CN111607585A - Immobilization method of recombinant escherichia coli and application of recombinant escherichia coli in synthesis of glutamine dipeptide - Google Patents
Immobilization method of recombinant escherichia coli and application of recombinant escherichia coli in synthesis of glutamine dipeptide Download PDFInfo
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Abstract
The invention discloses an immobilization method of overexpression trans-esterase recombinant escherichia coli and application thereof in glutamine dipeptide biocatalytic synthesis. By adopting the immobilization time sequence, the invention discloses a using method of immobilized cells, and experimental results show that the reaction can be continuously carried out for 600h, and the yield is kept not to be reduced; the production is continuously carried out for more than 10 batches, and the concentration of the glutamine dipeptide in the collected reaction liquid is between 70 and 76 mM. The immobilized cells have high activity and can be recycled, continuous reaction can be realized, and the conversion rate is basically maintained unchanged. The method disclosed by the invention is simple to operate, high in product conversion rate, easy to separate, high in production efficiency, low in cost, easy to realize large-scale production and good in industrial application prospect.
Description
Technical Field
The invention relates to the technical field of an immobilized production method of recombinant bacteria, in particular to an immobilized method of overexpressed transesterification enzyme recombinant escherichia coli and application of the immobilized method in the biocatalytic synthesis of glutamine dipeptide.
Background
Glutamine dipeptide (L-Ala-Gln), also known as N (2) -L-alanyl-L-glutamine, has the advantages of high solubility, and strong water solubility and thermal stability, and has gradually replaced glutamine (L-Gln) to become a main parenteral nutrition medicament. At present, the synthesis of glutamine dipeptide mainly adopts chemical synthesis method, combination of chemical synthesis and biological enzyme catalysis method, and microbe enzyme production synthesis method. The chemical synthesis method for producing the glutamine dipeptide has the advantages of multiple reaction steps, multiple byproducts, high reagent toxicity and non-environmental friendliness (Tangguo, N (2) -L-alanyl-L-glutamine dipeptide synthesis and reaction research, Xiamen university, 2004; Chinese patent, alanine-glutamine dipeptide synthesis method, CN 1392156A). The chemical and biological enzyme catalysis method has the defects of large enzyme consumption, low peptide productivity and the like. (in freshening, research on enzymatic synthesis of glutamine dipeptide. university of Tianjin, 2010). The microbial enzyme-producing synthesis method is to screen bacterial strain to obtain transesterifying enzyme for catalyzing alanyl-glutamine synthesis, and to apply the separated and purified enzyme or directly apply the microbial bacterial strain to catalyze alanine and glutamine to synthesize Ala-Gln dipeptide. (Kenzo Yokozeki. A novel and effective enzyme catalysis for the production of peptides from untreated starting materials. journal of Biotechnology 115 (2005). 211-220). The disadvantages of this method are that it is time-consuming and labor-intensive to select suitable strains, the amount of enzyme expression is insufficient, which causes a problem of long enzyme catalysis time, and the process of enzyme purification is complicated. Researchers have also obtained an amino acid ligase (Lal) from Bacillus subtilis to develop a patented technology for producing dipeptides by fermentation, but this catalytic synthesis process of glutamine dipeptide requires exogenous supply of ATP, which results in still high dipeptide production cost and cannot be widely popularized in the market (Tabata K, Hashimoto S. fermentation production of L-alkyl-L-glutamine by a metabolic enzyme engineered Escherichia coli strain expressing L-amino acid α -ligand. applied and environmental microbiology, 2007, 73(20): 6378. Chinese patent No. 2017100517990). CN104480075A and CN105274174A disclose two methods for the catalytic synthesis of glutamine dipeptide by using transesterification, which adopt enzyme freeze-dried powder or liquid enzyme buffer solution for catalytic reaction, but because the cost of enzyme separation and purification is high, and the enzyme is difficult to recycle after the reaction, the method is not suitable for practical industrial production.
