and 50 mgmLÀ1 kanamycin
was added directly to the culture plate for 8 h at 308C with shak-
ing for protein expression. The cell pellets were harvested and
washed with 50 mm potassium phosphate buffer (pH 7.4, 400 mL)
and centrifuged for 10 min at 3200 g and 48C. Then, the cell pel-
lets were resuspended in the same buffer (400 mL) with DNase I
(6 U) and lysozyme (1 mgmLÀ1) to break the cell at 308C for 1 h
with shaking. The crude lysate was centrifuged for 30 min at
3200 g and 48C. The supernatant (300 mL) was transferred into
new deep-well plates for reaction with substrate 1 (5 mm), NADP+
(50 mm) and 10% propan-2-ol for 14–16 h at 308C and 800 rpm
(INFORS); the final volume was 400 mL. The product and remaining
substrate were extracted by using equal volumes of EtOAc for GC
analysis on a chiral column. The GC conditions reference to our
previous publications.[3b,6c]
further comparison, we also evaluated the use of the consen-
sus approach that picks up conserved residues as building
blocks upon choosing the triple code. However, the consensus
method failed to obtain higher S-selective mutants than strat-
egies 1 and 2. The reason may be that the consensus method
is not directly correlated with the codon chosen in the tested
substrate.
To shed light on the distinct stereoselectivity of the best mu-
tants (SZ2150 and SZ2162) obtained from strategy 2, docking
analysis and molecular dynamics simulations on these two mu-
tants as catalysts in the asymmetric reduction of substrate 1
were performed. Following the Bꢀrgi–Dunitz model, coupled
with near-attack conformation calculations, theoretical analysis
suggested that SZ2150 and SZ2162 are able to produce highly
enantioenriched (S)-2 and (R)-2, respectively, as a result of a
proper angle (trajectory) and distance during nucleophilic
attack.
Protein expression and purification: E. coli BL21 (DE3) cells carry-
ing the recombinant plasmid were cultivated in LB medium (5 mL)
containing kanamycin (50 mgmLÀ1) overnight at 378C. The over-
night culture was inoculated in TB medium (500 mL) containing ka-
namycin (50 mgmLÀ1) and was grown at 378C. The culture was in-
duced by the addition of IPTG with a final concentration of 0.2 mm
when OD600 reached 0.6 and was then allowed to grow for an addi-
tional 12 h at 308C. After centrifugation for 15 min at 6000g and
48C, the bacterial pellet was washed with phosphate buffer
(50 mm, pH 7.4) and was resuspended in a phosphate buffer
(20 mm pH 7.4) containing NaCl (0.5m) and imidazole (20 mm). The
cells were lysed by sonication, and the supernatant was collected
by centrifugation for 60 min at 10000g and 48C. Protein purifica-
tion was performed at 238C by using an AKTA Purifier 10 system
with UNICORN 5 software (GE Healthcare). The WT and mutants
was purified by using affinity chromatography with a HisTrap FF
crude column (GE Healthcare). The column was pre-equilibrated
with equilibrium buffer (50 mL; 20 mm phosphate buffer, 0.5m
NaCl, 20 mm imidazole, pH 7.4). The sample (30 mL) was loaded
with a flow rate of 2.0 mLminÀ1. After washing with equilibrium
buffer (50 mL), the bound target protein was washed with 10–
500 mm imidazole solution containing NaCl (500 mm) and sodium
phosphate (20 mm; pH 7.4). The target protein fraction was collect-
ed and desalted through a Model HiPrep 26/10 desalting column
(GE Healthcare) against phosphate buffer (20 mm, pH 7.4) contain-
ing NaCl (100 mm). The protein concentration was measured by
using the Bradford method.
Experimental Section
Materials: KOD Hot Start DNA Polymerase was obtained from No-
vagen. Restriction enzyme DpnI was bought from NEB. The oligo-
nucleotides were synthesized by Life Technologies. The plasmid
preparation kit was ordered from Zymo Research, and the PCR gel
extraction kit was bought from QIAGEN. DNA sequencing was con-
ducted by GATC Biotech. All commercial chemicals were purchased
from Sigma–Aldrich, Tokyo Chemical Industry (TCI), or Alfa Aesar.
Lysozyme and DNase I were purchased from AppliChem.
PCR-based methods for library construction: Libraries were con-
structed by using the overlap PCR and mega primer approach with
KOD Hot Start polymerase. Reaction mixtures (50 mL) typically con-
tained water (30 mL), KOD hot-start polymerase buffer (10ꢂ, 5 mL),
MgSO4 (3 mL, 25 mm), dNTPs (5 mL, 2 mm), DMSO (2.5 mL), template
DNA (0.5 mL, 50–100 ng), primers mix (100 mm, 0.5 mL each), and
KOD hot start polymerase (1 mL). The PCR conditions for short frag-
ment: 958C, 3 min (958C, 30 s; 568C, 30 s; 688C, 40 s)ꢂ32 cycles;
688C, 120 s; and 168C, 30 min. For mega-PCR: 958C, 3 min (958C,
30 s; 608C, 30 s; 688C, 5 min 30 s)ꢂ24 cycles; 688C, 10 min; and
168C, 30 min. The PCR products were analyzed on agarose gel by
electrophoresis and were purified by using a Qiagen PCR gel ex-
traction kit. NEB CutSmart Buffer (2 mL) and DpnI (2 mL) were
added to the PCR reaction mixture (50 mL), and the digestion was
performed at 378C for more than 3 h. After DpnI digestion, the
PCR products (1 mL) were directly transformed into electrocompe-
tent Escherichia coli BL21(DE3) to create the final library for quick
quality control and screening.
Determination of kinetic parameters and thermostability: The ki-
netic parameters were obtained by measuring the initial velocities
of the enzymatic reaction and curve fitting according to the Mi-
chaelis–Menten equation. The activity assay was performed in a
mixture containing varying concentrations of substrate 1 (1–
50 mm) and 0.7 mm NADPH in phosphate buffer (0.2 mL, 50 mm,
pH 7.4) containing substrate 1, NADPH, and the purified enzyme.
The substrate was dissolved in acetonitrile, which did not exceed
5% of the total volume. The reaction was initiated by the addition
of the enzyme and was monitored for 2 min with SPECTRAMAX
M5 (Molecular Devices) at 308C. The activity was determined by
measuring NADPH oxidation from the decrease in the absorbance
Primer design and library creation: Library C was created accord-
ing to the following steps (Scheme S1): 1) Primers F1/R1 and F2/R2
were used to amplify WT of ADH separately. The PCR products
were purified by a gel extraction kit to remove the original plas-
mid. 2) Step 1 products were used as a template with primers F1/
R2 to do overlap PCR. Then, the PCR fragments were purified to
remove any remaining primers. 3) Step 2 products were used as
mega primers to amplify plasmid pRSFduetADHwt.[20] The PCR
products were digested by DpnI to remove the template plasmid
and 1 mL aliquot to transform E. coli BL21 (DE3) to create the var-
iants plasmid library for screening. Library D was constructed fol-
lowing the similar protocols as library C. The list of primers is
shown in Table S8.
at l=340 nm by using
a molar absorption coefficient of
6.22 mmÀ1 cmÀ1. One unit of enzyme activity was defined as the
amount of enzyme catalyzing the conversion of 1 mmol NADPH
&
ChemBioChem 2018, 19, 1 – 9
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ꢁ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!
Qu, Ge
