Purification, characterization, molecular cloning, and expression of a new aminoacylase from streptomyces mobaraensis that can hydrolyze N-(Middle/Long)-chain-fatty-acyl-L-amino acids as well as N-Short-chain-acyl-L- amino acids

Article abstract of DOI:10.1271/bbb.90081

We report here on the purification, characterization, molecular cloning, and expression of a new aminoacylase, initially isolated from the supernatant of Streptomyces mobaraensis (Sm-AA). Purified wild-type Sm-AA was found to be a monomeric protein with a molecular mass of 55 kDa. The cloned gene of Sm-AA contained an ORF of 1,383 bp, encoding a polypeptide of 460 amino acids. A BLAST search revealed that Sm-AA belongs to the peptidase M20 family, with identities to a hypothetical protein from Streptomyces pristinaespiralis, a putative peptidase from Streptomyces avermitilis, peptidase M20 from Frankia sp., succinyl-diaminopimelate desuccinylase from Hemophilus influenzae, and aminoacylase-1 from porcine kidney at 89, 88, 67, 29, and 25% respectively. The Sm-AA gene was subcloned into an expression vector, pSH19, and was expressed in Streptomyces lividans TK24. The amount of the recombi- nant Sm-AA expressed in the S. lividans cells was approximately 42-fold higher than that of Sm-AA found in the supernatant of S. mobaraensis. Sm-AA showed high hydrolytic activity towards various N-acetyl-L-amino acids and N-(middle/long)-chain-fatty-acyl-L- amino acids, with a preference for the acyl derivatives of L-Met, L-Ala, L-Cys, etc. with an optimum pH and temperature for reaction of about 7.5 and 50 °C (at pH 7.5).

Full text of DOI:10.1271/bbb.90081

Biosci. Biotechnol. Biochem., 73 (9), 1940–1947, 2009
Purification, Characterization, Molecular Cloning, and Expression
of a New Aminoacylase from Streptomyces mobaraensis
That Can Hydrolyze N-(Middle/Long)-chain-fatty-acyl-L-amino Acids
as Well as N-Short-chain-acyl-L-amino acids
Mayuko KOREISHI, Yasuyuki NAKATANI, Manaymi OOI, Hiroyuki IMANAKA,
Koreyoshi IMAMURA, and Kazuhiro NAKANISHI
Division of Chemistry and Biochemistry, Graduate School of Natural Science and Technology, Okayama University,
3-1-1 Tsushima-naka, Okayama 700-8530, Japan
Received February 2, 2009; Accepted May 29, 2009; Online Publication, September 7, 2009
[doi:10.1271/bbb.90081]
We report here on the purification, characterization,
molecular cloning, and expression of a new amino-
acylase, initially isolated from the supernatant of
Streptomyces mobaraensis (Sm-AA). Purified wild-type
Sm-AA was found to be a monomeric protein with a
molecular mass of 55 kDa. The cloned gene of Sm-AA
contained an ORF of 1,383 bp, encoding a polypeptide of
460 amino acids. A BLAST search revealed that Sm-AA
belongs to the peptidase M20 family, with identities to a
hypothetical protein from Streptomyces pristinaespiralis,
a putative peptidase from Streptomyces avermitilis,
peptidase M20 from Frankia sp., succinyl-diaminopime-
late desuccinylase from Hemophilus influenzae, and
aminoacylase-1 from porcine kidney at 89, 88, 67, 29,
and 25% respectively. The Sm-AA gene was subcloned
into an expression vector, pSH19, and was expressed in
Streptomyces lividans TK24. The amount of the recombi-
nant Sm-AA expressed in the S. lividans cells was approxi-
mately 42-fold higher than that of Sm-AA found in the
supernatant of S. mobaraensis. Sm-AA showed high
On the other hand, some other isolated aminoacylases
specifically hydrolyze N-long-chain-acyl amino acids,
such as those from Mycobacterium smegmatis17,18) and
carboxypeptidase G3 from Pseudomonas sp.21) Amino-
acylase from Pseudomonas diminuta specifically hydro-
lyzes N-long-chain-acyl glutamic acids,19,20) but the
genes of these N-long-chain-acyl aminoacylases are yet
to be identified. In addition to the short-chain-acyl
aminoacylase from S. mobaraensis, as above,14) we
recently isolated two other acylases: "-lysine acylase,
with a substrate specificity to various N"-acyl-L-
lysines;22,23) and penicillin V acylase, which efficiently
hydrolyzes N-lauroyl-L-amino acids, N-lauroyl-peptides,
and capsaicin (8-methyl-N-vanillyl-6-nonenamide) in
addition to Penicillin V.24,25) During the course of the
purification of these enzymes from the culture super-
natant of S. mobaraensis, we isolated another new
aminoacylase (hereafter Sm-AA), which can efficiently
hydrolyze N-(middle/long)-chain-acyl-L-amino acids,
as well as N-acetyl-L-amino acids, with a preference
for the acyl derivatives of L-Met, L-Ala, L-Cys, etc. An
aminoacylase that can hydrolyze both N-(middle/long)-
chain-fatty-acyl-L-amino acids and N-short-chain-fatty-
acyl-L-amino acids is yet to be reported, with the
exception of carboxypeptidase G3 from Pseudomonas
sp.21) Therefore, in this study, we purified and charac-
terized Sm-AA. On the basis of the N-terminal amino
acid sequence of the purified enzyme, we cloned the
Sm-AA gene, and sequenced and expressed Sm-AA in
S. lividans TK24 cells for characterization.
hydrolytic activity towards various N-acetyl-L-amino acids
and N-(middle/long)-chain-fatty-acyl-L-amino acids,
with a preference for the acyl derivatives of L-Met,
L-Ala, L-Cys, etc. with an optimum pH and temperature
for reaction of about 7.5 and 50 ꢀC (at pH 7.5).
