Construction of expression vectors for l-arginine oxidase mutants and transformation of E. coli

Based on the amino acid sequence of ArgOX from Pseudomonas sp. TPU 7192 (Additional file 1: Fig. S1), a sequence optimized for Escherichia coli codons was obtained through artificial gene synthesis(GenBank accession number LC771590). The synthesized genes were amplified using specific primers. DNA fragments were purified using a QIAquick Gel Extraction kit (QIAGEN, Venlo, Netherlands) and inserted into the NdeI/BamHI position at the multi-cloning site of the expression vector pET-22b ( +) (EMD Millipore Sigma, Burlington, MA, USA) using an In-Fusion HD Cloning Kit (Clontech Laboratories, Inc., Mountain View, CA, USA). Expression vectors for ArgOX mutants were generated using a KOD-Plus-Mutagenesis Kit (Toyobo, Osaka, Japan) and introduced into E. coli BL21(DE3) (Nippon Gene, Tokyo, Japan). The primer sequences for ArgOX mutants are listed in Table 1.

Table 1 List of primer sequences for ArgOX mutantsDetermination of oxidase activity for l-arginine or l-citrulline

Oxidase activity for l-arginine and l-citrulline was assayed in a reaction mixture containing 25 mM of each substrate, 0.74 mM of 4-aminoantipyrine, 15 mM of N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, 7.5 U/mL of peroxidase, 200 mM of potassium phosphate buffer (KPB) (pH 6.0), and the enzyme, in a final volume of 750 μL according to a partially modified method from a previous study (Sakaue et al. 2002). The production of H2O2 was spectrophotometrically evaluated at 37 ℃ for 3 min by measuring absorbance at 555 nm using a U-3900 spectrophotometer (Hitachi High-Tech Fielding Corporation, Tokyo, Japan). One unit of enzymatic activity was defined as the amount of enzyme that produces 1 μmol of H2O2 and 0.5 μmol of quinoneimine dye per minute at 37 ℃.

Determination of dehydrogenase activity for l-arginine or l-citrulline

Dehydrogenase activity for l-arginine and l-citrulline was assayed in a reaction mixture containing 25 mM of each substrate, 1.8 mM of 2,6-dichlorophenolindophenol (DCIP), 15 mM of phenazine methosulfate (PMS), 200 mM of KPB (pH 6.0), and the enzyme, in a final volume of 1.5 mL at 37 ℃ according to a partially modified method from a previous study (Masakari et al. 2020). The activity was calculated by monitoring the decrease in the absorbance of DCIP at 600 nm using a U-3900 spectrophotometer. One unit of enzymatic activity was defined as the amount of enzyme that caused the reduction of 1 μmol of DCIP per minute under the assay conditions. The Michaelis constant (Km) values were determined according to the above method with 0–200 mM concentrations of l-citrulline and by fitting the results to the Michaelis–Menten equation.

Enzyme production and purification

Recombinant BL21(DE3) E. coli were cultured in shake flasks containing 250 mL of ZYP-5052 medium (0.5% glycerol, 0.05% glucose, 0.2% lactose, 50 mM (NH4)2SO4, 50 mM KH2PO4, 50 mM Na2HPO4, and 1 mM MgSO4) at 30 ℃ for 24 h (Studier 2005). Cells were harvested by centrifuging at 6000 rpm at 4 ℃ for 10 min, disrupted by ultrasonication in 20 mM potassium phosphate buffer (pH 6.0), then centrifuged at 9,000 rpm for 30 min at 4 ℃. The supernatant was heated for 40 min at 60 ℃ and centrifuged at 9,000 rpm for 30 min at 4 ℃. The clear supernatant was purified via anion-exchange chromatography using a Q Sepharose Fast Flow column (Cytiva, Tokyo, Japan). The column was washed with 20 mM KPB, and the adsorbed enzyme was eluted stepwise with 20 mM KPB containing 100–500 mM NaCl. The active fraction was collected and dialyzed against 20 mM KPB (pH 7.5) using Amicon® Ultra-15 Centrifugal Filters Ultracel®—30 K (Millipore, Billerica, MA, USA). The dialyzed enzyme solution was purified via size exclusion chromatography using a HiLoad 26/600 Superdex 200 column (Cytiva, Tokyo, Japan) equilibrated with 20 mM KPB (pH 7.5) containing 150 mM NaCl. The purified enzyme solution was dialyzed against 20 mM KPB (pH 7.5).

Quantitation of l-citrulline using dehydrogenase assay

Quantitation of l-citrulline was assayed in a reaction mixture containing various concentrations of l- citrulline, 1.8 mM of DCIP, 15 mM of PMS, 200 mM of KPB (pH 6.0), and 2.7 μM of the enzyme, in a final volume of 1.5 mL at 37 ℃. The activity was calculated by monitoring the decrease in the absorbance of DCIP at 520 nm using a U-3900 spectrophotometer. One unit of enzymatic activity was defined as the amount of enzyme that caused the reduction of 1 μmol of DCIP per minute under the assay conditions.

3D model construction

A 3D structural model of ArgOX was generated via homology modeling using SWISS-MODEL [https://swissmodel.expasy.org/interactive] (Arnold et al. 2006). The crystal structure used as a template was an ancestral l-lysine oxidase K387A variant complexed with l-lysine (PDB ID:7EII). A substrate l-arginine molecule was converted from an l-lysine molecule in the resultant ArgOX homology model to avoid collision with other amino acids using the Mutagenesis Wizard of PyMOL 0.99rc6.