CN110372773B - Production method of high-purity glutamine dipeptide - Google Patents
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Abstract
The production method of high-purity glutamine dipeptide comprises the step of post-treating the reaction liquid of enzymatic glutamine dipeptide synthesis reaction, wherein the post-treatment comprises the step of removing impurity acid by inoculating candida tropicalis mutant strain thalli into the enzymatic glutamine dipeptide synthesis reaction liquid for culture; the preservation number of the candida tropicalis mutant strain is as follows: CGMCC No. 17928. In the whole process, seed crystals and crystal washing agents are not required to be added, and a recrystallization step is not required, so that the separation, purification and refining processes are effectively simplified, and the production cost is saved; the yeast can be repeatedly used for more than three batches, the waste yeast can be prepared into feed protein, no large amount of solid waste and waste water are generated, and the process is green and environment-friendly. The whole production process is easy to control, the production process is pollution-free, the product purity is high (more than 99 percent), the yield is high (more than 95 percent), and the method is suitable for large-scale industrial application.
Description
Background
Glutamine dipeptide is an important amino acid parenteral nutrition agent, and is mainly applied to injections, powder injections, water injections and tablets at present. The microbial enzyme method for synthesizing the glutamine dipeptide has the advantages of mild reaction conditions, less environmental pollution, high yield, low cost and the like, so that in recent years, a plurality of enterprises adopt the alpha-amino acid ester acyltransferase to prepare the glutamine dipeptide raw material.
The substrates adopted by the alpha-amino acid ester acyltransferase synthesis method are L-alanine ester derivatives and glutamine, wherein the L-alanine ester derivatives are very easily degraded into L-alanine (L-Ala) in a reaction system, and the product yield and purity are influenced because the physicochemical properties of the L-alanine ester derivatives are close to those of the product. In order to obtain high-purity glutamine dipeptide, ion exchange method or recrystallization method is usually adopted to refine the glutamine dipeptide crude product in the separation and extraction process. CN103623777A adopts active adsorbent to adsorb and play the role of decolorizing, removing impurities and removing endotoxin, the active adsorbent comprises 30-45% of white carbon black, 50-65% of black carbon black and 0.5-3% of ion exchange resin, the solid waste and the waste water are more, and the subsequent treatment cost is high. CN104163849A adopts continuous chromatographic separation equipment to carry out continuous chromatographic separation on impurities and inorganic salts, the cost is high, and the cost of high-purity glutamine dipeptide is high and is one of the reasons for limiting the application range of products. CN104480075A adopts methanol to refine glutamine dipeptide, the product yield is about 85%, the process is relatively environment-friendly, but the addition of methanol also brings solvent residue risk.
In view of the current state of the art, the product separation and purification technology is still further needed in the preparation process of high-purity glutamine dipeptide.
Disclosure of Invention
The invention aims to provide a method for improving the purity of glutamine dipeptide products by specifically eliminating free L-alanine (L-Ala) and L-glutamine (L-Gln) in a production system. Based on Candida tropicalis (CICC number 1253) as an original strain, a mutant strain IBEC-16 is obtained by mutagenesis and screening, and the mutant strain can specifically remove free amino acids L-Ala and L-Gln in a reaction system and does not use dipeptide when the L-Ala and the L-Gln exist. The Candida tropicalis mutant strain IBEC-16 is preserved in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms in Beijing, China, and has a preservation number of CGMCC No.17928 and a preservation date of 2019, 06 and 16 days.
Based on the candida tropicalis mutant strain, the invention aims to provide a production method of high-purity glutamine dipeptide, which comprises the step of post-treating reaction liquid of enzymatic glutamine dipeptide synthesis reaction, wherein the post-treatment comprises the step of removing impurity acid by inoculating candida tropicalis mutant strain thalli into the enzymatic glutamine dipeptide synthesis reaction liquid for culture; the preservation number of the candida tropicalis mutant strain is as follows: CGMCC No. 17928.
