Method for synthesizing glycine by catalyzing oxalic acid through multiple enzymes in vitro

Abstract

The invention provides a method for synthesizing glycine by catalyzing oxalic acid through multiple enzymes in vitro, the oxalic acid is used as a substrate, an enzyme catalysis reaction is carried out under the action of a multi-enzyme reaction system, the glycine (Glycine) is generated, and the multi-enzyme reaction system comprises oxalyl-CoA ligase, OCS, EC 6.2. 1.8), oxalyl-CoA reductase, OCR, EC 1.2. 1.17) and / or alanine dehydrogenase, ALD, EC 1.4. 1.1). The method provided by the invention has the advantages of simple synthesis method, mild reaction conditions, short synthesis route, strong controllability, high conversion rate, capability of avoiding accumulation of byproducts and the like, and is beneficial to industrial production of glycine.

Description

Technical Field

[0001] The invention relates to the technical field, and in particular to a method for synthesizing glycine from oxalic acid catalyzed by multiple enzymes in vitro. Background Art

[0002] Glycine is widely used in the fields of food, medicine, pesticides, etc. as an important fine chemical. The main methods for synthesizing glycine include chloroacetic acid ammonolysis, Streeck method, aminoacetonitrile method, direct hyne method and biosynthesis method (Zhu Shiying, Research on Synthesis Process of Glycine, Zhejiang Chemical Industry, 1990 (3): 40-44). The chloroacetic acid ammonolysis method commonly used in China has the problems of serious environmental pollution, high cost, low product purity and poor product quality. The Streeck method used abroad has the problems of toxic raw materials, unfriendly environment and geographical restrictions. Compared with traditional chemical synthesis methods, biosynthesis methods have the advantages of mild conditions, high selectivity and environmental friendliness. Nitto Chemical Industry Co., Ltd. of Japan reacts microorganisms such as Pseudomonas, Casein, and Alcaligenes with glycineamide for 45 hours at a temperature of 30°C and a pH value of 7.9-8.1, so that glycineamide is hydrolyzed and converted into glycine, with a conversion rate of up to 99% (JP04304892). Li Zhiruan et al. used genetic means to mutate ATP phosphoribosyltransferase (His G), which improved the ability of Corynebacterium microorganisms to produce glycine by about 1.6 times (CN111433366B). The biosynthesis method has certain development potential due to its advantages such as mild conditions, high selectivity of target products, no environmental pollution and low energy consumption, but it is still in the research and development stage and has not yet been industrialized.

[0003] Oxalic acid is a common organic acid that is corrosive, cheap, and has many uses, such as in the production of dyes, detergents, and metal chelators. Oxalic acid can be directly synthesized using carbon dioxide and electricity. Compared with the traditional fossil fuel method, the electrochemical method can reduce dependence on fossil fuel resources, achieve sustainable use of energy, and provide key technologies for "carbon neutrality" and the green innovation and development of the social economy.

[0004] In vitro multi-enzyme catalysis technology is a process that imitates the metabolic pathways in microorganisms and uses purified multiple enzymes and cofactors to efficiently convert specific substrates into target products in an in vitro environment. In vitro biotransformation is not limited by cell activity and complexity, and has the advantage of strong controllability. The yield of the target product can be increased by controlling and optimizing reaction conditions such as temperature, pH value, substrate concentration, and enzyme concentration. In vitro multi-enzyme catalysis has been successful in the synthesis of PHB, inositol, and starch. However, in the process of in vitro multi-enzyme catalysis to synthesize glycine, multiple steps and different enzymes are required, and there are often problems such as cumbersome reaction steps and low synthesis efficiency. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for synthesizing glycine from oxalic acid using in vitro multi-enzyme catalysis. The method is based on in vitro multi-enzyme catalysis and is a glycine synthesis method with a simple method, few catalytic reaction steps, strong controllability, and environmental protection.

[0006] In one aspect, the present invention provides a method for synthesizing glycine from oxalic acid by multi-enzyme catalysis in vitro, wherein oxalic acid is used as a substrate, and an enzyme catalyzed reaction is carried out under the action of a multi-enzyme reaction system to generate glycine.