Key words: aminoacylase; actinomycete; Streptomyces
mobaraensis; N-acyl amino acids
Aminoacylase (N-acyl-L-amino-acid amidohydrolase,
EC 3.5.1.14) exists in a wide variety of sources,
including animal tissues,1–3) plants,4) molds,5,6) and
bacteria.7–20) Most isolated aminoacylases show a sub-
strate specificity towards N-acetyl-L-amino acids, in-
cluding ones isolated from hog kidney,1) Aspergillus
oryzae,5) Alcaligenes denitrificans DA181,7) Bacillus
stearothermophilus,10) Lactococcus lactis MG1363,11)
and Streptomyces mobaraensis, as reported in our
previous paper.14) In practice, aminoacylases, such as
from A. oryzae, are utilized in the production of L-amino
acids from the corresponding acetyl-DL-amino acids.15,16)
Materials and Methods
Materials (chemicals). DEAE Sephadex A-50 and Octyl Sepharose
4 Fast Flow or Octyl Sepharose CL-4B were obtained from GE
Healthcare Bio-Sciences AB (Uppsala, Sweden). Hydroxyapatite
(Fast Flow Type) and Hi-Trap Chelating HP (5 ml) were from Wako
Pure Chemical Industries (Osaka, Japan) and GE Healthcare Bio-
Sciences AB respectively. Various Nꢀ-acetyl-amino acids were from
either Wako or Sigma-Aldrich (St. Louis, MO). N-Butyryl-L-Met,
N-hexanoyl-L-Met, N-octanoyl-L-Met, N-decanoyl-L-Met, N-lauroyl-L-
Met, N-myristoyl-L-Met, and N-palmitoyl-L-Met were prepared by a
y
To whom correspondence should be addressed. Tel: +81-86-251-8200; Fax: +81-86-251-8264; E-mail: kazuhiro@cc.okayama-u.ac.jp
Abbreviations: ACY1, aminoacylase-1 from porcine kidney; BSA, bovine serum albumin; DTT, dithiothreitol; Fs-PM, peptidase M20 from
Frankia sp.; NBRC, National Institute of Technology and Evaluation Biological Resource Center; PCMB, p-chloromercuribenzoic acid; SDAP,
succinyl-diaminopimelate desuccinylase from Hemophilus influenzae; Sa-PP, putative peptidase from S. avermitilis; Sm-AA, aminoacylase from
S. mobaraensis; Sp-HP, hypothetical protein from Streptomyces pristinaespiralis

New Aminoacylase from Streptomyces mobaraensis
1941
10
11
Sm-AA gene (1.4 kb)
0.41-kbp PCR product
1
2
SalI-fragment (3.2 kb)
4
3
BssHII-fragment (1.2 kb)
4
5
6
9
7
8
SmaI-fragment (0.95 kb)
Fig. 1. Scheme for Cloning of the Sm-AA Gene.
Arrows 1–12 show the primers used, which correspond to the numbers shown in Table 1.
method reported elsewhere.26) Briefly, the corresponding acid chloride
Table 1. Primers Used in This Study
(Tokyo Chemical Industry, Tokyo) was added drop-wise to 0.6 M
L-Met dissolved in 2 N NaOH on ice with vigorous stirring to obtain
the product as a precipitate. Then the precipitate was put in contact
with n-hexane to extract acids, followed by rinsing with pure water,
and finally freeze-drying.
Number
Sequence
1
2
3
4
5
6
7
8
9
50-TCGACATCTGCCGCGACCTGATCC-30
50-GTTGACGGTGAAGGAGAACCCGCC-30
50-ATCCACGGGCACACGGACGTCGTC-30
50-GGCGAGCTTCTCGGCCACGTACTC-30
50-CCGCTACCTGGTGGACAAGCACCC-30
50-TTCCTCGCCGACCTGGACCGGATC-30
50-CGTCCACTCCGTGGAACATCCCGG-30
50-CCGGGATGTTCCACGGAGTGGACG-30
50-GATCCGGTCCAGGTCGGCGAGGAA-30
We used pSH1927) as an expression vector, a gift from Dr. M.
Kobayashi, University of Tsukuba. Restriction endonucleases and T4
DNA ligase were from either TAKARA BIO (Ohtsu, Japan) or Toyobo
(Osaka). An Advantageꢀ-GC genomic PCR Kit (BD Biosciences, San
Jose, CA) and KOD Plus DNA Polymerase (Toyobo) were used to
amplify fragments including the partial and whole Sm-AA sequences
respectively. Colony PCR was performed using Go Taqꢁ Green Master
Mix (Promega, Madison, WI). Agarose-LE (Classic Type) was from
Nacalai Tesque (Kyoto, Japan). A Quantum Prepꢁ Plasmid Miniprep
Kit (Bio-Rad, Hercules, CA) was used in the purification of plasmids.
All other reagents were of analytical grade and were purchased from
either Wako, Sigma-Aldrich, or Nacalai Tesque.
10 50-GACCAGCGGGTTCTTGCGGGAGTG-30
11 50-CGACACGGAGGGAATCGGGCTACG-30
12 50-AAGCTTAGCAACGGAGGTACGGACATGGGAGT-
CATCTTGCGCTTGC-30
13 50-TGCGGGTGGTGCGGGTCGCCCAACGATAGGAA-
TTC-30
Buffers. The following buffers were used: buffer A, 50 mM Tris–
HCl buffer, pH 7.5; buffer B, 25 mM Tris–HCl buffer, pH 7.5; and
buffer C, 20 mM sodium phosphate buffer, pH 7.0. Buffer A was
mainly used in enzyme assays. Buffer B was an elution buffer used in
chromatographic separation of the enzyme and suspension of the cells.
