Characterization of a recombinant arginine deiminase from Enterococcus faecalis SK32.001 for L-citrulline production

Accepted Manuscript Title: Characterization of a recombinant arginine deiminase from Enterococcus faecalis SK32.001 for L-citrulline production Authors: Hangyu Jiang, Kai Huang, Wanmeng Mu, Bo Jiang, Tao Zhang PII: DOI: Reference:

S1359-5113(17)30309-4 http://dx.doi.org/doi:10.1016/j.procbio.2017.06.006 PRBI 11064

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

17-2-2017 2-6-2017 3-6-2017

Please cite this article as: Jiang Hangyu, Huang Kai, Mu Wanmeng, Jiang Bo, Zhang Tao.Characterization of a recombinant arginine deiminase from Enterococcus faecalis SK32.001 for L-citrulline production.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2017.06.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Characterization of a recombinant arginine deiminase from Enterococcus faecalis SK32.001 for L-citrulline production Hangyu Jiang, Kai Huang, Wanmeng Mu, Bo Jiang, Tao Zhang* State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China *Corresponding author: Tao Zhang Telephone number: 86-510-85919161 Fax number: 86-510-85919161 E-mail: [email protected]

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Graphical abstract Arginine deiminase

Citrulline

Arginine f1 origin

Xho¢ñ (159) His tag ADI

Kan Sma¢ñ (5494)

T7 terminator

BamH¢ñ (1392)

pET28a(+)-ADI

6562bp ori

lacl

Eco RV(2767)

The map of pET28a(+)-ADI

Structure of arginine deiminase

Highlights 

The arginine deiminase gene of Enterococcus faecalis was expressed in Escherichia coli for the first time.



The effects of amino acid residues of the enzyme were explored.

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The resting recombinant cells have high L-citrulline productivity and molar yield.

Abstract: Arginine deiminase is an enzyme used to biosynthesize L-citrulline from L-arginine. The arginine deiminase gene of Enterococcus faecalis SK32.001 was expressed in Escherichia coli with a specific activity of 131.2 U mg-1. The expressed enzyme was a dimer with a subunit molecular weight of 49.1 kDa. It was stable in a pH range of 5.0 to 5.5 and in a temperature range of 20 °C to 25 °C. The optimum pH and temperature of the enzyme were 5.5 and 55 °C, respectively. Fe3+ enhanced its enzymatic activity. The chemical modifiers and the three-dimensional structural model of the recombinant enzyme indicated that Lys, Trp and Cys were very important amino acid residues to the enzyme. It’s Km and Vmax for L-arginine were 10.1 mM and 378.1 μmol min-1 mg-1, respectively. The bioproductivity of L-citrulline in the resting recombinant cells was 48.5 g L-1 h-1, and the molar yield was 96.9%. Keywords: arginine deiminase; cloning; L-citrulline; biocatalysis; Enterococcus faecalis 2

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1. Introduction The arginine hydrolysis pathway (ADI pathway) is important for argininedependent prokaryotes because it catabolizes arginine to generate ATP[1]. This pathway involves three enzymes: arginine deiminase (ADI, EC 3.5.3.6), ornithine transcarbamylase (OTCase) and carbamate kinase (CK). ADI hydrolyzes L-arginine into L-citrulline as part of energy generation in many microorganisms. It also has potent anticancer activities. L-citrulline is a non-essential amino acid that plays a vital role in the urea cycle, and it has many health care and pharmaceutical applications. In health care, Lcitrulline can prevent oxidative damage to DNA and polymorphonuclear leukocytes (PMNs)[2, 3], maintain the normal function of the cardiovascular system[4, 5], improve splanchnic perfusion, reduce gut injury during exercise[6] and stimulate muscle protein synthesis[7]. Additionally, serum citrulline is a latent marker of acute cellular rejection[8, 9] and citrulline supplementation can improve sexual function in both men and women[10]. Due to these multiple functions, L-citrulline has a huge potential market in the pharmaceutical and fine chemical industries in the future. There are three ways to produce L-citrulline: chemical synthesis, microbial fermentation, and an enzymatic method. Chemical synthesis is a multistep approach in which D- and L-citrulline are generated by the hydrolysis of L-arginine under alkaline conditions[11]. Microbial fermentation has been a recent focus because of its safety, its economical raw materials, its mild processing conditions, and its higher productivity than other methods. Okumura et al. first discovered that L-citrulline 4

