L-ARGININE-GLYCINE AMIDINOTRANSFERASE AND USE THEREOF IN THE PRODUCTION OF GUANIDINOACETIC ACID

Abstract

The present invention discloses an L-arginine-glycine amidinotransferase and use thereof in the production of guanidinoacetic acid. In the present invention, through combined multi-site amino acid mutation, a technical effect of significantly improved enzyme activity of the mutant AkAGAT.sub.T225Q/A258P/L278K than that of the wild-type strain is achieved, providing an application value for large-scale production of guanidinoacetic acid in industry. When the L-arginine-glycine amidinotransferase mutant constructed in the present invention is used in the production of guanidinoacetic acid, by optimizing the conversion conditions, the yield of guanidinoacetic acid is up to 21.4 g/L and the conversion rate is 90.4%, after 24 hrs of reaction in a 1 L reaction system. Compared with the production of guanidinoacetic acid with the raw enzyme, the yield is increased by 49.6%.

Claims

1. An L-arginine-glycine amidinotransferase, having a sequence obtained by a mutation of the amino acid at position 225 in the enzyme having an amino acid sequence as shown in SEQ ID NO. 4 into glutamine.

2. The L-arginine-glycine amidinotransferase according to claim 1, wherein in the L-arginine-glycine amidinotransferase the alanine at position 258 is mutated into proline.

3. The L-arginine-glycine amidinotransferase according to claim 1, wherein in the L-arginine-glycine amidinotransferase the leucine at position 278 is mutated into lysine.

4. A gene encoding the L-arginine-glycine amidinotransferase according to m claim 1.

5. A recombinant expression vector carrying the gene according to claim 4.

6. A recombinant strain expressing the L-arginine-glycine amidinotransferase according to claim 1.

7. The recombinant strain according to claim 6, wherein E. coli BL21 (DE3) is used as a host and pET-28a plasmid is used as an expression vector.

8. A method for producing guanidinoacetic acid, comprising catalytically reacting L-arginine with glycine to produce guanidinoacetic acid using the the recombinant strain according to claim 6 as a catalyst.

9. The method according to claim 8, wherein in a catalytic reaction system with the recombinant strain as a catalyst, 0.08-0.12 M Tris-HCl is used as a buffer, and the recombinant strain is added in such an amount that OD.sub.600 is 30-50.

10. The method according to claim 9, wherein in the catalytic reaction system, the L-arginine content is 50-200 mM, the glycine content is 50-200 mM, and L-arginine:glycine=1:2-2:1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 is an electrophoretogram showing the construction verification results of pET-28a-AkAGAT, pET-28a-AcAGAT, and pET-28a-CrAGAT;

[0021] FIG. 2 is an SDS-PAGE electrophoretogram showing L-arginine-glycine amidinotransferase AGAT of various sources in Example 1, in which Lanes 1 and 2 are respectively a precipitate and a crude enzyme solution of pET-28a plasmid, Lanes 3 and 4 are respectively a precipitate and a crude enzyme solution of pET-28a-AkAGAT, Lanes 5 and 6 are respectively a precipitate and a crude enzyme solution of pET-28a-AtAGAT, and Lanes 7 and 8 are respectively a precipitate and a crude enzyme solution of pET-28a-CrAGAT;

[0022] FIG. 3 shows the effect of temperature on AkAGAT enzyme activity in Example 2;

[0023] FIG. 4 shows the effect of various metal ions on AkAGAT in Example 2;

[0024] FIG. 5 shows the exploration on stability against temperature of AkAGAT enzyme in Example 2;

[0025] FIG. 6 shows the exploration on stability against pH of AkAGAT enzyme in Example 2;

[0026] FIG. 7 is an SDS-PAGE electrophoretogram of AkAGAT mutant in Example 3, in which Lanes 1, 2, 3, and 4 are respectively AkAGAT, AkAGAT.sub.T225Q, AkAGAT.sub.T225Q/A258P, AkAGAT.sub.T225Q/A258P/L278K pure enzyme;

[0027] FIG. 8 compares the production of guanidinoacetic acid by whole cell conversion with various AkAGAT mutants;

[0028] FIG. 9 shows the effect of different substrate concentrations on the production of GAA in the conversion system.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention will be further described below in connection with specific examples, so that those skilled in the art can better understand and implement the present invention; however, the present invention is not limited thereto.

