Development of a rapid and simple glycine analysis method using a stable glycine oxidase mutant

Analytical Biochemistry 587 (2019) 113447

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Development of a rapid and simple glycine analysis method using a stable glycine oxidase mutant

T

Moemi Tatsumia, Wataru Hoshinoa, Yuya Kodamaa, Techawaree Ueatrongchitb, Kazutoshi Takahashia, Hiroki Yamaguchia, Uno Tagamia, Hiroshi Miyanoa, Yasuhisa Asanob, Toshimi Mizukoshia,∗ a b

Institute for Innovation, Ajinomoto Co., Inc., Kawasaki, Kanagawa, 210-8681, Japan Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan

A R T I C LE I N FO

A B S T R A C T

Keywords: Enzymatic analysis Glycine oxidase Human plasma analysis Amino acid measurement Protein engineering

Glycine analysis is important in research fields such as physiology and healthcare because the concentration of glycine in human plasma has been reported to change with various disorders. Glycine oxidase from Bacillus subtilis (GlyOX) is useful for quantitative analysis of glycine. However, GlyOX is not sufficiently stable for use in physiology-based research or clinical settings. In this report, site-directed mutagenesis was used to engineer a GlyOX mutant suitable for glycine analysis. The GlyOX triple-mutant (T42 A/C245 S/L301V) retained most of its enzymatic activity during storage for over a year at 4 °C. A colorimetric enzyme analysis protocol was established using the GlyOX triple-mutant to determine glycine concentrations in human plasma. The analysis showed high accuracy (−5.4 to 3.5% relative errors when compared with the results from an amino acid analyzer, and 96.0–98.7% recoveries) and high precision (< 4% between-run variation). Sample pretreatments of deproteinization and derivatization were not required. Therefore, this novel enzymatic analysis offers an effective and useful method for determining glycine concentrations in physiology related research and the healthcare field.

1. Introduction Amino acids are essential components in the body and their quantitation is important in various fields such as physiology research and healthcare. In particular, quantitation of amino acids has recently been the focus of attention because amino acid imbalances are associated with various human disorders and measuring their concentrations is a useful biomarker. For example, the molar ratio of plasma free branchedchain amino acids (BCAA: valine, leucine and isoleucine) to aromatic amino acids (phenylalanine and tyrosine, Fischer's ratio) or tyrosine (BTR ratio) is important for assessing liver function [1–3]. Quantitation of phenylalanine, leucine and methionine in blood are well-established methods for diagnosing inborn error of amino acid metabolism, e.g., phenylketonuria, maple syrup urine disease and homocystinuria [4–8]. Moreover, the concentrations of amino acids in blood are related to certain cancers and inflammatory bowel disease [9,10]. Thus, accurate and simple measurement of amino acids is important for diagnosing

disease states. Glycine is the only achiral natural α-amino acid and an essential component of proteins and neurotransmission in the body [11]. Ingestion of glycine improves sleep quality [12]. The concentration of glycine has been reported to fluctuate with various disorders. Patients with glycine encephalopathy, an autosomal recessive disorder, have increases in cerebrospinal fluid and plasma glycine levels [13]. The fluctuation of glycine concentrations in plasma have also been linked to schizophrenia, gout, obesity and diabetes [14–16]. Currently HPLC and LC-MS methods are used to quantify glycine concentrations [17]. These instrument-based methods quantify the glycine concentration accurately and precisely. However, these methods require expensive equipment, skilled operation and complicated sample pretreatments such as deproteinization and derivatization. Additionally, these methods are time consuming when analyzing a large number of samples. In contrast, enzymatic analysis is simple and can rapidly analyze

Abbreviations: GlyOX, glycine oxidase; BCAA, branched-chain amino acids; BTR, BCAA/tyrosine molar ratio; HPLC, high-performance liquid chromatography; LCMS, liquid chromatograph - mass spectrometer; FAD, flavin adenine dinucleotide; PCR, polymerase chain reaction; WT, wild-type; LB, Luria-Bertani; Tris, tris (hydroxymethyl)aminomethane; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; TOOS, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline; sodium salt, dehydrate; CV, coefficient of variation; PDB, Protein Data Bank ∗ Corresponding author. Institute for Innovation, Ajinomoto Co., Inc., Kawasaki, Kanagawa, 210-8681, Japan. E-mail address: [email protected] (T. Mizukoshi). https://doi.org/10.1016/j.ab.2019.113447 Received 29 January 2019; Received in revised form 6 September 2019; Accepted 19 September 2019 Available online 25 September 2019 0003-2697/ © 2019 Elsevier Inc. All rights reserved.

