Process for the preparation of l-tyrosine and fermentation product

Abstract

The present application relates to the technical field of fermentation, in particular to a preparation method of L-tyrosine and a fermentation product. The preparation method of L-tyrosine comprises: mixing an induced culture solution of recombinant Escherichia coli genetic engineering bacteria, lactic acid and phenol to perform fermentation, wherein the fermentation conditions comprise a temperature of 30-40 DEG C, a pH of 8.0-9.0, a phenol flow rate of 5-12 g / (L*h), and a ratio of phenol to lactic acid of 1:0.9-1.1. The preparation method can improve the utilization rate of pyruvic acid, reduce the inactivation of related enzymes, obtain L-tyrosine with high purity and high yield, and reduce the purification cost in the rear end. Moreover, the raw materials lactic acid and phenol are cheap and easy to obtain, and the expensive pyruvic acid is no longer used as a substrate, thereby reducing the fermentation cost, and the method can be applied to large-scale industrial production to meet market demand.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fermentation, in particular to a preparation method of L-tyrosine and a fermentation product. BACKGROUND

[0002] L-tyrosine, also known as 2-amino-3-p-hydroxyphenylpropionic acid, is an aromatic polar alpha-amino acid containing phenolic hydroxyl group. Tyrosine is a conditionally essential amino acid and a ketogenic and glucogenic amino acid in human body, which has the effects of stimulating the nervous system and regulating mood. It can help the human body to speed up metabolism and treat chronic fatigue and other diseases. Tyrosine can also be used as a food additive and a precursor for the synthesis of thyroxine, tyrosine sulfite, etc. At present, the preparation methods of tyrosine include extraction method, chemical synthesis method, etc.

[0003] The tyrosine extraction method mainly separates tyrosine from natural protein resources such as pig hair and feather through hydrolysis, extraction, purification and other steps. However, due to the low content of tyrosine in natural protein resources and the presence of many other amino acids, it is difficult to separate and purify, and the yield and purity of tyrosine obtained are very low, which is not conducive to large-scale acquisition.

[0004] The chemical synthesis method of tyrosine mainly includes the steps of hydroxylation of L-phenylalanine or condensation of p-hydroxybenzaldehyde and hydantin, alkaline hydrolysis and transamination, etc. The reaction steps are more, and the synthesized product is racemic DL-tyrosine, which needs to be racemized to obtain L-tyrosine. The process is complex and the efficiency is very low.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The purpose of the present application is to provide a preparation method of L-tyrosine and a fermentation product. The preparation method provided by the embodiments of the present application can improve the utilization rate of pyruvic acid, reduce the inactivation of related enzymes, and obtain L-tyrosine with high purity and high yield.

[0007] The present application is realized as follows:

[0008] In a first aspect, the present application provides a preparation method of L-tyrosine, comprising: mixing an induced culture solution of a recombinant Escherichia coli genetically engineered bacteria, lactic acid and phenol for fermentation,

[0009] The fermentation conditions include a temperature of 30-40℃, a pH of 8.0-9.0, and a phenol feeding rate of 5-12g / (L*h), and the addition amount ratio of the phenol to the lactic acid is 1:0.9-1.1.

[0010] In an optional embodiment, the addition amount of the phenol in the mixed solution of the lactic acid and the phenol is 80-120g / L;

[0011] Preferably, the fermentation conditions include: temperature of 30-32℃;

[0012] Preferably, the pH is 8.0-8.5;

[0013] Preferably, the feeding rate of the phenol is 10-12 / (L*h);

[0014] Preferably, the ratio of the added amount of the phenol to the added amount of the lactic acid in the mixed solution is 1:0.95-1.1;

[0015] Preferably, the added amount of the phenol in the mixed solution is 105-110g / L.

[0016] In an optional embodiment, the construction process of the recombinant E. coli genetically engineered bacteria includes: transferring pETDuet-1 and pCDFduet-1 into E. coli, and then performing plate screening on positive clones, wherein pETduet-1 is loaded with lactic acid oxidase gene and catalase gene, and pCDFduet-1 is loaded with tyrosine phenol-lyase gene.