Chinese patent CN 106754985B discloses a gene coding glutamine dipeptide biosynthetic enzyme and application thereof, and provides recombinant escherichia coli containing the gene and a method for synthesizing glutamine dipeptide by biotransformation. The gene and the application of the recombinant engineering bacteria thereof have the advantages of high molar conversion rate, high reaction rate, easy separation, low cost and the like, and lay a foundation for the industrial production of glutamine dipeptide. But also has the defects that the cells can be reused after being centrifugally collected, the operation steps are more, the intermittent period of production operation is needed, and the like.
Disclosure of Invention
The invention designs a method for immobilizing cells in a logarithmic growth phase and a novel process for inducing immobilized cells, namely, the cells are immobilized when growing to the middle and later logarithmic phases, the immobilized cells can be subjected to catalytic synthesis reaction after induction, the immobilized cells can be recycled, and the reaction effect is better than that of the immobilized cells after induction.
In one aspect, the invention provides an immobilization method of overexpression esterase-transferring recombinant escherichia coli, which comprises the following steps:
(1) culturing recombinant Escherichia coli with overexpression of trans-esterase until cell density is OD6000.4～1.0；
(2) Embedding the recombinant Escherichia coli obtained in the step (1) by using an immobilization material, and then performing induction culture.
In the above technical solution, preferably, the immobilization material is selected from soybean oil, agar powder, soybean oil, agarose, CaCl2At least one of sodium alginate and carrageenan.
In the technical scheme, preferably, the immobilization material is embedded by adding 1-2% of the immobilization material into the thalli, uniformly mixing, and then dropwise adding the mixture into a cross-linking agent for embedding, wherein the cross-linking agent is selected from soybean oil, 2-3% of KCl and 2-3% of CaCl2。
In the above technical solution, preferably, the conditions of the induction culture are: using isopropyl thiogalactoside (IPTG) with the final concentration of 0.2-2.0 mM for induction culture; a more preferred final concentration of IPTG is 0.3 mM.
In the above technical solution, preferably, the conditions of the induction culture are: culturing at low temperature for at least 12h, and preferably at 16-20 ℃; more preferably, the induction culture time is 16 hours.
In the above technical solution, preferably, the recombinant escherichia coli overexpressing the transesterase is a recombinant escherichia coli containing glutamine dipeptide biosynthetic enzyme. The cells in the step (1) are selected from yeast, escherichia coli or bacillus subtilis; the recombinant Escherichia coli containing glutamine dipeptide biosynthetic enzyme is E.coli BL21-pET29 a-AET. In fact, the recombinant bacteria in the present application are not limited to the recombinant escherichia coli e.coli BL21-pET29a-AET disclosed in the examples, and other recombinant bacteria or cells with inducible characteristics are all adapted to the technical scheme of "embedding and fixing first and then inducing and culturing" described above in the present application, and can also achieve better technical effects.
In the above technical solution, preferably, the culture medium used for cell culture in step (1) is a microbial culture medium containing a carbon source and a nitrogen source; the preferred medium is LB medium supplemented with antibiotics; wherein the antibiotic is kanamycin, the concentration of the kanamycin in the culture medium is 30-150 mu g/mL, and the more preferable concentration of the antibiotic is 50 mu g/mL.
On the other hand, the invention protects the application of the immobilization method in the biological catalytic synthesis of glutamine dipeptide.
In the above-mentioned applications, it is preferable that the substrate used in the biological catalytic synthesis of glutamine dipeptide is a mixture of L-alanine methyl ester hydrochloride (L-Ala-OMe) and L-glutamine (L-Gln); the molar ratio of L-alanine methyl ester hydrochloride (L-Ala-OMe) and L-glutamine (L-Gln) in the mixture was 2: 1-1: 3.
in the above applications, preferably, the concentration of L-alanine methyl ester hydrochloride in the buffer solution is in the range of 0.1-1.0M, preferably 0.1-0.6M; the concentration range of L-glutamine in the buffer solution is 0.1-1.0M, preferably 0.2-0.8M.