accumulates in the culture medium of an arginine-requiring mutant of Bacillus subtilis[12]. Subsequently, Pseudomonas putida (P. putida) was found to produce high levels of L-citrulline from L-arginine due to its ornithine transcarbamylase deficiency[13]. However, the purification of L-citrulline from P. putida is costly because of the complex composition of its fermentation broth. In contrast, when an enzyme catalyzes the hydrolysis of L-arginine to L-citrulline and ammonia, the ammonia by-product can be easily removed by vacuum concentration during the downstream process. Therefore, L-citrulline production by enzymatic synthesis with ADI was investigated in free-living and immobilized cells[14] and with enzymatic expression in Escherichia coli (E. coli.)[15]. However, the commercialization of enzymatic L-citrulline production was delayed due to the low enzyme activity of the wild-type strains. Despite this evident progress, many researchers remain focused on enhancing the enzyme’s activities and properties and on improving the bioconversion efficiency through overexpressing the ADI-encoding gene[15], random mutagenesis, and other approaches. In this investigation, a plasmid with the arginine deiminase gene from Enterococcus faecalis SK32.001 was constructed and expressed in E. coli. The enzyme’s characteristics and kinetic parameters were also investigated. 2. Materials and methods 2.1 Materials and chemicals

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Analytical-grade L-arginine and other chemicals were obtained from Sinopham Chemical Reagent Co., Ltd. (Shanghai, China). L-citrulline, bovine serum albumin (BSA), Coomassie brilliant blue R250, 2-mercaptoethanol (2-ME), diethylpyrocarbonate (DEPC), n-bromosuccinimide (NBS), 5,5’-dithiobis (2nitrobenzoic acid) (DTNB), N-acetylimidazole (NAI), 2,3-butanedione (BD) and 1ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) , trinitro-benzene-sulfonic acid (TNBS) were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). 2.2 Culture media and growth conditions Enterococcus faecalis SK32.001 was stored in our laboratory. It was cultured in seed medium and then in the fermentation medium for 16 h at 37 °C, respectively. The inoculum concentration was 3%. The expression host of Escherichia coli BL21 (DE3) was incubated in Luria-Bertani (LB) broth at 37 °C for 12 h. The seed broth had an initial pH of 6.0 - 6.5 and contained (in g L-1): glucose 10, L-arginine 5, yeast powder 5, peptone 5, K2HPO4 1, NH4Cl 1.5, and NaCl 0.1. The fermentation broth has an initial pH of 6.0 - 6.5 and contains (in g L-1): glucose 10, Larginine 10, yeast powder 5, peptone 5, K2HPO4 1, and NaCl 0.1. The LB broth contained (in g L-1): tryptone 10, yeast powder 5, and NaCl 10. All culture media were sterilized at 115 °C for 30 min. 2.3 Cloning and sequence analysis of arcA The arcA gene from Enterococcus faecalis SK32.001 was amplified via PCR using two primers (forward primer, 5’-CGCGGATCCATGTCCAATTAATGT-3’; 6

reverse primer, 5’-CCGCTCGAGTTAAAGATCTTCACGGT-3’). The PCR amplification was performed using Ex Taq DNA polymerase (TaKaRa, Dalian, China). The PCR conditions were as follow: pre-denaturing at 95 °C for 3 min, followed by 30 cycles of denaturing at 95 °C for 30 s, annealing at 55 °C for 30 s and elongating at 72 °C for 3.5 min, and a final elongating at 72 °C for 2 min. The amplified fragment was digested using BamHI and XhoI (Takara, Dalian, China) and was inserted into the pET-28a(+) (Novagen, Darmstadt, Germany) expression vector with a 6 × histidine tag at the C-terminus, generating the plasmid pET-28a(+)-ADI. Then, the pET-28a(+)-ADI was transformed into E. coli BL21 (DE3) to express the enzyme. The target gene sequence was analyzed by the dideoxy chain-termination method[16], and the homologies of the nucleotide sequences and amino acid sequences were determined via NCBI BLAST. 2.4 Expression of ADI Escherichia coli BL21 (DE3) harboring pET-28a(+)-ADI was cultured at 37 °C in 200 mL of LB broth containing 50 μg mL-1 kanamycin. ADI expression was induced with 1.0 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) for 6 h at 28 °C when the optical density at 600 nm (OD600) was 0.6 – 0.8. The cells were collected by centrifugation at 4 °C and 8,000 × g for 10 min. The sediments were washed twice with 20 mM PBS buffer (pH 6.0). The washed pellets were resuspended in the same buffer and disrupted using a VibraCellTM 72405 Sonicator (Sonics, Newtown, CT, USA) at 4 °C for 6 min (pulsations of 3 seconds, amplification 90 seconds). The cell