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taattccatggctaaaaggcattatgaagcagtggtgattg-3′) and the antisense primer (5′-taatactcgagtatctgaaccgcctccttgcgatc-3′) were used. The amplified PCR product and the pET-28a-vector (Merck, Darmstadt, Germany) were digested with NcoI and XhoI (Takara Bio). Each digested product was purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and ligated using Ligation high Ver.2 (Toyobo, Osaka, Japan) to obtain pET28a-GlyOX (WT). GlyOX mutants were created by sitedirected mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA).

multiple samples simultaneously. Various enzymes for analyzing amino acids have been reported [8,18]. In regards to enzymatic method for glycine, application of a glycine biosensor that uses a glycine oxidase (GlyOX) variant from Bacillus subtilis and detects glycine by a fluorescence response has been reported [19]. GlyOX (EC 1.4.3.19) is a flavoprotein that is a homotetramer of 180 kDa and contains one molecule of non-covalently bound FAD per monomer [19–21]. Crystal structures of GlyOX have been reported and display structural similarity with monomeric sarcosine oxidase and D-amino acid oxidase [22–24]. GlyOX is essential for the biosynthesis of the thiazole moiety of thiamine pyrophosphate [22] and catalyzes the oxidative deamination of various primary and secondary amines (such as glycine, sarcosine and Nethylglycine) and D-amino acids (such as D-alanine and D-proline) to form the corresponding α-keto acid and hydrogen peroxide [20,25]. The application of GlyOX has been explored, e.g., agricultural biotechnology [24]. However, long-term storage of GlyOX at 4 °C remains an issue that hampers the use of this enzyme in commercial biological assays. In this report, site-directed mutagenesis was used to engineer a stable GlyOX mutant suitable for long-term refrigerated storage and the development of an enzymatic method to analyze the glycine concentration in human plasma samples. The engineered GlyOX mutant exhibited long-term stability for over a year during storage at 4 °C, which is sufficient for practical use in solution kits. A colorimetric enzyme analysis using the GlyOX mutant was established to assess the reliability of the enzyme to measure glycine concentration.

2.3. Heterologous expression and purification of GlyOX Escherichia coli BL21 (DE3) cells were transformed with the plasmids and colonies were inoculated into LB medium containing 25 μg/ml kanamycin at 37 °C. His-tag fusion proteins were expressed by induction using 0.2 mM isopropyl-β-D-thiogalactopyranoside when the optical density at 660 nm reached 0.8 to 1.0. The culture temperature during induction was kept at 37 °C. The cells were incubated overnight and harvested by centrifugation. The cells were washed using 0.9% (w/ v) NaCl, the NaCl solution was removed by centrifugation, cells were resuspended in lysis buffer [20 mM Tris-HCl (pH 8.0) and 0.2 μM FAD] and disrupted for 20 min at 180 W with an ultrasonic disintegrator (ISONATOR 201 M, Kubota, Tokyo, Japan). The extract was centrifuged for 30 min at 15,000×g. The supernatant was loaded onto an open column packed with 5 ml of Ni Sepharose 6 Fast Flow resin (GE Healthcare, Buckinghamshire, England) and the resin washed with 50 mM HEPES (pH 7.5), 500 mM NaCl, 50 mM imidazole and 0.2 μM FAD to elute unbound proteins. The bound proteins were eluted with 50 mM HEPES (pH 7.5), 500 mM NaCl, 500 mM imidazole and 0.2 μM FAD. The eluted protein solution was loaded onto a HiPrep 26/10 desalting column (GE Healthcare) equilibrated with 100 mM sodium phosphate (pH 7.5) and 0.02 μM FAD using an ÄKTA Explorer 10 S system (GE Healthcare). Target proteins eluting from the column were stored in eluting buffer at 4 or –80 °C until use.

