1
80
A. Li et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 179–187
stereoselective enzymes and microorganisms is of great signifi-
cance.
The plasmid pET30a-EbSDR8 was transformed into E. coli
BL21(DE3) which was then spread on LB agar plates containing
50 g/mL kanamycin. The single colony with recombinant plasmid
pET30a containing the Ebsdr8 gene was confirmed by DNA sequenc-
In our previous study, a novel whole-cell biocatalyst, Empe-
dobacter brevis ZJUY-1401, was isolated to catalyze asymmetric
reduction of various prochiral ketones into the corresponding
anti-Prelog alcohols with excellent enantioselectivity [19]. This
biocatalyst has advantages such as noticeable tolerance against
ethanol, remarkable pH adaptability, utilization of cheaper coen-
zyme and broad substrate spectrum, which make it a promising
biocatalyst for the preparation of anti-Prelog chiral alcohols. The
shared bioinformatics from genome database provides us an
opportunity to take insights into the information hidden in the
microorganic genome and help us to hunt novel enzymes with
potential applications [20]. In the present study, a short-chain
dehydrogenase/reductase from E. brevis ZJUY-1401 (EbSDR8) with
excellent anti-Prelog stereoselectivity was cloned, heterologously
expressed, purified, and characterized based on the sequence
reported from National Center for Biotechnology Information
◦
ing. The recombinant cells were grown at 37 C in LB medium
containing 50 g/mL kanamycin until the optical density at 600 nm
(OD600) reached 0.6–0.8, then gene expression was induced with
0.1 mM isopropyl -d-1-thiogalactopyranoside (IPTG). The cells
◦
were subsequently cultured at 25 C for 5 h and then harvested by
centrifugation. The pellet was washed and resuspended in 100 mM
Na HPO –NaH PO buffer (pH 8.0) and disrupted by ultrasoni-
2
4
2
4
cation for 10 min. The cell lysate was centrifuged at 12,000 rpm
for 10 min, and the supernatant was applied to a Ni-NTA affinity
chromatography column that had been equilibrated with binding
buffer (20 mM sodium phosphate, 0.5 M NaCl and 20 mM imida-
zole, pH 7.4). The bound enzyme was eluted with elution buffer
(20 mM sodium phosphate, 0.5 M NaCl and 0.5 M imidazole, pH
7.4), and then the fractions containing EbSDR8 were desalted with
100 mM Na HPO –NaH PO (pH 7.0) by a HisTrap desalting col-
(
NCBI). The catalytic effectiveness of EbSDR8 toward a series of
2
4
2
4
◦
prochiral ketones was emphatically discussed.
umn and stored at 4 C for further use. SDS-PAGE was used to verify
EbSDR8 expression and purification. Protein concentrations were
quantified via the method of Bradford [21] using BSA as the protein
standard.
2
. Materials and methods
2.1. Materials
2.4. Enzyme assay and kinetic characterization
Aromatic ketones and their corresponding chiral alcohols of
analytical grade were purchased from J&K Scientific Ltd. (Beijing,
China) or Sigma–Aldrich (Shanghai, China). NADPH and NADH dis-
The catalytic activity of EbSDR8 was assayed spectropho-
◦
tometrically at 30 C by monitoring the change in absorbance
at 340 nm. One unit of enzyme activity was defined as the
amount of enzyme catalyzing the oxidation of 1 mol of
NAD(P)H per minute under measurement conditions. Enzyme
kinetic parameters were determined using different substrates
and coenzymes. Various substrate concentrations were used for
determining the corresponding activities when concentrations
of corresponding coenzymes were kept constant. Substrates and
coenzymes used were NADH (0–0.3 mM), NADPH (0–1.0 mM), iso-
propanol (0–25.0 mM), acetone (0–27.5 mM), DL-1-phenylethanol
odium salt were purchased from Sangon (Shanghai, China). The
enzymes used in molecular operation such as PrimeSTARTM HS
DNA polymerase, Taq DNA polymerase, restriction endonucleases
BamHI and XhoI) and T4 DNA ligase were purchased from TaKaRa
Dalian, China). Genomic DNA purification kit was obtained from
Dongsheng, China. Gel extraction kit and plasmid miniprep kit were
obtained from Axygen, China. Primers were synthesized by Invit-
rogen, China. Ni-NTA column were purchased from GE Healthcare
Bio-Science, Sweden. The other chemical reagents used were all of
analytical grade and obtained from local companies.
