Application of L-threonine aldolase in efficient synthesis of L-serine
The preparation of L-serine by catalyzing formaldehyde and glycine reaction using L-threonine aldolase, the problems of complex production process and high cost in the prior art are solved, and efficient and environmentally friendly L-serine synthesis is achieved, and the yield and purity meet industrial needs.
Patent Information
- Application Number
- CN202510475265.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-16
- Publication Date
- 2025-08-15
AI Technical Summary
The prior art has problems such as cumbersome reaction steps, high costs, serious environmental pollution and difficult to meet industrial demands in the production methods of L-serine. In particular, the existing enzymatic method catalytic process is complex and requires coenzyme tetrahydrofolate, resulting in an increase in production costs.
L-threonine aldolase from Chelativorans petroleitrophicus, Aureimonas sp.AU20, Pseudohoeflea suaedae was prepared by catalyzing the aldol condensation reaction of formaldehyde and glycine, and pyridoxal phosphate was used as coenzyme to simplify the catalytic process and proceed under mild conditions to avoid the generation of by-products.
It has achieved efficient and environmentally friendly L-serine synthesis, significantly improved yield and yield, high optical purity of the product, and has industrial application potential, and simplified subsequent separation steps.
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Abstract
Description
(1) Technical field
[0001] The invention belongs to the fields of chemical engineering and enzyme engineering, and particularly relates to the application of three L-threonine aldolases from Chelativorans petroleitrophicus, Aureimonas sp.AU20 and Pseudohoeflea suaedae in the efficient synthesis of L-serine. (2) Background technology
[0002] L-serine (chemical formula C3H7NO3), also known as L-2-amino-3-hydroxypropionic acid, is an important intermediate metabolite in organisms with numerous important physiological functions. It serves as a precursor for the synthesis of various amino acids, including glycine, nucleotides, choline, and phospholipids. It is widely used in the pharmaceutical, food, and cosmetic industries. For example, L-serine can be added to high-end cosmetics to enhance their moisturizing properties. It also has certain antibacterial and surfactant properties. L-serine can react with sugars to produce a Maillard reaction at high temperatures, giving foods a distinctive flavor.
[0003] L-serine production methods include protein hydrolysis and extraction, chemical synthesis, conversion, and microbial fermentation. The main disadvantage of the chemical synthesis method is the cumbersome reaction steps and the need for multiple purification steps to obtain optically pure chiral L-serine. The protein hydrolysis method is a relatively cumbersome process, the hydrolysis endpoint is difficult to accurately determine, the L-serine loss rate is high, and it also poses serious environmental pollution. Currently, the main method for producing L-serine is the precursor fermentation method, but this method is difficult to separate later, resulting in high costs and high requirements for production equipment. The bioenzymatic method has the advantages of mild reaction conditions, high stereoselectivity, and environmental friendliness, and has been widely used in the industrial production of chiral chemicals such as pharmaceutical intermediates and fine chemicals. Currently, the enzyme involved in the biosynthesis of L-serine is mainly serine hydroxymethyltransferase (SHMT). Its catalytic process is relatively complex, requiring not only PLP as a coenzyme but also tetrahydrofolate to catalyze the reaction to produce L-serine, which increases production costs.
[0004] Therefore, there is still a need to find a relatively simple and efficient method for producing L-serine. In recent years, many researchers at home and abroad have achieved L-serine production through microbial fermentation using renewable raw materials by modifying Escherichia coli and Corynebacterium glutamicum. However, key indicators such as output and yield still fail to meet the requirements of industrial production.
[0005] L-Threonine aldolase (L-TA) is ubiquitous in nature, present in bacteria, fungi, and mammalian species. Approximately 5,000 sequences annotated as LTA are currently available in databases (e.g., NCBI, Swiss-Prot). However, only a few genes have been structurally, mechanistically, and biochemically characterized.
[0006] Threonine aldolase (TA; EC4.1.2.5) is a glycine-dependent aldolase. Its defining characteristic is its ability to react with amino acids as donors in the presence of the cofactor pyridoxal phosphate (PLP) to produce β-hydroxy-α-amino acids. Threonine aldolase catalyzes the cleavage of threonine into glycine and acetaldehyde. Under certain conditions, it can also catalyze the synthesis of threonine from glycine and acetaldehyde. Studies have shown that this enzyme exhibits a wide substrate tolerance for its aldehyde acceptor, including various substituted aromatic and aliphatic aldehydes. To date, the most studied enzyme for L-serine production is serine hydroxymethyltransferase, which has a complex reaction process and is primarily used for fermentation-based L-serine production. Furthermore, other reported L-serine production methods involve multiple enzymes in a complex enzymatic cascade, resulting in relatively low yields.