2. Materials and methods 2.1. GlyOX mutant screening The amino acid sequence of GlyOX from Bacillus subtilis strain 168 (UniProt ID: O31616) was used as the source of GlyOX. The wild-type GlyOX and mutants were expressed using a cell-free system and purified by contract manufacturing services (RIKEN Yokohama Branch, Kanagawa, Japan) [26–28]. Enzymes were prepared at 0.5 mg/ml in 50 mM potassium phosphate (pH 8.0) buffer containing 0.02 μM FAD, stored at 4 °C, and used within three days after purification. The following colorimetric analysis was performed to evaluate the stability of each GlyOX mutant. Each GlyOX solution was incubated at 4 and 60 °C for 60 min before being added to the reaction mixture. Reaction mixtures contained 49 mM potassium phosphate (pH 8.0), 2 mM phenol, 0.5 mM 4-aminoantipyrine, 5 U/ml horseradish peroxidase (Wako, Osaka, Japan), 10 mM glycine and 0.1 mg/ml of each GlyOX solution in a total volume of 100 μl. The absorbance at 500 nm was measured at 25 °C by SpectraMax® M2e microplate reader (Molecular Devices, San Jose, CA USA) to detect the production of hydrogen peroxide. Protein concentrations were determined by the Quick Start™ Bradford Protein Assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as the standard. Discovery Studio 4.5 (BIOVIA, San Diego, CA, USA) was used for estimating minimum distances between any atom pair of amino acids and FAD or N-acetylglycine, and the side-chain solvent accessibility of cysteine residues.

2.4. Evaluation of GlyOX activity Reaction mixtures for evaluation of kinetic parameters contained 50 mM potassium pyrophosphate (pH 8.5), 2 mM phenol, 0.5 mM 4aminoantipyrine, 5 U/ml horseradish peroxidase, 4.5 × 10−3 units of GlyOX and serially diluted glycine solutions in a total volume of 200 μl. The concentrations of glycine used were 0, 50, 100, 250, 500, 750, 1000 and 3000 μM. One unit of GlyOX activity is defined as the amount of enzyme that produces 1 μmol of hydrogen peroxide per min at 37 °C and pH 8.5. The molar absorption coefficient of the chromogen with 4aminoantipyrine at 500 nm is 1.3 × 104 M−1 cm−1. An absorbance change at 500 nm every 20 s was measured at 37 °C by the microplate reader. Results are averages of three enzyme preparations, each assayed in triplicate. Amino acid mixtures with and without glycine were used as substrates to investigate the substrate selectivity of GlyOX. The amino acid mixture contained 25 amino acids (glycine, L-alanine, Lisoleucine, L-leucine, L-valine, L-proline, L-tyrosine, L-phenylalanine, Lcysteine, L-aspartic acid, L-glutamic acid, L-lysine, L-arginine, L-histidine, L-methionine, L-asparagine, L-glutamine, L-serine, L-threonine, L-tryptophan, L-α-amino-n-butyric acid, taurine, L-citrulline, L-cystine and Lornithine). The final concentration of each substrate in the reaction mixture was 100 μM. Reaction mixtures contained 196 mM HEPES (pH 8.0), 2 mM phenol, 0.5 mM 4-aminoantipyrine, 5 U/ml horseradish peroxidase, 4.5 × 10−3 units of GlyOX and the substrate solution in a total volume of 100 μl. Absorbance changes at 500 nm were measured at 37 °C by the microplate reader. Each Tm value was calculated by determining residual activity when the wild-type or the T42 A/C245 S/ L301V mutant GlyOX solution was incubated for 60 min at 4, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 and 90 °C. Enzymes were prepared in 0.5 mg/ml in 50 mM potassium phosphate (pH 8.0) buffer containing 0.02 μM FAD. The residual activity was measured using the same