[0007] The multi-enzyme reaction system comprises oxalyl-CoA ligase (OCS, EC6.2.1.8), oxalyl-CoA reductase (OCR, EC 1.2.1.17) and alanine dehydrogenase (ALD, EC 1.4.1.1).

[0008] In one embodiment of the present invention, the source of the oxalyl-CoA ligase includes, but is not limited to, Arabidopsis thaliana, Lathyrus sativus L., Medicagotruncatula, Glycine max, Rosa chinensis, Rosellinianecatrix, Vigna umbellata, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. In a specific embodiment of the present invention, the oxalyl-CoA ligase is derived from Lathyrus sativus L., and its protein sequence number is QID21531.1.

[0009] In one embodiment of the present invention, the source of the oxalyl-CoA reductase includes but is not limited to Altererythrobacter epoxidivorans, Haliotis discusdiscus, and Methylobacterium extorquens AM1. In a specific embodiment of the present invention, the oxalyl-CoA reductase is derived from Methylobacterium extorquens AM1, and its protein sequence number is WP_012752770.1.

[0010] In one embodiment of the present invention, the source of the alanine dehydrogenase includes but is not limited to Staphylococcus aureus, Bacillus subtilis, Bilophilawadsworthia, Lysinibacillus sphaericus, Mycobacterium tuberculosis, Oceanobacillus iheyensis, Staphylococcus epidermidis, Streptococcuschenjunshii, Bacillus tropicus. In a specific embodiment of the present invention, the alanine dehydrogenase is derived from Mycobacterium tuberculosis, and its protein sequence number is NP_217296.

[0011] In one embodiment of the present invention, in the enzyme catalyzed reaction, the amount of oxalyl-CoA ligase added is 0.5 mg / mL, the amount of oxalyl-CoA reductase added is 0.5 mg / mL, and the amount of alanine dehydrogenase added is 0.25 mg / mL.

[0012] In one embodiment of the present invention, in the enzyme-catalyzed reaction, the concentration of oxalic acid is 10 mM.

[0013] In one embodiment of the present invention, the time of the enzyme catalytic reaction is 0-7 hours, preferably 0.5-4 hours, and specifically 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, or 7 hours.

[0014] In one embodiment of the present invention, the temperature of the enzyme catalyzed reaction is 37° C. and the pH value is 6.0.

[0015] In one embodiment of the present invention, the multi-enzyme reaction system further comprises a buffer, a magnesium salt, an ammonium salt, and a cofactor.

[0016] In one embodiment of the present invention, the buffer is a HEPES buffer. Preferably, the pH of the buffer is 6.0. Preferably, the concentration of the buffer in the reaction system is 100 mM.

[0017] In one embodiment of the present invention, various magnesium salts can be used in the present invention, such as magnesium sulfate, magnesium chloride, etc. Preferably, the magnesium salt is magnesium sulfate. Preferably, the concentration of the magnesium salt in the reaction system is 5 mM.

[0018] In one embodiment of the present invention, various ammonium salts can be used in the present invention, such as ammonium sulfate, ammonium chloride, etc. Preferably, the ammonium salt is ammonium sulfate. Preferably, the concentration of the ammonium salt in the reaction system is 50 mM.

[0019] In one embodiment of the present invention, the cofactors are NADH, NADPH, and ATP. Preferably, the concentrations of the cofactors NADH, NADPH, and ATP in the reaction system are 15 mM, respectively.

[0020] In one embodiment of the present invention, the multi-enzyme reaction system further comprises polyphosphate kinase (PPK) and pyrophosphate. In the present invention, the polyphosphate kinase (PPK) and pyrophosphate are used for ATP energy regeneration.

[0021] In one embodiment of the present invention, the multi-enzyme reaction system further comprises glucose, glucose dehydrogenase (GDH), or the multi-enzyme reaction system further comprises formate, formate dehydrogenase (FDH). In the present invention, the glucose, glucose dehydrogenase (GDH), or the formate, formate dehydrogenase (FDH) is used for the regeneration of NADH and NADPH.

[0022] In one embodiment of the present invention, the NADH and NADPH can also be regenerated by electrochemical method.