[0017] In an optional embodiment, the preparation process of the induction culture solution includes: inoculating the recombinant E. coli genetically engineered bacteria into LBG culture medium to form recombinant E. coli seed solution;

[0018] Then, the recombinant E. coli seed solution is inoculated into TB culture medium for culture, and then an inducer is added for induction culture.

[0019] In an optional embodiment, the LBG culture medium includes: peptone, yeast extract, sodium chloride, glycerol and water.

[0020] In an optional embodiment, the culture conditions for forming the recombinant E. coli seed solution include: 30-40℃, and pH of 7.0-8.0.

[0021] In an optional embodiment, the TB culture medium includes: peptone, yeast powder, glycerol, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and water, and the pH of the TB culture medium is 6.8-7.2.

[0022] In an optional embodiment, the culture conditions in the TB culture medium include: 30-40℃, and pH of 7.0-8.0.

[0023] In an optional embodiment, the induction culture includes: using the TB culture medium to culture to OD 600After 10 nm, the temperature is reduced to 25-28℃, and the final concentration of 0.1-0.5mM IPTG is added for induction, and induction is performed for 10-14 hours, and then 0.1%-0.3% Trion X-100 and 0.1-0.5g / L PLP are added, and the culture is continued for 1-1.5 hours.

[0024] In a second aspect, the present application provides a fermentation product, which is obtained by fermenting L-tyrosine prepared by the method of any one of the preceding embodiments.

[0025] The present application has the following beneficial effects: the embodiments of the present application use recombinant E. coli fermentation engineering bacteria to ferment L-tyrosine, and control the conditions of fermentation, such as the ratio and flow rate of lactic acid and phenol, fermentation temperature, and pH, to improve the utilization rate of pyruvate, reduce the inactivation of related enzymes, and then improve the utilization rate of lactic acid and phenol, so that the utilization rate of lactic acid reaches more than 95%, and the utilization rate of phenol reaches more than 99%, reducing the purification cost of the rear end. At the same time, the high purity and high yield of L-tyrosine can be improved, so that the yield of L-tyrosine is more than 200g / L. Moreover, the raw materials lactic acid and phenol are cheap and easy to obtain, and the expensive pyruvate is no longer used as a substrate, reducing the fermentation cost and being conducive to industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

[0027] Figure 1 The L-tyrosine biological synthesis route map provided by the embodiments of the present application;

[0028] Figure 2 The standard curve map of tyrosine provided by the embodiments of the present application;

[0029] Figure 3 The standard curve map of phenol provided by the embodiments of the present application;

[0030] Figure 4 The standard curve map of lactic acid provided by the embodiments of the present application. DETAILED DESCRIPTION

[0031] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. If specific conditions are not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturers are adopted. If the manufacturers of the reagents or instruments are not specified, the conventional products that can be purchased in the market are adopted.

[0032] The embodiments of the present application provide a preparation method of L-tyrosine, and a biosynthesis route chart of the L-tyrosine is shown in Figure 1 , and the specific operation process comprises the following steps:

[0033] Firstly, a recombinant Escherichia coli genetically engineered bacterium is constructed, and the specific process is as follows:

[0034] In the embodiments of the present application, three genes are co-expressed by using pETDuet-1 and pCDFduet-1 double plasmids, the pETduet-1 is loaded with a lactate oxidase gene and a catalase gene, wherein the lactate oxidase gene can be a gene HN015_RS05890 (NCBI-Gene ID: 57366060) from Pediococcus acidilactici, and the catalase gene can be a gene ECs_1652 (NCBI-Gene ID: 913226) from Escherichia coli; and the pCDFduet-1 is loaded with a tyrosine phenol-lyase gene, which can be a gene BAACE33A1D18_RS03985 (NCBI-Gene ID: 66567973) from Fusobacterium mortiferum.

[0035] The above two plasmids are transformed into Escherichia coli BL21 (DE3), and positive clones are screened by using ampicillin and streptomycin sulfate plates, so as to obtain the recombinant Escherichia coli.

[0036] The inventors find that the mechanism of fermenting and synthesizing L-tyrosine is mainly as follows: in the Escherichia coli, lactate is converted into pyruvate by using lactate oxidase, at the same time, byproduct hydrogen peroxide is degraded by using catalase to reduce the damage to the bacterial strain, so as to maintain the fermentation efficiency of the bacterial strain. Finally, L-tyrosine is synthesized by using tyrosine phenol-lyase to produce pyruvate, ammonium and phenol. Therefore, the engineering bacterium is constructed by using the above method, so that the recombinant Escherichia coli genetically engineered bacterium can be fully fermented to form L-tyrosine.