2.2. Cloning and site-directed mutagenesis Bacillus subtilis strain 168 was purchased from Bacillus Genetic Stock Center (BGSC), Columbus, OH, USA. The gene encoding wild-type GlyOX from Bacillus subtilis strain 168 was amplified by the polymerase chain reaction (PCR) using Bacillus subtilis strain 168 genome as the template and the following two primers: the sense primer (5′-ggaattcatgaaaaggcattatgaagcagt-3′) and the antisense primer (5′-ggaattctcatatctgaaccgcctccttgc-3′). The amplified fragment was inserted into the pT7 blue-T-vector (Novagen) to yield the vector pT7blue-GlyOX (WT). DNA fragments encoding full-length GlyOX were amplified by PCR using pT7blue-GlyOX (WT) as the template. The sense primer (5′2

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mixture at a 10, 15 or 30-fold dilution. The recovery was determined by measuring added glycine using plasma A that was mixed at a 15-fold dilution.

procedure as evaluation of substrate specificity. 2.5. Evaluation of GlyOX storage stability Storage stability was evaluated using GlyOX solutions placed in the dark at 4 °C. The activity was measured using the same procedure described in Section 2.4. Reaction mixtures for analysis of the wild-type GlyOX contained 49 mM sodium phosphate (pH 8.0), 2 mM phenol, 0.5 mM 4-aminoantipyrine, 5 U/ml horseradish peroxidase, 10 mM glycine and 2.3 × 10−3 units of GlyOX in a total volume of 100 μl. Reaction mixtures for analysis of the GlyOX T42 A/C245 S/L301V mutant were the same as the reaction mixture for the wild-type GlyOX, except that 49 mM sodium pyrophosphate (pH 8.5) was used instead of 49 mM sodium phosphate (pH 8.0).

3. Results and discussion 3.1. Screening of stable mutant GlyOX The stability of an enzyme over a long period is an important factor to consider when developing an enzymatic analysis method [29]. In particular, for commercial use in ready-to-use solution reagent kits the enzyme should be stable and active under refrigerated conditions for months. Therefore, we attempted to improve the storage stability of wild-type GlyOX by site-directed mutagenesis because its storage stability is inadequate [21,30,31]. We designed GlyOX mutants by two approaches using the reported structure (PDB ID: 1NG3). The first approach focused on the cysteine residues in GlyOX. Previous reports showed that mutation of cysteine residues located in solvent-accessible areas improves enzyme stability because this circumvents issues such as thiol oxidation and disulfide bond formation, which likely cause protein aggregation [32–36]. Additionally, it was also reported that cysteine-to-serine substitutions of cysteine residues located on the surface of the protein improve stability [32–36]. GlyOX has four cysteine residues (Cys56, Cys154, Cys 226 and Cys245), and Cys245 has the highest side-chain solvent accessibility value of 27.1% (Cys56, Cys154 and C226 have solvent accessibility values of 6.6, 0.0 and 0.7%, respectively). Thus, we designed a C245S mutant. Thermostability of the mutant was examined as a surrogate parameter to evaluate long-term storage stability [37] because evaluation of long-term storage stability was not practical for screening mutants. The residual activity of wild-type GlyOX after incubation at 60 °C for 60 min was almost absent (i.e., 1.0%), whereas that of the C245S mutant was 4.4% (Fig. 1). This improvement in thermostability by substitution of the most solvent exposed cysteine to serine is consistent with previous research [32–36]. In another approach, we focused on amino acid residues around the cofactor-binding site of GlyOX. Mutation of residues around this site have been reported to affect the stability of the protein, probably because of changes in the interactions between the cofactor and protein,