Buffer C was used during separation of the enzyme from the cell-free
extract on a Hi Trap Chelating HP column.
We purified Sm-AA from the cell-free extract using several
chromatographic columns. Briefly, first we disrupted the cells
suspended in buffer B using Branson Sonifier 250 (Branson Ultra-
sonics, Danbury, CT). The supernatant of the suspended cells was first
subjected to ammonium sulfate (70% saturation) precipitation, and
subsequently the enzyme was separated by DEAE Sephadex A-50
chromatography in a way similar to that used in the separation of the
enzyme from the culture supernatant, as explained above. The active
fraction obtained from the DEAE Sephadex column was loaded on a
hydroxyapatite column (30 ꢁ 1:6 cm i.d.) and eluted by changing the
sodium phosphate buffer (pH 7.5) concentration from 10 to 200 mM.
The active fraction eluted at about 50 mM sodium phosphate buffer was
further purified by Octyl Sepharose CL-4B chromatography in a way
similar to that used above. Finally, the enzyme, dissolved in buffer B
containing 1 M NaCl, was loaded on a Hi-Trap Chelating HP column
and eluted using 50 mM EDTA in buffer C to obtain the purified
enzyme.
Enzyme assays. The reaction was carried out at 37 ꢀC using 15 mM
N-acetyl-L-Met or 3 mM N-lauroyl-L-Met as the substrate in buffer A.
The L-Met produced was assayed by the ninhydrin method. One unit of
enzyme activity was defined as the amount of enzyme that produces
1 mmol of L-Met at 37 ꢀC at pH 7.5 in 1 min. Most of the data obtained
were represented as mean values of triplicate experiments. Maximum
S.D. values were 7% of the mean values.
Microorganisms, cultivation of wild-type cells, and enzyme purifi-
cation. S. mobaraensis NBRC (IFO) 13819, a type culture of the
NBRC (National Institute of Technology and Evaluation Biological
Resource Center, Chiba, Japan) was cultured using a method reported
previously.22) The culture supernatant obtained was used in the
purification of the enzyme. All the purification procedures were carried
out at 4 ꢀC. First, ammonium sulfate was added to the culture
supernatant from S. mobaraensis (1,000 ml) to attain 60% saturation in
order to precipitate the enzyme. The precipitate, which was recovered
by centrifugation at 18;000 ꢁ g for 20 min, was dissolved in 50 ml of
buffer B containing 50 mM NaCl, and was dialyzed against the same
buffer. Then the dialyzed solution was loaded on a DEAE Sephadex
A-50 column (30 ꢁ 2:6 cm i.d.) and eluted by a linear increase in the
NaCl concentration in buffer B from 50 to 500 mM at a flow rate of
0.24 ml/min. The active fractions were collected and then placed on an
Octyl Sepharose 4 Fast Flow column (34 ꢁ 1:6 cm i.d.) for elution by a
linear decrease in the NaCl concentration in buffer B from 1 to 0 M,
followed by elution with buffer B at a flow rate of 0.27 ml/min. The
active fractions were collected, concentrated, and dialyzed against
buffer A. The N-terminal amino acid sequence of the purified enzyme
was analyzed by the method reported previously using a protein
sequencer (Applied Biosystems, Model 491, Foster City, CA).25)
Cloning and sequencing of the Sm-AA gene. Total genomic DNA
was isolated from the S. mobaraensis cells by a method reported by
Kieser et al.28) The E. coli DH5ꢀ cells, which were used as a host
strain in DNA manipulation, were grown in LB medium, as described
previously.25) The scheme for the cloning of the Sm-AA gene is shown
in Fig. 1 and the primers used are listed in Table 1. To identify an
internal partial sequence of the Sm-AA gene, PCR was carried out
using genomic DNA as a template with oligonucleotides 1 and 2 as
sense and anti-sense primers respectively. Oligonucleotide 1 was
designed on part of the revealed N-terminal amino acid sequence, and
oligonucleotide 2 was designed on the internal amino acid sequence of
a
hypothetical protein from S. avermitilis (GenBank accession
no. BAB69369)—the N-terminal amino acid sequence, which, accord-
ing to a BLAST search, shows a high homology (70%) to that of
Sm-AA, as explained below. The amplified PCR product of 0.41 kbp
was purified, inserted into the pUC118 HincII/BAP vector, and used in
the transformation of the E. coli DH5ꢀ cells. The positive clones were
isolated and sequenced using an ABI Prism 310 DNA sequencer
(Perkin-Elmer, Wellesley, MA).

1942
M. KOREISHI et al.
For hybridization, 15 mg of genomic DNA isolated from S. mobar-
The cell-free extract was used in purification of the recombinant
Sm-AA. All purification procedures were carried out at 4 ꢀC. The
protein precipitate, obtained by adding ammonium sulfate at 50%
saturation, was dissolved in 30 ml of buffer B containing 1 M NaCl,
and it was then dialyzed against the same solution and subjected
to Octyl Sepharose 4 Fast Flow gel chromatography to elute the
enzyme in a way similar to that for the purification of wild-type
Sm-AA.
aensis cells was digested using MluI, BssHII, NcoI, BamHI, SalI, PstI,
SacI, KpnI, SphI, and SmaI, and was transferred to a Hybond-Nþ
membrane (Amersham Biosciences, Little Chalfont, UK). For the first
hybridization, a probe was prepared by PCR reaction with a PCR DIG
Labeling Mix (Roche Diagnostics, Basel, Switzerland) using the
plasmid containing the sequence of the 0.41-kbp PCR product as a
template and oligonucleotides 1 and 2 as the primers.