[0037] Secondly, induction culture, and the specific process is as follows:

[0038] The above-mentioned recombinant E. coli genetically engineered bacteria are inoculated into LBG culture medium for culture, wherein the LBG culture medium comprises: peptone, yeast extract, sodium chloride, glycerol and water, for example, the LBG culture solution comprises peptone 10 g, yeast extract 5 g, NaCl 10 g, glycerol 5 g, and pure water is added to 1.0 L. The overnight culture is carried out at 30-40°C (for example, any value between 30-40°C such as 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C and 40°C, etc.), and pH is 7.0-8.0 (for example, any value between 7.0-8.0 such as 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 and 8.0, etc.). The recombinant E. coli seed solution is obtained.

[0039] Then the recombinant E. coli seed solution is inoculated into TB culture medium and cultured at 30-40°C (for example, any value between 30-40°C such as 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C and 40°C, etc.), and pH is 7.0-8.0 (for example, any value between 7.0-8.0 such as 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 and 8.0, etc.).

[0040] The TB culture medium comprises: peptone, yeast powder, glycerol, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and water, and specifically the TB culture medium components are as follows: 12 g / L peptone, 24 g / L yeast powder, 30 g / L glycerol, 2.31 g / L potassium dihydrogen phosphate, 12.54 g / L dipotassium hydrogen phosphate, sterilized at 121°C, and pH is adjusted to 7.0 with ammonia water.

[0041] When the bacterial culture is cultured to OD 600After 10 nm, the temperature is reduced to 25-28°C (for example, 25°C, 26°C, 27°C, and 28°C, or any value between 25-28°C), and 0.1-0.5 mM (for example, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, and 0.5 mM, or any value between 0.1-0.5 mM) IPTG is added for induction, and the induction is performed for 10-14 hours (for example, 10 hours, 11 hours, 12 hours, 13 hours, and 14 hours), and then 0.1%-0.3% (for example, 0.1%, 0.15%, 0.2%, 0.25%, and 0.3%, or any value between 0.1%-0.3%) Trion X-100 and 0.1-0.5 g / L (for example, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, and 0.5 g / L, or any value between 0.1-0.5 g / L) PLP are added, and the culture is continued for 1-1.5 hours (for example, 1 hour, 1.2 hours, and 1.5 hours), and the induction culture is obtained.

[0042] Then, the induction culture, lactic acid, and phenol are mixed for fermentation. The lactic acid and the phenol can be mixed and then the mixture is fed, or the lactic acid and the phenol can be fed simultaneously to the induction culture, as long as the ratio of the two is as required.

[0043] Further, the fermentation conditions include a temperature of 30-40°C, for example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 8°C, 39°C, and 40°C, or any value between 30-40°C; a pH of 8.0-9.0, for example, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, and 9.0, or any value between 8.0-9.0; a ratio of the addition amount of the phenol to the lactic acid of 1:0.9-1.1, for example, 1:0.9, 1:0.95, 1:1, 1:1.05, 1:1.1, or any value between 1:0.9-1:1.1; an addition amount of the phenol in the mixture of the lactic acid and the phenol of 80-120 g / L, for example, 80 g / L, 85 g / L, 90 g / L, 95 g / L, 100 g / L, 105 g / L, 110 g / L, 115 g / L, and 120 g / L, or any value between 80-120 g / L; and a feeding rate of the phenol of 5-12 g / (L*h), for example, 5 g / (L*h), 6 g / (L*h), 7 g / (L*h), 8 g / (L*h), 9 g / (L*h), 10 g / (L*h), 11 g / (L*h), and 12 g / (L*h), or any value between 5-12 g / (L*h).

[0044] If the lactic acid and phenol are fed into the induction culture solution at the same time, the feeding rate of the lactic acid can be calculated according to the ratio thereof. If the mixture of the two is fed, the feeding rate of the mixture can also be calculated according to the ratio of the two. The above calculation is well known to those skilled in the art, and the inventor will not go into the details of the calculation process and results.

