Purification and characterization of Stenotrophomonas maltophilia-derived l-amino acid ester hydrolase for synthesizing dipeptide, isoleucyl-tryptophan

Article abstract of DOI:10.1007/s13205-018-1195-1

In the present study, we purified α-amino acid ester hydrolase (AEH) from cell-free extracts of the Stenotrophomonas maltophilia strain HS1. The approximately 70-kDa AEH from S. maltophilia HS1 (SmAEH) was homogeneous in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analyses, and was present as a tetramer in gel-filtration experiments. The activity of the SmAEH enzyme was then determined by monitoring the synthesis of the antihypertensive agent dipeptide isoleucyl-tryptophan (Ile-Trp) from isoleucyl methyl ester (Ile-OMe) and tryptophan (Trp). In these experiments, SmAEH had wide substrate specificity for acyl donors, such as Gly-OMe, β-Ala-OMe, Pro-OMe and Trp-OMe and Ile-OMe, and maximal activity were observed under conditions of pH 9.0 and 30?°C. SmAEH also showed the greatest stability at pH 9.0, whereas its activity was reduced by 40% after 10-min incubation at approximately 50?°C. In subsequent activity assays in the presence of various metal ions, Ag⁺ strongly inhibited enzyme activity. Finally, SmAEH activity was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), suggesting that the protein is a serine protease.

Full text of DOI:10.1007/s13205-018-1195-1

3 Biotech (2018) 8:173
https://doi.org/10.1007/s13205-018-1195-1
ORIGINAL ARTICLE
Purifcation and characterization of Stenotrophomonas
maltophilia‑derived l‑amino acid ester hydrolase for synthesizing
dipeptide, isoleucyl‑tryptophan
Md Saddam Hossain1 · Takahiro Tanaka1 · Kazuyoshi Takagi2 · Junji Hayashi1 · Mamoru Wakayama1
Received: 5 May 2017 / Accepted: 3 March 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
In the present study, we purifed α-amino acid ester hydrolase (AEH) from cell-free extracts of the Stenotrophomonas
maltophilia strain HS1. The approximately 70-kDa AEH from S. maltophilia HS1 (SmAEH) was homogeneous in sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analyses, and was present as a tetramer in gel-fltration
experiments. The activity of the SmAEH enzyme was then determined by monitoring the synthesis of the antihypertensive
agent dipeptide isoleucyl-tryptophan (Ile-Trp) from isoleucyl methyl ester (Ile-OMe) and tryptophan (Trp). In these experi-
ments, SmAEH had wide substrate specifcity for acyl donors, such as Gly-OMe, β-Ala-OMe, Pro-OMe and Trp-OMe and
Ile-OMe, and maximal activity were observed under conditions of pH 9.0 and 30 °C. SmAEH also showed the greatest
stability at pH 9.0, whereas its activity was reduced by 40% after 10-min incubation at approximately 50 °C. In subsequent
activity assays in the presence of various metal ions, Ag+ strongly inhibited enzyme activity. Finally, SmAEH activity was
completely inhibited by phenylmethanesulfonyl fuoride (PMSF), suggesting that the protein is a serine protease.
