Novel Acylase from Streptomyces mobaraensis
J. Agric. Food Chem., Vol. 54, No. 1, 2006 73
Purification of Capsaicin-Hydrolyzing Enzyme. All purification
procedures were carried out at 4 °C. Ammonium sulfate was first added
to the culture supernatant (800 mL) to 60% saturation to precipitate
soluble proteins. The resulting precipitate was separated by centrifuga-
tion at 20000g for 30 min and dissolved in 100 mL of buffer B
containing 50 mM NaCl and dialyzed against the same buffer. The
dialyzed solution was placed on a CM Sephadex C-50 gel column (1.6
cm i.d. × 35 cm) equilibrated with the same buffer as was used for the
dialysis. Elution was accomplished by a linear increase in the NaCl
concentration in buffer B from 50 to 500 mM at a flow rate of 0.35
mL/min. Fractions showing capsaicin-hydrolyzing activity were col-
lected, concentrated, and dialyzed against buffer C. The enzyme solution
was applied to a hydroxyapatite gel column (1.6 cm i.d. × 15 cm) and
eluted by linearly increasing the concentration of potassium phosphate
buffer, pH 7.5, from 40 to 400 mM at a flow rate of 0.18 mL/min.
Active fractions were collected, concentrated, and finally dialyzed
against buffer A.
Sodium Dodecyl Sulfate-Polyaclylamide Gel (SDS-PAGE) and
Native PAGE. SDS-PAGE was performed using a 12.5% gel (Bio-
Rad Laboratories, Mini-Protean III Ready Gels J, Hercules, CA) by
the method of Laemmli (11). The protein bands were stained with
Coomassie Brilliant Blue R-250 (CBB R-250, Sigma-Aldrich Co.).
Phosphorylase b (94 kDa), BSA (67 kDa), ovalbumin (43 kDa),
carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa),
and R-lactalbumin (14.4 kDa) (Amersham Pharmacia Biotech, LMW
Electrophoresis Calibration Kit) were used as standard proteins. Native
PAGE was carried out using a 5-20% nondenatured gradient gel (Bio-
Rad Laboratories, Mini-Protean III Ready Gels J) in which an acetic
acid/â-alanine buffer, pH 4.5, was used as a reservoir buffer according
to Reisfeld et al. (12) to estimate the molecular mass of the enzyme.
As marker proteins for native PAGE, glyceraldehydes-3-phosphate
dehydrogenase from rabbit muscle (145 kDa), alcohol dehydrogenase
from equine liver (79.5 kDa), avidin from egg white (60 kDa), trypsin
from bovine pancreas (23 kDa), and ribonuclease A from bovine
pancreas (14 kDa) were used, all of which were obtained from Sigma-
Aldrich Co. Protein bands were stained with CBB R-250. The molecular
mass of the enzyme was determined from a calibration curve prepared
from the mobility of the marker proteins vs the logarithms of their
molecular masses.
Effects of Reagents and Metals on the Stability of Capsaicin-
Hydrolyzing Enzyme. A solution of the enzyme in buffer A was
preincubated at 37 °C for 15 min in the presence of a final 1 mM
concentration of reagents and metals [p-chloromercuribenzoic acid
(PCMB), iodoacetamide, dithiothreitol (DTT), MgSO4, FeSO4, CaCl2,
AgNO3, ZnCl2, CuSO4, ethylenediaminetetraacetate (EDTA), reduced
glutathione, CoCl2, L-cysteine, (NH4)6Mo7O24, and phenylmethylsul-
fonyl fluoride (PMSF)] and a final 10 mM concentration of 2-mer-
captoethanol. As a control, buffer A was used in place of the reagent
solution. The activity of the treated enzyme was assayed by the same
method described above.
pH Dependency of Capsaicin-Hydrolyzing Activity. Buffer A and
a 50 mM acetate buffer containing 0.5 mM CoCl2 were used over the
pH range from 5.9 to 10.4 and from 4.5 to 6.2, respectively. A 20 µL
aliquot of enzyme solution (about 10 µg/mL) was added to 80 µL of
a final 130 µM capsaicin solution at different pH values and incubated
for 1 h at 37 °C, and the remaining capsaicin concentration was then
determined by HPLC as described above.
Table 1. Capsaicin-Hydrolyzing Activities for 21 Strains of
Streptomyces sp.