[0045] Since the lactic acid is acidic, the pH of the fermentation solution will gradually decrease during the feeding of the lactic acid. The present embodiment uses ammonia water to maintain the pH of the fermentation solution and also provides the necessary ammonium ions for the subsequent catalytic reaction.

[0046] Secondly, during the fermentation of E. coli, high concentration of lactic acid can accelerate the catalytic reaction rate of lactic acid oxidase, thereby producing a large amount of pyruvic acid and hydrogen peroxide. Excessive hydrogen peroxide cannot be timely decomposed by catalase, which not only causes the deactivation of related enzymes, but also causes the reaction of hydrogen peroxide and pyruvic acid to synthesize acetic acid, resulting in the waste of pyruvic acid. In addition, high concentration of pyruvic acid accumulation can cause pyruvic acid to polymerize itself, causing the degradation of pyruvic acid, which is related to the catalytic temperature and pH. High concentration of phenol can cause the deactivation of lactic acid oxidase, catalase and tyrosine phenol-lyase, and the catalytic reaction rate of L-tyrosine synthesis is significantly reduced. Therefore, reasonable control of the feeding of lactic acid and phenol is a key factor for improving substrate utilization and obtaining high-yield L-tyrosine.

[0047] In summary, the present embodiment can improve substrate utilization and reduce enzyme deactivation by controlling the ratio, feeding rate, fermentation temperature and pH of lactic acid and phenol, thereby obtaining high-purity and high-yield L-tyrosine, which can be used for large-scale industrial production.

[0048] Preferably, the fermentation conditions include that the temperature is 30-32℃;

[0049] Preferably, the pH is 8.0-8.5;

[0050] Preferably, the feeding rate of the phenol is 10-12 / (L*h);

[0051] Preferably, the ratio of the phenol to the lactic acid in the mixture is 1:0.95-1.1;

[0052] Preferably, the addition amount of the phenol in the mixture is 105-110g / L.

[0053] Further optimization of the fermentation conditions can produce higher yield and higher substrate utilization rate under the above fermentation conditions.

[0054] The present embodiment also provides a fermentation product obtained by the above-mentioned method for preparing L-tyrosine.

[0055] The features and performances of the present application are further described in detail below in combination with examples.

[0056] Example 1

[0057] The present example provides a method for preparing L-tyrosine, comprising:

[0058] The two plasmids are transformed into Escherichia coli BL21(DE3), and positive clones are screened by ampicillin and streptomycin plates, i.e. to obtain a recombinant Escherichia coli genetically engineered bacteria. Among them, pETduet-1 is loaded with lactic acid oxidase gene (Pediococcus acidilactici) and catalase gene (Escherichia coli), and pCDFduet-1 is loaded with tyrosine phenol-lyase gene (Fusobacterium mortiferum).

[0059] The above strains and plasmids are purchased from Novagen company, including pETduet-1 plasmid, pCDFduet-1 plasmid, Escherichia coli BL21(DE3) and Escherichia coli TOP10 (which is used for cloning plasmid).

[0060] The recombinant Escherichia coli genetically engineered bacteria is placed in LBG culture medium, which includes 10g of proteose peptone, 5g of yeast extract, 10g of NaCl, 5g of glycerol, and pure water to 1.0L. The culture is incubated at 37℃ and pH 7.0 overnight to obtain a recombinant Escherichia coli seed solution.

[0061] The above recombinant Escherichia coli seed solution is transferred to a modified TB culture medium (12g / L proteose peptone, 24g / L yeast powder, 30g / L glycerol, 2.31g / L potassium dihydrogen phosphate, 12.54g / L dipotassium hydrogen phosphate, sterilized at 121℃, and pH adjusted to 7.0 with ammonia water) for culture. The fermentation conditions are 37℃ and pH 7.0. When the bacterial culture in the fermentation broth reaches OD 600nm = 10, the temperature is lowered to 28℃ and pH 7.0, and 0.5mM IPTG is added for induction. After 12h of induction, 0.1% TrionX-100 and 0.25g / L PLP are added, and the culture is continued for 1h to form an induction culture. The induction culture is used for fermentation in the following examples.