Keywords α-Amino acid ester hydrolase · Stenotrophomonas maltophilia · Antihypertensive dipeptide · Ile-Trp
Introduction
Multiple methods are available for synthesising peptides,
and peptide bonds between amino acids are widely produced
using chemical and chemo-enzymatic techniques (Guzmán
et al. 2007; Kurochkina et al. 2013; Yagasaki and Hashi-
moto 2008). However, these methods require the introduc-
tion and elimination of protective groups, or the synthesis of
intermediates, and therefore, have limited industrial utility
(Yokozeki and Suzuki 2010). As alternatives, methods using
complete enzymatic reactions exploit molecular recogni-
tion between enzymes and their substrates (Gill et al. 1996;
Guzmán et al. 2007), and peptide-hydrolysing enzymes, such
as proteases and esterases, are used to trigger peptide-bond
formation between amino acids (Yagasaki and Hashimoto
2008). Peptide synthesis using proteases has been exten-
sively performed by protecting or freeing either one or both
N- and C-terminal ends of acyl donors and acceptors, respec-
tively, and peptide bonds have been formed using Nα-free
amino acid esters with Cα-protected amino acids (Gill and
Vulfson 1993; Sheldon et al. 2001), and using α-protected
amino acid esters and Nα with Cα-free amino acids (Hansler
and Jakubke 1996). However, proteases fail to meet indus-
trial requirements of productivity, reaction rates, yields, pro-
portions of acyl acceptors to acyl donors, and numbers of
Peptides are important sources of nutrition, and are increas-
ingly considered for biomedical applications (Santos et al.
2012). Peptides are used as pharmaceuticals, favour-active
ingredients in natural health products, nutraceuticals, and
functional foods (Mine et al. 2011; Santos et al. 2012), and
dipeptides and tripeptides attract considerable attention
because of their cost efectiveness and potential oral activ-
ity, and because they are easily characterised in structural,
molecular and quantitative functional studies (Kurochkina
et al. 2013).
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s13205-018-1195-1) contains
supplementary material, which is available to authorized users.
* Mamoru Wakayama
wakayama@sk.ritsumei.ac.jp
1
2
Department of Biotechnology, College of Life Sciences,
Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
Department of Applied Chemistry, College of Life Sciences,
Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
Vol.:(0123456789)
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3 Biotech (2018) 8:173
peptides that can be synthesised using this approach. In addi-
tion, these methods are hampered by temporary accumula-
tion of dipeptides and degradation of dipeptides by proteases
(Yagasaki and Hashimoto 2008). In contrast, α-amino acid
ester hydrolase (AEH) can be used to synthesise dipeptides
efciently and irreversibly using Nα-free amino acid ester
as an acyl donor and Nα- and Cα-free amino acids as acyl
acceptors (Barends et al. 2003; Yokozeki and Hara 2005).
Several studies report the use of AEH to synthesise
dipeptides (Abe et al. 2011; Hirao et al. 2013; Sugihara
et al. 2001; Yokozeki and Hara 2005). Herein, we identifed
microorganisms that produced AEH efciently for use in
the synthesis of the dipeptide isoleucyl-tryptophan (Ile-Trp),
which is an important antihypertensive agent. Recently, the
antihypertensive efects of dipeptides have attracted con-
siderable attention (Sato et al. 2002), and hydrolysates of
fsh meat, seaweed and mushrooms have been shown to
lower blood pressure. Among active components of pro-
tein hydrolysates, the dipeptides Ile-Trp, Ile-Tyr, Lys-Trp
and Val-Tyr have been identifed (Yagasaki and Hashimoto
2008), and their antihypertensive efects follow inhibition
of angiotensin-I-converting enzyme (ACE), which regulates
blood pressure (Kitts and Weiler 2003; Sato et al. 2002).
Extracts and hydrolysates containing these dipeptides have
been approved as foods for specifed health uses in Japan
(Yagasaki and Hashimoto 2008), and represent healthier
natural alternatives to ACE inhibitor drugs. In particular,
the potent ACE-inhibitory activities of Ile-Trp have been
studied comprehensively (Ono et al. 2006; Enari et al. 2008),
and when administered to spontaneously hypertensive rats
at its IC50 value of 4.7 µM, Ile-Trp exerted antihypertensive
activities within 2 h (Iroyukifujita et al. 2000). However,
methods for the synthesis of Ile-Trp have not been reported
previously. Moreover, few enzymatically produced dipep-
tides are currently available due to the lack of cost-efective
manufacturing processes, and no standard protocols have
been established for scaling up and optimising dipeptide
production (Yagasaki and Hashimoto 2008). Because ef-
cient dipeptide production will facilitate the identifcation of
novel applications, we optimised the production, purifcation
and characterisation of AEH from the bacterial strain Steno-
trophomonas maltophilia HS1.