[0007] Therefore, it is crucial to discover enzymes that can catalyze L-serine synthesis relatively simply and efficiently. (3) Summary of the invention
[0008] The present invention aims to provide an application of L-threonine aldolase in the efficient synthesis of L-serine. The enzyme can effectively catalyze the aldol condensation reaction of formaldehyde and glycine to prepare L-serine. The catalytic process is simple, no by-products are generated in the reaction, and subsequent separation is simple. This solves the problem that the yield of L-serine synthesized by the existing precursor fermentation method cannot fully meet market demand.
[0009] The technical solution adopted in the present invention is:
[0010] The invention provides an application of L-threonine aldolase in efficiently synthesizing L-serine. The L-threonine aldolase comprises threonine aldolase family proteins derived from Chelativorans petroleitrophicus, Aureimonas sp.AU20 and Pseudohoefleasuaedae.
[0011] Furthermore, the L-threonine aldolase is one of the following: derived from Chelativorans petroleitrophicus, denoted as Cp-LTA, with an amino acid sequence as shown in SEQ ID No.2, and a nucleotide sequence of the encoding gene as shown in SEQ ID No.1; derived from Aureimonas sp.AU20, denoted as AU20-LTA, with an amino acid sequence as shown in SEQ ID No.4, and a nucleotide sequence of the encoding gene as shown in SEQ ID No.3; derived from Pseudohoeflea suaedae, denoted as Ps-LTA, with an amino acid sequence as shown in SEQ ID No.6, and a nucleotide sequence of the encoding gene as shown in SEQ ID No.5.
[0012] Furthermore, the application method comprises the following steps: using a crude enzyme solution obtained by ultrasonically crushing wet cells obtained by induction culture of a recombinant genetically engineered bacterium expressing L-threonine aldolase as a catalyst, using formaldehyde and glycine as substrates, using pyridoxal phosphate (PLP) as a coenzyme, adding KCl and mercaptoethanol, and using a pH 3-11 buffer as a reaction medium to form a reaction system, and reacting at 20-70° C. and 200-500 rpm (preferably 60° C. and 400 rpm) to obtain a reaction solution containing L-serine after the reaction is completed.
[0013] Furthermore, in the reaction system, the catalyst is added to a final concentration of 20-80 g / L (preferably 50 g / L) based on the weight of the wet cells before crushing; formaldehyde is added to a final concentration of 1-300 mM (preferably 100 mM); glycine is added to a final concentration of 10-250 g / L (preferably 50 g / L); the pyridoxal phosphate is added to a final concentration of 0.1-50 g / L (preferably 0.625 g / L); KCl is added to a final concentration of 10-50 g / L (preferably 37.5 g / L); and mercaptoethanol is added to a final concentration of 0.5-70 mL / L (preferably 17.5 mL / L).
[0014] Furthermore, the buffer solution preferably has a pH of 9-10. More preferably, when the L-threonine aldolase is Cp-LTA, the buffer solution is a 0.1 M Na2CO3-NaHCO3 buffer solution at a pH of 10. When the L-threonine aldolase is AU20-LTA, the buffer solution is a 0.1 M Na2CO3-NaHCO3 buffer solution at a pH of 9. When the L-threonine aldolase is Ps-LTA, the buffer solution is a 0.1 M Tris-HCl buffer solution at a pH of 9.
[0015] Furthermore, the host cell of the recombinant genetically engineered bacteria expressing L-threonine aldolase can be any conventional host cell in the art, as long as the recombinant expression vector can stably replicate on its own and can effectively express the target protein after being induced by an inducer. Escherichia coli is preferred, and E. coli BL21 (DE3) is preferred.
[0016] Furthermore, the recombinant genetically engineered bacteria expressing L-threonine aldolase are constructed according to the following steps: the coding gene fragment of L-threonine aldolase is inserted between the NdeⅠ and HindⅢ restriction sites of the expression plasmid pET-21b to obtain a recombinant plasmid; and the recombinant plasmid is transformed into the expression host Escherichia coli BL21 (DE3) to obtain a recombinant genetically engineered bacteria.