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debris was separated by centrifugation (8,000 × g, 10 min, 4 °C), and the supernatant was used for enzyme purification. 2.5 Purification of ADI The supernatant was loaded onto a column packed with Ni2+-chelating Sepharose Fast Flow resin (GE Healthcare, Uppsala, Sweden) and equilibrated with binding buffer (20 mM sodium phosphate buffer, 500 mM NaCl, pH 7.0). The unbound proteins and the recombinant enzyme were eluted separately from the column with the elution buffer (20 mM sodium phosphate buffer, 500 mM NaCl, 50 or 500 mM imidazole, pH 7.0). The fraction containing ADI was dialyzed against 20 mM sodium phosphate buffer (pH 6.0), 20 mM sodium phosphate buffer containing 10 mM ethylenediamine tetraacetic acid (EDTA), and 20 mM EDTA-free sodium phosphate buffer (pH 6.0) to remove imidazole, metal ions and EDTA, respectively. The Lowry method[17] was used to analyze the protein concentration with BSA as a standard. 2.6 Analytical methods 2.6.1 Subunit molecular mass determination The molecular weight of the expressed ADI subunit was evaluated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with premixed protein markers (Takara, Dalian, China) as reference proteins. All protein bands were stained with Coomassie brilliant blue R250 for analysis. 2.6.2 ADI activity determination

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ADI activity was evaluated based on L-citrulline production from L-arginine. Briefly, 0.01 mL of ADI protein was incubated with 0.5 mL of 100 g L-1 arginine in 20 mM potassium phosphate buffer (pH 6.0) and 0.49 mL of the same buffer for 10 min at 45 °C. The reaction was terminated in boiling water for 10 min. One unit of enzymatic activity was the amount of ADI needed to produce 1 μmol of L-citrulline per minute. The contents of L-citrulline and L-arginine were determined by high pressure liquid chromatography (HPLC) with gradient elution at 40 °C and a flow rate of 1 mL min-1. A C18 ODS HYPERSIL (Agilent 1200, Agilent Technologies, Palo Alto, CA, USA) was used as the column. The mobile phase gradient was composed of buffer A (6.5 g L-1 CH3COONa•3H2O including 0.16 mg L-1 triethylamine and 4.4 mg L-1 tetrahydrofuran, pH 7.2) and buffer B (24 g L-1 CH3COONa•3H2O, pH 7.2/acetonitrile/methanol (1:2:2 by volume)). The A/B ratios were 92:8, 62:38, 0:100, 0:100 and 92:8 at run times of 0, 20, 24, 25.5 and 28.5 min, respectively. oPhthaldialdehyde was used as the precolumn derivation reagent, and the products were monitored by a UV detector (model LC-9A, Shimadzu, Kyoto, Japan) at 338 nm with excitation at 262 nm. 2.6.3 Effects of temperature and pH The optimum temperature of ADI was investigated in 20 mM potassium phosphate buffer (pH 6.0) for 10 min at temperatures ranging from 10 °C to 70 °C. The highest enzymatic activity was set as 100%. Thermostability was determined by 9