Blue (CBB) were purchased from Nacalai Tesque (Kyoto,
Japan). DEAE-Cellufne and Butyl-Cellufne columns were
purchased from JNC (Tokyo, Japan), and Superdex 200 HR
10/30 columns were obtained from GE Healthcare (Tokyo,
Japan). All other reagents were of analytical grade.
Screening of AEH‑producing bacterial strains
Samples of soil were collected from a hill-tract forest area
in Shiga prefecture, Japan. Briefy, one loop of soil was sus-
pended in 1 mL of sterile water. Subsequently, one loop of
the resulting soil suspension was then transferred to 1 mL of
liquid medium 1 containing 0.1% KH2PO4, 0.1% K2HPO4,
0.05% MgSO4·7H2O, and 0.2% L-Ile-OMe (pH 7.0) and was
incubated at 30 °C for 48–72 h with shaking at 165 rpm.
The mixture was then suspended in 1 mL of fresh liquid
medium 1 and was incubated as above, and pure colonies
were isolated after loop streaking the resulting suspension
onto agar plates containing 0.5% glucose, 0.5% polypeptone,
0.5% yeast extract, 0.1% MgSO4·7H2O and 1.5% agar (pH
7.0) and incubating at 30 °C for 16–20 h. One loop of pure
colony was then transferred to 5 mL of liquid medium 2
containing 0.5% glucose, 0.5% yeast extract, 0.5% polypep-
tone, 0.2% (NH4)2SO4, 0.05% KH2PO4, 0.15% K2HPO4 and
0.05% MgSO4·7H2O (pH 7.0) and was incubated at 30 °C
at 250 rpm for 20–22 h. The resulting culture was then cen-
trifuged at 10,000g for 2 min, and intact cells were mixed
with reaction mixture containing 50-mM L-Ile-OMe (pH
7.0), 100-mM L-Trp (pH 9.0) and 100-mM Tris–HCl bufer
(pH 9.0). After 1 h at 30 °C, synthesised Ile-Trp was quan-
titatively analysed as described below.
Identifcation of isolates and production of SmAEH
Isolates were identifed using 16S ribosomal RNA sequenc-
ing and phylogenetic analyses were performed using mul-
tiple sequence alignments in the MUSCLE server (Edgar
2004). A phylogenetic tree was created and visualised using
the Wagner parsimony method in the PHYlip package
(Felsenstein 1989), and all tools were used in their default
settings. To produce SmAEH, seed cultures were prepared
by incubating freshly isolated colonies in 5-mL aliquots of
liquid medium 2 at 30 °C with shaking at 250 rpm for 20 h.
Seed cultures were then inoculated into 500 mL of the same
media in a 2-L fask, and were incubated at 30 °C with shak-
ing at 100 rpm for 20 h. Cells were then harvested by cen-
trifugation at 10,000g for 20 min, were washed two times
and dissolved in 50-mM Tris–HCl bufer (pH 8.0), and were
then treated with lysozyme (0.5 mg/mL) at 4 °C for 30 min.
Sonication was performed with optimised parameters, and
cell-free extracts with AEH activity were obtained by cen-
trifugation at 12,000g for 30 min followed by removal of
cell debris.
Materials and methods
Reagents
l-Isoleucine methyl ester (L-Ile-OMe) and l-tryptophan
(L-Trp) were purchased from Wako Pure Chemicals (Tokyo,
Japan). Isoleucyl-tryptophan (Ile-Trp) was obtained from
Watanabe Chemical Industry (Hiroshima, Japan) and acetic
acid, acetonitrile, tetrahydrofuran and Coomassie Brilliant
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3 Biotech (2018) 8:173
Page 3 of 8 173
Diferences were identifed using Student’s t test and were
considered signifcant when P<0.05.
