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Supplementary Material
A mutant Streptomyces leucine aminopeptidase with enhanced L-aspartyl L-amino acid
methyl ester synthetic activity
Jiro Arima*, Mirai Kono, Manami Kita, and Nobuhiro Mori
Department of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture,
Tottori University, Tottori
680-8553, Japan
*Corresponding author: Jiro Arima
E-mail: arima@muses.tottori-u.ac.jp
Preparation of purified enzymes
Purified wild-type and D198K SSAPs were obtained as follows. E. coli BL21(DE3) cells
harboring the expression vector for wild-type or mutant SSAP production were cultivated at
25°C for 18 h in 3 ml of LB medium containing 50 μg·ml-1 kanamycin. The cells were then
inoculated into 100 ml of synthetic medium containing 0.5% KH2PO4, 0.5% K2HPO4, 0.44%
Na2HPO4, 0.3% (NH4)2SO4, 0.5% glucose, 0.3% MgSO4·7H2O, 0.004% FeSO4·7H2O,
0.004% CaCl2, 0.00029% CoCl2·6H2O, 0.0003% CuSO4·5H2O, 0.000036% Na2MoO·2H2O,
0.001% H3BO3, 0.001% ZnSO4·7H2O, 0.2% glycerol, and 50 μg·ml-1 kanamycin (Mishima et
al. 1997), and cultivated at 22°C for 12 h. Isopropyl-β-D-thiogalactopyranoside was added at a
final concentration of 0.5 mM. Then cultivation was continued for another 24 h under identical
conditions. The culture was centrifuged to remove the cells, and the resultant supernatant was
brought to 80% saturation with ammonium sulfate. The precipitate, obtained by centrifugation,
was dissolved in 10 mM Tris–HCl containing 1 mM CaCl2 (pH 8.0). It was then heated at 60°C
for 30 min with occasional stirring. After centrifugation to remove the precipitate, the solution
was dialyzed against 20 mM Tris–HCl (pH 8.0). The dialysate was then loaded onto a VivapureQ spin column (Millipore 2 Corp.) equilibrated with the same buffer. The bound protein was
eluted with 0.2 M NaCl in 20 mM Tris–HCl (pH 8.0). The eluates were pooled and dialyzed
against 10 mM Tris–HCl (pH 8.0). The dialysate was used as a purified enzyme preparation. The
purity of recombinant enzymes was then confirmed using 12% SDS–PAGE under denaturing
conditions (Laemmli 1970).
LC/MS analysis of the products of enzyme reaction
Before evaluation of the L-Asp-L-Phe-OMe synthetic activity of the wild-type and mutant
SSAP, we analyzed the products using UPLC-ESI TOF MS analysis. The enzyme reaction was
performed under the condition of 90% MeOH, 50 Mm L-aspartate (acyl donor), 250 mM L-PheOMe (acyl acceptor) at 30°C for 10 min. As depicted in
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Supplementary Fig. 1, synthesized L-Asp-L-Phe-OMe was detected on the retention time of
approximately 3.7 min. In addition to L-Asp-L-Phe-OMe, a hydrolysate of L-Phe-OMe, free LPhe, and by-product of reverse reaction, L-Phe-L-Phe-OMe, were detected, respectively, for
retention times of approximately 1.7 and 4.9 min (Supplementary Fig. 1).
Supplementary Fig. 1 UPLC-ESI–TOF MS analysis of product synthesized with L-Asp and LPhe-OMe as substrates: A, Extracted ion chromatograms of 166.09 (L-Phe), 295.13 (L-Asp-LPhe-OMe), and 327.17 (L-Phe-L-Phe-OMe); B, MS of products. (a), MS chart of peak (a) in the
extracted ion chromatograms of m/z = 166.09 (L-Phe). (b), MS chart of peak (b) in the extracted
ion chromatograms of m/z = 295.13 (L-Asp-L-Phe-OMe). (c), MS chart for peak (c) in the
extracted ion chromatograms of m/z = 327.17 (L-Phe-L-Phe-OMe).