measuring the residual ADI activity after pre-incubation at 20 °C to 45 °C for various times and then immediate cooling on ice. The enzymatic activity of a control sample that was incubated on ice was set as 100%. The optimum pH of ADI was evaluated in a pH range of 4.0 to 8.5 using HAcNaAc buffer (20 mM, pH 4.0-5.5), potassium phosphate buffer (20 mM, pH 6.0-7.0), and Tris-HCl (20 mM, pH 7.5-8.5). The highest enzymatic activity was assumed to be 100%. The pH stability was assayed by measuring the residual enzymatic activities when the enzyme was pre-incubated in 20 mM potassium phosphate buffer in a pH range from 4.0 to 7.0 at 4 °C for 12 h. The enzymatic activity after pre-incubation at 4 °C was assumed to be 100%. 2.6.4 Effects of metal ions The effect of various metal ions on ADI activity was investigated using 1 mM of Cu2+, Co2+, Ba2+, Mg2+, Mn2+, Zn2+, Ni2+, Ca2+, Fe2+ and Fe3+. The relative residual activities were evaluated after preincubation in 20 mM potassium phosphate buffer with each metal ion at 4 °C for 12 h. The residual enzymatic activity was measured at 45 °C and pH 6.0 for 10 min. The relative activity assayed in the absence of chemicals under the above conditions was assumed to be 100%. 2.6.5 Effects of inactivation reagents on ADI To evaluate the effects of chemical modification reagents on ADI activity, the enzyme was preincubated for 1 h at 45 °C with 0.5 mM DTNB (phosphate buffer, pH 8.0), NBS (citrate buffer, pH 4.5), DEPC (phosphate buffer, pH 7.0), EDC (citrate 10

buffer, pH 4.5), TNBS (borate buffer, pH 9.0), 2-ME (phosphate buffer, pH 8.0), BD (borate buffer, pH 9.0) or NAI (phosphate buffer, pH 8.0). These modifiers act on the Cys, Trp, His, carboxyl groups (Asp or Glu), Lys, disulfide bonds, Arg and Tyr residues, respectively[18]. The reaction was initiated by the addition of L-arginine as a substrate. The residual enzymatic activity was evaluated under standard conditions. The activity of the enzyme in the absence of modification reagents was assumed to be 100%. 2.6.6 Kinetic parameters of the enzyme To evaluate the kinetics of ADI, its enzymatic activity was estimated with Larginine solutions of 1, 2, 4, 6, 8, 10, 20 and 30 mM under the standard conditions of 45 °C, pH 6.0 for 10 min. The Lineweaver-Burk method was used to calculate the Km and Vmax. 2.7 Bioproduction of L-citrulline by ADI L-citrulline was produced in a 55 °C water bath with shaking at 150 rpm for 2 h under the optimum conditions: substrate 100 g L-1 L-arginine, pH 5.5, 4 U mL-1 of the resting cells containing ADI and 0.03 g L-1 CTAB. 2.8 Statistical analysis The experiments were repeated 3 times, and statistical analyses were conducted with the Origin 9.0 program. The online software SWISS-MODEL (https://swissmodel.expasy.org) was used to build a three-dimensional structural

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model of the recombinant enzyme. The molecular model was visualized using PyMOL software. 3 Results and discussion 3.1 Gene cloning and sequence analysis A 1260-bp fragment including the arcA gene obtained from Enterococcus faecalis SK32.001 was deposited in the GenBank database. The theoretical isoelectric point and the molecular weight of the enzyme, calculated using ExPASy (www.expasy.org/tools/protparam.html), were pH 5.1 and 100.5 kDa, respectively. The homology of the hypothetical protein was analyzed with the DNAMAN7 program and was in accordance with the amino acid sequence of the ADI family (Fig. 1). It shared the highest (56.5%) and the lowest (41.3%) sequence identity with the ADIs of Borrelia burgdorferi B31[19] and Pseudomonas aeruginosa PAO1[20], respectively. Additionally, Mycobacterium tuberculosis H37Rv[21] and Pseudomonas putida KT2440[22] had 45.7% and 43% amino acid sequence similarity with ADI from Enterococcus faecalis SK32.001, respectively. The homologies of ADI with the amino acid sequences of 4bof.1.A[23] and 1lxy.1.A[24] were 68.4% and 40.4%, respectively. These homologies are over 40% and have conserved domains related to enzymatic activity, so they are isozymes. Based on previous publications[23, 24], the active site of ADI is probably composed of residues D162, E216, H271, D273, and C398, as shown in Fig. 1. 3.2 Expression and Purification of ADI 12