Purifcation of SmAEH
All SmAEH purification steps were performed at 4 °C
unless otherwise stated. Cell-free extracts were loaded onto
a DEAE-Cellufne column that was pre-equilibrated with
20-mM Tris–HCl bufer (pH 8.0) after dialysing against
20-mM Tris–HCl bufer (pH 8.0). Prior to elution, nonbind-
ing proteins were washed from the column using equilibra-
tion bufer, and bound proteins were eluted stepwise using
0–0.3-M NaCl in the same bufer at a fow rate of 1 mL/min.
Active fractions were then collected and dialysed against
20-mM Tris–HCl bufer (pH 8.0) and were assayed for AEH
activity. Prior to Butyl-Cellufne chromatography, enzymes
and columns were equilibrated with 20-mM Tris–HCl
bufer (pH 8.0) containing 2-M ammonium sulphate, and
the enzyme was then eluted with a linear gradient of 2–0 M
ammonium sulphate. Active fractions were then collected
and dialysed against 20-mM Tris–HCl bufer (pH 8.0) and
the enzyme was then applied to Superdex 200 HR 10/30
gel-fltration columns in 20-mM Tris–HCl bufer contain-
ing 150-mM NaCl. Active fractions were then collected and
dialysed against the same bufer, and enzyme samples were
concentrated by ultrafltration and were stored at 4 °C.
Characterisation of SmAEH
The thermal stability of SmAEH was determined after expo-
sure to 20–55 °C for 10 min followed by cooling for 2 min
on ice. Enzyme reactions were then allowed to proceed for
1 h. The efects of varying pH on AEH activity were deter-
mined after adjusting pH to between pH 7.0–10.5 using
bufer and allowing reactions to proceed at 30 °C for 1 h. In
further experiments, pH stability of AEH was determined by
incubating 100 µL aliquots of enzyme for 12 h with 100 µL
aliquots of various bufers with a pH range of 4.0–10.0 at
4 °C. Reactions were then performed at 30 °C for 1 h, and
residual enzyme activity was determined as described above.
To investigate the acyl donor specifcity of SmAEH,
hydrolysis reactions were performed with various amino
acid methyl ester substrates and hydrolytic activities of
SmAEH were assayed according to methanol release from
acyl donors at 30 °C for 1 h. In separate experiments, AEH
activity was assayed spectrophotometrically using assay
mixtures comprising 0.25 U of alcohol oxidase and 1 U of
horseradish peroxidase in 100-mM potassium phosphate
buffer (pH 7.0). Subsequently, SmAEH reaction solu-
tion (10 µL) was added to the assay mixture, was supple-
mented with 1-mM o-phenylenediamine and was incubated
at 30 °C for 30 min. These analyses showed that alcohol
oxidase catalyses the oxidation of methanol to the corre-
sponding carbonyl compound formaldehyde and hydrogen
peroxide. Finally, o-phenylenediamine signals were ampli-
fed by hydrogen peroxide and horseradish peroxidase, and
concentrations of the resulting intermediate were determined
at 420 nm.
Analysis
Protein concentrations were determined using the Lowry
method with egg albumin as a standard (Lowry et al. 1951).
The molecular mass of denatured enzyme was estimated
using SDS-PAGE and gels were stained with Coomassie
Brilliant Blue. Synthesised Ile-Trp in reaction mixtures was
quantitatively measured and monitored using high-perfor-
mance liquid chromatography (HPLC) with a COSMOSIL
5C18-MS-II column (4.6×250 mm; Nacalai Tesque, Inc.,
Kyoto, Japan) equilibrated with 0.1-M acetate bufer (pH
6.0) containing 7% (v/v) acetonitrile and 3% (v/v) tetrahy-
drofuran. The derivative Ile-Trp was eluted using a linear
gradient of acetonitrile (7–47%) at a fow rate of 1 mL/
min. Before analysis, samples (5 µL) were quenched and
diluted 100-fold by the addition of derivatized fuorescence
compound using 10-mM Boc-Cys-OH, 20-mM phthalalde-
hyde in 400-mM borate bufer (pH 9.0) and 5-µL aliquots
of sample mixtures were then applied to HPLC columns.