[0030] Unless otherwise stated, the experimental methods given in examples below are all conventional methods.

[0031] The materials and reagents used in examples below are commercially available, unless otherwise specified.

[0032] For the quantitative experiments in the following examples, three repeats are set and the results were averaged.

[0033] The host of the expression vector in the following examples is E. coli BL21 (DE3), purchased from Bena Bio, and the pET-28a plasmid is purchased from BioVector Plasmid Vector Strain Cell Gene Collection Center.

Preparation and Chemical Transformation of Escherichia coli Competent Cells Involved in Examples:

[0034] Escherichia coli competent cells for transformation are prepared using the Competent Cell Preparation Kit from TakaRa. For detailed operations, refer to the instruction. The E. coli BL21 cells are transformed by thermal shock at 42? C., and screened on an antibiotic resistant plate to obtain a positive transformant. The plasmid is extracted for verification by PCR and sent to GENEWIZ for sequencing.

Extraction Method of Related Plasmids Involved in Examples:

[0035] When the plasmid is extracted from a recombinant E. coli strain, the cell culture incubated to have an appropriate concentration is centrifuged, the supernatant is removed, and then the plasmid is extracted using a Plasmid Mini Preparation Kit from Generay. For detailed operations, refer to the instruction.

Culture Media Involved in Examples:

[0036] LB liquid medium (g/L): 5 yeast extract, 10 tryptone, 10 sodium chloride.

[0037] LB solid medium: On the basis of LB liquid medium, 1.5-2.0% agar powder is added.

Determination Method of L-Arginine-Glycine Amidinotransferase Enzyme Activity:

[0038] (1) Enzyme activity determining system: 100 mM Tris-HCl (pH=7), 120 mM L-arginine, 120 mM glycine, in which an appropriate amount of enzyme solution is added. [0039] (2) Enzyme activity determining method: The reaction is continued at 35? C. and 200 rpm for 1 hr, then terminated by adding 750 ?L HClO.sub.4, left to stand for 10 min, and centrifuged to remove the precipitate. 500 ?L reaction solution+500 ?L acetic acid+500 ?L ninhydrin solution are taken, and reacted at 100? C. for 1 hr. After cooling, the absorbance is measured at 515 nm.

[0040] Definition of enzyme activity: Under standard reaction conditions, the amount of enzyme needed to catalyze the production of 1 ?mol guanidinoacetic acid every 1 min is 1 unit of enzyme activity.

[0041] Determination method of specific enzyme activity of L-arginine-glycine amidinotransferase: The enzyme activity of purified L-arginine-glycine amidinotransferase (U.Math.mL.sup.?1) is determined, and the protein content of purified L-arginine-glycine amidinotransferase (mg.Math.mL.sup.?1) is determined by the Bradford method, to calculate the specific enzyme activity of L-arginine-glycine amidinotransferase.

[0042] The specific enzyme activity of L-arginine-glycine amidinotransferase is calculated by the formula:

Specific enzyme activity (U.Math.mg.sup.?1) of L-arginine-glycine amidinotransferase=enzyme activity of purified L-arginine-glycine amidinotransferase (U.Math.mL.sup.?1)/protein content of purified L-arginine-glycine amidinotransferase (mg.Math.mL.sup.?1).

Detection Methods Involved in Examples:

[0043] Detection of protein concentration: The protein concentration is determined by using the Bradford kit, with bovine serum protein as a standard.

[0044] Sample detection method: Guanidinoacetic acid is detected by Agilent high performance liquid chromatograph (equipped with an ultraviolet absorber). The chromatographic column is Waters XBridge BEH Amide 5 ?m column (4.6 mm?250 mm). The mobile phase is 30% acetonitrile aqueous solution, the detection wavelength is 210 nm, and the flow rate is 0.6 mL/min.