[0017] Furthermore, the crude enzyme solution was prepared as follows: the recombinant genetically engineered bacteria expressing L-threonine aldolase were inoculated into a test tube of LB liquid culture medium containing 100 mg / L ampicillin, and cultured overnight at 37°C and 220 rpm in a constant temperature shaker; the bacterial solution was inoculated into LB liquid culture medium containing 100 mg / L ampicillin at an inoculum concentration of 1% by volume, and cultured at 37°C and 220 rpm in a constant temperature shaker until the OD 600 When the pH value reaches 0.6, add IPTG to a final concentration of 0.1 mM and induce expression in a constant temperature shaker at 28°C and 220 rpm for 16 h. After the induction, centrifuge the bacterial solution at 4°C and 4000 rpm for 10 min, remove the supernatant, and store the wet bacteria in a refrigerator at -20°C for later use.
[0018] The wet cells were resuspended in PBS buffer (0.1 M, pH = 7), and then 1% CTAB (cetyltrimethylammonium bromide) was added. Ultrasonic disruption was performed at 300W for 15 min, with a working time of 2 s and an interval of 3 s, at 4°C and 12000 rpm. The cells were centrifuged for 15 min, and the supernatant was collected to obtain the crude enzyme solution.
[0019] The catalyst of the present invention can also be crude enzyme powder obtained by drying crude enzyme liquid or pure enzyme liquid.
[0020] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:
[0021] The present invention screened three new L-threonine aldolases for synthesizing L-serine. Compared with existing enzymes for synthesizing L-serine, the L-threonine aldolase of the present invention uses PLP as a coenzyme but does not require the presence of tetrahydrofolate to catalyze the production of L-serine from formaldehyde and glycine. This simplifies the catalytic process, produces no by-products, and has mild reaction conditions, environmental friendliness, and high atom economy.
[0022] The L-threonine aldolase of the present invention can effectively catalyze the aldol condensation reaction of formaldehyde and glycine to prepare L-serine. Under optimal conditions, 0.1M formaldehyde can be converted into 4.14g / L-6.17g / L of L-serine in a reaction of 2 hours, with a yield of 39.43%-58.76%. Key indicators such as yield and yield indicate that the L-threonine aldolase has industrial application potential. In addition, the product has high optical purity, showing good application prospects in the synthesis of L-serine. (IV) Description of the accompanying drawings
[0023] Figure 1 , agarose gel electrophoresis diagram of the bacterial liquid, supernatant and precipitate after induced expression of the engineered bacteria; A represents Cp-LTA engineered bacteria; B represents AU20-LTA engineered bacteria; C represents Ps-LTA engineered bacteria.
[0024] Figure 2 , schematic diagram of the principle of L-threonine aldolase catalyzing the condensation reaction of formaldehyde and glycine to produce L-serine.
[0025] Figure 3 , bar chart showing the yield of L-serine, the product, in the condensation reaction of formaldehyde and glycine catalyzed by L-threonine aldolase.
[0026] Figure 4 , L-serine peak area curve in the condensation reaction solution of formaldehyde and glycine catalyzed by Cp-LTA at different temperatures.
[0027] Figure 5 , L-serine peak area curve in the condensation reaction solution of formaldehyde and glycine catalyzed by Cp-LTA at different pH.
[0028] Figure 6 , L-serine peak area curve in the condensation reaction solution of formaldehyde and glycine catalyzed by Cp-LTA after formaldehyde incubation for different times.
[0029] Figure 7 , Curve diagram of the L-serine peak area in the condensation reaction solution of formaldehyde and glycine catalyzed by AU20-LTA at different temperatures.
[0030] Figure 8 , Curve diagram of the L-serine peak area in the condensation reaction solution of formaldehyde and glycine catalyzed by AU20-LTA at different pH.
[0031] Figure 9 , curve diagram of the L-serine peak area in the condensation reaction solution of formaldehyde and glycine catalyzed by AU20-LTA after formaldehyde incubation for different times.
[0032] Figure 10 , curve diagram of the L-serine peak area in the condensation reaction solution of formaldehyde and glycine catalyzed by Ps-LTA at different temperatures.
[0033] Figure 11 , curve diagram of L-serine peak area in the condensation reaction solution of formaldehyde and glycine catalyzed by Ps-LTA at different pH.
[0034] Figure 12 , curve diagram of the L-serine peak area in the condensation reaction solution of formaldehyde and glycine catalyzed by Ps-LTA after formaldehyde incubation for different times.