NBRC no.
activity (U/mL)
1.2 0.1
S. mobaraensis
S. ardus
S. blastmyceticus
S. cacaoi
S. caespitosus
S. cinnamoneus
S. exfoliatus
S. griseinus
S. lividoclavatus
S. lividus
S. luteoreticuli
S. mobaraenis
S. olivaceus
S. roseoverticillatus
S. scabiei
S. sioyaensis
S. spheroides
S. toyocaensis
S. tuirus
S. venezuelae
S. violaceoruber
13819
13430
12747
13813
13490
12852
12319
12869
13870
13787
13422
13476
12805
12817
13767
12820
12917
12824
15617
13097
13385
±
1.0
Aldrich Co. or the Peptide Institute. All other reagents were of analytical
grade and purchased from either Wako or Nacalai Tesque, Inc. (Kyoto,
Japan).
Buffers. The following buffers were used in this study. Buffer A:
50 mM Tris-HCl, pH 7.8; buffer B: 25 mM Tris-HCl, pH 7.5; buffer
C: 40 mM potassium phosphate, pH 7.5; buffer D: 100 mM Tris-HCl
containing 0.5 mM CoCl2, pH 6.9; and buffer E: 100 mM Tris-HCl
containing 0.5 mM CoCl2, pH 7.5. Buffer A was used for the enzyme
assay, and buffers B and C were used, respectively, as elution buffers
in CM Sephadex C-50 and hydroxyapatite gel chromatographies.
Buffers D and E were utilized in synthetic reactions of capsaicin
analogues and N-lauroyl-L-amino acids/N-lauroyl-peptides, respectively.
Bacteria and the Cultivation of Cells. S. mobaraensis NBRC13819
(National Institute of Technology and Evaluation Biological Resource
Center, Chiba, Japan) is typically used for the production of an acylase
enzyme. In addition, we tested 20 Streptomyces strains listed in Table
1. Each strain was aseptically transferred to an agar plate (4 g of yeast
extract, 10 g of malt extract, 4 g of glucose, and 20 g of agar in 1 L of
water, adjusted to pH 7.3) and statically incubated for 7 days at 30 °C.
A loopful of the agar culture was then incubated in a 300 mL shaking
flask containing 30 mL of preculture medium [10 g of glucose, 10 g
of dextrin, 5 g of NZ amine (type A), 5 g of yeast extract, and 1 g of
CaCO3 in 1 L of water, adjusted to pH 6.5] and incubated at 30 °C
with reciprocal shaking at 120 strokes/min. A 1.8 mL aliquot of the
preculture was added to 500 mL shaking flasks containing 50 mL of
medium (40 g of beef extract, 40 g of soluble starch, 20 g of Polypepton,
2 g of K2HPO4, and 20 g of MgSO4 in 1 L of water, adjusted to pH
7.0) for the main culture, which was the optimal medium found. The
cells were grown at 30 °C with reciprocal shaking at 120 strokes/min
for 8 days. After cultivation, the culture broth was recovered by
centrifugation at 20000g for 30 min at 4 °C.
Enzyme Assay. The capsaicin hydrolytic activity was measured as
follows. A 20 µL aliquot of the enzyme solution was added in 180 µL
of capsaicin solution dissolved in buffer A at a final concentration of
130 µM and incubated for 20 min at 37 °C. The remaining capsaicin
concentration in the reaction mixture was determined by high-
performance liquid chromatography (HPLC) (Shimadzu, Corp., Kyoto)
using a 5C18-AR-II column (4.6 mm i.d. × 150 mm, Nacalai Tesque,
Inc.) with detection at 280 nm. Elution was carried out using 50% (v/
v) acetonitrile solution containing 0.075% phosphoric acid as the mobile
phase at a flow rate of 0.8 mL/min at room temperature. One unit of
capsaicin-hydrolyzing activity was arbitrarily defined as the amount
of the enzyme required to hydrolyze 1 µmol of capsaicin in 1 h at 37
°C at pH 7.8. Protein concentrations were determined using a BCA
Protein Assay Reagent Kit (Pierce Chemical Co., Rockford, IL) with
bovine serum albumin (BSA) as a standard.
Thermal Stability of Capsaicin-Hydrolyzing Enzyme. The enzyme
solution was incubated in buffer A with or without 0.5 mM CoCl2 for
1 h at 4, 25, 37, 45, 50, 55, and 60 °C, and the remaining activity was
then determined as described above.
Optimum Reaction Temperature. To determine the optimum
temperature for the enzyme, it was added to a final 130 µM solution
of capsaicin and incubated for 1 h at 30, 37, 45, 50, 55, and 60 °C.
The residual capsaicin concentration was determined by HPLC as
described above.
pH Stability. The enzyme solution was added to 50 mM Tris-HCl
buffers at different pH values and incubated at 37 °C for 1 h, after
which the residual activity at pH 7.8 was determined.
Substrate Specificity. Various N-acetyl-L-amino acids, N-lauroyl-
L-amino acids, and N-lauroyl-L-dipeptides were used as the substrates