The production method of high-purity glutamine dipeptide provided by the invention adopts the characteristic that candida tropicalis mutant strains specifically eliminate free amino acids L-Ala and L-Gln, yeast is inoculated into glutamine dipeptide reaction liquid for reaction, and the filtrate after filtration and sterilization is subjected to membrane filtration to remove salt, concentration and crystallization and drying to obtain glutamine dipeptide products. In the whole process, seed crystals and crystal washing agents are not needed, and a recrystallization step is not needed, so that the separation, purification and refining processes are effectively simplified, and the production cost is saved; the yeast can be repeatedly used for more than three batches, the waste yeast can be prepared into feed protein, no large amount of solid waste and waste water are generated, and the process is green and environment-friendly. The whole production process is easy to control, the production process is pollution-free, the product purity is high (more than 99 percent), the yield is high (more than 95 percent), and the method is suitable for large-scale industrial application.
Drawings
The invention is shown in figure 3:
FIG. 1 is a graph showing the results of L-Ala, L-Gln and glutamine dipeptide consumption by Candida tropicalis mutant strain IBEC-16.
FIG. 2 is a graph showing the L-Ala, L-Gln and glutamine dipeptide utilization by Candida tropicalis mutant strain IBEC-16.
FIG. 3 shows the results of experiments on differences in L-alanine and glutamine utilization by Candida tropicalis CICC NO.1253 and its mutant strain IBEC-16.
Detailed Description
The invention relates to a production method of high-purity glutamine dipeptide, which is suitable for treating a reaction system after synthesizing glutamine dipeptide by enzyme catalysis (particularly microbial enzyme), and comprises the step of post-treating reaction liquid of the synthesis reaction of the enzyme-catalyzed glutamine dipeptide, wherein the post-treatment comprises the step of removing impurity acid by inoculating candida tropicalis mutant strain thalli into the reaction liquid of the synthesis reaction of the enzyme-catalyzed glutamine dipeptide for culture; the preservation number of the candida tropicalis mutant strain is as follows: CGMCC No. 17928.
In the reaction solution of the enzymatic glutamine dipeptide synthesis reaction (also referred to herein as the reaction solution), there is a residue of a hetero acid including L-alanine (L-Ala) and/or L-glutamine (L-Gln). The embodiment of the scheme is to remove the two kinds of mixed acid.
In a more specific embodiment, the step of removing the heteropolyacid is: adjusting the pH value of an enzymatic glutamine dipeptide synthesis reaction solution to 6.0, and inoculating candida tropicalis mutant strain thalli to enable the initial OD to be 8-12; then culturing at 30 +/-2 ℃ and 300-500rpm, wherein the Dissolved Oxygen (DO) is controlled to be 30-50% and the pH value is 5.5-6.0 in the culturing process; after the completion of the culture, the culture was centrifuged to collect the filtrate and the cells. In the reaction process, sampling every hour to measure the content of amino acid and glutamine dipeptide until the reaction is complete, centrifuging, respectively collecting filtrate and thalli, and recycling the thalli. Wherein the reaction is completed when the L-alanine content in the system is consumed to be less than 0.01g/L (concentration). The content detection of the amino acid and the glutamine dipeptide refers to high performance liquid chromatography of appendix V D of the second part of Chinese pharmacopoeia 2010 edition.
The candida tropicalis mutant strain thallus in any technical scheme is prepared by the following method: inoculating a ring of activated Candida tropicalis mutant strain (IBEC-16) into a conical flask filled with 50ml of YPD medium, and culturing in a shaking table at 30 ℃ and 220rpm for about 16 h; then 1ml of the culture solution is inoculated into another conical flask containing 50ml of YPD medium, and the conical flask is placed in a shaking table at 30 ℃ and 220rpm for about 20 hours, and the thalli are collected by centrifugation.
YPD culture medium is used for activated culture of candida tropicalis mutant strain (IBEC-16) preserved on slant, and the specific mode is as follows: inoculating 100 μ L of Gannan tube bacterial liquid into YPD culture medium, culturing in shaking table at 30 deg.C and 220rpm for about 16h, streaking, and inoculating single clone into slant for storage.