Hybridization was performed at 55 ꢀC for 4 h with the DIG-labeled
probe, and immunological detection was then performed according to
the manufacturer’s protocol (Roche Diagnostics). The 3.2-kbp SalI-
digested DNA fragments, which showed strong signal intensity, were
isolated, self-ligated, and used as a template for inverse PCR using
Characterization of Sm-AA. The enzyme properties of Sm-AA were
studied primarily using purified recombinant Sm-AA at a final
concentration of 1.5 mg/ml by a method similar to that reported in
our previous work.14) When wild-type Sm-AA was used, its final
concentration was usually 0.4 mg/ml.
oligonucleotides
3 and 4 as the sense and anti-sense primers
respectively. The obtained PCR product of 3 kbp was cloned, and its
nucleotide sequence, including the initiation codon, was determined by
the method described above. Similarly, the 1.2-kbp BssHII-digested
DNA fragments, which also showed strong signal intensity, were
isolated, self-ligated, and used as a template for inverse PCR using
The activity dependency on pH for the wild-type and recombinant
Sm-AAs was studied at 37 ꢀC using 50 mM acetate buffer (pH 4.0–6.0),
50 mM Tris–HCl buffer (pH 6.0–8.0), and 50 mM borate buffer
(pH 8.0–10.0). The optimal reaction temperature was determined by
conducting reactions for 10–20 min using purified wild-type and
recombinant Sm-AAs in buffer A containing 15 mM N-acetyl-L-Met as
the substrate in a temperature range of 16 to 70 ꢀC. The pH stability of
the recombinant enzyme was studied by incubating the recombinant
enzyme for 1 h at 37 ꢀC at various pH levels. In order to measure
thermal stability, the recombinant enzyme was incubated in buffer A
for 1 h in a temperature range of 16–70 ꢀC. To determine the effects
of the reagents on stability, the recombinant Sm-AA was incubated
in buffer A at 37 ꢀC for 15 min containing the following: 1 mM
p-chloromercuribenzoic acid (PCMB); iodoacetamide; ꢁ-mercapto-
ethanol; dithiothreitol (DTT); GSH; L-Cys; 1, 10-phenanthroline;
EDTA; and various 0.5 mM metal ions (ZnCl2, NiCl2, CoCl2, CaCl2,
CuSO4, MgSO4, FeSO4, and MnSO4). Then the remaining enzyme
activity was assayed at 37 ꢀC using 15 mM N-acetyl-L-Met as the
substrate. The effects of metal ions on the reactivation of the enzyme
were studied as follows: First the recombinant Sm-AA was dialyzed
against buffer A containing 10 mM of 1, 10-phenanthroline for 6 h at
4 ꢀC to prepare a metal-free enzyme, followed by dialysis against a
sufficiently large amount of buffer A for 36 h with five changes of the
buffer. Then the resulting metal-free enzyme was pre-incubated with
various metal ions (ZnCl2, NiCl2, CoCl2, CaCl2, CuSO4, MgSO4,
FeSO4, and MnSO4), usually at a final concentration of 0.005 mM for
1 h at 37 ꢀC, and the enzyme activity was measured by adding a final
dose of 15 mM N-acetyl-L-Met as the substrate. The metal-free enzyme
was also pre-incubated in buffer A containing final concentrations of
0.0005, 0.005, 0.05, 0.5, and 2.5 mM ZnCl2 for 1, 20, and 55 h at 37 or
4 ꢀC before the measurement of enzyme activity.
oligonucleotides
5 and 4 as the sense and anti-sense primers
respectively, and then the 0.85-kbp PCR product was obtained and
sequenced. Two primers, oligonucleotides 6 and 7, were designed by
the sequence that exists in the vicinity of the 30-end of the BssHII
fragment. These primers were used in the preparation of a DIG-labeled
probe by PCR reaction using the 0.85-kbp PCR product as a template
for the second hybridization. The 0.95-kb SmaI-digested fragments
with a strong signal intensity were separated, self-ligated, and used as a
template for inverse PCR using oligonucleotides 8 and 9 as the sense
and anti-sense primers respectively and a 0.75-kbp fragment was
amplified and sequenced. Finally, to confirm the overall ORF
sequence, the 1.4-kbp PCR product, including the full sequence of
the Sm-AA gene, was amplified using oligonucleotides 10 and 11,
which were designed by the DNA sequence of the upstream 50-end and
downstream 30-end of the Sm-AA gene. The PCR product was inserted
into pUC118 HincII/BAP, and the resulting plasmid, pUC118-SmAA,
was sequenced.
Nucleotide sequence data analysis and sequence alignment were
done using a DNASISꢁ Pro (Hitachi Software Engineering, Tokyo)
program. Sequence similarity to Sm-AA was analyzed using NCBI
BLAST for protein sequences. The signal peptide sequence was
predicted using SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/).
Construction of the expression plasmid. The Sm-AA gene was
amplified by PCR using pUC118-SmAA as a template and oligonu-
cleotides 12 and 13 as the sense and antisense primers respectively.
Oligonucleotide 12 was designed to add the HindIII site and the
ribosomal binding site to the 50-end of the Sm-AA gene. Oligonucleo-
tide 13 was designed to add the EcoRI site to the 30-end of the Sm-AA
gene. The amplified 1.4-kbp fragment was inserted into pUC118
HincII/BAP and sequenced. The 1.4-kbp fragment was inserted into
the HindIII and EcoRI sites of an expression vector, pSH19.27) The
resulting plasmid, pSH19-SmAA, was introduced into S. lividans
protoplasts, and the positive clones were selected by a method
described by Fukatsu et al.29)
The substrate specificity of Sm-AA was studied using recombinant
Sm-AA towards various 15 mM N-acetyl-amino acids, 3 mM N-lauroyl-
amino acids, and 3 mM N-saturated-acyl-L-Mets with carbon chain
lengths of C2, C4, C6, C8, C10, C12, C14, and C16.