Standard reaction mixtures for Ile-Trp synthesis contained
100-mM Tris–HCl bufer (pH 9.0), 50-mM L-Ile-OMe, 100-
mM Trp (pH 9.0) and enzyme in a fnal volume of 0.1 mL.
After 1 h at 30 °C, reactions were terminated by boiling for
10 min and one unit of enzyme activity was defned as the
amount of enzyme required to produce 1 μmol of Ile-Trp per
min under standard assay conditions. Data were presented
as means standard errors of the mean of at least three
independent experiments, or as noted in the fgure legends.
Results
Screening and identifcation of AEH‑expressing
bacterial isolates
A total of 200 soil samples were screened according to rates
of Ile-Trp synthesis from Ile-OMe and Trp. From these anal-
yses, ten microbial strains were selected as Ile-Trp producers
and four strains with higher Ile-Trp-synthetic activity were
identifed using 16S ribosomal RNA sequencing. S. malt-
ophilia HS1 was a remarkably efcient producer of Ile-Trp
(2 mM) under the present screening conditions (Supplemen-
tary data Table 1), and was selected for further experiments.
Phylogenetic analyses placed S. maltophilia HS1 in distinct
position on the tree, although sequence identity with relevant
strains was more than 96% (Supplementary data Fig. 1).
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3 Biotech (2018) 8:173
Optimisation of culture conditions for SmAEH
production
Purifcation of SmAEH
Enzymes were purifed using DEAE-Cellufne and Butyl-
Cellufne chromatography columns followed by gel fltration.
During these purifcation steps, specifc activity increased
and fnally reached approximately 5.34 U/mg at a purity of
116 fold (Table 2). Although the enzyme yield was 1.90%,
the purifed enzyme was homogeneous in SDS-PAGE analy-
ses (Fig. 1), as indicated by a single band of 70 kDa under
denaturing conditions. In addition, the molecular mass of the
purifed enzyme was approximately 300 kDa in gel-fltration
analyses, suggesting that the enzyme is active as a tetramer.
The effects of various carbon and nitrogen sources on
SmAEH production were examined (Table 1), and the
highest specific activity (approximately 0.12 U/mg) was
observed in medium-containing glucose, casamino acid
and yeast extract. Conversely, media with casamino acid
led to the lowest specific activity (0.007 U/mg). No activ-
ity or growth was observed in media supplemented with
glucose–(NH4)2SO4, glucose–NH4Cl and glucose–urea.
The cultivation period for optimal production of AEH
by S. maltophilia HS1 was 20 h (Supplementary data
Fig. 2) and production was highest during the exponen-
tial cell growth phase. Moreover, at 22 h, SmAEH pro-
ductivity was decreased and after 26 h, enzyme activity
was greatly decreased, although cell numbers continued
to increase.
Characterisation of SmAEH
We determined optimal physicochemical conditions for
purifed enzyme activity, and showed that SmAEH is most
active at 30 °C. Our data also show that the enzyme is sta-
ble for 10 min between 20 and 35 °C (Fig. 2a), and 60% of
enzyme activity remained after 10-min incubation at 50 °C.
Table 1 Efects of carbon
C source
N source
Total activity (U) Total protein (mg) Specifc
and nitrogen sources on AEH
productivity
activity (U/
mg)
d-Glucose
d-Glucose
d-Glucose
d-Glucose
d-Glucose
d-Glucose
d-Glucose
Glycerol
Polypeptone Yeast extract
Polypeptone (NH4)2SO4
Polypeptone Casamino acid
–
–
Yeast extract
Polypeptone
(NH4)2SO4
NH4Cl
Urea
Casamino acid
0.281
0.121
3.31
3
No growth
No growth
0.084
0.040
No growth
0.234
3.2
3.21
3
3.3
2.9
3.15
1.1
2
0.07
0.12
0.11
0.017
0.057
0.072
0.007
0.012
Casamino acid +0.05% yeast extract 0.395
Casamino acid
0.347
0.059
0.166
0.229
0.008
Casamino acid
Casamino acid +0.05% yeast extract 0.025
Cells were grown in a test tube containing 5-mL culture medium on a reciprocal shaker at 30 °C for 24 h.