As YPD medium, the following were mentioned in the present invention unless otherwise specified: 20g/L glucose, 10g/L yeast extract, 20g/L peptone and 20min at 121 ℃.
In the application of the Candida tropicalis mutant strain, the strain can be repeatedly used. The method for reusing the thalli is the same as the method for reusing the thalli for the first time, and the same thalli can be reused for 3 times.
In a further specific embodiment, after the reaction for removing the heteropolyacid, the method further comprises the steps of carrying out nanofiltration membrane desalination and evaporative crystallization on the filtrate obtained after the step for removing the heteropolyacid. Specifically, desalting with nanofiltration membrane with molecular weight cutoff of 400Da, distilling under reduced pressure until a large amount of solid is separated out, cooling, filtering, and drying the solid at 50 deg.C under reduced pressure to obtain pure glutamine dipeptide.
More specifically, the method comprises the following steps of treating the glutamine dipeptide reaction solution:
(1) removing heteropolyacid
Adjusting the pH of a reaction solution of enzymatic glutamine dipeptide synthesis reaction to 6.0, inoculating candida tropicalis mutant strain thalli, and enabling the initial OD to be 10; then culturing at the temperature of 30 +/-2 ℃, wherein the Dissolved Oxygen (DO) is controlled to be 30-50% and the pH value is 5.5-6.0 in the culturing process; after the culture is finished, centrifuging and respectively collecting filtrate and thalli;
(2) desalting and crystallizing
Desalting the filtrate obtained in the step (1) by a nanofiltration membrane with the molecular weight cutoff of 400Da, distilling under reduced pressure until a large amount of solids are separated out, cooling, filtering, and drying the solids under reduced pressure at 50 ℃ to obtain the pure glutamine dipeptide product.
On the other hand, the present invention is not particularly limited to the glutamine dipeptide reaction solution, and it can be selected and determined by those skilled in the art. The reaction solution of glutamine dipeptide may be obtained by a method according to the related art described in the prior art, or may be a reaction step coupled to the treatment step of the present invention, and non-limiting examples thereof include:
enzymatic synthesis of glutamine dipeptide: dissolving L-alanine methyl ester hydrochloride and L-glutamine in phosphate buffer solution, adding glutamine dipeptide synthetase, carrying out enzymatic reaction at 25-30 ℃ and under the condition of pH value of 8.0-9.0, stopping the reaction after 20 minutes, and collecting reaction liquid; wherein: the concentration of the reaction substrate L-alanine methyl ester hydrochloride is 200-300 mM; the concentration of the reaction substrate L-glutamine was 200-300 mM; the concentration of the glutamine dipeptide synthetase is 0.1-1.0% (v/v); phosphate buffer 0.2M, pH8.7; pH control during the enzymatic reaction was performed using 6M sodium hydroxide solution.
The reaction solution obtained after the reaction can be directly used for the step of removing the hydrochloric acid post-treatment.
The present invention will be further described with reference to the accompanying drawings and examples. These non-limiting examples should not be construed as limiting the present disclosure in any way.
Example 1 Yeast obtaining and culturing
Candida tropicalis (CICC number 1253) is used as an initial strain, a mutant strain IBEC-32 is obtained by ultraviolet mutagenesis screening, the yeast mutant strain can specifically remove L-Ala and L-Gln, and glutamine dipeptide is not used when L-Ala and L-Gln exist.
The media used consisted of the following:
slant culture medium: 20g/L glucose, 10g/L yeast extract, 20g/L peptone and 15-20g/L agar, and sterilizing at 121 ℃ for 20 min.
YPD medium: 20g/L glucose, 10g/L yeast extract and 20g/L peptone, and sterilizing at 121 ℃ for 20 min.