The initial reaction kinetics for hydrolysis of N-acetyl-L-Met was
determined as follows: A 10-ml aliquot of purified recombinant Sm-AA
solution was added to 0.99 ml of a substrate solution at various final
concentrations of 2–20 mM at 37 ꢀC. At appropriate times, a 10-ml
portion of the reaction mixture was withdrawn, and the concentration
of L-Met formed was assayed as described previously to determine the
initial reaction rate.
Cultivation of recombinant cells and purification of Sm-AA. The
transformed S. lividans cells were cultivated and expressed by a
method reported by Fukatsu et al.29) Recombinant S. lividans cells
prepared as above were pre-cultured in a 100-ml shaking flask
containing 30 ml of TSB medium (Kanto Chemical, Tokyo) that was
supplemented with 20 mg/ml of thiostrepton at 30 ꢀC with shaking at
120 strokes/min for 1 d. Then 1 ml of the pre-culture was inoculated
into 50 ml of the same culture medium in two 100-ml shaking flasks,
followed by cultivation at 30 ꢀC with shaking at 120 strokes/min.
Following a 2-d cultivation, isovaleronitrile or "-caprolactam was
added to the culture medium as an inducer at final concentrations of
0.1% (v/v) and 0.1% (w/v) respectively. Then cultivation was allowed
to continue for another day, after which the cells were recovered by
centrifugation at 5;000 ꢁ g for 15 min at 4 ꢀC. Then approximately
1 g of wet S. lividans cells was washed with 10 mM Tris–HCl buffer
(pH 7.5) and suspended in 20 ml of buffer A. The cell suspension
was sonicated using an Ultrasonic Disruptor UD-201 (Tomy Seiko,
Tokyo), followed by centrifugation at 8;000 ꢁ g for 20 min at 4 ꢀC.
Analytical methods. Protein concentration was determined using
a BCA Protein Assay Reagent Kit (Pierce Chemical Company,
Rockford, IL) with bovine serum albumin (BSA) as the standard. SDS–
PAGE was performed by the Laemmli method.30) The protein bands
were stained with Quick-CBB Plus (Wako Pure Chemical Industries).
Blue Native PAGE was performed using a NativePAGEꢀ NOVEXꢁ
Bis-Tris Gel System (Invitrogen, Carlsbad, CA), according to the
manufacturer’s protocol.
Results and Discussion
Purification of wild-type Sm-AA from culture super-
natant
The culture supernatant showed enzyme activity of
approximate 8.4 U/ml in terms of N-acetyl-L-Met

New Aminoacylase from Streptomyces mobaraensis
1943
Table 2. Purification Summary of Wild-Type Aminoacylase from
S. mobaraensis
A
B
1
2
3
4
5
6
1 2 3 4 5
Total
activity
(U)
Specific
activity
(U/mg)
Protein
(mg)
Yield
(%)
Purification
(fold)
(kDa)
(kDa)
Purification step
94
67
94
67
Culture supernatant 9418
Ammonium sulfate 1037
precipitation
8429 100
4953
0.89
4.8
1.0
5.4
59
43
30
43
30
DEAE Sephadex
A-50
94.8 2507
30
27
30
Octyl Sepharose 4
Fast Flow
1.8 806
9.7 440
494
20.1
14.4
20.1
14.4
Fig. 2. Results of SDS–PAGE Analysis of the Protein Fractions
Obtained at Various Purification Steps of Wild-Type and Recombi-
nant Sm-AAs.
Cloning of the Sm-AA gene
The complete ORF of Sm-AA contained a DNA
sequence of 1,383-bp encoding 460 amino acids (data
not shown). The ORF started with an ATG codon and
terminated at the TGA codon. The nucleotide sequence
of the Sm-AA gene was submitted to the DDBJ database
(accession no. AB478961). The deduced amino acid
sequence is shown in Fig. 3. In Fig. 3, the N-terminal
amino acid sequence determined from Sm-AA purified
from the culture supernatant and cell-free extract is
located in the deduced amino acid sequence and is
double-underlined. As Fig. 3 shows, the N-terminal
peptide of Sm-AAs purified from the supernatant and
cell-free extract was truncated at the 29th position.
Signal P 3.0 analysis suggested that Sm-AA is not a
secretory protein, due to lack of the signal peptide.
These findings suggest that Sm-AA is an intracellular
enzyme. The reason that Sm-AA was secreted in the
supernatant of S. mobaraensis is not known at present.
One possible reason is that Sm-AA, an intracellular
enzyme, was leaked during cultivation in a way similar
to S. mobaraensis "-lysine acylase, as reported in our
previous paper.22,23)
The whole amino acid sequence of Sm-AA was used
to search for protein sequence homology by the BLAST
program using a Genbank database. The Sm-AA gene
sequence showed 89, 88, 67, 29, and 25% identity with
those for a hypothetical protein from Streptomyces
pristinaespiralis (Sp-CHP, Genbank accession no.
EDY63492), a putative peptidase from S. avermitilis
(Sa-PP, Genbank accession no. BAB69369), peptidase
M20 from Frankia sp. (Fs-PM, Genbank accession
no. ABD12016), succinyl-diaminopimelate desucciny-
lase from H. influenzae (SDAP, Genbank accession
no. P44514), and aminoacylase-1 from porcine kidney
(ACY1, Genbank accession no. P37111), all of which
belong to the peptidase M20 family. In Fig. 3, the
alignment of the deduced amino acid sequence of
Sm-AA with those of the enzymes belonging to the
peptidase M20 family mentioned above is shown.