2.6. Construction of glycine standard curves by enzymatic analysis Reaction mixtures contained 100 mM Tris-HCl (pH 8.0), 3 mM TOOS (Dojindo laboratories, Kumamoto, Japan), 1 mM 4-aminoantipyrine, 15 U/ml horseradish peroxidase, 25 U/ml ascorbate oxidase (Wako, Osaka, Japan), glycine solution as substrate and 1.1 × 10−1 units of GlyOX in a total volume of 300 μl. The range of glycine concentrations used was 0–600 μM and these were mixed into the reaction mixture at a 10-fold dilution. The reaction was initiated by the addition of the enzyme to the reaction. Absorbance changes at 555 and 800 nm were measured at 37 °C by the microplate reader for 5 min to reach the endpoint of the reaction. The absorbance change (ΔAbsorbance) was determined as the final absorbance at 555 nm minus the final absorbance at 800 nm. The detection limit of the analysis procedure was calculated as an average value plus three standard deviations of the reaction mixture with 0 μM glycine (n = 3 for the same purified sample). 2.7. Instrumental analysis of glycine Instrumental analysis was performed using an L-8900 automated amino acid analyzer (Hitachi High-Technologies, Tokyo, Japan) that included a guard column and an analytical column packed with Hitachi custom ion exchange resin. Physiological amino acid analysis was carried out according to a previous report [17]. 2.8. Human plasma analysis Five samples of human plasma were purchased from C–C Biotech Corp. (San Diego, CA, USA), KAC (Kyoto, Japan) and Kohjin Bio (Saitama, Japan), and used in the current work (Table 1). Frozen plasma was thawed and centrifuged to remove precipitates immediately before use. The procedure for enzymatic analysis was based on Section 2.6 described above. For quantification of the glycine concentration, plasma samples were mixed with the reaction mixture at a 10-fold dilution. The intra-assay coefficient of variation (CV) was determined by measuring glycine in plasma A in a single run (n = 3 for the same purified sample), and the inter-assay (between-run) CV was calculated from six independent runs. Plasma A was mixed with the reaction Table 1 Plasma samples used in this study. Plasma

Sales company

Donor(s)

Lot number

A

C–C Biotech

B C D E

KAC KAC Kohjin Bio Kohjin Bio

Multiple donors Single donor Single donor Single donor Single donor

mixture of R175714, R175715, R175716, R175719 and R175721 PLA012706-0050 PLA012708-0050 BRH816526 BRH816534

Fig. 1. Thermostability of GlyOX mutants. The residual activity after incubation at 60 °C for 60 min (n = 3 for the same purified sample). Error bars indicate standard errors. 3

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Fig. 3. Long-term storage stability of the wild-type GlyOX and the T42 A/ C245 S/L301V mutant. Open circles and filled squares are the residual activity of the wild-type GlyOX and the T42 A/C245 S/L301V mutant, respectively (n = 3 for the same purified sample). The storage stability was investigated at 4 °C in the dark.

Table 2 Relative substrate specificity of the wild-type GlyOX and the T42 A/C245 S/ L301V mutant. Fig. 2. Structure of the GlyOX monomer (PDB ID: 1NG3 [22]). Ribbon representation of the GlyOX monomer (PDB ID: 1NG3, chain A) with FAD and Nacetylglycine as the inhibitor shown as stick models (cyan), and the three mutated residues (Thr42, C245 and Leu301) shown as stick models in green.

Relative activity (%)

Glycine Amino acid mixture Amino acid mixture without glycine

e.g., hydrogen bonds, hydrophobic interactions and salt-bridges [38]. Interaction with FAD has been reported to affect the stability of GlyOX [39]. Therefore, residues around the cofactor FAD molecule were targeted for mutation. We designed several conservative mutants for each residue to ensure that the size of the amino acid mutation did not affect the three-dimensional structure of GlyOX. Mutants T42S, T42A and L301V showed the largest improvement in residual activity (31.5, 18.7 and 12.9%, respectively) (Fig. 1). Thr42 and Leu301 are located near to FAD (2.9 Å and 4.2 Å, respectively) (Fig. 2). Consequently, mutation of these amino acids may have favorably changed the interaction with FAD, leading to the observed improvement in GlyOX thermostability.

Wild-type

T42 A/C245 S/L301V

100 107 1

100 101 0

Enzyme activity with glycine as the substrate was taken as 100%. The amino acid mixture contained L-alanine, L-isoleucine, L-leucine, L-valine, L-proline, Ltyrosine, L-phenylalanine, L-cysteine, L-aspartic acid, L-glutamic acid, L-lysine, Larginine, L-histidine, L-methionine, L-asparagine, L-glutamine, L-serine, L-threonine, L-tryptophan, L-α-amino-n-butyric acid, taurine, L-citrulline, L-cystine and L-ornithine.