[0062] The lactic acid and phenol mixture was added to the above induction medium, the total amount of phenol in the mixture was 80 g / L, the ratio of the amount of phenol to lactic acid was 1:1, the flow rate of phenol was controlled at 10 g / (L*h), the fermentation temperature was 30°C, and the pH was adjusted to 8.0 by ammonia.

[0063] After the substrate flow was completed, the fermentation was continued until the L-tyrosine yield no longer changed, and the residual amount of lactic acid, the residual amount of phenol, and the L-tyrosine yield were detected by HPLC.

[0064] Examples 2-17 and Comparative Examples 1-3

[0065] Examples 2-17 and Comparative Examples 1-3 respectively provide a method for preparing L-tyrosine, which is similar to Example 1, except that the induction medium and the fermentation conditions of lactic acid and phenol are different, and the specific conditions are as follows:

[0066] Example 2: The fermentation temperature was 30°C, the pH was adjusted to 8.0 by ammonia, the lactic acid and phenol mixture was added, the total amount of phenol in the mixture was 120 g / L, the ratio of the amount of phenol to lactic acid was 1:1, and the flow rate of phenol was controlled at 10 g / (L*h).

[0067] Example 3: The fermentation temperature was 30°C, the pH was adjusted to 9.0 by ammonia, the lactic acid and phenol mixture was added, the total amount of phenol in the mixture was 110 g / L, the ratio of the amount of phenol to lactic acid was 1:1, and the flow rate of phenol was controlled at 10 g / (L*h).

[0068] Example 4: The fermentation temperature was 30°C, the pH was adjusted to 8.0 by ammonia, the lactic acid and phenol mixture was added, the total amount of phenol in the mixture was 110 g / L, the ratio of the amount of phenol to lactic acid was 1:1, and the flow rate of phenol was 5 g / (L*h).

[0069] Example 5: The fermentation temperature was 40°C, the pH was adjusted to 8.0 by ammonia, the lactic acid and phenol mixture was added, the total amount of phenol in the mixture was 110 g / L, the ratio of the amount of phenol to lactic acid was 1:1, and the flow rate of phenol was 10 g / (L*h).

[0070] Example 6: The fermentation temperature was 30°C, the pH was adjusted to 8.0 by ammonia, the lactic acid and phenol mixture was added, the total amount of phenol in the mixture was 110 g / L, the ratio of the amount of phenol to lactic acid was 1:0.9, and the flow rate of phenol was 10 g / (L*h).

[0071] Example 7: The fermentation temperature was 30°C, the pH was adjusted to 8.0 by ammonia, the lactic acid and phenol mixture was added, the total amount of phenol in the mixture was 110 g / L, the ratio of the amount of phenol to lactic acid was 1:1.1, and the flow rate of phenol was 10 g / (L*h).

[0072] Example 8: Fermentation temperature is 30℃, ammonia water to adjust pH = 8.0, flow lactic acid and phenol mixture, the total amount of phenol in the mixture is 110g / L, the ratio of the amount of phenol and lactic acid is 1:1, the flow rate of phenol is 10g / (L*h).

[0073] Example 9: Fermentation temperature is 30℃, ammonia water to adjust pH = 8.0, flow lactic acid and phenol mixture, the total amount of phenol in the mixture is 110g / L, the ratio of the amount of phenol and lactic acid is 1:0.95, the flow rate of phenol is 10g / (L*h).

[0074] Example 10: Fermentation temperature is 32℃, ammonia water to adjust pH = 8.0, flow lactic acid and phenol mixture, the total amount of phenol in the mixture is 110g / L, the ratio of the amount of phenol and lactic acid is 1:1, the flow rate of phenol is 10g / (L*h).

[0075] Example 11: Fermentation temperature is 32℃, ammonia water to adjust pH = 8.5, flow lactic acid and phenol mixture, the total amount of phenol in the mixture is 110g / L, the ratio of the amount of phenol and lactic acid is 1:1, the flow rate of phenol is 10g / (L*h).

[0076] Example 12: Fermentation temperature is 32℃, ammonia water to adjust pH = 8.5, flow lactic acid and phenol mixture, the total amount of phenol in the mixture is 110g / L, the ratio of the amount of phenol and lactic acid is 1:0.95, the flow rate of phenol is 12g / (L*h).

