A glutaminase mutant, immobilized glutaminase and its applications

Abstract

This invention belongs to the fields of genetic engineering and enzyme engineering technology, and discloses a glutaminase mutant, immobilized glutaminase, and their applications. The glutaminase mutant is based on the amino acid sequence shown in SEQ ID NO.1, with amino acid mutations at the following sites: E113K, R136M, and H223V, and its amino acid sequence is shown in SEQ ID NO.3. Immobilized glutaminase prepared using the glutaminase mutant exhibits 5-10 times increased enzyme activity compared to the wild type. The substrate concentration in the synthesis of L-theanine catalyzed by the immobilized glutaminase reaches 80-150 g / L, with a conversion rate greater than 99%. It can be recovered through simple filtration and reused more than 5 times, with the conversion rate remaining essentially the same during repeated use, demonstrating high potential for industrial application.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, specifically to a glutaminase mutant, immobilized glutaminase, and their applications. Background Technology

[0002] L-Theanine (as shown in Formula I), chemical name: N-ethyl-L-glutamine, International Union of Pure and Applied Chemistry (IUPAC) name: 2-amino-4-ethylcarbamoyl. Molecular formula: C7H14N2O3, molecular weight: 174.20.

[0003]

[0004] L-Theanine is a non-protein amino acid found in tea plants. It is synthesized in the roots of the tea plant and accumulates in the buds and leaves through transport. It has a variety of important physiological functions, such as calming and relaxing, improving sleep, fighting fatigue, and enhancing learning and memory. It has important application value in the fields of food, health products, and medicine.

[0005] Currently, the main methods for producing L-theanine include tea extraction, chemical synthesis, bio-fermentation, and bio-enzyme catalysis. Tea extraction is unsuitable for large-scale industrial production due to limited raw material sources, complex processes, low yield, and high costs. While chemical synthesis allows for large-scale production, it involves harsh reaction conditions, numerous side reactions, and severe environmental pollution. Bio-fermentation, although offering certain advantages, suffers from complex metabolic pathways, numerous influencing factors, and relatively low efficiency.

[0006] In recent years, bio-enzyme catalysis has attracted widespread attention due to its advantages such as high efficiency and environmental friendliness. However, existing bio-enzyme catalysis methods mostly use free enzymes, which cannot be recovered and reused during the catalytic process and are easily affected by factors such as temperature and pH, leading to a decrease in enzyme activity. In addition, the use of free enzymes may introduce impurities, increasing the complexity of post-processing. To overcome these problems, immobilized enzyme technology is considered an effective solution, but its application in the production of L-theanine has not yet been reported. Therefore, developing a highly efficient and stable immobilized enzyme catalysis technology is of great significance for realizing the industrial production of L-theanine. Summary of the Invention

[0007] To address the aforementioned problems in the existing technology, this application provides a glutaminase mutant, immobilized glutaminase, and its applications.

[0008] To address the above problems, the present invention provides the following technical solution:

[0009] In a first aspect, this application provides a glutaminase mutant based on the amino acid sequence shown in SEQ ID NO.1, with the following amino acid mutations: E113K, R136M, and H223V.

[0010] In one embodiment of this application, the amino acid sequence of the glutaminase mutant is shown in SEQ ID NO.3.

[0011] Secondly, this application provides a gene encoding a glutaminase mutant, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0012] In one embodiment of this application, the primers for the E113K mutation are as follows:

[0013] E113K-F: 5'-CCGACTCGGAATAGCCAAGGGCATGCAAGC-3';

[0014] E113K-R: 5'-CCTCGGCGTAAGCTTGCATGCCCTTGGCTATTC-3'.

[0015] In one embodiment of this application, the primers for the R136M mutation are as follows:

[0016] R136M-F: 5'-GGTGGCGCGGGCCCGATTGCGCATGCGCGC-3';

[0017] R136M-R: 5'-GCAGGTGCGTCGGCCGCGCATGCGCAATC-3'.

[0018] In one embodiment of this application, the primers for the H223V mutation are as follows:

[0019] H223V-F: 5'-ATGGGGATCGTATTCTCCCTGGGTGTTGCTTTG-3';

[0020] H223V-R: 5'-GATCTCCCGGGGCTACCAAAGCAACACCAGG-3'.