Shake flask culture medium: glutamine dipeptide 20g/L, L-alanine 20g/L, glutamine 10g/L, KH2PO4·3H2O2.5g/L，MgSO4·7H2O 1.0g/L，KCl 0.5g/L，pH6.0。
1. The breeding process of the mutant strain IBEC-16 comprises the following steps:
(1) preparation of mutant strain IBEC-16 bacterial suspension
A ring of activated Candida tropicalis (CICC No. 1253) was inoculated into a conical flask containing 50ml YPD medium and cultured in a shaker at 30 ℃ and 220rpm until logarithmic phase. Centrifuging the culture solution in a sterile centrifuge tube at 8000rpm for 10min, discarding supernatant, washing with physiological saline, resuspending the cells with physiological saline, shaking for 10min, and adjusting cell concentration to 1 × 108And the strain per ml is a suspension of mutant strain IBEC-16.
(2) Ultraviolet mutagenesis and acclimatization
And (2) adding 5mL of the bacterial suspension obtained in the step (1) into a sterile plate, placing the sterile plate on a magnetic stirrer for stirring, irradiating 30-180 s at a position of 30cm under 20W ultraviolet light, sampling, diluting and coating the plate, selecting a single bacterial colony which grows fast and is large in bacterial colony size, inoculating the single bacterial colony to a slant culture medium, and performing slant storage.
Inoculating the strain on a ring inclined plane into a conical flask filled with 50ml of YPD culture medium, placing the conical flask in a shaking table at 30 ℃ and 220rpm for culturing until the strain reaches logarithmic growth phase, then inoculating the strain into an acclimatization culture medium, and selecting the strain which can tolerate the highest concentration of L-alanine for shake flask re-screening.
(3) Shake flask rescreen
And (3) taking the strain preserved on the inclined plane in the step (2), inoculating the strain into a conical flask filled with 50ml of YPD culture medium, culturing for 20h in a shaking table at 30 ℃ and 220rpm, centrifugally collecting thalli, inoculating the thalli into a shaking culture medium, culturing for 24h at 30 ℃ and 220rpm, sampling every hour during the culture process, controlling the pH to be about 6.0, and analyzing the content of various amino acids in the reaction solution by liquid chromatography. Among all the strains tested, the strain consuming the most L-alanine for 24 hours was the target mutant strain IBEC-16.
(4) Detection of genetic stability of mutant strain IBEC-16
Continuously carrying out slant subculture on the Candida tropicalis IBEC-16 obtained in the step (3) for 10 times, respectively inoculating each generation into a conical flask surrounded by a YPD culture medium, culturing in a shaking table at 30 ℃ and 220rpm until logarithmic phase, then inoculating into a shaking culture medium, carrying out shaking table fermentation at 30 ℃ and 220rpm for 24 hours, sampling every hour in the culture process, controlling the pH to be about 6.0, and determining the content of amino acid in a reaction solution. Meanwhile, Candida IBEC-16 of passage 1 was used as a control.
Example 2 obtaining high purity glutamine dipeptide Using mutant strain IBEC-16
The glutamine dipeptide reaction solution used in this example was obtained by the following method: the glutamine dipeptide reaction solution was prepared according to the method described in the introduction of L-alkyl-L-glutamine by recycling E.coli expression a-amino acid ester acyl transferase, Bioresource technology, 2017.
Taking a ring-activated mutant strain IBEC-16, inoculating the strain into a conical flask filled with YPD medium, culturing for 20h in a shaking table at the temperature of 30 ℃ and the rpm of 220, and centrifuging to collect thalli. Adjusting the pH of the reaction solution (containing 41.9g/L glutamine dipeptide, 4.3g/L alanine and 7.8g/L glutamine) to 6.0, inoculating thallus (OD is about 10 at the beginning of the reaction), culturing at 30 ℃ and 300-500rpm, controlling the DO at 30-50% and the pH at 5.5-6.0, sampling every hour to measure the content of amino acid and glutamine dipeptide, ending the sampling for about 8-10h, and centrifuging to collect the filtrate. And (3) carrying out nanofiltration on the filtrate to remove salt, carrying out reduced pressure distillation until a large amount of solid is separated out, cooling and filtering, and drying the solid at 50 ℃ under reduced pressure to obtain 40g of a pure glutamine dipeptide product, wherein the yield is 95% and the purity is 99%.