[0035] Figure 13 , bar graph of the product L-serine yield in the condensation reaction solution of formaldehyde and glycine catalyzed by L-threonine aldolase at the optimal temperature and pH. (V) Specific implementation methods
[0036] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:
[0037] Example 1: Construction of recombinant genetically engineered bacteria
[0038] Proteins annotated as threonine aldolase were screened from NCBI, artificially synthesized and expressed, and used as catalysts for L-serine synthesis using formaldehyde and glycine as substrates, pyridoxal phosphate (PLP) as a coenzyme, and KCl and mercaptoethanol. Only 6 of the 12 proteins had the ability to synthesize L-serine. Finally, 3 proteins with higher synthesis abilities were screened and expressed, as follows:
[0039] 1. E.coli BL21(DE3)-pET21b-Cp-LTA
[0040] A sequence annotated as "threonine aldolase family protein" from Chelativorans petroleitrophicus (NCBI accession number WP_261514694.1) was cloned from NCBI, and the gene fragment was designated L-threonine aldolase Cp-LTA. The gene sequence is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2. The fragment was inserted between the NdeⅠ and HindⅢ restriction sites of the expression plasmid pET-21b to obtain the recombinant plasmid pET21b-Cp-LTA. After sequencing verification, pET21b-Cp-LTA was transformed into the expression host Escherichia coli BL21 (DE3) to obtain the recombinant genetically engineered bacteria E. coli BL21 (DE3)-pET21b-Cp-LTA (also denoted as L-TA-Cp-BL21 (DE3) or Cp-BL21 (DE3)), which was used for the subsequent expression of the recombinase, and the recombinase was denoted as L-TA-Cp or Cp.
[0041] SEQ ID No.1:
[0042]
[0043] 2. E.coli BL21(DE3)-pET21b-AU20-LTA
[0044] The sequence annotated as "threonine aldolase family protein" from Aureimonas sp. AU20 in NCBI (NCBI accession number WP_061974969.1) was cloned, and the gene fragment was named L-threonine aldolase AU20-LTA. The gene sequence is shown in SEQ ID No. 3, and the amino acid sequence is shown in SEQ ID No. 4. It was inserted between the NdeⅠ and HindⅢ restriction sites of the expression plasmid pET-21b to obtain the recombinant plasmid pET21b-AU20-LTA. After sequencing verification, pET21b-AU20-LTA was transformed into the expression host Escherichia coli BL21 (DE3) to obtain the recombinant genetically engineered bacteria E. coli BL21 (DE3)-pET21b-AU20-LTA (also recorded as L-TA-AU20-BL21 (DE3)), which was used for the subsequent expression of the recombinase. The recombinase was recorded as L-TA-AU20 or AU20.
[0045] SEQ ID No.3:
[0046]
[0047] 3. E.coli BL21(DE3)-pET21b-Ps-LTA
[0048] A sequence from Pseudohoeflea suaedae annotated as a "threonine aldolase family protein" (NCBI accession number WP_133285361.1) was cloned and designated L-threonine aldolase (Ps-LTA). The gene sequence is shown in SEQ ID No. 5, and the amino acid sequence is shown in SEQ ID No. 6. The fragment was inserted between the NdeI and HindIII restriction sites of the expression plasmid pET-21b, generating the recombinant plasmid pET21b-Ps-LTA. After sequencing verification, pET21b-Ps-LTA was transformed into the expression host E. coli BL21(DE3), generating the recombinant engineered strain E. coliBL21(DE3)-pET21b-Ps-LTA (also designated L-TA-Ps-BL21(DE3)). This recombinant enzyme was subsequently expressed, designated L-TA-Ps or Ps.
[0049] SEQ ID No.5:
[0050]
[0051] Using the same method, sequences annotated as "threonine aldolase family protein" from Pimelobacter simplex and CandidatusMethylobacter favarea in NCBI (NCBI accession numbers WP_151578920.1 and WP_174626747.1, respectively) were used to construct recombinant genetically engineered bacteria, and the recombinant enzymes were labeled As-LTA (or As) and CMf-LTA (or CMf), respectively.
[0052] Example 2: Strain culture
[0053] The recombinant genetically engineered bacteria constructed in Example 1 were inoculated into a test tube containing 5 mL of LB liquid medium containing 100 mg / L ampicillin and cultured overnight at 37°C, 220 rpm. 1 mL of the overnight culture was then added to a 250 mL shake flask containing 100 mL of LB liquid medium containing 100 mg / L ampicillin and cultured at 37°C, 220 rpm for about 2.5 hours until the OD600 reached 0.6. IPTG was added to a final concentration of 0.1 mM and expression was induced in a shaker at 28°C, 220 rpm for 16 hours. After induction, the bacterial solution was poured into a 50 mL centrifuge tube and centrifuged at 4°C, 4000 rpm for 10 minutes. The supernatant and wet bacterial pellet were obtained, the supernatant was removed, and the wet bacterial pellet was stored in a -20°C refrigerator for later use. The bacterial solution (whole bacteria) after induction expression, the supernatant after centrifugation and the wet bacterial precipitate were analyzed by agarose gel electrophoresis. The results are shown in Figure 1 As shown, it shows that the engineered bacteria containing the target gene were successfully obtained.