[0021] Thirdly, this application provides a recombinant plasmid containing a gene encoding a glutaminase mutant.

[0022] In one embodiment of this application, the recombinant plasmid is selected from the pET series vectors.

[0023] In one embodiment of this application, the pET series vector may be selected from the pET28a vector.

[0024] Fourthly, this application provides a recombinant genetically engineered bacterium containing a gene encoding a glutaminase mutant or containing the aforementioned recombinant plasmid.

[0025] In one embodiment of this application, the recombinant genetically engineered bacteria are selected from Escherichia coli.

[0026] Fifthly, this application provides a method for preparing immobilized glutaminase using a glutaminase mutant, comprising the following steps:

[0027] The crude enzyme solution of glutaminase mutant was mixed with chitosan microspheres, glutaraldehyde solution was added, and the mixture was shaken for cross-linking, filtered, washed with water, and immobilized glutaminase was obtained.

[0028] Chitosan microspheres are obtained by dissolving chitosan in acetic acid solution, injecting it into sodium hydroxide solution, stirring, and washing with water until neutral.

[0029] In one embodiment of this application, the ratio of chitosan to acetic acid solution is 1g:80-120mL.

[0030] In one embodiment of this application, the ratio of chitosan to acetic acid solution is 1g:100mL.

[0031] In one embodiment of this application, the concentration of the acetic acid solution is 0.5%-2%.

[0032] In one embodiment of this application, the ratio of chitosan to sodium hydroxide solution is 1g:80-120mL.

[0033] In one embodiment of this application, the ratio of chitosan to sodium hydroxide solution is 1g:100mL.

[0034] In one embodiment of this application, the concentration of sodium hydroxide is 0.5%-2%.

[0035] In one embodiment of this application, the volume ratio of acetic acid solution, sodium hydroxide solution, crude enzyme solution of carbonyl reductase mutant and glutaraldehyde solution is 1:1:1:0.1.

[0036] In one embodiment of this application, the concentration of glutaraldehyde is 4%-8%.

[0037] In one embodiment of this application, the temperature for oscillatory crosslinking is 20-30°C; the time for oscillatory crosslinking is 1-3 hours.

[0038] In one embodiment of this application, the stirring temperature is 20-30°C and the stirring time is 0.5-3 hours.

[0039] In one embodiment of this application, recombinant genetically engineered bacteria are induced and cultured using an inducer, and the resulting bacterial cells are broken to obtain a crude enzyme solution of glutamine mutant.

[0040] In one embodiment of this application, the inoculum amount of recombinant genetically engineered bacteria is 1%-10%.

[0041] In one embodiment of this application, the culture medium used is LB medium.

[0042] Fifthly, this application provides immobilized glutaminase prepared by the above method.

[0043] In one embodiment of this application, the enzyme activity of the immobilized aminoamidase is >200 U / g.

[0044] Sixthly, this application provides the application of immobilized glutaminase in the catalytic preparation of L-theanine, wherein the immobilized glutaminase acts on the substrates glutamine and ethylamine to carry out an enzymatic reaction to obtain L-theanine.

[0045] In one embodiment of this application, the mass ratio of ethylamine, glutamine, and immobilized glutaminase is 0.3-0.4:1:10.

[0046] In one embodiment of this application, the concentration of ethylamine is 25-50 g / L.

[0047] In one embodiment of this application, the concentration of ethylamine is 35 g / L.

[0048] In one embodiment of this application, the concentration of glutamine is 80-150 g / L.

[0049] In one embodiment of this application, the concentration of glutamine is 100 g / L.

[0050] In one embodiment of this application, the concentration of immobilized glutaminase is 1-6 g / L.

[0051] In one embodiment of this application, the concentration of immobilized glutaminase is 1.5-3 g / L.

[0052] In one embodiment of this application, the temperature of the enzymatic reaction is 20-40°C; the time of the enzymatic reaction is 1-24 hours.

[0053] In one embodiment of this application, the temperature of the enzymatic reaction is 30°C.

