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ARTICLE IN PRESS
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S. He et al. / Process Biochemistry xxx (2014) xxx–xxx
K. rhizophila, which could catalyze the asymmetric reduction of
adrenalone to (R)-(−)-epinephrine with an enantiomeric excess
(e.e value) of more than 99%. It was named (R)-(−)-epinephrine
dehydrogenase (EPID). Moreover, this enzyme shows great selec-
tivity for aromatic -amino ketones/alcohols, and very poor or
almost no affinity for aliphatic ketones or amino acids.
2.6. Enzyme characterization
2.6.1. Cofactor specificity and enzymatic enantioselectivity
Cofactor specificity was detected on the basis of the standard enzyme activity
determination. When the dehydrogenase catalyzed a reduction reaction, NADH or
NADPH was selected as the cofactor and adrenalone was the substrate in the reac-
tion system. Analogously, NAD+ or NADP+ was the cofactor and (R)-(−)-epinephrine
was the corresponding substrate when it was a oxidation reaction. The reactions
were carried out at 40 ◦C for 2 min, and then each system’s enzymatic activity was
examined.
2. Materials and methods
Separation of enantiomers was performed on an automated P/ACETM MDQ
CE system (Beckman Instruments, Fullerton, CA, USA) equipped with fused-silica
capillaries (I.D. 75 m, efficient length 50 cm). Separation solutions were prepared
by dissolving methylol--cyclodextrin (20 mM) as the chiral selector in the back-
ground electrolyte (55 mM Tris–H3PO4, pH 3.0). The capillaries were conditioned
according to the following procedure before every run: UP (ultra pure) water for
5 min; 0.1 M NaOH for 5 min; UP water for 5 min. Separations were then started
thermostated at 25 ◦C using 10 kV, and UV absorbance was monitored at 214 nm.
The reaction system, which was used to determine enzymatic enantioselectivity,
comprising 2.5 mM substrates (adrenalone), 1 mM coenzyme NADH (or NADPH),
and 0.5 ml pure enzyme solution. 50 mM phosphate buffer was then added to the
system above to obtain a final volume of 1 ml. The enzymatic reaction was carried out
at 40 ◦C for 30 min, and then reaction products were detected with high performance
capillary electrophoresis (HPCE).
2.1. Chemicals
(R)-epinephrine and adrenalone were purchased from Sigma (USA). Coenzymes
(NADH, NADPH) were purchased from ROCHE (Germany). DEAE Sepharose F. F, Butyl
SephroseTM 4 Fast Flow and SephacrylTM S-200 High Resolution were purchased
from Amersham Biosciences (GE, USA). Standard proteins for gel filtration and SDS-
gel electrophoresis were purchased from Sangon (ShangHai, PRC). All other reagents
were of analytical grade.
2.2. Production of (R)-epinephrine dehydrogenase
K. rhizophila was cultured as described previously to obtain mycelia [11].
2.2.1. Freeze/thaw
Air-dried mycelia (1 g) were suspended in buffer A (5 ml). The mixture were
frozen at −196 ◦C for 1 min and then thawed at 37 ◦C for 1 min. The freeze/thaw
processes were repeated for three cycles. Finally, the mixture was centrifuged at
12,000 × g for 20 min and the supernatants were recovered as crude enzymatic
extracts.
2.6.2. Effects of pH and temperature on enzyme activity
The effect of pH on activity of (R)-epinephrine dehydrogenase was estimated
on the basis of standard activity assays carried out at 45 ◦C and over pH range
between 3.0 and 10.0 buffers of Different pH values were used. The pH stability of the
purified enzymes was determined by measuring residual activity after protein pre-
incubation (for 4 h at 45 ◦C) in same buffers with various pH values. The optimum
temperature of (R)-epinephrine dehydrogenase was determined by measuring rela-
tive activity in 100 mM phosphate buffer (pH 6.0) over a temperature range between
20 and 70 ◦C. Thermostability of purified enzymes was evaluated by their incubation
for 60 min at temperature varying from 20 to 60 ◦C in 100 mM phosphate buffer (pH
6.0) followed by residual activity assays under standard conditions.
2.2.2. Ammonium sulfate fractionation precipitation
The ammonium sulphate was added to the samples (20 ml) to give a saturation
concentration of 40%, and the resulting precipitate was removed by centrifugation
(12,000 × g for 20 min at 4 ◦C). Then, the ammonium sulphate concentration was
increased stepwise to 60% saturation, and precipitate was collected by centrifuga-
tion.
