Glutaminase mutant, immobilized glutaminase and application of immobilized glutaminase

Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, and discloses a glutaminase mutant, immobilized glutaminase and application thereof. The glutaminase mutant is based on an amino acid sequence as shown in SEQ ID NO.1 and is subjected to amino acid mutation at the following sites: E113K, R136M and H223V, and the amino acid sequence of the glutaminase mutant is as shown in SEQ ID NO.3. The immobilized glutaminase is prepared by using the glutaminase mutant, the enzyme activity is increased by 5-10 times compared with that of a wild type, the substrate concentration in the L-theanine catalyzed and synthesized by the immobilized glutaminase reaches 80-150g / L, the conversion rate is greater than 99%, the L-theanine can be recycled through simple filtration, and the L-theanine can be repeatedly used for more than five times after being recycled. And the conversion rate during repeated use is basically the same as that during primary use, so that the method has relatively high industrial application potential.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering and enzyme engineering, and in particular to a glutaminase mutant, immobilized glutaminase and applications thereof. Background Art

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

[0003]

[0004] L-theanine is a non-protein amino acid found in tea trees. It is synthesized in the roots of tea trees and accumulated in buds and leaves through transportation. It has many important physiological functions, such as calming and relaxing, improving sleep, resisting fatigue, and improving learning and memory abilities. It has important application value in the fields of food, health products, and medicine.

[0005] At present, the production methods of L-theanine mainly include tea extraction, chemical synthesis, biological fermentation and bio-enzyme catalysis. Tea extraction is not suitable for large-scale industrial production due to limited raw material sources, complex procedures, low yield and high cost. Although chemical synthesis can be produced on a large scale, its reaction conditions are harsh, there are many side reactions and serious environmental pollution. Although biological fermentation has certain advantages, its metabolic route is complex, there are many influencing factors, and the efficiency is relatively low.

[0006] In recent years, bio-enzyme catalysis has attracted wide attention due to its advantages such as high efficiency and environmental protection. However, the existing bio-enzyme catalysis methods mostly use free enzymes, which cannot be recycled during the catalytic process and are easily affected by factors such as temperature and pH, resulting in decreased enzyme activity. In addition, the use of free enzymes may also introduce impurities, increasing the complexity of post-processing. In order to overcome these problems, immobilized enzyme technology is considered to be an effective solution, but there has been no relevant application report in the production of L-theanine. Therefore, the development of an efficient and stable immobilized enzyme catalysis technology is of great significance for realizing the industrial production of L-theanine. Summary of the invention

[0007] In view of the above problems existing in the prior art, the present application provides a glutaminase mutant, immobilized glutaminase and applications thereof.

[0008] To solve the above problems, the present invention provides the following technical solutions:

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

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

[0011] In a second aspect, the present 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 the present 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 the present 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 the present application, the primers for the H223V mutation are as follows:

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

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

[0021] In a third aspect, the present application provides a recombinant plasmid comprising a gene encoding a glutaminase mutant.

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

[0023] In one embodiment of the present application, the pET series vectors can be selected from the pET28a vector.

[0024] In a fourth aspect, the present application provides a recombinant genetically engineered bacterium comprising a gene encoding a glutaminase mutant or comprising the above-mentioned recombinant plasmid.

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

[0026] In a fifth aspect, the present application provides a method for preparing immobilized glutaminase using a glutaminase mutant, comprising the following steps:

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

[0028] The chitosan microspheres are obtained by dissolving chitosan in an acetic acid solution, injecting the solution into a sodium hydroxide solution, stirring the solution, and washing the solution with water until the solution is neutral.

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

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

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

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

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

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

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

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

[0037] In one embodiment of the present application, the temperature of the oscillating cross-linking is 20-30° C.; and the time of the oscillating cross-linking is 1-3 h.

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

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

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

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

[0042] In a fifth aspect, the present application provides immobilized glutaminase prepared by the above method.

[0043] In one embodiment of the present application, the enzyme activity of the immobilized aminoacidase is greater than 200 U / g.