Concentration of carbon and nitrogen sources was 0.5%. However, the composition and amount of salt
in each medium remained constant, and only the efects of carbon and nitrogen in AEH production were
tested. The resulting culture was harvested and activity data were determined. Experiments were performed
in duplicate
Table 2 Steps of purifcation
Steps
Total Pro- Total activity (U) Specifc
Yield (%) Purifcation fold
tein (mg)
activity (U/
mg)
Cell-free extract
DEAE-cellufne (pH 8.0) 145
Butyl-cellufne (pH 8.0)
Gel fltration
1804
84.1
51.75
7
0.046
0.35
2.5
100
61
8.32
1.90
–
7.60
54.34
116.08
2.8
0.3
1.6
5.34
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3 Biotech (2018) 8:173
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(a) 120
(KDa)
204
M
1
2
3
4
100
80
60
40
20
0
114
84
62
46
~ 70 KDa
20
25
30
35
40
45
50
55
60
Temperature (°C)
27
120
(b)
100
80
60
40
20
0
Fig. 1 Sodium dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS-PAGE) analyses of SmAEH. Cell-free extracts from S. malt-
ophilia HS1 (lane 1), DEAE-Cellufne (lane 2), Butyl-Cellufne (lane
3) and gel fltration (lane 4) are shown. The SmAEH band is indi-
cated. Molecular mass markers were loaded in lanes labelled M, and
their masses (KDa) are shown on the left
However, enzyme activity was decreased to approximately
75% after 10-min treatment at 55 °C.
4
5
6
7
8
9
10
pH
SmAEH was highly active in the pH range of 7.0–10.0,
and was most active at pH 9.0 (Fig. 2b). The enzyme was
also stable at pH between 5.0 and 10.0, although approxi-
mately 25 and 10% decreases in enzyme activity were
observed following treated at pH 4.0 and 10.0, respectively.
Substrate specifcity for acyl donors was investigated
using a colorimetric method with 10 difering l-amino acid
esters. In these experiments, all tested acyl donors were
rapidly hydrolysed by SmAEH and the enzyme accepted a
wide range of amino acid methyl ester substrates, including
β-Ala-OMe (Fig. 3).
Fig. 2 Temperature stability and efects of pH on SmAEH enzyme
activity and stability. a Temperature stability of SmAEH; b efects of
pH on SmAEH activity and stability; reactions were adjusted to vari-
ous pH values using potassium phosphate bufer (pH 7.0–8.0; open
diamond), Tris–HCl bufer (pH 8.0–9.5; open square) and borate
bufer (pH 9.0–10.5; open circle). SmAEH stability was also exam-
ined in the presence of citrate bufer (pH 4.0–5.5; closed triangle),
potassium phosphate bufer (pH 6.0–7.0; closed diamond), Tris–HCl
bufer (pH 7.0–9.0; closed square) and borate bufer (pH 9.0–10.0;
closed circle)
To investigate the efects of metal ions on enzyme activi-
ties, reactions were performed in the presence of various
metal ions. These activity assays showed little preference of
SmAEH for specifc metals (Fig. 4), although Ag+ abolished
SmAEH activity completely and Na+ slightly enhanced
SmAEH activity. Finally, SmAEH activity was completely
abolished after 1-h treatment with 1-mM PMSF (Fig. 4).