[0054] In one embodiment of this application, the substrate is dissolved in PBS buffer.

[0055] In one embodiment of this application, the concentration of the PBS buffer is 50-300 mmol / L.

[0056] In one embodiment of this application, the concentration of the PBS buffer is 100 mmol / L.

[0057] In one embodiment of this application, the pH value of the PBS buffer is 6.0-9.0.

[0058] In one embodiment of this application, the pH value of the PBS buffer is 7.0.

[0059] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0060] This invention provides a glutaminase and its mutant derived from Pseudomonas aeruginosa. The immobilized glutaminase exhibits 5-10 times higher enzyme activity than the wild type. The substrate concentration in the synthesis of L-theanine catalyzed by the immobilized glutaminase reaches 80-150 g / L, with a conversion rate greater than 99%. It can be recovered through simple filtration and reused more than 5 times after recovery. Moreover, the conversion rate during repeated use is basically the same as that during the first use, demonstrating high potential for industrial application. Detailed Implementation

[0061] The technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments.

[0062] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of the particular range. The range defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range.

[0063] Unless otherwise stated, when this invention relates to percentages between liquids, the percentage is volume / volume percentage; when this invention relates to percentages between liquids and solids, the percentage is volume / weight percentage; when this invention relates to percentages between solids and liquids, the percentage is weight / volume percentage; and the remainder is weight / weight percentage.

[0064] The present invention will be further described below with reference to specific embodiments. Molecular biology experimental methods not specifically described in the following embodiments can be performed according to the methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition) or conventional methods in the art, or according to the kit and product instructions.

[0065] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0066] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0067] The room temperature described in the following examples is the conventional room temperature in the art, ranging from 20 to 40°C; E. coli DH5a and E. coli BL21(DE3) competent cells were purchased from Beijing Tiangen Biotech Co., Ltd.

[0068] Example 1: Construction of glutaminase gene and its mutants

[0069] Based on the gene sequence (Gene ID: Q9I6V9.1) of *Pseudomonas aeruginosa* glutaminase from GenBank, the gene was synthesized by Anhui General Biotechnology Co., Ltd., with BamHI and XhoI restriction endonuclease sites added to both ends of the coding region. The target gene fragment was digested with BamHI and XhoI restriction endonucleases, then ligated into a pET28a(+) vector that had undergone the same double digestion. The resulting vector was transformed and screened to obtain positive plasmids. Thus, an in vitro heterologous expression system for this glutaminase was constructed and named pET28a-RN025. The amino acid sequence of its glutaminase RN025 is shown in SEQ ID NO.1.

[0070] The obtained full-length glutaminase RN025 gene sequence (SEQ ID NO.2) was subjected to site-directed base mutations. The mutation positions were: E at position 113 of the glutaminase gene coding sequence was mutated to K, R at position 136 was mutated to M, and H at position 223 was mutated to V. The resulting mutant gene sequence is shown in SEQ ID NO.4, which is the RN025 enzyme mutant, and its amino acid sequence is shown in SEQ ID NO.3.

[0071] Mutant primers were designed, and PCR amplification was performed using the positive plasmid as a template. Amplification was performed using Takara Primestar max high-fidelity polymerase (purchased from Takara). The PCR program was: 98℃ pre-denaturation for 3 min, 30 amplification cycles (98℃ 30 s, 55℃ 30 s, 72℃ 360 s), 72℃ 10 min. The template plasmid was removed by DpnI digestion, and the digestion product was transformed into E. coli DH5α strain. Positive clones were selected for sequencing verification. Clones with correctly sequenced mutations were then used for subsequent expression.

[0072] The mutation primers are shown below:

[0073] E113K-F: 5'-CCGACTCGGAATAGCCAAGGGCATGCAAGC-3';

[0074] E113K-R: 5'-CCTCGGCGTAAGCTTGCATGCCCTTGGCTATTC-3'.

[0075] R136M-F: 5'-GGTGGCGCGGGCCCGATTGCGCATGCGCGC-3';

[0076] R136M-R: 5'-GCAGGTGCGTCGGCCGCGCATGCGCAATC-3'.