[0045] L-arginine, glycine, and L-ornithine are detected by using Agilent C18, 5 ?m, 4.6?250 mm chromatographic column. The flow rate is 1.0 mL.Math.min.sup.?1; the column temperature is 40? C.; and the detection wavelength is 338 nm. Mobile phase: phase A: 8.0 g sodium acetate (13.3 g sodium acetate trihydrate) is dissolved in 1000 mL water, 225 ?L triethyl amine is added, the pH value is adjusted to 7.20?0.05 with 5% acetic acid, and finally 5 mL tetrahydrofuran is added and mixed; phase B: 6.0 g sodium acetate is weighed and dissolved in 200 mL water, and the pH value is adjusted to 7.20?0.05 with 5% acetic acid. This solution was added with 400 mL HPLC grade methanol and 400 mL HPLC grade ethanol, and mixed.

Example 1: Screening and Expression of Heterologous AGAT with High Enzyme Activity

[0046] The specific steps were as follows.

1. Construction of Recombinant Strains BL21/pET-28a-AkAGAT, BL21/pET-28a-AcAGAT, and BL/pET-28a-CrAGAT

[0047] In order to obtain AGAT with higher enzyme activity, gene sequences encoding AGAT (as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 respectively) derived from Amycolatopsis kentuckyensis, Actinokineospora terrae, and Cylindrospermopsis raciborskii AWT205 were obtained respectively by searching Brenda database and NCBI BLAST, and synthesized by GENEWIZ. The agat gene fragments obtained were respectively ligated to the linearized plasmid pET-28a (amplification with P1/P2) by the homologous recombination enzyme ClonExpress II One Step Cloning Kit (Vazyme), and transformed into E. coli BL21(DE3) competent cells, to obtain transformants. The transformants were coated in an LB solid medium containing kanamycin with a concentration of 50 ?g/mL, and cultured at 37? C. for 12 hrs. The positive clone was picked up, and the single colony was verified by colony PCR using P3/P4 as primers and using Taq DNA polymerase (FIG. 1). 12 hrs after the positive single colony with the target band size was inoculated in a vial containing LB liquid medium, the plasmid was extracted, and sequenced to be correct by GENEWIZ. The successfully constructed recombinant strains were designated as BL21/pET-28a-AkAGAT, BL21/pET-28a-AcAGAT, and BL21/pET-28a-CrAGAT respectively. Primer sequences involved:

TABLE-US-00001 P1: (SEQIDNO:5) 5-AGCAAATGGGTCGCGGATCC-3 P2: (SEQIDNO:6) 5-GTGGTGCTCGAGTGCGGCCG-3 P3: (SEQIDNO:7) 5-CATGACTGGTGGACAGCAAAT-3 P4: (SEQIDNO:8) 5-GCTTTGTTAGCAGCCGGAT-3

2. Expression and Purification of L-Arginine-Glycine Amidinotransferase

[0048] The single colony of the recombinant strain successfully constructed above was inoculated into 10 mL of LB liquid medium, and cultured at 37? C. for 12 hrs. Then 1% of the cell culture was inoculated into 50 mL of LB liquid medium, and cultured to an OD.sub.600 of about 0.8. IPTG was added, and the cells were cultured at 16? C. for 16 hrs. The cells were washed three times with PBS. The collected cells were resuspended in PBS, and homogenized by an ultrasonic homogenizer, for a total time of 15 min with 3 s break after 1 s homogenization, and then centrifuged at 10000 rpm, for 20 min. The supernatant was subject to gel electrophoresis. All the proteins are shown to be solublely expressed, as shown in FIG. 2. The crude enzyme solution was purified by passing through a nickel column for protein purification, and the pure enzyme was verified by gel electrophoresis.