250
225
200
175
150
125
100
75
50
25
0
Analysis of the reaction products
In HPLC analyses of SmAEH reaction mixtures (Fig. 5),
products were formed in a time-dependent manner. In par-
ticular, the Ile-Trp (peak no. 3) was detected in 30-min reac-
tions and increased further after 1-h reactions. Moreover,
in comparisons with standards and zero-min HPLC peaks,
time-dependent reaction peak profles showed a new peak
with a retention time of 34 min, presumably refecting the
presence of Il-Trp at this time point. After 1 h reactions,
Acyl donors
Fig. 3 Substrate specifcity of SmAEH in hydrolysis reactions. Acyl
donor specifcity; hydrolysis on SmAEH was performed in 100-µL
aliquots of reaction bufer containing 50-mM acyl donor and 100-mM
Tris–HCl bufer (pH 9.0) at 30 °C for 1 h. Reaction mixtures without
enzyme were used as controls
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3 Biotech (2018) 8:173
Fig. 4 Efects of metal ions and
phenylmethane sulfonyl fuoride
(PMSF) on SmAEH activity.
Reactions were performed
140
120
100
80
separately with 50-mM Ile-
OMe, 100-mM Trp, individual
metal ions at 5 mM and PMSF
at 1 mM in Tris–HCl bufer (pH
9.0) at 30 °C for 1 h. Rela-
60
tive activity of SmAEH was
then determined and data were
presented as means standard
errors of the mean from at least
two independent experiments
40
20
0
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Fig. 5 High-performance liquid chromatography (HPLC) profles of
SmAEH reactions over time. HPLC analyses of the reaction prod-
ucts were classifed in the following four sections: a standard, b
0-min reactions, c 30-min reactions, and d 1-h reactions. Peaks 1, 2
and 3 correspond with Ile-OMe, Trp and Ile-Trp, respectively. Stand-
ard concentrations for Ile-OMe, Trp and Ile-Trp were 50, 100 and
10 mM, respectively
product concentrations reached approximately 2 mM, indi-
cating that SmAEH produces Ile-Trp.
isolated and characterised AEH from Xanthomonas citri and
demonstrated hydrolysis reactions of l-amino acid ester, but
could show no caseinolytic activity (Kato et al. 1980).
In the present study, we isolated S. maltophilia HS1 by
screening AEH activities in more than 200 soil samples.
We also found AEH activity in Pseudomonas spp. (Sup-
plementary data Table 1), and previous studies show
AEH activity in the bacterial strains Empedobacter brevis
(Yokozeki and Hara 2005), A. turbidans (Polderman-Tijmes
et al. 2002), Bacillus mycoides (Sugihara et al. 2001) and
Sphingobacterium siyangensis (Abe et al. 2011), and in yeast
Discussion
AEH was frst discovered by Takahashi et al. in Acetobacter
turbidans (Takahashi et al. 1974) and was shown to catalyse
amino-acyl transfer from α-amino acid esters to 7-amino-
3-deacetoxy-cephalosporanic acid, along with the hydroly-
sis of corresponding α-amino acid esters. Later, Kato et al.
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3 Biotech (2018) 8:173
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Saccharomyces spp. (Yokozeki and Hara 2005). Biochemi-
cal, physicochemical and catalytic properties of AEHs have
been studied previously and by 2013, dipeptide-synthetic
activities were documented in ten wild-type and mutant
strains, which are known to express AEHs comprising mul-
tiple subunits (Kurochkina et al. 2013). The present data
show that AEH from S. maltophilia is a tetramer, whereas
other studies show that AEH from Pseudomonas melano-
genum comprises two identical subunits of 72 kDa (Kim
and Byun 1990). Enzymes from the Xanthomonas family
comprise four identical subunits of 70–72 kDa, with total
molecular weights of 260–280 kDa (Kato et al. 1980). How-
ever, in A. turbidans, AEH reportedly comprises a tetramer
of two 70-kDa subunits and two 72-kDa subunits, with a
total molecular weight of 280–290 kDa (Ryu and Ryu 1988).