[0023] In one embodiment of the present invention, the multi-enzyme reaction system is in the form of intact microbial cells, permeabilized microbial cells, one or more components of microbial cell extracts, partially purified enzymes or purified enzymes.

[0024] In one embodiment of the present invention, the multi-enzyme reaction system exists in the form of partially purified enzymes or purified enzymes.

[0025] In one embodiment of the present invention, glycine is synthesized by a one-pot method.

[0026] In one embodiment of the present invention, the glycine concentration can reach 9.6 mM, and the molar conversion rate of glycine to oxalic acid is as high as 96%.

[0027] In one embodiment of the present invention, the method comprises the following reaction steps: S1, under the action of oxalyl-CoA ligase, oxalic acid and CoA combine to generate oxalyl-CoA; S2, under the action of oxalyl-CoA reductase, oxalyl-CoA is reduced to glyoxylate; S3, under the action of alanine dehydrogenase, glyoxylate undergoes a reductive amination reaction to generate glycine.

[0028] In one embodiment of the present invention, the reaction step S1 is performed in the presence of ATP.

[0029] In one embodiment of the present invention, the reaction steps S2 and S3 are carried out in the presence of NAD(P)(H).

[0030] In a second aspect, the present invention provides an application of a multi-enzyme reaction system in the preparation of glycine.

[0031] In one embodiment of the present invention, the multi-enzyme reaction system includes oxalyl-CoA ligase (OCS, EC 6.2.1.8), oxalyl-CoA reductase (OCR, EC 1.2.1.17) and / or alanine dehydrogenase (ALD, EC 1.4.1.1).

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The present invention can use oxalic acid as a substrate to synthesize glycine through a one-pot method using three enzymes.

[0034] 2. The method of the present invention has the advantages of simple synthesis method, mild reaction conditions, short synthesis pathway, strong controllability, high conversion rate, and ability to avoid accumulation of by-products, and is conducive to the industrial production of glycine.

[0035] 3. In the multi-enzyme catalyzed oxalate-to-glycine synthesis system constructed in the present invention, the regeneration of ATP, NADH and NADPH can be achieved, thereby reducing the dosage and use cost of ATP, NAD+ and NADP+. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The present invention provides a design diagram for the one-pot synthesis of glycine using oxalate as substrate via oxalyl-CoA ligase, oxalyl-CoA reductase and alanine dehydrogenase.

[0037] Figure 2 It is SDS-PAGE to detect OCS, OCR and ALD, and M is Marker.

[0038] Figure 3This is the HPLC test result of producing glycine from oxalic acid by one-pot method using oxalyl-CoA ligase, oxalyl-CoA reductase and alanine dehydrogenase. DETAILED DESCRIPTION

[0039] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0040] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0041] Materials used in the embodiments of the present invention:

[0042] pET28a vector, Novagen, Madison, WI; Escherichia coli expression bacteria BL21 (DE3), Invitrogen, Carlsbad, CA; β-nicotinamide adenine dinucleotide (NAD+), Aladdin, Shanghai, China; the enzymes in the present invention were all obtained by prokaryotic expression according to genetic engineering methods.

[0043] Example 1 Expression of key enzymes in the glycine biosynthesis pathway

[0044] The present invention constructs a catalytic pathway for converting oxalic acid into glycine through an in vitro multi-enzyme catalytic system. Figure 1 The key enzymes involved in this pathway include: ① Oxalyl-CoA ligase (OCS, EC 6.2.1.8), used to convert oxalate into oxalyl-CoA; ② Oxalyl-CoA reductase (OCR, EC1.2.1.17), used to convert oxalyl-CoA into glyoxylate; ③ Alanine dehydrogenase (ALD, EC 1.4.1.1), used to convert glyoxylate into glycine. The information of the enzymes used in this example is shown in Table 1.