[0049] The pure enzyme was added into a reaction solution containing the same concentration of substrate, and the activity of L-arginine-glycine amidinotransferase from various sources was determined according to the above enzyme activity determination method. The result shows that AkAGAT derived from Amycolatopsis kentuckyensis has higher catalytic performance, and has a specific enzyme activity of 2.23?0.03 U/mg. The enzyme was selected for subsequent experiments.

TABLE-US-00002 TABLE 1 Comparison of specific enzyme activity of L-arginine- glycine amidinotransferase from various sources Enzyme Specific enzyme activity, U/mg AkAGAT 2.23 ? 0.07 AtAGAT 1.44 ? 0.04 CrAGAT 1.23 ? 0.02

Example 2: Activity Analysis of AkAGAT Pure Enzyme

[0050] In the following experiment, purified AkAGAT enzyme was used, which was obtained by inducing the strain obtained in Step 2 in Example 1 to express at 16? C. and then purifying.

[0051] The enzyme activity was studied at various reaction temperatures (20-60? C.), at various pH values (3-10), and in the presence of various metal ions (Na.sup.+, K.sup.+, Mg.sup.2+, Mn.sup.2+, Zn.sup.2+, Ca.sup.2+, Cu.sup.2+, Fe.sup.3+, EDTA), and the stability against temperature and the stability against pH were determined. The results are shown in FIGS. 3-6. The optimum reaction temperature is 35? C., the optimum pH is 7, metal ions have no effect on the enzyme activity, the thermal stability is poor, and the enzyme is resistant to acids, but not bases.

Example 3: Mining and Selection of Mutable Sites in Gene Encoding L-Arginine-Glycine Amidinotransferase

[0052] Based on the research in Example 1 and Example 2, the thermal stability of AkAGAT is poor, the enzyme activity needs to be improved, and the enzyme needs to be modified.

[0053] The specific steps were as follows.

1. Preparation of AkAGAT Enzyme Mutant

[0054] In the present invention, L-arginine-glycine amidinotransferase (AkAGAT) derived from Amycolatopsis kentuckyensis was used as a parent and modified. The mutant proposed in the present invention has at least multiple mutations at sites 225, 258, and 278 compared with the amino acid sequence of AkAGAT as shown in SEQ ID NO: 4. The mutant has at least one of the following mutations: T225Q, A258P, L278K.

[0055] PCR amplification system: Primer F 1.0 ?L, Primer R 1.0 ?L, Template 1.0 ?L, PhantaR Max (p515) DNA polymerases 25 ?L, Nuclease-free water 22 ?L Procedure for PCR amplification: pre-denaturation at 95? C. for 5 min; 30 cycles of denaturation at 95? C. for 30 s, annealing generally at a temperature of 58-60? C., for 30-60 s, and extension at 72? C. for a time set according to the amplification rate of 1500 bp per minute; and extension at 72? C. for 5 min; The product was stored at 4? C. By using P5 and P6 as the primers and pET-28a-AkAGAT constructed in Example 1 as a template, pET-28a-AkAGAT was reversely amplified by PCR using the above PCR amplification system and PCR amplification conditions to obtain the AkAGAT.sub.T225Q mutant. The amplification product was separated by 1.5% agarose gel electrophoresis and then recovered by using a commercial kit. The plasmid pET-28a-AkAGAT.sub.T225Q mutant was obtained, which was then transformed into E. coli BL21 (DE3) competent cells. A cloned colony having L-arginine-glycine amidinotransferase activity were screened out on a Luria broth (LB) plate (containing 50 mg/L kanamycin). DNA of the plasmid pET-28a-AkAGAT.sub.T225Q mutant was extracted from the cloned colony, and sequenced to determine that the introduced site mutations were correct. Thus, a recombinant strain with the enzyme mutant was obtained.