(
(
(
0–27.5 mM), and acetophenone (0–25.0 mM). The constants were
calculated from the Lineweaver-Burk double-reciprocal plot.
2.2. Bacterial strains, plasmids and culture conditions
2.5. Characterization of recombinant EbSDR8
E. brevis ZJUY-1401 was isolated from soil and has been
deposited in the China Center for Type Culture Collection (CCTCC M
The catalytic properties of EbSDR8 was assayed with ace-
tophenone as substrate. The standard reaction mixture contained
2
014520, Wuhan, China). Cultures of the organism were grown rou-
1
7
0 mM acetophenone, Na HPO –NaH PO buffer (100 mM, pH
2 4 2 4
◦
tinely in Luria-Bertani (LB) medium at 30 C for 24 h. The plasmid
pET30a expression vector from Novagen (USA), was used for over-
expression of the EbSDR8-encoding gene, and the Escherichia coli
strain BL21(DE3) was used as the host. Recombinant E. coli was
.0), 2.5 mM NADH and an appropriate amount of the enzyme in a
total volume of 1.0 mL. The biotransformations were carried out at
◦
3
0 C with shaking at 200 rpm. Samples were extracted with ethyl
acetate and the organic layer was dried over anhydrous Na SO . The
2
4
◦
◦
cultivated at 37 C or 25 C in LB medium containing 50 g/mL
conversion and enantiomeric excess of product (eep) were deter-
mined by chiral gas chromatography (GC) analyses. All experiments
were conducted in triplicate if not specified.
kanamycin.
2
.3. Cloning, expression and purification of recombinant EbSDR8
The coenzyme specificity of EbSDR8 was examined by mea-
suring enzyme activity using 2.5 mM NADH or NADPH as the
coenzyme. The effect of cosubstrate was determined by using sev-
eral sugars and alcohols as cosubstrate at a concentration of 10%
(w/v or v/v). The intracellular concentration of NADH and NAD+ in
E. coli was determined using the method developed by Bernofsky
Genomic DNA was extracted from E. brevis ZJUY-1401 using
genomic DNA purification kit described above. The gene encoding
EbSDR8 was amplified by PCR using PrimeSTARTM HS DNA
polymerase with the following primers: EbSDR8F-BamHI:
ꢀ
ꢀ
◦
5
-GCTGAGGATCCATGTCAATATTAAAAGATAAGGTAGC-3
and
and Swan [22]. The optimal pH of EbSDR8 was determined at 30 C
ꢀ
EbSDR8R-XhoI 5 -GCATCCTCGAGTTAAACTGCTGTATATCCTCCATC-
3
using a set of 100 mM buffers: citrate buffer (pH 5.5–6.5); sodium
phosphate buffer (pH 6.5–8.5); glycine-NaOH buffer (pH 8.5–10.5).
The effect of temperature on the enzyme activity and stereose-
ꢀ
. The PCR product was double-digested with BamHI and XhoI,
followed by ligation into the expression vector pET30a. The
resulting plasmid, harboring the Ebsdr8 gene, was designated
pET30a-EbSDR8. The nucleotide sequence of EbSDR8 has been
submitted to the GenBank database under accession number
KT003817.
◦
lectivity was determined at temperatures ranging from 20 C to
◦
65 C. For evaluating thermostability, aliquots of the enzyme were
◦
◦
◦
assayed at 35 C after incubation at 35 C and 45 C for various peri-
ods of time. The half-life of enzyme activity was determined by