[0077] H223V-F: 5'-ATGGGGATCGTATTCTCCCTGGGTGTTGCTTTG-3';

[0078] H223V-R: 5'-GATCTCCCGGGGCTACCAAAGCAACACCAGG-3'.

[0079] Example 2 Expression and preparation of glutaminase and its mutants

[0080] The plasmid obtained in Example 1 was transformed into Escherichia coli BL21(DE3) host bacteria and cultured overnight at 37°C with the culture inverted position. It was then inoculated into 5 mL of LB medium containing kanamycin (5 g yeast extract; 10 g peptone; 10 g NaCl, pH 7.0) and cultured overnight at 37°C with shaking. Finally, 1% (w / w) inoculation was added to a 500 mL Erlenmeyer flask containing 100 mL of LB medium and cultured at 37°C with shaking at 200 rpm. 600 When the expression level was 0.6-0.8, IPTG was added to a final concentration of 0.5 mmol / L as an inducer. After induction at 18°C ​​for 16 h, the cells in the induced LB medium were collected by centrifugation and washed three times with PBS at 4°C. The cell pellet was then dissolved in PBS buffer at a ratio of 1:9 and sonicated (30 min total, 3 s sonication, 7 s pause, 50% power) to obtain crude glutaminase solution. SDS-PAGE was used to identify the expression status.

[0081] Example 3: Preparation of chitosan microspheres immobilized with glutaminase

[0082] 2g of chitosan was dissolved in 200mL of 1% acetic acid solution, and injected into 200mL of 1% sodium hydroxide solution using a syringe. The mixture was stirred at 25°C for 1 hour, and then washed with water until neutral to obtain chitosan microspheres. Then, 200mL of crude glutaminase solution obtained in Example 2 was mixed with the chitosan microspheres, and 20mL of 6% glutaraldehyde solution was gradually added dropwise while stirring at 25°C. The mixture was then crosslinked by shaking at 180rpm for 2 hours at 25°C. After filtration, the mixture was washed three times with water to obtain immobilized glutaminase.

[0083] Example 4: Preparation of LXTE-705 amino resin immobilized glutaminase

[0084] 2g of LXTE-705 amino resin was weighed and dissolved in 200mL of 1% acetic acid solution. This solution was then injected into 200mL of 1% sodium hydroxide solution using a syringe. The mixture was stirred at 25°C for 1 hour, and washed with water until neutral to obtain activated LXTE-705 amino resin. Next, 200mL of crude glutaminase solution obtained in Example 2 was mixed with the activated LXTE-705 amino resin. While stirring at 25°C, 20mL of 6% glutaraldehyde solution was gradually added dropwise. The mixture was then crosslinked at 25°C and 180rpm for 2 hours. After filtration, the mixture was washed three times with water to obtain immobilized glutaminase.

[0085] Example 5: Determination of the activity of immobilized glutaminase

[0086] Weigh 100 mg of substrate glutamine and 35 mg of ethylamine and dissolve them in 40 mL of 100 mM pH 7.0 PBS buffer. Then add 1 mg of immobilized glutaminase, mix quickly, and immediately place in a UV spectrophotometer for absorbance measurement at 210 nm. After three minutes, calculate the absorbance difference ΔA. Calculate the enzyme activity (U / g) using the formula [ΔA × 3000 / (6.28 × 30)] × 200. The results are shown in Table 1.

[0087] Table 1 Results of enzyme activity assay

[0088] enzyme name Enzyme activity U / g Enzyme activity recovery rate RN025 crude enzyme solution 0.83 100.00％ RN025 chitosan microspheres immobilized enzyme 15.20 18.31％ RN025 amino resin immobilized enzyme 10.33 12.45％ RN025 mutant crude enzyme solution 10.25 100.00％ RN025 mutant chitosan microsphere immobilized enzyme 205.30 20.03％ RN025 mutant amino resin immobilized enzyme 158.42 15.46％

[0089] Example 6: Preparation of L-theanine by Immobilizing Glutaminase

[0090] The immobilized glutaminase obtained in Example 4 was used for substrate-catalyzed reactions. 50.0 g of glutamine substrate was dissolved in 40 mL of 100 mM pH 7.0 PBS buffer, followed by the addition of 17.5 g of ethylamine. After stirring and dissolving, 500.0 mg of the immobilized glutaminase mutant supernatant was added to bring the volume to 500 mL. The reaction mixture was placed in a 30°C water bath and magnetically stirred. After 1 hour of reaction, a sample was taken for HPLC analysis, showing a substrate conversion rate of 30%. The reaction was continued for another 24 hours, and HPLC analysis showed a substrate conversion rate of 99.78%.