[0056] By using P7 and P8 as the primers and the constructed pET-28a-AkAGAT.sub.T225Q as a template, pET-28a-AkAGAT.sub.T225Q was reversely amplified by PCR using the above PCR amplification system and PCR amplification conditions to obtain the AkAGAT.sub.T225Q/A258P mutant. The amplification product was separated by 1.5% agarose gel electrophoresis and then recovered by using a commercial kit. The plasmid pET-28a-AkAGAT.sub.T225Q/A258P mutant was obtained, which was then transformed into E. coli BL21 (DE3) competent cells. A cloned colony having L-arginine-glycine amidinotransferase activity were screened out on a Luria broth (LB) plate (containing 50 mg/L kanamycin). DNA of the plasmid pET-28a-AkAGAT.sub.T225Q/A258P mutant was extracted from the cloned colony, and, sequenced to determine that the introduced site mutations were correct. Thus, a recombinant mutant strain was obtained.

[0057] By using P9 and P10 as the primers and the constructed pET-28a-AkAGAT.sub.T225Q/A258P as a template, pET-28a-AkAGAT.sub.T225Q/A258P was reversely amplified by PCR using the above PCR amplification system and PCR amplification conditions to obtain the AkAGAT.sub.T225Q/A258P/L278K mutant. The amplification product was separated by 1.5% agarose gel electrophoresis and then recovered by using a commercial kit. The plasmid pET-28a-AkAGAT.sub.T225Q/A258P/L278K mutant was obtained, which was then transformed into E. coli BL21 (DE3) competent cells. A cloned colony having L-arginine-glycine amidinotransferase activity were screened out on a Luria broth (LB) plate (containing 50 mg/L kanamycin). DNA of the plasmid pET-28a-AkAGAT.sub.T225Q/A258P/L278K mutant was extracted from the cloned colony, and sequenced to determine that the introduced site mutations were correct. Thus, a recombinant strain with the enzyme mutant was obtained.

TABLE-US-00003 P5: (SEQIDNO:9) 5CTTTGGTTCCAGGTGGCGTTGCA-3 P6: (SEQIDNO:10) 5-CGCCACCTGGAACCAAAGG-3 P7: (SEQIDNO:11) 5-TCAGGACCAGGCCAGGGC-3 P8: (SEQIDNO:12) 5-GGCCTGGTCCTGACCAACCC-3 P9: (SEQIDNO:13) 5-GAACTCCCAATCGTTTGCCTTGAA-3 P10: (SEQIDNO:14) 5-GCAAACGATTGGGAGTTCGTGA-3

2. Expression and Determination of Enzyme Activity of AkAGAT Enzyme Mutant

[0058] The single colony of AkAGAT mutant strain constructed above was inoculated into 10 mL of LB liquid medium (containing 50 mg/L kanamycin), and cultured at 37? C. for 12 hrs. Then 1% of the cell culture was inoculated into 50 mL of LB liquid medium, and cultured to an OD.sub.600 of about 0.8. IPTG was added, and the cells were cultured at 16? C. for 16 hrs. The cells were washed three times with PBS. The collected cells were resuspended in PBS, and homogenized by an ultrasonic homogenizer. The crude enzyme solution was purified by passing through a nickel column for protein purification, to obtain purified AkAGAT enzyme mutant. The activity of the AkAGAT enzyme mutant was determined according to the above enzyme activity determination method. The results are shown in Table 2.

[0059] The mutant enzyme is expressed in a soluble form, and at a higher level than that of wild type (see FIG. 7). Moreover, the thermal stability of the mutants AkAGAT.sub.T225Q, AkAGAT.sub.T225Q/A258P, and AkAGAT.sub.T225Q/A258P/L278K were determined at 30? C., 35? C., 40? C., and 45? C. It is found that at 40? C. and 45? C., the stability of the three mutants is improved. After 6 hrs, 30%-35% of the enzyme activity of the three mutants remains.

[0060] The enzyme activities of the mutants AkAGAT.sub.T225Q, AkAGAT.sub.T225Q/A258P, and AkAGAT.sub.T225Q/A258P/L278K are improved by 21.1%, 32%, and 36.7% respectively, compared to the wide type. The catalytic activity of AkAGAT.sub.T225Q/A258P/L278K is improved most obviously, and the specific enzyme activity can be up to 3.52?0.21 U/mg. BL21/pET-28a-AkAGAT.sub.T225Q/A258P/L278K is used in subsequent experiments.