E. coli harboring pET-28a(+)-ADI induced by IPTG was purified with Ni2+chelating Sepharose Fast Flow column. The molecular weight of the purified enzyme was approximately 49.1 kDa as detected and calculated by SDS-PAGE (Fig. 2, lane 2). The enzyme is most likely a dimer with two identical subunits, based on its calculated molecular weight of 100.5 kDa. The specific activity of purified ADI was 131.2 U mg-1 protein. These results showed that the recombinant enzyme was similar to ADI from Lactococcus lactis ssp. lactis[25] and Lactobacillus sanfranciscensis[26]. 3.3 Characterization of the purified enzyme 3.3.1 Effects of temperature, pH and metal ions The effects of temperature on ADI activity are shown in Fig. 3A. ADI activity increased slowly as the temperature increased to 55 °C and then decreased rapidly to zero as temperatures rose higher than 60 °C. It retained over 60% activity from 30 °C to 60 °C. The optimum temperature (55 °C) was lower than that (60 °C) of Lactococcus lactis ssp. lactis ATCC 7962[25] and higher than those of most ADIs [25-32]. The thermostability of ADI was evaluated at 20, 25, 30, 35, 40 and 45 °C. From Fig. 3B, we can see that ADI was stable at 20 and 25 °C. The enzyme retained over 50% activity at 30 °C for 120 min. After that, the relative enzymatic activity decreased rapidly with increasing temperature and incubation time. These results indicated that the thermostability of this enzyme is lower than those from other 13

sources[25]. However, some approaches could be used to enhance its thermostability, such as directed evolution[27] and immobilization[28]. The results in Fig. 3C indicate that this enzyme has a relatively narrow pHdependent spectrum. The enzymatic activity increased when the detection pH varied from 4 to 5.5, and then it decreased when the pH was raised continually. Therefore, the enzyme’s optimum pH was at 5.5. Most sources also observed that the optimum pH for ADI was acidic [29-31]. To evaluate pH stability, residual activity was assayed after the enzyme was pre-incubated with a pH of 4.0 to 7.0 at 4 °C for 12 h. The enzyme retained over 50% activity at pH 4.5 to 5.5, as shown in Fig. 3C. To facilitate its use as an antitumor drug, some researchers[32] have tried to increase the enzyme’s optimum pH and pH stability to 7.4 (physiological pH). However, acidic conditions are appropriate for L-citrulline biosynthesis. The enzymatic activity was enhanced by metal ions of Al3+, Cu2+, Ni2+, Zn2+, Ba2+, Fe3+ and Fe2+ (Fig. 3D). Notably, the presence of Fe3+ metal ions improved enzymatic activity by more than 50%. Enzymatic activity was inhibited by Mn2+, Mg2+, and Co2+. In general, Fe3+ participates in the electron transfer pathways to/from the active sites of enzymes. However, Andreini[33], using Metal-MACiE, demonstrated that Fe2+/Fe3+ can be used by enzymes for its Lewis acid properties instead of its redox properties. 3.3.2 Effects of chemical modification

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The effects of chemical modifiers were used to explore the enzyme’s active sites. As shown in Table 1, the chemical modifiers DEPC (for His), NAI (for Tyr) and BD (for Arg) had little effect on enzymatic activity. EDC (for carboxyl groups, Asp and Glu) and 2-ME (for disulfide bonds) had mild effects on enzymatic activity. However, TNBS (for Lys), NBS (for Trp) and DTNB (for Cys) strongly inhibited enzymatic activity, which indicates that the lysine, tryptophan and cysteine residues are essential to enzymatic activity. The X-ray crystal structure of ADI from Streptococcus pyogenes showed that the amino acid residues D166, E220, H275, D277 and C401 compose the catalytic activity center of ADI[23]. The recombinant ADI model had the highest homology (67.57%) with the enzyme from Streptococcus pyogenes. There are approximately 21 Lys residues in the recombinant enzyme, and they are all distributed on the molecular surface (Fig. 4A). With an increase in the TNBS concentration, enzymatic activity decreased gradually. Therefore, the Lys residues probably impacted enzymatic activity by influencing the electrostatic interactions or the protein’s hydrophobic interactions. NBS influenced the activity of Trp residues on the benzene ring of a substitution reaction. As shown in Table 1, enzymatic activity decreased with an increase in NBS concentration. The result of structural analysis showed that the only Trp residue is at residue 347. Although it is far away from the catalytic activity center, this residue is located in the substrate’s entrance to the enzyme molecule (Fig. 4B and 4C). After NBS modified the Trp residue, the entrance was blocked or reduced, which influenced the activity of enzyme. On the other hand, NBS probably influences the hydrophobic interactions of the protein because the 15