[0091] Example 7

[0092] After reacting for 24 hours in Example 6, the immobilized enzyme was separated from the conversion solution by filtration. The filter cake was washed three times with 200 mL of water to obtain the recovered immobilized glutaminase. Using the recovered immobilized glutaminase, catalytic conversion was continued according to the conversion scheme of Example 6. That is, after each conversion, the immobilized enzyme obtained by filtration was reused for the next catalytic reaction. The enzyme activity was stable, as shown in Table 2, indicating that the immobilized glutaminase can be recycled multiple times and maintains good stability.

[0093] Table 2 Reusability of Immobilized Glutaminase

[0094] Conversion count Substrate conversion rate relative conversion rate 1 (First Time) 99.28％ 100.00％ 2 (One-time recycling) 99.10％ 99.82％ 3 (Secondary recycling) 99.18％ 99.89％ 4 (Three recycling cycles) 99.04％ 99.76％ 5 (Four recycling sessions) 99.15％ 99.87％ 6 (Five recyclings) 99.20％ 99.92％

[0095] The present application has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present application. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and implementation methods of the present application without departing from the spirit and scope of the present application, and all such modifications and improvements fall within the scope of the present application.

Claims

1. A glutaminase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.1, the following amino acid mutations were performed at the following sites: E113K, R136M, and H223V; the amino acid sequence of the glutaminase mutant is shown in SEQ ID NO.

3.

2. A gene encoding the glutaminase mutant of claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

4.

3. A recombinant plasmid, characterized in that, It contains the gene encoding the glutaminase mutant as described in claim 2.

4. A recombinant genetically engineered bacterium, characterized in that, It contains the recombinant plasmid as described in claim 3.

5. A method for preparing immobilized glutaminase using the glutaminase mutant according to claim 1, characterized in that, Includes the following steps: The crude enzyme solution of glutaminase mutant was mixed with chitosan microspheres, glutaraldehyde solution was added, and the mixture was shaken for cross-linking, filtered, washed with water, and immobilized glutaminase was obtained. The chitosan microspheres are obtained by dissolving chitosan in acetic acid solution, injecting it into sodium hydroxide solution, stirring, and washing with water until neutral.

6. The method for preparing immobilized glutaminase using a glutaminase mutant according to claim 5, characterized in that, The ratio of chitosan, acetic acid solution, and sodium hydroxide solution is 1g:80-120mL:80-120mL; the volume ratio of acetic acid solution, sodium hydroxide solution, crude carbonyl reductase mutant enzyme solution, and glutaraldehyde solution is 1:1:1:0.1; the temperature for oscillation crosslinking is 20-30℃; and the time for oscillation crosslinking is 1-3h.

7. The immobilized glutaminase prepared by the method according to any one of claims 5 to 6, characterized in that, The immobilized glutaminase has an enzyme activity >200 U / g.

8. The application of the immobilized glutaminase according to claim 7 in the catalytic preparation of L-theanine, characterized in that, L-theanine was prepared by immobilized glutaminase acting on the substrates glutamine and ethylamine to carry out an enzymatic reaction.

9. The application of the immobilized glutaminase according to claim 8 in the catalytic preparation of L-theanine, characterized in that, The mass ratio of ethylamine, glutamine, and immobilized glutaminase is 0.3-0.4:1:10; the temperature of the enzymatic reaction is 20-40℃; and the time of the enzymatic reaction is 1-24h.

10. The application of the immobilized glutaminase according to claim 8 in the catalytic preparation of L-theanine, characterized in that, The substrate is dissolved in PBS buffer; the concentration of the PBS buffer is 50-300 mmol / L; the pH of the PBS buffer is 6.0-9.0.

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