2.6.3. Effects of various compounds on enzyme activity
The effect of various metals ions (Ag2+, Ca2+, Co2+, Cu2+, Fe2+, Hg2+, Mg2+, Mn2+
,
2.2.3. DEAE-sepharose fast flow (ion-exchange column chromatography)
The DEAE-sepharose FF column (1.6 cm × 50 cm) was equilibrated with 20 mM
Tris–HCl buffer at pH 7.2 and eluted with a linear gradient of sodium chloride
(0–0.5 M, pH 7.0) in the equilibrating buffer. The fractions showing activity were
collected and used for the next step.
Pb2+, Zn2+, NH4+) and EDTA on the activity of (R)-epinephrine dehydrogenase was
investigated by their pre-incubation with these compounds for 30 min at 37 ◦C fol-
lowed by measuring residual activity under standard conditions.
2.6.4. Substrate specificity and enzyme kinetics
2.2.4. Butyl-sepharose 4 fast flow (hydrophobic chromatography)
The butyl-sepharose 4 fast flow column (1.6 cm × 50 cm) was equilibrated and
eluted by 50 mM potassium phosphate buffer (pH 7.0).
For determination of substrate specificity of the purified enzyme, vari-
ous substrates such as norepinephrine, isoproterenol, ephedrine, phenylephrine,
acetaldehyde, acetone, 2-amino-acetophenone, l-tyrosine, ethanol, acetanilide and
other chemicals were used. Kinetic constants (Km and Vmax) for (R)-epinephrine,
adrenalone, NADH and NAD+ were determined by measurement of initial velocities
at various concentrations of one substrate at fixed concentrations of other substrates
under the standard assay condition. The data was plotted on a Lineweaver–Burk
double-reciprocal plot. Reactions were then performed at the optimal pH and tem-
perature of the dehydrogenase. All experiments were done in triplicates and the
2.2.5. Sephacryl S-200 HR (gel chromatography)
Sephacryl S-200 HR column (1.6 cm × 50 cm) was equilibrated and eluted with
50 mM potassium phosphate buffer (pH 7.0), containing 0.15 M NaCl. The active
fractions were pooled and dialyzed against buffer E. The enzyme purity was assessed
by SDS–PAGE.
average data were used to calculate the KM and Vmax
.
2.3. Determination of enzymatic activities
3. Results and discussion
The determination of enzyme activity was described in Vallee and Hoch method
[15]: coenzymes NAD(P)H absorbs at 340 nm while oxidized form NAD(P)+ does
not. Thus, coenzymes’ oxidation or reduction would cause absorbance rise or fall
at 340 nm respectively. Enzyme activity, therefore, could be measured through the
determination of absorbance changes at 340 nm in the reaction process.
3.1. Purification of enzymes
As shown in Fig. 1C, only one protein peak (fractions from 29
dehydrogenase activities (Fig. 1A). The third step of purification
gave one main peak of proteins that displayed (R)-epinephrine
dehydrogenase activity (Fig. 1B). Results of the three-step purifi-
cation procedure are summarized in Table 1. (R)-epinephrine
dehydrogenase was purified 226-fold with 32% recovery of its activ-
ity. The specific activity of (R)-epinephrine dehydrogenase was
190 U/mg.
The purified enzyme showed a single peak in Sephadex G-100
gel filtration chromatography, and the molecular mass was esti-
mated to be 67 kDa in comparison with the elution volume of
standard proteins. The purified enzyme migrated as a single band
with a size of about 33 kDa on SDS–PAGE (Fig. 2). The results of gel
2.4. Protein assay and electrophoresis
albumin as the standard [16]. Protein concentration in fractions derived by column
chromatography was estimated by measurements of absorbance at 280 nm.
The purity and molecular mass of the separated proteins were determined by
sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) according
to the method of Laemmli [17].
2.5. Native molecular weight determination of the purified enzyme
The native molecular weight of the purified enzyme was determined using
Sephadex G-100 column pre-equilibrated with 50 mM sodium phosphate buffer
(pH 7.0). The elution rate was 0.3 ml/min with the same buffer. Gel filtration
chromatography was performed at room temperature and a gelfiltration protein
marker comprising myoglobin (17.8 kDa), ovalbumin (45 kDa), bovine serum albu-
min (66 kDa) and ovalbumin-dimer (90 kDa) was used.
Please cite this article in press as: He S, et al. Purification and characterization of a novel carbonyl reductase involved in oxidoreduction