Sm-AA possesses fully conserved residues of metal-
binding sites, H107, D139, E174, E201, and H435, and
catalytic sites, D109 and E173 (shaded in Fig. 3), for the
peptidase M20 family, which requires Zn2þ for the
expression of enzyme activity. In addition, the three
conserved amino acid motifs in the peptidase M20
family were also found: (i) [L, F, V]-x-[S, G, A]-H-x-D-
x-V, and (ii) [G, D]-x-x-[Y, W, F]-[G, A]-x-G-x-x-D;
and, (iii) x-E-E (boxed in Fig. 3).
A, Wild-type Sm-AA. Lanes 1 and 6, marker proteins; lane 2,
culture filtrate; lane 3, ammonium sulfate precipitate; lane 4, the
active fraction from DEAE Sephadex A-50 column chromatogra-
phy; lane 5, that from Octyl Sepharose 4 Fast Flow column
chromatography. B, Recombinant Sm-AA. Lanes 1 and 5, marker
proteins; lane 2, cell-free extract; lane 3, ammonium sulfate
precipitate; lane 4, the active fraction from Octyl Sepharose 4 Fast
Flow column chromatography. As marker proteins, phosphorylase
(94 kDa), BSA (67 kDa), ovalbumin (43 kDa), carbonic anhydrase
(30 kDa), trypsin inhibitor (20.1 kDa), and ꢀ-lactalbumin (14.4 kDa)
were used.
hydrolyzing activity. On DEAE Sephadex A-50 column
chromatography, the active fraction of wild-type Sm-AA
appeared at a NaCl concentration of 480 mM in buffer B;
Octyl Sepharose 4 Fast Flow column chromatography,
the enzyme was eluted at an NaCl concentration of
200 mM in buffer B. According to the SDS–PAGE
results for the active fraction collected from the final
column, as shown in Fig. 2A, the enzyme was homoge-
neous, with an apparent molecular mass of approxi-
mately 50 kDa. Blue Native PAGE analysis showed a
single band with an estimated molecular weight of
approximately 55 kDa (data not shown). These findings
indicate that the enzyme was monomeric. Table 2 lists a
purification summary. The enzyme was purified approx-
imately 500-fold, with a yield of 10%. The specific
activity of the purified enzyme was 440 U/mg, with
15 mM N-acetyl-L-Met dissolved in buffer A at 37 ꢀC as
the substrate. The purified enzyme also showed hydro-
lytic activity towards 3 mM N-lauroyl-L-Met, with a
specific activity of 65 U/mg, under the same conditions.
Aminoacylases that exhibit high hydrolytic activity
towards both N-acetyl-L-Met and N-lauroyl-L-Met have
not yet been reported, as far as we know, and this
motivated us to identify its gene and characteristics of
the enzyme in detail. The first 15 N-terminal amino acid
residues of the purified enzyme were determined to be
GAENEVVDICRDLIR, and then were used in cloning
the gene.
Purification of wild-type Sm-AA from the cell-free
extract
Sm-AA purified from the cell-free extract was
homogeneous on the SDS–PAGE gel (data not shown).
The N-terminal amino acid residue of Sm-AA extracted
from the SDS–PAGE gel was GAENEVVDICRDLIR,
the same as the first 15 residues of the enzyme purified
from the culture supernatant.

1944
M. KOREISHI et al.
Fig. 3. Comparison of the Amino Acid Sequence of Sm-AA with Those of Enzymes Belonging to the Peptidase M20 Family.
The enzymes shown are Sm-AA, isolated in this study, a hypothetical protein from Streptomyces pristinaespiralis (Sp-HP, Genbank
accession no. EDY63492), a putative peptidase from S. avermitilis (Sa-PP, Genbank accession no. BAB69369), peptidase M20 from Frankia sp.
(Fs-PM, Genbank accession no. ABD12016), succinyl-diaminopimelate desuccinylase from H. influenzae (SDAP, Genbank accession
no. P44514), and aminoacylase-1 from porcine kidney (ACY1, Genbank accession no. P37111). The N-terminal amino acid sequence of the
purified Sm-AA is double-underlined.
Expression of recombinant Sm-AA and purification
S. lividans cells harboring pSH19-SmAA were cul-
tured in TSB medium containing 20 mg/ml of thiostrep-
ton, and Sm-AA was expressed by the addition of either
0.1% (v/v) isovaleronitrile or 0.1% (w/v) "-caprolac-
tam, as described in ‘‘Materials and Methods.’’ No
Sm-AA activity was detected in the culture supernatant,
in contrast to that from S. mobaraensis, as mentioned
previously, and the whole enzyme was recovered from
the cell-free extract. The amounts of the recombinant
Sm-AA obtained following induction with "-caprolac-
tam and isovaleronitrile were 33 U/ml-culture and
136 U/ml-culture respectively. The Sm-AA activity
obtained with isovaleronitrile was approximately 42-fold
higher than that of the wild-type Sm-AA recovered from
the culture supernatant of S. mobaraensis 3.2 U/ml-
culture. It should be noted here that the culture super-
natant contained some other enzymes in addition to
Sm-AA that might have hydrolyzed N-acetyl-L-Met,
such as aminoacylase, reported in our previous paper.14)
Therefore, the amount of wild-type Sm-AA in the
culture supernatant discussed above was evaluated from
the N-lauroyl-L-Met hydrolyzing activity of the culture
supernatant (0.47 U/ml) with the specific activities
measured for the purified enzyme towards N-acetyl-L-
Met (440 U/mg) and N-lauroyl-L-Met (65 U/mg). No
activity was detected when wild-type S. lividans cells or
those harboring pSH19 were used. The cell-free extract
was first subjected to Octyl Sepharose 4 Fast Flow
column chromatography, in which the active enzyme
fraction was eluted near a NaCl concentration of 0 M.