[0045] Oxaloyl-CoA ligase is derived from Lathyrus sativus L., and its protein sequence number is QID21531.1; oxalyl-CoA reductase is derived from Methylobacterium extorquensAM1, and its protein sequence number is WP_012752770.1; alanine dehydrogenase is derived from Mycobacterium tuberculosis, and its protein sequence number is NP_217296. After obtaining the protein sequences of oxalyl-CoA ligase, oxalyl-CoA reductase, and alanine dehydrogenase on the NCBI website (https: / / www.ncbi.nlm.nih.gov), they were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for codon optimization and gene synthesis, and cloned into the pET28a expression vector. Then, these plasmids were transformed into Escherichia coli BL21 (DE3) (Invitrogen, Carlsbad, CA) for protein expression and purification. The results of protein purification are shown in Figure 2. Figure 2 shown.

[0046] Table 1 Information of enzymes used in the present invention

[0047]

[0048] Example 2 One-pot synthesis of glycine

[0049] The method for synthesizing glycine by using oxalyl-CoA ligase, oxalyl-CoA reductase, and alanine dehydrogenase to catalyze oxalic acid is as follows:

[0050] A 1.0 mL reaction system contained 100 mM HEPES buffer (pH 6.0), 10 mM oxalic acid, 5 mM magnesium sulfate, 15 mM ATP, 15 mM NAD+, 15 mM NADP+, 2 mM CoA, 50 mM ammonium sulfate, 0.5 mg / mL oxalyl-CoA ligase, 0.5 mg / mL oxalyl-CoA reductase, and 0.25 mg / mL alanine dehydrogenase. The catalytic reaction was carried out at 37° C., and the reaction sampling time was 0 hour, 0.5 hour, 1 hour, 2 hours, and 4 hours.

[0051] The sample extracted from the reaction solution was first stopped by adding 1.88M HClO4, and then the pH was adjusted to neutral with 5M NaOH. The synthesis of glycine was determined by HPLC using an Agilent 1260 HPLC, an Agilent ZORBAX Eclipse AAA column (4.6×150mm, 3.5μm) and a UV detector. The sample was pre-column derivatized with boric acid and o-phthalaldehyde (OPA). Mobile phase A was NaH2PO4 (40mM, pH7.8), and mobile phase B was acetonitrile: methanol: water = 45:45:10 (v / v / v). The HPLC gradient program was: 2% B, 1 min; 2%-30% B 6 min; 30% B, 5 min; 30%-100% B 0.5 min; 100% B 5 min; 100%-30% B 0.5 min; 30% B 4 min; 30% B 2% 0.5 min; 2% B 2.5 min. The detection wavelength was 338 nm, the column oven temperature was 40° C., and the flow rate was 1 mL / min.

[0052] Detected by high performance liquid chromatography, Figure 3 As shown, at 4 hours, the glycine concentration was 9.6 mM. Therefore, the molar conversion of glycine to oxalate was 96%.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for synthesizing glycine from oxalic acid using multi-enzyme catalysis in vitro, characterized in that: With oxalic acid as substrate, glycine is generated through enzyme catalysis under the action of multi-enzyme reaction system. The multi-enzyme reaction system comprises oxalyl-CoA ligase (OCS, EC 6.2.1.8), oxalyl-CoA reductase (OCR, EC 1.2.1.17) and alanine dehydrogenase (ALD, EC 1.4.1.1).

2. The method for synthesizing glycine from oxalic acid using multi-enzyme catalysis in vitro as claimed in claim 1, characterized in that: The sources of the oxalyl-CoA ligase include, but are not limited to, Arabidopsis thaliana, Lathyrus sativus L., Medicago truncatula, Glycine max, Rosa chinensis, Rosellini necatrix, Vigna umbellata, Schizosaccharomyces pombe, and Saccharomyces cerevisiae; preferably, the oxalyl-CoA ligase is derived from Lathyrus sativus L., and its protein sequence number is QID21531.

1.

3. The method for synthesizing glycine from oxalic acid using multi-enzyme catalysis as claimed in claim 1, characterized in that: The sources of the oxalyl-CoA reductase include but are not limited to Altererythrobacter epoxidivorans, Haliotis discus discus, and Methylobacterium extorquens AM1; preferably, the oxalyl-CoA reductase is derived from Methylobacterium extorquens AM1, and its protein sequence number is WP_012752770.

1.