[0044] In a sixth aspect, the present application provides the use of immobilized glutaminase in the catalytic preparation of L-theanine, wherein the immobilized glutaminase acts on substrates glutamine and ethylamine to carry out an enzymatic reaction to produce L-theanine.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] The invention provides a glutaminase derived from Pseudomonas aeruginosa and a mutant thereof. The enzyme activity of the immobilized glutaminase is increased by 5-10 times compared with that of the wild type. The substrate concentration of L-theanine catalyzed by the immobilized glutaminase reaches 80-150 g / L, and the conversion rate is greater than 99%. The immobilized glutaminase can be recovered by simple filtration and can be repeatedly used for more than 5 times after recovery. The conversion rate during repeated use is substantially the same as that of the initial use, and the immobilized glutaminase has high industrial application potential. DETAILED DESCRIPTION

[0061] The following is a clear and complete description of the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments.

[0062] The "range" disclosed in this application is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range.

[0063] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0064] The present invention is further described below in conjunction with specific examples. Molecular biology experimental methods not specifically described in the following examples can be performed with reference to the methods listed in the book Molecular Cloning Laboratory Manual (3rd Edition) by J. Sambrook or conventional methods in the art, or according to the kits and product instructions.

[0065] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0066] Unless otherwise specified, the test 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, and the room temperature range is 20-40° C.; E. coli DH5a and E. coli BL21 (DE3) competent cells were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

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

[0069] According to the gene sequence of Pseudomonas aeruginosa glutaminase (Gene ID: Q9I6V9.1) included in Genbank, it was synthesized by Anhui General Biological Co., Ltd., and BamHI and XhoI restriction endonuclease sites were added at both ends of the coding region. After the target gene fragment was digested by restriction endonucleases BamHI and XhoI, it was connected, transformed and screened with the pET28a (+) vector that had been digested by the same double enzymes, and the positive plasmid was screened. Thus, the in vitro heterologous expression system of the glutaminase was constructed, named pET28a-RN025. The amino acid sequence of its glutaminase RN025 is shown in SEQ ID NO.1.

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

[0071] Design mutation primers, use positive plasmid as template for PCR amplification, use Takara Primestar max high-fidelity polymerase (purchased from Takara), and the PCR program is: 98℃ pre-denaturation for 3min, 30 amplification cycles (98℃30s, 55℃30s, 72℃360s), 72℃10min. DpnI enzyme digestion removes the template plasmid, and the enzyme digestion product is transformed into E.coli DH5a strain. The positive clones are selected for sequencing verification, and the clones with correct mutations are expressed later.

[0072] The mutation primers are as follows:

[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 transferred into the host bacterium Escherichia coli BL21 (DE3), inverted and cultured at 37°C overnight, inoculated into 5 mL of LB medium (5 g yeast powder; 10 g peptone; 10 g NaCl, pH 7.0) containing kanamycin, and cultured at 37°C overnight with shaking. The inoculum was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of LB medium at a rate of 1% (w / w), and cultured on a shaking table at 37°C and 200 rpm. When the OD of the culture solution reached 600 =0.6-0.8, add IPTG with a final concentration of 0.5mmo1 / L as an inducer, and after induction at 18°C ​​for 16h, collect the cells induced by expression in the LB medium by centrifugation, and wash with PBS for 3 times at 4°C; then the cell precipitate is placed in a ratio of cell precipitate: PBS buffer = 1:9, and is broken by ultrasound (working time 30min, supersonic 3s, stop 7s, power 50%) to obtain crude glutaminase enzyme solution, and the expression is identified by SDS-PAGE.

[0081] Example 3 Preparation of chitosan microspheres immobilized glutaminase

[0082] Weigh 2g of chitosan and dissolve it in 200mL of 1% acetic acid solution, inject it into 200mL of 1% sodium hydroxide solution with a syringe, stir at 25°C for 1h, wash with water until neutral, and obtain chitosan microspheres. Then mix 200mL of the crude glutaminase solution obtained in Example 2 with the chitosan microspheres, stir at 25°C, and gradually add 20mL of 6% glutaraldehyde solution. Oscillate and crosslink at 25°C, 180rpm for 2h, filter with suction, and wash with water three times to obtain immobilized glutaminase.