glycine in a similar manner to that of the wild-type GlyOX (Table 2) and maintains substrate specificity toward glycine. The structural distances between each mutation and the substrate were farther than the distance from the FAD (the distances to T42, C245 and L301 are 7.9, 6.5 and 8.4 Å, respectively) (Fig. 2). Thus, substrate recognition by the GlyOX T42 A/C245 S/L301V mutant is considered not to affect the determination of glycine concentration in human plasma. Finally, we evaluated enzyme activity. The Km, kcat and kcat/Km values of wild-type GlyOX toward glycine were reported to be 0.99 mM, 1.3 s−1 and 1.3 mM−1 s−1, respectively [25]. The Km, kcat and kcat/Km values of the T42 A/C245 S/L301V mutant were 0.047 ± 0.005 mM, 0.30 ± 0.03 s−1 and 6.4 ± 0.4 mM−1 s−1, respectively. Thus, the kcat/Km of GlyOX T42 A/C245 S/L301V mutant is five times greater than that of the wild-type. From a structural viewpoint, substitution of amino acid residues around the cofactor would affect enzymatic activity as well as improve protein stability because of the change in the type of interactions [38,42]. Determining the structure of the T42 A/ C245 S/L301V mutant in complex with FAD should provide detailed information and aid in explaining the observed changes in enzyme kinetics when compared with that of the wild-type protein. This represents a future study.

3.2. Evaluation of the GlyOX T42A/C245 S/L301V mutant The single mutations identified to improve thermostability compared with that of the wild-type protein were combined to generate triple mutant constructs. Both the T42 S/C245 S/L301V and T42 A/ C245 S/L301V mutants showed remarkable improvement in residual activity when compared with the wild-type protein (55.7 and 74.0%, respectively) (Fig. 1). We selected the T42 A/C245 S/L301V mutant with the highest residual activity. The Tm value for the T42 A/C245 S/L301V mutant was dramatically increased to 55 °C from 42 °C relative to that of the wildtype enzyme. Furthermore, we evaluated its storage stability. The longterm storage stability of the GlyOX T42 A/C245 S/L301V mutant was evaluated by measuring the activity of this mutant over 370 days in 100 mM sodium phosphate buffer (pH 7.5) and 0.02 μM FAD at 4 °C in the dark (Fig. 3). The activity of wild-type GlyOX was reduced by 50% after a month of storage. In contrast, the T42 A/C245 S/L301V mutant maintained its activity over 350 days. Thus, the storage stability of GlyOX was dramatically improved by these three mutations. This improvement in long-term storage may be sufficient for use as a commercial enzyme, which is the case for Aspergillus niger glucose oxidase [40,41]. We evaluated the substrate specificity of the T42 A/C245 S/L301V mutant by focusing on the reaction with various amino acids each at a concentration of 100 μM, which is the approximate concentration of glycine in human plasma. The triple-mutant showed reactivity toward

3.3. Standard curve for glycine quantitation using the GlyOX T42A/ C245 S/L301V mutant We used the TOOS reagent for enzymatic analysis instead of phenol because the molar extinction coefficient of the chromogen using TOOS is higher than that of phenol [43] (Fig. 4). Glycine concentrations between 0 and 600 μM were added to the reaction mixtures and the 4

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Fig. 4. Scheme for the colorimetric glycine quantitation analysis. HRP, horseradish peroxidase.

Table 4 Recovery of glycine added to human plasma by GlyOX (T42 A/C245 S/L301V) enzymatic analysis. Added Gly (μM)

Detected (μM)

Recovered (μM)

Recovery (%)

0 150 225 375

310 454 532 677

144 222 367

96.0 98.7 97.9

The recovered concentrations were calculated by subtracting the value of the blank sample to which no glycine was added.

the addition of glycine to plasma A. The recoveries of glycine were 96.0–98.7% in human plasma (Table 4), and this result supported the accuracy of this analysis approach. Moreover, CVs of this analysis were determined by using three concentrations of plasma A without deproteination to evaluate the precision of this analysis. All CV values (%) were within 4% (Table 5). These are equivalent to result obtained with an instrument-based method [17]. Thus, this analysis showed high precision even with samples analyzed on different days. The wild-type GlyOX has been reported to react with sarcosine, Nethylglycine, D-alanine and D-proline [20,25], and the reactivity of T42 A/C245 S/L301V mutant shows similar tendency toward these substrates (Table S1). Even so, it was assumed that these metabolites did not affect glycine determination in human plasma because of their low concentration of approximately less than 1% of the glycine concentration [44–46]. Compared with instrumental methods [17], the advantages of this enzymatic method is that pretreatment of samples before analysis is not required for accurate and precise glycine quantitation. Additionally, enzymatic analysis takes only 5 min and many samples can be analyzed simultaneously using a microplate reader; thus, it is possible to analyze samples in a high-throughput manner. Therefore, this is a rapid and simple method for glycine quantitation and enables analysis of glycine for physiology-based research and clinical applications.