In the above-mentioned application, preferably, the buffer solution of the substrate is a borate buffer solution; more preferably, the concentration of the buffer solution is 0.1-1.0 mM, and the pH value is 7.5-9.5; the most preferred buffer solution is borate buffer at pH 9 at a concentration of 0.2 mM.
In the above applications, preferably, the embedded cells after collection and culture are subjected to batch biosynthesis or continuous biosynthesis.
In the above application, preferably, the batch biosynthesis method is: (3-1) adding a substrate into a buffer solution to prepare a reaction solution containing the substrate, adding the immobilized cells obtained by the method in the steps (1) and (2), adjusting the pH value to 8.0-9.0, reacting the system at 20-30 ℃ until the pH value is not reduced any more, and removing the reaction solution; adding new reaction substrate, repeating the step (3-1), and obtaining the final product from the collected reaction solution.
In the above applications, preferably, the continuous biosynthesis method is: (3-2) loading the immobilized cells obtained in the steps (1) and (2) into a hollow column, adding the substrate into a buffer solution to prepare a reaction solution containing the substrate, and adding the reaction solution containing the substrate into the column containing the immobilized cells at a certain flow rate to carry out continuous production of glutamine dipeptide.
In the above application, preferably, the hollow column is selected from any aspect ratio of (12-16): the organic glass column, the PVC column, the metal column and the like of 1 can be filled in the column.
In the application, the flow rate is preferably 1.5-3 BV/h, and the flow rate is preferably 2 BV/h.
Has the advantages that:
1. immobilization timing of recombinant E.coli containing glutamine dipeptide biosynthetic enzyme. The cells are embedded by using an immobilized material, and then the immobilized cells are induced and expressed, so that the production efficiency of the glutamine dipeptide is improved more favorably than the traditional immobilization time sequence of firstly inducing and then embedding. According to the experimental results of the examples, it is shown that with the immobilization sequence according to the invention, the concentration of the reaction product, glutamine dipeptide, is increased by 20% compared to the traditional immobilization sequence of induction followed by entrapment, and in the technical field of glutamine dipeptide production, generally speaking, the conversion rate is only 60%, from which it can be seen that the invention achieves unexpected technical effects.
2. On the basis of determining the optimized immobilization time sequence, the invention also optimizes the immobilization material and the use concentration, and finally determines that the immobilization material is preferably sodium alginate, and the concentration is 1-2%, CaCl2The concentration of the glutamine dipeptide is 2-3%, and the high production efficiency of the glutamine dipeptide is obtained.
3. The invention discloses a using method of immobilized cells, namely the immobilized cells can be filled in a column to be made into a plug flow reactor. The substrate enters from the bottom, the product flows out from the top, and the reaction can continue. The experimental results according to the examples show that the reaction can be continuously carried out for 600h, and the yield is kept from decreasing. Continuously performing the reaction for more than 10 batches, wherein the concentration of the glutamine dipeptide in the collected reaction liquid is between 70 and 76 mM. The immobilized cells can keep high activity at the temperature of 20-35 ℃ and under the environment of pH 8.0-9.0, can be recycled, can realize continuous reaction, and basically maintains the conversion rate. The immobilized cell is immobilized when the cell grows to the middle and later logarithmic phases, the immobilized cell can perform dipeptide synthesis reaction after induction, the reaction effect and the immobilized cell after induction are more excellent, and the production efficiency of the glutamine dipeptide reaches 2.79mg/(min mL-CV).
4. The method disclosed by the invention is simple to operate, high in product conversion rate, easy to separate, high in production efficiency, low in cost, easy to realize large-scale production and good in industrial application prospect.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments.
The LB culture medium used in the specific embodiment of the invention has the following components in proportion: 5g/L of yeast extract powder, 10g/L of tryptone and 10g/L of NaCl, sterilizing for 15min at 121 ℃, cooling, and adding kanamycin with the final concentration of 10-100 mu g/mL; the LB medium in the specific examples was: 0.5% yeast powder, 1% peptone, 1% NaCl, 50. mu.g/mL kanamycin.