[0077] Example 13: Fermentation temperature is 30℃, ammonia water to adjust pH = 8.5, flow lactic acid and phenol mixture, the total amount of phenol in the mixture is 110g / L, the ratio of the amount of phenol and lactic acid is 1:1, the flow rate of phenol is 12g / (L*h).

[0078] Example 14: Fermentation temperature is 30℃, ammonia water to adjust pH = 8.0, flow lactic acid and phenol mixture, the total amount of phenol in the mixture is 110g / L, the ratio of the amount of phenol and lactic acid is 1:1, the flow rate of phenol is 12g / (L*h).

[0079] Example 15: Fermentation temperature is 30℃, ammonia water to adjust pH = 8.5, flow lactic acid and phenol mixture, the total amount of phenol in the mixture is 110g / L, the ratio of the amount of phenol and lactic acid is 1:0.95, the flow rate of phenol is 10g / (L*h).

[0080] Example 16: Fermentation temperature is 30℃, ammonia water to adjust pH = 8.5, flow addition of lactic acid and phenol mixed solution, the total amount of phenol in the mixed solution is 105g / L, the ratio of the amount of phenol and lactic acid is 1:1, and the flow rate of phenol is 10g / (L*h).

[0081] Example 17: Fermentation temperature is 32℃, ammonia water to adjust pH = 8.0, flow addition of lactic acid and phenol mixed solution, the total amount of phenol in the mixed solution is 105g / L, the ratio of the amount of phenol and lactic acid is 1:1, and the flow rate of phenol is 12g / (L*h).

[0082] Comparative Example 1: Fermentation temperature is 30℃, ammonia water to adjust pH = 6.0, flow addition of lactic acid and phenol mixed solution, the total amount of phenol in the mixed solution is 110g / L, the ratio of the amount of phenol and lactic acid is 1:1, and the flow rate of phenol is 10g / (L*h).

[0083] Comparative Example 2: Fermentation temperature is 30℃, ammonia water to adjust pH = 8.0, flow addition of lactic acid and phenol mixed solution, the total amount of phenol in the mixed solution is 110g / L, the ratio of the amount of phenol and lactic acid is 1:1, and the flow rate of phenol is 20g / (L*h).

[0084] Comparative Example 3: Fermentation temperature is 25℃, ammonia water to adjust pH = 8.0, flow addition of lactic acid and phenol mixed solution, the total amount of phenol in the mixed solution is 110g / L, the ratio of the amount of phenol and lactic acid is 1:1, and the flow rate of phenol is 10g / (L*h).

[0085] The determination methods of the residual amount of lactic acid, the residual amount of phenol and the yield of L-tyrosine in Examples 1-17 and Comparative Examples 1-3 are as follows:

[0086] (1) Preparation of L-tyrosine standard curve

[0087] Take 0.1g of L-tyrosine standard sample and move it to a 50mL volumetric flask. Dissolve with dilute hydrochloric acid and make up to volume. Then take 0.5mL, 1mL, 2mL, 4mL and 5mL respectively and move them to five 10mL volumetric flasks. Make up to volume with dilute hydrochloric acid to obtain concentrations of 0.1g / L, 0.2g / L, 0.4g / L, 0.8g / L and 1g / L respectively. Detect by HPLC. Chromatographic conditions: C18 column, mobile phase A: 0.1% formic acid, mobile phase B: methanol, mobile phase A: mobile phase B (v:v) = 40:60, flow rate: 1mL / L, column temperature: 30℃, wavelength: 275nm, injection volume: 10μL, retention time: 11min, L-tyrosine peak time is about 3.5min. Draw the L-tyrosine standard curve with concentration (g / L) as abscissa and peak area as ordinate.

[0088] The results are as follows: Figure 2As shown, y = 1 × 10 7 x-428227, R 2 =0.9999(R) 2 (It is a linear fitting constant).

[0089] (2) Plotting the standard curve of phenol

[0090] Weigh 0.1 g of phenol standard sample and transfer it to a 50 mL volumetric flask. Dissolve and dilute with water. Take 0.5 mL, 1 mL, 2 mL, 4 mL, and 5 mL of the standard sample and transfer them to six 10 mL volumetric flasks. Dissolve and dilute with water to obtain standard samples with concentrations of 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.8 g / L, and 1 g / L, respectively. Detect these standard samples by HPLC. Chromatographic conditions: C18 column, mobile phase A: 0.1% formic acid water, mobile phase B: methanol, mobile phase A:mobile phase B (v:v) = 40:60, flow rate: 1 mL / L, column temperature: 30 ℃, wavelength: 271 nm, injection volume: 10 μL, retention time: 11 min, phenol peak elution time: approximately 8.2 min. Plot a phenol standard curve with concentration (g / L) on the x-axis and peak area on the y-axis.