Fig. 5. Glycine standard curve for the T42 A/C245 S/L301V mutant. To construct a standard curve, the reaction mixture contained 0–600 μM glycine. The vertical axis shows the absorbance change at 555 nm per 300 s determined by the enzymatic method using the T42 A/C245 S/L301V mutant (n = 3 for the same purified sample).

absorbance was monitored to evaluate the linearity of this analysis. Linear standard curves were obtained with absorbance endpoints at 300 s (Fig. 5). The correlation coefficient was calculated to be 0.9999, demonstrating that glycine can be precisely quantified using the T42 A/ C245 S/L301V mutant. The limit of detection is 2.2 μM from the standard deviations of the blank sample to which no glycine was added. This standard curve covered the range of glycine concentrations found in human plasma (150–370 μM) [44], and the observed sensitivities are sufficient to analyze human plasma samples (see below). 3.4. Glycine quantification in human plasma Five plasma samples (A-E) were added to reaction mixtures to measure glycine concentrations without deproteination and derivatization pretreatments, and these results were compared with those determined by an amino acid analyzer (Table 3) [17]. The results clearly showed that enzymatic analysis with the T42 A/C245 S/L301V mutant could correctly determine glycine concentrations of human plasma (−5.4 to 3.5% relative errors when compared with the results from an amino acid analyzer). Additionally, we confirmed spike recovery with

4. Conclusions We have successfully engineered by site-directed mutagenesis a GlyOX T42 A/C245 S/L301V mutant, which has significantly improved long-term storage stability when compared with the wild-type GlyOX. This variant retained most of its enzymatic activity over 350 days of

Table 3 Quantitation of glycine in human plasma by GlyOX (T42 A/C245 S/L301V) enzymatic analysis. Plasma

Glycine concentration by an amino acid analyzer (μM) Glycine concentration by enzymatic analysis (μM) CV (%) of enzymatic analysis Relative error (%)

A

B

C

D

E

310

238

349

334

356

321

234

334

316

347

7.4 3.5

4.0 −1.7

2.5 −4.3

0.6 −5.4

0.6 −2.5

Table 5 CV values of plasma analysis by GlyOX (T42 A/C245 S/L301V) enzymatic analysis. Dilution ratio of plasma

10 15 30

The CV values (%) from enzymatic analysis (n = 3 for the same purified sample) are shown. The relative error (%) was calculated from the difference in glycine concentrations estimated by enzymatic analysis and the amino acid analyzer. Plasma A was pooled plasma and B–E were individual samples.

CV (%) Intra-assay

Inter-assay

1.6 1.5 1.9

2.2 3.8 3.1

The CV values (%) of the intra-assay were calculated by analysis of plasma A in a single run (n = 3 for the same purified sample). The CV values (%) of the inter-assay (between-run) were calculated from six independent runs. 5

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storage at 4 °C, whereas the activity of wild-type GlyOX was reduced by 50% after a month of storage. A colorimetric enzyme analysis was developed using this mutant and the performance of this mutant was confirmed using human plasma samples. The results clearly showed that our analysis method could correctly and precisely determine glycine concentrations in human plasma. Moreover, this analysis is easier than previously reported methods because it does not depend on sample pretreatment such as deproteinization and derivatization. Therefore, this analysis method would be suitable for physiology-based research and clinical applications.