In the specific embodiment of the invention, the buffer solution is: 0.1 to 1.0mM borate buffer solution with pH of 7.5 to 9.5; the buffer in the specific example is borate buffer at a concentration of 0.2mM, pH 9.
The recombinant E.coli containing glutamine biosynthetic enzyme used in the present embodiment is E.coli BL21-pET29a-AET, and the information on this species is disclosed in the following documents, Li Yimin, Yuan Wenjie, GaoJoqi et al.production of l-alkyl-l-glutamine by recycling E.coli x-expression α -amino acid ester acyl transfer enzyme [ J ]. Bioresource. Technol. 2017,245:1603 1609.
Example 1
Recombinant Escherichia coli containing glutamine dipeptide biosynthetic enzyme is cultured in LB culture medium at 37 deg.C and 200rpm until cell density OD600At 0.5 deg.C, centrifuge at 6000rpm for 5 min. The cells were mixed with 10mL of 1.5% agarose, and the mixture was added dropwise to soybean oil for embedding. The immobilized cells were placed in a fresh LB medium, added with 0.5mM IPTG to the final concentration, and cultured overnight at 18 ℃ to induce the expression of glutamine dipeptide biosynthetic enzyme. The immobilized cells after overnight incubation induction were removed, washed with sterile water, added to a buffer solution containing 100mM L-alanine methyl ester hydrochloride (L-Ala-OMe) and 200mM L-glutamine (L-Gln), the temperature was controlled at 20 to 30 ℃ and the pH was maintained at 8.0 to 8.5 with 6M NaOH solution, and the reaction was completed in 20 min. The concentration of glutamine dipeptide was 65 mM.
Comparative example 1
Recombinant Escherichia coli containing glutamine dipeptide biosynthetic enzyme is cultured in LB culture medium at 37 deg.C and 200rpm until cell density OD600At 0.5, 0.5mM IPTG, 1 was addedThe enzyme was expressed by induction at 8 ℃ overnight. Centrifuge at 6000rpm for 5min at 4 ℃. The cells were mixed with 10mL of 1.5% agarose, and the mixture was added dropwise to soybean oil for embedding. After the immobilization, the cells were washed with sterile water, added to a buffer solution containing 100mM L-alanine methyl ester hydrochloride (L-Ala-OMe) and 200mM L-glutamine (L-Gln), the temperature was controlled at 20 to 30 ℃, and the pH was maintained at 8.0 to 8.5 with 6M NaOH solution, and the reaction was completed for 20 min. The concentration of glutamine dipeptide was 55 mM.
Example 2
Recombinant Escherichia coli containing glutamine dipeptide biosynthetic enzyme is cultured in LB culture medium at 37 deg.C and 200rpm until cell density OD600At 0.5 deg.C, centrifuge at 6000rpm for 5 min. The cells were mixed with 10mL of 1.5% agar powder, and the mixture was added dropwise to soybean oil for embedding. The immobilized cells were placed in a fresh LB medium, 0.5mM IPTG was added thereto, and cultured overnight at 18 ℃ to induce the expression of glutamine dipeptide biosynthetic enzyme. Taking out the immobilized cells after overnight culture induction, washing with sterile water, adding a solution containing 100mM L-alanine methyl ester hydrochloride (L-Ala-OMe) and 200mM L-glutamine (L-Gln) and dissolving in a buffer solution, controlling the temperature to be 20-30 ℃, maintaining the pH to be 8.0-8.5 by using a 6M NaOH solution, and finishing the reaction for 20 min. The concentration of glutamine dipeptide was 64 mM.
Comparative example 2
Recombinant Escherichia coli containing glutamine dipeptide biosynthetic enzyme is cultured in LB culture medium at 37 deg.C and 200rpm until cell density OD600At 0.5, 0.5mM IPTG was added and the cells were cultured overnight at 18 ℃ to induce expression of the enzyme. Centrifuge at 6000rpm for 5min at 4 ℃. The cells were mixed with 10mL of 1.5% agar powder, and the mixture was added dropwise to soybean oil for embedding. After the immobilization, the cells were washed with sterile water, added to a buffer solution containing 100mM L-alanine methyl ester hydrochloride (L-Ala-OMe) and 200mM L-glutamine (L-Gln), the temperature was controlled at 20 to 30 ℃, and the pH was maintained at 8.0 to 8.5 with 6M NaOH solution, and the reaction was completed for 20 min. The concentration of glutamine dipeptide was 52 mM.