TABLE-US-00004 TABLE 2 Comparison of enzyme activities of different AkAGAT mutants Enzyme Specific enzyme activity, U/mg AkAGAT 2.23 ? 0.07 AkAGAT.sub.T225Q 2.70 ? 0.08 AkAGAT.sub.T225Q/A258P 2.94 ? 0.20 AkAGAT.sub.T225Q/A258P/L278K 3.52 ? 0.21

Example 4: Production of Guanidinoacetic Acid Using AkAGAT Mutant Enzyme

[0061] The specific steps were as follows.

1. Comparison of Production of Guanidinoacetic Acid by Whole Cell Catalysis with Various AkAGAT Mutants

[0062] The recombinant strains E. coli BL21/pET28a-AkAGAT, E. coli BL21/pET28a-AkAGAT.sub.T225Q, E. coli BL21/pET28a-AkAGAT.sub.T225Q/A258P, and E. coli BL21/pET28a-AkAGAT.sub.T225Q/A258P/L278K were induced to express as described in Example 1. The cells were washed three times with PBS. The collected cells were resuspended in 100 mM PBS. The catalytic reaction was carried out at an initial reaction pH controlled to 7.0, a reaction temperature of 30? C., a rotational speed of 200 rpm, and a substrate concentration of 120 mM. During the reaction, samples were taken at 0, 6, 12, 18, and 24 hrs. The sample was centrifuged at 12000 g/min for 10 min. The supernatant was collected, and passed through a 0.22 ?m filter membrane, and the yield of guanidinoacetic acid was determined by HPLC.

[0063] By determining the yield of guanidinoacetic acid in the samples taken in various periods, it is found that there is no related enzyme degrading guanidinoacetic acid in Escherichia coli. With the extension of conversion time, the yield of guanidinoacetic acid increases constantly. The results are shown in FIG. 8. After 24 hrs of reaction, 7.06 g/L guanidinoacetic acid can be obtained in the reaction system involving AkAGAT.sub.T225Q/A258P/L278K enzyme, and the conversion rate is 50.28%, which is 49.6%, 29.4%, and 17% higher than that in the systems involving AkAGAT, AkAGAT.sub.T225Q, and AkAGAT.sub.T225Q/A258P respectively. Therefore, the mutant enzyme AkAGAT.sub.T225Q/A258P/L278K can significantly improve the catalytic efficiency, save the reaction cost, and improve the yield of guanidinoacetic acid.

2. Optimization of Conditions for Producing Guanidinoacetic Acid by Mutant Enzyme AkAGAT.SUB.T225Q/A258P/L278K

[0064] In order to achieve the optimum conversion effect, the conversion conditions need to be optimized, mainly with respect to the type of buffer, cell concentration, substrate concentration and the amount of permeabilizer in the conversion system.

[0065] Effects of various buffers on GAA production: After the cells were collected, the cells were suspended and concentrated into PBS buffer/Tris-HCl buffer/MOPS buffer. The same volume of cell concentrate was added into a 50 mL triangular flask, and the final cell concentration in the reaction system was 30. The reaction was continued for 24 hrs in a whole cell catalytic system of 0.1M PBS/Tris-HCl/MOPS (pH=7.4), with a substrate concentration of 120 mM, at a reaction temperature of 35? C., and a rotational speed of 200 rpm. Samples were taken at 0, 6, 12, 18, and 24 hrs, and centrifuged at 12000 rpm for 10 min. The supernatant was collected, 10-fold diluted, and filtered through a 0.22 ?m filter membrane. The yield of GAA was determined by HPLC. The result shows that 0.1 M Tris-HCl is more suitable for this conversion system, where the yield of GAA reaches 8.41 g/L, which is increased by 19.7% and 12% compared with 0.1M PBS/MOPS respectively.