4. The method for synthesizing glycine from oxalic acid using multi-enzyme catalysis in vitro as claimed in claim 1, characterized in that: The sources of the alanine dehydrogenase include, but are not limited to, Staphylococcus aureus, Bacillus subtilis, Bilophila wadsworthia, Lysinibacillus sphaericus, Mycobacterium tuberculosis, Oceanobacillus iheyensis, Staphylococcus epidermidis, Streptococcus chenjunshii, and Bacillus tropicalus; preferably, the alanine dehydrogenase is derived from Mycobacterium tuberculosis, and its protein sequence number is NP_217296.

5. The method for synthesizing glycine from oxalic acid using multi-enzyme catalysis in vitro as claimed in claim 1, characterized in that: In the enzyme catalytic reaction, the amount of oxalyl-CoA ligase added was 0.5 mg / mL; the amount of oxalyl-CoA reductase added was 0.5 mg / mL; the amount of alanine dehydrogenase added was 0.25 mg / mL; the concentration of oxalic acid was 10 mM; Preferably, the enzyme catalyzed reaction time is 0-7h, preferably 0.5-4h; Preferably, the temperature of the enzyme catalyzed reaction is 37° C. and the pH value is 6.

0.

6. The method for synthesizing glycine from oxalic acid using multiple enzymes in vitro as claimed in claim 1, characterized in that: The multi-enzyme reaction system also includes a buffer, a magnesium salt, an ammonium salt, and a cofactor; Preferably, the buffer is HEPES buffer; pH is 6.0; Preferably, the concentration of the buffer in the reaction system is 100 mM; Preferably, the magnesium salt includes but is not limited to magnesium sulfate and magnesium chloride; more preferably, the magnesium salt is magnesium sulfate; Preferably, the concentration of magnesium salt in the reaction system is 5 mM; Preferably, the ammonium salt includes but is not limited to ammonium sulfate, ammonium chloride, etc. Preferably, the ammonium salt is ammonium sulfate; Preferably, the concentration of ammonium salt in the reaction system is 50 mM; Preferably, the cofactor is NADH, NADPH, ATP; Preferably, the concentrations of the cofactors NADH, NADPH and ATP in the reaction system are 15 mM respectively.

7. The method for synthesizing glycine from oxalic acid using multi-enzyme catalysis in vitro as claimed in claim 1, characterized in that: The multi-enzyme reaction system also includes polyphosphate kinase (PPK) and pyrophosphate; Preferably, the multi-enzyme reaction system further comprises glucose and glucose dehydrogenase (GDH), or the multi-enzyme reaction system further comprises formate and formate dehydrogenase (FDH); Preferably, the NADH and NADPH can also be regenerated by electrochemical method.

8. The method for synthesizing glycine from oxalic acid using multiple enzymes in vitro as claimed in claim 1, characterized in that: The multi-enzyme reaction system is in the form of intact microbial cells, permeabilized microbial cells, one or more components of microbial cell extracts, partially purified enzymes or purified enzymes; Preferably, the multi-enzyme reaction system is in the form of partially purified enzymes or purified enzymes; Preferably, glycine is synthesized using a one-pot process.

9. The method for synthesizing glycine from oxalic acid using multi-enzyme catalysis in vitro as claimed in claim 1, characterized in that: The method comprises the following reaction steps: S1, under the action of oxalyl-CoA ligase, oxalic acid and CoA are combined to generate oxalyl-CoA; S2, under the action of oxalyl-CoA reductase, oxalyl-CoA is reduced to glyoxylate; S3, under the action of alanine dehydrogenase, glyoxylate undergoes a reductive amination reaction to generate glycine; Preferably, the reaction step S1 is carried out in the presence of ATP; Preferably, the reaction steps S2 and S3 are carried out in the presence of NAD(P)(H).

10. Use of a multi-enzyme reaction system in the preparation of glycine; the multi-enzyme reaction system comprises oxalyl-CoA ligase (OCS, EC 6.2.1.8), oxalyl-CoA reductase (OCR, EC 1.2.1.17) and / or alanine dehydrogenase (ALD, EC 1.4.1.1).

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