benzene ring of the amino acid Trp is modified. The Cys amino acid residues play a crucial role in the process of Arg catalysis [24]. Calkin[34] and Ding[35] transformed the Cys residue at position 406 into another amino acid by site-specific mutagenesis to reduce the lost activity of arginine deiminase. Therefore, adding a trace amount of DTNB will have an enormous effect on enzyme activity. 3.3.3 Determination of kinetic parameters The Km value was 10.1 mmol L-1 for arginine as the substrate, with a maximum reaction rate (Vmax) of 378.1 μmol min-1 mg-1 (Fig. 5). The Km value is higher than those of ADIs in other organisms according to BRENDA (http://www.brendaenzymes.org/). The recombinant enzyme showed a lower affinity for arginine. In spite of its high Km, the Vmax was higher than those from ADIs in other organisms[25]. Therefore, the recombinant enzyme is still a potential candidate for converting Larginine to L-citrulline. 3.4 Bioconversion of L-citrulline Based on the results above, L-citrulline was produced by the recombinant ADI under optimum conditions (Fig. 6). After incubation for 2 h, L-citrulline production reached 96.9 g L-1, and the molar yield was 96.9%. These results indicated great potential for further scale-up and industrial applications. 4 Conclusions ADI from Enterococcus faecalis SK32.001 was successfully expressed in E. coli. The enzyme displayed distinct properties compared to ADIs from other sources. 16

Meanwhile, Lys, Trp and Cys residues may be affect the enzyme activity in various ways. The biosynthesis of L-citrulline by recombinant cells expressing ADI is a good candidate for commercialization. Acknowledgement This work was financially supported by the 863 project of China (No. 2013AA102102), the Science and Technology Infrastructure Program of Jiangsu (No. BM2014051) and the Scientific Research and Technological Development Program of Guangxi (GKH14251003). References [1]

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Figures legends Fig.1 Amino acid sequences and their homologous arginine deiminase family enzymes. The images were created with the DNAMAN program. The origin and GenBank accession number of enzymes are as follows: BB-ADI (Borrelia burgdorferi B31), MT-ADI (Mycobacterium tuberculosis H37Rv), FE-ADI (Enterococcus faecalis SK23.001), PA-ADI (Pseudomonas aeruginosa PAO1), PPADI (Pseudomonas putida KT2440). Black represents identical amino acid residues, gray indicates strongly conserved residues, and light gray denotes weakly conserved residues. The potential active-site residues of the enzymes are highlighted with black boxes. Fig.2 SDS-PAGE of purified arginine deiminase (lane 2) and protein marker (lane 1), stained with Coomassie brilliant blue R250. Fig. 3 Characteristics of ADI. (A) Effects of temperature. (B) Thermostability of the arginine deiminase at 20 °C (◆), 25 °C (△), 30 °C (■), 35 °C (●), 40 °C (▲) and 45 °C (▼). (C) The optimum pH (■) and pH stability (▼) of the recombinant arginine deiminase. (D) Effects of metal ions on the arginine deiminase. Fig.4 Structure of ADI (A) Stereo view of the structure of ADI. The active-site residues (D162, E216, H271, D273 and C398) are shown in red. (B) A view of the substrate binding pocket of the enzyme. The active-site residues and Tyr residue are shown in red and green, respectively. The black loop represents the putative entrance to the pocket. (C) A zoomed-in view of the substrate binding pocket of ADI rotated approximately 90° about the horizontal axis compared to Figure 4B. The red residues 23

are the active-site residues, and the green residue is the Tyr amino acid residue. The black arrow represents the putative entrance to the pocket. Fig.5 Kinetic parameters of the recombinant arginine deiminase. Fig.6 L-citrulline production (▲) curve with the recombinant resting cells. Table Table 1 Effect of inactivation agents on recombinant arginine deiminase activity Modifying agent DEPC EDC NAI TNBS NBS

2-ME BD DTNB

Concentration(mM) 0.5 0.5 0.5 0.1 0.5 1 0.1 0.5 1 5 0.5 0.5 0.05 0.1 0. 5

control

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Relative activity (%) 95.77±3.78 90.47±1.74 104.64±3.13 29.06±1.43 2.85±0.65 0±0.13 87.32±3.83 75.23±2.35 71.14±2.26 11.78±1.93 83.51±1.48 103.35±2.47 6.11±2.55 3.51±0.85 1.18±0.43 100±2.49

Figures Fig.1

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Fig.2

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Fig.3

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Fig. 4

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Fig. 5

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Fig. 6

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