Although the SDS–PAGE result appeared to indicate
that the Sm-AA fraction recovered from the Octyl
Sepharose 4 Fast Flow column was homogeneous, as
shown in Fig. 2B, detailed protein sequence analysis
later revealed that it was actually composed of a mixture
of two proteins of Sm-AA—N-terminal amino acid
residue A11 and V23—and was different from that of
the wild-type Sm-AA, G30. The reason for the differ-
ence in the length of the N-terminal peptides truncated
between purified wild-type and recombinant Sm-AAs is
not known at present and remains, as a future project.
We subjected the mixture of two recombinant Sm-AAs
to column chromatography using Macro-Prep Ceramic

New Aminoacylase from Streptomyces mobaraensis
1945
B
A
100
80
60
40
20
0
100
80
60
40
20
0
4
5
6
7
8
9
10
20
40
60
80
Temperature (°C)
pH
C
D
100
80
60
40
20
0
100
80
60
40
20
0
10 20 30 40 50 60 70
4
6
8
10
12
Temperature (°C)
pH
Fig. 4. Some Properties of Sm-AA.
A, pH Dependency of hydrolysis activity towards N-acetyl-L-Met at 37 ꢀC for recombinant Sm-AA (closed keys) and wild-type enzyme (open
keys). B, Optimal reaction temperature for hydrolysis of 15 mM N-acetyl-L-Met at pH 7.5 using recombinant Sm-AA (closed keys) and wild-type
enzyme (open keys) incubated at 37 ꢀC for 1 h. C, pH Dependency of stability for Sm-AA. D, Thermal stability of the enzyme at pH 7.5 using
recombinant Sm-AA.
Table 3. Purification Summary of Recombinant Aminoacylase from
S. lividans TK24
and B for comparison. The optimal pH for the reaction
was in a range of 7 to 8 at 37 ꢀC for both recombinant
and wild-type Sm-AAs (Fig. 4A), although the tendency
was slightly different between the two Sm-AAs in the
acidic pH region. The optimal reaction temperature
was approximately 50 ꢀC (Fig. 4B) for both Sm-AAs.
Recombinant Sm-AA was stable in a pH range of 8 to 9
at 37 ꢀC (Fig. 4C) at temperatures below 40–45 ꢀC at
pH 7.5 (Fig. 4D).
Total
activity
(U)
Specific
activity
(U/mg)
Protein
(mg)
Yield
(%)
Purification
(fold)
Purification step
Cell-free extract
142
13600 100
7088
96
129
1.0
1.3
Ammonium sulfate 55
precipitation
52
Octyl Sepharose 4
Fast Flow
4.6
2438
18
530
5.5
As shown in Table 4, the remaining activity for the
enzyme treated by 1, 10-phenanthroline was approx-
imately 10% as compared to that for the native enzyme,
suggesting that Sm-AA is a metalloenzyme. EDTA,
which is known to show weaker affinity for Zn2þ than 1,
10-phenanthroline, did not affect enzyme stability. The
effects of metal ions on the stability of the enzyme were
studied at a final concentration of 0.5 mM. Metal ions
such as Ca2þ and Mg2þ did not affect the remaining
activity, while Fe2þ decreased it by 90%. The remaining
activity of the enzyme was slightly decreased by the
addition of Zn2þ, Ni2þ, Co2þ, Cu2þ and Mn2þ, and
strongly by Fe2þ (data not shown).
hydroxyapatite Type II (Bio-Rad), then eluted it, and
this linearly increased the phosphate buffer concentra-
tion from 5 to 200 mM. The two Sm-AA enzymes,
however, eluted at the same 40 mM phosphate buffer
concentration and could not be separated. In Table 3, the
purification summary of the recombinant Sm-AA from
S. lividans is presented. The specific activities of the
purified recombinant Sm-AA towards substrates of
15 mM N-acetyl-L-Met and 3 mM N-lauroyl-L-Met were
530 and 72 U/mg respectively, and were slightly higher
than those for the purified wild-type enzyme, as
mentioned previously, 440 and 65 U/mg. The difference
in the length of the N-terminal peptides truncated
above might have caused the difference in specific
activities between the purified wild-type and recombi-
nant Sm-AAs.
The effects of metal ions on reactivation of the metal-
free enzyme were studied under various pre-incubation
conditions. Table 5 shows the effects of pre-incubation
for 1 h at 37 ꢀC in the presence of various metal ions at
a final concentration of 0.005 mM on recovery of the
activity of metal-free recombinant Sm-AA. Metal ions
such as Zn2þ and Ni2þ reactivated the enzyme to some
extent, taking the remaining activity of the enzyme
incubated in buffer A as control (100%). With increas-
ing the final metal ion concentrations to 0.5 mM, the
remaining activity was slightly decreased by pre-
Some properties of recombinant Sm-AA
Figure 4A–D shows the pH dependency of activity,
optimal reaction temperature, pH stability, and thermal
stability of the recombinant enzyme, of which the results
from the wild-type Sm-AA are also shown in Fig. 4A
incubation with Zn2þ, Ni2þ, Co2þ, Cu2þ, and Mn2þ
,