Example 3 degradation test of the mutant strain IBEC-16 on different amino acids
The one-cycle activated mutant strain IBEC-16 was inoculated into a conical flask containing 50ml of YPD medium and cultured in a shaker at 30 ℃ and 220rpm for 20 hours. Centrifuging to collect thallus, inoculating into shake flask culture medium (glutamine dipeptide 20g/L, L-alanine 20g/L, glutamine 10g/L, KH)2PO4·3H2O 2.5g/L，MgSO4·7H2O1.0 g/L, KCl 0.5g/L), culturing at 30 deg.C and 220rpm for 24 hr, sampling and controlling pH to 5.5-6.0 every hour during the culture, and analyzing the contents of L-alanine, glutamine and glutamine dipeptide in the reaction solution by liquid chromatography. The results are shown in FIG. 1. As can be seen from FIG. 1, glutamine is completely utilized within about 6h, L-alanine is completely degraded within about 20h, and the content of glutamine dipeptide is basically unchanged.
Example 3 application test of mutant Strain IBEC-16 in fermenter
The glutamine dipeptide reaction solution used in this example was obtained by the following method: a reaction solution of glutamine dipeptide was prepared by the method described in "Production of L-alkyl-L-glutamine by immunological catalysis GS115expressing a-amino acid ester acyl transferase", microbiological Cell industries, 2019.
Inoculating the one-ring activated mutant strain IBEC-16 into a conical flask filled with YPD medium, and culturing in a shaker at 30 deg.C and 220rpm for 20 h; inoculating into 50L fermentation tank containing YPD medium at an inoculation amount of 10%, culturing at 30 deg.C and 150-400rpm with initial aeration of 0.7VVM and DO of 20% and pH of 5.0 for about 6-8 h. 300L of the reaction solution (containing glutamine dipeptide 60g/L, alanine 12.5g/L, glutamine 4.5g/L) was adjusted to pH6.0, cells were inoculated (OD at the start of the reaction was about 10), initial aeration was 0.7VVM, the cells were cultured at 30 ℃ and 350rpm at 150-. As shown in FIG. 2, glutamine and L-alanine were completely utilized in about 4 hours and 12 hours, respectively. And after the reaction is finished, centrifuging and collecting filtrate, desalting the filtrate by nanofiltration, distilling under reduced pressure until a large amount of solid is separated out, cooling and filtering, and drying the solid at 50 ℃ under reduced pressure to obtain 58.2g of the pure glutamine dipeptide product with the yield of 97 percent and the purity of 99 percent.
Example 4 bacterial cell reuse test of mutant strain IBEC-16
The glutamine dipeptide reaction solution used in this example was obtained by the following method: the glutamine dipeptide reaction solution was prepared according to the method described in the introduction of L-alkyl-L-glutamine by recycling E.coli expression a-amino acid ester acyl transferase, Bioresource technology, 2017.
Inoculating the one-ring activated mutant strain IBEC-32 into a conical flask filled with YPD medium, culturing at 30 deg.C and 220rpm shaking table for 20h, and centrifuging to collect thallus. Adjusting pH of the reaction solution to 6.0, inoculating thallus (OD at initial reaction is about 10), culturing at 30 deg.C and 500rpm under 300-. And (3) carrying out nanofiltration on the filtrate to remove salt, carrying out reduced pressure distillation until a large amount of solid is separated out, cooling and filtering, and drying the solid at 50 ℃ under reduced pressure to obtain the pure glutamine dipeptide product. The recovered yeast cells were reused by referring to the above steps, and the results of removing the heteropolyacid by recycling the yeast are shown in Table 1 below.