Effect of MeOH and substrate concentration on L-Asp-L-Phe-OMe synthesis by wild-type SSAP
The effect of the substrate and MeOH concentration on L-Asp-L-Phe-OMe synthesis by wild
type SSAP was investigated. In terms of the MeOH concentration, L-Asp-L-Phe-OMe was
synthesized at MeOH concentrations of 70–99% with the maximum reaction rate of 90% MeOH
(Supplementary Fig. 2A). In contrast to the production of L-Asp-L-Phe-OMe, the production of
the by-product, L-Phe-L-Phe-OMe, was increased following the decrease of MeOH. As depicted
in Supplementary Fig. 2B, the production of L-Asp-L-Phe-OMe by wild-type SSAP was
increased following the increase of L-Phe-OMe (up to 300 mM) when the L-Asp concentration
was maintained at a steady level of 50 mM. Similarly, the production of the by-product
increased. In contrast, L-Asp-L-Phe-OMe was synthesized at L-Asp concentrations of 10–160
mM with maximum reaction rate at 20 mM L-Asp (Supplementary Fig. 2C). In terms of the
production of the by-product, L-Phe-L-Phe-OMe was decreased following the increase of L-Asp.
The dependence curves for syntheses of all products by wild-type SSAP were all similar to those
by D198K SSAP. In almost all cases, free L-Phe existed around 10–15 mM in the reaction
mixture. However, the relation between the condition described above and liberation of L-Phe
could not be ascertained (data not shown).
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Supplementary Fig. 2 Effects of the substrate and MeOH concentration on synthesis of L-AspL-Phe-OMe (upper panel) and L-Phe-L-Phe-OMe (lower panel) by wild-type SSAP: A, Effect of
the MeOH concentration. The reaction was performed at 30°C for 10 min under conditions of
250 mM L-Phe-OMe, 50 mM L-Asp, and 0.1 mg·ml-1 enzyme. B, Effect of L-Phe-OMe
concentration. The reaction was performed at 30°C for 10 min under conditions of 90% MeOH,
50 mM L-Asp, and 0.1 mg·ml-1 enzyme. C, Effect of L-Asp concentration. The reaction was
performed at 30°C for 10 min under conditions of 90% MeOH, 250 mM L-Phe-OMe, and 0.1
mg·ml-1 enzyme. A–C, the y-axis of the lower panels show the area of ion intensity measured
using UPLC-ESI–TOF MS. Each value is the average of three independent experiments ± the
standard deviation.
Effect of Enzyme concentration on L-Asp-L-Phe-OMe synthesis by wild-type and mutant SSAP
We examined the effect of enzyme concentration on L-Asp-L-Phe-OMe synthesis to bring this
enzymatic L-Asp-L-Phe-OMe synthesis to reaction equilibrium. The reaction was performed for
1 h. As depicted in Supplementary Fig. 3, although the concentration of D198K SSAP (over 0.2
mg·ml-1) affected the quantity of synthesized L-Asp-L-Phe-OMe only slightly, the production of
L-Asp-L-Phe-OMe by wild-type SSAP increased following the increase of the enzyme
concentration (up to 1.6 mg·ml-1). In terms of the by-product synthesis, the curves of both
enzymes were almost identical, and the quantity of L-Phe-L-Phe-OMe increased following the
increase of the enzyme concentration.
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Supplementary Fig. 3 Effects of enzyme concentration on synthesis of L-Asp-L-Phe-OMe by
wild-type (open circles) and D198K (closed circles) SSAPs: Effects of enzyme concentration on
L-Asp-L-Phe-OMe (upper panel) and L-Phe-L-Phe-OMe (lower panel) synthesis. The reaction
was performed at 30°C for 1 h under conditions of 90% MeOH, 250 mM L-Phe-OMe and 50
mM L-Asp. The y-axis of lower panel shows the area of ion intensity measured using UPLCESI–TOF MS. Each value represents the average of three independent experiments ± the
standard deviation.
Synthesis of various L-aspartyl L-amino acid-OMe
The syntheses of various L-aspartyl L-amino acid-OMes using both SSAPs were examined.
Supplementary Fig. 4 shows data obtained from the LC and MS analyses.
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Supplementary Fig. 4 Synthesis of various L-aspartyl L-amino acid-OMes by D198K and wildtype SSAPs. In all panels, the left shows extracted ion chromatograms of products; the right
shows the MS chart for peak in the extracted ion chromatograms of product synthesized using
D198K SSAP. The reaction was performed at 30°C for 2 h under the condition of 250 mM acyl
donor, 50 mM L-Asp, and 1 mg·ml-1 enzyme. L-tert-Leu-OMe, L-2-amino-3,3dimethylbutanoic acid, L-Asp(Me)-OMe, L-aspartate dimethyl ester.
References
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