[0091] The results are as follows Figure 3 As shown, y = 2 × 10 7 x-106369, R 2 =0.9999(R) 2 (It is a linear fitting constant).

[0092] (3) Plotting the standard curve of lactic acid

[0093] Weigh 0.1 g of lactic acid standard sample and transfer it to a 50 mL volumetric flask. Dissolve and dilute with water. Take 0.5 mL, 1 mL, 2 mL, 4 mL, and 5 mL of the standard sample and transfer them to six 10 mL volumetric flasks. Dissolve and dilute with water to obtain standard samples with concentrations of 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.8 g / L, and 1 g / L, respectively. Detect these standard samples by HPLC. Chromatographic conditions: H column, mobile phase A: 0.0257% sulfuric acid water, flow rate: 0.5 mL / L, column temperature: 50 ℃, wavelength: 210 nm, injection volume: 10 μL, retention time: 20 min, lactic acid peak elution time: approximately 16 min. Plot a lactic acid standard curve with concentration (g / L) on the x-axis and peak area on the y-axis.

[0094] The results are as follows Figure 4 As shown, y = 2 × 10 7 x-106369, R 2 =0.9999(R) 2 (It is a linear fitting constant).

[0095] (4) Determination of the residual amount of lactic acid, the residual amount of phenol and the yield of L-tyrosine in Examples 1-17 and Comparative Examples 1-3

[0096] 1 mL of the final fermentation product obtained in Examples 1-17 and Comparative Examples 1-3 was taken, 9 mL of dilute hydrochloric acid was added to dissolve the tyrosine precipitate, and then centrifugation was performed, 1 mL of the supernatant was taken, diluted with water to different multiples, and then HPLC detection analysis was performed to obtain the concentrations of L-tyrosine, phenol and lactic acid in the corresponding examples and comparative examples.

[0097] The yield of L-tyrosine, the residual amount of phenol and the residual amount of lactic acid were obtained according to the corresponding standard curve; the utilization rate of phenol = (the yield of L-tyrosine * the relative molecular mass of phenol) / (the added amount of phenol * the relative molecular mass of L-tyrosine).

[0098] The utilization rate of lactic acid = (the yield of L-tyrosine * the relative molecular mass of lactic acid) / (the added amount of lactic acid * the relative molecular mass of L-tyrosine).

[0099] The specific results are shown in the following table:

[0100]

[0101]

[0102] From the above results, it can be seen that the changes in fermentation conditions such as temperature, pH, raw material flow rate directly affect the utilization rate of raw materials, i.e. the lactic acid utilization rate and the phenol utilization rate in the determination results, and then indirectly affect the yield of the product L-tyrosine through the utilization rate of the raw materials; and the added amount of the raw materials is the index that can directly affect the yield of the product L-tyrosine.

[0103] The specific analysis is as follows:

[0104] In the comparison between Example 1 and Example 2, the added amount of the raw materials was changed under the same raw material ratio and other fermentation conditions, which resulted in the increase of the yield in Example 2 with more raw materials added. Meanwhile, in terms of raw materials, the added amount of the raw materials also needs to match the corresponding rate for flow addition. In the comparison between Example 1 and Example 2, the added amount of the raw materials was increased while the flow rate was still maintained, which reduced the utilization rate of the raw materials and increased the cost, and did not meet the industrial economic demand.

[0105] The index for the comparison between Comparative Example 1 and Example 3 is pH. When the pH is within the range of the present application, the utilization rate of the raw materials in the fermentation process is high, and the yield of the product is high; otherwise, if the pH of Comparative Example 1 is acidic (6.0), it will lead to incomplete fermentation, thereby reducing the utilization rate of the raw materials and the yield of the product.

[0106] The index for comparing example 4 and comparative example 2 is the raw material flow rate, and the flow rate of comparative example 2 is too fast, which results in incomplete fermentation process, and greatly reduces the raw material utilization and product yield.