[0054] Wet cells collected from 50 mL of induced expression culture were resuspended in 15 mL of PBS buffer (0.1 M, pH 7). CTAB (cetyltrimethylammonium bromide) was then added at a 1% volume concentration. Ultrasonic disruption was performed at 300W for 15 minutes, with a 2-second on-time interval and a 3-second rest interval. The disrupted culture was centrifuged at 12,000 rpm at 4°C for 15 minutes, and the supernatant was collected to obtain a crude enzyme solution. Both the disrupted culture solution and the crude enzyme solution were used as recombinant enzyme to catalyze the reaction of formaldehyde and glycine to produce L-serine.
[0055] Example 3: Comparison of the reaction of formaldehyde and glycine catalyzed by L-TA to produce L-serine
[0056] Reference Figure 2The reaction system 10 mL of L-TA-catalyzed condensation reaction of formaldehyde and glycine to produce L-serine was composed of the following components: the crude enzyme solution (As, Cp, Ps, AU20, CMf) prepared by the method of Example 2 was added with a final concentration of 50 g / L, 0.1 M formaldehyde, 50 g / L glycine, 0.625 g / L PLP, 37.5 g / L KCl, 17.5 mL / L mercaptoethanol, and Tris-HCl buffer (0.1 M, pH 9.0) based on the weight of the wet cells before crushing.
[0057] The reaction solution was placed at 60°C and 400 rpm for 2 hours. After the reaction, a sample was taken and derivatized with DNFB (2,4-dinitrofluorobenzene). The peak area of L-serine was detected by HPLC. The amount of L-serine produced was calculated based on the standard curve of L-serine standard concentration and peak area detected under the same conditions. The results are shown in Figure 3 The results showed that Cp, Ps, and AU20 could produce L-serine, and Cp-LTA catalyzed 0.1M formaldehyde to produce 2.22g / L of L-serine; AU20-LTA catalyzed 0.1M formaldehyde to produce 2.44g / L of L-serine; and Ps-LTA catalyzed 0.1M formaldehyde to produce 6.17g / L of L-serine.
[0058] HPLC detection conditions: Thermo Fisher liquid chromatograph, using a C18 column (4.6*250 mm, 5 μm), injection volume 10 μL, flow rate 0.8 mL / min, column temperature 30°C, UV detection wavelength 260 nm, gradient elution as shown in Table 1, mobile phase A: pure acetonitrile; mobile phase B: 826 mL ddH2O, 170 mL acetonitrile, 2 mL triethylamine, 2 mL acetic acid.
[0059] Table 1 Liquid chromatography gradient elution program
[0060]
[0061]
[0062] Example 4: Optimization of conditions for the reaction of formaldehyde and glycine to produce L-serine catalyzed by Cp-LTA
[0063] 1. Optimum temperature of Cp-LTA
[0064] Temperature has a great influence on the catalytic ability of enzymes. The optimal temperature of Cp-LTA was optimized by investigating under different temperature conditions (28℃-60℃).
[0065] The reaction system 10 mL of Cp-LTA catalyzes the condensation reaction of formaldehyde and glycine to produce L-serine, and the final concentration composition is as follows: the crude enzyme solution prepared by the method of Example 2 is added with a final concentration of 50 g / L, 0.1 M formaldehyde, 50 g / L glycine, 0.625 g / L PLP, 37.5 g / L KCl, 17.5 mL / L mercaptoethanol and Tris-HCl buffer (0.1 M, pH 9.0) based on the weight of the wet bacteria before crushing.
[0066] The reaction solution was placed at different temperatures (28°C, 37°C, 42°C, 50°C, 60°C) and a rotation speed of 400 rpm for 2 hours. After the reaction, a sample was taken and derivatized with DNFB (2,4-dinitrofluorobenzene). The peak area of L-serine was then detected using the HPLC described in Example 3. The results are shown in FIG. Figure 4 The optimum catalytic temperature of Cp-LTA is 60℃.
[0067] 2. Optimal pH of Cp-LTA
[0068] Different pH values of the reaction solution have a great influence on enzyme activity, thereby affecting the catalytic efficiency of the enzyme.