In contrast, more recent data suggest that purifed native
AEH from A. turbidans is a multimer of 70-kDa subunits,
and that both dimers and tetramers have enzymatic activity
(Polderman-Tijmes et al. 2002). These AEHs have been con-
sidered for industrial enzyme production of semi-synthetic
β-lactam antibiotics (Barends et al. 2003; Polderman-Tijmes
et al. 2002), but were not used for peptide synthesis until
2001 (Sugihara et al. 2001), refecting the lack of data con-
frming that the enzyme synthesises and hydrolyses peptides.
Accordingly, Sugihara et al. was the frst to demonstrate that
AEH from Bacillus mycoides catalyses the formation of
dipeptides (Sugihara et al. 2001). Among other enzymatic
methods for synthesising dipeptides, l-amino acid ligase
(Lal)-catalysed synthesis of dipeptides leads to the forma-
tion of unprotected l-amino acids in an ATP-dependent
manner, and several Lals have been used for the synthesis
of dipeptides such as Ala-Gln, Phe-Ala, Met-Gly and Leu-
Gly (Kino et al. 2008a, b; Tabata et al. 2005). However,
Lals sufer from low accumulation and productivity of the
target dipeptide (Yagasaki and Hashimoto 2008), refecting
the technological nascence of dipeptide formulations. How-
ever, Ile-Trp potently inhibits ACE activity, reduces matrix
metalloproteinase-2 activity and improves coronary fow
reserves (Sato et al. 2002; Li et al. 2004; Murray and Fitz
Gerald 2007), and could be administered as a food additive.
Because Ile-Trp is an important ACE inhibitor with
applications as a drug pressure lowering drug, we investi-
gated the synthesis of Ile-Trp by AEH from S. maltophilia.
Subsequently, we characterised the enzyme for industrial
application. AEHs from diferent microbial sources show
similar biochemical, physicochemical and catalytic prop-
erties (Kurochkina et al. 2013), and the present data show
that SmAEH has high activity under conditions of pH 9.0 at
30 °C. Although AEH from S. siyangensis showed maximal
activity at pH 8.5 and 25 °C (Hirao et al. 2013), optimal
pH and temperature values may difer between substrates
(Malathi and Chakraborty 1991). We also examined pH
and temperature stability of the enzyme and found that
SmAEH activity decreases dramatically after exposure to
temperatures above 50 °C, whereas the enzyme remained
stable between pH 5.0 and 10.0. In agreement, Kato et al.
isolated AEH from X. citri and showed complete inactiva-
tion after incubation at 50 °C for 15 min. These authors also
report a broad range of pH stability from 4.0 to 9.0 (Kato
1980). Finally, because serine residues play active roles in
the catalytic activities of α-β hydrolases (Ollis et al. 1992),
we performed experiments with the serine protease inhibitor
PMSF and confrmed that serine residues are critical to the
activity of SmAEH. Similarly, Sugihara et al. showed that
PMSF strongly inhibits AEH from B. mycoides (Sugihara
et al. 2001).
Colorimetric assays of acyl donor hydrolysis by SmAEH
showed relatively high substrate preference for non-bulky
amino acid methyl esters, and Ala-OMe was preferred over
the other substrates. However, further studies with standard
dipeptide stocks are required to confrm the preference of
SmAEH for acceptor substrates. Such studies of acceptor
preferences are of high priority, particularly because these
will inform the synthesis of other biologically important
dipeptide derivatives.
The present findings indicate noteworthy utility of
SmAEH for the synthesis of Ile-Trp and suggest that other
dipeptide derivatives may be biologically signifcant. How-
ever, enzyme production and dipeptide yields were inad-
equate under the present conditions. Hence, future studies
are required to clone the SmAEH gene for the production of
dipeptides in large scale with minimum production costs
and high yields. These aims will be facilitated by the cur-
rent development of an expression system for recombinant
SmAEH in Escherichia coli.
Compliance with ethical standards
Conflict of interest The authors declare that there is no confict of in-
terests.
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