Example 3
Recombinant Escherichia coli containing glutamine dipeptide biosynthetic enzyme is cultured in LB culture medium at 37 deg.C and 200rpm until cell density OD600At 0.5 deg.C, centrifuge at 6000rpm for 5 min. The thallus is added with 10mL of 1.5 percent carrageenan, mixed evenly and dripped into 2.0 percent KCl solution for embedding. The immobilized cells were placed in a fresh LB medium, 0.5mM IPTG was added thereto, and cultured overnight at 18 ℃ to induce the expression of glutamine dipeptide biosynthetic enzyme. The immobilized cells after overnight incubation induction were removed, washed with sterile water, added to a buffer solution containing 100mM L-alanine methyl ester hydrochloride (L-Ala-OMe) and 200mM L-glutamine (L-Gln), the temperature was controlled at 20 to 30 ℃ and the pH was maintained at 8.0 to 8.5 with 6M NaOH solution, and the reaction was completed in 20 min. The concentration of glutamine dipeptide was 61 mM.
Comparative example 3
Recombinant Escherichia coli containing glutamine dipeptide biosynthetic enzyme is cultured in LB culture medium at 37 deg.C and 200rpm until cell density OD600At 0.5, 0.5mM IPTG was added and the cells were cultured overnight at 18 ℃ to induce expression of the enzyme. Centrifuge at 6000rpm for 5min at 4 ℃. The thallus is added with 10mL of 1.5 percent carrageenan, mixed evenly and dripped into 2.0 percent KCl solution for embedding. After the immobilization, the cells were washed with sterile water, added to a buffer solution containing 100mM L-alanine methyl ester hydrochloride (L-Ala-OMe) and 200mM L-glutamine (L-Gln), the temperature was controlled at 20 to 30 ℃, and the pH was maintained at 8.0 to 8.5 with 6M NaOH solution, and the reaction was completed for 20 min. The concentration of glutamine dipeptide was 50 mM.
Example 4
Recombinant Escherichia coli containing glutamine dipeptide biosynthetic enzyme is cultured in LB culture medium at 37 deg.C and 200rpm until cell density OD600At 0.5 deg.C, centrifuge at 6000rpm for 5 min. The thalli is added with 10mL of 1.5 percent sodium alginate and mixed evenly, and is dripped into 2.5 percent CaCl2Carrying out embedding. The immobilized cells were placed in a fresh LB medium, IPTG was added to the medium to a final concentration of 0.5mM, and cultured overnight at 18 ℃ to induce the expression of glutamine dipeptide biosynthetic enzyme. And taking out the immobilized cells after overnight culture induction, washing the cells with sterile water, adding a reaction solution containing 100mM L-alanine methyl ester hydrochloride (L-Ala-OMe) and 200mM L-glutamine (L-Gln) into a borate buffer solution, controlling the temperature to be 20-30 ℃, maintaining the pH to be 8.0-8.5 by using a 6M NaOH solution, and finishing the reaction after 20 min. Glutamine dipeptideHas a concentration of 76 mM.
Comparative example 4
Recombinant Escherichia coli containing glutamine dipeptide biosynthetic enzyme is cultured in LB culture medium at 37 deg.C and 200rpm until cell density OD600At 0.5, 0.5mM IPTG was added and the cells were cultured overnight at 18 ℃ to induce expression of the enzyme. Centrifuge at 6000rpm for 5min at 4 ℃. The thalli is added with 10mL of 1.5 percent sodium alginate and mixed evenly, and is dripped into 2.5 percent CaCl2Carrying out embedding. After the immobilization, the cells were washed with sterile water, added to a buffer solution containing 100mM L-alanine methyl ester hydrochloride (L-Ala-OMe) and 200mM L-glutamine (L-Gln), the temperature was controlled at 20 to 30 ℃, and the pH was maintained at 8.0 to 8.5 with 6M NaOH solution, and the reaction was completed for 20 min. The concentration of glutamine dipeptide was 62 mM.