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

[0084] Weigh 2g of LXTE-705 amino resin and dissolve it in 200mL of 1% acetic acid solution, inject it into 200mL of 1% sodium hydroxide solution with a syringe, stir at 25°C for 1h, wash with water until neutral, and obtain activated LXTE-705 amino resin. Then mix 200mL of crude glutaminase obtained in Example 2 with activated LXTE-705 amino resin, stir at 25°C, and gradually add 20mL of 6% glutaraldehyde solution. Oscillate and crosslink at 25°C, 180rpm for 2h, filter with suction, and wash with water three times to obtain immobilized glutaminase.

[0085] Example 5 Determination of enzyme activity of immobilized glutaminase

[0086] 100 mg of substrate glutamine and 35 mg of ethylamine were weighed and dissolved in 40 mL of 100 mM pH 7.0 PBS buffer, and then 1 mg of immobilized glutaminase was added. After rapid mixing, the mixture was immediately placed in a UV spectrophotometer for absorbance detection at a wavelength of 210 nm. After three minutes, the absorbance difference ΔA was calculated, and the enzyme activity was calculated according to the formula, in units of U / g, and the calculation formula was [ΔA×3000 / (6.28×30)]×200. The results are shown in Table 1.

[0087] Table 1 Enzyme activity determination results

[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％ Immobilization of enzyme on RN025 mutant chitosan microspheres 205.30 20.03％ RN025 mutant amino resin immobilized enzyme 158.42 15.46％

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

[0090] The immobilized glutaminase obtained in Example 4 was used for substrate catalysis reaction. 50.0g substrate glutamine was dissolved in 40mL 100mM pH7.0 PBS buffer, followed by adding 17.5g ethylamine, and after stirring and dissolving, 500.0mg immobilized glutaminase mutant supernatant was added to make up the volume to 500mL. The reaction solution was placed in a 30°C constant temperature water bath and stirred magnetically. Samples were taken after 1 hour of reaction, and HPLC detection was performed, and the substrate conversion rate reached 30%; the reaction was continued for 24 hours, and samples were taken for HPLC detection, and the substrate conversion was 99.78%.

[0091] Example 7

[0092] After Example 6 was reacted for 24 hours, the immobilized enzyme was separated from the conversion solution by suction filtration, and the filter cake was washed 3 times with 200 mL of clean water to obtain the recovered immobilized glutaminase. The recovered immobilized glutaminase was used to continue the catalytic conversion according to the conversion scheme of Example 6, that is, after each conversion was completed, the immobilized enzyme was obtained by suction filtration and continued to be used for the next catalytic reaction. The enzyme activity was stable as shown in Table 2, indicating that the immobilized glutaminase can be recycled for many times and maintains good stability.

[0093] Table 2 Reusability of immobilized glutaminase

[0094] Conversions 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 times recycling) 99.04％ 99.76％ 5 (four times recycling) 99.15％ 99.87％ 6 (five times recycling) 99.20％ 99.92％

[0095] The present application is described in detail above in conjunction with specific implementation methods and exemplary embodiments, but these descriptions cannot be understood as limiting the present application. Those skilled in the art understand that, without departing from the spirit and scope of the present application, the technical solution of the present application and its implementation methods may be subjected to a variety of equivalent substitutions, modifications or improvements, all of which 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 amino acid mutations at the following sites were performed: 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 according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

4.

3. A recombinant plasmid, characterized in that: It comprises the gene encoding the glutaminase mutant according to claim 2.

4. A recombinant genetically engineered bacterium, characterized in that: It comprises the recombinant plasmid according to claim 3.

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

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

7. The immobilized glutaminase prepared by the method according to any one of claims 5 to 6, characterized in that: The enzyme activity of the immobilized aminoacidase is greater than 200 U / g.

8. Use of the immobilized glutaminase according to claim 7 in catalytic preparation of L-theanine, characterized in that: Immobilized glutaminase is used to act on substrates glutamine and ethylamine to carry out enzymatic reaction to obtain L-theanine.

9. The use of the immobilized glutaminase according to claim 8 in 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° C.; and the time of the enzymatic reaction is 1-24 hours.

10. The use of the immobilized glutaminase according to claim 8 in 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; and the pH value of the PBS buffer is 6.0-9.0.

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