[0069] The buffer in the reaction system of step 1 was changed to citric acid-disodium hydrogen phosphate buffer (3.0, 4.0, 5.0), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer (5.0, 6.0, 7.0, 8.0), Tris-HCl buffer (8.0, 9.0), sodium carbonate-sodium bicarbonate buffer (9.0, 10.0, 11.0), the reaction temperature was fixed at 60 ° C, and other operations were the same. The L-serine peak area was shown in FIG. Figure 5 As shown, the optimum pH value is 10.0, and the corresponding buffer solution is 0.1 M Na2CO3-NaHCO3 buffer solution.
[0070] 3. Formaldehyde tolerance of Cp-LTA
[0071] Using the reaction system of step 1, the crude Cp-LTA enzyme solution was first added to formaldehyde and Tris-HCl buffer (0.1M, pH 9.0), and incubated in an ice bath for 0h, 0.5h, 1h, 2.5h, 3h, 5h, and 6h, respectively. Then, using the conditions of step 1, the reaction temperature was fixed at 60°C, and the L-serine peak area was observed. Figure 6 As shown, the results showed that the crude Cp-LTA enzyme solution was basically inactivated after incubation in formaldehyde for 1 hour.
[0072] Example 5: Optimization of conditions for the reaction of formaldehyde and glycine to produce L-serine catalyzed by AU20-LTA
[0073] 1. Optimum temperature of AU20-LTA
[0074] Temperature has a great influence on the catalytic ability of enzymes. The optimal temperature of AU20-LTA was optimized by investigating under different temperature conditions (28℃-60℃).
[0075] The final concentration composition of the 10 mL reaction system was as follows: the crude enzyme solution prepared by the method of Example 2 was added with a final concentration of 50 g / L, 0.1 M formaldehyde, 50 g / L glycine, 0.625 g / L PLP, 37.5 g / L KCl, 17.5 mL / L mercaptoethanol, and Tris-HCl buffer (0.1 M, pH 9.0) based on the weight of the wet cells before disruption.
[0076] The reaction solution was placed at different temperatures (28°C, 37°C, 42°C, 50°C, 60°C) and a rotation speed of 400 rpm for 2 hours. After the reaction, a sample was taken and derivatized with DNFB (2,4-dinitrofluorobenzene). The peak area of L-serine was then detected using the HPLC described in Example 3. Figure 7 As shown in the figure, the optimum catalytic temperature of AU20-LTA is 60℃.
[0077] 2. Optimal pH of AU20-LTA
[0078] Different pH values of the reaction solution have a great influence on enzyme activity, thereby affecting the catalytic efficiency of the enzyme.
[0079] The buffer in the reaction system of step 1 was changed to citric acid-disodium hydrogen phosphate buffer (3.0, 4.0, 5.0), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer (5.0, 6.0, 7.0, 8.0), Tris-HCl buffer (8.0, 9.0), sodium carbonate-sodium bicarbonate buffer (9.0, 10.0, 11.0), the reaction temperature was fixed at 60 ° C, and other operations were the same. The L-serine peak area was shown in FIG. Figure 8 As shown, the optimum pH value is 9.0, and the corresponding buffer solution is 0.1 M Na2CO3-NaHCO3 buffer solution.
[0080] 3. Formaldehyde tolerance of AU20-LTA
[0081] The reaction system of step 1 was used. The crude enzyme solution was first added to formaldehyde and Tris-HCl buffer (0.1 M, pH 9.0). After incubation in an ice bath for 0 h, 0.5 h, 1 h, 2.5 h, 3 h, 5 h, and 6 h, the reaction was carried out using the reaction conditions of step 1. The reaction temperature was fixed at 60 ° C. The L-serine peak area was Figure 9 As shown, the results showed that the crude AU20-LTA enzyme solution was basically inactivated after incubation in formaldehyde for 1 hour.
[0082] Example 6: Optimization of conditions for the reaction of formaldehyde and glycine to produce L-serine catalyzed by Ps-LTA
[0083] 1. Ps-LTA catalyzes the reaction of formaldehyde and glycine to produce L-serine
[0084] Temperature has a great influence on the catalytic ability of enzymes. The optimal temperature of Ps-LTA was optimized by investigating under different temperature conditions (28℃-60℃).
[0085] The final concentration composition of the 10 mL reaction system was as follows: the crude enzyme solution prepared by the method of Example 2 was added with a final concentration of 50 g / L, 0.1 M formaldehyde, 50 g / L glycine, 0.625 g / L PLP, 37.5 g / L KCl, 17.5 mL / L mercaptoethanol, and Tris-HCl buffer (0.1 M, pH 9.0) based on the weight of the wet cells before disruption.