[0066] Effects of various cell concentrations on GAA production: After the cells were collected, the cells were suspended and concentrated to an appropriate volume, and the cell concentration was determined (OD.sub.600). Various volumes of the cell concentrates were respectively added to a 50 mL triangular flask, and the final cell concentrations in the reaction system were 10, 20, 30, 40, 50 and 60 respectively. The reaction was continued for 24 hrs in a whole cell catalytic system of 0.1M Tris-HCl (pH=7.4), with a substrate concentration of 120 mM, at a reaction temperature of 35? C., and a rotational speed of 220 rpm. Samples were taken at 0, 6, 12, 18, and 24 hrs, and centrifuged at 12000 rpm for 10 min. The supernatant was collected, 10-fold diluted, and filtered through a 0.22 ?m filter membrane. The yield of GAA was determined by HPLC. The result shows that when OD.sub.600=40, the yield of GAA is the highest and is 9.12 g/L.

TABLE-US-00005 TABLE 3 Effects of various cell concentrations on GAA production Cell concentration (OD.sub.600) 10 20 30 40 50 60 GAA concentration (g/L) 6.60 8.30 7.59 9.12 8.60 8.71

[0067] Effect of substrate concentrations on GAA synthesis: With the optimum cell concentration, various concentrations of substrates were respectively added into the system, including {circle around (1)} Arg 50 mM, Gly 50 mM, {circle around (2)} Arg 100 mM, Gly 100 mM, {circle around (3)} Arg 150 mM, Gly 150 mM, {circle around (4)} Arg 200 mM, Gly 200 mM, {circle around (5)} Arg 50 mM, Gly 100 mM, and {circle around (6)} Arg 100 mM, Gly 50 mM. After the reaction, samples were taken, the yield of GAA was determined, and the maximal conversion rate was calculated. The result is shown in FIG. 9. When OD.sub.600 is 40, it is found that when arginine:glycine=1:1 is used as the substrate and the substrate concentration is 150 mM, the yield is the highest, but the conversion rate is less high. When the substrate concentration is 50 mM, the conversion rate is the highest, but the yield is low. When the substrate concentration is 100 mM, the yield and the conversion rate are both high, the yield is 9.31 g/L, and the conversion rate is 80%. In addition, when arginine:glycine is 1:2 and arginine:glycine is 2:1, the yield is lower than that when the ratio is 1:1.

[0068] In summary, in the subsequent experiments, 0.1M Tris-HCl was used as the buffer system, the cells were added to give an OD.sub.600 of 40, and L-arginine and glycine were added at a ratio of 1:1 and have a concentration of 100 mM respectively, for production of guanidinoacetic acid by whole cell conversion.

3. Scaling Up of Conversion to Guanidinoacetic Acid

[0069] In order to efficiently produce guanidinoacetic acid by conversion by recombinant E. coli BL21/pET28a-AkAGAT.sub.T225Q/A258P/L278K, the conversion by the recombinant strain in a large-scale system was studied under the optimized whole-cell catalytic conditions. The cells were suspended in 1 L 0.1M Tris-HCl buffer, the reaction temperature was controlled to 35? C., the initial pH was 7.4, the substrates L-arginine and glycine were each 200 mM, 1% Triton was added, and the cell concentration was such that OD.sub.600 was 40. The conversion was carried out under these conditions. The content of each component in the conversion solution was detected by liquid chromatography.

[0070] The result shows that the reaction proceeds smoothly with time, during which L-arginine and glycine are consumed to produce guanidinoacetic acid, and the conversion system gradually becomes stable after 24 hrs of catalytic reaction. At a substrate concentration of 200 mM, a total of 21.4 g.Math.L.sup.?1 guanidinoacetic acid is produced, with a conversion rate of 90.4%.

[0071] The above-described embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions or modifications can be made by those skilled in the art based on the present invention, which are within the scope of the present invention as defined by the claims. The scope of the present invention is defined by the appended claims.