1946
M. KOREISHI et al.
Table 4. Effects of Various Reagents on the Enzyme Activity of
Recombinant Sm-AA
Table 6. Effects of Zn2þ Ion on Recovery of the Activity for Metal-
Free Recombinant Sm-AA
Reagentsa
Controlb
PCMB
Relative activity (%)
Metal ions
Relative remaining activity (%)
100
108
85
Control
2.5 mM ZnCl2
100a
39b
55b
84b
115b
120b
136c
157d
Iodoacetamide
ꢁ-Mercaptoethanol
DTT
0.5 mM ZnCl2
81
0.05 mM ZnCl2
0.005 mM ZnCl2
0.0005 mM ZnCl2
0.0005 mM ZnCl2
0.0005 mM ZnCl2
105
106
95
GSH
L-Cys
1, 10-Phenanthroline
EDTA
10
106
aThe activity of the metal-free enzyme pre-incubated in buffer A without the
addition of metal ions under the same conditions in the corresponding
experiment in the presence of ZnCl2 was taken as the control (100%).
b,c,dThe metal-free enzyme was pre-incubated in buffer A, containing
different final ZnCl2 concentrations, at 37 ꢀC for 1 h (b), 20 h (c), and 55 h
(d) before measurement of enzyme activity.
aThe concentration of the reagents at the first incubation was 1 mM.
bThe activity of the enzyme in the absence of reagents was taken to be
100%.
Table 5. Effects of Various Metal Ions on Recovery of the Activity
of Metal-Free Recombinant Sm-AA
Table 7. Substrate Specificity of Recombinant Sm-AA towards
Various N-Acetyl- and N-Lauroyl-amino Acids
Metal ionsb
Relative remaining activity (%)
Specific activity (Ua/mg)
Amino acid
Control
ZnCl2
NiCl2
100a
120
110
82
N-Acetyl-amino acidb
N-Lauroyl-amino acidc
L-Arg
L-His
L-Lys
L-Asp
L-Glu
L-Ala
L-Asn
L-Cys
L-Gln
Gly
66
59
4
6
CoCl2
CaCl2
86
93
0
14
—
—
96
6
CuSO4
MgSO4
FeSO4
MnSO4
8
96
86
0
194
88
31
412
aThe remaining activity for the metal-free enzyme pre-incubated in buffer A
without the addition of metal ions, at 37 ꢀC for 1 h was taken as the control
(100%).
bThe metal-free enzyme was pre-incubated in buffer A, containing a final
concentration of 0.005 mM metal ions at 37 ꢀC for 1 h before the measure-
ment of enzyme activity.
17
—
34
0
14
8
L-Ile
—
60
530 (173c)
L-Leu
L-Met
L-Phe
L-Pro
L-Ser
L-Trp
L-Tyr
L-Val
0
72
0
19
0
0
and strongly with Fe2þ (data not shown) as reported by
some investigators,13,31) while with decreasing metal ion
concentrations, the remaining activity of the metal-free
enzyme was increased, in particular by pre-incubation
with Zn2þ. Table 6 shows the effects of the Zn2þ
concentration on the reactivation of Sm-AA. The relative
remaining activity was decreased to 39% by incubation
with 2.5 mM Zn2þ, while the enzyme was re-activated
markedly after 20-h and 55-h pre-incubation at 4 ꢀC in
the presence of 0.0005 mM Zn2þ, indicating that Zn2þ is
a requisite metal ion for the expression of enzyme
activity. The enzyme activity was not fully restored by
the addition of Zn2þ, probably because the removal
process of Zn2þ by 1, 10-phenanthroline includes an
irreversible process32,33) although the details are not
known at present.
Table 7 summarizes the substrate specificity towards
various N-acetyl-amino acids and N-lauroyl-amino acids
using recombinant Sm-AA. Here, the substrate concen-
trations for N-acetyl-amino acids were 15 mM, while
those for N-lauroyl-amino acids were 3 mM, taking into
consideration the low solubility. The enzyme efficiently
catalyzed N-acetyl-amino acids with a preference for the
derivatives of L-Met, L-Cys, L-Ala, etc., as well as N-
lauroyl-amino acids, with a preference for the deriva-
tives of L-Ala, L-Met, L-Ser, and Gly. The specific
activities towards N-lauroyl-Gly and N-lauroyl-L-Lys
were higher than those towards N-acetyl-Gly and N-
acetyl-L-Lys. Furthermore, as shown in Table 8, Sm-AA
—
0
36
0
0
24
0
0
aOne unit was defined as the amount of enzyme required to hydrolyze
1 mmol of substrate in buffer A in 1 min at 37 ꢀC.
bSubstrate concentration, 15 mM.
cSubstrate concentration, 3 mM.
Table 8. Substrate Specificity of Recombinant Sm-AA towards
Various N-Acyl-L-methionines
Substratea
Specific activity (Ub/mg)
N-Acetyl-L-Met
173
169
175
251
89
N-Butyryl-L-Met
N-Hexanoyl-L-Met
N-Octanoyl-L-Met
N-Decanoyl-L-Met
N-Lauroyl-L-Met
N-Myristoyl-L-Met
N-Parmitoyl-L-Met
72
106
105
aSubstrate concentration, 3 mM.
bOne unit was defined as the amount of enzyme required to hydrolyze
1 mmol of the substrate in buffer A in 1 min at 37 ꢀC.
efficiently hydrolyzed 3 mM N-acyl-L-Met, which has a
carbon chain length of C2 to C16. The specific activity
for hydrolysis towards 3 mM N-acetyl-L-Met, shown in
Table 8, was approximately 1/3, that towards 15 mM
N-acetyl-L-Met, shown in Table 7. The kinetics of

New Aminoacylase from Streptomyces mobaraensis
1947
the hydrolysis of N-acetyl-L-Met was studied to make
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