[0086] The reaction solution was placed at different temperatures (28°C, 37°C, 42°C, 50°C, 60°C) and a rotation speed of 400 rpm for 2 hours. After the reaction, a sample was taken and derivatized with DNFB (2,4-dinitrofluorobenzene). The peak area of L-serine was then detected using the HPLC described in Example 3. Figure 10 As shown in the figure, the optimum catalytic temperature of Ps-LTA is 60℃.
[0087] 2. Optimal pH of Ps-LTA
[0088] Different pH values of the reaction solution have a great influence on enzyme activity, thereby affecting the catalytic efficiency of the enzyme.
[0089] The buffer in the reaction system of step 1 was changed to citric acid-disodium hydrogen phosphate buffer (3.0, 4.0, 5.0), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer (5.0, 6.0, 7.0, 8.0), Tris-HCl buffer (8.0, 9.0), sodium carbonate-sodium bicarbonate buffer (9.0, 10.0, 11.0), the reaction temperature was fixed at 60 ° C, and other operations were the same. The L-serine peak area was shown in FIG. Figure 11 As shown, the optimum pH value is 9.0, and the corresponding buffer is 0.1 M Tris-HCl buffer.
[0090] 3. Formaldehyde tolerance of Ps-LTA
[0091] The reaction system of step 1 was used. The crude Ps-LTA enzyme solution was first added to formaldehyde and Tris-HCl buffer (0.1 M, pH 9.0). After incubation in an ice bath for 0 h, 0.5 h, 1 h, 2.5 h, 3 h, 5 h, and 6 h, the reaction was carried out using the reaction conditions of step 1. The reaction temperature was fixed at 60 ° C. The L-serine peak area was Figure 12 As shown, the results showed that the crude Ps-LTA enzyme solution was basically inactivated after incubation in formaldehyde for 5 hours.
[0092] Example 7: Comparison of the reaction of formaldehyde and glycine to produce L-serine catalyzed by L-TA under optimal conditions
[0093] 1. Cp-LTA
[0094] The reaction system 10mL of Cp-LTA catalyzes the condensation reaction of formaldehyde and glycine to produce L-serine has the following final concentration composition: the crude enzyme solution prepared by the method of Example 2 is added with a final concentration of 50g / L, 0.1M formaldehyde, 50g / L glycine, 0.625g / L PLP, 37.5g / L KCl, 17.5mL / L mercaptoethanol and Na2CO3-NaHCO3 buffer (0.1M, pH10.0) based on the weight of the wet bacteria before crushing.
[0095] The reaction solution was placed at 60°C and 400 rpm for 2 h, and the content of L-serine was detected by the method of Example 3. The results are shown in FIG. Figure 13 Under the conditions of optimal temperature and optimal pH, Cp-LTA catalyzes 0.1 M formaldehyde to produce 4.24 g / L L-serine.
[0096] 2. AU20-LTA
[0097] The final concentration composition of the 10 mL reaction system is as follows: the crude enzyme solution prepared by the method of Example 2 is added with a final concentration of 50 g / L, 0.1 M formaldehyde, 50 g / L glycine, 0.625 g / L PLP, 37.5 g / L KCl, 17.5 mL / L mercaptoethanol and Na2CO3-NaHCO3 buffer (0.1 M, pH 9.0) based on the weight of the wet cells before crushing.
[0098] The reaction solution was placed at 60°C and 400 rpm for 2 h, and the content of L-serine was detected by the method of Example 3. The results are shown in FIG. Figure 13 Under the conditions of optimal temperature and optimal pH, AU20-LTA catalyzed 0.1 M formaldehyde to produce 4.14 g / L L-serine.
[0099] 3. Ps-LTA
[0100] The final concentration composition of the 10 mL reaction system is as follows: the crude enzyme solution prepared by the method of Example 2 is added with a final concentration of 50 g / L, 0.1 M formaldehyde, 50 g / L glycine, 0.625 g / L PLP, 37.5 g / L KCl, 17.5 mL / L mercaptoethanol and Na2CO3-NaHCO3 buffer (0.1 M, pH 9.0) based on the weight of the wet cells before crushing.
[0101] The reaction solution was placed at 60°C and 400 rpm for 2 h, and the content of L-serine was detected by the method of Example 3. The results are shown in FIG. Figure 13Under the conditions of optimal temperature and optimal pH, Ps-LTA catalyzes 0.1 M formaldehyde to produce 6.17 g / L L-serine.
