A Mutant Streptomycesleucineaminopeptidase with Enhanced L-Aspartyl L-Amino Acid Methylester

Supplementary Material

A mutant Streptomycesleucineaminopeptidase with enhanced L-aspartyl L-amino acid methylester synthetic activity

JiroArima*, MiraiKono, Manami Kita, and Nobuhiro Mori

Department of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture, Tottori University, Tottori

680-8553, Japan

*Corresponding author: JiroArima

E-mail:

Preparation of purified enzymes

Purified wild-type and D198KSSAPs were obtained as follows. E. coli BL21(DE3) cells harboring the expression vectorfor wild-type or mutant SSAP production were cultivated at 25°C for 18 h in 3 ml of LB medium containing 50 μg·ml-1kanamycin. 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 wasadded at a final concentration of 0.5 mM. Then cultivation was continued for another 24 h under identical conditions. Theculture was centrifuged to remove the cells, and the resultant supernatant was brought to 80% saturation with ammoniumsulfate. The precipitate, obtained by centrifugation, was dissolved in 10 mMTris–HCl containing 1 mMCaCl2 (pH 8.0). Itwas then heated at 60°C for 30 min with occasional stirring. After centrifugation to remove the precipitate, the solution wasdialyzed against 20 mMTris–HCl (pH 8.0). The dialysate was then loaded onto a Vivapure-Q spin column (Millipore2Corp.) equilibrated with the same buffer. The bound protein was eluted with 0.2 M NaCl in 20 mMTris–HCl (pH 8.0). Theeluates were pooled and dialyzed against 10 mMTris–HCl (pH 8.0). The dialysate was used as a purified enzymepreparation. The purity of recombinant enzymes was then confirmed using 12% SDS–PAGE under denaturing conditions(Laemmli1970).

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 productsusing UPLC-ESITOF MS analysis. The enzyme reaction was performed under the condition of 90% MeOH, 50 MmL-aspartate (acyl donor), 250 mM L-Phe-OMe (acyl acceptor) at 30°C for 10 min. As depicted in Supplementary Fig. 1,synthesized L-Asp-L-Phe-OMe was detected on the retention time of approximately 3.7 min. In addition toL-Asp-L-Phe-OMe, a hydrolysate of L-Phe-OMe, free L-Phe, and by-product of reverse reaction, L-Phe-L-Phe-OMe, weredetected, 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 L-Phe-OMe as substrates: A,Extracted ion chromatograms of 166.09 (L-Phe), 295.13 (L-Asp-L-Phe-OMe), and 327.17 (L-Phe-L-Phe-OMe); B, MS ofproducts. (a), MS chart of peak (a) in the extracted ion chromatograms of m/z = 166.09 (L-Phe). (b), MS chart of peak (b) inthe extracted ion chromatograms of m/z = 295.13 (L-Asp-L-Phe-OMe). (c), MS chart for peak (c) in the extracted ionchromatograms 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 themaximum reaction rate of 90% MeOH (Supplementary Fig. 2A). In contrast to the production of L-Asp-L-Phe-OMe, theproduction of the by-product, L-Phe-L-Phe-OMe, was increased following the decrease of MeOH. As depicted inSupplementary Fig. 2B, the production of L-Asp-L-Phe-OMe by wild-type SSAP was increased following the increase ofL-Phe-OMe (up to 300 mM) when the L-Asp concentration was maintained at a steady level of 50 mM. Similarly, theproduction of the by-product increased. In contrast, L-Asp-L-Phe-OMe was synthesized at L-Asp concentrations of 10–160mM 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 bywild-type SSAP were all similar to those by D198KSSAP. In almost all cases, free L-Pheexisted around 10–15 mM in thereaction mixture. However, the relation between the condition described above and liberation of L-Phe could not beascertained (data not shown).

Supplementary Fig. 2 Effects of the substrate and MeOH concentration on synthesis of L-Asp-L-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 performedat 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 ofL-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 conditionsof 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 ionintensity measured using UPLC-ESI–TOF MS. Each value is the average of three independent experiments ± the standarddeviation.

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-OMesynthesis to reaction equilibrium. The reaction was performed for 1 h. As depicted in Supplementary Fig. 3, although theconcentration of D198KSSAP (over 0.2 mg·ml-1) affected the quantity of synthesized L-Asp-L-Phe-OMe only slightly, theproduction of L-Asp-L-Phe-OMe by wild-type SSAP increased following the increase of the enzyme concentration (up to1.6 mg·ml-1). In terms of the by-product synthesis, the curves of both enzymes were almost identical, and the quantity ofL-Phe-L-Phe-OMe increased following the increase of the enzyme concentration.

Supplementary Fig. 3 Effects of enzyme concentration on synthesis of L-Asp-L-Phe-OMe by wild-type (open circles) andD198K (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-OMeand 50 mM L-Asp. The y-axis of lower panel shows the area of ion intensity measured using UPLC-ESI–TOF MS. Eachvalue 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 showsdata obtained from the LC and MS analyses.

Supplementary Fig. 4 Synthesis of various L-aspartyl L-amino acid-OMes by D198K and wild-type SSAPs. In all panels,the left shows extracted ion chromatograms of products; the right shows the MS chart for peak in the extracted ionchromatograms of product synthesized using D198KSSAP. The reaction was performed at 30°C for 2 h under thecondition of 250 mMacyl donor, 50 mM L-Asp, and 1 mg·ml-1 enzyme. L-tert-Leu-OMe, L-2-amino-3,3-dimethylbutanoicacid, L-Asp(Me)-OMe, L-aspartatedimethyl ester.

References

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophageT4. Nature227:680–685

Mishima N, Mizumoto K, Iwasaki Y, Nakano H, Yamane T (1997) Insertion of stabilizing loci in vectors of T7 RNApolymerase-mediated Escherichia coli expression systems: a case study of the plasmids involving foreignphospholipase D gene. BiotechnolProg 13:864–868