TABLE 1
Example 5 differential test of L-alanine and Glutamine utilization by Candida tropicalis CICC No.1253 and mutant IBEC-16
One-ring activated Candida tropicalis CICC NO.1253 and mutant IBEC-16 were inoculated into 100ml YPD medium conical flask, and cultured in shaking table at 30 deg.C and 220rpm for 20 h. Centrifugally collecting bacteriaThen the reaction solution (glutamine dipeptide 20g/L, L-alanine 20g/L, glutamine 10g/L, KH) was inoculated2PO4·3H2O2.5g/L，MgSO4·7H2O1.0 g/L, KCl 0.5g/L), the initial OD of the reaction620The reaction mixture was incubated at 30 ℃ and 220rpm for 24 hours at about 10 ℃ with pH controlled at about 5.5 to 6.0, and the reaction mixture was sampled every 8 hours and analyzed for the amino acid content by liquid chromatography. The results are shown in FIG. 3.
As seen from the results in FIG. 3, the two strains did not utilize glutamine dipeptide (Ala-Gln) substantially, but utilized L-alanine (L-Ala) and glutamine (Gln) to different degrees. The two strains firstly utilize glutamine, and the utilization rate of the mutant strain IBEC-16 is slightly higher than that of Candida tropicalis CICC NO. 1253; the starting strain CICC NO.1253 can not completely utilize L-alanine after being cultured for 24 hours, while the mutant strain IBEC-16 can completely utilize about 20g/L, and the utilization efficiency of L-alanine is improved by 100 percent.
Claims (7)
1. The production method of high-purity glutamine dipeptide includes the step of post-treatment of reaction liquid of enzymatic glutamine dipeptide synthesis reaction, and is characterized in that the post-treatment includes the step of removing impurity acid by inoculating candida tropicalis mutant strain thalli into the enzymatic glutamine dipeptide synthesis reaction liquid for culture;
the preservation number of the Candida tropicalis mutant strain is as follows: CGMCC No. 17928.
2. The method of claim 1, wherein said heteroacid comprises L-alanine and/or L-glutamine.
3. The method of claim 2, wherein the step of removing the heteropolyacid is: adjusting the pH value of an enzymatic glutamine dipeptide synthesis reaction solution to 6.0, and inoculating candida tropicalis mutant strain thalli to enable the initial OD = 8-12; then culturing at 30 +/-2 ℃ and 300-500rpm, wherein the dissolved oxygen content is controlled to be 30-50% and the pH value is 5.5-6.0 in the culturing process; after the completion of the culture, the culture was centrifuged to collect the filtrate and the cells.
4. The method according to claim 3, wherein the bacterial cells are reused.
5. The method as claimed in claim 3, wherein the post-treatment further comprises the steps of nanofiltration membrane desalination and evaporative crystallization of the filtrate obtained after the deacidification step.
6. The method of claim 2, comprising the steps of:
(1) removing heteropolyacid
Adjusting the pH of a reaction solution of enzymatic glutamine dipeptide synthesis reaction to 6.0, and inoculating candida tropicalis mutant strain thalli to ensure that the initial OD = 10; then culturing at the temperature of 30 +/-2 ℃, wherein the dissolved oxygen is controlled to be 30-50% and the pH value is 5.5-6.0 in the culturing process; after the culture is finished, centrifuging and respectively collecting filtrate and thalli;
(2) desalting and crystallizing
Desalting the filtrate obtained in the step (1) by a nanofiltration membrane with the molecular weight cutoff of 400Da, distilling under reduced pressure until a large amount of solids are separated out, cooling, filtering, and drying the solids under reduced pressure at 50 ℃ to obtain the pure glutamine dipeptide product.
7. The method according to claim 1, wherein the Candida tropicalis mutant strain is prepared by: inoculating a ring of activated Candida tropicalis mutant strain thallus into a conical flask filled with 50ml of YPD culture medium, and culturing for 16h in a shaking table at 30 ℃ and 220 rpm; then 1ml of the culture solution is taken and inoculated into another conical flask containing 50ml of YPD medium, the conical flask is placed in a shaking table at the temperature of 30 ℃ and the rpm of 220 for culturing for 20 hours, and the thalli are collected by centrifugation.
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