Example 5
An organic glass column with the height-diameter ratio of 15 is taken as a filling column, and the sodium alginate-CaCl obtained by the method of the embodiment 4 is used2The embedded immobilized cells are packed into a packed column. A buffer solution containing 100mM L-alanine methyl ester hydrochloride (L-Ala-OMe) and 200mM L-glutamine (L-Gln) was passed through a packed column at 20 to 30 ℃ at a flow rate of 2BV/h, and the concentration of glutamine dipeptide exiting the packed column was 72 mM. Can be continuously used for 600h without reducing the yield.
Example 6
The immobilized cells obtained in example 4 were washed with sterile water, added to a buffer solution containing 100mM L-alanine methyl ester hydrochloride (L-Ala-OMe) and 200mM L-glutamine (L-Gln), the temperature was controlled to 20 to 30 ℃, the pH was maintained at 8.0 to 8.5 with 6M NaOH solution, and the reaction was completed in 20 min. Removing the reaction solution, collecting the immobilized cells, repeating the reaction steps for more than 10 times continuously, wherein the concentration of the glutamine dipeptide in the collected reaction solution is 70-76 mM.
Comparative example 5
Recombinant Escherichia coli containing glutamine dipeptide biosynthetic enzyme is cultured in LB culture medium at 37 deg.C and 200rpm until cell density OD600At 0.5, 0.5mM IPTG was added and the cells were cultured overnight at 18 ℃ to induce expression of the enzyme. Centrifuge at 6000rpm for 5min at 4 ℃. Adding into a solution containing 100mM L-alanineThe reaction was completed in 10min at a temperature of 25 ℃ in a buffer solution of methyl ester hydrochloride (L-Ala-OMe) and 200mM L-glutamine (L-Gln) with a concentration of 81mM by maintaining pH at 8.5 with 6M NaOH solution.
Example 7
Recombinant Escherichia coli containing glutamine dipeptide biosynthetic enzyme is cultured in LB culture medium at 37 deg.C and 200rpm until cell density OD600At 0.5 deg.C, centrifuge at 6000rpm for 5 min. The thalli is added with 10mL of 1.5 percent sodium alginate and mixed evenly, and is dripped into 2.5 percent CaCl2Carrying out embedding. The immobilized cells were placed in a fresh LB medium, IPTG was added to the medium to a final concentration of 0.5mM, and cultured overnight at 18 ℃ to induce the expression of glutamine dipeptide biosynthetic enzyme. Taking out the immobilized cells after overnight culture induction, washing with sterile water, adding reaction solution containing 600mM L-alanine methyl ester hydrochloride (L-Ala-OMe) and 800mM L-glutamine (L-Gln) to dissolve in buffer solution, controlling the temperature to be 20-30 ℃, maintaining the pH to be 8.0-8.5 by 6M NaOH solution, and finishing the reaction for 20 min. The concentration of glutamine dipeptide was 413 mM.
Comparative example 7
Recombinant Escherichia coli containing glutamine dipeptide biosynthetic enzyme is cultured in LB culture medium at 37 deg.C and 200rpm until cell density OD600At 0.5, 0.5mM IPTG was added and the cells were cultured overnight at 18 ℃ to induce expression of the enzyme. Centrifuge at 6000rpm for 5min at 4 ℃. The thalli is added with 10mL of 1.5 percent sodium alginate and mixed evenly, and is dripped into 2.5 percent CaCl2Carrying out embedding. The immobilized cells were washed with sterile water, added to a buffer solution containing 600mM L-alanine methyl ester hydrochloride (L-Ala-OMe) and 800mM L-glutamine (L-Gln), controlled at 20 to 30 ℃ and maintained at a pH of 8.0 to 8.5 with 6M NaOH solution, and the reaction was completed for 30 min. The concentration of glutamine dipeptide was 341 mM.