[0102] 4. As-LTA, CMf-LTA
[0103] The final concentration composition of the 10mL reaction system is as follows: the crude enzyme solution (As-LTA, CMf-LTA) prepared by the method of Example 2 is added with a final concentration of 50g / L, 0.1M formaldehyde, 50g / L glycine, 0.625g / L PLP, 37.5g / L KCl, 17.5mL / L mercaptoethanol and Na2CO3-NaHCO3 buffer (0.1M, pH 9.0) based on the weight of the wet bacteria before crushing.
[0104] The reaction solution was placed at 60°C and 400 rpm for 2 h, and the content of L-serine was detected by the method of Example 3. The results are shown in FIG. Figure 13 , As-LTA or CMf-LTA catalyzed 0.1 M formaldehyde did not produce L-serine.
Claims
1. An application of L-threonine aldolase in the efficient synthesis of L-serine, characterized in that: The L-threonine aldolase includes threonine aldolase family proteins derived from Chelativorans petroleitrophicus, Aureimonas sp. AU20, and Pseudohoefleasuaedae.
2. The use according to claim 1, characterized in that The L-threonine aldolase is one of the following: derived from Chelativorans petroleitrophicus, denoted as Cp-LTA, with an amino acid sequence as shown in SEQ ID No. 2; derived from Aureimonas sp. AU20, denoted as AU20-LTA, with an amino acid sequence as shown in SEQ ID No. 4; derived from Pseudohoefleasuaedae, denoted as Ps-LTA, with an amino acid sequence as shown in SEQ ID No.
6.
3. The use according to claim 1, characterized in that The application method comprises the following steps: using a crude enzyme liquid obtained by ultrasonically crushing wet bacteria obtained by induction culture of a recombinant genetically engineered bacterium expressing L-threonine aldolase as a catalyst, using formaldehyde and glycine as substrates, using pyridoxal phosphate as a coenzyme, adding KCl and mercaptoethanol, and using a pH 3-11 buffer as a reaction medium to form a reaction system, and reacting completely at 20-70° C. and 200-500 rpm to obtain a reaction liquid containing L-serine.
4. The use according to claim 3, characterized in that In the reaction system, the catalyst is added to a final concentration of 20-80 g / L based on the weight of the wet cells before crushing; formaldehyde is added to a final concentration of 1-300 mM; glycine is added to a final concentration of 10-250 g / L; the pyridoxal phosphate is added to a final concentration of 0.1-50 g / L; KCl is added to a final concentration of 10-50 g / L; and mercaptoethanol is added to a final concentration of 0.5-70 mL / L.
5. The use according to claim 3, characterized in that pH is 9-10.
6. The use according to claim 5, characterized in that When the L-threonine aldolase is Cp-LTA, the buffer is 0.1M Na2CO3-NaHCO3 buffer with a pH of 10; when the L-threonine aldolase is AU20-LTA, the buffer is 0.1M Na2CO3-NaHCO3 buffer with a pH of 9; when the L-threonine aldolase is Ps-LTA, the buffer is 0.1M Tris-HCl buffer with a pH of 9.
7. The use according to claim 3, characterized in that The recombinant genetically engineered bacteria expressing L-threonine aldolase are constructed according to the following steps: inserting the coding gene fragment of L-threonine aldolase between the NdeⅠ and HindⅢ restriction sites of the expression plasmid pET-21b to obtain a recombinant plasmid; and transforming the recombinant plasmid into the expression host Escherichia coli BL21 (DE3) to obtain the recombinant genetically engineered bacteria.
8. The use according to claim 3, characterized in that The crude enzyme solution was prepared as follows: recombinant genetically engineered bacteria expressing L-threonine aldolase were inoculated into a test tube of LB liquid culture medium containing 100 mg / L ampicillin, and cultured overnight at 37°C and 220 rpm in a constant temperature shaker; the bacterial solution was inoculated into LB liquid culture medium containing 100 mg / L ampicillin at an inoculum concentration of 1% by volume, and cultured at 37°C and 220 rpm in a constant temperature shaker until the OD 600 When the protein expression reaches 0.6, add IPTG to a final concentration of 0.1 mM, place in a constant temperature shaker at 28 ° C and 220 rpm to induce expression for 16 hours. After the induction expression is completed, the bacterial solution is centrifuged at 4 ° C and 4000 rpm for 10 minutes, the supernatant is removed, the wet bacteria are taken and resuspended in PBS buffer, and then 1% volume concentration of hexadecyltrimethylammonium bromide is added, and ultrasonic disruption is carried out at 300W for 15 minutes, working for 2 seconds and with an interval of 3 seconds, and centrifuged at 4 ° C and 12000 rpm for 15 minutes. The supernatant is collected to obtain the crude enzyme solution.