[0107] The index for comparing comparative example 3 and example 5 is temperature, when the temperature is within the range of the present application, the raw material utilization of the fermentation process is higher, and the product yield is higher; otherwise, if the temperature of comparative example 3 is too low (25℃), it will result in incomplete fermentation, thereby reducing the raw material utilization and product yield.

[0108] The index for comparing example 6, example 7, example 8 and example 9 is the raw material ratio, when the lactic acid ratio is low, it reduces the raw material utilization and product yield to a certain extent, but overall, the influence on the fermentation is not as great as other indexes, so example 6 can be considered as an example with slightly poor effect. Similarly, through the four examples, it can be seen that when the lactic acid content in the raw material is excessive within the range of the present application, it can promote the fermentation, and the raw material utilization and product yield can achieve good results.

[0109] Examples 7-17 are all within the range of the present application to change the fermentation conditions to explore the fermentation effect, and from the results, the raw material utilization and product yield can achieve good results.

[0110] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing L-tyrosine, characterized in that, include: The induction culture medium of recombinant Escherichia coli, lactic acid, and phenol were mixed and fermented. The fermentation conditions include a temperature of 30-40℃, a pH of 8.0-9.0, a phenol flow rate of 5-12 g / (L*h), and a ratio of phenol to lactic acid of 1:0.9-1.

1. The construction process of the recombinant Escherichia coli genetically engineered bacteria includes: transforming pETDuet-1 and pCDFduet-1 into Escherichia coli, followed by plate screening for positive clones. pETduet-1 carries a lactate oxidase gene and a catalase gene. The lactate oxidase gene is the gene HN015_RS05890 from Pediococcus lactis; the NCBI-Gene ID of HN015_RS05890 is 57366060. The catalase gene is the gene ECs_1652 from Escherichia coli; the NCBI-Gene ID of ECs_1652 is 913226. pCDFduet-1 is loaded with a tyrosine phenol lyase gene; the tyrosine phenol lyase gene is derived from the gene BAACE33A1D18_RS03985 of *Clostridium perfringens*; the NCBI-Gene ID of BAACE33A1D18_RS03985 is 66567973.

2. The method for preparing L-tyrosine according to claim 1, characterized in that, The fermentation conditions include: the amount of phenol added to the mixture of lactic acid and phenol is 80-120 g / L; The temperature is 30-32℃; pH is 8.0-8.5; The flow rate of the phenol is 10⁻¹² / (L*h). The ratio of phenol to lactic acid added in the mixture is 1:0.95-1.

1.

3. The method for preparing L-tyrosine according to claim 1 or 2, characterized in that, The amount of phenol added to the mixture is 105-110 g / L.

4. The method for preparing L-tyrosine according to claim 1 or 2, characterized in that, The preparation process of the induction culture medium includes: inoculating the recombinant Escherichia coli genetically engineered bacteria into LBG medium for culture to form a recombinant Escherichia coli seed culture; The recombinant Escherichia coli seed culture was then inoculated into TB medium and cultured, followed by the addition of an inducer to induce further culture.

5. The method for preparing L-tyrosine according to claim 4, characterized in that, LBG medium consists of peptone, yeast extract, sodium chloride, glycerol, and water.

6. The method for preparing L-tyrosine according to claim 4, characterized in that, The culture conditions for forming the recombinant Escherichia coli seed culture include: 30-40℃ and pH 7.0-8.

0.

7. The method for preparing L-tyrosine according to claim 4, characterized in that, TB medium consists of peptone, yeast extract, glycerol, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and water. The pH of TB medium is 6.8-7.

2.

8. The method for preparing L-tyrosine according to claim 4, characterized in that, The conditions for culturing in TB medium include: 30-40℃ and pH 7.0-8.

0.

9. The method for preparing L-tyrosine according to claim 4, characterized in that, Induction culture includes: culturing to OD using TB medium. 600 After nm=10, cool to 25-28℃, add IPTG to a final concentration of 0.1-0.5mM for induction, and incubate for 10-14 hours. Then add 0.1%-0.3% Triton X-100 and 0.1-0.5g / L PLP, and continue culturing for 1-1.5 hours.

Images