Desalting column

The PLP-form of AmicoTA was obtained by incubation with an excess of both PLP and α-ketoglutarate for 1 h at 25 °C, followed by transfer into 50 mM CHES buffer, pH 9.0, using a 5 mL Desalting column (Cytiva, USA). Half-transamination reactions of the AmicoTA PLP form (35–40 µM) with D-amino acids were followed spectrophotometrically at 408 nm by measuring a decrease in the aldimine concentration in presence of different concentrations of D-amino acids in 50 mM CHES buffer, pH 9.0 at 40 °C in UV-transparent microtiter plates (UV-Star, Greiner, Germany) using SPECTROstar Omega plate reader (BMG Labtech, Germany). The rate constants of half-reactions were determined by fitting Equation (2): $$A_t=A_{\infty }+\Delta Aexp\left(-{\mathrm{k}}_{\mathrm{obs}}t\right)$$
where  $A_t$  is the absorbance at time t,  $\Delta A$  is the difference between absorbance at t = 0 and t =  $\infty$ ,  $A_{\infty }$  is the final absorbance, and  ${\mathrm{k}}_{\mathrm{obs}}$  is the observed rate constant. The dissociation constant for the enzyme–substrate complex  ${\mathrm{K}}_{\mathrm{D}}$ , the maximal rate constant  ${\mathrm{k}}_{\max }$ , the rate constant of the reverse reaction  ${\mathrm{k}}_{\mathrm{r}}$ , and the specificity constant  $\raisebox{1ex}{${\mathrm{k}}_{\max }$}\!\left/ \!\raisebox{-1ex}{${\mathrm{K}}_{\mathrm{D}}$}\right.$  were obtained by fitting Equation (3): $${\mathrm{k}}_{\mathrm{obs}}=\frac{{\mathrm{k}}_{\max }\left[\mathrm{S}\right]}{{\mathrm{K}}_{\mathrm{D}}+\left[\mathrm{S}\right]}+{\mathrm{k}}_{\mathrm{r}}$$
All measurements were performed at least in triplicates. The data were analyzed using Origin 8.0 software.

Genes were heterologously expressed in Escherichia coli BL21(DE3) harboring the plasmid pET28a. The mutants were created using the site-directed mutagenesis kit from Tiangen Biotech. The primer pairs used for site-directed mutagenesis are presented in Supplementary Table S1. E. coli BL21(DE3) was grown in lysogeny broth at 37°C with 200 rpm shaking until the optical density (OD₆₀₀) of the cultures reached 0.8. The cultures were then cooled to 16°C and isopropyl β-d-1-thiogalactopyranoside was added to a finial concentration of 0.5 mM. The cells were further incubated at 16°C with 200 rpm shaking for 20 h. The cultures were centrifuged at 12 000 g for 10 min at 4°C. The cell pellets were resuspended in 50 mM Tris–HCl buffer (pH 8.0, 150 mM NaCl). The suspension was subjected to ultrasonication to disrupt the cells. The cell lysate was then centrifuged at 12 000 g for 20 min at 4°C. The supernatants were filtered through a 0.45-μm membrane (Merck Millipore) and then loaded onto AKTA pure (Cytiva, MA, USA) coupled with a HisTrap HP column (5 ml, Cytiva). The product fraction was eluted with imidazole-containing (50–500 mM) elution buffer (10 mM Tris–HCl, 150 mM NaCl, pH 8.0) at a flow rate of 1 ml/min. The collected product fractions were further subjected to a desalting column (5 ml, Cytiva) with elution buffer to remove imidazole.

Procedures to obtain nanobody Nb6B9 were as previously reported^{19 (link)}. Briefly, expression was carried out in BL21-RIL cells grown in LB supplemented with kanamycin (50 μg mL⁻¹). Once cultures achieved an OD₆₀₀ of 0.8 at 37 °C, expression was induced posthaste by addition of 1 mM IPTG and temperature was reduced to 25 °C for 16 h with shaking at 200 rpm, after which cells were harvested by centrifugation. Cells were resuspended in 20 mM TrisHCl, pH 8.0, 150 mM NaCl, and 1 × Roche protease inhibitor cocktail, and lysed with 3 passes through an EmulsiFlex-C5 operating at ~500 bar. The lysate was clarified at 75,000 g for 20 min and sonicated on ice at 40% amplitude for a total of 2 min with pulse on and off times of 5 s. The lysate was passed over a 5 mL NiNTA column and washed with 20 mM TrisHCl, pH 8.0, 150 mM NaCl, then washed with 20 mM TrisHCl, pH 8.0, 150 mM NaCl, and 6 mM imidazole. Nb6B9 was eluted from the column with 20 mM TrisHCl, pH 8.0, 150 mM NaCl, and 250 mM imidazole. The eluted Nb6B9 was dialysed against 2 L of 50 mM sodium acetate, pH 4.8, and 75 mM NaCl, then applied to a Resource-S cation exchange column. Bound protein was eluted with a linear NaCl gradient up to 1 M. Purified Nb6B9 was then buffer exchanged into 20 mM TrisHCl, pH 8.0 and 150 mM NaCl using a desalting column (Cytiva) and concentrated to ~1.2 mM.