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Declaration of competing interest The authors declare no competing interests. Acknowledgements This work was supported by grant from the Hokuriku Innovation Cluster for Health Science (MEXT, Japan) to Y. Asano. Protein expression and purification were performed by RIKEN as a member of the NMR platform in Japan. We are grateful to Yoko Miyama, Sayaka Kitahara and Shunsuke Onishi for their technical assistance with experiments. We thank the Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ab.2019.113447. References [1] T. Ishikawa, Branched-chain amino acids to tyrosine ratio value as a potential prognostic factor for hepatocellular carcinoma, World J. Gastroenterol. 18 (2012) 2005–2008. [2] T. Ishikawa, Early administration of branched-chain amino acid granules, World J. Gastroenterol. 18 (2012) 4486–4490. [3] R. Yanagisawa, M. Kataoka, T. Inami, Y. Momose, T. Kawakami, M. Takei, M. Kimura, S. Isobe, M. Yamakado, K. Fukuda, H. Yoshino, M. Sano, T. Satoh, Usefulness of circulating amino acid profile and fischer ratio to predict severity of pulmonary hypertension, Am. J. Cardiol. 115 (2015) 831–836. [4] D.H. Chace, S.L. Hillman, D.S. Millington, S.G. Kahler, C.R. Roe, E.W. Naylor, Rapid diagnosis of maple syrup urine disease in blood spots from newborns by tandem mass spectrometry, Clin. Chem. 41 (1995) 62–68. [5] D.H. Chace, S.L. Hillman, D.S. Millington, S.G. Kahler, B.W. Adam, H.L. Levy, Rapid diagnosis of homocystinuria and other hypermethioninemias from newborns' blood spots by tandem mass spectrometry, Clin. Chem. 42 (1996) 349–355. [6] K. Nakamura, T. Fujii, Y. Kato, Y. Asano, A.J.L. Cooper, Quantitation of L-amino acids by substrate recycling between an aminotransferase and a dehydrogenase: application to the determination of L-phenylalanine in human blood, Anal. Biochem. 234 (1996) 19–22. [7] S. Kawana, K. Nakagawa, Y. Hasegawa, S. Yamaguchi, Simple and rapid analytical method for detection of amino acids in blood using blood spot on filter paper, fastGC/MS and isotope dilution technique, J. Chromatogr. B 878 (2010) 3113–3118. [8] Y. Asano, Screening and development of enzymes for determination and transformation of amino acids, Biosci. Biotechnol. Biochem. 83 (2019) 1402–1416. [9] Y. Miyagi, M. Higashiyama, A. Gochi, M. Akaike, T. Ishikawa, T. Miura, N. Saruki, E. Bando, H. Kimura, F. Imamura, M. Moriyama, I. Ikeda, A. Chiba, F. Oshita, A. Imaizumi, H. Yamamoto, H. Miyano, K. Horimoto, O. Tochikubo, T. Mitsushima, M. Yamakado, N. Okamoto, Plasma free amino acid profiling of five types of cancer patients and its application for early detection, PLoS One 6 (2011) e24143. [10] T. Hisamatsu, S. Okamoto, M. Hashimoto, T. Muramatsu, A. Andou, M. Uo, M.T. Kitazume, K. Matsuoka, T. Yajima, N. Inoue, T. Kanai, H. Ogata, Y. Iwao, M. Yamakado, R. Sakai, N. Ono, T. Ando, M. Suzuki, T. Hibi, Novel, objective, multivariate biomarkers composed of plasma amino acid profiles for the diagnosis and assessment of inflammatory bowel disease, PLoS One 7 (2012) e31131. [11] R.Y. Gundersen, P. Vaagenes, T. Breivik, F. Fonnum, P.K. Opstad, Glycine - an important neurotransmitter and cytoprotective agent, Acta Anaesthesiol. Scand. 49 (2005) 1108–1116. [12] M. Bannai, N. Kawai, New therapeutic strategy for amino acid Medicine: glycine improves the quality of sleep, J. Pharmacol. Sci. 118 (2012) 145–148. [13] D.A. Applegarth, J.R. Toone, Nonketotic hyperglycinemia (glycine encephalopathy): laboratory diagnosis, Mol. Genet. Metab. 74 (2001) 139–146. [14] N. Guy, M. Blanaru, B. Bloch, I. Kremer, M. Ermilov, D.C. Javitt, U. Heresco-Levy, Relation of plasma glycine, serine, and homocysteine levels to schizophrenia symptoms and medication type, Am. J. Psychiatry 162 (2005) 1738–1740.

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