From the data analysis of the above examples and comparative examples, it can be seen that: the optimum reaction temperature and the optimum pH of the immobilized cells are wider than those of free cells, can keep high activity at the temperature of 20-35 ℃ and under the environment of pH 8.0-9.0, can be recycled, can realize continuous reaction, and basically maintains the conversion rate unchanged. The immobilized cell is immobilized when the cell grows to the middle and later logarithmic phases, the immobilized cell can perform dipeptide synthesis reaction after induction, the reaction effect and the immobilized cell after induction are more excellent, and the production efficiency of the glutamine dipeptide reaches 2.79mg/(min mL-CV).
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. An immobilization method of recombinant escherichia coli overexpressing transesterase, which is characterized by comprising the following steps:
(1) culturing recombinant Escherichia coli with overexpression of trans-esterase until cell density is OD6000.4～1.0；
(2) Embedding the recombinant Escherichia coli obtained in the step (1) by using an immobilization material, and then performing induction culture.
2. The method according to claim 1, wherein the immobilizing material is at least one selected from agar powder, agarose, sodium alginate, and carrageenan.
3. The method according to claim 1, wherein the immobilization material is embedded by adding 1-2% of the immobilization material into the bacteria, mixing the immobilization material uniformly, and then dropping the mixture into a cross-linking agent for embedding, wherein the cross-linking agent is selected from soybean oil, 2-3% of KCl and 2-3% of CaCl2。
4. The method of claim 1, wherein the recombinant escherichia coli overexpressing the transesterase is recombinant escherichia coli BL21-pET29 a-AET.
5. Use of the immobilization method of claim 1 for the biocatalytic synthesis of glutamine dipeptide.
6. The use according to claim 5, wherein the substrate used in the biocatalytic synthesis of glutamine dipeptide is a mixture of L-alanine methyl ester hydrochloride and L-glutamine, and the molar ratio of L-alanine methyl ester hydrochloride to L-glutamine in the mixture is 2: 1-1: 3.
7. the use according to claim 6, wherein the concentration of L-alanine methyl ester hydrochloride in the buffer is 0.1-1.0M; the concentration of L-glutamine in the buffer solution is 0.1-1.0M.
8. Use according to claim 5, wherein the embedded cells are biosynthesized in batch or continuous mode.
9. The use of claim 8, wherein the batch biosynthesis process comprises: (3-1) adding a substrate into a buffer solution to prepare a reaction solution containing the substrate, adding the immobilized cells obtained by the method in the steps (1) and (2), adjusting the pH to 8.0-9.0, and reacting the system at 20-30 ℃ until the pH value is not reduced any more to serve as a reaction end point; and (4) removing the reaction solution, adding new reaction substrates, and repeating the step (3-1) to obtain the final product from the collected reaction solution.
10. The use of claim 8, wherein the continuous biosynthesis process comprises: (3-2) filling the immobilized cells obtained by the method in the steps (1) and (2) into a hollow column, adding the substrate into a buffer solution to prepare a reaction solution containing the substrate, adding the reaction solution containing the substrate into the column containing the immobilized cells at a certain flow rate, and carrying out continuous production of the glutamine dipeptide, wherein the hollow column is selected from an organic glass column, a PVC column or a metal column.
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| CN113817790A (en) * | 2021-10-18 | 2021-12-21 | 精晶药业股份有限公司 | Method for preparing alanyl glutamine by immobilized enzyme |
| CN113862249A (en) * | 2021-10-20 | 2021-12-31 | 徐州工程学院 | Preparation and application of recombinant beta-1, 4-endoglucanase immobilized cell |
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Cited By (3)
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| CN113862249A (en) * | 2021-10-20 | 2021-12-31 | 徐州工程学院 | Preparation and application of recombinant beta-1, 4-endoglucanase immobilized cell |
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